Periodontology 2000

Environmental and other
modifying factors of the

periodontal diseases
DENI S F. KI NANE, MELANI E PETERSON & PANAGI OTA G. STATHOPOULOU
The aim of this review is to address how environ-
mental factors modify periodontal disease. Our cur-
rent understanding is that the environment and
genetics govern whether an individual develops per-
iodontitis. Innate genetic differences are revealed
following plaque accumulation, resulting in inam-
mation that may manifest and remain as gingivitis or
go on to form periodontitis in certain subjects. This
chronic inammatory condition is modied by
smoking, hormones, diabetes, drugs and in rare cases
by systemic diseases with periodontal manifesta-
tions, i.e. by environmental factors. Thus the envi-
ronment inuences healthy and diseased periodontal
tissues and may modify preexisting gingival inam-
mation and result in more or less severe gingivitis
responses or altered periodontitis.
Chronic gingivitis is seen commonly in individuals
who stop toothbrushing for between 10 and 20 days
(43). The clinical signs are exaggerated and the gin-
giva is more edematous and inamed in individuals
undergoing hormonal disturbance, such as children
during puberty and females during pregnancy. Cer-
tain drug therapies such as nifedipine (a calcium
channel blocker used in hypertensive patients),
phenytoin (used to control epilepsy) and cyclospo-
rine (an immunosuppressive drug) can result in gin-
gival overgrowth in approximately 30%of individuals
taking these medications. The gingival overgrowth is
an exaggerated response to microbial plaque. Gingi-
vitis is also affected by smoking. Smoking tends to
reduce gingival inammation, possibly by the effect
of nicotine in causing vascular constriction and thus
reduced tissue edema and reduced gingival crevicular
uid ow.
Periodontitis, in contrast to gingivitis, is seen in only
a subset of the population (1015%). It is variable in
that it does not affect all teeth evenly, but has both a
subject and site predilection. Recent epidemiologic
studies of periodontal disease suggest that relatively
few subjects in each age group suffer from advanced
periodontal destructionand only specic sites in these
individuals are affected (65, 89, 106). When consider-
ing changes in attachment level over time, it is also
peculiar that only relatively few sites actually undergo
extensive periodontal destruction during any given
observationperiod. Studies (65, 84, 85) have foundthat
70%of the sites deterioratedby 3 mmor more duringa
2-year monitoring period, but this occurred in only
12%of the subjects examined.
Periodontal disease is considered to have multiple
risk factors. The term risk factor refers to an aspect
of personal behavior or lifestyle, an environmental
exposure, or an inherited characteristic, which on the
basis of epidemiologic evidence is known to be
associated with a health related condition (80). Risk
factors are part of the causal chain for a particular
disease or can lead to the exposure of the host to a
disease (15). The presence of a risk factor implies a
direct increase in the probability of a disease occur-
ring. Although specic microorganisms have been
considered as potential periodontal pathogens, it has
become apparent that pathogens are necessary but
not sufcient for disease activity to occur (128).
Destructive periodontal disease is a consequence of
the interaction of genetic, environmental, host and
microbial factors (154). The presence of micro-
organisms is a crucial factor in inammatory perio-
dontal disease, but the progression of the disease is
related to host based risk factors such as genetics,
age, gender, smoking, socioeconomic factors, and
certain systemic diseases. Examples of microbes
implicated as risk factors in periodontitis are
numerous. Carlos et al. (25) found that the presence
of Prevotella intermedia, along with gingival bleeding
107
Periodontology 2000, Vol. 40, 2006, 107119
Printed in the UK. All rights reserved
2006 The Authors.
Journal compilation 2006 Blackwell Munksgaard
PERIODONTOLOGY 2000
and calculus, was correlated with attachment loss in
a group of Navajo adolescents aged 1419. Grossi
et al. (50) found that Porphyromonas gingivalis and
Tannerella forsythia were associated with increased
risk for attachment loss as a measure of periodontal
disease after adjustment for age, plaque, smoking,
and diabetes. Susceptibility to periodontitis will un-
doubtedly have both genetic and environmental
components and these modifying factors will be ad-
dressed in this review.
The microbial plaque biolm
Short-term clinical studies have shown that microor-
ganisms quickly colonize tooth surfaces when an
individual stops oral hygiene procedures; within a few
days, microscopic and clinical signs of gingivitis be-
come apparent. The inammatory changes can be
resolved when adequate oral hygiene is resumed (89,
145). Microorganisms which form dental plaque and
cause gingivitis do so by various means including the
release of bacterial products that induce tissue
inammation. Clinical trials emphasize the need to
remove supra- and subgingival microbial plaque in
the treatment of gingivitis and periodontitis. Further-
more, animal experiments have indicated that gingi-
vitis only develops in animals that accumulate
bacterial deposits. Clearly, gingivitis is a prerequisite
for the development of periodontitis and thus pre-
vention of gingivitis is also a primary preventive
measure for periodontitis. As stated earlier, not all
patients develop periodontitis and for those who do, it
is due to a mixture of environmental and genetic fac-
tors which affect their host response to microbial
plaque. This provides a research challenge for those
interested in the pathogenesis of this multifactorial
disease. The site specicity and predilection in perio-
dontitis and gingivitis probably relate to the retention
of plaque in specic areas such as restoration over-
hangs, poor crownmargins, etc. The type of plaque, i.e.
the specic organisms present, and its quantity, may
be a crucial environmental inuence in periodontal
disease, but at the same time it could be the individual
host response or most likely a combination of the two,
but the weight of these predisposing and modifying
factors needs to be tested experimentally.
Systemic modiers of periodontitis
Microbial dental plaque is the initiator of periodontal
disease but whether it affects a particular subject,
what form the disease takes, and how it progresses
are all dependent on the host defenses to this chal-
lenge. Systemic factors modify all forms of perio-
dontitis principally through their effects on the
normal immune and inammatory defenses. Some
good examples of this effect are when a reduction in
number or function of polymorphonuclear leuko-
cytes occurs; this may result in an increased rate and
severity of periodontal destruction. Many other sys-
temic factors are much less clear cut and are difcult
to link causally to periodontitis. In many cases the
literature is insufcient to make denitive statements
on links between systemic factors and periodontitis.
It is also at times difcult to be precise regarding the
causative agent in systemic exposures such as smo-
king and even pharmaceutically with prescribed drug
therapy.
The possible role of systemic diseases and systemic
exposures in initiating or modifying the progress of
periodontal disease is clearly a complex issue. It is,
however, generally agreed that several conditions
may give rise to an increased prevalence, incidence,
or severity of gingivitis and periodontitis. The categ-
orization of the systemic modifying factors causing
periodontitis and the evidence to support the role of
these factors are the focus of this review. An attempt
has been made to consider the conditions under
broad headings, but it will be clear that many con-
ditions fall within more than one category and that
for several conditions, only case reports exist,
whereas in other areas an extensive literature is pre-
sent.
Diabetes mellitus
Periodontal disease has been characterized as the
sixth complication of diabetes (88), a view supported
by several reviews which conclude that the bulk of
evidence indicates a direct relationship between
diabetes mellitus and periodontal disease (73, 111).
Another report (102) concluded that the preponder-
ance of evidence from studies conducted throughout
the world suggests that some diabetics are at in-
creased risk of periodontitis.
In a cross-sectional study of 1426 subjects (50),
diabetes mellitus was the only systemic disease
positively associated with attachment loss, with an
odds ratio of 2.32. The relationship appears to be
very strong within special populations; in a 6-year
longitudinal study in Pima Indians, the age- and sex-
adjusted incidence of periodontal disease, as meas-
ured by alveolar bone levels, was 2.6 times higher in
noninsulin dependent diabetic (NIDDM) subjects
108
Kinane et al.
than in those without NIDDM. However, some cau-
tion has to be adopted in this interpretation, as there
are numerous conicting studies which do not sup-
port this association between periodontal disease and
diabetes (129), suggesting that that there are differ-
ences in susceptibility to periodontitis among popu-
lations of diabetics. Insulin (IDDM) and noninsulin
dependent diabetics appear to be equally at risk of
periodontitis (138), and, with effective maintenance,
their response to surgical and nonsurgical perio-
dontal treatment is as favorable as in nondiabetics
(149).
A study (138) involving 75 diabetic patients (IDDM
and NIDDM) aimed to determine the association
between long-term control of diabetes, as evaluated
by glycosylated hemoglobin levels, and periodontitis.
In that study, the prevalence, severity, and extent of
periodontitis increased with poor control of diabetes
when calculus was present; in the absence of calcu-
lus, however, the level of control did not affect the
severity of periodontitis. A 1995 report (4) conrmed
that metabolic control may be the most important
factor between periodontal health and IDDM. These
data support the hypothesis that diabetes and the
level of metabolic control are important modiers of
periodontitis.
Thorstensson & Hugoson (140) compared period-
ontal disease status, as measured by alveolar bone
levels, in 83 adult long-duration IDDM patients and
99 age- and sex- matched controls. They concluded
that diabetics aged 4049 years had more extensive
bone loss and suggested that the age of onset was an
important risk factor for future periodontal destruc-
tion. A more recent review (129), however, concluded
that the duration of diabetes does not inuence
periodontal severity, a view supported by a 5-year
longitudinal study (149), as well as others that failed
to show an association between the duration of
IDDM and NIDDM and the severity of periodontitis
(138).
Thus it would appear that diabetes (IDDM and
NIDDM) is associated with an increased risk of
periodontitis and that the association may vary
depending on differences in susceptibility to perio-
dontitis among populations; that the level of diabetic
control is an important factor in this relationship and
may modify the response to dental plaque; that the
duration of diabetes per se does not appear to be
important; and that diabetics with periodontitis can
be successfully treated, surgically or nonsurgically,
and maintained. Further studies have suggested that
periodontal treatment inuences diabetic control
positively and that diabetics with severe periodontal
disease were much more at risk of renal and cardio-
vascular complications (139).
Medications
Phenytoin is an anticonvulsant drug strongly associ-
ated with gingival overgrowth. The onset of gingival
overgrowth is generally 3 months after the com-
mencement of phenytoin therapy (37) and approxi-
mately 50% of patients on phenytoin develop some
degree of overgrowth (139), although this incidence is
higher in institutionalized epileptics (59). It occurs
primarily in young individuals and is reported to be
seen rarely in persons over 40 years of age (1, 70), and
it appears to affect the anterior teeth more severely
than the posterior teeth (127, 139). More recent
studies have implicated alternative antiepileptic
drugs, such as valproic acid (7) and vigabatrin (71), as
also causing gingival overgrowth.
Calcium channel blockers are drugs that block the
slow calcium channels in human cell membranes
and are used in the management of arrhythmias, the
treatment of angina and the control of hypertension.
They clearly affect gingival overgrowth; however, the
prevalence of gingival overgrowth related to calcium
channel blockers is relatively low. In a study in 911
English patients medicated with nifedipine, amlodi-
pine or diltiazem for more than 6 months, only nif-
edipine was associated with signicant gingival
overgrowth, with a prevalence greater than 6% (38).
The overgrowth associated with nifedipine is clinic-
ally and histopathologically similar to phenytoin-
induced overgrowth and is considered to be due to an
increase in ground substance secreted by gingival
broblasts when stimulated by gingival inammation
following plaque accumulation (38). Although it
could be argued that gingival overgrowth and
pseudo-pocketing may be plaque retentive and thus
might be local modiers of periodontitis, this has not
been shown in the literature. Thus, although there is
evidence supporting the effects of these drugs on
gingival overgrowth, there is currently no evidence of
an association between calcium channel blockers
and periodontitis.
Cyclosporine is an immunosuppressant which acts
solely on the cell-mediated immune responses (21)
and is used in post-transplant patients. Gingival
overgrowth is a widely recognized side-effect of
cyclosporine (16, 115) and it resembles phenytoin-
induced overgrowth clinically and histopatho-
logically. It tends to appear within 3 months of
commencing the therapy, occurs in approximately
30% of individuals (122), although an incidence as
109
Modifying factors of periodontal diseases
high as 77% has been reported (107), and the extent
of the overgrowth is related to the serum concentra-
tion of the drug as well as the presence of plaque.
Other immunosuppressants, such as azathioprine,
have been shown to exhibit a lower risk for gingival
overgrowth (107, 122).
Although it could be argued that gingival over-
growth and pseudo-pocketing may be plaque
retentive and thus might act as local modiers of
periodontitis, this has not been shown in the litera-
ture. Thus, although there is evidence supporting the
effects of these drugs on gingival overgrowth, there is
currently no evidence of an association between
anticonvulsants, calcium channel blockers, or
immunosuppressants and periodontitis.
Studies associating steroid therapy with periodon-
tal disease and alveolar bone loss are conicting.
Animal studies in rats have shown that hydrocorti-
sone acetate signicantly decreased the gingival
concentrations of hyaluronic acid, chondroitin sul-
fate, and heparin (75) and induced periodontal
breakdown by impairing collagen and mucopolysac-
charide synthesis in bone (86). In contrast, clinical
studies have failed to show any association between
steroid treatment and periodontal disease. One study
reported that, although long-term prednisone ther-
apy may predispose to osteoporosis, no loss of
alveolar bone was observed (72). In another study, a
group of patients receiving prednisone for the treat-
ment of multiple sclerosis for up to 4 years was
compared with a group of patients on nonsteroidal
therapy and a group of healthy controls; no differ-
ences in either the frequency or severity of perio-
dontal disease were shown, indicating the lack of
inuence of steroids on periodontal disease (116).
Sex hormones
Elevations in plasma levels of sex hormones during
pregnancy cause a modication in the hosts
response to dental plaque but this is largely conned
to the soft tissues and manifests as an increase in
inammation severity in chronic gingivitis. Several
studies have shown that the incidence and severity of
gingival redness, edema, bleeding, and exudation
increase from the second month of gestation to the
eighth month and then decrease (30, 63, 87). These
changes do not appear to be due to an increase in
plaque but rather to an increase in the anaerobe to
aerobe ratio and more specically in P. intermedia.
In a control study of 20 pregnant and 11 nonpregnant
women, bleeding on probing and gingival indices
peaked between 21 and 24 weeks of gestation and
this correlated with an increase in the anaerobe to
aerobe ratio (76). In the same study there was a
positive correlation between P. intermedia and
estradiol and progesterone levels at 2124 and 25
28 weeks, respectively. Fluctuations in gingivitis with
phases of the menstrual cycle and the effects of oral
contraceptives on the gingiva further document the
effect of sex hormones on the periodontal tissues (61,
69, 83). Moreover, puberty is often accompanied by
increased gingival inammation and this increased
response to plaque has been attributed to the con-
centration of sex hormones in the plasma (134). An
alternative explanation for gingivitis observed during
puberty is that this is a period of mixed dentition,
where erupting and exfoliating teeth present many
sites for plaque retention. The decrease in gingivitis
after puberty may reect the fact that adolescents
have improved dexterity and also become more
aware of oral hygiene.
There is strong evidence that sex hormone levels
may alter the inammatory response to plaque and,
although this predominantly results in gingivitis
alone, an increased risk of periodontitis in these pa-
tients cannot be ignored (101, 146). However, to date,
there are no published studies which implicate peri-
odontitis as a sequelae to sex hormone-induced
chronic gingivitis.
Osteoporosis
Several recent papers have drawn attention to a
possible link between osteoporosis and periodontal
disease (64). An animal study in sheep with estrogen
deciency suggests that reduced estrogen levels
may inuence periodontal disease progression (68)
although an earlier study (90) in hamsters showed that
hormones did not inuence alveolar bone loss in this
model. In a cross-sectional study of 28 women aged
between 23 and 78 years of age, subjects were divided
into two groups, an older postmenopausal group on
estrogen replacement therapy and a younger pre-
menopausal group (133). The older group had
reduced alveolar bone density, from which the
authors concluded that menopause may cause
reductions in alveolar bone density. Age was not
controlled for in this study and since aging could
clearly inuence the results, the choice of the control
group is highly questionable. Another study of human
subjects with osteopenia and osteoporosis has sug-
gested that the severity of osteopenia is related to loss
of alveolar crestal height and tooth loss in postmen-
opausal women (147). Large and, ideally, longitudinal
studies or carefully controlled cross-sectional studies
110
Kinane et al.
are needed to elucidate the possible relationship be-
tween chronic periodontitis and osteoporosis.
Immunosuppression
The role of immunologic processes in the pathogen-
esis of chronic periodontal disease is illustrated by
studies involving individuals with primary immuno-
deciencies or those receiving immunosuppressive
therapy. Cross-sectional studies on patients receiving
immunosuppressive therapy (135, 142) failed to show
differences between these patients and healthy con-
trols in the prevalence or severity of periodontitis.
These reports suggest that immunologic deciencies
do not predispose to periodontal disease, but it must
be remembered that patients on immunosuppressive
therapy often take repeated and intensive anti-
microbial therapy, which may compensate for the
reduced immune response.
HIV infection
Although many HIV-infected individuals do not have
any form of periodontitis, they may frequently pre-
sent with oral manifestations, several of which are
found in the periodontium. Periodontal ndings in
HIV-positive patients include linear gingival ery-
thema, necrotizing ulcerative gingivitis, severe
localized periodontitis and severe destructive necro-
tizing stomatitis affecting the gingiva and bone
(similar to noma or cancrum oris) (119, 150, 152,
153). It is possible that these lesions are not HIV or
AIDS specic, but that they are necrotizing or com-
plicated forms of periodontal disease which may be
more exaggerated in immunosuppressed patients.
Interestingly, HIV-infected individuals with CD4
+
cell
counts <200 cells mm
3
present with more severe
and extensive chronic periodontitis-related attach-
ment loss (14, 113). This suggests that in immuno-
compromised HIV patients, preexisting periodontitis
may be exacerbated. Thus HIV infection can be
considered a modier of periodontitis.
Smoking
The relationship between smoking and periodontal
diseases has been studied extensively over the past
15 years and both cross-sectional and longitudinal
studies provide strong epidemiologic evidence of a
positive association between smoking and clinical
and radiographic signs of periodontitis, as well as an
increased risk of periodontitis in smokers (2, 17, 51
53, 62, 109).
In a 10-year longitudinal radiographic study of
alveolar bone loss, smoking was a signicant pre-
dictor of future bone loss in those subjects who had
at least 20 teeth at the beginning of the study (22). In
a 5-year study of attachment loss in 800 community
dwelling adults, smokers were found to be at an
increased risk of attachment loss. In a further 12-
month longitudinal study, smokers exhibited both
greater attachment loss and bone loss when com-
pared with their nonsmoking counterparts. Smokers
were shown to be at signicantly greater risk for
further attachment loss when compared to non-
smokers, the odds ratio being quoted as 5.4 (94).
In one of the largest studies of risk factors for
periodontal disease with 1361 subjects from Erie
County, NY, aged 2574 years, it was shown that
smokers were at greater risk of experiencing severe
bone loss than nonsmokers, with odds ratios ranging
from 3.25 to 7.28 for light and heavy smokers,
respectively (49). In a study of 155 Swedish patients
with periodontal disease, a signicantly higher per-
centage were found to be smokers than in the pop-
ulation at large and the risk ratio was reported as 2.5
(17). Another study of 540 Swedish adults 2070 years
of age has revealed that three variables smoking,
greater age and higher mean plaque levels were
potential risk factors for severe periodontitis (100).
The risk for periodontitis is considerably greater for
tobacco users, with estimated ratios in the range of
2.57.0 or even higher for smokers as compared with
nonsmokers (118). Even when the levels of plaque
accumulation and gingival inammation were not
signicantly different between smokers and non-
smokers, smokers exhibited an increase in prevalence
as well as severity of destructive disease (17, 53). A
case-control study of the relationship between life-
events and periodontitis has shown smoking to be
statistically associated with periodontal disease, after
controlling for oral health behavior and sociodemo-
graphic variables (53).
The relationship between smoking and periodonti-
tis appears to be dose-dependent; the odds for more
severe attachment loss range from 2.05 for light
smokers to 4.75 in heavy smokers (50), and there is a
signicant correlation between probing depth and
smoking pack-years (5). Furthermore, years of expo-
sure to tobacco products have been shown to be a
statistically signicant risk factor for periodontal dis-
ease in 1156 community dwelling NewEngland elders,
regardless of other social and behavioral factors (66).
In a study of 889 Spanish patients, gingival
recession, probing depth, and clinical attachment
level were signicantly associated with smoking
111
status. It was further noted that smoking one cigar-
ette per day, up to 10, and up to 20, increased clinical
attachment loss by 0.5%, 5% and 10%, respectively.
However, only in the last group did loss of attach-
ment differ signicantly from that of nonsmokers.
The authors concluded that tobacco use increases
disease severity, and that this effect is clinically
evident above a certain threshold (95). These data
support those of Wouters et al. (155), who found
signicantly less alveolar bone in individuals smo-
king more than 5 g of tobacco per day than in those
smoking between 1 and 5 g of tobacco per day, as
well as those of Norderyd & Hugoson (100), who
examined 547 Swedish adults and found that mod-
erate to heavy smoking (greater than or equal to 10
cigarettes per day) was associated with severe peri-
odontitis but that light smoking (less than 10 cigar-
ettes per day) was not.
The response to surgical and nonsurgical period-
ontal treatment has been shown to be less favorable
in smokers compared to nonsmokers in terms of
probing depth reduction and clinical attachment
gain, even in the presence of ongoing, effective sup-
portive therapy (2, 109). Furthermore, in a group of
refractory periodontitis patients, 90% of the subjects
reported to be smokers (92).
The mechanisms by which smoking leads to loss of
attachment are not well-understood (51). It has been
noted that the occurrence, relative frequency, or
combinations of microorganisms associated with
periodontitis were not different between smokers and
nonsmokers (110). It has been suggested that smo-
king affects the vasculature, the humoral immune
system, the cellular immune and inammatory sys-
tem and has effects throughout the cytokine and
adhesion molecule network.
Smokers with periodontal disease present with
reduced signs of clinical inammation (41) and
bleeding on probing (18) compared with nonsmok-
ers. Although in the past it was hypothesized that this
is due to the property of nicotine to exert local
vasoconstriction reducing blood ow, research
results have been contradictory. Recent studies sug-
gest that inamed sites in smokers have reduced
vascular density and angiogenesis compared to in-
amed sites in nonsmokers, thus impairing inam-
matory response and wound healing (19, 108, 112).
Emotional stress
The incidence of necrotizing ulcerative gingivitis
increases during periods of physiologic (39) and
emotional stress (46) and, as a result, stress has long
been recognized as one of the contributing factors for
necrotizing ulcerative gingivitis. The negative effect
of stress on the periodontium can be due either to
altered behaviors, such as poor oral hygiene and
smoking, and or to impaired immune function,
leading to increased susceptibility to infection (124).
Another mechanism through which stress may affect
the periodontium is an increase in levels of circula-
ting corticosteroids (114). Although stress is not an
easily measured factor, corticosteroid levels in urine
can be measured and were found to be higher in
necrotizing ulcerative gingivitis patients (32). Maupin
& Bell (96) found a signicant elevation of 17-
hydroxycorticosteroids in necrotizing ulcerative gin-
givitis patients and a signicant decrease when the
disease was resolved.
Recent studies suggest that there is also an asso-
ciation between emotional stress, usually measured
as negative life events, and chronic periodontitis.
Green et al. (48) studied individual life events such
as divorce and bereavement and concluded that
increased stressful events led to a greater prevalence
of periodontal disease. These conclusions are sup-
ported by a more recent study (33) which looked at
the relationship between life events and chronic
periodontitis and found that both negative life events
and oral health risk behaviors, such as poor oral hy-
giene and smoking, clustered together as important
determinants of periodontitis. Genco et al. (44) eval-
uated the association of stress, distress, and coping
behaviors with periodontal disease in 1426 subjects,
aged 2574. They found that psychosocial measures
of stress associated with nancial strain are signi-
cant risk indicators for periodontal disease in adults.
Psychologically depressed human subjects who
smoked and had high titers of IgG against T. forsythia
were found to have more severe and extensive chro-
nic periodontitis; the authors explained this by the
negative inuence of depression on the immune
system (99). Another study of chronic periodontitis
patients found that those resistant to therapy were
more stressed than those who responded to therapy
(10). Further studies on experimental gingivitis
volunteers also suggested that proinammatory
cytokine levels are increased in stressed subjects (36).
These data strongly suggest that stress may be a
contributing factor not only for necrotizing ulcerative
gingivitis, but also for other periodontal diseases,
such as gingivitis and chronic periodontitis, and may
also modify the response to periodontal treatment.
The intervening physiologic mechanisms between
stress and increased susceptibility to periodontal
disease are not well documented but are probably
112
Kinane et al.
related to impaired immune function and altered oral
health behaviors.
Hematologic disorders
The linkage between periodontal disease and hema-
tologic disorders is variable depending upon the
nature of the disorder. For example, in a series of 50
patients with histiocytosis syndromes, 36% had oral
involvement; 16% of these patients were rst diag-
nosed by a dentist (125). Adults, children, and infants
can all be affected by histiocytosis syndromes, which
are characterized clinically by punched out necrotic
ulcers with granulation tissue, tissue necrosis and
signicant bone loss. Because the lesions may clin-
ically mimic necrotizing ulcerative periodontitis
lesions, denitive diagnosis must be conrmed by
biopsy of the granulation tissue. Familial erythro-
phagocytic lymphohistiocytosis is the only form that
appears to have a genetic component. Early hema-
tologic and immunologic investigations, along with
possible biopsy of the associated granulation tissue,
should be initiated at an early stage to facilitate
sound diagnostic management (24, 123). The extent
of the disease may also be determined with chest
radiographs and skeletal surveys.
Although it has been demonstrated that polymor-
phonuclear leukocytes may cause tissue damage in
periodontal disease (42, 79), there is a growing body
of evidence that polymorphonuclear leukocytes
actually play a protective role in hematologic diseases
where there is a greater susceptibility to periodontitis.
To be effective in this role, polymorphonuclear
leukocytes are integrated in chemotaxis, phagocyto-
sis, and destruction of the ingested organism or
substance. Individuals exhibiting polymorphonuclear
leukocyte deciencies, either quantitative (neutro-
penia or agranulocytosis) or qualitative (chemotactic
or phagocytic), exhibit severe destruction of the
periodontal tissues.
Quantitative polymorphonuclear leukocyte deci-
encies are generally accompanied by destruction of
the periodontium. Patients with neutropenia present
with a variety of periodontal manifestations such as
the malignant form, where there is ulceration and
necrosis of the marginal gingiva with associated
bleeding and occasional involvement of the attached
gingival (9). More protracted forms of the disease such
as cyclic, chronic, and familial benign neutropenia
exhibit lesions that are frequently severe, with deep
periodontal pockets and extensive, generalized bone
loss involving the permanent dentition (12, 81, 126).
The primary dentition may in some cases be charac-
terized by bone resorption (29, 78). Periodontitis does
not always occur in familial benign chronic neu-
tropenia (35) and not all subjects are affected by either
recurrent infections or by periodontal disease. Vari-
able expression of the disorder between siblings or the
impact of the environment (e.g. oral hygiene) on this
disorder may help explain these ndings.
Leukemia is another quantitative polymorpho-
nuclear leukocyte deciency where affected individu-
als often exhibit periodontal lesions. Acute forms of
leukemia are associated with more severe periodontal
lesions; 36% of individuals with acute and 10% of
those with chronic forms (91) of leukemia exhibited
generalized gingival enlargement due to inltration by
leukemic cells. This phenomenon is usually a feature
of acute monocytic leukemia, although it has been
reported as a feature of other forms, including chronic
lymphocytic leukemia (136). Thrombocytopenia has
been linked with gingival bleeding in both acute and
chronic leukemia because gingival bleeding is a com-
mon occurrence in both forms (131).
Qualitative polymorphonuclear leukocyte decien-
cies associated with neutrophil function are also
associated with severe forms of periodontal destruc-
tion. Qualitative defects are often associated with
localized destruction affecting only the periodontium
of certain teeth (151). ChediakHigashi syndrome is a
rare genetic disease transmitted as an autosomal
recessive trait. Generalized, severe gingivitis, extensive
loss of alveolar bone and premature loss of teeth (137)
characterize affected individuals due to an extreme
susceptibility to bacterial infections that appears to
be related to alterations in the functional capacity of
the neutrophil. ChediakHigashi syndrome, Chronic
granulomatous disease and Leukocyte adhesion
deciency syndrome are other functional deciencies
of leukocytes and are discussed within the genetic
diseases section of this review.
Other blood disorders such as those involving red
blood cells, platelets and clotting disorders also
inuence the management of periodontal disease;
however, there is no evidence that these conditions
increase susceptibility to periodontal disease (72).
Genetic disorders
A number of genetic disorders increase susceptibility
to chronic periodontitis. Microbial plaque, modied
by levels and duration of accumulation, environ-
mental factors (e.g. smoking), diabetes, systemic
health, and individual genetic make-up all contribute
to susceptibility. Genetic aspects are covered else-
where in this volume.
113
Downs syndrome is characterized by a generalized
early periodontitis that manifests itself in the primary
dentition (31, 34, 67) and continues into adulthood.
The prevalence and severity of periodontal disease in
individuals with Downs syndrome is extremely high
when compared to either their siblings (103) or other
mentally handicapped individuals (67, 120). Early
onset periodontitis is evidenced by pocket formation
in 36% of Downs syndrome children less than
6 years of age (67, 120). Older age groups are char-
acterized by an increased prevalence and severity of
periodontal disease (31, 34, 67) as reported in cross-
sectional studies. Longitudinal studies indicate that
the progression of periodontal disease is very rapid
(98). The most frequent sites of periodontal destruc-
tion are the incisor and molar areas (31, 121). Pre-
mature loss of the mandibular incisors is associated
with short roots (23) and loss of bone in this area (67).
One study calculated a mean annual incidence which
predicted that the entire dentition would be lost
9 years after the onset of periodontal disease.
Leukocyte adhesion deciency syndrome is a rare
autosomal recessive disease characterized by neutro-
phils with defects in several cell-cell adhesion
receptors. Defects in these receptors may lead to
increased susceptibility to infectious diseases such as
periodontitis (6). Two studies that describe severe
inammatory periodontal disease in young patients
with the Leukocyte adhesion deciency syndrome
(105) indicate that the disease is often fatal. Children
with deciencies in expression of the lymphocyte
function-associated antigen (LFA) family of adhesions
have been reported as suffering from severe perio-
dontal infections (6), but data relating adult perio-
dontitis to this condition are not available. The
importance of the adhesion molecules to the function
of the immune and inammatory systems is never-
theless fundamental and even small variations or
deciencies could contribute to a depressed host
response and an increased risk of periodontitis.
Chronic granulomatous disease is a rare condition
that manifests as either autosomal recessive or
x-linked recessive. Phagocytic cells (both poly-
morphonuclear leukocytes and monocytes) are
unable to kill through utilizing the oxidative pathway
after ingestion. Kinane & Davies (74) described a
family with the X-linked recessive form of Chronic
granulomatous disease where the female carriers
suffered from photosensitivity dermatoses and had
severely lower nitroblue tetrazolium reducing ability
within their phagocytes. All female carriers in this
series were examined for oral and periodontal lesions
and no periodontal manifestations were attributed to
this condition, although gingival erythema and occa-
sional ulceration were noted.
PapillonLefe`vre syndrome is a disease exhibiting
autosomal recessive inheritance (97) and character-
ized by the presence of hyperkeratotic skin lesions.
Individuals with this disease exhibit diffuse palmar-
plantar keratosis associated with a severe generalized
periodontitis that commonly occurs before puberty
and is characterized by early loss of primary and
permanent teeth (13, 23, 28, 40, 47, 55, 148). Teeth are
generally lost in the order of eruption (55) and as yet
there is no general agreement on the success of
dental therapy. The general population frequency of
this disease is reported as 1 in 4 million (13), with
25%having an increased susceptibility to infection. A
history of consanguinity is also noted in 33%of those
affected (55). Another related disease characterized
by palmar-plantar keratosis and severe early onset
periodontitis is Haim Munk syndrome. Preliminary
genetic studies of these two diseases suggests that the
gene defect in Haim Munk syndrome is not genetic-
ally linked to the other more common forms of pal-
mar-plantar keratosis (57). It does appear that there
is a high degree of consanguinity in these families
and that they are probably part of the same syndrome
(130), although this work has been questioned.
Hypophosphatasia is a condition in which patients
exhibit a decreased serum alkaline phosphatase level.
Affected individuals exhibit a severe loss of alveolar
bone and premature loss of the primary teeth (11, 20,
26, 141).
ChediakHigashi syndrome is inherited as an
autosomal recessive trait associated with severe
periodontitis (27, 45, 54, 104, 137). Affected patients
exhibit defective neutrophil chemotaxis and abnor-
mal bactericidal functions.
The EhlersDanlos syndrome encompasses a group
of autosomal dominant connective tissue disorders
that is characterized by defective collagen synthesis.
The disorders are classied into 10 types on the basis
of inheritance and clinical symptoms. The joints and
skin are the most affected sites. Patients with Types
IV and VIII have an increased susceptibility to
periodontitis (58). Type VIII is particularly associated
with fragile oral mucosa and blood vessels along with
severe generalized periodontitis that has the clinical
appearance of generalized early onset periodontitis
(82). The EhlersDanlos syndrome type VIII was rst
recognized by McKusick (97) in a family with skin
fragility, abnormal scarring, early tooth loss and
severe periodontitis. Another family with the Ehlers
Danlos syndrome type VIII exhibited joint laxity, skin
fragility and extensive periodontal destruction (8). It
114
Kinane et al.
appears that there is considerable interfamilial vari-
ability in the EhlersDanlos syndrome type VIII but
the distinguishing nding is periodontitis clinically
resembling the early onset form and leading to pre-
mature loss of permanent teeth (132).
There are other rare genetic disorders that deserve
mention here. Glycogen storage disease 1b is an
autosomal recessive condition in which there is faulty
carbohydrate metabolismand an association with low
neutrophil numbers, impaired neutrophil function
and periodontal disease (56, 104). Infantile genetic
agranulocytosis is an extremely rare autosomal reces-
sive disorder that features severe neutropenia and an
associated periodontitis resembling the early onset
form (77, 117). Cohens syndrome is an autosomal
recessive disease characterized by nonprogressive
mental and motor retardation, obesity, dysmorphia
and neutropenia (3). Individuals with Cohens syn-
drome manifest more frequent and extensive alveolar
bone loss thanmatchedmentally retardedcontrols (3).
Age
The prevalence of periodontal disease increases with
age. However, it is not clear if becoming older is
related to an increased susceptibility to periodontal
disease; that is, if it changes our host response
capability, or if the cumulative effects of disease over
a lifetime explain the increased prevalence of disease
in older people. Horning et al. (62) stated that age is a
risk factor for periodontitis, although loss of attach-
ment and alveolar bone with age is dependent on the
presence of plaque and calculus. Holm-Pederson
et al. (60) and Machtei et al. (93) suggest that, up
until the age of 70, the rate of periodontal destruction
is the same throughout adulthood and that age per se
is not a risk factor, at least for those under the age of
70 (43).
Summary
Microbial dental plaque initiates periodontal disease
but the form and severity of the disease is dependent
on the environmental, genetic and host defenses to
this challenge. Systemic disorders or variations and
environmental exposures may modify the normal
defenses and inuence the resultant periodontal
disease. A reduction in number or function of poly-
morphonuclear leukocytes results in increased sever-
ity of periodontal destruction. Many drugs such as
phenytoin, nifedipine, and cyclosporine predispose to
gingival overgrowth in conjunction with microbial
plaque andhost response characteristics andthus may
modify pre-existing periodontitis. Changes in circula-
ting hormone levels may result inanincreasedseverity
of plaque-induced gingival inammation but not
typically in any increased susceptibility to periodontal
attachment or bone loss. Hormonal changes as seen
during and after menopause have been associated
with osteoporosis but there is a lack of studies linking
menopause or an estrogen-decient state to a higher
susceptibility to periodontal disease. Immunosup-
pressive drug therapy and any disease resulting in
suppression of the normal inammatory and immune
processes, such as HIV infection, may predispose the
individual to periodontal destruction. It is difcult,
however, to determine the precise causative agent in
these conditions and it is particularly complex when
immunosuppressive drugs are prescribed together
with antibiotics for variable periods of time. The evi-
dence for smoking having a deleterious inuence on
periodontal health is convincing. Nutritional deci-
encies in animals have been shown to affect the perio-
dontal tissues but epidemiologic data do not support
the suggestion that such deciencies play an import-
ant role in chronic periodontal disease. Gingival
bleeding is the most consistent oral feature of vitamin
Cdeciency, or scurvy, but there is also some evidence
to suggest that avitaminosis-C may aggravate estab-
lished chronic periodontitis (143, 144). Stress and
other psychosomatic conditions which may have
direct anti-immune effects or indirect, behavior-
mediated effects on the bodys defenses may prove to
be important in the etiology of periodontitis and
necrotizing ulcerative gingivitis and periodontitis. The
role or relative importance of these mechanisms has
yet to be fully elucidated but the evidence that stress,
neural factors, and depression can inuence the im-
mune system is increasing. Many genetic disorders
have numerous host response modications, which
may render the individual susceptible to periodontal
disease. Many genetic conditions inuence the perio-
dontium during childhood and the periodontal man-
ifestations of the disease may resemble the early onset
forms of periodontitis; the effects of such diseases may
persist into adulthood. Although systemic diseases
such as diabetes will aggravate all forms of periodon-
titis, chronic or adult periodontitis is the most preval-
ent andthus will be the most commonformpresenting
with diabetes-induced modications. Currently,
numerous genetic polymorphisms relevant to inam-
matory and immune processes have been suggested
and are being investigated for their modifying effects
on periodontal disease. The literature on how perio-
dontitis is modied by systemic factors, with the
115
exception of the link with diabetes, HIV infection, and
smoking, is as yet sparse and clearly well controlled
cross-sectional and longitudinal studies are needed to
fully elucidate the relationship between environmen-
tal modiers and genetic inuences and periodontitis.
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119
Effects of medications on the
periodontal tissues in health and
disease
ROBI N A. SEYMOUR
It is now well recognized that the adult population is
living longer and retaining their teeth into old age. A
major part of this increase in life expectancy is
attributed to an expansion in our understanding of
disease processes and the subsequent explosion in
drug treatments. Some of these drugs will have an
impact on the periodontium and its response to
bacterial plaque. This paper reviews the various
possible interactions between a patients medication
and their periodontium in both health and disease.
The effect of systemic drug therapy on the perio-
dontium can be categorized as follows:
an adverse effect on the periodontal tissues;
affording some degree of protection against peri-
odontal breakdown;
causing an increased risk of periodontal break-
down.
Adverse effects on systemic
medication on the periodontal
tissues
Drug-induced gingival overgrowth remains the most
widespread unwanted effect of systemic medication
on the periodontal tissues. Three drugs are most
frequently implicated; phenytoin, cyclosporine and
the calcium channel blockers. Case reports have
implicated other drugs (e.g. sodium valproate and
erythromycin) but these are rare incidents (60).
Prevalence of drug-induced
gingival overgrowth
The prevalence of drug-induced gingival overgrowth
varies signicantly and, as with most epidemiolog-
ical studies, depends upon the population investi-
gated. Many authors have used hospital-based
patients, which is entirely appropriate for cyclo-
sporine. For phenytoin and the calcium channel
blockers, again many authors have frequently
examined hospital-based patients. Bearing this in
mind, it has nonetheless been estimated that 50%
of dentate patients on phenytoin experience gingi-
val changes, whereas the gures for cyclosporine
and the calcium channel blockers are 30% and
10%, respectively (1, 2, 15, 63).
The above gures do not take into account the
severity of gingival changes and that the term clinical
signicant overgrowth is more appropriate to epi-
demiologic studies. This term is applied to those
patients whose gingival overgrowth requires surgical
excision to correct the gingival contour (59).
Risk factors for drug-induced
gingival overgrowth
A variety of risk factors for drug-induced gingival
overgrowth have been identied and these have been
recently reviewed (59). Essentially, they are age and
other demographic variables, drug variables, con-
comitant medication, periodontal variables, and
genetic factors.
Age and demographic variables
Age is now recognized as an important risk factor for
both cyclosporine- and phenytoin-induced gingival
overgrowth (10, 17, 24, 28, 57). For calcium channel
blockers, age is not applicable since the use of these
drugs is mainly conned to the middle-aged and
elderly. Children and teenagers are more susceptible
to phenytoin and cyclosporine-induced gingival
overgrowth. This could suggest that a hormonal
120
Copyright Blackwell Munksgaard 2006
PERIODONTOLOGY 2000
component contributes to broblast sensitivity to the
challenging drugs. Phenytoin has been shown to
enhance the ability of gingival broblast to meta-
bolize testosterone to the more active 5a dihyd-
rotestosterone (69). Similar results were found for
broblast, obtained from both cyclosporine- and
nifedipine-induced gingival overgrowths (68). Ado-
lescents will have higher levels of circulating andro-
gens which could stimulate further gingival broblast
to increase collagen synthesis and or decrease col-
lagenase activity. There is also the additional action
of increased circulatory sex hormones on the
expression of gingival inammation. Any increase in
inammation will compound the expression of
gingival overgrowth.
Gender
A few studies have investigated whether gender is a
risk factor for drug-induced gingival overgrowth. This
is in part due to a lack of reporting, and as far as
organ transplant patients are concerned, the out-
come is limited by a preponderance of males in the
patient population. Gender does not appear to be a
risk factor for phenytoin-induced gingival overgrowth
(21), but seems to be relevant in cyclosporine and
nifedipine cases (15, 74, 75). Whether such ndings
relate to existing periodontal variables, pharmacolo-
gical factors or a hormonal cofactor remains to be
determined.
Drug variables
There is considerable controversy concerning the
relationship between a variety of drug variables and
expression of drug-induced gingival overgrowth. A
range of pharmacokinetic variables have been stud-
ied and include peak and trough serum concentra-
tion, drug dosage, drug concentration in saliva and
gingival crevicular uid. There is no consistent nd-
ing and, as such, these parameters do not appear to
be a useful prognostic indicator as to how a patients
gingival tissue would respond to one of the implica-
ted drugs (59). Despite these reservations, most
workers would concur that some baseline or thresh-
old concentration of the drug is required to induce
the gingival changes. Such a threshold concentration
may vary from drug to drug and from individual to
individual.
Drug dosage tends to be a poor predictor of
gingival changes (60, 61, 65). It would be more
appropriate to correlate dose with body weight in
order to obtain a more meaningful interpretation of
doses and their relationship to gingival overgrowth.
Phenytoin and calcium channel blockers obtain
steady state therapeutic drug levels 710 days after
the initiation of therapy, thus for these two drugs a
serum sample at any time point is likely to be a
true reection of the drug concentration. Cyclo-
sporine measures are often taken as trough con-
centrations. Whereas such single measures are
useful for checking compliance, the level of anti-
convulsant activity or immunosuppression, they
only reect one aspect of the drugs pharmaco-
kinetic prole. Other pharmacokinetic measures
that may have more potential in relation to the
expression of gingival overgrowth include bioavail-
ability, degree of protein binding, volume of dis-
tribution, and an overall assessment of the drug
concentration in relation to time. This later method
is referred to as an area under the plasma serum
concentration curve (AUC) and measures the total
concentration of the drug over a specic time
period. Such a measurement requires repeated
sampling, which is impractical in large population
investigations. Single serum measures are easy to
obtain and often available as part of the patients
ongoing medical care. Thus the lack of any clear
relationship between blood concentrations of the
drug with the expression of gingival overgrowth
may be a reection of the shortfall of the sampling
technique or a lack of investigation into more
appropriate pharmacokinetic variables.
Concomitant medication
The three major drugs implicated in gingival over-
growth are seldom the only medications prescribed
to the patient. The effect of additional drugs on
the expression of gingival overgrowth has been
investigated with respect to both cyclosporine and
phenytoin.
Nifedipine and other calcium channel blockers are
used extensively in organ transplant patients medic-
ated with cyclosporine. As these drugs also cause
gingival overgrowth, it is not surprising that the pre-
valence of this unwanted effect increases signicantly
in transplant patients (4, 34, 45, 74, 75, 86, 88). It has
been suggested that combined therapy may increase
the prevalence of the condition, but not the severity
(50).
Other drugs taken by organ transplant patients
could likewise inuence the expression of the gin-
gival changes. Prednisolone and azathioprine
appear to afford adult transplant patients some
degree of protection against the development of
121
Periodontal tissues in health and disease
gingival overgrowth (21, 66, 86). The so-called
protective effects of these two drugs on the gingival
tissues may arise from their anti-inammatory
action on plaque-induced gingival inammation
which will be discussed later.
Other anti-epileptics can affect the hepatic
metabolism of phenytoin, which in turn can impact
upon the gingival tissue response. Phenobarbitone,
primidone and carbamazepine induce the hepatic
enzyme P450. The latter has a greater stimulatory
effect on the gingival broblast, which may explain
the increased prevalence of gingival overgrowth in
patients receiving multiple anti-epileptic therapy.
Periodontal variables
Any plaque-induced inammatory changes within
tissues are going to exacerbate the expression of drug-
induced gingival overgrowth. This nding suggests
causality, with a patients oral hygiene being a signi-
cant risk factor for both the development and the
expression of drug-induced gingival overgrowth (15,
30, 49, 66, 7476), althoughreports tothe contrary have
also appeared (57, 63, 87). Furthermore, most of the
evidence supporting the relationship between bac-
terial plaque and drug-induced gingival overgrowth
has been derived from cross-sectional studies, and in
such studies it is difcult to determine whether plaque
is a contributory factor to or a consequence of the
gingival changes. In circumstances where other addi-
tional structures such as orthodontic appliances
interfere withcleaning, the prevalence of overgrowthis
high (11).
Although there may be some debate as to the role
of plaque and gingival inammation in drug-induced
gingival overgrowth, there is no doubt that improving
a patients oral hygiene and reducing the inamma-
tory component in the gingival tissue by nonsurgical
means does have an impact on this unwanted effect.
Despite such measures there still remains a cohort of
patients who develop overgrowth irrespective of their
oral hygiene or periodontal condition (62). In such
patients, other risk factors may be more signicant.
A patients underlying periodontal status may also
be a signicant risk factor for drug-induced gingival
overgrowth (49, 82). Of particular concern is the
extent of inammation present in the gingival tissue
prior to dosing. Organ transplant patients are more
likely to develop overgrowth prior to transplantation
and dosing with cyclosporine when they exhibit sig-
nicant gingival inammation. A similar situation
arises with the calcium channel blockers (6, 40, 72). It
is interesting to note that gingival changes are more
pronounced in patients taking nifedipine for cardio-
vascular disorders than in patients taking these drugs
for other reasons. This may be due to the impact of
other cardiovascular drugs on the gingival vascula-
ture.
Genetic factors
The variable gingival response seen in patients
following drug challenge has been attributed to
broblast heterogeneity. Although this may be a
useful in vitro explanation, it has little value in
determining at-risk patients. Much interest has
focused on drug metabolizing enzymes and the
expression of gingival overgrowth. Phenytoin,
cyclosporine and nifedipine are all metabolized by
the hepatic cytochrome P450 enzymes. Cytochrome
P450 genes exhibit considerable polymorphism,
which results in interindividual variation in drug
levels. This inherited variation in metabolism of
either drug may inuence patient serum and tissue
concentrations and hence their gingival response.
Whereas cytochrome P450 variation may be a risk
factor for drug-induced gingival overgrowth, it is
totally impractical to assess this on a clinical basis
(59). Other studies have investigated P-glycoprotein
drug-transporter MDR1 gene polymorphisim and
CYP2C polymorphism in relation cyclosporine- and
phenytoin-induced gingival overgrowth, respectively
(14, 64). Neither of these investigations showed a
direct correlation between these two genetic
markers and gingival overgrowth.
Other genetic markers for gingival overgrowth have
focused on human lymphocyte antigen expression
(HLA), because HLA phenotype is determined prior
to organ transplantation. There is evidence that
patients who express HLA-DR1 are afforded some
degree of protection against the development of
drug-induced gingival overgrowth, whereas those
who express HLA-DR2 may be more susceptible to
this unwanted effect (7, 48).
Further studies have shown that HLA-A19 expres-
sion may increase susceptibility and HLA-B37 is a
signicant risk factor (34, 75).
The mechanisms that link HLA expression to
gingival overgrowth are unclear. The concept of
molecular mimicry in the wider eld of periodontal
disease (4) has been postulated, via an effect on
lymphocyte function. The apparent HLA associa-
tions may represent nothing more than a tight
linkage disequilibrium between HLA and non-HLA
genes in the MCH region of human chromosome 6
(32).
122
Seymour
Pathogenesis of drug-induced
gingival overgrowth
The main histopathological feature of drug-induced
gingival overgrowth is a brotic or expanded con-
nective tissue with various levels of inammation
and an enlarged gingival epithelium. As a conse-
quence, many of the investigations into the path-
ogenesis of this unwanted effect have been focused
on drug interactions or challenge to various aspects
of connective tissue metabolism. Many investiga-
tions have also considered roles of a variety of
inammatory cytokines and growth factors on the
effect of the drug on gingival broblast. An excel-
lent paper (78) has reviewed the extensive literature
of connective tissue metabolism and gingival over-
growth and little further can be added in this
review.
The pathogenesis of drug-induced gingival over-
growth remains multifactorial, different drugs having
separate impacts on the range of cytokines and
growth factors involved in connective tissue meta-
bolism. It is hoped that a greater understanding of
the pathogenesis of this unwanted effect could lead
to improved management strategies for either its
prevention or its treatment.
Treatment of drug-induced
gingival overgrowth
Gingival surgery (invariably a gingivectomy) remains
the main treatment option for correcting drug-
induced gingival overgrowth. Various different
surgical approaches and techniques have been
employed for removing the excess gingival tissues,
but few studies have demonstrated consistent
advantages over the standard 45 gingivectomy exci-
sion (58). Lasers have also been employed for gingival
excision, but the cost of using these instruments
often precludes their routine use.
Although surgery remains the main option for
treatment of drug-induced gingival overgrowth,
alternative strategies have been investigated to either
prevent the unwanted effect or reduce the incident of
recurrence.
For cyclosporine-induced gingival overgrowth,
attention has focused on the use of systemic or topical
(local) antibiotics to reduce the severity of the
condition or prevent recurrence after surgery. Such
investigation followed on from a small study in
renal transplant patients (89) where four cases of
drug-induced gingival overgrowth responded to a 7-
day course of systemic metronidazole. This uncon-
trolled study did not take into account the patients
underlying periodontal condition, and hence it was
difcult to discern whether the gingival changes were
drug-induced or secondary to underlying gingival
inammation.
Azithromycin is another antibiotic that has been
evaluated in the management of cyclosporine-
induced gingival overgrowth. A review of the clinical
trials that have been completed on the efcacy of the
compound (71) suggests that there are some benets
of systemic azithromycin in the management of
cyclosporine-induced gingival overgrowth. Azithro-
mycin appears to be more effective than metroni-
dazole (8) for this condition. Two investigations have
speculated on the mode of action azithromycin in
cyclosporine-induced gingival overgrowth. A clinical
study (37) concluded that a 7-day course of azithro-
mycin does not affect the remission of drug-induced
gingival overgrowth but does act on concomitant
bacterial over-infection and hence reduces inam-
mation. A study in rats showed that cyclosporine
decreases collagen degradation by lowering phago-
cyte activity of gingival broblasts. Azithromycin
increases this activity (47).
It remains to be determined what are the extent
and benets of azithromycin in the management of
drug-induced gingival overgrowth. Short-term cour-
ses have been employed and such courses may bring
about some reduction in overgrowth. Since drug-
induced gingival overgrowth is a recurrent and con-
tinuous problem, there will be concerns about the
use of repeated doses of antibiotics in the manage-
ment of this unwanted effect, especially in immu-
nosuppressed patients.
There have been other chemotherapeutic applica-
tions in the management of drug-induced gingival
overgrowth, especially to reduce the incidence of
recurrence after surgery. Chlorhexidine mouthwash
(0.2% w v) has been shown to be of benet in pre-
venting phenytoin-induced gingival overgrowth,
especially after surgery (43). Long-term use of this
plaque inhibitory agent has not been evaluated for
this purpose.
Phenytoin inhibits folic acid metabolism, but the
mechanism is uncertain. There is some evidence that
folic acid (1 mg ml mouthwash) appears to be more
efcacious than systemic administration (51). It has
been suggested that topical folate may reduce gingi-
val inammation by binding to the plaque-derived
endotoxins. This action may, in turn, reduce gingival
overgrowth. Patients with a low baseline plasma and
123
red blood cell folate show a better gingival response
to topical folic acid than patients with normal levels
(13).
Good plaque control, removal of plaque retentive
factors and treatment of any underlying periodontal
condition will reduce gingival inammation and
hence the severity of any drug-induced gingival
overgrowth. However, in some patients these meas-
ures alone will not reduce the occurrence or recur-
rence of overgrowth and surgical excision remains
the only option.
One obvious solution in the management drug-
induced gingival overgrowth is to change medication.
For many years this was not an option for cyclospo-
rine. However, new immunosuppressant (e.g. tacr-
olimus) alternative medication is now available, and
it has been shown that converting from cyclosporine
to tacrolimus does reduce the severity of overgrowth
and the need for surgical intervention (16, 25).
Although such a change in medication may improve
the gingival tissue, it does not completely resolve the
overgrowth (15).
Although phenytoin usage is declining and there
are more anti-epileptics available, changing the
medication for these patients can be a challenge.
Carbamazepine, ethosuximide and sodium valproate
are alternatives to phenytoin. If a change in anti-
convulsant therapy is contemplated, this should be
accomplished gradually over a period of 23 months.
During this time there should be monitoring of serum
levels of the anti-epileptics and the occurrence of
seizures.
Systemic medication can afford the
patient some degree of protection
against periodontal breakdown
It is well established that activation of the hosts
inammatory and immune responses are pivotal in
the pathogenesis of periodontal breakdown. As a
consequence, systemic medication that affects these
processes are of interest to the periodontist. This
interest is twofold. Firstly, these drugs can be used
as tools to identify the particular roles of immune
and inammatory responses in the periodontal
breakdown process. Secondly, the drugs may have
an additional therapeutic role in the management of
periodontal disease. Drugs that can potentially affect
the periodontium and its response to plaque include
immunosuppressants, corticosteroids and nonster-
oid anti-inammatory drugs. Antibiotics and the
host modulating agent doxycycline will also impact
on the periodontal breakdown process; however,
such drugs will not be considered as part of this
review.
Immunosuppressants
Many studies have investigated the effect of systemic
immunosuppressant medication on the various
parameters of periodontal diseases (60).
Azathioprine and prednisolone reduce the
inammatory responses of the periodontal tissue to
bacterial plaque (56). This nding was conrmed by
Kardachi & Newcomb (29), who compared plaque
scores and gingival indices in renal transplant
patients taking immunosuppressants with those
of otherwise healthy subjects. Both groups had
similar plaque scores, but the gingival index in the
renal transplant patients was signicantly lower
(P < 0.01).
There is evidence to suggest that uraemic patients
are in a state of reduced immunocapacity (5) and the
ndings reported above may be related to the previ-
ous disease, or to the drug therapy or both. In some
of the subsequent studies (3, 44, 77) various perio-
dontal measures were compared in renal transplant
patients taking immunosuppressants, patients on
hemodialysis and healthy controls. Gingival inam-
mation, periodontal destruction and plaque accu-
mulation were similar in all three groups, but in the
transplant group there was again a lack of correlation
between plaque levels and both gingival inamma-
tion and periodontal destruction.
Immunosuppressants do affect the response of
gingival and periodontal tissues to bacterial plaque.
They do not abolish the reaction of the tissues to
plaque, but appear to dampen down inamma-
tory reactions. The specic pharmacodynamics of
immunosuppressants may provide further insight
into the pathogenesis of periodontal inammation
and breakdown.
Corticosteroids
This group of drugs is used in virtually every aspect of
clinical medicine. They are for the most part used for
their anti-inammatory and immunosuppressant
properties (60). Prolonged therapy with corticoster-
oids may favor osteoporosis, which is now regarded
as a risk factor for periodontal disease. Several animal
studies have conrmed that systemic steroids have
adverse effects on the periodontium and its response
to bacterial plaque. Clinical studies are somewhat
124
Seymour
equivocal with respect to a signicant anti-inam-
matory action. An early study in bed-ridden patients
on long-term corticosteroids showed that regardless
of steroid therapy, inammatory changes were more
severe in those subjects with poor oral hygiene.
Therefore, gingival inammation and periodontal
destruction were more dependent on plaque control
than on any anti-inammatory action attributable to
systemic steroids (31). Topical application of corti-
costeroids to inamed marginal gingiva of patients
with periodontal disease resulted in a reduction of
inammation and bleeding with no effect on the
progression of periodontitis (20, 70). However, when
steroids are injected directly into the gingival tissue,
they cause a histological reduction in capillary per-
meability, a reduction in plasma cells and granula-
tion tissue, an inhibition of collagen synthesis and a
clinical improvement in hemorrhagic and hypoplas-
tic gingivitis (26).
The effect of systemic prednisolone upon gingival
inammation and periodontal bone destruction has
been studied in a group of patients suffering from
multiple sclerosis (55). Comparisons were made
between this group, a group of patients who suffered
from neurological disorders but were receiving no
steroids, and healthy controls. There were no differ-
ences between the groups and it was concluded that
long-term systemic steroid therapy had no inuence
on the measures of periodontal disease.
It would appear that despite observations from
animal studies, systemic and topical corticosteroids
have little impact on the periodontuim and its
response to bacterial plaque. The reasons for these
inconsistent ndings may include insufcient dosage
as well as inadequate interactions among the com-
bination of drugs that various patient groups are
taking. Despite their recognized anti-inammatory
and immunosuppressive properties, corticosteroids
have no application in the management of perio-
dontal diseases.
Non-steroidal anti-inammatory drugs
(NSAIDs)
In the early 1970s it was recognized that prostaglan-
dins (PGs) were important mediators in the patho-
genesis of periodontal destruction and activation of
mechanisms of bone resumption (19). At the same
time, Vane and coworkers were identifying the mode
of action of cyclo-oxygenase and subsequent pros-
taglandin synthesis and their inhibition by aspirin
(80). As a consequence of these ndings, a signicant
interest emerged in the possible application of these
drugs in the control and management of periodontal
diseases.
Evidence that such drugs may have an application
in the management of periodontal diseases came
from controlled studies on patients who had been on
long-term NSAID therapy for musculoskeletal rea-
sons (18, 23, 83). In the rst of these studies (83)
patients on long-term NSAIDs had reduced amounts
of gingival inammation and probing depths com-
pared with age- and plaque-matched controls. In a
further study, it was demonstrated that NSAIDs
afforded patients some degree of protection against
alveolar bone loss (18). By contrast, a cross-sectional
study showed no signicant differences for a variety
of periodontal measures between patients on long-
term NSAIDs and a control group (23). However,
patients from the NSAID group had signicantly
lower gingival crevicular uid ow rate than controls.
This was attributed to a possible effect of the NSAID
medication on the vascularity and permeability of
small blood vessels.
Animal studies have conrmed benecial effects of
NSAIDs in ligature-induced periodontitis (41, 42).
Another animal study showed that systemic urbi-
profen signicantly resolved bone loss in beagle dogs
undergoing either surgical or nonsurgical manage-
ment of periodontitis (85).
Clinical studies on NSAIDs when used as adjuncts
in the management of periodontal disease have
been somewhat equivocal with respect to outcomes.
Systemic urbiprofen taken for 12 months reduces
the rate of alveolar bone loss (84) and is benecial
for the resolution of experimental gingivitis (22).
Systemic administration of the drugs in asthmatics
is associated with an increased risk of unwanted
effects including peptic ulceration, interference with
platelet aggregation and increased risk of broncho-
constriction. Concerns over these unwanted effects
directed interest to the topical application of
NSAIDs for adjunctive management of periodontal
disease. Such agents could be delivered in the form
of a mouthrinse or incorporated into toothpaste. A
ketorolac rinse may be of benet in the treatment of
adult periondontitis and appears to have advantages
over systemic urbiprofen in reducing alveolar bone
loss (27). By comparison, a 1% w w urbipropfen
toothpaste was shown to exert a small yet signicant
effect on bone metabolism when used over a 12-
month period as an adjunct to root surface instru-
mentation (22).
Whereas systemic NSAIDs appear to afford a
patient some degree of protection, their use as a
therapeutic measure in the treatment of periodontal
125
disease is limited. In part this is due to the high
prevalence of unwanted effects, which subsequently
is encouraging the development of topical prepara-
tions. The success of these agents is also somewhat
limited and their use as a means of controlling peri-
odontal disease has been surpassed by other more
effective compounds.
The new COX-2 inhibitors have all the attributes of
NSAIDs with a reduced risk of unwanted effects.
These drugs have been evaluated as an adjunct to
root surface instrumentation in patients with chronic
periodontitis (81). The results showed little clinical
benet of COX-2 inhibitors in the management of
such patients, but signicant reductions in gingival
tissue levels of PGE
2
and PGF
2
. The recent scare over
the long-term use of these drugs will probably
exclude their use in the management of periodontal
diseases.
Drugs which can increase the
expression of periodontal diseases
Sex hormones
The effects of sex hormones on the gingival tissues
are well established and distinct changes in relation
to puberty and pregnancy are well documented (35).
Such changes are brought about by increased levels
of circulating estrogen and progesterone. Both sex
hormones are constituents of the contraceptive pill
and are also used in hormone replacement therapy.
The oral contraceptive can mimic the gingival and
periodontal changes observed in pregnancy. These
include an increased tendency for gingivitis and
increased probing depths (33, 38, 39), increased
susceptibility to infection (9), decreased neurophil
chemotaxis (52, 67) and an increased number of
periodontopathogens (79). Long-term use of the oral
contraceptive can lead to acceleration in the pro-
gression of periodontal disease, but much would
depend upon the concentration of hormones used in
the pill. However, low doses of estrogen and prog-
esterone are now widely used in contraceptive pills
and these lower doses have little impact on the per-
iodontal tissues and their response to plaque (39).
The impact of hormone replacement therapy on
the periodontium is difcult to dene. It is now
recognized that osteoporosis is a risk factor for peri-
odontal disease and reduction in bone mineral den-
sity is a major health problem in postmenopausal
woman. Estrogen is the main hormone used in hor-
mone replacement therapy and is of value in pre-
venting bone loss and the need for dentures in the
older woman (46). Patients on estrogen replacement
had less tooth loss and hence less need for dentures
than control patients. This would suggest some pro-
tective action of hormone replacement therapy,
possibly medicated by the prevention of osteo-
porosis.
In a further study it was reported that a 2-year
course of estrogen replacement therapy benets a
patients periodontal condition, with reduced levels
of inammation and a reduced frequency of clinical
attachment loss (53). These two studies (46, 53)
suggest that patients on hormone replacement ther-
apy may receive some periodontal benet. Again,
such benets may be medicated via reduction in the
risk of osteoporosis.
Drug-induced desquamative gingivitis
Several drugs can cause oral lichenoid reactions that
can present as desquamative gingivitis. Drugs most
commonly implicated include beta-adrenoceptor
blockers (e.g. propranolol, atenolol), antidiabetic
drugs (e.g. chlorporpamide and tolbutamide), gold
salts and nonsteroidal anti-inammatory drugs. Most
examples have been presented as case reports and a
detailed list of all the drugs implicated in this
unwanted effect can be found in specialized texts
(36). The diagnosis of drug-induced lichenoid erup-
tions (desquamative gingivitis) can be problematic
and often involves stopping the suspected drug and
re-challenging the patient. Most of the drugs fre-
quently implicated can be substituted by an alter-
native medication with the same therapeutic goals.
Drug-induced depression of bone
marrow
Drug-induced depression of bone marrow is the most
serious and potentially life-threatening adverse effect
of systemic medication. Fortunately, this unwanted
effect is rare, but it can result in aplastic anaemia,
agranulocytosis and thrombocytopenia. Again, a list
of drugs that have been implicated in this unwanted
effect can be found in specialist textbooks (12). Drug-
induced depression of bone marrow will affect the
periodontal tissues. There may be a rapid increase in
the rate of periodontal destruction following a
reduction in white blood cell numbers or activity.
Thrombocytopenia will manifest in the gingival tis-
sues, especially if these are inamed. Excessive
bleeding on probing and prolonged bleeding may be
secondary to a drug-induced thrombocytopenia.
126
Seymour
Other unwanted effects that can follow a drug-
induced depression of bone marrow include oral
ulceration, swollen gingiva and an increased risk of
opportunistic infections (e.g. herpes and Candida).
Drug-induced bone marrow often accompanies
chemotherapy used in the treatment of malignant
diseases. Careful monitoring of bone marrow activity
is part of any chemotherapy regimen, but periodontal
manifestation may occur during the course of treat-
ment. Whenever possible, patients about to start
chemotherapy should have a thorough oral screening
and prevention measures should be put in place to
reduce the effect of these drugs on the periodontal
tissues.
Increased bleeding on probing has also been
reported in patients taking aspirin for the prevention
of thrombo-embolic disorders (54). This relates to the
ability of aspirin to reduce platelet aggregation via
blocking cyclo-oxygenase enzymes. The impact of
aspirin upon hemostasis should be taken into
account when assessing the response of the gingival
tissues to gentle probing.
Conclusions
It is evident that periodontal tissue is susceptible to a
range of systemic medications. Such drug therapy
can produce unwanted effects (e.g. gingival over-
growth), and reduce or increase the expression of
periodontal disease. The periodontium may also be
the target of adverse reactions. This emphasizes the
importance of regular medical and drug histories and
thorough oral and periodontal screening for all
patients, especially the elderly.
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129
Structure of periodontal tissues
in health and disease*
ANTONI O NANCI & DI ETER D. BOSSHARDT
The periodontium, dened as those tissues support-
ing and investing the tooth, comprises root cemen-
tum, periodontal ligament, bone lining the tooth
socket (alveolar bone), and that part of the gingiva
facing the tooth (dentogingival junction). The wide-
spread occurrence of periodontal diseases and the
realization that lost tissues can be repaired and,
perhaps, regenerated has generated considerable
interest in the factors and cells regulating their for-
mation and maintenance. It is important to under-
stand that each of the periodontal components has
its very specialized structure and that these structural
characteristics directly dene function. Indeed,
proper functioning of the periodontium is only
achieved through structural integrity and interaction
between its components.
In recent years, a number of detailed descriptions
of the structural and compositional features of
periodontal tissues have been published (3, 57, 9, 15,
17, 46, 50, 56, 58, 61); we refer the reader to these
for a comprehensive description of the develop-
ment, formation, and structure of periodontal tissues.
The present review will focus on structurefunction
relationships pertinent to understanding periodontal
tissue breakdown and the repair regeneration of
affected structures.
Healthy periodontal tissues
Dentogingival junction
The dentogingival junction (gingiva facing the tooth)
is an adaptation of the oral mucosa that comprises
epithelial and connective tissue components. The
epithelium is divided into three functional com-
partments gingival, sulcular, and junctional
epithelium and the connective tissue into super-
cial and deep compartments. The junctional epi-
thelium plays a crucial role since it essentially seals
off periodontal tissues from the oral environment.
Its integrity is thus essential for maintaining a
healthy periodontium. Periodontal disease sets in
when the structure of the junctional epithelium
starts to fail, an excellent example of how structure
determines function.
The junctional epithelium
The junctional epithelium arises from the reduced
enamel epithelium as the tooth erupts into the oral
cavity. It forms a collar around the cervical portion of
the tooth that follows the cementoenamel junction
(Fig. 1). The free surface of this collar constitutes the
oor of the gingival sulcus. Basically, the junctional
epithelium is a nondifferentiated, stratied squa-
mous epithelium with a very high rate of cell turn-
over. It is thickest near the bottom of the gingival
sulcus and tapers to a thickness of a few cells as it
descends apically along the tooth surface. This epi-
thelium is made up of attened cells oriented paral-
lel to the tooth that derive from a layer of cuboidal
basal cells situated away from the tooth surface that
rest on a basement membrane. Suprabasal cells have
a similar ultrastructure and, quite remarkably,
maintain the ability to undergo cell division. The cell
layer facing the tooth provides the actual attachment
of the gingiva to the tooth surface by means of a
structural complex called the epithelial attachment.
This complex consists of a basal lamina-like structure
that is adherent to the tooth surface and to which the
supercial cell layer is attached by hemidesmosomes.
The basal lamina-like structure is a specialized
extracellular matrix in which typical basement
membrane constituents have not been immuno-
detected in any signicant quantity but which is *Parts of this article are adapted from Reference 50.
11
PERIODONTOLOGY 2000
enriched in glycoconjugates and contains laminin 5.
The latter matrix protein mediates cell adhesion and
regulates the polarization and migration of kera-
tinocytes (27).
Junctional epithelial cells differ considerably from
those of the gingival epithelium. They contain more
cytoplasm, rough endoplasmic reticulum, and Golgi
bodies. They exhibit fewer tonolaments and des-
mosomes, and wider intercellular spaces. The latter
uid-lled spaces normally contain polymorpho-
nuclear leukocytes and monocytes that pass from the
subepithelial connective tissue through the junc-
tional epithelium and into the gingival sulcus. The
mononuclear cells, together with molecules they
secrete and others originating from junctional epi-
thelial cells, blood and tissue uid represent the rst
line of defense in the control of the perpetual
microbial challenge. Among these molecules are
a- and b-defensins, cathelicidin LL-37, interleukin
(IL)-8, IL-1a and -1b, tumor necrosis factor-a, inter-
cellular adhesion molecule-1, and lymphocyte func-
tion antigen-3.
Connective tissue compartment
The connective tissue supporting the junctional epi-
thelium is structurally different from that supporting
the oral gingival epithelium. Even in clinically normal
circumstances, it shows an inammatory cell inl-
trate. The gingival connective tissue adjacent to the
junctional epithelium contains an extensive vascular
plexus. Inammatory cells such as polymorphonu-
clear leukocytes and T-lymphocytes continually
extravasate from this dense capillary and postcapil-
lary venule network, and migrate across the junc-
tional epithelium into the gingival sulcus and
eventually the oral uid. The vascular distribution in
the gingival lamina propria is described in detail in
Schroeder & Listgarten (58).
One point of view considers the junctional epi-
thelium as an incompletely developed stratied
squamous epithelium. Alternatively, it may be viewed
as a structure that evolves along a different pathway
and produces the components of the epithelial
attachment instead of progressing further into a
keratinized epithelium. The special nature of the
junctional epithelium is believed to reect the fact
that the connective tissue supporting it is function-
ally different than that of the sulcular epithelium, a
difference with important implications for under-
standing the progression of periodontal disease and
the regeneration of the dentogingival junction after
periodontal surgery. The subepithelial connective
tissue (lamina propria) is believed to provide
instructive signals for the normal progression of
stratied squamous epithelia (36, 38). Such signaling
presumably is absent from deeper connective tissues
so that epithelium in contact with it does not attain
the same degree of differentiation.
Thus the sulcular epithelium, in marked distinc-
tion to the gingival epithelium, is nonkeratinized,
yet both are technically supported by a similar
lamina propria. Indeed, this difference in epithelial
expression may be attributed to inammation. Even
under normal clinical conditions, the connective
tissue associated with the dentogingival junction is
slightly inamed. If the inammatory process is
removed by implementation of a strict regimen of
oral hygiene combined with antibiotic coverage in
experimental animals, the sulcular epithelium kera-
tinizes (21, 22).
Cementum
Cementum is the hard, avascular connective tissue
that coats the roots of teeth and that serves primarily
Fig. 1. Backscattered scanning electron micrograph of a
decalcied tissue section showing the cervical region of a
rat tooth with the junctional epithelium (JE), the enamel
space (ES), and the cemento-enamel junction (CEJ).
Numerous blood vessels (BV) are present in the connect-
ive tissue (CT) of the lamina propria. Note how the enamel
space extends between cementum and dentin (arrow-
head), a situation which may give the impression that
there is an intermediate layer between them. D, dentin.
12
Nanci & Bosshardt
to invest and attach the principal periodontal liga-
ment bers. There are basically two varieties of ce-
mentum distinguished on the basis of the presence or
absence of cells within it and the origin of the colla-
gen bers of the matrix.
Cementum varieties
Acellular extrinsic ber cementum (primary cemen-
tum or acellular cementum) is found on the cervical
half to two thirds of the root (Fig. 24). It develops
very slowly and is considered to be acellular since the
cells that form it remain on its surface. The very high
number of principal periodontal ligament bers
inserting into the AEFC (where they are called
Sharpeys bers) points to its important function in
tooth attachment. The overall degree of mineraliza-
tion of AEFC is about 4560%, but soft X-ray
examination reveals that the innermost layer is less
mineralized and that the outer layers are character-
ized by alternating bands of more and less mineral
content that run parallel to the root surface.
Cellular intrinsic ber cementum (secondary
cementum, cellular cementum) is distributed along
the apical thirdor half of the root andinfurcationareas
(Fig. 5). As cellular intrinsic ber cementum is also
produced as a repair tissue that lls resorptive defects
and root fractures, it may also be found further coro-
nally. Collagen produced by cementoblasts (intrinsic
collagen bers) and the presence of cementoblasts
entrapped in lacunae within the matrix they produce
(cementocytes) are the characteristic features of cel-
lular intrinsic ber cementum. The heterogeneous
collagen organization, its rapid speed of formation,
and the presence of cells and lacunae may be the
reason why this cementum variety is less well miner-
alized than acellular extrinsic ber cementum.
Cellular intrinsic ber cementum constitutes the
intrinsic component of cellular mixed stratied
Fig. 2. Backscattered scanning electron micrographs
showing the development of acellular extrinsic ber ce-
mentum (AEFC) in a human premolar from apical (A) to
cervical (B, C). A) Following disintegration of Hertwigs
epithelial root sheath (HERS), cells on the exposed root
surface implant a collagenous ber fringe (FF) into the not
yet mineralized dentin matrix (PD predentin). B) The
ber fringe is oriented perpendicular to the root surface
and engulfs the adjacent cells. C) When the cementum
layer has attained a thickness of approximately 10 lm,
most of the fringe bers are still short, while others have
elongated into the periodontal ligament (PL) space. D,
dentin; MF, mineralization front; P, pulp; Od, odonto-
blasts.
13
cementum, which possesses a stratication that is
derived from consecutively deposited, alternating
layers of acellular extrinsic ber cementum and
cellular intrinsic ber cementum. Cellular mixed
stratied cementum is not found in rodent molars
but is always present in human teeth. Because the
Fig. 3. Transmission electron micro-graph illustrating the
cervical root surface of a human tooth. acellular extrinsic
ber cementum (AEFC), which prevails in this root region,
is characterized by densely packed periodontal ligament
bers (PLF) that enter the cementum layer at the miner-
alization front (MF). Cb, cementoblast; N, nucleus.
Fig. 4. Electron micrographs of acellular extrinsic ber
cementum (AEFC) from tissue sections following im-
munogold labeling for bone sialoprotein (BSP). A) Perio-
dontal ligament bers (PLF) enter the cementum layer at
the mineralization front (MF). Labeling predominates over
the interbrillar cementum matrix. B) In the region of
the dentino-cemental junction, cemental and dentinal
collagen brils overlap and interdigitate. noncollagenous
proteins like bone sialoprotein ll the wide interbrillar
spaces. D, dentin.
14
Nanci & Bosshardt
intrinsic cementum variety can be formed very rap-
idly and focally, it may serve as a means to adjust the
tooth position to new requirements.
Biochemical composition of cementum
The composition of cementum resembles that of
bone. As a bulk, it contains about 50% mineral
(substituted apatite) and 50% organic matrix. Type I
collagen is the predominant organic component,
constituting up to 90% of the organic matrix. Other
collagens associated with cementum include type III,
a less cross-linked collagen found in high concen-
trations during development and repair regener-
ation of mineralized tissues, and type XII, a
bril-associated collagen with interrupted triple
helices (FACIT) that binds to type I collagen and also
to noncollagenous matrix proteins. Trace amounts of
other collagens, including type V, VI, and type XIV,
are also found in extracts of mature cementum;
however, these may be contaminants from the peri-
odontal ligament region associated with bers
inserted into cementum. Almost all noncollagenous
matrix proteins identied in cementum are also
found in bone (7). These include bone sialoprotein
(Fig. 4), dentin matrix protein 1 (DMP-1) (20, 24, 44),
dentin sialoprotein (1), bronectin, osteocalcin,
osteonectin, osteopontin, tenascin (47, 69), proteo-
glycans, proteolipids, and several growth factors
including cementum growth factor that appears to be
an insulin-like growth factor (IGF)-like molecule.
Enamel proteins have also been suggested to be
present in cementum. It has been reported that
Hertwigs epithelial root sheath (HERS) cells may
synthesize amelogenins that accumulate on the
forming root surface to form a layer, referred to as
intermediate cementum (4042, 60). To date, how-
ever, there is no conclusive evidence that either
amelogenins or nonamelogenins accumulate in nor-
mal cementum matrix constituents or even form a
distinct layer between dentin and cementum.
Whenever enamel matrix proteins are found on the
root, their presence is limited to a very short, cervical
region which likely represents the cervical extremity
of the crown onto which cementum is deposited
(11, 12). Sporadic expression of enamel proteins has
also been reported along the root in porcine teeth
Fig. 5. Backscattered scanning electron micrographs
showing the development of cellular intrinsic ber
cementum (CIFC) in a human premolar from apical (A) to
a coronal direction (B, C). A) Following disintegration of
Hertwigs epithelial root sheath (HERS), cementoblasts
(Cb) on the exposed root surface rapidly deposit the
cementum matrix onto the not yet mineralized dentin
matrix (PD = predentin). B) Some cementoblasts become
embedded as cementocytes (Cc) in their own matrix. C) In
a more mature, thicker cementum layer, the cementocytes
lodge in lacunae. D, dentin; Od, odontoblasts; PGE
2
,
prostaglandin E
2
.
15
(Fig. 6) (13), and in rodent molars in association with
epithelial cells entrapped in cellular intrinsic ber
cementum (12, 25, 26, 63). Finally, an apparently
unique cementum attachment protein has also been
identied in cementum (64).
Cementum development
Cementum formation takes place along the entire
root and during the entire life of the tooth. However,
its initiation is limited to the advancing root edge
during root formation. At this site, Hertwigs epithe-
lial root sheath, which derives from the apical
extension of the inner and outer enamel epithelium,
is believed to send an inductive message, possibly by
secreting some enamel matrix proteins, to the facing
ectomesenchymal pulp cells. These cells differentiate
into odontoblasts and produce a layer of predentin.
Soon after, HERS becomes fragmented and ectome-
senchymal cells from the inner portion of the dental
follicle can now come in contact with the predentin.
Some cells from the fragmented root sheath form
discrete masses surrounded by a basement mem-
brane, known as epithelial rests of Malassez that
persist in the mature periodontal ligament. Following
these events, cementoblasts will differentiate and
deposit cementum matrix onto the forming radicular
dentin. The origin of cementoblasts and series of
events that culminates in their differentiation is still
unresolved and will be discussed below.
Cementum formation
Acellular extrinsic ber cementum: during root
development in human teeth, the rst cells that align
along the newly formed, but not yet mineralized,
mantle dentin surface exhibit broblastic character-
istics (Fig. 2). These cells deposit collagen within the
unmineralized dentin matrix so that brils from both
matrices interdigitate. Mineralization of the mantle
dentin starts internally and does not reach the sur-
face until blending of collagen brils from both layers
has occurred. It then spreads across into cementum
matrix, thereby establishing the dentincementum
junction. Initial acellular extrinsic ber cementum
thus consists of a thin mineralized layer with a short
fringe of collagen bers implanted perpendicular to
the root surface. The cells on the root surface con-
tinue to deposit collagen so that the ber fringe
lengthens and thickens. At the same time, they also
Fig. 6. Transmission electron micro-graph showing the
cervical root surface at the beginning of root formation in
a porcine tooth; the tissue section was processed for
immunogold labeling with anti-amelogenin antibody.
Matrix masses containing amelogenin (arrowheads) are
sporadically observed along the dentin (D) surface. They
co-localize with the collagenous pre-cementum (PC)
matrix. AMEL; amelogenin; N, nucleus.
16
Nanci & Bosshardt
secrete noncollagenous matrix proteins that ll in the
spaces between the collagen bers and regulate
mineralization of the forming cementum layer
(Fig. 4). This activity continues until about 1520 lm
of cementum has been formed, at which time the
intrinsic brous fringe becomes connected to the
developing periodontal ligament ber bundles
(Fig. 3). Thereafter, acellular extrinsic ber cemen-
tum formative cells will be essentially engaged in
synthesis of noncollagenous matrix proteins; collagen
brils that embed in it will be formed by periodontal
ligament broblasts. No morphologically distinct
layer of cementoid, akin to osteoid or predentin,
exists on the surface of acellular extrinsic ber
cementum. Although this cementum variety is classi-
ed as having extrinsic bers, one may question
whether its initial part should rather be classied as
having intrinsic bers. As described above, the col-
lagenous matrix of the rst-formed cementum is the
result of cementum-associated cells and is elaborated
before the periodontal ligament forms; therefore,
the collagen is of local origin and thus of intrinsic
derivation.
Cellular intrinsic ber cementum: after at least half
of the root has been formed, cementoblasts start
forming a less mineralized variety of cementum that
is distinctive in that its constituent collagen brils are
produced by the cementoblasts themselves (Fig. 5).
In all cases, the rst collagen is deposited onto the
unmineralized dentin surface such that brils from
both layers intermingle. As for acellular extrinsic ber
cementum, cellular intrinsic ber cementum-form-
ing cementoblasts also manufacture a number of
noncollagenous matrix proteins that ll in the spaces
between the collagen brils, regulate mineral
deposition and impart cohesion to the mineralized
layer. A layer of unmineralized matrix, termed
cementoid, is established at the surface of the min-
eralized cementum matrix, with the mineralization
front at the interface between the two layers. In
contrast to osteoid, cementoid is not as regular and
readily discernible. As the process proceeds, some
cementoblasts become trapped in the matrix they
form. These entrapped cells, with reduced secretory
activity, are called cementocytes and sit in lacunae.
The structural organization of the matrix and the
presence of cells in it give cellular intrinsic ber
cementum a bone-like appearance. Collagen brils
are produced rapidly and deposited haphazardly
during the initial phase; however, subsequently the
bulk of brils organize as bundles oriented mostly
parallel to the root surface. When the periodontal
ligament becomes organized, cementum may form
around some of the periodontal ligament ber bun-
dles they are thus incorporated into cementum and
become partially mineralized. In human teeth,
incorporation of periodontal ligament bers into
cellular intrinsic ber cementum occurs only rarely,
essentially in the acellular extrinsic ber cementum
component of cellular mixed stratied cementum.
How does cementum hold onto dentin?
The attachment mechanism of cementum to dentin
is both of biological interest and of clinical relevance,
since pathological alterations and clinical interven-
tions may inuence the nature of the exposed root
surface and hence the quality of the new attachment
that forms when repair cementum is deposited. The
mechanism by which these hard tissues bind
together is essentially the same for acellular extrinsic
ber cementum and cellular intrinsic ber cemen-
tum. Mineralization of the mantle dentin starts
internally and does not reach the surface until the
collagen brils of dentin and cementum have had
time to blend together. It then spreads through the
surface layer of dentin, across the dentincementum
junction and into cementum, essentially resulting in
an amalgamated mass of mineral. Whereas dentin
mineralization is initiated by matrix vesicles, the
subsequent spread of mineral deposition is under the
regulatory inuence of noncollagenous matrix
proteins. From a biomechanical perspective, this
arrangement appears optimal for a strong union
between dentin and cementum. In acellular extrinsic
ber cementum of rodent teeth, cementum is
deposited onto mineralized dentin, making amalga-
mation of dentin and cementum impossible and
establishing a weakened interface. Indeed, histologi-
cal sections of rodent teeth often show a separation
between dentin and cementum in the cervical third
of the root. Although tissue processing is commonly
held responsible for tissue separation in histologic
sections, arguments have been raised to question this
generalized interpretation (16). Interestingly, repair
cementum adheres very well to the root surface if
a resorptive phase precedes new matrix deposition
(14, 8), implying that odontoclasts favorably precon-
dition the root surface. Chemical preconditioning of
the root surface with acids or chelators (Fig. 7B) is an
often-applied step in periodontal therapy (43, 45).
Following various regenerative procedures, tissue
separation between repair cementum and the treated
root surface is frequently observed, implying poor
attachment quality (8, 16) and suggesting that there
17
still is room for improvements in chemical root sur-
face conditioning of periodontitis-affected teeth.
Origin of cementoblasts and periodontal ligament
broblasts
There are still several fundamental issues that need to
be resolved and whose clarication is not only
essential to understand the process of cemento-
genesis but, most importantly, to devise targeted
therapeutic approaches for the prevention and
treatment of periodontal diseases. These include
determining the following:
the precursors of cementoblasts;
whether cementoblasts are a distinct cell popula-
tion that expresses unique gene products;
whether acellular and cellular cementum are dis-
tinct tissues;
what regulates formation and maintenance of
periodontal ligament vs. cementum, thus pre-
venting fusion of the root to the alveolar bone
(ankylosis).
The long-standing view is that precursors of
cementoblasts and periodontal ligament broblasts
reside in the dental follicle and that factors within the
local environment regulate their ability to function as
cementoblasts that form root cementum, broblasts
of the periodontal ligament or osteoblasts forming
bone tissue (32). It is widely held that inltrating
dental follicle cells receive a reciprocal inductive
signal from the forming dentin and differentiate into
cementoblasts. However, there is increasing evidence
that HERS cells may undergo epithelialmesenchy-
mal transformation into cementoblasts during
development (7). This is a fundamental process in
developmental biology that occurs, among others, as
ectodermal cells migrate away from the neural crest
and during medial edge fusion of the palatal shelves.
Structural and immunocytochemical data support
the possibility that cementoblasts derive, at least in
part, from transformed epithelial cells of HERS. In
rodents, initial formation of acellular cementum
takes place in the presence of epithelial cells and it
has been shown that enamel organ cells are capable
of producing typical mesenchymal products such as
type I collagen, bone sialoprotein, and osteopontin
(10, 12, 13, 18, 48).
There is still debate as to whether acellular (pri-
mary) and cellular (secondary) cementum are pro-
duced by distinct populations of cells expressing
spatio-temporal behaviors that result in the charac-
teristic histological differences between these tissues.
The possibility has been raised that acellular extrinsic
Fig. 7. Transmission electron micrographs of human
teeth affected by periodontitis and conservatively treated
with root scaling and planing, and root surface deminer-
alization using EDTA. A) A long junctional epithelium
(LJE) may establish on the treated root. Note the presence
of bacteria (arrowheads) among the epithelial cells. B) The
EDTA treatment aims at exposing collagen brils (CF).
AEFC, acellular extrinsic ber cementum.
18
Nanci & Bosshardt
ber cementum is formed by HERS-derived cells,
whereas cellular intrinsic ber cementum is pro-
duced by cells that derive from the dental follicle (68).
Experimental evidence supports the concept that
the periodontal ligament is a repository for cells
involved in the formation of cementum, periodontal
ligament itself, and alveolar bone (37); however, the
nature and precise location of progenitor cells remain
to be determined. It is also not known whether dis-
tinct precursor cell lines exist for each of the three
support tissues or whether periodontal ligament
broblasts, cementoblasts, and osteoblasts arise from
a common precursor. The complexity of the perio-
dontal ligament is enhanced by the fact that it con-
tains several cell types (broblastic subpopulations,
osteoblasts, cementoblasts, endothelial cells, peri-
vascular cells, blood-borne cells, and epithelial cells).
In addition, recent ndings also suggest the presence
of cells with stem cell characteristics (59). A com-
prehensive review of the literature on cell origin
and differentiation has recently been published by
Bosshardt (7). This review highlights several lines of
evidence that support the concept that cemento-
blasts producing both acellular extrinsic ber
cementum and cellular intrinsic ber cementum are
unique phenotypes that differ from osteoblasts, and
proposes a model that may explain how cell diversity
evolves in the periodontal ligament. This new theory
proposes that cells derived from HERS play an
essential role in tissue development and mainten-
ance, and that periodontal regeneration recapitulates
tooth development. Cells descending from HERS may
give direct rise to cells that form new cementum and
periodontal ligament tissues, or play an indirect role
by producing the necessary signaling molecules for
cell recruitment and differentiation. Understanding
cell origin and cell differentiation mechanisms within
the periodontal ligament is mandatory for the
development of more effective therapies aimed at
achieving true and signicant periodontal regener-
ation.
Molecular factors regulating cementogenesis
Bone morphogenetic proteins: Bone morphogenic
proteins are members of the transforming growth
factor b (TGF-b) superfamily that act through
transmembrane serine threonine protein kinase
receptors. These signaling molecules have a variety
of functions during morphogenesis and cell differ-
entiation and, in teeth, they are considered to be
part of the network of epithelialmesenchymal
signaling molecules regulating crown development.
The roles for bone morphogenic proteins in root
development, including whether they are involved
in epithelialmesenchymal signaling, and the sign-
aling pathways and transcription factors involved in
modulating their behavior remain to be dened.
However, it is known that several of the bone
morphogenic proteins, including BMP-2, -4, and -7,
promote differentiation of preosteoblasts and
putative cementoblast precursor cells. In this con-
text, bone morphogenic proteins have been suc-
cessfully used to induce periodontal regeneration in
a number of experimental models but their clinical
use is still lagging behind.
Epithelial factors: The same two populations of
cells involved in crown morphogenesis, i.e. enamel
epithelium and ectomesenchymal cells, also take part
in root formation. It would thus not be surprising if
some of the same signaling molecules implicated in
crown morphogenesis were also active during
development of the root. Prospective candidates
include enamel matrix proteins, parathyroid hor-
mone-related protein and basement membrane
constituents. In the case of enamel matrix proteins,
the debate centers on the fact that they have not
been consistently detected along the root, in every
species and in all teeth. However, this inconsistency
does not rule out their participation in root forma-
tion. Some proteins may still be transiently secreted
in limited amounts at early stages of root formation
by HERS cells to inuence odontoblast or cemento-
blast differentiation; such a limited expression would
be difcult to detect.
Matrix proteins: as indicated above, bone
sialoprotein and osteopontin are fundamental con-
stituents of cementum matrix, during both its
development and repair. Present data suggest that
osteopontin is involved in regulating mineral growth,
whereas bone sialoprotein promotes mineral forma-
tion on the root surface (18, 49). They may also
be involved in cellular events through their RGD
cell-binding motifs. Since no tooth root develop-
mental anomalies have to date been reported in
osteopontin knockout mice, it is likely that other
noncollagenous matrix proteins compensate for the
absence of osteopontin in these animals.
Bone Gla protein (osteocalcin) is a marker for
maturation of osteoblasts, odontoblasts and
cementoblasts that may regulate the extent of
mineralization. No root development and forma-
tion problems have so far been reported in knockout
mice.
Transcription factors: Runx-2 (runt-related tran-
scription factor 2), also known as Cbfa1 (core binding
factor alpha 1), and osterix, downstream from Cbfa1,
19
have been identied as master switches for differen-
tiation of osteoblasts. Runx-2 has now been found
in dental follicle cells, periodontal ligament cells,
cementoblasts, cementocytes, odontoblasts, and
ameloblasts. Based on proposed similarities with
osteoblasts, they may also be involved in cemento-
blast differentiation. However, the expression of
Runx-2 in fully differentiated cells suggests additional
roles. The exact factors triggering expression and or
activation of these key transcription factors are cur-
rently being investigated, however, bone genetic
proteins have already been identied as factors
promoting expression of Runx-2.
Other factors: Other molecules that may have a
regulatory function in cementoblast differentiation
and activity and that are found within the devel-
oping and mature periodontal tissues include alka-
line phosphatase, several growth factors (e.g. IGF,
TGF-b and platelet-derived growth factor), metal-
loproteinases, and proteoglycans. The latter are
important in the formation of mineralized tissues,
although a specic role related to promoting
inhibiting differentiation of the cementoblasts has
not been established. An accumulation of proteo-
glycans has been observed at the dentincementum
junction and it has been proposed that, together
with other noncollagenous matrix proteins such as
bone sialoprotein and osteopontin, they may be
associated with initial mineralization and ber
attachment (65).
The signicance of alkaline phosphatase for
cementum formation has long been appreciated,
particularly with regard to the potential cellular and
formative distinctiveness between acellular extrinsic
ber cementum and cellular intrinsic ber cemen-
tum. In mice null for tissue nonspecic alkaline
phosphatase gene or rats treated with bisphospho-
nates, acellular cementum formation is signicantly
affected whereas cellular cementum appears to
develop normally. This suggests differences in cell
types and or factors controlling the development of
these two varieties of cementum. In the human
counterpart, hypophosphatasia, characterized by
very low levels of alkaline phosphatase, there appears
to be limited or no cementum formation. In contrast,
mice with mutations in genes that maintain extra-
cellular pyrophosphate levels, such as ank and PC-1,
resulting in limited levels of pyrophosphate, exhibit
more cellular cementum when compared with wild-
type littermates, even at early stages of root devel-
opment (51). These ndings suggest an important
role for phosphate in controlling the rate of cemen-
tum formation.
Periodontal ligament
The bulk of the periodontal ligament is that soft,
specialized connective tissue situated between the
cementum covering the root of the tooth and the
bone forming the socket wall (alveolo-dental liga-
ment). It ranges in width from 0.15 to 0.38 mm,
with its thinnest portion around the middle third of
the root, showing a progressive decrease in thick-
ness with age. It is a connective tissue particularly
well adapted to its principal function, supporting
the teeth in their sockets and at the same time
permitting them to withstand the considerable for-
ces of mastication. In addition, the periodontal
ligament has the capacity to act as a sensory
receptor necessary for the proper positioning of the
jaws during mastication and, very importantly, it
is a cell reservoir for tissue homeostasis and
repair regeneration.
Periodontal ligament formation
Apart from the recognition that the periodontal
ligament forms within the dental follicle region,
the exact timing of events associated with the
development of an organized periodontal ligament
varies among species, with individual tooth families
and between primary and permanent teeth. At the
beginning, the ligament space is occupied by an
unorganized connective tissue extending between
bone and cementum. This tissue is then remodeled
and the provisional extracellular matrix is converted
into a ber system organized as bundles that
extend between the bone and cementum surfaces.
The reorganized tissue can now establish continuity
across the ligament space and thereby secure
the attachment of the tooth to bone. Eruptive
tooth movement and the establishment of occlu-
sion then further modify this initial attachment
system.
Periodontal ligament cells and extracellular matrix
constituents
Similar to all other connective tissues, the periodontal
ligament consists of cells and an extracellular com-
partment comprising collagenous and noncollagen-
ous matrix constituents. The cells include osteoblasts
and osteoclasts, broblasts, epithelial cell rests of
Malassez, monocytes and macrophages, undifferen-
tiated mesenchymal cells, and cementoblasts and
odontoclasts. The extracellular compartment consists
mainly of well-dened collagen ber bundles
embedded in an amorphous background material,
known as ground substance.
20
Nanci & Bosshardt
Fibroblasts: The principal cells of the periodontal
ligament are broblasts. Although all broblasts look
alike microscopically, heterogeneous cell popula-
tions exist between different connective tissues and
also within the same connective tissue. The bro-
blasts of the periodontal ligament are characterized
by their rapid turnover of the extracellular compart-
ment, in particular, collagen. Periodontal ligament
broblasts are large cells with an extensive cytoplasm
containing an abundance of organelles associated
with protein synthesis and secretion. They have a
well-developed cytoskeleton and show frequent
adherens and gap junctions, reecting the functional
demands placed on the cells. Ligament broblasts
are aligned along the general direction of the ber
bundles and extend cytoplasmic processes that wrap
around them. The collagen brils of the bundles are
continuously being remodeled by the broblasts,
which are capable of simultaneously synthesizing
and degrading collagen.
Epithelial cells: The epithelial cells in the perio-
dontal ligament are remnants of HERS and known as
the epithelial cell rests of Malassez. They occur close
to the cementum as a cluster of cells that form an
epithelial network, and seem to be more evident or
abundant in furcation areas. The function of these
rests is unclear but they could be involved in
periodontal repair/regeneration (discussed in [7]).
Undifferentiated mesenchymal cells: An important
cellular constituent of the periodontal ligament is the
undifferentiated mesenchymal cell, or progenitor
cell. The fact that new cells are being produced for
the periodontal ligament whereas cells of the liga-
ment are in a steady state means that selective
deletion of cells by apoptosis must balance the pro-
duction of new cells. In periodontal wound healing,
the periodontal ligament contributes cells not only
for its own repair but also to restore lost bone and
cementum (5, 37). Recently, cells with stem cell
characteristics have been isolated from the human
periodontal ligament (59).
Fibers: The predominant collagens of the perio-
dontal ligament are type I, III, and XII, with
individual brils having a relatively smaller average
diameter than tendon collagen brils, a difference
believed to reect the relatively short half-life of
ligament collagen, and hence less time for brillar
assembly. The vast majority of collagen brils in the
periodontal ligament are arranged in denite and
distinct ber bundles, and these are termed principal
bers. Each bundle resembles a spliced rope; indi-
vidual strands can be continually remodeled while
the overall ber maintains its architecture and func-
tion. In this way the ber bundles are able to adapt to
the continual stresses placed on them. The extrem-
ities of collagen ber bundles are embedded in
cementum or bone. The embedded portion is
referred to as Sharpeys bers. Sharpeys bers in
primary acellular cementum are fully mineralized;
those in cellular cementum and bone are generally
only partially mineralized at their periphery.
Other ber bundles (gingival ligament bers) are
found extending from the cervical region of a tooth to
that of the adjacent tooth (transseptal ligament
bers), and in the lamina propria of the gingiva.
These, together with the main alveolo-dental liga-
ment bers, constitute the periodontal ligament-ber
system.
Elastic bers: There are three types of elastic bers:
elastin, oxytalan, and elaunin. Only oxytalan bers
are present within the periodontal ligament; how-
ever, elaunin bers may also be found in association
with ber bundles in the gingival ligament.
Oxytalan bers are bundles of microbrils that run
more or less vertically from the cementum surface,
forming a three-dimensional branching meshwork
that surrounds the root and terminates in the apical
complex of arteries, veins, and lymphatics. They are
also associated with neural elements. Although their
function has not been fully determined, they are
thought to regulate vascular ow in relation to tooth
function. Because they are elastic, they can expand in
response to tensional variations, with such variations
then registered on the walls of the vascular struc-
tures.
Noncollagenous matrix proteins: Several noncolla-
genous matrix proteins produced locally by resident
cells or brought in by the circulation are found in the
periodontal ligament, these include alkaline phos-
phatase (31), proteoglycans (33), and glycoproteins
such as undulin, tenascin, and bronectin (69).
Ground substance: The periodontal ligament
ground substance has been estimated to be 70%
water and is thought to have a signicant effect on
the tooths ability to withstand stress loads. There is
an increase in tissue uids within the amorphous
matrix of the ground substance in areas of injury and
inammation.
Periodontal ligament homeostasis and adaptation
to functional demand
A remarkable capacity of the periodontal ligament is
that it maintains its width more or less over time,
despite the fact that it is squeezed in between two
21
hard tissues. Compelling evidence exists indicating
that populations of cells within the periodontal liga-
ment, both during development and during regen-
eration, secrete molecules that can regulate the
extent of mineralization and prevent the fusion of
tooth root with surrounding bone, e.g. ankylosis.
Among these molecules, balance between the activ-
ities of bone sialoprotein and osteopontin may
contribute to establishing and maintaining an un-
mineralized periodontal ligament region. Matrix Gla
protein is also present in periodontal tissues; based
on its role as an inhibitor of mineralization, it may
also act to preserve the periodontal ligament width.
At the cell level, it has been reported that Msx2 pre-
vents the osteogenic differentiation of periodontal
ligament broblasts by repressing Runx2 Osf2
transcriptional activity (67). Indeed, Msx2 may play a
central role in preventing ligaments and tendons, in
general, from mineralizing (67). It has also been
suggested that glycosaminoglycans (39) or RGDce-
mentum attachment protein, a collagen-associated
protein (52) may also play a role in maintaining the
unmineralized state of the periodontal ligament. At
this point, the issue of how the periodontal ligament
stays uncalcied while being trapped between two
calcied tissues remains unresolved and will require
more attention.
The periodontal ligament has also the capacity to
adapt to functional changes. When the functional
demand increases, the width of the periodontal
ligament can increase by as much as 50%, and the
ber bundles also increase markedly in thickness.
Conversely, a reduction in function leads to narrow-
ing of the ligament and a decrease in number and
thickness of the ber bundles. These functional
modications of the periodontal ligament also
implicate corresponding adaptive changes in the
bordering cementum and alveolar bone.
Alveolar bone
The alveolar process is that bone of the jaws con-
taining the sockets (alveoli) for the teeth. It consists of
outer cortical plates (buccal, lingual, and palatal) of
compact bone, a central spongiosa, and bone lining
the alveolus (alveolar bone). The cortical plate and
bone lining the alveolus meet at the alveolar crest. The
bone lining the socket is specically referred to as
bundle bone because it provides attachment for the
periodontal ligament ber bundles.
The cortical plates consist of surface layers (lamel-
lae) of ne-bered bone supported by Haversian
systems. They are generally thinner in the maxilla and
thickest on the buccal aspect of mandibular premo-
lars and molars. The trabecular (or spongy) bone
occupying the central part of the alveolar process also
consists of bone disposed in lamellae, with Haversian
systems present in the larger trabeculae. Yellow
marrow, rich in adipose cells, generally lls the int-
ertrabecular spaces, although sometimes there can
also be some red or hematopoietic marrow. Trabe-
cular bone is absent in the region of the anterior teeth
and, in this case, the cortical plate and alveolar bone
are fused together. The important part of this com-
plex, in terms of tooth support, is the bundle bone,
which consists of successive layers of intrinsic ber
bundles running more or less parallel to the socket.
Embedded within this bundle bone, almost perpen-
dicular to its surface, are the extremities (Sharpeys
bers) of the extrinsic collagen ber bundles of the
periodontal ligament (which, as in cellular intrinsic
ber cementum cellular mixed stratied cementum,
are mineralized only at their periphery). Because the
tooth is constantly making minor movements and
alveolar bone must respond to the functional demand
placed on it by the forces of mastication, the bone of
the socket wall is constantly remodeled and its
structural organization varies along the wall (56). The
presence of an alveolar bone along the entire tooth
socket separates the support bone anatomically and
functionally from the periodontal ligament. The
organization of the alveolar process is yet another
example of structurefunction relationship in the
periodontium.
Whereas the overall formationandregulatory events
in alveolar bone are the same as at other anatomical
sites, alveolar bone is distinctive because it turns over
very rapidly and it is lost in the absence of a tooth.
These two characteristics suggest that local regula-
tory mechanisms are particularly important in the
case of alveolar bone. They also clearly demonstrate
the interdependence of the periodontal tissues and
underlines the important fact that the periodontal
tissues function together as a unit.
The remodeling process of alveolar bone is essen-
tially similar to that of bone in general (56). However,
resorption is asynchronous, so that periodontal
ligament attachment is lost only focally and for short
periods of time. During tooth migration, the distri-
bution of force is such that bone lost by resorption on
one surface of the tooth socket is balanced by bone
formation along the opposite surface. This bone
balance together with the continued deposition of
cementum throughout life act to maintain a more or
less constant relationship between the root surface
and that of the alveolar socket. The factors that
22
Nanci & Bosshardt
trigger the various events in periodontal homeostasis
still need to be ascertained. In elucidating this
question, perhaps we could take advantage of events
during orthodontic treatment, which essentially rep-
resents a circumstance where the limits of the normal
physiology are stretched.
As was mentioned above, there are progenitor cells
in the periodontal ligament that can differentiate into
osteoblasts for the physiological maintenance of
alveolar bone and, most likely, for its repair as well.
Since the evidence points towards a common pre-
cursor residing in the periodontal ligament for
cementoblasts, periodontal ligament broblasts and
bone cells (7), an important issue for devising
targeted regenerative therapies is identication of the
signals that guide cell differentiation along each of
these pathways.
Pathological structure-function
alterations of periodontal tissues
Gingivitis and periodontitis (Fig. 7 and 8) are infec-
tious diseases that afict a high percentage of the
population, even at younger ages. In its FY2003 Fact
Sheet, the American Association for Dental Research
reports that 48% of adults aged 3544 years of age
have inammation of the gingiva (gingivitis), and
22% destructive periodontal disease a major cause
of tooth loss. In addition, evidence has been
mounting that chronic periodontal diseases are
linked with major systemic diseases such as cardio-
vascular and pulmonary diseases (4, 23, 28, 53).
Although bacteria are essential for periodontitis to
develop, the fact that it develops to variable degrees
in different individuals suggests a multifactorial eti-
ology. All forms of periodontitis, however, appear to
have a common series of underlying events leading to
tissue breakdown and tooth attachment loss.
The junctional epithelium, by virtue of its struc-
tural and functional uniqueness, provides a very
efcient barrier against periodontal pathogens and
their products. However, periodontal pathogens, in
particular Porphyromonas gingivalis, may perturb its
integrity allowing subgingival spread of bacteria and
their antigens (9, 19, 35, 55). The ensuing inamma-
tory response leads to the degradation of the under-
lying connective tissue, rst around blood vessels and
Fig. 8. Light (A) and transmission electron (B) micro-
graphs of human teeth affected by periodontitis. A) The
periodontal pocket (PP) occupies the space between the
plaque bacteria (PB) adhering to the root surface and the
epithelium (PE) lining the pocket space. Note the large
number of blood vessels (BV) in the pocket epithelium. (B)
After root scaling and planing, periodontal pathogens may
re-establish a bacterial biolm within a few days on the
exposed dentin (D) surface.
23
then spreading into adjacent regions, resulting in the
structural and functional disintegration of the gin-
giva.
One of the rst changes of periodontitis is the
migration of the junctional epithelium along the root
surface and its elongation, resulting in the formation
of a long junctional epithelium and a gingival pocket.
This structural alteration is accompanied by a num-
ber of functional changes. The direction of neutrophil
migration and of the crevicular exudate ow across
the epithelium changes drastically as the free surface
of the epithelium is now displaced from the sulcus
bottom to the root surface. Also, the free surface
increases in size and is therefore exposed to more
bacterial plaque.
Just as the nature of the connective tissue with
which the junctional epithelium is in contact is
believed to inuence its development, one may
explain the formation of a long junctional epithelium
along the same lines. The junctional epithelium
needs a certain connective tissue environment to
establish itself whose specic characteristics have
not yet been dened but which, in a healthy perio-
dontium, is usually found near the cervical portion of
the tooth. When gingivitis sets in, the connective
tissue bordering the junctional epithelium is con-
tinuously altered by the inammatory response.
Thus, it needs to migrate deeper along the root sur-
face to nd a connective tissue structure that is intact
enough and capable of signaling the epithelium to
stop its downward movement, form a functional
epithelial attachment, and attach to the tooth
surface.
Bacteria cause tissue destruction indirectly by
exacerbating the hosts immune response. Recent
advances in the acquired immune responses invol-
ving B-lymphocytes, T-lymphocytes, and inamma-
tory mediators in the context of periodontal disease
progression have been reviewed by Teng (62) and
Yamazaki et al. (66). A number of pro-inammatory
cytokines and growth factors, in particular IL-1 and
tumor necrosis factor-a (TNF-a), are known to be
associated with bone resorption (Fig. 9). Normal
bone remodeling depends on a delicate balance
Fig. 9. Schematic illustration of the RANK-RANKL-osteoprotegerin system.
24
Nanci & Bosshardt
between bone formation and bone resorption.
Receptor of nuclear factor-kappa (RANK) and its lig-
and RANK-L, members of the tumor necrosis factor
family of receptors, are directly involved in the dif-
ferentiation of osteoclast precursors and activation
and survival of osteoclasts (Fig. 9). RANK-L is ex-
pressed by bone marrow stromal cells, osteoblasts,
and broblasts, whereas RANK is expressed by oste-
oclasts precursors and mature osteoclasts. The
binding of RANK and RANK-L induces osteoclast
differentiation and activity. Osteoprotegerin, which is
produced by bone marrow stromal cells, osteoblasts,
and periodontal ligament broblasts, however, com-
petes for this binding and functions as a soluble de-
coy receptor for RANK-L. Thus, osteoprotegerin is a
natural inhibitor of osteoclast differentiation and
activation. Any interference with this system can shift
the balance towards increased bone formation or
resorption. It has been shown that pro-inammatory
cytokines such as IL-1 and TNF-a, two very important
players in periodontal bone loss, regulate the
expression of RANK-L and osteoprotegerin. More-
over, T-cells also express RANK-L, which by binding
directly to RANK on the cell membrane of osteclast
progenitors, preosteoclasts, and osteoclasts, stimu-
lates both cell differentiation and activation of cells of
the osteoclast lineage. Thus, the shift of bone
homeostasis toward bone resorption in periodontitis
may be driven by pro-inammatory cytokines that
regulate the expression of RANK-L on both mesen-
chymal cells and specic activated T-cells. In this
context, it has been demonstrated that there is
diminished alveolar bone loss after oral infection
with P. gingivalis in mice lacking T-lymphocytes (2).
The discovery of this regulatory system linking bone
biology with immune cell biology (29, 30, 34) has
opened new therapeutic possibilities such as the
inhibition of RANKRANKL interaction through the
local application of osteoprotegerin.
Because of the exceptionally high rate of turnover
of collagen in the periodontal ligament, any inter-
ference with broblast function by disease rapidly
produces a loss of the tooths supporting tissue.
Importantly, in inammatory situations, such as
those associated with periodontal diseases, there is
an increased expression of matrix metalloproteinases
that aggressively destroy collagen (54). Thus,
attractive therapies for controlling tissue destruction
may include host-modulators that have the capacity
to inhibit matrix metalloproteinases.
When inammation reaches the root surface,
resorption may occur, resulting in its excavation. It is
likely that this destructive process involves some
imbalance in the RANK RANKL osteoprotegerin
system (Fig. 9) (7). Root resorption occurs quite
commonly with hyperplastic gingivitis and less fre-
quently adjacent to inammatory lesions in the per-
iodontal ligament. Fractures and microcracks in the
supercial root portion may facilitate invasion of
bacteria or diffusion of bacterial products into the
root. Damaged cementum and dentin may also serve
as a bacterial reservoir from which recolonization of
scaled and planed root surfaces can occur. On the
positive side, exposure to the oral environment often
leads to the establishment of a hypermineralized
zone in the supercial cementum layer. The mineral
crystals in this supercial layer are very resistant to
acid demineralization, slowing down the progression
of carious lesions.
Concluding remarks
In the preface of Periodontology 2000 (57), editor
Schroeder states the following: Insight into the
architecture of (human) tissues is necessary to enable
a creative mind to ask pertinent biological questions.
Indeed, knowledge of how tissue structure develops
and how it relates to function is fundamental for
understanding the disease process, and for devising
effective therapeutic strategies, particularly in the
case where tissue destruction, and hence a concom-
itant loss of function, ensues. The present chapter
has been assembled with this in mind, and with the
hope of providing investigators with solid bases for
regenerative therapies. One particularly good exam-
ple of induced tissue repair that may not give optimal
functional results is the stimulation of cellular
intrinsic ber cementum formation for root repair. As
was discussed, cellular intrinsic ber cementum
deposited onto periodontitis-affected roots that were
treated in various ways is not a major medium for
tooth attachment and, when deposited onto a min-
eralized surface, it does not interface with the
preexisting calcied matrix and is thus subject to
detachment. This may, however, be related to inap-
propriate root surface properties.
The rst line of defense is the junctional epithe-
lium. Its structural alteration is clearly the rst step
towards the progression of disease. Comparatively
little attention has been given to understanding
what triggers its formation and the composition of
the basal lamina-like structure that mediates its
attachment to the tooth surface. Could the constit-
uents of this adhesive layer be used to slow down
the downgrowth of the junctional epithelium and its
25
detachment from the tooth surface? Once disease
has progressed beyond the epithelial seal and
spreads to the connective tissue elements of the
periodontium, regeneration is complicated by the
fact that three tissues are now involved cementum,
the periodontal ligament, and bone. Although any of
these can be in principle rebuilt, one must
remember that tooth attachment function requires
that the architecture of all three tissues be restored
to corresponding degrees. In the case of cementum,
in addition to quantity, the type of cementum will
also be critical.
We still do not know whether the precursor to
periodontal cells is ectodermal or ectomesenchy-
mal, or whether there is more than one precursor.
This may be an issue of semantics, however, since
ectomesenchymal cells are also of ectodermal ori-
gin. The fact that current periodontal therapies
mainly stimulate cellular intrinsic ber cementum
formation and that acellular extrinsic ber cemen-
tum and cellular intrinsic ber cementum can be
distinctively affected by disease or experimental
conditions, certainly suggests that specic cell
pathways can be elicited and that different signa-
ling pathways exist. For effective and targeted per-
iodontal regeneration, it is thus most important to
recognize this.
Over the past few years, enamel matrix proteins
have generated much attention for periodontal
repair and shown promising clinical results. Vari-
ability in clinical outcomes and the benet with
respect to open-ap debridement or guided tissue
regeneration are still issues that need to be
resolved. Relatively close clinical results can be
obtained with very distinct therapeutic approaches,
suggesting that these enamel matrix proteins may
function indirectly. Indeed, there is still no evidence
that they really are major players in root formation.
Certainly, no major root defect has yet been
reported in amelogenin and ameloblastin knockout
and transgenic mice. This is not to say that enamel
matrix proteins should not be used for periodontal
treatment but rather that if we understood better
the mechanism by which they exert their inuence,
we could use them or other proteins more
efciently.
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28
Nanci & Bosshardt
Periodontology 2000, Vol. 31, 2003, 1231 Copyright C Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
PERIODONTOLOGY 2000
ISSN 0906-6713
Structure and function of the
toothepithelial interface in
health and disease
MnnJn T. PoIInNrN, Juxxn I. SnIoNrN & VrIi-Juxxn Ui11o
Introduction
Three types of mucous membranes (masticatory, lin-
ing, and specialized) line the oral cavity and form
the structural boundary between the body and the
external environment. Although each type of mu-
cosa protects against mechanical and microbial
damage, the epithelia exhibit considerable differ-
ences in their histology, thickness and differentiation
suitable for the functional demands of their location.
Furthermore, the structure of different epithelia re-
ects their effectiveness as a barrier to the penetra-
tion of microbes and noxious agents into the deeper
tissues (141,160). Mucosal epithelia are composed of
continuously dividing and shedding populations of
keratinocytes whose proliferation is conned to the
basal layer. The teeth, passing through the gingival
masticatory mucosa, create a unique environmental
challenge to the protective continuity. At the inter-
face where the healthy gingiva meets the tooth sur-
face the structural continuity is secured by the junc-
tional epithelium attached to the tooth surface by a
distinct mechanism known as the epithelial attach-
ment apparatus (143). As opposed to the constantly
renewing epithelia, teeth are units of nonshedding
surfaces, which provide a solid substratum not only
for the attachment of the junctional epithelial cells
but also for bacterial colonization and spreading in
the oral cavity. At the dentogingival junction the bac-
terial colonies, exhibiting a variety of virulence fac-
tors (68), pose a potential threat to the epithelial
attachment. The attachment may be affected directly
by bacteria or indirectly through their ability to acti-
vate inammatory and immune processes, which
contribute to the composition of the gingival crevice
uid (GCF) and thus to the conditions under which
12
the epithelial attachment apparatus is formed and/
or maintained. In addition to the attachment, the re-
newal rate and reparative capacity of the junctional
epithelial cells are equally important for the health
of the dentogingival junction. Accordingly, any de-
generative episodes that involve the cells responsible
for the attachment may gradually lead to the detach-
ment of the junctional epithelium from the tooth
surface and compromise the protective mucosal
continuity.
Periodontal diseases are associated with bacterial
plaque infecting the dentogingival margin and caus-
ing inammation in the underlying tissues (53,87).
Logically, one of the main concerns of periodontal
research has been to describe the inammatory re-
action in the adjacent tooth-supporting tissues and
the consequences it may have at the alveolar bone
margin (70,121,147). Less attention has been focused
on the effects of factors released from bacteria and/
or from the inammatory reaction on the junctional
epithelial cells. The precise mechanisms of epithelial
degeneration and/or activation leading to the de-
tachment and apical migration of the junctional epi-
thelial cells and consequent conversion of the gingi-
val sulcus into an infected periodontal pocket have
not yet been discovered. This article deals with fac-
tors associated with periodontal tissue protection
and destruction with special reference to the junc-
tional epithelial cells.
Junctional epithelium
Schroeder and Listgarten rst claried the anatomy
and histology of the dentogingival junction in their
monograph: Fine structure of developing epithelial
Toothepithelial interface in health and disease
Fig. 1. Schematic illustration of the different epithelia at
the dentogingival junction. The junctional epithelium (JE)
exhibits a distinct phenotype that allows the tissue to at-
tach to the tooth surface and participate in the host de-
fense in a number of ways.
attachment of human teeth (143). Since then,
knowledge of the junctional epithelium has been re-
viewed in numerous articles (90,142,144,161,168). It
is commonly accepted that the junctional epithel-
ium exhibits several unique structural and func-
tional features that contribute to preventing patho-
genic bacterial ora from colonizing the subgingival
tooth surface. First, junctional epithelium is rmly
Fig. 2. Photomicrograph demonstrating the junctional ium into the sulcus. At the apical part of the junctional
epithelial DAT cells on the tooth surface (a). When the epithelium, cells (arrow) are seen to grow/proliferate into
gingiva is displaced laterally the DAT cells are left on the the connective tissue (CT). A higher magnication (c) of
enamel (E) surface. The degeneration of the DAT cells ap- the apical part of the junctional epithelium. Note the
pears to be a prerequisite for the apical advancement of dark-stained DAT cells along the cementum surface, the
bacterial plaque (P). D dentin. The junctional epithel- epithelial nger proliferating apically and the absence of
ium (JE) attached to tooth (to enamel or cementum (C) inammatory inltrate. Gingival epithelial cells grown on
as in (b) forms a structural barrier against the bacterial decalcied dentin matrix (d) show apparent ability to
plaque. Polymorphonuclear leukocytes that cover the extracellular collagenolysis (e).
plaque (P) have migrated through the junctional epithel-
13
attached to the tooth and thus forms an epithelial
barrier against the plaque bacteria (Fig. 1 and 2a,b).
Second, it allows the access of GCF, inammatory
cells and components of the immunological host de-
fense to the gingival margin. Third, junctional epi-
thelial cells exhibit rapid turnover, which contributes
to the hostparasite equilibrium and rapid repair of
damaged tissue (48). Although the importance of the
junctional epithelium in host defense is well recog-
nized, an exact understanding of its role in the
pathogenesis of periodontal diseases is largely miss-
ing (Fig. 3). A major part of the older literature on
junctional epithelium describes its histological fea-
tures at different stages of development or disease
(113,141). More recent studies report on the distri-
bution of cytoplasmic keratin laments in the junc-
tional epithelial cells, on different cell surface anti-
gens and receptors, intercellular adhesion molecules
and details of the nonepithelial cells within the junc-
tional epithelium and its nerve supply [for a review
see (142)]. These results consistently support the
view that to be able to form a successful junction
between two dissimilar tissues and to respond in a
exible manner to the external environment, the
epithelial cells responsible must have a distinct un-
differentiated phenotype (160) (Fig. 4). The pheno-
type of junctional epithelium, at least partly, reects
a self-instructed adaptation of oral mucosa to the
Pllnen et al.
existence of a solid tooth surface penetrating it
(134,168). It is pertinent to suggest that the pheno-
type required for epithelial adaptability may also
render the cells vulnerable to the action of various
exogenous and endogenous agents. Since our under-
standing of the pathogenic mechanisms that lead to
failure in junctional epithelium defense is limited,
there is a clear need for fundamental research into
the junctional epithelial cells and their functions
under different clinical conditions.
Epithelial attachment apparatus
The attachment of the junctional epithelium to the
tooth is mediated through an ultramicroscopic
mechanism dened as the epithelial attachment ap-
paratus (143). It consists of hemidesmosomes at the
plasma membrane of the cells directly attached to
the tooth (DAT cells) and a basal lamina-like extra-
cellular matrix, termed the internal basal lamina, on
the tooth surface [Fig. 5 (80)]. By morphological cri-
teria the internal basal lamina between the junc-
tional epithelial DAT cells and the enamel is quite
similar to the basement membrane between the epi-
thelium and the connective tissue. However, by bio-
chemical criteria the internal basal lamina differs
essentially from the established basement mem-
brane composition and thus from the external basal
lamina.
Fig. 3. The junctional epithelium (JE) is repeatedly or con-
tinually exposed to bacterial challenges, which may lead
to the failure of the junctional epithelium and eventually
to subgingival plaque formation (brown), conversion of
the gingival sulcus into a periodontal pocket and increase
in the size of the inammatory focus in the connective
tissue (gray).
14
The components of the internal basal lamina are
synthesized by the DAT cells in the absence of the
immediate vicinity of connective tissue (80,142).
Internal basal lamina proteins include laminin and
type VIII collagen (128,130). Laminin, identied as
type 5, is localized mainly to the optically electron-
dense part of the internal basal lamina and it seems
to be associated with hemidesmosomes (69,93,100).
Characteristically, the internal basal lamina lacks la-
minin-1 and type IV collagen, which are components
of true basement membranes (129,130). In addition,
the internal basal lamina structure may involve
other molecules that are unique to this structure (L.
Hkkinen, unpublished results).
Hemidesmosomes have a decisive role in the rm
attachment of the cells to the internal basal lamina
on the tooth surface. Recent data suggest that the
hemidesmosomes may also act as specic sites of
signal transduction and, thus, participate in regula-
tion of gene expression, cell proliferation and cell
differentiation (71). The intracellular part of hemi-
desmosomes consists of at least two distinct pro-
teins, the 230kDa bullous pemphigoid antigen
(BP230) and plectin, which is a high molecular
weight cytomatrix protein (Fig. 5). These proteins
mediate the attachment of the epithelial cell cyto-
plasmic keratin laments to two transmembrane
components of the hemidesmosome known as the
180kDa bullous pemphigoid antigen (BP180) and
a
6
b
4
integrin (71,174). The a
6
b
4
integrin plays an im-
portant role in the interaction of epithelial cells with
the extracellular matrix (101,127). This interaction
utilizes the intracellular plectin connected through
the b
4
-domain of the integrin to laminin-5 (ligand
for the a
6
b
4
integrin) in the internal basal lamina
(66,69,169). In general, the interaction between the
different components of the extracellular matrix and
the cell surface molecules linked to the intracellular
cytoskeleton is fundamental for cell adhesion, cell
motility, synthetic capacity, tissue stability, regenera-
tion and responses to external signals (175). Since
the biochemical composition of internal basal lam-
ina matrix differs from that of the true basement
membrane, the behavior of the attached DAT cells
cannot be directly deduced from the data reporting
on the behavior of basal cells that grow adjacent to
the external basal lamina (167).
Turnover of the junctional epithelial cells
The junctional epithelium is a stratied epithelium
composed of two strata, the basal layer facing the
connective tissue and the suprabasal layer extending
to the tooth surface (Fig. 2 and 4). Coronally, close to
the sulcus, junctional epithelium is about 15 cell
layers thick and narrows towards the apical part of
the tissue. The turnover rate of junctional epithelium
is exceptionally rapid. In nonhuman primates it is
about 5days and approximately twice the rate of the
oral gingival epithelium (153). Previously it was
thought that only epithelial cells facing the external
basal lamina were rapidly dividing. However, recent
evidence indicates that a signicant number of the
DAT cells are, like the basal cells along the connec-
tive tissue, capable of synthesizing DNA, which dem-
Fig. 4. Haematoxylin-eosin staining
of a clinically healthy human dento-
gingival junction (a) shows an in-
ammatory inltrate in the connec-
tive tissue (CT) lateral to the junc-
tional epithelium (JE). The
junctional epithelium exhibits wider
intercellular spaces as compared to
the sulcular epithelium (SE). The
wide intracellular spaces allow the
access of the components of the im-
munological defense into the sulcus.
DAT cells directly attached to the
tooth are seen as a string lateral to
the enamel space (ES). Note the most
coronal DAT cell () that appears to
be supported only by the tooth and
thus forms the medial wall of the sul-
cus (see also schematic illustration
in Fig. 8). Reaction with a mono-
clonal antibody to keratin 19 (b)
shows that all the suprabasal junc-
tional epithelial cells express this
protein, as do the undifferentiated
basal cells. The reaction with the
antibody to keratin 10 shows that
terminal differentiation is a charac-
teristic feature of the oral epithelium
(OE) but not of the sulcular or junc-
tional epithelia. The DAT cells along
the tooth surface (d) are especially
rich in K19.
15
onstrates their mitotic activity (133,135). At the co-
ronal part of the junctional epithelium, the DAT cells
typically express a high density of transferrin recep-
tors (131), which supports the idea of their active
metabolism and high turnover (172). The ndings
suggest that the DAT cells have a more important
role in tissue dynamics and reparative capacity of
the junctional epithelium than has previously been
thought (143). Based on these data, alternative
models for the turnover of DAT cells can be pro-
posed (Fig. 6). The existence of a dividing population
of epithelial cells (DAT cells) in a suprabasal loca-
Pllnen et al.
Fig. 5. A schematic illustration of a DAT cell shows the
structural and molecular composition of the epithelial
attachment apparatus (EAA). N nucleus of a DAT cell,
IF cytoplasmic keratin laments (intermediate size
laments). The hemidesmosomes at the plasma mem-
brane are associated with the a
6
b
4
integrin that communi-
cates with Ln-5 laminin 5 located mainly in the internal
basal lamina, the extracellular domain (?) for BP180 is a
collagenous protein (perhaps type VIII), that has not yet
been denitely characterized. LL lamina lucida, LD
lamina densa, SLL sublamina lucida, IBL internal
basal lamina.
tion, several layers from the connective tissue, is a
unique feature of the junctional epithelium. The dis-
tinct phenotype may result from specic permissive
or instructive signals provided by the internal basal
lamina matrix on the tooth surface. Therefore, any
structural or molecular changes in the internal basal
lamina can potentially inuence the vital functions
of the DAT cells and contribute to the effectiveness
or failure of the junctional epithelial defense or vice
versa; changes in the cell metabolism, etc. may affect
the internal basal lamina.
Morphological studies of the internal basal lamina
of teeth extracted because of advanced periodontitis
have shown that remnants of the internal basal lam-
ina can be detected even adjacent to severely de-
generated DAT cells (111). Although the internal
basal lamina morphologically appears to be rela-
tively resistant to external challenges its molecular
structures may still be altered, leading to changes of
the DAT cell function. Indeed, it has been shown that
certain matrix metalloproteinases from eukaryotic
cells cleave laminin-5, exposing a cryptic molecular
16
site that triggers cell migration (49). It has not yet
been shown, however, if bacterial proteinases can
perform directly the same selective cleavage of lami-
nin-5. Bacterial agents may thus indirectly trigger
mechanisms that lead to modulation of host cell be-
havior. Hypothetically, even minor changes in cell
metabolism, biosynthetic activity or ability to divide
and migrate may eventually lead to degeneration
and detachment of the junctional epithelium/DAT
cells and allow pathogenic ora to grow on the ex-
posed subgingival tooth surface. A wide variety of
bacterial species and their products, such as lipo-
Fig. 6. The mechanism of DAT cell turnover is not fully
understood. Considering the fact that the DAT cells are
able to divide and migrate, three possible mechanisms
can be proposed. These are (1) the daughter cells pro-
duced by dividing DAT cells replace degenerating cells on
the tooth surface, (2) the daughter cells enter the exfoli-
ation pathway and gradually migrate coronally between
the basal cells and the DAT cells to eventually break off
into the sulcus, or (3) epithelial cells move/migrate in the
coronal direction along the tooth surface and are replaced
by basal cells migrating round the apical termination of
the junctional epithelium.
polysaccharides, lipoteichoic acids, short-chain fatty
acids, and phospholipases, have been shown to af-
fect adversely the metabolism of epithelial cells in
cultures, leading to changes in proliferation and/or
production of cytokines and matrix metalloprotein-
ases (43,44,64,118120,156). Understanding of the
potential signicance of these events in relation to
the pathogenesis of periodontal pocket formation
calls for further studies.
Junctional epithelium in the
antimicrobial defense
Junctional epithelium consists of active populations
of cells and antimicrobial functions, which together
form the rst line of defense against microbial in-
vasion into tissue (Fig. 7). Even though junctional
epithelial cell layers provide a barrier against bac-
teria many bacterial substances, such as lipopolysac-
charide, pass easily through the epithelium but have
only limited access through the external basal lam-
ina into the connective tissue (146). Both the inter-
nal and external basal laminas act as barriers against
infective agents.
Rapid turnover, as such, is an important factor in
the microbial defense of junctional epithelium (see
below). Also, because the area covered by the divid-
ing cells in the junctional epithelium is at least 50
times larger than the area through which the epi-
thelial cells desquamate into the gingival sulcus,
there is a strong funnelling effect that contributes to
the ow of epithelial cells (145). Rapid shedding and
effective removal of bacteria adhering to epithelial
cells is therefore an important part of the anti-
microbial defense mechanisms at the dentogingival
junction.
There is increasing evidence indicating that sev-
eral specic antimicrobial defense systems exist in
the oral mucosa. Many epithelial cell types, includ-
ing junctional epithelium, have been found to con-
tain enzyme-rich lysosomes. Their fusion with
plasma membrane is triggered by elevation of the
intracellular calcium concentration (122,145). In
rats, the lysosomes have been demonstrated to con-
tain cysteine proteinases (cathepsin B and H) active
at acidic pH (190). Porcine epithelial cells of Malas-
sez share several characteristics with junctional epi-
thelial cells and are able to produce several neutral
proteinases, including collagen-degrading enzymes
and a chymotrypsin-like proteinase (44). The role of
these enzymes in the antibacterial mechanism has
not yet been studied. Recently, it has been found
that the junctional epithelial cells lateral to DAT cells
17
produce matrilysin (matrix metalloproteinase-7)
(176). In Paneth cells of the mouse intestine this en-
zyme is able to activate the precursor peptide of a-
defensin, an important antimicrobial agent of mu-
cosal epithelium (184). It is possible that a similar
active matrilysin/defensin system exists in junc-
tional epithelium, as in other mucosa exposed to
bacteria such as intestine, lungs and urogenital
tissues (88). Another possible effect by which matri-
lysin contributes to the mucosal defense is the re-
lease of bioactive molecules from the cell surfaces
which play a role in the inammatory reaction. Such
effects are currently being actively explored.
Fig. 7. Several antimicrobial mechanisms exist in the
junctional epithelium. In the coronal part of the junc-
tional epithelium quick cell exfoliation (1) because of
rapid cell division (2) and funnelling of junctional epi-
thelial cells towards the sulcus hinder bacterial coloniza-
tion (see text). Laterally, the (external) basement mem-
brane forms an effective barrier against invading mi-
crobes (3). Active antimicrobial substances are produced
in junctional epithelial cells. These include defensins and
lysosomal enzymes (4). Epithelial cells activated by mi-
crobial substances secrete chemokines, e.g. interleukin-
8 and cytokines, e.g. interleukins -1 and -6, and tumour
necrosis factor-a that attract and activate professional de-
fense cells, such as lymphocytes (LC) and polymorpho-
nuclear leukocytes (PMN). Their secreted product, in turn,
cause further activation of the junctional epithelial cells
(5). CT connective tissue.
Pllnen et al.
Despite the selective barrier formed by the gingi-
val basal laminas, components of the inammatory
and immunological defense pass easily through the
basement membrane and epithelium into the sul-
cus. Here they play an important role in restricting
bacterial access into the subgingival tissues (for a re-
view see [81]) (Fig. 2a,b, 4a). Leukocytes, especially
the polymorphonuclear leukocytes that migrate
through the junctional epithelium, comprise prob-
ably the most important defense mechanism at the
gingival margin (112). The cell surface carbohydrates
(1) expressed by the junctional epithelial cells are
thought to respond to extracellular molecular
changes in a manner which allows the cells to com-
municate with their environment. The cells respond
actively to bacterial infection by producing cell ad-
hesion molecules (intercellular adhesion molecule-
1) and chemotactic substances (chemokines such as
C5a, leukotriene B
4
, lymphocyte function-associated
antigen-3 and interleukin-8) that facilitate the mi-
gration of leukocytes through the junctional epithel-
ium (16,28,99,171).
In the subsequent line of defense inammatory
mediators and antibodies produced by macro-
phages, lymphocytes and plasma cells in the gingival
tissues restrict the spreading of bacterial infection
into the connective tissue and systemic circulation.
Signicant amounts of lymphocytes may be present
also within the junctional epithelium, thus contribu-
ting to the protective functions of the tissue
(148,149). Recently, it has been suggested that
supplementary to system-derived antibodies and
antibodies produced locally by plasma cells, the
junctional epithelial cells may also have a secretory
function (77).
The detachment of the DAT cells
from the tooth surface (host vs.
bacteria; battle for surface)
Role of the gingival crevice uid
GCF is an exudate of varying composition found in
the sulcus/periodontal pocket between the tooth
and marginal gingiva. GCF contains components of
serum, inammatory cells, connective tissue, epi-
thelium, and microbial ora inhabiting the gingival
margin or the sulcus/pocket (26, 37, 41, and articles
in this issue) (Fig. 8). In the healthy sulcus the
amount of GCF is very small. However, its constitu-
ents participate in the normal maintenance of func-
tion of the junctional epithelium throughout its lat-
18
eral and vertical dimensions, including the most co-
ronal DAT cells. During inammation the GCF ow
increases and its composition starts to resemble that
of an inammatory exudate (26). The increased GCF
ow contributes to host defense by ushing bacterial
colonies and their metabolites away from the sulcus,
thus restricting their penetration into the tissue.
The main route for GCF diffusion is through the
(external) basement membrane and then through
the relatively wide intercellular spaces of the variable
thickness junctional epithelium into the sulcus. Al-
though all the junctional epithelial cells are con-
stantly exposed to the GCF and its various constitu-
ents, the nutritional and other vital conditions in the
different parts of the junctional epithelium depend
on a large number of local factors. Clearly, the
changes in the composition of the GCF caused either
by bacteria, bacterial metabolites/enzymes or other
factors, or the inammatory reaction is likely to be
Fig. 8. The GCF passing through the junctional epithelium
determines the environmental conditions and provides
sufcient nutrients for the DAT cells to grow. At the gingi-
val margin the GCF may become contamined so that
agents from the oral cavity and/or the plaque bacteria
challenge the most coronal DAT cells. Obviously, the con-
ditions for DAT cell survival and adequate function at the
coronal part of the JE are different and more susceptible
of compromises than those for the basal cells living in
the vicinity of the connective tissue (CT) and the blood
circulation.
largest at the most coronal junctional epithelial cells.
A considerable number of bacteria and host-de-
rived products found in the GCF have been associ-
ated with the initiation and progression of peri-
odontal disease. The bacterial agents include endo-
toxins, hydrogen sulde, butyric and propionic
acids, bacterial collagenases and other proteases
(e.g. trypsin-like), and a variety of enzymes, such as
hyaluronidase and neuraminidase (37). Host-derived
agents include components associated with the in-
ammatory reaction, such as factors of the comple-
ment system, prostaglandins, different cytokines, in-
tracellular enzymes, and products of tissue break-
down such as lactate dehydrogenase, aspartate
aminotransferase, polyamines, and collagen pep-
tides (37,41,82). Furthermore, antimicrobial agents
and leukocyte-derived enzymes such as lysozyme,
alkaline phosphatase, b-glucuronidase, cathepsin D,
elastase, collagenase, and lactoferrin as well as os-
teonectin and bronectin are also found in the GCF.
Indeed, the GCF contains a wide variety of biologic-
ally active molecules with the potential capacity to
affect the growth of junctional epithelium/DAT cells
as well as oral bacteria, both competing for the tooth
surface at the dentogingival interface (Fig. 8 and 9,
Table1).
Role of the polymorphonuclear
leukocytes
Polymorphonuclear leukocytes form the most im-
portant line of defense against bacterial plaque at the
gingival margin (112). When the polymorphonuclear
leukocytes reach the bacteria, they release the con-
tents of their granules and may adhere to individual
bacteria and phagocytose them. The polymorpho-
nuclear leukocytes do not, however, appear to have
the capacity to remove dental plaque but rather form
a protective wall against it. Therefore, polymorpho-
nuclear leukocytes are a major contributor in the
hostparasite equilibrium but have a limited capacity
to reclaim any tooth surface once lost to the plaque
bacteria. On the other hand, activated polymorpho-
nuclear leukocytes can cause tissue damage as a re-
sult of a variety of enzymes, oxygen metabolites, and
other components that are released from their gran-
ules during the battle against microbes (3,179,187).
The polymorphonuclear leukocytes have two main
types of granules that contain agents that are effective
inkilling the bacteria. The azurophilic (primary) gran-
ules contain myeloperoxidase, lysozyme, elastase, ca-
thepsin G, urokinase, acid hydrolases, and defensins,
whereas the specic (secondary) granules contain
19
lactoferrin, elastase, and lysozyme (30). Activated
polymorphonuclear leukocytes also generate hydro-
gen peroxide (H
2
O
2
) and highly reactive oxygen rad-
icals with the potential to destroy bacteria and gingi-
val cells (21,30). The polymorphonuclear leukocytes
are most effective in aerobic conditions close to the
gingival margin (30), suggesting a different role for
them in anaerobic periodontal lesions. While di-
recting their effects to the invading microbes the
powerful polymorphonuclear leukocyte substances
also potentially affect the structural cells andextracel-
lular matrix. Polymorphonuclear leukocytes may also
become activatedininammatory lesions andrelease
specically their secondary granule contents during
their chemotactic migration (63,189). This phenom-
enon may also take place in tissues such as the junc-
tional epithelium. Therefore, the effects of the sec-
ondary granule contents on gingival epitheliumare of
special interest in research into the failure of the junc-
tional epithelium and the formation of periodontal
pockets.
Lactoferrin is an important antimicrobial protein
present in the secondary granules of polymorpho-
nuclear leukocytes. It has high afnity for iron
(5,32,85) and it acts on bacteria by causing iron de-
Fig. 9. The degeneration and detachment of DAT cells ex-
poses the tooth surface and creates a subgingival niche
suitable for the colonization of anaerobic gram-negative
bacteria and apical growth of dental plaque.
Pllnen et al.
Table1. Dental plaque and gingival crevice uid (GCF) components associated with periodontal diseases
Dental plaque in vivo Human GCF/GCW
Cultured Periodontal Periodontal
Compound plaque bacteria Healthy disease Healthy disease
Alkaline phosphatase 60mIU/site (25) 120mIU/site (25)
Ammonia 1486mr (176)
Butyric acid 9.1mM (151) 00.2mr 2.614mr 0.08mr (19) 0.331.14mr
(104,105) (104,105) (19)
Hydrogen sulde 539nr (115) 150p.p.m. (98) 0.8ng/mL (155) 4.2ng/mL (155)
Interleukin-1b 23150ng/mL 86882ng/mL
(95,117) (95,117)
Lactoferrin 600mg/mL (46) 1500mg/mL (46)
Lipoteichoic acid 50mg ml (181)
Lipopolysaccharide 0.83.6mg/mL 9.636mg/mL
(42,173) (42,173)
Prostaglandin E
2
5ng/mL (103) 10.5ng/mL
(103)
Propionic acid 113mr (151) 0.80.9mr 9.544mr 0.75mr (19) 0.98mr (19)
(104,105) (104,105)
Transforming growth factor-a 226pg/mL (95) 54pg/mL (95)
Tumor necrosis factor-a 0.113ng/mL
(126)
GCW, gingival crevicular washing.
pletion, and thus reduction in bacterial cell division
rate, glucose metabolism and macromolecular syn-
thesis (7,178). Besides bacteriostatic activity, lacto-
ferrin also has bactericidal effects, at least in vitro.
Binding of lactoferrin to bacteria and their sub-
sequent lysis has been reported in a number of
studies (7,8,12,36). Furthermore, lactoferrin may ex-
ert antimicrobial effects through interfering with
bacterial attachment and colonization in the oral
cavity (4,159,178). In addition to the effects de-
scribed above, lactoferrin may also interfere with the
host defense not only by blocking complement acti-
vation and inhibiting hydroxyl radical formation but
also by enhancing it by stimulating polymorpho-
nuclear leukocyte recruitment to the infected sites
(12). In the GCF from sites exhibiting gingival in-
ammation or periodontal pockets the amount of
lactoferrin is signicantly elevated (10001500mg/
ml) as compared to the healthy sites (500mg/ml)
(2,46). While microbial multiplication is already sig-
nicantly inhibited at low lactoferrin concentrations
(50mg/ml), even high amounts of lactoferrin (500
mg/ml) seem to have no effect on epithelial cell divi-
sion in model systems simulating the junctional epi-
20
thelium in vivo (118). High concentrations of lacto-
ferrin do, however, hamper epithelial cell growth by
interfering with their adhesion and spreading. The
molecule may, thus, have a role in delaying the re-
pair of the junctional epithelium/DAT cell popula-
tion during severe inammation.
Role of host proteinases and
inammatory mediators
Degradation of extracellular matrix during peri-
odontal inammation is a multistep process that in-
volves several proteolytic enzymes. Different cell
types of periodontal tissue produce matrix metallo-
proteinases (collagenases, stromelysins, gelatinases,
membrane-type metalloproteinases), plasminogen
activator, cathepsins and elastase (17,165). In re-
sponse to the bacteria and inammatory cytokines,
broblasts, junctional epithelial cells, osteoblasts/
osteoclasts, macrophages, and polymorphonuclear
leukocytes release proteinases that are involved in
the defense against microbes. At the same time they
also contribute to periodontal tissue destruction by
degrading extracellular matrix and basement mem-
brane components (17,60,132,158). In concert, ma-
trix metalloproteinases are able to degrade all extra-
cellular matrix proteins (17). Collagenases degrade
interstitial type collagen brils (I, II, III), whereas
gelatinases, stromelysins, and membrane-type
metalloproteinases have the ability to degrade
bronectin and gelatin (denatured collagen), and
basement membrane components including type IV
collagen, entactin, nidogen, and laminin (17,72,102).
Neutrophil elastase and cathepsin G are capable of
degrading basement membrane type IV collagen and
laminin, and also type VIII collagen, found in the
internal basal lamina (61,78). Proteinases of host ori-
gin are thus capable of degrading all known extracel-
lular components of connective tissue and epithel-
ium including components of both the external
basal lamina (basement membrane at the connec-
tive tissuejunctional epithelium interface) and the
internal basal lamina at the epitheliumtooth inter-
face. Therefore, these enzymes seem to have the po-
tential to contribute to the lateral and apical prolifer-
ation of the junctional epithelium into the connec-
tive tissue (Fig. 2) as well as to epithelial
disintegration through degradation of the internal
basal lamina and increase in epithelial permeability.
However, electron microscopic studies on DAT cells
attached to teeth extracted because of advanced
periodontitis do not support the idea that enzymatic
degradation of the epithelial attachment apparatus
precedes the degeneration of DAT cells (111). On the
contrary, the DAT cells in this material seemed to be
more severely affected than the epithelial attach-
ment apparatus once produced and maintained by
the degenerating cells. This implies that it might be
more rewarding to focus the studies of pocket forma-
tion on mechanisms that disturb the vital functions
of the DAT cells rather than on the destruction of
the matrix components of the epithelial attachment
apparatus.
As described elsewhere in this issue, regulation of
proteinase activities is a complex process involving
activation of latent precursor molecules as well as
inhibition of the active enzymes. Therefore, the ac-
tual damage caused by, for example, polymorpho-
nuclear leukocyte proteinases may be limited in the
presence of proteinase inhibitors, such as a2 macro-
globulin, a1 antitrypsin and tissue inhibitors of
metalloproteinases, found in the junctional epithel-
ium and in the gingival crevice. In fact, a recent
study has demonstrated that there is only a limited
amount of active metalloproteinases in the pocket
epithelium obtained from sites of periodontitis
(140). Since the degradation of the extracellular ma-
21
trix by metalloproteinases plays a major role in the
inamed connective tissue, it is pertinent to ask how
signicant is the failing support of the connective
tissue to the integrity of the junctional epithelium.
Obviously, a less effective connective tissue support
predisposes the tissue to intraepithelial splits and
contributes to the lateral and apical proliferation of
the epithelium. Together with increased per-
meability of the junctional epithelium, this may alter
the nutritional conditions of the DAT cells on the
tooth surface and expose them to agents from the
dental plaque. Another area of junctional epithelium
biology that has not been addressed in depth is the
composition of the epithelial interstitial matrix and
how its degradation affects the behavior of junc-
tional epithelial cells. Also, a limited proteolytic
cleavage of matrix molecules, e.g. laminin, bronec-
tin and proteoglycan, may expose cryptic molecular
sites with biological activity not possessed by the in-
tact molecule (39). These active tissue fragments
may regulate cell adhesion, migration and prolifer-
ation in inamed tissue (175). For instance, while in-
tact laminin-5 promotes formation of hemidesmo-
somes and inhibits cell migration, its fragment pro-
duced by a proteolytic cleavage promotes epithelial
cell migration (49). Even though fragments of
bronectin and collagen have been demonstrated in
GCF their effects on junctional epithelium/pocket
epithelium are unknown (38). In future, extracellular
matrix molecules and their fragments can be ex-
pected to provide useful tools for prevention and
management of periodontal disease. For example,
laminin-5 treatment of teeth or titanium dental im-
plants may enhance long-term stability of epithelial
attachment (35).
Cytokines, especially the interleukins -1 and -6,
and tumor necrosis factor-a, and the arachidonic
acid metabolite prostaglandin E
2
, have been strongly
associated with periodontal disease [for a review see
(47)]. These inammatory mediators are secreted
into the GCF by both leukocytes and activated junc-
tional epithelium cells and their amounts have been
shown to increase at sites exhibiting periodontal
tissue destruction [Table1; (95,103,109,117)]. In-
terleukin-1, interleukin-6 and prostaglandin E
2
stimulate bone resorption and contribute to peri-
odontal tissue destruction also by inducing matrix
metalloproteinase (collagenase) production (47). In-
terleukin-1 has been shown to promote broblast
proliferation and production of other cytokines by
periodontal cells, and to activate the arachidonic
acid pathway and thus production of prostaglandin
E
2
(27,34,110). Prostaglandin E
2
in turn has an over-
Pllnen et al.
all catabolic effect on gingival broblasts, as shown
by suppressed DNA synthesis and collagen synthesis
(6). Studies reporting the effects of inammatory
mediators on the junctional epithelium and its func-
tions are not available today.
Role of bacterial products
It is plausible that several products released from
bacteria during periodontal infection have junc-
tional epithelium as their major target tissue. Even
though there is ample evidence that bacterial sub-
stances have a multitude of effects on several cell
types, ranging from activation of cell functions to
cell death, the importance of specic substances in
initiation and progression of periodontal diseases is
not yet understood. We review here the literature of
the main bacterial products and their potential ef-
fects on the junctional epithelium.
In the early phase of plaque formation gram-posi-
tive bacteria accumulate on the tooth surface close
to the most coronal junctional epithelium/DAT cells
which may thus be exposed to the cell wall compo-
nents of these typically supragingival bacteria;
namely the peptidoglycan matrix, surface antigens,
teichoic and lipoteichoic acids. Lipoteichoic acids
are genus- and species-specic molecules (45) that
are synthesized especially abundantly from sucrose
at neutral pH (79,124). The precise functions of
lipoteichoic acids in bacteria have not yet been es-
tablished. However, they have been implicated as
carriers in cell wall synthesis, inhibitors of autolytic
activity and as reservoirs of cations (45,125). In the
oral cavity lipoteichoic acids are thought to mediate
bacterial adhesion to human cells and teeth
(13,67,123).
If the bacterial plaque is allowed to grow, the
amount of gram-negative bacteria increases. The cell
wall of these bacteria is composed of a thin peptido-
glycan layer and a bilayered outer membrane, which
contains the major surface antigens including the
porin proteins, and lipopolysaccharides (lipopolys-
accharides/endotoxins) (55). The variation in the
lipopolysaccharides between bacterial genera, spe-
cies, and even within species (54,180,181) may ac-
count for the reported differences in host responses
to lipopolysaccharides derived from different bac-
terial populations.
From the periodontal point of view both lipotei-
choic acids and lipopolysaccharides are interesting
molecules. They are released from the bacteria into
the extracellular environment during bacterial dis-
ruption, and also during normal cell wall turnover
22
of living bacteria (42,45,55,56,68,150,173,181). Thus,
varying amounts of lipoteichoic acids and lipopolys-
accharides are present at the gingival margin where
they may stimulate leukocyte function, increase
cytokine and inammatory mediator production
and activate the complement system as shown in
studies in vitro (15,86,89,96). In addition to their role
as inammation modulators lipoteichoic acids and
lipopolysaccharides have been shown to affect peri-
odontal tissues, e.g. by stimulating bone resorption
(9,59,94). Furthermore, lipopolysaccharides appear
to have the ability to increase epithelial permeability
and penetrate healthy gingival sulcular epithelium
(120,138,154). Lipopolysaccharides from Salmonella
enteritidis, Escherichia coli, Actinobacillus actino-
mycetemcomitans, Porphyromonas gingivalis, Prevo-
tella intermedia and Porphyromonas asaccharolytica
have been shown to stimulate human gingival
broblast proliferation at low (1mg/mL) concen-
trations and suppress their proliferation at higher (
10mg/mL) concentrations (11,31,65,74,83,84). High
concentrations of lipopolysaccharides from non-oral
bacteria (E. coli, 5000mg/mL) have been shown to
increase the proliferation of basal cells of the junc-
tional epithelium in an animal model (167). In hu-
man epithelial cell and junctional epithelial tissue
cultures lipopolysaccharides from oral pathogens
show only slight or no effects on the growth and mi-
totic activity of the cells (120). This indicates that
lipopolysaccharides, despite their established role as
modulators of inammation, may not signicantly
harm the epithelial cells at the concentrations found
in dental plaque and GCF (below 50mg/mL). There-
fore, lipopolysaccharides do not appear to have a key
role in DAT cell degeneration and detachment from
the tooth surface.
When epithelial cells in different culture systems
are exposed to lipoteichoic acids from gram-positive
oral bacteria (Streptococcus sanguis and Streptococ-
cus mutans, 1050mg/mL) their growth and mitotic
activity are consistently reduced (120). According to
these results, the lipoteichoic acids appear to have
the potential to interfere with the renewal of various
types of epithelial cells. As discussed before, a pro-
longed inhibition of the renewal of the coronal junc-
tional epithelium/DAT cells would most likely lead
to their degeneration and detachment, and eventu-
ally to subgingival colonization by gram-negative
periodontal pathogens.
Periodontopathogenic bacteria release numerous
proteolytic enzymes that are a prerequisite for the
normal metabolism of amino acids and for the sur-
vival of these mainly asaccharolytic bacteria in the
oral environment (for a review see [68]). Bacterial
collagenases, gelatinases, and trypsin- and chymo-
trypsin-like enzymes have been shown to degrade
host extracellular matrix macromolecules and base-
ment membrane components in vitro suggesting
that also they have the potential to contribute to
periodontal tissue destruction, as do the host-de-
rived proteinases (17,116). This is especially true in
the junctional epithelium, where the bacterial en-
zyme concentrations are much higher than in the
connective tissue. Degradation of immunoglobulins
and complement proteins by bacterial proteinases
may result in an incomplete host defense and thus
facilitate bacterial colonization and growth. Some
bacterial proteinases are able to activate host matrix
metalloproteinases and thereby increase the total
proteolytic activity in the infected tissue (29,157).
The complex interactions of bacterial and host-de-
rived proteinases and their inhibitors, as well as the
presence of bacteria capable of degrading the pro-
teinase inhibitors (24,107,139) make the exact role
of the bacterial proteloytic enzymes in periodontal
tissue destruction difcult to study. It appears, how-
ever, that the role, if any, of the bacterial enzymes
on epithelial detachment and DAT cell viability/de-
generation is primarily indirect and because of
changes in the living conditions of these cells rather
than of direct effects on the cells or on the epithelial
attachment apparatus (111).
The oral cavity provides bacteria with a large num-
ber of ecologic niches and substrates that can be
metabolized to different end products depending on
the bacterial population and the prevailing environ-
mental conditions. Therefore, the composition of a
bacterial population and its virulence, associated
with specic pathogenic mechanisms, reects the
interaction of a large number of constantly changing
variables determined not only by the oral microora
itself but also by the host. For example, the met-
abolism of carbohydrates in dental plaque depends
on the pH, the oxygen gradient, the amount and
quality of available substrates, and most importantly,
the bacterial composition of the plaque (92,185). As
a principal rule, cariogenic, supragingival plaques
contain a high percentage of streptococci, whereas
subgingival plaques growing in periodontal pockets
contain high proportions of gram-negative an-
aerobes (22,23,185). The main metabolic end prod-
uct of the streptococcal plaque is lactic acid, whereas
the gram-negative bacteria produce butyric acid
more abundantly (68,91,106,164,185) (Fig. 10). It is
noteworthy that certain bacteria can take advantage
of the metabolic end products released by other bac-
23
teria and thereby contribute to the production of
agents that are increasingly detrimental to the host
tissues. Anaerobic plaque bacteria, especially the as-
accharolytic bacteria, utilize amino acids and pep-
tides, derived either from diet or tissue/cell break-
down, for their sources of energy. This requires the
presence of proteolytic enzymes that rst degrade
the macromolecules (proteins) into small peptides
or amino acids which are then further metabolized
by the bacteria (68,185). When amino acids are util-
ized as an energy source, ammonia, sulfur-contain-
ing compounds (hydrogen sulde) and short-chain
fatty acids, especially butyric and propionic acids,
are formed (23,68,164).
Butyric and propionic acids are short-chain fatty
acids containing four and three carbon atoms, re-
spectively. They are produced by periodontopathog-
enic bacteria, such as Porphyromonas, Fusobacteri-
um, Prevotella and Treponema (1820,22,51,97,164).
As described above, the production of these acids
depends on a variety of environmental factors in-
cluding the proteolytic activity and the pH in the
pocket. The maximal activity of proteolytic enzymes
produced by periodontal pathogens is at pH7.08.0.
Some periodontal bacteria (e.g. Prevotella interme-
dia) are, however, capable of surviving and func-
tioning over a much broader pH range and even of
raising the pH by producing ammonia, and thereby
making the environment more suitable for bacterial
populations adapted to alkaline conditions (68). It is
of particular interest that the concentrations of bu-
tyric and propionic acids found in human plaque
and GCF correlate directly with the degree of gingival
inammation and periodontal pocket depth (Table1
[19,104,105]). Furthermore, the application of milli-
molar concentrations of these short-chain fatty acids
onto the healthy gingiva of beagle dogs has been re-
ported to produce a marked gingival inammatory
response (152). More recently, human studies have
shown that both food, which supports bacterial gen-
eration of high levels of short-chain fatty acids (50
mr), and short-chain fatty acids (100mr) applied
directly to healthy human gingiva elicit an inam-
matory response in the tissue. This response can be
demonstrated by increased GCF ow, subgingival
temperature, polymorphonuclear leukocyte emi-
gration, and elevated levels of inammatory cyto-
kines (interleukin-8) in the GCF (75,106,191). Short-
chain fatty acids have also been shown to activate
leukocytes to release inammatory cytokines and ex-
tend their lifetime (163,166). The activation of the
inammatory response and simultaneous inhibition
of the polymorphonuclear leukocyte function
Pllnen et al.
(phagocytosis and degranulation) (106) can alter and
prolong the inammatory response and lead to more
severe tissue destruction.
In cell cultures, butyric and propionic acids, ap-
plied in concentrations found in human plaque and
GCF, have been shown to inhibit the proliferation of
both broblasts and epithelial cells (33,119,151,156).
Microbial populations producing these short-chain
fatty acids may thus signicantly impair the rapid
renewal of the junctional epithelium/DAT cells and
thereby counteract one of the tissues main host pro-
tective functions. Taken together, the current litera-
ture suggests that butyric and propionic acids play a
role in the initiation and progression of periodontal
pocket formation by triggering and modifying the in-
ammatory response and by hampering the turn-
over and repair of the tissues at the dentogingival
junction.
Utilization of amino acids by bacteria for their en-
ergy needs, results in the formation of short-chain
fatty acids, hydrogen sulde and ammonia as by-
products. Like the short-chain fatty acids, am-
monium and hydrogen sulde have potentially det-
rimental effects on periodontal cells. Ammonium
(2080mr) has been shown to cause cell vacuoliz-
ation in chondrocytes and to inhibit (210mr am-
monium) collagen secretion by human gingival
broblasts in vitro (62,177). Whether or not the am-
Fig. 10. Oral bacteria produce short-chain fatty acids. En- glucose is abundant in aerobic (and low pH) conditions
vironmental factors such as the oxygen gradient, pH, sub- the metabolites on the left are the main products. In an-
strate quality and quantity, and the presence of bacterial aerobic conditions at high pH and when lactose starch or
enzyme inhibitors have a strong effect on the survival of amino acids are used as substrates, the metabolites on the
bacteria and the metabolites produced. When sucrose or right hand side of the gure are increased.
24
monium levels produced by plaque bacteria signi-
cantly inuence epithelial cells is not known.
Hydrogen sulde is a highly toxic compound that
causes adverse effects on human tissues (eyes and
respiratory tract) at concentrations of 50p.p.m. (50
mg/mL) and above (14). Hydrogen sulde has been
detected in the GCF collected from gingival sulcus/
pocket and its amount has been shown to increase
in gingival inammation (155). In the concentrations
found in dental plaque (150ppm) hydrogen sulde
has been shown to be toxic to HeLa cells (98). Inter-
estingly, oxidation of hydrogen sulde results in sul-
fate formation and detoxication of the compound,
whereas its toxicity is increased in the presence of
short chain hydrocarbons, ethanol and/or proteins
(14). Considering the protein-rich, short-chain fatty
acid-containing and anaerobic conditions in the
subgingival space, hydrogen sulde appears to be a
potential candidate to cause signicant damage to
the junctional epithelium/DAT cells on the tooth
surface during periodontal disease progression.
Role of risk factors for periodontal
disease
It is clear that periodontal diseases are primarily
caused by bacterial infections and that a number of
risk factors contribute to the susceptibility of indi-
viduals to these infections, and to the pathogenesis
and severity of the disease. These factors include
smoking, diabetes, immunosuppression, genetic fac-
tors, stress and age (136). Studies on how the risk
factors inuence disease progression have mainly
been focused on the inammatory reaction
(10,52,57,58,76,108,114,137,162,186,188). The con-
clusion from these studies is that a sound inam-
matory host response is needed for successful peri-
odontal defense. Factors that modify this response
may either cause an overwhelming reaction or an
inadequate reaction, both of which may accelerate
tissue destruction. It is interesting that periodontal
risk factors, such as hyperglycaemia and chemical
compounds released from tobacco, have harmful ef-
fects on the renewing capacity of both broblasts
and bone cells (40,50,170) However, very little is
known about the inuences of any of the risk factors
on oral gingival/sulcular or junctional epithelium
(64). From the defense point of view, it would be im-
portant to examine whether these factors also impair
the turnover or other defense mechanisms of the
junctional epithelium and thus contribute to the de-
generation of the dentoepithelial junction.
It is also quite possible that a specic response of
junctional epithelial cells to bacterial or host sub-
stances is a key factor in the pathogenesis of certain
forms of periodontal diseases. One example could be
localized juvenile periodontitis, where a rapid apical
growth of the junctional epithelium is associated
with a typically distributed pocket formation. A local
junctional epithelial cell defect might in this case re-
sult, for example, in a decreased ability of the DAT
cells to form hemidesmosomes. In fact, in Kindler
syndrome a defect in the formation of hemidesmo-
some-associated bers consisting of type VII colla-
gen leads to detachment of skin and gingival epithel-
ium and to early onset periodontitis (183).
Conclusions
The precise mechanisms that lead to the degenera-
tion and detachment of the junctional epithelium
from the tooth surface and to the conversion of a
gingival sulcus into an infected periodontal pocket
have not been established. The destructive mechan-
isms may involve many of the modalities of both the
host defense and microbial virulence. Studies of the
degeneration process of the junctional epithelial
cells themselves are, however, largely missing. The
junctional epithelium consists of distinct popula-
25
tions of cells at different anatomical locations. All
these cells have a responsive phenotype that allows
them to exhibit specic functions in periodontal de-
fense and to take or lose an active role during peri-
odontal disease progression. The junctional epithel-
ium is rmly attached to the tooth by the suprabasal
DAT cell/internal basal lamina structure, and to the
connective tissue by the basal cell/external basal
lamina complex. Environmental conditions differ
essentially in these two locations and even similar
pathogenic challenges may become modied and
cause considerably different cellular responses.
While the coronal DAT cells grow close to the bac-
terial plaque the apical and lateral parts of the junc-
tional epithelium are likely to be inuenced by the
connective tissue and the inammatory reaction.
The GCF provides the most coronal DAT cells with
the necessary conditions to survive but it also con-
tains a variety of biologically active molecules with
the potential capacity to affect their vital functions
and behavior. Although the signicance of the vari-
ous components of the GCF to the failure of the
junctional epithelium and its DAT cells calls for
further studies there are a few candidates that are of
particular interest today. Among these are the poly-
morphonuclear leukocyte products that may have
direct inuences on DAT cell survival and/or their
adhesion and bacterial lipoteichoic acids and meta-
bolic end products, such as propionic and butyric
acids that may interfere with cell division and thus
the turnover and reparative capacity of the junc-
tional epithelium. Similarly, inammatory factors
and components associated with periodontal risk
factors may reach high concentrations in the GCF
and severely interfere with the junctional epithel-
iums normally protective functions.
When the physiology of the junctional epithelium
and its molecular reactions during different external
challenges are known in more detail the tissues role
in the failure of the healthy dentogingival junction
and the reasons that make some individuals suscep-
tible to periodontal diseases will be better under-
stood.
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1996.
PERIODONTOLOGY 2000
ISSN 0906-6713
Formation, collection and
signicance of gingival crevice
uid
Gnnr1n S. Gnirri1ns
Although the existence of gingival crevice uid
(GCF), a uid that emerges between the surface of
the tooth and the epithelial integument, has been
recognized for over 100years (8), the exact nature of
the uid, its origins and composition, has been the
subject of controversy. This may be a result of vari-
ations in the amount and/or nature of the uid pro-
duced under different clinical conditions and the use
of a wide variety of sampling methods. The principal
questions that remain unanswered are whether the
initial uid produced represents a transudate or is
an exudate, and whether any GCF may be found at
a clinically healthy site. These two questions are in-
extricably linked, and although they appear to be of
no more than academic interest, they could yield
therapeutic approaches. An increase in GCF ow
may have physical protective effects through ush-
ing the pocket, as well as facilitating the passage of
immunoglobulins. Such treatment approaches
would aim to enhance the hosts natural specic and
non-specic responses and therefore a better under-
standing of the production of GCF would be desir-
able.
How is GCF formed?
Is GCF an inammatory exudate?
The initial investigations of GCF attempted to relate
its formation to the inammatory changes in the
connective tissues underlying the sulcular and junc-
tional epithelia. These changes were primarily an in-
creased permeability of the blood vessels, which was
induced by chemical or mechanical means. These
early studies monitored the appearance in GCF of
32
substances introduced into the parenteral circula-
tion. The effect on the tissues was also monitored by
studying the histology in gingival biopsies.
Early experiments (15) showed that systemically
administered uorescein appeared in the GCF col-
lected from healthy gingival crevices in dogs. The
samples were collected using lter paper strips held
in place for three minutes and these experiments
were extended to human volunteers (14). Since the
other oral epithelia had not allowed the passage of
the uorochrome it was concluded that differences
in permeability must exist between these oral epi-
thelia and the epithelium lining the gingival pocket.
The transport of uid across the epithelium in the
gingival crevice was also discussed; whether it
should be viewed as a physiological or pathological
process. Subsequent experiments in dogs showed
that the ow of gingival uid increased markedly fol-
lowing stimulation of the gingivae by tooth brushing
or by chewing (16), or after intravenous injection of
histamine or the development of inammation (9).
This led to the conclusion that some irritation,
whether chemical or mechanical, was necessary to
induce the production of GCF and that it should
therefore be considered as a pathological phenom-
enon. Even so, the studies of Brill (9) emphasized the
possible benecial effects of GCF and he postulated
that GCF was an important component of the pro-
tective mechanisms of the crevicular region. This
concept was supported by the ushing effect of GCF,
which was shown to be capable of removing carbon
particles and bacteria which had been introduced
into the gingival crevice (11). In addition, the import-
ant role that GCF may have in transporting antibac-
terial substances, either of host origin or those intro-
duced into the circulation such as antibiotics, to the
Formation, collection and signicance of gingival crevice uid
crevicular space was appreciated (10). He concluded
that stimulation of the gingival margin was import-
ant for the maintenance of gingival health (16).
This extensive work (916) was further supported
by the equally comprehensive studies of Egelberg
(2730) who investigated the histology of the vas-
culature underlying the sulcular and junctional epi-
thelia. In his experiments a suspension of carbon
particles, of known particle size, was injected intra-
venously into dogs which were then killed and their
gingival tissue was examined histologically. In tissue
samples which were healthy and served as control
specimens, the carbon particles remained within the
capillaries and small venules (28). In the presence of
acute inammation, however, the particles could be
seen in the intercellular spaces (29). Changes in the
capillary permeability of healthy gingivae induced
either mechanically by massage of the gingivae with
a ball-ended burnisher, or chemically by the topical
action of histamine were studied for their effect (27).
These stimuli produced a greater ow of GCF and an
increase in vascular permeability, as shown by pene-
tration through the endothelium of the carbon par-
ticles. An indication of the susceptibility of the gingi-
val tissues to mild trauma was provided by the ob-
servation that extravascular carbon particles
accumulated only in the region corresponding to the
width of the lter paper used for uid collection (28).
In a further series of experiments (30), the different
susceptibilities of chronically inamed and healthy
gingivae to extraneous stimuli were demonstrated,
with chronically inamed gingivae showing an in-
crease in GCF production in response to air drying
and systemic histamine, whereas healthy gingivae
only occasionally responded to these stimuli.
Is GCF a transudate of interstitial uid?
The work of both Brill and Egelberg seemed to sug-
gest that the production of GCF was primarily a re-
sult of an increase in the permeability of the vessels
underlying junctional and sulcular epithelium. An
alternative theory arose from the work of Alfano (2)
and from the hypothesis postulated by Pashley (52)
which suggested that the initial uid produced could
simply represent interstitial uid which appears in
the crevice as a result of an osmotic gradient. This
initial, pre-inammatory uid was considered to be
a transudate, and, on stimulation, this changed to
become an inammatory exudate.
The rst studies by Alfano (2) suggested that, at
a clinically healthy gingival crevice, bacterial plaque
would result in the accumulation of high molecular
33
weight molecules. These would permeate the inter-
cellular regions of the epithelium, but would then be
limited by the basement membrane. Here they
would accumulate and produce an osmotic gradient
which would induce the ow of interstitial uid from
the connective tissue to the gingival sulcus. This hy-
pothesis was supported experimentally (3), by the
application of phosphate-buffered saline containing
10mg/ml of homologous serum albumin which re-
sulted in a 100% increase in the volume of GCF pro-
duced. In contrast, the application of phosphate-
buffered saline alone did not enhance the volume of
GCF produced.
A mechanistic analysis of gingival uid production
which analyzed the physiology of the gingival crevice
area with respect to the factors described originally
by Starling was made by Pashley (52). In his paper
he pointed out the lack of essential data on factors
such as periodontal capillary pressure and tissue
compliance. The lack of such information restricted
this study to a theoretical analysis, as approxi-
mations and assumptions had to be made. However,
data are now available on interstitial hydrostatic
pressure and colloid osmotic pressure in rats (1) and
rabbits (26) which largely support the Starlings prin-
ciples applied by Pashley. The model proposed by
Pashley (Fig. 1), predicted that GCF production is
governed by the passage of uid from capillaries into
the tissues (capillary ltrate) and the removal of this
uid by the lymphatic system (lymphatic uptake).
When the rate of capillary ltrate exceeds that of
lymphatic uptake, uid will accumulate as edema
and/or leave the area as GCF. Factors modulating the
respective ltration and uptake processes include
the ltration coefcients of the lymphatic and capil-
lary endothelium as well as the osmotic pressure
within the different compartments. Thus, even in
health, if the osmotic pressure of the sulcular com-
partment exceeds that of the tissue uid, possibly
because of accumulation of plaque-derived mol-
ecules, there will be a net increase in the ow of GCF.
The models proposed by Alfano (2) and Pashley
(52) would account for the effects of histamine and
mechanical stimulation on GCF production as de-
scribed by Brill (916) and Egelberg (2730) as well as
some experimental ndings subsequent to the rst
postulation of these hypotheses. Recent work on
GCF protein concentration suggested that inamed
gingivae had a protein concentration similar to that
of serum (5, 22). Most proteins were signicantly
lower in GCF, but with a strong co-variation between
the proteins studied in the two uids, suggesting
that GCF represents an inammatory exudate of
Grifths
serum (59). However, the small volumes of uid col-
lected from healthy gingival crevices (Fig. 2) had a
protein concentration similar to interstitial uid (22)
with a different range of proteins (23). This would be
consistent with the hypothesis of Alfano (2) that the
initial uid accumulation represents a transudate of
interstitial uid produced by an osmotic gradient
and that the later uid represents a true exudate.
Fig. 1. Mathematical model of Pashley, illustrated within
the gingival crevice. Cross-section of two capillaries and
longitudinal section of lymph vessel with arrows showing
the passage of uid. (a) Absence of inammation: low vas-
cular permeability and low permeability of the basement
membrane results in low GCF ow and high % uptake by
lymph vessels. (b) Macromolecules of plaque result in an
osmotic gradient, increased vascular permeability and
basement membrane changes, resulting in increased pas-
sage of uid into the tissues and increased GCF produc-
tion.
34
This means studies with data showing consistently
lower concentrations of individual proteins in GCF
compared to serum (59), are open to a different
interpretation, as GCF transudate would be expected
to have lower concentrations, whereas the transfer
to an exudate approaches serum concentrations.
Methods of collection and
measurement of the volume of GCF
Several techniques have been employed for the col-
lection of GCF and the technique chosen will de-
pend upon the objectives of the study as each tech-
nique has advantages and disadvantages. The tech-
niques can be divided into three basic strategies,
subject to various modications in their application
by different authors.
Gingival washing methods
In this technique the gingival crevice is perfused
with an isotonic solution, such as Hanks balanced
salt solution, usually of xed volume. The uid col-
lected then represents a dilution of crevicular uid
and contains both cells and soluble constituents
such as plasma proteins.
Two different techniques have been used. The
simplest (56) involved the instillation and re-aspir-
ation of 10ml of Hanks balanced salt solution at the
interdental papilla (Fig. 3). This process was repeated
12 times to allow thorough mixing of the transport
solution and GCF. This technique could therefore be
applied either to individual interdental units or to
Fig. 2. Protein concentration of bodily uids: serum, in-
terstitial uid (IF), lymph, gingival crevice uid from
healthy sites (GCF H) and gingival crevice uid from sites
with pockets (GCF P).
multiple units which were then pooled. A more com-
plicated method (51) involved the construction of a
customized acrylic stent which isolated the gingival
tissues from the rest of the mouth. The tissues were
then irrigated for 15min, with a saline solution,
using a peristaltic pump, and the diluted GCF was
removed.
The washing technique is particularly valuable for
harvesting cells from the gingival crevice region.
However, the production of customized acrylic
stents is complicated and technically demanding. It
is therefore a technique of limited application and
has been restricted to the study of GCF obtained
from only a few individuals. It has also usually only
been applied to the maxillary arch, presumably be-
cause of the difculties of producing a technically
satisfactory appliance for the mandibular arch. It is
also a disadvantage that GCF from individual sites
cannot be analyzed. In contrast, the simpler tech-
nique (56) may be applied to individual sites or
groups of sites which may be categorized as healthy
or inamed. The major disadvantage of this tech-
nique is that all uid may not be recovered during
the aspiration and re-aspiration procedure. Thus ac-
curate quantication of GCF volume or composition
is not possible as the precise dilution factor cannot
be determined.
Capillary tubing or micropipettes
Following the isolation and drying of a site, capillary
tubes of known internal diameter are inserted into
the entrance of the gingival crevice (Fig. 4). GCF from
the crevice migrates into the tube by capillary action
and because the internal diameter is known the vol-
ume of uid collected can be accurately determined
Fig. 3. Collection of crevicular uid by means of gingival
washings; 10ml of uid is ejected from a microsyringe and
re-aspirated,
35
by measuring the distance which the GCF has mi-
grated (58). This technique appears to be ideal as it
provides an undiluted sample of native GCF whose
volume can be accurately assessed. However, it is
difcult to collect an adequate volume of GCF in a
short period, unless the sites are inamed and con-
tain large volumes of GCF. To collect a reasonable
volume of uid may, in some instances, mean that
collection times from an individual site may exceed
30min and, even then, adequate samples from
healthy crevices may be impossible to obtain. It is
difcult to conceive that holding a capillary tube at
the entrance to a gingival crevice for such lengthy
periods ensures an atraumatic collection. A further
complication of this technique is the difculty of re-
moving the complete sample from the tubing. This
has either been forced out with a jet of air or, by
passing a larger xed volume of a diluting solution
through the capillary or, more usually, by centrifuga-
tion of the tube.
Absorbent lter paper strips
There are considerable variations in the application
of the lter paper strip method of collection (Fig. 5).
The principal variations are with respect to not only
the method and timing of sample collection, but also
the means of estimating the volume of sample col-
lected. The advantages of the technique are that it is
quick and easy to use, can be applied to individual
sites and, possibly, is the least traumatic when cor-
rectly used.
Fig. 4. Collection of crevicular uid by means of capillary
tubing. Capillary tubing of known diameter is placed at
the entrance of the crevice and the uid migrates up the
tube by capillary action.
Grifths
Fig. 5. Collection of crevicular uid by means of lter
paper strips. A Periopaper
A
strip is shown at the entrance
of the crevice and the uid migrates up the paper by capil-
lary action.
Methods of collection
The methods of collection may be broadly divided
into the intracrevicular and the extracrevicular tech-
niques (Fig. 6). The former depends on the strip
being inserted into the gingival crevice, whereas in
the latter the strips are overlaid on the gingival crev-
ice region in an attempt to minimize trauma. The
intracrevicular method is the method used most fre-
quently and can be further subdivided depending
upon whether the strip is inserted just at the en-
trance of the crevice or periodontal pocket (47) or
whether the strip is inserted to the base of the pocket
or until minimum resistance is felt (13). In shallow
Fig. 6. Illustration of the positioning of papers for the l-
ter paper strip method of collection: (a) extracrevicular
method; (b) intracrevicular method supercial [Le &
Holm-Pederson (47)], (c) intracrevicular method deep
[Brill (13)].
36
pockets or healthy crevices they probably represent
the same thing.
Methods of estimating the volume
collected
In many early studies the only purpose of the inves-
tigation was to determine the amount of GCF pro-
duced at a given site. This relegated the sampling
of GCF to an additional clinical measurement. The
amount of GCF collected on a strip was assessed by
the distance the uid had migrated up the strip. This
was often taken as a simple linear measurement, but
a more accurate value was achieved by assessing the
area of lter paper wetted by the GCF sample.
Further accuracy was achieved by staining the strips
with ninhydrin to produce a purple color in the area
where GCF had accumulated (20). A similar result
was shown with 2g uorescein given systemically to
each patient 2hours prior to the collection of GCF,
following which the strips were examined under
ultraviolet light (61). Although they used this tech-
nique to determine presence or absence of uid,
rather than to quantify the volume collected, they
found that uorescein labeling was 100 times more
sensitive than ninhydrin for staining protein.
The staining techniques discussed above have a
number of disadvantages; rstly, they are not easily
applied at the chairside. The inevitable delay in
measuring the strip may result in increased variation
in the reported volume as a result of evaporation.
Secondly, the staining of the strips for protein label-
ing prevents further laboratory investigations of the
components of GCF, effectively limiting the tech-
nique to that of volume determination. The intro-
duction of an electronic measuring device, the Per-
iotron
A
, has allowed accurate determination of the
GCF volume and subsequent laboratory investiga-
tion of the sample composition. The instrument
measures the affect on the electrical current ow of
the wetted paper strips. It has two metal jaws which
act as the plates of an electrical condenser. If a dry
strip is placed between the jaws, the capacitance is
translated via the electrical circuitry and registers
zero on the digital readout. A wet strip will increase
the capacitance in proportion to the volume of uid
and this can be measured as an increased value in
the readout. The technique is rapid and has no dis-
cernible affect upon the GCF sample.
Three models of Periotron
A
have been produced
(the 600, 6000 and now the 8000) and each one has
been shown to be an efcient means of measuring
the volume of uid collected on lter paper strips.
However, although a linear t has been described as
satisfactory for calibration over very small volume
ranges (6, 41) other authors have described various
forms of curve as a better t (18, 33, 54) and indeed
different curves or lines have been described for dif-
ferent aspects of the volume range (17). One of the
limitations of the Periotron
A
has been its inability to
measure volumes of GCF greater than 1.0ml, al-
though when using lter strips such as Whatman
3MM strips, the strips themselves are capable of ab-
sorbing much larger volumes. Volumes greater than
1.0ml may be recovered from severely inamed sites
which may be the samples of most interest in any
study of volume, ow rate, or composition of GCF.
The newer Periotron
A
8000 has a wider range, par-
ticularly if the Sialo scale is used, this being usually
reserved for its role as a sialometer. The calibration
of the Periotron
A
8000 cannot be resolved by linear
regression, because it has signicant curved compo-
nents which are best described by a fourth order
polynomial (18). This is in agreement with the
manufacturers prescribed conversion programme
rIcoNvrn1, but it is clear that the calibration curve
generated needs to span the whole range of possible
readings as extrapolating beyond these data values
would lead to inaccuracies. Most studies which have
described calibration of the Periotron
A
describe the
magnitude of error between repeat samplings. How-
ever, it should be remembered that they are usually
measuring the total error in the model and this may
be attributed to the Periotron
A
itself, uid evapor-
ation, the syringe used to dispense replicate vol-
umes, and the dispensing method. These small
errors become magnied in percentage terms when
the original volumes are small, and this is especially
so when the lower end of the calibration curve is
generated. For this reason the lower end of the cali-
bration curve is considered to be suspect and it is
probably best to view it as a machine which can ac-
curately detect volumes between 0.1 and 1.2ml.
From most of the studies on calibration of the Per-
iotron
A
the following recommendations can be
made:
O each machine needs its own calibration, as ma-
chines differ markedly in their range.
O serum is a suitable material to generate a cali-
bration curve.
O an accurate syringe and some form of standard-
ization of dispensing duplicate volumes are essen-
tial.
O duplicate volumes are required, but it is not
necessary to add additional replicates.
37
O a full range of volumes from 0.1 to 1.2ml is necess-
ary to generate an accurate curve.
An alternative approach, involving the weighing of
strips before and after sample collection, has been
adopted by some workers (21, 60, 61). This has been
successful but requires a very sensitive balance to
estimate the very small amounts of uid which may
be collected from a healthy crevice. As with the pro-
tein staining and assessment of wetted area tech-
niques described earlier, evaporative losses because
of delays in determining the volume may distort the
volumes obtained. Indeed, if a lter paper strip with
either GCF or a known volume of serum is placed
on a balance without being included within a sealed
container evaporation can be observed by following
the decrease in weight recorded on the balance.
Association of GCF with health or
disease
Controversy still exists as to whether GCF is present
at sites designated as clinically healthy. This dispute
probably arises, at least in part, as a result of the
different techniques used to sample the GCF. The
initial experiments in healthy crevices used the in-
tracrevicular sampling technique (inserting strips
until resistance is felt) and yielded detectable levels
of GCF (15). In contrast, if lter paper strips were
placed just at the entrance to the gingival crevice
GCF was seldom detected (47, 50). The proponents
of the latter technique concluded that the deep in-
tracrevicular technique induced trauma, producing
an artefactual ow of uid into the healthy crevice.
Although these arguments were derived from the l-
ter paper strip method of collection, similar argu-
ments of the effect of trauma during sampling apply
equally to the other sampling techniques, such as
those using capillary tubes.
The association of an increased volume of GCF
with an increase in the severity of inammation is
well supported by evidence from the literature (12,
47, 48, 50, 55). This probably formed the basis for
the criteria in the brochure from the original Per-
iotron
A
600 which gave guidelines on interpreting
the meaning of Periotron readings obtained in clin-
ical terms. (Table1).
These associations of crevicular uid production
with clinical and histological signs of inammation
have been thoroughly investigated in both cross-
sectional and longitudinal studies (24, 31, 40, 45,
Grifths
Table1. Translation of Periotron values to clinical
conditions and Gingival Index with which they may
be associated
Periotron Level of gingival Gingival
reading inammation Index
020 healthy 0
2140 mild 1
4180 moderate 2
81200 severe 3
Data supplied by manufacturers adapted from research investigations,
see text.
47, 48, 55). However, the manufacturers produced
a chart which further extended the interpretation of
the Periotron readings to express detailed underly-
ing pathological mechanisms which may be taking
place and also detailing a prognosis for the tooth.
Thus Periotron readings of 80200 were allegedly
associated with the invisible symptoms of wide-
spread inammation. In contrast, sites designated
clinically healthy may yield volumes as large as 0.71
ml (approximately 120Periotron Units), whereas
some clinically inamed sites may yield volumes
1.0ml (approximately 20Periotron Units) (35). This
suggests that either the clinical indices are too cru-
de an assessment of inammation, or that the
method for sampling and determining the volume
of GCF is imprecise. As in vitro studies of volume
determination suggest a robustness to the method
of GCF collection and measurement, the authors
suggest that the quantitative measurement of GCF
volume and ow rate may be better measures of
the early signs of inammation than the subjective
measures of color, bleeding and the composite na-
ture of the Gingival Index.
It has been suggested by other workers that in-
creases in the ow of GCF, together with an in-
creased tendency to bleed is one of the earliest signs
of inammation of the gingivae (19, 32, 49). Many
workers have suggested that increased GCF volumes
may be a sign of subclinical inammation. Sites
classied as healthy by clinical criteria would poss-
ibly vary in their degree of subclinical inammation.
This explanation is supported by histological obser-
vations (4) which show that a gingival connective
tissue, totally free of inammatory cells, probably
does not exist (nor can it be achieved). If this argu-
ment is extended to the production of GCF, all sites
would yield GCF, even those classied as healthy by
clinical criteria.
The preceding discussion highlights the potential
38
role of GCF volume and ow rate measurements in
the diagnosis of gingival inammation. Little refer-
ence has been made to their potential for the diag-
nosis of the destructive forms of periodontal disease.
This aspect has seldom been investigated, presum-
ably because other assessments of inammation
such as color and bleeding show no association with
destructive disease (38, 39). Associations between
GCF volume or ow rate and pocket depths have
been difcult to interpret A good correlation be-
tween GCF ow rate and the level of gingival in-
ammation and an even higher correlation between
ow rate and pocket depth has been demonstrated
(48). In contrast, only a weak correlation between
GCF ow and pocket depths has been described
(60). This discrepancy may once again be attributed
to variations in the method of sampling; where one
study used the deep intracrevicular technique (48)
whilst the other (60) inserted strips into the crevice
to a depth of 1mm. Thus, the area of the strip in
contact with the pocket epithelium would vary be-
tween the two studies. When allowance was made
for this variation in the area available for uid ab-
sorption in the deep pockets, only a weak correlation
between GCF ow and pocket depth was described
(48). This raises another interesting problem as it is
not clear what the explanation for differing GCF vol-
umes collected from patients or indeed sites within
a patient means. Clearly repeated samples from the
same site have value in determining progress of in-
ammation. However, a larger volume collected
from one site compared to another may have little
meaning. The variation is partly a result of variations
in the inammation present, but is also attributed to
the sample area from which they were collected,
which may be a result of pocket depth or simply ana-
tomical variation; a molar giving a much larger po-
tential pool than a lower incisor. It is also not clearly
established what the role of gravity on collection
would be, although an effect for uid migration onto
lter paper strips with or against gravity has been
demonstrated (46). However, in a general linear
models procedure the major sources of variation
came from the subject and the site, rather than from
associations between any of the clinical variables
(35).
A further explanation for the failure to detect any
consistent associations between clinical parameters
and GCF volume and ow rate, has been attributed
to the differences between the sensitivities of the dif-
ferent parameters being tested. GCF volume and
ow rate are indicators of changes in vascular per-
meability, which occurs in the early stages of in-
ammation. The standard clinical measurements
used to record inammation assess changes in color,
contour and the tendency of the tissues to bleed.
Whilst these may also be representative of the early
signs of inammation, they are categorized variables
which are more prone to subjective assessments and
may therefore, be less reliable than the ratio meas-
urements obtained by determining volume and ow
rate of GCF.
Problems with GCF collection and
data interpretation
Contamination
The major sources of contamination of GCF samples
would be blood, saliva, or plaque. Frank blood con-
tamination is usually dealt with by discarding the
sample and removing the data from analysis. How-
ever, detailed reporting of how many samples this
affects is seldom recorded.
The presence of dental plaque on lter paper
strips used for collecting GCF has been shown to
have a marked effect on the volume recorded (57).
Experiments where dental plaque was applied di-
rectly to lter paper strips (37) showed that a large
plaque mass contained a considerable amount of
uid which would inuence volume determinations,
which would be expressed as volume of GCF. This
has been supported by other studies which showed
that failure to remove plaque adequately from the
site prior to sampling has a major effect on the vol-
ume determined (25).
Careful isolation should be performed in an effort
to minimize the potential for saliva contamination.
Occasionally this good isolation breaks down and
samples obviously contaminated should be dis-
carded. Alpha-amylase was used in an assay to con-
rm, or refute, the presence of the contamination of
GCF samples with saliva (37). Application of the as-
say to saliva samples collected using a good isolation
and drying technique conrmed that the likelihood
of a signicant contribution from saliva was small.
Sampling time
The early literature from the Periotron
A
suggested
that lter paper strips should be left in place for 5
seconds. Alternative approaches to sampling tech-
niques have been developed in an effort to increase
the volume of GCF sample available for subsequent
laboratory analysis. This has either included leaving
39
the strip in place for a longer period (this runs the
risk of going off-scale on the Periotron
A
and there-
fore precluding accurate volume determination) or
the use of a sequence of repeated strips, with poss-
ible recovery periods in between. Alternative ap-
proaches have also included collecting until a mini-
mum volume has been recovered. This latter ap-
proach has sometimes resulted in collection times of
2030min.
The problem with prolonged collection times is
that the nature of the GCF sample collected is likely
to change with the protein concentration of the ini-
tial GCF collected comparable to interstitial uid,
whereas prolonged sampling at the site resulted in
protein concentrations approaching those of serum
(22). Furthermore, analysis of GCF samples by so-
dium dodecyl sulphatepolyacrylamide gel electro-
phoresis (23) showed detectable electrophoretic
bands in the early samples of GCF which were not
present in serum samples. These bands disappeared
in samples of GCF collected over longer periods.
Similarly, there were bands present in serum which
were not evident during the early samples of GCF,
but which appeared in the samples collected during
prolonged sampling. Further support for the inu-
ence of sampling technique on the composition of
GCF comes from an experimental gingivitis study,
which showed that IgM was infrequently detected in
GCF samples at a normal healthy crevice, but was
found increasingly when inammation was present
and especially if a prolonged sampling protocol had
been adopted (36).
Volume determination
As highlighted earlier, evaporation is considered to
be a signicant problem in accurate volume deter-
mination of GCF samples. This is particularly the
case as the total volumes collected are usually less
than 1ml and more often than not are less than 0.5
ml. Concerns are also expressed regarding the accu-
racy of the calibration graph produced for the Per-
iotron
A
, particularly with respect to small volumes.
Whilst both these factors will result in small errors
of volume determination, because the total sample
of GCF collected is small the percentage of error is
considered to be of more major signicance. In prac-
tical terms it may be necessary to determine a par-
ticular volume (for example 0.2ml) below which it is
not possible to analyze the sample. This would then
place the Periotron readings at a point in the cali-
bration graph at which we may have more con-
dence in the values obtained.
Grifths
Recovery from strips
Having collected the GCF sample and determined its
volume the samples are usually then required for
some investigation of the composition of GCF. To
achieve this it is necessary to recover the GCF from
the lter paper strips and initial work indicated that
protein recovery was close to 100% using a centrifu-
gal elution technique (21). A variety of other
methods of elution have been employed, but it is es-
sential in all instances to determine the percentage
recovery from the original samples. This is particu-
larly so in light of recent ndings which have de-
scribed signicant differences in the percentage re-
covery of proteins from lter papers, which were de-
pendent on both the type of paper and the
concentration of the original protein sample (42). In-
deed, this may be a reason for conicting results de-
scribed between different research groups, possibly
because of entrapment within, or binding of GCF
proteins to the lter papers.
Data reporting
Constituents found within GCF samples have either
been reported as absolute amount (mg), concen-
trations (mg/ml) or either of these two measure-
ments with reference to pocket depth or duration of
sample collection.
A study comparing four methods of data analysis
for lysosomal enzyme activity in GCF indicated that,
because of the inherent problems of accurate deter-
mination of GCF volume, concentration was not an
appropriate method of data presentation (43). Their
analysis suggested that the total amount of enzyme
activity was the preferred method of data expression.
Nevertheless, earlier work (7, 44), indicated that data
presented as concentration and total enzyme activity
can be used to identify differences between disease
activity within patients. These studies indicate that
patients exhibiting disease activity tend to have an
increased enzyme activity and an increased uid vol-
ume, the combination of which may ultimately re-
sult in a lower concentration in comparison with pa-
tients not exhibiting disease progression. This dis-
cussion was extended to consider whether an
increased volume of GCF would be diluting the con-
centration, as would be the case when the constitu-
ent examined is primarily being produced locally
(34, 43), or whether the concentration would be in-
creased because of a signicant inclusion from the
systemic circulation, as may be the case with, for ex-
ample, immunoglobulins (36). In view of the exten-
40
sive laboratory studies, in a wide variety of elds,
which report signicant doseresponse effects it
would seem sensible to report GCF data as both total
amount and concentration. The inclusion of the vol-
ume data would then allow the reader a clear view
of what is occurring. Furthermore, some estimation
of the biological effect of the amount or concen-
tration of the constituent examined would appear to
be desirable.
Composition of GCF
The failure of simple estimations of GCF volume or
ow rate to indicate adequately the current disease
activity or to predict the future disease activity has
led to more detailed investigations in the laboratory
of the composition of GCF. Nevertheless, whilst con-
sidering potential markers in GCF it is important to
remember that the method of collecting GCF may
have a signicant effect upon the nature of the
sample collected and will therefore prejudice results
assessing diagnostic markers.
The failure of current clinical methods accurately
to diagnose existing active periodontal disease
makes treatment an empiric exercise and accurate
evaluation of the success of various treatment re-
gimes is therefore impossible. This has led to the op-
timistic comment (in a review of the status and fu-
ture needs of periodontal diagnosis): The compo-
sition of gingival uid seems promising as a
potential medium for the detection of early changes
which could indicate the onset of disease (53). How-
ever, this is contrasted by the more pessimistic view
that, despite considerable progress in our knowledge
of crevicular uid, Up to now, for instance, none of
the multiple components analyzed in the uid has
improved clinical judgement of the rate of progress
of gingivitis and periodontitis or of the rate of repair
of these conditions (20).
The major attraction of GCF as a diagnostic
marker (53) is the site-specic nature of the sample.
This allows laboratory investigations of GCF con-
stituents to be linked to clinical assessments at the
site of sample collection. However, the small vol-
umes collected from individual sites mean that re-
searchers must often restrict themselves to a single
analyte or pool samples and data with a resultant
loss in the site-specic nature of the sample. It may
be possible, however, with more sensitive techniques
to estimate more than one analyte.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Gingival crevice uid ow
J. M G
Introduction
Gingival crevice uid (GCF) ow is an important de-
terminant in the ecology of the periodontal pocket
or sulcus. It creates a ushing action and an isolation
effect. In addition, it probably determines the growth
level of subgingival microorganisms and is a poten-
tial marker for periodontal disease activity.
The rst important characteristic associated with
GCF ow is its ushing action. Substances put into
the periodontal pocket are rapidly washed out. Even
before the importance of GCF was recognized, clear-
ance of carbon particles (33) and of amalgam par-
ticles (19) from the gingival sulcus had been re-
ported. As GCF became more intensively studied, in-
stilled charcoal particles and even bacteria (2) were
found to be rapidly removed from the periodontal
pocket. The introduction of controlled release anti-
bacterial therapy directed to the periodontal en-
vironment was conceived as compensating for the
rapid removal of substances from the gingival sulcus
(15). Indeed, this has been the primary rationale for
the use of intrapocket controlled drug delivery sys-
tems for the administration of medication to peri-
odontal tissues.
The second important characteristic associated
with GCF ow is the isolation effect. Substances
from the outside do not easily penetrate the peri-
odontal pocket. Several examples lead one to con-
clude that GCF is relatively isolated from saliva. Con-
siderable evidence suggests that antibacterial mouth
rinses do not penetrate the periodontal pocket (14).
Although salivary amylase can be measured in GCF
samples, it appears to be a sampling contaminant
because the majority of GCF samples (70%) have
undetectable levels of salivary amylase (17). The
concentration of immunoglobulin G (IgG) found in
GCF from periodontal pockets is approximately 100
times that found in saliva (3). Clearly, this could not
be the case if saliva gained ready access to the peri-
odontal pocket. One may reasonably conclude that
43
the net outward GCF ow inhibits retrograde salivary
ow and that salivary contents do not generally en-
ter the periodontal pocket. This creates a relative iso-
lation of the periodontal pocket from the rest of the
oral cavity.
The nutrient effect of GCF ow has been sug-
gested by associations between bacterial numbers or
composition and GCF ow in several studies (18, 27).
Although it is widely suggested that accumulation of
bacteria leads to increased GCF ow, it has not been
so widely appreciated that increased GCF ow may
support larger bacterial plaque masses.
The diagnostic potential of GCF has long been rec-
ognized (13). The specic importance of GCF ow,
however, has received little emphasis.
It is now widely appreciated that GCF is formed as
a blood ultraltrate but accumulates elements of the
metabolism from both bacterial and host cells from
the gingival crevice environment. Because of this
characteristic of accumulation, GCF composition
has become a logical focus of methods to diagnose
disease in the periodontal environment. Analysis of
GCF constituents has allowed us to probe many of
the mysteries of the relationship between the human
host and oral bacteria (12, 23, 30). The emphasis of
this article, however, will be on the GCF ow rate
rather than its composition. The purpose of this re-
view is to focus attention on the importance of GCF
ow and the effect that small streams of uid owing
out of the periodontal pocket have on the peri-
odontal environment. Thus, GCF ow will be de-
ned, methods to measure it are described and its
signicance is considered.
Denition
GCF ow (or ow rate) is the process of uid moving
into and out of the gingival crevice or pocket (Fig. 1).
It is a small stream, usually only a few microliters
per hour. GCF in a gingival sulcus or periodontal
Goodson
pocket is like a spring-fed pond. Fluid enters the
pond from an unseen source and leaves by owing
out at the margin. Fluid ow is a rate measure. It is
the volume that crosses a dened boundary over a
given time, mathematically symbolized as dV/dt, the
rst derivative of volume with respect to time (Fig.
1). In the gingival environment at equilibrium, the
inux (f
i
dV
i
/dt) should equal the efux (f
o
dV
o
/
dt) so that measurement of either could be con-
sidered the GCF ow. As a practical matter, however,
only the inux is measured. The gingival sulcus or
pocket also has a resting volume (V
r
) through which
the GCF ows. The washout ratio (f
i
/V
r
) is the num-
ber of times the pocket volume is replaced in unit
time.
Methods of measurement
The volume of a GCF sample is most commonly
measured by placing a calibrated lter paper strip
at the opening of the gingival crevice or periodontal
Fig. 1. The three components of gingival crevice uid
(GCF) ow are the resting volume, the inux and the ef-
ux. Since the resting volume is constant over the meas-
urement period and losses from evaporation or absorp-
tion are minimal, GCF ow can be measured by consider-
ing either the inux or efux. Measurement of GCF inux
is the most easily accomplished.
44
pocket and allowing uid to accumulate for a rela-
tively short period of time, usually in the order of 30
seconds, and measuring the volume non-destruc-
tively by a chair-side instrument based on the dia-
lectric change of the wetted lter paper (Periotron
A
).
Assuming that all GCF at the sampling site has been
removed, this measurement (V
s
), the sample volume,
is neither the inux (f
i
dV
i
/dt) nor the efux (f
o
dV
o
/dt) but the sum of the resting volume and the
inux increment, V
r
f
i
Dt over the collection period
Dt (i.e. V
s
V
r
f
i
Dt).
The resting volume (V
r
) is the result of forming a
pool of uid in the crevice or pocket. The increment
in volume as a result of GCF ow (f
i
Dt) results be-
cause the GCF ow cannot be turned off while taking
the measurement. Many investigators have assumed
that the resting volume of the gingival sulcus or peri-
odontal pocket is negligible (i.e. V
r
0), hence the
volume of any GCF sample collected divided by the
time over which it was collected is the GCF ow rate.
This can only be true if the resting volume is zero,
which appears never to be the case. Since the GCF
sample itself is usually only a fraction of a microliter,
it may seem unreasonable that such a small number
should be further divided into a resting volume and
a ow component. This division, however, is both
signicant and important to understanding the ef-
fects of GCF ow on the environment.
How can the inux be measured separately from
the resting volume? To explain the methods consider
rst the leaky faucet problem (Fig. 2). In this ex-
ample, water slowly drips into an unknown volume
Fig. 2. The leaky faucet problem. How does one measure
the ow of water from a leaky faucet into a cup without
removing the cup or changing the ow?
and overows to the sink. Since it is a leak, we can-
not stop the ow while we make a measurement. In
addition, we cannot remove the cup. How does one
measure this ow?
There are at least two approaches to measuring
GCF ow using conventional methods for measure-
ment of GCF volume (Fig. 3). Both approaches col-
lect the liquid sample and measure its volume. The
difference between approaches is in the timing of
the collection and consequently, the underlying as-
sumptions.
In the rst approach (Fig. 3, Method 1) samples are
collected rapidly under a strict timing protocol so that
the resting volume is removed by the rst sample and
the volume of the newly formedGCFcanbe measured
in subsequent samples. The key to this measurement
is to select a sampling time (Dt) that is small enough
so that the resting volume of the pocket is not allowed
to re-establish. Under these conditions, the resting
Fig. 3. Two methods of measuring gingival crevice uid
ow (f
i
) and resting pocket volume (V
r
). The rst method
depends on removing all uid from the pocket or sulcus
and measuring the amount that forms over a specied
time (Dt). The second method depends on allowing vol-
ume equilibrium to be re-established before each sample
and measuring total resting volume plus the volume that
entered the pocket during the sampling period for varying
periods of time.
45
volume is rapidly depleted. Ideally, one would remove
the entire contents of the pocket with the rst sample
so that all subsequent samples would be repetitive es-
timates of the GCF volume ow over the sampling
time. Once depleted, the ow would be computed as
DV/Dt where DV is the volume that accumulates over
the sampling time Dt. Note, that the sampling time in-
cludes both the time the sampling paper is in place
and any preceding rest interval during which uid is
still accumulating.
The second approach (Fig. 3, Method 2) is to col-
lect samples after equilibrium has been re-estab-
lished in the pocket over several different time inter-
vals. In this case, the rest interval should be much
greater than the time to ll so that equilibrium con-
ditions have had time to re-establish. In general, in-
tervals of 10min or more should be sufcient. In this
case, each GCF sample includes both the resting vol-
ume (V
r
) and the volume of GCF that entered the
pocket over the sampling period. This approach
lends itself directly to linear regression analysis in
which the volume and time of each sample contrib-
ute to the analysis to determine the slope which is
the GCF inux (f
i
) and the intercept which is the
resting volume of the pocket (V
r
).
Method 1: Remove resting volume
This method has been used to estimate GCF ow (1,
14). In this protocol, GCF samples were taken from
four slightly inamed periodontal sites with an aver-
age pocket depth of 3.8mm using a strictly timed
protocol. Filter paper strips were placed for 20s and
25s was allowed before the next 20-s sample was
taken. This entire sequence was repeated ve times
for each subject visit. The sites were identically
measured at nine visits over a 2-month period and
all values for each sample time were averaged. This
experiment produced the results illustrated in Fig. 4.
In the analysis as originally presented, it was as-
sumed that the volume of the rst sample was the
best estimate of the resting volume V
r
. The termin-
ology of resting volume as applied to the rst GCF
sample taken was introduced in studies of GCF en-
zymatic activity (24). In the analysis of Fig. 4, the
total volume measured is reduced by the amount
that would have entered the pocket in the 20-s sam-
pling period using the computed ow.
In a study of healthy subjects, the GCF samples
were taken from two sites in 32 adolescent (1719
years) male subjects with little or no periodontitis
(9). The protocol was to take four, 5-s samples with
a 1-min rest interval. As suggested in Fig. 5, one may
Goodson
estimate the GCF ow by averaging volumes of the
last three samples and the resting volume from the
volume of the rst sample. In this case, the average
of GCF samples 2, 3 and 4 was 0.0873ml. From this,
the GCF ow(0.0873/60) 36005.24ml/h. The
Fig. 4. Gingival crevice uid (GCF) sample volumes col-
lected for 20s with 25-s intervals between collections in a
subject with mild periodontal disease (14). Computational
data include the volume of the initial sample and the
equilibrium sample. By this analysis, gingival crevice uid
ow was 20ml/h and the resting volume was 0.4ml.
Fig. 5. Volume of gingival crevice uid (GCF) samples
from 17- to 19-year-old healthy males. The protocol in-
volved 5-s samples taken every minute (9).
46
resting volume can then be calculated as the volume
of sample 1 minus the GCF ow occurring in the 5-
s sampling period 0.168(0.001455) 0.16ml.
In a second study by the same group, the GCF
samples were taken at two sites (maxillary left rst
molar and mandibular right cuspid) in 102 18-year-
old, healthy male subjects with little or no peri-
odontitis (average pocket depth 3mm) and mild gin-
givitis (average gingival index 1.6) (16). The protocol
of this study was the same as illustrated in Fig. 5. The
GCF ow obtained by averaging volumes of the last
three samples was 2.4 (cuspids) to 6 (molars) ml/h
(Table1). The resting volume estimated from the vol-
ume of the rst sample was 0.040.1ml. As would be
expected, the rst GCF sample had a signicantly
greater volume than the following three samples.
Measurements were repeated in this study 1year
later with no signicant changes in either GCF ow
or resting GCF volume. This observation suggests
that within a healthy population, GCF ow measure-
ments may not change appreciably over time.
Another study of periodontally healthy subjects
suggests effects that may occur as a result of irri-
tation (5). In this study, two subjects were selected
with gingival index and plaque index of zero and no
periodontal pockets. Filter paper strips were placed
for 30s and 30s was allowed before the next 30-s
sample was taken. This entire sequence was re-
peated ve times for each subject visit. The sites
were identically measured at ve visits and all values
for each sample time were averaged. This experi-
ment produced the results illustrated in Fig. 6. By
this analysis, the GCF ow was 3ml/h and the resting
volume was 0.055ml.
This study indicates that although measurable
GCF ow occurred at healthy sites, the process of
sampling shallow sites may have irritated the sulcus.
This might be expected because the lter paper strip
and air syringe blasts could more easily reach the
base of the sulcus at healthy sites than at sites with
pockets. The increased variability seen in samples 4
and 5 suggests that trauma as a result of sample tak-
ing may have occurred at some but not all sites.
If one interprets the second sample in this study
as being a reasonable measure of inux volume over
60s, then an inux rate of 3ml/h can be calculated.
This is not unreasonable because a single sampling
of a healthy gingival sulcus would be expected to re-
move most if not all of the residual volume V
r
. If the
later samples 4 and 5 represent ow in response to
gingival irritation, one can similarly compute that
the inux rate more than doubles in response to
mechanical irritation.
Table1. Average gingival crevice uid (GCF) ow and resting volume of two teeth in 120 healthy adolescent
males (16) measured twice over a 1-year interval (sample 1 versus sample 2)
Tooth Sample Sample 1 Average volume GF ow Resting
(ml) of sample 24 (ml) (ml/ h) volume (ml)
Maxillary left rst molar 1 0.20 0.10 6.0 0.100
2 0.16 0.10 6.0 0.100
Mandibular right cuspid 1 0.11 0.05 3.0 0.050
2 0.07 0.04 2.4 0.040
Method 2: Measure combined resting
volume and inux for varying time
periods
A second method of measuring GCF ow (Fig. 3) is
to measure the resting volume plus the inux for dif-
fering time periods. It is assumed in this approach
that the resting volume remains constant and that
a sufciently long time between measurements has
been allowed to elapse so that the pocket or sulcus
volume will have returned to a steady state. Each
sample would represent the resting volume (V
r
) plus
varying amounts of GCF from inux (f
i
) over the
period collected (Dt). The ow would be computed
Fig. 6. Gingival crevice uid (GCF) sample volumes col-
lected for 30s with 30-s intervals between collections in
two healthy subjects (no plaque, gingivitis or periodontal
disease). Whiskers represent standard deviations. The vol-
ume of sample 1 was signicantly greater than that of 2
or 3 but there was no difference between sample 1 and
samples 4 or 5 (5).
47
by linear regression assuming that each observation
is an estimate of the function VV
r
f
i
Dt.
As an example of this approach published data
(29) were used for analysis. In this study, GCF collec-
tion for varying durations (5, 10, 20 and 30s) were
permuted in sequence and repeated four times with
a 10-min (600-s) equilibration between sequences.
For the purposes of this analysis, only the rst
sample of each sequence was considered. This pro-
vided samples of each duration with a 10-min equili-
bration period (Fig. 7). Samples were taken from the
mesiofacial line angle of each of four adjacent teeth
of 18 subjects with untreated adult periodontitis. The
average GCF volume values were estimated from
Fig. 7. Average volume of samples taken for 5, 10, 20 and
30s immediately following a 10-min equilibration period
from 18 subjects with periodontal disease (upper panel).
By regression analysis (lower panel), the gingival crevice
uid (GCF) ow was 14.4ml/h and the resting volume was
0.12ml (29).
Goodson
Fig. 8. Method 3. A marker substance pumped into the
reservoir at a constant rate will establish an equilibrium
concentration dependent on the uid ow rate.
these data assuming that 1 crevicular uid unit
0.005ml (20).
Method 3: Measure the equilibrium
concentration of a marker substance
pumped into a pocket at a constant rate
This approach describes a method and illustrates an
important application to which knowledge of GCF
ow rate may be applied. The concept is that by
pumping a marker substance into a periodontal
pocket at a constant rate, an equilibrium concen-
tration will be established which is the result of the
uid ow rate and the pump delivery rate (Fig. 8).
An intrapocket drug delivery system is a small
pump that can deliver a marker substance into the
periodontal pocket. Although many devices of this
type have been developed (6), only one, the tetracy-
cline ber, establishes and maintains the constant
concentration required for this method. When
placed in a periodontal pocket, this pump will estab-
lish and maintain an equilibrium concentration (C
e
)
of approximately 1590mg/ml 1.6mg/ml (32) for 10
days.
The in vitro delivery rate of this pump has an early
burst over the rst 12h and begins to fall off after
200h (Fig. 9). Between 12 and 200h, however, the re-
lease is relatively constant at 2mg/cm/h. Since each
23cm-long ber is sufcient to treat approximately
two teeth, the approximate length of ber to treat a
tooth is 11.5cm. Hence, the rate of tetracycline deliv-
ery is:
dq/dt 2mg/cm/h 11.5cm 23mg/h
and the GCF ow rate is:
f
i
[(dq/dt)/C
e
] [(23mg/h)/(1.6mg/ml)] 14.4ml/h
48
Application of this method requires that a measured
length of tetracycline ber be placed into a peri-
odontal pocket and the equilibrium concentration
be measured at a later time. To date, no published
study has been conducted to evaluate GCF ow in
this manner. The measurement of tetracycline con-
centration requires two measurements, the GCF vol-
ume of the sample and the amount of tetracycline
in the sample. Several methods for both have been
described (7, 32).
Signicance
GCF ow and experimental gingivitis
The data of Lamster et al. (24) describe changes in
GCF sample volumes taken during experimental gin-
givitis that are directly amenable to analysis by
method 1. In the GCF sampling protocol of this
study (Fig. 10), a lter paper strip was placed in the
sulcus for 30s and removed for volume determi-
nation (V
1
). Thirty seconds was allowed to elapse
Fig. 9. In vitro release rate of tetracycline bers. Constant
release of 2mg/cm/h occurs between 12 and 200h.
Fig. 10. Sampling protocol for determination of gingival
crevice uid (GCF) sample volumes V
1
and V
2
. The sam-
pling interval for GCF ow determination was 33s.
Fig. 11. Gingival crevice uid (GCF) sample volumes V
1
and V
2
during the development of experimental gingivitis.
At all sampling times, V
1
was signicantly greater than V
2
.
The average volume of V
2
was signicantly greater than
baseline at 3 and 4weeks (*P0.01). The volume of V
1
became signicantly greater than baseline only at 4weeks
(*P0.05) (24).
and the GCF volume was determined by introducing
a second lter paper strip into the site for 3s (V
2
).
Eight male dental students (ages 2329) partici-
pated in the study. After a period of intensive oral
hygiene, they suspended oral hygiene in the maxil-
lary right quadrant for 4weeks. GCF samples were
taken each week from four sites in each subject: the
mesiobuccal crevicular region of the cuspid, rst and
second bicuspids and rst molar of the uncleaned
quadrant. The average values for each of the two
samples (V
1
and V
2
) are illustrated in Fig. 11.
The results of this study clearly indicated that the
two sample volumes collected increased linearly
over the period of development of experimental
Table2. Computation of the gingival crevice uid inux (f
i
) and the resting volume (V
r
), and the time to form 1
ml (T) from gingival crevice uid samples V
1
and V
2
(24)
Week V
1
V
2
f
i
f
i
f
i
V
r
T
(ml) (ml) (ml/s) (ml/min) (ml/h) (ml) (min)
0 0.151 0.097 0.0029 0.18 10.6 0.063 5.6
1 0.182 0.117 0.0035 0.21 12.7 0.076 4.7
2 0.192 0.124 0.0038 0.23 13.5 0.079 4.4
3 0.199 0.154 0.0047 0.28 16.8 0.059 3.6
4 0.250 0.179 0.0054 0.32 19.5 0.087 3.1
Formula V
2
/33 60f
i
3600f
i
V
1
30f
i
1/f
i
(ml/s) (ml/s)
49
Fig. 12. Gingival crevice uid (GCF) ow and resting vol-
ume during experimental gingivitis analyzed by method 1.
By this analysis, the resting volume of the sulcus remains
relatively constant but the GCF ow increases linearly as
experimental gingivitis develops (24).
gingivitis. As identied in the published study, V
2
is
closely related to GCF ow. The actual ow, how-
ever, is the collected volume divided by the collec-
tion interval (f
i
V
2
/33s). Also, as identied in the
published study, V
1
is closely related to the resting
volume. The actual resting volume, however, is the
measured sample volume minus the amount of
uid that entered the sulcus during the sampling
period (V
r
V
1
30f
i
). Values taken from this
study and the associated computations are illus-
trated in Table2.
By this analysis (Fig. 12), the GCF inux extrapo-
lated initially to 10.2ml/h and increased by 2.2ml/h
each week during the development of experimental
gingivitis (r
2
0.98). By contrast, the resting volume
of the sulcus was initially 0.07ml and remained rela-
tively constant over the period of developing gingi-
vitis, increasing by only 0.003ml/week.
These results are more in accord with expec-
tations. The resting volume of gingival sulci should
be relatively constant because it is clearly related to
Goodson
Fig. 13. Distribution of gingival crevice uid (GCF) ow
measurements of 68 sites in 17 subjects before treatment
(T0 and bars) and at 90 and 150days following treat-
ment. Values ranged from 1.8 to 137ml/h.
the depth of the sulcus and anatomical features that
should not change appreciably during experimental
gingivitis. The gingival ow, however, would be ex-
pected to increase dramatically as inammation be-
comes more severe and vascular permeability in-
creases.
GCF ow and therapeutic response
Deep pockets with periodontal disease can exhibit
high GCF ow rates. It is therefore reasonable to ex-
pect that effective therapy should substantially re-
duce the ow rate bringing the pocket closer to the
GCF ow rates measured at healthy sites. To test this
hypothesis, a study was conducted on 17 subjects to
determine the effect of combined scaling and root
planing followed by placement of the tetracycline
ber local delivery system (21). All sites with pocket
depth greater or equal to 5mm were treated and
monitored longitudinally for 150days. GCF ow was
measured in addition to the evaluation of changes
in pocket depth, attachment level and bleeding on
probing. The sampling protocol for this study was to
place a lter paper strip for 2min, discard that strip
and place another for 20s measuring the volume of
the second strip using the Periotron 6000. The GCF
ow rate was computed by multiplying the Periotron
units measured on the second strip by 1806060/
20) times the calibration factor (approximately 0.005
ml/unit) to obtain GCF ow in ml/h.
The distribution of GCF ow rates measured be-
fore therapy in these subjects varied from 1.8 to 137
ml/h with an average of 45.735.7 (SD) ml/h (Fig.
13). All sites were selected to have initial pocket
50
depths of 4mm or greater with an average of 5.4
0.9mm (SD), the correlation between initial pocket
depth and GCF ow was low (r
2
0.005).
On average, the GCF ow progressively decreased
in parallel with changes in other clinical responses
from an average rate of 45ml/h to 10ml/h (Fig. 14).
The variability of GCF ow relative to the mean
change was greater than that for either pocket depth
or attachment level. The coefcient of variation (SD/
mean) of the change from baseline to 150days for
pocket depth was 0.7, attachment level was 1.2, and
GCF ow was 1.9. These results indicate that as an
outcome variable for therapy studies, GCF ow does
not have the statistical power of either pocket depth
reduction or attachment level gain.
GCF ow and clinical status
A simple protocol that can give reasonable estimates
of GCF ow is based on the collection of three
sequential 30-s samples. The average volume of the
last two samples can be used to compute GCF ow.
This strategy was used to evaluate GCF ow under
four clinical categories (10). One site from each of 56
systemically healthy subjects with periodontal dis-
ease was assigned to each clinical category (Table3).
The data of Fig. 15 indicate that broadly dened
categories of health and disease can be dis-
tinguished by differences in GCF ow. By this analy-
sis, the GCF ow at healthy sites was signicantly
less than all other categories. The GCF ow at sites
with gingivitis was signicantly less than the GCF
Fig. 14. Comparison of gingival crevice uid (GCF) ow
changes with probable pocket depth (PD), clinical attach-
ment level (AL) and bleeding on probing (BOP) following
periodontal therapy by scaling and root planing with te-
tracycline ber placement.
Table3. Denitions of clinical status
Clinical status Probing Bone Gingival
depth Loss Index
Health 13mm None 0
Gingivitis 25mm None 1
Moderate periodontitis 45mm 530% 2
Advanced periodontitis 69mm 3560% 2
ow at advanced periodontitis sites. The difference
between the GCF ow at gingivitis sites and sites
with moderate periodontitis was not statistically sig-
nicant. The ability, using GCF ow as a chair-side
measure, to differentiate healthy sites from sites with
mild disease within the same mouth was a note-
worthy nding in this study.
Measurement of GCF efux
Since all GCF ow nds its way into the saliva, meas-
urement of a marker substance specic for GCF in
the saliva should estimate the total volume of gingi-
val uid excreted. An early suggested marker is IgG
(3). The reported concentration of IgG is 14.8mg/
100ml in mixed saliva and 120mg/100ml. in gingival
uid. Assuming that about 600ml of saliva are se-
Fig. 15. Gingival crevice uid (GCF) ow and clinical sta-
tus. Bar heights represent median values and whiskers
represent the range of values obtained (10). GCF ow
values were obtained from the average volume of the last
two of three sequential 30-s samples.
51
creted daily, one can compute that 524ml of GCF
are secreted daily. Unfortunately, analysis of salivary
levels of the IgG in diseased and healthy subjects do
not show a difference in the direction expected (11).
Salivary levels of several other substances thought
to be uniquely present in GCF have been investi-
gated as marker substances, such as bronectin (22),
osteocalcin (25) and pseudocholinesterase (31).
None of these have been found to be elevated to a
level that would suggest they could be used as a
measure of GCF efux. The failure in these cases
does not, however, suggest that GCF products do not
nd their way to saliva but that the oral cavity is rich
in non-specic binding and destructive processes
that obscure the simple dilution kinetics proposed
in this measurement. It seems unlikely that there
exists a GCF marker substance that is specic, non-
reactive and maintains a constant concentration in
GCF. Hence, measurement of GCF efux will clearly
be a difcult procedure. It is possible that the use of
an appliance (28) would facilitate this type of deter-
mination.
Pitfalls in experimental design
It is assumed in any GCF sampling study that three
basic tenets are observed:
O the instrument for measuring volume is cali-
brated.
O measured sites are isolated from saliva.
O the isolated area is dried by gentle air ow care-
fully avoiding irritation.
In addition to these general considerations, there are
specic considerations relating to the method used.
The most important experimental design con-
dition under which method 1 will not work is that
the sampling time is too long. Under this condition,
the pocket volume will be re-established and the col-
lected volume essentially will not change with re-
peated sampling. For experimental design purposes,
the values of Table4 can be used to provide reason-
able range estimates from selected literature values.
Clearly shallow sulci have lower GCF ow rates (as
low as 3ml/h) but also have lower resting vol-
ume(0.05ml). Deep pockets have a greater resting
volume (up to 1.5ml) and also higher GCF ow rates
(up to 44ml/h). Table1 suggests that these factors
tend to average out so that irrespective of pocket
depth, equilibrium volumes are re-established
Goodson
Table4. Nominal estimates of gingival crevice uid
(GCF) ow and resting volume and effects on lling
time of the pocket or crevice
Clinical GF ow Resting Time to Ref.
characteristic (ml/h) volume (ml) ll (min)
Healthy crevice 3 0.05 1 5
Intermediate pocket 20 0.4 1.2 14
Deep pockets 44 1.5 2 10
within 12min Hence, this method depends on se-
lecting a sampling time (Dt) that is considerably less
than 1min.
The most important experimental design con-
dition under which method 2 would not work is if
the capacity of the sampling device were exceeded.
In general, the lter paper collection system cannot
be used to collect volumes greater than 1ml. If the
resting pocket volume is as large as 1.5ml, as has
been suggested for some deep pockets, then this
method could not be used. For intermediate pockets
however, with a resting volume of 0.4ml and a GCF
ow of 20ml/h, it would take 1.7min for the volume
to reach 1ml. Hence, any sampling interval up to 1
min would be within the range of the sampling de-
vice. With higher ow rates, this method could re-
quire the use of capillary tube sampling devices. A
second condition under which method 2 would not
work is if sufcient time has not been allowed for
the resting volume to return to equilibrium levels.
This is particularly true of studies in which resting
intervals of 1min are selected. This is the worst de-
sign. Under these conditions, shallow healthy sites
will have lled to overowing whereas deeper sites
will have not yet achieved equilibrium. Averaging
these data will produce uninterpretable results.
Conclusions
In reviewing the currently available data in which
GCF ow measurements can be inferred, there is
clearly a disease-related spectrum of values. Shallow
sulci in healthy subjects have GCF ow rates of 38
ml/h. Pockets with intermediate periodontal disease
have GCF ow rates of approximately 20ml/h. GCF
ow at sites with advanced periodontal disease as
high as 137ml/h have been observed. The evidence
from experimental gingivitis studies that GCF ow
can increase dramatically with no change in resting
52
volume suggests that this may be a sensitive early
indicator of gingival inammation. Evidence from
treatment studies indicates that GCF ow can also
be used to evaluate treatment response.
The currently available data in which GCF resting
volume values can be inferred, indicate this too has
a disease-related spectrum of values. Shallow sulci
in healthy subjects have resting volumes in the order
of 0.06ml. Pockets with periodontal disease have
resting volumes from 0.4 to 1.5ml. This agrees with
values estimated by radioactive isotope dilution to
be in the range of 0.241.56ml (3, 4).
As has been illustrated, many sampling protocols
that provide an estimate of GCF ow have been
tested. It is suggested that a simple sampling proto-
col for routine clinical use measures the volume in
Periotron units of two 30-s GCF samples (Fig. 16).
The volume of the second sample (V
2
) in Periotron
units multiplied by 0.6 (the calibration factor, ap-
proximately 0.005ml/unit, multiplied by 6060/30)
is the ow rate in ml/h. The rst volume (V
1
) minus
the second volume (V
2
) multiplied by 0.005 (the cali-
bration factor) is the resting volume V
r
. Given the
evidence that change in GCF ow may be a sensitive
measure of local inammation, this type of evalu-
ation could increase the diagnostic potential of this
Fig. 16. Proposed simplied protocol to provide a chair-
side measure of gingival crevice uid (GCF) ow (f
i
) and
resting volume (V
r
). The sampling protocol requires taking
two sequential 30-s GCF samples.
chair-side measure. Because of the time required for
a single measurement, it is unlikely that this would
compete with probe measurements for full-mouth
evaluation. Nevertheless, when it becomes necessary
to focus on treatment of an individual problem
tooth, GCF ow measurement could provide added
benet in establishing a diagnosis and monitoring
the response to therapy.
It is instructive to think broadly about the nature
of GCF ow and the ecology of the periodontal en-
vironment. If one considers that the standard lter
paper strip used for sampling is 2mm wide, how
much of the tooth pocket or sulcus volume is being
sampled? If one imagines a sample that is 2mm wide
by 6mm long and the adherent plaque being 0.1mm
thick (34), the plaque volume adhering to the site
would be 260.11.2mm
3
1.2ml. Since ap-
proximately 34% of wet plaque is uid (26), uid as-
sociated with this plaque would 1.2ml 0.340.4ml,
a volume approximately that of the resting volume
(V
r
) of GCF. These simple calculations would lead
one to suspect that the GCF ows as a thin lm sur-
rounding and percolating through the adherent sub-
gingival dental plaque. This is similar to the ow of
saliva over oral surfaces where a saliva lm of 0.1
mm has been suggested (8).
The number of times a volume is replaced in unit
time is a measure of uid exchange and is computed
as the washout ratio f
i
/V
r
. This ratio for a healthy
sulcus (Fig. 5) would be approximately 5.24ml/h/0.16
ml 33 times per hour or approximately once every
2min. In this case, both the GCF ow and the resting
volume are small. For a gingivitis site (Table2) this
could be 19.5ml/h/0.087ml 224 times per hour, al-
most four times per minute. In this case, the resting
volume is still small but the GCF ow is much
greater than at the healthy site. This ratio for a mod-
erate periodontal pocket (Fig. 4) where both the GCF
ow and resting volume are increased could be 20
ml/h/0.4ml 50 times per hour, or almost once per
minute. Irrespective of the type of periodontal site,
it is clear that the small resting volume is replaced
at a surprisingly rapid rate. This clearly accounts for
the ushing and isolation effects of GCF ow.
GCF ow is often referenced but seldom meas-
ured. This comes from a common misconception
that a sample volume measured by inserting a lter
paper strip into a sulcus or periodontal pocket is the
same as gingival uid ow. In preparing this review,
a literature search for the key word gingival crevice
uid and the text word ow revealed that 85% of
papers claiming to measure GCF ow did not. If only
a single GCF sample is taken or if a duration of 10
53
min or more occurs between samples, a study can-
not be truly a measure of GCF ow. Before and after
GCF sample differences (as in prepost-therapy
comparisons) could be indicative of changes in GCF
ow (for example decreased inammation) but they
could also indicate a change in resting volume (as,
for example, the pocket became shallower). Clearly
there is a potential for ambiguity here. For that rea-
son, it is recommended that the terminology GCF
ow be strictly reserved for protocols that measure
GCF ow and that studies which take single samples
be referred to simply as GCF samples.
References
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4. Challacombe SJ, Russell MW, Hawkes JE, Bergmeier LA,
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24. Lamster IB, Vogel RI, Hartley LJ, DeGeorge CA, Gordon JM.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Potential for gingival crevice
uid measures as predictors of
risk for periodontal diseases
Cn1nrniNr M. E. CnnrncNr, WiIIinr BucnnNnN, MicnnrI S. Rrnnv,
JonN S. Pnrissrn, Jnrrs D. Brcx & S1rvrN OrrrNuncnrn
The composition of the gingival crevice uid (GCF)
is the result of the interplay between the bacterial
biolm adherent to the tooth surfaces and the cells
of the periodontal tissues. The collection of GCF is
a minimally invasive procedure and the analysis of
specic constituents in the GCF provides a quanti-
tative biochemical indicator for the evaluation of the
local cellular metabolism that reects a persons
periodontal health status. Many studies have looked
at the association between total amount or concen-
tration levels of different constituents of GCF and
periodontal health status. Since host response is a
critical determinant in periodontal disease patho-
genesis, the measure of inammatory mediator
levels in the GCF has been used to evaluate risk: risk
for a tooth, or more precisely a site, to lose clinical
attachment and alveolar bone, or risk for an individ-
ual to develop periodontal disease. Results from a
pilot longitudinal study indicate that GCF inam-
matory mediator levels increase with time in peri-
odontitis patients, both in sites with increased bone
loss and stable sites with no bone loss. This nding,
along with other evidence, suggests that the risk is
more patient-based than site-specic. Findings from
diabetic patients illustrate how the measure of GCF
inammatory mediator levels seems to be highly
correlated with the overall individuals systemic in-
ammatory response.
Pathogenesis of periodontitis
To evaluate the usefulness of a marker of risk for a
disease, it is helpful to have an understanding of the
current concepts of the disease pathogenesis. The
167
consensus report of the American Academy of Peri-
odontology provides a model of periodontal disease
pathogenesis and denes the critical model pathway
for disease expression (48). Periodontal diseases are
caused by a localized inammatory reaction in re-
sponse to a bacterial infection of the teeth, and are
manifested by an alteration of the integrity of the
tissues supporting the teeth. Gingivitis is a form of
periodontal disease in which gingival tissues present
with inammation but in which tissue destruction is
mild and reversible. Gingivitis affects more than 90%
of the population, but only 715% of the adult popu-
lation is affected by a more severe form of disease,
periodontitis (7). Periodontitis is a chronic inam-
matory response to the subgingival bacteria, produc-
ing irreversible periodontal tissue destruction and
tooth loss. The progression of periodontitis is
chronic, with cyclic periods of exacerbation and re-
mission, and may remain unnoticed with minimal
symptoms in the early stages. Periodontitis is diag-
nosed clinically by loss of attachment between the
tooth and the supporting tissues (clinical attach-
ment loss), by deepening of the pocket between the
root of the tooth and the supporting tissues (pocket
depth), and/or by radiographic evidence of bone
loss. Periodontitis is a multifactorial disease, with the
presence of pathogenic bacteria being necessary but
not sufcient. The host immune and inammatory
response to the microbial challenge is a critical de-
terminant of susceptibility to develop the destructive
disease, under the inuence of multiple behavioral,
environmental, and genetic factors. Hence, although
disease progression is episodic in nature on a tooth
site level, the risk of developing periodontal disease
is principally patient-based rather than site-based.
Healthy periodontal sites are characterized by the
Champagne et al.
presence of a microbial plaque, composed of mainly
gram-positive microorganisms. In this situation, the
GCF represents a serum exudate, owing from the
gingival tissues into the gingival crevice. Gingivitis is
characterized by a change in the composition of the
microbial plaque, with an increased presence of
gram-negative microorganisms (74). These gram-
negative bacteria trigger a localized host response,
that produces gingival erythema, edema, loss of stip-
pling, pocketing, and bleeding on probing. Gingivitis
occurs naturally in most individuals, although its se-
verity varies. The histological presentation includes
vascular changes with increased vasopermeability
and vasodilatation, and the presence of an exudate
of polymorphonuclear neutrophils, migrating from
the tissue into the gingival crevice (53, 75). Neutro-
phils have highly specialized antimicrobial mechan-
isms that form the rst line of defense against bac-
teria. Bacterially activated neutrophils also produce
chemotactic and vasoactive mediators that perpetu-
ate the host inammatory response. Gingivitis is
thought to be a neutrophil-dominated response, as
mostly neutrophil mediators are identied in GCF,
including leukotriene B
4
, platelet activating factor,
thromboxane B
2
, elastase, and collagenase (matrix
metalloproteinase-8; 10, 19, 24, 32, 59). Tonetti et al.
demonstrated that increases in the GCF level of the
neutrophil chemoattractant interleukin-8 is one of
the earliest changes associated with transition from
health to gingivitis (75). Low GCF levels of monocytic
products such as interleukin-1 or tumor necrosis
factor are detected in gingivitis, indicating a low acti-
vation level of cells associated with chronic in-
ammation. In periodontitis, the gram-negative mi-
crobial plaque evolves and colonizes deeply into the
gingival crevice (subgingival plaque), and propagates
a chronic inammatory response. The presence of
specic subgingival pathogens is another indicator
of disease, being necessary for, but not sufcient to
cause, disease (5, 68). Plaque scores (visual measure
of supragingival plaque amount accumulating at a
site) are not strongly associated with clinical signs of
periodontal disease (51). Plaque control however, is
extremely important in patients and at sites of dis-
ease, to slow down disease progression. Several peri-
odontal pathogens have been identied, and classi-
ed into different cluster groups according to their
association with health, gingivitis, or periodontitis
(69). As the plaque matures, becoming more patho-
genic, in parallel, the host inammatory response
evolves from an acute to a chronic one. Gram-nega-
tive periodontal pathogens can evade the host clear-
ance mechanisms (complement, antibodies, and
168
neutrophils), while shedding vesicles containing mi-
crobial toxins, proteases, and endotoxin (lipopoly-
saccharide). Lipopolysaccharide penetrates the
tissues, stimulating monocytes which secrete me-
diators of inammation, including prostaglandin E
2
,
thromboxane B
2
, interleukins -1, -6 and -8, tumor
necrosis factor, and collagenase. These mediators of
inammation then activate vascular smooth muscle
cells, broblasts, more monocytes, and osteoclasts
to produce matrix metalloproteinases and stimulate
bone resorption. This inammatory cascade pro-
duces clinical inammation, attachment loss, pock-
eting and bone loss, and eventually, tooth loss.
Alongside the monocytic synthesis of inammatory
mediators, antigen presentation also occurs. This
arm of the host response triggers the adaptive im-
mune response, with an initial T helper type 1 re-
sponse (pro-inammatory, interleukin-2, tumor ne-
crosis factor, and interferon-g) and a later shift to a
T helper type 2-dominant response (anti-inamma-
tory, interleukins -4, -5, -6, -10, and -13 and produc-
tion of immunoglobulins) (16, 17). This is consistent
with a shift from T helper type 1 to type 2 between
the transition from gingivitis to periodontitis as de-
scribed by Seymour & Gemmell (65). However, other
studies (62) suggest that during periods of active
periodontal disease progression there is a dominant
T helper type 1 response versus a T helper type 2
response, with the type 2 response being consistent
with periodontal disease stability (nonprogression).
This shift from a a type 2 to a type 1 prole during
periods of active periodontal disease progression in
human is consistent with animal models of disease
progression (34). Thus, inammatory cytokines can
be detected within the GCF and serve as an indicator
of local immunoregulatory and inammatory status.
In addition, collagen breakdown products such as
hydroxyproline and pyridinoline cross-linked car-
boxy-terminal telopeptide fragments of type I colla-
gen are found in GCF, and may serve as direct meas-
ures of connective tissue catabolism for both soft
and hard tissues (18). Furthermore, the superimpo-
sition of acute episodes of disease progression in a
chronic periodontitis state undoubtedly involves
neutrophil activation and resultant increases in GCF
neutrophil by-products. The periodontal abscess is
perhaps the most dramatic indicator of how neutro-
philic inltration produces local reductions in tissue
pH, release of oxidative metabolites, interleukin-8,
matrix metalloproteinases and neutrophilic cathep-
sins can lead to rapid loss of attachment structures.
However, the signals provided to neutrophils in
chronic lesions appear considerably different to
GCF markers of risk for periodontal diseases
those in early gingivitis lesions, resulting in more
tissue destruction rather than vasoactive molecules
promoting phagocytosis and neutrophil-mediated
killing (32, 36). Thus the magnitude and quality of
the host mediator response to oral pathogens re-
mains a major determinant of disease severity. The
objective biochemical assessments by analysis of
GCF constituents can provide a critical insight into
the cellular events that are transpiring at a site level.
Furthermore, the host inammatory tone with re-
gards to total whole body stress will also affect im-
mune and phagocytic cell function locally. Thus,
GCF can provide a patient-level indicator of im-
mune/inammatory status, especially if the mi-
crobial burden is known(see below) (50).
Determination of risk markers
The observation that periodontal diseases affect only
715% of individuals means that not all individuals
are at equal risk for this disease. Therefore, it is im-
portant to establish who is at risk and what charac-
teristics can be used to identify those individuals at
the greatest risk. According to the type of study in-
vestigating the association of a characteristic (or fac-
tor) to a disease, this characteristic should be desig-
nated with the appropriate nomenclature (51). A fac-
tor is considered a risk factor if it increases the
probability of the disease occurring. A risk factor
may be part of what causes the disease or may bring
someone into contact with a causal chain of events.
Observational longitudinal study designs are needed
to demonstrate that the exposure to the risk factor
occurs prior to the onset of disease. The association
between the risk factor and the disease should be
consistent with current understanding of the disease
process, and the factor should remain associated
with the disease after controlling for other known
risk factors and background variables. Such a factor
could be used to screen individuals successfully and
classify them accurately into high- and low-risk
groups, and if this factor is targeted by specic inter-
ventions in high-risk individuals the probability of
disease occurrence in these individuals should be
lowered.
Because GCF composition reects the nature and
amplitude of the host response to the microbial
plaque challenge, and because periodontal disease
progression is highly dependent upon the host re-
sponse, determination of GCF constituent levels rep-
resents a putatively good approach to the evaluation
169
of a persons risk for the disease. This risk might re-
late to incident disease, that is, new disease in a sub-
ject or site with periodontal health. This would apply
to subjects who are healthy undergoing a transition
from health to gingivitis, patients undergoing a tran-
sition from gingivitis to periodontitis (72), or the risk
for progression at sites with pre-existing disease. Fi-
nally, the levels of GCF constituents might relate to
response to therapy or prognosis.
Use of GCF inammatory
mediators as indicators of risk for
periodontal disease
Recent ndings from epidemiological and molecular
studies have brought a paradigm change in peri-
odontal disease pathogenesis (52). These ndings in-
dicate that the microorganisms associated with dis-
ease are also found at healthy or nonprogressing
sites (albeit at lower levels) (72, 73, 81); the level of
plaque control (oral hygiene) is not associated with
an individuals disease severity or extent (20, 22, 60);
the presence or a certain level of specic pathogens
(such as Porphyromonas gingivalis) makes a statisti-
cally signicant but small contribution in multivari-
ate models of disease (78); studies of the host re-
sponse indicate that in twin pairs, 48% of the vari-
ability in disease expression is a result of genetic
inuences (42); stress, tobacco smoking, and dia-
betes are risk factors for periodontal diseases (51); a
large variability exists in the interpatient inamma-
tory response to a standard microbial challenge (15,
44, 50, 66); and anti-inammatory drugs are effective
in preventing clinical attachment or bone loss, even
in the presence of subgingival microbial plaque (8,
26, 30, 54).
Lipopolysaccharide is a key microbial stimulus
that will trigger the host response at periodontal dis-
ease sites. It is a cell-wall component of gram-nega-
tive bacteria, shed out of the biolm in membrane
vesicles. Lipopolysaccharide in circulation can in-
duce all the symptoms of septic shock, including fe-
ver, vascular collapse and death. Locally, lipopoly-
saccharide triggers monocytes to release inamma-
tory mediators (prostaglandin E
2
, thromboxane B,
interleukins -1, -6 and -8, tumor necrosis factor, and
collagenase) that increase local destruction of the
connective tissues structural elements. Therefore,
levels of monocytic inammatory mediators (includ-
ing prostaglandin E
2
, interleukin-1, and tumor ne-
crosis factor) in GCF may well represent the ideal
Champagne et al.
markers of disease activity at a site level. Elevated
GCF levels of neutrophil markers (including neutro-
phil elastase, b-glucuronidase, and leukotriene B
4
)
may reect acute episodes of localized tissue de-
struction. Taken together, these monocytic and neu-
trophilic mediator levels in the GCF may also give
an indication of the quality of the host response, and
of the level of risk for the individual to develop peri-
odontal disease.
GCF inammatory mediator levels
in health, gingivitis, and
increasingly severe forms of
periodontitis
Many studies have reported that GCF prostaglandin
E
2
levels are signicantly elevated in patients suffer-
ing from severe forms of diseases (juvenile peri-
odontitis and refractory periodontitis) compared to
healthy controls or patients suffering from a mild
form of the disease (gingivitis or chronic adult peri-
odontitis). For example, in a study conducted in our
laboratory including 21 healthy controls, 28 gingi-
vitis patients, 21 cases of adult periodontitis, 15
cases of juvenile periodontitis and 15 cases of refrac-
tory periodontitis, GCF prostaglandin E
2
levels (ng/
ml) were 20.37.9 (controls), 49.44.8 (gingivitis),
42.312.3 (adult periodontitis), 81.620.3 (juvenile
periodontitis), and 90.015.9 (refractory peri-
odontitis), respectively. It seems that GCF prosta-
glandin E
2
levels are signicantly elevated in gingi-
vitis patients compared to controls (64). This nding
was conrmed in a human model of experimental
gingivitis where GCF prostaglandin E
2
levels in-
creased at 4weeks following the cessation of oral hy-
giene procedures (24). This would indicate that the
determination of GCF prostaglandin E
2
levels is a
good indicator of inammatory activity. Patients
with mild forms of adult periodontitis did not have
much higher levels than gingivitis patients, although
GCF prostaglandin E
2
levels seemed to increase with
increasing disease severity. Studies by others have
conrmed that GCF prostaglandin E
2
levels are elev-
ated in periodontitis patients compared to controls
and gingivitis patients (25, 56, 75). In addition, vari-
ous periodontal treatment therapies induced a de-
crease in GCF prostaglandin E
2
levels (2, 37, 56).
GCF interleukin-1 levels are also signicantly elev-
ated in all forms of periodontitis compared to health
or gingivitis. In the Salvi et al. study (63), GCF in-
terleukin-1 levels were 16.85.3 for healthy controls
170
(n21), 115.852.6 for gingivitis patients (n22),
263.273.7 for adult periodontitis patients (n17),
836.8284.2 for juvenile periodontitis patients (n
13), and 457.9157.9 for refractory periodontitis pa-
tients (n17), expressed in ng/ml. Studies by others
have conrmed an association between elevated
GCF interleukin-1 levels and gingival inammation
(19, 35), as well as a relationship between the sever-
ity of periodontitis and elevated GCF interleukin-1
levels (10, 38, 40, 43). However, within a group of
patients with a similar level of disease, differences
were detected in GCF interleukin-1 levels between
groups of patients with different interleukin-1 gene
polymorphisms (11, 66). This may explain some of
the variability observed within patient groups. An-
other explanation for the variability of GCF interleu-
kin-1 levels within a disease group could be because
of sampling strategy, permitting inclusion of both
diseased and nondiseased periodontal sites.
Evaluation of GCF inammatory
mediator levels at nondiseased
versus diseased periodontal sites
in periodontitis patients
Results presented in the previous section were all re-
ported as patient mean or median values of in-
ammatory mediator levels measured at several sites
within each patient. Selection of sites for GCF evalu-
ation of inammatory mediators varied among
studies, but in general, it was thought that one or
two sites per quadrant or a similar design would be a
representative sample of each patient. However, one
might argue that because not all sites have active
disease at all times, it might be interesting to look at
sites separately. Reddy and co-workers proposed to
identify potential site-specic predictive factors for
periodontal disease progression by following se-
lected periodontal sites over time in periodontal pa-
tients. A subset of 10 patients who exhibited sites
with periodontal disease progression, as identied
by radiographic bone loss over 6months was studied
in a pilot manner. The goal was to determine
whether there were differences in GCF mediator
levels at sites with radiographic bone loss as com-
pared to paired sites with no bone loss. Patients with
adult periodontitis were followed for 6months with
recall visits every 2months. At each visit, clinical
data and GCF samples were collected from several
sites. Stable and active periodontal sites were iden-
tied at the end of the 6-month period within each
patient using radiographic subtraction techniques.
For the pilot data set discussed here, 10 patients
were selected who provided at least one site exhibit-
ing bone loss (active diseased sites) and one site with
no change in bone level (stable sites). GCF samples
from these selected sites were then analyzed blindly
in other research laboratories for lipopolysaccharide
content (Limulus lysate method) and for prosta-
glandin E
2
and interleukin-1 levels (enzyme-linked
immunosorbent assay). As shown in Fig. 1(A), the
log-transformed GCF prostaglandin E
2
levels in-
creased with time, in both stable and bone-loss sites,
except at the 2-month visit. Patients in this study
were treated with dental prophylaxis only, and the
decrease in GCF prostaglandin E
2
levels at 2months
was accompanied by a transient improvement in
their periodontal disease status and an improvement
in plaque scores (data not shown). This transient
clinical improvement at 2months illustrates the so-
called Hawthorne effect of participating in a study
and a response to dental prophylaxis. It is note-
worthy that there was a general increase in GCF
prostaglandin E
2
levels at later time-points at both
stable and progressing sites. The same was true for
the log transform of GCF interleukin-1 levels (Fig.
1B), increasing with time at both stable and bone-
loss sites. lipopolysaccharide levels were also meas-
ured in GCF samples taken from the same sites (Fig.
1C). Within the rst 4months, GCF lipopolysacchar-
ide levels increased dramatically with time at bone-
loss sites. GCF lipopolysaccharide levels also in-
creased at stable sites with time.
Statistical analysis for GCF prostaglandin E
2
, in-
terleukin-1, and lipopolysaccharide levels was based
upon tting linear models to the logarithmic trans-
formations of these variables from the 10 patients.
The primary model included a dichotomous factor
for bone-loss vs. no-bone-loss site, a linear trend for
visit, the interaction of bone-loss status and linear
trend, and a dichotomous factor for the baseline visit
to allow for the effect of entry into the study. Infer-
ence focused on the trend from 2months to 6
months. In all models, intraperson correlation be-
cause of repeated measures (up to four repeated
measures on each of two sites), was accounted for
with covariance structures, consistent with having
two repeated factors in a longitudinal design (13).
For each of the three variables, prostaglandin E
2
, in-
terleukin-1, and lipopolysaccharide, a model with
linear trend was supported with likelihood ratio
goodness-of-t tests conducted with respect to an
expanded two factor interaction model that treated
the visits as a qualitative variable (P0.91, 0.62, and
171
0.34, respectively). First, we investigated whether
diseased sites had higher GCF marker levels than
nondiseased sites. The log GCF prostaglandin E
2
, log
GCF interleukin-1, and log GCF lipopolysaccharide
values were higher at bone-loss sites compared to
no-bone-loss sites but the differences were not stat-
istically signicant (P0.095, 0.463, and 0.337, re-
spectively). This could be because the standard
errors of the GCF measures are large as a result of
the small sample size of 10 patients (or to high assay
variability, as for lipopolysaccharide). Next, we in-
Fig. 1. Kinetics of gingival crevice uid prostaglandin E
2
(A), interleukin-1 (B), and lipopolysaccharide (C) levels in
sites exhibiting bone loss vs. sites exhibiting no bone loss.
Champagne et al.
vestigated whether or not there was a signicant
change in GCF marker values over time, for all sites,
diseased and nondiseased. The increases in log GCF
prostaglandin E
2
and log GCF interleukin-1 values
over time from 2 to 6months were highly signicant
(P0.001 and 0.004, respectively). Although log GCF
lipopolysaccharide increased over time, the change
was not statistically signicant (P0.162). Finally,
we investigated whether the rate of change in GCF
markers over time were different at bone-loss and
no-bone-loss sites. For each of the three variables,
the difference in rates of change in the log GCF
values between diseased and nondiseased sites was
estimated to be near zero and nonsignicant [t ratios
(b/SE) of 0.09, 0.24, and 0.03, with corresponding
P-values of 0.93, 0.82, and 0.97, for prostaglandin E
2
,
interleukin-1, and lipopolysaccharide, respectively].
This nding indicates that both bone-loss and no-
bone-loss sites display similar increases in GCF
levels of prostaglandin E
2
, interleukin-1 and lipo-
polysaccharide over time.
The next interesting question was to look at corre-
lations and associations between the different GCF
markers evaluated. Overall, the measured GCF levels
of prostaglandin E
2
and interleukin-1 were highly
correlated, with R
2
0.5634 (Fig. 2). This means that
a site exhibiting an elevated level of one mediator
has a high probability to exhibit an elevated level of
the other mediator, and vice versa (sites with low
prostaglandin E
2
levels have a high probability of
also having a low interleukin-1 level). This is not too
surprising because cells within gingival tissues can
be triggered to synthesize prostaglandin E
2
by in-
terleukin-1 or vice versa (interleukin-1 inducing the
Fig. 2. Correlation of gingival crevice uid inammatory
mediator levels; IL-1, interleukin-1; PGE
2
, prostaglandin
E
2
.
172
synthesis of prostaglandin E
2
). Using similar statisti-
cal analysis as described earlier, both log GCF
prostaglandin E
2
and log GCF interleukin-1 were
statistically signicantly correlated with log GCF
lipopolysaccharide (P0.017 and 0.002, respec-
tively). There is marginally signicant evidence that
the positive association of log GCF prostaglandin E
2
(and log GCF interleukin-1) with log GCF lipopoly-
saccharide decreased over time (P0.059 and P
0.058, respectively). However, the relationship be-
tween either log GCF prostaglandin E
2
or log GCF
interleukin-1 with log GCF lipopolysaccharide did
not differ signicantly by disease status of the site
(P0.0801 and 0.593, respectively).
At baseline, the median GCF lipopolysaccharide
value was 121.15, and 70% of sites with GCF lipopoly-
saccharide values above the median were the sites
that experienced bone loss. In addition, the mean
GCF prostaglandin E
2
value at baseline was higher in
sites with GCF lipopolysaccharide values above the
median compared to sites with GCF lipopolysacchar-
ide values below the median (120.0123.78 vs. 28.29
5.89ng/ml). The same was true for the mean GCF
interleukin-1-values(134.1717.59 vs. 21.546.20
ng/ml). This pattern of ndings could be interpreted
as an exposure (GCF lipopolysaccharide) leading to
an elevated host inammatory response (GCF prosta-
glandin E
2
and interleukin-1), resulting in the disease
outcome (bone loss). Within our data set, we could
detect signicant associations between log GCF
prostaglandin E
2
or log GCF interleukin-1 values with
log GCF lipopolysaccharide values, which did not
change over time or according to site status (diseased
vs. nondiseased). Results fromlarger data sets, involv-
ing more patients, more sites, and the actual clinical
measures (including attachment loss, pocket depth,
and bone loss) would be necessary to clarify the as-
sociation between measures of exposure (GCF lipo-
polysaccharide levels), measures of host response
(GCF prostaglandin E
2
and interleukin-1 levels), and
clinical status. Published reports addressing this issue
lack consistency, suffering fromlownumbers of study
subjects, and grouping of sites with similar disease
status (healthy, gingivitischaracterized by inam-
mationand periodontitischaracterized by clinical
attachment loss) taken from different patient groups
(healthy sites from healthy individuals and peri-
odontitis patients). Some studies report positive cor-
relations between GCF inammatory mediators
levels and site clinical status (14, 29, 41, 46, 57), while
other studies report poor correlations between GCF
inammatory mediator levels and site clinical status
(12, 19, 47, 78).
The fact that even stable sites exhibit increasingly
higher GCF levels of inammatory mediators with
time is not too surprising, because these stable sites
are sampled in patients that do have progressing sites
within their mouth. This is probably a result of the lo-
cal host responses being reective of systemic
changes in inammatory responsiveness (50). During
periods of disease progression, the release of in-
ammatory mediators increases at all sites within the
patients mouth, both at progressing and at stable
sites. In addition, clinically healthy sites in patients
with progressive disease are probably different from
healthy sites in healthy patients, harboring different
microbial plaque (80), and exhibiting different host
response (71). Although the disease progresses dif-
ferently at each local site because of site-specic
characteristics (71), the host response is more global,
and follows the same pattern at all sites within an in-
dividuals mouth. This was conrmed by Figueredo
et al. (12) who found that levels of GCF interleukin-1
were increased in samples from periodontitis pa-
tients, regardless of the severity of disease at the
sample site. These authors suggest that GCF interleu-
kin-1 levels are characteristic of the patient. Hence,
periodontal disease is a disease of the whole mouth,
not just of some sites within the mouth. This implies
that in terms of patient-level risk, normal sites within
a periodontitis patient are more likely to develop dis-
ease and show tissue breakdown than diseased sites
or sites with signs of previous tissue breakdown. For
example, in the Oral Condition and Pregnancy Study
conducted in our laboratory, disease incidence/pro-
gression in enrolled pregnant mothers consisted en-
tirely of increasing pockets in previously normal sites
(49); in a representative study of older adults (Pied-
mont study), most disease progression in subjects
with periodontitis occurred in nondiseased sites (4).
Fig. 3. (A) Gingival crevice uid prostaglandin E
2
levels (B) Correlation between gingival crevice uid prosta-
and basal prostaglandin E
2
secretion of isolated periph- glandin E
2
levels and basal peripheral bloodmonocytic
eral blood monocytes from different patient populations. prostaglandin E
2
secretion levels.
173
This observation has consequences in terms of
GCF sampling strategy. Rather than trying to identify
stable and progressing sites within each patient,
which is difcult and involves longitudinal follow-
up, several sites can be sampled within the mouth of
an individual regardless of their disease progression
status. GCF inammatory mediator levels can then
be determined at each sampled site, and mean
values can be reported for each individual. These
mean values are representative of the individuals in-
ammatory response at a certain time, and can be
related to a patient-level disease status and possibly
a patient-level disease risk.
Notion of host susceptibility and
its association to GCF
inammatory mediator levels
Overall individual response to bacterial
challenge
One way to investigate the idea that GCF inamma-
tory mediator levels reect the overall ability of an
individual to produce inammatory mediators is to
evaluate the individuals ability to produce inam-
matory mediators. This can be accomplished by
measuring the levels of inammatory mediators re-
leased from isolated peripheral blood monocytes.
Figure 3 shows prostaglandin E
2
levels produced by
unstimulated peripheral blood monocytes isolated
from healthy subjects and from patients suffering
from different forms of periodontitis in relation to
GCF prostaglandin E
2
levels measured in those same
patients. Clearly, monocytes isolated from peri-
odontitis patients produced higher levels of prosta-
glandin E
2
than healthy controls, and monocytes iso-
Champagne et al.
lated from patients suffering from severe forms of
periodontal diseases produced even higher levels of
prostaglandin E
2
than monocytes isolated from pa-
tients suffering from milder forms of the disease.
Interestingly, levels of prostaglandin E
2
released by
isolated peripheral blood monocytes were highly
correlated to the levels of prostaglandin E
2
measured
in the GCF (Fig. 3B). Another approach consists of
measuring levels of inammatory mediators re-
leased by isolated peripheral blood monocytes
stimulated with increasing doses of stimulant. We
and others have reported previously on differences
in production levels of prostaglandin E
2
by periph-
eral blood monocytes isolated from individuals with
various degrees of periodontal disease severity in re-
sponse to increasing doses of lipopolysaccharide (27,
39, 55, 65). Remarkably, the ranges of differences in
prostaglandin E
2
levels produced between peri-
odontitis patient groups remain fairly constant at all
doses of lipopolysaccharide used as stimulant (50).
It is likely then that the amount of inammatory me-
diator produced by an individuals monocytes
(whether locally in the GCF or from isolated periph-
eral blood monocytes) in response to increasing
doses of bacterial challenge (lipopolysaccharide) is a
characteristic of that individuals host response.
Systemic response driving local response:
example of diabetes
Some of the most convincing data in support of a cor-
relation between peripheral blood monocytic release
of inammatory mediators and levels of the same in-
ammatory mediators measured in GCF samples
have been brought forward with the investigation of
diabetic patients. Diabetes is a systemic condition af-
Fig. 4. Host response characteristics as a function of chal-
lenge level; LPS, lipopolysaccharide.
174
fecting more than 12 million individuals in the USA,
and is a recognized risk factor for periodontitis, with
odds ratios of 2.13.0 (62). Our laboratory conducted
a study examining GCF mediator levels, monocytic
secretion of inammatory mediators, and peri-
odontal clinical presentation from 39 diabetic and 64
nondiabetic patients with various degrees of peri-
odontal health status (60). The rst remarkable nd-
ing was that diabetics had signicantly higher GCF
levels of prostaglandinE
2
andinterleukin-1 compared
to nondiabetic patients with similar periodontal sta-
tus. The second nding was that when stimulated
with different doses of lipopolysaccharide, mono-
cytes isolated from diabetic patients released higher
2
, interleukin-1 and tumor
necrosis factor than monocytes isolated fromnondia-
betic patients. Peripheral blood monocytes isolated
from diabetic patients behaved as if they were up-
regulatedor hyperresponsive. Finally, there was a high
degree of correlation between the GCF prostaglandin
E
2
levels and the levels of prostaglandin E
2
released by
monocytes upon lipopolysaccharide challenge (50).
Incremental changes in both prostaglandin E
2
levels
were observed in groups of patients with increasingly
severe condition, from healthy subjects, to nondia-
betic patients with increasing severity of periodontal
disease, to diabetic patients with increasing severity
of periodontal disease. These ndings may be ex-
plained by an overall individual hyperresponsive
monocytic trait, which manifests with elevated levels
of inammatory mediators released by monocytes in
response to bacterial challenge, systemically and loc-
ally in the gingival tissues. Since prostaglandin E
2
and
interleukin-1 trigger molecular events leading to sup-
portive gingival tissue degradation, the hyperrespon-
sive monocytic trait would then explain why diabetic
patients are at higher risk of developing periodontal
breakdown.
Notion of host susceptibility
We propose the schematic representation of Fig. 4 to
illustrate the differences among individuals ability
to synthesize inammatory mediators (prosta-
glandin E
2
, interleukin-1 and tumor necrosis factor)
in response to increasing doses of challenge (bac-
terial lipopolysaccharide). Individuals with a normal
response would be represented by the middle line.
In response to a certain microbial challenge, these
individuals would have a sufcient level of inam-
mation, as reected by high levels of inammatory
mediators. Hyperresponsive individuals (top line),
would have a characteristic doseresponse curve
shifted to the left. These individuals would produce
much higher levels of inammatory mediators than
normal individuals at the same given challenge, and
would exhibit disease expression at a lower mi-
crobial load threshold. This could be the case of dia-
betic patients who have elevated advanced glycated
end-products serum levels. These advanced glycated
end-products have clearly been shown to up-regu-
late isolated peripheral blood monocyte responses
in vitro (76), and are the suspected reason for the
hyperresponsive monocytic trait observed in mono-
cytes isolated from diabetic patients. On the other
hand, there may be some circumstances under
which individuals have their doseresponse curve
shifted to the right (hyporesponsive individuals),
producing lower inammatory mediator levels than
normal individuals for a similar challenge. Several
factors might inuence an individuals monocyte re-
sponsiveness, including genetic factors and environ-
mental factors. Interindividual differences in mono-
cytic synthesis of interleukin-1 and tumor necrosis
factor have been reported in relation to different
genetic backgrounds. Men that were HLA-DR2-posi-
tive were found to be low responders (44). Other
genetic factors, such as interleukin-1 or tumor ne-
crosis factor gene polymorphisms, will inuence the
cell expression levels for interleukin-1 and tumor ne-
crosis factor (11, 66). These genetic factors are intrin-
sic to the individual and do not vary with time. Other
factors may also affect the cells expression levels for
inammatory mediators, including smoking (6, 70),
and advanced glycated end-products (76). These be-
havioral and environmental factors may change over
time and affect the individuals doseresponse curve
differently at different time-points.
Individuals response characteristics and
periodontal disease threshold
Since the individuals ability to produce monocytic
inammatory mediators is highly correlated to the
levels of inammatory mediators measured in GCF,
a similar host challengeresponse curve can illus-
trate the localized periodontal inammatory re-
sponse to subgingival plaque (Fig. 5). This curve rep-
resents the quality of the host response of one indi-
vidual at one given time. For a certain level of
challenge (subgingival periodontal bacteria) the host
will produce a nite amount of inammatory me-
diators such as prostaglandin E
2
, interleukin-1 and
tumor necrosis factor. These mediators will have
benecial effects up to a certain threshold to ght
the infection, and will not be sufcient to trigger
175
periodontal tissue breakdown. However, if the mi-
crobial burden increases, the levels of inammatory
mediators produced will also increase. The amount
of inammatory mediators produced may then be
sufcient to overwhelm the system, trigger a de-
structive inammatory response, leading to peri-
odontal tissue breakdown. Therefore, a disease
threshold exists, representing the level of inamma-
tory mediators produced above which there will be
disease progression and clinical measures of in-
creased pocket depth, loss of attachment and bone.
Fluctuations in hostresponse curve,
modulating local periodontal disease
breakdown
As discussed earlier, the hostresponse curve may
vary over time, as a result of behavioral and environ-
mental factors, and experience small shifts towards
an increased responsiveness, as illustrated in Fig. 6.
This shift in the hostresponse curve will or will not
have consequences in terms of disease progression,
depending on the change (D) in the inammatory
mediator levels produced. For example, if the
pathogenicity (load and composition) of subgingival
plaque present at a specic site within an individual
is low, the increase in inammatory mediators pro-
duced will remain below the disease threshold and
will not have consequences in terms of disease (blue
arrows in Fig. 6). In contrast, if the pathogenicity of
subgingival plaque is high, the increase in inam-
matory mediator levels produced at a specic site in
that same individual are likely to be sufcient to
cross the disease threshold and induce periodontal
tissue breakdown (red arrows in Fig. 6). This simple
model of disease provides an explanation of why
Fig. 5. Characteristic individual host response curve in re-
lation to periodontal disease threshold; lipopolysacchar-
ide, lipopolysaccharide.
Champagne et al.
periodontitis appears as a site-specic disease pro-
cess. Changes that inuence the patient at a sys-
temic level, such as diabetes would cause increases
in inammation at all periodontal sites, but only
sites with higher levels of pathogenic microbes
would demonstrate clinical disease progression.
These levels of microbes are typically at the level of
10
6
per sample (80). Therefore, the microbial bur-
den determines site-specic risk within an individ-
ual, but the overall disease activity appears to be
tightly coupled to the patients overall systemic in-
ammatory responsiveness. Thus, it is not unreason-
able to propose that when a patient undergoes an
acute episode of periodontal disease progression
only selected sites (those with the highest microbial
and lipopolysaccharide burden) demonstrate clinical
disease progression, whereas other sites with lower
levels of microbial challenge show biochemical
changes, but do not reach the inammatory me-
diator level threshold necessary to demonstrate
overt clinical disease progression.
Are GCF inammatory mediator
levels good indicators of risk for
periodontal disease at the
individual level?
The inammatory mediators discussed in the pre-
vious sections, prostaglandin E
2
, interleukin-1 and
tumor necrosis factor, are not the only mediators
that can be detected in GCF as a result of the inter-
play between the microbial plaque and the host re-
sponse. Other mediators that have been evaluated
include leukotriene B
4
, thromboxane B
2
, T helper
type 1 (interleukin-2, interferon-g) and type 2 cyto-
Fig. 6. Periodontal disease pro-
gression as a consequence of a shift
in the host response to given mi-
crobial plaque challenges; LPS, lipo-
polysaccharide.
176
kines (interleukins -4, -6, -10, and -13), chemokines
(interleukin-8, RANTES, monocyte chemoattractant
protein-1, macrophage inammatory protein-1, and
interferon-inducible protein-10) and their receptors
(CCR5), other receptors (sCD14, lipopolysaccharide-
binding protein) and adhesion molecules (selectins
and soluble intercellular adhesion molecule), and
enzymes (neutrophil elastase, neutrophil aspartate
transaminase, neutrophil collagenase matrix
metalloproteinase-8, and other collagenasesmatrix
metalloproteinase-3and their inhibitorsTIMP)
(3, 9, 14, 21, 23, 3133, 58, 79). This list will probably
grow as new mediators are identied and new detec-
tion assays are developed. Also, in addition to look-
ing at levels detected in GCF, investigators some-
times look at expression levels in cells present in gin-
gival tissues (broblasts, epithelial cells, neutrophils,
monocytes, dendritic cells, lymphocytes, and endo-
thelial cells) from biopsy samples (1, 28, 45). Several
investigators chose to study multiple mediators in
parallel, and look at either individual or combined
associations between these mediator levels and clin-
ical signs of disease. The results are not always con-
cordant, but this can be explained as these types of
investigations are facing multiple hurdles. The rst
problem may come from the choice of the denition
of disease, as periodontal diseases can have several
clinical presentations. Over the years, clinicians have
been battling the problem of periodontal disease
classication. This reects in the literature with peri-
odontitis patients being grouped differently, based
on different clinical criteria. Another problem that
researchers are being confronted with is that clinical
signs of disease (clinical attachment loss or radio-
graphic bone loss) represent historical measures of
disease, or past episodes of disease activity. However,
clinical signs of inammation may be easier to de-
tect, such as in gingivitis. Hence, it is conceivable
that some markers, or groups of markers, might be
more associated with the early acute inammatory
phase, dealing with the elimination of the infectious
agents, while other markers might be more associ-
ated with chronic inammatory stages, during which
tissue breakdown occurs.
If we look again at the denition of a risk factor,
several criteria need to be fullled. The rst is the
ability to demonstrate that the exposure of the indi-
vidual to the risk factor occurs prior to the onset of
disease. This requires the design of large observa-
tional longitudinal studies, to detect the rare occur-
rence of new disease in a subject, undergoing a tran-
sition from health to gingivitis, or gingivitis to peri-
odontitis. Although the detection methods have
evolved and are becoming easier to use (commer-
cially available enzyme-linked immunosorbent assay
kits), they remain fairly expensive, which is probably
one of the main factors that has limited their appli-
cation to relatively small studies. Another limiting
factor is the necessity to control for other known risk
factors, which either limits the number of subjects
that may qualify for enrolment in the study or in-
creases the number of subjects to be enrolled to
reach statistical signicance. Classically recognized
controlling factors include age, gender, race, smok-
ing status, diabetes, and socio-economic status.
Newer controlling factors are being suggested, such
as body mass, for example, and should be taken into
consideration. Another criterion for the recognition
of a risk factor is for it to be consistent with the cur-
rent understanding of the disease process. Since
periodontal diseases result from the level of host re-
sponse to microbial challenge, it seems important to
also evaluate the microbial burden. This is becoming
possible with the development of detection and
analysis methods such as the checkerboard DNA
DNA hybridization technique (69). Finally, the cri-
teria of specic targeting of the risk factor concord-
ant with the lowering of disease occurrence is some-
what easier to address, as pharmaceutical compan-
ies are usually keen to design such studies, for
obvious reasons. Nonsteroidal anti-inammatory
drugs can be used to lower levels of prostaglandin
E
2
produced, and several blocking agents are avail-
able for interleukin-1 and tumor necrosis factor.
Despite the complexity of measuring the relevant
components of periodontal disease, several inves-
tigative teams have gathered the means to conduct
large-scale longitudinal studies, combining the in-
vestigation of clinical presentation of disease, mi-
crobial burden, and multiple aspects of the host re-
177
sponse that include GCF measures. Such studies are
currently under way, and partial or interim results
are being presented at various meetings. Although
the current available published ndings on the topic
of GCF markers and risk for periodontal diseases are
still limited, we anticipate that the role of GCF
markers will be better specied in the near future.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Structure and function of the
toothepithelial interface in
health and disease
MnnJn T. PoIInNrN, Juxxn I. SnIoNrN & VrIi-Juxxn Ui11o
Introduction
Three types of mucous membranes (masticatory, lin-
ing, and specialized) line the oral cavity and form
the structural boundary between the body and the
external environment. Although each type of mu-
cosa protects against mechanical and microbial
damage, the epithelia exhibit considerable differ-
ences in their histology, thickness and differentiation
suitable for the functional demands of their location.
Furthermore, the structure of different epithelia re-
ects their effectiveness as a barrier to the penetra-
tion of microbes and noxious agents into the deeper
tissues (141,160). Mucosal epithelia are composed of
continuously dividing and shedding populations of
keratinocytes whose proliferation is conned to the
basal layer. The teeth, passing through the gingival
masticatory mucosa, create a unique environmental
challenge to the protective continuity. At the inter-
face where the healthy gingiva meets the tooth sur-
face the structural continuity is secured by the junc-
tional epithelium attached to the tooth surface by a
distinct mechanism known as the epithelial attach-
ment apparatus (143). As opposed to the constantly
renewing epithelia, teeth are units of nonshedding
surfaces, which provide a solid substratum not only
for the attachment of the junctional epithelial cells
but also for bacterial colonization and spreading in
the oral cavity. At the dentogingival junction the bac-
terial colonies, exhibiting a variety of virulence fac-
tors (68), pose a potential threat to the epithelial
attachment. The attachment may be affected directly
by bacteria or indirectly through their ability to acti-
vate inammatory and immune processes, which
contribute to the composition of the gingival crevice
uid (GCF) and thus to the conditions under which
12
the epithelial attachment apparatus is formed and/
or maintained. In addition to the attachment, the re-
newal rate and reparative capacity of the junctional
epithelial cells are equally important for the health
of the dentogingival junction. Accordingly, any de-
generative episodes that involve the cells responsible
for the attachment may gradually lead to the detach-
ment of the junctional epithelium from the tooth
surface and compromise the protective mucosal
continuity.
Periodontal diseases are associated with bacterial
plaque infecting the dentogingival margin and caus-
ing inammation in the underlying tissues (53,87).
Logically, one of the main concerns of periodontal
research has been to describe the inammatory re-
action in the adjacent tooth-supporting tissues and
the consequences it may have at the alveolar bone
margin (70,121,147). Less attention has been focused
on the effects of factors released from bacteria and/
or from the inammatory reaction on the junctional
epithelial cells. The precise mechanisms of epithelial
degeneration and/or activation leading to the de-
tachment and apical migration of the junctional epi-
thelial cells and consequent conversion of the gingi-
val sulcus into an infected periodontal pocket have
not yet been discovered. This article deals with fac-
tors associated with periodontal tissue protection
and destruction with special reference to the junc-
tional epithelial cells.
Junctional epithelium
Schroeder and Listgarten rst claried the anatomy
and histology of the dentogingival junction in their
monograph: Fine structure of developing epithelial
Fig. 1. Schematic illustration of the different epithelia at
the dentogingival junction. The junctional epithelium (JE)
exhibits a distinct phenotype that allows the tissue to at-
tach to the tooth surface and participate in the host de-
fense in a number of ways.
attachment of human teeth (143). Since then,
knowledge of the junctional epithelium has been re-
viewed in numerous articles (90,142,144,161,168). It
is commonly accepted that the junctional epithel-
ium exhibits several unique structural and func-
tional features that contribute to preventing patho-
genic bacterial ora from colonizing the subgingival
tooth surface. First, junctional epithelium is rmly
Fig. 2. Photomicrograph demonstrating the junctional ium into the sulcus. At the apical part of the junctional
epithelial DAT cells on the tooth surface (a). When the epithelium, cells (arrow) are seen to grow/proliferate into
gingiva is displaced laterally the DAT cells are left on the the connective tissue (CT). A higher magnication (c) of
enamel (E) surface. The degeneration of the DAT cells ap- the apical part of the junctional epithelium. Note the
pears to be a prerequisite for the apical advancement of dark-stained DAT cells along the cementum surface, the
bacterial plaque (P). D dentin. The junctional epithel- epithelial nger proliferating apically and the absence of
ium (JE) attached to tooth (to enamel or cementum (C) inammatory inltrate. Gingival epithelial cells grown on
as in (b) forms a structural barrier against the bacterial decalcied dentin matrix (d) show apparent ability to
plaque. Polymorphonuclear leukocytes that cover the extracellular collagenolysis (e).
plaque (P) have migrated through the junctional epithel-
13
attached to the tooth and thus forms an epithelial
barrier against the plaque bacteria (Fig. 1 and 2a,b).
Second, it allows the access of GCF, inammatory
cells and components of the immunological host de-
fense to the gingival margin. Third, junctional epi-
thelial cells exhibit rapid turnover, which contributes
to the hostparasite equilibrium and rapid repair of
damaged tissue (48). Although the importance of the
junctional epithelium in host defense is well recog-
nized, an exact understanding of its role in the
pathogenesis of periodontal diseases is largely miss-
ing (Fig. 3). A major part of the older literature on
junctional epithelium describes its histological fea-
tures at different stages of development or disease
(113,141). More recent studies report on the distri-
bution of cytoplasmic keratin laments in the junc-
tional epithelial cells, on different cell surface anti-
gens and receptors, intercellular adhesion molecules
and details of the nonepithelial cells within the junc-
tional epithelium and its nerve supply [for a review
see (142)]. These results consistently support the
view that to be able to form a successful junction
between two dissimilar tissues and to respond in a
exible manner to the external environment, the
epithelial cells responsible must have a distinct un-
differentiated phenotype (160) (Fig. 4). The pheno-
type of junctional epithelium, at least partly, reects
a self-instructed adaptation of oral mucosa to the
Pllnen et al.
existence of a solid tooth surface penetrating it
(134,168). It is pertinent to suggest that the pheno-
type required for epithelial adaptability may also
render the cells vulnerable to the action of various
exogenous and endogenous agents. Since our under-
standing of the pathogenic mechanisms that lead to
failure in junctional epithelium defense is limited,
there is a clear need for fundamental research into
the junctional epithelial cells and their functions
under different clinical conditions.
Epithelial attachment apparatus
The attachment of the junctional epithelium to the
tooth is mediated through an ultramicroscopic
mechanism dened as the epithelial attachment ap-
paratus (143). It consists of hemidesmosomes at the
plasma membrane of the cells directly attached to
the tooth (DAT cells) and a basal lamina-like extra-
cellular matrix, termed the internal basal lamina, on
the tooth surface [Fig. 5 (80)]. By morphological cri-
teria the internal basal lamina between the junc-
tional epithelial DAT cells and the enamel is quite
similar to the basement membrane between the epi-
thelium and the connective tissue. However, by bio-
chemical criteria the internal basal lamina differs
essentially from the established basement mem-
brane composition and thus from the external basal
lamina.
Fig. 3. The junctional epithelium (JE) is repeatedly or con-
tinually exposed to bacterial challenges, which may lead
to the failure of the junctional epithelium and eventually
to subgingival plaque formation (brown), conversion of
the gingival sulcus into a periodontal pocket and increase
in the size of the inammatory focus in the connective
tissue (gray).
14
The components of the internal basal lamina are
synthesized by the DAT cells in the absence of the
immediate vicinity of connective tissue (80,142).
Internal basal lamina proteins include laminin and
type VIII collagen (128,130). Laminin, identied as
type 5, is localized mainly to the optically electron-
dense part of the internal basal lamina and it seems
to be associated with hemidesmosomes (69,93,100).
Characteristically, the internal basal lamina lacks la-
minin-1 and type IV collagen, which are components
of true basement membranes (129,130). In addition,
the internal basal lamina structure may involve
other molecules that are unique to this structure (L.
Hkkinen, unpublished results).
Hemidesmosomes have a decisive role in the rm
attachment of the cells to the internal basal lamina
on the tooth surface. Recent data suggest that the
hemidesmosomes may also act as specic sites of
signal transduction and, thus, participate in regula-
tion of gene expression, cell proliferation and cell
differentiation (71). The intracellular part of hemi-
desmosomes consists of at least two distinct pro-
teins, the 230kDa bullous pemphigoid antigen
(BP230) and plectin, which is a high molecular
weight cytomatrix protein (Fig. 5). These proteins
mediate the attachment of the epithelial cell cyto-
plasmic keratin laments to two transmembrane
components of the hemidesmosome known as the
180kDa bullous pemphigoid antigen (BP180) and
a
6
b
4
integrin (71,174). The a
6
b
4
integrin plays an im-
portant role in the interaction of epithelial cells with
the extracellular matrix (101,127). This interaction
utilizes the intracellular plectin connected through
the b
4
-domain of the integrin to laminin-5 (ligand
for the a
6
b
4
integrin) in the internal basal lamina
(66,69,169). In general, the interaction between the
different components of the extracellular matrix and
the cell surface molecules linked to the intracellular
cytoskeleton is fundamental for cell adhesion, cell
motility, synthetic capacity, tissue stability, regenera-
tion and responses to external signals (175). Since
the biochemical composition of internal basal lam-
ina matrix differs from that of the true basement
membrane, the behavior of the attached DAT cells
cannot be directly deduced from the data reporting
on the behavior of basal cells that grow adjacent to
the external basal lamina (167).
Turnover of the junctional epithelial cells
The junctional epithelium is a stratied epithelium
composed of two strata, the basal layer facing the
connective tissue and the suprabasal layer extending
to the tooth surface (Fig. 2 and 4). Coronally, close to
the sulcus, junctional epithelium is about 15 cell
layers thick and narrows towards the apical part of
the tissue. The turnover rate of junctional epithelium
is exceptionally rapid. In nonhuman primates it is
about 5days and approximately twice the rate of the
oral gingival epithelium (153). Previously it was
thought that only epithelial cells facing the external
basal lamina were rapidly dividing. However, recent
evidence indicates that a signicant number of the
DAT cells are, like the basal cells along the connec-
tive tissue, capable of synthesizing DNA, which dem-
Fig. 4. Haematoxylin-eosin staining
of a clinically healthy human dento-
gingival junction (a) shows an in-
ammatory inltrate in the connec-
tive tissue (CT) lateral to the junc-
tional epithelium (JE). The
junctional epithelium exhibits wider
intercellular spaces as compared to
the sulcular epithelium (SE). The
wide intracellular spaces allow the
access of the components of the im-
munological defense into the sulcus.
DAT cells directly attached to the
tooth are seen as a string lateral to
the enamel space (ES). Note the most
coronal DAT cell () that appears to
be supported only by the tooth and
thus forms the medial wall of the sul-
cus (see also schematic illustration
in Fig. 8). Reaction with a mono-
clonal antibody to keratin 19 (b)
shows that all the suprabasal junc-
tional epithelial cells express this
protein, as do the undifferentiated
basal cells. The reaction with the
antibody to keratin 10 shows that
terminal differentiation is a charac-
teristic feature of the oral epithelium
(OE) but not of the sulcular or junc-
tional epithelia. The DAT cells along
the tooth surface (d) are especially
rich in K19.
15
onstrates their mitotic activity (133,135). At the co-
ronal part of the junctional epithelium, the DAT cells
typically express a high density of transferrin recep-
tors (131), which supports the idea of their active
metabolism and high turnover (172). The ndings
suggest that the DAT cells have a more important
role in tissue dynamics and reparative capacity of
the junctional epithelium than has previously been
thought (143). Based on these data, alternative
models for the turnover of DAT cells can be pro-
posed (Fig. 6). The existence of a dividing population
of epithelial cells (DAT cells) in a suprabasal loca-
Pllnen et al.
Fig. 5. A schematic illustration of a DAT cell shows the
structural and molecular composition of the epithelial
attachment apparatus (EAA). N nucleus of a DAT cell,
IF cytoplasmic keratin laments (intermediate size
laments). The hemidesmosomes at the plasma mem-
brane are associated with the a
6
b
4
integrin that communi-
cates with Ln-5 laminin 5 located mainly in the internal
basal lamina, the extracellular domain (?) for BP180 is a
collagenous protein (perhaps type VIII), that has not yet
been denitely characterized. LL lamina lucida, LD
lamina densa, SLL sublamina lucida, IBL internal
basal lamina.
tion, several layers from the connective tissue, is a
unique feature of the junctional epithelium. The dis-
tinct phenotype may result from specic permissive
or instructive signals provided by the internal basal
lamina matrix on the tooth surface. Therefore, any
structural or molecular changes in the internal basal
lamina can potentially inuence the vital functions
of the DAT cells and contribute to the effectiveness
or failure of the junctional epithelial defense or vice
versa; changes in the cell metabolism, etc. may affect
the internal basal lamina.
Morphological studies of the internal basal lamina
of teeth extracted because of advanced periodontitis
have shown that remnants of the internal basal lam-
ina can be detected even adjacent to severely de-
generated DAT cells (111). Although the internal
basal lamina morphologically appears to be rela-
tively resistant to external challenges its molecular
structures may still be altered, leading to changes of
the DAT cell function. Indeed, it has been shown that
certain matrix metalloproteinases from eukaryotic
cells cleave laminin-5, exposing a cryptic molecular
16
site that triggers cell migration (49). It has not yet
been shown, however, if bacterial proteinases can
perform directly the same selective cleavage of lami-
nin-5. Bacterial agents may thus indirectly trigger
mechanisms that lead to modulation of host cell be-
havior. Hypothetically, even minor changes in cell
metabolism, biosynthetic activity or ability to divide
and migrate may eventually lead to degeneration
and detachment of the junctional epithelium/DAT
cells and allow pathogenic ora to grow on the ex-
posed subgingival tooth surface. A wide variety of
bacterial species and their products, such as lipo-
Fig. 6. The mechanism of DAT cell turnover is not fully
understood. Considering the fact that the DAT cells are
able to divide and migrate, three possible mechanisms
can be proposed. These are (1) the daughter cells pro-
duced by dividing DAT cells replace degenerating cells on
the tooth surface, (2) the daughter cells enter the exfoli-
ation pathway and gradually migrate coronally between
the basal cells and the DAT cells to eventually break off
into the sulcus, or (3) epithelial cells move/migrate in the
coronal direction along the tooth surface and are replaced
by basal cells migrating round the apical termination of
the junctional epithelium.
polysaccharides, lipoteichoic acids, short-chain fatty
acids, and phospholipases, have been shown to af-
fect adversely the metabolism of epithelial cells in
cultures, leading to changes in proliferation and/or
production of cytokines and matrix metalloprotein-
ases (43,44,64,118120,156). Understanding of the
potential signicance of these events in relation to
the pathogenesis of periodontal pocket formation
calls for further studies.
Junctional epithelium in the
antimicrobial defense
Junctional epithelium consists of active populations
of cells and antimicrobial functions, which together
form the rst line of defense against microbial in-
vasion into tissue (Fig. 7). Even though junctional
epithelial cell layers provide a barrier against bac-
teria many bacterial substances, such as lipopolysac-
charide, pass easily through the epithelium but have
only limited access through the external basal lam-
ina into the connective tissue (146). Both the inter-
nal and external basal laminas act as barriers against
infective agents.
Rapid turnover, as such, is an important factor in
the microbial defense of junctional epithelium (see
below). Also, because the area covered by the divid-
ing cells in the junctional epithelium is at least 50
times larger than the area through which the epi-
thelial cells desquamate into the gingival sulcus,
there is a strong funnelling effect that contributes to
the ow of epithelial cells (145). Rapid shedding and
effective removal of bacteria adhering to epithelial
cells is therefore an important part of the anti-
microbial defense mechanisms at the dentogingival
junction.
There is increasing evidence indicating that sev-
eral specic antimicrobial defense systems exist in
the oral mucosa. Many epithelial cell types, includ-
ing junctional epithelium, have been found to con-
tain enzyme-rich lysosomes. Their fusion with
plasma membrane is triggered by elevation of the
intracellular calcium concentration (122,145). In
rats, the lysosomes have been demonstrated to con-
tain cysteine proteinases (cathepsin B and H) active
at acidic pH (190). Porcine epithelial cells of Malas-
sez share several characteristics with junctional epi-
thelial cells and are able to produce several neutral
proteinases, including collagen-degrading enzymes
and a chymotrypsin-like proteinase (44). The role of
these enzymes in the antibacterial mechanism has
not yet been studied. Recently, it has been found
that the junctional epithelial cells lateral to DAT cells
17
produce matrilysin (matrix metalloproteinase-7)
(176). In Paneth cells of the mouse intestine this en-
zyme is able to activate the precursor peptide of a-
defensin, an important antimicrobial agent of mu-
cosal epithelium (184). It is possible that a similar
active matrilysin/defensin system exists in junc-
tional epithelium, as in other mucosa exposed to
bacteria such as intestine, lungs and urogenital
tissues (88). Another possible effect by which matri-
lysin contributes to the mucosal defense is the re-
lease of bioactive molecules from the cell surfaces
which play a role in the inammatory reaction. Such
effects are currently being actively explored.
Fig. 7. Several antimicrobial mechanisms exist in the
junctional epithelium. In the coronal part of the junc-
tional epithelium quick cell exfoliation (1) because of
rapid cell division (2) and funnelling of junctional epi-
thelial cells towards the sulcus hinder bacterial coloniza-
tion (see text). Laterally, the (external) basement mem-
brane forms an effective barrier against invading mi-
crobes (3). Active antimicrobial substances are produced
in junctional epithelial cells. These include defensins and
lysosomal enzymes (4). Epithelial cells activated by mi-
crobial substances secrete chemokines, e.g. interleukin-
8 and cytokines, e.g. interleukins -1 and -6, and tumour
necrosis factor-a that attract and activate professional de-
fense cells, such as lymphocytes (LC) and polymorpho-
nuclear leukocytes (PMN). Their secreted product, in turn,
cause further activation of the junctional epithelial cells
(5). CT connective tissue.
Pllnen et al.
Despite the selective barrier formed by the gingi-
val basal laminas, components of the inammatory
and immunological defense pass easily through the
basement membrane and epithelium into the sul-
cus. Here they play an important role in restricting
bacterial access into the subgingival tissues (for a re-
view see [81]) (Fig. 2a,b, 4a). Leukocytes, especially
the polymorphonuclear leukocytes that migrate
through the junctional epithelium, comprise prob-
ably the most important defense mechanism at the
gingival margin (112). The cell surface carbohydrates
(1) expressed by the junctional epithelial cells are
thought to respond to extracellular molecular
changes in a manner which allows the cells to com-
municate with their environment. The cells respond
actively to bacterial infection by producing cell ad-
hesion molecules (intercellular adhesion molecule-
1) and chemotactic substances (chemokines such as
C5a, leukotriene B
4
, lymphocyte function-associated
antigen-3 and interleukin-8) that facilitate the mi-
gration of leukocytes through the junctional epithel-
ium (16,28,99,171).
In the subsequent line of defense inammatory
mediators and antibodies produced by macro-
phages, lymphocytes and plasma cells in the gingival
tissues restrict the spreading of bacterial infection
into the connective tissue and systemic circulation.
Signicant amounts of lymphocytes may be present
also within the junctional epithelium, thus contribu-
ting to the protective functions of the tissue
(148,149). Recently, it has been suggested that
supplementary to system-derived antibodies and
antibodies produced locally by plasma cells, the
junctional epithelial cells may also have a secretory
function (77).
The detachment of the DAT cells
from the tooth surface (host vs.
bacteria; battle for surface)
Role of the gingival crevice uid
GCF is an exudate of varying composition found in
the sulcus/periodontal pocket between the tooth
and marginal gingiva. GCF contains components of
serum, inammatory cells, connective tissue, epi-
thelium, and microbial ora inhabiting the gingival
margin or the sulcus/pocket (26, 37, 41, and articles
in this issue) (Fig. 8). In the healthy sulcus the
amount of GCF is very small. However, its constitu-
ents participate in the normal maintenance of func-
tion of the junctional epithelium throughout its lat-
18
eral and vertical dimensions, including the most co-
ronal DAT cells. During inammation the GCF ow
increases and its composition starts to resemble that
of an inammatory exudate (26). The increased GCF
ow contributes to host defense by ushing bacterial
colonies and their metabolites away from the sulcus,
thus restricting their penetration into the tissue.
The main route for GCF diffusion is through the
(external) basement membrane and then through
the relatively wide intercellular spaces of the variable
thickness junctional epithelium into the sulcus. Al-
though all the junctional epithelial cells are con-
stantly exposed to the GCF and its various constitu-
ents, the nutritional and other vital conditions in the
different parts of the junctional epithelium depend
on a large number of local factors. Clearly, the
changes in the composition of the GCF caused either
by bacteria, bacterial metabolites/enzymes or other
factors, or the inammatory reaction is likely to be
Fig. 8. The GCF passing through the junctional epithelium
determines the environmental conditions and provides
sufcient nutrients for the DAT cells to grow. At the gingi-
val margin the GCF may become contamined so that
agents from the oral cavity and/or the plaque bacteria
challenge the most coronal DAT cells. Obviously, the con-
ditions for DAT cell survival and adequate function at the
coronal part of the JE are different and more susceptible
of compromises than those for the basal cells living in
the vicinity of the connective tissue (CT) and the blood
circulation.
largest at the most coronal junctional epithelial cells.
A considerable number of bacteria and host-de-
rived products found in the GCF have been associ-
ated with the initiation and progression of peri-
odontal disease. The bacterial agents include endo-
toxins, hydrogen sulde, butyric and propionic
acids, bacterial collagenases and other proteases
(e.g. trypsin-like), and a variety of enzymes, such as
hyaluronidase and neuraminidase (37). Host-derived
agents include components associated with the in-
ammatory reaction, such as factors of the comple-
ment system, prostaglandins, different cytokines, in-
tracellular enzymes, and products of tissue break-
down such as lactate dehydrogenase, aspartate
aminotransferase, polyamines, and collagen pep-
tides (37,41,82). Furthermore, antimicrobial agents
and leukocyte-derived enzymes such as lysozyme,
alkaline phosphatase, b-glucuronidase, cathepsin D,
elastase, collagenase, and lactoferrin as well as os-
teonectin and bronectin are also found in the GCF.
Indeed, the GCF contains a wide variety of biologic-
ally active molecules with the potential capacity to
affect the growth of junctional epithelium/DAT cells
as well as oral bacteria, both competing for the tooth
surface at the dentogingival interface (Fig. 8 and 9,
Table1).
Role of the polymorphonuclear
leukocytes
Polymorphonuclear leukocytes form the most im-
portant line of defense against bacterial plaque at the
gingival margin (112). When the polymorphonuclear
leukocytes reach the bacteria, they release the con-
tents of their granules and may adhere to individual
bacteria and phagocytose them. The polymorpho-
nuclear leukocytes do not, however, appear to have
the capacity to remove dental plaque but rather form
a protective wall against it. Therefore, polymorpho-
nuclear leukocytes are a major contributor in the
hostparasite equilibrium but have a limited capacity
to reclaim any tooth surface once lost to the plaque
bacteria. On the other hand, activated polymorpho-
nuclear leukocytes can cause tissue damage as a re-
sult of a variety of enzymes, oxygen metabolites, and
other components that are released from their gran-
ules during the battle against microbes (3,179,187).
The polymorphonuclear leukocytes have two main
types of granules that contain agents that are effective
inkilling the bacteria. The azurophilic (primary) gran-
ules contain myeloperoxidase, lysozyme, elastase, ca-
thepsin G, urokinase, acid hydrolases, and defensins,
whereas the specic (secondary) granules contain
19
lactoferrin, elastase, and lysozyme (30). Activated
polymorphonuclear leukocytes also generate hydro-
gen peroxide (H
2
O
2
) and highly reactive oxygen rad-
icals with the potential to destroy bacteria and gingi-
val cells (21,30). The polymorphonuclear leukocytes
are most effective in aerobic conditions close to the
gingival margin (30), suggesting a different role for
them in anaerobic periodontal lesions. While di-
recting their effects to the invading microbes the
powerful polymorphonuclear leukocyte substances
also potentially affect the structural cells andextracel-
lular matrix. Polymorphonuclear leukocytes may also
become activatedininammatory lesions andrelease
specically their secondary granule contents during
their chemotactic migration (63,189). This phenom-
enon may also take place in tissues such as the junc-
tional epithelium. Therefore, the effects of the sec-
ondary granule contents on gingival epitheliumare of
special interest in research into the failure of the junc-
tional epithelium and the formation of periodontal
pockets.
Lactoferrin is an important antimicrobial protein
present in the secondary granules of polymorpho-
nuclear leukocytes. It has high afnity for iron
(5,32,85) and it acts on bacteria by causing iron de-
Fig. 9. The degeneration and detachment of DAT cells ex-
poses the tooth surface and creates a subgingival niche
suitable for the colonization of anaerobic gram-negative
bacteria and apical growth of dental plaque.
Pllnen et al.
Table1. Dental plaque and gingival crevice uid (GCF) components associated with periodontal diseases
Dental plaque in vivo Human GCF/GCW
Cultured Periodontal Periodontal
Compound plaque bacteria Healthy disease Healthy disease
Alkaline phosphatase 60mIU/site (25) 120mIU/site (25)
Ammonia 1486mr (176)
Butyric acid 9.1mM (151) 00.2mr 2.614mr 0.08mr (19) 0.331.14mr
(104,105) (104,105) (19)
Hydrogen sulde 539nr (115) 150p.p.m. (98) 0.8ng/mL (155) 4.2ng/mL (155)
Interleukin-1b 23150ng/mL 86882ng/mL
(95,117) (95,117)
Lactoferrin 600mg/mL (46) 1500mg/mL (46)
Lipoteichoic acid 50mg ml (181)
Lipopolysaccharide 0.83.6mg/mL 9.636mg/mL
(42,173) (42,173)
Prostaglandin E
2
5ng/mL (103) 10.5ng/mL
(103)
Propionic acid 113mr (151) 0.80.9mr 9.544mr 0.75mr (19) 0.98mr (19)
(104,105) (104,105)
Transforming growth factor-a 226pg/mL (95) 54pg/mL (95)
Tumor necrosis factor-a 0.113ng/mL
(126)
GCW, gingival crevicular washing.
pletion, and thus reduction in bacterial cell division
rate, glucose metabolism and macromolecular syn-
thesis (7,178). Besides bacteriostatic activity, lacto-
ferrin also has bactericidal effects, at least in vitro.
Binding of lactoferrin to bacteria and their sub-
sequent lysis has been reported in a number of
studies (7,8,12,36). Furthermore, lactoferrin may ex-
ert antimicrobial effects through interfering with
bacterial attachment and colonization in the oral
cavity (4,159,178). In addition to the effects de-
scribed above, lactoferrin may also interfere with the
host defense not only by blocking complement acti-
vation and inhibiting hydroxyl radical formation but
also by enhancing it by stimulating polymorpho-
nuclear leukocyte recruitment to the infected sites
(12). In the GCF from sites exhibiting gingival in-
ammation or periodontal pockets the amount of
lactoferrin is signicantly elevated (10001500mg/
ml) as compared to the healthy sites (500mg/ml)
(2,46). While microbial multiplication is already sig-
nicantly inhibited at low lactoferrin concentrations
(50mg/ml), even high amounts of lactoferrin (500
mg/ml) seem to have no effect on epithelial cell divi-
sion in model systems simulating the junctional epi-
20
thelium in vivo (118). High concentrations of lacto-
ferrin do, however, hamper epithelial cell growth by
interfering with their adhesion and spreading. The
molecule may, thus, have a role in delaying the re-
pair of the junctional epithelium/DAT cell popula-
tion during severe inammation.
Role of host proteinases and
inammatory mediators
Degradation of extracellular matrix during peri-
odontal inammation is a multistep process that in-
volves several proteolytic enzymes. Different cell
types of periodontal tissue produce matrix metallo-
proteinases (collagenases, stromelysins, gelatinases,
membrane-type metalloproteinases), plasminogen
activator, cathepsins and elastase (17,165). In re-
sponse to the bacteria and inammatory cytokines,
broblasts, junctional epithelial cells, osteoblasts/
osteoclasts, macrophages, and polymorphonuclear
leukocytes release proteinases that are involved in
the defense against microbes. At the same time they
also contribute to periodontal tissue destruction by
degrading extracellular matrix and basement mem-
brane components (17,60,132,158). In concert, ma-
trix metalloproteinases are able to degrade all extra-
cellular matrix proteins (17). Collagenases degrade
interstitial type collagen brils (I, II, III), whereas
gelatinases, stromelysins, and membrane-type
metalloproteinases have the ability to degrade
bronectin and gelatin (denatured collagen), and
basement membrane components including type IV
collagen, entactin, nidogen, and laminin (17,72,102).
Neutrophil elastase and cathepsin G are capable of
degrading basement membrane type IV collagen and
laminin, and also type VIII collagen, found in the
internal basal lamina (61,78). Proteinases of host ori-
gin are thus capable of degrading all known extracel-
lular components of connective tissue and epithel-
ium including components of both the external
basal lamina (basement membrane at the connec-
tive tissuejunctional epithelium interface) and the
internal basal lamina at the epitheliumtooth inter-
face. Therefore, these enzymes seem to have the po-
tential to contribute to the lateral and apical prolifer-
ation of the junctional epithelium into the connec-
tive tissue (Fig. 2) as well as to epithelial
disintegration through degradation of the internal
basal lamina and increase in epithelial permeability.
However, electron microscopic studies on DAT cells
attached to teeth extracted because of advanced
periodontitis do not support the idea that enzymatic
degradation of the epithelial attachment apparatus
precedes the degeneration of DAT cells (111). On the
contrary, the DAT cells in this material seemed to be
more severely affected than the epithelial attach-
ment apparatus once produced and maintained by
the degenerating cells. This implies that it might be
more rewarding to focus the studies of pocket forma-
tion on mechanisms that disturb the vital functions
of the DAT cells rather than on the destruction of
the matrix components of the epithelial attachment
apparatus.
As described elsewhere in this issue, regulation of
proteinase activities is a complex process involving
activation of latent precursor molecules as well as
inhibition of the active enzymes. Therefore, the ac-
tual damage caused by, for example, polymorpho-
nuclear leukocyte proteinases may be limited in the
presence of proteinase inhibitors, such as a2 macro-
globulin, a1 antitrypsin and tissue inhibitors of
metalloproteinases, found in the junctional epithel-
ium and in the gingival crevice. In fact, a recent
study has demonstrated that there is only a limited
amount of active metalloproteinases in the pocket
epithelium obtained from sites of periodontitis
(140). Since the degradation of the extracellular ma-
21
trix by metalloproteinases plays a major role in the
inamed connective tissue, it is pertinent to ask how
signicant is the failing support of the connective
tissue to the integrity of the junctional epithelium.
Obviously, a less effective connective tissue support
predisposes the tissue to intraepithelial splits and
contributes to the lateral and apical proliferation of
the epithelium. Together with increased per-
meability of the junctional epithelium, this may alter
the nutritional conditions of the DAT cells on the
tooth surface and expose them to agents from the
dental plaque. Another area of junctional epithelium
biology that has not been addressed in depth is the
composition of the epithelial interstitial matrix and
how its degradation affects the behavior of junc-
tional epithelial cells. Also, a limited proteolytic
cleavage of matrix molecules, e.g. laminin, bronec-
tin and proteoglycan, may expose cryptic molecular
sites with biological activity not possessed by the in-
tact molecule (39). These active tissue fragments
may regulate cell adhesion, migration and prolifer-
ation in inamed tissue (175). For instance, while in-
tact laminin-5 promotes formation of hemidesmo-
somes and inhibits cell migration, its fragment pro-
duced by a proteolytic cleavage promotes epithelial
cell migration (49). Even though fragments of
bronectin and collagen have been demonstrated in
GCF their effects on junctional epithelium/pocket
epithelium are unknown (38). In future, extracellular
matrix molecules and their fragments can be ex-
pected to provide useful tools for prevention and
management of periodontal disease. For example,
laminin-5 treatment of teeth or titanium dental im-
plants may enhance long-term stability of epithelial
attachment (35).
Cytokines, especially the interleukins -1 and -6,
and tumor necrosis factor-a, and the arachidonic
acid metabolite prostaglandin E
2
, have been strongly
associated with periodontal disease [for a review see
(47)]. These inammatory mediators are secreted
into the GCF by both leukocytes and activated junc-
tional epithelium cells and their amounts have been
shown to increase at sites exhibiting periodontal
tissue destruction [Table1; (95,103,109,117)]. In-
terleukin-1, interleukin-6 and prostaglandin E
2
stimulate bone resorption and contribute to peri-
odontal tissue destruction also by inducing matrix
metalloproteinase (collagenase) production (47). In-
terleukin-1 has been shown to promote broblast
proliferation and production of other cytokines by
periodontal cells, and to activate the arachidonic
acid pathway and thus production of prostaglandin
E
2
(27,34,110). Prostaglandin E
2
in turn has an over-
Pllnen et al.
all catabolic effect on gingival broblasts, as shown
by suppressed DNA synthesis and collagen synthesis
(6). Studies reporting the effects of inammatory
mediators on the junctional epithelium and its func-
tions are not available today.
Role of bacterial products
It is plausible that several products released from
bacteria during periodontal infection have junc-
tional epithelium as their major target tissue. Even
though there is ample evidence that bacterial sub-
stances have a multitude of effects on several cell
types, ranging from activation of cell functions to
cell death, the importance of specic substances in
initiation and progression of periodontal diseases is
not yet understood. We review here the literature of
the main bacterial products and their potential ef-
fects on the junctional epithelium.
In the early phase of plaque formation gram-posi-
tive bacteria accumulate on the tooth surface close
to the most coronal junctional epithelium/DAT cells
which may thus be exposed to the cell wall compo-
nents of these typically supragingival bacteria;
namely the peptidoglycan matrix, surface antigens,
teichoic and lipoteichoic acids. Lipoteichoic acids
are genus- and species-specic molecules (45) that
are synthesized especially abundantly from sucrose
at neutral pH (79,124). The precise functions of
lipoteichoic acids in bacteria have not yet been es-
tablished. However, they have been implicated as
carriers in cell wall synthesis, inhibitors of autolytic
activity and as reservoirs of cations (45,125). In the
oral cavity lipoteichoic acids are thought to mediate
bacterial adhesion to human cells and teeth
(13,67,123).
If the bacterial plaque is allowed to grow, the
amount of gram-negative bacteria increases. The cell
wall of these bacteria is composed of a thin peptido-
glycan layer and a bilayered outer membrane, which
contains the major surface antigens including the
porin proteins, and lipopolysaccharides (lipopolys-
accharides/endotoxins) (55). The variation in the
lipopolysaccharides between bacterial genera, spe-
cies, and even within species (54,180,181) may ac-
count for the reported differences in host responses
to lipopolysaccharides derived from different bac-
terial populations.
From the periodontal point of view both lipotei-
choic acids and lipopolysaccharides are interesting
molecules. They are released from the bacteria into
the extracellular environment during bacterial dis-
ruption, and also during normal cell wall turnover
22
of living bacteria (42,45,55,56,68,150,173,181). Thus,
varying amounts of lipoteichoic acids and lipopolys-
accharides are present at the gingival margin where
they may stimulate leukocyte function, increase
cytokine and inammatory mediator production
and activate the complement system as shown in
studies in vitro (15,86,89,96). In addition to their role
as inammation modulators lipoteichoic acids and
lipopolysaccharides have been shown to affect peri-
odontal tissues, e.g. by stimulating bone resorption
(9,59,94). Furthermore, lipopolysaccharides appear
to have the ability to increase epithelial permeability
and penetrate healthy gingival sulcular epithelium
(120,138,154). Lipopolysaccharides from Salmonella
enteritidis, Escherichia coli, Actinobacillus actino-
mycetemcomitans, Porphyromonas gingivalis, Prevo-
tella intermedia and Porphyromonas asaccharolytica
have been shown to stimulate human gingival
broblast proliferation at low (1mg/mL) concen-
trations and suppress their proliferation at higher (
10mg/mL) concentrations (11,31,65,74,83,84). High
concentrations of lipopolysaccharides from non-oral
bacteria (E. coli, 5000mg/mL) have been shown to
increase the proliferation of basal cells of the junc-
tional epithelium in an animal model (167). In hu-
man epithelial cell and junctional epithelial tissue
cultures lipopolysaccharides from oral pathogens
show only slight or no effects on the growth and mi-
totic activity of the cells (120). This indicates that
lipopolysaccharides, despite their established role as
modulators of inammation, may not signicantly
harm the epithelial cells at the concentrations found
in dental plaque and GCF (below 50mg/mL). There-
fore, lipopolysaccharides do not appear to have a key
role in DAT cell degeneration and detachment from
the tooth surface.
When epithelial cells in different culture systems
are exposed to lipoteichoic acids from gram-positive
oral bacteria (Streptococcus sanguis and Streptococ-
cus mutans, 1050mg/mL) their growth and mitotic
activity are consistently reduced (120). According to
these results, the lipoteichoic acids appear to have
the potential to interfere with the renewal of various
types of epithelial cells. As discussed before, a pro-
longed inhibition of the renewal of the coronal junc-
tional epithelium/DAT cells would most likely lead
to their degeneration and detachment, and eventu-
ally to subgingival colonization by gram-negative
Periodontopathogenic bacteria release numerous
proteolytic enzymes that are a prerequisite for the
normal metabolism of amino acids and for the sur-
vival of these mainly asaccharolytic bacteria in the
oral environment (for a review see [68]). Bacterial
collagenases, gelatinases, and trypsin- and chymo-
trypsin-like enzymes have been shown to degrade
host extracellular matrix macromolecules and base-
ment membrane components in vitro suggesting
that also they have the potential to contribute to
periodontal tissue destruction, as do the host-de-
rived proteinases (17,116). This is especially true in
the junctional epithelium, where the bacterial en-
zyme concentrations are much higher than in the
connective tissue. Degradation of immunoglobulins
and complement proteins by bacterial proteinases
may result in an incomplete host defense and thus
facilitate bacterial colonization and growth. Some
bacterial proteinases are able to activate host matrix
metalloproteinases and thereby increase the total
proteolytic activity in the infected tissue (29,157).
The complex interactions of bacterial and host-de-
rived proteinases and their inhibitors, as well as the
presence of bacteria capable of degrading the pro-
teinase inhibitors (24,107,139) make the exact role
of the bacterial proteloytic enzymes in periodontal
tissue destruction difcult to study. It appears, how-
ever, that the role, if any, of the bacterial enzymes
on epithelial detachment and DAT cell viability/de-
generation is primarily indirect and because of
changes in the living conditions of these cells rather
than of direct effects on the cells or on the epithelial
attachment apparatus (111).
The oral cavity provides bacteria with a large num-
ber of ecologic niches and substrates that can be
metabolized to different end products depending on
the bacterial population and the prevailing environ-
mental conditions. Therefore, the composition of a
bacterial population and its virulence, associated
with specic pathogenic mechanisms, reects the
interaction of a large number of constantly changing
variables determined not only by the oral microora
itself but also by the host. For example, the met-
abolism of carbohydrates in dental plaque depends
on the pH, the oxygen gradient, the amount and
quality of available substrates, and most importantly,
the bacterial composition of the plaque (92,185). As
a principal rule, cariogenic, supragingival plaques
contain a high percentage of streptococci, whereas
subgingival plaques growing in periodontal pockets
contain high proportions of gram-negative an-
aerobes (22,23,185). The main metabolic end prod-
uct of the streptococcal plaque is lactic acid, whereas
the gram-negative bacteria produce butyric acid
more abundantly (68,91,106,164,185) (Fig. 10). It is
noteworthy that certain bacteria can take advantage
of the metabolic end products released by other bac-
23
teria and thereby contribute to the production of
agents that are increasingly detrimental to the host
tissues. Anaerobic plaque bacteria, especially the as-
accharolytic bacteria, utilize amino acids and pep-
tides, derived either from diet or tissue/cell break-
down, for their sources of energy. This requires the
presence of proteolytic enzymes that rst degrade
the macromolecules (proteins) into small peptides
or amino acids which are then further metabolized
by the bacteria (68,185). When amino acids are util-
ized as an energy source, ammonia, sulfur-contain-
ing compounds (hydrogen sulde) and short-chain
fatty acids, especially butyric and propionic acids,
are formed (23,68,164).
Butyric and propionic acids are short-chain fatty
acids containing four and three carbon atoms, re-
spectively. They are produced by periodontopathog-
enic bacteria, such as Porphyromonas, Fusobacteri-
um, Prevotella and Treponema (1820,22,51,97,164).
As described above, the production of these acids
depends on a variety of environmental factors in-
cluding the proteolytic activity and the pH in the
pocket. The maximal activity of proteolytic enzymes
produced by periodontal pathogens is at pH7.08.0.
Some periodontal bacteria (e.g. Prevotella interme-
dia) are, however, capable of surviving and func-
tioning over a much broader pH range and even of
raising the pH by producing ammonia, and thereby
making the environment more suitable for bacterial
populations adapted to alkaline conditions (68). It is
of particular interest that the concentrations of bu-
tyric and propionic acids found in human plaque
and GCF correlate directly with the degree of gingival
inammation and periodontal pocket depth (Table1
[19,104,105]). Furthermore, the application of milli-
molar concentrations of these short-chain fatty acids
onto the healthy gingiva of beagle dogs has been re-
ported to produce a marked gingival inammatory
response (152). More recently, human studies have
shown that both food, which supports bacterial gen-
eration of high levels of short-chain fatty acids (50
mr), and short-chain fatty acids (100mr) applied
directly to healthy human gingiva elicit an inam-
matory response in the tissue. This response can be
demonstrated by increased GCF ow, subgingival
temperature, polymorphonuclear leukocyte emi-
gration, and elevated levels of inammatory cyto-
kines (interleukin-8) in the GCF (75,106,191). Short-
chain fatty acids have also been shown to activate
leukocytes to release inammatory cytokines and ex-
tend their lifetime (163,166). The activation of the
inammatory response and simultaneous inhibition
of the polymorphonuclear leukocyte function
Pllnen et al.
(phagocytosis and degranulation) (106) can alter and
prolong the inammatory response and lead to more
severe tissue destruction.
In cell cultures, butyric and propionic acids, ap-
plied in concentrations found in human plaque and
GCF, have been shown to inhibit the proliferation of
both broblasts and epithelial cells (33,119,151,156).
Microbial populations producing these short-chain
fatty acids may thus signicantly impair the rapid
renewal of the junctional epithelium/DAT cells and
thereby counteract one of the tissues main host pro-
tective functions. Taken together, the current litera-
ture suggests that butyric and propionic acids play a
role in the initiation and progression of periodontal
pocket formation by triggering and modifying the in-
ammatory response and by hampering the turn-
over and repair of the tissues at the dentogingival
junction.
Utilization of amino acids by bacteria for their en-
ergy needs, results in the formation of short-chain
fatty acids, hydrogen sulde and ammonia as by-
products. Like the short-chain fatty acids, am-
monium and hydrogen sulde have potentially det-
rimental effects on periodontal cells. Ammonium
(2080mr) has been shown to cause cell vacuoliz-
ation in chondrocytes and to inhibit (210mr am-
monium) collagen secretion by human gingival
broblasts in vitro (62,177). Whether or not the am-
Fig. 10. Oral bacteria produce short-chain fatty acids. En- glucose is abundant in aerobic (and low pH) conditions
vironmental factors such as the oxygen gradient, pH, sub- the metabolites on the left are the main products. In an-
strate quality and quantity, and the presence of bacterial aerobic conditions at high pH and when lactose starch or
enzyme inhibitors have a strong effect on the survival of amino acids are used as substrates, the metabolites on the
bacteria and the metabolites produced. When sucrose or right hand side of the gure are increased.
24
monium levels produced by plaque bacteria signi-
cantly inuence epithelial cells is not known.
Hydrogen sulde is a highly toxic compound that
causes adverse effects on human tissues (eyes and
respiratory tract) at concentrations of 50p.p.m. (50
mg/mL) and above (14). Hydrogen sulde has been
detected in the GCF collected from gingival sulcus/
pocket and its amount has been shown to increase
in gingival inammation (155). In the concentrations
found in dental plaque (150ppm) hydrogen sulde
has been shown to be toxic to HeLa cells (98). Inter-
estingly, oxidation of hydrogen sulde results in sul-
fate formation and detoxication of the compound,
whereas its toxicity is increased in the presence of
short chain hydrocarbons, ethanol and/or proteins
(14). Considering the protein-rich, short-chain fatty
acid-containing and anaerobic conditions in the
subgingival space, hydrogen sulde appears to be a
potential candidate to cause signicant damage to
the junctional epithelium/DAT cells on the tooth
surface during periodontal disease progression.
Role of risk factors for periodontal
disease
It is clear that periodontal diseases are primarily
caused by bacterial infections and that a number of
risk factors contribute to the susceptibility of indi-
viduals to these infections, and to the pathogenesis
and severity of the disease. These factors include
smoking, diabetes, immunosuppression, genetic fac-
tors, stress and age (136). Studies on how the risk
factors inuence disease progression have mainly
been focused on the inammatory reaction
(10,52,57,58,76,108,114,137,162,186,188). The con-
clusion from these studies is that a sound inam-
matory host response is needed for successful peri-
odontal defense. Factors that modify this response
may either cause an overwhelming reaction or an
inadequate reaction, both of which may accelerate
tissue destruction. It is interesting that periodontal
risk factors, such as hyperglycaemia and chemical
compounds released from tobacco, have harmful ef-
fects on the renewing capacity of both broblasts
and bone cells (40,50,170) However, very little is
known about the inuences of any of the risk factors
on oral gingival/sulcular or junctional epithelium
(64). From the defense point of view, it would be im-
portant to examine whether these factors also impair
the turnover or other defense mechanisms of the
junctional epithelium and thus contribute to the de-
generation of the dentoepithelial junction.
It is also quite possible that a specic response of
junctional epithelial cells to bacterial or host sub-
stances is a key factor in the pathogenesis of certain
forms of periodontal diseases. One example could be
localized juvenile periodontitis, where a rapid apical
growth of the junctional epithelium is associated
with a typically distributed pocket formation. A local
junctional epithelial cell defect might in this case re-
sult, for example, in a decreased ability of the DAT
cells to form hemidesmosomes. In fact, in Kindler
syndrome a defect in the formation of hemidesmo-
some-associated bers consisting of type VII colla-
gen leads to detachment of skin and gingival epithel-
ium and to early onset periodontitis (183).
Conclusions
The precise mechanisms that lead to the degenera-
tion and detachment of the junctional epithelium
from the tooth surface and to the conversion of a
gingival sulcus into an infected periodontal pocket
have not been established. The destructive mechan-
isms may involve many of the modalities of both the
host defense and microbial virulence. Studies of the
degeneration process of the junctional epithelial
cells themselves are, however, largely missing. The
junctional epithelium consists of distinct popula-
25
tions of cells at different anatomical locations. All
these cells have a responsive phenotype that allows
them to exhibit specic functions in periodontal de-
fense and to take or lose an active role during peri-
odontal disease progression. The junctional epithel-
ium is rmly attached to the tooth by the suprabasal
DAT cell/internal basal lamina structure, and to the
connective tissue by the basal cell/external basal
lamina complex. Environmental conditions differ
essentially in these two locations and even similar
pathogenic challenges may become modied and
cause considerably different cellular responses.
While the coronal DAT cells grow close to the bac-
terial plaque the apical and lateral parts of the junc-
tional epithelium are likely to be inuenced by the
connective tissue and the inammatory reaction.
The GCF provides the most coronal DAT cells with
the necessary conditions to survive but it also con-
tains a variety of biologically active molecules with
the potential capacity to affect their vital functions
and behavior. Although the signicance of the vari-
ous components of the GCF to the failure of the
junctional epithelium and its DAT cells calls for
further studies there are a few candidates that are of
particular interest today. Among these are the poly-
morphonuclear leukocyte products that may have
direct inuences on DAT cell survival and/or their
adhesion and bacterial lipoteichoic acids and meta-
bolic end products, such as propionic and butyric
acids that may interfere with cell division and thus
the turnover and reparative capacity of the junc-
tional epithelium. Similarly, inammatory factors
and components associated with periodontal risk
factors may reach high concentrations in the GCF
and severely interfere with the junctional epithel-
iums normally protective functions.
When the physiology of the junctional epithelium
and its molecular reactions during different external
challenges are known in more detail the tissues role
in the failure of the healthy dentogingival junction
and the reasons that make some individuals suscep-
tible to periodontal diseases will be better under-
stood.
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Periodontology 2000, Vol. 24, 2000, 5672 Copyright C Munksgaard 2000
Printed in Denmark All rights reserved
PERIODONTOLOGY 2000
ISSN 0906-6713
Role of physical forces in
regulatingthe formandfunctionof
the periodontal ligament
CHRISTOPHER A. G. MCCULLOCH, PREDRAG LEKIC & MARC D. MCKEE
The periodontal ligament is a physically small but
functionally important tissue in tooth support, pro-
prioception and regulation of alveolar bone volume.
There is a long-standing and widespread interest in
the periodontal ligament as a model connective
tissue because of its rapid matrix turnover and its
ability to adapt to alterations of mechanical loading.
These features are mediated in part by heterogen-
eous cell populations that enable the roots of teeth
to maintain dynamic yet strong attachments to bone
in spite of highly variable applied force levels. The
remarkably precise maintenance of periodontal liga-
ment width in spite of these force levels or the res-
toration of the ligament space after surgical ablation
indicates the existence of highly effective regulatory
systems for measuring tissue domains and for
initiating localized matrix resorption and synthesis.
Constitutive adaptation to applied forces is mediated
in part by specic structural and regulatory proteins
expressed by periodontal ligament cells. As an ex-
ample of these adaptations we review recent evi-
dence of how cytoskeletal proteins mediate protec-
tive responses to applied force and how these re-
sponses may enable the cells to survive in a
mechanically active environment.
Background
The rapid growth of interest in endosseous implants
as surrogates for natural teeth could indicate that the
demise of the periodontal ligament is imminent. As
many types of implants do not employ a gomphosis
to provide support and attachment to the jaw bone,
what is the rationale for continued study of the peri-
odontal ligament? We suggest that the many inter-
esting functional and biological features of this
tissue, its basic role in the development and main-
56
tenance of the periodontium and its core function
in the healing of periodontal wounds underlines its
fundamental importance. Further, the rapid re-
modeling of extracellular matrix proteins in the peri-
odontal ligament is the basis for its utility as a model
system to study connective tissue homeostasis and
remodeling. This chapter considers two discrete yet
interrelated aspects of periodontal ligament physi-
ology that underline its unique biological character-
istics and its central role in tooth support: 1) homeo-
static mechanisms for preservation of tissue do-
mains; and 2) adaptational features to high load
bearing. For denitive reviews on the general fea-
tures of the periodontal ligament, we refer you to
Schroeder (71), Berkovitz & Moxham (10) and Berko-
vitz (11). As in vivo data on the regulation of peri-
odontal ligament function in humans are limited,
much of the work discussed in this chapter relates
to teeth of limited eruption in rodents or in non-
human primates and in vitro work on human peri-
odontal cells. While these models provide interesting
insights into the human periodontal ligament in
vivo, they are by no means perfect surrogates and,
consequently, it is not appropriate or desirable to ex-
trapolate directly from these data to the human situ-
ation.
Periodontal ligament structure and
organization
The periodontal ligament is a complex, vascular, and
highly cellular soft connective tissue that attaches
the tooth roots to the inner wall of the alveolar bone.
In general, all ligaments and tendons consist of par-
allel bundles of collagen bers; some contain an ad-
ditional network of elastic bers (such as ligamenta
Periodontal ligament homeostasis
nuchae and ava) (86). The mechanical strength of
tendons and ligaments derives largely from the mol-
ecular structure of the type I collagen molecule and
its ordered arrangement into bers (47). The bers
of the periodontal ligament form a meshwork similar
to a stretched shing net that extends between the
cementum and the bone. The bers are anchored by
their insertion into bone or cementum as Sharpeys
bers. The mechanism by which collagen bers are
tethered at their extremities is likewise important in
dening the overall mechanical properties of the
tissue system as a whole (16).
The periodontal ligament is unique among the
various ligament and tendon systems of the body in
that it is the only ligament to span two distinct hard
tissues namely, tooth cementum and bone (Fig. 1).
While tooth cementum has been likened to bone in
terms of function and morphology, it nevertheless
possesses unique anatomical and structural prop-
erties and is positioned at a soft tissuehard tissue
interface that is absolutely critical to the process of
mastication. Such an important anchoring tissue for
attachment of the periodontal ligament to the tooth
is mirrored on the wall of the alveolus in that bone
exhibits similar features in regard to structure and
composition; likewise, the periodontal ligament has
the ability to attach periodontal ligament bers to
the alveolar process of the mandible and maxilla (15,
37, 43). Collectively, this arrangement forms a sus-
pensory complex involving two hard tissues and the
intervening soft connective tissue comprising the
periodontal ligament.
As might be expected for any biological system
routinely receiving repetitive biomechanical strains,
here associated with mastication, there are special
metabolic requirements and an architectural tissue
design that facilitate the function of the periodontal
ligament. Not surprisingly, such a unique and dy-
namic connective tissue system involving multiple
tissues requires exquisite regulation at the cellular
level. Maintenance and remodeling of periodontal
ligament collagen bers (2123, 81), together with
the embedding and calcication of their extremities
to form Sharpeys bers (37, 43), requires the con-
certed action of numerous cell types (25) and
multiple, synchronized signaling mechanisms to co-
ordinate these activities. Central to these integrated
activities is the periodontal ligament broblast,
whose responsibilities include the formation and re-
modeling of the periodontal ligament bers, and
presumably a signaling system to maintain peri-
odontal ligament width and thickness across the soft
tissue boundary dened by this ligament (53). Such
57
a central role in the physiology of the periodontium
dictates the need for precise cellular organization
and cellular signaling. In the periodontal ligament,
cellular signals are, in part, mediated by the forces
transmitted to the broblasts via collagen brils with
which they are in direct contact (25). Although not
particularly well characterized at the molecular level
specically for periodontal ligament broblasts,
some reports are available (33, 39, 78). Cell-matrix
interactions between broblasts and the extracellu-
lar matrix have been extensively studied and are re-
viewed elsewhere (69).
At the tissue level, periodontal ligament bro-
blasts are rather regularly dispersed throughout the
ligament and are generally oriented with their long
axes parallel to the direction of the collagen brils
(Fig. 1). By virtue of their ability to both synthesize
and shape the proteins of the extracellular matrix,
periodontal ligament broblasts generate an organ-
izational tissue pattern in which collagen brils form
bundles that insert into the bone and tooth ce-
mentum as Sharpeys bers (16, 43, 56). This struc-
ture conforms to the three-dimensional architecture
of the periodontal ligament in a very precise manner
(20). At the level of the broblast cell body, the nu-
cleus occupies a large percentage of the volume of
the cell, but the surrounding cytoplasm contains the
full complement of organelles necessary to effect
protein secretion (7) (Fig. 2). During development
and the initial formation of the periodontal liga-
ment, the cytoplasm-to-nucleus ratio is high, and
broblasts appear very active in terms of having an
extensive network of rough endoplasmic reticulum,
a well-developed Golgi apparatus and abundant se-
cretory granules containing predominantly type I
collagen molecules destined for export. The cells
also develop long and thin cytoplasmic extensions
that form three-dimensional veils that compart-
mentalize the collagen brils into bers. At all levels
of the cell, intimate connections are established be-
tween the plasma membrane and individual colla-
gen brils. Presumably, these sites represent contact
points for integrin-matrix linkages such that strain
occurring in the ligament is transmitted to the cell,
and appropriate cell signaling cascades are activated
(27). Typically, the collagen brils of the periodontal
ligament are quite uniform in size but show some
minor variability with age and at different anatomi-
cal locations within the periodontal ligament.
Several studies have indicated that the extracellu-
lar matrix collagens of the periodontal ligament have
an extremely high turnover and remodeling rate,
much higher than in gingiva, skin and bone (77).
McCulloch et al.
Fig. 1. Light micrographs illustrating hard and soft tissue osteoblasts (Ob) at the crest of an inter-radicular alveolar
relationships in the periodontium. A. Low-magnication bone septum. D: dentin. DF. Age-related changes in peri-
image showing the histology of the periodontal ligament odontal ligament structure and organization. Early in its
(PL) and surrounding tissues. The periodontal ligament formation and just prior to tooth eruption (D), peri-
intervenes between the alveolar bone (AB) and the tooth odontal ligament formation by broblasts (F) forms co-
root, the latter consisting of dentin (D) and cementum. incident with extensive bone formation by osteoblasts
Other nearby tissues include the pulp (P) of the tooth, (Ob) in each surrounding alveolus of the alveolar process
Hertwigs epithelial root sheath (HRS), marrow elements of the mandible and maxilla. Osteoblasts are positioned
(M) situated in the alveolar bone and the mandibular at the bone surface, whereas broblasts are dispersed
nerve (N). B, C. Higher magnication images showing throughout the developing periodontal ligament (PL).
periodontal ligament (PL) relationships with alveolar Collagen bers are relatively small in diameter at this
bone (AB) and tooth. The collagen bers of the peri- point, and their structure and insertions are not readily
odontal ligament span these two hard tissues, inserting visible. Capillaries (asterisks) are abundant at all stages of
into the matrices of bone on one side, and the cementum periodontal ligament development. At the time of tooth
(CEM) of the tooth on the other. Fibroblasts are abun- eruption (E), the periodontal ligament develops an obvi-
dantly dispersed throughout the periodontal ligament, as ous tissue organization such that collagen bers are
are numerous capillaries. C illustrates bone formation by clearly dened and can be observed to insert as Sharpeys
58
Turnover and remodeling in the periodontal liga-
ment imply synthesis and breakdown of matrix com-
ponents, particularly the collagenous ber mesh-
work that extends between cementum and bone. As
indicated above, the collagen bers of the peri-
odontal ligament form are critical for tooth support
and attachment to bone. They form a meshwork of
smaller bers, each of which is composed of un-
branched collagen brils that may run from one
ber strand into another. It seems improbable that
one single bril extends the entire dimensions of
tooth to bone, although there is no denitive evi-
dence for or against this view. Early work (72) sug-
gested that remodeling of the ligament is conned
largely to the mid-region of the periodontal ligament
where bers from the bone and bers from the tooth
interdigitate in an intermediate plexus. More re-
cent evidence suggests that this idea may not
necessarily be correct. Turnover and remodeling ac-
tivity in teeth of limited eruption, like the molars of
rodents, are found throughout the width of the peri-
odontal ligament from cementum to bone (6, 8, 66).
To adapt to changes of tooth position, the ber sys-
tems in the periodontal ligament must be degraded
and new bers synthesized. Since the periodontal
ligament is not made up of single strands of straight
collagen bers but consists instead of a complex
meshwork, remodeling does not necessarily occur at
all sites synchronously. There is apparently some
bers (arrows) intothe alveolar bone (AB). Osteoblasts con-
form to this arrangement, and are clustered between the
insertion sites. Fibroblasts at this stage are abundant and
showa large amount of perinuclear cytoplasmhousing the
extensive synthetic and secretory organelles expected for
such an active, collagen-producing cell. In association with
the organization of the periodontal ligament, bone re-
modeling occurs, as evidenced by the presence of cement
lines (CL) in the bone. In adult, mature periodontal liga-
ment of an erupted tooth (F), broblasts are more stellate
in appearance, and the volume of the periodontal ligament
that they occupy is less than at earlier stages, with extra-
cellular matrix (collagen bers) being the predominant
component of the periodontal ligament. Although some
sites in the alveolus show obvious insertions of Sharpeys
bers (arrow) into bone, other surfaces of the alveolar bone
appear not to have obvious insertions of collagen bers
into the bone. This observation may be related to the
timing of the bone remodeling cycle and the ability of resi-
dent broblasts and osteoblasts to act synergistically to
create locally new insertion sites. Nevertheless, a sufcient
number of biomechanically sound Sharpeys bers exist at
any one time to accommodate the forces of mastication. D:
dentin. All samples are from post-natal rat periodontium
embedded in LR White and stained with toluidine blue.
Bars equal 50 mm.
59
exibility in the system to permit adaptational
changes by breaking down short stretches of colla-
gen ber bundles or single brils while leaving
others intact. This highly localized remodeling pro-
cess is undoubtedly facilitated by the phagocytosis
of collagen. Unlike the bulk removal of collagen that
is effected by extracellular matrix metalloprotein-
ases, collagen phagocytosis enables periodontal liga-
ment broblasts to very precisely remove collagen
brils at specic sites (23).
While their roles in the periodontal ligament are
not yet clear, a number of reports have identied ad-
ditional extracellular matrix components including
collagen types V and VI, chondroitin sulfate, proteo-
glycans, bronectin, tenascin and undulin (38, 48,
49, 91). An arborizing network of oxytalan bers has
also been demonstrated in the periodontal ligament
and is most prominent in its occlusal half (7). In re-
lation to other ligaments and tendons, the peri-
odontal ligament is a highly vascularized tissue (13),
and almost 10% of periodontal ligament volume in
rodent molar comprises blood vessels (53). This rela-
tively high blood vessel content may provide hydro-
dynamic damping to applied forces as well as pro-
vide high perfusion rates to this tissue.
Of particular importance to the function of the
periodontal ligament are its attachment points to
bone and tooth cementum (Fig. 3). At both sites, the
actual insertion of periodontal ligament bers into
bone and cementum occurs as Sharpeys bers, an
anatomical arrangement that represents the most
obvious means by which a tooth is retained in the
alveolus and in its occlusal plane. Once embedded in
either the wall of the alveolus or the tooth, Sharpeys
bers calcify to a signicant degree (36) and are as-
sociated with an abundance of noncollagenous pro-
teins commonly found in bone but also recently
identied in tooth cementum (17, 56). Notable
among these proteins are osteopontin and bone sial-
oprotein. Ultrastructural immunolabeling studies
using colloidal gold have demonstrated that high
levels of osteopontin accumulate at the insertion site
within the interbrillar volume of the tissue (56), and
it is thought that osteopontin and other proteins
contribute to the regulation of mineralization and to
tissue cohesion at these sites of elevated biomechan-
ical strain. Indeed, recent data directly demonstrate
that osteopontin is rapidly induced in alveolar bone
shortly after application of orthodontic forces to
teeth (84). A high concentration of noncollagenous
proteins relative to collagen could conceivably en-
dow unique and advantageous physical properties to
this critical hard tissuesoft tissue interface. Particu-
McCulloch et al.
Fig. 2. Transmission electron micrographs and immuno- extensions (arrows) that envelop and dene collagen ber
cytochemical preparations illustrating the ultrastructure (CF) bundles. Nu, nucleus. B. Cross-sectional proles of
of periodontal ligament broblasts, collagen brils, and the collagen brils (Coll) of mature periodontal ligament
cell-matrix relationships, and the intracellular pathway show them to be relatively uniform in diameter. C. Peri-
for collagen secretion. A. A transverse section through the odontal ligament broblasts show an extensive network of
periodontal ligament showing the stellate nature of peri- rough endoplasmic reticulum (rER) and a well-developed
odontal ligament broblasts (Fb) and their cytoplasmic Golgi apparatus with abundant stacked Golgi saccules
60
larly for bone, remodeling activity successively
severs Sharpeys bers to variable degrees as older
alveolar bone is replaced by new bone (37, 43). Con-
comitant with osteoclastic resorption, new connec-
tions are made in an asynchronous fashion such that
periodontal ligament bers are continuously being
embedded in the alveolar wall.
Importantly, another architectural arrangement by
which periodontal ligament bers interface with the
bony surface has been identied. Here, Sharpeys
bers are not readily identiable as distinct bundles
within bone, but periodontal ligament bers can be
observed to abruptly terminate at the bone surface
within a dark-staining band of variable, but generally
thin dimensions (37, 43). This band is rich in noncol-
lagenous proteins but is relatively poor in collagen
(Fig. 3). Based on its ultrastructure and protein com-
position as determined by immunogold cytochem-
istry, this band appears to represent a modied form
of cement line and is present at sites where collagen
bers of the periodontal ligament are directly ap-
posed to the bone surface and partly inserted into
this material. In all cases, this band of matrix is min-
eralized and rich in osteopontin, and may represent
a means by which a rapid connection of periodontal
ligament with the bone surface is established, al-
though it probably possesses less mechanical
strength. Full incorporation of the extremities of
periodontal ligament bers at a later time would
likely require the concerted action of osteoblasts lin-
ing the alveolar wall.
(GS) showing typical, collagen-containing spherical dis-
tensions (SD) at their periphery. Transfer vesicles (TV) are
also common in this region of the cell. The plasma mem-
brane (PM) is in direct apposition with the collagen brils
(Coll) of the extracellular matrix. D, E. After post-embed-
ding, colloidal-gold immunocytochemistry on thin sec-
tions of periodontal ligament using antibodies raised
against mouse N-terminus collagen a1(I) to visualize in-
tracellular pathways for the production of type I collagen
by periodontal ligament broblasts (Fb), gold particle im-
munolabeling indicates the presence of this protein in the
Golgi (G) region and secretory granules (SG) of these cells.
Ultimately, these a chains form the basis of the collagen
molecule, which assembles as collagen brils (Coll) in the
extracellular matrix of the periodontal ligament. rER:
rough endoplasmic reticulum; TV: transfer vesicles. Nu,
nucleus. Epon (AC) and LR White (D, E) sections of rat
periodontium stained with uranyl acetate and lead citrate.
Bars equal 5 mm (A) and 0.5 mm (B-E). Collagen antibody
courtesy of Paul Bornstein, Department of Biochemistry,
University of Washington, Seattle, USA.
61
Periodontal ligament cell
populations
The healthy periodontal ligament contains several
discrete cell populations including broblasts, endo-
thelial cells, epithelial cell rests of Malassez, sensory
cells (such as Runi-type end organ receptors),
osteogenic and osteoclastic cells and cementoblasts.
The predominant cell type is the broblast, which
occupies about 30% of the volume of the periodontal
ligament space in rodents (8). The broblasts of the
periodontal ligament originate in part from the ecto-
mesenchyme of the investing layer of the dental pa-
pilla and from the dental follicle (82) and are differ-
ent from cells in other connective tissues in a num-
ber of respects. For example, the rapid degradation
of collagen by broblast phagocytosis is the basis for
the very fast turnover of collagen in the periodontal
ligament (23).
Although periodontal ligament cells are frequently
considered as a homogeneous population, there are
some data indicating that the periodontal ligament
contains a variety of broblast populations with dif-
ferent functional characteristics (52). Whether these
subsets are derived from a single type of progenitor
cell is unknown. For example, the broblasts on the
bone side of the periodontal ligament exhibit more
abundant alkaline phosphatase activity than those
on the tooth side (32). Developmental differences
may also exist: Freeman & Ten Cate (24) and Ten
Cate (80) demonstrated that periodontal ligament
broblasts near the cementum are derived from the
ectomesenchymal cells of the investing layer of the
dental papilla, while broblasts near the alveolar
bone are derived from perivascular mesenchyme.
Cell kinetic experiments in rodent molar teeth
have shown that periodontal ligament cells comprise
a renewal system in steady state (51, 54). The rate of
cell renewal in the periodontal ligament is notable
for its rapidity and for the precise degree of regula-
tion in spatially discrete compartments. Periodontal
ligament progenitor cell populations undergo exten-
sive turnover in the maintenance of the steady state
and, in spite of extensive tooth drift or wounding,
many of these cells remain precisely located within
a narrow zone in the vicinity of small blood vessels
(30) and in the endosteal spaces of the adjacent al-
veolar bone (55). These cells proliferate, migrate and
ultimately produce more differentiated cells that can
synthesize bone, cementum and the extracellular
matrix of the periodontal ligament as has also been
shown in the developing periodontal ligament (62).
The generation of highly specialized cell popula-
McCulloch et al.
Fig. 3. Immunocytochemical preparations selected to il- gen bers (CF) insert as Sharpeys bers (SF) into alveolar
lustrate the distribution of the noncollagenous protein os- bone, where they are tethered into a mineralized matrix
teopontin (OPN) at periodontal ligament collagen ber in- rich in noncollagenous protein and containing abundant
sertion sites into alveolar bone and tooth cementum. osteopontin. Intense immunolabeling for osteopontin
A, B. The extremities of periodontal ligament (PL) colla- commences where the collagen brils insert (arrows) into
62
tions that can remodel and heal damaged tissues in a
temporally and spatially appropriate manner is
thought to be essential for the repopulation and dif-
ferentiation responses in healing periodontium fol-
lowing extirpation of the periodontal ligament (58).
The signals that regulate these processes include cell-
matrix and direct cell-cell interactions which are
known to control cell proliferation, differentiation
and cell function (34). For example, periodontal liga-
ment cells produce cell adhesion proteins like vi-
tronectin, tenascin and undulin as well as several
integrin subunits (78). Adhesive and cell-to-cell inter-
actions may be conducted through systemically
acting regulators that act onspecic cell types that ex-
press the appropriate cognate receptor (83) or
through intercellular functions such as gap-junction-
mediated calcium uxes (88). Indeed, the broblasts
of the periodontal ligament are connected by special-
ized junctional complexes which include as gap junc-
tions (7, 9, 76). While paracrine and autocrine regula-
tion of periodontal ligament function is undoubtedly
important in mechanical stress-induced differen-
tiation of periodontal ligament broblast function
(50), recent evidence on mechanical stress-induced
DNA synthesis (41) suggests that autocrine function
may not be as important as other systems that may in-
clude electrical coupling. Thus the connectivity of
cells in the periodontal ligament may be of consider-
able importance in terms of the propagation of mech-
anically induced signals. Notably, mechanical stimu-
lation of the periodontal ligament stimulates the ex-
an otherwise mineralized bone matrix, which here has
been decalcied to highlight the underlying organic ma-
trix. At the insertion site, osteopontin appears to accumu-
late throughout the inter-brillar spaces and is likely in-
volved in regulating calcication at these sites and/or par-
ticipating in the maintenance of tissue cohesion. CL,
cement line. C. Where periodontal ligament (PL) collagen
bers (CF) do not obviously insert for any distance into
the alveolar bone, they frequently abruptly terminate on
the bone surface in an osteopontin-rich, layer of organic
material (arrows) that resembles a cement line typically
found in bone. This arrangement may serve as an alterna-
tive attachment mechanism for the adhesion of collagen
bers to bone in the absence of Sharpeys bers. Fb,
broblast. D. On the tooth surface, Sharpeys bers (SF)
exist where collagen bers (CF) of the periodontal liga-
ment (PL) insert into cementum (CEM). Like for bone, os-
teopontin is a prominent constituent of these insertion
sites in cementum, with immunolabeling commencing at
the edge of the mineralized cementum (arrows) and con-
tinuing internally. Fb, broblast. LR White sections of rat
periodontium stained with uranyl acetate and lead citrate.
Bars equal 1 mm (A) and 0.5 mm (BD).
63
Fig. 4. Photomicrographs of rat periodontium including
the periodontal ligament showing the preservation of the
ligament width in young (A: 6 weeks) and old (B: 1 year)
rats. The sections were immunostained for bone sialop-
rotein (brown staining). AB: alveolar bone; PL: peri-
odontal ligament; C: cementum; P: pulp. Bar equals 100
mm.
pression of connexin 43, an important protein in the
formation of gap junctions in periodontal ligament
cells (79).
Regulation of periodontal ligament
width
The cells, vascular elements and extracellular matrix
proteins of the periodontal ligament function collec-
tively to enable mammalian teeth of limited erup-
tion to adjust their position while remaining rmly
attached to the bony socket. Indeed some of the
most interesting features of the periodontal ligament
are its adaptability to rapidly changing applied force
levels and the capacity to maintain its width at con-
stant dimensions throughout the lifetime of the ani-
mal (53) (Fig. 4). This preservation of periodontal
ligament width throughout mammalian lifetime is
an important measure of periodontal ligament
homeostasis and gives insight into the function of
McCulloch et al.
biological mechanisms that tightly regulate the met-
abolism and spatial locations of the cell populations
involved in the formation of bone, cementum and
the periodontal ligament. Cytokines and growth fac-
tors are important locally-acting regulators of cell
function and periodontal ligament cells are capable
of synthesizing and secreting some of these factors
(14, 18, 26). The ability of periodontal ligament cells
to synthesize and secrete a wide range of regulatory
molecules is an essential component of tissue re-
modeling and periodontal ligament homeostasis.
Notably, some of the transforming growth factor-b
isoforms synthesized by periodontal ligament cells
can induce mitogenic effects but can also dose-de-
pendently down-regulate osteoblastic differentiation
of periodontal ligament cells (18). On the other
hand, prostaglandins, which are also produced by
periodontal ligament cells, can inhibit mineralized
bone nodule formation and prevent mineralization
by periodontal ligament cells in vitro (60, 61). Peri-
odontal ligament cells are also capable of regulating
bone formation by producing paracrine factors that
inhibit bone resorption (26). Conceivably, these mol-
ecules may modulate the osteogenic activity of peri-
odontal ligament cell populations and contribute to
the preservation of periodontal ligament width.
These types of cellular signaling systems may, there-
fore, be capable of accurately measuring and
maintaining the width of the periodontal ligament
in spite of high-amplitude physical forces during
mastication and despite the presence of osteogenic
cells within the whole width of the periodontal liga-
ment. Further, recent evidence has shown that the
pro-inammatory cytokine interleukin-1 (75) and
one of the isoenzymes responsible for prostaglandin
synthesis (cyclooxygenase 2) (74) are induced by ap-
plied mechanical force on periodontal ligament cells
in vitro. As prostaglandins and interleukin-1 can
strongly induce matrix degradation, there is evi-
dently an important relationship between mechan-
ical forces, cytokine production and regulation of the
periodontal ligament space. The appropriate regula-
tion of these signaling systems is clinically important
since the failure of homeostatic mechanisms to
regulate periodontal ligament width may lead to
tooth ankylosis and/or root resorption.
Experimental disruption of
periodontal ligament homeostasis
Various experimental perturbations including des-
iccation (2), heat (46) and bisphosphonates (64, 87)
64
have been used to study homeostasis of periodontal
ligament width. These interventions rely in part on
the depletion of periodontal ligament cell popula-
tions or an apparent alteration of the differentiation
repertoire of periodontal ligament cells (44), disrup-
tion of periodontal ligament homeostasis and the
transient or permanent ingrowth of bone. Experi-
ments in dogs (40, 59), monkeys (1) and rodents (87)
have shown that when periodontal ligament cells are
physically removed from the cementum or are per-
turbed by drugs such as the bisphosphonate 1-hy-
droxyethylidene-1,1-bisphosphonate, bone grows
into the periodontal ligament space and ankylosis
may occur. Although ankyloses can persist for long
periods of time, the tendency of the tooth root to
be resorbed and replaced by bone usually leads to
complete resorption over the long-term. As the peri-
odontal ligament is replaced with bone, propriocep-
tion is lost because pressure receptors in the peri-
odontal ligament are deleted or do not function cor-
rectly. Further, the physiological drifting and
eruption of teeth can no longer occur and conse-
quently the ability of the teeth and periodontium to
adapt to altered force levels or directions of force is
greatly reduced.
To understand how periodontal ligament cell
populations restore their cellular and tissue do-
mains, appropriate model systems are required that
can provide insight into the origin of cells, their
regulation and differentiation. For this reason we
have used the rat periodontal window wound model
(Fig. 5) developed by Melcher (57) and modied by
Gould et al. (30). This model facilitates studies of
periodontal ligament homeostasis since precise por-
tions of the alveolar bone and the periodontal liga-
ment can be reproducibly deleted. Selective deletion
of the periodontal ligament and alveolar bone
causes a transient (60-day) disruption of the cellular
domains required to preserve homeostasis, thereby
providing a system to study the regulation of osteo-
genic cells by the adjacent periodontal ligament
cells.
To assess repopulation and differentiation of peri-
odontal ligament cells in healing periodontal tissues,
we used combined
3
H-thymidine labeling (31) and
immunostaining with a-smooth muscle actin, osteo-
pontin, alkaline phosphatase and bone sialoprotein
as differentiation markers of soft and mineralizing
connective tissue cell populations (45, 65). These
studies have shown that in contrast to ablation of
the periodontal ligament, preservation of the peri-
odontal ligament in the window wound model pro-
motes healing of the alveolar bone. Regardless of the
type of wounding method a subset of periodontal
ligament cells express osteopontin, a widely recog-
nized but not wholly specic differentiation marker
of early bone formation. Immunostaining of peri-
odontal ligament cells for bone sialoprotein, a
marker of differentiated osteoblasts and cemento-
blasts, shows no staining reaction in periodontal
ligament cells under physiological or wounding con-
ditions. The absence of this late marker of osteoblast
differentiation in repopulating periodontal ligament
demonstrates that, while a signicant portion of
periodontal ligament cells may have osteogenic
characteristics (45, 67), these cells are blocked from
differentiating into osteoblasts. Consequently peri-
odontal ligament width is restored during the initial
healing phase (710 days, Fig. 6A) because osteo-
genic cells are unable to enter the mineralization
phase of osteogenic differentiation.
We have used bone morphogenetic protein-7 im-
plants in rat periodontal window wounds to probe
the effect of this potent osteoinductive agent on
periodontal ligament cell differentiation (65). In
spite of the known ability of bone morphogenetic
proteins to induce ectopic bone formation in other
tissues (muscle) (85), periodontal ligament width is
preserved in healing periodontal tissues after
wounding. Bone morphogenetic protein-7 implants
are selective in promoting the proliferation and dif-
ferentiation of osteogenic cells but do not apparently
affect brogenic periodontal ligament cell popula-
tions, since the periodontal ligament width is un-
changed after administration of bone morphogen-
etic protein-7. However, administration of a bisphos-
phonate (1-hydroxyethylidene-1,1-bisphosphonate)
(44) reduces periodontal ligament width; this loss of
homeostasis could be the result of altered differen-
tiation of precursor cells in the periodontal ligament
and the recruitment of these cells into the osteo-
genic lineage. Notably, administration of 1-hydroxy-
ethylidene-1,1-bisphosphonate inhibits periodontal
ligament cell proliferative activity, reduces cell
counts and induces bone sialoprotein expression in
the body of the periodontal ligament, implying a dis-
ruption of subsequent periodontal ligament cell dif-
ferentiation (Fig. 6B). Periodontal ligament width is
perturbed only after treatment with 1-hydroxyethyli-
dene-1,1-bisphosphonate for 2 weeks and occurs
after the reduction of periodontal ligament cell
counts, suggesting that a relatively small proportion
of non-osteogenic periodontal ligament cells is re-
quired for the maintenance of periodontal ligament
width.
To assess in more detail the requirement for speci-
65
Fig. 5. Diagrammatic representation of periodontal win-
dow wound through the buccal surface of the rat man-
dible. The wound model was originally developed by
Melcher (57) and modied later by Gould et al. (30). The
wound (W) extirpates either alveolar bone alone or bone
and the periodontal ligament (PL). The regeneration of
the periodontal tissues following wounding provides an
excellent model to study the origin of the cells recoloniz-
ing extirpated periodontium and has been used exten-
sively for phenotyping of periodontal cells involved in re-
generation (44, 45) without microbial contamination from
the oral environment and without involvement of gingival
cells. AB: alveolar bone; C: cementum; P: pulp; D: dentine.
c cell populations in tissue remodeling and the
preservation of periodontal ligament homeostasis,
we transplanted Lac-Z-positive murine periodontal
ligament cells into periodontal wounds of a Lac-Z-
negative animal (submitted manuscript). By trans-
planting previously characterized periodontal liga-
ment cells that express b-galactosidase (Lac-Z-posi-
tive cells) into periodontal wounds of rats not ex-
pressing this marker, we have examined the
differentiation in vivo of cells with a known initial
McCulloch et al.
Fig. 6. A. Longitudinal section through rat periodontium ministered 1-hydroxyethylidene-1,1-bisphosphonate be-
including periodontal ligament (PL) and alveolar bone fore wounding. 1-hydroxyethylidene-1,1-bisphosphonate
(AB) stained for osteopontin. The periodontal ligament reduces cell counts in the body of the periodontal liga-
and bone were extirpated 10 days before sectioning. Note ment and causes the periodontal ligament width to
the staining for osteopontin in the newly formed bone shrink. Notably, periodontal ligament cells express bone
(NFB) and the restoration of normal periodontal ligament sialoprotein (blue staining in cells). The normal patterns
width. C: cementum. Bar equals 50 mm. B. In situ hybridi- of periodontal ligament homeostasis following wounding
zation for bone sialoprotein in a longitudinal section are lost, presumably because of switch of the periodontal
through rat periodontium as in A but animals were ad- ligament cell differentiation repertoire to an osteogenic
66
phenotype. Interestingly, at the rst post-wounding
time (7 days), transplanted cells are present through-
out the entire periodontal ligament and at bone re-
modeling sites (Fig. 6C). Transplanted cells located
in the periodontal ligament exhibit the same pheno-
typic expression as had been determined in in vitro
cell cultures (osteopontin-positive, bone sialoprot-
einnegative). However, at day 14 and 28 after
wounding, transplanted periodontal ligament cells
are not found in the periodontal ligament but in-
stead are located at bone remodeling sites (day 14)
or in the outer layer of the regenerating bone (day
28, Fig. 6D, E). At these later time periods, the Lac-
Z-positive cells express bone sialoprotein, a marker
of a differentiated osteogenic cell. These data show
that transplanted Lac-Z-positive/bone sialoprotein
negative cells can, when embedded in the peri-
odontal ligament, differentiate into osteogenic cells
and migrate into appropriate bony sites. Evidently,
there are well-regulated systems that ensure cells
with the capacity to differentiate into osteoblasts are
restricted to existing bony sites. This regulation may
depend in part on physical loading of the peri-
odontal ligament, since unloaded teeth exhibit a
narrow periodontal ligament space.
Force distribution and
mediators of periodontal ligament
remodeling
Periodontal ligament and alveolar bone cells are ex-
posed to physical forces in vivo in response to masti-
cation, parafunction, speech and orthodontic tooth
movement. Physiological loading of teeth or ortho-
dontically induced tooth movements involve re-
modeling of the periodontal and gingival connective
tissue matrices. Although the histological and some
of the biochemical effects of orthodontic force appli-
cation have been described, the mechanisms by
which applied forces produce reactive changes in
periodontal ligament and bone cells are poorly
phenotype. Bar equals 20 mm. CE. Transplanted Lac-Z-
positive periodontal ligament cells transplanted into
wounded rat periodontium. At 7 days after wounding (C),
transplanted cells marked with Lac-Z by histochemistry
are present throughout the periodontal ligament. At 14
days after wounding (D) or 28 days after wounding (E),
cells are associated with the margin of the alveolar bone
(AB) and begin to express bone sialoprotein. Bar equals
20 mm.
67
understood. Thus, while it is known that applied
mechanical force leads to more rapid bone remodel-
ing in vivo (68), knowledge of exactly how force dis-
tribution from the periodontal ligament to the al-
veolar bone regulates bone remodeling is meager. In
spite of these limitations, morphological obser-
vations of bone and periodontal ligament after ap-
plication of applied forces to mammalian teeth have
lead to the following general suppositions: 1) the
periodontal ligament distributes applied forces to
the contiguous alveolar bone; 2) the direction, fre-
quency, duration and size of the forces determines
in part the extent and rapidity of bone remodeling;
3) when forces are applied to teeth devoid of a peri-
odontal ligament, the rate and extent of bone re-
modeling is very limited. These conclusions suggest
that the periodontal ligament may be both the me-
dium of force transfer and the means by which al-
veolar bone remodels in response to applied forces.
Progress over the last 10 years on force transduc-
tion in biological systems has now been applied to
bone remodeling and to the role of the periodontal
ligament in force adaptation. Komatsu et al. (42)
have used a simple animal model system to examine
stress-strain functions in which root sections from a
variety of animal species are extruded from the al-
veolar bone. They found that the organization of
periodontal ligament collagen at particular sites in
the periodontal ligament is closely related to the
load characteristics in vitro. Andersen et al. (3)
examined stress and strain levels and their distri-
bution within the periodontium in a model system
based on human autopsy material. This model sys-
tem permitted an estimate of the stress levels that
may be distributed across the periodontal ligament
under applied loads. Mechanical forces can induce
bronectin and collagen synthesis by periodontal
ligament cells in a strain magnitudedependent
fashion (35). These studies show in a reasonably di-
rect way that the metabolism and the organization
of the soft connective tissues of the periodontal liga-
ment are indeed modied by applied physical forces.
While applied loads may induce reactive changes
in cells of the periodontium because of secondary
vascular and inammatory effects, current evidence
suggests that periodontal ligament cells have a
mechanism to respond directly to mechanical forces
by activation of a wide variety of mechanosensory
signaling systems including adenylate cyclase,
stretch activated ion channels and by changes in
cytoskeletal organization. These alterations result in
the generation of intracellular second messengers
such as [Ca
2
]
i
, inositol 1,4,5-triphosphate and cyclic
McCulloch et al.
adenosine monophosphate. For example, [Ca
2
]
i
os-
cillations are generated in periodontal cells re-
sponding to substrate tension (4), and increased in-
ositol 1,4,5-triphosphate has been observed after
stretching of osteoblasts (19). Intermediate-term re-
sponses to applied force may include generation of
arachadonic acid metabolites. Indeed, the recent
demonstration of increased COX-2 expression by
stretching of periodontal ligament cells in vitro (74)
suggests a mechanism by which increased prosta-
glandin levels may be generated. Interleukin-1 may
also be involved in periodontal ligament regulation
of bone remodeling in that cyclic-tension force
causes increased interleukin-1 production by human
periodontal ligament cells (75). Aging may exert a
modulating effect on interleukin-1 production since
in vitro aged periodontal cells produce more in-
terleukin-1 when stretched than younger cells (73).
Longer-term responses to mechanical loading in
vitro may include stimulation of cell division, al-
though this response in periodontal ligament bro-
blasts is apparently not due to an autocrine regula-
tory mechanism (41). Increased collagen synthesis
(35) and stimulation of alkaline phosphatase activity
(89) are also force-induced downstream changes that
likely impact on altering the form and function of
loaded periodontal ligament. These data indicate
that there are many potential routes by which ap-
plied loads to the periodontal ligament may lead
either directly or indirectly to alveolar bone remodel-
ing. Currently, much of the ongoing work on mech-
anotransduction has focused on signaling mechan-
isms, and there is great interest in determining the
nature of the mechanosensors in periodontal liga-
ment and bone cells.
Some of the most rapid responses in periodontal
broblasts subjected to mechanical strain in vitro in-
volve an elevation in intracellular calcium ions
([Ca
2
]
i
) (4), and changes in actin lament poly-
merization (63), which implies a fundamental role
for their modulation of subsequent intracellular
events. An increase in calcium-channel or nonspec-
ic cation-channel conductance would permit a
rapid elevation in [Ca
2
]
i
due to ion inux down a
strong electrochemical gradient. While earlier
studies investigating the mechanisms of physical
force transduction in periodontal tissues concen-
trated on the role of piezoelectric charges, the vas-
culature, cytokines and inammatory mediators in
regulating the response of bone cells and broblasts
to mechanical forces, more recent studies have in-
vestigated the ability of these cell types to respond
directly to membrane perturbation. In periodontal
68
ligament broblasts, stretch of the cell membrane
induced by hypoosmolar cell volume increase can
activate stretch activated calcium permeable ion
channels, leading to an inux of calcium ions (12).
The inux of calcium ions can then strongly induce
other effectors including those proteins that regulate
the cytoskeleton.
The ability of actin laments to rapidly reorganize
in response to diverse external signals has been
demonstrated in cultured stromal cells in vitro. In
relation to physical stimuli, mechanical strain of at-
tached periodontal cells via a exible substrate re-
duces lamentous actin content within 10 seconds,
which is followed by rapid polymerization (63). The
polymerization state of the sub-membrane cortical
actin meshwork may then affect the mobility and
function of cell surface receptors and could also me-
diate stretch-activated cation channel current (70).
Mechanoprotection
The actin-dependent sensory and response elements
of stromal cells that are involved in mechanical sig-
nal transduction are beginning to be claried. To
study the role of actin in mechanotransduction we
have described a collagen-magnetic bead model in
which application of well-dened forces to integrins
induces an immediate (1 second) calcium inux
(28). We used this model to determine the role of
calcium ions and tyrosine-phosphorylation in the
regulation of force-mediated actin assembly and the
resulting change in membrane rigidity (27). Colla-
gen-beads were bound to periodontal cells through
the focal adhesion-associated proteins talin, vincul-
in, a
2
-integrin and b-actin, indicating that force ap-
plication was mediated through cytoskeletal ele-
ments. When force (2 N/m
2
) was applied to collagen
beads, confocal microscopy showed a marked verti-
cal extension of the cell, which was counteracted by
an actin-mediated retraction. Immunoblotting
showed that force application induced F-actin ac-
cumulation at the bead-membrane complex, but
vinculin, talin and a
2
-integrin remained unchanged.
Atomic force microscopy showed that membrane
rigidity increased 6-fold in the vicinity of beads ex-
posed to force. Force also induced tyrosine phos-
phorylation of several cytoplasmic proteins, includ-
ing paxillin. The force-induced actin accumulation
was blocked in cells loaded with the intracellular cal-
cium chelator BAPTA/AM or in cells pre-incubated
with genistein, an inhibitor of tyrosine phosphoryla-
tion. Repeated force application progressively inhib-
ited the amplitude of force-induced calcium ion ux.
As force-induced actin reorganization was depend-
ent on calcium and tyrosine phosphorylation, and
as progressive increases of lamentous actin in the
submembrane cortex were correlated with increased
membrane rigidity and dampened calcium inux,
we suggest that cortical actin regulates stretch-acti-
vated cation permeable channel activity and pro-
vides a desensitization mechanism for cells exposed
to repeated long-term mechanical stimuli. Thus, the
actin response may be cytoprotective since it
counteracts the initial force-mediated membrane ex-
tension and potentially strengthens cytoskeletal in-
tegrity at force-transfer points.
It seems self-evident that to survive in a mechan-
ically active environment, cells must adapt to vari-
ations of applied membrane tension. Part of this ad-
aptation involves sensing increases in extracellular
tension, maintaining contact with extracellular ma-
trix ligands and preventing irreversible membrane
disruptions. Again with the use of the collagen-
coated magnetic bead model to apply forces directly
to the actin cytoskeleton through integrin receptors,
we investigated how the cytoskeleton reorganizes in
response to increased membrane tension. We found
that by a calcium-dependent mechanism, human
periodontal broblasts reinforce locally their con-
nection with extracellular adhesion sites (collagen-
coated beads) by recruiting actin binding protein-
280 into the cortical adhesion complexes (29). Actin
binding protein-280 was phosphorylated on serine
residues as a result of force application. This phos-
phorylation and the force-induced actin reorganiza-
tion were inhibited by bisindoylmaleimide, indi-
cating a role for protein kinase C isoforms. In a hu-
man myeloma cell line that does not express actin
binding protein-280, actin accumulation could not
be induced by force, while in stable transfectants ex-
pressing actin binding protein-280, force induced
actin accumulation similarly to human broblasts.
Cortical actin assembly evidently played an import-
ant role in regulating the activity of stretch-activated,
calcium permeable channels since sustained force
application desensitized these channels to sub-
sequent force applications and the decrease in
stretch sensitivity was reversed after treatment with
cytochalasin D. Further, in comparison to actin
binding protein-280-positive cells, actin binding
protein-280-decient cells exhibited an almost two-
fold increase in stretch-activated channel activity
and signicantly less (50% compared to 90% in actin
binding protein-280-positive cells) channel desensit-
ization following prolonged force application. Actin
69
binding protein-280-decient cells showed a 90%
increase in cell death compared to actin binding
protein-280-positive cells (30% increase) after force
application, indicating a potential mechanoprotec-
tive role for force-induced actin binding protein-
280/actin reorganization. We suggest that actin bind-
ing protein-280 plays an important role in mechano-
protection by: 1) reinforcing the membrane cortex
and thereby preventing force-induced membrane
disruption; 2) increasing the strength of cytoskeletal
links to the extracellular matrix; and 3) desensitizing
stretch activated ion channel activity.
Conclusions
Collectively, these data, which are largely from in vi-
tro investigations, indicate that stromal cells, and in
particular the broblasts and osteoblasts that popu-
late the periodontal ligament, have the necessary
signaling and effector mechanisms to both sense ap-
plied physical force and to mount a stream of re-
sponses which serve to maintain periodontal liga-
ment width and preserve cell viability. In the in-
stance of the periodontal ligament and the alveolar
bone these cellular characteristics have an important
consequence: the periodontal ligament is an abso-
lute requirement for rapid remodeling of alveolar
bone when physical forces are applied to teeth. This
requirement may be critically important for the
maintenance of alveolar bone volume following
tooth extraction since the loss of the periodontal
ligament terminates the mechanotransduction sig-
nals required for bone homeostasis.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Development and general
structure of the periodontium
MOON-IL CHO & PHILIAS R. GARANT
The periodontium is a collective term describing
tooth supporting and investing tissues including the
root cementum, periodontal ligament, alveolar bone
and gingiva. These periodontal tissues develop and
function as a unit (60, 114). The majority of peri-
odontal tissues develop along with the formation of
the roots of teeth and tooth eruption and have an
origin from the dental follicle that is derived from
neural crest. This chapter discusses the development
of these tissues by emphasizing the origin and lin-
eage of the cells responsible for formation of their
structural components.
Development of the dental follicle
During the past decade, developmental biologists
have made numerous studies of gene expression in
tooth formation. Most of these studies were focused
on the earliest stages of tooth bud development,
with emphasis on the interaction between the en-
amel organ and the dental papilla. Less attention has
been given to the role of gene activation and growth
factor regulation in the development of the peri-
odontium. Information on gene expression in the
dental follicle and the developing periodontium may
be obtained in the Web site http://honeybee
.helsinki./toothexp.
Following the development of the neural tube by
invagination of the overlying ectoderm, migratory
pluripotent neuroepithelial cells, the neural crest
cells, migrate from the dorsal midline region of the
neural tube to invade the developing branchial arch-
es (22). In exiting from the neural tube, neural crest
cells lose their epithelioid nature and assume a mes-
enchymal phenotype capable of directed cell mi-
gration. By tracing the movement of dye-injected
neural crest cells in organ cultures of developing
dental arches, it was shown that neural crest cells
from the posterior midbrain, and to a lesser extent
from the anterior hindbrain, formed dental ectome-
9
senchyme (25, 61). The failure of the normal mi-
gration of neural crest ectomesenchymal cells to ap-
propriate sites during craniofacial development
leads to serious developmental defects, including
the absence of teeth (anodontia) and underdevel-
oped jawbones (micrognathia). Neural crest ectome-
senchymal cells interact in a series of reciprocal in-
ductive interactions with early oral epithelium to
form tooth primordia (81). Subsets of cranial neural
crest cells give rise to chondrocytes, osteoblasts,
periodontal ligament broblasts, cementoblasts and
odontoblasts. Final phenotype differentiation is
regulated by interaction of the ectomesenchymal
cells with extrinsic factors, such as growth factors, in
the local microenvironment (69).
The dental papilla and dental follicle, the non-ec-
todermal components of the tooth buds, are formed
by concentration of neural crest ectomesenchymal
cells (Fig. 1). Schroeder (114) and Moxham & Grant
(99) have reviewed the classical descriptive and ex-
perimental studies that described the development,
histology and the fate of the dental follicle in form-
Fig. 1. Diagrammatic view of a developing tooth at the cap
stage
Cho & Garant
ing the tissues of the periodontium. It has been sug-
gested that the interaction between cell surface syn-
decan and tenascin, an extracellular matrix adhesion
molecule, reduces migration and promotes aggre-
gation of the ectomesenchymal cells to form the
dental papilla and dental follicle (136, 140, 141).
During tooth development, the dental papilla
gives rise to odontoblasts and the dental pulp, while
the dental follicle gives rise to cementum, peri-
odontal ligament and alveolar bone. Anatomically,
the dental follicle consists of the dental follicle
proper, a rather well-dened band of cells juxta-
posed to the dental papilla and the convex outer sur-
face of the enamel organ; and the perifollicular mes-
enchyme, a more loosely dened population of cells
bordering the developing bony trabeculae which
partly surround the tooth bud. A poorly populated
zone of loose connective tissue separates these
layers. The loose connective tissue interface creates
a natural cleavage plane during tooth bud extir-
pation; the dental follicle proper remains attached to
the tooth bud, while the perifollicular mesenchyme
remains associated to the bony trabeculae.
The developmental potential of the dental follicle
was studied in numerous tooth transplantation ex-
periments. Hoffman demonstrated the formation of
all elements of the periodontium following tooth
bud transplantation to subcutaneous sites (60). Al-
though he was quite certain that cementum and
periodontal ligament originated from the trans-
planted tissue, he was less certain about the origin
of the surrounding bone. He speculated that bone
cells might have been induced in host cells by the
epithelial components of the tooth bud. Ten Cate et
al. studied the fate of transplanted tooth buds pre-
viously labeled with tritiated thymidine (132). Ce-
mentoblasts and periodontal ligament broblasts in
the developing tooth were clearly labeled, indicating
their origin from the transplanted tooth bud. Since
osteoblasts were only weakly labeled, their origin
was less certain. Ten Cate et al. noted that since only
the dental follicle proper was transplanted along
with the tooth bud, it must be the source of progeni-
tor cells for cementum, alveolar bone and peri-
odontal ligament broblasts (132, 133). Palmer &
Lumsden conrmed these results but cautioned that
it was difcult to exclude cellular contamination
from the perifollicular mesenchyme (104).
The role of the dental follicle was claried by
studies of the development of tooth buds trans-
planted to sites known to have an inability to form
mineralized tissue. When tooth buds were trans-
planted into the anterior chamber of the eye, root
10
formation with cementum and alveolar bone forma-
tion were noted (104, 155). These and other trans-
plantation experiments led to the conclusion that
the tooth bud developed as a biological unit, capable
of giving rise to all components of the mature tooth.
Yoshikawa & Kollar (155) also demonstrated that the
dental papilla and the dental follicle had similar de-
velopmental potentials, and could be substituted for
one another in reconstituting a fully developed
tooth.
Despite the fact that the dental follicle proper
contains all the precursors needed for cementum,
bone and periodontal ligament formation, migrating
broblasts from the perifollicular mesenchyme pro-
liferate during root development to contribute to the
pool of periodontal ligament broblasts. The perifol-
licular mesenchyme and perivascular cells may also
give rise to osteoblasts of the alveolar bone.
Cementum
Types of cementum
Cementum is an avascular mineralized tissue cover-
ing the entire root surface. It forms the interface be-
tween root dentin and the periodontal ligament. Tra-
ditionally, cementum has been classied as cellular
and acellular cementum depending on the presence
and absence of cementocytes in cementum, further
grouped into intrinsic and extrinsic ber cementum
depending on the presence of collagen bers formed
by cementoblasts or by broblasts, respectively (63,
115). Acellular abrillar cementum is located over
cervical enamel at the cementoenamel junction (77,
79, 115, 119). Its major structural components are
glycosaminoglycans (115) and its functional signi-
cance is unknown. Cellular intrinsic ber cementum
contains cementocytes embedded in a collagenous
matrix of intrinsic collagen bers. These collagen
bers are oriented mostly parallel to the root surface
and course in a circular fashion around the root
(115). Cellular intrinsic ber cementum is found in
old resorption lacunae and in root fracture sites. Cel-
lular mixed stratied cementum is located primarily
on the apical one third of the root and in the fur-
cation area of multirooted teeth. It is composed of
alternating layers of acellular extrinsic ber ce-
mentum and cellular intrinsic ber cementum/acel-
lular intrinsic ber cementum, and is covered by a
thin layer of acellular extrinsic ber cementum for
attachment to the periodontal ligament (115). Cellu-
lar mixed stratied cementum serves to reshape the
root surface in order to compensate for physiological
Development and general structure of the periodontium
Fig. 2. A. Light micrograph of the developing root of the
rst maxillary rat molar which shows the dental follicle
proper (DFP) (I) and precementoblast (II) stages. The dot-
ted line separates the areas showing these two stages. At
the cervical end of the developing root, the cells of both
the inner (i) and outer (o) epithelial layers of Hertwigs
root sheath are intact and arranged parallel to one an-
other. Adjacent to the outer layer of the root sheath, there
are several layers of elongated DFP cells, whose long axis
is parallel to the root sheath epithelial cells. Note the per-
ifollicular mesenchymal cells (PFM) with a stellate shape
in the PF area bordered by the DFP and the alveolar bone
(AB). Also, note precementoblasts (arrowheads) that in-
vade and push their way toward the newly formed preden-
tin surface. The outer and inner epithelial cells of the root
11
drift and nonphysiological shifting of teeth in their
alveolar sockets (21, 115, 118). Acellular extrinsic
ber cementum covers 40% to 70% of the root sur-
face and is comprised of collagen bers and glycosa-
minoglycans. It serves the exclusive function of an-
choring the root to the periodontal ligament. The
monograph of H.E. Schroeder should be consulted
for the further information on the classical histologi-
cal and ultrastructural aspects of cementum (115).
Since information on the development of acellular
afbrillar cementum, cellular intrinsic ber ce-
mentum and cellular mixed stratied cementum is
limited, our discussion of cementogenesis will focus
on acellular extrinsic ber cementum formation, a
topic of resurgent study.
Acellular extrinsic ber cementum formation in
animals and humans
Animals. Formation of acellular extrinsic ber ce-
mentum has been studied during root formation in
rodents (27, 31, 45, 72, 8285, 102, 120, 130, 151,
152), dogs and humans (12, 19, 101, 103, 116, 121).
Such studies have informed us about the develop-
ment, biological potential, microanatomy and
physiological responsiveness of cementum in ani-
mals. Not all of the knowledge gained from such
studies is applicable to humans (20, 21, 118). But,
based upon what we have learned from other organ
systems, it is reasonable to expect that there is a sig-
nicant carry-over. Apparent differences may be-
come resolved as we come to a better understanding
of the molecular events in cementogenesis.
Cementoblast differentiation and cementogenesis
are closely related with root formation. Previous
studies have noted two different mesenchymal cell
populations adjacent to the apical end of developing
roots (Fig. 2, 3). There are the cells of the dental fol-
licle proper, located nearest to the odontogenic cells
and developing dentin surface, and the cells of per-
sheath become discontinuous as the cells of the DFP in-
vade. B. Light micrograph showing cementoblasts (III)
and postcementoblast (IV) stages. Note fully differentiated
cementoblasts (C) on the dentin surface (D) and postce-
mentoblastic cells (arrowheads) migrating away from the
dentin surface. Cementoblasts are characterized by an
oval shape and a high degree of cellular polarization to-
ward the dentin surface. They are more intensely stained
with toluidine blue than PDL broblasts. Post cemento-
blastic broblasts are elongated in shape and maintained
an intensely stained. The dotted line separates the areas
occupied by these cell types. Od: odontoblast. PD: Pre-
dentin.
Cho & Garant
Fig. 3. Electron micrograph showing Hertwigs root sheath DFP have a close and parallel arrangement to the outer
at the cervical end of the developing root. Note both the epithelial cells. In contrast, the mesenchymal cells (thin
inner basal lamina (thick arrows) under the inner epi- arrows) in the PF area located next to the cells of DFP are
thelial cells (IE) and external basal lamina (arrowheads) randomly oriented. Pod: Preodontoblast.
adjacent to the outer epithelial cells (OE). Cells (*) in the
ifollicular area, located more peripherally (47, 72,
102, 103, 120).
The most apical portion of the developing root
contains an intact epithelial root sheath, located be-
tween the preodontoblasts of the dental papilla and
the dental follicle proper (Fig. 2, 3). The inner and
outer epithelial cells of the Hertwigs root sheath are
closely parallel to one another with a minimum of
intervening intercellular space. The internal basal
lamina separates the root sheath cells from the pre-
odontoblasts, while the external basal lamina separ-
ates them from the cells of the dental follicle proper.
Elongated broblast-like dental follicle proper cells
are oriented parallel to the external basal lamina,
which forms a continuous intact surface all along the
outer root sheath cells. These follicle cells have long
cytoplasmic processes, inactive Golgi complexes,
numerous free ribosomes, and a small number of
rough endoplasmic reticulum cisternae. The cells of
the perifollicular area are stellate-shaped and ran-
domly oriented. In contrast to the dental follicle
12
proper, the cells of the perifollicular area were rather
widely separated.
The cells of dental follicle proper project cytoplas-
mic processes from their leading edge towards and
eventually into the intercellular space between the
root sheath cells. The dental follicle proper cells ap-
pear to invade the intercellular spaces of the root
sheath. These cells are identied as precemento-
blasts on the basis of their enlarged cell bodies that
contain numerous proles of rough endoplasmic
reticulum, lysosomes and a moderately developed
Golgi complex (26, 27) and by their ability to syn-
thesize matrix components (27). The unidirectional
migration of precementoblasts towards the preden-
tin surface appears to contribute to the break up of
the root sheath and the formation of Sharpeys
bers. Precementoblasts synthesize and deposit col-
lagen brils while moving towards the predentin sur-
face, thereby establishing Sharpeys bers (27, 31).
Upon contact with the predentin surface, the
elongated precementoblasts become cuboidal in
shape and differentiate into cementoblasts. They are
characterized by a well-developed Golgi complex po-
larized toward the root surface, and by numerous
cisternae of rough endoplasmic reticulum (27, 31).
The cells responsible for depositing the rst layer of
acellular extrinsic ber cementum exhibit a high
level of basophilia consistent with a well-developed
rough endoplasmic reticulum (27). A high level of al-
kaline phosphatase (10, 56) also characterizes these
cells. Specic collagen secretory granules are formed
in a large and conspicuous Golgi complex. Acellular
extrinsic ber cementum matrix secretory activity
has been documented by electron microscopic auto-
radiography of tritiated mannose utilization as an in-
dicator of glycoprotein synthesis during acellular ex-
trinsic ber cementum formation in rat molars (27).
Following a brief period of cementogenesis, the
cementoblasts appear to detach from the newly
formed cementum surface and join the broblast
population in the periodontal ligament. During this
process, cementoblasts lose their cuboidal shape
and assume a more elongated shape. Thus, during
root formation, cementoblasts originate primarily
from the cells of dental follicle proper, and ce-
mentogenic cells become a part of the periodontal
ligament broblast population (27, 31).
Human. In human acellular extrinsic ber ce-
mentum formation, Hertwigs epithelial root sheath
does not remain in contact with the root surface fol-
lowing odontoblast differentiation (18, 20, 27, 31,
117). Hertwigs epithelial root sheath detaches from
the dentin surface very close to the apical edge of
the developing root. After the detachment and disin-
tegration of Hertwigs epithelial root sheath, acellular
extrinsic ber cementum forms at the growing root
tip when broblasts of the dental follicle make con-
tact with the predentin matrix. According to Bossh-
ardt & Schroeder, broblasts secreting in a unipolar
direction deposit and bundle collagen brils at the
dentin surface to form a thin layer of perpendicu-
larly oriented fringe bers (18). The collagen brils
of the fringe bers appear to interdigitate and there-
by become linked with the unmineralized dentin
collagen bers at the dentinocemental junction.
Acellular extrinsic ber cementum-forming cells
have sheath-like cytoplasmic processes that delin-
eate extracellular compartments within which the
fringe bers are assembled (17, 18, 152). Acellular ex-
trinsic ber cementum formation proceeds along
the developing root at a rate of about 5 to 7 mm per
day, requiring 43 to 65 months for completion in hu-
man premolars (117). As the mineralization front ad-
13
vances to reach of the most peripheral mantle den-
tin, it contacts the fringe bers and they undergo
slow mineralization to complete the process of acel-
lular extrinsic ber cementum formation. The rst
evidence of mineralization in the fringe bers ap-
pears in the central core of each ber bundle, pre-
sumably by epitaxy from the mineralized dentin (18).
With time, the mineralization spreads across the en-
tire width of the fringe bers, and the resulting uni-
form mineralization front subsequently advances in
proportion to the growth of the acellular extrinsic
ber cementum. Whether or not the acellular extrin-
sic ber cementumforming broblasts deposit
special glycoproteins and/or glycosaminoglycans
needed for the supramolecular organization of colla-
gen bers, or for supporting mineralization, remains
to be established. After the fringe bers reach a
length of about 20 mm they become associated and
continuous with the principal bers developing in
the periodontal ligament (117). During the life of the
tooth, the acellular extrinsic ber cementum con-
tinues to grow in thickness at a slow rate of 1.5 to
3.0 mm per year. Closely spaced incremental (ce-
ment) lines suggest that the growth of acellular ex-
trinsic ber cementum is episodic. Presumably the
periodontal ligament cells adjacent to the root sur-
face respond to appropriate environmental signals
calling for an increase in acellular extrinsic ber ce-
mentum matrix and its mineralization.
When root development is about two thirds com-
pleted and the tooth is about to enter its functional
stage, cementum formation converts from acellular
extrinsic ber cementum to a cellular mixed strati-
ed cementum (cellular intrinsic ber cementum
and acellular intrinsic ber cementum) type (18, 21).
The conditions and factors responsible for this tran-
sition are unknown. The formation of cellular intrin-
sic ber cementum closely resembles bone forma-
tion. Cementoblasts and cementocytes are involved
in the secretion of intrinsic bers (in contrast to the
extrinsic bers that are a product of periodontal liga-
ment broblasts). The rate of apposition of cellular
mixed stratied cementum (about 0.1 to 0.5 mm per
day) is less than that of bone (13). The intrinsic colla-
gen bers are assembled in bundles that follow a
spiral course along and around the root. These bers
are best observed in scanning electron micrographs
of the root surface (114).
Mature cementoblasts are relatively large cells
with a highly basophilic cytoplasm. During cellular
intrinsic ber cementum formation, they secrete in
a relatively rapid multipolar mode and become en-
trapped in the matrix as cementocytes (16, 114, 117,
Cho & Garant
153, 154). Slow matrix deposition is thought to occur
in a unipolar fashion during acellular intrinsic ber
cementum formation, permitting the cementoblasts
to escape entombment in the matrix. Cementoblasts
share similar morphological features with osteo-
blasts, suggesting that these two cell types might
originate from a common progenitor pool located in
the periodontal ligament and the marrow spaces of
the adjacent alveolar bone.
Cementoblasts express parathyroid hormone re-
ceptors, but unlike osteoblasts and bone-lining cells,
they do not retract in response to parathyroid hor-
mone to expose the root surface to preosteoclasts
(73). Also, rat cementoblasts (like osteoblasts) and
their precursors express growth hormone receptors
(157). Growth hormone receptors are expressed in
precementoblasts adjacent to the Hertwigs epi-
thelial root sheath. Receptor expression increases
during cementum formation and thereafter declines
in cementocytes. Periodontal ligament cells next to
acellular extrinsic ber cementum do not express
growth hormone receptors. Excessive amounts of
growth hormone cause hypercementosis (5). In con-
trast, hypophysectomy leads to reduced amounts of
cellular cementum. In humans, growth hormone de-
ciency causes some teeth to fail to form and others
to undergo delayed eruption.
The role of the epithelial root sheath of Hertwig in
root formation and cementogenesis
Hertwigs epithelial root sheath has an origin from
the inner and outer enamel epithelial cells. Hertwigs
epithelial root sheath consists of a double layer of
epithelial cells continuous with and extending apic-
ally from the apical rim of the enamel organ. At this
stage, the sheath forms a circumferential structure
encompassing pulpal ectomesenchyme, separating
it externally from dental follicular and perifollicular
ectomesenchyme (Fig. 2, 3). Apical growth of Hert-
wigs epithelial root sheath occurs by proliferation of
the epithelial cells of the sheath. Continuity between
the enamel organ and Hertwigs epithelial root
sheath is lost soon after root formation begins. The
apical region of the developing root contains ecto-
mesenchymal progenitor cells that give rise to
broblasts, preodontoblasts and precementoblasts.
The coordinated proliferation of the epithelial and
ectomesenchymal cells at the apical site gives rise
to cells needed for root elongation and mineralized
tissue formation. Preodontoblasts differentiate ad-
jacent to the inner layer of the root sheath and its
basal lamina (Fig. 3). The inner layer of the root
14
sheath appears to perform the same inductive func-
tions attributed to the inner enamel epithelium dur-
ing coronal odontoblast development. Slavkin and
colleagues have reported that Hertwigs epithelial
root sheath secrets polypeptides that are related to,
but different from, enamelin and amelogenin pro-
teins (126, 127, 129). The inductive effects that these
matrix polypeptides may have on adjacent bro-
blasts and precementoblasts have not been estab-
lished. However, an epithelio/mesenchymal interac-
tion between cultured cells of Hertwigs epithelial
root sheath and broblasts has been demonstrated
in vitro (138). When broblasts were grown with cells
of Hertwigs epithelial root sheath they showed in-
creases in rough endoplasmic reticulum, Golgi
membranes and associated secretory granules and
increased secretion of collagen.
The role of Hertwigs epithelial root sheath in root
development and especially as it relates to the initia-
tion of cementogenesis has become a focus of con-
siderable attention (138). Since the epithelial cells of
the inner layer of Hertwigs epithelial root sheath are
analogous to the preameloblasts, it was suggested
early on that they might secrete enamel matrix pro-
teins over the newly deposited root dentin (101, 102,
125, 129, 142). Based on various studies, it is now
generally accepted that there is a transient period
of secretion of proteins, including bone sialoprotein,
osteopontin and amelin by the cells of Hertwigs epi-
thelial root sheath (14, 15, 44, 74). In addition to
these matrix proteins there are also the components
of the epithelial basement membrane, such as lami-
nin and collagen type IV, which are included in the
narrow band of matrix juxtaposed to the dentin ma-
trix. This layer is sometimes identied as intermedi-
ate cementum, a misleading term since the matrix
in question is a product of epithelial and dentinog-
enic cells (102). According to Schroeder (114) and
Bosshardt & Selvig (21), there is no such layer inter-
posed between cementum and dentin in human
teeth. This layer becomes highly mineralized as de-
velopment continues (59). The potential role for
these epithelial matrix molecules in triggering the
differentiation of cells capable of forming acellular
extrinsic ber cementum and cellular intrinsic ber
cementum is a primary question that remains most-
ly unanswered. Yet, the concept that epithelial (en-
amel organ) proteins stimulate cementogenesis has
found clinical application in experimental tissue re-
generation protocols. It has been reported that the
application of hydrophobic amelogenin peptides to
denuded root surfaces promotes new cementum for-
mation (58).
It is generally accepted that the breakup of the in-
ner and outer cell layers of Hertwigs root sheath
along the predentin surface is due to epithelial cell
degeneration (4, 47, 72, 101, 103, 125). In rat molar
root development acellular extrinsic ber cementum
formation occurs only after cells of the adjacent fol-
licular ectomesenchyme invade the Hertwigs epi-
thelial root sheath (102). The follicular cells appear
to migrate to the dentin surface concomitant with
the breakup of the root sheath. In migrating to the
predentin the cells of the dental follicle proper may
be responding to a chemoattractant present in the
dentin matrix or to one produced by the inner epi-
thelial layer of the root sheath.
The fate of Hertwigs epithelial root sheath follow-
ing the onset of cementogenesis is also a subject of
debate that remains unresolved. Traditional thinking
proposed that Hertwigs epithelial root sheath disin-
tegrated into small clusters and/or strands of epi-
thelial cells that survived indenitely in the peri-
odontal ligament. More recent studies have sug-
gested that epithelial cells might undergo epithelial/
mesenchymal transition into broblasts and ce-
mentoblasts that deposit acellular and cellular ce-
mentum respectively (137). The possibility that some
epithelial cells of the root sheath undergo epithelial-
mesenchymal transformation and subsequently se-
crete cementum matrix must be investigated further.
There is evidence that cells of the inner layer of the
root sheath become incorporated in cellular ce-
mentum or trapped between cementum and dentin
during formation of the apical part of the root (1, 46,
72). However, the evidence that many of the cells of
the root sheath retain an epithelial phenotype, and
survive in the periodontal ligament as the epithelial
rests of Malassez, is incontrovertible (114, 143).
Periodontal ligament collagen ber attachment to
the root surface: implications of cell migration and
cytoplasmic polarization
The attachment of the principal bers of the peri-
odontal ligament to the root surface provides an in-
formative example of the role that cells play in or-
ganizing and orienting extracellular bers into func-
tional networks.
Prior to the onset of cementogenesis, the dental
follicle proper cells nearest to the root sheath are
aligned parallel to the epithelial cells (26, 152). Colla-
gen bundles that lie parallel to the root sheath are
partly enveloped in cytoplasmic grooves formed by
the dental follicle proper cells. Cytoplasmic microtu-
bules and collagen secretory granules are oriented in
15
the same direction as the extracellular collagen
bers. With the onset of the disruption of the root
sheath, the dental follicle proper cells assume an
elongated prole with polarity toward the dentin
matrix. The cells appear to move toward the dentin
in the spaces created by the disruption of the root
sheath. During shifting of the dental follicle proper
cells the collagen bundles that were initially parallel
to the root sheath are reorganized, so that they come
to lie in the lateral intercellular between the dental
follicle proper cells, oriented perpendicular to the
root surface (26, 152). Many small cytoplasmic pro-
cesses extend from the leading edge of the dental
follicle proper cells. Collagen brils secreted from
these leading edge processes intermingle with the
dentin matrix collagen. Although many of the small
collagen bers appear to have no preferred orien-
tation, most are aligned perpendicular to the root by
the microtubule-secretory-granule apparatus in the
cytoplasmic processes (152).
In a later stage of acellular extrinsic ber ce-
mentum formation, broblasts (or dental follicle
proper cells) extend thin cytoplasmic sheets that
partially surround the developing fringe bers, or
Sharpeys bers. These sheets or veils of cytoplasm
are best developed on the part of the cell nearest the
periodontal ligament. Examination of the cytoskel-
eton in the cytoplasmic sheets reveals that the
microtubules and collagen secretory granules are
aligned mostly parallel to the fringe bers, indicating
that fringe bers grow in circumference by secretion
from the surface of cytoplasmic sheets (152). In con-
trast, small cytoplasmic processes that give rise to
intrinsic bers characterize the end of the cell near
the dentin (or the previously deposited cementum).
In the early development of cellular intrinsic ber
cementum, cementoblasts appear to deposit ber
bundles parallel to the surface of the root. Subse-
quently, the cementoblasts engage in binding these
bers with smaller intrinsic bers deposited from
cytoplasmic processes extending from the end of the
cell facing the dentin. Transmission electron micro-
scopic analysis of the small cell processes show that
microtubules align collagen secretory granules par-
allel to the developing intrinsic bers. In the forma-
tion of cellular intrinsic ber cementum, these ce-
mentoblasts are eventually surrounded by matrix as
new waves of cementoblasts differentiate at the ce-
mental surface.
Although the full story has yet to be developed,
preliminary evidence suggests that cells orient newly
deposited collagen by aligning secretory granules
parallel to microtubules within the cytoplasmic pro-
Cho & Garant
cesses and sheets that demarcate extracellular
microcompartments (11, 29). Cell migration and/or
movements of cell processes, controlled by the cyto-
plasmic actin contractile network, could also play a
role in organizing the bers of the extracellular ma-
trix. This has been observed in developing tendons
as well as in the periodontal ligament. The cell sur-
face receptors and cytoplasmic signaling steps that
control the ow of cell membrane components and
secretory granules to the cell surface and the sub-
sequent cell/matrix interactions needed to construct
the brous architecture of a specic tissue are un-
doubtedly complex.
Periodontal ligament
Development of the periodontal ligament
The development of the periodontal ligament begins
with root formation prior to tooth eruption. The
continuous proliferation of the inner and external
enamel epithelium forms the cervical loop of the
tooth bud. This sheath of epithelial cells grows apic-
ally, in the form of Hertwigs epithelial root sheath,
between the dental papilla and the dental follicle.
At this stage, the sheath forms a circumferential
structure encompassing dental papilla separating it
externally from dental follicle cells. The dental fol-
licle cells, located between the alveolar bone and the
epithelial root sheath, are composed of two sub-
populations with distinct morphological character-
istics and locations; mesenchymal cells of the dental
follicle proper and the perifollicular mesenchyme
perifollicular mesenchyme (Fig. 2). The morpholog-
ical features and their differentiation into cemento-
blasts during acellular extrinsic ber cementum for-
mation were discussed earlier. The mesenchymal
cells of the perifollicular mesenchyme bounded by
the dental follicle proper and the developing alveolar
bone are stellate-shaped, small, and randomly
oriented. In contrast to the dental follicle proper, the
cells of the perifollicular mesenchyme are rather
widely separated. Ultrastructural study of these cells
indicates that they contain an euchromatic nucleus
and small cytoplasm that contains a small number
of short cisternae of rough endoplasmic reticulum,
mitochondria, free ribosomes, and an inactive Golgi
area. These cells also have several long and thin
cytoplasmic processes that connect with those from
neighboring cells. A small number of short collagen
brils are located close to the cell surface (27, 31,
47). As the root formation continues, cells in the per-
ifollicular area gain their polarity, increased cellular
16
volume and synthetic activity. These cells become
elongated and contain increased numbers of rough
endoplasmic reticulum and mitochondria and an ac-
tive Golgi complex. As a result, they actively synthes-
ize and deposit collagen brils and glycoproteins in
the developing periodontal ligament (27, 31, 47).
The developing periodontal ligament, as well as
the mature periodontal ligament, contains undiffer-
entiated stem cells that retain the potential to differ-
entiate into osteoblasts, cementoblasts and bro-
blasts (90, 91, 93). Experimental studies suggest that
stem cells occupy perivascular sites in the peri-
odontal ligament and in adjacent endosteal spaces
(54, 71, 93). Stem cell progeny undergo further matu-
ration during migration to the bone and cemental
surfaces (92, 110). Whether or not osteoblasts, ce-
mentoblasts and broblasts originate from a com-
mon ancestor or from a specic line of progenitor
cells remains to be claried.
Development of the principal bers
Development of the major collagen bundles, the
principal bers of the periodontal ligament, is
closely correlated to root formation. Fiber bundles
originate at the surface of the newly formed root
dentin in close relation to elongated and highly po-
larized broblasts. These nascent ber bundles
(fringe bers) are tightly packed (bundled) by the ac-
tion of cementoblasts during the initial development
of acellular extrinsic ber cementum. During tooth
eruption, as the periodontal ligament matures, the
fringe bers merge across the width of the ligament
to form the principal ber bundles. In the middle of
the periodontal ligament the collagen ber bundles
are less tightly packed. In general, the majority of the
principal bers course in a coronal direction from
cementum to bone, forming the oblique ber group.
During the development of the fringe bers, bro-
blasts exhibit cytoplasmic polarity toward the root
and alveolar bone surfaces respectively. The ultra-
structural appearance of these cells is consistent
with directed cell migration toward each of these
surfaces, concurrent with the deposition of a colla-
gen and proteoglycan-rich extracellular matrix (26).
A specic cementum attachment protein may favor
periodontal ligament broblast attachment to the
cementum surface (107).
With continued development of the root, major
collagen bundles, the principal bers, are estab-
lished as continuous structures embedded as Shar-
peys bers in bone and cementum. Sloan & Carter
have reviewed the subject of the structural organiza-
tion of the bers of the ligament (128). In histological
sections the following distinct groups of principal
bers are noted: dentogingival, alveolar crest, trans-
septal, interradicular, horizontal, oblique and apical
ber bundles. The oblique bers, occupying nearly
two thirds of the ligament, are inserted into bone
coronal to their insertion into cementum. This geo-
metric arrangement of the oblique bers is ideally
suited to absorb intrusive forces generated during
mastication. In order to attach the tooth in its al-
veolus, the bers must be embedded in mineralized
bone and cementum. A nonbrillar matrix that
stains with ruthenium red appears to cement the
terminus of the fringe bers (Sharpeys bers) at
their insertion in newly formed acellular extrinsic
ber cementum and bone, and in the case of fully
developed specimens on a reversal line deep within
cementum or bone. Immunocytochemical studies
have shown that osteopontin is a signicant compo-
nent of the matrix of the reversal line. The mature
periodontal ligament can be subdivided into three
regions (Fig. 4): a) a bone-related region, rich in cells
and blood vessels, b) a cementum-related region
characterized by dense well-ordered collagen
bundles, and c) a middle zone containing fewer cells
and thinner collagen brils (128).
Cellular components
Fibroblasts are the most abundant cells in the peri-
odontal ligament, and are responsible for met-
abolism of extracellular matrix components. The
periodontal ligament is known to have heterogen-
eous population of broblasts. A subpopulation of
osteoblast-like broblasts, rich in alkaline phospha-
tase, has been identied in the periodontal ligament
(34, 52, 65, 80). These cells have the capacity to give
rise to bone cells and cementoblasts. They are also
responsible for the production of acellular extrinsic
ber cementum in the mature periodontal ligament
(56, 57). Periodontal ligament broblasts are also
needed to maintain the normal width of the peri-
odontal ligament by preventing the encroachment of
bone and cementum into the periodontal ligament
space (94, 95). The factors responsible for this activ-
ity have yet to be identied.
Morphology of periodontal ligament broblasts
Mechanical strain applied to periodontal ligament
broblasts appears to determine their shape, syn-
thetic activity and adhesive interaction with the sur-
rounding extracellular matrix. Most periodontal liga-
17
Fig. 4. Histological section showing the periodontal liga-
ment (PDL), dentin (D), cementum (C) and alveolar bone
(AB)
ment broblasts are highly active cells, exhibiting an
elongated well-polarized cytoplasm with extensive
areas of contact to collagen bers (8, 49). These
broblasts contain large amounts of rough endo-
plasmic reticulum and well-developed Golgi com-
plexes, indicative of a high rate of protein synthesis
(7, 29). The Golgi complex of the periodontal liga-
ment broblast contains several Golgi stacks com-
prised of cisternae and terminal saccules. Each Golgi
stack is made up of ve cisternae, about 2 mm in
length, terminating at each end in an expanded sac-
cule (30). Immature cisternae at the cis surface of
the Golgi complex are slightly dilated and in routine
preparations devoid of any stainable content. The
saccules associated to these cisternae contain ne,
loosely arranged laments. The cisternae of the trans
surface contain dense material and their associated
saccules contain rod-like structures with globular
terminal elements, resembling segment-long-spa-
cing collagen aggregates. These saccules are released
to form presecretory granules that quickly associate
to microtubules (29). Autoradiographic and bio-
chemical studies have conrmed a high rate of pro-
tein secretion in the periodontal ligament (29, 66).
Proline is incorporated into collagen polypeptides in
Cho & Garant
the rough endoplasmic reticulum of periodontal
ligament broblasts within minutes of its exit from
the bloodstream. At 10 minutes, newly synthesized
procollagen molecules are present inside Golgi ves-
icles, and by 20 minutes are ready for secretion
within secretory granules associated with microtu-
bules. In less than 30 minutes, newly synthesized
collagen brils are present in the immediate extra-
cellular vicinity of broblasts (29). At 60 minutes,
newly secreted collagen brils are heavily labeled
with tritiated proline. An intact microtubular net-
work is required for movement of collagen secretion
granules from the trans-Golgi network to the se-
cretory pole of the cell (29, 30). During trans-
migration, the secretory pole of the periodontal liga-
ment broblast is also its leading edge.
Polarization of cellular organelles
The anatomical and functional polarization of
broblasts in the periodontal ligament was clearly
established in animals fed a diet containing beta-
aminopropionitrile, a substance that blocks the for-
mation of intermolecular cross-links in the collagen
molecules. Autoradiographic analysis of collagen se-
cretion in the periodontal ligament of these animals
showed that secretion of new (-labeled) collagen oc-
curred from the end of the cell that was also the
leading edge. The secretion of extracellular matrix at
the leading edge of a transmigrating cell may have
signicance in modeling the construction of three-
dimensional ber networks. Chemotactic migration
of matrix forming cells toward bone and cementum
could establish the orientation of fringe-bers dur-
ing periodontal ligament development and sub-
sequent repair. The presence of actin networks and
stress bers endow the periodontal ligament bro-
blast with a high degree of contractility, with which
it can exert tractional forces on the extracellular ma-
trix (2, 43, 51). Highly developed stress bers have
been described in broblasts of the transseptal bers
(50).
Intracellular collagen brils
The broblast is not only responsible for the forma-
tion of collagen ber networks but also in the re-
moval of collagen brils (6, 42, 48, 96). With the ad-
vent of electron microscopy, striated collagen brils
were observed inside vesicles of broblasts, particu-
larly abundant in the periodontal ligament bro-
blasts (48, 78, 96). Localization of acid phosphatase
inside the same vesicles that contained intracellular
18
collagen brils gave added support to the idea that
broblasts were involved in lysosomal digestion of
collagen brils (39, 48). In vitro studies have demon-
strated that broblasts are eminently capable of
phagocytozing collagen brils from the extracellular
environment and degrading them inside phagolyso-
mal bodies (9, 131). Additional study of this activity
indicates that collagenase (matrix metalloprotein-
ase-1) is not involved in the intracellular phase of
the degradation of collagen brils (41). Lysosomal
cysteine proteinases (cathepsins B, L and N) of the
lysosomal granules are capable of rapid degradation
of internalized collagen brils. It has been suggested
that cell surface matrix metalloproteinases and inte-
grin collagen receptors localized in phagocytic clefts
may regulate the initial steps in bril internalization
(70). However, in our previous studies, we observed
that secretory granules formed in the Golgi complex
are translocated to the periphery of periodontal liga-
ment broblasts on microtubules prior to their fu-
sion with the cell membrane. In some cases, several
secretory granules translocated on the same micro-
tubule fuse together and undergo polymerization in-
tracellularly. These observations strongly suggest a
possibility that some intracellular collagen brils
may represent those in the process of secretion
rather than phagocytosed ones (29, 117).
Fibroblast-to-matrix adhesion and traction
Fibroblasts attach to the substratum of the extracel-
lular matrix via surface receptors for collagen and
bronectin. Attachment to the substratum is essen-
tial for cell migration and for organization of the
extracellular brillar matrix. The focal adhesion and
its mature form, the bronexus, has received a great
deal of attention over the past decade (37, 124). In
the formation of these adherent contacts, the cell
membrane integrin a5b1 attaches to the RGD se-
quence (arginine-glycine-aspartic acid) of bronec-
tin (97). Fibronectin molecules can polymerize to
form pericellular matrices. Assembly is initiated by
binding soluble bronectin molecules to cell surface
integrin receptors (a5b1 and avb3) (98, 149). The
cytoplasmic domain of the integrin receptor attaches
to the peripheral cytoplasmic protein, talin, which in
turn interacts with a protein called vinculin. Confor-
mational changes in vinculin cause it to bind to actin
microlaments in the cortical cytoplasm, thereby
completing a molecular bridge between the cells
contractile apparatus and bronectin in the extracel-
lular matrix (3). With the binding of bronectin to
collagen brils, the molecular linkage extends from
the cytoplasmic contractile apparatus to an extracel-
lular collagen ber network, establishing a mechan-
ism for exerting traction on the collagen bers.
Through such cell-to-matrix contacts, the extracellu-
lar matrix can exert an effect on cell shape and be-
havior. Tension in the extracellular matrix is trans-
mitted to broblast integrin receptors, leading to sig-
naling events that alter the activity of the cell.
Human periodontal ligament broblasts respond to
increased tension by upregulating the expression of
interleukin-1a and interleukin-1b (38) and by secret-
ing prostaglandin E
2
(150). In this outside-in type
of signaling, tension transmitted to the broblast
causes a rise in the activity of several small gua-
nosine triphosphatases, which regulate the enzyme
cascades that lead to changes in cell shape and func-
tion. Cells can also alter the binding strength of their
integrin receptors through cytoplasmic signaling
pathways. This represents a form of inside-out signal
transduction (100). The linkage between the cell sur-
face and the immediate extracellular matrix serves
as a node whereby the cell can receive regulatory in-
formation from the outside, as well as providing a
mechanism for exerting an organizing inuence on
the adjacent matrix (64, 89, 112, 156).
On highly mobile broblasts these receptors are
diffusely distributed, while in stationary cells they
are arranged in linear arrays, codistributed with
cytoplasmic actin and extracellular bronectin ag-
gregates. Immunocytochemical localization of
bronectin in the periodontal ligament has shown
that it forms aggregates about 90 nm thick on the
surface of broblasts (32). These aggregates are
usually codistributed with intracellular components
of the bronexus contact (106).
Morphology and roles of undifferentiated cells
Progenitor broblasts are smaller, less polarized, and
contain less rough endoplasmic reticulum and Golgi
saccules (53). Exceptions are noted around blood
vessels and near the surface of the cementum where
broblast-like cells appear smaller and less active.
Cells of the osteoblastic subtype can be identied
by their high level of alkaline phosphatase and by
their ability to bind a newly discovered cementum
attachment protein (80). Cells of this subtype pro-
duce mineralized nodules in vitro, while maintaining
a broblast phenotype. Analysis of the mineralized
matrix revealed the presence of osteopontin and
bone sialoprotein, characteristics shared with osteo-
blasts and cementoblasts (108). Transmission elec-
tron microscopy of these mineralized nodules re-
19
veals a tissue with similarities to acellular extrinsic
ber cementum. These results demonstrate the im-
portant role of periodontal ligament broblasts in
anchoring collagen brils to the mineralized matrix
of the root surface.
In repair, new broblasts are derived from perivas-
cular progenitor cells in the adjacent normal peri-
odontal ligament. Migration of broblasts into the
area to be repaired is facilitated by the presence of
brin and bronectin networks. New collagen bers
are laid down rapidly and often without functional
orientation or attachment to the adjacent hard
tissues. Reorganization of the initial collagen matrix
into oriented principal ber bundles requires con-
tinued cell activity over several weeks.
Studies of potential progenitor-cell pools have
shown that the marrow spaces of the alveolar bone,
particularly along lateral communications between
the periodontal ligament and the marrow, are sites
of cell proliferation. Of interest is the nding that
newly divided cells from these sites appear to con-
tribute to new cementum formation as well as to the
deposition of new periodontal ligament collagen. Al-
though there is still uncertainty surrounding the ori-
gin of cementoblasts and osteoblasts in the peri-
odontal ligament, evidence suggests that cells of the
osteoblastic subtype develop from perivascular cells
in the periodontal ligament proper, as well as from
the progenitor cells arising from adjacent marrow
compartments. After extensive damage to the peri-
odontal ligament connective tissue, the periodontal
ligament compartment is populated by an increased
number of bone-forming cells, and ankylosis of the
tooth to the alveolar bone usually results.
Role of epidermal growth factor receptor in
stabilization of periodontal ligament broblast
phenotype
The regulatory mechanism by which periodontal
ligament broblasts maintain their phenotype and
differentiate into cementoblasts or osteoblasts re-
mains largely unknown. We demonstrated for the
rst time that numerous epidermal growth factor re-
ceptor are expressed on the cell membrane of bro-
blastic cells in the functional periodontal ligament
(periodontal ligament broblasts, paravascular cells
and preosteoblasts), whereas no epidermal growth
factor receptor is expressed on fully differentiated
cementoblasts and osteoblasts (28, 31). Also, peri-
odontal ligament broblasts express many epider-
mal growth factor receptors during differentiation.
For example, epidermal growth factor receptors are
Cho & Garant
found on the precursor cells of periodontal ligament
broblasts (parafollicular cells), throughout their dif-
ferentiation, as well as on mature periodontal liga-
ment broblasts with full synthetic activity (28, 31,
33). During differentiation of osteoblasts and chon-
drocytes, however, a large number of epidermal
growth factor receptors is expressed only on undif-
ferentiated preosteoblasts and prechondrocytes. The
number of epidermal growth factor receptors on
these cells falls dramatically as their differentiation
progresses, with fully differentiated osteoblasts and
chondrocytes not expressing epidermal growth fac-
tor receptors (31, 33). Epidermal growth factor re-
ceptors are also known to be associated with pro-
liferation of various cell types, including dental fol-
licle cells (105, 134, 135, 139). The need for the
continued expression of a high density of epidermal
growth factor receptors on periodontal ligament
broblasts cannot be suitably explained by the
known physiological functions of epidermal growth
factor. Epidermal growth factor does not show sig-
nicant mitogenic and chemotactic effects on peri-
odontal ligament broblasts, as observed with plate-
let-derived growth factor-BB and insulin-like growth
factor-1 (88). However, periodontal ligament bro-
blasts of the functional periodontal ligament dem-
onstrate an extremely low
3
H-thymidine-labeling
index ranging from 0.5% to 3% under normal physio-
logical conditions (62, 92, 109). On the basis of these
observations, we proposed that the expression of
epidermal growth factor receptor on periodontal
ligament broblasts is associated with maintaining
the cells in an undifferentiated state, while the loss
of epidermal growth factor receptor is related to dif-
ferentiation of the cells. Our recent in vitro study
with periodontal ligament broblastic cells sup-
ported this notion (87). Untreated periodontal liga-
ment cells showed a gradual increase in spon-
taneous alkaline phosphatase activity, a osteogenic
cell differentiation marker, from 2 days to 7 days of
culture. Alkaline phosphatase activity was further in-
creased at 7 days after treatment with dexametha-
sone, an osteogenic cell differentiation inducer,
whereas epidermal growth factor treatment reduced
it to a lower level than the baseline. Interestingly, un-
treated periodontal ligament cells have both high-
and low-afnity forms of epidermal growth factor re-
ceptor, while fully differentiated rat osteosarcoma
cells (ROS 17/2.8) did not have any detectable epi-
dermal growth factor receptor. Treatment with dexa-
methasone for 2 days decreased the number of epi-
dermal growth factor receptors, the synthesis of epi-
dermal growth factor receptor protein and the
20
expression of epidermal growth factor receptor
messenger RNA by 50% compared with control. On
the other hand, epidermal growth factor treatment
increases the expression of epidermal growth factor
receptor messenger RNA. These observations
strongly indicate that epidermal growth factor recep-
tor on periodontal ligament broblastic cells is re-
sponsible for maintenance of their relative undiffer-
entiated state functioning as a negative regulator.
Gingiva
The gingiva, comprised of gingival epithelium and
connective tissue, is a portion of the oral mucosa
that covers the tooth-bearing part of the alveolar
bone and the cervical neck of the tooth. The epi-
thelial component of gingiva shows regional
morphological variations that are a reection of
tissue adaptation to the tooth and alveolar bone
(113). These include the oral gingival epithelium,
oral sulcular epithelium and junctional epithelium.
The gingiva evolves as the crown enters the oral cav-
ity by breaking through the oral epithelium. As the
development of gingiva prior to tooth eruption into
the oral cavity has not been studied (113), our de-
scription on the formation of gingival structural
components in association with the tooth is limited
to the stage of mucosal penetration of the crown and
subsequent differentiation.
Development of gingival epithelium
As an erupting tooth approaches the oral epithelium,
the enamel epithelium rapidly proliferates, forming
the thick reduced enamel epithelium. As the crown
erupts further, the reduced enamel epithelium over-
lying the enamel fuses with the oral epithelium, un-
dergoes transformation, and establishes the dento-
gingival junction forming the junctional epithelial
cells.
The junctional epithelium maintains a direct
attachment to the tooth surface. During eruption,
contact is established between the reduced enamel
epithelium and the oral gingival epithelium. Since
epithelial cells of the junctional epithelium contact
with the tooth surface, they produce an internal
basal lamina and are anchored to this basal lamina
by numerous hemidesmosomes (75, 76, 114, 119).
The basal cells of the junctional epithelium, how-
ever, are separated from the connective tissue by the
external basal lamina. The interface between the
junctional epithelium and the underlying connective
tissue is relatively smooth, unlike the condition
found in the oral gingival epithelium. Although junc-
tional epithelium does not exhibit true phenotypic
stratication, the outermost cells tend to be elon-
gated and to lie with their long axis parallel to the
tooth surface. Suprabasal cells of the junctional epi-
thelium express markers typically found in basal
cells and simple epithelia. The junctional epithelium
tapers from its coronal end, which may be 10 to 20
cells wide, to 1 or 2 cells at its apical termination,
located at the cementoenamel junction in healthy
tissue. Of interest is the observation that keratin
tonolaments are not inserted into the hemidesmo-
somes along the internal basal lamina. The internal
basal lamina is approximately three times thicker
than the external basal lamina. It contains laminin
and proteoglycans. Cells in contact with the internal
basal lamina express the a6b4 integrin, a laminin re-
ceptor. The cells in contact with the internal basal
lamina contain a relatively well-developed rough en-
doplasmic reticulum, and numerous Golgi compo-
nents. Several investigators indicate that the cells of
the junctional epithelium have an endocytotic ca-
pacity equal to that of macrophages and neutrophils
and that this activity might be protective in nature.
Cells leave the external basal lamina and migrate
to the free surface of the junctional epithelium
located at the base of the gingival sulcus, where they
are exfoliated. As measured in nonhuman primates,
the rate of cell turnover in the junctional epithelium
(46 days) is signicantly faster than in the oral sul-
cular epithelim. The oral gingival epithelium has the
slowest rate of proliferation of all three regions, with
a turnover time of 9 to 12 days. It appears that differ-
ences in cell proliferation among the three regions
of gingival epithelium may be due to their respon-
siveness to the growth inhibitory effect of trans-
forming growth factor-b and stimulatory effect of
epidermal growth factor.
In healthy teeth, the junctional epithelium (epi-
thelial attachment) ends at the cementoenamel
junction. Densely packed collagen bundles are an-
chored to the acellular extrinsic ber cementum just
below the terminal point of the junctional epithel-
ium. These collagen bundles form the connective
tissue attachment. The stability of this connective
tissue attachment is a key factor in limiting the mi-
gration of the junctional epithelium.
Development of gingival connective tissue
Prior to emergence of the crown, the connective
tissue in the future eruption pathway shows alter-
21
ations including the degeneration of collagen bers,
cells and blood vessels. Formation of this eruption
pathway accelerates the rate of tooth eruption.
Gingival connective tissue broblasts originate
from perifollicular mesenchyme, a derivative of the
stomodeal mesoderm. During normal development
of the periodontium, gingival broblasts do not
come into contact with the tooth surface. In con-
trast, the broblasts of the periodontal ligament be-
come juxtaposed to the tooth surface soon after the
disruption of the root sheath. New broblasts are de-
rived from the proliferation of undifferentiated peri-
vascular cells. Gingival collagen turns over more
rapidly than that of skin and bone, but slower than
that of the periodontal ligament.
Gingival broblasts show considerable variation in
morphology. In general they contain an abundance
of rough endoplasmic reticulum, well developed
Golgi complexes and mitochondria. Other bro-
blasts may show signs of swelling and degeneration
depending on site-to-site microenvironmental vari-
ations in cytokines and other biological mediators of
inammation.
The collagen matrix of gingival connective tissue
is well organized into ber bundles, which constitute
the gingival supra-alveolar ber apparatus. It is
made up of the transseptal, circular, semicircular,
transgingival, and intergingival bers, which connect
and link the adjacent teeth of one arch. These bers
secure the teeth against rotation and maintain tooth
linkage during mesial drift. Tractional forces in the
extracellular matrix produced by broblasts are be-
lieved to be the motive forces responsible for gener-
ating tension in the collagen, keeping the teeth
tightly bound to each other and to the alveolar bone.
Alveolar bone
The maxilla and mandible of the adult human can
be subdivided into two portions: (a) the alveolar pro-
cess that involves in housing the roots of erupted
teeth and (b) the basal body that does not involve in
housing the roots (114). The alveolar processes con-
sist of the thin alveolar bone proper that forms the
alveolar wall of the tooth socket, the inner and outer
cortical plates, and spongy bone between the al-
veolar bone proper the cortical plates. Since the al-
veolar processes develop and undergo remodeling
with the tooth formation and eruption, they are
tooth-dependent bony structures (116). Therefore,
the size, shape and location and function of the teeth
determine their morphology.
Cho & Garant
The alveolar bone proper is 0.1 to 0.4 mm thick
and is consisted of a Harversian system and lamel-
lated and bundle bone (114). The coronal and apical
regions of the alveolar bone proper have a sieve-like
structure. These openings connect the periodontal
ligament to the bone marrow spaces and correspond
to Volkmanns canals through which blood vessels,
lymphatic vessels and nerve bers pass.
Development of the alveolar bone proper
Since the development of the alveolar bone proper
has not been systemically studied in animals and
humans, the information on this subject is very
much fragmented. Tooth germs develop within bony
structures. At the late bell stage, bony septa and
bony bridge start to form, and separate the individ-
ual tooth germs from another, keeping individual
tooth germs in clearly outlined bony compartment
(114). At this stage, the dental follicle surrounds each
tooth germ, which is located between a tooth germ
and its bony compartment. Even prior to root forma-
tion, the tooth germs within bony compartments
show continued bodily movement in various direc-
tions to adjust to the growing jaws. This movement
causes minor remodeling of bony compartment
through bone resorption and deposition of new
bone.
The major changes in the alveolar processes begin
to occur with the development of the roots of teeth
and tooth eruption. As the roots of teeth develop, the
alveolar processes increase in height. Also, cells in
the dental follicle start to differentiate into peri-
odontal ligament broblasts and cementoblasts re-
sponsible for the formation of the periodontal liga-
ment and cementum, respectively. At the same time,
some cells in the dental follicle also differentiate into
osteoblasts and form the alveolar bone proper (60,
114, 132, 133). The formation of the alveolar bone
proper is closely related to the formation of the peri-
odontal ligament and cementum during root forma-
tion and tooth eruption (114). Thus, the size and
shape of the individual developing tooth roots deter-
mine the overall structure of the alveolar bone
proper. On the other hand, the rest of bony struc-
tures in the alveolar process are achieved by perios-
teal bone formation (114).
Remodeling of the alveolar processes during tooth
eruption
The tooth germs develop within the alveolar pro-
cesses, and when the root formation begins, the al-
22
veolar processes have already grown over the occlusal
plane of the developing tooth. Thus, for successful
tooth eruption, there must be bone remodeling. In
order for the developing tooth to escape from the
alveolar bone, a gubernacular canal must be widened
by osteoclastic bone resorption (23, 86). At the same
time, new bone formation at the base of the bony
crypt is believed to be important in producing an
outward eruption force directed against the erupting
tooth. Morphological studies and experimental surgi-
cal interventions have provided evidence that the
post-secretory enamel organ and the highly vascular-
ized dental follicle connective tissue coordinate the
eruption of teeth of limited eruption (24, 145, 146).
The presence of the dental follicle was found to be
essential for bone resorption during the formation of
the eruptive pathway as well as for new bone forma-
tion apical to the erupting tooth (40, 68). Supporters
of the concept that the dental follicle is the key struc-
tural component responsible for regulating eruption
of teeth point to the fact that proteinase activity in
the follicular connective tissue peaks at initiation of
tooth eruption (123). The observation that rootless
teeth undergo eruption (24, 55) is further convincing
proof for the integrated activity of bone resorption
and bone formation under the control of the dental
follicle during eruption. Monocytes containing tar-
trate-resistant acid phosphatase, an indicator of lys-
osomal activity, invade the connective tissue of the
dental follicle early in tooth development and during
tooth eruption (111, 144). These cells are believed to
be osteoclast precursors that play an important role
in alveolar bone resorption during tooth eruption.
Recent evidence shows that colony-stimulating fac-
tor-1 and epidermal growth factor are involved in
tooth eruption (35, 36, 67, 122, 145, 148). Isolated
cells of the dental follicle secrete colony-stimulating
factor-1, a substance involved in the recruitment and
differentiation of preosteoclasts. Epidermal growth
factor upregulates the production of colony-stimulat-
ing factor-1 via its ability to stimulate the cells of the
reduced enamel organ to make interleukin-1a (145
148).
Functions of the alveolar processes
The general functions of the alveolar bone processes
are to house the roots of teeth and to absorb and
distribute occlusal pressures generated during tooth
contacts. Their most important and unique function
is to anchor the roots of teeth to the alveoli, which
is achieved by the insertion of Sharpeys bers into
the alveolar bone proper.
Conclusion
Not long ago knowledge of the development and
general structure of the periodontium was limited to
morphological information. Over the years, exten-
sive use has been made of transmission electron
microscopy, histochemistry, cytochemistry and
radioautography to document the cell and tissue
structure of the periodontium. We have attempted a
brief review of this topic. During the last decade,
rapid progress has been made in understanding of
the molecular and cellular biology of periodontal
tissue development. However, there remain many in-
triguing aspects of this story that remain to be fully
explored at the tissue, cell and molecular level. This
subject is of special importance, for we now know
that periodontal tissue healing and regeneration
share many, if not all, of the events that take place
during normal development. Further studies of how
cells interact with their neighbors and with the extra-
cellular matrix, in vivo as well as in vitro, during de-
velopment, will help to create a rmer foundation
upon which periodontal regeneration techniques
can be developed and implemented clinically.
Acknowledgment
This work was supported by USPHS grant DE 04898.
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Introduction to microbial aspects
of periodontal biolm
communities, development and
treatment
ANNE D. HAFFAJ EE & SI GMUND S. SOCRANSKY
In the rst part of the Microbiology of Periodontal
Diseases, published in Periodontology 2000, vol. 38,
2005, we provided an update of some of the advances
in our understanding of the role of oral organisms in
periodontal infections that had been made between
1994 and 2005. These included an examination of the
relationship of spirochetes and viruses to the etiology
of periodontal diseases (5, 16), the inuences of
genomic approaches on our understanding of the
pathogenesis of subgingival species (4), the examina-
tion of virulence factors produced by the red complex
species (11), the detection of important virulence
factors by using the hosts immunological response
(10), and a description of ecological relationships
among bacterial species and between bacterial spe-
cies and the host (18). In the current issue, we extend
these ndings by examining genetic, phylogeny and
species interactions in dental biolms, the role of
specic periodontal pathogens in disease etiology,
and the effect of treatment on biolm composition
and clinical outcomes. It is anticipated that the two
parts will provide a reasonably comprehensive update
of the microbiology of periodontal diseases. However,
it must be recognized that research in this area is
moving at a rapid pace and thus our understanding of
periodontal diseases continues to evolve.
Molecular genetics analyses of
dental biolm formation
One of the important paradigm shifts that has taken
place in the last decade has been the recognition and
acceptance that dental plaque is a biolm and that
dental biolms have many properties in common
with biolms that occur in other locations in nature.
This seemingly inconsequential change in termin-
ology has actually had profound effects on the
approaches taken by periodontal microbiologists.
Less emphasis is being placed on pure culture
studies and a greater effort is being directed towards
studying bacterial species in community structures
that mimic dental biolms. A number of different
systems have been described for studying biolm
development in vitro. Most of these systems have
studied aerobic or facultative species. Davey des-
cribes three biolm model systems used to study
in vitro biolm development by the anaerobic
periodontal pathogen, Porphyromonas gingivalis,
and some of the factors that inuence biolm
development of different strains of this species (2).
The clinical importance of the intricate biolm
ecosystem is the appreciation that one is trying to
control living lms (i.e. microbial communities that
have the ability to change as a result of therapeutic
intervention or host modication). Of biological
consequence is the recognition that a remarkably
complex series of processes take place in the estab-
lishment and maintenance of these biolms and that
far more communication/interaction takes place
among bacterial species and between species and
their environment than had been previously recog-
nized. Early studies of biolm development empha-
sized changes in the structure and spatial location of
mixed-species biolms. Davey and Costerton (3)
present an overview of the current understanding of
the genes that regulate dental biolm formation.
They describe genes that are responsible for adhesin
formation, which is necessary for cellsurface or
7
Printed in Singapore. All rights reserved
2006 The Authors.
PERIODONTOLOGY 2000
cellcell attachment. They emphasize the important
role that the production of signaling molecules may
play in regulating genetic activity within a species, as
well as the intriguing, recently described, signaling
molecules that can provide interspecies communi-
cation during biolm development.
Genetic basis of horizontal gene
transfer among oral bacteria
The close proximity of bacterial cells in oral biolms
provides abundant opportunity for bacterial cells to
acquire genetic information from other cells of the
same or different species residing in the same hab-
itat. This exchange of genetic information may ac-
count, in part, for the remarkable ability of species
in oral biolms to adapt to changes in their envi-
ronment. One clinically important example of this
adaptability is the acquisition of antibiotic resist-
ance genes by a previously antibiotic-sensitive oral
strain from an antibiotic-resistant strain in the same
or different species. However, this is only one of
many traits that may be transmitted from cell to cell
by horizontal gene transfer. Roberts and Mullany
(15) describe the major mechanisms for the hori-
zontal transfer of genetic information, including
plasmids, conjugated transposons and bacterio-
phage, as well as environmental factors that may
affect gene transfer.
Bacterial interactions and
successions during plaque
development
The rst time that an investigator uses dark-eld or
phase-contrast microscopy to examine a subgingival
biolm sample, he or she is probably impressed by
the teeming numbers of organisms, the complexity of
the morphotypes and the fact that so many diverse
organisms live together in an obviously very complex
community. At rst glance, the community structure
appears utterly random with little order to the species
that accumulate, their physical location and inter-
species relationships. However, this is clearly not the
case. Certain species are early colonizers in biolm
development. They set the stage and act as recep-
tors for the colonization of other organisms. There-
fore, certain species are found to be frequently
associated with a dened set of other species. What
are the mechanisms that govern the specicity of
these interspecies interactions? Kolenbrander et al.
(13) examined this question in detail. They high-
lighted the critical role of co-aggregation in inter-
species organization and provided examples of
specic interactions at the species level. They pro-
ceeded to describe the molecular basis of these
interactions for dened pairs of species, describing
the nature of specic adhesins and receptors that are
involved. They showed that many species have a
limited set of species partners, while other possible
bridging species, such as Fusobacterium nucleatum,
adhere to a wide range of species. While initial
adhesion to a solid surface and co-aggregation may
be critical in the selection of colonizing species for a
given biolm, other factors play an important role in
the development of these structures. For example,
within-species and between-species signaling appear
to be important regulators of the genes that control
biolm development. The authors go on to propose a
sequence of mechanisms and specic interspecies
events that may lead to microbial succession and
ultimately to the mature biolm.
The breadth of bacterial diversity
in the oral cavity
An article in the 1994 issue of Periodontology 2000
described the sequencing of the 16S rRNA genes of
cultured and uncultured bacterial species directly
from biolm samples (20). This approach has been
remarkably fruitful and has provided a much im-
proved estimate of the range of bacterial species that
may colonize the subgingival area and the oral soft
tissues, as well as a clearer picture of the nature of the
taxa in these locations. Indeed, the data, to date,
suggest that there is a location specicity for colon-
ization of specic species (i.e. certain species that
colonize the teeth are different from the species that
colonize primarily the soft tissues). Current estimates
suggest that 700 species can colonize the oral cavity
and 400 different species may colonize the subgin-
gival biolms. The sequencing of the 16S rRNA genes
has also led to the development of DNA probes for
culturable and as-yet-uncultured species. In addi-
tion, high-throughput checkerboard hybridization
and microarray techniques have been developed to
detect large numbers of bacterial species in large
numbers of biolm samples. For example, micro-
array techniques permit the rapid detection of as
many as 600 bacterial species in individual biolm
samples. The status of work in this area, and the
8
Haffajee & Socransky
potential of these new techniques in the next decade,
will be examined by Paster et al. (14).
The role of Tannerella forsythia in
the etiology of periodontitis
About 30 years ago, Anne Tanner carried out a un-
ique study. She dispersed, diluted and plated biolm
samples (plaque samples in those days) from subjects
with chronic periodontitis onto nonselective blood
agar plates. After 7 days of anaerobic incubation, she
photographed the plates and re-incubated them for
another 721 days. By comparison with the photo-
graphs, she detected tiny colonies that appeared on
the blood agar plates after prolonged incubation. The
cellular morphology of most of the isolates was fus-
iform, with unusual swellings in parts of the cells.
These slow-growing isolates, which were described in
the early days as gelatin-loving Bacteroides, would
grow better when streaked adjacent to other subgin-
gival species such as F. nucleatum. This novel species
is now known as Tannerella forsythia. Difculty in
culturing this species hampered analysis of its clinical
importance. However, in 1989, Gmur et al. (8) used
immunological techniques to identify the species in
plaque samples and revealed a strong association
with pocket depth and periodontitis. Development of
a medium for this species also facilitated its investi-
gation (22). This species is a consensus periodontal
pathogen (1) and we are fortunate to have Tanner
and Izard (19) to provide an update on the role of this
species in periodontal diseases and other oral infec-
tions, as well as the biology of the species during its
colonization.
Current understanding of the role
of Actinobacillus
actinomycetemcomitans in
aggressive forms of periodontitis
In the late 1970s, the relationship of Actinobacillus
actinomycetemcomitans to localized aggressive peri-
odontitis was considered to be the Rosetta stone of
periodontal disease. Indeed, this species was the rst
of the consensus periodontal pathogens to be recog-
nized, initially by its association with lesions of locali-
zed aggressive periodontitis and later by its decrease
in levels in successfully treated subjects. However,
association and treatment studies were not the sole
reason for indicting, and later convicting, this species
as a possible pathogen, as the hosts antibody re-
sponse, animal studies and the species wide range of
possible virulence factors lent credence to its prob-
able role in periodontal disease etiology. The patho-
genic potential and virulence genes of this species
have been studied in much greater detail in the last
decade. A comprehensive review of current under-
standing of the mechanisms of virulence of A. actin-
omycetemcomitans from its colonization of the soft
tissues and the tooth through its evasion of host
defense mechanisms and to the destruction of sup-
porting tissues is provided by Fine et al. (6). The
detective story presented by Fine et al. (6) took a
unique twist when it was recognized that there were
multiple clonal types of A. actinomycetemcomitans
and that certain clonal types were more virulent than
others. Indeed, one clonal type of A. actinomyce-
temcomitans serotype b (strain JP2), with a 530-base
pair deletion in the promoter region of the leukotoxin
gene, appears to be uniquely virulent and may ac-
count for a large proportion of localized aggressive
periodontitis in certain populations. The fascinating
story of the detection, biological signicance and
clinical implications of specic virulent clonal types
of A. actinomycetemcomitans has been outlined by
Kilian et al. (12), as well as the usefulness of popu-
lation genetic analysis in the hunt for pathogens of
infectious diseases.
Effect of periodontal treatment on
the subgingival microbiota
The long-term objective of the work described in this
issue of Periodontology 2000 is to improve the diag-
nosis, treatment and prevention of periodontal
infections. This will not be an easy process. Earlier
papers in this issue of Periodontology 2000 describe
the complexity of subgingival biolms, their plasti-
city, in terms of changing in response to their envi-
ronment, and the probability that the clinician will
have to control multiple species in biolms of dif-
ferent individuals or even in the same individual. At
present, the clinician has a limited set of tools to shift
the subgingival biolm microbiota to one that is
host-compatible. Some of the studies, using different
microbiological techniques to determine the effect of
different periodontal therapies on the subgingival
microbiota, are described by Teles et al. (21). Major
efforts to determine the microbial shifts in the sub-
gingival microbiota brought about by treatment have
been initiated only in the last decade. The same
improvements in microbial assessment that were
9
Microbial aspects of periodontal biolm communities, development and treatment
useful in examining ecological relationships of the
subgingival microbiota have also permitted exam-
ination of the effect of various treatment approaches
on the composition and long-term control of the
subgingival microbiota. The changes that occur in the
subgingival microbiota, and concomitant clinical
changes that occur as a result of different forms
of periodontal therapy, will be described in some
detail (9).
Comment
The past decade has been characterized by sustained,
substantial progress in our understanding of the
microbiology of periodontal diseases. Much of this
progress has been a result of improvement in our
techniques, particularly the widespread use of the
techniques of molecular biology. These techniques,
including genomic sequencing, 16S rRNA phylogeny
and identication of virulence determinants, have
permitted studies that otherwise could not be carried
out, or could only be performed on a limited scale,
such as those on ecology and treatment. The high
throughput of some of these techniques suggests a
large joint clinical/microbiological activity in the
eld, when in fact the data are generated by small
numbers of investigators.
While there are a number of excellent investiga-
tors examining the basic aspects of biolm forma-
tion, virulence factors and their genetic regulation,
there are few investigators willing to undertake
the translational research that bridges the basic
ndings with clinical implementation. The few
researchers in this area are aging and there appears
to be a limited number of individuals willing to
take their place. Who, for example, will write the
2016 issue of Periodontology 2000 on the Micro-
biology of Periodontal Diseases? There are several
reasons for this lack of enthusiasm for the trans-
lational aspect of research, although we are told
that it is a high-priority emphasis of funding
agencies. Typically, investigators in translational
research have to be dually trained in both clinical
and basic skills and require a working knowledge of
experimental design, data management and analy-
sis. Few individuals are willing to undertake the
extensive training required to be effective. Fur-
thermore, with clinical training, these individuals
can opt to go into practice if the research envi-
ronment presents unreasonable barriers for con-
tinued productivity. Currently, funding is limited
and it is particularly difcult to obtain funding for
clinical/laboratory investigations. In its wisdom, the
National Institute for Dental and Craniofacial
Research no longer funds the incredibly useful tools
of randomized clinical trials (except for Phase III
multicenter trials). The randomized clinical trial has
been, and continues to be, a major tool in bio-
medical research, providing answers to specic
questions and suggesting new avenues of research.
A second problem is that clinical studies are dif-
cult to carry out. They are expensive, labor inten-
sive and burdened by regulatory issues not affecting
basic research. Given this scenario, it is difcult to
maintain the infrastructure, in terms of personnel,
facilities and equipment, necessary to perform
clinical, laboratory and translational studies. If you
were a young investigator with probably 15 years of
training after high school, and were given the
choice of a relatively modest salary, with the
opportunity to be hassled by granting agencies,
institutional promotion committees, space com-
mittees, department chairs, regulatory agencies and
biostatisticians, or to go into practice with at least
three to four times the income, which would you
choose?
The loss of senior investigators, and the reluctance
of young individuals to pursue careers in oral
microbiology, particularly as it relates to clinical
investigation, is a major concern to us and should be
a major concern to the readers of Periodontology
2000. This challenge to our eld is important,
because it will dene the nature and scope of the next
generation of studies to be carried out by periodontal
microbiologists in association with clinical investi-
gators, biostatisticians and individuals interested in
the host response. This issue of Periodontology 2000
indicates that it is entirely feasible to understand the
nature of periodontal biolms, to determine which
microbial agents and which virulence mechanisms
can lead to tissue damage and to design methods to
alter biolm composition that improve and sustain
improved clinical status. It would be unfortunate if
such momentum were lost.
A note of caution
While we are as enthralled as any with the potential
provided by developing new technologies, they raise
new concerns. Will we become so enamored by our
techniques that the techniques become an end in
themselves? Will our enthusiasm for metagenomic
and proteomic approaches provide vast arrays of
information about virtually nothing (i.e. more and
10
more information about fewer and fewer samples)? A
recent example from veterinary microbiology may
prove instructive. In this study (7), the authors used a
novel metagenomic approach to characterize the
bacterial and Archaeal contributions to the total 16S
rRNA of a rumen sample from each of two steers
housed together and fed identically. They detected
unexpectedly large differences in the Archaeal com-
munity structure between the two rumen popula-
tions. If you are a technophile, you would probably
say wow, look at all those species detected and all of
the gene sequences that were revealed. If you are a
cynic, you might say wow, what a waste of money.
At one time, this type of research would have been
denigrated as descriptive or a shing expedition.
Now, because of our developing love for, for example,
gene chips, with in some instances, 22,000 genes on
them, or our fondness for looking at a very wide range
of species or messenger RNAs or other markers in a
small number of samples, we may be falling into the
trap of nding 200+ signicant differences between
ve to 10 samples in each of two groups. (As an aside,
the authors wonder why they were castigated, virtu-
ally ogged, by biostatisticians for the multiple
comparison issue when they examined 40 bacterial
species in hundreds of plaque samples, while the
same statisticians cast a benevolent, or blind, eye to
evaluating 22,000 variables in ve to 10 samples per
group. We were just wondering.) The point of this
discourse is that vast amounts of information des-
cribing the content of small numbers of samples is
unlikely to provide answers to any but the most
trivial of questions, for example the range of species
in an unstudied habitat. Given the improvement in
technology, it is hoped that the questions will drive
the technique, and not the techniques drive the
questions.
Finally, in the 1994 issue of Periodontology 2000
(17), the authors included a footnote to the next
generation of periodontal research workers, trying to
explain why the research problem was taking so long
to solve. Among the concerns were the (mis)direction
of funds to good science, studies that while scien-
tically laudable had no chance of leading to thera-
peutic or preventive benet, and the engulfment of
investigators by the paperwork needed to write, re-
view or describe progress for the endless grant
applications needed to fund research. In addition, the
authors lamented the need to account to virtually
every regulatory agency and interest group on Earth
as a signicant barrier to performing studies in a
timely and efcient manner. We are happy to report
to this future generation that the research problem
will still be unsolved and available to them. The same
limitations on productivity described in 1994 are still
present in 2006, but they have been enhanced by a
new generation of more imaginative administrators
who have found new absolutely essential busy work
for investigators to perform.
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12
Molecular genetics analyses of
biolm formation in oral isolates
MARY E. DAVEY & JOHN W. COSTERTON
Its clinical importance and accessibility for in vivo
research make dental plaque one of the most studied
and best-understood biolm communities. Research
has shown that the development of this microbial
community is a process that involves cooperation
and competition among an extremely diverse com-
munity of organisms (78). Although our knowledge of
dental plaque is substantial at the microbiological
level, we are only just beginning to dissect the
development and function of this community at the
molecular level. The intent of this review is to sum-
marize the progress made in determining the
molecular genetic basis of biolm formation of oral
isolates using data from in vitro studies on either
single species or multi-species systems. The currently
available data on relevant non-oral model organisms
are used to compare and contrast these ndings. The
development of genetic systems and the sequencing
of entire bacterial genomes have made these analyses
possible (27, 66). To date, Streptococcus gordonii and
Streptococcus mutans, as well as Actinobacillus
actinomycetemcomitans have been the primary
models for molecular genetic biolm studies, so they
are the focus of much of this review. However,
investigation of other key players in oral health
(Porphyromonas gingivalis, Treponema denticola,
Fusobacterium nucleatum, and Tannerella forsythia)
is now beginning and their involvement in the
development of multi-species biolms will also be
discussed.
Background
The human oral cavity supports a remarkably diverse
microbial community. At the last count, 30 genera
representing at least 500 different species have been
identied (81, 129), and all of these bacteria persist by
adhering to surfaces such as the enamel of the tooth,
the epithelial cells of the mucosa, and to one another.
Without secure attachment bacteria are washed away
with saliva; hence attachment and development of a
surface-attached community is fundamental to per-
sistence in the oral cavity. The complexity of the
periodontal microora combined with a host of
variable environmental parameters make this system
particularly challenging to study, yet in vivo accessi-
bility and the development of new technologies has
allowed signicant progress in our understanding of
this microbial community. Research over the past
several decades has provided a strong foundation of
knowledge as to the types of organisms that colonize
the tooth surface. Clearly both outside inuences and
in-biolm factors contribute to the complexity of the
system. The periodontal habitat experiences con-
stant changes in pH, oxygen tension, and nutrient
availability, in response to variations in diet and
microora, and a variety of physical and nutritional
interactions within the biolm community itself
contribute to the dynamic nature of this system. In
essence, changes in habitat by outside inuences
affect the microbial community, and, in turn, the
microora affects the habitat. Describing the com-
plex ecology of the oral cavity is beyond the scope of
this article; we refer the reader to a recently published
comprehensive review of the microbial ecology of the
oral cavity (148).
Recent studies using confocal microscopy have
shown that the architecture of supragingival plaque,
like other biolm communities, is heterogeneous,
consisting of areas of high cell density encased in an
extracellular matrix surrounded by open areas that
transverse from the top down to the tooth surface
(shown in Fig. 1). This heterogeneous structure
results in the development of focal points of chemical
gradients (e.g. metabolic end-products, toxins, sub-
strates, signaling molecules, as well as pH and oxy-
gen), which inuences microbial distribution and
13
2006 The Authors.
PERIODONTOLOGY 2000
activity. Many studies on oral microbiology have
focused on determining the spatial organization and
development of plaque by examining the succession
of organisms in the growing biolm. A number of
excellent reviews have covered the structure and
composition of oral communities (8, 24, 75, 77, 103,
147, 165). Clearly, plaque is not simply an organism-
containing layer of slime on the tooth surface;
instead, plaque represents a structured, inter-
dependent, microbial community.
Although development of plaque on the tooth
surface is complex and has both spatial and temporal
inuences, it can be described in three distinct steps:
formation of the conditioning lm or pellicle on
the tooth enamel,
subsequent cell-to-pellicle or cell-to-surface
attachment of bacteria (primary colonizers), and
cell-to-cell interactions of the mid- and late-colo-
nizers with one another (as well as with primary
colonizers).
The early events of plaque formation have been
reviewed in some detail (45, 5557). In brief, after
professional cleaning of the tooth surface, the sur-
face rapidly becomes coated with a variety of saliv-
ary constituents including albumin, glycoproteins,
acidic proline-rich proteins, mucins, cell debris,
exoproducts (such as a-amylase and lysozyme), and
sialic acid (26, 38, 40, 143, 144), thus providing
receptors that are recognized by colonizing bacteria.
As a consequence of attachment it is likely that a
change in gene expression occurs. Consequently,
primary colonizers alter the surface not only by their
physical presence but also they are likely to repre-
sent a new surface-attached phenotype with dis-
tinct metabolic activity and surface properties, thus
altering their surroundings and creating new niches
for other bacteria to colonize. Hence, dental plaque
formation should be viewed as a distinct type of
biolm development. Unlike single species biolm
systems in which individual cells attach to the sur-
face and then grow into large mushroom-shaped
colonies, plaque is formed by mixed-species inter-
actions (coaggregation) followed by growth on the
surface (79).
The complexity of the oral microora has made
identication of specic organisms responsible for
diseases extremely challenging (43). In essence, the
cause of dental caries and periodontitis should be
considered polymicrobial in nature, with certain
virulent organisms linked to the disease state. Clearly
the presence of pathogens is necessary for disease,
however potential pathogens are often detected in
the absence of disease. It is likely that expression of
virulence determinants occurs in response to specic
signals, for instance a shift in community structure in
response to outside inuences may be enough to
trigger the expression of genes that are detrimental to
the host. As described above, the development of this
multi-species community creates specic associa-
tions between metabolically cooperative organisms.
Their close proximity in the biolm facilitates inter-
species substrate exchange and removal or distribu-
tion of metabolic products, hence the loss of a
benecial organism may cause an opportunistic
pathogen to acquire its nutrients elsewhere, resulting
in damage to the host. Although conclusive evidence
as to which organisms are responsible for disease is
still being investigated, certain virulent organisms are
repeatedly found to be associated with disease and
these bacteria have become model organisms for
biolm studies. Research has shown that many dif-
ferent types of genes are required for biolm devel-
opment (see reviews 20, 82). The focus of this article
is to review the advances in understanding the
molecular mechanisms required for the formation of
supra- and sub-gingival plaque using data obtained
from in vitro studies. The studies discussed include
both single species and mixed species systems. Genes
with a variety of functions have been discovered
including quorum-sensing systems, environmental
sensing two-component systems, general stress
response pathways, and those encoding surface
adhesins involved in cellcell or cell-to-surface
interactions.
a v i g n i G
a v i g n i G
Fig. 1. Diagrammatic representation of the oral biolm
community showing the diversity of organisms growing in
large clusters (macro-colonies) extending out from the
surface of the tooth into the gingival crevice. Structurally,
the biolm is very heterogeneous. Depicted are areas of
high cell density encased in an extracellular matrix sur-
rounded by open areas that transverse from the top down
to the tooth surface. Detached clusters of bacteria, as well
as bacteria attached to the gingival epithelial cells are also
shown.
14
Davey and Costerton
Streptococci
Viridan streptococci, such as S. gordonii, Strepto-
coccus sanguinis, and Streptococcus parasanguis
make up a large portion (5080%) of the com-
mensals that initially colonize the tooth surface (122).
These species form the physical foundation of dental
plaque (121). The importance of streptococci in pla-
que development and disease has brought them to
the forefront of biolm research. The use of poly-
styrene dishes as models for an abiotic surface has
been instrumental in assessing biolm formation in a
variety of gram-positive bacteria. This system was
initially used to investigate biolm development in
two non-oral human pathogens, Staphylococcus au-
reus and Staphylococcus epidermidis, which cause
persistent life-threatening infections through biolm
formation on indwelling medical devices (4648, 101,
102, 172). Ganeshkumar and colleagues used a sim-
ilar microtiter plate system to determine the inu-
ence of various environmental factors on biolm
formation by the oral isolate S. gordonii, and to
search for genetic loci associated with biolm
development (97). In these studies, they showed that
biolm formation in S. gordonii is inuenced by a
number of parameters, including shifts in osmolarity,
pH, and oxygen availability. A library of 25,000
transposon-insertion mutants was generated and
screened, and 18 biolm-defective mutants were
identied. Among the genes identied in this screen
were those that code functions required for com-
petence (comD), peptidoglycan biosynthesis (PBP 2B,
PBP 5, glmM, and bacA), oligopeptide transport
(appC), and DNA replication/repair (mutT).
Interestingly, comD encodes a sensor kinase that is
required for the development of competence for
genetic transformation in Streptococcus (100) and the
regulation of this two-component system is mediated
by a cell-density-dependent (quorum-sensing) sys-
tem, which depends on a competence-stimulating
peptide signaling system (see reviews 19, 152). Like
other quorum-sensing systems, the extracellular
signaling molecule (in this case the competence-sti-
mulating peptide) is expressed continuously, and as
cell density increases the concentration of compet-
ence-stimulating peptide increases and this, in turn,
activates the transcription of a specic set of genes.
This process allows bacteria to distinguish between
low and high cell density and to coordinately control
gene expression in a population of cells. Genetic dis-
section of biolm development in both gram-negative
and gram-positive species [e.g. Pseudomonas aerugi-
nosa (21) and S. aureus (170, 171)] has demonstrated
that quorum sensing is required, indicating that the
involvement of this process in biolm development is
widespread. Yet, the study on S. gordonii was the rst
report of a quorum-sensing two-component system
being important in both genetic competence and
biolm formation. Furthermore, the connection be-
tween quorum sensing and competence during bio-
lm growth has only been demonstrated in gram-
positive bacteria. Recent studies in S. gordonii have
further developed these ndings. Biolm formation
and competence for genetic transformation also ap-
pear to be linked to the uptake of metals. Mutations in
the adc operon, which shows high similarity to a
metal uptake system of Streptococcus pneumoniae,
resulted in a S. gordonii strain defective in biolm
formation and competence (98, 115). Since trace
elements are essential for growth and survival it is not
surprising that sensing levels of available metals and
biolm development are linked. However, the authors
propose an intriguing hypothesis that high levels of
available metals, such as zinc or manganese, may be a
signal for the cells to switch from surface-attached
growth to planktonic growth and dissemination. In
addition, these researchers discovered that another
metal uptake system, a copper-transport operon,
copYAZ, which is located immediately downstream of
the adc locus, is also involved in biolm development.
Inactivation of this locus does not inhibit biolm
formation, however mutation of either copZ or copY
affects detachment from the biolm (114). These
studies indicate that trace elements play an important
role in the life cycle of this organism.
In addition to the factors described above, a
putative oxidative stress response (osr) operon in
S. gordonii has been found to play a role in biolm
formation (99). The osr operon consists of three genes
nosX, and two Qor genes, qor1 and qor2. Although the
function of nosX is unknown, the qor genes found in
other bacteria are involved in aerobic respiration and
are important in the response to oxidative stress.
Since strict anaerobes persist in subgingival plaque, it
is typically viewed as anaerobic, however aerobic
metabolism does occur, therefore it may be that
these genes are required to maintain a reduced
environment which directs this facultative anaerobe
to form a biolm. Further characterization of this
locus is needed to understand the connection
between biolm formation and oxygen tolerance.
Since bacteria selectively attach to the conditioning
lm or pellicle on the tooth enamel during the
initial stages of biolm formation, determining the
interactions between cell surface proteins and
15
Molecular genetics analyses of biolm formation in oral isolates
constituents of the salivary lm or pellicle that coat
the tooth has been a primary focus of oral biolm
research. Surface protein peptides of S. gordonii have
been found to bind to components of saliva (88), and
a variety of genes encoding adhesins have been dis-
covered, including those for SsaB, Hsa, AbpA, AbpB,
SspA, and SspB. SsaB is involved in adhesion to
saliva-coated hydroxyapatite (36, 37). The sialic acid-
binding protein Hsa has been shown to bind glyco-
conjugates found in salivary mucins (92, 153), which
are major pellicle constituents, and bind to platelets
(154). AbpA and AbpB are two amylase-binding pro-
teins (96, 136). Protein A (AbpA) has been shown to
function as an adhesin to amylase-coated hydroxy-
apatite, and to play a role in human saliva-supported
biolm formation (137). In addition, since amylase
hydrolyzes starch, binding of bacterial cells to the
enzyme may also facilitate their acquisition of
nutrients. Clearly, modulation in expression of sur-
face proteins and structures can direct interactions
with surfaces. This will continue to be a key area of
investigation, especially in determining how expres-
sion of these surface proteins and peptides is
coordinated.
The sanguis streptococci are also primary colo-
nizers of the tooth surface and thus play a key role in
the development of dental plaque. Like S. gordonii,
bacterial surface structures have also been shown to
be involved in S. sanguinis (formerly Streptococcus
sanguis) biolm formation. Inactivation of fap1,
which is a glycoprotein essential for mbriae forma-
tion, results in a strain defective in attachment and in
the formation of cell aggregates or microcolony for-
mation (35, 167). Interestingly, these bacteria are also
uniquely capable of colonizing native and prosthetic
heart valves resulting in infective endocarditis. It has
been shown that once S. sanguinis colonizes the valve
surface, small surface cell aggregates begin to form
and eventually colonies with a high density of tightly
packed cells are created. Over time a layer of platelets
and brin is deposited over the bacteria, thus estab-
lishing a structured surface-attached vegetative bio-
lm. Like other biolm-based infections, these
infections are life threatening and often persistent,
even after repeated antibiotic treatment. Under-
standing the molecular genetic basis of biolm
development in this organism is the key to the
development of therapies to prevent or treat these
costly, life-threatening infections.
S. mutans, which is considered to be a principal
etiological agent in human dental caries formation,
is another streptococcus commonly found to inhabit
the oral cavity. This species has the ability to fer-
ment an array of sugars producing acid end-prod-
ucts that result in demineralization of tooth enamel
and caries formation. Hence biolm formation and
the ability to adjust to unfavorable conditions, such
as low pH, are key factors in the cariogenicity of this
organism. Like the viridans streptococci, S. mutans
cells growing in a biolm have been shown to
incorporate foreign DNA much more efciently (10-
to 600-fold higher) than the same strain growing in
liquid culture (93). The development of this com-
petence was shown to be dependent on growth rate,
pH, and the age of the biolm. A proteomics study
clearly demonstrated a role for the maintenance of
transformation during biolm growth (134). Studies
have shown that S. mutans, like S. gordonii, relies on
the comCDE quorum-sensing system to form a
biolm, at least under certain growth conditions
(95), and for acid tolerance (93). In addition, in
S. mutans another quorum-sensing system (luxS)
affects biolm formation (108). The auto-inducer
(AI-2) signaling molecule encoded by the gene luxS
is a novel type of chemical signaling molecule. This
molecule is a boron-containing furanone that is
involved in both intra- and inter-species commu-
nication (11, 145). Unlike the quorum-sensing
signaling molecule discussed above that allows
communication within a certain cell population,
AI-2 is a non-specic, universally recognized signal
that has the potential to communicate cell-density
to a mixed community of bacteria. The role of
quorum sensing and this novel signaling molecule is
further discussed below in the section Intra- and
inter-species interactions.
A link between sucrose-dependent biolm forma-
tion and competence with bacteriocin (also referred
to as mutacin) production has recently been estab-
lished (80, 130, 132), and production has been shown
to be controlled by quorum sensing, via luxS (109).
Bacteriocins are peptides with antibacterial activity.
Although the function of expression at high cell
density has not been established, antimicrobial pep-
tide activity results in cell lysis, which liberates DNA.
This extracellular DNA can then be taken-up and
recombined into the chromosomes of the remaining
bacteriocin-resistant cells within the biolm com-
munity. Thus, a linkage between competence and
bacteriocin production would be advantageous. It
has already been established that DNA is a constitu-
ent of the extracellular biolm matrix of P. aeruginosa
biolms (150), and to be required for biolm stability
during the early stages of development (163). The
increased incidence in antibiotic resistance through
gene transfer makes understanding the role of extra-
16
Davey and Costerton
cellular DNA in the function and persistence of bio-
lms a priority.
Another two-component regulatory system (HK/
RR11) in S. mutans was recently shown to play an
important role in biolm development and acid
resistance (94). Deletion of either the sensor or the
response regulator in this system resulted in a strain
that formed a biolm with reduced biomass and a
sponge-like architecture that was more readily washed
off the surface. Also, besides being more sensitive to
acid, the chain length was on the average twice as long
in the mutant, indicating that HK/RR11 affects a
number of physiological parameters. Inanother study,
inactivation of a putative transcriptional regulator
designated brpA (for biolm regulatory protein A)
resulted in a strain with an altered biolm and chain
length phenotype surprisingly similar to that demon-
strated by the HK/RR11 mutant (161). Hence, this two-
component system may intersect with the BrpA
pathway. Further studies are required to determine if
there is interaction between the comDE system, the
BrpA regulatory protein, and the HK/RR11system.
S. mutans is adept at synthesizing exopolysaccha-
rides from dietary sucrose via the enzymatic activity
of glucosyltransferases and fructosyltransferases. The
production of these extracellular polymers facilitates
adherence (155) and biolm formation (6, 104, 139),
and enhances the virulence of this organism (118,
119, 169). Recently the two-component signal trans-
duction system vicRK was found to control the
expression of glucosyltransferases and fructosyl-
transferases (146). A mutation in the histidine kinase
sensor vicK resulted in a strain that produced less
dextran and formed less stable biolms than the
parental strain. Also, transformation efciency was
altered, indicating that this two-component system,
like comCDE, regulates both biolm formation and
competence. However, addition of the signal peptide
competence-stimulating peptide, failed to restore
competence in the vicK mutant, hence the data
indicate that the vicK-initiated signaling pathway has
a distinct regulatory effect on the competence
development pathway. Another regulatory protein
(regM) has also been shown to control expression of
glucosyltransferases and fructosyltransferases (7),
and to affect biolm formation (161). Although the
regulatory mechanism has not been determined for
regM, this protein shows similarity to the catabolite
control protein A (CcpA), which is found in a variety
of bacteria. Catabolite repression (inactivation of
certain sugar-metabolizing operons in the presence
of specic metabolic intermediates or other carbon
sources) has been shown to be involved in biolm
development in P. aeruginosa (124), Escherichia coli
(54), and Bacillus subtilis (149), hence nutrient
availability and biolm formation are linked. Another
gene, designated ropA, encoding a putative trigger
factor (a molecular chaperone associated with the
50S ribosomal subunit and central to protein bio-
genesis and bacterial survival) has also been shown to
regulate the expression of glucosyltransferases (162).
In fact, a RopA-decient mutant is defective in acid
and oxidative stress tolerance, genetic competence,
and biolm formation, again demonstrating a link
between biolm development, competence, and
stress tolerance. Furthermore, using a relA-decient
strain, a link between the stringent response
(a translational control mechanism that represses
tRNA and rRNA synthesis during amino acid starva-
tion) and pathways involved in biolm formation, as
well as acid tolerance, has been established (91).
Like the other streptococcal species, a variety of
surface adhesins in S. mutans play a role in biolm
formation. For example, surface protein P1, also
known as antigen I/II and SpaP, is an adhesin that
interacts with a high-molecular-weight salivary
agglutinin and has been shown to be critical for
sucrose-independent adherence of S. mutans to the
tooth surface (3). Furthermore, a gene (srtA) enco-
ding a sortase enzyme has been shown to be
responsible for anchoring P1 to the cell surface and
thus plays a key role in modulating the surface
properties and cariogenicity of S. mutans (89).
Actinobacillus actinomycetemcomitans
A. actinomycetemcomitans is a virulent gram-negat-
ive periodontal pathogen associated with human
infections, including localized aggressive periodonti-
tis and systemic diseases, such as infective endocar-
ditis and brain abscesses [see review (110)]. This
bacterium expresses a wide variety of virulence fac-
tors [see review (33)], and like other oral isolates, it is
extremely adept at adherence and biolm formation
(31, 58). It is able to bind to and invade both epi-
thelial and endothelial cells (110112) and surface
structures and surface proteins involved in these
processes are being identied. Studies using electron
microscopy have shown that these cells produce long
thick mbrils composed of elaborate bundles of pili
(31, 32, 49, 138, 142). Through genetic analyses, a
gene cluster (p-rcp-tad) involved in the synthesis of
these surface structures has been identied (59, 60),
and studies have shown that these brils modulate
cellsurface, cellcell interactions, and biolm
development (42, 53, 59, 60). During invasion of the
17
host, A. actinomycetemcomitans migrates through
epithelial cells and comes in contact with collagen
and connective tissue. In a recent study, a reverse
genetics approach was used to identify genes
involved in adherence to components of underlying
connective tissue. A total of 2300 transposon-inser-
tion mutants were screened for changes in adhesion
to collagen and/or bronectin and 11 different loci
were identied. Among the genes identied in this
screen are those that are predicted to code functions
required for the biosynthesis of a molybdenum
cofactor MoCo (moaA and moeA), global regulatory
proteins of sugar metabolism (cAMP receptor protein
(Crp) also known as catabolite activator protein
(CAP) and Mlc), the heat modiable surface protein
(OMP34), putative membrane protein (YbjE), a
putative N6-adenine-specic DNA methylase, and
adenylate cyclase (CyaA) which generates cAMP a
global regulatory molecule in a variety of biological
systems (113). One gene, emaA, which has high
sequence similarity to the collagen-binding protein
YadA of Yersinia enterocolitica, was characterized and
found to be a collagen-specic surface-localized
adhesin protein. Further characterization is required
to determine the exact role of this protein in adher-
ence.
The extracellular polysaccharide matrix is essential
to the development and maintenance of biolm
structure. Genetic and biochemical studies on the
matrix of A. actinomycetemcomitans biolms have
shown that PGA, a linear polymer of N-acetyl-D-
glucosamine residues in b(1,6)-linkage, is a major
component of the extracellular matrix (63). This
polymer is also a major constituent of E. coli biolms
(157). Further studies by Kaplan et al. identied the
genes (pgaABCD) involved in the synthesis of PGA in
A. actinomycetemcomitans (63). Interestingly, they
also found that overexpression of the gene dspB,
which encodes dispersin B (a glycoside hydrolase),
causes dissolution of the exopolysaccharide matrix
and detachment of A. actinomycetemcomitans bio-
lm cells from the surface (6163, 133).
Porphyromonas gingivalis
P. gingivalis is a gram-negative anaerobe that resides
within subgingival plaque and is associated with se-
vere and chronic cases of periodontal disease, a con-
dition characterized by destruction of the tissue
supporting the teeth and, ultimately, tooth loss (12,
29, 41, 116). Like other oral isolates, P. gingivalis has
been implicated in systemic disease, specically this
bacterium is associated with atherosclerosis (13).
A number of factors are associated with the virulence
of this oral anaerobe, including a variety of proteases,
endotoxins, and collegenases; and the production of
surface structures such as mbriae and capsular
polysaccharide (50). This organism, like A. actino-
mycetemcomitans, attaches to and invades human
epithelial cells (28, 8587), connective tissue, and
endothelial cells (23, 25, 84), and invasion is mediated
by the expression of mbriae and a variety of surface
adhesins. It is evident that the growth of P. gingivalis
within the subgingival plaque is central to the disease
process, however, since genetic manipulation of
P. gingivalis became possible only a decade ago
[see review (83)], reports on the molecular genetic
basis of biolm formation in this organism are relat-
ively few. It has been shown that defects in poly-
phosphate kinase activity result in a strain that is
attenuated in biolm formation, indicating that the
synthesis of this storage polymer (short-chain polyP)
affects biolm development (10). In another more
recent study, it was found that a mutation in gftA,
which is a putative glycosyl-transferase, inhibits auto-
aggregation, attachment to epithelial cells, and the
maturation of mbriae on the surface (120). Also, the
pili of P. gingivalis have been found to bind to salivary
proteins that are part of the acquired pellicle on the
tooth surface, such as proline-rich salivary protein 1
and statherin (1, 90). This result is particularly inter-
esting since many gram-negative organisms also
require mbriae (or pili) for initial interactions with a
surface (123, 131, 158).
Intra- and inter-species
interactions
In vivo, maturation of plaque relies on select physical
interactions between a broad spectrum of distinctly
different bacteria. Coaggregation has been dened as
the recognition and adhesion between genetically
distinct bacteria (165). This physical interaction of
bacterial cells was rst described over 30 years ago by
Gibbons and Nygaard (39). Since then, researchers
have been dissecting the numerous cellcell interac-
tions that are the foundation of this elaborate biolm
community. An example of this type of study has been
performed by Kolenbrander and colleagues in which
pairs of organisms were mixed and assayed for
coaggregation by monitoring the rapid settling of
strains out of suspension (65, 67, 69, 73, 74). As is the
case for initial adhesion events, there are some
excellent reviews of studies describing coaggregation
in oral bacteria (71, 75, 135, 165). These physical
18
Davey and Costerton
interactions have been shown to occur between
organisms of the same or different genera, and can be
disrupted by the addition of certain sugars (15, 38, 64,
7274, 106). The results of these coaggregation assays
have been used to develop a model for bacterial
interactions before and during the initial stages of
plaque biolm, as well as in the accretion of late
colonizers to pioneer species already on the tooth
surface. As discussed above, S. gordonii are especially
adapted to primary colonization of saliva-coated
surfaces, and this species can coaggregate with
F. nucleatum, yet not with A. actinomycetemcomitans,
which is a late colonizer (68). Therefore, F. nucleatum
plays a unique role, since it can interact with both
species and is therefore able to bridge early and late
colonizers. In fact, F. nucleatum has been shown to
coaggregate with a variety of early and late colonizing
bacteria (4, 78). This includes T. forsythia and the
interaction has been shown to be mediated via the
surface-associated (and secreted) BspA protein (52).
Besides the temporal considerations inuencing cell
cell interaction during the maturation of plaque, the
surroundings clearly affect contact. Bacteria that
inhabit certain sites within the oral cavity tend to
interact with other bacteria isolated from the same
location [e.g. bacteria isolated from the tongue coag-
gregate best with other tongue-localized bacteria
(51)], suggesting a direct spatial organization in the
formation of oral biolms. Also, a number of lines of
evidence indicate that coaggregation is directed
by species-specic interactions. For example, the
adherence of P. gingivalis to S. gordonii involves a
discrete region of the streptococcal surface polypep-
tide SspB, designated BAR (22). However, P. gingivalis
does not adhere to S. mutans, even though this spe-
cies has a putative homologue of SspB with high
sequence similarity, which is designated SpaP. Hence,
subtle differences in the sequence of this surface
polypeptide eliminate this interaction, thus support-
ing the hypothesis that cellcell interactions are
highly selective. Also, as mentioned above, specic
interactions can be disrupted by the addition of
sugars (such as lactose or galactose), also indicating
specic receptorligand interactions (9, 70, 160).
Genetic and molecular techniques have been used
to identify genes and dene mechanisms required for
these cellcell interactions. Both spontaneous and
transposon-insertion mutants defective in coaggre-
gation within and between species have been isolated
(2, 5, 64, 164). Coaggregation between P. gingivalis
and T. denticola, which are anaerobic colonizers of
the subgingival area and associated with severe forms
of periodontal disease, has been studied. Interaction
between these two organisms was investigated by
using both T. denticola and P. gingivalis mutants and
evaluating the effect of mutations on cellcell
adherence and, in addition, biolm formation (156,
168). Interestingly, T. denticola was unable to form a
biolm in pure culture; however, it was able to attach
to and grow with pre-attached P. gingivalis cells,
demonstrating a cooperative, synergistic effect of
mixed cultures. In fact, several studies have shown
that mutualistic associations among oral bacteria
have enhanced the growth of one or both of the
bacterial partners (34, 105, 126). Among the T. den-
ticola genes identied in this study, were those that
code functions involved in the synthesis of the cyto-
plasmic lament (cfpA), motility (gE), and cell sur-
face proteins (Msp, PrtP). Also, in another study it
was reported that P. gingivalis mbriae and T. den-
ticola dentilisin play a role in coaggregation between
these two organisms (44). These studies provide data
indicating that T. denticola surface proteins and
structures are involved in cellcell interactions and
synergistic biolm formation. In another genetic
study, S. gordonii mutants were identied that were
defective in coaggregation with other streptococci,
but these mutants were fully procient in coaggre-
gation with organisms of other genera (16, 17, 166),
supporting the idea that oral bacteria have multiple
adhesins for interaction with different bacteria.
Examples of identied adhesins include FimA (125),
ScaA (2, 69), ScbA (18), PsaA (140), and SsaB (36, 37).
Studies have shown that many of these adhesins are
lipoproteins and, interestingly, are part of ABC
transporter systems. ScaA is predicted to be a sur-
face-localized lipoprotein that is part of a manganese
uptake ABC transporter system required for growth
on low manganese concentrations (76). ScbA from
Streptococcus crista is also an ABC transporter and
although the substrate transported by this system has
not yet been determined, based on its sequence
similarity to ScaA (93% at the amino acid level),
ScbA may also play a role in metal transport (2). Also,
the FimA protein, which is a component of mbriae,
was also shown to play a role in cellcell interactions
(30). All of these lipoproteins belong to the LraI
(lipoprotein receptor antigen I) family of proteins and
have been shown to be involved not only in coag-
gregation (69), but also, as discussed above, in
binding components of the salivary pellicle as well
(55). In addition, there is evidence that some of these
lipoproteins are found in the extracellular medium
and therefore may be secreted (30, 125), indicating
that essential cellular functions play an additional
role in cellcell interactions. Recent studies of
19
proteins like SspA and SspB of P. gingivalis have gone
a step further and identied a 100 amino acid
domain conserved between these proteins that is
required for binding to the partner bacterium
S. gordonii (5). Another surface protein, encoded by
gbpC, was identied in a mutant hunt for strains that
no longer aggregated in the presence of dextran (141).
GbpC is one example of a class of probable surface
proteins required for glucan-dependent binding.
These adhesins may recognize glucan-like mole-
cules on the surfaces of bacteria with which they
coaggregate.
High cell density and microbial diversity make the
oral biolm a unique and extremely complex biolo-
gical system. As we have discussed throughout this
review, both cooperative and competitive interactions
are involved in the maturation and function of this
complex community, hence interspecies signaling
(communication) is a key aspect. Clearly, the close
proximity of bacterial cells in this community facili-
tates cellcell communication. In essence, cells con-
vey their presence to one another by secreting and
sensing the accumulation of metabolic end-products
and signaling molecules. Density-dependent signa-
ling (quorum sensing) has been shown to be one key
mechanism of communication and, as discussed
above, this type of signaling pathway is involved in
many aspects of gene regulation during biolm
development. Type 2 autoinducer (AI-2) is a density-
dependent signaling molecule produced by a number
of different bacteria. It is important to note, AI-2 does
not represent a family of highly similar molecules,
instead the AI-2 molecule from different bacteria is
identical, and many different species sense this signal.
Hence, AI-2 is a non-specic, signal that communi-
cates cell-density between bacterial genera. Although
studies on the role of AI-2 during development of
plaque are only just beginning, it has already been
shown that inactivation of luxS (which is required for
AI-2 production) in S. gordonii and P. gingivalis, does
not affect mono-species biolm formation in either of
these organisms. However, a LuxS-null strain of
S. gordonii was shown to be unable to form a mixed
species biolm with a LuxS-null strain of P. gingivalis
(14, 107), indicating that the AI-2 signaling molecule
may play a more signicant role during the develop-
ment of multi-species biolm communities.
Conclusions
Historically, microbiologists have been limited to
studies of pure cultures isolated from natural and
pathogenic ecosystems. All of the research sum-
marized in this review has involved in vitro analysis
of planktonic or biolms cells growing in dened
liquid media. Because we know that environmental
factors and the presence of other species of bacteria
greatly inuence the production of signals, and the
response elicited from receiving cells; the ne details
of signal communication will likely differ when we
can analyze these processes by direct observation in
living mixed species communities. However, these
in vitro studies have an intrinsic value and a vital
role in opening up this eld, because they reveal the
basic machinery of cellcell signaling in microbial
communities. From this work we know what signals
are produced by cells of certain species, which
receptors they activate in cells of the same and other
species, and the types of cellular activities that are
regulated by these signals. While signal communi-
cation may actually operate differently in different
parts of the natural biolms that occupy the gingival
space, in health and disease, the basic nuts and
bolts of the signal machinery they use are identied
and dened in this exciting body of work. The future
challenge lies in the ability of the dental research
community to develop research programs that
Fig. 2. Fluorescent in situ hybridization (FISH) of plaque.
A subgingival biolm sample was taken with a curette
from an 8 mm periodontal pocket of a patient with rapid
progressive periodontal disease. The sample was imme-
diately xed with 4% paraformaldehyde, and hybridized
with a (green) FITC-labeled EUB338 oligonucleotide probe
(a universal probe for detection of bacteria), and a fuso-
bacteria (Cy3-labeled) FISH probe (red). The sample was
analyzed with Confocal Laser Scanning Microscopy.
Shown are the long rod-shaped fusobacteria cells
throughout the biolm matrix tightly associated with a
morphologically diverse community of bacteria (Courtesy
of Dr Christoph Schaudinn, Center for Biolms, School of
Dentistry, University of Southern California).
20
Davey and Costerton
integrate in vitro ndings with in vivo studies. For-
tunately, the development of new technologies has
made studies on the microbiology of the human oral
cavity quite amenable to this merger. One most
notable advance is the use of uorescence in situ
hybridization (FISH) to identify and localize partic-
ular species within biolm communities. FISH is a
well-established technique in environmental micro-
biology studies. It is typically based on specic
hybridization of uorescently labeled oligonucleo-
tide probes to rRNA target sequences within per-
meabilized intact bacterial cells. Figure 2 is an image
of a FISH experiment performed on a subgingival
biolm sample, clearly demonstrating the potential
of this technique. In this analysis, the biolm sample
was treated with two separate probes. One probe
was designed to selectively hybridize to fusobacteria
(red) and the other was used to detect all bacteria
(green). The large rod-shaped fusobacteria located
throughout the biolm matrix are shown associated
with numerous morphologically diverse bacterial
cells. FISH has also been combined with new dental
techniques (such as the use of retrievable enamel
chips or other retrievable carriers) to study microbial
interactions during plaque maturation in vivo (117,
127, 128, 151, 159). Needless to say, data obtained
from in vitro studies on cellcell associations, signal
communication, and culture-based identication of
bacteria are essential in the design of these in vivo
studies. Furthermore, the concerted efforts of inno-
vative scientists from a variety of disciplines have led
to the development of these techniques. The future
of oral biolm discoveries will certainly rely on the
synthesis of data from different research strategies
and such collaborative efforts.
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26
Davey and Costerton
Techniques for the growth of
Porphyromonas gingivalis
biolms
MARY E. DAVEY
Periodontal disease is a biolm-mediated disease, and
Porphyromonas gingivalis is an anaerobic bacterium
that resides within the biolm community in the
subgingival crevice of the oral cavity and is regarded
as a major causative agent in the initiation and pro-
gression of severe forms of this disease. As an oppor-
tunistic oral pathogen, the ability to proliferate within
and disseminate from subgingival biolm (plaque) is
central to its virulence. Here we report techniques for
the growth and analysis of P. gingivalis biolms. We
showthat three different wild-type strains (W83, ATCC
33277, and 381) differ greatly in their ability to form a
biolm, and we demonstrate the difference of biolm
development in a static system versus a system where
nutrients are constantly owing.
Although culturing bacteria in broth has been
instrumental to the study of microbial biology, pure-
culture planktonic growth is rarely how bacteria exist
in nature. It is now widely recognized that most
bacteria found in their natural environments persist
in complex communities of microorganisms attached
to surfaces or associated with interfaces (biolms),
not as free-oating organisms, and not alone (12, 14).
In fact, biolms throughout nature represent struc-
tured biological systems where interspecies interac-
tions and metabolic cooperation are fundamental to
the development and persistence of the community
(7, 49). Although this basic aspect of microbiology
has long been recognized (31, 6062), because of
technical limitations it is only within the past few
decades that researchers have begun to explore the
structure and function of biolms. Advances in
techniques for growing biolms as well as in
molecular methods for monitoring the location,
growth, and metabolic activities of bacteria within
biolms, are now providing the means to study this
fascinating aspect of microbiology (4143, 54).
Although many species-specic behaviors exist
that reect the unique requirements of each organ-
ism, some general concepts for the formation of a
variety of bacterial biolms have been observed (49).
Biolm formation can be viewed as one part of a
developmental cycle involving a series of distinct
steps. To form a biolm, the bacteria rst attach to a
surface and adjust to this surface-attached state. If
the environment is conducive to growth, the bacteria
then grow on the surface and further develop into a
mature matrix-encased biolm. To complete the
cycle the bacteria detach, disseminate, and colonize a
new surface. The complexity of this developmental
process indicates that many genes are likely required.
During the past decade, a series of studies has
addressed the regulation and molecular genetic basis
of biolm development in a variety of disease-caus-
ing model organisms, including Pseudomonas sp. (46,
47), Escherichia coli (23, 24, 51, 56), Staphylococcus
aureus (29, 30), and Vibrio cholerae (5, 33, 45, 58, 59).
The environmental parameters that control this
switch vary greatly among bacteria (17, 47, 51).
Studies have shown that nutrition plays a critical role
in biolm development (6). Interestingly, organisms
with distinctly different planktonic growth rates can
form a mixed species biolm and coexist (52, 55).
Besides nutrient availability, other environmental
signals have been found to affect biolm formation,
including osmolarity, pH, iron availability, oxygen
tension, and temperature (21, 46, 47, 50, 51, 53).
Biolms can be composed of a phenotypically
complex population of cells that develop from a
single species or a community of organisms derived
from a mixture of microbial species, and they can
form on a vast array of abiotic or biotic surfaces.
Structural organization is clearly a hallmark of bio-
lm communities that differentiates this mode of
27
2006 The Author.
PERIODONTOLOGY 2000
growth from liquid cultures. The application of con-
focal scanning laser microscopy to biolm research
radically altered our perception of biolm structure
and function (40). Confocal scanning laser micro-
scopy allows the visualization of fully hydrated sam-
ples. Having a system that could microscopically
examine hydrated biolms in situ was the key to
revealing their elaborate three-dimensional structure
(14, 10, 40). Interestingly, biolms from mono-spe-
cies laboratory simulations or those formed in nature
exhibit similar overall structural features. Most bio-
lms have been found to show some level of
heterogeneity in that patches of cell aggregates
(microcolonies) are interspersed throughout an
extracellular matrix that is variable in density, cre-
ating open areas where highly permeable water
channels are formed (11, 13, 58). These water chan-
nels provide an effective means of exchanging
nutrients and metabolites with the bulk water phase,
thereby enhancing nutrient availability and poten-
tially the removal of toxic end products (12). The
metabolic characteristics of bacteria within a biolm
community are distinctly different from their
independent planktonic counterparts. Microenvi-
ronments are formed within these spatially well-
organized systems; consequently, bacteria located in
different positions within the biolm are exposed to
distinct environmental signals. For instance, cells
situated near the center of a microcolony may
experience lower oxygen tensions or nutrient avail-
ability than the cells located near the periphery.
One of the best-studied biolm communities is the
oral biolm. Studies on bacterial colonization of the
tooth surface have shown that the early colonizers are
typically gram-positive organisms, while the late or
secondary colonizers are the more fastidious gram-
negative anaerobes (34, 35). It is likely that late
colonizers take advantage of an altered environment
created by their predecessors, e.g. a decrease in redox
potential or the availability of substrates. Early colo-
nizers can also provide new cellcell attachment
sites, and recent in vitro studies using saliva-condi-
tioned ow cells have clearly demonstrated the
importance of coaggregation between different gen-
era in the development of this complex community
(20, 22). P. gingivalis is recognized as one of the late
colonizers during subgingival plaque development.
This anaerobe is fastidious, requiring nutritional
supplements (hemin and menadione) and an anaer-
obic environment for growth. Therefore, this bacter-
ium likely requires the colonization and metabolic
activity of other organisms to become established
within the biolm (57). Coaggregation studies have
shown specic interactions between P. gingivalis and
a variety of other microbial species, such as Strepto-
coccus species and Treponema denticola (28), thus
supporting the model that early colonizers provide
attachment sites and possibly a niche for secondary
colonizers to become established.
Periodontal disease is best described as a microbial
shift disease. The progression of the disease is
strongly associated in a changeover of the microbial
ora to anaerobic metabolism. Although the disease
is polymicrobial in nature, P. gingivalis is one of the
key anaerobes associated with the microbial shift and
progression of the disease (26, 36). Interestingly,
P. gingivalis is also one of the primary oral pathogens
associated with systemic disease, such as athero-
sclerosis (9). A number of factors are associated with
the virulence of this organism, including a variety of
proteases, endotoxins, and collegenases; and the
production of surface structures such as mbriae
and capsular polysaccharide (32). This opportunistic
pathogen also attaches to and invades human epi-
thelial cells (19, 3739), connective tissue, and
endothelial cells (16, 18, 36), and invasion is medi-
ated by the expression of mbriae and a variety of
surface adhesins. As an oral opportunistic pathogen,
biolm growth is central to its virulence. Hence,
studying P. gingivalis physiology and gene expression
during biolm development is fundamental to our
understanding of the pathogenicity of this organism.
Here we report the development of techniques for the
growth and analysis of P. gingivalis biolms.
Systems to grow P. gingivalis
biolms
Static 96-well microtiter plate assay
A 96-well plate assay to study P. gingivalis biolm
formation was developed, based on previous reports
(25, 46). Three different media supplemented with
hemin and menadione, including Trypticase Soy
Broth, Todd Hewitt Broth, and a chemically dened
medium developed for P. gingivalis (44) were tested
under various conditions. In addition, the phase of
growth and the size of the inoculum were tested. For
these studies, P. gingivalis strains were maintained
in a COY anaerobic chamber on trypticase soy
agar plates supplemented with 5% debrinated
sheep blood (North-East Laboratory, Waterville, ME),
1 lg/ml hemin and 1 lg/ml menadione (BAPHK).
The strains used in this study were 33277 (American
Type Culture Collection); W83 (Obtained from
28
Davey
Christian Mouton, Laval University, Quebec City,
Quebec, Canada), and 381 (obtained from Howard
Kuramitsu, State University of Buffalo, Buffalo, NY).
Although P. gingivalis does form biolms in both
Trypticase Soy Broth and Todd Hewitt Broth, chem-
ically dened medium supplemented with 1% tryp-
tone provided the most robust and reproducible
biolms; in addition, a chemically dened medium is
advantageous for nutritional studies. We therefore
chose to work with chemically dened medium
supplemented with 1% tryptone.
To perform this assay, a Todd Hewitt broth culture
was inoculated from a fresh agar plate of P. gingivalis
that had been grown in a COY anaerobic chamber for
48 h. After 24 h, the full density THB broth culture
was diluted 1 : 10 in chemically dened medium
supplemented with 1% tryptone, aliquots were then
placed into 96-well polystyrene at-bottom plates.
We found that the test medium did not have to be
pre-reduced. However, we did nd that a high con-
centration of inocula (1 : 10 dilution) reproducibly
insured growth after subculture. For biolm devel-
opment, the microtiter plate was incubated at 37C
under anaerobic conditions for 48 h. The culture
supernatant was then removed and the plate was
washed twice by immersion in distilled water. The
biolm-containing plates were air-dried for 1 h and
then the biolm was stained with 0.1% safranin for
15 min (100 ll/well), washed again twice in distilled
water. The amount of biolm was then assessed
macroscopically and, to quantify the amount of bio-
lm, 95% ethanol (100 ll/well) was added for 5 min
to solubilize the safranin. The ethanol was transferred
to a new microtiter dish and semi-quantitative data
were obtained by determining the absorbance
(492 nm) with a plate reader.
Static 12-well microtiter plate assay
As a further step in the development of assays to study
P. gingivalis biolm formation, a method to grow and
microscopically examine P. gingivalis biolms using
12-well polystyrene plates (Falcon 353225) was devel-
oped. To assay biolm formation in this system, the
culture was diluted, as described above in the 96-well
assay. Samples (750 ll/well) were aliquoted into the
wells and incubated at 37C under anaerobic condi-
tions for 48 h. The plate was removed from the incu-
bator and the wells were washed twice with fresh
medium. CDM (1 ml) containing the uorescent stain
Syto-9 (Molecular Probes, Eugene, OR) was added to
the well to stain the bacterial cells, and the plate was
incubated for 15 min in the dark. The wells were then
washed twice more with fresh medium. The biolms
were examined by Confocal Laser Scanning Micros-
copy (Leica TCSSP2 with AOBS) using a 40 water
immersion objective.
Flow-cell system
We constructed a ow-cell system similar to that
used during our previous research on Pseudomonas
aeruginosa biolms (15). The system was assembled
and sterilized by ushing the chamber and inlet/
outlet tubing with a solution of 0.4%hypochlorite for
3 h. The ow-cell was rinsed with sterile water then
run dry (three times), followed by rinsing overnight
(1216 h) with sterile water to remove the residual
hypochlorite. Water was then removed from the
system, and the pre-reduced anaerobic medium
(CDM supplemented with 1% tryptone) was intro-
duced and allowed to ow through the plumbing and
ow cell chamber for 1 h. To inoculate the ow cell,
the ow through the system was stopped and the
tubing upstream of the chamber was clamped (to
prevent the back-ow of the bacteria inoculated into
the system). The inoculum was injected through the
tubing (just upstream of the chamber) with a tuber-
culin ne-needle syringe and the hole in the tubing
was sealed with liquid silicone. The inoculum was
300 ll of an overnight grown culture, washed, and
diluted 1 : 10 into fresh CDM containing L-cysteine
(0.02%). After the bacteria were given 1 h to initiate
biolm formation, the ow through the system was
resumed. The entire ow cell apparatus was incu-
bated at 37C for 4 days, in a portable glove bag
(Instruments for Research and Industry Inc., Chel-
tenham, PA) inated with nitrogen. To visualize the
bacteria with confocal scanning laser microscopy, the
bacterial were stained with uorescent dye, Syto-9
(Molecular Probes, Eugene, OR). The cells were
stained with Syto-9 for 1 h without ow and then the
ow was resumed to wash away the dye. The ow
was continuous while the images were collected.
Findings from the different growth
systems
96-well microtiter plate assay
Three different wild-type strains of P. gingivalis (W83,
ATCC 33277, and 381) were evaluated in the 96-well
microtiter plate assay. As shown in Fig. 1A, the
amount of biolm produced by these three strains
varied greatly. Strain W83 was decient in biolm
29
Techniques for the growth of Porphyromonas gingivalis biolms
formation, yet strain 381 formed a robust biolm (as
indicated by the amount of safranin incorporated
into the biolm). Strain 33277 was found to be less
efcient than 381, yet was able to form a modest
biolm. Quantitative data of this strain comparison
are shown in Fig. 1B. Again, the data indicate that
W83 was unable to form a biolm, strain 33277
formed a modest biolm (absorbance of 0.09 0.01),
while strain 381 was the most procient (absorbance
of 0.17 0.03).
12-well microtiter plate system
For microscopic examination, biolms of the three
different P. gingivalis strains were grown in 12-well
microtiter plates. Representative top-down views of
biolms grown in this system are shown in Fig. 2A. As
was shown in the 96-well assay, W83 was decient in
biolm formation. Very few cells were observed on
the surface, indicating that this strain is unable to
initiate biolm formation. Small microcolonies were
visible in the 381 biolm, however, strain 33277
appeared to form more of a monolayer of cells, with
very few cell aggregates (microcolonies). Figure 2B
shows an xz plane of a reconstructed three-dimen-
sional image (i.e. from the side looking horizontal) of
the biolm formed by strain 381. This image shows
that even with strain 381, which forms the most
developed biolms of the three strains, biolm
development was limited. The biolm was only
approximately 25 lm in thickness and little topology
was evident in this static (non-owing) system.
Flow-cell system
To obtain cells at different stages of biolm devel-
opment requires a owing system that can be used
to grow and monitor P. gingivalis biolm develop-
ment over time. The system we have developed is
based on a design reported by Christensen et al. (8).
The ow cell was adapted for anaerobic conditions,
because previous studies had shown that P. gingi-
valis does not initiate biolm formation under aer-
obic conditions (27). The pump that we used was a
Peri-Star high-performance digital peristaltic pump
(World Precision Instruments, Sarasota, FL) which
provides a steady ow of media through the cham-
ber with minimal pulsing and thus minimal distur-
bance to the biolm. In this system, cells growing on
the surface are exposed to a continuous ow of fresh
nutrients (chemically dened medium supplemen-
ted with 1% tryptone). Because it was the best bio-
lm former, strain 381 was used for these analyses.
Figure 3A shows a top-down view of a biolm grown
for 4 days. A view from the side looking horizontally
at the same biolm formed by strain 381 is shown in
Fig. 3B. The biolm produced by this non-motile,
anaerobe is remarkably complex. It demonstrates
mature biolm characteristics, i.e. large macrocolo-
nies that are surrounded by open areas. These
characteristics have been observed in biolms
formed by other bacteria, such as the aerobic
motile bacterium, P. aeruginosa. In addition,
and most importantly, the biolms formed in this
owing system show much more heterogeneity than
the biolm formed in the static system described
above. The large macrocolonies in the biolm were
found to be almost 200 lm in thickness, which is
approximately 10-fold thicker than the biolm
formed by this strain in the same medium in the
static system.
Summary
We have described three systems used to grow
P. gingivalis biolms, two static systems and one
ow-cell system. The rst was a 96-well microtiter
dish assay, based primarily on a protocol developed
by Denis Mayrand and colleagues (57). This assay
W83
33277
381
0
0.05
0.1
0.15
0.2
0.25
W83 33277 381
A
b
s
o
r
b
a
n
c
e

(
4
9
2

n
m
)
A
B
Fig. 1. Biolm formation phenotype of three different
P. gingivalis wild-type strains (W83, 33277, and 381) in
96-well microtiter plates. The biolms were grown as
described for 48 h: (A) safranin-stained biolms and (B)
the corresponding quantitative data. The encapsulated
strain W83 was unable to form a biolm in this assay.
Strain 33277 formed a modest biolm, and strain 381 grew
to the highest density, as indicated by the amount of
safranin incorporated into the biolm.
30
Davey
can be used as a rst step in assessing biolm for-
mation by different strains of P. gingivalis. Further-
more, by modifying the CDM used in this assay by
the addition of different carbon and energy sources,
one can assess nutritional effects on biolm forma-
tion. Although many studies on biolm development
use saliva as a preconditioning lm or nutrient
source, we propose that serum is more relevant for
the study of P. gingivalis, because it resides in the
subgingival crevice and is therefore more likely to be
exposed to gingival crevicular uid. It has been
shown that P. gingivalis can grow on human serum
diluted 1 : 10 in water that is supplemented with
0.01% hemin (57), and our preliminary studies indi-
cate that P. gingivalis can make a biolm when
growing in CDM supplemented with 10% human
serum (unpublished data). The development of our
protocol using a CDM will be instrumental in future
nutritional studies.
The second system we describe is a 12-well assay
that can be used to microscopically examine biolms.
Since it is not a owing system, this system cannot be
used to study the different stages of biolm devel-
opment over time, however it is a good method to
inspect the biolms visually at an early time point.
Using this assay, we were able to obtain data showing
that the three different wild-type strains used in this
study varied greatly in their ability to form a biolm.
Most striking is our nding that strain W83 is unable
to initiate biolm formation. This strain is distinct
from the other two strains. Unlike 33277 and 381,
strain W83 is encapsulated and does not typically
produce mbriae. Since both of these structures af-
fect the surface properties of the cell, and thus affect
cellcell and cellsurface interactions, we propose
that this is likely to contribute to the inability of this
strain to attach to the surface and initiate biolm
formation.
W83 33277 381
40.0 m
40.0 m 40.0 m
A
B
Fig. 2. Microscopic examination of biolms formed by
three different P. gingivalis wild-type strains (W83, 33277,
and 381) grown in 12-well microtiter plates. The biolms
were grown for 48 h and images were obtained with a
confocal scanning laser microscope, as described. Top-
down views of the biolms (A) and an xz plane (i.e. from
the side looking horizontally) of the biolm formed by
strain 381 (B). The three wild-type strains demonstrate
distinctly different biolm phenotypes. Strain W83 was
unable to initiate biolm formation. However, strain
33277 formed a modest biolm and strain 381 developed
into a biolm that covered the surface and demonstrated
the development of microcolonies (cell aggregates). In (B)
the biolm formed by strain 381 in this static system was
approximately 25 lm in thickness.
31
We also describe the adaptation of a ow-cell sys-
tem to grow biolms of P. gingivalis anaerobically.
The data presented are the rst report of a fully
developed P. gingivalis biolm (constant ow of
nutrients for 4 days). As described above, a 4-day-old
P. gingivalis biolm demonstrates characteristics that
are typically associated with a mature biolm, i.e. a
heterogeneous architecture consisting of large
macrocolonies surrounded by open areas not a
dense monolayer of cells on the surface. Also, the
thickness of the biolm was found to be approxi-
mately 200 lm, which is comparable to measure-
ments obtained in studies on other model organisms.
This indicates that although there are probably many
differences in the nutritional and environmental
parameters that regulate biolm development by
different bacteria, there are some characteristics
(such as biolm structure) that are conserved. It has
200.0 m
B
A
Fig. 3. Three-dimensional reconstruction of a 4-day-old
P. gingivalis strain 381 biolm using confocal scanning
laser microscopy. The large macrocolonies in the biolm
were found to be almost 200 lm in thickness. A top-down
view of the biolm (A), and the xz plane (i.e. from the side
looking horizontally) (B). The biolm demonstrates ma-
ture biolm characteristics, i.e. large macrocolonies that
are surrounded by open areas. Scale bar on (A) 200 lm.
32
Davey
been proposed that this architecture supports survi-
val by facilitating the delivery of nutrients and the
removal of end products (5), thus the development of
a biolm into this architecture may be a conserved
characteristic. Further studies on biolm develop-
ment will likely enlighten us to other strategies that
are used by bacteria to survive under different growth
parameters.
This ow-cell apparatus can be used to examine
biolm development under different growth condi-
tions and to obtain biomass for chemical analyses or
for the isolation of RNA or protein. The system is
based on the principle that fresh medium is con-
tinuously supplied to a ow cell chamber. The walls
of the chamber provide surfaces on which biolm
develops. One side of the chamber is a glass cover-
slip, and the chamber itself is small enough to be
mounted on the stage of a microscope, so the biolm
can be imaged by either epiuorescent or confocal
microscopy. In addition, each channel in the four-
channel ow cell can be inoculated with a different
strain by using a ne-gauge needle and syringe. The
advantage of the four-channel system is that a single
media reservoir and a single pump can be used to
feed the four channels simultaneously, helping to
maintain consistency from channel-to-channel and
allowing the analysis of up to four different strains
per experiment. Flow cells such as the one described
here have been an instrumental tool in the study
of biolm morphology, chemistry, and physical
parameters using a variety of model organisms (48,
49). Now that we have a system to grow P. gingivalis
biolms, we can begin to study the nutritional and
environmental parameters that regulate develop-
ment, as well as identify the genes that are expressed
during different stages of biolm growth. Since bio-
lm formation is essential to the persistence of this
organism, such studies should lead to a greater
understanding of how to control this opportunistic
oral pathogen.
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35
The effect of periodontal therapy
on the composition of the
subgingival microbiota
ANNE D. HAFFAJ EE, RI CARDO P. TELES & SI GMUND S. SOCRANSKY
Periodontal diseases are infections caused by bac-
teria residing in subgingival biolms. The treatment
of the infectious component of periodontal diseases,
and the replacement of damaged or lost periodontal
tissues, are typically achieved using different
approaches. Mechanical therapies, including scaling
and root planing, and surgery, are aimed at improv-
ing clinical conditions by lowering the microbial load
either by physical removal of plaque or by radical
alteration of the subgingival habitat. Antimicrobial
approaches, including the use of systemically and
locally administered antibiotics, directly target sub-
gingival species residing in the plaque biolm or in
the adjacent epithelial tissues lining the periodontal
pocket. Finally, replacement of destroyed tissues and
teeth can be achieved by the use of gingival grafting,
osseous replacement and tooth implant techniques.
There are literally hundreds of publications in the
literature (some of these are reviewed in Ref. 35)
examining the effects of different therapeutic
approaches for the treatment of periodontal infec-
tions. By and large, the majority of these studies
report an improvement in clinical parameters post-
therapy for differing periods of time depending on
the study design. Often, adjunctive test agents pro-
vide a better therapeutic response than the control,
which is typically scaling and root planing. However,
because the results from such studies are presented
as mean values for the different treatment groups, it
is difcult to know if a successful therapy was in-
deed successful for all or most subjects in that group.
Despite the number of studies evaluating different
therapies in the literature, periodontal therapy has
changed little over the past 50 years. Typically, a
patient will receive a clinical evaluation followed by
full-mouth scaling and root planing and oral hygiene
instruction. Deep periodontal pockets that remain
after this initial treatment phase will be treated by
surgery with the goal of reducing or eliminating
pockets in order to reduce the bacterial load and
facilitate good oral hygiene. Failure of these standard
therapies to halt disease progression may lead to
other treatment strategies, including the use of sys-
temic and/or local antibiotic therapy. At this stage,
some clinicians may employ microbial testing of
plaque samples from deteriorating sites to guide
antibiotic therapy.
The use of microbial parameters to determine the
most appropriate periodontal treatment for a patient
has appeal. Given the infectious nature of periodontal
diseases, specic knowledge of the bacterial species
in plaque samples of a patient should be useful to
guide periodontal therapy. However, this approach
may be hampered by the need to analyze multiple
plaque samples from multiple teeth so that a com-
prehensive assessment of the subgingival microbiota
can be made. With currently available microbial
testing procedures, this would be expensive. Fur-
thermore, even if full-mouth microbial assessments
were made, the most benecial therapy for a specic
microbial prole is not known (35).
Many of the studies examining the effect of dif-
ferent periodontal therapies on clinical outcomes and
the subgingival microbiota have been reviewed by
Teles et al. (35) in this issue of Periodontology 2000.
The reported studies typically focused on one or a
small number of species and often, for technical
reasons, were limited in the numbers of samples and
subjects that could be examined. In order to provide
a broader description of the nature of changes in-
duced by various periodontal therapies on the com-
position of the subgingival microbiota, we employed
high-throughput techniques that permitted exam-
ination of large numbers of samples within a subject,
219
2006 The Authors.
PERIODONTOLOGY 2000
7 1 6 1 5 1 4 1 3 1 2 1 1 1 0 1 9 8 7 6 5 4 3 2 1
p a l f n a m d i W d e i f i d o M
p a l f d e n o i t i s o p e r y l l a c i p A
e l o z a d i n o r t e M & n i l l i c i x o m A c i m e t s y S
e l o z a d i n o r t e M c i m e t s y S
n i l l i c i x o m A c i m e t s y S
T l a c o L e n i l c y c a r t e
g n i n a e l c l a n o i s s e f o r P
n i c y m o r h t i z A c i m e t s y S
e n i l c y c y x o D c i m e t s y S
e n i l c y c y x o D e s o d w- o L
3 9 4 = s t c e j b u s n
P R S D E V I E C E R S T C E J B U S L L A
s p u o r g t n e m t a e r T
9 4 2 9 1 2 6 3 8 1 4 1 0 2 7 5 1 1 1 7 2 6 2 3 3 3 2 3 2 8 1 4 2
Fig. 1. Treatment groups included in the analyses. The columns represent 17 treatment groups. The rows indicate the
treatments employed for each group, as indicated by the red boxes. The number of subjects in each treatment group is
presented in row 1. SRP, scaling and root planing.
0 4
7 4
5 5
2 6
Percentage of
n o i t a l u m u c c a e u q a l P
0 4
7 4
5 5
2 6
0 7
s s e n d e r l a v i g n i G
0 2
8 2
5 3
3 4
0 5
g n i b o r p n o g n i d e e l B
0
0 . 1
5 . 1
0 . 2
n o i t a r u p p u S
8 . 2
0 . 3
2 . 3
4 . 3
h t p e d t e k c o p n a e M
2 . 3
3 . 3
4 . 3
5 . 3
6 . 3
l e v e l t n e m h c a t t a n a e M
6 . 3
1 0 0 . 0 < P
1 0 0 . 0 < P 1 0 0 . 0 < P
1 0 0 . 0 < P
1 0 0 . 0 < P
1 0 0 . 0 < P
5 . 0
m m m m
s e t i s
Percentage of
s e t i s
0 7
2 1 6 3 0 2 1 6 3 0 2 1 6 3 0
s h t n o M
Fig. 2. Mean ( standard error of the mean) for percent-
age of sites with plaque accumulation, gingival redness,
bleeding on probing and suppuration, as well as mean
pocket depth and attachment level, at baseline, and at 3, 6
and 12 months. The data for each parameter, measured at
up to 168 sites in each subject, was averaged within a
subject and then across subjects at each time-point sep-
arately. The signicance of differences over time was
determined using the Friedman test.
220
Haffajee et al.
large numbers of subjects and a broad range of bac-
terial species at multiple time-points. The microbio-
logical changes were related to changes in the clinical
status of the adjacent periodontal tissues. Thus, this
article will provide a synthesis of data from various
longitudinal studies of periodontal therapy that have
been conducted at The Forsyth Institute during the
last decade, in order to demonstrate the changes that
occur in the subgingival microbiota and the rela-
tionship with changes in periodontal status.
Overall design, subject population,
monitoring protocols and
treatment groups
A long-term goal of the clinical research performed in
the Department of Periodontology at The Forsyth
Institute is to dene the most appropriate perio-
dontal therapy for an individual patient based on the
nature of that individuals periodontal infection. To
achieve this, a number of longitudinal clinical studies
have been performed in the Department of Period-
ontology at The Forsyth Institute to evaluate the ef-
fects of different treatment protocols on clinical and
microbiological parameters. Clinical measurements
were made before, and at multiple time-points post-
therapy, at six sites per tooth in each subject. Sub-
gingival plaque samples were also taken at the same
time-points from 28 subgingival mesiobuccal sites in
each subject. The difculty and expense of making
a full-mouth assessment of a patients subgingival
microbial prole has been overcome by development
of the checkerboard DNADNA hybridization tech-
nique. Using this technique, up to 28 plaque samples
(one sample from each tooth in a subject) can be
Table 1. Mean ( standard error of the mean) clinical parameters for the 493 subjects at baseline, and at 3, 6 and
12 months post-therapy
Baseline 3 months 6 months 12 months
Mean pocket depth (mm) 3.44 0.04 2.96 0.03 2.87 0.02 2.85 0.02
Mean attachment level (mm) 3.54 0.05 3.31 0.05 3.34 0.05 3.34 0.05
Percentage of sites with
Plaque accumulation 64 2 47 1 50 2 50 1
Gingival redness 61 1 42 1 43 1 44 1
Bleeding on probing 40 1 23 1 23 1 21 1
Suppuration 2 0 0 0 0 0 0 0
Changes over time were signicant at P < 0.001 for all parameters, according to the Friedman test.
3
6
9
B
a
s
e
l
i
n
e

p
o
c
k
e
t

d
e
p
t
h

(
m
m
)
2 1 6 3 0
F
i
n
a
l

p
o
c
k
e
t

d
e
p
t
h

(
m
m
)
0
3
6
9
2 1
m m 9 5 . 0 = n o i t c u d e r D P n a e M
) 3 6 6 , 6 = n (
) 9 0 6 , 9 1 = n (
) 4 8 7 , 9 1 = n (
) 1 3 5 , 9 = n (
) 2 2 2 , 6 = n (
) 7 1 6 , 3 = n (
) 4 6 4 , 1 = n (
) 6 8 7 = n (
) 2 1 4 = n (
) 7 8 1 = n (
) 0 1 1 = n (
1
2
4
5
7
8
0 1
0 1 >
s h t n o M
Fig. 3. Mean ( standard error of the
mean) pocket depths at baseline,
and at 3, 6 and 12 months. The data
were subset into baseline pocket
depth categories at 1-mm intervals,
ranging from 1 to 10 mm as well as
>10 mm. The mean overall full-
mouth pocket depth at each time-
point for all subjects is indicated in
red. Mean full-mouth pocket depth
averaged across all sites for all
subjects was reduced by 0.59
0.03 mm. PD, pocket depth.
221
Effect of periodontal therapy on the subgingival microbiota
0
7 1
4 3
1 5
8 6
1 D P B
2 D P B
3 D P B
4 D P B
5 D P B
6 D P B
7 D P B
8 D P B
0 1 D P B
0 1 > D P B
9 7 5 3 1 1 1
) m m ( D P l a n i F
9 D P B
r e p e e D
e g n a h c o N
r e w o l l a h S
% 1 9
% 6 9
% 0 9
% 9 8
% 0 9
% 7 8
% 2 8
% 0 7
% 0 4
% 8 1
) 3 6 6 , 6 = n (
) 9 0 6 , 9 1 = n (
) 4 8 7 , 9 1 = n (
) 1 3 5 , 9 = n (
) 2 2 2 , 6 = n (
) 7 1 6 , 3 = n (
) 4 6 4 , 1 = n (
) 6 8 7 = n (
) 2 1 4 = n (
) 7 8 1 = n (
) 0 1 1 = n (
%
Fig. 4. Percentage of sites with different pocket depths at
12 months post-therapy for sites with different baseline
pocket depths. Each bar graph represents a different ini-
tial pocket depth category, ranging from 1 to >10 mm.
The x-axis on each graph represents the nal pocket depth
and the y-axis the percentage of sites. The color of the bars
indicates whether the sites got shallower, deeper or
remained unchanged at 12 months. The brackets indicate
the percentage of sites in each baseline pocket depth
category that improved at 12 months. The data were de-
rived from 68,385 baseline sites in 493 subjects (i.e.
273,540 measurements for all four visits). BPD, baseline
pocket depth; PD, pocket depth.
0
5 . 3
7
5 . 0 1
4 1
2 1 6 3 0
m m 0 2 . 0 = n i a g L A n a e M
3
6
9
B
a
s
e
l
i
n
e

a
a
t
a
c
h
m
e
n
t

l
e
v
e
l

(
m
m
)
F
i
n
a
l

a
t
t
a
c
h
m
e
n
t

l
e
v
e
l

(
m
m
)
1
2
4
5
7
8
0 1
s h t n o M
0
1 1
1 1 > ) 8 5 1 = n (
) 6 6 1 = n (
) 0 6 3 = n (
) 0 8 6 = n (
) 9 5 2 , 1 = n (
) 4 7 1 , 2 = n (
) 2 9 6 , 3 = n (
) 1 5 4 , 6 = n (
) 4 9 3 , 9 = n (
) 9 7 9 , 4 1 = n (
) 5 2 1 , 7 1 = n (
) 5 2 0 , 1 1 = n (
) 6 7 7 = n (
Fig. 5. Mean ( standard error of
the mean) attachment level meas-
urements at baseline, and at 3, 6
and 12 months post-therapy. The
data were subset into baseline
attachment level categories at
1-mm intervals, ranging from 0 to
11 mm, as well as >11 mm. The
mean full-mouth attachment level
at each time-point for all subjects is
indicated in red. The mean full-
mouth attachment level averaged
across all sites for all subjects was
reduced by 0.20 0.02 mm. AL,
attachment level.
222
Haffajee et al.
0 . 0
5 . 3 1
0 . 7 2
5 . 0 4
0 . 4 5
e s a e r c n I
e g n a h c o N
e s a e r c e D
0 L A B
0 1 8 6 4 2 0
1 L A B
2 L A B
3 L A B
4 L A B
5 L A B
6 L A B
7 L A B
8 L A B
9 L A B
0 1 L A B
1 1 L A B
1 1 > L A B
) m m ( L A L A N I F
% 9 7
% 0 8
% 9 7
% 6 7
% 2 7
% 0 7
% 2 6
% 3
% 1 2
% 7 3
% 8 4
% 7 5
) 8 5 1 = n (
) 6 6 1 = n (
) 0 6 3 = n (
) 0 8 6 = n (
) 9 5 2 , 1 = n (
) 4 7 1 , 2 = n (
) 2 9 6 , 3 = n (
) 1 5 4 , 6 = n (
) 4 9 3 , 9 = n (
) 9 7 9 , 4 1 = n (
) 5 2 1 , 7 1 = n (
) 5 2 0 , 1 1 = n (
) 6 7 7 = n (
%
Fig. 6. Percentage of sites with different clinical attach-
ment levels at 12 months post-therapy for sites with dif-
ferent baseline attachment levels. Each bar graph
represents a different initial attachment level category,
ranging from 0 to >11 mm. The x-axis on each graph
represents the nal attachment level and the y-axis the
percentage of sites. The color of the bars indicates
whether the percentage of sites decreased, increased or
remained unchanged at 12 months. The brackets indicate
the percentage of sites in each baseline attachment level
category that showed an improvement at 12 months. The
data were derived from 68,239 baseline sites in 493 sub-
jects (i.e. 272,956 measurements for all four visits). AL,
attachment level; BAL, baseline attachment level.
0
0 2
5
0 1
5 1
Percentage
f o
s e t i s
e n i l e s a B
t e k c o p
) m m ( h t p e d
1
2
3
4
5
6
7
8
9
0 1
0 1 >
0 1 >
0 1
9
8
7
6
5
4
3
1
2
1-year
t e k c o p
) m m ( h t p e d
r e p e e D
e g n a h c o N
r e w o l l a h S
Fig. 7. Three-dimensional bar chart
demonstrating the percentage of
sites exhibiting different pocket
depths at baseline and at 12 months
post-therapy. The x-axis represents
the 12-month pocket depth cate-
gory, the y-axis represents the base-
line pocket depth category and the
z-axis represents the percentage of
sites. The bars represent the per-
centage of sites in each baseline/12-
month pocket depth category. The
color of the bars indicates whether
the sites got shallower, deeper or
remained unchanged at 12 months.
223
evaluated for their content of 40 bacterial species on
a single membrane, providing 1,120 bacterial counts.
The checkerboard technique has been described in
detail previously (33, 34). Its major strength is that it
allows the rapid and relatively inexpensive quantita-
tive analysis of multiple plaque samples from mul-
tiple sites in multiple subjects.
Figure 1 presents the different treatment groups
and the number of subjects receiving these treat-
ments. Complete clinical data from baseline to
12 months post-therapy were available from a total of
493 subjects in 17 different treatment groups, while
complete microbiological data were available for 461
subjects. All subjects received scaling and root pla-
Fig. 9. Plots of mean baseline pocket depth vs. mean
pocket depth change (left panel) and mean baseline
attachment level vs. mean attachment level change (right
panel) from baseline to 12 months. Each circle represents
the mean baseline pocket depth or attachment level and
the mean change in that parameter from baseline to
12 months in each of the 493 subjects. The circles above
the horizontal lines represent subjects whose mean pocket
depth worsened (n 56) or exhibited attachment level
loss (n 160). Circles below the horizontal lines represent
subjects who showed improvement in these parameters at
12 months (pocket depth, n 437; attachment level,
n 333). Regression analysis indicated a signicant
relationship between baseline levels of disease and sub-
sequent improvement.
0 . 0
0 . 4 1
5 . 3
0 . 7
5 . 0 1
Percentage
f o
s e t i s
0
e n i l e s a B
t n e m h c a t t a
) m m ( l e v e l
1
2
3
4
5
6
7
8
9
0 1
1 1 1 1
0 1
9
8
7
6
5
4
3
1
2
r a e y 1-
t n e m h c a t t a
) m m ( l e v e l
0
1 1 >
1 1 >
e s a e r c n I
e g n a h c o N
e s a e r c e D
Fig. 8. Three-dimensional bar chart
demonstrating the percentage of
sites exhibiting different clinical
attachment levels at baseline and at
12 months post-therapy. The x-axis
represents the 12-month attach-
ment level category, the y-axis rep-
resents the baseline attachment
level category and the z-axis repre-
sents the percentage of sites. The
bars represent the percentage of
sites in each baseline/12-month
attachment level category. The color
of the bars indicates whether the
percentage of sites decreased, in-
creased or remained unchanged at
12 months.
224
Haffajee et al.
ning. The treatments included modied Widman ap
surgery, apically repositioned ap surgery, systemic-
ally administered antibiotics (including amoxicillin,
azithromycin, metronidazole and doxycycline, and
low-dose doxycycline), local tetracycline bers and
repeated professional supragingival plaque removal.
These treatments were given alone or in conjunction
with other treatments. Thus, for example, group 1
subjects received scaling and root planing, modied
Widman ap surgery, systemically administered
amoxicillin plus metronidazole and locally delivered
tetracycline at sites with an initial pocket depth of
>4 mm. Group 8 subjects received scaling and root
planing only and comprised the combined control
subjects from the various treatment studies. The
studies were performed from 1994 to date at The
Forsyth Institute.
The comparison of data from subjects participa-
ting in different studies over a 10-year span presents
certain difculties. The 493 subjects were not ran-
domly assigned to the 17 treatment groups in a
single randomized clinical trial. Rather, the data
were from a series of randomized clinical trials either
completed or ongoing during the past 10 years. For
example, treatment groups 17 and a subset of the
scaling and root planing subjects from group 8
constituted a single randomized clinical trial. Simi-
larly, groups 10 and 11 and subsets of groups 8 and 9
constituted a second randomized trial. For this rea-
son, the number of subjects in each of the treatment
groups differed substantially. Thus, this overview is
not intended to represent a head-to-head compar-
ison of 17 therapies, but rather an indication of the
effects that periodontal therapy had overall on the
subgingival microbiota, and consideration of some
of the effects that might be attributed to specic
adjuncts.
In spite of these limitations, there were a number
of features in common among the different studies.
All subjects were clinically monitored in the same
manner. Clinical measures of plaque accumulation,
suppuration, gingival redness, bleeding on probing,
pocket depth and attachment level were made at six
sites per tooth (mesiobuccal, midbuccal, distobuc-
cal, distolingual, midlingual and mesiolingual), at all
teeth, excluding third molars, using a North Carolina
probe. The pocket depth and attachment level
measurements were repeated at each visit, and the
means of the pairs of measurements were used in
the analyses. Subjects were measured pretreatment
at baseline and at 3, 6 and 12 months post-therapy.
In some studies there were also monitoring visits at
18 and 24 months. One clinician was responsible for
all measurements taken for a given subject. There
was a modest turnover of clinicians during the 10-
year period. However, new examiners were calibra-
0 . 0
3 . 4 1
6 . 8 2
9 . 2 4
2 . 7 5
s h t n o M
2 1 6 3 e n i l e s a B
C
o
u
n
t
s

x

1
0
5
1 0 0 . 0 < P
Fig. 11. Plot of mean total DNA probe counts (10
5
,
standard error of the mean) in subgingival plaque sam-
ples taken from 461 chronic periodontitis subjects at
baseline and at 3, 6 and 12 months post-therapy. Mean
values were computed by summing the DNA probe counts
for each species at each site, averaging up to 28 samples in
each subject, and then averaging across subjects at the
four time-points separately. Signicance of differences
over time was determined using the Friedman test.
Fig. 10. Plot of the riskburden of sites with different
baseline pocket depths 12 months after periodontal ther-
apy. The x-axis presents the risk, as dened by the per-
centage of sites exhibiting 3 mm clinical attachment loss
(AL) 12 months post-therapy. The y-axis presents the
burden, which was dened as the number of sites per
10,000 total sites that exhibited disease progression of
3 mm. The numbers in the gure indicate the pocket
depth pretherapy and their location in the plot represents
the riskburden for that pocket depth category.
225
ted with current clinicians before taking measure-
ments on subjects, and all examiners were recali-
brated at 6-month intervals. Furthermore, the
inclusion and exclusion criteria were comparable for
each study. All subjects were >20 years of age,
systemically healthy and had chronic periodontitis,
dened as having at least eight teeth with a pocket
depth of >4 mm.
The subjects were also monitored microbiologi-
cally in the same manner. Subgingival plaque sam-
ples were taken, using individual, sterile Gracey
curettes, from the mesial aspect of all teeth (exclu-
ding third molars) at each monitoring visit and were
individually analyzed for their content of 40 bacterial
species using checkerboard DNADNA hybridization
(33, 34). Finally, treatment protocols were standard-
ized, so that a treatment such as scaling and root
planing was performed in the same manner (i.e.
quadrants were scaled at weekly intervals under local
anesthetic).
Overall effect of the employed
periodontal therapies on clinical
parameters
The rst question asked was what was the effect of
periodontal therapy, irrespective of the nature of the
therapy on clinical, and as described later, micro-
biological outcomes. The overall effect of the differ-
ent therapies on clinical parameters is presented in
Fig. 2 and Table 1. Data for each parameter were
averaged within a subject and then across subjects
at each time-point. The results indicated that there
was a signicant reduction in all clinical parame-
ters over time. The major effect was from baseline
Fig. 12. Plots of mean counts (left panel), percentage
values of the total DNA probe count (middle panel) and
percentage of sites colonized by 40 bacterial species at
counts >10
5
(right panel) in subgingival plaque samples
taken from 461 chronic periodontitis subjects at baseline
and at 12 months post-therapy. The bands represent the
mean values standard error of the mean. Mean values
for each species were computed by averaging up to 28
samples in each subject, and then averaging across sub-
jects at the two time-points. Signicance of differences
between groups was sought using the nonparametric
Wilcoxon signed ranks test; *P < 0.05, **P < 0.01,
***P < 0.001 after adjusting for multiple comparisons (32).
The species were ordered and grouped according to the
complexes described by Socransky et al. (31). The red
proles represent baseline data and the yellow proles
represent data at 12 months.
226
Haffajee et al.
(pretherapy) to 3 months post-therapy and, for some
parameters, such as percentage of sites with bleeding
on probing and mean pocket depth, there was a
continued modest improvement in these parameters
to 12 months post-therapy. The percentage of sites
with gingival redness and plaque, as well as mean
clinical attachment level, showed slight increases
from 3 to 12 months, but the 12-month data were still
signicantly lower than pretreatment values.
The effect of therapy on mean pocket depth and
attachment level change at sites with different base-
line pocket depths and clinical attachment levels was
examined (Fig 36). The mean full-mouth pocket
depth reduction at 12 months was 0.59 mm. This was
achieved by major reductions in pocket depths at
sites with initially deep pockets and with lesser
reductions or modest increases at sites with initially
shallower pockets. For example, sites with initial
pocket depths of >7 mm showed a mean reduction
of >3 mm, while sites with an initial pocket depth of
1 mm increased by 0.3 mm (Fig. 3). The seemingly
modest overall mean reduction in pocket depth of
0.59 mm occurred because of the larger number of
sites with shallower initial pocket depths where the
pocket depth reduction was less. Figure 4 presents
the percentage of sites at different pocket depths at
12 months for the sites subset according to pocket
depth at entry. It is observed that >80%of sites with
a baseline pocket depth of >4 mm exhibited a
reduction in pocket depth at 12 months, while the
sites with initially shallow pockets (12 mm) showed
either no change or pocket deepening at 12 months.
Figures 5 and 6 present similar data for attachment
level. Once again, the major improvements in clinical
attachment level were seen at sites with greater initial
attachment loss, while sites with baseline attachment
0 . 0 1 . 1 3 . 2 6 . 4 4 . 3
* * *
* * *
* * *
* * *
* * *
* * *
* *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
e a i r e s c n e r e g . A
i i l e a r s i . A
1 i i d n u l s e a n . A
s u c i t y l o t n o d o . A
a l u v r a p . V
i i n o d r o g . S
s u i d e m r e t n i . S
s i t i m . S
s i l a r o . S
s i n i u g n a s . S
s n a t i m o c m e t e c y m o n i t c a . A
s i l a v i g n i g . C
a e c a r h c o . C
a n e g i t u p s . C
s n e d o r r o c . E
s i l i c a r g . C
s u t c e r . C
e a w o h s . C
m u t a d o n . E
m u t a e l c u n s s m u t a e l c u n . F
m u h p r o m y l o p s s m u t a e l c u n . F
i i t n e c n i v s s m u t a e l c u n . F
m u c i t n o d o i r e p . F
s o r c i m . P
a i d e m r e t n i . P
s n e c s e r g i n . P
s u t a l l e t s n o c . S
a i h t y s r o f . T
s i l a v i g n i g . P
a l o c i t n e d . T
m u e r r u b a s . E
m u r o l l i b r o m . G
s i l a c c u b . L
a s o c u m . N
s e n c a . P
a c i n e g o n i n a l e m . P
s u s o n i g n a . S
a i x o n . S
i i k s n a r c o s . T
s e c y m o n i t c A
e l p r u P
n e e r G
w o l l e Y
e g n a r O
d e R
r e h t O
s h t n o m 3 e n i l e s a B s h t n o m 6 s h t n o m 2 1
4 . 1
7 . 1
1 . 2
5 . 2
8 . 2
9 . 0
3 . 1
8 . 1
2 . 2
6 . 2
3 . 1
5 . 1
8 . 1
0 . 2
2 . 2
0 1 x s t n u o C
5
C
o
u
n
t
s

x

1
0
5

2 1 6 3 0 s h t n o M
Fig. 13. Proles of mean counts (10
5
) of 40 taxa in sub-
gingival plaque samples from 461 chronic periodontitis
subjects at baseline, and at 3, 6 and 12 months post-
therapy. Plaque samples were taken from the mesial
aspect of each tooth and analyzed separately for their
content of 40 species. Data for each species were averaged
within a subject, and then across subjects, at the different
time-points separately. Signicance of differences over
time was sought using the Friedman test and adjusted
for multiple comparisons (32); *P < 0.05; **P < 0.01;
***P < 0.001. Species were ordered according to microbial
complexes (31). The three panels to the right represent the
mean counts ( standard error of the mean) at each time-
point for Prevotella intermedia, Tannerella forsythia and
Fusobacterium nucleatum ss vincentii, indicating different
patterns of recolonization post-therapy.
227
levels of 02 mm showed a mean attachment loss at
12 months (Fig. 5).
The effect of the different numbers of sites in dif-
ferent baseline pocket depth or attachment level
categories is reinforced in Fig 7 and 8. Figure 7 pre-
sents the percentage distribution of sites according to
baseline and 12-month pocket depth, and Fig. 8
provides similar data for clinical attachment level. It
is clear that most sites at baseline exhibited relatively
modest pocket depths or clinical attachment levels
and that these sites showed less change than sites
with initially more periodontal destruction. Further-
more, the small numbers of sites of interest to the
clinician (i.e. that exhibited either deep pocket depths
or increased attachment level at baseline) received
the most benet from therapy.
Mean changes in clinical parameters for a popu-
lation, as presented above, can provide insight into
the general effects of therapy. The mean changes
presented in Fig. 2 suggested overall clinical
improvement. However, examination of changes in
individual subjects may indicate that therapy was not
necessarily effective in all individuals. This is illus-
trated by the data in Fig. 9 where the mean change in
pocket depth and attachment level for each subject is
presented. While 437 of 493 (88.6%) subjects showed
a reduction in mean pocket depth at 12 months post-
therapy, a small percentage of subjects (11.4%)
showed a mean increase. Similarly, about two-thirds
of the subjects showed an improvement in mean
clinical attachment level at 12 months, but the
remainder showed loss of attachment. Nonetheless,
0 . 0 6 . 1 3 . 3 9 . 4 5 . 6
i i l e a r s i . A
a l u v r a p . V
i i n o d r o g . S
s i t i m . S
s i l a r o . S
a e c a r h c o . C
s i l i c a r g . C
s u t c e r . C
e a w o h s . C
m u t a d o n . E
s o r c i m . P
s i l a c c u b . L
a s o c u m . N
s e n c a . P
a i x o n . S
0 . 0 6 . 1 3 . 3 9 . 4 5 . 6 0 . 0 6 . 1 3 . 3 9 . 4 5 . 6
*
* * *
* * *
*
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* *
* * *
* *
* * *
* * *
*
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* *
* * *
*
* * *
* * *
* *
*
* * *
* * *
* * *
* *
* *
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
* *
0 1 x s t n u o C
5
m m 6 > D P e n i l e s a B m m 6 4 D P e n i l e s a B m m 4 < D P e n i l e s a B
e l p r u P
n e e r G
w o l l e Y
e g n a r O
d e R
r e h t O
5
subjects at baseline, and at 3, 6 and 12 months post-
therapy, at sites subset into baseline pocket depth categ-
ories of <4, 46 and >6 mm. Plaque samples were taken
from the mesial aspect of each tooth and analyzed sepa-
rately for their content of 40 species of bacteria. Data for
each species were averaged within each pocket depth
category in each subject and then across subjects in the
different pocket depth categories for each time-point se-
parately. Signicance of differences over time was sought
using the Friedman test and adjusted for multiple com-
parisons (32); *P < 0.05; **P < 0.01; ***P < 0.001. Species
were ordered according to the complexes described by
Socransky et al. (31). PD, pocket depth.
228
Haffajee et al.
the mean reduction in pocket depth and gain in
attachment in subjects showing improvement was
greater than the mean pocket depth increase and
attachment level loss in the subjects whose treatment
response was less favorable. Not surprisingly, the
data in Fig. 9 also indicated that, in general, subjects
with more disease before therapy showed a better
clinical response.
Riskburden for disease progression at
sites with different baseline pocket depth
The above data may be somewhat overwhelming and
difcult to interpret in a practical sense. The clinician
is concerned, in large part, that a treated site (or
subject) may show disease progression after therapy
and on the burden that this disease progression will
place on his/her practice. We estimated the risk
burden for 67,413 treated sites with different baseline
pocket depths in the 493 subjects. Disease progres-
sion was dened as suggested by the 5th European
Workshop on Periodontology Etiology and Patho-
genesis Leading to Preventive Concepts (36) as an
increase in clinical attachment level of >2.5 mm,
and the time frame of this analysis was 12 months
post-therapy. Figure 10 presents a plot of the result-
ing data. It is seen that the risk of disease progression
post-therapy was somewhat higher at periodontal
sites with initially deeper pockets, but the burden to
the periodontal therapist occurred primarily at the
shallow sites. The burden was larger, as mentioned
earlier, because there were far more shallow pockets
at risk in a periodontally diseased population than
deep pockets.
Overall effect of the employed
periodontal therapies on
microbiological parameters
When examining the effect of periodontal therapy on
the subgingival microbiota, the data may be ex-
pressed in various ways [see Fig. 5 in Teles et al. (35)].
3 . 0
0 . 1
7 . 1
4 . 2
1 . 3
m u t a d o n . E
8 . 0
4 . 2
0 . 4
5 . 5
1 . 7
6 . 0
3 . 2
0 . 4
7 . 5
4 . 0
2 . 1
9 . 1
7 . 2
4 . 3
4 . 7
C
o
u
n
t
s

x

1
0
5
m m 4 < D P e n i l e s a B
m m 6 4 D P e n i l e s a B
m m 6 > D P e n i l e s a B
s h t n o M 2 1 6 3 0 2 1 6 3 0
Fig. 15. Mean counts ( standard error of the mean) for
Eubacterium nodatum, Tannerella forsythia, Porphyro-
monas gingivalis and Treponema denticola at baseline, and
3, 6 and 12 months post-therapy, at sites subset according
to baseline pocket depth categories of <4, 46 and >6 mm
in 461 chronic periodontitis subjects. Data averaging and
statistical testing were as described in Fig. 13. All four
species showed signicant changes over time in each
pocket depth category at P < 0.001, after adjusting for
multiple comparisons (32). PD, pocket depth.
229
These include changes in counts, proportions or
percentage of sites colonized at greater than a
selected threshold (or the detection limit of the
method). The overall effect of 17 therapies taken to-
gether was to reduce the total subgingival bacterial
counts signicantly at 3 months and the reduced
levels were maintained to 12 months (Fig. 11). Mean
counts (10
5
standard error of the mean) were
54.6 2.6, 39.3 2.0, 39.1 2.4 and 39.3 2.5 at
baseline, and at 3, 6 and 12 months respectively
(P < 0.001). Figure 12 presents the changes that oc-
curred for individual species in the subgingival
microbiota from baseline to 12 months in the 461
subjects described above. The majority of species
decreased in counts and percentage of sites colonized
at >10
5
, particularly members of the red and orange
complexes (31), which contain many of the presumed
periodontal pathogens. The proportions of Prevotella
intermedia, Prevotella nigrescens, Tannerella forsy-
thia, Porphyromonas gingivalis and Capnocytophaga
0 . 0 3 . 1 6 . 2 9 . 3 2 . 5
* *
* *
* * *
* *
* *
* * *
*
*
* * *
* * *
* * *
* * *
* *
i i l e a r s i . A
a l u v r a p . V
i i n o d r o g . S
s i t i m . S
s i l a r o . S
a e c a r h c o . C
s i l i c a r g . C
s u t c e r . C
e a w o h s . C
m u t a d o n . E
s o r c i m . P
s i l a c c u b . L
a s o c u m . N
s e n c a . P
a i x o n . S
P O B P O B o N
e l p r u P
n e e r G
w o l l e Y
e g n a r O
d e R
r e h t O
Fig. 16. Proles of baseline mean counts (10
5
) of 40 taxa
in subgingival plaque samples from 461 chronic perio-
dontitis subjects at sites that did or did not bleed on
probing at baseline. Plaque samples were taken from the
mesial aspect of each tooth and analyzed separately for
their content of 40 species of bacteria. Data for each
species were averaged, within each subject, at bleeding
and nonbleeding sites separately, and then across subjects
for the two site categories. Signicance of differences be-
tween bleeding and nonbleeding sites was determined
using the Wilcoxon signed ranks test and adjusted
for multiple comparisons (32); *P < 0.05; **P < 0.01;
***P < 0.001. Species were ordered according to microbial
complexes (31). BOP, bleeding on probing.
230
Haffajee et al.
gingivalis were signicantly reduced, while the pro-
portions of Streptococcus gordonii, Streptococcus
mitis, Streptococcus oralis, Streptococcus sanguinis,
Eikenella corrodens, Gemella morbillorum and
Neisseria mucosa increased after treatment.
Although there was a signicant reduction in the
mean counts of many species at 12 months (Fig. 12,
left panel), the data presented in Fig. 13 indicate that
the patterns of reduction and recolonization over
time differed among species. For example, the pro-
portion of species such as P. intermedia decreased at
3 months and remained at the reduced levels to
12 months (Fig. 13 and inset). T. forsythia showed a
marked decrease at 3 months and slowly increased at
6 and 12 months, although not to baseline values.
Other species, such as Fusobacterium nucleatum ss
vincentii, were decreased by therapy at 3 months but
returned to baseline values by 12 months.
Sampled sites were subset into the baseline pocket
depth categories of <4, 46 and >6 mm. Not sur-
prisingly, the lowest mean baseline counts of most
species were observed at pockets of <4 mm at base-
line, while the highest counts were seen at the sites
with baseline pocket depth of >6 mm (Fig. 14).
Nonetheless, there were signicant decreases in the
levels of red and orange complex species at all initial
pocket depth categories. Figure 15 emphasizes the
observation that the counts of periodontal pathogens,
Eubacterium nodatum, T. forsythia, P. gingivalis and
Treponema denticola, were higher at the deeper
pockets at baseline. Although reduction of these
species was greatest at the deeper sites, the mean
levels of the four presumed periodontal pathogens
after therapy at the deep sites (>6 mm) were still
higher than the mean counts of these species in the
shallow pockets (<4 mm) at all time-points.
0 . 0 2 . 1 4 . 2 6 . 3 8 . 4
* *
* *
*
* * *
*
* * *
* * *
* * *
* *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
*
i i l e a r s i . A
a l u v r a p . V
i i n o d r o g . S
s i t i m . S
s i l a r o . S
a e c a r h c o . C
s i l i c a r g . C
s u t c e r . C
e a w o h s . C
m u t a d o n . E
s o r c i m . P
s i l a c c u b . L
a s o c u m . N
s e n c a . P
a i x o n . S
0 . 0 2 . 1 4 . 2 6 . 3 8 . 4
* * *
* *
* * *
* * *
* * *
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
*
* * *
* *
* * *
positive P O B e n i l e s a B negative P O B e n i l e s a B
0 1 x s t n u o C
5
e l p r u P
n e e r G
w o l l e Y
e g n a r O
d e R
r e h t O
5
subjects at baseline, and 3, 6 and 12 months post-therapy,
at sites that did or did not bleed on probing at baseline.
Plaque samples were taken from the mesial aspect of each
tooth and were analyzed separately for their content of 40
species of bacteria. Data for each species were averaged
within and then across subjects at bleeding and non-
bleeding sites separately for each time-point. Signicance
of differences over time was sought using the Friedman
test and adjusted for multiple comparisons (32); *P < 0.05;
**P < 0.01; ***P < 0.001. Species were ordered according
to the complexes described by Socransky et al. (31). BOP,
bleeding on probing.
231
The proportions of species also related to other
clinical parameters. Figure 16 presents the baseline
counts of the 40 test species at sites that did or did
not exhibit bleeding on probing at baseline, as a
measure of periodontal inammation. Sites that bled
on probing had signicantly higher counts of 13 of 40
test species, including all members of the red com-
plex, nine members of the orange complex and
Treponema socranskii. The change in mean counts of
the 40 test species at initially bleeding and non-
bleeding sites over time is presented in Fig. 17. As
demonstrated in the previous gure, the bleeding on
probing sites exhibited higher mean counts at base-
line compared with the nonbleeding sites, and both
groups showed signicant reductions of multiple
species at 3 months. However, there was a tendency
0 4 . 2
0 2 . 1
0 0 . 0
0 2 . 1
0 4 . 2
0 . 8 2 5 . 2 1 0 . 3 5 . 8 1 0 . 4 3 8 2 7 1 6 5 6 1 0 . 1 5 . 0 0 . 0 5 . 0 0 . 1
t n u o c e b o r p A N D Percentage
A
L

c
h
a
n
g
e

f
r
o
m

b
a
s
e
l
i
n
e

t
o

1
2

m
o
n
t
h
s

(
m
m
)
d e z i n o l o c s e t i Percentage of s 0 1 x s t n u o C
5
n i e g n a h C a i h t y s r o f . T s h t n o m 2 1 to e n i l e s a b m o r f
7 1 . 0 = r
1 0 0 . 0 < P
8 1 . 0 = r
1 0 0 . 0 < P
2 2 . 0 = r
1 0 0 . 0 < P
Fig. 19. Plots of mean change in attachment level vs.
mean change in counts (10
5
, left panel), percentage DNA
probe counts (middle panel) and percentage of sites col-
onized (right panel) of Tannerella forsythia from baseline
to 12 months. Each circle represents the mean change in
attachment level and the mean change in T. forsythia from
baseline to 12 months in each of the 461 subjects. The
circles in the bottom left hand segment of each panel
represent subjects who exhibited both a decrease in mean
attachment level and a mean decrease in counts, per-
centage or prevalence of T. forsythia. By all analyses, the
largest number of subjects was located in that cell.
Regression analysis indicated a signicant relationship
between a decrease in mean attachment level and a
decrease in counts, percentage and prevalence of T. for-
sythia.
0 0 . 3
5 8 . 1
0 7 . 0
5 4 . 0
0 6 . 1
0 . 8 2 5 . 2 1 0 . 3 5 . 8 1 0 . 4 3
t n u o c e b o r p A N D Percentage
8 2 7 1 6 5 6 1 P
D

c
h
a
n
g
e

f
r
o
m

b
a
s
e
l
i
n
e

t
o

1
2

m
o
n
t
h
s

(
m
m
)
0 . 1 5 . 0 0 . 0 5 . 0 0 . 1
d e z i n o l o c s e t i s Percentage of 0 1 x s t n u o C
5
n i e g n a h C a i h t y s r o f . T s h t n o m 2 1 to e n i l e s a b m o r f
6 2 . 0 = r
1 0 0 . 0 < P
8 2 . 0 = r
1 0 0 . 0 < P
9 2 . 0 = r
1 0 0 . 0 < P
Fig. 18. Plots of mean change in pocket depth vs. mean
change in counts (10
5
, left panel), percentage DNA probe
counts (middle panel) and percentage of sites colonized
(right panel) of Tannerella forsythia from baseline to
12 months. Each circle represents the mean change in
pocket depth and the mean change in T. forsythia from
baseline to 12 months in each of the 461 subjects. The
circles in the bottom left hand segment of each panel
represent subjects who exhibited both a decrease in mean
pocket depth and a mean decrease in counts, percentage
or prevalence of T. forsythia. By all analyses, the largest
number of subjects was located in that cell. Regression
analysis indicated a signicant relationship between a
decrease in mean PD and a decrease in counts, percentage
and prevalence of T. forsythia.
232
Haffajee et al.
for species (including T. forsythia, P. gingivalis, P.
intermedia and P. nigrescens) to return more rapidly
at the sites that initially bled on probing, while Ve-
illonella parvula, Campylobacter gracilis and Seleno-
monas noxia exceeded baseline values at 12 months
at these sites. This is in accord with the notion that
sites which are more inamed are likely to be recol-
onized more quickly by species of the red and orange
complexes (29, 30).
Relationship between change in clinical
parameters and change in
microbiological parameters
The data presented thus far have indicated that
periodontal therapy, on average, provides a signi-
cant improvement in clinical parameters and a
reduction in the levels of many subgingival species.
The expectation would be that the improvement in
clinical parameters should be related to a decrease
in periodontal pathogens. Indeed, this was the
case. Regression analysis was used to examine the
relationship between change in mean pocket depth
and change in the 40 test species. It was found that
decreases in the counts of T. forsythia, P. gingivalis
and C. gingivalis, decreases in the proportions of T.
forsythia, C. gingivalis and the red complex species
combined, and decreases in the percentage of sites
colonized by Actinomyces odontolyticus, Actinoba-
cillus actinomycetemcomitans, C. gingivalis, E. no-
datum, F. nucleatum ss vincentii, Fusobacterium
periodonticum, P. intermedia, P. nigrescens, T. for-
Fig. 20. Plots of mean counts (10
5
gingival plaque samples at baseline and at 12 months
post-therapy at sites that exhibited attachment level gain
of >2 mm, (left panel), change of 2 mm (middle panel)
or loss of >2 mm (right panel) from baseline to
12 months. Counts of each species at sites in each of the
three attachment level change categories were deter-
mined, averaged within a subject and then averaged
across subjects in the three site categories at baseline and
12 months separately. Signicance of differences between
counts at baseline and 12 months was determined using
the Wilcoxon signed ranks test and adjusted for multiple
comparisons (32) (*P < 0.05; **P < 0.01; ***P < 0.001).
Species were ordered according to the complexes de-
scribed by Socransky et al. (31). AL, attachment level.
233
sythia, P. gingivalis and T. denticola were signi-
cantly related to a decrease in mean pocket depth
at 12 months post-therapy. Fewer species related to
gain of attachment, but decreases in the counts of
T. forsythia and P. gingivalis, in the percentage of
T. forsythia and red complex species combined and
in the percentage of sites colonized by T. forsythia
and P. gingivalis, were signicantly associated with
attachment level gain at 12 months.
It is clear from these data that decreases in red
complex species, and to a lesser extent orange com-
plex species, are most frequently found to be asso-
ciated with clinical improvement. Figures 18 and 19
illustrate the relationship between the change in
counts, proportions and prevalence of one of the red
complex species, T. forsythia, from baseline to
12 months, and change in mean pocket depth
(Fig. 18) and in mean attachment level (Fig. 19), in
individual subjects.
Relationship between change in clinical
parameters and change in
microbiological parameters at
individual sites
The relationship between the change in microbial
species and the change in clinical parameters at
individual sites between baseline and 12 months
was examined (Fig 2023). Figure 20 presents the
mean counts of the 40 test species at baseline and at
12 months post-therapy at sites that showed a gain
in attachment level of >2 mm (left panel), a loss of
attachment of >2 mm (right panel) and change
between these two thresholds (middle panel). The
Fig. 21. Plots of mean change in counts (10
5
) of 40 taxa
in subgingival plaque samples from baseline to 12 months
at sites that exhibited attachment level gain of >2 mm,
(left panel), change of 2 mm (middle panel) or loss of
>2 mm (right panel) from baseline to 12 months post-
therapy. Change in counts of each species at sites in each
of the three attachment level change categories were
determined averaged within a subject and then across
subjects in the three site categories. Signicance of dif-
ferences among the three site categories was determined
using the KruskalWallis test and adjusted for multiple
comparisons (32); *P < 0.05; **P < 0.01; ***P < 0.001.
Species were ordered according to the complexes de-
scribed by Socransky et al. (31). AL, attachment level.
234
Haffajee et al.
counts were averaged within a subject at sites in the
three attachment level change categories and then
averaged across subjects at baseline and 12 months
separately. The greatest reductions in mean counts
of the test species at 12 months were seen at sites
exhibiting attachment level gain and those sites
showing a change of 2 mm. Major reductions were
seen in the red and orange complex species, as well
as for certain members of the genera Actinomyces
and Capnocytophaga, and for Prevotella melanino-
genica, Eubacterium saburreum, and T. socranskii. At
sites exhibiting an increase in attachment level at
12 months, there were no signicant changes be-
tween baseline and 12 months, with the exception
of decreases in the mean counts of Actinomyces
naeslundii genospecies 2, C. gingivalis and P. ni-
grescens. Figure 21 emphasizes the data from the
previous gure and presents the mean change pro-
les of the 40 test species at sites in the three
attachment level change categories. The greatest
reductions in mean counts of the test species at
12 months were seen at sites exhibiting attachment
level gain. This was particularly noticeable for the
red complex species. In contrast, at sites that lost
attachment, there was less of a reduction in mean
counts at 12 months, and several species (partic-
ularly those of the orange complex) showed modest
mean increases. Sites that exhibited little change in
attachment showed the least mean change in spe-
cies counts.
Figures 22 and 23 present similar data for sites
subset according to a decrease in pocket depth of
>2 mm, an increase in pocket depth of >2 mm and
a change between these two thresholds. Similarly to
the ndings for the attachment level change categ-
ories, the greatest reductions in the mean counts of
the test species occurred at sites that showed a de-
crease in pocket depth or a change of 2 mm at
12 months. These reductions were primarily for
members of the red and orange complexes.
5
gingival plaque samples at baseline to 12 months post-
therapy at sites that exhibited pocket depth reduction of
>2 mm, (left panel), change of 2 mm (middle panel) and
increase of >2 mm (right panel) from baseline to
12 months. The averaging of the data and the statistical
testing were as described for Fig. 19. PD, pocket depth.
235
Changes in mean microbial counts at
periodontal sites exhibiting attachment
level gain or loss during periodontal
maintenance therapy
The above data indicated that the counts of many
subgingival species were altered by active periodontal
therapy and that the clinical changes which occurred
in subjects or at periodontal sites were related to the
changes that occurred in the subgingival microbiota.
The present section examined the relationship of
changes that occurred in periodontal attachment
level and subgingival species during maintenance
(supportive) periodontal therapy (312 months).
Figure 24 presents mean subgingival microbial pro-
les at 3 months (i.e. immediately after active
periodontal therapy) and at 12 months post-therapy
for the subjects receiving various forms of perio-
dontal therapy as outlined in Fig. 1. The data
indicate that sites which gained or lost >2 mm of
attachment, or exhibited changes of 2 mm, had
comparable microbial proles at 3 months. However,
sites that gained attachment showed mean reduc-
tions for many species or continued low levels for
others from 3 to 12 months. Sites that showed new
attachment loss exhibited mean increases in sub-
gingival taxa, particularly those of the red and orange
complexes. Sites that exhibited little change in
periodontal attachment level during the maintenance
phase exhibited little change in mean subgingival
microbial proles. In contrast, there was a tendency
for the proportion of species to increase at sites that
showed an increase in attachment level during the
maintenance phase.
Effect of different periodontal
therapies on clinical parameters
Up to this point, we have evaluated the overall clin-
ical and microbiological effects of different perio-
dontal therapies. The following sections will examine
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i i l e a r s i . A
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5
Fig. 23. Plots of mean change in counts (10
5
) of 40 taxa
in subgingival plaque samples from baseline to 12 months
post-therapy at sites that exhibited pocket depth reduc-
tion of >2 mm, (left panel), change of 2 mm (middle
panel) or pocket depth increase of >2 mm (right panel)
from baseline to 12 months. The averaging of the data and
the statistical testing were as described for Fig. 20. PD,
pocket depth.
236
Haffajee et al.
the effect of individual therapies on clinical and
microbiological parameters. Figure 25 presents the
mean change in clinical attachment level from
baseline to 12 months post-therapy in each of the 17
treatment groups separately. The changes were or-
dered with the best response to the left and the worst
response to the right. All treatment groups, except
scaling and root planing in conjunction with apically
repositioned ap surgery or modied Widman ap
surgery, showed a gain in mean clinical attachment
level 12 months post-therapy. The loss of attachment
in the surgical groups was not surprising as such an
outcome would be expected following this procedure
(20, 25, 39). There appeared to be a better mean
attachment level gain in subjects receiving adjunctive
systemically administered antibiotics than in subjects
who received mechanical debridement without such
agents. Figure 26 presents the reduction in mean full-
mouth pocket depth in subjects in the 17 treatment
groups. All groups showed reductions in mean pocket
depth at 12 months post-therapy. The treatment
groups that exhibited the greatest clinical attachment
level gain were not the same as those that exhibited
the greatest pocket depth reduction. This was par-
ticularly apparent for the groups receiving surgery.
Comparison of responses to periodontal
therapies that included or did not
include systemically administered
antibiotics
Besides examining the outcome after individual
treatment strategies, the data could also be subset
according to broader treatment categories. One
5
gingival plaque samples, at 3- and 12-month time-points,
at sites that exhibited attachment level gain of >2 mm,
(left panel), change of 2 mm (middle panel) or loss of
>2 mm (right panel) from 3 to 12 months post-therapy.
Counts of each species at sites in each of the three
attachment level change categories were determined,
averaged within a subject and then averaged across sub-
jects in the three site categories at 3 and 12 months sep-
arately. Signicance of differences between counts at 3
and 12 months was determined using the Wilcoxon signed
ranks test and adjusted for multiple comparisons (32)
(*P < 0.05; **P < 0.01; ***P < 0.001). Species were ordered
according to the complexes described by Socransky et al.
(31). AL, attachment level.
237
question of interest was whether subjects receiving a
systemic antibiotic responded better than those not
receiving such agents. Given the conclusions of two
recent systematic reviews (10, 12), and the data pre-
sented in Fig 25 and 26, one would surmise that the
subjects receiving systemically administered antibi-
otics would show a better clinical response post-
therapy. Indeed, at 12 months post-therapy, subjects
showed a mean decrease ( standard deviation) in
pocket depth of 0.54 (0.57) and 0.66 (0.65)
(P < 0.05) and a mean gain in clinical attachment
level of 0.08 (0.57) and 0.31 (0.46) (P < 0.001) in the
nonantibiotic and antibiotic groups, respectively.
Mean pocket depth and attachment level measure-
ments at baseline and at 12 months post-therapy in
each subject, receiving or not receiving systemically
administered antibiotics, is highlighted in Fig 27 and
28. There was considerable variability among indi-
vidual subjects in the treatment response in both
groups. However, as illustrated by the mean change
for the groups, more subjects receiving systemically
administered antibiotics responded positively to
therapy, particularly in terms of the attachment level
response (Fig. 28). In both groups the majority of the
subjects showed a decrease in mean pocket depth
post-therapy, with 12.2% and 10% of subjects
exhibiting an increase in mean pocket depth at
12 months. The difference between the two treat-
ment groups was more marked for attachment level
change at 12 months, with 42.4% and 23.5% of
subjects losing attachment in the no-antibiotic and
antibiotic groups, respectively (P < 0.001, chi-square
analysis).
Riskburden for disease progression of
sites in subjects who did or did not
receive adjunctive systemically
administered antibiotics
The above data suggested that systemically admin-
istered antibiotics were of clinical benet in the
treatment of periodontitis in the tested subjects. The
riskburden for subjects who did or did not receive
systemically administered antibiotics is presented in
7 . 0
4 . 0
1 . 0
1 . 0
4 . 0
5 1
5
9
6
4 1
1
1 1
6 1
7
0 1
2
3 1
8
3
7 1
2 1
4
m m
M
e
a
n

A
L

c
h
a
n
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e

(
m
m
)
c i t o i b i t n a c i m e t s y S
c i t o i b i t n a l a c o L
e n i l c y c y x o d e s o d - w o L
e n o N
y p a r e h t e v i t c n u j d A
s s o L
Gain
1 0 0 . 0 < P
Fig. 25. Bar plots of the change in mean attachment level
from baseline (pretherapy) to 12 months post-therapy in
subjects in the 17 treatment groups. Attachment level was
measured at six sites per tooth for each tooth at baseline
and at 12 months. The mean change in attachment level
was computed for each subject and then averaged across
subjects in the different treatment groups. The bars have
been ordered from the best attachment level change on
the left to the least on the right. The bars represent the
mean values and the whiskers represent the standard er-
ror of the mean. The numbers under the bars represent
the different treatment groups. Signicance of difference
among treatment groups was determined using analysis of
covariance (ANCOVA), adjusting for baseline pocket depth.
AL, attachment level.
238
Haffajee et al.
Fig. 29. The data indicate that systemically adminis-
tered antibiotics reduced the risk of a site showing
disease progression and decreased the burden on the
clinician in terms of reducing the number of sites that
showed disease progression measured as attachment
level loss of 3 mm. This was particularly noticeable
at the vast majority of sites with initial pocket depths
of <8 mm. The burden to the clinician of progressing
sites was cut essentially in half by adding adjunctive
systemic antibiotic therapy to mechanical debride-
ment, even at the shallow and moderate pockets.
Effect of baseline pathogen levels on
clinical outcomes after different
therapies
Analysis of samples of subgingival biolm from
different periodontal sites in different subjects with
periodontitis revealed a wide range of counts of
individual species. For example, before therapy,
4,100 sampled sites exhibited no detectable levels
of P. gingivalis, while 6,667 sampled sites revealed
levels of P. gingivalis ranging from 10
4
to 2 10
7
.
The question was raised as to what the therapeutic
response would be at sites that harbored different
levels of this species and whether the addition of
either periodontal surgery or systemically adminis-
tered antibiotics would alter this response. Fig-
ure 30 presents the pocket depth change from
baseline to 12 months at sites that harbored dif-
ferent levels of P. gingivalis at baseline and the
effect that either systemically administered antibi-
otic(s) or periodontal surgery had on pocket depth
reduction. Clearly, sites with greater baseline levels
of P. gingivalis exhibited greater pocket depth
reduction in subjects in any treatment group. This
was probably a result of the fact that deeper
periodontal pockets harbored higher levels of
P. gingivalis and thus were more likely to show
greater pocket depth reduction. Furthermore,
treatment that included periodontal surgery
appeared to reduce pocket depth at sites with the
different levels of P. gingivalis more than treatment
which included systemically administered antibi-
otic(s), although signicantly better reductions were
observed for both adjunctive therapies.
9 . 0
7 . 0
5 . 0
2 . 0
0 . 0
1
2
6
4 1
4
3
9
5
0 1
2 1 6 1
5 1
8
1 1
7
3 1
7 1
M
e
a
n

A
L

c
h
a
n
g
e

(
m
m
)
2 0 0 . 0 = P
c i t o i b i t n a c i m e t s y S
c i t o i b i t n a l a c o L
e n i l c y c y x o d e s o d w- o L
e n o N
y p a r e h t e v i t c n u j d A
Fig. 26. Bar plots of the change in mean pocket depth
subjects in the 17 treatment groups. Pocket depth was
measured at six sites per tooth for each tooth at baseline
and at 12 months. The mean change in pocket depth was
computed for each subject and then averaged across
subjects in the different treatment groups. The bars have
been ordered from the best pocket depth reduction on the
left to the least on the right. The bars represent the mean
values and the whiskers represent the standard error of
the mean. The numbers under the bars represent the
different treatment groups. Signicance of difference
among treatment groups was determined using analysis of
covariance (ANCOVA), adjusting for baseline pocket depth.
AL, attachment level.
239
0 . 1
2 . 3
3 . 5
5 . 7
6 . 9
0 . 1
2 . 3
3 . 5
5 . 7
6 . 9
S N
c i t o i b i t n a o N
l e v e l t n e m h c a t t A
c i t o i b i t n A
s t c e j b u S
m m m m
Fig. 28. Plots of mean attachment level values at baseline
and at 12 months post-therapy in subjects who did not
(left panel) or did (right panel) receive systemic antibiotics
as part of their periodontal therapy. The description of the
gure is as described for Fig. 27 except that the subjects
were ordered according to pretherapy mean baseline
attachment level measurements. The baseline and 12-
month distributions differed signicantly only for the
subjects receiving systemic antibiotics.
Fig. 27. Plots of mean pocket depth values at baseline and
at 12 months post-therapy in subjects who did not (left
panel) or did (right panel) receive systemic antibiotics as
part of their periodontal therapy. The blue circles repre-
sent the mean baseline pocket depth (measured at six sites
per tooth for up to 28 teeth) for each subject and have
been ordered from the subject with the lowest mean
pocket depth value to the subject with the highest value
pretherapy. The red and green circles represent the 12-
month mean pocket depth data for each subject. The red
circles represent subjects who exhibited a mean increase
in pocket depth, and the green circles represent subjects
who exhibited a mean decrease in pocket depth at
12 months. The distributions were signicantly different
for both treatment groups (KolmogorovSmirnov test).
240
Haffajee et al.
Figure 31 presents similar data for attachment level
change. Once more, in general, greater attachment
level reduction occurred at sites with higher baseline
levels of P. gingivalis, although this was not as con-
sistent as observed for pocket depth reduction
(Fig. 30). In contrast to the previous gure, the
greatest clinical benet observed was for the im-
proved attachment level reduction in subjects
receiving adjunctive systemic antibiotic(s). Surgery
had a mixed effect on attachment level change,
leading to greater attachment level reductions at
some microbial thresholds, but more commonly,
fewer attachment level reductions or even losses of
attachment at others.
Effect of different periodontal
therapies on microbiological
parameters
The above sections indicated that, in general, the
overall effects of periodontal therapy were benecial
in that mean clinical parameters were improved and
species that were thought to be periodontal patho-
gens were reduced in number by therapy. It was also
demonstrated that subjects receiving therapies which
included adjunctive systemically administered anti-
biotics often showed a better therapeutic response
than subjects who did not receive these adjunc-
tive agents (Fig 2731). The mean changes in the
subgingival microbial proles from baseline to
12 months post-therapy, which took place in subjects
who did or did not receive systemically administered
antibiotics, are shown in Fig. 32. The data indicate
that the group which did not receive systemically
administered antibiotics showed a reduction in mean
counts of the red complex species, T. forsythia, P.
gingivalis and T. denticola, as well as certain orange
complex species, such as Peptostreptococcus micros,
P. intermedia and P. nigrescens. The counts of A.
naeslundii genospecies 1 and 2, as well as C. gingi-
valis, E. saburreum and P. melaninogenica, were also
signicantly reduced by therapies that did not in-
clude systemically administered antibiotics. Subjects
whose therapy included adjunctive systemically
administered antibiotics demonstrated a somewhat
wider spectrum of statistically signicant reductions
in mean counts of individual species than subjects
who did not receive these adjuncts. This was in ac-
cord with the greater clinical benet observed for
such subjects (Fig 27 and 28). All species of the red
complex, and nine of 12 orange complex species,
were signicantly reduced in the subjects receiving
systemically administered antibiotics at 12 months
(Fig. 32).
Clinical and microbiological effects
of periodontal therapies 24 months
post-therapy
The data presented above indicated that periodontal
therapy had an effect on the subgingival microbiota
and that this effect was reected in clinical
improvement. The data also indicated that certain
therapies had a more profound effect on the sub-
gingival microbiota, particularly treatments that
included systemically administered antibiotics. What
was not clear from the above information was
whether the changes in the subgingival microbiota
and clinical response would persist beyond 1 year.
Within the 17 treatment groups outlined in Fig. 1,
eight were part of a randomized clinical study of
subjects recruited in Boston (MA, U.S.A.) and
0
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0 3
5 4
0 6
0 2 4
3 <
3
4
5
6
7
8
9
3 <
3
4
5
6
7 8
9
k s i r d e s a e r c n I
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L
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t
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t
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0
0
,
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r
e
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m
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s
i
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t
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s
e
t
i
s
l
a
t
o
t
> L A s h t n o m 2 1 o t e n i l e s a b m o r f m m 3
I
n
c
r
e
a
s
e
d

b
u
r
d
e
n
Percentage of sites exhibiting
Fig. 29. Plot of the riskburden at 12 months post-ther-
apy of sites with different baseline pocket depths in sub-
jects receiving or not receiving systemically administered
antibiotics. The x-axis presents the risk as dened by the
percentage of sites exhibiting 3 mm clinical attachment
loss (AL) 12 months post-therapy. The y-axis presents the
burden dened as the number of sites per 10,000 total
sites that exhibited disease progression of 3 mm. The
numbers in the gure indicate the pocket depth pre-
therapy, and their location in the plot represents the risk
burden for that pocket depth category. The black numbers
represent the sites in subjects not receiving antibiotics,
while the red numbers represent sites in subjects who
received systemically administered antibiotics.
241
Sweden, examining the effects of eight combinations
of periodontal treatment on clinical and microbial
outcomes over a 2-year post-therapy period. The
study employed a 2
3
factorial design in which, after
baseline monitoring, all subjects received full-mouth
scaling and root planing and were randomly as-
signed to one of eight groups who did or did not
receive: (a) Widman ap surgery, (b) systemically
delivered amoxicillin + metronidazole for 2 weeks,
or (c) locally delivered tetracycline at sites with an
initial pocket depth of >4 mm. Groups 18 in Fig. 1
indicate the treatment groups and the therapies re-
ceived. It should be noted that the large number of
subjects in the scaling and root planing-only group
(group 8) in Fig. 1 represents not only the 21 sub-
jects in this randomized study, but also the control
subjects for other studies (groups 917). Complete
clinical and microbiological data for baseline, and at
3, 6, 12, 18 and 24 months post-therapy, were
available for 169 subjects. Some of the results of this
ongoing study are presented below in order to
demonstrate that many of the benecial clinical and
microbial effects of the tested therapies persist for at
least 24 months. The complete ndings and details
of the methodology will be published when all data
from that study have been analyzed.
Overall effects of periodontal therapy on
clinical parameters and the subgingival
microbiota to 2 years
There was a decrease in mean clinical parameters
over time for the entire group of subjects. All of the
decreases were statistically signicant, with the
exception of mean clinical attachment level change
(Fig. 33). The major reductions occurred between the
pretherapy visit and 3 months post-therapy. The
percentage of sites with plaque accumulation, sup-
puration and bleeding on probing, as well as mean
pocket depth, remained at the lowered levels to
24 months, while the percentage of sites with gingival
redness and mean attachment level increased slightly
during this time period.
The effects of therapy on the subgingival microb-
iota of the complete group of 169 subjects were
impressive in that mean reductions in species that
had occurred from 3 to 12 months were maintained,
or even continued to decrease, at 18 and 24 months
Fig. 30. Mean pocket depth changes 12 months post-
therapy at sites with different baseline levels of Porphy-
romonas gingivalis. Samples were taken from 10,767
periodontal sites in 461 subjects with periodontitis prior to
therapy and individually analyzed for their content of
P. gingivalis. A total of 4,100 sites exhibited no detectable
P. gingivalis. The remaining 6,667 sites were subset into
eight equal groups, with cut-off points varying from
<0.17 10
5
to >8.32 10
5
, as indicated in the gure. The
changes in pocket depth from baseline to 12 months, at
sites in each P. gingivalis category, were averaged within a
subject and then averaged across subjects who were
subset according to receiving systemic antibiotic(s) or not
(left panel), or receiving periodontal surgery or not (right
panel). Signicance of differences in mean pocket depth
change between subjects receiving or not receiving
adjunctive antibiotic(s) (left panel) or surgery (right panel)
was determined using the MannWhitney test.
242
Haffajee et al.
(Fig. 34). The pattern of red and orange complex
species showing the greatest reductions post-therapy
that was observed in Fig 1115 and 17 was main-
tained until the 2-year time-point. In addition,
members of the green complex and the Actinomyces
species also showed signicant reductions in mean
counts at 2 years. Total DNA probe counts declined,
over time, to 40% of the mean original value
(Fig. 35, P < 0.001). The mean counts of likely or
consensus periodontal pathogens, including P. gin-
givalis, T. forsythia, T. denticola and E. nodatum,
were also maintained at lowered levels over the
2-year monitoring period (Fig. 35).
Reduction in specic bacterial species was associ-
atedwithgain inattachment level (Fig. 36). The mean
counts of E. nodatum, T. forsythia, P. gingivalis and T.
denticola were reduced more at sites that gained
2 mm in attachment from baseline to 24 months
post-therapy. Sites that exhibited no change or loss of
attachment of 2 mm exhibited less of a reduction in
these bacterial species at 24 months. These data sug-
gest that failure to reduce periodontal pathogens after
therapy leads to a diminished therapeutic benet.
Effects of individual therapies on clinical
parameters and the subgingival
microbiota to 2 years
The effects of different combinations of periodontal
therapies on pocket depth and attachment level
change from pretherapy to 24 months post-therapy
are presented in Fig. 37. In general, all treatment
groups exhibited a signicant reduction in mean
pocket depth that was maintained to 24 months.
However, there were signicant differences among
groups in pocket depth reduction at 6 and
12 months, and differences approached signicance
at 3 and 24 months. The greatest pocket depth
reduction occurred in subjects receiving surgery
together with systemically administered amoxicillin
and metronidazole, with and without locally deliv-
ered antibiotics. The least pocket depth reduction
occurred in subjects receiving scaling and root
planing only, or scaling and root planing and
adjunctive locally delivered tetracycline. Mean (
standard error of the mean) full-mouth pocket
depth reductions for all groups in this study were
Fig. 31. Mean attachment level changes 12 months post-
therapy at sites with different baseline levels of Porphy-
romonas gingivalis. Samples were taken from 10,767
periodontal sites in 461 subjects with periodontitis before
therapy and individually analyzed for their content of
P. gingivalis. A total of 4,100 sites exhibited no detectable
P. gingivalis. The remaining 6,667 sites were subset into
eight equal groups with cut-off points varying from
<0.17 10
5
to >8.32 10
5
, as indicated in the gure. The
changes in attachment level from baseline to 12 months
at sites in each P. gingivalis category were averaged within
a subject and then averaged across subjects who were
subset according to receiving systemic antibiotic(s) or not
(left panel), or receiving periodontal surgery or not (right
panel). Signicance of differences in mean attachment
level change between subjects receiving or not receiving
adjunctive antibiotic(s) (left panel) or surgery (right panel)
was determined using the MannWhitney test.
243
remarkably good, ranging from 0.81 0.08 (scaling
and root planing and local antibiotics) to 1.13
0.09 mm (surgery plus local and systemic antibiot-
ics) at 2 years.
Attachment level change over the 24-month period
was less consistent among treatment groups (Fig. 37).
Six of the treatment groups exhibited a mean de-
crease in attachment level at 24 months, with the
greatest improvement observed in the subjects
receiving surgery plus systemically administered
antibiotics. Subjects receiving scaling and root
planing only, and scaling and root planing and ad-
junctive surgery, exhibited loss of attachment at
24 months. Signicant differences among treatment
groups was observed at 6, 12 and 18 months and
approached signicance at 24 months. Mean (
standard error of the mean) full-mouth attachment
level change ranged from a loss of 0.01 0.12 (scaling
and root planing + surgery and scaling and root
planing alone) to a gain of 0.43 0.13 mm in the
subjects receiving surgery plus systemically admin-
istered amoxicillin and metronidazole. A mean full-
mouth attachment level gain of 0.4 mm is quite
impressive 2 years after completion of therapy.
The 2
3
factorial design permitted the evaluation
of main effects of the three types of adjunctive
therapies surgery, systemically administered anti-
biotics and locally delivered antibiotics as well as
the interaction among treatments. The expectation
was that the treatment effects would be additive (i.e.
there would be no interactions among or between
treatments). This proved to be the case (data not
shown), indicating that additive effects did occur as a
result of combining two or more treatments, as
shown in Fig. 38. The least mean pocket depth
reduction occurred in the subjects who received
neither adjunctive surgery nor adjunctive systemic-
ally administered antibiotics. The addition of
0 . 0 6 . 1 2 . 3 8 . 4 4 . 6
*
* *
* * *
*
* *
* * *
* * *
* * *
*
* *
* *
i i l e a r s i . A
a l u v r a p . V
i i n o d r o g . S
s i t i m . S
s i l a r o . S
a e c a r h c o . C
s i l i c a r g . C
s u t c e r . C
e a w o h s . C
m u t a d o n . E
s o r c i m . P
s i l a c c u b . L
a s o c u m . N
s e n c a . P
a i x o n . S
0 . 0 6 . 1 2 . 3 8 . 4 4 . 6
* *
* * *
* *
* * *
* *
* * *
* * *
* *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
*
* *
s c i t o i b i t n a c i m e t s y s o N
s c i t o i b i t n a c i m e t s y S
e l p r u P
w o l l e Y
n e e r G
e g n a r O
d e R
r e h t O
0 1 x s t n u o C
5
y p a r e h t - t s o p s h t n o m 2 1 e n i l e s a B
5
gingival plaque samples taken at baseline and 12 months
post-therapy from subjects who did not (left panel) or did
(right panel) receive systemic antibiotics as part of their
periodontal therapy. Plaque samples were taken from the
mesial aspect of each tooth and analyzed separately for
their content of 40 species of bacteria. Data for each
species were averaged within each subject and then across
subjects in the two treatment groups for each time-point
separately. Signicance of differences over time was
sought using the Wilcoxon test and adjusted for multiple
comparisons (32); *P < 0.05; **P < 0.01; ***P < 0.001.
Species were ordered according to microbial complexes
(31).
244
Haffajee et al.
either adjunct resulted in increased pocket
depth reduction, and the addition of both adjuncts
resulted in the largest reduction in mean pocket
depth.
The main effects of adjunctive surgery or adjunc-
tive systemic antibiotics at the different time-points
are shown in Fig. 39. From 6 to 24 months there was
a greater mean reduction in pocket depth for subjects
who received Widman ap surgery. However,
attachment level reduction was greater in the group
who did not receive periodontal surgery, although the
difference between groups at each time-point was
not statistically signicant. In contrast, subjects who
received systemically administered amoxicillin and
metronidazole exhibited signicantly greater pocket
depth reduction and signicantly greater clinical
attachment level gain from 6 to 24 months than
subjects not receiving these agents. Similar ndings
were observed at sites with initial pocket depth values
of >6 mm (Fig. 40). Systemic amoxicillin and met-
ronidazole provided signicantly better attachment
level gain and pocket depth reduction from 6 to
24 months, while surgery provided signicantly bet-
ter pocket depth reductions during the same time
period.
The changes in mean counts of the likely or con-
sensus periodontal pathogens P. gingivalis, T. forsy-
thia, T. denticola and E. nodatum, in subjects
receiving or not receiving adjunctive surgery or
adjunctive systemic antibiotics, are presented in
Figs 41 and 42. Although there were signicant
reductions over time in these four species in subjects
who did or did not receive adjunctive surgery, there
were no signicant differences between groups from
6 to 24 months, and the mean counts of E. nodatum
and T. denticola showed an increase by 24 months in
both groups (Fig. 41). In contrast, the mean counts of
the four bacterial species were reduced more in
subjects receiving systemically administered antibi-
otics compared with subjects not receiving these
agents (Fig. 42). In the antibiotic-treated subjects, the
reduction in counts achieved by therapy was main-
tained to 24 months, while counts of E. nodatum and
T. denticola had increased in the nonantibiotic-
5 2
1 3
7 3
3 4
8 4
n o i t a l u m u c c a e u q a l P
9 2
5 3
2 4
8 4
5 5
s s e n d e r l a v i g n i G
2 2
9 2
7 3
5 4
2 5
g n i b o r p n o g n i d e e l B
0
5 7 . 0
0 5 . 1
5 2 . 2
0 0 . 3
n o i t a r u p p u S
8 9 . 2
6 2 . 3
5 5 . 3
4 8 . 3
h t p e d t e k c o P
2 6 . 3
5 7 . 3
9 8 . 3
3 0 . 4
6 1 . 4
3 1 . 4
1 0 0 . 0 < P 1 0 0 . 0 < P 1 0 0 . 0 < P
1 0 0 . 0 < P 1 0 0 . 0 < P
Percentage of
s e t i s
Percentage of
s e t i s
m m m m
4 2 8 1 2 1 6 3 0
4 2 8 1 2 1 6 3 0
4 2 8 1 2 1 6 3 0
s h t n o M
Fig. 33. Mean ( standard error of the mean) for per-
centage of sites with plaque accumulation, gingival red-
ness, bleeding on probing and suppuration, as well as
mean pocket depth and attachment level at baseline, and
at 3, 6, 12, 18 and 24 months in 169 subjects with data to
24 months post-therapy. The data for each parameter
measured at up to 168 sites in each subject was averaged
within a subject and then across subjects at each time-
point separately. The signicance of differences over time
was determined using the Friedman test.
245
treated group by 24 months. Indeed, there was a
signicant between-group difference for the counts
of all four species at 24 months.
The data from this 2-year post-therapy study are
quite intriguing. There appears to be a consensus
in the eld that treatment of the infectious com-
ponent of periodontal diseases achieves its maxi-
mum benet from 3 to 6 months post-therapy and
that clinical and microbiological benets slowly
reverse thereafter. This clearly was not the case in
the present study where there was, in large part,
periodontal stability or even continuing benet over
2 years. The benecial effects differed among
treatment groups, with individuals who received
systemically administered antibiotics showing the
greatest and most sustained improvement up to
2 years. The mean counts of P. gingivalis, T. forsy-
thia, T. denticola and E. nodatum were signicantly
lower at 24 months in the subjects receiving sys-
temically administered antibiotics (Fig. 42). These
results are in agreement with the ndings of others
who have reported a sustained benecial effect on
the subgingival microbiota when systemically
administered antibiotics were employed as part of
the treatment (3, 6, 11, 16, 21, 26, 27, 38). The
reason for the sustained benet of these adjunctive
agents is not clear. A rapid reduction in the four
test and other species would be expected immedi-
ately after therapy, involving mechanical removal of
the biolm and systemic antibiotic administration.
It may be surmised that the changes in the
microbiota led to a reduction in inammation and
pocket depth in the adjacent tissues. This change in
habitat may have resulted in an environment less
conducive to the regrowth of the pathogenic spe-
cies, thereby sustaining a habitat which was bene-
cial to the host, but not to the pathogens. The
ndings also question whether clinical trials that
evaluate therapies for the treatment of periodontitis
should be ended at 6 months, as recommended by
the Task Force on Design and Analysis in Dental
and Oral Research (13).
0 . 0 7 . 2 3 . 5
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* *
* * *
* * *
i i l e a r s i . A
a l u v r a p . V
i i n o d r o g . S
s i t i m . S
s i l a r o . S
a e c a r h c o . C
s i l i c a r g . C
s u t c e r . C
e a w o h s . C
m u t a d o n . E
s o r c i m . P
s i l a c c u b . L
a s o c u m . N
s e n c a . P
a i x o n . S
0 7 . 2 3 . 5 0 7 . 2 3 . 5 0 7 . 2 3 . 5 0 7 . 2 3 . 5 0 7 . 2 3 . 5
4 2 8 1 2 1 6 3 0
e l p r u P
w o l l e Y
n e e r G
e g n a r O
d e R
r e h t O
s h t n o M
0 1 x s t n u o C
5
5
subjects at baseline (prior to therapy) and at 3, 6, 12, 18
and 24 months post-therapy. Plaque samples were taken
from the mesial aspect of each tooth at each time-point
and analyzed separately for their content of 40 species.
Data for each species were averaged within and then
across subjects separately for each time-point. Signi-
cance of differences over time was sought using the
Friedman test and adjusted for multiple comparisons (32);
*P < 0.05; **P < 0.01; ***P < 0.001. Species were ordered
according to microbial complexes (31). The dashed red
prole in the 24-month panel represents the pretherapy
(0 months) prole to facilitate direct comparison between
these 2 time points.
246
Haffajee et al.
An initial attempt to relate the
nature of polymicrobial
complexes to the effect of
different periodontal therapies
There is a growing recognition that subgingival
microbial species often occur in specic associations
in subgingival biolms. Some of these associations
(often termed microbial complexes or communities)
may occur a result of specic interspecies co-aggre-
gations, or of nutritional interdependence (14). Given
the frequent occurrence of specic combinations of
pathogens in the same diseased periodontal site, it
seems likely that the initiation and progression of
periodontitis might often be polymicrobial in nature,
rather than caused by a single species. It is recog-
nized that each individual has an unique subgingival
microbial prole (14, 35). In the present section, we
provide an initial attempt to relate the nature of a
subjects subgingival microbial prole, as measured
by the levels of 40 bacterial species, to the effect of
periodontal therapy. Without knowledge of the cor-
rect polymicrobial proles to use in this analysis, we
employed cluster analysis to provide an unbiased
grouping of subjects with different subgingival
microbiotas.
Relationship between subgingival
microbial proles and response to
different periodontal therapies
It was pointed out previously (35) that there were
differences in mean subgingival microbial proles
among subjects with chronic periodontitis. However,
subjects could be grouped into clusters (Fig. 3 in
Ref. 35) in which they showed similar mean sub-
gingival microbial proles. Figure 43 reproduces
Fig. 4 from Teles et al. (35), and presents the mean
baseline microbial proles of the subjects in the 10
cluster groups and those subjects who were not in
the clusters. Not surprisingly, the microbial proles
of the different clusters were quite distinct and
characterized by different species and/or complexes.
0 . 0
6 . 7 1
2 . 5 3
8 . 2 5
4 . 0 7
s h t n o M
8 1 2 1 6 3 0
4 2
1 0 0 . 0 < P
2 . 0
4 . 0
5 . 0
6 . 0
7 . 0
m u t a d o n . E
3 . 1
1 . 2
9 . 2
6 . 3
4 . 4
9 . 0
7 . 1
5 . 2
3 . 3
1 . 4
4 . 0
6 . 0
8 . 0
0 . 1
2 . 1
l a t o T 0 1 x s t n u o c e b o r p A N D
5
C
o
u
n
t
s

x

1
0
5
1 0 0 . 0 < P
1 0 0 . 0 < P
1 0 0 . 0 < P
1 0 0 . 0 < P
Fig. 35. Mean total DNA probe counts and counts of
selected species (10
5
, standard error of the mean) in
subgingival plaque samples from 169 chronic periodon-
titis subjects at baseline (prior to therapy) and at 3, 6, 12,
18 and 24 months post-therapy. Plaque samples were
taken from the mesial aspect of each tooth at each time-
point and analyzed separately for their content of 40
species. Data for each species were averaged within and
then across subjects separately for each time-point. Sig-
nicance of differences over time was sought using the
Friedman test and adjusted for 40 comparisons (32).
247
For example, clusters 3, 4, 5 and 6 exhibited elevated
levels of red complex species, while other clusters
had lower levels of this complex. Cluster 1 was
predominated by E. nodatum, while clusters 9 and
10 exhibited high levels of A. naeslundii genospecies
1 and 2. P. intermedia was present in high mean
levels in clusters 4 and 7, P. melaninogenica was
present in high levels in cluster 4 and V. parvula was
dominant in cluster 8. The mean change in pocket
depth and attachment level at 12 months post-
therapy in the subjects in the different microbial
cluster groups is presented in Fig. 44. All cluster
groups showed an improvement in full-mouth mean
pocket depth, ranging from 0.27 to 0.95 mm, and all
groups showed an improvement in mean clinical
attachment level post-therapy, with the exception of
cluster group 10. Mean attachment level change
ranged from a loss of 0.04 mm for cluster group 10
to a gain of 0.45 mm for cluster group 5. It is worth
noting that cluster group 5 was dominated by the
red complex species.
An initial attempt was made to relate clinical
attachment level change post-therapy to the nature
of the baseline subgingival microbiota and the ther-
apy employed (Fig. 45). Cluster 10, the group that
showed loss of attachment post-therapy, as described
above, was dominated by A. naeslundii genospecies 1
and 2, but had low levels of the other test species.
Subjects in this cluster showed a poor response to a
number of different therapies, including scaling and
root planing alone, surgery alone (both apically
repositioned ap and modied Widman ap), or
scaling and root planing with either systemically
administered metronidazole or azithromycin. How-
ever, treatment of subjects with this microbial prole
by professional cleaning provided a modest response,
while scaling and root planing with systemically
administered metronidazole and amoxicillin, or sca-
0 6 . 0
1 4 . 0
3 2 . 0
4 0 . 0
5 1 . 0
m u t a d o n . E
1 9 . 5
3 4 . 4
6 9 . 2
8 4 . 1
8 2 . 5
6 9 . 3
4 6 . 2
2 3 . 1
0 0 . 0
3 0 . 1
4 7 . 0
6 4 . 0
8 1 . 0
5 0 . 0 < P 1 0 0 . 0 < P
5 0 . 0 < P
0 0 . 0
0 0 . 0
C
o
u
n
t
s

x

1
0
5
m m 5 . 1 > s s o l L A
m m 2 < e g n a h c L A
m m 5 . 1 > n i a g L A
Fig. 36. Mean change in counts (10
5
, standard error of
the mean) of Eubacterium nodatum, Tannerella forsythia,
Porphyromonas gingivalis and Treponema denticola at
sites that gained attachment of 2 mm, lost attachment
of 2 mm, or exhibited changes between these values,
from baseline to 24 months post-therapy. Plaque samples
were taken from the mesial aspect of each tooth at each
time-point and analyzed separately for their content of 40
species. Differences in counts between baseline and
24 months for each species were averaged within a site-
type category in each subject and then across subjects
separately for each site-type category. Signicance of
differences among site-type categories was sought using
the KruskalWallis test and adjusted for 40 comparisons
(32). AL, attachment level.
248
Haffajee et al.
ling and root planing plus systemically administered
doxycycline, provided a good response. Cluster 1
subjects, who showed the second worst clinical re-
sponse and who had very high levels of E. nodatum,
showed a poor response to multiple therapies, but a
good response to therapies that included systemically
administered metronidazole, with and without
amoxicillin (treatments 9 and 16). Cluster 5, charac-
terized by high counts of species, particularly red
complex species, and the group showing the largest
mean gain in attachment level post-therapy, was
effectively treated by a number of different thera-
peutic modalities. Certain treatments, including
those in groups 1, 2, 5, 6 and 16, exhibited a good
response for multiple microbial proles.
Similar data for pocket depth change are presented
in Fig. 46. As expected from the data presented in
Fig. 26, the vast majority of cluster/treatment groups
showed pocket reduction at 12 months post-therapy.
However, cluster groups 1, 9 and 10 tended to show
less pocket depth reduction, than other cluster
groups, to a wide range of periodontal therapies. The
clusters had low levels of red complex species, but
high levels of E. nodatum (cluster 1) or Actinomyces
species (clusters 9 and 10). As expected, the treat-
ments that included periodontal surgery (treatment
groups 14 and 12) provided good pocket depth
reductions.
There are many caveats to this section. This is a
retrospective analysis of data from a number of
clinical investigations examining the microbiological
and clinical effects of different periodontal therapies.
Thus, there was no overall randomization of subjects
according to treatment and cluster group. For some
4 2 . 1
3 9 . 0
2 6 . 0
1 3 . 0
0 0 . 0
0 6 . 0
0 4 . 0
9 1 . 0
2 0 . 0
2 2 . 0
1 0 . 0 < P
5 0 . 0 < P
9 0 . 0 = P
5 0 . 0 < P
1 0 . 0 < P
5 0 . 0 < P
8 0 . 0 = P
6 0 . 0 = P
l e v e l t n e m h c a t t A h t p e d t e k c o P
s h t n o M 4 2 8 1 2 1 6 3 0
) 3 2 , 1 G ( t c A M A g r u S
) 8 1 , 2 G ( M A g r u S
) 1 2 , 3 G ( t c A g r u S
) 2 2 , 4 G ( g r u S
) 4 2 , 5 G ( t c A M A
) 5 2 , 6 G ( M A
) 6 2 , 7 G ( t c A
) 1 2 , 8 G ( y l n o P R S
m m m m
4 2 8 1 2 1 6 3 0
Fig. 37. Mean change ( standard error of the mean) in
pocket depth (left panel) and attachment level (right pa-
nel) from baseline (pretherapy) to 3, 6, 12, 18 and
24 months post-therapy in subjects in the eight treatment
groups. Clinical measurements were made at six sites per
tooth at up to 28 teeth in each subject at each time-point.
The measurements were averaged within a subject, aver-
aged at each time-point and then the change from base-
line was computed for each parameter. Signicance of
differences among treatment groups was determined
using analysis of covariance (ANCOVA), adjusting for base-
line pocket depth, baseline attachment level, country,
current smoking status and age. Signicance of differ-
ences among treatment groups at each time-point is
indicated on the graphs. Change over time for each
treatment group for both pocket depth and attachment
level was signicant at P < 0.001 (Friedman test). The
number of subjects in each group is indicated in par-
entheses next to the group label. All subjects received
scaling and root planing (SRP). Adjunctive therapies in-
cluded: Widman ap surgery (Surg); systemically
administered amoxicillin (500 mg three times daily) and
metronidazole (250 mg three times daily), each for
14 days (AM); and locally delivered tetracycline bers at
sites with initial pocket depth >4 mm (Act).
249
0 0 . 0
0 2 . 1
0 3 . 0
0 6 . 0
0 9 . 0
4 1 . 1
1 0 . 1
1 0 . 1
4 8 . 0
y r e g r u S
s e Y
o N
t e M + x o m A
o N
s e Y
5 0 . 0 < P
m m
5 0 . 0 < P
Fig. 38. Mean pocket depth reduc-
tion from baseline to 24 months in
subjects who did or did not receive
adjunctive Widman ap surgery
and in subjects who did or did not
receive systemically administered
amoxicillin (AMOX) and metroni-
dazole (MET). There were signi-
cant differences between pocket
depth reduction in the subjects who
did or did not receive surgery
(P < 0.05) and between the subjects
who did or did not receive systemic
antibiotics (P < 0.05; analysis of
covariance (ANCOVA), adjusting for
baseline pocket depth, baseline
attachment level, country, age and
current smoking status).
2 1 . 1
4 8 . 0
6 5 . 0
8 2 . 0
1 4 . 0
1 3 . 0
0 2 . 0
0 1 . 0
5 0 . 0 < P
1 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
0 0 . 0
0 0 . 0
1 1 . 1
4 8 . 0
6 5 . 0
8 2 . 0
0 0 . 0
5 4 . 0
4 3 . 0
2 2 . 0
1 1 . 0
5 0 . 0 < P
1 0 . 0 < P
1 0 0 . 0 < P
1 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
0 0 . 0
m m
4 2 8 1 2 1 6 3 0
s h t n o M
4 2 8 1 2 1 6 3 0
4 2 8 1 2 1 6 3 0 4 2 8 1 2 1 6 3 0
m m
4 8 = n y r e g r u s
6 9 = n y r e g r u s o N
0 9 = n t e M + x o m A
0 9 = n t e M + x o m A o N
s h t n o M
Fig. 39. Mean change ( standard error of the mean) in
pocket depth (top two panels) and attachment level
(bottom two panels) at different time-points in subjects
who did or did not receive surgery (left panels) and in
subjects who did or did not receive systemic antibiotics
(right panels). The mean values represent the main effects
for the treatment comparison after adjusting for baseline
pocket depth, baseline attachment level, country, age and
smoking status using analysis of covariance (ANCOVA).
AMOX, amoxicillin, MET, metronidazole.
250
Haffajee et al.
of the cells, there were small numbers of subjects and
some cells were empty. In addition, baseline clinical
characteristics were not necessarily identical in each
cluster/treatment group, and no attempt was made
to adjust for baseline differences, although all sub-
jects exhibited chronic periodontitis. Nonetheless,
the data may provide the most comprehensive effort
at linking broad microbial proles to the outcome of
different periodontal treatments. It seems clear that
certain microbial proles, such as those dominated
by species such as A. naeslundii genospecies 2 or
E. nodatum, may respond poorly to many periodon-
tal therapies and might require quite specic anti-
microbial adjuncts. Other proles might be more
easily treated using mechanical debridement proce-
dures, with or without antimicrobial agents. The data
suggest that no single treatment was effective for
every microbial prole encountered in this popula-
tion. Clearly, prospective studies that link the deter-
mination of the composition of the subgingival
microbiota with the most appropriate therapeutic
modalities should be of high priority.
Concluding thoughts
The treatment of periodontal infections has evolved
for well over 100 years, largely on an empirical basis.
It has long been recognized that removal of deposits
on the teeth has a benecial effect on the adjacent
periodontal tissue. This removal, whether by scaling
and root planing or oral hygiene procedures, has
become the cornerstone of periodontal therapy.
However, the effect of this treatment on the subgin-
gival microbiota is not fully understood. It is apparent
that physical removal of subgingival biolms results
in only a brief reduction in the numbers of colonizing
organisms and that the total bacterial mass of dental
4 4 . 3
8 5 . 2
2 7 . 1
6 8 . 0
0 0 . 0
7 8 . 1
1 4 . 1
4 9 . 0
7 4 . 0
0 0 . 0
1 0 0 . 0 < P
1 0 0 . 0 < P
1 0 . 0 < P
5 0 . 0 < P
0 3 . 3
8 4 . 2
5 6 . 1
3 8 . 0
0 0 . 0
2 0 . 2
1 5 . 1
1 0 . 1
0 5 . 0
1 0 0 . 0 < P
8 0 . 0 = P
1 0 . 0 < P
1 0 0 . 0 < P
1 0 . 0 < P
1 0 . 0 < P
8 0 . 0 = P
1 0 . 0 < P
5 0 . 0 < P
0 0 . 0
m m
4 2 8 1 2 1 6 3 0
s h t n o M
4 2 8 1 2 1 6 3 0
4 2 8 1 2 1 6 3 0 4 2 8 1 2 1 6 3 0
m m
1 8 = n y r e g r u s
3 9 = n y r e g r u s o N
6 8 = n t e M + x o m A
8 8 = n t e M + x o m A o N
s h t n o M
Fig. 40. Mean change ( standard error of the mean), at
sites with baseline pocket depth >6 mm, in pocket depth
(top two panels) and attachment level (bottom two panels)
at different time-points in subjects who did or did not
receive surgery (left panels) and in subjects who did or did
not receive systemic antibiotics (right panels). The mean
values represent the main effects for the treatment com-
parison after adjusting for baseline pocket depth, baseline
attachment level, country, age and smoking status using
analysis of covariance (ANCOVA). AMOX, amoxicillin; MET,
metronidazole.
251
biolms may be restored in as little as 27 days.
However, two important phenomena occur as a re-
sult of the mechanical removal of supragingival and
subgingival microorganisms. The rst is a shift in
proportions of the species during the recolonization
period. The second is habitat modication. These are
the key features of treatments that involve mechan-
ical debridement.
Recolonization of bacteria on oral surfaces after
physical removal is a rapid event because small
numbers of bacteria, when provided with relatively
abundant nutrients, can multiply quite rapidly. In
laboratory culture, for example, many species of
bacteria exhibit generation times of under 60 min.
In the oral cavity, species do not return initially in
the proportions that they made up in the mature
dental plaque removed by the therapist. There is
clear evidence of microbial succession in the return
of species colonizing the teeth and periodontal
tissues. Certain Streptococcus and other species
appear to colonize quite rapidly, setting the stage
for subsequent colonization by other taxa (14, 15,
30). Selective adhesion, co-aggregation and nutri-
tional interdependence appear to be important
determinants of the rst wave of colonization (14).
Importantly, from a therapeutic perspective, many
of the species that return slowly after mechanical
debridement are periodontal pathogens, such as the
red complex species, T. forsythia, P. gingivalis and
T. denticola. Unfortunately, these species are usu-
ally not eliminated by mechanical debridement.
They return slowly post-therapy, making it neces-
sary for the patient to return for repeated main-
tenance visits.
In addition to the slow return of many periodontal
pathogens post-therapy, habitat modication occurs
1 . 0
3 . 0
4 . 0
5 . 0
m u t a d o n . E
0 . 1
8 . 1
5 . 2
3 . 3
0 . 4
7 . 0
5 . 1
3 . 2
0 . 3
3 . 0
5 . 0
6 . 0
8 . 0
0 . 1
5 0 . 0 < P
y r e g r u S
y r e g r u s o N
s h t n o M 4 2 8 1 2 1 6 3 0 4 2 8 1 2 1 6 3 0
6 . 0
8 . 3
C
o
u
n
t
s

x

1
0
5
5 0 . 0 < P
5 0 . 0 < P
monas gingivalis and Treponema denticola, at baseline,
and at 3, 6, 12, 18 and 24 months post-therapy, in subjects
who did or did not receive adjunctive Widman ap sur-
gery. Plaque samples were taken from the mesial aspect of
each tooth at each time-point and analyzed separately for
their content of 40 bacterial species. Counts for the four
species were individually averaged within a subject, and
then across subjects, in the two treatment groups for each
time-point separately. Signicance of differences between
treatment groups at each time-point was sought using
analysis of covariance (ANCOVA), adjusting for baseline
counts. Signicance of differences over time was sought
using the Friedman test.
252
Haffajee et al.
in the form of reduced periodontal inammation and
pocket depth reduction. Habitat controls microbial
colonization. If a species depends on the presence of
a deep pocket or products resulting from periodontal
inammation in order to ourish in an environment,
then that species will exist at low numbers or be
eliminated from a successfully treated periodontal
site. Return to pretherapy levels of the pathogenic
species will depend on repeated iterations of the
pathogen(s) or other species colonizing that site
affecting the habitat (host tissues), the habitat
responding typically by inammation, which may in
turn favor pathogen growth, etc. The re-establish-
ment of the original climax community, which
included high levels of pathogens, could occur
quickly in some individuals and slowly in others.
Individuals who have a hyperinammatory pheno-
type might be more likely to react to low levels of
periodontal pathogens, leading to local inammation
that favors pathogen regrowth.
Habitat modication and slowing of recolonization
by pathogenic species that may be achieved by sca-
ling and root planing can be augmented by other
physical procedures. Periodontal surgery can slow
the return of periodontal pathogens by reducing
periodontal pockets, namely the habitats that most
favor the overgrowth of certain species, particularly
members of the red complex. Scrupulous, repeated
removal of supragingival plaque by the patient or by
a dental professional can also profoundly affect
subgingival colonization of shallow to moderate
periodontal pockets. This may be a result, in part, of
slowing the recolonization of pathogens in supra-
gingival plaque before extension to subgingival areas,
of reduction in gingival inammation and of
decreasing the levels of supragingival biolm species
1 . 0
3 . 0
4 . 0
5 . 0
7 . 0
m u t a d o n . E
9 . 0
7 . 1
5 . 2
3 . 3
0 . 4
6 . 0
4 . 1
2 . 2
0 . 3
2 . 0
4 . 0
6 . 0
8 . 0
0 . 1
5 0 . 0 < P
8 . 3
t e M + x o m A
t e M + x o m A o N
s h t n o M 4 2 8 1 2 1 6 3 0 4 2 8 1 2 1 6 3 0
C
o
u
n
t
s

x

1
0
5
5 0 . 0 < P
1 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
5 0 . 0 < P
monas gingivalis and Treponema denticola, at baseline,
and at 3, 6, 12, 18 and 24 months post-therapy, in subjects
who did or did not receive adjunctive systemically
administered amoxicillin and metronidazole. Plaque
samples were taken from the mesial aspect of each tooth
at each time-point and analyzed separately for their con-
tent of 40 species. Counts for the four species were indi-
vidually averaged within a subject and then across sub-
jects in the two treatment groups for each time-point
separately. Signicance of differences between treatment
groups at each time-point was sought using analysis of
covariance (ANCOVA), adjusting for baseline counts. Signi-
cance of differences over time was sought using the
Friedman test. AMOX, amoxicillin; MET, metronidazole.
253
0 4 8
i i l e a r s i . A
a l u v r a p . V
i i n o d r o g . S
s i t i m . S
s i l a r o . S
a e c a r h c o . C
s i l i c a r g . C
s u t c e r . C
e a w o h s . C
m u t a d o n . E
s o r c i m . P
s i l a c c u b . L
a s o c u m . N
s e n c a . P
a i x o n . S
0 4 8 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8
9 . 2 1
5 . 0 1
3 . 1 1
8 . 3 1
1 3 0 2 9 1 0 2 2 1 9 4 7 3 3 4 1 1 3 3 1 6 8
C I N
0 1 x s t n u o C
5
0 1 9 8 7 6 5 4 3 2 1
s p u o r g r e t s u l C
e l p r u P
w o l l e Y
n e e r G
e g n a r O
d e R
r e h t O
s e x e l p m o C
Fig. 43. Mean subgingival microbial proles from sub-
gingival biolm samples from 461 chronic periodontitis
subjects. The subjects were clustered on the basis of their
mean counts of each of 40 species averaged across 28
sampled sites in each subject taken before therapy. The
percentage similarity between subject proles was com-
puted using the chord coefcient (18) and the resulting
values were sorted using an average unweighted linkage
sort (28). The cluster analysis dendrogram was presented
in Fig. 3 in an accompanying chapter (35). The data in this
gure provide the mean counts 10
5
for the mean micro-
bial proles in the resulting clusters and the not in cluster
(NIC) group. The species have been arranged according to
the complexes described by Socransky et al. (31). The
numbers at the bottom of each panel represent the num-
ber of subjects in the cluster. The proles for clusters 1, 4, 6
and 10 have been truncated for certain species with high
mean counts to permit better visualization of the proles.
The numbers in these proles represent the actual mean
counts (10
5
) for truncated species.
0 1 . 1
3 8 . 0
5 5 . 0
8 2 . 0
0 0 . 0
6 5 . 0
9 3 . 0
3 2 . 0
6 0 . 0
0 1 . 0
5 0 . 0 < P 1 0 0 . 0 < P
m m m m
9 8 7 6 5 4 3 2 1
n pocket depth a e M n a e M s h t n o m 2 1 to 0 m o r f e g n a h c l e v e l t n e m h c a t t a s h t n o m 2 1 to 0 m o r f e g n a h c
0 1 C I N
Fig. 44. Bar plots of the mean pocket depth change (left
panel) and mean attachment level change (right panel)
subjects in the 10 cluster groups and those not in cluster
(NIC), described in Fig. 43. The pocket depth and attach-
ment levels were measured at six sites per tooth for each
tooth at baseline and at 12 months. The mean change in
pocket depth and attachment level was computed for each
subject and then averaged across subjects in the different
cluster groups. The bars represent the mean values and
the whiskers represent the standard error of the mean.
Signicance of difference among cluster groups was
determined using the KruskalWallis test.
254
Haffajee et al.
that might contribute to the growth of subgingival
taxa.
The addition of systemically administered che-
motherapeutic agents to the physical local removal
of microorganisms enhanced the therapeutic
response, as measured by clinical and microbio-
logical outcomes. The agents were administered
over a 2-week period, usually in conjunction with
mechanical debridement. The question then
becomes, how did these agents, provided for a
2-week period, lead to greater attachment level
gain, pocket depth reduction and long-term
reduction of subgingival pathogens? One factor may
have been the rapid reduction of sensitive species at
a local level (7, 17) and perhaps contributions to the
full-mouth disinfection described by the Leuven
group (4, 5, 19, 2224, 37). A second factor may
have been the reduction of pathogens in the soft
tissues lining the periodontal pocket and conceiv-
ably microorganisms in the roots of the teeth (1, 2,
8). A third possibility may have been a greater effect
than mechanical debridement on certain probable
periodontal pathogens, such as E. nodatum
(Fig. 42). Whatever the reason, the relatively inex-
pensive addition of chemotherapeutic agents to
therapy can enhance treatment outcome.
However, from the data presented above, it is clear
that subjects differ in the composition of their
subgingival microbiota because they are colonized
by different species and/or levels of periodontal
pathogens. It is also clear that individual subjects
respond quite differently to the same periodontal
therapy. It may be surmised that these differences in
clinical response are a result partly of the hosts
ability to cope with the infection and partly of the
nature of the colonizing subgingival species. The goal
of the next wave of periodontal studies should
probably be to match treatment to the specic col-
onizing microbial prole of the individual subject: a
rather complex task.
7 1 6 1 5 1 4 1 3 1 2 1 1 1 0 1 9 8 7 6 5 4 3 2 1
C I N
1
2
3
4
5
6
7
8
9
C
l
u
s
t
e
r

g
r
o
u
p
s
0 1
s h t n o m 2 1 0 e g n a h c L A
) n a i d e m ( m m 7 1 . 0 >
m m 7 1 . 0 0
) s s o l ( m m 0 <
a t a d o N
Fig. 45. Grid plot showing the mean attachment level
change for subjects subset according to cluster groups and
periodontal treatment groups. The rows indicate the 10
cluster groups and the not in cluster (NIC) group, and the
columns the 17 treatment groups (described in Fig. 1).
The boxes represent the mean change in attachment
level from baseline to 12 months for the subjects in a
given cluster/treatment group. Yellow boxes represent a
decrease in the mean attachment level measurement of
greater than the median value for all 461 subjects
(0.17 mm); green boxes represent reductions in mean
attachment level from 0 to 0.17 mm; red boxes represent
an increase in attachment level measurements (i.e. further
attachment loss). The white boxes represent cluster/
treatment groups for which no data were available. AL,
attachment level.
255
Given that systemically administered and perhaps
locally administered antibiotics can be of benet in
the treatment of periodontitis, the clinician is left
with the same critical questions. These include:
which patients should receive a systemic antibiotic;
which antibiotic(s) should be employed for which
infections; what should the doses and duration be of
antibiotic administration; and what is the optimal
time of delivery of these agents in the overall treat-
ment regime? Furthermore, do the risks of antibiotic
administration outweigh the benets? Masked in the
last question is, if antibiotics provide a more com-
plete elimination/suppression of pathogenic species,
would there be no risks, either locally or systemically,
in the incomplete elimination by other (nonantibi-
otic) treatment strategies?
In a 1988 publication, we discussed the different
possible microbiological outcomes of periodontal
therapy, and these thoughts appear relevant today
(9). We noted that the least desirable outcome would
be either no change or an increase in the levels of
periodontal pathogens, while the most desirable
effect would be the elimination of these species. The
data of the 1988 publication, using culture tech-
niques, and the data provided in this chapter, using
checkerboard DNADNA hybridization, indicate that
the most frequent microbiological outcome of peri-
odontal therapy appears to be a signicant reduction
in the levels, proportions and percentage of sites
colonized at detectable levels by many periodontal
pathogens. However, total elimination of the target
species was seldom achieved in these studies.
Although the reduction in periodontal pathogens was
associated with signicant clinical improvements, an
increase in these species may occur over time, lead-
ing to disease progression, unless frequent mainten-
ance care is provided. Thus, the major outcome of
most forms of periodontal therapy appears to be the
establishment of an uneasy equilibrium with lowered
numbers of pathogens in the new climax community,
a situation which provides lowered, but still signi-
cant, risk for disease recurrence. At this time, perio-
dontal therapy is, for the most part, arbitrary and not
necessarily optimally matched to the infection of the
individual subject. Although we and others have
made attempts to guide therapy based on the
7 1 6 1 5 1 4 1 3 1 2 1 1 1 0 1 9 8 7 6 5 4 3 2 1
C I N
1
2
3
4
5
6
7
8
9
C
l
u
s
t
e
r

g
r
o
u
p
s
0 1
s h t n o m 2 1 0 e g n a h c D P
) n a i d e m ( m m 7 4 . 0 >
m m 7 4 . 0 0
) g n i n e p e e d ( m m 0 <
a t a d o N
Fig. 46. Grid plot showing mean pocket depth change
from baseline to 12 months for 461 chronic periodontitis
subjects subset according to microbial cluster group and
periodontal treatment groups. The layout of the plot is as
described in Fig. 45, except that the threshold for pocket
depth change was 0.47 mm (median for the entire group).
PD, pocket depth. NIC, not in cluster.
256
Haffajee et al.
subgingival microbial prole of the subject, pros-
pective randomized clinical trials are essential to
establish therapeutic guidelines.
Acknowledgments
This work was supported, in part, by research grants
DE-10977, DE-12108, DE-12861, DE-13232 and DE-
14242 from the National Institute of Dental and
Craniofacial Research.
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Biology and principles of
periodontal wound
healing/regeneration
GI USEPPE POLI MENI , ANDREAS V. XI ROPAI DI S & ULF M. E. WI KESJ O
The native periodontium includes cementum, a

functionally oriented periodontal ligament, alveolar
bone and gingiva. Pathologic and/or traumatic events
may lead to the loss or damage of this anatomical
structure. Since the 1970s, a number of procedures
have been investigated in an attempt to restore such
lost tissues. Numerous clinical trials have shown
positive outcomes for various reconstructive surgical
protocols. Reduced probing depths, clinical attach-
ment gain, and radiographic bone ll have been
reported extensively for intrabony and furcation de-
fects following scaling and root planing, open ap
debridement, autogenous bone grafting, implanta-
tion of biomaterials including bone derivatives and
bone substitutes, guided-tissue regeneration (GTR)
procedures, and implantation of biologic factors,
including enamel matrix proteins.
Histological studies have shown that various
surgical periodontal procedures can lead to differ-
ent patterns of healing. Healing by formation of a
long junctional epithelium (epithelial attachment) is
characterized by a thin epithelium extending apically
interposed between the root surface and the gingival
connective tissue (4, 23). Connective tissue repair
(new attachment) is represented by collagen bers
oriented parallel or perpendicular to a root surface
previously exposed to periodontal disease or other-
wise deprived of its periodontal attachment. In con-
trast, periodontal regeneration is characterized by
de novo formation of cementum, a functionally ori-
ented periodontal ligament, alveolar bone, and gin-
giva (restitutio ad integrum). Nevertheless, it would
be naive to expect these to occur as distinctly sep-
arate biologic outcomes following reconstruction of
the periodontal attachment. For example, periodon-
tal regeneration should be expected to include ele-
ments of a new, as well as an epithelial, attachment.
Predictability of outcomes following surgical pro-
cedures is of fundamental importance in medicine.
As periodontal-regenerative procedures are time
consuming and nancially demanding, there is
increasing interest by clinicians to learn of factors
that may inuence the clinical outcome following
periodontal reconstructive surgery in order to pro-
vide the best possible service to patients. This goal
can only be achieved if biological aspects of wound
healing and regeneration are taken into considera-
tion. The objectives of the present article are to
provide an overview of wound healing following
periodontal surgical procedures, to discuss the basic
principles of periodontal regeneration, and to illus-
trate the factors that inuence this process.
Wound healing
The healing of wounds in nonoral sites has been
studied in great detail (Fig. 1) (5). The general prin-
ciples of healing, and the cellular and molecular
events observed in nonoral sites, also apply to the
healing processes that take place following perio-
dontal surgery. Traumatic injury causes capillary
damage and hemorrhage and, as a result, a blood clot
is formed. The formation of a clot is the immediate
response to any trauma. The clot has two functions: it
temporarily protects the denuded tissues; and it
serves as a provisional matrix for cell migration (5).
The blood clot consists of all cellular components of
blood (including red and white blood cells and
platelets) in a matrix of brin, plasma bronectin,
vitronectin, and thrombosporin (24).
Clot formation is followed by an early stage of
inammation. Within hours of injury, inammatory
cells (predominantly neutrophils and monocytes)
30
PERIODONTOLOGY 2000
populate the clot. These cells cleanse the wound of
bacteria and necrotic tissue through phagocytosis
and release of enzymes and toxic oxygen products.
Within 3 days, the inammatory reaction moves into
its late phase. Macrophages migrate into the wound
area and, in addition to wound debridement, secrete
polypeptide mediators targeting cells involved in the
wound-healing process (5, 45).
The macrophage plays an important role in the
formation of granulation tissue. Growth factors and
cytokines secreted by macrophages are involved in
the proliferation and migration of broblasts,
endothelial cells, and smooth muscle cells into the
wound area. The cell-rich granulation tissue next
undergoes maturation and remodeling. Fibroblasts
responsible for the replacement of the provisional
extracellular matrix produce a new collagen-rich
matrix. Approximately 1 week following wounding,
and once the collagen matrix has been synthesized,
some broblasts undergo transformation into myo-
broblasts and express a-smooth muscle actin. This
transformation and synthesis is responsible for
wound contraction. Endothelial cells, responsible for
angiogenesis, migrate into the provisional wound
matrix to form vascular tubes and loops, and as the
provisional matrix matures, the endothelial cells un-
dergo programmed cell death (apoptosis) and the
number of vascular units is reduced (1, 24).
Epithelization of the wound is initiated within hours
of injury. Epithelial cells from the basal layer prolif-
erate and migrate through the brin clot and eventu-
ally the breach in the epithelium is sealed. The
epithelial cells in normal gingival tissues use surface
receptors, knownas integrins, to bind to laminininthe
basal lamina. In order to initiate migration, the kera-
tinocytes dissolve this attachment to start expressing
integrins suitable for the wound environment (1).
Maturation of the granulation tissue will lead to the
regeneration or repair (scar formation) of the injured
tissues. Whether the damaged tissues heal by regen-
eration or repair depends upon two crucial factors: the
availability of cell type(s) needed; and the presence or
absence of cues and signals necessary to recruit and
stimulate these cells (8). This summary represents
an oversimplied explanation of the wound-healing
process. The stages described may overlap consider-
ably and the time needed for completion of each stage
may vary, depending on local and systemic factors.
Periodontal wound healing
A more complex situation presents itself when a
mucoperiosteal ap is apposed to an instrumented
root surface deprived of its periodontal attachment.
In this case, the wound margins are not two opposing
vascular gingival margins but comprise the rigid
nonvascular mineralized tooth surface, on the one
hand, and the connective tissue and epithelium of
the gingival ap, on the other. The periodontal
wound also includes tissue resources from the
alveolar bone and the periodontal ligament. Clot
formation at the interface between the tooth and a
gingival ap is initiated as blood elements are im-
posed onto the root surface during surgery and at
wound closure in a seemingly random manner, much
like a Jackson Pollock dripped and poured style
canvas (http://www.nga.gov/feature/pollock/pollock
home.html [accessed 16 February 2006]). This
represents the very rst healing event at the tooth
gingival ap interface (i.e. the absorption and adhe-
sion of plasma proteins onto the root surface) (Fig. 2)
(49). Within minutes, a brin clot attached to the root
surface is developed. Within hours, one may observe
the early phase of inammation as inammatory
cells, predominantly neutrophils and monocytes,
accumulate on the root surface, and within 3 days
the late phase of inammation dominates the healing
picture as macrophages migrate into the wound fol-
lowed by the formation of granulation tissue. At
7 days, a connective tissue attachment may be seen
at the root surface; however, areas of the brin clot in
various stages of maturation may also be observed,
depending on wound volume and tissue resources.
Early
phase
Late
phase
Fig. 1. Phases of wound healing (epidermal incisional
wounds), including an early (within hours) and a late
(within days) phase of inammation dominated by poly-
morphonuclear neutrophils and macrophages, respect-
ively. The magnitude of wound contraction parallels the
phase of granulation tissue formation. Collagen accumu-
lation is rst observed during the phase of granulation
tissue formation, continuing through the phase of matrix
formation and remodeling. Redrawn with permission
from Dr Richard AF Clark.
31
Biology and principles of periodontal wound healing/regeneration
Fig. 2. Early healing events at the toothgingival ap
interface. (A) Red blood cells (RBC) in a granular preci-
pitate adhering to the dentin surface, shown immediately
(10 min) upon wound closure; an artifactual split (arrows)
between the red blood cells and the dentin-adhering
precipitate veries the absorption/adhesion of blood ele-
ments to the dentin surface (transmission electron
micrograph, 4000). (B) Red blood cells in a brin network
in close proximity to the root surface observed within 1 h
of wound closure (photomicrograph 450). (C) Early
phase of inammation: RBC aggregates are loosely inter-
spersed in an organized brin network at 6 h. The brin
clot appears to be attached to the dentin surface.
Numerous polymorphonuclear cells are observed lining
the dentin surface (photomicrograph 450). (D) Late
phase of inammation, 3 days following wound closure,
showing macrophages lining the dentin surface (trans-
mission electron micrograph, 10,500). (E) Granulation
tissue formation, including broblasts in the maturing
brin clot at the dentin surface (photomicrograph, 450).
(F) Cell-rich connective tissue closely adapted to the
dentin surface at 7 days following wound closure (pho-
tomicrograph, 450). D, Dentin/root surface; F, brin; FB,
broblast; M, macrophage; P, plasma precipitate; PMN,
polymorphonuclear neutrophils; RBC, red blood cells;. For
detail see: Wikesjo et al. 1991 (49). These gures are
copyrighted by and modied with permission from the
American Academy of Periodontology.
32
Polimeni et al.
The studies described above investigated the
absorption, adhesion, and structural maturation of
the brin clot in periodontal wound healing, but have
not taken into consideration the functional integrity
of the toothgingival ap interface. Only a few
experimental studies have as such evaluated the
functional integrity of a maturing periodontal wound.
Hiatt et al. (11) examined the tensile strength of the
toothgingival ap interface following reconstructive
surgery of relatively small surgical dehiscence defects
over the maxillary canine teeth in the dog. They
found that the tensile strength increases from200 g
at 3 days postsurgery to 340 g at 57 days postsur-
gery, and to >1700 g at 2 weeks postsurgery. In other
words, they found that a relatively limited perio-
dontal wound might not reach functional integrity
until 2 weeks postsurgery. These data suggest that
wound integrity during the early healing phase rests
primarily on the stabilization of the gingival aps
offered by suturing. In consequence, the choice of
suture material, placement and removal, for perio-
dontal surgical procedures aimed at regeneration
should be dictated by such observations as should
postoperative protocols be instituted aimed at pro-
tecting the surgical site from trauma from oral hy-
giene procedures, and plaque colonization and
infection (21, 37).
As mentioned above, the regeneration of lost tis-
sues depends on the availability of the cell type(s)
needed and the presence or absence of cues and
signals necessary to recruit and stimulate these cells.
The extracellular matrix regulates how cells respond
to these signals. Stem cells responsible for regener-
ation of the periodontal tissues reside within the
periodontal ligament (8). The innate regenerative
potential of the periodontium has been investigated
extensively and clearly appears to be dependent on
wound management (see below). Current research
focuses on identifying biologic factors that favor
migration and proliferation of periodontal tissues
and to use those to alter the microenvironment of the
wound, favoring unimpeded healing and regener-
ation of the periodontium.
Biological factors at the base of
periodontal regeneration
Melcher (25) postulated biological concepts at the
base of periodontal regeneration. Accordingly,
periodontal structures are subdivided in four
compartments (gingival corium, periodontal liga-
ment, cementum, and bone) and the nature of the new
attachment following periodontal surgery is deter-
mined by the cells repopulating the root surface.
Karring, Nyman et al. (14) corroborated these
concepts in a series of experiments. They asked: Can
a new connective tissue attachment be established to
a root surface previously exposed to the oral envi-
ronment and implanted into bone? (13). In a dog
model, they extracted and crown-resected perio-
dontitis-affected teeth, scaled and root planed the
portion of the root that was affected with periodon-
titis, and implanted the roots into cavities created in
the alveolar bone. While connective tissue repair did
not occur at the periodontitis-affected portion of the
roots, the portion where the periodontal ligament
was preserved showed an attachment with func-
tionally oriented periodontal bers.
A second study answered the question: Can a new
connective tissue attachment establish to a perio-
dontitis-affected root implanted into gingival con-
nective tissue? (26). Using the dog model, they
implanted teeth oriented in such a way that one side
of the tooth faced alveolar bone while the other faced
the gingival connective tissue. Similarly to the previ-
ous study, no connective tissue attachment was
observed at the periodontitis-affected portion of
the roots. Conversely, the half of the root where
periodontal ligament was preserved showed a con-
nective tissue attachment.
They next asked: Can a new connective tissue
attachment establish to root surface deprived of its
periodontal attachment giving preference to cells
from the periodontal ligament? (27). Periodontal
fenestration defects were created at the maxillary
lateral incisors and mandibular canines in nonhu-
man primates. A Millipore lter was placed to cover
the fenestration defects with the aim of preventing
gingival connective tissue from contacting the root
surface. New cementum with a functionally oriented
periodontal ligament was observed within a 6-month
healing interval. Using the same rationale and tech-
nology, they subsequently provided the rst evidence
that periodontal regeneration can be obtained at a
periodontitis-affected tooth in a human (28).
This series, and several associated studies by
Karring, Nyman et al., established that cells from
periodontal ligament have the capacity to regenerate
the periodontal attachment, while the alveolar bone
and gingival connective tissue do not possess this
ability (14). As postulated by Melcher, these ndings
suggest that if preference is provided to cells origin-
ating from the periodontal ligament, periodontal
regeneration may consistently occur (25). It also
appears from these studies that occlusion of cells
33
originating from the gingiva by means of tissue bar-
riers, also known as GTR techniques, is of paramount
importance in achieving periodontal regeneration.
Although Karring, Nyman et al. elegantly pioneered
and elucidated biological concepts at the base of
periodontal regeneration, a review of additional
studies investigating wound-healing dynamics and
maturation in periodontal defects suggest that addi-
tional factors play a role. Early observations by
Linghorne & OConnell (22) suggest that a lack of
mechanical stability of the wound is the main factor
in the formation of a long junctional epithelium.
Hiatt et al. (11) demonstrated a fundamental role of
the root surface-adhering brin clot as a preventive
measure to apical migration of the gingival epithe-
lium. Polson & Proye (34), using a monkey model,
pointed to the importance of the unimpeded ab-
sorption, adhesion and maturation of a brin clot in
periodontal wound healing. In brief, teeth were re-
implanted after root planing of the coronal third of
the root (control) or root planing followed by root
surface demineralization with citric acid. While
healing by long junctional epithelium occurred in the
controls, the root surface-demineralized teeth ex-
hibited a brin linkage maturing into a connective
tissue attachment. Apparently, root surface demin-
eralization provided a stable anchorage of the brin
clot over that of the root planed-only teeth, allowing
its maturation into a connective tissue attachment.
Collectively, these studies all appear to support the
vital importance of unimpeded absorption, adhesion
and maturation of the brin clot for formation of a
connective tissue attachment over a long junctional
epithelium, as discussed above.
Our laboratories have developed and characterized
a preclinical model, designated as the Critical-size
Supraalveolar Periodontal Defect Model (Figs 3 and 4)
(17, 18, 44, 50). This animal model does not sponta-
neously regenerate following reconstructive surgery
without adjunctive measures. In addition, it allows
clinically relevant periodontal regeneration, induced
or supported by implanted biologics, biomaterials, or
devices over that in a surgical control. We initially
used this model to evaluate the signicance of brin
clot absorption, adhesion and maturation to the root
surface. In a rst study, root surfaces were coated
with heparin, to potentially interfere with brin clot
formation/absorption/adhesion. It was shown that
root surfaces coated with heparin exhibited forma-
tion of an epithelial attachment (long junctional
epithelium) whereas at control sites conditioned with
saline, the epithelium was arrested at or immediately
apical to the cementoenamel junction (Fig. 5) (48).
Apparently, the heparin coating compromised local
brin clot formation/absorption/adhesion, either by
compromising the clotting cascade, or by some
Fig. 3. The critical-size, supraalveolar periodontal defect
model. The alveolar bone and periodontal attachment,
including the cementum, are surgically reduced circum-
ferentially around the third and fourth mandibular pre-
molar teeth to a level 56 mm from the cementoenamel
junction. The rst molar is reduced to the level of the
reduced alveolar bone and the rst and second premo-
lars are extracted. Experimental treatments are applied
immediately upon defect induction. Wound closure for
primary intention healing may be transgingival, leaving
the tooth structure intact, or submerged following
reduction of the clinical crowns. Examples of histometric
parameters evaluated in the critical-size, supraalveolar
periodontal defect model are shown: The green line and
arrowheads represent the base of the surgically created
defect and the yellow arrowheads represent the cemento-
enamel junction. The defect height (vertical green ar-
row), bone regeneration height (vertical yellow arrow),
defect area (blue lines) delineated by an expanded
polytetrauoroethylene (ePTFE) membrane in this
example (membrane height: vertical blue arrow), and
bone regeneration area (orange lines) are shown. The
white irregular ghost structures within the wound area
and regenerated alveolar bone represent a bone bioma-
terial evaluated in this example. For detail see: Wikesjo
and Nilveus 1991 (44); Wikesjo et al. 1994 (50); Koo et al.
2004 (18); Koo et al. 2004 (17). These gures are copy-
righted by and modied with permission from Blackwell
Munksgaard.
34
Polimeni et al.
nonspecic surface action, or by a combination of
these effects. This single experimental manipulation
apparently prevented the maturation of the brin clot
into a connective tissue attachment but resulted in
epithelial migration and proliferation along the root
surface, probably as a consequence of exposure of
the compromised brin clot to wound-rupturing
forces acting on the gingival margins. In contrast,
when a polylactic acid implant or expanded poly-
tetrauoroethylene (ePTFE) membranes supported
the gingival aps in heparin-coated defects, the
toothgingival ap interface healed by formation of
connective tissue rather than by forming an epithelial
attachment (Fig. 6) (9, 43). In wound sites stabilized
by means of the polylactic acid implant or ePTFE
membranes, the epithelium was arrested coronally at
some distance from the implant or membrane, in
itself indicating that wound stability, and not tissue
occlusion, played a fundamental role in the outcomes
of healing. Apparently the polylactic acid implant and
the ePTFE membrane stabilized the wound, protect-
ing the compromised fragile brin clot from wound-
rupturing forces acting on the gingival margins.
These studies suggest that provided adequate wound
stability, periodontal wound healing may result in the
formation of a connective tissue attachment rather
than an epithelial attachment (long junctional epi-
thelium), which in turn should be considered a
consequence of wound failure. In perspective,
modication of the root surface by application of
etching and chelating agents may enhance brin clot
adhesion (2, 3) and promote a connective tissue
attachment (34, 47). In contrast, conditioning the
root surface with protein constructs may compro-
mise brin clot adhesion and, consequently, perio-
dontal regeneration (3, 46).
Altogether, the evidence suggests that wound sta-
bility is essential for the establishment of a new con-
nective tissue attachment to a root surface deprived of
its periodontal attachment, and that tissue resources
originating from the periodontal ligament represent a
single source for periodontal regeneration, providing
Fig. 4. The critical-size, supraalveolar periodontal defect
model. The photomicrograph shows a representative
section of a sham-surgery control following transgingival
wound closure and a 4-week healing interval. The green
arrowhead identies the apical extension of the defect
(see Fig. 3) and the red arrowhead delineates the extent
of alveolar regeneration. The schematic illustration
shows healing, expressed as a percentage of the defect
height, in the critical-size, supraalveolar periodontal de-
fect model following a 4-week healing interval and
transgingival wound closure, and following an 8-week
healing interval and submerged wound closure. Note that
the epithelium is arrested at or immediately below the
cementoenamel junction in sham-surgery control sites.
There is limited, if any, regeneration of the periodontal
attachment, as evaluated by regeneration of cementum
or a cementum-like tissue extending from the apical
extension of the defect. Bone regeneration is limited to
<25% of the defect height following a 4- or 8-week
healing interval, indicating that in control sites bone
regeneration is exhausted within 4 weeks. These char-
acteristics provide a discriminating critical-size model for
evaluation of the clinical potential of implantable/
injectable devices, biomaterials, biologics, and cell con-
structs, with or without root surface biomodications.
Substantial regeneration in this discriminating model
warrants clinical pursuit. Limited regeneration appears
less deserving. For detail see: Wikesjo and Nilveus 1991
(44); Wikesjo et al. 1994 (50); Koo et al. 2004 (18); Koo
et al. 2004 (17). These gures are copyrighted by and
modied with permission from the American Academy of
Periodontology.
35
cells with the ability to differentiate into cemento-
blasts, broblasts, and osteoblasts. Detachment of the
maturating brin clot from the root surface owing to a
lack of wound stabilization will inexorably compro-
mise periodontal wound healing, ultimately jeopard-
izing the regenerative process.
Clinical and biologic variables
affecting periodontal regeneration
Kornman & Robertson (20) classied factors that may
inuence the successful management of periodontal
osseous defects. Their classication includes:
Bacterial contamination.
Innate wound-healing potential.
Local site characteristics.
Surgical procedure/technique.
Cortellini & Tonetti (6) suggested decision trees,
along these lines, to provide clinicians with direction
in their treatment of periodontal intrabony defects.
Again, patient factors and defect morphology appear
to be crucial for the direction of therapy. In the fol-
lowing we use biologic observations in the Critical-
size Supraalveolar Periodontal Defect Model to
elucidate factors, including wound maturation, tissue
occlusion, primary intention healing, wound failure
and membrane exposure, defect characteristics,
space provision, and innate regenerative potential,
that clinicians may need to consider in the regener-
ative treatment of periodontal defects.
Wound maturation
Haney et al. (9) evaluated periodontal wound healing
associated with GTR membranes in supraalveolar
Fig. 5. Critical-size, 5-mm, supraalveolar periodontal
defect including coating the root surfaces with a heparin
solutionimmediately prior towoundclosurewiththeintent
to interfere with local brin clot formation/absorption/
adhesion. The clinical series shows the defect, the root
surfaces isolated with a rubber dam for the heparin appli-
cation, transgingival wound closure, and healing at
4 weeks. The left photomicrographs show sites that have
received the heparin coating. The green arrowheads indi-
cate the base of the defects and the blue arrowhead the
apical termination of the epithelial attachment (long
junctional epithelium) formed in these sites. Apparently,
the heparin coating compromised the brin clot in the
toothgingival ap interface to such an extent that allowed
apical migration and proliferation of cells fromthe gingival
epitheliumrather than maturation into a connective tissue
attachment. In contrast, the epithelium is arrested at the
cementoenamel junction in control sites (right) treated
with saline, leaving the entire denuded root surface with a
new connective tissue attachment. This singular manipu-
lation aimed at interfering with coagulum formation/
absorption/adhesion points to the critical importance of
the provisionary matrix of the brin clot in periodontal
wound healing and ultimately periodontal regeneration.
Healing interval 4 weeks. For detail see: Wikesjo et al. 1991
(48). These gures are copyrighted by and modied with
permission from Blackwell Munksgaard.
Fig. 6. Critical-size, 5-mm, supraalveolar periodontal de-
fect including coating of the root surfaces with a heparin
solutionimmediately prior towoundclosurewiththeintent
to interfere with local brin clot formation/absorption/
adhesion. The clinical series shows the defect, the root
surfaces isolated with a rubber dam for the heparin appli-
cation, placement of an expanded polytetrauoroethylene
(ePTFE) membrane with the intent to stabilize the wound,
and transgingival wound closure. Control defects were
coated with heparin but did not receive ePTFE membranes.
The photomicrographs showthe epitheliumarrested at the
cementoenamel junction at some distance from the cor-
onal extension of the ePTFE membrane (blue arrow) in
heparin-coated defects. The controls (not shown) exhibited
formation of an epithelial attachment (long junctional
epithelium). Similar observations were made in heparin-
coated defects implanted with a polylactic acid block bio-
material. Collectively, these observations suggest that the
implanted device or biomaterial provided some stability to
the heparin-compromised toothgingival ap interface,
allowing the provisionary matrix (i.e. the brin clot) to
mature into a connective tissue attachment rather than
migration and proliferation of cells from the gingival epi-
thelium, resulting informationof anepithelial attachment.
Healing interval 4 weeks. For detail see: Haney et al. 1993
(9); Wikesjo and Nilveus 1990 (43). These gures are copy-
righted by and modied with permission from the Amer-
ican Academy of Periodontology.
36
Polimeni et al.
periodontal defects and observed that most of the
space adjacent to the teeth underneath the mem-
branes lled with alveolar bone within a 4-week
healing interval (Fig. 7) (9). However, there was lim-
ited, if any, appreciable regeneration of cementum
and a functionally oriented periodontal ligament, as
evaluated by incandescent light microscopy, also
observed in subsequent studies using a 4-week
healing interval (19, 54). In contrast, evaluations of
periodontal regeneration in supraalveolar periodon-
tal defects using incandescent light microscopy and
healing intervals of 8 or 24 weeks demonstrated that
the observed bone formation is accompanied by the
regeneration of cementum and a functionally orien-
ted periodontal ligament (Fig 810) (15, 38, 5153). As
experimental conditions were similar among these
studies, these observations point to the possibility of
a delayed structural maturation of the periodontal
attachment compared with that of the alveolar bone
following regenerative procedures.
Tissue occlusion
Design criteria for GTR membranes include bio-
compatibility, cell occlusion, space maintenance,
tissue integration, and ease of use (10, 36). Although
biocompatibility, space maintenance, tissue integra-
tion, and ease of use have been evaluated extensively,
the concept of tissue occlusion has received limited
attention. Karaki et al. (12) evaluated bone formation
in periodontal sites using surgically created con-
tralateral horizontal periodontal defects in the
mandibular premolar region in dogs. A tissue-
expanding gold mesh was applied on one side, while
the contralateral side served as a sham-surgery con-
trol. Compared with the surgical control, bone for-
mation was enhanced in defects receiving the gold
mesh. Evidently, osteogenesis in a periodontal envi-
ronment may proceed in the presence of space pro-
vision without strict occlusion of the gingival
connective tissues. A concept of regeneration util-
izing space provision without connective tissue
occlusion emerges from this observation. Thus, a
study was initiated to evaluate the possibility of
periodontal regeneration without gingival tissue
occlusion (52). Structurally reinforced, spaceprovi-
ding, macroporous ePTFE membranes were sur-
gically implanted into supraalveolar periodontal
defects and compared with occlusive membranes
(Fig. 10). These observations clearly demonstrate that
tissue occlusion is not an absolute requirement for
periodontal regeneration, as sites receiving the por-
ous membrane showed signicant regeneration of
cementum, a functionally oriented periodontal liga-
ment and alveolar bone similar to that observed at
sites receiving the occlusive membrane. There were,
defect, including coating of the root surfaces with a
heparin solution immediately prior to wound closure,
with the intent to interfere with local brin clot forma-
tion/absorption/adhesion. The clinical series shows the
defect, the root surfaces isolated with a rubber dam for
heparin application, placement of an expanded poly-
tetrauoroethylene (ePTFE) membrane with the intent to
stabilize the wound, and transgingival wound closure.
Control defects were coated with heparin but did not
receive ePTFE membranes. The left photomicrograph
shows a site where the ePTFE membrane allows a space
at the root surface, resulting in complete ll with newly
formed alveolar bone. The center photomicrograph
shows a membrane collapsed or compressed onto the
root surface, obstructing any regeneration of periodontal
structures. There was a signicant correlation between
the space provided by the membrane and the newly
formed alveolar bone (r 0.997; P 0.002). The right
photomicrograph (yellow arrow) shows an experimental
site with wound failure, membrane exposure, infection,
inammation and necrosis. The green arrowheads
delineate the apical extension of the defects. Healing
interval 4 weeks. For detail see: Haney et al. 1993 (9).
These gures are copyrighted by and modied with
permission from the American Academy of Period-
ontology.
37
however, remarkable clinical differences between the
experimental conditions. Whereas all sites receiving
the porous membrane remained submerged for pri-
mary intention healing, 50% of sites receiving the
occlusive membrane exhibited wound failure and
membrane exposure. Obviously the porous mem-
brane supported ap survival, probably being less of
a challenge to the vascular support of the gingival
aps than the occlusive membrane. The results of
this study ultimately support a concept of periodon-
tal regeneration following gingival ap surgery,
including primary intention healing and space pro-
vision without barrier membranes.
Primary intention healing vs. wound
failure and membrane exposure
Wound failure including membrane exposure is a
calamity of periodontal-regenerative therapy utilizing
GTR techniques, making the procedure unpredicta-
ble in clinical practice (35, 41). The membrane can be
difcult to submerge completely by gingival tissues at
Fig. 9. Critical-size, 5-mm, supraalveolar periodontal de-
fect implanted with an occlusive, space-providing expan-
ded polytetrauoroethylene (ePTFE) membrane. The
high-magnication photomicrographs from the apical,
mid, and coronal aspect of the defect show regeneration of
the periodontal attachment, including cellular cementum,
a functionally oriented periodontal ligament, and alveolar
bone. Note the gradual thinning of the regenerated cel-
lular cementum in a coronal direction. Healing interval
8 weeks. For detail see: Sigurdsson et al. 1994 (38). These
gures are copyrighted by and modied with permission
from the American Academy of Periodontology.
defect implanted with an occlusive, space-providing
expanded polytetrauoroethylene (ePTFE) membrane.
The green arrow points to newly regenerated bone
reaching from the apical aspect of the defect to the ce-
mentoenamel junction, the ePTFE membrane provides
a suitable space for periodontal regeneration, and the
green arrowheads delineate the apical aspect of the
supraalveolar periodontal defect. The second photomi-
crograph shows the membrane collapsed or compressed
onto the root, with minimal regeneration as a conse-
quence. The third photomicrograph shows a sham-sur-
gery control also with minimal regeneration, the muco-
gingival ap being collapsed or compressed onto the
root. Finally, the fourth photomicrograph with a yellow
arrow shows a site where the membrane has been ex-
posed to the oral cavity, resulting in infection and nec-
rosis without any regeneration of periodontal tissues.
This study points to the critical importance of primary
intention wound healing and unobstructed space provi-
sion for periodontal regeneration. Healing interval
8 weeks. For detail see: Sigurdsson et al. 1994 (38). These
from the American Academy of Periodontology.
38
Polimeni et al.
wound closure, or it may exhibit subclinical exposure
or poor ap retention, even following the best
intentions for primary intention healing, and thus
becomes exposed during the healing sequel. Clinical
experience and histologic evaluations of periodontal
wound healing in supraalveolar periodontal defects
demonstrate that GTR membranes frequently
become exposed, possibly as a consequence of
compromised nutritional support to the overlaying
gingival tissues (38, 52). Oral bacteria, provoking an
inammatory reaction within the regenerate under-
neath the membrane, in turn colonize the exposed
sites. In early studies, animals experiencing mem-
brane exposure received systemic antimicrobial
therapy and daily rinses with a chlorhexidine gluco-
nate solution throughout the healing sequel. Al-
though this treatment maintains optimal gingival
health in nonexposed sites, exposed sites exhibit
large inammatory inltrates and necrotic tissues
with limited, if any, signs of periodontal regeneration
in the histological evaluation (Fig 7 and 8). In con-
trast, when GTR membranes were removed imme-
diately upon exposure followed by wound closure
over the exposed regenerate, the newly formed tis-
sues matured into alveolar bone, cementum, and a
functionally oriented periodontal ligament, even in
sites where the wound failure/membrane exposure
occurred as early as 1 week postsurgery (52). In sites
where periodontal regeneration is allowed to pro-
gress unobstructed under conditions for primary
intention healing, complete, or almost complete,
regeneration of the periodontal attachment becomes
an imminent possibility (Fig 810) (38, 5153). The
clinical signicance of these biologic observations
have been demonstrated in a retrospective evaluation
of GTR therapy in 38 healthy patients receiving
treatment of intrabony periodontal defects with a
defect depth averaging 6.5 1.6 mm and probing
depth averaging 7.6 1.5 mm (41). Probing bone
level gain in sites without membrane exposure aver-
aged 4.1 2.3 mm, in contrast to 2.2 2.3 mm for
sites with membrane exposure. These observations
likely apply to all membrane technologies until
shown otherwise. The observations demonstrate the
critical signicance of primary (unexposed) intention
healing for periodontal regeneration.
Defect characteristics, space provision,
and innate regenerative potential
Defect conguration is considered to be a critical
factor inuencing the outcome of periodontal-
regenerative therapy in clinical practice. Deep,
narrow intrabony defects appear to be favorable
candidates for regenerative surgery compared with
wide, shallow defects (6), as do three-wall intrabony
defects compared with two- and one-wall intrabony
defects. Supracrestal periodontal regeneration is
generally not considered a clinical possibility. From
a conceptual point of view, it appears logical that
deep, narrow, three-wall intrabony defects should
react favorably over shallower, wider, and more
open sites. The relative abundance of tissue re-
sources contributing to the regeneration in three-
wall intrabony defects, the defect area being more or
less circumscribed by the residual periodontal liga-
ment, should enhance the regenerative potential of
these sites over that in two- and one-wall intrabony
defect implanted with occlusive and macro-porous, space-
providing expanded polytetrauoroethylene (ePTFE)
membranes; the photomicrographs show a site implanted
with the porous membrane. Note signicant periodontal
regeneration, including a functionally oriented perio-
dontal ligament, cellular cementum, and alveolar bone,
approaching the cementoenamel junction (green arrow).
Similar results were found in sites implanted with the
occlusive membrane, clearly suggesting that tissue
occlusion is not a critical requirement for periodontal
regeneration. Healing interval 8 weeks. For detail see:
Wikesjo et al. 2003 (52). These gures are copyrighted by
and modied with permission from Blackwell Munks-
gaard.
39
defects, providing that conditions for primary inten-
tion healing are maintained. However, observations
of periodontal wound healing and regeneration in
this text, based on the Critical-size Supraalveolar
Periodontal Defect Model, demonstrate the biologic
possibility of extensive, if not complete, regeneration
of the periodontal attachment, including alveolar
bone, in supracrestal, zero-wall periodontal defects
(Fig 810) (38, 5153).
Early reports, evaluating GTR technology using
barrier membranes and supraalveolar periodontal
defects, point to a key role of space provision in
periodontal-regenerative therapy. Haney et al. (9)
reported a signicant correlation (r 0.997; P
0.002) between the space provided by the membrane
and the newly formed bone (Fig. 7). Sigurdsson et al.
(38) showed that sites subject to space provision
exhibited extensive bone regeneration compared
with that in controls (Fig. 8). In other words, a large
wound area resulted in increased bone regeneration.
Sigurdsson et al. (38) reported greater bone regener-
ation compared with that reported by Haney et al. (9),
although the space underneath the GTR membrane
was not completely lled with alveolar bone. Rather,
the newly formed bone assumed a physiologic form
along the root surface, much like the preceding
resident bone, and the remaining space underneath
the membrane was occupied by dense connective
tissue (Figs 8 and 9). There were, however, meth-
odological variations between the studies that may
account for the differences observed in the magni-
tude of alveolar bone regeneration. Haney et al. (9)
used transgingival wound closure and positioned
individual clinical GTR membranes around the neck
of the teeth, while Sigurdsson et al. (38) positioned a
structurally reinforced space-providing membrane in
such a manner that the teeth and membrane became
completely submerged, suturing the aps over the
top of the membrane. The wound area delineated by
the membrane was obviously increased in the latter
study. It can be speculated that variation in posi-
tioning of the membrane inuenced space provision
and that this, in turn, inuenced the regenerative
potential of the defect sites. Similar observations
have been reported by Cortellini et al. (7) and Tonetti
et al. (40) in clinical studies aimed at investigating
factors affecting the healing response of intrabony
defects following GTR and access ap surgery. Space
provision and wound stability have also been
advocated as main factors inuencing the magnitude
of bone regeneration in three-wall compared with
two- and one-wall intrabony defects in a dog model
(16).
The effect of defect characteristics and space pro-
vision, and innate regenerative potential has received
further analysis using the Critical-size Supraalveolar
Periodontal Defect Model. Polimeni et al. (2933) used
the height of the regenerated alveolar bone along the
root surface as a parameter for periodontal regener-
ation to evaluate the biologic potential for regener-
ation under various conditions. Other parameters
included the width of the alveolar crest at the base of
the defect and the wound area delineated by the base
of the defect, the lateral extension of a GTR mem-
brane, the cementoenamel junction, and the tooth
surface (Fig. 11). The use of the height of the regen-
erated alveolar bone as a surrogate parameter for
periodontal regeneration was based on observations
suggesting a signicant correlation between the
height of newly formed bone along the root surface
and regeneration of the periodontal attachment
extending just coronally of the alveolar crest in supra-
alveolar periodontal defects (r 0.96; P 0.001; G.
Polimeni & C. Susin 2005, unpublished). These
observations as such suggest that the periodontal
ligament provides the leading edge for the regene-
rating periodontal tissues.
A rst study evaluated the role of space-provision
for periodontal regeneration. Supraalveolar, period-
ontal defects, having received either a space-provid-
ing, porous ePTFE membrane or sham-surgery, were
subject to histometric analyses (including investiga-
Fig. 11. Photomicrograph showing parameters evaluated
by Polimeni et al., including defect area (orange) under-
neath the membrane delineated by the base of the defect
apically and the cementoenamel junction coronally,
bone regeneration height (green arrow), the height of
the newly formed alveolar bone along the root surface
representing a surrogate parameter for regeneration
of the periodontal attachment (G. Polimeni & C. Susin
unpublished); and the width of the alveolar crest (yellow
arrow) at the base of the defect. For detail see: Polimeni
et al. 2004 (32); Polimeni et al. 2004 (29); Polimeni et al.
2004 (30); Polimeni et al. 2004 (31); Polimeni et al. 2005
(33). This gure is copyrighted by and published with
permission from Blackwell Munksgaard.
40
Polimeni et al.
tion of vertical regeneration of the alveolar bone and
analysis of the width of the alveolar crest at the base
of the defect) following an 8-week healing interval
(33). Bone regeneration at sites receiving the space-
providing membrane was signicantly greater than
that at sites receiving sham-surgery (P 0.0003). A
signicant relationship between the width of the
alveolar crest at the base of the defect and bone
regeneration was observed, with no signicant dif-
ference between sites receiving different treatments
(P 0.84). It can be concluded therefore that space
provision has a signicant effect on periodontal
regeneration. Notably, the width of the alveolar crest
at the base of the defect appears to inuence space
provision effectively, supporting regeneration. Sites
providing a wide alveolar base showed enhanced
regeneration, whereas sites exhibiting a narrow base
showed limited regeneration for both treatment
conditions. One may speculate that in the presence of
a wide alveolar base, the mucoperiosteal ap serves
the same mechanical function as the space-provi-
ding, porous ePTFE membrane, whereas in the
presence of a narrow base, the ap and the mem-
brane-supported ap collapse onto the tooth surface,
providing limited space for regeneration. In other
words, the characteristics of the mucoperiosteal ap
alone, or supported by the space-providing, porous
ePTFE membrane, are not different, from a wound
mechanical point of view.
A clinical approach to space provision for perio-
dontal regeneration has included the placement of
bone biomaterials to support GTR membranes.
Trombelli et al. (42) evaluated regeneration following
GTR procedures, including the placement of a
nonresorbable bone biomaterial in supraalveolar
periodontal defects (Fig. 12). They found a signi-
cantly positive correlation between space provision
by the membrane and alveolar regeneration, and a
signicantly negative correlation between the density
of biomaterials and alveolar regeneration (P < 0.01).
In other words, the biomaterial obstructed the space,
thus preventing regeneration. Polimeni et al (32).
evaluated periodontal regeneration following GTR
procedures, including placement of a resorbable
bone biomaterial (Fig. 13). Bilateral, supraalveolar,
periodontal defects, having received a clinical ePTFE
membrane with (cGTR) or without (GTR) the coral-
derived biomaterial, were subject to histometric
analysis, including vertical regeneration of alveolar
bone relative to space provision by the ePTFE
membrane, following a 4-week healing interval. Sig-
nicantly greater bone regeneration was observed at
sites receiving cGTR compared with GTR alone
(P < 0.0001). Sites providing larger wound areas
showed greater bone regeneration compared with
sites exhibiting smaller wound areas (P < 0.0001).
However, grouping the sites by wound area thres-
holds showed that bone regeneration was not sig-
nicantly different in sites receiving cGTR compared
with GTR alone, irrespective of the size of the wound
area (P > 0.5). This study showed that the coral-de-
rived biomaterial inuences space provision by en-
hancing the wound area. The physical structure of
the biomaterial appeared to prevent the GTR mem-
brane from collapsing onto the root surface. This
overall effect supported enhanced bone formation in
defect implanted with an occlusive expanded polytetra-
uoroethylene (ePTFE) membrane and an osteoconduc-
tive biomaterial. Note signicant regeneration of alveolar
bone in the left photomicrograph in the absence of the
biomaterial. It is shown that less tissue regeneration
occurs as more biomaterial is used, raising the question of
any benet of using slowly resorbing or nonresorbable
bone biomaterials in conjunction with periodontal-
regenerative surgery. This study points to the critical
importance of unobstructed space provision for perio-
dontal regeneration. Healing interval 4 weeks. For detail
see: Trombelli et al. 1999 (42). These gures are copy-
righted by and modied with permission from Blackwell
Munksgaard.
41
sites receiving cGTR compared with sites receiving
GTR alone. However, when adjusted for the effect of
wound area, a two-way analysis of variance did not
show statistically signicant differences between the
protocols. Consistent with this observation, strati-
cation of the wound area into subgroups did not re-
veal signicant differences between the protocols.
This should be interpreted to indicate that the coral-
derived biomaterial did not exhibit osteoconductive
properties. In other words, it did not enhance the
osteogenic potential of the site. These observations
corroborate histopathological evaluations of this
biomaterial in periodontal sites (19). On the other
hand, the coral-derived biomaterial did not appear to
obstruct regeneration, in contrast to that observed for
other particulate biomaterials used to support space
provision or to serve as osteoconductive conduits
in conjunction with GTR (39, 42). Future research,
evaluating osteoconductive properties of biomateri-
als to be used for space provision/regeneration,
should take into consideration the native osteogenic
potential. Proper methodology and analysis should
be applied to distinguish this effect from any osteo-
conductive effects of the biomaterial.
In a separate evaluation, Polimeni et al. (29) esti-
mated the effect of cell occlusion and space provision
on periodontal regeneration. Space-providing occlu-
sive and porous ePTFE membranes were implanted
to provide for GTR in supraalveolar periodontal de-
fects (Fig. 14). The gingival aps were advanced for
primary intention healing that was allowed to pro-
gress for 8 weeks. The histometric analysis assessed
regeneration of alveolar bone relative to space pro-
vision by the ePTFE membranes. The bivariate ana-
lysis showed that space provision and membrane
occlusivity signicantly enhanced bone regeneration.
Sites receiving the occlusive GTR membrane, and
sites with enhanced space provision, showed signi-
cantly greater bone regeneration than sites receiving
the porous GTR membrane (P 0.03) or exhibiting
more limited space provision (P 0.0002). Never-
theless, a signicant relationship was observed be-
tween bone regeneration and space provision for
sites receiving the occlusive (b 0.194; P < 0.02) and
the porous (b 0.229; P < 0.0004) GTR membranes,
irrespective of treatment (P 0.14). In other words,
the relationship between space provision and re-
generation was signicant for both the porous and
the occlusive GTR membranes. Regeneration fol-
lowed similar patterns in both groups. It may be
speculated that the healing process supported by
these different membranes is similar, or at least
similarly inuenced by space provision. Nevertheless,
the magnitude of regeneration was signicantly in-
creased at sites receiving the occlusive GTR mem-
branes compared with that at sites receiving the
defects implanted with an occlusive expanded polytetra-
uoroethylene (ePTFE) membrane for guided tissue
regeneration (GTR) in the presence (cGTR) or absence
(GTR) of a coral-derived biomaterial. The green arrow-
heads delineate the apical aspect of the supraalveolar
periodontal defect. The left photomicrograph shows a site
receiving cGTR where the membrane has been com-
pressed onto the root, with minimal regeneration as a
consequence. The left center photomicrograph shows a
site receiving the same treatment protocol with enhanced
space provision allowing increased bone regeneration.
The right and right center photomicrographs show sites
receiving GTR alone. Similarly to that observed for cGTR
sites, bone regeneration appears to be inuenced by space
provision in the GTR sites. This study showed that the
coral-derived biomaterial inuences space provision by
enhancing the wound area. The physical structure of the
biomaterial appeared to prevent the GTR membrane from
collapsing onto the root surface. This overall effect sup-
ported enhanced bone formation in sites receiving cGTR
compared with sites receiving GTR alone. Importantly,
when adjusted for the effect of wound area, a two-way
analysis of variance (ANOVA) did not show statistically
signicant differences between the protocols. Healing
interval 4 weeks. For detail see: Polimeni et al. 2004 (32).
These gures are copyrighted by and modied with per-
mission from Blackwell Munksgaard.
42
Polimeni et al.
porous GTR membrane, when adjusted for the effect
of wound area. Thus, even if space provision appears
to be a critical factor for regeneration, membrane
occlusivity appears to provide adjunctive effects.
While it may not be legitimate to consider cell oc-
clusion as an absolute prerequisite for periodontal
regeneration (52), it appears that the use of cell-
occlusive membranes may optimize the magnitude
of regeneration.
The inuence of the resident alveolar bone on bone
regeneration in conjunction with GTR, in the pres-
ence or absence of cell occlusivity, was evaluated in a
subsequent analysis (31). Space-providing, occlusive
or porous ePTFE membranes were implanted into
contralateral supraalveolar periodontal defects to
assist GTR under conditions for primary intention
healing (Fig. 14). The healing interval was 8 weeks,
after which block sections were collected for histo-
metric analysis, including analysis of regeneration of
alveolar bone relative to space provision by the GTR
membrane and width of the alveolar crest at the base
of the defect. There were no signicant differences in
mean alveolar regeneration between sites receiving
the porous GTR membrane with a narrow vs. a wide
alveolar base after adjusting for wound area (2.2 vs.
2.5 mm, respectively; P 0.36). In contrast, analysis
using sites receiving the occlusive GTR membrane
revealed signicantly greater bone regeneration at
sites with a wide compared with a narrow alveolar
base (3.3 vs. 2.5 mm, respectively; P 0.02). Regres-
sion analysis showed a signicant relationship
(P 0.05) between space provision and bone regen-
eration for all groups, except for sites with a wide
alveolar base receiving the occlusive GTR membrane
defect implanted with an occlusive, space-providing
expanded polytetrauoroethylene (ePTFE) membrane (A)
and with a porous ePTFE membrane (B). Green arrow-
heads delineate the apical aspect of the supraalveolar
periodontal defects. Green lines approximate the coronal
aspect of the regenerated bone. Notably, bone regener-
ation is inuenced by space provision under the mem-
branes. This study showed that space provision and
membrane occlusivity signicantly enhanced bone
regeneration. Sites receiving the occlusive guided tissue
regeneration (GTR) membrane and sites with enhanced
space provision showed signicantly greater bone regen-
eration compared with sites receiving the porous GTR
membrane (P 0.03) or exhibiting more limited space
provision (P 0.0002). Nevertheless, the relationship be-
tween space provision and regeneration was signicant
for both occlusive and porous GTR membranes. Regen-
eration followed similar patterns for both groups. It may
be speculated that the healing process supported by these
different membranes may be similar to, or at least be
similarly inuenced by, space provision. Healing interval
8 weeks. For detail see: Polimeni et al. 2004 (29). These
from Blackwell Munksgaard.
43
(P 0.5). The present study (undertaken in the
presence of tissue occlusion and controlling for
wound area) established that sites exhibiting a
wide alveolar base might have a greater osteogenic
potential than sites with a narrow base. This obser-
vation suggests that the osteogenic potential of the
resident bone plays a role in periodontal regener-
ation. On the other hand, in the absence of tissue
occlusion (porous GTR membranes) and controlling
for wound area, sites exhibiting a wide alveolar base
did not show an enhanced osteogenic potential
compared to sites with a narrow base. One may
speculate that this might be a consequence of the
porous space-providing GTR membrane allowing the
gingival connective tissue access to the wound area.
Consequently, tissue resources, including molecules,
cells, and vascularity originating from the gingival
connective tissue, may have an inhibitory effect on
osteogenesis, and/or migration and proliferation of
tissue elements from the gingival connective tissue
competitively occupied the space for bone to form
into. Thus, the resident alveolar bone might signi-
cantly inuence the magnitude of alveolar bone
regeneration, while the relative presence of cells from
the gingival connective tissue may attenuate this
effect.
Subsequently, Polimeni et al. (30) evaluated the
inuence of alveolar bone and space provision on
bone regeneration at teeth and titanium implants,
comparing observations at supraalveolar periodontal
and supraalveolar peri-implant defects (Fig. 15). The
experimental sites had been subject to GTR using
space-providing porous ePTFE membranes. The
healing interval was 8 weeks. The histometric analy-
sis assessed alveolar bone regeneration (height) rel-
ative to space provision by the membrane and the
width of the alveolar crest at the base of the defect.
Statistical analysis used the linear mixed models. The
results revealed a signicant correlation between
bone width and wound area (r 0.56, P < 0.0001).
Generally, bone width and wound area had statisti-
cally signicant effects on the extent of bone re-
generation (P < 0.0005 and P < 0.0001, respectively).
Bone regeneration was linearly correlated with the
bone width at periodontal (P < 0.001) and implant
(P 0.04) sites, and with the wound area at period-
ontal (P < 0.0001) and implant (P 0.03) sites. The
relationships of bone regeneration with these two
variables were not signicantly different between
teeth and implants (bone width, P 0.83; wound
area, P 0.09). When adjusted for wound area, bone
regeneration was signicantly greater at periodontal
than at implant sites (P 0.047). Thus, the histo-
metric analysis suggested similar patterns of bone
regeneration at periodontal and implant sites. Simi-
larities in the behavior of factors inuencing bone
regeneration were observed for both periodontal and
implant sites. The width of the alveolar crest and the
space provided by the porous ePTFE membrane
resulted in a signicant relationship with the extent
of alveolar bone regeneration for both sites. Adjusting
for the effect of wound area, the periodontal sites
Fig. 15. Critical-size, 5-mm, supraalveolar peri-implant
defect including three titanium implants and a space-
providing porous expanded polytetrauoroethylene
(ePTFE) membrane. The green arrowheads delineate the
apical aspect of the supraalveolar peri-implant defect. The
red line approximates the coronal aspect of the newly
formed bone. Notably, the amount of bone regeneration is
inuenced by space provision under the membrane. This
study showed that the width of the alveolar crest and the
space provided by the porous ePTFE membrane signi-
cantly inuences alveolar bone regeneration. Adjusting
for the effect of wound area, periodontal sites exhibit
signicantly increased bone regeneration compared with
that in alveolar (peri-implant) sites. This observation
suggests critical biologic differences between periodontal
and alveolar (peri-implant) sites. Healing interval 8 weeks.
For detail see: Polimeni et al. 2004 (30). These gures are
copyrighted by and modied with permission from
Blackwell Munksgaard.
44
Polimeni et al.
exhibited signicantly greater bone regeneration than
the implant sites. This observation suggests critical
biologic differences between periodontal and implant
sites. While bone regeneration in periodontal sites
may be induced by vascular and cellular elements
sequestered in the periodontal ligament, or by
synergistic effects between the periodontal attach-
ment and the resident alveolar bone, regeneration at
implant sites appears to be solely dependent on the
evidently limited regenerative potential of the alveo-
lar bone. In consequence, principles valid for GTR in
periodontal sites may not necessarily immediately
apply to implants. Moreover, from a clinical per-
spective, these biologic observations suggest that
bone-regenerative procedures at implant sites may
be considerably more challenging than at periodontal
sites.
Conclusions
Current scientic evidence points to the presence of
cells originating from the periodontal ligament,
wound stability, space provision, and primary inten-
tion healing, as fundamental biologic and clinical
factors that must be met to obtain periodontal
regeneration.
Only a profound understanding of biological and
clinical variables affecting the outcome of perio-
dontal-regenerative procedures will allow clinicians
to manipulate biological and clinical factors effect-
ively in order to optimize the clinical result and in-
crease the predictability of periodontal-regenerative
therapy.
It is evident that the search for novel regenerative
therapies requires preclinical evaluation in well-
characterized rodent screening models to determine
biologic potential and safety, and analysis in
discriminating large animal models (designated as
Critical-size Defect Models) to establish clinical
potential and efcacy. Clinical evaluation should be
limited to therapies showing promising results in
these preclinical models.
Acknowledgment
Earlier versions of this text have been published
for reviews in journals and book chapters. The text
is continuously subject to revisions and updating
as new information becomes available in our
laboratory.
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a bioresorbable space-providing macro-porous membrane
with recombinant human bone morphogenetic protein-2.
J Periodontol 2003: 74: 635647.
54. Wikesjo UME, Lim WH, Razi SS, Sigurdsson TJ, Lee MB,
Tatakis DN, Hardwick WR. Periodontal repair in dogs: a
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2003: 74: 955962.
47
Effects of growth factors and
cytokines on osteoblast
differentiation
FRANCI S J. HUGHES, WENDY TURNER, GEORGI OS BELI BASAKI S &
GI ANLUCA MARTUSCELLI
The therapeutic management of new bone formation
remains one of the key issues in periodontology and
dental implantology. A full understanding of the
nature of the bone-forming cells (osteoblasts) and
their lineage, the factors that may regulate osteoblast
behavior and bone formation locally, and how these
different regulatory mechanisms could interact, has
been the subject of extensive study for many years,
particularly with the aspiration that such an under-
standing will lead to the development of novel bio-
logically based therapies for the management of bone
regeneration, not only in dental applications but also
for many other disciplines, such as orthopedic sur-
gery, and maxillofacial and craniofacial surgery.
The aim of this review was as follows.
to describe the nature of the osteoblast phenotype,
its lineage and the genetic regulation of osteoblast
differentiation and function.
to discuss the role of a range of cytokines and
growth factors in regulating osteoblast differenti-
ation and function.
to review how some of these cytokine pathways
may be regulated and interact with each other in
the co-ordinated control of osteoblast function.
to review, briey, studies that have investigated
the pharmacological application of cytokines for
therapeutic management of bone formation locally
in vivo.
Cytokines are soluble, secreted glycoproteins which
act as local signaling molecules to control and
co-ordinate cellular behavior and function. They
function as ligands which bind to cell-surface
receptors, triggering a series of intracellular signaling
events, ultimately resulting in the modulation of gene
expression in their target cells. Growth and differen-
tiation factors are generally considered to be a subset
of cytokines with anabolic physiological and patho-
physiological roles in the regulation of tissue growth
and healing. Although this terminology is widely
used, the distinction between growth factors and
cytokines is probably an articial one, particularly
given the different complex roles that these mole-
cules may all have on cell and tissue function.
However in keeping with convention, here we have
used the term growth factors to describe this distinct
subgroup of cytokines, and have separately consid-
ered the role of cytokines which are particularly
associated with inammation on bone cell regula-
tion.
Osteoblast lineage and function
The osteoblast phenotype
Osteoblasts are specialized mesenchymal-derived
cells whose function is the deposition and mainten-
ance of skeletal tissues. As with any cell type, they are
dened ultimately by their expression of genes that
are a subset of the total genome and which dene
their unique function. Specically, osteoblasts
express the genes coding for the bone matrix pro-
teins, high levels of the tissue nonspecic form of
alkaline phosphatase and are also frequently char-
acterized by their expression of parathyroid hor-
mone/parathyroid hormone receptors, which results
in a typical cAMP response on stimulation with the
osteotropic parathyroid hormone.
The bone matrix proteins include type I collagen
and a number of other matrix proteins, which to a
greater or lesser extent are characteristic or specically
expressed in bone. Collagen type I makes up 90%of
48
PERIODONTOLOGY 2000
the organic matrix of bone and provides the strength,
structure and elasticity of the mature bone tissue. The
noncollagenous proteins of bone include osteocalcin
(bone GLA protein) osteopontin, osteonectin and
bone sialoprotein. In addition, the proteoglycans
decorin and biglycan are signicant components of
the bone organic matrix. Although a full description
of the function of the different matrix constituents
of bone is beyond the scope of this review, these pro-
teins collectively have calcium-binding activity, which
is probably responsible for the exquisite regulation of
hydroxyapatite crystal deposition, orientation and
specic crystal size seen in the mineralized bone
matrix. In addition, it is known that bone sialoprotein
and osteopontin contain the ArgGlyAsp (RGD)
peptide cell-attachment sequences and can mediate
attachment of both osteoblasts and osteoclasts to the
bone matrix.
Of the noncollagenous proteins of bone, osteocal-
cin is considered to be the most bone-specic.
Expression of osteocalcin in the mature organism is
restricted to bone, dentin and cementum. Evidence
particularly from mouse knockout models suggest
that the bone matrix proteins play an important role
in regulating the overall control of bone mass. For
example, the osteocalcin knockout mouse exhibits an
overall increase in bone mass as its main phenotype
(54).
The expression of bone-specic genes and proteins
are valuable markers for demonstration of bone
osteoblast phenotype in vitro, and understanding of
the transcriptional regulation of these genes is of
fundamental importance in the regulation of osteo-
blast differentiation and function.
The osteoblast lineage
Osteoblasts are derived from multipotent mesen-
chymal stem cells that give rise to progenitor cells of
restricted osteoblast lineage and which may then
undergo a series of proliferation/amplication stages
before expressing recognizable specic osteoblastic
markers. Early studies of the kinetics of normal
osteoblast turnover using pulse-chase
3
H-thymidine
labeling demonstrate the recruitment of osteopro-
genitor cells from the surrounding connective tissues
toward the periosteal layer on the surface of the bone
(188, 189). The periosteum consists of two layers in
these studies: a broperiosteum on the outside most
distant from the bone surface; and an inner osteo-
genic periosteum where the rst directly recognizable
cell of the differentiating osteoblast lineage, the pre-
osteoblast, is found. Pre-osteoblasts retain a prolif-
erative capcacity but express many of the proteins
associated with the mature osteoblast phenotype,
including alkaline phosphatase and osteopontin. The
mature osteoblast layer lies immediately adjacent to
the bone and is a post-mitotic cell expressing all of
the features of the mature phenotype (189).
These descriptions give rise to the concept of
recruitment of proliferating osteoprogenitor cells
from adjacent connective tissues, which undergo
further differentiation, resulting in the expression of
the mature phenotype. These studies also suggest
that mature osteoblasts have an average lifespan of
1 month, after which either they undergo apopto-
sis, to be replaced by newly differentiated osteoblasts
or alternatively about one-third of these cells may be
incorporated into deposited bone matrix as osteo-
cytes.
The osteocyte is thus the most differentiated cell of
the osteoblast lineage and may persist in bone matrix
for very lengthy periods of time. Although its func-
tions are not fully understood, there is compelling
evidence that the osteocyte has a critical role in the
maintenance of bone mass and is the principal bone
cell responsible for mechanotransduction. It is well
established that maintenance of bone mass relies on
regular mechanical stimulation. It is thought that the
subtle exing seen in bones in response to any
loading causes pulsatile uid ow through osteocyte
canaliculi, resulting in signal transduction by osteo-
cytes in order to retain bone mass (24, 128, 228). The
precise molecular mechanisms whereby this occurs
are unknown. However, mechanical strain and pul-
satile uid ow are able to induce signaling mole-
cules, such as nitric oxide, by osteocytes in vitro (127,
194). Interestingly, the recently described bone
morphogenetic protein (BMP) inhibitor, sclerostin,
has also been localized specically to osteocytes and
may contribute to the regulation of overall bone mass
(264).
Around the periodontal tissues, similar pulse
chase
3
H-thymidine-labeling experiments demon-
strate the continual recruitment of proliferating cells
from bone marrow stroma through vascular channels
that communicate with the periodontal ligament.
These cells appear to preferentially migrate to the
osteoblast (and cementoblast) surfaces, consistent
with the recruitment of osteoprogenitor cells from
stem cells located in the bone marrow stroma
through to the periodontal ligament and ultimately
undergo osteoblastic differentiation, akin to the kin-
etics described in other bony tissues (159). Thus, the
periodontal ligament is constantly repopulated by
relatively undifferentiated mesenchymal cells and is a
49
Effects of growth factors and cytokines on osteoblast differentiation
rich source of progenitor cells capable of giving rise
to new bone formation (see Fig. 1).
Mesenchymal stem cells
Stem cells are dened as undifferentiated self-
renewing cells. In the mature organism they tend to
occur with relatively low frequency and although, by
denition, they have extremely high proliferative
potential, they are typically not continually cycling.
In addition, many (but not all) stem cells have
multiple potentials, that is they have the capacity to
give rise to a range of differentiated phenotypes. The
existence of undifferentiated self-renewing cells is
clearly essential for the organism to replenish con-
tinually differentiated cells which have reached the
end of their normal lifespan. Classic studies by
Owen & Fredenstein were the rst to describe the
existence of multipotent mesenchymal stem cells
isolated from bone marrow stromal cell populations
(66, 67, 190). In these experiments it was demon-
strated that colonies from bone marrow stroma (and
therefore of clonal origin) could give rise to a wide
range of connective tissues, including bone, carti-
lage, muscle, brous and adipose tissues, when
re-implanted into a suitable host animal. More for-
mal demonstration of the true multipotential nature
of mesenchymal stem cells was subsequently dem-
onstrated in clonally derived cell lines in vitro
including the murine embryonic broblastic cell
line, C3H10T1/2 (132), and the rat calvaria-derived
line, RCJ3.1 (84). These and other multipotent
mesenchymal cell lines have been valuable tools for
using to investigate the role of a range of different
factors that may regulate the commitment of
multipotent stem cells to cells of restricted lineages,
such as osteoprogenitor cells.
In recent years there has been considerable debate
about the plasticity of stem cells, that is, the ability
of committed progenitor cells to dedifferentiate and
subsequently redifferentiate along a different lineage.
There is considerable evidence for the existence of
bipotent osteoblastadipocyte cells from bone mar-
row in both human and other species (4, 18, 42) and
these also exhibit extensive potential for undergoing
cell renewal. These data suggest the possibility that
restriction to specic differentiation pathways may
not occur as a single event but it is possible for
multipotent stem cells to exhibit increasing lineage-
restricted specication. Further evidence of this is
provided by studies of C3H10T1/2 cells, where sub-
clones have also been shown to exhibit tripotent
properties (132) and these potentials have also been
Fig. 1. Demonstration of continual recruitment of pro-
genitor cells from bone marrow to the periodontal liga-
ment. Cycling cells were labeled by a bromodeoxyuridine
(BrdU) pulse on day 0 and followed-up for periods of up to
10 days in Wistar rats. (A) Day 0. A bone marrow space is
seen within the bone, communicating directly with the
PDL via a vascular channel. Labeled cycling cells (red) are
largely restricted to the bone marrow stroma. (B) Day 6.
Labeled cells have now migrated into the periodontal
tissue spaces and are seen adjacent to vascular channel
openings into the ligament. (C) Day 10. Labeled cells are
relatively evenly distributed throughout the ligament,
tending toward the bony surface. B, bone; T, tooth; PDL,
periodontal ligament. Original magnication 100 [King
GN, King N, Hughes FJ, unpublished, based on experi-
ments described by McCulloch et al. (159)].
50
Hughes et al.
reported in primary marrow stromal cells, partic-
ularly as osteoblast/chondrocyte/adipocyte stem
cells (2, 187, 195). There is much less clear evidence
of cells at an advanced stage of differentiation
exhibiting true dedifferentiation and redifferentiation
along distinct pathways (132). However, studies
suggest that this phenomenon may occur in osteo-
blasts redifferentiating into chondrocytes. In support
of the possibility of a differentiated cell subsequently
redifferentiating as a distinct phenotype, it has been
shown that osteoblastic cells, when transfected with
the tissue-specic transcription factor, PPAR-c 2, can
be converted to express an adipocytic phenotype (4).
In addition to these complexities of the stem cell
system, it has proved extremely difcult to identify
specic cells of the mesenchymal lineages with labels
that specically reect a particular stage in the stem
cell/early commitment phases of connective tissue
cell lineages. Typically, in stem cell studies using
culture methods, in vitro differentiation potentials
are elucidated by directly demonstrating differenti-
ation outcomes and ascribing those to cultured cells
in retrospect. In addition, at least one antibody (Stro-
1) has proved extremely useful in identifying the cells
in marrow stromal cell cultures with osteogenic
potential (225). Stro-1 staining in combination with
alkaline phosphatase expression using uorescence-
activated cell sorting has demonstrated the presence
of early osteogenic precursor cells in a Stro-1-posit-
ive/alkaline phosphatase-negative population of
marrow stromal cells (51, 85, 222). During differen-
tiation it has been shown that Stro-1 expression is
lost, whilst alkaline phosphatase expression is
upregulated (257). Despite these observations and
many other attempts, it has still proven difcult and
impossible at the present time to dene a marker or
markers that would unequivocally identify a single
multipotent mesenchymal stem cell in a mixed
stromal cell population.
Transcriptional regulation of osteoblastic
commitment and differentiation
Lineage-specic gene expression is ultimately under
the control of transcription factors that act to regulate
specic gene expression. They act as the key
switching mechanisms to induce gene transcription.
Considerable progress has been made in identifying
those transcription factors which act as master
switches during commitment of multipotent cells to
specic lineages. Basic helix-loop-helix genes of the
myoD family are known to specify commitment and
differentiation of mesenchymal stem cells to the
myoblast lineage (260). Similarly, the transcription
factor, ppar-c 2, can specify adipocyte differentiation
(209), and sox-9 expression is required for chondro-
cyte differentiation (44). A major breakthrough in the
understanding of genetic regulation of osteoblast
differentiation was made with the identication
of the role of the transcription factor core binding
factor 1 (Cbfa-1/Runx-2) (55, 130). Cbfa-1/Runx-2
expression is an absolute requirement for osteoblast
differentiation. In cbfa-1 knockout mice there is a
normal cartilaginous skeleton seen but a complete
absence of bone (and tooth) formation. Cbfa-1/Runx-
2 is known to interact directly with the osteocalcin
promoter to induce its expression (56). However an
additional transcription factor, osterix, which is a
downstream target for Cbfa-1/Runx-2, has also been
shown to be an absolute requirement for normal
osteoblast differentiation in knockout mice experi-
ments (170). More recent studies have shown the
existence of distinct isoforms of Cbfa-1, which may
have subtly different roles during normal tissue for-
mation, including regulation of cartilage expression
in addition to bone.
The expression of Cbfa-1/Runx-2 occurs quite late
in the osteoblast lineage scheme and physiologically
probably does not specify osteoblastic commit-
ment events in multipotent stem cell populations.
Recently, the role of the transcription factor, TAZ, has
been reported, which appears to act by specifying
osteoblastic cell fate in bipotent osteoblast/adipocyte
stem cells, by activating Cbfa-1/Runx-2 whilst sim-
ultaneously repressing the adipocyte transcription
factor, ppar-c 2 (95), Experimental overexpression of
TAZ in primary mesenchymal stem cells appears to
preferentially specify osteoblastic differentiation in
mesenchymal stem cells.
A number of other transcription factors, whilst not
being absolute requirements for osteoblast differen-
tiation, are also known to regulate osteoblast differ-
entiation. These include Msx-2, Dlx-5 and the AP-1
family of transcription factors formed by hetero-
dimers of members of fos and jun families. Although
a full review of these molecules is beyond the scope
of this review, msx-2 appears to act upstream of
Cbfa-1/Runx-2 (91) whilst Dlx-5 may be a transcrip-
tional regulator of late stage osteoblast differentiation
acting downstream of Cbfa-1/Runx-2.
Summary
In summary, the osteoblast lineage consists of
mesenchymal stem cells which exhibit self-renewal
and may include both multipotent cells and those of
51
more restricted potentials. Commitment of these
cells to the osteoblast lineage is likely to be mediated
by the transcription factor TAZ, resulting in com-
mitted monopotent osteoprogenitor cells with
extensive proliferative capacity. Differentiation of the
mature osteoblast is ultimately regulated by tran-
scription factors, including Cbfa-1/Runx-2 and
osterix. A diagramatic schema of the osteoblast lin-
eage and its features is shown in Fig. 2.
The effects of growth factors on
osteoblast differentiation
Huge advances have been made in the understanding
of cellular and molecular control of bone formation
in the past decade. The establishment of in vitro
models of osteoblast differentiation and formation
(11) have been essential for determining the effects of
specic growth factors and growth factor-induced
transcription factors on osteogenesis. These models
include the use of primary cell cultures and estab-
lished cell lines from a range of different species and
with distinct properties (98). Interpretation of the
large amount of data from these in vitro models can
sometimes be difcult owing to the variations seen in
different model systems and the fact that cells may
show different responses according to the precise
stage of target cells through the lineage at different
stages of differentiation. For example, as reviewed
below, broblast growth factor-2 treatment of oste-
oprogenitor cells in vitro results in a mitotic response
but suppression of the expression of osteoblast dif-
ferentiation markers, but paradoxically, when applied
in vivo, may result in an overall stimulation of bone
formation. This can be explained by the suggestion
that broblast growth factor-2 results in the expan-
sion of relatively undifferentiated cells, which is seen
as a suppression of differentiation in vitro but
ultimately results in increased mature osteoblast
numbers in vivo.
Tissue engineering aims to produce tissues that are
both structurally and functionally identical to the
original tissues they are replacing, and strategies for
growing bone therapeutically are emerging based on
the knowledge of the physiological role of different
Fig. 2. Schematic diagram of stages in the osteoblast lineage and some phenotypic features seen at different stages of the
lineage. Cbfa-1, transcription factor expression. AIP, alkaline phosphatase; AP-1, activator-protein-1; BSP, bone sialo-
protein; OSX, osterix; PTH, parathyroid hormone; PTHrP R, PTH-related protein receptor.
52
Hughes et al.
signaling molecules in the bone-forming process.
The potential therapeutic application of growth fac-
tors in bone tissue engineering is considered further
below.
Bone formation is directed by the co-ordinated
expression of many molecules, including growth
factors, BMPs and specic transcription factors,
which utilize developmentally derived signals to
induce cellular and molecular stimuli to guide cel-
lular commitment and differentiation in the proper
spatial and temporal sequence. It is envisaged that
future periodontal tissue engineering may involve the
use of appropriate gene expression systems for con-
trolled delivery of regenerative growth factors within
the appropriate biological scaffold.
Growth factors are soluble proteins that act as
signaling agents for cells and inuence critical
functions, such as cell division, matrix synthesis and
tissue differentiation, by receptorligand binding.
Results of experimental studies have established that
growth factors play many important roles in bone
formation and bone repair. Within the periodontal
environment, growth factors found in bone,
cementum and healing tissues include transforming
growth factor-b, basic broblast growth factor,
insulin-like growth factors, platelet-derived growth
factor and BMPs. Recently, with the advent of
recombinant growth factor proteins there has been
considerable interest in their use as therapeutic
agents in the treatment of bone disorders (including
periodontal disease), and as growth factors become
available as therapeutic agents, it is essential that we
understand their biological characteristics and bio-
logical potential for periodontal tissue engineering.
During bone formation, a number of candidate
growth factors and their respective signaling path-
ways have been delineated, although there is a pau-
city of understanding of the complexities in the way
that these growth factors interact to regulate bone
formation and repair. It seems biologically sensible
that there is involvement of multiple bioactive factors
in osteoblast development and bone formation, with
factors acting in a sequential manner. A number of
growth factors, and their downstream molecular
targets, have been characterized during osteoblast
differentiation. These ndings suggest that at least
some of the growth factors reviewed in this article, or
their genetic effectors, may be potential therapeutic
targets for regenerating bone. Growth factors that
are known to affect osteogenic cells include trans-
forming growth factor-b, broblast growth factors,
platelet-derived growth factor and insulin-like growth
factor.
Transforming growth factor-b
Transforming growth factor-b belongs to a large
superfamily of related proteins that also includes
BMPs, growth and differentiation factors, activins, in-
hibitins and anti-Mullerian hormone. All members
playimportant roles inregulatingcell proliferationand
differentiation and the production of extracellular
matrix. There are ve isoforms of transforming growth
factor-b (transforming growth factor-b1 to trans-
forming growth factor-b5). Most cells synthesize and
respond to transforming growth factor-b, but high
levels are found in bone, platelets and cartilage.
Transforming growth factor-b1 is the most abundant
isoform at the protein level (for a recent comprehen-
sive review see Janssens et al. (111)).
The activation of transforming growth factor-b is
highly regulated, and once activated it interacts with
transmembrane serine/threonine kinase receptors.
Pivitol genetic studies identied that intracellular
transforming growth factor-b signaling is mediated
through mothers against dpp (Mad) in Drosophila
melanogaster and subsequently the homologous
genes in vertebrates (Smads) have been identied
[reviewed in Massague and Chen (158)]. Although
Smads are the key mediators in the transforming
growth factor-b signaling pathway, BMPs have also
been shown to signal through the Ras/mitogen-acti-
vated protein kinase (MAPK)/activator-protein-1
(AP-1) pathway [for review see Nohe et al. (176)].
During the early stages of bone formation, the
action of transforming growth factor-b is to recruit
and stimulate osteoprogenitor cells to proliferate,
providing a pool of early osteoblasts (36, 206). In
contrast, during later phases of osteoblast differenti-
ation, transforming growth factor-b blocks differen-
tiation and mineralization (154). These effects appear
to be highly dependent on bone cell source, dose
applied and the local environment, which may be
a result of the inhibition of DNA synthesis at
high transforming growth factor-b concentrations
(35). Additionally, transforming growth factor-b
inhibits the expression of the Runx2 and osteocalcin
genes, whose expression is controlled by Cbfa1/
Runx2 in osteoblast-like cell lines, and this was found
to be mediated by Smad3 (5).
Transforming growth factor-b interacts with a
range of other growth factors in bone with a resulting
complex response. Further work is needed to clarify
the role of transforming growth factor-b during per-
iodontal regeneration and wound healing and to
determine the inter-relationship between transform-
ing growth factor-b and other growth factors that
53
have effects during different stages of osteoblast dif-
ferentiation.
Bone morphogenetic proteins
Bone morphogenetic proteins are secreted signaling
molecules which have a variety of functions during
development (92) and cell differentiation (266). They
were discovered for their remarkable ability to induce
cartilage and bone formation from nonskeletal
mesenchymal cells (252, 253) by recapitulating the
entire sequence of events occurring during endo-
chondral ossication. They have been subsequently
cloned, sequenced and recombinant proteins
manufactured (219, 268). These recombinant pro-
teins have been the subject of many studies in vivo
and in vitro and it is likely that these recombinant
proteins will be used therapeutically in the near
future for a range of applications in orthopedics,
craniofacial surgery and dentistry (267).
Like transforming growth factor-b, BMPs are
expressed during embryonic development as well as
into adulthood. Bone morphogenetic proteins are
synthesized as a precursor molecule, and the active
homodimeric or heterodimeric forms of BMP follow
release of the C terminal by proteolysis (268).
More than 20 BMP-related proteins have been
identied, and there is diversity of biological activity
as each BMP binds to their receptor with different
afnities. Bone morphogenetic proteins bind to two
distinct type I and II serine/threonine kinase recep-
tors, both of which are required for signal trans-
duction, although the type I receptor determines
specicity of intracellular signals. Once activated,
signals are transmitted intracellularly via Smad-
dependent and Smad-independent pathways [for
review see Miyazono et al. (161)].
Among the BMP family, BMP-2, BMP-4 and BMP-7
have key roles during osteoblast commitment and
differentiation. The primary effect of BMPs is on the
pluripotent cells that are capable of differentiating
into other mesenchymal cell types (10, 119, 258), and
BMP-2 can direct these cells to commit to an osteo-
blastic pathway, as can BMP-4 and BMP-6 (102). Bone
morphogenetic proteins can also increase the differ-
entiation of committed cells to the osteoblast lineage,
with the formation of bone nodules and expression
of markers of the mature osteoblast phenotype (39,
102). Gene-expression studies, directly delivering an
adenovirus with BMP-7 to gingival or dermal bro-
blasts, leads to a robust osteogenic response (61).
In recent years there has been an explosion of
interest in potential molecular targets of BMPs, both
from the basic molecular pathway and potential
therapeutic perspectives. This has given considerable
insight into the genetic cascade leading to bone for-
mation. Among these specic targets are the tran-
scription factors Cbfa1/Runx2 (55), osterix (170) and
TAZ (95). Bone morphogenetic proteins can upregu-
late Cbfa1/Runx2 under certain conditions during
osteoblast differentiation; therefore, this is a candi-
date downstream target of BMPs, although Smad
complexes can also directly interact and activate
target genes independently of Cbfa1/Runx2 (115).
Evidence is also emerging to suggest that some as-
pects of BMP-2-induced differentiation may be
mediated through Wnt/b-catenin signaling, although
co-operation between b-catenin and Smads are
needed to activate late osteoblast gene expression
(13). b-catenin is the classical effector of Wnt protein
signaling, suggesting a role for this group of proteins
in BMP-2 signal transduction.
Fibroblast growth factor
The broblast growth factors are a family of struc-
turally related polypeptides that are known to play a
critical role in angiogenesis and mesenchymal cell
mitogenesis.
To mediate their range of effects, broblast growth
factor proteins signal via membrane-spanning tyro-
sine kinases (FGFR) and there are a wide variety of
mechanisms for receptor regulation and availability.
Mutations in these receptors are associated with
abnormalities in ossication and activating muta-
tions in FGFR2 cause several craniosynostosis
syndromes by affecting the proliferation and differ-
entiation of osteoblasts (185), highlighting a key role
for these molecules in the control of bone formation.
In normal adult tissues, the most abundant proteins
are broblast growth factor-1 and broblast growth
factor-2. Fibroblast growth factor-2 is expressed by
osteoblasts and is generally more potent than bro-
blast growth factor-1 (29), although the expression of
other broblast growth factors are not nearly as ubi-
quitous. Although broblast growth factor signaling
has been implicated in bone development, studies on
null mutant mice have not yet fully shown the role of
this family in skeletal development (166). Fibroblast
growth factor-1 and broblast growth factor-2 in vitro
stimulate osteoblast proliferation (in calvarial cells,
ROS 17/2.8 and MC3T3-E1) but do not increase col-
lagen production or alkaline phosphatase in differ-
entiated osteoblasts (28, 106, 207), although these
effects may be differentiation stage-specic as con-
stitutive broblast growth factor signaling inhibits
54
Hughes et al.
osteoblastic differentiation (49) and dramatically
increases apoptosis when cells are exposed to differ-
entiating conditions (157). Fibroblast growth factors
are strongly mitogenic to bone marrow stromal cells
and are able to maintain the self-renewal of these cells
in culture (135).
It seems that short-term treatment with broblast
growthfactor, followedbyits removal fromculture, has
an overall stimulatory effect on osteoblasts, and these
growth factors may have potential as an adjunctive
agent to increase bone formation. Their general effects
in vitro are consistent with them acting mainly to
stimulate proliferation in immature cells (whilst con-
comitantly inhibiting differentiation), resulting in
expansion of the osteoblast progenitor pool.
Platelet-derived growth factor
Platelet-derived growth factor is secreted by platelets
during the early phases of fracture healing, but its
presence has been found in various tissues, including
bone. Owing to its expression by a range of tissues, it
is thought to have both systemic and local actions.
Platelet-derived growth factor is composed of two
polypeptide chains and can exist in three different
isoforms of two gene products (AA,BB,AB) and these
bind to two separate a and b receptors.
Platelet-derived growth factor is a powerful mitogen
for connective tissue cells, and although it can sti-
mulate, and is synthesized by, mesenchymal cells and
osteoblast like cells (83), it does not have powerful
bone-induction properties. Platelet-derived growth
factor isoforms have a strong chemotactic effect on
osteoblasts and other connective tissue cells (100,
101), and may act to recruit mesenchymal cells during
bone development and remodeling. Platelet-derived
growth factor may also have direct and indirect effects
on bone resorption by the upregulation of collagenase
transcription (215) and an increase in interleukin-6
(IL-6) expression (64) in osteoblasts. In addition to
platelet-derived growth factor autoregulation (214) in
osteoblasts, there is paracrine regulation by other
growth factors, such as transforming growth factor-b
(213). Overall, the main physiological actions for
platelet-derived growth factor in bone regulation are
as nonspecic mitogens, but their effects on cell
chemotaxis and neovasculogenesis may be partic-
ularly important during would healing.
Insulin-like growth factors
Growth hormone and insulin-like growth factors play
critical roles in skeletal development. Growth hor-
mone participates in the regulation of skeletal growth
and triggers the release of insulin-like growth factor in
target cells. The insulin-like growth factors are bound
to binding proteins, adding another crucial tier to
modulate the activity of insulin-like growth factor.
Two insulin-like growth factors have been identi-
ed insulin-like growth factor-1 and insulin-like
growth factor-2 both of which are found in high
concentration in serum. In bone, whilst insulin-like
growth factor-2 is more abundant, insulin-like growth
factor-1 may be more potent, although this might be
different both between and within species (208).
The regulation of insulin-like growth factor is
complex, and the growth hormone mode of action
in skeletal cells is largely unknown. Of the major
hormones that regulate the skeleton, all have signi-
cant effects on skeletal insulin-like growth factor,
as do many growth factors, such as BMP-2 (27),
transforming growth factor- (181) and broblast
growth factor (70). Insulin-like growth factors in-
crease proliferation and play a major role in sti-
mulating mature osteoblast function. As with other
growth factors detailed in this section, the way that
osteoblasts respond to insulin-like growth factor
signals may well depend on both the differentiation
status of the cell and cell type. At the molecular
level, insulin-like growth factor-1 upregulates the
osteoblast-associated transcription factor, osterix,
but not Cbfa1/Runx2. In addition, insulin-like
growth factor-1, in combination with BMP-2, acts
synergistically on osterix expression (34).
Although it is widely accepted that insulin-like
growth factors have a dening role in bone remode-
ling, their actual role is still unclear and needs to be
understood within the complex inter-relationships of
the components of the insulin-like growth factor
system that evidently occur in vivo. Overall, the evi-
dence suggests that the major effects of insulin-like
growth factors are to promote the late-stage differ-
entiation and activity of osteoblasts.
Wnt signaling
There is increasing evidence for the role of Wnt
signaling in the control of early stages of the osteo-
blast lineage. Wnts are a group of over 15 related
extracellular signaling molecules that show ligand
binding with their receptor, frizzled, and co-receptors
LRP5/6. Ligand binding sets off a series of intra-
cellular reactions, resulting in stabilization of the
protein b-catenin (200). This is then translated to
the nucleus, where it acts as a co-factor with other
DNA-binding proteins to regulate gene transcription.
55
This Wnt/b-catenin signaling pathway is also known
as the canonical Wnt signaling pathway. In addition,
Wnt ligand binding can regulate gene expression by
noncanonical pathways without the involvement of
b-catenin. The canonical Wnt signaling pathway has
been shown to have important roles in the main-
tenance of self-renewal of stem cells in epidermal
and hematopoietic cells, and there is increasing evi-
dence for its role in the regulation of bone formation
(31, 200, 262). First, it has been shown that canonical
Wnt signaling can directly promote osteogenesis
through actions on the Cbfa-1/Runx-2 gene (72).
Second, genetic defects and polymorphisms of the
Wnt co-receptor, LRP5, are associated with an
osteoporotic phenotype (14, 129). The relationship
between Wnt signaling and other growth factor-
mediated osteoblast effects is, to date, confusing,
with evidence for Wnt signaling acting through the
induction of BMPs, and, conversely, BMPs acting
through the induction of Wnt signaling in mesen-
chymal cells (13, 199, 265). At present there is little or
no information about the role of Wnt signaling in
local bone formation during wound healing, although
it is likely that this signaling pathway may play an
important role in the early events of local bone
formation.
Other growth factors
There are many other growth factors that can interact
with cells of the osteoblast lineage and may play a
role in the local regulation of bone formation. For
example, osteoprogenitor cells are responsive to
epidermal growth factor stimulation, although epi-
dermal growth factor receptors may be downregu-
lated in mature osteoblastic cells (48). Treatment of
cell cultures in vitro with epidermal growth factor
results in increased cell proliferation and suppression
of bone module formation (7). Epidermal growth
factor utilizes many of the signal transduction path-
ways in common with platelet-derived growth factor
although, using a proteomic approach, subtle differ-
ences in intracellular signaling have recently been
reported in these two signaling mechanisms (137).
Sonic hedgehog (Shh) is another growth factor-like
protein which binds to a specic cell-surface recep-
tor, patched (Ptc). Shh can induce osteoblastic com-
mitment in cultured multipotent mesenchymal stem
cells and promote osteoblast differentiation (126, 230,
271). These actions can be blocked with BMP
antagonists, suggesting that Shh is an upstream
regulator of BMP production and the Shh-activated
intracellular signaling molecule, Gli, can upregulate
BMP expression by direct interaction with bmp gene
promoters (121).
Enamel matrix derivatives
Enamel matrix derivatives (EMD) are the major
component of commercially available Emdogain
and are discussed here in order to compare infor-
mation on their biological activity with that presen-
ted for growth factors above. The main biological
effects of EMDs have been attributed to their
predominant protein, amelogenin (37), with the
remaining fraction comprising less-characterized
factors. Amelogenin is not a classic growth factor, but
rather a cell-adhesion matrix-bound protein, and
specic amelogenin gene products are thought to
have activity as epithelialmesenchymal signaling
molecules. In addition, there is evidence that the
alternate splice variant of the amelogenin gene, leu-
cine-rich amelogenin peptide may also have direct
signaling activities on cementoblasts, and, thus at
least by implication, osteoblasts (20).
Enamel matrix derivative has been used clinically
for periodontal regeneration, and its therapeutic
effectiveness has been variously attributed to amel-
ogenin, nonamelogenin enamel matrix proteins and
growth factors. While EMDs may induce periodontal
regeneration, the precise mechanism of this is not
known. In vitro studies show that EMDs can increase
matrix production (86), proliferation and bone nod-
ule formation of periodontal ligament cell cultures
(73) and the differentiation of human and murine
osteoblast cell lines (221), with the stimulation of
phenotypic bone markers in some osteoblast cell
lines (270). In contrast, some studies show no effect
of EMDs on osteoblastic differentiation, although
other growth factors were stimulated (183). In addi-
tion, noncommercial EMDs have been shown to
contain both transforming growth factor-b and BMP-
like growth factors (233). The osteogenic activity of
EMDs may be mediated by the induction of BMP-like
molecules, as EMDs induce Cbfa1/Runx2 expression
and the phosphorylation of Smad1, both of which can
be blocked by the BMP inhibitor, noggin (237).
The search for downstream target genes has also
revealed EMD response elements in the rat bone
sialoprotein gene promoter that may mediate the
effects of EMDs on bone sialoprotein gene tran-
scription (223). A cDNA microarray study examining
EMD-mediated changes in gene expression in
periodontal ligament cells in vitro has reported the
downregulation of genes involved in the early inam-
matory phases of wound healing, while simulta-
56
Hughes et al.
neously upregulating genes encoding growth and
repair-promoting molecules (193). The in vitro treat-
ment of cementoblasts with EMDs was found to
decrease osteocalcin expression and to increase
osteopontin expression (249). Overall, the data sup-
port the positive effect of EMDs on osteoblast differ-
entiation, although further studies are needed to
clarify which molecules in EMDs stimulate osteogen-
esis and to dene their precise modes of action.
Summary
Considerable advances have been made in describing
the regulation of osteoblasts by growth factors and in
identifying some of the molecular mechanisms
involved in these processes. Although the data are at
times complex and sometimes contradictory, an
overall pattern emerges of a carefully regulated,
temporal sequence of co-ordinated growth factor
expression, which can be modulated at any stage of
the differentiation cycle. A schematic representation
of this is shown in Fig. 3.
Effects of inammatory cytokines
on osteoblast differentiation
Apart from responding to growth and differentiation
factors, osteoblasts are also responsive to a number of
locally acting cytokines, which are molecules that
regulate cellular behavior of bone under situations of
inammation, infection and wound healing. The
outcome of these cellular responses may be both
anabolic and catabolic. This section will provide
an insight into the capacity of the major pro-inam-
matory cytokines to regulate osteoblast differentiation
and function. Finally, the coupling of the osteoblast to
the regulation of bone resorption will be introduced.
Interleukin-1
Interleukin-1 is a multipotent cytokine comprising
two individual peptides, namely IL-1a and IL-1b,
which exhibit similar biological activities (52). Both
hematopoietic and mesenchymal/osteoblastic cells
can produce IL-1 (89). There are two IL-1 receptors,
namely type-I and type-II. All cellular responses eli-
cited by IL-1 are known to be mediated through the
type-I receptor (227), whereas type-II acts as a non-
signaling decoy receptor for the cytokine (41). Inter-
leukin-1 also has a natural inhibitor, termed IL-1
receptor antagonist (IL-1ra). This binds to, but does
not activate, IL-1 receptors (88), thus blocking the
biological activity of the cytokine (9). The signaling
events downsteam of the IL-1 receptor are mediated
by IL-1 receptor-associated kinase (IRAK), and
include the recruitment of phosphatidylinositol
3-kinase (PI3K), phosphorylation of MAPKs, and
ultimately activation of the transcription factor,
nuclear factor kappa B (NF-jB) (12).
Inthe context of bone, IL-1 is knowntoregulate both
resorption (149) and formation (25). However, the
outcomes of different studies on the effects of IL-1 on
osteoblast function are rather divergent. On the one
Fig. 3. Simplied schematic diagram showing the main
stages of the osteoblast lineage where different growth
factors may act. BMP, bone morphogenetic protein; IGF,
insulin-like growth factor; FGF, broblast growth factor;
PDGF, platelet-derived growth factor; Shh, Sonic hedge-
hog; TGF-b, transforming growth factor-b; Wnts, a group
of >15 related extracellular signaling molecules.
57
hand, both IL-1a and IL-1b have been shown to inhibit
osteoblast proliferation and enhance bone formation,
as demonstrated by enhanced alkaline phosphatase
activity and bone nodule formation (50, 87, 179). On
the other hand, depending on the differentiation stage
of the cell, prolongation of the culture period and
concentration of the cytokines, IL-1a and IL-1b, may
stimulate osteoblast proliferation, including DNA and
protein synthesis, and inhibit bone formation (25, 57,
202, 241), and inhibit osteocalcin and type I collagen
production (231, 232). Interleukin-1 can also stimu-
late osteoblasts to produce other pro-inammatory
cytokines, such as IL-6 (38, 110), IL-7 (261) and tumor
necrosis factor-a (TNF-a) (259), or other inammatory
mediators, such as prostaglandin E
2
and nitric oxide
(104, 105, 120, 197, 198).
Interleukin-6
The IL-6 family of cytokines includes IL-6, IL-11,
leukemia inhibitory factor, oncostatin M, cardiotro-
phin-1, and ciliary neurotrophic factor (216). Inter-
leukin-6 is a pleiotropic cytokine crucial for the
regulation of bone metabolism, reportedly more so
than other members of the IL-6 family. The major
source of this cytokine in bone is osteoblasts and
stromal cells (94). Its involvement in bone remode-
ling is particularly evident in situations of increased
bone turnover, rather than in physiological condi-
tions (65, 93, 134, 156). It was initially demonstrated
in vitro that IL-6 does not affect, or may slightly
downregulate osteoblast differentiation (99, 110, 146,
147). These ndings were further strengthened by the
absence of any pathological bone phenotype in IL-6
knockout mice (133). However, it is now established
that the cellular expression of IL-6R, the cognate
receptor to this cytokine, occurs at low levels.
Therefore, the presence of the soluble receptor, sIL-
6R, an agonist for IL-6, is imperative for the activation
of the downstream effects of this cytokine, in a pro-
cess called trans-signaling (59, 65, 117).
In the light of this requirement, studies on the
osteoblast demonstrate that the IL-6/sIL-6R complex,
IL-11, leukemia inhibitory factor and oncostatin M
inhibit proliferation and stimulate osteoblastic cell
differentiation via a cascade of intracellular events
mediated by gp130, a signal-transducing receptor
subunit (17, 58, 235). This molecule is more ubiqui-
tously expressed than IL-6R (216). Binding of IL-6 to
either its membrane-bound or its soluble receptor,
results in homodimerization of gp130 and initiation
of downstream signaling pathways, including the
activation of the Janus Kinases and subsequently the
extracellular signal-regulated protein kinase 1/2
MAPK, as well as the signal transducers and activa-
tors of transcription 1 and 3 (STAT-1 and STAT-3) (59,
65). Desensitization of IL-6 signaling is accomplished
by endocytosis of the receptorligand complex, a
process dependent on gp130. However, attenuation
of IL-6-induced STAT-1 and STAT-3 expression, is
independent of IL-6/IL-6R, or gp130 endocytosis
(247, 248). Apart from the direct effects on osteoblast
proliferation and differentiation, it is also possible
that the IL-6/sIL-6R complex may indirectly regulate
the expression of other factors known to control these
cellular events. These include insulin-like growth
factors (63, 62), BMPs (269), and a number of other
paracrine and endocrine regulators of osteoblasts.
Tumor necrosis factor-a
Tumor necrosis factor-a is a member of the TNF
family of inammatory cytokines that share common
signaling pathways. It has a pivotal role in bone
pathophysiology, because it has been shown to sti-
mulate bone resorption and inhibit bone formation
(19, 171). The major source of TNF-a is the T cell, and
the cognate receptors to TNF-a are TNFR-1 (or p55)
and TNFR-2 (or p75) (171). The former is required to
initiate cell signaling, whereas the latter may con-
tribute to the sensitivity of the cell response to the
cytokine (77). Ligandreceptor binding activates TNF
receptor-associated factors (TRAFs), mainly TRAF-1
and TRAF-2, in osteoblasts (16, 33), which in turn
evoke a series of interconnected events, ultimately
leading to the activation of NF-jB. In brief, NF-jB is a
set of ve polypeptide transcription factors, called
p50, p52, p65 (or Rel A), Rel B and c-Rel, which form
heterodimers. The NF-jB heterodimers form an
inactive complex with their I-jB inhibitors. Once
phosphorylated by the I-jB kinases, I-jB is ubiquiti-
nated and degraded in proteosomes. This results in
the liberation and translocation of NF-jB into the
nucleus, where it activates the transcription of a
number of genes (21). Optimal induction of the
respective target genes also requires phosphorylation
of the NF-jB proteins (254). It should be noted that
NF-jB knockout mice develop osteopetrosis as a
result of a defect in osteoclast differentiation (108).
Tumor necrosis factor-a is known to inhibit
osteoblast function and differentiation. Suppression
of the osteoblastic phenotype is characterized by the
inhibition of cell proliferation and decreased pro-
duction of a number of extracellular matrix compo-
nents, such as type I collagen (82, 172, 210, 229).
Indeed, genetic analyses have identied TNF-a-
58
Hughes et al.
responsive regions in the promoter of the respective
genes (COL1A1 and COL1A2), indicating that NF-jB
mediates the inhibition of collagen transcription
(109, 116, 136, 163). Tumor necrosis factor-a also
inhibits alkaline phosphatase and osteocalcin
expression. However, the nature of osteocalcin inhi-
bition is different from that of type I collagen,
because this does not require binding of NF-jB to the
promoter of the osteocalcin gene (173, 171). Apart
from suppressing extracellular matrix production by
osteoblasts, TNF-a also inhibits the differentiation of
pre-osteoblasts as a result of the suppression of
Cbfa1/Runx2 (1, 76) and osterix (171). In addition,
TNF-a stimulates IL-6 production by osteoblasts (47,
110, 203) in an NF-jB-dependent mechanism (139).
However, recent evidence suggests that other path-
ways such as p38 MAPK, and protein kinase C may
also be involved in this event (138, 167). Tumor
necrosis factor-a also induces prostaglandin E
2
pro-
duction by osteoblasts, an event at least partially
mediated by nitric oxide (104).
Regulation of osteoclastogenesis and
bone resorption by the osteoblast
In recent years it has become evident that osteoblasts
have a global role in orchestrating the bone
remodeling process. Their function is not restricted
solely to bone formation, but it is now rmly estab-
lished that they are responsible for initiating bone
resorption. In cellular terms, apart from forming the
mineral and organic extracellular compartment of
bone, the osteoblast provides the essential and suf-
cient stimuli that control the behavior of the oste-
oclast, an event that occurs via cellcell interaction.
The molecular determinants of this interaction are
two factors produced by osteoblasts/stromal cells,
namely M-CSF and RANKL, the former secreted and
the latter mainly cell-membrane bound (244). M-CSF
binds to c-Fms on the surface of osteoclast precur-
sors, and this event enhances their proliferation and
survival. RANKL, a member of the TNF ligand
superfamily, is the factor that actually triggers the
differentiation of osteoclast precursors, into mature
osteoclasts and activates bone resorption, and is an
absolute requirement for osteoclast formation (131,
140). Therefore, upon binding of RANKL to its
cognate RANK receptor on the surface of the M-CSF-
triggered osteoclast precursors, a number of down-
stream signaling pathways are activated, committing
the cell to the osteoclast lineage. These are initially
mediated by TRAF-6, subsequently leading to the
activation of NF-jB, the AP-1 transcription factor
complex, and ultimately nuclear factor of activated T
cells (NFAT) c1 (238, 245). The effects of RANKL can
be blocked by the soluble nonsignaling decoy
receptor, osteoprotegerin (OPG), a member of the
TNFR superfamily with high homology to RANK
(226). OPG is produced and secreted by osteoblasts
and stromal cells, and by binding to RANKL it pre-
vents RANKL/RANK interaction and consequently
the downstream events described. RANKL-decient
mice lack functional osteoclasts and develop osteo-
petrosis, whereas OPG-decient mice develop early-
onset osteoporosis, demonstrating the indispensable
role of a balanced RANKL/OPG expression in phy-
siological bone remodeling (23, 131). An increased
RANKL/OPG expression ratio has been demonstrated
in diseases involving inammation-induced bone
loss, including periodontitis (148, 243, 246) and
rheumatoid arthritis (60).
Interleukin-1, IL-6 and TNF-a are all potent
inducers of osteoclast differentiation and bone
resorption. It is postulated that their catabolic effects
on bone are mediated, to a great extent, through
osteoblasts/stromal cells, and more specically
through the regulation of RANKL and OPG expression
in these cells (144, 148, 256). Indeed, it has been
demonstrated that IL-6/sIL-6R, IL-11, leukemia
inhibitory factor and oncostatin M increase RANKL
and OPG expression in osteoblasts, in a manner
dependent on gp130 and its downstream effector
STAT-3 (177, 178, 191). Similarly, both IL-1 and TNF-
a induce RANKL expression, the former possibly
being dependent on activation of STAT-3, whereas
the latter is dependent on phosphorylation of p38
(47, 90, 259). OPG expression is also enhanced by
these two cytokines (22, 255). Although both RANKL
and OPG are upregulated in these systems, the
overall outcome is an increased RANKL/OPG
expression ratio, resulting in stimulation of osteo-
clastogenesis and bone resorption.
Other inammatory cytokines
A number of other cytokines have been reported to
regulate bone cell behavior. Interleukin-7 (78) and
IL-10 (71) have an inhibitory action on osteoblast
differentiation. Interleukin-4 and IL-13 are known to
inhibit osteoblast differentiation (251), and mice
over-expressing IL-4 appear to have a decreased bone
formation, owing to a reduction in the number and
activity of bone-lining osteoblasts (145). Interleukin-
18 is structurally and functionally homologous to
IL-1, but it employs differential signal-transducing
pathways, preferentially activating p38 MAPK rather
59
than NF-jB (53, 143). It is produced by osteoblasts/
stromal cells, to which it acts as a mitogen, and is also
known to inhibit osteoclast differentiation (43, 250).
This may, in part, account for its capacity to stimu-
late osteoprotegerin, but not affect RANKL expression
in osteoblasts/stromal cells (155). Interferon-c is a
lymphokine produced by activated T cells, with a
strong potency to inhibit osteoclastogenesis (80, 81,
239), It also inhibits osteoblast proliferation, as well
as collagen and noncollagen protein synthesis (82,
172, 229). These effects appear to be dependent on
nitric oxide production (103, 152, 153), rather than on
prostaglandin E
2
synthesis (229).
Summary
In summary, inammatory cytokines may have both
anabolic and catabolic effects on osteoblast function,
and consequently on bone physiology. However
there is relatively little information on how these
effects might also interact directly with growth factor
pathways, but these interactions are likely to be very
important. Studies have shown that both IL-1 and
TNF-a may inuence the effects of BMPs on osteo-
blasts, but the nature of these interactions have not
been explored at a mechanistic level (169, 240).
Therefore, any tissue engineering approach targeting
osteoblasts for bone regeneration must take into
consideration the putative cellular responses elicited
by cytokines upon them during inammation. This
should be a concern, not only in the case of exo-
genously administered cytokines as a direct treat-
ment strategy. It is highly probable that several other
primary molecular agents may induce secondary
inammatory responses by the target cells, inuen-
cing, in turn, the cellular dynamics of bone in an
autocrine or paracrine manner. This issue is partic-
ularly important in the instance of catabolic effects,
which may occur either as inhibition of bone for-
mation, or, most reportedly, as enhancement of bone
resorption. Tackling efciently the potential side-
effects of these cytokines may help to ensure that
the bone-remodeling balance would tend favorably
toward bone formation, ultimately resulting clinically
in bone regeneration.
Regulation and interaction of
growth factor pathways
Most of the information regarding the mechanisms of
action of growth factors and cytokines on their target
cells have been derived from studies carried out
in vitro or, on some occasions, from transgenic and
null mutant (knockout) mouse models. These meth-
odologies are extremely powerful for dissecting out
specic signaling pathways as it is possible to control
many of the extrinsic variables, thus facilitating the
study of, for example, the role of individual specic
molecules and signaling pathways. However, cells
in vivo are exposed to a wide range of these signaling
pathways simultaneously, and it is the total sum of
these interactions which results in the carefully
co-ordinated control of osteoblast function that is
seen in vivo. Although there are some data on how
different pathways may interact during the co-ordi-
nated regulation of bone growth, at the present time
it is only possible to provide examples of such
interactions, rather than give a complete picture of
the complex interactions in cytokine signaling path-
ways during bone growth.
There are a number of mechanisms which can
regulate different growth factor pathways. Different
signaling pathways can also interact with each other
in order to ne tune the nal observed outcome.
These mechanisms are listed below.
the regulation of growth factor activity by the
secretion of soluble antagonists which are often
regulated in both autocrine and paracrine fashions.
the regulation of receptor expression on specic
cells
the interaction between intracellular signaling
mechanisms in both antagonistic and synergistic
ways.
Growth factor antagonists and binding
proteins
Growth factors may bind to common proteins, such
as a-2 macroglobulin, and be sequestered, for
example, in matrices. Bone matrix is a rich source of
many growth factors, including transforming growth
factor-b and platelet-derived growth factor (45). In
addition, many growth factors also appear to have
specic binding proteins, which bind with their target
factor with high afnity to inactivate them, or in
some cases to increase ligand binding. Regulation of
expression of growth factor antagonists and the bal-
ance of factor to antagonist is a clear mechanism for
growth factors to control each others function in a
sequential cascade, such as that described above.
The activity of BMPs is regulated by a number of
inhibitor proteins, including noggin, gremlin, follist-
atin and sclerostin, which bind to BMPs with high
afnity in order to inactivate them (30). In addition,
a novel BMP inhibitor, ectodin, has recently been
60
Hughes et al.
described, whose expression is reportedly restricted
to ectodermal-derived tissues in regulating epithe-
lialmesenchymal interactions that are mediated by
BMPs (142). The important actions that these inhib-
itors may have are suggested by a number of obser-
vations. First, mutations in the SOST gene (which
codes for sclerostin) result in the rare bone disease,
sclerosteosis, which is characterized by increased
bone mass (15). Second, the overexpression of noggin
in transgenic mice results in blocking the produc-
tion of BMP-4-induced heterotopic bone formation
(79). Third, evidence suggests that BMP-4-induced
expression of noggin and gremlin is dysfunctional in
patients with brodysplasia ossicans progressiva, a
multilating condition where heterotopic bone forms
in subdermal tissues (3). Finally, normal expression
of ectodin in mice blocks enamel formation on one
surface of the mouse incisor, and mice lacking ecto-
din have a number of defects in tooth formation,
including an altered pattern of cusp formation and
the formation of extra teeth (118, 142).
Distinct BMP inhibitors may have tissue-specic
activities and may also interact with different BMPs.
For example, sclerostin expression is largely restric-
ted to bone cells, particularly osteocytes, and may be
involved in the osteocyte-mediated regulation of
bone mass (264). In general, the expression of BMP
antagonists is transcriptionally regulated by BMP
signaling, giving rise to a negative feedback loop to
regulate BMP signaling (30, 180). In addition, other
pathways may regulate the expression of BMPs. In
the case of ectodin, broblast growth factor and Shh
signaling downregulates its expression, providing a
mechanism whereby other growth factor signaling
pathways may effectively upregulate BMP responses
(142).
The insulin-like growth factors are regulated by a
series of at least six different binding proteins (insu-
lin-like growth factor-binding proteins). As with other
antagonists, insulin-like growth factor-binding pro-
teins bind to the insulin-like growth factors with high
afnity and typically may inactivate activity by pre-
venting ligand binding. However, it is known that
insulin-like growth factor-binding proteins may also
act to mediate or regulate the ligand binding of
insulin-like growth factors to their receptors on some
occasions (26, 160). Insulin-like growth factors have
been shown to differentially regulate insulin-like
growth factor-binding protein gene expression (69).
Insulin-like growth factor-binding proteins may also
be regulated by a number of other osteotropic growth
factors, including BMPs and transforming growth
factor-b (68, 107, 160).
Fibroblast growth factors have complex interac-
tions with a range of proteins and matrix molecules
that regulate their function. First, it is known that
broblast growth factors are normally matrix bound
to heparan sulphate-containing proteoglycans and
that heparan sulphate-containing proteoglycan
binding is required for normal broblast growth
factor ligandreceptor interactions (217). Alternat-
ively spliced broblast growth factor receptor genes
can give rise to broblast growth factor soluble
receptors, which inactivate broblast growth factor
by preventing normal receptorligand binding. Fi-
nally, there are additional broblast growth factor-
binding proteins that also interact with broblast
growth factors to control their function (141). The
presence of these complex different levels of regula-
tion together suggest a nely balanced set of mech-
anisms to control physiological broblast growth
factor activity, although the specic functional sig-
nicance of broblast growth factor antagonists re-
mains to be determined in bone.
Transforming growth factor-b is also regulated
closely by its association with binding proteins.
Transforming growth factor-b is secreted in an inac-
tive form bound to a specic binding peptide known
as latency associated peptide. This latent transform-
ing growth factor-b complex is then normally bound
to one of at least four latent transforming growth
factor-b-binding proteins (LTBPs) (182, 213). Latent
transforming growth factor-b-binding proteins are
extracellular matrix proteins found in bone matrix,
and may be responsible for the large amounts of la-
tent transforming growth factor-b sequestered in
bone matrix. They may act as a delivery and release
vehicle for transforming growth factor-b, and active
transforming growth factor-b can be released from
the complex by the enzymatic action of the plasmi-
nogen-activating system (6). In addition, a number of
other proteins, including matrix metalloproteinases
and thrombospondin, can release active transform-
ing growth factor-b from latency associated peptide.
The importance of these proteins is supported by the
reports that LTBP-2 null mice die in utero at around
stage e3.5; LTBP-3 knockout mice have a range of
bone and craniofacial defects associated with a low
turnover of bone (46).
In contrast to most other growth factors, specic
binding proteins have not been described for plate-
let-derived growth factor. However, platelet-derived
growth factor binds to the matrix protein osteonectin
(SPARC) and this may promote sequestering of
platelet-derived growth factor in bone matrix and
other sites (196).
61
Overall, it is clear that the production of growth
factor-binding proteins is a mechanism whereby the
activity of these proteins can be carefully controlled,
and represents one level of regulation allowing
careful cross-talk between different factors in a
carefully co-ordinated way.
Interaction of growth factor intracellular
signaling pathways
There are a number of examples described where
different growth factor pathways regulate each other
during intracellular signaling. Of particular interest,
studies have demonstrated the regulation of SMAD
signaling following BMP stimulation by additional
growth factor signaling pathways (161). Recent
results have demonstrated the downregulation of
BMP-activated SMAD signaling by both transforming
growth factor-b, via stimulating the expression of
inhibitory SMAD (154), and by extracellular signal-
regulated protein kinase and PI3 kinase pathways,
mediated by insulin-like growth factor-1 and other
growth factor signals (186). These observations sug-
gest many mechanisms whereby different growth
factors regulate responsiveness to other signaling
pathways. In this way it is possible that specic
growth factor expression and signaling activity can be
very delicately regulated to balance proliferation and
differentiation events within cells of the osteoblast
lineage.
Overall, it is clear that although different cytokines
may have distinct regulatory functions, during the
control of bone formation, the interaction of these
different pathways can be exquisitely regulated at a
number of points. These observations, taken
together, are consistent with a model of cytokine
signaling consisting of sequential and overlapping
expressions of factors to regulate nal bone-forming
outcomes.
Therapeutic application of growth
factors for bone regeneration
The topic of growth factor-induced bone and peri-
odontal regeneration has been discussed extensively
in other recent reviews (74, 234, 267), and it is not
the intention in this article to revisit the detail of
these experiments. However, here we consider some
of the basic issues surrounding the use of exogen-
ously applied growth factors to modulate bone
growth, particularly in the periodontal tissues, and
briey describe the general ndings of some of these
studies.
Given the limitations of currently used clinical
techniques, it is unsurprising that there is great
interest in the possible utility of recombinant growth
factors for tissue regeneration therapies. From a
theoretical standpoint, the pharmacological applica-
tion of growth factors results in a local single dose of
the factor, which may be present at a concentration
thousands of times higher than that normally seen
physiologically. The pharmacological effects of the
factor may be distinct from its physiological activity,
for example because the normal regulatory feedback
mechanisms which control that factor are tempor-
arily overwhelmed by such a large dose. It seems
quite likely that therapeutic application of any one of
a number of growth factors could perturb the normal
bone-forming process in a positive way and, indeed,
preclinical studies support this idea. For example,
based on the knowledge of physiological function,
discussed above, BMPs may increase osteoblastic
stem cell recruitment and commitment, broblast
growth factor may stimulate early progenitor cell
number expansion, platelet-derived growth factor
may also increase progenitor cell recruitment and
proliferation, and insulin-like growth factors might
act to increase nal osteoblast differentiation and
function. Any one of these actions may result in the
desired nal outcome of increased bone formation.
With this in mind, it is likely that one of the main
critical determinants of success for these treatments
is achieving sufcient substantivity for the factor to
provide a continual therapeutic dose over a lengthy
period of time (122). Although there is little pub-
lished data on the pharmacokinetics of growth
factor delivery, one study demonstrated that
delivery of platelet-derived growth factor with
insulin-like growth factor-I in a collagen matrix had
a half-life of <4 h, with complete loss of activity by
96 h (151). Thus, in order to address this issue a
number of different delivery systems have been
used for these applications, including collagen
matrix (123, 212), cross-linked gelatin pellets (124),
calcium phosphate ceramics (112, 174), and poly-
lactic-acid (PLA)-based resorbable polymers (192).
In a different approach to provide persisting growth
factor delivery, somatic cell gene transfer of plate-
let-derived growth factor has also been tested
in vitro and in animal models (8, 61, 114, 113, 272).
This approach might prove particularly successful
in providing a sustained active concentration of
growth factor to the site being treated. However, at
present, serious concerns about the safety of the
62
Hughes et al.
adenovirus vectors used for gene transfer, and other
ethical and practical issues, will need to be over-
come before this could be thought of as a viable
clinically applicable technique.
Platelet-derived growth factor and
periodontal regeneration
A number of studies have tested the potential for
tissue regeneration stimulated by platelet-derived
growth factor and also with platelet-derived growth
factor combined with insulin-like growth factor-1 or
dexamethasone (40, 97, 112, 150, 151, 211, 212). In
general terms, these studies (carried out in animals)
have demonstrated an enhanced rate and an
increased total amount of regenerated tissue com-
pared with controls. However, there is little evidence
of the efcacy of insulin-like growth factor-1, either
on its own or in combination with platelet-derived
growth factor (75). Most interestingly, a large multi-
centre human study (involving 180 patients) of
platelet-derived growth factor has recently been
reported (175). In that study, recombinant human
platelet-derived growth factor-BB, with a b-tricalcium
phosphate carrier, demonstrated improved bone ll
and enhanced rate of clinical attachment gain com-
pared with carrier-only controls. This is probably the
rst large human study to assess the role of recom-
binant growth factor therapy and these interesting
results will need further evaluation over time (201).
Other growth factors
Bone morphogenetic proteins have also been widely
tested for their ability to be used pharmacologically
to enhance new bone formation. Bone morpho-
genetic proteins can powerfully promote bone for-
mation in critical-size segmental defects in animals
and have shown some promise for their ability to
regulate bone and periodontal regeneration (123, 125,
205, 204, 224, 263). As these studies are the subject of
a further separate review in this volume, they will not
be considered further here.
The effects of broblast growth factor-2 on animal
models of periodontal regeneration have also been
tested in animal models. Results of these studies are
again promising, with broblast growth factor-2
showing a stimulation of parameters of bone and
periodontal regeneration in surgically created defects
(96, 164, 165, 168, 236). Other growth factors to have
been tested include transforming growth factor-b
where initial results of studies were disappointing
when compared with the effects of these other
growth factors (162, 242). There is also clinical
interest in the application of platelet-rich plasma, an
autologous preparation obtained from a patients
own platelets, which is rich in a number of growth
factors, including platelet-derived growth factor and
trefoil factor family-b, and which has been applied
clinically in both periodontal and implant applica-
tions (32, 184, 218, 220). However, disappointingly, to
date there is very little proper scientic evidence
which evaluates the efcacy of this procedure, and
well-controlled clinical studies of platelet-rich plas-
ma would be valuable in ascertaining whether this
treatment has a similar potential, for example, as the
pharmacological use of recombinant growth factors.
Summary
The explosion of knowledge and the understanding of
the role of growth factors, their mechanisms of action
and molecular signaling pathways, which have been
reviewed in this article, suggest the potential for
many novel therapeutic targets, not only for applying
growth factors but also for the potential use of growth
factor inhibitors or agents that target specic parts of
the intracellular signaling pathways. There remains
an enormous challenge to convert some of the
knowledge from basic studies of bone cell physiology
to therapeutically useful techniques for the future.
We are optimistic that such novel approaches may
result in real qualitative improvements in clinical
outcomes over currently available techniques.
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Bone morphogenetic proteins
and the induction of periodontal
tissue regeneration
UGO RI PAMONTI & LOUI SE RENTON
Bone: formation by induction
Since antiquity, bone has been known to have a
remarkable potential for repair and regeneration (28).
Tissue engineering, dened as the science of fabri-
cation of new tissues for replacement and the
regeneration of lost or destroyed tissues, has learned
and is still learning, the secrets of its principles from
bone repair and regeneration, and it is likely that
more secrets still remain to be learned from the
principles of bone tissue engineering (27, 28).
What are the rules that sculpt tissue morphology
and engineer the architecture of mineralized tissues?
The three critical ingredients for optimal tissue
engineering are soluble molecular signals, respond-
ing cells with associated cell-surface receptors, and
assembly of the extracellular matrix (27, 28). The
accrued knowledge can now be applied not only to
bone regeneration, but also to alveolar bone with
associated cementum and periodontal ligament
regeneration, tissues that are known to be recalcit-
rant to heal and regenerate (2, 38, 39).
The complex tissue morphologies of the perio-
dontal tissues are a superb example of design archi-
tecture and engineering. The supportive alveolar
bone consists of cortical or compact bone and can-
cellous or trabecular bone. The periodontal ligament
system, epitomized by inserting Sharpeys bers into
the cementum, provides a gomphosis that uniquely
articulates the tooth to the alveolar bone and permits
mechanical function and adaptation to changing
mechanical environments and signals additionally
modulated by the avascular mineralized root
cementum (2, 3739).
Substantial knowledge has now been gained about
the molecular signals that determine the emergence
of the complex tissue morphologies during regener-
ation of the periodontal tissues (2, 17, 39, 44, 51). The
molecular mechanisms for such regeneration are the
osteogenic proteins of the transforming growth fac-
tor-b superfamily (26, 32, 33, 35, 39, 50, 51), of which
the bone morphogenetic and osteogenic proteins
(BMPs/OPs) are a class of powerful inducers of
endochondral bone differentiation (Fig. 1) (25, 31, 35,
36, 40, 71).
Pioneering experiments gave rise to the fascinating
phenomenon of bone formation by induction (64,
65). Unexpectedly, the ligature of the renal artery in
rabbits induced transformation of the kidney into
bone with marrow (53). Acid alcohol bone extracts,
implanted intramuscularly in rabbits, induced
de novo bone formation (13), and Lacroix hypothes-
ized that bone contains substances, which he named
osteogenins, that initiate bone growth and differen-
tiation (13). Urist (64) made the key discovery that
demineralized bone matrix, when implanted hetero-
topically in intramuscular sites of allogeneic rat
recipients, induces de novo endochondral bone
formation by induction (64, 65). Reddi and Huggins
(29) showed that subcutaneous implantation of de-
mineralized bone matrix results in de novo local
endochondral bone differentiation, with the associ-
ated molecular and biochemical changes correlating
with each morphological step of bone differentiation
by induction (23, 24).
There is a direct relationship between differenti-
ation in early development and regeneration in
postnatal life. Fracture repair recapitulates events that
occur during the normal course of embryonic bone
development (6, 23, 24, 26, 28, 67). The tissue
response elicited by heterotopic implantation of
demineralized bone matrix is reminiscent of embry-
onic bone development (Fig. 1) (6, 23, 24, 26, 28, 32,
67). Unlike the epiphyseal growth plate, however,
73
PERIODONTOLOGY 2000
A
B
C
D
E
F
Fig. 1. Soluble molecular signals and the initiation of
endochondral bone formation as a recapitulation of
embryonic bone development. (A) and (B) Endochondral
bone formation in the subcutaneous space of the rodent
after implanting 520 lg of naturally derived bone mor-
phogenetic and osteogenic proteins (BMPs/OPs), puried
from baboon bone matrices after gel-ltration chroma-
tography on Sephacryl S-200. Islands of cartilage anlages
(white arrows) between particles of collagenous matrix,
vascular invasion and bone differentiation by induction
(black arrows) are shown. (C) and (D) Angiogenesis and
induction of bone after implantation of 0.10.5 lg doses of
the nal gel-eluted osteogenin fractions puried to
apparent homogeneity after electroendosmotic elution
(52). (C) Chondroblastic differentiation attached to the
matrix (white arrows), vascular invasion and differenti-
ation of osteoblast-like cells (black arrows) secreting bone
matrix. (D) Capillary invasion and angiogenesis surfacing
the implanted matrix as carrier with the endothelium
almost touching the osteoblast-like layer attached to the
matrix (white arrows), conrming the intimate and
exquisite relationships between invading capillaries and
osteoblastic cell differentiation. The black arrow points
to an endothelial cell migrating from the vascular com-
partment to the extracellular matrix compartment. (E)
Induction of cartilage anlages (white arrows) as a reca-
pitulation of embryonic development with angiogenesis,
osteoblastic cell differentiation and bone matrix depos-
ition 11 days after subcutaneous implantation of 2.5 lg of
human recombinant osteogenic protein-1 (hOP-1). (F)
Bone formation by induction in heterotopic extraskeletal
sites of the nonhuman primate, Papio ursinus, 30 days
after implantation of 100 lg of hOP-1 per gram of gamma-
irradiated bovine collagenous bone matrix as carrier.
Newly formed mineralized bone is shown in blue, surfaced
by thick osteoid seams in orange/red. (AF) Undecalcied
sections, cut at 5 lm, stained free-oating with Goldners
trichrome. Original magnications: 90 (A), 125 (B), 275
(C), 275 (D), 125 (E) and 90 (F).
74
Ripamonti & Renton
where a continuum of cartilage and bone differenti-
ation is observed, in the demineralized matrix-
induced bone differentiation, a single cycle of
endochondral bone formation is evident (23). The
sequential developmental cascade (23, 24, 26, 29) in-
cludes: activation and migration of undifferentiated
mesenchymal cells by chemotaxis; anchorage-
dependent cell attachment to the matrix via bro-
nectin; mitosis and proliferation of responding
mesenchymal cells; differentiation of chondroblasts
and deposition of cartilage; mineralization of the
cartilage; angiogenesis, vascular invasion and chon-
drolysis; differentiation of osteoblasts and bone mat-
rix deposition; and, nally, mineralization of the
newly induced bone and differentiation of hemopoi-
etic marrow in the newly formed ossicles (23, 24, 28).
Bone morphogenetic and osteogenic proteins
(BMPs/OPs) were originally identied from extracts
of bone matrix after incisive work on the develop-
ment of the heterotopic bioassay in subcutaneous
sites of rodents (23, 24, 2729). Intact demineralized
bone matrix induces endochondral bone differenti-
ation by induction when implanted in heterotopic
extraskeletal sites of animal models (23, 24, 29, 64,
65). The dissociative extraction of the demineralized
bone matrix with chaotropic agents, such as urea and
guanidinium hydrochloride, yielded an insoluble
component, mainly type I collagen, i.e. the insoluble
collagenous bone matrix, and a solubilized protein
extract containing the putative osteogenic proteins
(54). Neither the insoluble collagenous bone matrix
nor the lyophilized protein extract, when implanted
singly in the subcutaneous space of the rodent,
induced endochondral bone differentiation, high-
lighting that the osteogenic activity of intact demin-
eralized bone matrix has been abolished by the
dissociative extraction of the matrix components
(54). A single step of gel ltration to remove high-
molecular-weight contaminants was instrumental in
restoring the osteogenic activity when the soluble
signals of the extracted matrix were recombined and
reconstituted with the insoluble signal of the insol-
uble collagenous bone matrix (54).
The operational reconstitution of the inactive and
insoluble collagenous bone matrix with protein
extracts after a single step of gel-ltration chroma-
tography (54) restored the biological activity, yielding
endochondral bone differentiation by induction after
recombining the insoluble with the soluble signals
(54). The dissociative extraction and the operational
reconstitution of the matrix components were instru-
mental to purify BMPs/OPs to homogeneity, yielding
amino acid sequences for cloning of the human
proteins (Fig. 1) (4, 9, 25, 56, 71). The operational
reconstitution of the soluble and insoluble compo-
nents of the extracellular matrix of bone pointed to the
requirement of an insoluble signal or substratum to
deliver the osteogenic activity of the soluble molecular
signal (28, 32, 35, 52, 54). Following increasingly
rened purication procedures of the solubilized
proteins extracted from demineralized bone matrices,
BMPs/OPs were isolated and puried in sufcient
quantities to provide amino acid sequence informa-
tion (4, 35, 56, 71). Four important technical develop-
ments were instrumental for the characterization of
the family of the BMPs/OPs: the development of a
functional bioassay in the subcutaneous space of the
rat to monitor the biological activity of osteogenic
proteins (23, 24, 29); purication schemes utilizing
heparin-afnity chromatography (14); the use of
electroendosmotic elution techniques after prepara-
tive sodium dodecyl sulfate electrophoresis to achieve
nal purication to homogeneity (14); and the use of
recombinant DNA technologies to clone and express
several human BMPs/OPs (28, 35, 49, 50, 71).
Endochondral bone induction is the result of the
combinatorial action of BMPs/OPs and the comple-
mentary substratum that delivers the osteogenic
activity of the soluble molecular signal (28, 32, 35, 49,
50, 71). After initiation of the rst wave of bone dif-
ferentiation, including the clonal expansion of oste-
oprogenitor stem cells, the osteogenic cascade is
promoted and maintained by a variety of other
morphogens of the transforming growth factor-b
superfamily, including the transforming growth
factor-b isoforms per se. In the matrix-induced
endochondral bone formation, transforming growth
factor-b was detected from day 9 onwards after
heterotopic subcutaneous implantation in rodents
(3). An increasing concentration correlated with the
induction of angiogenesis and the calcication of
cartilage (3). Transforming growth factor-b was found
to be compartmentalized in the newly formed and
mineralized bone matrix as a mechanism for storage
of the latent or processed morphogen (3).
Pleiotropism of BMPs/OPs: from
bone induction to cementogenesis
and periodontal ligament
regeneration
More than 40 related proteins with BMP/OP-like
sequences and activities have been sequenced and
cloned. By default, each protein either puried to
75
BMPs and the induction of periodontal tissue regeneration
homogeneity from natural sources or cloned and
expressed by recombinant DNA technology induces
endochondral bone formation, by induction, in
heterotopic sites of a variety of animal models, inclu-
ding adult primates (28, 35). Highly puried naturally
derived BMPs/OPs, extracted and puried from
baboon and bovine bone matrices, induce complete
regeneration of calvarial defects in the adult baboon
Papio ursinus (35, 42, 43, 46, 52). The endogenous
mechanisms of bone repair and regeneration may
require the concerted action of several of the BMPs/
OPs present within the natural milieu of the bone
matrix. Complete regenerationmay be the result of the
sumof a plurality of BMP/OP activities, or the result of
synergistic interaction amongst the partially puried
BMPs/OPs from bone matrices (33, 35, 46, 49).
In addition to bone induction, BMPs/OPs are
expressed during early development and organo-
genesis in Drosophila melanogaster, nematodes and
Xenopus laevis, as well as in mammals, indicating
that BMPs/OPs and related members play critical
roles as soluble mediators of epithelialmesenchymal
interactions and inductive events unrelated to bone
induction (10, 12, 17, 35, 52). In situ hybridization,
immunolocalization and in vivo studies in mammals,
including human primates, have indicated that
fruit ies, frogs and fractures share common mor-
phogenetic and inductive mechanisms (i.e. the
pleiotropic cascade of activities of BMP/OP gene
products) (Fig. 2) (6, 28, 35). DPP and 60A gene
products in the fruit y D. melanogaster are homo-
logues of human BMPs/OPs, so much so that
heterotopic implantation of recombinant hDPP and
h60A in heterotopic sites of rodents induce endo-
chondral bone differentiation (57). Nature has had a
powerful lesson to teach: to initiate the emergence of
the skeleton and thus of the vertebrates, Nature has
found it easier to usurp gene products and amino
acid sequences already in existence more than a
billion years in the fruit y D. melanogaster rather
A B
C
D
Fig. 2. Bone induction in the nonhuman primate, Papio
ursinus, and in the primate, Homo sapiens, 30 (A, B) and
90 (C, D) days after implantation of highly puried oste-
ogenic fractions from baboon and bovine bone matrices,
respectively. (A) Mineralized bone is stained blue, sur-
faced by osteoid seams, 30 days after implantation of
280 lg of naturally derived osteogenic fractions (puried
from baboon bone matrices) in calvarial defect. (B) High-
power view showing osteoid seams populated by conti-
guous osteoblasts. (C) Newly formed mineralized bone,
surfaced by osteoid seams, 90 days after implantation of
highly puried bone-derived bovine bone morphogenetic
and osteogenic proteins (BMPs/OPs) in a mandibular
defect of a human patient. (D) High-power view showing
mineralized bone in blue, surfaced by osteoid seams.
(AD) Undecalcied sections, cut at 5 lm, stained free-
oating with Goldners trichrome. Original magnica-
tions: 35 (A), 125 (B), 35 (C) and 125 (D).
76
Ripamonti & Renton
than devise new gene products responsible for the
emergence of the skeleton. Specic amino acid vari-
ations yielded several homologous morphogenetic
proteins, all of which were endowed with the striking
prerogative of initiating bone formation by induction
sculpting the architecture of the skeleton, a unique
construct assembling the vertebrate animal (28, 35).
Ultimately, predictable bone regeneration in pre-
clinical and clinical contexts requires information
concerning the expression and cross-regulation of
gene products of the osteogenic proteins of the
transforming growth factor-b superfamily elicited by
a single application of a recombinant morphogen (31,
35, 50). Using the calvarium and the rectus abdominis
muscle of adult baboons as a model for tissue
induction and morphogenesis, a study investigated
the induction of bone morphogenesis by gamma-
irradiated hOP-1 delivered by gamma-irradiated bo-
vine-insoluble collagenous bone matrix, the hOP-1
osteogenic device, for bone induction in heterotopic
and orthotopic sites of the primate P. ursinus (34).
Tissue induction and morphogenesis were found to
be correlated to the expression patterns of OP-1,
collagen type IV, BMP-3 and transforming growth
factor-b1 mRNAs, elicited by increasing the dose of
single applications of the hOP-1 osteogenic devices
(0.0, 0.1, 0.5, and 2.5 mg hOP-1/g of matrix) applied
heterotopically in the rectus abdominis muscle and
orthotopically in 48 calvarial defects of 12 adult
baboons (34). Histology and histomorphometry
analyses performed on serial undecalcied sections
prepared from the specimens harvested on days 15,
30 and 90 showed that the 0.1, 0.5 and 2.5 mg hOP-1
of the hOP-1 osteogenic device induced bone for-
mation, culminating in complete calvarial regener-
ation by day 90. Expression of type IV collagen
mRNA, a marker of angiogenesis, was strongly
expressed in both heterotopic and orthotopic tissues.
High expression levels of OP-1 mRNA demonstrated
autoinduction of OP-1 mRNAs. Expression levels of
BMP-3 mRNA varied from tissues induced in
heterotopic vs. orthotopic sites with high expression
in rapidly forming heterotopic ossicles together with
high expression of type IV collagen mRNA. The
temporal and spatial expressions of transforming
growth factor-b1 mRNA indicate a specic temporal
transcriptional window during which expression of
transforming growth factor-b1 is mandatory for suc-
cessful and optimal osteogenesis (34). The induction
of bone by hOP-1 in P. ursinus develops as a mosaic
structure with distinct spatial and temporal patterns
of gene expression of members of the transforming
growth factor-b superfamily that singly, synergisti-
cally and synchronously initiate and maintain tissue
induction and morphogenesis (31, 35, 50, 52).
The expression of OP-1, type IV collagen, BMP-3
and transforming growth factor-b1 mRNAs by Nor-
thern blot analyses showed a temporal and spatial
pattern of expression, indicating progressing stages of
osteogenic differentiation during the initiation of
bone formation by the hOP-1 osteogenic device (34).
The continuous temporal and spatial high-expression
patterns of type IV collagen mRNA indicates the
critical role of hOP-1 in the induction of angiogenesis
(22). Angiogenesis is a prerequisite for osteogenesis;
continuous vascularization explains mechanistically
the sustained osteogenesis induced by the hOP-1
osteogenic device supported by prominent vascular
invasion (34). The biosynthesis and supramolecular
assembly of the perivascular extracellular matrix of
invading sprouting capillaries during the initiation
of bone formation will ultimately provide the extent
of regeneration of the treated calvarial defects and, in
addition, will play pivotal physiological roles by
sequestering both angiogenic and osteogenic pro-
teins (7, 2022, 49, 66).
To sculpt tissue morphogenesis, nature relies on
common (yet limited) molecular mechanisms tailored
to initiate the emergence of specialized tissues and
organs, including the periodontal tissues (28, 31, 35,
52). Natures parsimony in sculpting tissue constructs
is epitomized by the deployment of a restricted family
of molecularly different, but functionally homolog-
ous, proteins with minor variations in amino acid
motifs within highly conserved C-terminal regions, all
endowed with the striking prerogative of initiating
endochondral bone formation by induction in addi-
tion to specialized pleiotropic functions (Fig. 3) (28,
31, 35, 50, 52). We have learned that signaling net-
works deployed in both Drosophila and mammalian
development, including development of the limbs and
the emergence of osteogenesis and thus skeletogen-
esis, are also deployed in tooth morphogenesis (1, 10,
12, 17) during epithelialmesenchymal interactions in
tooth development (1, 10, 6163).
BMPs/OPs are involved in tooth morphogenesis at
different stages of development as temporally and
spatially connected events (Fig. 4) (1, 10, 12, 61, 62).
During the later developmental stages of tooth
morphogenesis, the induction of cementogenesis,
periodontal ligament and alveolar bone differenti-
ation, is regulated by the co-ordinated expression of
BMPs/OPs family members (Fig. 4) (1, 10, 12, 6163).
Tissue induction and morphogenesis of the complex
tissue morphologies of the periodontal tissues in
postnatal life recapitulate embryonic development.
77
The mosaicism of the expression of gene products
during embryonic development is recapitulated
postnatally by the exogenous application of highly
puried, naturally derived, BMPs/OPs and of single
recombinant osteogenic proteins (Fig. 5 and 6) (5, 31,
3335, 50).
A systematic analysis of the expression of six dif-
ferent Bmps in tooth morphogenesis has shown that
whilst the expression patterns of each Bmp is differ-
ent, there is co-distribution between specic family
members (1, 61, 62). Root morphogenesis is a clas-
sical example of epithelialmesenchymal interactions
(1, 61). In this context, the localization of BMP-3 and
OP-1 during morphogenesis of the mouse root (from
the developmental stages of mantle dentine depos-
ition) suggests that these proteins play a role during
cementogenesis and the assembly of a functionally
oriented periodontal ligament ber system (63). The
localization of BMP-2 in alveolar bone only, and of
BMP-3 and OP-1 in all three components of the
periodontium (Fig. 4), indicates that the morpho-
genesis of periodontal tissues may involve a com-
posite pattern of co-ordinated expression of different
signaling isoforms (63), each endowed with the
striking prerogative of initiating bone formation by
induction. The mosaicism of BMP/OP expression
during root morphogenesis indicates that optimal
therapeutic regeneration and tissue engineering may
entail binary combinations of osteogenic gene prod-
ucts based on recapitulation of embryonic develop-
ment (32, 33, 35, 51, 63).
Structureactivity prole and
therapeutic signicance of
redundancy
BMPs/OPs are involved in inductive events that
control pattern formation during morphogenesis in
A B
C D
Fig. 3. Pleiotropic activity of bone morphogenetic and
osteogenic proteins (BMPs/OPs), unrelated to bone
induction, in mammalian tissues. (A) In situ hybridization
localizes the messenger RNA of osteogenic protein-1
(OP-1) in the epidermis of an 18-day-old mouse pup. (B)
Immunolocalization of OP-1 in the cochlea of a 16-day-
old pup. (C) and (D) Immunolocalization of BMP-3 in the
murine cerebrum indicates that BMP-3 is neurotrophic
during the development and maintenance of the nervous
system. (C) Immunolocalization of BMP-3 between
cerebral white and gray matters of the developing brain of
16-day-old mouse pups. Arrows indicate delineation of
developing neurite axonal patterns within the cerebrum
by BMP-3 immunolocalization. (D) BMP-3 expression and
immunolocalization in the cytoplasm of Purkinjes cells
within the cortex of the cerebellum. Original magnica-
tions: 90 (A), 175 (B), 90 (C) and 375 (D).
78
Ripamonti & Renton
A B
C
D E F
Fig. 4. Immunolocalization and mosaicism of expression
of bone morphogenetic proteins (BMPs) during embry-
onic development and morphogenesis of murine perio-
dontal tissues. (A) Developing mandibular molar of a
13-day-old mouse pup, showing immunolocalization of
BMP-3 in the alveolar bone, periodontal ligament and
cementum. Arrows indicate expression of BMP-3 in the
inferior alveolar nerve. (B) BMP-3 expression in all three
components of the periodontal tissues during tooth mor-
phogenesis of a molar of an 11-day-old mouse pup. Note
BMP-3 immunolocalization in the periodontal ligament
bers uniting the alveolar bone to the newly forming ce-
mentum (arrow). (C) Developing root of a mandibular
molar in a 16-day-old mouse pup: strong expression of
osteogenic protein-1 (OP-1) is observed during cemento-
genesis in cementoblasts and cementoid matrix and in
developing periodontal ligament bers. (D) Developing
root of a mandibular molar in a 16-day-old mouse pup.
Strong localization of OP-1 during cementogenesis and
developing bers inserting into the newly deposited
cementoid. Conspicuous staining in predentine and
mantle dentine (arrow). (E) Developing root of a molar
tooth in a 140-day-old mouse pup: OP-1 signals strongly
in cementum and in the periodontal ligament space,
delineating the assemblage of the periodontal ligament
system together with cementogenesis. (F) Immunolocali-
zation of BMP-2 in the furcation area of a 16-day-old
mouse pup. BMP-2 signals in alveolar bone and preden-
tine. Note the lack of staining in the periodontal ligament
and cementum. Original magnications: 90 (A), 90 (B),
145 (C), 125 (D), 90 (E) and 75 (F).
79
A B C
D E F
Fig. 5. Tissue induction, morphogenesis and regeneration
as a recapitulation of embryonic development in furcation
defects of mandibular molars of the nonhuman primate,
Papio ursinus, implanted with naturally derived bone
morphogenetic and osteogenic proteins (BMPs/OPs),
extracted and puried from bovine bone matrices greater
than 70,000-fold and predominantly containing BMP-3
(30, 33, 56). (A) Newly formed Sharpeys bers coursing
from regenerated alveolar bone in blue inserting into
newly formed mineralized cementum 60 days after the
implantation of 250 lg of highly puried osteogenic
fractions. (B) High-power detail of the previous section,
showing the assemblage of the newly formed periodontal
ligament system inserting into mineralized cementum in
blue (arrows). (C) Periodontal tissue regeneration in
another furcation defect, treated with bovine BMP/OPs
delivered by allogeneic collagenous matrix as a carrier,
showing newly formed mineralized alveolar bone surfaced
by osteoid seams (arrows) and Sharpeys bers uniting the
newly formed bone to the regenerated cementum. (D) In
another specimen of a furcation defect treated with
naturally derived BMPs/OPs, angiogenesis and vascular
invasion is observed, with formation of cellular conden-
sations around the invading capillaries and osteoid-like
tissue deposition with foci of mineralization and the
induction of bone formation as a ripple-like cascade of
events centered around the invading capillaries during
angiogenesis as a prerequisite for osteogenesis. (E)
Deposition of cemental matrix, as cementoid yet to be
mineralized, on the planed dentinal surface after
implantation of 250 lg of naturally derived bovine BMPs/
OPs. White arrows indicate foci of mineralization within
the yet-to-be-mineralized cemental collagenic matrix.
Black arrows indicate generating Sharpeys bers from the
newly deposited cementoid. (F) Higher magnication of
the coronal area of (A) showing the generation of Shar-
peys bers (open arrows) and the mineralization of newly
formed cementoid. (AF) Undecalcied sections cut at
5 lm and stained free-oating with Goldners trichrome.
Original magnications: 75 (A), 125 (B), 75 (C), 175
(D), 175 (E) and 175 (F).
80
Ripamonti & Renton
such disparate tissues and organs as the kidney, eye,
nervous system, lung, skin, heart and teeth (Fig. 3
and 4) (1, 10, 12, 17, 6163). BMPs/OPs induce
de novo endochondral bone formation and act as
soluble signals of tissue morphogenesis, sculpting the
multicellular mineralized structures of the perio-
dontal tissues with functionally oriented periodontal
ligament bers inserted into newly formed cemen-
tum (Fig. 5 and 6) (35, 39, 44, 5052).
Amino acid sequence variations in the C-terminal
domain confer specialized and pleiotropic activities
to each isoform, the molecular basis of the structure
activity prole of each morphogenetic protein (32, 35,
5052). In addition, the strikingly pleiotropic effects
of each isoform are also conferred by the transduc-
tion of distinct signaling pathways by individual
Smad proteins (human homologues of mothers
against decapentaplegic) after activation of the
transmembrane serinethreonine kinase receptor
(31, 35, 5052). The presence of multiple forms of
BMPs/OPs has therapeutic signicance, and the
choice of a suitable isoform is a formidable challenge
for the practicing skeletal reconstructionist and
periodontologist alike (35, 38, 51, 52). Amino acid
sequence variations in the active C-terminal domain
of each morphogenetic protein confer specialized
activities to a BMP/OP isoform, and this is the
molecular basis that determines the structureactiv-
ity prole of single BMPs/OPs (i.e. specic amino
acid structure/sequences that govern specialized and
pleiotropic activities during tissue induction and
morphogenesis) (35, 45, 5052). This is the biological
signicance of redundancy and its therapeutic
implication rests on developing a structureactivity
prole amongst the members of the BMP/OP family
(i.e. to study in vivo the morphogenetic impulse of
each single and structurally different recombinant
hBMP/OP in primates only, in order to identify the
pleiotropic activities of different protein isoforms
based on specic amino acid sequences) (35, 5052).
Periodontal tissue regeneration by
naturally derived and recombinant
human BMPs/OPs in the
nonhuman primate P. ursinus
To induce the cascade of bone differentiation, the
soluble osteogenic molecular signals of the trans-
forming growth factor-b superfamily must be recon-
stituted with an insoluble signal or substratum that
triggers the bone differentiation cascade (28, 35, 36,
49, 50, 52, 54). Different BMPs/OPs, and combina-
tions thereof, have been implanted in furcation de-
fects of the mandibular rst and second molars of
adult baboon, P. ursinus, delivered by insoluble col-
lagenous bone matrices as a carrier (35, 39, 44, 51,
52).
Naturally derived BMPs/OPs, puried more than
50,000-fold with respect to the initial crude guanidi-
nium hydrochloride extract of bovine bone matrices,
induced cementum, periodontal ligament and
alveolar bone regeneration in surgically created class
II furcation defects of mandibular molars of P. ursi-
nus (Fig. 5) (44). Osteogenic fractions were prepared
by extracting bovine demineralized bone matrices
with 4 M guanidinium-hydrochloride and were puri-
ed greater than 70,000-fold by heparinSepharose,
hydroxyapatiteUltrogel and gel-ltration chroma-
tography on Sephacryl S-200 (44). Importantly, for
research experiments in preclinical and clinical con-
texts, far-reaching experiments by Sampath & Reddi
(55) have shown homology of mammalian bone
inductive proteins, and that bovine BMPs/OPs, in
conjunction with the allogeneic baboon collagenous
matrix, induce bone differentiation, comparable with
that of baboon BMPs/OPs, in extraskeletal sites of the
baboon (41).
Undecalcied sections of the treated furcation
defects showed the induction of cementogenesis as a
yet-to-be-mineralized collagenic material and the
synthesis of Sharpeys bers with foci of mineraliza-
tion within the newly formed cementoid (Fig. 5E,F)
(44). Sharpeys bers were shown to unite the newly
formed cementum to the regenerated mineralized
alveolar bone surfaced by osteoid seams populated
by contiguous osteoblasts. It is noteworthy that
Sharpeys bers were inserted perpendicularly into
the newly formed cementum covered by a thin layer
of cementoid (Fig. 5) (44).
The source(s) of responding cells that initiate
cementogenesis and periodontal ligament regener-
ation are still not well understood. Recent studies
have indicated that the periodontal ligament system
contains stem cells that have the potential to regen-
erate cementum and periodontal ligament in vivo
(58). Exogenous applications of selected BMPs/OPs
initiate cementogenesis and regulate the assembly of
a functionally oriented periodontal ligament system
(44) by transforming specic stem cells, capable of
differentiation, towards cementoblasts and osteo-
blasts (44, 58).
In class II furcation defects of mandibular molars
of P. ursinus, relatively low doses of hOP-1 (100
and 500 lg/g of xenogeneic bovine collagenous
bone matrix as a carrier) induced preferentially
81
cementogenesis (45), indicating that BMPs/OPs have
multiple and pleiotropic functions in vivo that are not
limited to endochondral bone formation by induc-
tion and that there is a structureactivity prole
amongst BMPs/OPs in controlling tissue morpho-
genesis and regeneration (30, 33, 35, 45, 51). hOP-1,
A
B
C
D
E F
82
Ripamonti & Renton
when in contact with dentine extracellular matrix, is
preferentially cementogenic, with limited, if any,
alveolar bone induction in short-term experiments in
P. ursinus at relatively low doses of the recombinant
morphogen (Fig. 6) (30, 45). On the other hand, in
long-term experiments in P. ursinus, using higher
doses of the hOP-1 osteogenic device, regeneration of
cementum was observed, with coursing Sharpeys
bers uniting the cementum to the newly induced
alveolar bone (Fig. 6C,D) (48).
Previous studies in P. ursinus assessed healing on
root surfaces that had been surgically exposed and
planed (39, 44). While the regeneration of the con-
nective tissue attachment and alveolar bone seemed
to be independent of the character of the root surface
if the exposed root had been adequately decontam-
inated (19), more challenging was the demonstration
of cementogenesis and alveolar bone regeneration in
periodontally induced furcation defects with root
surfaces exposed for a relatively long time to perio-
dontal pathogens. Although nonhuman primates,
such as P. ursinus, experience naturally occurring
site-specic gingivitis (48), the contention still exists
that novel regenerative procedures should be tested
in animal models with root surfaces exposed by dis-
ease (68). A pathogenic human strain of Porphyro-
monas gingivalis was inoculated into the furcations
areas of the rst and second mandibular molars of
four adult P. ursinus, twice a month for 12 months,
and chronic periodontitis was induced in all four
animals, as assessed by probing periodontal pocket
depths, intra-oral radiographs, and microbiological
analyses that conrmed the presence of P. gingivalis
(48). Two months after scaling, root planing and a
plaque-control regimen, with clinical resolution of
gingivitis, mucoperiosteal aps were elevated to ex-
pose class II furcation defects of the affected man-
dibular molars lled with granulation tissue (48).
After root planing and debridement, 12 furcation
defects were implanted with 0.5 or 2.5 mg of gamma-
irradiated hOP-1 per gram of xenogeneic bovine-
insoluble collagenous bone matrix as a carrier (48).
Serial undecalcied sections, prepared 6 months after
surgery, showed regeneration of alveolar bone and the
induction of cementogenesis, with Sharpeys bers
uniting the regenerated bone to the newly formed
cementum (Fig. 6C). Radiographic analysis showed
substantial alveolar bone regeneration, even with the
lower-dose hOP-1 osteogenic device (Fig. 6D). Doses
of 2.5 mg of the hOP-1 osteogenic device induce
complete regeneration and restitutio ad integrum of
the treated furcation defects (Fig. 6C) (48).
The induction of cementogenesis is clearly a crit-
ical pleiotropic function of hOP-1 in both primate
and canine models (30, 45, 48). The higher hOP-1
doses tested in periodontally induced furcation
defects of P. ursinus might have accounted for the
observed histological differences between the long-
term and the short-term experiments (33, 45, 48).
Moreover, the longer observation period in P. ursi-
nus, of up to 6 months (compared with the previous
short-term experiments), might have also been
critical in determining the spatially correct morpho-
genesis of the periodontal tissues within the perio-
dontally induced furcation defects (48).
Synchronous, but spatially distinct, OP-1 and
BMP-2 expression during murine root formation
points to specic functions of OP-1 and BMP-2 in
periodontal tissue morphogenesis and thus regener-
ation in postnatal life (6, 47, 48). The co-localization
ndings of OP-1 and BMP-2 during tooth morpho-
genesis (63) has suggested that co-administration of
OP-1 and BMP-2 in recombinant form would result
in synergistic tissue morphogenesis as a recapitula-
tion of memory of developmental events in the em-
bryo (47, 63).
Fig. 6. Induction of cementogenesis and periodontal
tissue regeneration by recombinant human osteogenic
protein-1 (hOP-1) and recombinant human bone
morphogenic protein 2 (hBMP-2). (A) Composite photo-
micrograph of furcation defects treated with 500 lg of
recombinant hOP-1 in surgically created furcation defects
of Papio ursinus. Arrows point to the foci of mineraliza-
tion within the newly formed, yet-to-be-mineralized
cementum. (B) Mineralized tissue in blue in the furcal
area (open arrows) of a defect treated with 500 lg of hOP-
1, interpreted as cementum deposition with scattered
remnants of collagenous matrix as the carrier (black
arrows). (C) Higher doses of the hOP-1 osteogenic device
(i.e. 2.5 mg of hOP-1 per gram of gamma-irradiated
bovine collagenous matrix as a carrier) show complete
regeneration of the inammatory-induced furcation
defect and cementum regeneration in the most coranal
furcal area of the exposed furcation. (D) Peri-apical radi-
ographs 6 months after implantation of the 0.5-mg hOP-1
device per gram of gamma-irradiated bovine collagenous
matrix as a carrier. Arrows indicate the supracrestal gain
of mineralized bone from the remaining alveolar bone at
the time of surgery. (E) Periodontal tissue regeneration
after implantation of 100 lg of hBMP-2 in a surgically
created furcation defect in P. ursinus. Prominent osteoid
deposition (black arrows) with limited cementogenesis
(white arrow). (F) Prominent osteogenesis (black arrows)
and superior cementogenesis (white arrow) after binary
applications of 100 lg of hOP-1 and BMP-2. (AC and EF)
Undecalcied sections, cut at 5 lm, and stained free-
oating with Goldners trichrome; original magnications
125, 90, 10, 75 and 75.
83
Twelve furcation defects, prepared in the rst and
second mandibular molars of adult P. ursinus, were
used to assess whether qualitative periodontal tissue
regeneration could be enhanced and tissue mor-
phogenesis modied by binary applications of hOP-1
and hBMP-2. Doses of recombinant proteins were
100 lg of each protein per gram of insoluble colla-
genous bone matrix as carrier. Approximately 200 mg
of carrier matrix was used per furcation defect.
Undecalcied sections were cut for histological and
histomorphometrical analyses on day 60 after heal-
ing. hOP-1-treated-furcation defects showed sub-
stantial cementogenesis, with scattered remnants of
the collagenous carrier (47). hBMP-2 applied singly
induced greater amounts of mineralized bone and
osteoid when compared to hOP-1 alone (Fig. 6F).
Binary applications of hOP-1 and hBMP-2 showed
superior cementogenesis, as induced by hOP-1
(Fig. 6G) (47).
The results of the above studies, which are the rst
to attempt to address the structureactivity relation-
ship amongst BMPs/OPs family members, indicate
that tissue morphogenesis induced by hOP-1 and
hBMP-2 is qualitatively different when the morpho-
gens are applied singly (47). hOP-1 induces sub-
stantial cementogenesis; hBMP-2-treated defects, on
the other hand, showed limited cementum forma-
tion but a temporal enhancement of alveolar bone
regeneration and remodeling (47). The results
achieved by hOP-1 and hBMP-2 in periodontal-
regenerative procedures, indicate that hOP-1 and
hBMP-2, as tested to date in both canine and primate
models, possess a structureactivity prole, as shown
by the morphological detail of healing and tissue
regeneration. While hBMP-2 was found to be more
osteogenic than cementogenic in both beagle dogs
and baboons (5, 8, 47, 59, 69), hOP-1 clearly modu-
lates the expression of the cementogenic phenotype
and the induction of cementogenesis in both animal
models (8, 30, 45, 48), and also on root surfaces
exposed by disease (48).
Although the majority of animal studies involved
the use of hOP-1 and hBMP-2 in a variety of clinical
settings and animal models (17, 35, 39, 51, 52),
hBMP-6 has also been investigated in a periodontal
fenestration defect model in rodents (11). The study
indicated that hBMP-6 induced signicantly more
new cementum formation as opposed to control
fenestration defects (11). hBMP-12 has also become
the focus of attention for periodontal regenerative
studies (70). Wikesjo et al. (69) evaluated hBMP-12
for periodontal tissue regeneration, particularly
periodontal ligament formation. hBMP-12 and
hBMP-2 were implanted on absorbable collagen
sponges in periodontal defects and the results were
compared after 60 days of healing (70). Greater bone
regeneration was observed in implants treated with
hBMP-2, but ankylosis was noted (70). Defects trea-
ted with hBMP-12 showed less bone regeneration,
but exhibited a functionally oriented periodontal
ligament system inserting into newly formed
cementum (70). In a tooth replantation study using
hBMP-12, Sorensen et al. noted that the topical
application of hBMP-12 to dentine that had been
previously denuded of periodontal ligament, failed to
have any effect on re-establishing a new periodontal
ligament apparatus (60).
Perspectives in periodontal tissue
engineering by BMPs
Periodontal tissue engineering foremost entails the
induction of cementogenesis and the genesis of
Sharpeys bers inserting into newly formed cemen-
tum (2, 17, 38, 39, 52). Several studies have high-
lighted that partially puried and puried extracts of
cementum contain a mitogenic growth factor as a
distinct molecular species (16, 18, 72). The presence
of mitogenic growth factors within the cemental
matrix indicates that cementum has the potential to
regulate the adjacent periodontal ligament space
(72). The extracellular matrix of the cementum may
provide a framework for the regeneration of the var-
ious tissue components of the periodontal ligament
and, in addition, may play important physiological
roles in sequestration of morphogenetic factors in-
volved in repair, regeneration and remodeling (20, 21,
25, 37, 66). It will be of importance to bioassay
cemental extracts after 6 M guanidinium or urea dis-
sociative extraction followed by heparinSepharose
afnity chromatography. Cemental extracts puried
by afnity chromatography may retain osteogenic
proteins embedded within the matrix as a memory of
developmental events, as highlighted by demineral-
ized dentine matrix of P. ursinus with osteogenic
activity in the rectus abdominis (15).
If cementum does induce endochondral bone dif-
ferentiation, it would be tempting to suggest that
bone induction modulated by cemental matrices may
be the result of a slow release of embryonic remnants
of osteogenic proteins that were required and
deployed during cementogenesis. The capacity of
mammalian BMPs/OPs to initiate a programmed
cellular cascade that results in the induction of bone is
a functionally conserved process utilized in embry-
84
Ripamonti & Renton
onic development, recapitulated in postfetal osteo-
genesis and can be re-exploited for the therapeutic
initiation of periodontal tissue regeneration. There
are several challenges that provide opportunities
to gain mechanistic insights into the regulation of
periodontal tissue regeneration; a challenge of great
molecular importance is the biological signicance of
apparent redundancy. The presence of the structure
activity prole amongst soluble osteogenic molecular
signals indicates a therapeutic signicance in clinical
contexts (35). Signicant advances in periodontal
tissue regeneration may be expected if ongoing and
future research is tailored to provide further mech-
anistic insights into the relevance of apparent
redundancy and the structureactivity prole of the
recombinant human osteogenic proteins.
Nonetheless, at the beginning of the 21st century, a
soluble osteogenic and recombinant molecular sig-
nal, when combined with an insoluble signal, triggers
periodontal tissue regeneration with the induction of
cementogenesis and insertion of Sharpeys bers,
essential ingredients to engineer periodontal tissue
regeneration (35, 48).
Acknowledgments
This work is supported by the South African Medical
Research Council, the University of the Wit-
watersrand, Johannesburg and the National Research
Foundation. For the molecular and surgical work we
thank Laura Yates, Karolina Kuun, Lenthsa Nathaniel
Ramoshebi and Manolis Heliotis. For the unique
undecalcied histological sections, we thank Barbara
van den Heever and June Teare. We thank Jamie
Kemler and David Rueger of Stryker Biotech for the
scientic collaboration and the supply of human
recombinant osteogenic protein-1 and bone mor-
phogenetic protein-2. Fishwicks Digital and Gilly
Haagensen for capturing and compiling digital ima-
ges of the undecalcied sections. Many thanks to
Daniella Bella, Paige, Ryan, Caitlyn and Kathleen for
understanding and encouragement.
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Molecular aspects of tissue
engineering in the dental eld
TANI DA SRI SUWAN, DANI EL J. TI LKORN, JEREMY L. WI LSON,
WAYNE A. MORRI SON, HAROLD M. MESSER, ERI K W. THOMPSON
& KEREN M. ABBERTON
Tissue engineering is a multidisciplinary eld with
the potential to replace tissues lost as a result of
trauma, cancer surgery, or organ dysfunction. The
successful production, integration, and maintenance
of any tissue-engineered product are a result of nu-
merous molecular interactions inside and outside the
cell. We consider the essential elements for success-
ful tissue engineering to be a matrix scaffold, space,
cells, and vasculature, each of which has a signicant
and distinct molecular underpinning (Fig. 1). Our
approach capitalizes on these elements. Originally
developed in the rat, our chamber model (Fig. 2) in-
volves the placement of an arteriovenous loop (the
vascular supply) in a polycarbonate chamber (pro-
tected space) with the addition of cells and an
extracellular matrix such as Matrigel or endogenous
brin (34, 153, 246, 247). This model has also been
extended to the rabbit and pig (J. Dolderer, M.
Findlay, W. Morrison, manuscript in preparation),
and has been modied for the mouse to grow adipose
tissue and islet cells (33, 114, 122) (Fig. 3).
Regenerative dentistry, including periodontics,
endodontics and maxillofacial surgery, is a new eld
that seeks to apply the concepts of tissue engineering
to the management of lost oral tissues using various
types of stem cells, growth factors, and scaffolds. In
the eld of periodontics, tissue engineering tech-
niques have already been applied to reconstruct the
damaged periodontal organ (12, 162, 166, 181), with
acceptable results reported in clinical practice (147,
175, 220, 237). Tissue engineering strategies are also
being applied in an attempt to regenerate the dentin
pulp complex. Stem cells, growth factors, and differ-
ent physical matrices have been used in conjunction
with human or animal dental pulp cells both in vitro
and in vivo (67, 66, 68, 105, 165, 258). The promising
results reported in animal models by several
researchers (97, 164, 203, 232), together with studies
demonstrating the regenerative capacity of human
dental pulp (11, 108), are positive steps toward suc-
cessful regenerative treatment in humans.
Tissue engineering is generally considered to
consist of three key components: (i) stem/progenitor
cells; (ii) signaling molecules; and (iii) a scaffold or
extracellular matrix. In addition, we believe that
growing tissues require an adequate vascular supply
to ensure viability and an unencumbered physical
space into which the growing tissue can expand.
Further research is needed to improve our under-
standing of the molecular aspects of cell behavior
during developmental and regenerative processes so
that engineering of tissues and organs may become
a clinical reality. In this chapter we will review
current concepts of tissue engineering at a
molecular level, including extracellular matrix and
its interactions, cells and intracellular events, and
also the importance of mechanical factors and the
vasculature in regulating and supporting tissue
growth.
Cells in dental tissue engineering:
odontoblast, cementoblast, and
osteoblast
The dental structure is a complex composition of
different specialized tissue and cell types consisting
of dentin-producing odontoblasts, ameloblasts and
periodontal structures such as cementum, perio-
dontal ligament, gingiva, and alveolar bone. These
structures are often affected by infectious disease
for example, periodontitis and caries as well as
trauma-causing pain and impaired function, which if
not treated adequately can result in early tooth loss.
At present the therapy widely relies on the use of
88
PERIODONTOLOGY 2000
implants, capping procedures, or root canal llings.
Tissue engineering holds the promise of one day
offering the true replacement of tooth structures.
Advanced knowledge of the molecular and cellular
aspects in the dental eld is essential for possible
application in this area.
A number of strategies have been developed to
support the healing process, such as:
inductive dental tissue engineering strategies
that activate and stimulate endogenous cells by
the incorporation of demineralized dentin frag-
ments.
use of various extracellular matrix scaffolds
(bronectin, collagen, brin, hydroxyapatite, etc).
the addition of cytokines such as bone morpho-
genic proteins and transforming growth factor-b
(158, 213, 259). Cell-based treatment has emerged
experimentally to be another promising option
(224, 251).
It is well established that hemopoietic stem cells
can differentiate into virtually all the cell types of the
blood cell lineage. Bone marrow transplantation and
stem cell transplantation for the treatment of
hemopoietic diseases have long been part of medical
ECM
Adjacent cell
Blood vessels
Cell-ECM
interaction
Cell-cell
interaction
Cell-vasculature
interaction
Cell differentiation,
proliferation and
other consequences
Fig. 1. An overview of cell and tissue growth regulators.
Various types of interactions affect cell proliferation,
differentiation, and other consequences. For example
cellextracellular matrix (ECM), cellcell, and cellvascu-
lature interactions are depicted. Unencumbered space
and adequate vascular supply also play a signicant role
in cell and tissue expansion leading to successful tissue
growth.
89
Molecular aspects of tissue engineering
practice (130, 179). A second population of stem cells,
well distinguished from the hemopoietic stem cell
pool, is also found within the bone marrow. The
presence of cluster-forming cells within the bone
marrow was rst described by Friedenstein (61).
These cells are able to differentiate into various tissue
lineages, and offer great potential for regenerative
procedures. The mesenchymal broblast-like cells
were separated from hemopoietic cells using their
property of adherence to tissue culture plastic. A
subpopulation of these mesenchymal bone marrow-
derived cells shares some of the important stem cell
characteristics, such as their ability to renew them-
selves, and their potential to differentiate in various
tissue lineages (20, 61, 126, 180). Further isolation of
these pluripotent stem cells according to their spe-
cic surface markers increased their harvest ef-
ciency and overall utility (68). Considerable ongoing
effort is being applied to increase the knowledge
concerning the specic properties of these cells, and
the mechanisms involved in their proliferation and
differentiation processes (67, 248). Stem cell popu-
lations have now been identied in many organs and
tissues in addition to teeth and bone marrow, such as
adipose tissue and brain. It has been well documen-
ted that stem cells and progenitor cells contribute to
various regenerative processes such as wound heal-
ing, angiogenesis or healing of bone fractures (66,
126, 225, 226). The local environment is thought to
play an important and specic role in the commit-
ment and differentiation of mesenchyme-derived
stem cells (63, 191). Several studies have investigated
their potential for use in the regeneration of cranio-
facial defects (1, 110, 126, 187, 198). For example, the
experiment by Nakahara et al. (163) demonstrated
improved regeneration of periodontal fenestration
defects in dogs after implantation of collagen sponge
scaffolds seeded with alkaline phosphatase-positive
periodontal ligament-derived cells, conrming their
potential in dental tissue engineering.
Unlike bone, dentin and cementum have no
physiological turnover. Indeed it has been indicated
that these structures have only a limited reparative
capacity to form tertiary dentin and new cementum,
Femoral vessels
Transparent
chamber
Venous graft
Artery
Vein
Hole of insertion
of vessel loop
Femoral
vessels
Leg
Fig. 2. Diagrammatic representation of arteriovenous
shunt loop model in the rat (34, 153). The femoral artery is
anastomosed to a femoral vein graft (from the contralat-
eral side) and thence to the femoral vein, to form the
vascular loop. A polycarbonate chamber with a hole is
used for placing the arteriovenous shunt loop.
90
Srisuwan et al.
as seen after orthodontic tooth movement (257).
These regenerations were thought to involve locally
derived uncommitted cell populations (149, 151).
Recently, however, stem cells have been identied
and isolated from the periodontal ligament and
dental pulp. Their cluster-forming and differentiation
abilities, as well as their cell surface markers, were
described by Gronthos and co-workers in 2005 (223,
227), and stem cells isolated from periodontal liga-
ments have been shown to form cementum-like
mineralized structures in vitro. Whether cemento-
cytes and osteoblasts share the same ancestor cells is
still uncertain (70, 71, 225); however, certain differ-
ences have been reported, including differing degrees
of organization of mineralized cell products and dif-
ferences in their alkaline phosphatase activity (23).
The dental pulp stem cells represent a clonogenic
and highly proliferative cell population generating
densely calcied nodules in vitro. After transplanta-
tion in vivo a dentin-like and dentin sialo-phospho-
protein-rich mineralization product was observed.
Unlike bone marrow stromal cells, dental pulp
stem cells do not give rise to hemopoietic precursor
cells (66).
Apoptosis and necrosis
Cell death occurs via one of two mechanisms, nec-
rosis or apoptosis. Necrosis is a pathological mech-
anism by which irreversibly damaged cells undergo
cell death and is a common diagnosis in dental pulp
disease. The changes in cells undergoing necrosis are
mitochondrial swelling and deposition of granular
calcium, followed by the loss of cellular membrane
integrity and lysis leading to the release of materials
such as proteases which may affect adjacent cells
causing an inammatory response (193, 236, 272,
273). In contrast, apoptosis or programmed cell death
can be observed under both physiological and
pathological conditions and plays an important role
in the process of morphogenesis and homeostasis.
Apoptosis is characterized by distinct morphological
changes. The apoptotic cell shrinks and a loss of cell
cell junctions results in detachment from the adja-
cent cells. The cytoplasm and the nucleus with its
chromatin condense, and the plasma membrane
forms so-called pseudopods, referred to as blebbing.
The apoptotic cell is fragmented leaving behind
Femoral vessels
Fat pad
Chamber
Leg
Femoral
vessels
Epigastric pedicle
Epigastric
vessels
Fig. 3. Diagrammatic representation of the ow-through loop pedicle tissue engineering chamber in the mouse (43).
91
fragments known as apoptotic bodies. Finally these
apoptotic bodies are phagocytosed by either macro-
phages or adjacent cells (115, 236, 272, 273). Apop-
tosis is essential for appropriate development, for
example in the formation of glandular ducts and re-
sorption of branchial arches, and is a key process in
oral development (81, 260). Under pathological con-
ditions it may contribute to the progression of oral
disease, periradicular lesion development, inam-
mation and immune responses, and wound healing
(10, 54, 98, 177, 193).
During apoptosis an imbalance in death-suppres-
sing and death-inducing signals activates phylo-
genetic cell death. Phylogenetic cell death plays a role
in the loss of vestigial structures for example in the
suppression of vestigial tooth buds (185, 186, 264)
during shaping of structures (morphogenetic cell
death), in the shaping of primordial of functional
teeth (116, 131, 185, 260, 264), and during cell dif-
ferentiation (histogenetic cell death) (26, 29, 42, 109,
143, 159, 192, 228, 270, 286). Activators of the phy-
logenetic cell death pathway include bacteria, dental
extracellular matrix changes, Ca
2+
levels, hormones,
and orthodontic movement (18, 52, 62, 111, 135, 142,
160, 195, 268, 269). The extrinsic pathway activates
the caspase cascade after the binding of appropriate
ligands to death receptors leading to apoptosis. These
ligands belong to the family of tumor necrosis fac-
tors, Fas and tumor necrosis factor-a being two
examples (9, 88). Other apoptotic stimulators include
external signals and DNA damage, causing mitoch-
ondrial stress such as cytochrome c which reacts with
apoptotic protease activating factor 1 (Apaf-1) which
is then released from the mitochondria. Agonists like
BAX and antagonists like B-cell lymphoma/leukemia-
2 (bcl-2) regulate this process.
One family member (CED-9/bcl-2) is localized or
associated with intracellular membranes of mito-
chondria, endoplasmic reticulum, and nuclei, and
was found to block apoptosis (201). It was proposed
that ced-9/bcl-2 might act through the regulation of
the redox system of cells and prevent cell death as a
result of oxidizing agents (125, 283). The overex-
pression of ced-3/ice was shown to cause apoptosis
(155, 280); ced-3/ice is a cysteine protease which
cleaves motifs possessing aspartic acid. Ice is the
rst-named member of the caspase family (144, 252),
and other proteases belonging to the same family
have been identied (ICH-I/Nedd-2, cpp 32b/yama,
Tx/ICH-2, Mch). Overexpression of any of these
seems to cause apoptosis in various cell types (144).
Both apoptotic pathways join with the activation of
caspase-3, resulting in complete cell destruction and
phagocytosis. Inhibitors of apoptosis proteins were
further identied as antagonists of the caspase-3
activation. These proteins in turn may be counter-
acted by Smac/DIABLO protein from mitochondria
(88, 172).
Structure and roles of the
extracellular matrix in tissue
engineering
The extracellular matrix is a mixture of proteins
including collagens, proteoglycans, and laminins
forming an elastic network surrounding most cells
and tissue structures (77, 208). The composition,
organization, and distribution of the extracellular
matrix is diverse, depending on tissue types and
developmental stage (37, 99, 129). The extracellular
matrix consists of proteins and glycoproteins such as
collagens, laminins, bronectin, and proteoglycans
and also the polysaccharide hyaluronic acid (77, 208).
Various growth factors and cytokines are conjugated
to the extracellular matrix, such as broblast growth
factor-2, interleukin-6, platelet-derived growth factor,
and transforming growth factor-b1, to name a few
(45, 55, 92, 218). This co-localization acts as a storage
pool of growth factors and may reduce growth factor
degradation (56), protecting them from the local
microenvironment, while facilitating the presentation
of the growth factors to cell surface receptors (205).
Several studies have reported on roles for the
extracellular matrix in cell proliferation, migration,
differentiation, and apoptosis through interaction
with cells and cell receptors (24, 51, 85, 103, 189, 190).
Bissell et al. (21) and others (24, 103) have presented
a model of dynamic reciprocity between the extra-
cellular matrix and the intracellular cytoskeleton
(Fig. 4). The extracellular matrix binds to cell surface
receptors, triggering changes in gene expression in
the nucleus resulting in feedback mechanisms, which
in turn modulate the extracellular matrix. Hood et al.
(95) proposed an area code hypothesis, the presence
of a recognition system that guides cell positioning.
Ruoslahti and co-workers studied area code mole-
cules in cancer, reporting the characteristics of
bronectin which correlated to cell attachment (51,
189, 190). Cell adhesion is mediated by the interac-
tions between the cells and extracellular matrix pro-
teins such as bronectin, vitronectin, laminin, and
collagen, initiating the expression of specic genes in
its nucleus (17, 239) and the subsequent events that
play vital roles in embryonic morphogenesis (171,
92
Srisuwan et al.
215). These interactions are mediated by cell surface
receptors (integrins) and specic extracellular matrix
protein motifs such as the RGD (arginine-glycine-
aspartate) domains in bronectin and vitronectin
(188), the YIGSR domain in laminin (2, 3, 176, 261),
and the GER domain in type I collagen (121). Inte-
grins are a family of heterodimeric transmembrane
proteins composed of a- and b-subunits (197). Cur-
rently there are 24 known integrins (100) with dif-
fering a- and b-subunit arrangements determining
the ligand-binding specicities and responses in
intracellular signal transduction. Horwitz et al. (96)
propose that once cell binding to the extracellular
matrix occurs through cell surface receptors, the
signal is transmitted by direct structural interaction
between the integrin and the cells internal cytoske-
leton. These events will upregulate gene expression,
which affects cell growth and differentiation (Fig. 4).
An alternative theory proposes that the integrin
receptor ties directly into the biochemical signaling
pathway, with several studies reporting the increas-
ing of tyrosine phosphorylation after integrin-ligand
binding (72, 124). Other cell surface receptors include
syndecan (19), CD44, thrombomodulin (82), laminin-
binding protein (150), CD36 (65), and matrikines.
These have a direct effect on intrinsic tyrosine kinase
activity affecting proliferation, migration, differenti-
ation, and dedifferentiation (230, 253, 266).
The extracellular matrix undergoes constant re-
modeling during development and during patholo-
gical states such as inammation, wound healing,
and cancer. The characteristics of each remodeling
process differ depending on the cause. Nevertheless,
several key processes are common to most etiologies
including the synthesis of and proteolytic breakdown
of the extracellular matrix, where matrix metallo-
proteinases play an essential role. Matrix metallo-
proteinases are a family of metalloendopeptidases
(200) rst studied in relation to tadpole tail dissolu-
tion (69). Several subclasses of matrix metallopro-
Cell surface
receptor
ECM
Receptor-ligand
binding
cytoskeleton
Gene
expression
Growth factors,
cytokines
Storage
Pool
Modulation
of ECM
Embryonic
morphogenesis
Feedback
to ECM
Dynamic
reciprocity
ECM
intracellular
?
Receptor-GF
binding
Presenting
Fig. 4. Interactions between extracellular matrix (ECM)
and cell surface receptors (integrin or non-integrin) play
important roles in embryonic morphogenesis (171, 215).
There are several pathways, only some of which have been
discovered, that are activated after cell surface receptors
bind to specic ligands in the extracellular matrix and/or
to growth factors. Specic gene expression in the cell
nucleus after these mechanisms leads to cell differenti-
ation and morphogenesis (96). However, the changes in
gene expression triggered in the nucleus also result in
feedback mechanisms which in turn modulate the extra-
cellular matrix a process referred to as dynamic reci-
procity (21, 24).
93
teinases have been identied, some situated in the
cell membrane while others are secreted. Most matrix
metalloproteinases are expressed at low levels, and
are regulated by cytokines, growth factors, and
physical cellular interaction (235). They are respon-
sible for the breakdown and remodeling of extracel-
lular matrix proteins which affect cell migration and
behavior.
For tissue engineering purposes, the extracellular
matrix is required to act as a scaffold for cell
attachment and to modulate cell proliferation and
differentiation. The ideal scaffold for generating new
tissue should also be biodegradable and non-toxic,
with two classes of extracellular matrix materials
used in tissue engineering natural and synthetic.
Naturally derived materials such as collagen and
Matrigel have some advantages over synthetic scaf-
folds. These materials are biodegradable and possess
known cell-binding sites. Potential disadvantages
include the level of immunogenicity, the speed of
degradation, and a perceived limited ability to tailor
native matrix for specic properties.
Matrigel, a well-known extract from the Engel-
brethHolmSwarm sarcoma that is rich in basement
membrane components (120), is widely used in basic
science for the study of cellmatrix interactions and
increasingly in tissue engineering, particularly in
areas such as nerves (169), adipose tissue (33), and
skeletal muscle (194). Collagens, which are large
proteins synthesized by several cell types (209), are
used in various formats such as gels, meshes, and
sponges or in combination with slow-release gelatin
microspheres as cell scaffolds for the engineering of
tissues such as adipose tissue (119), blood vessels
(86), and skin (73). The mechanical strength of col-
lagen scaffolds and the rate of absorption have been
of concern (209, 267), resulting in the use of cross-
linking agents to alter the biomedical, thermal, and
mechanical properties of collagen (267). Other
extracellular matrices used as scaffolds include brin
and brinogen (33, 93, 278). A recent study focusing
on the development of brin-based matrices repor-
ted that a modied brin/L1Ig6 matrix could induce
angiogenesis activity in human umbilical vein
endothelial cells in vitro (78). Studies looking at the
addition of bronectin to tissue engineering scaffolds
suggest that bronectin may be critical for scaffold
vascularization (25, 265).
Semi-synthetic and synthetic scaffolds can be made
to mimic the natural properties of the extracellular
matrix, but tailored to specic tissue outcomes with
specic mechanical properties such as strength, pore
size, and stability. Additional factors can be added to
induce tissue in-growth. The commonest synthetic
matrices are various degradable synthetic polymers
and the ceramic hydroxyapatite. Studies have been
conducted into improving synthetic matrices by
integrating integrin-binding oligopeptides (211, 271)
or oligopeptide metalloproteinase-cleavage sequences
(139) to regulate cell behavior through receptor-
mediated adhesion and growth factors (79, 140). Zisch
et al. (285) have produced modied hydrogels integ-
rating vascular endothelial growth factor-121 and
vascular endothelial growth factor-165 into the matrix
to develop a scaffold for new blood vessel formation.
Among these synthetic polymers, polyesters are the
most popular for use in tissue engineering because
they are easily degradable and the rate of degradation
can be adjusted (209).
Synthetics such as poly(glycolic acid), poly(lactic
acid) or poly(lactic acid)/poly(glycolic acid) and their
copolymers, poly(p-dioxanone) and copolymers tri-
methylene carbonate have been used in clinical
applications, as well as hydroxyapatite, calcium sul-
fate, and tricalcium phosphate, some of which have
already proven to be osteoconductive (145, 219, 245).
Bioactive glass, a synthetic scaffold based on a silicon
network structure, has the ability to bond with living
bone and form active hydroxycarbonate apatite. This
is chemically similar to inorganic bone matter and
has been used for bone repair and regeneration (64,
240, 263, 275).
The extracellular matrix in
dentistry
Many extracellular matrix proteins are involved in
tooth development. Type IV collagen, bronectin,
laminin, and nidogen are all expressed in the base-
ment membrane of the murine tooth germ layer (214,
217, 249, 250), with tenascin, laminin, and bronectin
also expressed in the odontoblastic layer (59, 138,
281). Tabata et al. (242) studied the effects of bro-
nectin on ameloblast cells in vitro, demonstrating
that it accelerated ameloblast differentiation. The
interactions between various types of extracellular
matrix, dental pulp broblasts, and ameloblasts have
been studied for decades, but the exact mechanisms
of each interaction are not yet determined. There is
strong evidence that they are integrin-related, with
Mizuno et al. (156) showing that the effects of colla-
gen I on dentin matrix protein-1 and osteocalcin gene
expression in rat dental pulp cells are mediated by
integrins. Fibronectin and laminin are also believed
94
Srisuwan et al.
to affect dental pulp cells during tooth development,
inuencing cell adhesion and tissue architecture via
integrin binding. Zhu et al. (284) showed that b
1
in-
tegrins played a role in dental pulp cell adhesion to
laminin but not bronectin, using monoclonal anti-
body blocking studies. Fibronectin and laminin have
also been shown to enhance gingival cell attachment
to the surfaces of dental implants (47), while bro-
nectin is proposed to mediate cytoskeletal re-
organization during odontoblast polarization (258).
Laminin is a potent neurite mitogen and has been
found in cultured tooth pulp broblasts, in particular
the subunits laminin a
1
, a
2
, a
4
, a
5
, b
1
, and a1, with the
laminin-binding integrins a
3
, a
6
, b
1
, and b
4
chain
expressed in the trigeminal nerve (60). When trige-
minal neurons were cultured on laminins 2 (a
2
b
1
c
1
)
and 8 (a
4
b
1
c
1
), strong neurite outgrowth was
observed (60).
Reelin, a large extracellular matrix glycoprotein, has
been associated with human odontoblasts in vivo and
in vitro (146). When trigeminal ganglion and odon-
toblasts were co-cultured, a polarized neurite out-
growth from the trigeminal ganglion towards
odontoblasts was positive for reelin. In addition, the
receptors for reelin apolipoprotein E receptor
(ApoER-2), very low density lipoprotein receptor
(VLDLR), and cadherin-related neuronal receptor
(CNR) are expressed by the trigeminal ganglion,
suggesting that reelin may be involved in the terminal
innervation of the dentinpulp complex, promoting
the adhesion of dental nerve ending and odontoblasts.
Osteoadherin, a small leucine-rich proteoglycan, is
synthesized by osteoblasts and ameloblasts (27, 136).
It has also been reported in the alveolar bone, pre-
dentin, and enamel matrices of rat and mouse teeth
(41). Lucchini et al. (137) reported strong expression
of osteoadherin in the whole odontoblast layer and in
predentin, while the a
v
b
3
integrin, a proposed osteo-
adherin receptor, is seen on odontoblast cell body
membranes and intercellular junctions. This suggests
that osteoadherin and a
v
b
3
integrin play a role in in-
ter-odontoblast adhesion and odontoblast binding to
the predentin/dentin/pulp matrix. Osteoadherin
labeling is strong at the beginning of predentin syn-
thesis whereas a
v
b
3
integrin staining is weaker, the
integrin increasing during odontoblast maturation.
a
v
b
3
integrin may also bind to other odontoblast-
derived RGD-containing proteins, such as dentin
sialoprotein and dentin matrix protein-1, controlling
other processes of odontoblast differentiation and
maturation. Another matrix protein, brodentin, is
thought to be essential for initiation of reparative
dentine during wound healing (13).
Tissue engineering scaffolds in
dentistry
Dental tissue engineering has been primarily utilized
in treating periodontitis, a chronic inammatory
disease resulting in the permanent destruction of
alveolar bone and gingival recession. Several studies
have investigated combinations of scaffolds and stem
cells to regenerate the periodontium and secure
teeth. Using a collagen sponge scaffold containing
gelatin microspheres loaded with broblast growth
factor-2 in alveolar bone defects, Nakahara et al. (162,
163) demonstrated vascularization, osteogenesis,
and recovery of periodontal ligament function after
4 weeks. Using collagen sponges seeded with perio-
dontal ligament cells in another study, they found
that cementum had uniformly regenerated along the
root surface of bony defects in dog teeth. Sculean
et al. (220), reported that the application of bovine-
derived xenograft and bioresorbable collagen in pa-
tients with periodontal defects resulted in signicant
improvement, with reduction of periodontal probing
depth and clinical attachment levels observed 1 year
post-treatment.
Dentin regeneration, especially in vital pulp
therapy, is another potential area for tissue engin-
eering applications. The extracellular matrix plays
an essential role in odontoblast differentiation (210)
and guides dentin repair. However, the ability of the
matrix to regenerate may be compromised in the
damaged area, requiring a replacement matrix to
encourage cell migration and differentiation. Syn-
thetic scaffolds, such as alginate hydrogels, have
been used in dentin generation (22, 48). Dobie et al.
used alginate hydrogels with and without trans-
forming growth factor-b in the dentinpulp complex
of tooth slices to support dentin regeneration (48),
while another study by Tsukamoto et al. (255) found
that pulp capping with calcium hydroxide induced
odontoblast differentiation and osteodentin pro-
duction. Dental pulp cell interactions with matrices
such as dentin, bronectin, ethylenediaminetetra-
acetic acid-soluble dentin components or recom-
binant transforming growth factor-b
1
absorbed onto
Millipore lters indicated that dental pulp cell
differentiation into odontoblasts occurred (256). The
exact mechanisms behind the odontoblast differ-
entiation were not determined, however, the pres-
ence of insoluble bioactive molecules seemed
important for pulp cell differentiation and the
results were promising for future tissue engineering
applications.
95
Inuence of mechanical factors in
tissue engineering
Current tissue-engineering strategies have largely
focused on identifying and manipulating the bio-
chemical factors that regulate tissue regeneration at a
predetermined site. However, this biochemical
paradigm does not address the fact that a large
number of tissues requiring replacement serve pre-
dominantly biomechanical functions, nor does it
address the important role that mechanical factors
play in regulating tissue regeneration within in vitro
and in vivo environments. We believe that mechan-
ical signals are key regulators of tissue regeneration
in vivo and play an important role in determining the
quality and quantity of tissue that can be generated at
a particular site. We also believe that physical forces
play an important role in determining the long-term
composition, quality, and volume of the tissue
construct once integrated with the surrounding
tissues.
Mechanical forces as regulators of
tissue growth and development
Recently there has been a resurgence of interest in
mechanical forces as biological regulators in an at-
tempt to understand the complexity of living tissues.
Physical forces are known to play an important role
in the shaping of an embryo (182), while numerous in
vitro studies have demonstrated that many of the cell
behaviors required for developmental control (e.g.
growth, stem cell lineage commitment and differen-
tiation, polarity, motility, contractility, and apoptosis)
are inuenced by the physical distortion of cells
through their extracellular matrix adhesions (53, 104,
148). The mechanism(s) by which mechanical force is
translated into a biological response is unknown, but
there is evidence that integrins, through linkages to
the actin cytoskeleton and various signal transduc-
tion proteins, play an important role (4). Critical to
the understanding of how cellular form and function
can be regulated by changes in extracellular matrix
tension is the observation that adherent cells exist
within a state of isometric tension. All living cells
generate tension within contractile microlaments in
their cytoskeleton, using an actinomyosin lament
sliding mechanism, and they exert this tension on
their surface membrane at the sites of their cell
extracellular matrix adhesions. These inward directed
forces are resisted by external adhesions to the
extracellular matrix, by internal molecular struts
within the cytoskeleton itself, and by the surface
membrane when stiffened by osmotic pressure (36).
Tensional forces generated in the actin cytoskeleton
and resisted by the extracellular matrix feed back to
alter cell shape and function, and different cellular
responses are produced depending on the ability of
extracellular matrix to physically balance this stress.
Rigid substrates that support high levels of isometric
tension in the cell promote cell spreading and growth
in the presence of soluble mitogens, whereas exible
extracellular matrix scaffolds that cannot resist cyto-
skeletal forces promote cell retraction, turn off
growth, and switch on differentiation in the same
medium (103). Tension-dependent changes in cell
shape are critical to this process. Planar substrates
that contain microfabricated extracellular matrix is-
lands the size of a single cell can also control whether
a cell will grow, differentiate, undergo directional
motility or die, depending on their ability to support
or restrict cell spreading (35). Thus, although
mechanical signals may be sensed and transduced
into biochemical signals at individual cellextracel-
lular matrix adhesion sites, it appears that mechan-
ical signal processing and integration proceed at the
level of the whole cell in response to changes in
cytoskeletal tension and cell shape. For example,
application of a mechanical stress to integrins pro-
duces the same intracellular increase in cAMP within
both round and spread endothelial cells (152). Yet,
the round cells integrate this signal with other inputs
and switch on an apoptosis response, whereas the
spread cells proliferate (104). This relationship makes
sense in situations such as tissue regeneration. For
example, if an epithelial or endothelial layer is dam-
aged, there are fewer cells covering the damaged
area, the remaining cells are able to spread, and this
stimulates them to proliferate until the tissue gap is
lled (212).
Functional tissue engineering
Functional tissue engineering is a novel eld of tissue
engineering which has developed with the aim of
improving understanding of the role that mechanical
factors play in tissue regeneration (28, 75, 76). The
tenets of functional tissue engineering are best
summarized (53) as:
understanding the native mechanical environment
of the tissue that is being replaced.
understanding the relevant mechanical properties
of the native undamaged tissue.
96
Srisuwan et al.
using a subset of these mechanical properties as
design parameters.
studying the inuence of mechanical factors on
cells in vivo as well as in vitro in bioarticial
matrices to affect an improved therapeutic out-
come.
A thorough understanding of the native biome-
chanical environment of the tissue to be replaced is
essential for a number of reasons. First, knowledge of
the sub-failure and failure properties of native tissues
will facilitate the design and engineering of repair
tissues that provide the appropriate functional
properties (76). Second, knowledge of the physical
properties of native tissues will provide insight into
the mechanical microenvironment that cells nor-
mally experience within a specic tissue. This infor-
mation can then be used to determine how cell
behavior is affected by changes in the mechanical
microenvironment, and whether desired cell behav-
iors can be induced by specic manipulation of the
physical properties of the tissue or engineered con-
struct in which they reside (53). As it is not possible to
completely reproduce the structure and material
properties of native tissue in an engineered construct,
tissue engineers will need to focus on identifying and
prioritizing specic biomechanical properties of
native tissues as design parameters for engineered
constructs (74). Tissue engineered constructs will
continue to be exposed to mechanical forces in vivo,
and researchers will need to determine the impact
that these physical forces will have on the function of
cells within the construct, its composition, and its
long-term integration with surrounding native tissues
(76). Finally, it is hoped that an improved under-
standing of the role played by mechanical factors in
tissue regeneration will lead to our ability to use
physical factors to enhance tissue regeneration
in vitro and in vivo. Evidence already exists that
physical factors can be used to improve or accelerate
tissue regeneration and repair in vitro (4, 74, 112, 222,
262). It has been postulated that in the near future an
in vitro model for mechanically induced cell growth
will be developed that excludes direct involvement of
soluble growth factors (262). In vivo, physical forces
have also been used to enhance tissue regeneration
of many tissues, including bone (102), skin (14),
skeletal muscle (117), bowel (118), lung (199), uro-
genital viscera (101), and nerve (233). Application of
tissue stretch (or distraction) is the basis for the
accepted clinical practice of skin expansion and dis-
traction osteogenesis (244).
In summary, functional tissue engineering strat-
egies have targeted mechanical signals as key regu-
lators of the cell behaviors required for successful
engineered tissue growth. Of particular interest is the
ability of specic mechanical signals to induce stem
cell lineage commitment and differentiation into a
particular phenotype in vitro (53, 148). Specically
designed bioreactors are already being developed
that are able to apply lineage-specic mechanical
signals to populations of stem cells. It is hoped that in
the near future engineered tissue growth will become
a reality as improvements are made in our under-
standing of the biomechanical regulation of tissue
regeneration. The application of such practices to
dental tissue engineering will offer new challenges
but may come with great rewards.
Role of vasculature and
neovascularization in tissue
engineering for dental applications
Cell survival depends on oxygen supply, nourish-
ment, and the disposal of metabolic waste products.
A vascular system is essential to deliver nutrients to
cells while removing metabolic products in a timely
manner (91, 106, 277). Without the existence of a
functional vascular network, cells have to rely on
diffusion alone. Indeed, the tissue quality within
cellular scaffolds declines steeply from the surface to
the interior (50, 113). Cells more than approximately
200 lm from a blood supply are found to be either
metabolically inactive or necrotic (39). Tissue cannot
be implanted in volumes >23 mm
3
because of
these limitations unless vascularization is facilitated
(57). Thus, tissue engineering approaches depend on
angiogenesis to form a new vascular network within
the matrix material of the new tissue construct.
An approach taken at our institution is to capitalize
on the neovascularization that occurs from an arte-
riovenous loop in a protected chamber (Fig. 4) (34,
153, 246, 247). This has been established in the rat,
but extended to the rabbit and pig (J. Dolderer, M.
Findlay, W. Morrison, manuscript in preparation).
The chamber lls with a provisional brin-rich matrix
and new vessels sprout from the loop within days.
These chambers provide an appropriate study venue
for the survival and differentiation of dental pulp
stem cells and other dental tissues (e.g. periodontal
ligament). Although these tissue engineering cham-
bers will have broad application in a number of sites
(e.g. liver, pancreatic islets, muscle, etc.), ultimately it
will not be possible to perform these vascular pro-
cedures in the tooth undergoing in situ repair, and we
97
will have to rely on new spontaneous vascularization
of any new dental pulp construct.
Areas within tissue engineering constructs and
scaffolds without a pre-existing capillary network
experience local hypoxia, a known stimulant for
angiogenesis (154, 221, 231). Hypoxia causes an
increase in hypoxia-induced transcription factors
(HIF-1a, HIF-2a) which in turn induce the expression
angiogenic factors such as vascular endothelial
growth factor, inducible nitric oxide synthase, ery-
thropoietin, angiotensin-2, hepatocyte growth factor,
placental growth factor, Tie-2, Flt-1, platelet-derived
growth factor-b, matrix metalloproteinases 2 (MMP-
2) and -13 (MMP-13), integrins, and various other
molecules resulting in vessel branching towards the
area of hypoxia (83, 132, 196, 229). Tissue engineering
approaches exploit this knowledge of vascular growth
in choosing matrix materials, and adding growth
factors either bound to the scaffold or included in
composite beads allowing a constant release in a
timely manner (46). The formation of new blood
vessels during inammation, wound healing and
tumor growth are examples for angiogenesis in the
adult organism, and recapitulation of this in the tooth
environment will greatly facilitate the use of any new
construct. Migration and co-option of existing
endothelium and recruitment of circulating endot-
helial progenitor cells are thought to be possible
sources for the newly formed endothelium in engin-
eered tissue (5, 80, 94). The presence of endothelial
progenitor cells in the peripheral blood was rst
discovered in 1997 (5). They express CD34, vascular
endothelial growth factor-R2, VE-cadherin and the
AC133 antigen, which is expressed specically on
endothelial progenitor cells (183). Hemangioblasts,
the precursor cells that gives rise to red blood cells as
well as endothelial cells in the embryo, have not been
identied in the adult organism (184). It was sug-
gested that CD34
+
FLK-1
+
cells resemble the post-
natal hemangioblast population because it has been
shown that this population is capable of giving rise to
endothelial cells as well as hematopoietic cells (5,
184). The contribution of endothelial progenitor cells
to neovascularization in the adult was documented in
several in vivo experiments but the numeric contri-
bution varied considerably depending on the model
used (32, 178). Endothelial progenitor cell mobiliza-
tion is likely to be triggered by a number of angio-
genic factors and chemokines such as vascular
endothelial growth factor, stromal cell-derived factor
1-a, hepatocyte growth factor, granulocyte colony-
stimulating factor, granulocytemacrophage colony-
stimulating factor, erythropoietin, angiopoietin, and
estrogen, as well as exercise (68, 31, 44, 84, 87, 107,
128, 168, 202, 238, 243, 276).
Much has been learned about the molecular and
cellular processes that induce and mediate new ves-
sel formation and survival, and understanding these
processes may assist in vascularization of dental tis-
sue engineered constructs in situ. The proteolytic
degradation of the basement membrane is the rst
step in the process of sprouting angiogenesis, sub-
sequently leading to an activation and liberation of
endothelial cells. A number of MMPs, particularly
MMP-2 and MMP-9, but also MMP-3, MMP-7, MMP-
12 and MMP-13, are involved in the degradation and
cleavage of laminin and collagen IV within the
basement membrane (49). Collagen IV cleavage leads
to the activation of a
v
b
3
integrin which regulates
endothelial cell migration (274). After migration and
proliferation of endothelial cells, lumen formation
occurs in a process regulated in part by vascular
endothelial growth factor and integrins a
v
b
3
and a
5
b
3
(167). Factors such as urokinase plasminogen acti-
vator, heparinase, chymases, tryptase, and cathepsins
are also engaged in the breakdown of extracellular
matrix molecules and therefore are important for
endothelial cell migration and invasion (30). The
presence of angiogenic growth factors such as vas-
cular endothelial growth factor-A, broblast growth
factor-2, broblast growth factor-1 or hepatocyte
growth factor, and the cytokine tumor necrosis fac-
tor-a are required to activate endothelial cells (123,
157). Degradation of the extracellular matrix is also
important in the angiogenic process. Tumor necrosis
factor-a induces urokinase plasminogen activator
and a number of MMPs in human endothelial cells.
The expression of these proteases allows the endo-
thelial cell to penetrate the extracellular matrix.
Successful endothelial cell invasion relies on the
interaction of the endothelial cell with the matrix. For
example, it has been shown that the structure of
brin inuences the neovascularization process (38,
170). To mature and stabilize, the structural support
of so-called mural cells, comprising pericytes and
vascular smooth muscle cells, is essential for these
newly formed vessels. A contribution of bone mar-
row-derived cells to this pool has also been implica-
ted (90, 133, 178).
When considering dental tissue engineering it may
be important to understand any specializations of
dental vasculature. Pulpal connective tissue is highly
vascularized (173, 282), with large arterioles entering
the tooth, passing through apical foramina towards
the coronal region and terminating in a capillary
plexus in the sub-odontoblastic region (40, 279).
98
Srisuwan et al.
Twenty percent of pulpal blood vessels appeared to
have a positive innervation (207) with large arterioles
receiving a prolic peptidergic innervation (282).
Vasculature plays many roles in pulp tissue such as in
maintaining tissue homeostasis and in odontoblast
development. A study by Trubiani et al. (254) pro-
posed that persistence of vasculogenesis in the pulp
connective tissue in adult life leads to continuous
adjustment of vessel and network structures in re-
sponse to functional needs in deciduous and per-
manent human teeth, playing a key role in the spatial
arrangement of dental pulp formation and mainten-
ance (254). Angiogenesis and neovascularization are
also crucial for dentin formation. During primary
dentinogenesis, active vascular development occurs
in the odontoblastic zone decreasing following dentin
production (279), suggesting that peripheral capillary
activity is closely related to odontoblast activity (206).
Local angiogenesis inside the pulp is also important
for tissue repair (13) with growth factors acting as
important angiogenesis regulators (58). The seques-
tration of several types of growth factors in the dentin
matrix is believed to play a central role in dentin re-
pair and the differentiation of new odontoblast-like
cells (16, 15, 258), and local increases in tooth vas-
culature were found after isolated dentin matrix
components were implanted into injury sites (234).
Neuropeptides such as substance P, neurokinin A,
and calcitonin gene-related peptide secreted from
pulpal nerves are proposed as mediators for pulpal
blood ow after sensory nerve stimulation (89).
The periodontium is also highly vascularized tis-
sue. The intraseptal artery supplies the periodontium
perforations of the alveolar sockets and anastomoses
with blood vessels from the periodontal ligaments,
creating a polyhedral network surrounding the tooth
like a stocking. Several studies using various growth
factors to induce angiogenic activity and the growth
of periodontal ligament cells (58, 127, 141, 161, 204)
have been conducted. A study by Murakami et al.
(161) demonstrated the upregulation of laminin in
periodontal ligament cells after the application of -
broblast growth factor-2, which was suggested to play
a role in angiogenesis. Another study on the effect of
hepatocyte growth factor on periodontal endothelial
cells and tissue repair proposed that hepatocyte
growth factor may promote the proliferation and
tubulogenesis of these endothelial cells in vitro (216).
Gingival tissue receives its blood supply primarily
from the supraperiosteal vessels, which are the ter-
minal branches of larger vessels such as the sublin-
gual, mental, and facial arteries. These vessels are
located in the gingiva, creating a subepithelial plexus
with capillary loops on the oral epithelium side and
dentinogingival plexus on the junctional epithelium
side (134). This vasculature not only maintains local
homeostasis but has an important role in providing
nutrition for newly engineered tissues implanted in
this area (241), and is crucial for the initiation of
periodontal tissue healing or regeneration when re-
quired (174).
Conclusions
In conclusion, a host of molecular processes can be
identied in relation to the various aspects pertinent
to dental tissue engineering. Identication, under-
standing, and characterization of these pave the
way for optimized methodology, such as coated
implants, use of stimulants, enhanced adhesion,
cellular recruitment, vascularization, survival, appro-
priate apoptosis, and differentiation. Considerable
intersection exists in the molecular classes associ-
ated with the various cellular aspects of tissue
engineering pertinent to dentistry cells, space,
vascularization such that the possibility exists for a
certain molecule to dramatically enhance the overall
outcomes.
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Srisuwan et al.
Regeneration of vascularized
bone
SUSAN X. HSI ONG & DAVI D J. MOONEY
Musculoskeletal diseases affect hundreds of millions
of people worldwide and are one of the leading
causes of long-term pain and physical disability (1).
Over 6 million bone fractures are reported annually
in the USA alone (88), and intra-oral bone loss caused
by periodontal disease is a major public health con-
cern. Approximately 80% of American adults have
some form of periodontal disease, and severe perio-
dontal disease can cause loss of cementum and other
supporting bone tissue. Novel therapeutic strategies
that enhance bone healing and regeneration, such as
tissue engineering, have the potential to improve the
quality of life for millions of people.
Traditional treatment methods for promoting bone
healing primarily utilize bone grafts or synthetic
materials to ll the defect and provide structural
support. Bone autografts are considered the gold
standard for treating bone defects owing to the low
risk of an adverse immune response (31). Allografts
involve harvesting and processing bone from a
cadaver, which is subsequently transplanted into the
patient. Xenografts, typically of bovine or porcine
origin, are also used to treat bone defects. The
drawbacks of using bone grafts include a lack of
viable bone tissue available for autograft and allograft
transplantations, and potential disease transmission
with allografts and xenografts. In cases where bone
grafts from human or animal sources are not feasible
(e.g. limited supply or insufcient bone volume
available), synthetic graft materials (alloplasts) are
used. In the past, alloplast materials were bioinert
materials, such as titanium or alumina alloys and
polyethylene polymers, which simply provided
mechanical support. However, limitations of these
synthetic bone-replacement materials include poor
integration with the surrounding tissue, a potential
need for future retrieval or replacement, and an
inability to adapt with the dynamic environment.
One-third to one-half of articial skeletal prosthesis
fail within 1025 years (37). A guided tissue-regen-
eration (GTR) approach has since evolved to employ
materials (e.g. hydroxyapatite, calcium phosphate,
bioactive glasses, collagen and biodegradable poly-
mers) that support natural bone-formation processes
(42). The success of GTR is not always predictable,
and this approach is limited in its application (19, 77),
probably as a result of its passive approach to bone
regeneration.
Bone tissue engineering has emerged as a new
therapeutic alternative to promote bone healing. This
approach aims to regenerate or repair bone tissue
with various combinations of polymeric scaffolds,
cells and inductive factors into a system that actively
stimulates tissue formation. Tissue engineering seeks
to mimic the bodys natural tissue formation pro-
cesses to form functional living tissue. One common
tissue-engineering strategy utilizes a small tissue
biopsy to isolate and expand appropriate cell popu-
lations in vitro, followed by their seeding onto
materials to fabricate custom-made articial tissue
(Fig. 1) (55). Bone is a highly vascular tissue that
relies on the blood vessels to supply essential
nutrients and oxygen. For tissue to grow beyond
100200 lm (the diffusion limit of oxygen), new
blood vessel recruitment is required (14). To suc-
cessfully engineer bone of signicant size and
volume, strategies that incorporate vascularization to
support the metabolic demands of the engineered
tissue are important. This review provides an
overview of bone development, describes how
this information is being used to develop bone-
engineering strategies and discusses approaches to
generate vascularized bony tissue.
Bone development and
regeneration
Knowledge of the developmental processes that
govern bone formation and fracture healing are often
used to guide bone tissue engineering strategies. This
109
PERIODONTOLOGY 2000
section provides general background on the process
of bone development, and discusses the roles of
osteoinductive factors and a key angiogenic factor in
bone development and fracture healing.
Bone is derived from multipotent cells that give rise
to the skeletal, lymphatic and hematopoietic elements
of the body (Fig. 2) (11). These cells differentiate to
form bone by one of two mechanisms: endochondral
ossication; or intramembranous ossication. During
endochondral ossication, bone forms via a cartilage
intermediate. Mesenchymal stem cells proliferate,
aggregate and differentiate into chondrocytes, which
produce cartilage-specic markers, such as aggrecan
and type II collagen. These chondrocytes subse-
quently hypertrophy, mineralize and become vascu-
larized by the invasion of blood vessels. Osteoblasts,
the primary bone-forming cells, migrate and replace
the cartilage with mineralized bone (20). Contrary to
endochondral ossication, intramembranous bone
forms as mesenchymal stem cells progress down a
sequential differentiation pathway to become mature
osteoblasts without undergoing a cartilage interme-
diate. An early step in this pathway occurs upon the
activation of Cbfa1/Runx2, a key transcription factor
that regulates mesenchymal stem cell commitment
towards the osteoblast lineage (21). These committed
mesenchymal stem cells, termed pre-osteoblasts,
proliferate and form mature osteoblasts that synthes-
ize and secrete the major bone matrix proteins
(e.g. collagen I, the predominant component of
bone organic matrix) and bone-specic proteins (e.g.
osteocalcin and osteopontin) that dictate bone struc-
ture and function.
Growth factors in bone
development
The various stages of bone formation and fracture
healing occur in a dened spatiotemporal sequence
of events that are regulated by many inductive factors
(Fig. 3) (10). Hormones, cytokines and morphogens
Inductive
factors
Fig. 1. A schematic diagram of a popular tissue-engin-
eering approach: cells obtained from a small biopsy
obtained from the patient are expanded in vitro, seeded
onto a polymeric matrix or encapsulated in a gel, cultured
with select inductive factors (e.g. growth factors) and
implanted or injected into the patient. Reprinted from
Lee & Mooney (55) with permission from the American
Chemical Society.
Fig. 2. Mesenchymal stem cells (MSCs) have the potential to follow varying pathways and differentiate into various cell
types. Reprinted from Caplan & Bruder (11), with permission from Elsevier.
110
Hsiong & Mooney
play a critical role in driving bone development (29).
In this section, we give a brief overview of several key
osteoinductive growth factors (Table 1) that inu-
ence bone development.
The most extensively studied osteoinductive factors
are members of the transforming growth factor-b
superfamily of morphogenetic proteins, comprising
structurally and functionally related proteins, such as
transforming growth factor-b and bone morpho-
genetic protein (BMP), which play signicant roles in
embryonic development and tissue repair as well as in
bone development (71, 86). Transforming growth fac-
tor-b, one of the major growth factors present in bone
matrix, is a polypeptide synthesized and secreted in
bone cultures. It is believed that the primary role of
transforming growth factor-b in bone formation is to
increase the pool of committed osteoblasts. BMPs
were identied and cloned after the discovery that
extracts from demineralized human bone matrix in-
duce ectopic bone formation (120, 126). Currently, 20
members of the BMPfamily have beenidentiedinthe
human genome (BMP-2, BMP-4, BMP-7, BMP-8, etc.)
(92). BMP-2 acts primarily as a differentiation factor
for bone and cartilage precursor cells during osteo-
genesis and bone regeneration. Other osteoinductive
factors present inbone are insulin-like growthfactors I
and II (peptide hormones synthesized primarily in the
liver, but also locally by osteoblasts). Insulin-like
growth factors mediate the effects of systemic hor-
mones (e.g. growth hormone), cytokines (e.g. inter-
leukin-1a) and morphogens (e.g. BMPs) in bone for-
mation and healing (23, 52, 61). Fibroblast growth
factors also play an important role in bone develop-
ment and repair. Fibroblast growth factors are auto-
crine/paracrine regulators of bone formation, which
stimulate chemotaxis, proliferation and matrix syn-
thesis of osteoblasts or osteoblast precursors, but do
not directly promote osteoblast differentiation (24, 34,
52, 86).
Role for vascular endothelial
growth factor in bone formation
and fracture healing
Bone is a highly vascularized tissue that relies onblood
vessels for the transport of essential nutrients and
oxygen, as well as the delivery of circulating osteogenic
factors and stem cells. The vasculature forms by two
mechanisms: vasculogenesis and angiogenesis. Vas-
culogenesis involves the de novo formation of blood
vessels in which endothelial progenitor cells assemble
to form vessels in early development. The primitive
vessel network subsequently expands and remodels to
form a more mature network via angiogenesis, a pro-
cess in which new blood vessels sprout from existing
blood vessels (13). During wound healing and repair of
ischemic tissues, endothelial cells andtheir precursors
actively participate in the healing process. The major
angiogenic factors include broblast growth factors,
platelet-derived growth factor and vascular endo-
thelial growth factor (VEGF). Fibroblast growth factors
and VEGF stimulate endothelial cell production of
proteases andplasminogenactivators that degrade the
vessel basement membrane and allowendothelial cell
proliferation and migration (18). Endothelial cells and
endothelial progenitor cells then assemble to form
new blood vessels and secrete key factors, such as
platelet-derived growth factor and angiopoietin-1, to
recruit smooth muscle cells and pericytes to stabilize
and support the newly formed blood vessels (60).
Growth factors, which stimulate angiogenesis also
drive bone repair and regeneration. The role of VEGF,
in particular, has been the subject of a number of
studies (25).
The VEGF family comprises six related proteins:
VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and pla-
centa growth factor. The most studied member of the
VEGF family is VEGFA, which exists as multiple splice
forms (VEGF121, VEGF165, VEGF189 and VEGF209 in
humans) (95). Although endothelial cells are the pri-
mary VEGF target, VEGF also modulates the recruit-
Fig. 3. A schematic diagram of the bone regeneration
process. Various cytokines and growth factors are pro-
duced locally after a bone fracture. These factors stimulate
bone regeneration by promoting osteoprogenitor migra-
tion to the defect site, proliferation and differentiation
into bone-forming osteoblasts. BMP, bone morphogenetic
protein; FGF, broblast growth factor; IGF, insulin-like
growth factor; IL-1b,interleukin-1b; PDGF, platelet-de-
rived growth factor; TGF, transforming growth factor;
TNF-a, tumor necrosis factor-a; VEGF, vascular endo-
thelial growth factor. Reprinted from Braddock et al. (10),
with permission from the American Physiological Society.
111
Regeneration of vascularized bone
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Hsiong & Mooney
ment, survival and activity of osteoblasts and osteo-
clasts (12). In addition to expressing VEGF receptors,
osteoblasts respond to and secrete VEGF (67). Thus,
VEGF acts directly on osteoblasts to promote osteo-
blast migration, proliferation and differentiation in an
autocrine manner (67, 68). VEGF is also a mediator of
many osteoinductive factors, including transforming
growth factor-b1, insulin-like growth factor-I and
broblast growth factor-2, which upregulate VEGF
expression in osteoblasts (30, 99, 122). In addition to
the direct effects of VEGF on osteoblasts, VEGF also
inuences osteoblasts indirectly via its effects on
endothelial cells. VEGF stimulates the production, by
endothelial cells, of anabolic (bone-forming) factors
for osteoblasts (68). Furthermore, endothelial cells and
osteoblasts also communicate via cellcell contact
(121). Therefore, direct and indirect effects of VEGF on
osteoblast differentiation may synergistically enhance
bone formation.
The importance of VEGF signaling in bone regen-
eration is demonstrated by the ndings that inhibi-
tion of VEGF in a mouse fracture and cortical defect
model resulted in impaired bone formation, while the
addition of exogenous VEGF-enhanced bone repair in
a rabbit critical-sized defect model (112). Treatment
with an anti-VEGF chimeric protein was found to
inhibit blood vessel formation during endochondral
ossication and led to compromised bone formation
(28). VEGF may be the major factor coupling osteo-
genesis and angiogenesis (28).
Strategies for bone tissue
engineering
Tissue-engineering strategies often seek to recapit-
ulate natural tissue-formation processes, and appro-
priate cell populations, scaffolds and inductive
factors are selected to facilitate the repair and
regeneration of bone.
Scaffolds
Scaffolds act as a delivery vehicle for cell transplan-
tation and as a three-dimensional template for tissue
regeneration, but also provide specic cues to regu-
late bone formation (35). Candidate biomaterials for
scaffolds must meet several criteria. First, they must
be biologically compatible to minimize adverse
inammatory responses. In addition, a biodegradable
scaffold is typically desired such that it degrades, over
time, to yield space for new tissue formation (87).
Furthermore, the mechanical, chemical and biologi-
cal properties of the scaffold should be suitable for
the specic application. Scaffold characteristics, such
as porosity, topography and material composition,
dictate certain of these features (43, 107). Scaffolds
which simply guide and support bone regeneration
are termed osteoconductive, whereas scaffolds which
actively stimulate bone regeneration via the delivery
of inductive factor(s) are osteoinductive. Scaffolds
utilized for bone tissue engineering may be osteo-
conductive, osteoinductive or both.
Osteoconductive and osteoinductive scaffolds are
often designed to mimic native bone, which is a
porous composite material composed mainly of
hydroxyapatite in a collagen I matrix (73). Hence,
collagen I, hydroxyapatite and other calcium phos-
phate biomaterials are commonly used for bone tissue
engineering. These scaffold materials promote osteo-
blast and osteoprogenitor attachment and differenti-
ation to enhance bone tissue formation (56). However,
naturally derived materials, such as collagen, have
limitations, including batch-to-batch variations and a
limited range of physical properties, and undesirable
degradation proles limit the utility of hydroxyapatite.
Other materials utilized for bone tissue engineering
include synthetic, biocompatible and biodegradable
polymers, such as poly(lactide-co-glycolide). The use
of poly(lactide-co-glycolide) and other synthetic pol-
ymers is benecial as one can vary the molecular
weight and composition to control the rate of degra-
dation and kinetics of growth factor release (94, 106).
The scaffoldcharacteristics canfrequently be precisely
tailored to the specic application.
For minimally invasive applications, injectable
polymers, such as alginate or poly(ethylene glycol)
hydrogels, are desired (87). Due to their easily
injectable and moldable nature, hydrogels may be
particularly well suited for lling small defects (e.g.
periodontal defects). Hydrogels can be designed to
prevent cell adhesion or protein adsorption as a
result of their high hydrophilicity (55). Thus, they can
act as a blank slate and can be ideal materials for
precise control of cellular interactions. Modication
of materials, such as alginate, with peptide sequences
commonly found in many extracellular matrix pro-
teins (e.g. arginineglycineaspartic acid), allows
precise control over cellular adhesion by varying the
peptide sequence, distribution and density (36, 93).
As cell adhesion to the extracellular matrix stimulates
signals that control many cellular processes, argin-
ineglycineaspartic acid peptide-modied materials
have been shown to improve cell adhesion, viability,
proliferation and differentiation for a variety of cell
types (4, 97). For example, transplantation of primary
113
rat calvarial osteoblasts in an arginineglycine
aspartic acid-modied alginate hydrogel promoted
signicantly more in vivo bone formation compared
with unmodied alginate scaffolds (3). For more
information on hydrogels in tissue engineering, see
Lee & Mooney (55).
Cells
Although solely materials-based approaches (e.g.
GTR) have been shown to enhance bone repair, these
approaches may be insufcient to heal large bone
defects. As the primary cell types involved in bone
formation, osteoblasts and osteoprogenitor cells are
important components of the bone-repair process
and have been incorporated into various scaffolds to
enhance bone repair. Scaffolds composed of hydro-
xyapatite, collagen I, poly(lactide-co-glycolide) or
arginineglycineaspartic acid peptide-modied
alginate, support osteoblast and preosteoblast differ-
entiationinvitro andinvivo by upregulating collagenI
production, osteocalcin expression and mineraliza-
tion (3, 104, 124). Implantation of cells on these types
of scaffolds enhances bone regeneration in vivo and is
generally superior to the implantation of scaffolds
alone (5, 85, 101).
In order to obtain autologous or allogeneic cells for
bone regeneration, osteoblasts and osteoprogenitors
must be obtainedfromthe patient or a donor. Isolation
of sufcient numbers of primary human osteoblasts is
difcult as it requires the sacrice of healthy bone
tissue. This has thus stimulated interest in the use of
osteoprogenitor cells as a source of bone regeneration.
Stemcells derived fromthe bone marrow, periosteum,
adipose tissue, skeletal muscle or even baby teeth, can
be induced to differentiate into diverse cell types such
as muscle, nerve, cartilage, bone andfat (11, 69, 74, 83).
As they can be isolated and expanded in culture, stem
cells have great potential for clinical utility to repair or
regenerate tissue (119). In fact, most bone tissue
engineering strategies focus on the delivery of osteo-
progenitor cell populations in lieu of mature osteo-
blasts as a result of their greater clinical relevance.
Many studies have demonstrated that these cells,
when seeded in a polymeric scaffold and implanted
in vivo, either subcutaneously or in an osseous defect,
stimulate bone formation (75, 101).
Osteoinductive factor delivery
Direct or gene-therapy approaches to the delivery of
osteoinductive factors are promising approaches for
bone regeneration. As the advent of recombinant DNA
technology enabled polypeptide growth factor pro-
duction, the utility of these factors in enhancing tissue
regeneration and repair has been widely investigated.
Conventional protein delivery methods involve direct
injection into the defect site. However, as a result of
the short half-life of many inductive proteins, this
method requires very high doses for therapeutic effect
and still may not permit the necessary concentration
of the factor to be maintained for the appropriate
period of time (54). Controlled release of proteins
from a polymeric scaffold allows localized, sustained
protein delivery and may be a more effective means to
enhance bone formation (17, 54). The properties of
the scaffold material (e.g. composition and chemistry)
may govern the efcacy of protein delivery on fracture
healing and bone regeneration.
Owing to its potent ability to induce bone forma-
tion, delivery of BMP-2 has been the focus of a variety
of preclinical and clinical models of bone regener-
ation. Delivery of BMP-2 protein from an absorbable
collagen sponge induces bone formation and heals
bony defects (58). In relation to intra-oral defects, the
repair of large critical-sized defects in the canine
mandible can be accomplished with BMP-2 protein
delivery froma collagen scaffold (125). Applications of
BMPs to dental tissue engineering have been reviewed
comprehensively (76). BMP-2 can also be delivered
from synthetic materials, such as poly(ethylene gly-
col) hydrogels, to promote the repair of bone defects
(62). Furthermore, such materials can be engin-
eered to mimic the ability of natural extracellular
matrix to undergo cell-triggered proteolysis and
matrix remodeling (e.g. with a combination of matrix
metalloproteinase substrates and cell adhesion
sites) (63, 62). US Food and Drug Administration
(FDA)-approved BMP products for bone regeneration
are now available. BMP-2 has been demonstrated to
be effective in human fractures, and several products
are FDA approved for this indication. One such
product is the InFUSE
TM
bone graft (Medtronic
Sofamor Danek, Memphis, TN), which promotes
lumbar interbody spinal fusion in humans. For the
treatment of established nonunions, OP-1
TM
(Stryker,
Kalamazoo, MI) was granted a humanitarian device
exemption and also received FDA approval. In addi-
tion to BMP, sustained release of other osteoinductive
factors, such as broblast growth factor-2 and insulin-
like growth factor-I, can also accelerate bone forma-
tion, as shown in calvarial defect and tibial fracture
models (49, 102). However, clinical applications of
these factors in bone regeneration have yet to be
achieved.
114
Hsiong & Mooney
Localized gene-therapy approaches to the delivery
of bone-inductive factors may circumvent limitations
of direct protein delivery, which include the frequent
requirement for supraphysiologic (mg) quantities
(96) and the challenge of maintaining protein bio-
activity (53). Delivery of genes encoding the inductive
factors allows sustained, localized protein production
and can be used for either short-term (e.g. transient
transfection of cells) or long-term (e.g. retroviral
transduction) delivery (81). In either case, gene
delivery takes advantage of the protein synthesis
machinery of the cells to produce the specic bio-
active factor. The gene of interest may be directly
introduced to the target cells in vivo (in vivo gene
therapy) or used to transduce target cells in vitro
prior to in vivo transplantation (ex vivo gene therapy)
(27). However, signicant immunogenic issues,
resulting from in vivo gene therapy with certain viral
vectors, have led to signicant interest in ex vivo gene
therapy (27). Adenoviral transduction of osteogenic
and nonosteogenic cells with the gene encoding
BMP-2 (33) and BMP-7 (50) have been shown to
promote bone repair and formation in vivo. The
concerns of potential mutagenesis, carcinogenesis
and immune response to viral infection (81) have also
motivated interest in nonviral methods of DNA
delivery. Nonviral methods, including liposome-
based or direct plasmid delivery, are clinically
appealing as a result of their potentially low toxicity
and immunogenicity. The localized, sustained deliv-
ery of a naked plasmid encoding a secreted peptide
fragment of human parathyroid hormone (hPTH
1-34) has demonstrated utility in a canine tibia and
femur metaphysis fracture model (8), as has locali-
zed, sustained delivery of a plasmid encoding BMP-4
from a poly(lactide-co-glycolide) scaffold in a critical-
size cranial defect (39). Although plasmid and other
nonviral delivery methods are currently limited by
low transfection efciencies, they have great poten-
tial clinical utility.
Combined delivery of cells and
growth factors
Although delivery of either cells or osteoinductive
factors has been shown to promote bone regener-
ation, an integrative approach delivering combina-
tions of osteoinductive factors and cells from an
appropriately designed scaffold may be more efcient
or yield greater quantities of regenerated bone.
Tissue-engineering strategies which adopt this
approach may better recapitulate the complex
microenvironment present in normal bone regener-
ation processes. Much of the research, to date, com-
bining growth factor delivery with cell populations,
focuses on the ex vivo transduction of the cells with
genes encoding osteogenic factors such as the BMPs
(50, 59, 105, 118). Direct growth factor delivery,
combined with osteogenic cell transplantation, is
promising (109). The process of bone formation is
driven by the complex interplay of various growth
factors that regulate bone development and regener-
ation in a spatially and temporally dened manner.
Thus, various studies have investigated the delivery of
osteoinductive factors in different combinations and
sequences. Simultaneous or sequential delivery of
BMP-2 and insulin-like growth factor-I from a gelatin
scaffold induced osteogenic differentiation of a mu-
rine multipotential cell line in vitro (91). Combined
delivery of transforming growth factor-b1 and BMP-2
with osteoprogenitor cell transplantation allowed
physiologic doses of these growth factors to enhance
bone formation in vivo (109). These studies support
the premise that multiple growth factors, working in
concert or sequentially, may signicantly enhance the
formation of functional tissue (111). Representative
studies, utilizing tissue-engineering constructs for
bone regeneration, are shown in Table 2.
Promoting tissue
neovascularization
Blood vessel formation and vascularization are crit-
ical in driving endochondral and intramembranous
ossication, as well as regenerative processes (e.g.
restoring function in injured and ischemic organs)
(90). During fracture healing, new blood vessels
sprout from existing blood vessels to restore blood
supply and to facilitate bone regeneration. Inad-
equate bone vascularity is associated with decreased
bone formation and bone mass (12). In this section,
we give an overview of tissue-engineering strategies
to promote neovascularization, and describe how
these approaches are being adapted to manipulate
angiogenesis and improve bone regeneration.
Endothelial cell transplantation
Transplantation of blood vessel-forming cells pro-
vides one direct approach to create new vascular
networks and mimics some aspects of the vasculo-
genesis process. Intravenous infusion or injection of
progenitor cells enhances angiogenesis, thereby
115
improving cardiac function and restoring limb func-
tion (7, 116). However, the potential tumorigenesis
resulting from injection of endothelial progenitors
into the systemic circulation has focused attention on
a more site-specic delivery of the cells. Scaffolds
that maintain cell localization, while also acting as
an extracellular matrix supporting endothelial cell
tubule formation, may be ideal. Poly(lactide-co-gly-
colide) scaffolds can be designed to allow trans-
planted endothelial cells to form functional vascular
networks in concert with the host vasculature (78,
84). A combined approach to promote neovascu-
larization via delivery of human umbilical vein
endothelial cells and VEGF from a poly(lactide-
co-glycolide) scaffold has been demonstrated (84).
Laminin-rich gels (44, 64), poly(propylene fumarate-
co-ethylene glycol) hydrogels (113), peptide-modied
alginates (26), brin (100), collagen (114) and colla-
gen-modied poly(l-lactic acid) (130) have also been
investigated. Endothelial precursors, such as circu-
lating endothelial progenitor cells, are perhaps a
more relevant cell source for transplantation. Derived
from the bone marrow, systemic vasculature or or-
gans, circulating endothelial progenitor cells home to
sites of neovascularization and differentiate into
endothelial cells (46, 64, 90). Although endothelial
progenitor cells are a more readily available source of
cells for therapeutic applications, the ability to sort
and identify a pure progenitor cell population for
transplantation is limited (90). An alternative cell
source to endothelial progenitor cells are embryonic
stem cells derived from the embryonic inner cell
mass (117). Embryonic stem cells have been dem-
onstrated to differentiate into endothelial cells (57,
129), and may provide the ideal cell source if their
differentiation to endothelial cells can be controlled.
Delivery of angiogenic factors
Delivery of angiogenic factors is the simplest and
most extensively studied approach to promote angi-
ogenesis (22). However, similar to the delivery of
osteogenic factors, direct injection of angiogenic
proteins requires delivery of supraphysiologic con-
centrations for a therapeutic effect owing to the
proteins short half-life (in the order of minutes) (15).
In addition, injection of excessive concentrations of
angiogenic factors may cause undesirable or poten-
tially dangerous side effects, such as leaky blood
Table 2. Representative studies of tissue engineering constructs for bone regeneration
Osteoinductive factor Cells Scaffold vehicle Conclusions Rep. Ref.
None rBMSC PLG foam PLG scaffold supports osteogenic
differentiation in vitro
(45)
None RCO RGD-modied
alginate
Preosteoblasts encapsulated in
RGD-modied alginate hydrogel
exhibited enhanced differentiation
in vitro and in vivo
(3, 5)
None Sheep
BMSC
Coral Scaffold and cell vehicle improved healing
in a sheep critical defect model
(85)
rhBMP-2 None Collagen sponge Signicant increase in bony union of
the rhBMP-2-treated groups in a rabbit
distraction osteogenesis model
(98)
rhBMP-2 None RGD, MMP-modied
PEG hydrogel
Enhanced bone repair in a rat cranial
defect model
(62)
BMP-4 (plasmid DNA) None Porous PLG Enhanced bone repair in a rat cranial
defect model
(39)
rhBMP-2 (adenoviral-
mediated gene delivery)
hBMSC Porous PLA Bone matrix production and mineralization
observed in in vivo diffusion chamber
(38)
rhTGF-b3
rhBMP-2
hBMSC Degradable
RGD-modied
alginate
Degradable scaffold and dual growth
factor delivery allow physiologic doses of
growth factor for bone regeneration
(109)
HA, hydroxyapatite; hBMSC, human bone marrow stromal cells; MMP, matrix metalloproteinase; OP-1, osteogenic protein 1 (also BMP-7); PEG, poly(ethylene
glycol); PLA, poly(lactide); PLG, poly(lactide-co-glycolide); rBMSC, rat bone marrow stromal cells; RCO, rat calvarial osteoblasts; rhBMP, recombinant human
bone morphogenetic protein; RGD, arginine-glycine-aspartic acid; TGF-b3, transforming growth factor-b3.
116
Hsiong & Mooney
vessels or hemorrhage (32). To minimize adverse ef-
fects, polymeric scaffolds, including alginate (32),
gelatin and poly(lactide-co-glycolide) (94), have been
developed for localized, controlled angiogenic growth
factor delivery. Sustained VEGF delivery from a por-
ous poly(lactide-co-glycolide) scaffold has been
shown to enhance angiogenesis and the survival of
transplanted cells (110). Co-delivery of endothelial
cells and angiogenic factors may be particularly
suitable for patients whose vasculature may not be
responsive to angiogenic factors alone. Although
VEGF initiates the formation of new blood vessels,
VEGF alone is unable to induce the formation of
mature, functional blood vessels (6). Other factors,
such as angiopoietin-1 (Ang1) and platelet-derived
growth factor (PDGF), are required to promote
vessel maturation and stabilization (Fig. 4) (128). To
engineer an organized network of mature, functional
blood vessels for tissue repair, delivering combina-
tions of angiogenic factors, such as VEGF, Ang1 and
PDGF, in the appropriate spatiotemporal sequence is
likely to be important. In support of this concept,
dual delivery of VEGF and platelet-derived growth
factor, each with distinct release kinetics, from a
single poly(lactide-co-glycolide) scaffold, stimulated
formation of mature, stable vasculature in a mouse
ischemic hind limb model (94). Delivery of VEGF or
platelet-derived growth factor, either alone or sim-
ultaneously, resulted in either decreased blood vessel
densities or less mature vessel networks.
Gene-therapy approaches to promote revasculari-
zation via delivery of angiogenic factors have also
been pursued. Clinical trials using adenoviral delivery
of VEGF121 in human patients with limb ischemia
(72) indicate that local adenovirus injection is well
tolerated and safe. Nonviral methods of angiogenic
growth factor delivery have also been investigated,
and delivery of a plasmid-encoding VEGF165 was
found to improve perfusion and myocardial function
in a porcine chronic myocardial ischemia model (89).
In another study, bolus injection of a plasmid-enco-
ding platelet-derived growth factor did not signi-
cantly affect tissue formation, whereas sustained
plasmid delivery from a polymer enhanced blood
vessel formation (103). Controlled, sustained release
may thus provide a more effective mode of gene
delivery, as previously noted for protein delivery.
Combining angiogenesis and
osteogenesis strategies
The clear synergy between bone formation and an-
giogenesis is prompting the development of strat-
egies that incorporate neovascularization approaches
into bone-regeneration systems (Table 3). A direct
approach to enhance vascularization during bone
formation is to grow tissue around a natural vessel
and subsequently transplant the tissue to the desired
site as a vascularized bone ap (2, 66).
The intimate association of angiogenesis with bone
formation and regeneration has prompted approa-
ches to deliver angiogenic factors to promote bone
formation and healing. Localized and sustained VEGF
delivery from osteoconductive scaffolds results in
greater bone regeneration in calvarial defects than
observed with scaffold implantation alone (73). Fur-
thermore, the combined delivery of angiogenic and
osteogenic factors has been observed to promote
bone formation and healing. Delivery of VEGF pro-
tein and a plasmid-encoding BMP-4 from a scaffold
transplanting mesenchymal stem cells synergistically
enhanced bone formation (40). Although the com-
bined delivery of angiogenic and osteogenic factors
improve bone regeneration, delivering these factors
in an appropriate ratio may be critical for bone
healing (83).
In addition to the indirect effects of endothelial
cells on bone formation, via their ability to form
vascular networks, it has also become clear that
endothelial cells can interact directly with bone-
forming cells to regulate their gene expression.
Endothelial cells stimulate alkaline phosphatase
activity of human osteoblasts in in vitro coculture
experiments (123), and this effect may be dependent
on gap junctional communication between the two
VEGF
PDGF,Ang1
Blood vessel
Angiogenic
sprouting
Fig. 4. Blood vessel formation via angiogenesis. Vascular
endothelial growth factor (VEGF) stimulates new blood
vessels to sprout from existing blood vessels, but the new
vessels are immature and lack associated pericytes or
smooth muscle cells. Angiopoietin 1 (Ang 1) and platelet-
derived growth factor (PDGF) promote the association of
pericytes and smooth muscle cells with the nascent
vessels, which leads to the formation of stable, mature
vessels. Reprinted from Yancopoulos et al. (128), with
permission from Nature.
117
cell types (121). Endothelial cells have also been
demonstrated to secrete BMP (9), and endothelial
cells and human bone marrow stromal cells exhibit
a reciprocal relationship, as human bone marrow
stromal cells also secrete VEGF (9, 47). Furthermore,
VEGF secretion by bone marrow stromal cells may
modulate BMP expression by endothelial cells (9).
These studies suggest that cotransplantation of
endothelial cells with osteoblasts or osteoprogenitor
cells may regulate bone regeneration at multiple
levels. One study demonstrated that cotransplanta-
tion of endothelial cells and human bone marrow
stromal cells in a mouse subcutaneous model
resulted in greater bone formation than transplan-
tation of human bone marrow stromal cells alone
(48). Endothelial cells may not only enhance the
development of new vasculature, which supplies
nutrients to newly forming bone, but also modulate
the osteogenic differentiation of osteoprogenitor
cells (48).
Conclusions and future directions
Tissue engineering holds great promise as a future
therapeutic alternative to regenerate or repair dam-
aged tissues. Strategies for cell transplantation and/
or growth factor delivery from a scaffold have suc-
cessfully enhanced tissue formation and healing in
many animal models and patients.
Studies, to date, have mainly investigated the
delivery of a single growth factor or transplantation of
a single cell type for tissue regeneration but may fail
to provide the complex signals present in the normal
physiologic environment. Tissue formation and
reparative processes rely on several growth factors
and cells that work in concert to form functional
tissue (111). To engineer successfully tissues and
organs of increasing complexity, appropriate combi-
nations of scaffolds, cells and inductive factors may
be necessary. Novel scaffolds will be designed to exert
precise control over tissue formation processes by
delivering the proper combinations and ratios of
growth factors in a specic spatial and temporal se-
quence to target cells for tissue regeneration. In
addition to controlling the kinetics of protein and
gene delivery, scaffolds guide and support tissue
regeneration by acting as a synthetic extracellular
matrix (35, 41, 103). As the natural extracellular
matrix has structural elements in the range of
nanometers, fabrication of scaffolds that present
information to the cells at the nanoscale level may
better approximate the natural extracellular matrix
(127). For example, nanobrous scaffolds have been
observed to enhance bone and blood vessel forma-
tion (108, 127). Nanoscale patterning of cell-adhesion
molecules on biomaterials may allow more precise
control of cell behavior (51). The discovery and use of
human multipotential stem cells holds great promise
for bone tissue engineering strategies, but the
mechanism by which the development and remode-
ling program occur must be fully understood. Finally,
the further development of strategies, which incor-
porate angiogenesis may enhance bone regeneration
and enable the engineering of large volumes of
functional tissues in the future.
Table 3. Representative approaches to enhance bone formation by promoting vascularization
Angiogenic factor Cells Vehicle/mode of delivery Model Ref.
None ECs hBMSC Porous PLG Rat subcutaneous implantation (129)
VEGF
(adenovirus)
None Intramuscular injection
around bone defect
Rat femur defect (115)
None rBMSC Coral Rat intramuscular implantation
with or without vascular pedicle
(82)
VEGF None Biomineral-coated
PLG scaffold
Rat cranial defect (73)
VEGF
BMP-4
(plasmid DNA)
hBMSC Porous PLG Mouse subcutaneous implantation (40)
VEGF*
BMP-4*
hMDSC Gelfoam scaffold Mouse cranial defect
Mouse femur defect
(83)
ECs, endothelial cells; hBMSC, human bone marrow stromal cells; hMDSCs, human muscle derived stem cells; HOBs, human osteoblasts; HUVECs, human
umbilical vein endothelial cells; PLG, poly(lactide-co-glycolide); rBMSC, rat bone marrow stromal cells, VEGF, vascular endothelial growth factor.
*Retroviral-mediated gene delivery.
118
Hsiong & Mooney
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122
Hsiong & Mooney
Advances in dental implant
materials and tissue
regeneration
JAN EI RI K ELLI NGSEN, PETER THOMSEN & S. PETTER LYNGSTADAAS
In most tissue engineering strategies a highly por-
ous articial extracellular matrix or scaffold is
required to accommodate mammalian cells and
direct their growth, and hence the tissue regener-
ation, in three-dimensions (185). When seeded with
specic cells like stem cells or genetically enhanced
cells, such tissue engineering bio-scaffolds are
regarded as very promising and powerful strategies
for tissue and organ regeneration (28, 33). In bone,
the use of three-dimensional scaffolds for skeletal
tissue engineering has so far been limited because
of their lack of mechanical strength and rm
structure, and an inherent instability when loaded
(9, 19).
Metal implants and prostheses on the other hand,
having both the structure and mechanical strength
necessary for direct function in heavily loaded posi-
tions, such as joints and jaws, lack important features
like osteogenic or osteoconductive capacity and act-
ive surfaces for specic cell binding and growth.
Moreover, implanted biomaterials often induce the
formation of a poorly vascularized collagenous cap-
sule that can eventually lead to implant failure. This
process, often referred to as brous encapsulation,
develops in response to almost all implanted bio-
materials with smooth surfaces and consists of
overlapping phases similar to those in wound healing
and tissue repair processes (102). Recently, the use of
micro-rough surface topography has reduced the
extent of brous encapsulation of implants, and in-
creased the biomechanical properties of the implant
bone interface (1, 139). However, even though
reduced in size, the problem of foreign body reac-
tions and brous encapsulation still has a major
clinical impact and is considered an important issue
in all implant treatment strategies, including ortho-
pedics and dentistry.
Other strategies for improving the biocompatibil-
ity and osteogenic capacity of metal implants in-
clude surface modication by inorganic mineral
coatings, plasma spraying, particulates, or cements
containing a diversity of calcium salts, mainly cal-
cium phosphates, sulfates or carbonates. Biocoat-
ings of implant surfaces to control peri-implant
tissue responses are also under development (121).
The idea behind all these strategies is to make the
metal surface more acceptable to bone cells and, by
doing so, trick the body into rapid integration of the
implanted structure rather than brous encapsula-
tion. The success criteria for this strategy is two-
sided. First, to be successful the surface coating in
question has to have the (bio-)chemical and struc-
tural characteristics suitable for eliciting the wanted
cell activity (i.e. osteogenesis), resulting in full os-
seointegration. Second, the resulting boneimplant
interface must have the internal strength and
bonding capacity to the underlying metal surface to
endure the forces produced by implant loading. The
major challenge in implantology today is therefore
to combine current knowledge in materials science,
tissue engineering, and biology to design metal im-
plant surfaces capable of optimal bone bonding and
at the same time providing epigenetic signals to
cells in the peri-implant tissues to elicit proper
biological responses that favor bone healing and
osseointegration over adverse effects and sub-opti-
mal treatment outcomes. Thus, to achieve an opti-
mal biological reaction in the bone following the
placing of an implant, and to develop new biomi-
metic strategies for improved implant performance
136
PERIODONTOLOGY 2000
in bone, three major factors must be optimized and
carefully controlled:
the surface structure and surface chemistry of the
implanted device.
the nature and quality of the tissueimplant
interface and the peri-implant environment.
the physiology and biology of the healing process
in the compromised bone.
Calcium phosphates
Calcium phosphate (CaPO
4
) materials have been
suggested to possess several interesting properties in
the context of implantable devices:
similarity in composition to bone mineral.
the ability to form carbonate hydroxyapatite on
their surfaces.
the ability to promote cellular function and
expression leading to formation of a strong bone
CaPO
4
biomaterial interface.
provision of an appropriate scaffold for bone for-
mation.
the ability to bind and concentrate endogenous
bone morphogenetic proteins (111).
Chemical analyses have shown that the biological
apatites in enamel, dentin, and bone are not pure
hydroxyapatite but contain ions such as CO
2
3
,
HPO
2
4
, F
)
, Cl
)
, Mg
2+
, Na
+
, or K
+
and some trace
elements (Sr, Zn). The bone mineral known as cal-
cium hydroxyapatite, [Ca
10
(PO
4
)
6
(OH)
2
] is indeed a
carbonate hydroxyapatite by the formula: (Ca,Mg,
Na)
10
(PO
4
HPO
4
CO
3
)
6
(OH)
2
. Pure synthetic calcium
hydroxyapatite was synthesized in the early 1970s
(113). Incorporation of different ions is associated
with changes in morphological features of crystal size
and shape and in the dissolution properties of the
apatite. Thus, substituted ions, such as Mg
2+
and
CO
2
3
, are suggested to cause reduction in the crystal
size of the apatite with enhanced dissolution rate of
the apatite (115, 130).
Hydroxyapatite and glass ceramics are considered
as bioactive materials whereas titanium is inert (133).
Bioactivity is referred to as the characteristic of an
implant material which allows it to form a bond with
living tissues (72). Bioactive materials are suggested
to be osteopromotive (class A) or osteoconductive
(class B) (74). While the former have been suggested
to allow colonization of their surfaces by osteogenic
stem cells at the implantation site subsequent to
insertion, the latter allow only bone ongrowth.
According to this classication the synthetic hy-
droxyapatite is designated as a class B bioactive
material and glass-ceramic is designated a class A
material.
Bioactivity has been associated with materials
allowing the formation of carbonate hydroxyapatite
on their surfaces when immersed in simulated body
uid in vitro (99, 111). Microcrystals identied as
carbonate hydroxyapatite have also been observed in
conjunction with CaPO
4
materials which have been
implanted in bone (112, 114, 171) as well as in soft
tissues (76, 97, 189, 190).
Bioactive biomaterials develop direct, adherent,
and strong bonding with the bone tissue (73, 87).
Several biomechanical investigations have shown
higher bond strength for hydroxyapatite-coated
implants than for non-coated controls (36, 70, 71). A
number of interfacial bonding mechanisms have
been proposed for CaPO
4
ceramics (88). Epitaxial
crystal growth (114), chemical bonds (128), and
mechanical interlocking of resorbable CaPO
4
ceramics with bone (127) have been suggested.
As a result of the brittleness and low fatigue
resistance, bioactive surface coatings for implants
were developed (34, 88). Several coating techniques
have been described, including ame spraying,
sputtering, electrophoretic coating, hot isostatic
pressing, and solution coating. Details on the differ-
ent coating methods, their advantages and disad-
vantages, and elds of application are found in Refs
(75, 186, 187).
Plasma-spraying has been the method of choice for
CaPO
4
coating on metal substrates used as clinical
implants. The plasma-sprayed CaPO
4
surfaces have
irregular surface topographies (175) and reported to
consist of a mixture of amorphous and crystalline
CaPO
4
phases (25, 85, 193).
A greater boneimplant contact and/or improved
biomechanical stability have been demonstrated for
plasma-sprayed CaPO
4
-coated implants compared to
non-coated Ti and Ti6Al4V implants (22, 50, 60, 65,
81, 183). In addition, a dissolution of plasma-sprayed
hydroxyapatite coatings has been observed in vivo
(22, 35, 98). However, the observations of a non-
uniform and partial degradation of CaPO
4
plasma-
sprayed coatings after implantation (61, 80, 81) and a
relationship between fragments from fractured coat-
ings and adverse cellular reactions in the interfacial
bone, have been suggested to adversely affect the
long-term integration (32, 61).
Another aspect is the propensity of materials to
contribute to established infection (132). So far there
are not enough studies reported which would allow
any denite conclusion as to the clinical performance
of oral CaPO
4
-coated implants in comparison with
137
Advances in dental implant materials and tissue regeneration
non-coated implants. A problem is the variability in
the composition and microstructure of both experi-
mental and commercially available hydroxyapatite-
coated oral implants (62). Nevertheless, the clinical
survival data reported for hydroxyapatite coatings on
oral implants were similar to those reported for non-
coated titanium (110) and retrieval of hydroxyapatite-
coated implants after 10 years of functional loading
in mandible have revealed maintained coating and
high implantbone contact (172).
The introduction of the concept of thin (in the
micrometer and sub-micrometer range) coatings of
CaPO
4
to the implant surfaces may provide an
alternative approach, providing a bioactive surface
for bone formation and possibly chemical bonding,
without increasing the risk for fragmentation of the
coating. Magnetron-sputtering, pulsed-laser depos-
ition, and ion beam-assisted deposition represent
such preparation techniques. For technical and bio-
logical results in this eld, the reader is referred to the
reviews by Jansen et al. (86) and Yang et al. (186).
Several results and issues related to thin CaPO
4
coatings remain controversial, contradictory and
hence, unsolved, including the optimal characteris-
tics of the underlying substrate, the optimal Ca:PO
4
ratio, the biological effects of the amorphous and
crystalline nature of the coatings and the fate of the
coating over time. Animal experiments have shown
that thin CaPO
4
coatings, in comparison with both
minimally or markedly roughened surfaces, stimulate
both increased short-term (30, 90, 125, 129, 131, 134)
and long-term (126) bone apposition and/or biome-
chanical strength (Fig. 1). Therefore, this line of re-
search appears promising also from an application
point of view. Hitherto, the major oral implant
manufacturers have been reluctant to modify the
substrates of their oral implant surfaces with such
thin micron and sub-micron coatings. To the authors
knowledge, no comparative data on currently avail-
able titanium surfaces and thin CaPO
4
-coated tita-
nium in humans have been reported. This stands in
contrast to the emerging clinical benets of ortho-
pedic prostheses with novel coatings [reviewed by
Lappalainen & Santavirta (105)].
The precise mechanisms for the bone-promoting
effects of CaPO
4
-coated titanium implants are not
understood. Although the chemistry of CaPO
4
ap-
pears to be crucial, the surface topography has been
suggested as a dominant factor (66). Future strategies
to induce bone formation and to circumvent infec-
tion, excessive bone resorption, and other adverse
events, are likely to involve pharmaceuticals, cells,
growth factors, extracellular matrix proteins, and
antibacterial peptides. Thin coatings of CaPO
4
rep-
resent interesting vehicles for such efforts. One
example is the immobilization of bisphosphonates, a
pharmaceutical used in the treatment of osteopor-
osis, which when combined with thin CaPO
4
coating
results in a higher bone contact than control blasted
and etched surfaces (188).
Advances in surface texture
technology
Albrektsson et al. (6) dened osseointegration as a
direct contact between living bone and implant on
the light microscopical level. He suggested six factors
Fig. 1. Imaging (annular dark-eld STEM) of the interface
between bone and titanium coated with 0.1-lm thick,
crystalline CaPO
4
coating. Specimen was retrieved
6 weeks after insertion in rabbit tibia, non-decalcied,
embedded, and processed using focused ion beam
microscopy. Ultrathin section was obtained after lift-out,
mounted on TEM grid and milled to electrotransparency
(100 nm thick). The gure reveals an intimate contact
between (from left to right) bone, a 0.1-lm CaPO
4
coating
(as revealed by elemental mapping of Ca and P; data not
shown), an approximately 0.5-lm thick titanium dioxide
and titanium metal. Electron diffraction (inserts) shows
(from left to right) crystalline hydroxyapatite, rutile tita-
nium dioxide and titanium metal. The image is the result
of the new focused ion beam microscopy preparation
technique for preparing intact metalbone interfaces and
strongly suggests a role of ultrathin CaPO
4
coatings for
improving the integration of titanium in bone. Repro-
duced by courtesy of John Wiley & Sons, Publishers from
Engqvist et al. A novel tool for high-resolution transmis-
sion electron microscopy of intact interfaces between
bone and metallic implants. J Biomed Mater Res, in press.
138
Ellingsen et al.
as prerequisites for establishing reliable osseointe-
gration: implant material, implant design, surface
quality, status of the bone, surgical technique, and
implant loading conditions. The role of material
properties for achieving a successful long-term clin-
ical performance is related to the type of local tissue
conditions and clinical needs. For the majority of
long-term implanted materials in bone, the inertness
of a material is usually the preferred characteristic
(181).
The surface properties of materials are regarded as
decisive for the tissue response in association with
materials. The physico-chemical aspects of surfaces
and techniques to characterize surfaces are discussed
in depth in the review by Voros et al (173). The sur-
face aspects of materials may be highlighted for
several reasons:
The surface of the material is different from the
bulk material. The traditional techniques for
analyzing bulk material properties are therefore
different from those employed for surface charac-
terization.
The surface of the material is reactive. Atoms lack
stabilizing bonds to neighboring atoms. The
surface tends to minimize its surface energy to
provide stability. For example, contamination of
surfaces reduces the interfacial energy.
The contact between implant material surfaces and
biological components, such as proteins and cells,
and the outcome of this contact (protein adsorp-
tion, cascade reactions, and cell behavior) are
highly dependent on the properties of the material
surface.
The characterization of surfaces using surface-
sensitive techniques has revealed a number of
parameters which may describe the surface
properties, including chemical composition,
structure, roughness, wettability, electro-optics
and mechanics.
The correlation between material surface proper-
ties and biological processes is a key topic for
future research, ultimately aiming towards the
engineering of surface features for specic, wanted
biological reactions.
Methods for surface texturing
Mechanical, chemical, and combined mechanical
chemical techniques are used to modify the surface
roughness (and chemistry) of materials.
The mechanical techniques induce the shaping of
material surfaces by physical forces. The most
commonly employed mechanical techniques are
machining, polishing, and blasting. The surface
topography of a machined/turned oral implant is
characterized by grooves and valleys more or less
oriented along the machining direction. Such surfa-
ces are represented by the oral titanium implant
surfaces pioneered by Professor P-I Branemark. Pol-
ishing techniques are often based on the removal of
material by a hard, abrasive medium while using a
lubricant. Grit blasting or abrasive blasting are fre-
quently used techniques for increasing the surface
roughness of oral implants. The surface roughness
may be varied depending on the particle size. It is
important to realize that any mechanical technique
applied for surface modication may also lead to
changes of the surface chemical properties.
Although machining, blasting, and etching tech-
niques constitute the most common production
methods for the currently available oral implants,
new techniques for surface chemical and topo-
graphical modications will appear within the eld of
oral implants. An emerging area, still at the experi-
mental stage, is the use of photolithography to pro-
duce microfabricated surfaces. Such surfaces, made
of silicon and titanium, incorporate intentional sur-
face chemical and topographical features in the
nano- and micrometer scales and provide far greater
opportunities to study cell behavior [the interested
reader is referred to the review by Jaeger and Bru-
nette (84)]. A crucial need is to scale up the produc-
tion of dened and controlled shapes, patterns, and
dimensions on more complex designs of medical
devices. An interesting technique for micromachin-
ing complex three-dimensional geometries such as
oral implants is the use of a high-energy laser.
Hitherto, few biological studies using this approach
have been published but there is a recent review on
technical details of lasers for potential biomedical use
by Kurella & Dahotre (101).
A crucial task is to correlate the surface topo-
graphical features with molecular and cellular reac-
tions at the surface or, as is usually the case for
in vivo implants, in the vicinity of the implant. A
necessity for such studies is a proper analysis of the
surface topography. The analyses include stylus
(mechanical stylus prolometry), optical (non-con-
tact laser prolometry, confocal laser scanning
microscopy, interference microscopy), scanning
electron microscopy, and scanning probe microscopy
(e.g. scanning tunneling microscopy and atomic
force microscopy) techniques. Each technique dis-
plays its advantages and limitations, e.g. standardized
or not, destructive or non-destructive, lateral and
vertical resolution, measurement area, suitability for
139
conductive or non-conductive surfaces, reective or
non-reective surface, time consumption, etc. For
example, the mechanical stylus tip, laser beam, and
electron beam approaches provide different possi-
bilities to resolve the surface.
Roughness parameters are divided into three
groups: amplitude, spatial, and hybrid parameters.
The surface topography consists of different wave-
lengths, as a result of the waviness, form, and
roughness of the surface. Since a selected Gaussian
cut-off lter separates the waviness and form from
the roughness, the interpretation of the roughness
parameters requires information about the lter and
size. Furthermore, because roughness parameters are
scale-dependent, data on roughness obtained with
different analytical techniques can only be compared
within the same spatial frequency domain.
For several reasons, it is therefore wise to select
complementary techniques, such as stylus, optical
and scanning probe microscopic techniques to
characterize oral implant topography.
The surface topography of oral implants has been
suggested to be characterized by at least three
parameters: one height parameter, one spatial
parameter, and one hybrid parameter. A compre-
hensive review of the different techniques, including
their advantages and limitations, is found in Voros
et al. (173).
Biological effect of microtextured
surfaces
Micro- and nanotextured surfaces have multiple
effects on cell behavior (31, 55). Material surface
topographical features from the nm to the mm range
are important for cellular responses as well as the
integrated tissue response to implants: dimensions in
the <1 lm range have an inuence on focal contacts,
cytoskeleton, adhesion, morphology, and orientation
of cells (15, 24, 179); cell adhesion, morphology, and
orientation, as well as bone formation, are inuenced
by dimensions in the 1100 lm range (24, 93, 179)
whereas the vertical and lateral dimensions in the
>100 lm range are important for mechanical inter-
locking (136).
It is generally considered that machined titanium
surfaces promote bone formation close to their sur-
face, however, this is not primarily via bone growth
directly on the surface but via bone growing toward
the surface. The adaptation of bone to the machined
surface includes the formation of mineralized bone
matrix close to the titanium oxide but usually inter-
cepted by an amorphous zone (106). The claim for a
roughened oral implant surface is usually not based
on evidence for bioactivity, chemical bonding or an
altered ultrastructural zone of contact but in general
is based on an improved micromechanical interlock
(136). This is also demonstrated in the majority of
papers, which compare the biomechanical strength
of the implantbone junction of roughened surfaces
with smoother surfaces.
It is generally agreed that an increase of surface
roughness promotes the incorporation of implants in
bone (17, 20, 58, 59, 117, 168, 169, 176, 177) although a
large number of experimental studies (reviewed in
Ref. 106) indicate that the responses in bone to in-
creased material surface roughness are far more
complex than is usually believed. In a majority of
studies it has been shown that the mechanical inter-
locking in bone is promoted by the increase of surface
roughness. The reader interested in the biological
in vivo results of machined and roughened titanium
surfaces is referred to the comprehensive reviews by
Esposito (47) and Buser (16). Recent review of the
literature concludes that minimally rough (average
height deviation from a mean plane [Sa] 0.51.0 lm)
oral implants have the longest clinical documentation
of all implants and that moderately rough (Sa 1.0
2.0 lm) implants have a tendency to better clinical
results than turned implants (5).
Are there negative effects of an increased surface
roughness? In a clinical setting, a risk analysis is
important before the introduction of new implant
surface modications. Apart from mechanical fail-
ures (fractures) there are no rm data establishing
that failures of oral titanium implants are ascribed to
material properties per se. In fact, early biological
failures are caused by infection, excessive surgical
trauma, micromotion, and impaired healing whereas
late failures are the result of overload and peri-
implantitis (46, 48).
The only negative clinical effect for titanium dental
implants with a rough surface shown in the scientic
literature through a meta-analysis of three random-
ized clinical trials (49) was a borderline statistically
signicant difference: implants with rougher surfaces
(specically titanium-plasma sprayed surface with a
Sa roughness equivalent to 2.35 lm) (no longer mar-
keted) showed an increased risk of 20% when com-
pared with turned surface Branemark implants for
developing peri-implantitis after 3 years in function.
Inammatory cascade systems are triggered by
materialsurface interactions. An important aspect of
the inammatory response is the role of the implant
surface for promoting and down-regulating leukocyte
accumulation, adhesion, and secretion of proin-
140
Ellingsen et al.
ammatory substances. Data indicate that inam-
matory cells respond to surface texture and the
expression and secretion of proinammatory cytok-
ines is increased on rough titanium surfaces com-
pared to relatively smoother surfaces (153). Under
in vivo conditions, relatively smooth, machined tita-
nium surfaces elicit a rapid and relatively strong
inammatory response, distinctly higher than that
resulting from the surgical trauma per se and initially
even higher that that demonstrated for cytotoxic
materials, like copper (164166). A crucial observa-
tion in these latter studies is that the inammation
has a transient character around machined titanium
surfaces (Fig. 2). An important area for future re-
search is therefore to identify the surface properties
of titanium that are decisive for this transient
appearance of inammation.
Titanium dioxide
The chemical qualities of the surface are of import-
ance for the biological reactions that take place after
inserting the biomaterial into living tissues such as
bone. The implants surface chemistry, together with
the surface texture on micro- and nanolevels, will
characterize the biomaterial and, depending on these
physical, chemical, and biochemical qualities, will
make the surface attractive for biological molecules
and ions to react and initialize the osseointegration
process. The natural or non-modied titanium sur-
face is highly reactive and an oxide lm forms
spontaneously in a few nanoseconds when exposed
to oxygen in air (108, 124, 137, 152). This oxide lm,
consisting of different oxide states, is considered
essential for the biological performance of the im-
plant and the chemical stability of this surface oxide
makes the corrosion resistance and repassivation
ability of titanium excellent (167). Spectroscopic
studies suggest that the oxide layer on commercially
pure (c.p.) titanium dental implants is amorphous,
morphologically homogeneous and approximately
217 nm thick. With increasing thickness, the oxide
will become increasingly crystalline with a texture
corresponding to the grain structure of the oxidized
metal. It is the naturally formed oxide lm that comes
in contact with the biological environment after an
implantation into bone. The biological reaction tak-
ing place after this implantation with ions, proteins,
and other molecules and cells will depend on the
qualities of this oxide. It is thus the titanium oxide
lm, with a thickness of only a few nanometers,
which denes the biological properties of the tita-
nium implants that are important for the healing
reaction and not the metallic titanium as such.
The surface oxide layer of titanium has many of the
qualities regarded as important for a reaction with
bone. The oxide has a low solubility and an isoelectric
point between 3.5 and 6.7 (178). This makes the
surface only slightly negatively charged at physiolo-
gical pH, which is thought to reduce the ability for
capsule formation after insertion into bone and is
also thought to lead to favorable reactions with bio-
molecules. The dielectric constant of titanium diox-
ide is comparable to that of water, which makes its
interaction with charged molecules similar to that of
water. The titanium dioxide also has low or no tox-
icity and the growth of osteoblast cells on titanium
dioxide-covered titanium surfaces demonstrates the
inertness of this substance (100, 156159, 180).
The naturally formed oxide lm is quickly estab-
lished, but increases slowly in thickness over time
and under stable conditions. Several factors, inclu-
ding temperature and humidity, inuence the rate
and thickness of the oxide formation and make the
surface passive in biological tissues.
Modication of the surface oxide
The surface oxide may be modied using different
techniques such as heat treatment, sol gel-derived
oxidation, and electrochemical oxidation. The inter-
pretation of the biological response to such implants
is, however, not straightforward. The surface oxide is
0
50
100
150
200
0 10 20 30 40 50
Ti
Cu
Sh
T
N
F
-

,

(
p
g
/
m
l
)
implantation time, (hours)
Fig. 2. Tumor necrosis factor-a (TNF-a) concentration

(pg/ml) in peri-implant exudates 148 h after implanta-
tion in soft tissues. Peri-implant exudates around tita-
nium had higher concentrations of TNF-a than exudates
around copper at early times (<18 h) after implantation in
this rat subcutaneous model. TNF-a concentrations de-
clined around titanium with time and became comparable
with the levels at sham-operated sites at 48 h. A similar
high, but transient TNF-a secretion pattern was observed
ex vivo. Reproduced by courtesy of Elsevier Science Ltd.
from Ref. 165.
141
usually altered with respect to several different
properties and it is difcult to isolate the specic role
of changes in surface chemistry, crystallinity, surface
roughness, and porosities on different scales for the
observed biological response.
The TiUnite implant is manufactured using anodic
oxidation, an electrochemical technique that in-
cludes the use of an electrolyte. The details of the
procedure and the electrolyte have not been revealed
in the published literature. It has been reported (4)
that the TiUnite surface contains phosphorus ions,
indicating the presence of phoshoric acid in the
electrolyte. The roughness of the surface was repor-
ted to be 0.51.0 lm for the upper, thin oxide layer
and Sa >2 lm in the apical region, having a thicker
oxide (in the range of more than 10 lm).
In vitro studies focusing on the early (minutes to
hours) response of peripheral blood leukocytes to
smooth or rough titanium dioxide surfaces with ei-
ther thin or thick oxides concluded that topography
had a greater impact on most cellular reactions
studies whereas oxide thickness appeared to have a
dampening effect on the responses (45).
Recent studies in vitro examined human monocyte
adhesion, viability, differentiation, and cytokine
secretion to smooth turned surfaces (Sa 0.19 lm),
surfaces blasted with 75-lm size Al
2
O
3
particles (Sa
1.02 lm), micro-arch electrochemically oxidized
surfaces containing magnesium (Sa 0.27 lm), and
electrochemically oxidized TiUnite (Sa 1.13 lm) sur-
faces (57). The oxidized surfaces had higher numbers
of adherent inammatory cells, irrespective of the
presence of lipopolysaccharide stimulation or not. It
is difcult to draw any conclusions as to the role of
specic titanium oxide properties, e.g. if roughness
and chemical properties counteract each other or
have synergistic effects on cells in these studies, be-
cause of the lack of description of the electrolytes
and/or surface chemical composition. It is also too
early to extrapolate from these studies to studies
in vivo although it is indicated that any potential
negative drawbacks of oxidized surfaces for the
regulation of inammatory cells have to be excluded,
particularly in local environments where bacteria and
other microorganisms would be allowed to interact
with the porosities and textures on the micro- and
nanolevels.
Studies in vivo on the biological effects of oxidized
titanium implants demonstrate contradictory results.
Several studies demonstrate no (91) or only early
(106) morphological and biomechanical differences
compared to control implants. Other experimental
studies on oxidized titanium implants show equal or
greater bone formation and/or biomechanical per-
formance in comparison with control implants
(usually machined/turned implants) (163, 162). It has
been suggested that the enhanced osseointegration
would be the result of either (an increased)
mechanical interlocking or a biochemical bonding.
The data published so far for oxidized implants
indicate ndings of strong anchorage but the current
data do not provide evidence for a biochemical
bonding.
Experimental studies in posterior maxillae have
suggested on the other hand that oxidized implants
(TiUnite) exhibit osteoconductive properties (79)
even exceeding those of calcium phosphate-coated
implants (184). The ultrastructural appearance of the
oxidized implantbone interface is not yet reported.
Furthermore, the relationships between specic
surface chemical and topographical features of the
different oxidized implants and the described osteo-
conductivity remain unanswered. Among such
surface characteristics, the elemental and phase
composition of the oxide are target areas for future
research. Hitherto, no randomized controlled clinical
trials have been published on the performance of
oxidized implants.
The sol-gel technique is another interesting meth-
od for the modication of oral implant surfaces.
Hydrophilic, roughened and partly porous sol-gel-
processed titanium alloy surfaces reveal higher
osteoblast-like cell adhesion and mineralization
in vitro (2).
The Turku group used early and late time
implantation periods in rodents for the biological
evaluation of sol-gel-coated, heat-treated, and laser-
treated, c.p. titanium implants (135). Light micro-
scopic evaluation revealed a close contact between
titania coated titanium implants and soft tissues. This
contact was referred to as bonding or adhesion.
Further work with sol-gel-derived non-resorbable
reactive titania coatings by this group resulted in an
initial, immediate contact with components of the
peri-implant gingival tissues, in contrast to control
titanium implants (7). Although not yet clinically
proven, the approach opens up new possibilities to
improve the integration and use of materials in soft
tissues, an area that, in comparison with bone im-
plants and substitutes, has been largely neglected.
Oral, machined titanium implants were introduced
using a two-stage, surgical procedure. This was the
early, empirical, and logical way of permanently
anchoring a load-bearing implant in bone without
jeopardizing the adaptation of bone and soft tissues
to the implant. The machined implant, having a
142
Ellingsen et al.
titanium dioxide and moderately roughened surface,
has an undisputed long-term, successful clinical
performance. For normal clinical indications this
approach would be sufcient. When appropriate
surgical techniques and treatment schedules are
employed, it appears that currently available oral
implants possess adequate surface properties to be-
come osseointegrated long term.
Which are the situations when implant surfaces
need to be rened and/or adjuvant therapy initiated?
Much interest is now focused on improving the
likelihood for success under early/immediate loading
and in locally impaired healing conditions.
Chemically modied titanium oxides and
bone response
The surface oxide layer of titanium implants can be
manipulated chemically and there has been specu-
lation whether the biological properties of the oxide
surface may then be changed and even improved as a
result. The signicance of surface chemistry can be
illustrated by the different cellular responses reported
for different titanium alloys (14), different grades of
c.p. titanium (3), and different bulk metals (146, 151).
Chemical modication of titanium surfaces by
treatment in simulated body uid (147), covalent
attachment of biological molecules (10), changes in
the surface ion content (78), glow discharge (8), or
alkali treatment (119) are reported to affect cellular
responses to the implant. The effect of biological
grafting with adhesive polymers (170) to biologically
activate the implant surface and alteration of surface
hydrophobicity (18) have also been exploited. These
different studies indicate that even modest changes
in the surface chemistry of c.p. titanium will inu-
ence culture cell responses.
Fluoride
By using hydrogen uoride at low concentrations it is
possible to manipulate the titanium dioxide without
changing the surface microtexture signicantly.
Small amounts of uoride will then be incorporated
into the crystal structure of the titanium dioxide.
Such an ion implantation into the supercial layer of
a biomaterial may then change the biological re-
sponses to the implant based on the biological
activity of the ions used. Fluoride has been selected
for supercial modication of titanium dioxide based
on the specic attraction uoride has for calcium and
phosphates and the known benecial effects of clin-
ical importance that can be observed when this ion is
in contact with calcied tissues (11, 56). Trabecular
bone density is increased and calcication of bone is
induced when uoride is present during bone re-
modeling (51, 141). Fluoride has a specic attraction
for skeletal tissue and has also a specic mitogenic
activity and attraction for cells of skeletal origin.
Fluoride seems to be an enhancer of bone cell growth
factors and seems also to require the presence of
appropriate growth factors to induce calcication
(107). Osteoprogenitor cells and/or undifferentiated
osteoblasts, synthesize an abundance of growth fac-
tors and there is evidence that uoride may act pri-
marily on these cells rather than stimulating the
proliferation of highly differentiated osteoblasts (12,
94, 95). Such an effect is important in bone healing
and bone regeneration because it will induce the
differentiation of osteoprogenitor cells and undiffer-
entiated precursor cells into osteoblasts. Local sti-
mulation of the natural formation of growth factors
by the tissues osteoprogenitor cells is an interesting
approach rather than introducing high doses of
recombinant growth factors into the patients tissues
(Fig. 3).
This effect on calcied tissue and attraction of the
activity of these cells is also induced because the
bone cell mitogenic action is improved and uoride
treatment of bone cells has been shown to trigger an
acute increase in intracellular calcium levels (191).
Increases in intracellular calcium have been associ-
ated with cell proliferation and it has been suggested
that this may be an effect of the action of uoride.
Fluoride may thus promote bone formation or
regeneration not only on a physicalchemical level
but also at the cellular level in its function as a
modulator for the intrinsic formation of growth fac-
tors by the osteoprogenitor cells, and through this
effect it may catalyze bone formation. In addition, on
a cellular level, uoride seems to be potentiated by
mechanical stresses, indicating that there is a syner-
gism between weight-bearing activity and uoride
action. Such an effect would be clinically important
for the integration and stability of dental implants.
The uoride-modied titanium dioxide has modi-
ed surface properties in reaction with calcium
phosphates compared with non-modied titanium
dioxide. The binding or nucleation ability of calcium
and phosphate ions to the implant surfaces indicate
how well this surface would perform after placement
in bone and indicate a possible clinical response.
This property of the uoride-modied surface was
evaluated in a nucleation test system using a super-
saturated solution of calcium and phosphate with
143
radioactively labeled phosphorous,
32
P (41). Titanium
dioxide blasted implants, F-modied/non-modied,
were immersed in the calcium-phosphate solution
and allowed to react. The radioactivity of the im-
plants exposed to the
32
P-labeled calcium phosphate
solutions was recorded with a Geiger counter as a
measure of the uptake of phosphate at the surface.
The uptake of phosphate was signicantly higher at
the uoride-modied implant surfaces than at the
non-modied titanium dioxide surfaces demonstra-
ted by an elevated level of radioactivity at these sur-
faces. Phosphate afnity was thus higher for the
uoride-modied titanium implants, an important
property of a bone xating biomaterial. This quality
will make the implant surface attractive to calcium
phosphate nucleation after implantation and may
thus permit the calcication and bone growth at the
implant surface shortly after the operation.
The quality of the modied titanium surface in
binding calcium and phosphate was further evalu-
ated by analyzing the implant surfaces by scanning
electron microscopy. This analysis showed that the
uoride-modied implants exhibited calcium phos-
phate precipitates all over the surfaces, demonstra-
ting an afnity for calcium phosphate and a further
crystallization on the uoride-modied titanium di-
oxide. This was not observed on the non-modied
titanium dioxide surfaces. This physicalchemical
reaction may be the result of changes in the crystal
structure of the titanium dioxide with incorporation
of uoride ions, or may be because of the small
change in nanostructure also observed after this
modication, or a combination of these (Fig. 4A,B).
Fig. 4. Fluoride modication of the titanium dioxide sur-
face changes the physico-chemical property and the
ability to react with calcium phosphate. Exposed to cal-
cium phosphate in solution a reaction takes place and a
nucleation of calcium phosphate crystals occurs on the
implant surface as can be seen on the SEM (A). This
reaction does not happen on non-modied implant sur-
faces (B). This nucleation promoting property may be
important for the initial healing after implantation. An
attraction of calcium phosphate to the surface in the ini-
tial healing phase will be important for a fast minerali-
zation at the implant surface. Reproduced by courtesy of
Applied Osseointegration Research.
Stem cell
Pre-osteoblast
Osteoblast
Osteoid
Old bone
New bone
Fig. 3. Fluoride is suggested to have an action on osteo-
progenitor cells and undifferentiated osteoblasts. This
may induce the differentiation of these precursor cells
into osteoblasts and subsequently new bone formation. In
a clinical situation this may give a faster bone healing
after implantation and more supporting bone surround-
ing the implant. Reproduced by courtesy of Applied
Osseointegration Research.
144
Ellingsen et al.
Osseointegration represents the processes of oste-
oinduction, osteoconduction, and osteogenesis (27).
The recruitment of precursor bone marrow stromal
cells to osteoblasts is regarded as a central step in
osseointegration and as important and signicant for
the biological effects of an implant surface. Masaki et
al. (122) compared the cell responses of osteoblasts
(human palatal mesenchymal cells, HEPM 1486,
American Type Culture Collection) grown on tita-
nium with different surfaces. They found that the
mRNA level of the transcription factor RUNX-2 was
signicantly (P < 0.01) increased on the uoride-
modied (Osseospeed) surfaces compared with the
controls. RUNX-2 plays an essential role in osteo-
genesis, osteoblast matrix formation, chondrocyte
differentiation, and bone resorption by osteoclasts
(77). This transcription factor regulates osteoblast
differentiation and expression of bone extracellular
matrix protein genes that encode for bone sialopro-
tein, osteocalcin, and collagen I (38, 69) and could
therefore be a downstream target of extracellular
matrix adhesion-mediated signaling pathways. A
second transcription factor, Osterix, has been sug-
gested to play a key role downstream of RUNX-2 in
which its expression is necessary for the ongoing
differentiation within the osteogenic pathway. The
expression of Osterix was also tested in this study and
these authors showed signicant increase (P < 0.05)
of Osterix in these osteoblast cells grown on the
uoride-modied titanium surfaces. The effect on
osteogenesis of uoride ion modication of titanium
dioxide grit-blasted c.p. titanium implants as mea-
sured in cell culture assays has also been investigated
by Cooper et al. (29). Titanium implant surfaces of
different roughness and with different levels of
uoride ion modication of the titanium dioxide
surface layer were tested on human mesenchymal
stem cells. The proliferation of adherent cells in this
test was distinguished among some of the surface
groups and the analysis showed that at 24 h, cells
adherent to uoride-modied surfaces displayed
greater proliferation than cells grown on the surfaces
that were not modied. Osteoblastic differentiation
of human mesenchymal stem cells cultured on dif-
ferent titanium surfaces indicated that small grit/
uoride and large grit/uoride surfaces supported
osteopontin expression and large grit/uoride
surfaces supported early osteocalcin at day 7. The
steady-state levels of bone sialoprotein mRNA were
greater on the uoride-modied surfaces than the
unmodied surfaces at day 14 of this experiment.
After 28 days of culture, osteopontin and osteocalcin
expression was notably higher in human mesenchymal
stem cells cultured on the uoride-modied surfaces.
An increased osteogenesis was demonstrated by
earlier and more abundant bone sialoprotein and
bone morphogenetic protein-2 expression of the
human mesenchymal stem cells adherent to uoride-
modied titanium dioxide grit-blasted c.p. titanium.
This was also associated with increased surface-
directed osteogenesis in vivo and bone formation
with the uoride-modied c.p. titanium endosseous
implants when tested in a rat model of osseointegra-
tion. Bone was more continuous in its apposition
along the implant surface and signicantly more bone
was formed directly on the uoride-modied im-
plants than the implants with non-modied surface.
This in vivo observation conrms earlier observations
with the uoride-modied titanium implants.
Ellingsen et al. (43) observed signicantly more
bone in contact with the uoride-modied implant
surfaces than with non-modied control implants
following a healing period of 1 month in rabbit tibial
bone. Already after 1 month the degree of bone-to-
implant contact with uoride-modied titanium
implants was signicantly higher than with non-
modied implant surfaces. After a 3-month healing
period the percentage of bone area surrounding the
uoride-modied implants was signicantly higher
than the bone surrounding the non-modied im-
plants. A tight contact between the bone and the
implant surface was reported indicating a faster bone
regeneration process in the areas close to the uoride
implant surfaces. The higher bone-to-implant con-
tact and binding strength was also demonstrated
through removal torque tests with an improved
retention of the uoride-modied implants. After a 3-
month healing period a mean removal torque of
85 N cm was recorded for the uoride-modied im-
plants in contrast to a mean removal torque of
54 N cm for the non-modied control implants. The
maximum torque level was as high as 119 N cm in
the uoride-modied group and only 79 N cm in the
group of implants with non-modied titanium diox-
ide at the surface. This effect of increasing torque has
been observed and documented for different healing
periods up to 12 months in rabbits, with a continu-
ous increase in the retention with longer healing
time. A 40% higher retention (removal torque) was
observed with the uoride-modied Osseospeed
implants than was recorded for the implants with the
TioBlast surface (89).
In the bone marrow area there is normally no min-
eralized bone, but precursor cells that can be guided
into osteoblast differentiation when given the
right stimuli and so form new bone. When the
145
uoride-modied implants were placed into the bone
marrow region new bone was formed on the implant
surfaces and this new bone seemed to cover these
surfaces (40, 42). This new bone formed on the im-
plant surfaces both in the cortical and in the marrow
regions; it had a very strong retention to the surfaces
and was not removed from the surface even in the
removal torque test. This new bone formation was not
observed when implants with non-modied and
blasted surfaces were used demonstrating that the
uoride-modied implants evoked a different
response from the osteoblast cells. The uoride
modication of blasted titaniumsurfaces causes small
changes in the titanium dioxide layer and crystal
structure with incorporation of small amounts of
uoride. It has been suggested that this modication
of the surface chemistry is the cause of the signicant
improvements in bone healing observed. An addi-
tional effect may also be induced by changes of the
surface topography at the nanoscale level as is
observed at high magnication of the surfaces
(Fig. 5AC).
Induction of peri-implant tissue
regeneration
Studies with modied implant surfaces have shown
that the interaction of the implant with its biological
environment, the formation of the implant mater-
ialtissue interface, and the long-term outcome of
implant integration are strongly dependant on the
surface properties of the implanted device, and the
ability of the organism to respond positively to such
surfaces (109). True integration of metal implants
in bone requires osteoinductive signals to be pro-
duced in the peri-implant tissues immediately after
implantation. Such osteoinductive signals are
mostly attributed to the bone morphogenetic pro-
teins, a subfamily within the transforming growth
factor-b superfamily of growth factors. These sig-
nals, believed initially to be expressed from macro-
phages during the initial bone healing, initiate a
cascade reaction that ideally should lead to osseo-
integration of the implanted structure (23). The
outcome of the implant procedure (i.e. failure of
A
B
C
Fig. 5. Six-year follow-up of a uoride-modied implant
(OsseoSpeed, Astra Tech) placed in a periodontitis-com-
promised area after extraction of upper left canine. A
porcelain fused to metal crown was cemented and the
implant was permanently loaded 4.5 weeks after place-
ment. The marginal bone has been stable from baseline
(A) to 6-year control (B, C).
146
Ellingsen et al.
osseointegration) is thus dictated by the initial tis-
sue response to the surface of the implant. All other
modes for success in osseointegration are secondary
to this single stage. With this in mind, it is clear that
the optimal implant surface should provide an in-
stant epigenetic signal for cells exposed to the sur-
face to express combinations of growth factors and
signal molecules optimized for rapid osseous heal-
ing and subsequent integration of the osseous im-
plant.
Growth factors in peri-implant healing
Bone-forming cells grow, differentiate, and maintain
their level of differentiation through a network of
epigenetic signals from other cells and from the
surrounding extracellular matrix. Growth factors
make up the predominant class of these intercellular
signal molecules. Growth factors are generally clas-
sied according to their ability to inuence prolifer-
ation or differentiation or both. However, it is now
well established that these effects are not constant for
each factor, but vary with concentration, cell type,
and developmental stage of the cells or tissue in
question (104). Moreover, most, if not all, growth
factors act in concert to vary differentiation and
proliferation of cells as part of interdependent mor-
phogenetic networks. This allows each growth factor
to exhibit multiple functions simply by up- or down-
regulating the expression of additional growth
factors.
A number of growth factors has been tested for
their effects on bone formation and implant osseo-
integration (23, 37, 53, 103, 143, 145, 148, 160). Of
signicant interest is the transforming growth factor-
b superfamily of polypeptide factors, including the
bone morphogenetic proteins, that control both cell
growth and differentiation, according to cell type and
state of differentiation (104), and has been shown to
directly affect expression of receptors involved in
surface recognition and cellular attachment to tita-
nium implants (145). The bone morphogenetic pro-
teins are multifunctional molecules that are able to
promote ectopic bone formation and modulate the
expression and organization of osteoblastic cell pro-
teins (192). For example, bone morphogenetic pro-
tein-2 treatment of osteoblastic cells has been shown
to signicantly affect the cytoskeletal and extracel-
lular matrix organization, and promote cellular
adhesion to titanium surfaces by increasing the
expression of bronectin and integrin receptor sub-
units with a subsequent rise in focal adhesion kinase
(p125FAK) activity (148). Moreover, studies have
shown that bone morphogenetic protein-2 can be
incorporated into biomimetic coatings on titanium
while retaining a biological activity benecial for
implant integration (118).
The importance of growth factors in general and
the bone morphogenetic proteins in particular, in
bone formation and bone repair can hardly be over-
estimated. Nevertheless, numerous attempts to use
growth factors for improving the osteogenic capacity
of implanted biomaterials have so far failed to reach
the clinic. The reason for this somewhat disappoint-
ing situation is most probably to be found in the
intrinsic nature of these molecules. Growth factors
are by nature free-oating, short-lived signals that
initiate or modulate small, incremental changes in
cells as part of cascade reactions. The effect of these
signals varies dramatically with cell-type, stage, and
location, with the condition of the surrounding tis-
sues, and with the presence of other growth factors.
Also, growth factors are temporal signals that are
mobilized substrates unsuitable for cell adhesion,
and their cellular receptors are not involved in cell
attachment. It should therefore not come as a sur-
prise that application of single growth factors alone
does not provide a signal potent enough to signi-
cantly improve implant integration in bone. Conse-
quently, to take full advantage of growth factors one
needs to attack the problem also at a more funda-
mental level where one can trigger an orchestrated
cascade of growth factor expression that provides a
full-scale, local signal for cells to kick-start and
maintain osteogenesis at a high level over a clinically
signicant time period.
Extracellular matrix components in
peri-implant healing
One possible route for triggering developmental-like
cascades of growth factor expression for bone
growth is to take advantage of the surface molecular
recognition apparatus for extracellular matrix lig-
ands. When an implant is brought into contact with
the body, the surface is instantly (within seconds)
covered by a proteinaceous lm that precipitates
out from tissue uids, ruptured cells, and blood.
The nature of this lm depends strongly on the
surface characteristics of the implanted device, in
particular the surface hydrophobicity, the zeta
potential and the surface charge density (96).
Investigations into the role of zeta potentials and
surface charge densities have shown that extracel-
lular organic matrix proteins are the main surface
charge regulators in hard tissues. In contrast to
147
mineral depositions, these organic matrix molecules
stabilize and pattern the mineraltissue interface in
bone and control the formation of the interfacial
structure and thus the stability of the tissuemineral
complex (96). In sum, the implant surface that is
exposed to cells is a composite bioactive surface
resulting partly from the chemical and structural
characteristics of the underlying implant, and partly
from the composition and thickness of the acquired
lm.
The cells, when attached to the implant surface, are
not blind, but see the surface and each other by
probing the surroundings with a complex mixture of
cell surface structures commonly known as receptors
and ligands that mediate cell adhesion and cell
signaling and communication (144) (Fig. 6). The cell
extracellular matrix interactions are analogous to a
compound cellular Velcro. Cells attach to the matrix,
to structures on the implant surface, and to each
other to form the structure of a tissue. The ligands
presented on the implant surface provide an address
code for a cell to adhere or to migrate, or an epi-
genetic signal for a certain cell to proliferate, grow,
differentiate, undergo structural alterations, or even
die. The signals from a ligated receptor may be
transmitted directly to the nucleus to activate or
inactivate gene transcription or mRNA translation, or
the signal might activate cellular kinases that can
affect a wide range of cellular processes including
secretion of growth factors and matrix molecules that
can replenish the extracellular ligand stocks and
reinforce the ongoing signal cascade. Alternatively, a
receptor ligated to the extracellular matrix may pro-
vide a direct link to the internal cytoskeleton that,
much like the skeleton of an organism, provides
structural integrity and dictates migratory behavior
and shape of the cell. This is the case for some inte-
grin receptors that connect the extracellular matrix
protein bronectin to the intracellular molecules
talin and vinculin, which in turn latch onto actin, a
major cytoskeleton component (144). In this way, an
integrin receptor binding to the extracellular matrix,
or to a bronectin coated implant, can directly affect
the shape and internal structure of the attached cell.
Integrins have also been suggested to play important
roles when cells adhere directly onto metals like
titanium, and it has been shown that the nature of
the metal alloy can inuence the expression pattern
of the involved integrins (150). In a third scenario,
ligands presented on the surface of one cell recruit a
second cell type into the tissue. This type of recruit-
ment is the basis for the immune system to function,
and is the way lymphocytes are recruited from the
circulatory system to initiate wound healing and re-
pair processes, including osseous healing around
implants.
Each type of cell has its own unique pattern and
combinations of surface recognition molecules,
and is genetically pre-programmed to launch its
responses when certain surface structures are
encountered. In addition, many different families of
receptors are present on the surface of a single cell,
and a large variety of ligands are displayed in the
extracellular environment. In fact, several extracel-
lular matrix proteins contain multiple domains that
make up a set of different ligands for different cells or
even different receptors on the same cell surface.
Such ligand clustering controls overall receptor
occupancy because the binding energy of a ligated
matrix receptor increases in the presence of neigh-
boring ligated receptors. This gives rise to a strong
ligand distribution effect where juxtaposition of
substrate-immobilized matrix ligands gives rise to a
non-linear positive receptor response to increasing
ligand density (82). This complexity in ligand pres-
entation, and the diversity of receptors on the cell
surfaces, makes it difcult to pinpoint the individual
roles of each ligandreceptor pair during intricate
processes like wound healing and histogenesis. It also
makes it difcult to improve the biological perform-
ance of implants by using free-oating ligands like
hormones or growth factors, that specically target
single receptors whose binding equilibria are little
inuenced by neighboring receptorligand com-
plexes. On the other hand, the redundancy and syn-
0.0
0.0
20.0 20.0
40.0
60.0
0.0 m
4.1 m
1.8
0.4
2.7
5.0
Fig. 6. Atomic force microscopy micrograph of a human
osteoblast cell attaching to a smooth titanium substrate.
The cell attaches to the substrate by focal adhesion. The
cytoskeleton of the cell, visible as ridges in the cell
structure, then assembles at the contact points and pro-
vides internal support for cell attachment and migration.
Once attached, the focal adhesion process also activates
cellular signal pathways, which allows the cell to respond
to the surface by migration, differentiation, secretion of
signal molecules or production of extracellular matrix
molecules for bone formation.
148
Ellingsen et al.
ergy in the receptorligand system for substrate-
immobilized ligands and surface recognition ensures
robustness and stability to the process of osseous
healing and repair. Thus, if properly utilized, a sub-
strate-immobilized multi-domain extracellular mat-
rix protein, a synthetic biomimetic material, or a
combination of such molecules, could form the basis
for new biomodied implant surfaces that takes
advantage of how the spatial distribution of ligands
controls cell responses and initiates focal contact
formation.
In mineralizing bone, the bulk of matrix molecules
are produced by the osteoblastic cells. Many of the
structural extracellular matrix molecules in bone have
an arginine-glycine-aspartic acid (RGD) sequence
where cells can attach via an integrin receptor (83).
Several of these molecules, including bronectin,
osteonectin, vitronectin, laminin, collagens, osteo-
pontin, bone sialoprotein, dentin sialoprotein, and
thrombospondin, have been tested for initiation of
bone growth and maintenance of bone integrity
(52, 63, 64, 92, 123, 143). Integrin receptor binding is
believed to represent the primary mechanism of the
osteoblastic cellextracellular matrix interactions that
control cell morphology, proliferation, and differen-
tiation (161). Studies have shown that coating tita-
nium implants with RGD-containing peptides is
possible, providing a functionalized surface that can
bind integrin receptors and improve osteblastic cell
attachment and function (13, 44, 142). However,
experiments also suggest that in bone, RGD-con-
taining molecules must work in concert with bone
morphogenetic proteins to be able to induce bone
growth. Thus, bone induction by implantation of
RGD-containing matrices is observed mainly when
bone morphogenetic proteins are supplied concom-
itantly, and is dened spatially by the volume of the
matrix, but limited temporally only to the time when
the morphogenetic proteins are present (39).
Other matrix components that do not express an
RGD motif are also directly involved in the formation
of mineralized tissues. The glycosaminoglycan hep-
aran sulfate and the proteoglycan lumican are
both closely associated with the differentiation of
osteoblastic cells (26, 140). Other extracellular matrix
molecules like members of the von Willebrand factor
A-domain superfamily play important roles in carti-
lage and bone structure and function (54). Also
matrix metalloproteinases and their corresponding
inhibitors, the tissue inhibitors of metalloproteinases,
are important components of the bone extracellular
matrix. These proteins are directly responsible for the
plasticity and integrity of the bone by providing a
system for controlled breakdown and remodeling of
insoluble organic matrix components like collagens.
Moreover, the matrix metalloproteinases provide a
secondary signal for bone cell differentiation and
function, as well as for angiogenesis during bone
healing, simply by the release of soluble signaling
peptides from the degrading matrix biomolecules.
This release of breakdown products and matrix-
bound growth factors provides a strong feedback
signal from the extracellular matrix to the bone cells,
which in turn can react by adjusting the rate of bone
formation or remodeling through altered expression
and secretion of extracellular matrix components
(174, 182).
Finally, enamel extracellular matrix proteins are also
possible candidates for bioactive implant coatings.
During tooth formation these molecules initiate and
templates enamel biomineralization, and induce root
cementum formation (42, 43, 120). If, as several
observations suggest (67, 68, 154, 155), enamel matrix
protein deposition directly precedes hard tissue
development in the jaw, enamel matrix to cell inter-
action at pretreated implant surface could be used to
restart dormant developmental programs in progen-
itor cells for (re-)generation of bone. Such responses
would typically involve sequential cascades of growth
factors that act on the multitude of cells needed to
completely reconstitute lost tissues or to build new
ones. This quality makes these extracellular matrix
molecules attractive potential therapeutic agents for
use with metal implants where direct formation of
functional bone or bone-related tissues is required for
a successful clinical outcome. Infact, several studies in
animals have now demonstrated that enamel extra-
cellular matrix proteins are capable of enhancing bone
formation around titanium implants (21, 149).
It is evident that the bone extracellular matrix is a
biologically active entity containing molecules that if
added to the implant surface, could represent
breakthroughs in guided interfacial osteogenesis
(116). Such molecules, when used for recruiting the
surface recognition apparatus and focal adhesion
mechanisms for cell-surface interactions, could pro-
vide a viable way for biomimetic induction of growth
factor cascades at the boneimplant interface that
promote attachment, growth and repair of hard tis-
sues over a time period consummate with a favorable
clinical outcome (138).
Future prospects
The rapidly progressing elds of biomedicine and
tissue engineering have so far failed to come up with
149
alternatives to metal implants to restore functional
structures in the adult skeletal tissues. Even though
titanium implants and joint prostheses have shown
considerable progress over the last three decades
they are still not ideal, and there are several patient
groups that can still not be satisfactorily reconstruc-
ted with todays materials. The surface modications
of titanium presented recently have brought us closer
to the main goal of restoring the patients safely and
efciently with short healing time and fewer com-
plications. To further improve metal implant per-
formance, strategies for making metal surfaces more
osteoinductive must be designed. Adding bioactive
surface layers to implants and prostheses could im-
prove implant biocompatibility, osseointegration,
and durability, all important qualities required for
long-term performance in the adult body. Of partic-
ular interest is the use of implant surfaces as carriers
for biomolecules that can control the early peri-im-
plant tissue response. Recruiting growth factor-pro-
ducing cells to the boneimplant interface through
focal attachment and molecular recognition may give
faster bone formation in areas with low bone quality
as well as in patients with reduced bone regenerative
potential. This route will thus possibly widen the uses
of implants and enlarge the possibilities for success-
ful help of our patients through bone regenerative
therapies.
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Prospects for tooth regeneration
SI LVI O E. DUAI LI BI , MONI CA T. DUAI LI BI , JOSEPH P. VACANTI &
PAMELA C. YELI CK
Regenerative dental medicine uses an integrated
sciences approach, involving developmental and
molecular/cellular biology, molecular genetics and
chemical engineering (5, 25, 28, 30, 41, 62, 65, 75).
Recent advances in the elds of dental tissue
engineering, materials science and stem cell biology,
suggest that tooth regeneration will be possible
in the foreseeable future. Published reports have
demonstrated that dental tissue progenitor cells
present in the pulp tissue of deciduous and adult
teeth can be used to generate dentin and alveolar
bone (59, 84), while those present in immature tooth
buds can be used to bioengineer small, anatomically
correct, whole tooth crowns consisting of enamel,
dentin and pulp tissue (13, 8183). These promising
results, along with many other studies reporting
dental tissue regeneration, suggest a means for the
eventual regeneration of replacement teeth in
humans. However, before this can occur, certain
obstacles must rst be overcome. Below, we will
review recent progress in tooth-regeneration efforts
to date, and identify challenges that must be met in
order for this approach to reach clinical relevance in
humans.
Tooth development as a model for
tooth regeneration
The interactions of the dental epithelium and
mesenchyme during natural tooth development
provide insight into how bioengineered tooth for-
mation may be facilitated. Although human incisor,
canine, premolar and molar teeth exhibit distinct
morphologies, (Fig. 1), they develop in basically the
same manner. Tooth development, the result of
reciprocal interactions between the dental epithe-
lium and the neural crest cell-derived ectomesen-
chyme, is initiated by the dental epithelium and
proceeds through ve distinct morphological stages:
bud, cap, bell, crown, and root (70, 71) (Fig. 2). The
coordinated development of tooth supporting struc-
tures, including periodontal ligament and alveolar
bone, begins around the bell stage (68).
The tooth germ is rst identiable as a localized
thickening and proliferation of the oral epithelium
(Fig. 2A). The dental epithelium forms a bud that
extends into the underlying dental mesenchyme,
marking the rst stage of tooth development. The
dental epithelium subsequently undergoes signi-
cant proliferative activity (Fig. 2B), extending around
the periphery to form a cap-like structure (Fig. 2C).
During this process, the nonproliferating enamel
knot signaling center (72) becomes identiable, as
epithelial cells organize themselves into three
distinct regions, namely the outer epithelium, the
inner epithelium, and central cell layers called
the stratum intermedium and stellate reticulum
(Fig. 2C,D). The ectomesenchymal cells of the dental
papilla condense beneath the invaginating dental
epithelium, eventually giving rise to dentin and pulp
tissues. The dental follicle forms around the enamel
organ and dental papilla, eventually forming the
periodontal tissues (Fig. 2C,D).
The bell stage is characterized by continued
proliferation and histodifferentiation of the dental
epithelium (Fig. 2D). Inner dental epithelial cells
assume a cuboidal shape and produce high levels
of glycogen, adjacent stratum intermedium pro-
duces high levels of alkaline phosphatase, and the
stellate reticulum assumes a distinctive star shape,
surrounded by the outer epithelial cell layer
(Fig. 2D).
As tooth development proceeds through differen-
tiation stages, dental mesenchyme-derived odonto-
blasts differentiate and elaborate the dentin matrix,
and epithelial cell-derived ameloblasts cells secrete
the enamel matrix for enamel production (Fig. 2E).
After the tooth crown has formed, tooth root struc-
tures develop from the rudimentary Hertwigs epi-
thelial root sheath, forming dentin, cementum,
177
PERIODONTOLOGY 2000
periodontal ligament and alveolar bone (Fig. 2F).
The co-ordinated processes of tooth root maturation
and tooth eruption proceed in an interdependent
manner that is not well understood at the present
time (68).
Human replacement teeth form as a localized
proliferation of the dental lamina of a pre-existing
deciduous tooth (68). Humans form only one set of
replacement teeth, which do not exhibit subsequent
regenerative capabilities. The molecular signaling
Deciduous Teeth Permanent Teeth
7 years
11 years
12 years
15 years
21 years
8 years
9 years
10 years
Fig. 1. Human deciduous and adult tooth from 7 to 21 years of age. Deciduous and permanent human teeth form and
erupt as shown. (Adapted from: Picosse M. Anatomia dentaria, 2nd edition. Sao Paulo: Sarvier, 1977: 6667.)
178
Duailibi et al.
cascades regulating the fascinating process of
replacement tooth formation remain largely unchar-
acterized as a result of the lack of a suitable animal
model.
Thus, natural primary and replacement tooth
formation provides a foundation upon which tooth-
regeneration strategies can be based. It is anticipa-
ted that tooth-regeneration strategies need not
necessarily be as complex as natural tooth devel-
opment, but rather can mimic certain aspects of
natural tooth formation to facilitate tooth-regener-
ation therapies.
Regenerative capabilities of naturally
formed dental tissues
Human deciduous and adult tooth tissues exhibit a
limited degree of regenerative capacity. Each type of
mineralized dental tissue enamel, dentin, cemen-
tum and alveolar bone exhibits distinct properties.
Enamel is formed by a process called amelogenesis,
dentin formation is termed dentinogenesis, cemen-
tum formation is termed cementogenesis, and alve-
olar bone forms by osteogenesis (26).
Enamel, the most highly mineralized dental tissue,
is composed of crystalline calcium phosphate, and is
approximately 96% mineral with the remaining 4%
consisting of organic components and water. Its
hardness, when allowed to dry, is comparable with
brittle steel. The basic structural unit of enamel is an
enamel rod, which is tightly packed and mechanically
adherent to other rods, providing high resistance to
stress fractures (68). The interwoven architecture of
enamel crystals provides both strength and protec-
tion for the tooth (43). The enamel-forming progen-
itor cells ameloblasts undergo apoptosis as they
elaborate the enamel matrix, such that by the time
the enamel is fully formed, no ameloblasts remain
(60). Enamel regeneration therefore is not possible in
erupted teeth because the progenitor cells are no
longer present.
Dentin, the mineralized tissue underlying enamel,
is characterized by distinctive uid-lled dentin tu-
bules (7, 68), and is approximately 74%mineral, with
the organic phase consisting mostly of type-1 colla-
gen, with small amounts of dentin proteins and water
(68). Primary dentin consists of two distinct miner-
alized types circumpulpal dentin, which surrounds
the pulp chamber, and mantle dentin, which is
located at the dentinenamel junction (44). Dentin is
composed of millions of tubules (approximately
60 000 tubules/mm
2
), which extend through a colla-
gen- and calcium-rich zone of intertubular dentin,
from the pulpal wall to the dentinenamel junction
(66). The tubule diameter at the dentinenamel
junction is 0.06 lm, and widens to approximately
3.0 lm at the pulpal wall. Dentin tubules are uid
Fig. 2. Human tooth developmental stages. (A) Tooth
bud forms as a proliferation of the dental epithelium (de)
into the underlying dental mesenchyme (dm). (B) The
dental epithelium continues to proliferate, and the
underlying dental mesenchyme undergoes condensation.
EK, enamel knot signaling center. (C) The cap stage tooth
exhibits distinct outer epithelium (oe), and inner epi-
thelium (ie) layers, surrounding the condensed dental
mesenchyme (dm). (D) The bell-stage tooth exhibits dif-
ferentiated enamel organ, consisting of distinct inner
enamel (ie), stratum intermedium (si) and stellate reti-
culum (sr) cell layers, which surround the dental papilla
(dp). (E) Differentiation-stage teeth exhibit polarized
odontoblasts (od) and ameloblasts (am), which elaborate
dentin and enamel, respectively. (F) Late differentiation-
stage teeth display rudimentary root structures, called
Hertwigs epithelial root sheath (hers), periodontal liga-
ment tissues (pdl), polarized odontoblasts (od), and
mineralized dentin (d) and enamel (e) layers. (Courtesy
of Katchiburian E, Arana VE. Histologia e Embriologia
Oral, 2nd edition. Guanabara Koogan: Medica Pan-
americana S.A.C.F., 2004. Reproduced with permission
from the editors.)
179
lled and may contain an odontoblast process, col-
lagen and nonmyelinated pulpal nerves (68). The
distinct architecture of the uid-lled dentin tubules
is thought to provide a means of communication
from the enamel layer to the tooth pulp. Tertiary
dentin consists of reactionary dentin (formed by pre-
existing odontoblasts) or reparative dentin (formed
from newly differentiated odontoblasts) (64). Once
erupted, teeth maintain a limited capacity to form
reparative (or tertiary) dentin, when progenitor cells
are recruited from the pulp to elaborate localized
dentin matrix at the site of injury (68).
Cementum, the thin layer of mineralized tissue
that covers the dentin of tooth roots, is also highly
mineralized, but softer than dentin, consisting of
approximately 45% inorganic material, 33% organic
material and 22% water. Cementum, formed by
cementoblasts, is sandwiched between the inner
dentin surface and the outer periodontal ligament
surface, and serves to secure the tooth, via the per-
iodontal ligament, to the alveolar bone. Cementum
consists of thin, plate-like hydroxyapatite crystals,
approximately 55-nm wide and 8-nm thick, and is
similar in chemical composition and physical prop-
erties to bone. Three types of cementum are present
on the tooth: acellular cementum, which covers one-
third to one-half of the tooth root adjacent to the
cemento-enamel junction (the area where cemen-
tum and enamel meet); abrillar cementum, which
is present at the cemento-enamel junction; and
cellular cementum, which typically covers the apical
one-half to two-thirds of the tooth root. Cementum
naturally regenerates, slowly forming throughout
the life of the tooth, allowing for continual re-
attachment of the periodontal ligament bers,
although the identication of the cementoblast
progenitor cells remains somewhat controversial at
the present time (6).
Alveolar bone, formed from neural crest cell-
derived dental mesenchymal cells, functions as the
primary support for teeth, and is composed of
bundles of bone that are built up in layers in a par-
allel orientation to the coronalapical direction of the
tooth. Alveolar bone exhibits rapid turnover in
response to mechanical stimulation (79). This char-
acteristic plasticity of alveolar bone distinguishes it
from other types of bone and allows for constant
minor accommodation of tooth movements during
mastication (68). Alveolar bone resorption following
tooth loss can be signicant estimated to be
4060% during the rst 3 years, with an additional
0.250.5% loss every year thereafter (1, 2) presum-
ably as a result of disuse atrophy, decreased blood
supply, localized inammation or unfavorable pros-
thesis pressure (1, 14).
In summary, in naturally formed teeth, enamel
the only mineralized tooth tissue derived from the
dental epithelium exhibits no regenerative proper-
ties, while the remaining mineralized periodontal
and dental tissues, including dentin, pulp, cemen-
tum, periodontal ligament and alveolar bone, all of
which are formed from neural crest-derived dental
ectomesenchyme, each exhibit a certain degree of
regenerative capability. Therefore, while the devel-
opment of clinically relevant regeneration strategies
for all types of dental tissues remains a challenging
task, that of enamel is the greatest, owing to the
absence of dental epithelial progenitor cells in
erupted teeth.
Postnatal dental stem cells and dental
tissue regeneration
Progenitor stem cells have been identied in hun-
dreds of human postnatal tissues (1012, 52, 54, 55,
85). Stem cells are dened as quiescent cell popula-
tions present in low numbers in normal tissue, which
exhibit the distinct characteristic of asymmetric cell
division, resulting in the formation of two distinct
daughter cells a new progenitor/stem cell, and an-
other daughter cell capable of forming differentiated
tissue (18, 32). In this way, stem cells are able to self-
renew and maintain themselves in an undifferenti-
ated state, while also giving rise to differentiating
daughter cells. Progenitor cells differ from stem cells
in that they exhibit a nite life span rather than
existing throughout the life of the organism, and
exhibit limited differentiative potential, with the
capacity to form only limited tissue types.
The characterization of dental progenitor/stem
cells has increased signicantly over the past 5
10 years. Dental mesenchymal progenitor cells have
been characterized using transgenic mouse reporter
models (3, 38), and mesenchymal stem/progenitor
cells have been identied and characterized in dental
pulp obtained from both deciduous and adult human
teeth (16, 42, 59). Evidence for both common and
distinct progenitor cells for periodontal tissues has
been reported (4, 6, 56, 67), as described in more
detail in other chapters of this volume. Preliminary
characterization of postnatal epithelial and mesen-
chymal dental stem/progenitor cells present in
immature tooth buds demonstrated the ability to
generate bioengineered, anatomically correct tooth
crowns containing enamel, dentin, pulp and alveolar
bone, as described below.
180
Duailibi et al.
Regenerative therapies for dental
tissues
Tissue-engineering approaches have proven to be
useful for dental tissue- and whole-tooth-regener-
ation strategies (33). Based on preclinical cell- and
gene-therapy strategies used for soft tissue organs,
reports of the emerging use of tissue-engineering
strategies for dentin, pulp and cementum, as an
alternative to commonly used root canal and crown
therapies, are becoming more numerous. Advances
in vital pulp therapies to regenerate the dentinpulp
complex without the removal of the whole pulp, in-
clude application of exogenous growth factors and/or
stem/progenitor cells (46, 68).
We have previously shown that tooth bud cells can
be used to bioengineer anatomically correct tooth
crowns (13, 19, 8183). Our approach used tooth bud
cells isolated from both rat and pig tooth buds, which
were cultured in vitro for 6 days (Fig. 3), seeded onto
biodegradable scaffolds, and implanted and grown in
the omenta of adult rat hosts. Cultured rat tooth bud
cells formeddistinctly mineralizedtissues in12 weeks,
while pig tooth bud cells formed tooth crowns in
approximately 2030 weeks (Fig. 4). Histologic and
immunohistochemical analyses revealed that bio-
engineered rat and pig dental tissues exhibited many
characteristics of naturally formed dental tissues (13,
8183). The fact that rat tooth bud cells were cultured
for up 6 days before being used to generate bioengi-
neered dental tissues suggests that progenitor dental
stem cells can be maintained in culture for at least
this long.
The formation of multiple small tooth crowns in
our bioengineered tooth constructs, rather than one
large tooth, reveals a number of challenges that
need to be addressed before this approach can
Fig. 3. Cultured tooth bud cells. (A) Rat tooth bud cells
cultured for 1 day contain broblastic dental mesenchy-
mal (dm) cells and small, rounded, dental epithelial (de)
cells. (B) After 5 days in culture, small epithelial colonies
are evident (de), surrounded by conuent dental mesen-
chymal (dm) cells.
Fig. 4. Bioengineered mammalian tooth crowns. (A) Bio-
engineered pig tooth exhibits distinct cell layers and tis-
sues observed in naturally formed teeth, including:
odontoblasts (od), predentin (pd), dentin (d), enamel (e),
ameloblasts (am) and stellate reticulum (sr). (B) Bioengi-
neered rat tooth crowns exhibits distinct pulp (pu), pre-
dentin (pd), dentin (d), and enamel (e) layers.
181
reliably be used for tooth-regeneration applications.
First, as bioengineered tooth crown formation re-
quires the interactions of both dental epithelial cell
progenitors and mesenchymal cell progenitors (as
in natural tooth formation), the ability to bioengi-
neer a tooth of specied size and shape will depend
on the ability to rst identify, and then guide, the
interactions of both types of cells. As the cultured
tooth bud cell populations used in our studies were
quite heterogeneous, methods to generate puried
dental stem cell populations must be developed.
Next, methods to guide the interactions of epithelial
and mesenchymal postnatal dental stem cells to
form dentin and enamel layers characteristic of
natural teeth, using modied scaffold materials and
designs, for example, must be developed. Finally, as
our bioengineered teeth consisted of fairly well-
developed tooth crowns, while the tooth roots were
relatively undeveloped, we also need to devise
methods to improve bioengineered tooth root for-
mation. Progress in each of these areas is discussed
below.
Generation of enriched epithelial
and mesenchymal dental stem cell
populations
We are currently using two methods to generate en-
riched postnatal dental stem cell populations. The
rst method uses the stem cell antibody, STRO-1
(61), to generate enriched, STRO-1-positive stem cell
populations from cultured tooth bud cell prepara-
tions. The second method uses side population
proling to generate enriched dental stem cell
populations, based on the demonstrated ability for
stem cells to efux Hoechst dye, while nonstem cell
populations cannot (15). Fluorescent-activated cell
sorting allows the separation of Hoechst-negative
stem cells from dye retaining nonstem cell popula-
tions. Clonal cell lines are being established from
cells sorted by both methods for future testing in
dental tissue engineering applications.
Scaffold materials and design for
whole tooth tissue regeneration
The importance of scaffold materials and design for
tissue engineering has long been recognized. Scaf-
fold porosity, biocompatibility and biodegradability,
the ability to support cell growth, and use as a
controlled gene- and protein-delivery vehicle (45)
are all highly signicant properties. A variety of
hydrophilic polymers have been synthesized that
provide cell support and guidance. Importantly,
scaffold materials provide a three-dimensional
macromolecular structure to guide the nal shape of
bioengineered tissues. Poly-L-lactic acid and poly
lactic co-glycolic acid co-polymers have been used
to generate composite scaffolds that degrade within
a period of a few weeks up to 1 year (37). Poly-L-
lactic acid sponges can support the growth of
chondrocytes in a uniform cellular distribution, their
utility has been demonstrated in cartilage tissue
regeneration (36), and polyglycolic acid and poly-
lactic acid have been shown to support the growth of
biopsied neonatal intestine cells into functional,
small intestinal tissue (9).
Optimized polymer fabrication techniques have
been used to generate three-dimensional structures
composed of an intercommunicating network of
pores, where the resulting morphology and mech-
anical properties of the scaffold walls were found to
inuence tissue engineering applications (29, 34).
Others authors (20) investigated the use of macro-
molecular materials of natural origin (i.e. collagen,
alginate, and agarose), derived from hyaluronic acid
and brin glue, to identify polymers that provide the
best guidance for cellular differentiation and prolif-
eration (with the objective of replacing tissues by
cellular transplantation). Natural silk proteins have
been successfully used to generate scaffolds suitable
for bone tissue engineering (35). Each type of scaffold
has unique features that provide exibility for a
variety of tissue-engineering applications. We are
currently testing a variety of scaffold materials and
designs for guided dentinenamel junction forma-
tion, and optimized whole tooth tissue-engineering
applications, based on morphologic cell movements
of natural tooth development.
Functional bioengineered tooth
root formation
Our current bioengineered tooth model exhibits
crowns that are much more developed than the tooth
roots. To improve bioengineered tooth root forma-
tion, we designed hybrid tooth/bone constructs to
test whether the co-ordinated alveolar bone forma-
tion could improve bioengineered tooth root devel-
opment (82). These studies demonstrated that
bioengineered tooth roots generated from the hybrid
tooth/bone constructs were, in fact, more developed
than those formed by tooth constructs alone, indi-
182
Duailibi et al.
cating that this approach is promising. Further
modications of our whole tooth tissue-engineering
methods are likely to reveal distinct properties of
epithelial and mesenchymal dental progenitor cells,
which can be manipulated to improve current tooth-
regeneration efforts.
Animal models for tooth
regeneration
Analyses of tooth development in a variety of animal
models have provided signicant insight into tooth-
regeneration strategies. Characterizations of epithe-
lial dental stem cell populations have largely been
limited to rodent models exhibiting continuous
incisor and molar eruption, including the rat, mouse,
rabbit and vole (17, 27, 73). These recent reports
demonstrate that the epithelial dental stem cell niche
is a specialized epithelial structure located at the
apical end of the tooth, termed the apical bud, and
provide models for identication of genes that
maintain the epithelial dental stem cell niche (17).
The vole, which exhibits continuously erupting mo-
lars as well as incisors, has proven useful for com-
paring molecular signaling cascades regulating tooth
crown versus tooth root cell fates (73). These studies
revealed that the notch signaling pathway, in partic-
ular, is important for maintaining dental stem cell
populations, and that the absence of notch signaling
results in the terminal differentiation of dental stem
cells to tooth root tissue fates.
The zebrash has also proven useful as a model for
tooth regeneration (21, 22, 31, 50, 51, 76, 78, 80). Al-
though the zebrash teeth are pharyngeal, located in
the pharynx rather than the jaw, their development is
remarkably similar to that of mammals (51). In
addition, zebrash continuously regenerate teeth
throughout their lives. Replacement tooth formation
in zebrash is morphologically very similar to adult
tooth formation in humans, where adult teeth form
as a dental laminar proliferation from a deciduous
tooth (Fig. 5). Although humans generate only two
sets of teeth, zebrash continuously regenerate teeth
throughout their lives. Replacement teeth form
approximately every 714 days in juvenile zebrash,
and more slowly in adults (77), providing the means
to study molecular signaling pathways that are per-
missive for continuous tooth regeneration. The
signicant nucleotide and amino acid sequence
identity, and genomic synteny shared between ze-
brash and humans, make the zebrash a pertinent
model for elucidation of molecular strategies for
human replacement tooth formation. These charac-
teristics, together with the molecular/genetic mani-
pulations possible in zebrash, make them a superb
model for in vivo analyses of replacement tooth
formation.
Autologous tissues for dental
tissue-regeneration applications
A major challenge for autologous dental tissue-
engineering applications in humans is the identi-
cation of suitable cell populations to use for these
applications. Reports documenting the successful
bioengineering of dental tissues include the following
provocative ndings. Human dental pulp tissues,
isolated from both adult and juvenile teeth, exhibit
the capacity to differentiate, in vitro and in subcu-
taneous implants, into dental mesenchyme-derived
tissues, including dentin, cementum and bone, as
well as nerve and vascular endothelium (8, 16, 39, 57,
58). Mesenchymal stem cells have been identied in
a variety of adult tissues as a population of pluripo-
tential self-renewing cells isolated from bone mar-
row, which exhibit the capacity to differentiate into
dl
pu
d
B A
Successional Tooth Germ
Dental Lamina
Dental Organ
Dental Papilla
Dental Follicle
Fig. 5. Human and zebrash
replacement tooth formation. (A)
Human successional tooth germs
form as a localized proliferation of
the dental lamina (arrow). (B) Simi-
larly, zebrash replacement teeth
form as a localized proliferation of
the dental lamina (dl, arrow) of an
existing functional tooth. Functional
zebrash teeth exhibit distinct pulp
(pu) and dentin (d).
183
bone, cartilage, muscle, tendon and adipose tissue.
Recent studies have identied and characterized
stem cell populations for cementum, dentine and
periodontal ligament (53). As a result of the suc-
cessful generation of bioengineered reparative den-
tin, it is anticipated that this technique may become
clinically relevant in the near future (47). Reports
demonstrating the use of embryonic stem cells for
dental tissue formation reveal the potential utility of
embryonic stem cells for tooth tissue-engineering
applications (40, 49). Whole tooth tissue-engineering
studies revealed that dissociated mammalian tooth
bud cells retain a cell-autonomous developmental
program, even when dissociated into single-cell
suspensions and grown in culture (13, 19, 81, 83).
These studies suggest the potential for human tooth
buds for dental tissue-engineering applications. The
recent identication and characterization of dental
stem cells suggests the potential use of dental stem
cells for tooth tissue-replacement therapies. Gene-
delivery techniques have demonstrated the potential
for periodontal ligament regeneration, as well as
for dentin and alveolar bone (24, 47). Methods to
generate enriched dental stem cell populations are
suggested by studies of transgenic mice expressing
green uorescent protein under the direction of the
collagen type I gene promoter, which exhibit a
population of green uorescent protein-expressing
dental pulp cells that exhibit stem cell-like charac-
teristics (8, 38).
In contrast to dental mesenchymal dental stem
cells, the identication and characterization of epi-
thelial dental stem cells has proven more elusive,
partly owing to the fact that human enamel does not
exhibit regenerative capabilities in vivo, reecting the
fact that epithelial dental stem cells are no longer
present in functional human teeth. The recent suc-
cessful bioengineering of whole tooth crowns con-
taining pulp, dentin and enamel, from immature pig
and rat tooth buds, provides the rst evidence that
postnatal epithelial and mesenchymal dental stem
cells may exhibit utility for whole tooth tissue
engineering (13, 82, 83).
The future of regenerative dentistry
Recent advances in the elds of tissue engineering
and surgery have merged to create a promising new
era in which it is possible that bioengineered tissues
and organs may routinely be used to replace con-
genitally missing tissues and organs, and/or those
lost to injury or disease. These advances also apply to
the highly mineralized tooth organ, the product of
reciprocal signaling events between the dental epi-
thelium and dental mesenchyme (23, 69). The need
for alternative dental tissue-replacement therapies
is evident in recent reports (48, 74), which reveal
startling statistics regarding the high incidence of
tooth decay and tooth loss in the USA. Recent
advances in the identication and characterization of
dental stem cells, and in dental tissue-engineering
strategies, suggest that within the next decade, bio-
engineering approaches may successfully be used to
regenerate dental tissues (25) and whole teeth (13,
8183).
Interest in dental tissue-regeneration applications
continues to increase as clinically relevant methods
for the generation of bioengineered dental tissues,
and whole teeth, continue to improve. Although
obvious practical obstacles remain to be overcome
before routine clinical treatments become com-
monly available, dental tissue regeneration research
efforts provide an example of how advances in basic
research can be translated into clinically relevant
dental therapies (63). Tissue engineering offers
exciting opportunities for innovative collaborative
research efforts, integrating the elds of medicine,
developmental biology, and physical sciences.
Clearly, the future application of regenerative and
tissue-engineering techniques to dentistry is one of
immense potential, capable of meeting a variety of
patient needs. High-quality basic dental research is
paramount to ensuring that the development of
novel clinical treatments is supported by robust
mechanistic data and that such approaches are
effective. These efforts reveal how successful inno-
vations in the eld of dentistry can be guided by
advances in basic research, highlighting the need for
close partnerships between basic research and clin-
ical scientists.
Acknowledgments
This work was supported by the Center for the In-
tegration of Medicine and Innovative Technology
(CIMIT), NIH grants R41DE015445, R21DE16370, and
R01DE016132-01A1, CAPES grant SAUX-PE 1295/
2005, and FAPESP grant 04/08924-08.
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187
Regeneration of periodontal
tissues: cementogenesis revisited
MARGARI TA ZEI CHNER-DAVI D
Virtually all types of periodontal disease are caused
by periodontal pocket infections, although several
other factors, including trauma, aging, systemic dis-
eases, genetics, etc., can contribute to the destruction
of the periodontium (1, 18, 31, 52, 60, 107, 128, 127,
194). Repair of the periodontium and the regener-
ation of periodontal tissues remains a major goal in
the treatment of periodontal disease and is an area
still in need of major research attention, as recently
stated by the American Academy of Periodontology
(260). In general, to achieve complete tissue regen-
eration and repair, it is necessary to recapitulate the
process of embryogenesis and morphogenesis in-
volved in the original formation of the tissue. In the
case of the periodontium, complete periodontal re-
pair entails de novo cementogenesis, osteogenesis
and the formation of periodontal ligament bers.
Current strategies for periodontal repair are based on
anti-infectious measures such as scaling and root
planing, guided tissue regeneration (with or without
bone grafts) or the use of growth factors, none of
which fully restore the architecture of the original
periodontium. Several different approaches involving
tissue engineering are currently being explored to
achieve complete, reliable and reproducible regen-
eration of the periodontium. As tissue engineering is
dened as the science that develops techniques
(based on principles of cell and developmental bio-
logy) for fabricating new tissues to replace or regen-
erate lost tissues (205), it is important to understand
the formation of specic tissues, the physico-chem-
ical characteristics of the tissues and the molecular
events leading to the normal function of the tissues.
Development of the periodontium
The periodontium can be dened as an intricate
mosaic of cells and proteins that is primarily
responsible for the attachment of teeth in the oral
cavity (144). Several excellent reviews have been
published describing the embryonic lineage of the
principal periodontal tissues (cementum, periodontal
ligament, gingiva and alveolar bone), as well as the
cells and extracellular matrix components of the
periodontium (10, 13, 14, 21, 19, 46, 45, 51, 71, 80, 82,
144, 158, 185, 186, 193, 212, 214, 243, 244, 245).
Formation of the periodontium is initiated with
the process of root formation where, following
crown formation, the apical mesenchyme continues
to proliferate to form the developing periodontium,
while the inner and outer enamel epithelia fuse
below the level of the cervical enamel to produce a
bilayered epithelial sheath, termed the Hertwigs
epithelial root sheath. As these cells divide, there is
an apical migration of the Hertwigs epithelial root
sheath cells through the underlying dental ectome-
senchymal tissues, dividing them into the dental
papilla and the dental follicle (Fig. 1). As the root
develops, the rst radicular mantle dentin is formed
and the epithelial sheath is fenestrated. It is believed
that cells of the Hertwigs epithelial root sheath
migrate away from the root into the region of the
future periodontal ligament where they re-associate
to form the Epithelial Rest of Malassez. However,
not all Hertwigs epithelial root sheath cells migrate
into the periodontal ligament site; a few undergo
apoptosis and some remain in the root surface
(108).
Although it is accepted that the Hertwigs epithelial
root sheath plays an important role in root develop-
ment, the precise nature of its role remains contro-
versial. In 1940, Schour & Massler suggested that the
major function of the Hertwigs epithelial root sheath
was to induce and regulate root formation, including
the size, shape and number of roots (244). Other
investigators suggested that the role of the Hertwigs
epithelial root sheath was to induce the differentiation
196
PERIODONTOLOGY 2000
of odontoblasts to form the root dentin (183, 182, 222,
243, 251), or to differentiate dental sac cells into
cementoblasts (181). The current notion states that
Hertwigs epithelial root sheath cells produce the
basement membrane containing chemotactic pro-
teins, which serve to direct the migration of prece-
mentoblast cells (140, 141, 182, 235, 251) and to
induce cementoblast differentiation (191, 232, 234).
Amongst the basement membrane molecules are
several extracellular matrix proteins, growth factors,
enamel proteins and adhesion molecules, such as a
collagenous-like protein, known as cementum
attachment protein (CAP), which has chemotactic
potential capable of recruiting putative cementoblast
precursors (11, 149, 156, 196, 275). In the second
stage of cementogenesis (when the tooth reaches
occlusion and cellular cementum is formed), the
proliferation of cells of the Hertwigs epithelial root
sheath is considerably reduced, and some cells are
entrapped in the newly formed mineral where they
may inuence phenotypic changes in the dental sac
cells (252). It is also suggested that Hertwigs epi-
thelial root sheath cells undergo epithelialmesen-
chymal transformation to become functional ce-
mentoblasts in charge of producing the acellular
cementum (251, 275).
The gingival tissues appear to be derived from both
the oral mucosa and the developing tooth germ (135).
It has been suggested that the dental follicle (con-
nective tissue surrounding the developing teeth)
gives rise to the broblasts forming the periodontal
ligament as well as to the alveolar bone and
cementoblasts (45, 136, 186, 243), all of which have
a common neural crest origin (34). Therefore, it is
postulated that there are different types of
cementoblasts: those originating from the Hertwigs
epithelial root sheath via epithelialmesenchymal
transformation and which form the acellular
cementum; and those derived from the dental follicle,
which form the cellular cementum (9, 19, 105, 251,
275). It is also believed that progenitors for perio-
dontal ligament, osteoblast and cementoblast cells
adopt a paravascular location in the periodontal
ligament, and these cells, which exhibit some fea-
tures of stem cells, can regenerate functional tissues
when the need arises (150153, 195). Periodontal
Fig. 1. Root development and periodontium formation.
Histological sections of 7-day postnatal mouse mandibu-
lar molars showing the initial development of the root by
formation of the Hertwigs epithelial root sheath. At the
14-day postnatal time-point, apical migration of the roots
continues, and there is formation of the periodontium
with cementum, periodontal ligament and bone. Am,
ameloblasts; C, cementum; D, dentin; Ds, dental sac;
HERS, Hertwigs epithelial root sheath; Od, odontoblasts;
PDL, periodontal ligament.
197
Regeneration of periodontal tissues: cementogenesis revisited
ligament stem cells have recently been isolated from
the human periodontium (162, 224, 225).
The Epithelial Rest of Malassez cells remain in the
periodontal ligament throughout life, suggesting that
they have important, although yet unknown, func-
tions, rather than just being leftover structures. Roles
attributed to the Epithelial Rest of Malassez cells
range from bad to good. The Epithelial Rest of
Malassez cells are held responsible for the formation
of periodontal cysts and tumors as a result of peri-
apical inammation associated with pulpal necrosis
(26, 57, 77, 176, 226, 242). It has also been suggested
that Epithelial Rest of Malassez cells contribute to the
formation of the periodontal pocket because of their
continuum with the junctional epithelium (176, 238).
Some studies report the ability of Epithelial Rest of
Malassez cells to resorb bone and extracellular mat-
rix, and thus implicate the cells in root resorption (15,
75, 122). On the other hand, it has also been sug-
gested that the cells of the Epithelial Rest of Malassez
may protect the root from resorption (259). The
nding of Epithelial Rest of Malassez cells being
closely associated with neural endings suggests that
they have a role in the development of periodontal
ligament innervation (126). Studies performed with
1-hydroxyethylidene-1,1-bisphosphonate, a drug that
interferes with homeostasis in the periodontal liga-
ment, showed a severe reduction in the width of the
periodontal ligament with the development of anky-
losis, which was repaired after discontinuing the
administration of 1-hydroxyethylidene-1,1-bisphos-
phonate (261). As the study did not detect a change in
the number of Epithelial Rest of Malassez cells post-
treatment, it was suggested that cells of the Epithelial
Rest of Malassez are unlikely to play an important
part in the homeostasis of, and may not be a prere-
quisite for, the repair and maintenance of the perio-
dontal ligament. On the other hand, the Epithelial
Rest of Malassez cells secrete hyaluronic acid, which
contributes to the formation of the loose connective
tissue characteristics of the periodontal ligament
(155). Cells of the Epithelial Rest of Malassez react to
mechanical stress, like that associated with ortho-
dontic tooth movement, by increasing their prolifer-
ation rate and cell size (27), and thereby help to
maintain the space between the periodontal bone
and cementum to avoid ankylosis (134). The in-
creased activity of the Epithelial Rest of Malassez
cells is consistent with their putative role on collagen
turnover in the periodontal ligament, which is
accelerated during tooth movement (241), and during
cementum repair in areas of root resorption (24). It is
suggested that the Epithelial Rest of Malassez cells
may negatively regulate root resorption and induce
acellular cementum formation (56). In addition, cells
of the Epithelial Rest of Malassez may help in ce-
mentum repair because of their ability to activate
matrix proteins, such as amelogenin, which are also
expressed during tooth development (76, 81).
In summary, based on the information presented, it
appears that the developed or adult periodontium
retains its potential for repair/regenerationinthe form
of cells of the Epithelial Rest of Malassez, progenitor
cells and stem cells, which can be induced to differ-
entiate into cementoblast, osteoblast or periodontal
ligament cells to regenerate periodontal tissues.
Molecular factors involved in
periodontal development
It is well knownthat toothdevelopment is regulated by
temporal- and spatial-restricted reciprocal epithelial
mesenchymal interactions. A number of genes that
play a crucial role in tooth development have been
identied and include growth factors and their
receptors, such as transforming growth factor b-1
and )2, bone morphogenetic protein-2 and )4
(BMP-2, )4), activins, broblast growth factor-4, )8
and )9 (FGF-4, )8, )9), hepatocyte growth factor, and
midkine and transcription factors, such as the home-
obox genes (Msx1, Msx2, Dlx1, Dlx2, Dlx3, Otlx2,
Barx1), Pax genes (Pax9 and Pax6), and Lef1, Gli2/Gli3
and Shh (40, 100, 192, 249, 274). It has been docu-
mented that growth factors are involved in establish-
ing the presence, number, site, size or shape of teeth.
The availability of knockout mice has provided critical
information on some growth factors that are deter-
minants of early tooth development. However, little
information is currently available on the growth and
transcription factors involved in the later stages of
tooth development, such as root development. Al-
though one can assume that the same epithelial
mesenchymal interactions will take place between the
Hertwigs epithelial root sheath and the underlying
root mesenchyme, andall or some of the same growth
factors will be involved in root formation, these issues
have been only minimally addressed. Transforming
growthfactor b-1andits receptors (58, 59), andBMP-2,
)3 and )7 (249), have been identied in cemento-
blasts, periodontal ligament and alveolar bone, and
BMP-2, )4 and MSX-2 have been reported in the
Hertwigs epithelial root sheath (266). Fibroblast
growth factor-2 (143), receptors for epidermal growth
factor (42) and growth hormone (270) have been
detected in periodontal tissues. However, the pub-
lished studies are all descriptive and do not provide
198
Zeichner-David
information as to the function of these growth factors
in periodontium development. Furthermore, the
transforming growth factor-b1-knockout mouse dis-
plays no apparent defects in tooth and root develop-
ment (39), thus excluding a role for this factor in these
processes. Onthe other hand, by using transgenic mice
that express the BMP inhibitor, noggin, driven by the
keratin 14 promoter (K14-noggin), we recently dem-
onstrated that BMPs are important for proper root
morphogenesis. When the function of BMPs is re-
pressed, the transgenic mice demonstrate a delay in
tooth development, lack of enamel formation and
abnormally shaped roots (198). Insulin-like growth
factor-I receptor has been demonstrated in the Her-
twigs epithelial root sheath, and in vitro experiments
suggest that insulin-like growth factor-I receptor plays
a role in the proliferation and elongation of the Her-
twigs epithelial root sheath, which is critical for root
development (55).
Transcription factors associated with root develop-
ment include two members of the homeobox family of
transcription factors: Dlx2 and Dlx3. The expression of
Dlx2by the Hertwigs epithelial root sheathduringroot
development was demonstrated using Dlx2/LacZ
transgenic mice (132). Although these studies are only
suggestive of a role of Dlx2 in root development, it was
of interest that the Dlx2 knockout mice showednormal
teeth, while the Dlx1/Dlx2 knockout mice lacked
maxillarymolars (253). Theinvolvement of Dlx3inroot
development comes fromthe phenotype expressed by
patients affected with the genetic disease, tricho-
dento-osseous syndrome, which presents root defects
as well as defects in hair, bone and enamel. A deletion
of 4 bp in the Dlx3 gene, which causes a frameshift
mutation and premature codon termination, resulting
in an altered protein, were identied in a family with
tricho-dento-osseous syndrome (199). We recently
reported the importance of the N-c transcription
factor in root development. N-c knockout mice ap-
pear normal, except that they exfoliate their teeth
shortly after eruption. These mice show a lack of roots
of both mandibular and maxillary teeth, and therefore
their teeth have no bone attachment. Histological
analysis indicated a normal crown, enamel and dentin
formation, and although there is initial formation of
the Hertwigs epithelial root sheath and a budding
root, no further development occurs of the roots, ce-
mentumand periodontal attachment apparatus (239).
Cementum composition
In order to understand the process of cementogenesis,
it is important to determine the composition of
cementum. As in bone and dentin, the major com-
ponent of cementum is collagen (16). The expression
of noncollagenous proteins that stimulate cell migra-
tion, attachment, proliferation, protein synthesis and
mineralization during root formation has been
reported by several investigators (38, 142, 147). In the
early stages of root development, immunohisto-
chemical techniques have shown the expression of
multifunctional proteins, such as laminin and
bronectin (140). These proteins, as well as other
proteins extracted from cementum (173), are initially
believed to function as chemo-attractants. Laminin
and bronectin can also function as adhesion pro-
teins, together with tenascin (137), bone sialoprotein
(38, 142), osteopontin (25), and a 55-kDa cementum-
attachment protein (196, 263). The presence of other
bioactive proteins, such as enamel-like proteins (235,
234), osteonectin/SPARC (201), and mitogenic factors
(157, 269), have also been reported in the cementum.
In addition to these proteins, cementoblasts synthes-
ize and secrete several glycosaminoglycans (such as
chondroitin-4-sulfate, chondroitin-6-sulfate and der-
matan sulfate, and collagen brils), which are present
in the cementodentinal junction (88, 264, 265).
It has been suggested that cementoblasts exhibit
an osteoblast-like, rather than an odontoblast-
like, phenotype (25). Odontoblast, osteoblast and
cementoblast cells express several matrix proteins,
such as osteopontin, bone sialoprotein (BSP), osteo-
nectin, osteocalcin, matrix Gla protein (208) and den-
tin-matrix-protein 1 (DMP-1) (106). The presence of
osteocalcin in cementum is more controversial.
Bronckers et al. (25), using immunohistochemistry,
reported the presence of osteocalcin on the cellular
intrinsic ber cementum (CIFC) and associated
cementoblasts (mature), but not in the acellular
cementum and its associated cementoblasts. Tenorio
et al. (246) reported the presence of osteocalcin in
acellular extrinsic ber cementum (AEFC) but not in
the associated cementoblasts, while CIFC and associ-
ated cementoblasts stained weakly. Bosshardt &Nanci
(20) used two different antibodies (OC1 and OC2),
which gave different results: OC1 showed reactivity
with acellular cementum, while OC2 was negative.
Similarly, the presence of DMP-1 has been associated
with acellular cementum (275) and cementocytes, but
not with cementoblasts (255). It has been suggested
that acellular cementum is a unique tissue, while cel-
lular cementum and bone share some similarities,
although there are still morphological, functional and
biochemical differences between the two tissues (19).
The presence of cementum-specic proteins
remains questionable, although some putative
199
cementum-specic proteins have been invoked: a
55-kDa CAP (263); a mitogenic factor (167); and a
72-kDa protein, CEM-1 (235). However, as the char-
acterization and the sole expression by cementoblasts
of these proteins have not been determined, the
possible existence of cementum-specic proteins
remains unknown. It has been reported that
cementoblasts and cementocytes produce high levels
of the GLUT-1 monosaccharide transporter, while
osteoblasts or osteocytes do not express this protein.
These data suggest that GLUT-1 may play a role in
cementogenesis and could serve as a biomarker to
differentiate between cells of cementoblastic and
osteoblastic lineage (124). However, the observed
differences in GLUT-1 are quantitative, and GLUT-1 is
present in many different cell types. Recently, we
reported the isolation of a cementoblastoma-derived
protein, CP-23, that is expressedby cementoblasts and
some precursor cells present in the periodontal liga-
ment, but not by osteoblasts. The functionof the CP-23
protein is currently unknown; however, given its
nuclear location, it may be required for cementoblast
differentiation and may be used as a marker for
cementoblast cells (3). The CP-23 protein is also ex-
pressed by Hertwigs epithelial root sheath cells (275).
Based on our current knowledge of the develop-
ment of periodontal tissues, several strategies exist
for targeting regenerative therapy, ranging from
inducing their own regenerative mechanisms using
molecular approaches, or utilizing cells to repopulate
and recapitulate the developmental process.
Strategies for periodontal
regeneration/repair
The process of periodontal tissue regeneration starts
at the moment of tissue damage by means of growth
factors and cytokines released by the damaged con-
nective tissue and inammatory cells. It is well
accepted that in order to improve periodontal healing,
root planing or root conditioning is a necessary
antecedent to mesenchymal cell migration and
attachment onto the exposed root surface. Acid
treatment, in particular with citric acid, has been
found to widen the orices of dentinal tubules,
thereby accelerating cementogenesis and increasing
cementum apposition and connective tissue attach-
ment. However, a systematic review performed by
Mariotti (145) suggested that the use of citric acid,
tetracycline or EDTA to modify the root surface pro-
vides no clinical signicant benet for regeneration in
patients with chronic periodontitis. Conversely, when
periodontal ligament cells are removed from the ce-
mentum or are unable to regenerate, bone tissue in-
vades the periodontal ligament space and establishes
a direct connection between the tooth and the wall of
the alveolar socket, resulting in ankylosis. The ankyl-
otic, nonexible type of tooth support can lead to loss
of function and resorption of the tooth root (13).
Can guided tissue regeneration and bone
grafting regenerate cementum?
Nyman et al. (174), using Millipore membranes,
introduced the concept of a membrane barrier, which
excludes the apical migration of gingival epithelial
cells and provides an isolated space for the inwards
migration of periodontal ligament cells, osteoblasts
and cementoblasts. Guided tissue regeneration was
successfully used to aid in the regeneration of lost
periodontal tissues caused by periodontitis (67). The
rst guided tissue regeneration membranes were
nonabsorbable and made of polytetrauoroethylene,
such as Gore-Tex. Studies on experimentally
induced periodontal defects in monkeys suggested
that guided tissue regeneration was capable of
inducing the formation of new bone and cementum
(4). The second generation of guided tissue regener-
ation used absorbable membranes made of collagen
or polylactic and citric acid (28, 159), which elimin-
ated the need for surgical membrane retrieval (66).
Recent systematic reviews indicate that, in the
treatment of intrabony and furcation defects, guided
tissue regeneration is more effective than open ap
debridement. Various barrier types yielded no sys-
tematic difference in clinical outcome, but barrier
types could explain some heterogeneity in the results.
Overall, guided tissue regeneration is consistently
more effective than open ap debridement in the
gain of clinical attachment and reduction of probing
depth in the treatment of intrabony and furcation
defects (99, 163). The use of grafting material in
combination with guided tissue regeneration seems
to improve clinical outcomes for furcation, but not
for intrabony defects, when compared with the use of
barrier membranes alone. It has also been questioned
whether guided tissue regeneration produces true
cementum regeneration or only cemental repair. The
newly formed cementum has been characterized as a
cellular cementum that is usually poorly attached to
the dentin surface (125). It is suggested that perio-
dontal healing with guided tissue regeneration ther-
apy occurs in two stages. The rst stage comprises an
initial healing phase with the formation of a blood
clot, transient root resorption/demineralization,
200
Zeichner-David
deposition of acellular cementum on the root surface
and formation of connective tissue. The second
phase comprises a remodeling process, which will
result in a regenerated cementum similar to pristine
cementum as maturation proceeds over time (69). In
conclusion, several clinical studies have demonstra-
ted that guided tissue regeneration is a successful
treatment modality for periodontal reconstructive
surgery and it has become an accepted procedure in
most periodontal practices, either by itself or in
combination with other treatment modalities.
Autologous bone grafts to repair periodontal osse-
ous defects have been used for many years and dif-
ferent approaches have been the subject of several
reviews (165, 209). Bone repair can also be achieved
using ceramic materials such as Bioglass, which is a
bone-bonding bioactive material that has been widely
used for bone healing (110). Studies in monkeys sug-
gested that PerioGlas (synthetic bone particulate)
can achieve superior bone repair and cementum
regeneration and retard epithelial down-growth
compared with other, similar materials (50, 109).
Additionally, these materials can be used as scaffolds
or todeliver other bioactivemolecules toenhance their
function. The use of bone grafts, powders or ceramics
is quite prevalent in many dental practices. A recent
systematic reviewon the efcacy of bone replacement
grafts compared with other interventions in the treat-
ment of periodontal osseous defects was performed by
Reynolds et al. (202). Meta-analysis indicated that for
the treatment of intrabony defects, bone grafts are
effective in reducing crestal bone loss, increasing bone
level, increasing clinical attachment level, and redu-
cing probing depth compared with open ap debri-
dement procedures. Histological studies showed that
demineralizedfreeze-driedbone allografts support the
formation of a newattachment apparatus in intrabony
defects; however, the available data indicate that
alloplastic grafts support periodontal repair rather
than regeneration, and that the best treatment is a
combination of bone grafts with barrier membranes.
Nevertheless, these strategies are directed mainly to
enhance alveolar bone and periodontal ligament
repair and have the problems that they do not address
cementogenesis and therefore do not completely
regenerate the architecture of the original periodon-
tium.
Molecular approaches for cementum
regeneration
Advances in our knowledge of developmental bio-
logy, and of the growth factors that initiate and
regulate tooth development and tissue repair, sug-
gests the use of some of these factors for periodon-
tium regeneration (37, 61, 68, 71, 118, 116, 117, 128,
170). Some attachment proteins, such as bronectin
(29, 206, 262) or CAP (156, 196), are able to enhance
broblast migration, attachment and orientation of
the connective tissue to the root surface. New
strategies, utilizing growth factors to induce cell
migration, proliferation and differentiation, were
developed to repopulate the damaged periodontal
tissues with periodontal ligament cells (32, 247). It is
believed that growth factors play important roles in
modulating the proliferation and/or migration and/
or differentiation of structural cells in the periodon-
tium (58, 86, 97, 197, 230). It is suggested that growth
factor molecules are produced during cementum
formation and then stored in the mature cementum
matrix with the potential to induce periodontal repair
or regeneration when needed (236). Large-scale pro-
duction of recombinant growth factors has facilitated
in vitro and in vivo studies to determine the efcacy
of growth factors in periodontal tissue regeneration.
Amongst the growth factors currently being used
are platelet-derived growth factor, insulin-like growth
factor (36, 63, 92, 138, 188, 210), transforming growth
factor-b1 (146), basic broblast growth factor (213),
dexamethasone (211) and BMPs (121, 205, 211).
However, problems in applying these growth factors
for periodontal repair include the nonspecic activity
of some factors on different cell lineages in time and
space, and the rapid loss of growth factors applied
topically (13, 138).
It has been shown that both platelet-derived
growth factor and insulin-like growth factor-1 can
stimulate the proliferation and chemotaxis of perio-
dontal ligament cells, and that the combination of
platelet-derived growth factor and insulin-like growth
factor-1 can further increase the mitogenic effect (23,
44, 175). In addition to the mitogenic activity, plate-
let-derived growth factor also appears to stimulate
collagen synthesis in periodontal ligament cells (146).
Furthermore, dexamethasone has been shown to
exert the same effect as insulin-like growth factor-1
on periodontal ligament broblasts, gingival bro-
blasts and pulp broblasts, and may substitute for
insulin-like growth factor-1 in the platelet-derived
growth factor stimulation of cell proliferation (210).
In addition to the previously described effects,
platelet-derived growth factor has the capacity to
signicantly negate and reverse the inhibitory effects
of lipopolysaccharide on the proliferation of human
gingival broblasts. Lipopolysaccharide from a vari-
ety of gram-negative bacteria is known to inhibit
201
gingival broblast proliferation and synthesizing
activity, has been implicated in periodontal inam-
mation and may also be responsible for delayed
wound healing following periodontal therapy (12).
In vivo studies using the beagle dog (natural perio-
dontal disease) and the nonhuman primate (ligature-
induced attachment loss) models showed that the
application of platelet-derived growth factor/insulin-
like growth factor-1 resulted in signicant amounts of
new bone and cementum formation (138, 210).
Treatment with insulin-like growth factor-1 alone did
not signicantly alter healing compared with controls,
while treatment with platelet-derived growth factor
alone showed signicant regeneration of attachment.
Although there are differences in the response to
platelet-derived growth factor/insulin-like growth
factor-1, depending on which animal model is used
(the osseous response in dogs appears to be greater
than that of the nonhuman primate, while new
attachment formation appears to be greater in the
nonhuman primate than in the dog), there is consis-
tency in promoting periodontal regeneration (63, 64).
Rutherford et al. (211) showed that platelet-derived
growth factor and dexamethasone, combined with a
collagen carrier matrix, induced regeneration of the
periodontiumin monkeys. It has also been shown that
the combination of platelet-derived growth factor and
guided tissue regeneration work better than either of
the two modalities alone (36, 188).
Clinical trials in humans using platelet-derived
growth factor/insulin-like growth factor to treat per-
iodontal osseous defects showed that only high doses
of these factors gave rise to a statistically signicant
increase in alveolar bone formation (92). When
platelet-derived growth factor was used in combina-
tion with bone allografts to treat Class II furcations
and interproximal intrabony defects, histological
evaluation showed regeneration of new alveolar
bone, cementum, and periodontal ligament (30, 171).
Platelet-rich plasma is a fraction of plasma that
contains platelet-derived growth factor and trans-
forming growth factor-b (180). An alternative to the
use of recombinant growth factors is the use of a
platelet gel in combination with demineralized
freeze-dried bone allografts (5, 43).
The limitations of topical protein delivery to peri-
odontal osseous defects include transient biological
activity and bioavailability of platelet-derived growth
factor at the wound site. To overcome these limita-
tions, studies have used genetic engineering to
transduce cells derived from the periodontium, using
adenovirus carrying the platelet-derived growth fac-
tor gene to promote sustained release and ensure
biological activity (7, 6, 65). The potential use of gene
therapy in vivo to stimulate periodontal tissue
regeneration has been studied in large tooth-associ-
ated alveolar bony defects in rats. The results showed
that the direct gene transfer of platelet-derived
growth factor-B stimulates the regeneration of
alveolar bone and cementum (104).
As stated above, some members of the BMPs are
normally expressed during the development of the
periodontium, such as BMP-3 and BMP-7/OP-1,
which have been localized immunologically in
alveolar bone, cementum, and periodontal ligament,
whereas BMP-2 was only localized in the alveolar
bone (249, 266). Although the exact role of BMPs in
the development of the periodontium has not yet
been determined, these proteins are good candidates
for stimulating periodontal regeneration because of
their ability to promote not only osteogenesis but
also cementogenesis. The expected role of BMPs in
stimulating intramembranous bone formation with-
out an endochondral intermediate may provide
greater osteogenic potential than autogenous bone or
other bone substitutes (121, 118, 119, 170, 205, 240).
Studies indicate that recombinant BMP-2 exerts no
effect on the growth and differentiation of human
periodontal ligament cells in vitro; however, BMP-2
stimulates alkaline phosphatase activity and para-
thyroid hormone-dependent 3
,5
-cyclic adenosine
monophosphate (cAMP) accumulation, which are
early markers of osteoblast differentiation. Never-
theless, BMP-2 produced no mature osteoblasts, as
measured by expression of osteocalcin, and also
inhibited 1,25(OH)2D3-induced osteocalcin synthesis
in these cells (123). In vitro studies using mouse-de-
rived dental follicle and periodontal ligament cells
suggest that BMP-2 induced dental follicle cells to
differentiate towards a cementoblast/osteoblast phe-
notype but had no effect on periodontal ligament cells
(278). Paradoxally, BMP-2 was found to inhibit ce-
mentoblast cell mineralization in vitro by decreasing
the expression of BSP and collagen type 1 (279). In
studies of BMP-2 on early wound healing in a rat
model of periodontal regeneration, the connective
tissue attachment was found to be similar in animals
receiving BMP-2 and in controls. However, BMP-2
induced bone formation at some distance from the
defect, which indicates the need for a suitable delivery
system to maintain the BMP-2 at the site of implan-
tation (120). Other studies suggest that the effects of
BMPs may be inuenced by certain factors, such as
root surface conditioning, delivery systems, mastica-
tory forces, etc., and that BMP-2 stimulates the pro-
liferation and migration of cells from the adjacent
202
Zeichner-David
periodontal ligament into the wounded area, pro-
moting new cementum formation (119).
The expression of both BMP-2 and BMP-7 during
periodontal tissue morphogenesis suggests that
optimal therapeutic regeneration may require the
combined use of the two BMPs. BMP-7-treated molar
furcation defects in baboons resulted in substantial
cementogenesis, while BMP-2 showed limited
cementum formation but greater amounts of miner-
alized bone and osteoid; however, the combined
application did not enhance alveolar bone regener-
ation or new attachment formation over and above
that obtained by separate applications of the two
BMPs (207). Recently, it was shown that the appli-
cation of a synthetic BMP-6 polypeptide to a perio-
dontal fenestration defect in rats resulted in
increased formation of new bone and cementum
(93). Perhaps the use of other members of the BMP
family, such as growth and differentiation factor-5,
)6, and )7, might provide better and more complete
regenerative outcomes. These factors have been
detected during the process of periodontal develop-
ment at the surfaces of alveolar bone, cementum and
periodontal ligament ber bundles (223).
Limitations for the regular use of BMPs are the
need for high doses, non-specic activity on different
cell lineages in time and space, and the rapid loss of
topically applied growth factors (13, 138). Some of
these problems can be overcome by the use of gene
transfer technology. Jin et al. (103) used adenoviruses
containing BMP-7 to transduce dermal broblasts
that were then used to treat mandibular alveolar
bone defects in a rat wound repair model. Their
results showed chrondrogenesis, with subsequent
osteogenesis, cementogenesis and bridging of the
periodontal bone defects, suggesting that this genetic
engineering approach may be useful in alveolar bone
regeneration. A recent literature review (62) conclu-
ded that although promising, there were insufcient
data at the present time to conduct a meta-analysis
on the effect of growth factors for periodontal repair,
and pointed to the need for more clinical trials.
Do enamel-associated proteins
regenerate cementum?
Based on the presence of enamel proteins in acellular
cementum (133, 235, 182, 233), it was thought that
these proteins may play a role in the repair/regen-
eration of periodontal tissues destroyed by perio-
dontal disease (78). This idea was tested by adding
enamel proteins or puried enamel matrix derivative
to surgically produced periodontal defects in mon-
keys, followed by histological analysis that showed
almost complete regeneration of acellular cementum,
rmly attached to the dentin and with collagenous
bers extending towards newly formed alveolar bone
(79). These studies resulted in a new therapeutic
preparation to treat periodontal disease, consisting of
hydrophobic enamel matrix proteins extracted from
porcine developing enamel, which has been marke-
ted by Biora, Inc., under the name of Emdogain. In
the past 8 years, the use of enamel proteins for
inducing the formation of cementum, bone and
dentin has generated numerous in vivo and in vitro
studies, as well as clinical trials, resulting in almost
300 publications. In vitro studies, animal studies and
clinical trials are all being conducted simultaneously
(60, 70, 83, 154).
In vitro studies, using periodontal-associated cells
such as periodontal ligament broblasts, osteoblasts,
cementoblasts, gingival broblasts, gingival epithelial
cells, etc., have been conducted in an attempt to
understand the molecular and cellular mechanisms
involved in the process of enamel matrix derivative-
induced tissue regeneration. In order for enamel
matrix derivative to regenerate periodontal tissues, it
will need to exert an effect on proliferation, migra-
tion, attachment and/or differentiation of the sur-
rounding periodontal cells, and most studies have
measured these parameters, as shown in Table 1.
Few studies have tested the effect of enamel matrix
derivative on cell migration, but available data sug-
gest an increased migration of periodontal ligament
cells, osteoblasts, gingival broblasts and dermal
broblasts in response to enamel matrix derivative,
with the exception of one study that found no effect
on periodontal ligament cells (184). Most studies on
the effect of enamel matrix derivative on cell
attachment, which generally included periodontal
ligament cells, found an increase in cell attachment
(184). However, one study found the enamel matrix
derivative to have no effect on cell attachment of
gingival broblasts (256). A number of studies, which
measured the effect of enamel matrix derivative on
cell proliferation, have found an increase in cell
proliferation in the presence of enamel matrix
derivative. However, the proliferative effect was not
found in two studies using periodontal ligament cells
(41, 256), in two studies using osteoblast cell lines
(215, 268) and in one study using gingival broblasts
(256). Several studies found an inhibition of cell
proliferation when epithelial cells were used (112,
139, 273). These data may explain the clinical
observation that application of enamel matrix
derivative suppresses the down-growth of junctional
203
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(
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204
Zeichner-David
T
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1
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205
epithelium onto dental root surfaces, a process that
frequently interferes with the formation of new con-
nective tissue attachments (79, 78).
The majority of available in vitro studies have
analyzed the effect of enamel matrix derivative on
gene expression and differentiation, and most of
these studies found either an increased or a de-
creased expression of certain transcription and
growth factors, extracellular matrix proteins or min-
eralization-associated proteins in the cells tested.
Where mineralization was measured, it was found
that enamel matrix derivative induced mineralization
of periodontal ligament cells (166), increased miner-
alization of osteoblasts (268) and gingival broblasts
(115), decreased mineralization of cementoblast cells
(254) and inhibited the mineralization of dental fol-
licle cells (74). Differences in results amongst studies
can be explained by differences in sources and con-
centrations of enamel matrix derivative and in the
cell preparations used. Most studies employed pri-
mary cell cultures derived from different patients,
which probably contained mixed populations of a
variety of cells present in the periodontium. Never-
theless, taken together, these studies suggest that
enamel matrix derivative can act as a multipurpose
growth factor capable of stimulating the proliferation
of mesenchymal cells while inhibiting the cell divi-
sion of epithelial cells, and can stimulate attachment
and phenotypical changes in some cells, while
inhibiting matrix production in others.
Given the widespread use of Emdogain, and the
fact that it is made from an extract of enamel pro-
teins, it is important to identify the actual protein
responsible for its function. Studies by Maycock et al.
(148) found that, in addition to amelogenin, Emdo-
gain contains metalloproteases and serine pro-
teases. Studies by Kawase et al. (114) demonstrated
that porcine enamel matrix derivative contains
transforming growth factor-b1 (or a transforming
growth factor-b-like substance), and that the action
of enamel matrix derivative is mediated by the smad-
2 signaling pathway. In addition, a neutralizing
anti-transforming growth factor-b immunoglobulin
blocked the action of enamel matrix derivative on
epithelial cells, although it failed to block completely
enamel matrix derivative-induced broblastic prolif-
eration, suggesting the presence of more than one
growth factor. Iwata et al. (98) isolated the inductive
activity of enamel matrix derivative by using chro-
matography and characterized it as being BMP-2 and
BMP-4 using specic antibodies. Furthermore, in the
presence of noggin (an inhibitor of BMPs), enamel
matrix derivative lost its inductive activity, indicating
that BMPs are the molecules responsible for enamel
matrix derivative activity. Although these studies
suggest that the action of Emdogain is a result of
the presence of contaminating growth factors, other
studies have shown that pure recombinant enamel
proteins indeed have activity as inducers. The results
obtained in our laboratory indicate that mouse
recombinant amelogenin can increase attachment
and proliferation of mouse periodontal ligament cells
in vitro (272, 273). Furthermore, a post-translational
modied recombinant ameloblastin, another enamel-
associated protein, had an effect similar to that of
amelogenin on periodontal ligament cells. Both
recombinant amelogenin and ameloblastin can
change the phenotype expressed by periodontal
ligament cells by inhibiting the expression of colla-
gen type I and inducing de novo expression of
osteocalcin. Amelogenin also induced the expression
of bone sialoprotein and BMP-2, while ameloblastin
induced the de novo expression of BMP-3 (273).
These results indicate that both enamel-associated
proteins have a modulatory effect on the expression
of BMPs, suggesting that perhaps these proteins exert
their signaling effect by means of BMPs. Recombin-
ant mouse amelogenin improved osteoblast adhe-
sion (90), and increased the expression of bone
sialoprotein and decreased the formation of miner-
alized nodules in cementoblasts (258). A leucine-rich
amelogenin peptide, which exhibited no effect on
cell proliferation, down-regulated osteocalcin and
up-regulated osteopontin in a dose- and time-
dependent manner, and inhibited the capacity to
form mineral nodules (17). Taken together, these
reports point towards a growth factor activity for
enamel proteins that may be of importance in
periodontal tissue regeneration.
Several clinical trials have shown an increase in
periodontal attachment and bone formation in indi-
viduals treated with Emdogain (54, 85, 87, 154, 179,
200, 217, 216, 218, 219, 277). However, in many of
these studies, the results were no better than those
obtained with other previously established treat-
ments, such as guided tissue regeneration, which
yields better outcomes in the management of deep
intrabony periodontal defects (84, 187, 218, 221, 231).
Histological studies revealed that treatment with
Emdogain is unpredictable, resulting in the forma-
tion of cellular cementum rather than acellular
cementum, and this cementum was only partially
attached to the root surface, similar to the cementum
formed with the use of guided tissue regeneration.
Furthermore, more bone regeneration occurred by
using a guided tissue regeneration procedure than
206
Zeichner-David
Emdogain (216, 219, 218). Other studies showed no
evidence of improvement in radiographic bone level,
and surgical re-entry found new tissue with a rubbery
consistency and that was not mineralized (189, 190).
Experiments in rats, using a wounded rat periodon-
tium model followed by immunohistochemical
analysis, showed that Emdogain does not affect the
expression of differentiation markers or bone matrix
protein synthesis in the repopulation response of
wounded rat molar periodontium (35).
Systematic studies, using literature reviews and
meta-analysis, suggest that treatment with enamel
matrix derivative results in signicant variations in
clinical outcomes (107). Although Emdogain is able
to signicantly improve probing attachment levels
and pocket depth reduction, some studies found no
evidence of clinically important differences between
guided tissue regeneration and Emdogain (47, 62)
and reported that guided tissue regeneration is more
predictable for cementum and bone regeneration
(257). Although animal histological studies with sur-
gically created defects suggest that enamel matrix
derivative induces the formation of acellular cemen-
tum and promotes attachment of the supporting
periodontal tissues, human histological studies have
questioned both the consistency of the histological
outcomes and the ability of enamel matrix derivative
to predictably stimulate the formation of acellular
cementum (107). It appears that following treatment
with enamel matrix derivative, a bone-like tissue
resembling cellular intrinsic brous cementum is
formed (22).
Despite the mixed results obtained from both
in vitro and in vivo studies, new applications of
Emdogain are continuously being reported. Some
studies suggest that it has the ability to induce the
formation of reparative dentin in pulpotomized teeth
(94, 96, 168, 169). It is being used to coat titanium
implants with mixed results; one study suggests that
there is enhanced formation of trabecular bone (229)
while the other found no effect (53). It has also been
suggested that enamel matrix derivative can combat
bacteria in postsurgical periodontal wounds, which
otherwise could hamper wound healing and reduce
the outcome of regenerative procedures (8, 172, 220,
237). More recently, an acceleration of skin wound
14 days
PLF PL-7 DPM
Control Control HERS-CM HERS-CM Control HERS-CM
21 days
28 days
35 days
Fig. 2. Effect of Hertwigs epithelial root sheath-condi-
tioned media (HERS-CM) on periodontium-associated cell
mineralization. HERS-CM was prepared by growing the
cells in Dulbeccos modied Eagles minimal essential
medium (DMEM) supplemented with 10%fetal calf serum
(FCS) and 100 U/ml of penicillin/streptomycin. Cells were
incubated at 39.5C in a humidied atmosphere of 95%
air and 5% CO
2
for 7 days, after which the media were
collected, the protein concentration determined and then
lyophilized. Periodontal ligament broblasts (PLF), ce-
mentoblasts (PL-7) and dental papillae mesenchyme
broblasts (DPM) were prepared from Immortomouse
(275). Cells were grown in differentiation conditions
(DMEM supplemented with 10% FCS, 100 U/ml of peni-
cillin/streptomycin, 50 mg/ml of ascorbic acid and 2 mM
sodium b-glycerophosphate), with or without (controls)
100 lg of HERS-CM proteins. At different time-points of
culture, cells were xed with 70% methanol and 30%
acetic acid and stained with Von Kossa to determine
mineralization.
207
healing in the presence of enamel matrix derivative
was reported (160).
Cellular tissue engineering for
cementum regeneration
It has long been recognized that a recolonization of
periodontal ligament cells onto the root surface is
necessary for periodontal ligament regeneration (129,
174). One therapeutic approach proposed the removal
of autologous cells from the patients periodontal
ligament, culture of the cells in vitro, to place them
back onto the exposed root coated with chemo-
attractant factors, and then to cover the area with an
articial basement membrane (247). A pilot study was
carried out with four patients, using hydroxyapatite as
a vehicle for cell delivery. After 6 months, the treated
patients exhibited greater pocket reduction and clin-
ical attachment gain, and less gingival recession, than
control patients; however, both groups showed good
ll of the osseous defects studied (48, 49, 91).
Lekic et al. (130) tracked the fate and differenti-
ation of rat periodontal cells and bone marrow cells
transplanted into periodontal wounds in rats using
cells constitutively expressing b-galactosidase as a
marker. Labeled cells were localized in the perio-
dontal ligament and regenerating alveolar bone and it
was suggested that, following a cyclical process of
growth and development, both cell types were able to
differentiate into periodontal ligament broblasts,
osteoblasts and cementoblasts, and to contribute to
periodontal regeneration (131). Regeneration of
cementum, periodontal ligament and alveolar bone
has also been observed using auto-transplantation of
bone marrow mesenchymal stem cells into perio-
dontal osseous defects in dogs (111). Similar results
have been observed after the application of perio-
dontal ligament cell sheets (2).
The ability of cementoblasts and dental follicle
cells to promote periodontal regeneration in a rodent
periodontal fenestration model was analyzed recently
(280). The results indicated that cementoblast-trea-
ted and carrier alone-treated defects showed com-
plete bone bridging and periodontal ligament
formation; however, no new cementum was formed
along the root surface in either group. Puzzling,
however, was the fact that no repair, or even osteo-
genesis, was seen within dental follicle cell-treated
defects, even though these cells are believed to be
precursors of cementoblasts and to be responsible for
alveolar bone formation.
As our laboratory has established immortal cell
lines for the Hertwigs epithelial root sheath (275) and
the Epithelial Rest of Malassez cells, we are exploring
the ability of these cells, or their secreted products, to
induce periodontal ligament cells to differentiate into
cementoblasts in vitro. When periodontal ligament
cells, which do not produce a mineralized extracel-
lular matrix, are grown in the presence of Hertwigs
epithelial root sheath conditioned media (HERS-CM),
these cells produce a mineralized extracellular mat-
rix, as determined by a positive Von-Kossa staining
Effect of HERS-CM on PLF
cell differentiation
P
BSP
OCN
OSN
OPN
AP
BMP4
Col1
Actin
21d 21d + HERS
Fig. 3. Effect of Hertwigs epithelial root sheath-condi-
tioned media (HERS-CM) on the phenotype of periodontal
ligament cells. HERS-CM was prepared as previously
described. Periodontal ligament cells were grown under
proliferation (P) conditions (in the presence of interferon-
c at 33C) or differentiation conditions [Dulbeccos
modied Eagles minimal essential medium (DMEM)
supplemented with 10% fetal calf serum (FCS), 100 U/ml
of penicillin/streptomycin, 50 mg/ml of ascorbic acid and
2 mM sodium b-glycerophosphate] with or without (con-
trols) 100 lg of HERS-CM proteins. Cells were collected
after 21 days in culture (media were changed every other
day), the media were removed, cells were rinsed in
phosphate-buffered saline (PBS) and total RNA was
extracted for determination of phenotype by using reverse
transcriptionpolymerase chain reaction (RTPCR). AP,
alkaline phosphatase; BMP-4, bone morphogenetic pro-
tein-4; BSP, bone sialoprotein; Col1, collagen type I; OCN,
osteocalcin; OPN, osteopontin; OSN, osteonectin.
208
Zeichner-David
(Fig. 2). This effect is specic for periodontal liga-
ment cells because other types of broblasts, such as
those derived from the dental pulp, do not produce a
mineralized extracellular matrix, even in the presence
of HERS-CM. When cementoblast cells, capable of
producing a mineralized extracellular matrix, were
grown in the presence of HERS-CM, an acceleration
in the formation of mineral was detected. Analysis of
the phenotype at the molecular level indicated a
de novo induction of the expression of bone sialo-
protein and osteocalcin, two markers of mineraliza-
tion (Fig. 3). These results support the concept that,
during root development, the secreted products of
the Hertwigs epithelial root sheath induce adjacent
cells of the periodontal ligament to differentiate and
produce new cementum. However, whether these
cells differentiate into cementoblasts or osteoblasts
awaits further in vivo experiments.
Conclusions
It is obvious that major progress has occurred in the
world of biology, medicine and dentistry in the past
30 years, and the management of periodontal disease
has beneted from these advances. New knowledge
about the etiology and pathogenesis of periodontitis,
the relationship of the disease to systemic problems,
and advances in genetics, molecular biology, cell
biology and biomaterials, have opened the door for
new regenerative techniques based upon the tissue
engineering approach. Treatment of periodontal
disease has evolved from just ghting bacteria to a
combined effort to eliminate the offending microor-
ganisms, to arrest the progression of tissue damage
and to regenerate lost tissues. Although some of the
regenerative techniques have been available for sev-
eral years, and some have shown promising results,
none of the techniques are without problems and
none have proven to be 100% effective. Many of the
regenerative approaches reviewed in this article are
still under assessment and further research is needed
to develop cell-based tissue strategies, perhaps using
stem cells and biomaterials for delivery of these cells.
New scaffold materials, which are being developed,
are also needed to address some of the delivery issues
(164). What may be concluded from the current sta-
tus of periodontal regeneration is that, as many
investigators have previously stated, there is not
going to be one magic solution that can be used to
treat all periodontal patients, but rather a combina-
tion of different approaches that can be adjusted to
t the specic need of individual patients.
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217
Challenges and potential in
tissue engineering
HAROLD C. SLAVKI N & P. MARK BARTOLD
During the last 50 years we have realized that science
is the fuel for the engine of technology. Scientic dis-
coveries from cellular, developmental, and molecular
biology have truly revolutionized our collective
understanding of biological processes, human genetic
variations, the continuity of evolution, and the etiol-
ogy and pathogenesis of thousands of human diseases
and disorders. This enormous accumulation of sci-
entic discovery (theory, principles, concepts, and
facts) provides the fuel for the clinical research and
translation revolution of the 21st century (15). This is
particularly evident when considering the opportun-
ities to understand the etiology, pathogenesis, treat-
ments, and outcomes related to periodontal diseases
and disorders, the science of dental and medical tissue
engineering, and comprehensive evidence-based
periodontics for the 21st century (7, 14, 16, 28, 30, 32,
4550, 52, 54, 60). Today the eld of tissue engineering
has established the essential foundations for the de-
sign and fabrication of neo-tissues in two or three
dimensions for transplantation (29, 30, 31).
Biology has become an
information science
The discovery of the structure of DNA transformed
biology, catalyzed the human genome project, and
engendered a new biology as an information science
(1114, 19, 2325, 39, 44, 59, 60). Two features of
DNA structure should be considered: the digital nat-
ure, and the complementary features (one strand of
the DNA helix binds perfectly with its complementary
strand of DNA). DNA has two types of digital infor-
mation: the genes, which encode the information that
directs protein content and synthesis, and the gene
regulatory networks, which specify the behavior of the
genes (e.g. on/off as well as levels of expression).
The recent completion of the Human Genome
Project provides a parts list of life or 30,009 discrete
structural and regulatory genes mapped within 23
pairs of human chromosomes (http://genome.
ucsc.edu and http://www.ncbi.nlm.nih.gov/genome/
guide/) and the mitochondrial DNA (mtDNA) (6, 8,
13, 2325, 33, 4244, 61, 62). The human genome
project was designed in 1988, ofcially started in
1990, and delivered the rst draft with 95% accuracy
in 2001 (24, 61). Functional genomics is the study of
human, as well as microbial gene function(s), as
discrete protein activities or within the orchestration
of thousands of different proteins. We now appreciate
that each gene can produce as many as eight or ten
different isotypes through a process of alternative
splicing that results in several hundred thousand
proteins. The eld of proteins and their various
structures and functions is termed proteomics. This
new post-genomic era provides the questions and
tools to better dene which modules of genetic
information are critically signicant in disease diag-
nosis and to better understand the pathogenesis of
disease biological processes (39). Moreover, we can
now dene human genetic variation; approximately
no more than 0.1%between any two people on Earth
(13, 25, 44). The recent draft and analysis of the
human genome has revealed signicant evolutionary
continuity (8, 13, 2325, 33, 42, 43, 59, 61, 62). More
recent progress has resulted in the completion of the
chimpanzee genome (5, 6, 21), a critical comparison
between the human and chimpanzee genomes (5, 6,
21), and a robust analysis of the conservation of
Y-linked genes during human evolution, including
the major gene amelogenin, which is required for
enamel matrix formation (5, 6, 21).
Genes control complex circuits or
networks
Human and microbial gene and protein functional
investigations reveal complex circuits, networks, or
9
PERIODONTOLOGY 2000
pathways that are required for biological processes
such as oral, dental, and craniofacial morphogenesis,
tissue regeneration, infection/virulence, and the
pathogenesis of inammatory diseases and disorders.
Complex networks of genes and their products (pro-
teins) are expressed to address growth and develop-
ment as well as multiple host responses to surgical
and loading trauma such as inammatory, endo-
crine, vascular, and neurological responses. Soft and
hard tissue wound healing involves many hundred
genes with multiple, yet complementary, functions.
Many hundreds of gene activities change under the
forces associated with tooth movements, and these
changes are further restricted by the different stages
of craniofacial growth and development. These and
other scientic and technological changes are already
having an impact on orthodontics, oral and maxillo-
facial surgery, oral and periodontal medicine,
implants and osseointegration, prosthodontics,
pediatric dentistry, public health dentistry, bioimag-
ing and radiology, and endodontics with new diag-
nostics, therapeutics, and biomaterials for the 21st
century (4, 7, 11, 15, 16, 30, 31, 39, 4553, 60). A major
derivative from the Human Genome Project is the
new era of molecular dentistry and medicine (8, 11,
14, 19, 33, 38, 39, 42, 4650, 58, 62).
Examples of genomics,
biomimetics, and tissue
engineering: tooth regeneration
During the last few years, new cellular, develop-
mental, and molecular knowledge has become
available for describing and understanding tooth
morphogenesis; this includes determination, initi-
ation, dental lamina, bud, cap, bell and crown sta-
ges, and root formations (3, 4, 27, 45, 48). These
molecular studies of dental biological processes have
yielded the identication of the combination of
regulatory and structural genes required for tooth
morphogenesis (4, 27). For example, tooth develop-
ment depends upon networks, or combinations, of
transcription factors (27). Gene networks that con-
trol signal transduction processes during tooth
morphogenesis have been discovered that belong to
several families including the hedgehog, the bone
morphogenetic proteins, the broblast growth
factors, and the Wnt family of signaling molecules
(3, 4, 27). The genes that control both when and
where, and tooth size and shape have been identi-
ed (3, 4, 27, 45, 48). In addition, the principles for
the possible design and fabrication of tooth organ
development have been discovered (9, 17, 26, 34, 37).
Bioengineering is a strategy that can mimic these
and other biological processes using biomimetics
(to mimic biology), and thereby design and fabri-
cate tooth organs for tooth replacements, perio-
dontal tissue engineering, and a host of new human
and microbial gene-targeted therapeutics (9, 16, 17,
26, 34, 37). Moreover, the recent discoveries of adult
stem cells, such as the mesenchymal stromal stem
cells that reside in dental pulp tissue, gingival dermis
tissue, and bone marrow tissue, provide numerous
opportunities for applications to tissue engineering
(9, 17, 26, 34, 37) (Fig. 1).
A paradigm shift involving
biological solutions to biological
problems
Biological solutions to biological problems is emer-
ging as a new paradigm in dentistry and medicine.
Diagnosis, treatment, therapeutics, and biomaterials
are all becoming biological and gene-based. We are
on the verge of shifting or evolving from mechanical
(e.g. surgical) to biological solutions for health pro-
motion, risk assessment, diagnostics, treatments,
therapeutics, and health care outcomes (7, 11, 1416,
19, 2931, 39, 45, 46, 5054, 55, 5760, 63) (Table 1).
The 21st century also appears to represent a time in
history when there is a convergence between clinical
dentistry and medicine, human genetics, develop-
mental and molecular biology, biotechnology, bio-
engineering, and bioinformatics (9, 11, 14, 17, 19, 26,
2931, 34, 37, 39, 48, 59, 63). In the context of this
perspective on tooth and bone regeneration, for
example, there appear to be connections between
clinical human genetics and craniofacialoraldental
dysmorphogenesis, clinical dentistry and medicine,
the human genome completion and molecular bio-
logy (functional genomics, proteomics), stem cell
biology, tissue engineering and nanotechnology,
and bioinformatics, that support the feasibility of
applying tissue engineering to clinical problems in
dentistry (9, 11, 14, 1618, 19, 26, 2832, 34, 35, 37, 39,
40, 4550, 52, 54, 57, 59). At this convergence is the
opportunity for bioengineering to design and fabri-
cate enamel, dentine, cementum, periodontal liga-
ment, and alveolar bone. The papers that have
contributed to this special issue demonstrate the
remarkable progress the application of tissue engin-
eering to periodontics.
10
Slavkin & Bartold
Meanwhile, for fundamental changes to take
place in clinical periodontics, a number of trans-
formations are needed to advance clinical research
enterprise (58), oral health care provisions, econo-
mic management, and training (60). It is currently
extremely difcult to predict the costbenet ratio
Table 1. Tissue engineering approaches in periodontics
Technique Advantages Complications
Cell injection Easy delivery
Injected stem or precursor cells
can induce the formations of extracellular
matrices and blood vessels
Low cell survival
Cells may not differentiate
Cultured tissues Easy to grow in the laboratory
Increased stability compared
with cell injection
Tend to be very small in size
without vasculature
Very fragile
Porous scaffolds Supports cell organization and
promotes vascularization
Delay between implantation and
vascularization
Three-dimensional printing Multiple cell types can be
precisely positioned
Inconsistent results
Injectable scaffolds Simple delivery
Can mediate regeneration by
providing biomedical cues
Inconsistent results
Fig. 1. Tooth morphogenesis advances from the initi-
ation of the dental lamina through tooth eruption using
an elaborate and complex network of signaling, signal
transduction (growth factors binding to complementary
receptors), and subsequent gene regulation through
transcription factors binding to specic regions of genes.
This scheme indicates the sequence of tooth morpho-
genesis and the sequence of genes expressed into the
orchestration of tooth formation (the scheme is modied
from Refs 25, 36, and 38). Growth factors are listed in
regular type and transcriptional factors are represented in
italics. Growth factor abbreviations: BMP, bone morpho-
genetic proteins; FGF, broblast growth factors; PDGF,
platelet-derived growth factors; SHH, Sonic hedgehog;
TGF-b, transforming growth factor-b; WNT, wingless-type
mouse mammary tumor virus integration site family.
11
Tissue engineering: challenges and potential
for translation of the new biology into clinical
practice. Many indicators suggest that molecular
dentistry and medicine will not be initially less
expensive than current practices. Very few dental
and medical schools adequately educate and train
their students to think about diseases and disorders
in terms of biological mechanisms. Importantly, all
too many dental and medical schools are following
a trend of pattern recognition rather than basic
or fundamental understanding of the molecular
pathogenesis of diseases and disorders. What this
suggests is that we may not yet be preparing the
next generation of clinicians to apply the know-
ledge and tools derived from the remarkable con-
tributions from the human genome and all that
follows. The incremental evolution in oral health
care that will incorporate these new molecular
principles of early diagnosis and individualized
therapy will be an incredible challenge as well as
an opportunity in an era of uncertainty regarding
U.S. health care systems (60).
Tissue engineering and the
periodontium
As detailed above, tissue engineering is a contem-
porary area of applied biomedical research aimed at
developing procedures and biomaterials for the fab-
rication of new tissues to replace damaged tissues
and is based on principles of cell biology, develop-
mental biology, and biomaterials science.
The main requirements for producing an engin-
eered tissue are the appropriate levels and sequen-
cing of regulatory signals, the presence and numbers
of responsive progenitor cells, an appropriate extra-
cellular matrix or carrier construct, and an adequate
blood supply. Recent advances in growth factor bio-
logy and biodegradable polymer constructs have set
the stage for successful tissue engineering of carti-
lage, bone, and related tissues. The periodontium
could be considered a prime candidate for such
procedures. Preliminary studies have indicated that
periodontal ligament and bone cells can be trans-
planted into periodontal sites with no adverse
immunological or inammatory consequences.
In this volume we have tried to distill the current
developments in the eld of tissue engineering by
inviting experts from around the world to share their
expertise and knowledge in the intricate and complex
processes of regenerating tissue via a tissue engin-
eering approach.
In this introductory chapter the scene is set with an
overview of how genomics, biomimetics, and pro-
teomics will play pivotal roles in the development of
this edgling eld. This chapter explores how the
post-genomic era now permits a more structured
approach to many biological problems. This chapter
highlights how new cellular, developmental, and
molecular knowledge has enabled a better under-
standing of tissue formation and organ morphogen-
esis. By copying these processes, biomimetic design
and fabrication of tissues and organs has become a
strategy for fabrication of tissue and organ replace-
ments. Thus an emerging paradigm of biological
solutions for biological problems is appearing in
both clinical dentistry and medicine. This allows
diagnosis, treatment, therapeutics, and biomaterials
to become biological and gene-based rather than
simply mechanical.
Recognizing that these new technologies are upon
us, Smith (55) presents a legal perspective to the
issues that will confront us now and in the future.
Issues such as patent protection, patient welfare and
protection, public health debate, integration of sci-
ence, clinical practice, and corporate nancing are
but a few of the issues confronting this new eld. Of
course no new development is free from govern-
ment scrutiny and intervention. Government agen-
cies are becoming increasingly involved in product
development from the funding of the science that
leads to its development, through consideration of
the ethical implications of such developments to
protecting the safety of the recipients (our patients)
of these products.
As already detailed above, understanding the basic
principles of embryogenesis and wound healing will
lead us to the principles of biologically based ther-
apies. After all, development and wound repair and
regeneration are not dissimilar processes. In the
chapter by Polimeni et al. (41) the complex processes
of periodontal wound healing and regeneration are
discussed in light of current technologies. The use of
barrier membranes as well as of potent biological
agents to induce and promote regeneration is
discussed. These studies clearly herald the com-
mencement of a tissue engineering approach for the
management of periodontal destruction.
The molecular aspects of tissue regeneration will
undoubtedly be of tremendous signicance. To nd
the right signaling molecules expressed and delivered
in the correct spatial and temporal sequence is a
daunting task. In the chapters by Hughes et al. (22),
Ripamonti & Rentin (43), and Srisuwan et al. (56), the
specic features of matrix molecules, growth factors,
12
Slavkin & Bartold
cytokines, and the bone morphogenetic proteins are
specically discussed. From these three chapters we
nd that signicant headway is being made in the
rational use of these agents and how they affect not
only cellcell interactions but also cellmatrix for-
mation.
Notwithstanding the importance of soluble
mediators for tissue engineering and tissue regen-
eration, a critical factor limiting success is neovas-
cularization of the implants. Without rapid and
effective vascularization of an implanted engineered
matrix the chances of successful tissue regeneration
are almost negligible. By using bone engineering as
an example Hsiong & Mooney (20) discuss new
approaches and strategies to develop adequately
vascularized bone tissue for tissue engineering
purposes.
In the remaining chapters various biomaterials and
approaches for tissue engineering are explored. The
principles and applications of cell delivery systems
for periodontal regeneration are presented by Bartold
et al. (1). Successful tissue engineering requires an
interplay between three components:
the implanted and cultured cells that will create the
new tissue.
a biomaterial to act as a scaffold or matrix to hold
the cells.
biological signaling molecules that instruct the
cells to form the desired tissue type.
This review focuses mainly on the use of scaffold
materials used to transplant cells as a means of
delivering either cells or proteins to a defect site.
The importance of biomaterial surface structure,
chemistry, and conguration is explored by Ellingsen
et al. (12) and Moradian-Oldak et al. (36). Here we see
how even minor, and often quite simple, changes to a
biomaterial can have a signicant impact on the
surrounding tissues and cell function. Many labor-
atories are producing data on the impact of surface
modication of dental implants and bone regener-
ation. Chemical and biomimetic strategies are
increasingly being developed to design improved
surface chemistry of implants. The utilization of
these coatings as carriers of different therapeutic
and bioactive agents, including amelogenins, is also
under active investigation.
Another surface often overlooked is that of the root.
In the chapter by Zeichner-David (64), the critical
role played by cementum in periodontal regeneration
is explored. This review provides an up-to-date
overview of advances in tissue engineering research
to recapitulate the developmental process involved
in cementogenesis, osteogenesis, and formation of
periodontal ligament bers leading to the regener-
ation of the damaged periodontium.
No current consideration of regeneration today
would be complete without mentioning stem cells.
While the use of embryonic stem cells is still very
controversial, the harvesting and use of postnatal
mesenchymal stem cells, which reside in most, if not
all, adult tissues, are receiving considerable attention.
The chapter by Duailibi et al. (10) details pioneering
work on the successful bioengineering of pig and rat
tooth tissues, including enamel, dentin, and pulp,
from cultured tooth bud cells seeded onto biode-
gradable scaffolds implanted and grown in the
omenta of adult rat hosts. These results signicantly
advance the practical application of tooth tissue
engineering strategies by demonstrating that whole
replacement teeth may eventually be grown at the
site of previously lost teeth. Bone marrow stromal
stem cells also possess potent regenerative potential.
Gronthos et al. (18) highlight that with an increasing
number of older people, the aging baby boomer
generation, and the increasing life expectancy in
developed countries, there is an increased clinical
need for effective bone regeneration treatment
options to repair skeletal defects caused by trauma
and disease. Current bone regenerative techniques,
such as autologous bone graft, allografts, and allo-
plastic materials, have limitations that hinder their
use in a wider range of clinical conditions. Hence, the
development of improved methods such as bone
marrow stromal stem cell-mediated bone regener-
ation is necessary for achieving future viable thera-
peutic alternatives.
We also present the exciting work of Baum & Tran
(2). Here the concepts of developing an articial
organ utilizing the synergy of genetic and tissue
engineering are presented. In this chapter the con-
cepts of the previous chapters come together to
highlight the trials and tribulations of tissue engin-
eering. This is not going to be an easy task but by
adhering to biological principles, together with a little
ingenuity, we see that it is possible to recreate a func-
tional organ in the form of an articial salivary gland.
In conclusion, the future appears exciting for tissue
engineering. However, it is clear that, as periodon-
tists, we will not be able to engineer any components
of the periodontium without enlisting the help of
many experts. This will be a team effort engaging the
expertise of biomaterials scientists/engineers, cell
biologists, matrix biologists, molecular biologists,
microbiologists, immunologists, pharmacologists,
nanotechnologists, and of course clinical perio-
dontists.
13
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15
Smoking and periodontal disease
Francisco Rivera-Hidalgo
The study of the relationship between periodontal
disease and smoking has received increased atten-
tion during the past few years. A search of published
papers indicates that more than 200 papers dealing
with this relationship have been written in the past
4 years. Recently, there have been excellent reviews
of this topic (33, 36), including a position paper by
the American Academy of Periodontology (51). This
position paper implicates smoking as a risk factor
affecting the prevalence and progression of period-
ontal diseases (adult periodontitis, refractory period-
ontitis, generalized early-onset periodontitis and
acute necrotizing ulcerative gingivitis) and that the
severity of periodontal disease is related to smoking
use. With some exceptions, this paper will review
recent publications (19992002) and draw opinions
based on the reported ndings.
Gingival bleeding and alveolar
bone loss
A study of co-twins (4) indicated that the degree of
alveolar bone loss and the number of teeth lost were
greater in the twins with a high lifetime smoking expo-
sure when compared to their twin partners with a low
life-time exposure. Also, that gingival bleeding was
less in the high lifetime smoking exposure group. Sub-
sequently, it was reported that even though smokers
had a signicantly greater plaque index, the average
number of bleeding sites in smokers (27%) was smal-
ler than in non-smokers (40%) (56). When reviewing
369 periodontal patients with moderate to severe per-
iodontitis, smokers reported fewer gingival bleeding
sites (25%) than did non-smokers (51%) (57). Further,
the investigators suggested that gingival inammatory
symptoms appeared to be suppressed in smokers.
This subject was further studied by looking at the
development of experimental gingivitis in a group of
20 dental students, 10 of whom had been smokers for
at least 4 years. The subjects were free of periodontitis
and not taking any medications. During the develop-
ment phase, patients were examined at 7, 14, 21 and
28 days, and afterwards at 35 and 42 days. This study
revealed the number of gingival bleeding sites, the
amount of gingival exudate and the number of gingi-
val sites with distinct redness to be signicantly less in
smokers during the development phase, essentially
returning to day 0 levels at the 35- to 42-day examina-
tions. Dental plaque accumulation revealed no signif-
icant difference between the two groups. This study
lends support to the contention that the inammatory
gingival response could be suppressed in smokers (5).
Another study reported that after 28 days of plaque-
induced gingivitis, evaluated with stereophotographs,
the intensity of the vascular reaction in smokers was
only 50% of that observed in non-smokers (8).
When looking at the vestibular gingival blood ves-
sel density using monoclonal antibody to CD34
molecule expressed on endothelial cells and hema-
topoietic progenitor cells, the authors report that
smokers had a higher proportion of small blood ves-
sels and a lower proportion of large blood vessels
compared to non-smokers, whereas the difference
in density was not signicant (45). The authors spec-
ulate that the difference in blood vessels may be
associated with a suppression of the inammatory
response. The hemorrhagic responsiveness in 243
patients with different levels of periodontal disease
was found to be suppressed in smokers, while in
non-smokers an exponential hemorrhagic response
to increasing plaque levels was suggested (2).
Bone loss may be associated with smoking even in
patients with good oral hygiene. In a group of 235
patients of whom 72 were smokers, radiographic
analysis of bone height expressed as a percentage
of root length, revealed lower mean bone levels for
the smokers (77.982.8%), suggesting that smoking is
a risk factor for periodontal health (3). In another
radiographic study, the distance from the cemento-
enamal junction to the interdental septum in 210
Swedish dental hygienists with good oral hygiene and
no periodontal disease was greatest in smokers, fol-
lowed by former smokers and then non-smokers (7).
50
Periodontology 2000, Vol. 32, 2003, 5058 Copyright
#
Blackwell Munksgaard 2003
PERIODONTOLOGY 2000
ISSN 0906-6713
A clinical and radiographic study of the condition
of 257 dentally aware adults aged 2069 years
revealed that the condition of former smokers was
intermediate between current smokers and non-
smokers, suggesting that former smokers who have
quit smoking have a better periodontal health con-
dition than current smokers, although worse than
that of non-smokers (6). It has been reported that
the periodontal health condition of former smokers
after 10 years remained stable (16). Also, it has been
reported that the progression of bone loss is signi-
cantly retarded in individuals who give-up smoking
(10). In postmenopausal women, smoking and parity
were reported as strong independent negative factors
for alveolar bone loss (30). In early onset periodonti-
tis, smokers (mean smoking experience of 9.2 pack
years) had signicantly more maxillary bone loss
than non-smokers (48).
More recent reports present similar ndings. Haf-
fajee & Socransky (26) studied 289 adults with peri-
odontitis and concluded that smokers had more
mean attachment loss, deeper periodontal pockets,
more missing teeth, fewer sites with gingival bleeding
on probing, and similar dental plaque levels and
gingival inammation than those who had never
smoked. The observed patterns of attachment loss
indicated more loss in the maxillary lingual area,
suggesting the possibility of a local effect. While look-
ing at cotinine levels in gingival crevicular uid, Chen
et al. (16) reported that smokers had increased prob-
ing depth, attachment loss and tooth loss at an ear-
lier age, with fewer bleeding sites. Smokers had
detectable cotinine levels; the levels in gingival cre-
vicular uid were approximately four times higher
than in saliva and were not correlated to probing
depth, attachment loss or tooth loss. A comparison
of bone levels from bitewing radiographs of 812 indi-
viduals retrospectively, revealed that smokers had a
higher mean bone loss than non-smokers and that
the bone loss suggested a threshold period before
changes become evident, leveling off after a number
of years in an S-shaped dose-response (9). Attach-
ment loss was exhibited more often in smokers than
in non-smokers when evaluating a rural Chinese
population over a period of 2 years (70). Increased
attachment loss, recession, probing depth, furcation
involvement, and tooth mobility were reported to be
worst in smokers. Smokers also exhibited fewer
molar teeth than non-smokers (35).
In summary, the preponderance of the evidence
suggests that smoking diminishes gingival bleeding
and that it may induce changes in the proportion of
small to large blood vessels in the gingiva. Alveolar
bone loss and periodontal attachment loss were
increased in smokers, with some studies suggesting
a dose-response relationship and a leveling of the
effect after many years.
Smoking as a risk factor of
periodontitis
In a case-control study of 155 patients admitted to
the dental school in Stockholm, the frequency of per-
iodontally diseased teeth, the frequency of sites (pro-
bing depth greater than 4 mm), gingival index, and
plaque index of smokers was compared to a control
group of a random sample of the Stockholm popula-
tion. In the patient sample, 56% were smokers, which
was signicantly greater than in the population at
large. Further, they had signicantly higher frequen-
cies of periodontally involved teeth and diseased
sites and more severe disease. The calculated risk
for smokers of having periodontal disease was 2.5 (1).
While looking at the effectiveness of using interleukin
(IL)-1 genotype knowledge to predict prognoses and
tooth survival in a group of patients in maintenance
care for 14 years, the authors reported that being IL-1
genotype-positive increased the risk of tooth loss by
2.7 times compared to 2.9 times for heavy smoking;
when both were present, the risk increased to 7.7
times. The authors concluded that this knowledge
should be used to target therapy for non-responding
areas (42).
In a recent report derived from data of the United
States Third National Health and Nutrition Exami-
nation Survey it was calculated that 41.9% of perio-
dontitis cases (6.4 million cases) in the adult
population were attributable to current cigarette
smoking and 10.9% (1.7 million cases) to former
smoking. The same study estimates that more than
one-half of the cases of periodontitis affecting adults
may be due to cigarette smoking. The relative risk for
smokers was 3.97 and the risk for former smokers
was 1.68. Among smokers the odds of periodontitis
increased with the number of cigarettes smoked per
day, from 2.79 for smoking nine or fewer cigarettes
per day to 5.88 for 31 or more cigarettes per day. After
quitting, the odds are 3.22 for the rst 2 years,
decreasing to 1.15 after 11 or more years (72). In a
study of 3,050 Mexican-Americans aged 6569 years
in ve southern states it was reported that tooth loss
risk in current smokers was 1.69 times higher, and in
diabetics 1.53 higher, than in other participants. The
authors state that a limitation of this study was
that they used self-reported data and there was no
51
information on the overall condition of the mouth
and gingiva (60). Using a longitudinal approach to
categorizing exposure to cigarette smoke, Hashim
et al. (28) reported that smoking persisting through
mid-adolescence and into adulthood will double the
likelihood of periodontitis occurring by the mid-
twenties. In a study of type 1 diabetes mellitus and
oral health, it was reported that cigarette smoking
was a signicant factor (odds ratio 9.73) in
explaining the majority of extensive periodontal dis-
ease in this group of diabetics (47).
One report stands apart from the others because of
its nding that attachment loss over 20 years in a
population of Sri Lankan male tea laborers was not
related to a history of smoking (50). These laborers
did not receive any professional care over the 20-year
period of the study. The data was collected longitud-
inally allowing for assessment of risk of disease pro-
gression. The authors speculate that the reason for
the ndings might be related to the way loss of
attachment was calculated (full-mouth means rather
than individual sites) and to not quantitating the
amount of tobacco smoked.
A statistical modeling analysis, taking into account
that progression of periodontal disease is not uni-
form and may have periods of regression (healing),
has shown that smoking has a signicant effect on
the rate of disease regression. The authors suggest
that smoking diminishes the capability for repair to
an equivalent level of a non-smoker 36 years older
(20). The concept of healing being interfered with by
smoking is one that has received attention and sup-
port from various investigators.
suggests that smoking is a signicant risk factor for
the development of periodontal disease and it may
further be responsible for a large number of the cases
reported. It may be that the primary effect induced
by smoking is one of interference with the normal
healing mechanisms.
Periodontal treatment in smokers
In one study, non-surgical periodontal treatment has
been shown to be effective in both smokers and non-
smokers, although probing depths are greater in
smokers (59). In refractory periodontitis patients,
where non-surgical therapy with systemic metroni-
dazole was given for 1 week and regular follow-up
visits were carried out for 5 years, smokers res-
ponded less favorably to treatment than non-smo-
kers (69). Similarly, in a 5-year study of non-surgical
treatment of 90 patients, those who were smokers at
baseline were more likely to develop new surgical
needs (surgery or extraction) than the non-smokers
(40). In a retrospective study of 35 smokers and 35
non-smokers, non-surgical treatment in non-smo-
kers was successful in most patients but less success-
ful in smokers, suggesting by decision analysis that
surgical treatment in these patients should not be
delayed by non-surgical treatment (52).
Smokers, when compared to non-smokers, were
reported to respond less favorably to ap debride-
ment surgery in terms of pocket depth reduction and
attachment level gains, especially in sites with deep
initial pocket depth (64). The use of enamel matrix
proteins (EMD) in conjunction with ap procedures
to enhance healing in patients that smoke has
received some attention. A reduction in regeneration,
after the use of EMD and guided tissue regeneration,
in smokers compared to non-smokers was reported
in a study of 90 patients at the 1-year follow-up (80).
Investigators looking at the healing after papilla pre-
servation aps have reported that smoking reduced
attachment level gains even when heavy smokers had
been excluded from the study (73). A reduced healing
response in smokers receiving ap treatment for
intrabony periodontal defects with EMD was re-
ported. In this study, smokers were found to have a
higher incidence of tooth hypersensitivity, tooth pain
and swelling when compared to non-smokers (29).
While looking at the survival of osseointegrated
implants in patients who were IL-1 genotype posi-
tive, investigators found that implant survival was
signicantly inuenced by the smoking status of
patients. Implant failure was 2.5 times greater in
smokers, whereas the genotype status was not a sig-
nicant factor (79). In a report of a long-term pro-
spective study over 15 years, periimplant bone loss in
smokers was ``great and signicant in the mandible''
but not in the maxilla when compared to non-smo-
kers (14). In a 3-year study, increased failure rates for
implants in smokers when compared to non-smo-
kers was interpreted as being the result of exposure
of the periimplant tissues to cigarette smoke. Overall,
the failure rate of hydroxyapatite-coated implants
was 4.8% in smokers compared to 2.4% in the
non-smokers, while the failure rate of non-coated
implants was 16% in smokers and 11.7% in non-
smokers (38). In a 35-year follow-up of patients with
severe resorbed maxilla rehabilitated with implants,
authors reported that the failure rate leveled off after
the rst 2 years and that smokers had a higher rate of
failures than non-smokers (78). Complications after
implant placement were reported to be higher in
52
Rivera-Hidalgo
smokers, particularly in implants with a high cover
screw, and were related to smoking duration. Most
complications did not lead to implant failure (67).
Smokers had a lower success rate (65.3%) than non-
smokers (82.7%) when implants were placed in
grafted maxillary sinuses (34).
Earlier studies looking at the healing associated
with connective tissue grafting has reported a nega-
tive inuence (49) and poorer root coverage in smo-
kers (74, 81). However, results obtained with guided
tissue regeneration procedures in Miller's class I or II
buccal recessions, were maintained over a 4-year
period irrespective of the smoking status (65). Similar
conclusions were reached after reviewing gingival
recession defects and guided tissue regeneration
(18). The success rate associated with the loading
of implants in augmented ridges using deproteinized
bone mineral after 46 years was reported to be 43%
for smokers and 100% for non-smokers (41).
suggests that surgical and non-surgical treatments
are less successful in smokers than in non-smokers.
Clinical studies suggest that the healing outcome is
less favorable in smokers.
Periodontal microbiology of
smokers
Investigators have looked at the relationship between
some bacterial strains associated with periodontal
disease and smoking. Preber, in a study of 145
patients where 83 were smokers, found that testing
for Actinobacillus actinomycetemcomitans, Porphyr-
omonas gingivalis, and Prevotella intermedia in
pockets of 6 mm or more failed to show any differ-
ence between the groups (58). Using DNA-hybridiza-
tion for P. gingivalis, P. intermedia, Prevotella
nigrescens, Bacteroides forsythus, A. actinomycetem-
comitans, Fusobacterium nucleatum, Treponema
denticola, Peptostreptococcus micros, Campylobacter
rectus, Eikenella corrodens, Selenomonas noxia, and
Streptococcus intermedius in a group of 33 smokers
and 31 non-smokers with moderate to severe perio-
dontal disease, investigators reported that both
smokers and non-smokers exhibited the same fre-
quencies of the 12 species investigated (11). In a
microbiologic study of 272 subjects divided into
never, past, and current smokers, the authors (27)
reported a difference in the prevalence (percent of
sites colonized) of species rather than in counts or
proportions with the prevalence of B. forsythus and
P. nigrescens being signicantly greater in the maxilla
than the mandible where greater pocket depth and
attachment loss was evident (26). They speculate that
this may explain the greater severity of periodontal
destruction in smokers. A similar distribution of
pockets was reported in a retrospective study of 183
patients in which pockets 5 mm were found to be
more prevalent in smokers and were mainly distri-
buted to maxillary anterior and premolar teeth (75).
In a study of the subgingival microbial ora of
468 patients with periodontal disease divided into
untreated smokers, treated smokers, untreated
non-smokers, and treated non-smokers, the authors
concluded that smoking was a determining factor for
the composition of the microbial ora and may select
for B. forsythus, P. micros, F. nucleatum and C. rectus
(76). In a study where bacteria were quantitated, the
authors concluded that smoking extends a favorable
habitat for bacteria such as P. gingivalis, P. interme-
dia and A. actinomycetemcomitans to shallow sites
with a pocket depth 5 mm (19). The authors pro-
pose several potential mechanisms by which smoke
could inuence the host control of bacteria. These
include the effects of carbon monoxide, enhancing
growth of bacteria, which that in turn provide growth
factors for anaerobes, and damaging cells involved in
the protection of the periodontal environment such
as neutrophils which could be affected by the forma-
tion of advanced glycation endproducts by smoke.
In summary, it is not yet clear if there is a consis-
tent effect from smoking in selecting the bacterial
population, although recent studies suggest such
an effect. Perhaps differences in the areas sampled
or in the recovery techniques account for some of the
disagreement. There is also a suggestion that smok-
ing through a local effect may play a role in the
distribution of pockets of 5 mm or more.
Gingival inflammatory response in
smokers
Inammatory components inthe gingival crevice uid
(GCF) have been studied in relation to smoking. In a
recent report evaluating young adults, it is proposed
that smoking may affect GCF by the release from neu-
trophils of proteases like collagenase and elastase or
the activity of protease inhibitors like apha-1-antitryp-
sin and alpha-2-macroglobulin. However, results
showed a reduced volume of GCF among smokers,
no difference in elastase activity and no difference in
concentrations of protease inhibitors (53). Neutrophil
elastase activity and matrix metalloproteinase-8 were
reported to be increased in smokers with refractory
53
periodontitis (69). In another study of 32 adults with
moderate to severe periodontitis, the results indicate
that for patients withsevere lesions only, boththe total
volumeandtheconcentrationof alpha-2-macroglobu-
lin in the GCF are lower in smokers; the levels of elas-
tase andlactoferrinwere not statistically different (54).
In a study of 40 periodontitis and 43 control patients,
smokers had a lower level of IgG2, which may impair
neutrophil function and in this way aggravate period-
ontal disease (22).
In another report (21) tumor necrosis factor-alpha
(TNF-a) and IL-8 were found to be depressed. How-
ever, smoking was not found to alter levels of IL-1b
and its receptor antagonist (IL-1ra) in smokers (13).
In an in vitro study of the neutrophils priming capa-
city for TNF-a, measured as the generation of oxygen
radicals from smokers and non-smokers with and
without periodontal disease, the smokers showed a
stronger effect than the non-smokers with the effect
in the periodontal disease group being even greater,
suggesting an additive response. The authors spec-
ulate that this increased effect could be important in
the aggravation of tissue destruction (25). In an ear-
lier study, Bostrom et al. had reported higher levels
of TNF-a in the GCF of current and former smokers
in comparison to non-smokers (12). In generalized
early onset periodontitis maintenance patients,
smokers appear to have reduced levels of antibody
to A. actinomycetemcomitans, P. intermedia and
T. denticola compared to non-smokers. The authors
suggest that this may indicate an interrupted matu-
ration of the immune response (46).
In summary, the role of pro-inammatory factors in
the overall picture of the immune response insmokers
is not clear. More research in elucidating the complex
interactions and cellular roles is required.
Genetic polymorphismand
smoking
Differences between individuals in the species may
result from usual genetic variability or polymorph-
isms. Individual susceptibility to conditions or indi-
vidual variations in response to treatments may also
reect individual genetic differences. Given this con-
cept, many investigators have looked at genetic
variability, its relationship with periodontal disease
and its interaction with smoking.
Studies looking at tooth loss reported that a positive
genotype for IL-1 increases the risk for toothloss by 2.7
times, while smoking increases the risk by 2.9 times.
When both were combined, the risk increased to 7.7
times (42). Another report failed to nd an association
between patients with generalized early onset period-
ontitis and IL-1 genotypes (31). Ina study of 323 main-
tenance patients, bleeding on probing was associated
witha positive genotype of IL-1inthose whohadnever
smoked but the effect was not signicant in smokers,
where the authors suggest that the effect is oversha-
dowed by the effect of smoking (39). In a prospective
longitudinal study (5 years) of the progression of per-
iodontal disease in295 subjects of whom25 were smo-
kers, theauthors concludethat IL-1genotypepositivity
is acontributingbut non-essential factor. There was an
increase in the number of probing depths of 3.5 mm
inthe genotype positive smokers as well as ingenotype
positive individuals with P. gingivalis in their plaque
(17). Inanother study, therewassignicant attachment
loss (percentageof sites > 4 mm) onlywhengenotype-
positive (IL-1 gene cluster) individuals were also smo-
kers (43).
In a study of 154 patients, the authors evaluated the
possible pharmacogenetic interaction of arylamines
produced in tobacco smoke and the N-acetyltransfer-
ase 2 (NAT2) prole of patients with periodontal dis-
ease (44). The inactivationof arylamines by acetylation
may be involved in detoxication of tobacco smoke.
The NAT2 polymorphismaffects the population, mak-
ing individuals into rapid or slow acetylators. Results
indicated that patients with the most severe period-
ontal condition were the slow acetylators (risk ratio of
more than 2). The authors speculate that the altered
metabolism of arylamines may inuence the immune
response and may act as immunosuppressants. In
another study, the NAT2 genotype-positive patients
were signicantly associated with the severity of
bone loss, with this being more striking in smokers
4055 years of age (37).
In a study to determine if polymorphism of the
transforming growth factor-beta-1 gene alters sus-
ceptibility to adult periodontitis, including smokers,
the authors failed to detect any effects (32).
In summary, polymorphisms tend to explain some
of the variation in disease observed in patients. How-
ever, more studies are necessary to elucidate the role
that these polymorphisms play. The study of the
interaction between polymorphisms and smoking
appears to be promising.
Effect of nicotine and smoke on
periodontal tissues
Several in vitro studies have examined the effects of
nicotine. These studies have reported that nicotine
54
Rivera-Hidalgo
adversely affected the proliferation, the attachment
and the chemotaxis of periodontal ligament cells
(23). Nicotine was reported to affect the attachment
by broblasts (71) and when epithelial cells were
treated with nicotine, both the collagen production
and the production of non-collagenous proteins by
broblasts were severely affected (24). While nicotine
can induce human gingival broblasts to produce
pro-inammatory cytokines IL-6 and IL-8, a syner-
gistic upregulation occurs when nicotine and either
Escherichia coli or P. gingivalis lipopolysaccharide
are together (77).
Cotinine from smoking was reported to enhance
the effects of toxins from periodontopathogens in a
chick embryo toxin assay, suggesting a mechanism
by which smoking contributes to the severity of per-
iodontal disease (63). Acrolein and acetaldehyde
volatile tobacco smoke components were found to
be toxic to cultures of human gingival broblasts,
affecting attachment and proliferation in a dose-
dependent response (15). These compounds affect
cell adhesion in culture by disruption of microtu-
bules and associated laments (55, 62).
Final remarks
Signicantly, the understanding of the relationship
between smoking and periodontal disease is the
result of the work by many groups of investigators.
Having reviewed the literature, we would be remiss
not to mention the signicant contribution to the
study of periodontal disease and smoking by the
group from the Karolinska Institutet in Stockholm,
Sweden. This group, which includes Bergstrom, Pre-
ber, Bostrom and others, has systematically studied
and reported on important questions of the smok-
ingperiodontal disease interaction for many years.
Many of the research reports reviewed here have
helped answer some of the questions posed in a
similar review of this topic 16 years ago (61).
Research in the intervening years has shed light not
only on previous questions but also on new issues
such as the host's response to tobacco smoking
(Table 1).
As we get closer to elucidating mechanisms,
we have accepted that smoking is a signicant
factor in the development and progression of period-
ontal diseases and that gingival bleeding is dimin-
ished in these patients. The epidemiologic studies
have established the signicant risk effect of smok-
ing on developing periodontal disease, while other
studies found that bone loss is greater in these
individuals. Treatment in smokers appears not to
be as effective as in non-smokers and the microbial
role is still being claried. Levels of inammatory
substances vary from the levels in non-smokers
but the signicance of this is not clear. The study
of genetic polymorphisms indicates that these
variations may play a role in susceptibility to the
disease, disease severity and in the patient's res-
ponse to treatment. Research in this eld during
the next 15 years should be even more exciting as
we get closer to a better understanding of the
mechanisms underlying the clinical manifestations
in smokers.
Table 1. Summary of ndings on smoking and periodontal disease
Parameters Smokers
Gingival bleeding Less gingival bleeding, higher proportion of small blood vessels
Alveolar bone loss Greater alveolar bone loss and periodontal attachment loss
Smoking as a risk factor
for periodontitis
Significant factor for development of periodontal disease,
primary effect may be interference with wound healing
Treatment in smokers: non-surgical
and surgical
Non-surgical and surgical treatment response lessened,
healing response lessened
Treatment in smokers: grafting Not clear if smoking affects connective tissue healing
Microbial factors Not clear if smoking selects specific bacterial populations
in periodontal pockets
Gingival inflammatory response Not clear what effects result from alterations in
pro-inflammatory factors due to smoking
Genetic polymorphism Not clear what role polymorphisms play
Effect of nicotine May affect cells involved in periodontal repair
Effect of smoke May affect cells involved in periodontal repair
55
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CJ. Rehabilitation of patients with severely resorbed max-
illae by means of implants with or without bone grafts: a 3-
to 5-year follow-up clinical report. Int J Oral Maxillofac
Implants 2001: 16: 7379.
79. Wilson TG Jr, Nunn M. The relationship between the inter-
leukin-1 periodontal genotype and implant loss. Initial
data. J Periodontol 1999: 70: 724729.
80. Zucchelli G, Bernardi F, Montebugnoli L, De SM. Enamel
matrix proteins and guided tissue regeneration with tita-
nium-reinforced expanded polytetrafluoroethylene mem-
branes in the treatment of infrabony defects: a comparative
controlled clinical trial. J Periodontol 2002: 73: 312.
81. Zucchelli G, Clauser C, De Sactis M, Calandriello M. Muco-
gingival versus guided tissue regeneration procedures in the
treatment of deep recession type defects. J Periodontol
1998: 68: 138145.
58
Rivera-Hidalgo
Hormonal inuences: effects of
diabetes mellitus and
endogenous female sex steroid
hormones on the periodontium
Brian L. Mealey & Alan J. Moritz
Hormonal inuences on the periodontium are many,
with widely varying clinical presentations and
degrees of effect. The primary hormonal factors dis-
cussed in this chapter are those associated with dia-
betes mellitus and female sex steroid hormones.
Effects of diabetes mellitus on the
periodontium
Diabetes mellitus is the term used to describe a group
of metabolic disorders distinguished by altered glu-
cose tolerance and impaired carbohydrate metabo-
lism (5). Diabetes is characterized by hyperglycemia
(elevated blood glucose) that results from defects in
secretion of the hormone insulin, or from impaired
insulin action, or both. Alterations in lipid and protein
metabolism are also seen. Chronic hyperglycemia is
associated with long-term dysfunction and damage to
numerous end-organs, with marked effects on the
eyes, kidneys, heart, nerves, and blood vessels.
It is important that the dentist and other oral health
care providers have a thorough knowledge of diabetes,
if for no other reason than its widespread prevalence.
More than 16 million Americans have the disease
(122). The prevalence has risen from 4.9% in 1990 to
almost 7% in 1999 (123). Thus, for every 1000 patients
in an average dental practice, 70 have diabetes. Unfor-
tunately, 40 to 50% of individuals with diabetes are
unaware of their disease and remain undiagnosed.
The prevalence of diabetes varies with ethnicity.
About 6.2% of whites have the disease, compared to
8.0% of Hispanics and almost 10% of blacks. Approxi-
mately 800,000 new cases of diabetes are diagnosed
every year, and the incidence continues to rise (122).
The increased incidence of diabetes correlates strik-
ingly with the rapid increase in obesity in the United
States, the prevalence of which has risen from 12.0%
in 1991 to 18.9% in 1999 (121, 123). Obesity is clearly
linked to an increased risk for developing diabetes
(50, 153). As the prevalence and incidence of diabetes
increase, so do the costs, both nancially and in
terms of morbidity and mortality. In 1997, the direct
and indirect costs associated with diabetes were esti-
mated at $98 billion (4). Diabetes is the seventh lead-
ing cause of death in the U.S., and is a major
contributor to hypertension, stroke, heart disease,
blindness, kidney failure, and amputations. With an
expected prevalence of over 9% by 2025, the effect of
diabetes on oral health and on the practice of den-
tistry will only increase in the future (6).
The most current classication of diabetes was pro-
vided by the American Diabetes Association in 2001
(5). The terms insulin-dependent and non-insulin
dependent are no longer used, nor are the terms
adult-onset, maturity-onset, and juvenile-onset dia-
betes. Currently accepted diagnostic categories
include: type 1 diabetes, type 2 diabetes, impaired glu-
cose tolerance, impaired fasting glucose, gestational
diabetes, and other specic types of diabetes such as
those secondary to diseases of the pancreas, drug ther-
apy, endocrinopathies, infections, and genetic disor-
ders (Table 1). Impaired glucose tolerance and
impaired fasting glucose are terms used to describe
an intermediate metabolic stage that lies between nor-
mal glucose metabolism and frank diabetes.
59
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PERIODONTOLOGY 2000
ISSN 0906-6713
Type 1 diabetes usually results from cellular-
mediated autoimmune destruction of the insulin
producing b cells of the pancreas (Table 2) (10). One
or more markers of autoimmune destruction are
generally present, such as autoantibodies to pancrea-
tic islet cells, insulin, glutamic acid decarboxylase, or
tyrosine phosphatases IA-2/IA-2b (5). Pancreatic b
cells are destroyed when genetically predisposed
individuals are exposed to a triggering event such
as a viral infection, which induces an autoimmune
response. There are also idiopathic forms of type 1
diabetes in which there is no evidence of autoimmu-
nity. Onset of type 1 diabetes is usually abrupt, its
management can be difcult, and it predisposes to
diabetic ketoacidosis if not well controlled.
Type 2 diabetes results from insulin resistance and
a relative insulin deciency, not an absolute lack of
insulin production, as occurs in type 1 (Table 2).
Autoimmune destruction of the pancreatic b cells
does not occur. The pancreas still produces insulin,
yet tissue resistance to insulin decreases its activity
and ability to facilitate glucose metabolism at the
cellular level (116). Most type 2 patients are over-
weight, and obesity itself often leads to insulin resis-
tance (19). The onset of type 2 diabetes is gradual; in
fact, type 2 usually goes undiagnosed for an
extended period of time, often years. Ketoacidosis
is uncommon in type 2 diabetes and usually occurs
in association with the stress of other illnesses such
as infection (196).
The terms impaired glucose tolerance and
impaired fasting glucose describe a metabolic state
lying between normal glycemia and diabetes. Many
people with impaired glucose tolerance have normal
Table 1. Classication of diabetes mellitus
Type 1 diabetes (formerly insulin-dependent diabetes)
Type 2 diabetes (formerly non-insulin-dependent diabetes)
Gestational diabetes
Other types of diabetes
Genetic defects in b-cell function
Genetic defects in insulin action
Diseases of or injuries to the pancreas Pancreatitis, neoplasia, cystic fibrosis, trauma,
pancreatectomy, others
Infections Congenital rubella, cytomegalovirus, others
Drug-induced or chemical-induced diabetes Glucocorticoids, thyroid hormone, dilantin,
thiazides, others
Endocrinopathies Acromegaly, Cushings syndrome, glucagonoma,
pheochromocytoma, hyperthyroidism
Other genetic syndromes with associated diabetes Downs syndrome, Huntingtons chorea,
myotonic dystrophy, others
Table 2. Characteristics of type 1 and type 2 diabetes
Type 1 diabetes Type 2 diabetes
Age at onset Generally < 30 years Generally in adulthood (prevalence in
young is increasing)
Racial preference White Black, Hispanic, American Indian, Pacific Islanders
Presence of family history Common More common than type 1
Most common morphotype Thin or normal stature Obese
Onset of Clinical Disease Abrupt Slow
Pathophysiology Autoimmune b-cell
destruction
Insulin resistance, impaired insulin secretion, increased
liver glucose production
Level of endogenous insulin
production
None Decreased, normal, or elevated (depends on
stage of disease)
Susceptibility to ketoacidosis High Low
Common treatment regimens Insulin, diet, exercise Diet (weight loss), exercise, oral agents, insulin
Mealey & Moritz
60
blood glucose levels most of the time, often manifest-
ing hyperglycemia only after challenge with a large
glucose load (5). Those with impaired fasting glucose
have elevated fasting glucose levels, but may be nor-
mal in a fed state. Impaired fasting glucose and glu-
cose tolerance are not considered to be clinical
entities in and of themselves. Instead, they are risk
factors for development of diabetes (24). They can be
seen as intermediate stages in all types of diabetes.
Endogenous insulin production is normal, and
remains so in the majority of these patients. How-
ever, about 3040% of patients with impaired fasting
glucose or glucose tolerance develop type 2 diabetes
within 10 years after initial diagnosis (116). In these
patients, insulin resistance increases and insulin
secretion is impaired. Eventually, the patient mani-
fests overt clinical and laboratory signs of diabetes.
Glucose intolerance during pregnancy may lead to
gestational diabetes. This condition usually develops
during the third trimester of pregnancy, but can
occur earlier. Like impaired glucose tolerance,
impaired fasting glucose, and type 2 diabetes, gesta-
tional diabetes is strongly associated with insulin
resistance (5). An increased prevalence of gestational
diabetes is seen in women who have a family history
of diabetes, are over 25 years old, are obese, and are
members of ethnic groups with higher prevalence
rates for type 2 diabetes (black, Hispanic, American
Indian) (40).
Diabetes may also be associated with various
genetic defects in the function of pancreatic b-cells
or defects in insulin action (Table 1) (5). Injuries to
the pancreas or pancreatic diseases may induce sec-
ondary diabetes, as can certain infections. Diabetes
may occur subsequent to other endocrine disorders.
One of the most common forms of secondary dia-
betes occurs due to use of certain drugs such as
corticosteroids.
Complications of diabetes
Diabetes has been classically associated with a group
of microvascular and macrovascular complications
(Table 3). The microvascular complications of reti-
nopathy, nephropathy and neuropathy are speci-
cally associated with diabetes, and the risk of
macrovascular disease is greatly increased in diabetic
patients (5). Diabetic patients have a dramatically
increased risk for visual impairment or blindness,
kidney failure, limb amputation, stroke, and myocar-
dial infarction (60, 84, 175, 176). Sustained hypergly-
cemia plays a primary role in both the onset and
progression of these complications.
Macrovascular complications are strongly asso-
ciated with the increased atherosclerosis common
in diabetes (5, 116). Hyperglycemia results in altera-
tions in lipid metabolism as well as nonenzymatic
glycation of proteins such as collagen. These changes
result in altered function of cell membranes and
changes in cellcell and cellmatrix interactions. This
may then lead to increased vessel wall thickness and
formation of atheromas and microthrombi in large
vessels, and alterations in endothelial cell function
and vascular permeability in the microvasculature.
The glycation of proteins, lipids and nucleic acids
in diabetic patients results in accumulation of these
glycated proteins in the small blood vessels of end-
organs such as the retina, glomerulus and endoneur-
ial region, and in the walls of large blood vessels (21,
201). These glycated proteins, known as advanced
glycation end-products (AGEs), form in diabetic and
non-diabetic individuals; however, their accumula-
tion is greatly increased in diabetic patients with
sustained hyperglycemia. The result of their accumu-
lation is increased basement membrane thickness in
the retina and around the nerves, thickening of the
mesangial matrix in the glomerulus, and accumula-
tion of collagen in larger vessels. The cumulative
effect is a progressive narrowing of the vessel lumen
and decreased perfusion of affected organs.
AGEs form on collagen, causing increased collagen
cross-linking, and resulting in formation of highly
stable collagen macromolecules that are resistant
to normal enzymatic degradation and tissue turnover
(21, 125, 201). In the blood vessel wall, AGE-modied
collagen accumulates, thickening the vessel wall and
narrowing the lumen. AGE formation occurs in both
central and peripheral arteries, and is thought to
contribute signicantly to the macrovascular compli-
cations of diabetes. AGE-modied collagen in vessel
walls covalently cross-links with circulating low
density lipoprotein, contributing to atherosclerosis.
AGE modication of collagen also occurs in the
basement membrane of small blood vessels. Again,
Table 3. Classic complications of diabetes mellitus
Retinopathy Blindness
Nephropathy Renal failure
Neuropathy Sensory
Macrovascular
disease
Autonomic
Peripheral vascular disease
Cardiovascular disease
(coronary artery disease)
Cerebrovascular disease (stroke)
Altered wound healing
61
Hormonal influences on periodontium
AGE-modied collagen accumulates and increases
basement membrane thickness, altering normal
homeostatic transport across the membrane.
AGEs have major effects at the cellular level. A
receptor for AGEs known as RAGE (receptor for
AGE) has been identied on the surface of endothe-
lial cells, neurons, smooth muscle cells, and mono-
cytes/macrophages (164, 165, 202). Hyperglycemia
results in increased expression of the receptor and
increased AGERAGE interaction. The effect on
endothelial cells is an increase in vascular perme-
ability and thrombus formation (42). AGEs are che-
motactic for monocytes. Interaction between AGEs
and the receptor, RAGE, on monocyte/macrophage
membranes induces increased cellular oxidant stress
and activates the transcription factor NF-kB. This
signals a change in the monocyte/macrophage phe-
notype and results in increased production of pro-
inammatory cytokines such as interleukin-1 (IL-1)
and tumor necrosis factor, and growth factors such
as platelet-derived growth factor and insulin-like
growth factor (82, 166, 200). These cytokines and
growth factors contribute to the chronic inamma-
tory process in atheroma formation.
Diabetes and oral diseases
Diabetes has been associated with several oral con-
ditions, including alterations in salivary ow and
constituents of saliva, increased incidence of oral
infection, burning mouth, changes in wound healing,
and increased prevalence and severity of periodontal
disease. Xerostomia and parotid gland enlargement
may occur in people with diabetes, and may be
related to the degree of glycemic control (31, 66,
173, 191). Xerostomia may be associated with the
sensation of burning mouth syndrome. Fungal infec-
tions such as candidiasis may increase on dry muco-
sal surfaces, although studies on the incidence of
candidiasis in diabetic subjects are not consistent
(49, 145). Guggenheimer et al. (62) found that
15.1% of 405 type 1 diabetes mellitus subjects had
candidiasis, compared to only 3.0% of 268 non-dia-
betes mellitus control subjects. The prevalence of
Candida hyphae on cytologic smears was 23.0% in
diabetes mellitus and 5.7% in non-diabetes mellitus
subjects. Multivariate regression analysis found the
presence of Candida hyphae to be signicantly
related to poor glycemic control.
The inuence of diabetes on dental caries is con-
troversial. Some studies have shown an increased
caries rates in diabetes mellitus (74); however, others
have demonstrated similar or lower caries incidence
(182, 184). Some authors have speculated that
decreased salivation or increased glucose concentra-
tions in saliva and gingival crevicular uid account
for an increased caries risk. However, most diabetic
patients limit their fermentable carbohydrate intake,
and this less cariogenic diet may decrease caries risk.
Autonomic neuropathy, a complication of diabetes
mellitus, may result in alteration of salivary secretion
(117). Many diabetic patients have conditions other
than diabetes for which they are medicated, and
many of these medications produce xerostomia as
a side effect. Xerostomia may therefore result not
from the diabetic condition itself, but from medica-
tions taken by the patient.
In studies of type 2 diabetes mellitus subjects and
non-diabetes mellitus controls, no signicant differ-
ences in salivary ow rates or organic constituents of
saliva were seen (117). Likewise, there were no differ-
ences in the prevalence of coronal caries or root caries
(29). The salivary counts of yeasts and of acidogenic
streptococci and lactobacilli were also similar
between the groups. However, the effect of xerostomic
medications on salivary ow rates was greater in dia-
betic individuals than in control patients.
Diabetes and periodontal diseases
Understanding the relationships between diabetes
and periodontal diseases is complicated by the varia-
bility in diagnostic parameters used to describe the
metabolic state in study populations. In addition,
study designs lack commonality in populations stu-
died, use or non-use of control populations, and
periodontal parameters assessed to describe the clin-
ical conditions present. Many studies nd no specic
relationship between periodontal parameters and
duration of diabetes mellitus, presence of various
diabetic complications, or degree of glycemic con-
trol. Conversely, other studies do nd such relation-
ships. Due to the absence of homogeneity in study
design, rm conclusions are difcult to make.
Diabetes has been associated with increased pre-
valence and severity of gingivitis. Gusberti et al. (63).
studied children with type 1 diabetes mellitus. Before
puberty, poorly controlled diabetes mellitus children
had a higher incidence and severity of gingival
inammation than did well controlled children. Dur-
ing puberty, there was a general increase in gingivitis,
independent of glycemia. Cianciola et al. (26). con-
rmed an increase in gingivitis in type 1 diabetes
mellitus children after the age of 11 when compared
to non-diabetes mellitus controls. In other studies,
poorly controlled diabetes mellitus children had
62
Mealey & Moritz
higher levels of gingival inammation than did well
controlled patients, regardless of plaque levels (48,
78). Improvement in glycemic control was associated
with decreased signs of gingival inammation (78,
161). DePommereau et al. (36) found a signicantly
greater percentage of sites with gingivitis in diabetes
mellitus children (48%) compared to non-diabetes
mellitus control children (26%) with similar plaque
levels, although gingivitis was not associated with the
level of glycemic control in the diabetic subjects.
Attachment loss was generally not seen in either
group.
Conversely, Firalti et al. (47) found no increase in
gingival inammation in diabetic children, but did
nd increased probing depth and clinical attachment
loss, when compared to age- and sex-matched non-
diabetes mellitus control subjects. There was a posi-
tive correlation between clinical attachment loss and
the duration of diabetes, with a greater prevalence
and severity of disease in children with long-standing
diabetes mellitus. Other authors have also found no
increase in gingivitis in children with diabetes melli-
tus (162, 163).
In adult type 1 diabetes mellitus patients consid-
ered as a single group, Ervasti et al. (41) found no
signicant difference in gingival inammation com-
pared to non-diabetes mellitus control subjects.
However, when diabetes mellitus patients were stra-
tied according to their level of glycemic control,
signicantly greater gingival bleeding was seen in
poorly controlled patients. Conversely, well con-
trolled patients had less gingival bleeding than did
non-diabetes mellitus control subjects. In general,
the number of bleeding sites decreased as glycemic
control improved. Guthmiller et al. (65) found greater
gingival inammation in pregnant women with
type 1 diabetes mellitus than in pregnant women
without diabetes mellitus. Cutler et al. (33) demon-
strated greater gingival inammation in type 2 dia-
betes mellitus adults than in non-diabetes mellitus
controls, with the highest level of inammation in
subjects with poor glycemic control. While gingival
inammation was greater in diabetes mellitus adults
than in non-diabetes mellitus controls, Bridges et al.
(20) found no relationship between gingivitis and the
level of glycemic control. These studies show that the
presence of diabetes mellitus is often, but not always,
associated with increased gingival inammation;
furthermore, the level of glycemic control may play
a role in the gingival response to bacterial plaque in
many individuals.
The relationship between diabetes and periodonti-
tis has been extensively studied, and is complicated
by the same factors as studies of gingivitis. In a thor-
ough meta-analysis examining this relationship,
Papapanou (142) concluded that the majority of
studies demonstrate a more severe periodontal con-
dition in diabetic adults than in adults without dia-
betes. These studies, including over 3,500 type 2
diabetes mellitus adults, clearly demonstrated a
signicant association between periodontitis and
diabetes.
In younger individuals, diabetes mellitus has been
associated with an increased risk of periodontitis. In
a group of 263 type 1 diabetes mellitus patients com-
pared to 59 non-diabetic siblings and to 149 non-
diabetic, unrelated controls, Cianciola et al. (26)
found no periodontitis among the diabetes mellitus
subjects under the age of 12. Between ages 13 and 18,
however, 13.6% of the individuals had periodontitis.
Individuals from 19 to 32 years old had a prevalence
of 39%. Periodontitis was not found in the non-dia-
betes mellitus siblings of the diabetes mellitus
patients, while a prevalence of 2.5% was noted in
the non-diabetic, unrelated control subjects. Dia-
betes mellitus subjects with periodontitis had a
longer duration of diabetes than did those without
periodontitis. Not all studies of diabetes mellitus
children nd an increase in risk for periodontitis.
In both cross-sectional and longitudinal studies,
Sbordone et al. (162, 163) found no differences in
probing depth or clinical attachment level between
type 1 diabetes mellitus children and their non-dia-
betes mellitus siblings.
Epidemiologic studies in adults have often shown
an increase in extent and severity of periodontitis in
individuals with diabetes mellitus (12, 39, 169, 186).
In the Pima Indian population of Arizona, a popula-
tion with the highest prevalence of type 2 diabetes in
the world, the prevalence of attachment loss and
bone loss was greater among diabetes mellitus sub-
jects than among non-diabetes mellitus control sub-
jects in all age groups (39, 169). These differences
were most pronounced in younger individuals. In
addition, the severity of periodontitis was greater in
diabetes mellitus patients, with greater mean bone
loss and attachment loss. Again, the differences in
disease severity were greatest in the younger age
groups. Diabetes mellitus subjects aged 1534 years
had mean attachment loss and bone loss scores
approximately twice as high as similarly aged non-
diabetes mellitus subjects. In a multivariate risk ana-
lysis, diabetic subjects had a 2.83.4-fold increased
risk of periodontitis compared to non-diabetes
mellitus subjects, after adjusting for the effects of
confounding variables such as age, sex, and oral
63
hygiene measures. The most signicant risk factors
for periodontitis were age, presence of calculus, and
presence of type 2 diabetes mellitus. Smaller cross-
sectional studies have generally conrmed greater
risk for attachment loss and bone loss in diabetes
mellitus adults (20, 30, 33, 65, 126, 189).
In addition to cross-sectional studies, longitudinal
research has shown an increased risk for progressive
periodontal destruction in people with diabetes mel-
litus. In a 2-year longitudinal study (183), type 2 dia-
betes mellitus Pima Indians had a signicantly
increased risk of progressive alveolar bone loss com-
pared to non-diabetes mellitus subjects, with an
odds ratio of 4.2. When compared to similarly aged
non-diabetes mellitus individuals, the greatest risk of
progressive bone loss occurred in those diabetes
mellitus subjects under the age of 34.
The relationship between metabolic control of dia-
betes and periodontal disease is unclear, although
many studies nd poorer glycemia associated with
increasedperiodontal destruction(137). Somediabetic
patients with poor glycemic control develop extensive
periodontal destruction, while others do not. Conver-
sely, many well controlled diabetic patients have
excellent periodontal health, but others develop peri-
odontitis. In this way, periodontal disease is similar
to the classic complications of diabetes, with signi-
cant heterogeneity of both onset and progression of
complications within the affected population.
Over 23 years, Seppala et al. (167) demonstrated
that poorly controlled diabetes mellitus subjects had
signicantly greater bone loss and attachment loss
than did well controlled diabetes mellitus subjects.
Tervonen & Oliver (186) showed that type 1 diabetes
mellitus subjects with poor metabolic control over
the preceding 25 years had a signicantly greater
prevalence of deep probing depths and advanced
attachment loss than did subjects with good glyce-
mic control. This trend has been conrmed by
others. In a study of 71 type 1 diabetes mellitus
patients with a 16.5-year mean duration of diabetes,
subjects with poor glycemic control had more inter-
proximal attachment loss and bone loss than well
controlled subjects (157). In the same patient popu-
lation in which Ervasti et al. (41) showed increased
gingivitis in poorly controlled versus well controlled
diabetes mellitus subjects, Tervonen & Knuuttila
(185) also showed increasing prevalence of deeper
pockets as metabolic control worsened.
In the longitudinal Pima Indian studies, poor gly-
cemic control of type 2 diabetes mellitus was asso-
ciated with signicantly increased risk of progressive
bone loss compared to better metabolic control
(183). Diabetes mellitus subjects with poor glycemic
control had a signicantly increased risk of progres-
sive bone loss, with an odds ratio of 11.4, compared
to non-diabetes mellitus controls. Conversely, well
controlled diabetes mellitus subjects had no signi-
cant increase in risk. Thus, metabolic control of dia-
betes may be an important variable in both the onset
and progression of periodontal disease, with well
controlled patients having a similar risk as non-dia-
betes mellitus individuals.
Some studies have given only marginal support to
the relationship between glycemic control and extent
or severity of periodontitis, while others have shown
no relationship. Tervonen et al. (189) found a trend
toward increasing prevalence of alveolar bone loss as
glycemic control worsened. The mean percentage of
sites with > 15% bone loss went from 28% in well
controlled type 1 diabetes mellitus subjects to 44% in
poorly controlled subjects. However, the difference
did not reach statistical signicance, perhaps due to
the small size of the study population. While Bridges
et al. (20) found deeper probing depths and greater
gingival inammation, bleeding on probing, and
attachment loss in 118 diabetes mellitus subjects
compared to 115 age-matched controls, the level of
glycemic control among the diabetes mellitus sub-
jects did not correlate to the periodontal parameters
measured. Some studies have found no evidence of a
relationship between glycemic control and period-
ontal status (14, 160). It is important to note that
periodontal disease prevalence and severity varies
greatly within the non-diabetic population, just as
it does in the population with diabetes mellitus. Pre-
sence of periodontal disease in some diabetic indi-
viduals may be related more to other risk factors for
periodontitis such as poor oral hygiene and smoking
than to the mere presence of diabetes.
Mechanisms of interaction
Over the years, many potential mechanisms have
been studied by which diabetes could affect the per-
iodontium (Table 4). These mechanisms may explain
the alterations in periodontal disease expression,
initiation, and progression that have been found by
numerous authors in individuals with diabetes.
Alterations in subgingival microbiota and gingival
crevicular fluid
Early studies demonstrated a shift in the subgingival
microbiota of animals in which diabetes was induced
chemically (115). Deepening of periodontal pockets
and a shift to a ora predominated by gram-negative
64
Mealey & Moritz
rods and laments was seen. In young type 1 dia-
betes mellitus subjects, Mashimo et al. (111) reported
an increase in proportions of Capnocytophaga spe-
cies, while Fusobacterium and Bacteroides species
remained at low levels. Sastrowijoto et al. (160) found
a high prevalence of Capnocytophaga in type 1 dia-
betes mellitus subjects, but the proportion of organ-
isms was low in both diseased and healthy sites. High
proportions of Prevotella intermedia were also found
in diseased sites, but this organism was frequently
detected in healthy sites as well. Other studies have
failed to show an increase in Capnocytophaga in
diabetes mellitus patients (105, 214).
In fact, most studies show very few differences in
the subgingival microbiota of periodontitis sites in
diabetes mellitus subjects compared to similar sites
with periodontitis in non-diabetes mellitus subjects
(63, 162, 163, 192, 214). When comparing type 1 dia-
betes mellitus children to their non-diabetes mellitus
siblings, Sbordone et al. (162, 163) found no signi-
cant differences in subgingival pathogens. One study
showed an increased prevalence of Porphyromonas
gingivalis in type 1 diabetes mellitus subjects com-
pared to non-diabetes mellitus controls (192). Type 2
diabetes mellitus subjects with periodontitis have a
fairly similar microbiota to non-diabetes mellitus
periodontitis patients, although Zambon et al. (214)
demonstrated a different serotype of P. gingivalis in
diabetes mellitus subjects. In diabetes mellitus
patients, the level of glycemic control does not
appear to alter the subgingival ora either. In type 1
diabetes mellitus subjects, similar species were
detected from poorly controlled and well controlled
subjects, with no change following improvement in
glycemic control through intensive insulin therapy
(160, 161). Similar ndings occurred in type 2 dia-
betes mellitus subjects (187). Overall, one can con-
clude that the differences in periodontal disease
expression often seen in individuals with diabetes
are not explained simply by differences in period-
ontopathic microorganisms.
Increased glucose levels in gingival crevicular uid
often accompany elevated blood glucose levels in
diabetes (46, 83). Nishimura et al. (134) showed
decreased chemotaxis of periodontal ligament bro-
blasts in response to platelet-derived growth factor
when cultured in a hyperglycemic environment,
compared to normoglycemic conditions. Elevated
glucose levels in the gingival crevicular uid of indi-
viduals with diabetes may, thus, adversely affect per-
iodontal wound healing and the local host response
to microbial challenge.
Collagen metabolism, advanced glycation
endproducts, and wound healing
Changes in collagen synthesis, maturation, and turn-
over are common in diabetes mellitus. Since the
periodontium is composed primarily of collagen,
these changes in collagen metabolism may contri-
bute to alterations in wound healing and to period-
ontal disease initiation and progression. Skin and
gingival broblasts from diabetic animals produce
decreased amounts of collagen and glycosaminogly-
cans (205, 209). The rate of collagen production can
be restored by administration of insulin to normalize
blood glucose levels (151). In addition to decreased
synthesis, newly formed collagen is susceptible to
degradation by collagenase, a matrix metalloprotei-
nase which is elevated in diabetic tissues, including
the periodontium (56, 58, 152, 156, 172). The pri-
mary source of collagenase in the gingival crevicular
uid of diabetes mellitus patients appears to be the
neutrophil (172). A greater percentage of this col-
lagenase is in active form in patients with diabetes
mellitus compared to non-diabetes mellitus pati-
ents (172).
Use of tetracycline antibiotics results in a reduc-
tion in collagenase production (58, 113). This is
Table 4. Mechanisms of interaction between diabetes mellitus and periodontium
Changes in subgingival environment Altered microbiota
Change in gingival crevicular fluid composition
Altered tissue homeostasis and wound healing Decreased collagen production
Increased matrix metalloproteinase activity
Accumulation of advanced glycation endproducts
Decreased tissue turnover
Changes in host immunoinflammatory response Decreased polymorphonuclear leukocyte chemotaxis,
adherence, phagocytosis
Elevated pro-inflammatory cytokine response from
monocytes/macrophages
Increased tissue oxidant stress
65
accomplished through mechanisms that are indepen-
dent of the antimicrobial properties of these agents.
Low dose tetracyclines and chemically modied tetra-
cyclines, which have no antimicrobial effect, have
been shown to signicantly decrease collagenase pro-
duction and collagen degradation as well (13, 59, 61).
Although chemically modied tetracyclines are not
yet available for routine use, tetracyclines such as
doxycycline, minocycline and tetracycline HCl have
been used for many years. Low dose doxycycline is
now available as well, although its use in diabetic
patients has not yet been reported (23). Due to their
anticollagenolytic effect, tetracyclines and chemically
modied tetracyclines have potential benets in inhi-
biting the onset and progression of periodontitis,
arthritis, and osteoporosis, among other conditions
(61). Since collagenase production is increased in
diabetes mellitus, these drugs may have benecial
effects by normalizing collagen metabolism and
wound healing events.
In addition to decreased collagen production and
increased collagenase activity, collagen metabolism
is altered by accumulation of AGEs in the period-
ontium. Changes affecting the blood vessels of the
glomerulus and retina also occur in the periodon-
tium, and these changes are related to AGE forma-
tion. Increased thickness of gingival capillary
endothelial cell basement membranes and the walls
of small blood vessels may be seen in diabetic indi-
viduals (51, 98, 168). This thickening may impair
exchange of oxygen and metabolic waste products
across the basement membrane. Schmidt et al. (166)
demonstrated a two-fold increase in accumulation of
AGEs in the periodontium of diabetes mellitus
patients, compared to non-diabetes mellitus indivi-
duals. Increased oxidant stress was also noted in
diabetic tissues. Elevated oxidant stress has been
suggested as the probable mechanism responsible
for the widespread vascular injury associated with
diabetes. AGE formation also stimulates proliferation
of arterial smooth muscle cells, increasing thickness
of vessel walls and decreasing the vessel lumen.
AGE accumulation results in increased cross-link-
ing of collagen, reducing collagen solubility and
decreasing turnover rate (21, 125, 201). Increased
collagenase activity in diabetes mellitus results in
greater degradation of newly formed, more soluble
collagen. Conversely, the accumulation of AGEs
causes greater cross-linking of mature collagen. The
net effect is a predominance of older, highly cross-
linked AGE-modied collagen. In the capillaries, this
accumulation of highly cross-linked collagen in the
basement membrane increases membrane thickness
(51, 98, 168). These events may play a role in altering
the tissue response to periodontal pathogens, result-
ing in increased severity and progression of period-
ontitis.
Changes in host immunoinflammatory response
With few major differences in the subgingival micro-
biota in diabetes mellitus patients, attention has
turned to differences in the host immunoinamma-
tory response to this bacterial challenge as a possible
explanation for the increased prevalence and severity
of periodontitis often seen in the diabetes mellitus
population. The polymorphonuclear leukocyte plays
a major role in maintaining a healthy periodontium
in the face of periodontopathic microorganisms. In
diabetes mellitus, numerous studies have shown a
reduction in polymorphonuclear leukocyte function,
including chemotaxis, adherence and phagocytosis
(68, 91, 106, 107, 114). Diabetes mellitus patients with
severe periodontitis have been shown to have
depressed polymorphonuclear leukocyte chemotaxis
compared to diabetes mellitus patients with mild to
moderate periodontitis (18, 106, 107). Depressed
polymorphonuclear leukocyte chemotaxis has been
found in non-diabetes mellitus siblings of diabetes
mellitus children, suggesting a defect with a genetic
component (91). Chemotaxis may be improved in
those with better glycemic control (57, 91). Defects
affecting polymorphonuclear leukocytes, the rst
line of defense against subgingival microbial agents,
may result in signicantly increased tissue destruc-
tion. Polymorphonuclear leukocyte function has
been demonstrated to be normal in many individuals
with diabetes mellitus. Oliver et al. (136) have even
suggestedhyper-responsivenessor increasednumbers
of polymorphonuclear leukocytes within the gingival
crevice of poorly controlled diabetic patients, as indi-
cated by elevated levels of the polymorphonuclear
leukocyte-derived enzyme b-glucuronidase.
In addition to the polymorphonuclear leukocyte,
another critical cell line in the periodontal immu-
noinammatory response to pathogens is the mono-
cyte/macrophage line. Studies suggest that many
diabetic patients possess a hyper-responsive mono-
cyte/macrophage phenotype in which stimulation by
bacterial antigens such as lipopolysaccharide results
in dramatically increased pro-inammatory cytokine
production (135). Salvi et al. (158) have demonstrated
signicantly increased production of pro-inamma-
tory cytokines such as tumor necrosis factor-a by
monocytes derived from patients with diabetes
mellitus. When challenged with lipopolysaccharide
from P. gingivalis, diabetic monocytes showed a
66
Mealey & Moritz
2432-fold increased production of tumor necrosis
factor-a compared to non-diabetic monocytes. Pro-
duction of prostaglandin E
2
and interleukin-1b was
also signicantly higher in diabetes mellitus subjects
than in non-diabetes mellitus subjects (159). In dia-
betes mellitus patients with periodontitis, the gingival
crevicular uid levels of prostaglandinE
2
andinterleu-
kin-1b were signicantly higher than in non-diabetes
mellitus subjects with a similar degree of periodontal
destruction.
Not all people with diabetes mellitus have a hyper-
responsive monocyte/macrophage phenotype, and it
is likely that there is a genetic component to this
phenomenon (135). AGE formation plays an impor-
tant role in upregulation of the monocyte/macro-
phage cell line. Accumulation of AGEs in the
periodontium stimulates migration of monocytes to
the site. Once in the tissue, AGEs interact with recep-
tors for AGEs (RAGE) on the cell surfaces of mono-
cytes (164, 165). This AGERAGE interaction results
in immobilization of monocytes at the local site.
AGE-RAGE interaction then induces a change in
monocyte phenotype, upregulating the cell and signi-
cantly increasing pro-inammatory cytokine pro-
duction. This provides another explanation for
increasedgingival crevicular uidproductionof tumor
necrosis factor-a, prostaglandin E
2
and interleukin-1b
notedindiabeticpatients withperiodontitis (159). This
interaction also increases oxidant stress within the tis-
sue, resulting in tissue destruction (166). Interestingly,
in diabetes mellitus animal models, blocking the
receptor RAGE decreases levels of the pro-inamma-
tory cytokines tumor necrosis factor-a and IL-6 in gin-
gival tissues, decreases levels of tissue-destructive
matrix metalloproteinases, lowers AGE accumulation
inperiodontal tissues and decreases alveolar bone loss
in response to P. gingivalis (89).
These alterations in the host immunoinamma-
tory response suggest that diminished polymorpho-
nuclear leukocyte function in some diabetes mellitus
individuals may prevent effective elimination of bac-
teria and bacterial products. The subsequent persis-
tence in bacterial challenge may then be met with an
elevated monocyte/macrophage response, which
results in increased tissue destruction.
Response to periodontal treatment in
patients with diabetes
Clinicians are often faced with managing periodontal
diseases in patients with diabetes mellitus. The ques-
tion arises, Do diabetic patients respond well to
periodontal therapy? In a study of well controlled
diabetes mellitus subjects with moderate to advan-
ced periodontal disease, scaling and root planing
resulted in similar clinical changes when compared
to non-diabetes mellitus subjects with similar levels
of periodontal disease (25). Conversely, poorly con-
trolled diabetes mellitus patients often have a less
favorable response to treatment than those with well
controlled diabetes. Tervonen et al. (188) found that
the initial response to scaling and root planing was
good in diabetes mellitus subjects, but the incidence
of disease recurrence over the subsequent 12 months
was greater in poorly controlled diabetes mellitus
individuals comparedto well or moderately controlled
subjects. Westfelt et al. (206) performed a longitudinal
assessment of periodontal therapy, including scaling
and root planing, modied Widman ap surgery, and
regular maintenance, in diabetes mellitus subjects
and non-diabetes mellitus controls with moderate to
advanced periodontitis. At the 5-year re-evaluation,
diabetes mellitus patients had a similar percentage of
sites gaining or losing attachment, and a similar per-
centage of sites with stable attachment levels, com-
pared to non-diabetes mellitus subjects. Most of the
diabetic patients in this study had well or moderately
controlled glycemia. These data suggest that indivi-
duals with well controlled diabetes mellitus respond
to periodontal therapy in a fashion similar to non-dia-
betes mellitus patients, but that poorly controlled
patients may have a less favorable response.
Few data have been collected examining the re-
sponse to dental implant therapy in diabetes mellitus
subjects. Animal studies have suggested decreased
bone-to-implant contact in diabetes mellitus (132,
180, 181). The animals in these studies had extremely
high blood glucose levels. In a human prospective
case series of 89 male type 2 diabetes mellitus sub-
jects, 178 implants were followed for 5 years after
loading (138). The survival rate of implants was
90%, leading the authors to conclude that implant
therapy is a viable option in type 2 diabetes mellitus
individuals. In a large multi-center study of 255
implants in type 2 diabetes mellitus patients com-
pared to 2632 implants in non-diabetes mellitus
patients, the failure rate was 7.8% in diabetes melli-
tus subjects compared to 6.8% in non-diabetes mel-
litus patients (127). Implants were followed for at
least 36 months following prosthetic loading. The
difference in failure rate was statistically signicant,
but the P value of 0.0498 led the authors to conclude
that the inuence of type 2 diabetes mellitus on
implant failure rates was only marginally signicant.
No data are presented on the level of glycemic con-
trol in diabetes mellitus patients in this study.
67
Conclusion
Diabetes mellitus is a risk factor for periodontal
diseases. As with other complications of diabetes,
periodontal status demonstrates signicant hetero-
geneity within the diabetic population. Just as many
diabetic individuals have minimal complications
such as retinopathy, nephropathy and neuropathy,
many have a healthy peridontium. However, others
demonstrate an increased propensity for gingival
inammation and an increased risk for destructive
periodontitis, just as they do the classic complica-
tions of diabetes. This may be particularly true in
patients with poor glycemic control. In addition,
there are clear biologically plausible mechanisms
by which diabetes inuences the periodontium,
including changes in the host immunoinammatory
response and tissue homeostasis. Many of the peri-
odontal changes seen in diabetes reect similar
changes seen in other end-organ systems such as
the retina and glomerulus. Scientic evidence sup-
ports the concept that diabetes truly is the sixth
complication of diabetes.
Effects of endogenous female sex
steroid hormones on the
periodontium
Currently accepted periodontal disease classication
acknowledges the inuence of endogenously pro-
duced female sex steroid hormones on the period-
ontium. These effects are primarily seen as gingival
manifestations (Table 5) (9). Under the broad cate-
gory of dental plaque-induced gingival diseases that
are modied by systemic factors, those associated
with the endocrine system are classied as puberty,
menstrual cycle, or pregnancy-associated gingivitis.
The pyogenic granuloma, or pregnancy tumor, is
a reactive lesion also classied under pregnancy-
associated gingival disease. Given that distinct
hormonally-related events such as menstruation
and pregnancy may result in endocrinotropic mani-
festations visible in the gingival tissues, it is under-
standable that such gender-specic observations
have come to light.
Exaggerated gingival responses during pregnancy
were rst described in the 1800s (37, 146) and further
evidence has since established that multifactorial
mechanisms involving the endocrine system are
involved in the homeostasis of the periodontium.
Female sex steroid hormones are neither neces-
sary nor sufcient to produce gingival changes by
themselves. However, they may alter periodontal tis-
sue responses to microbial plaque, and thus indir-
ectly contribute to periodontal disease (55). This
section reviews and discusses the various factors
and inuences involving endogenous sex steroid hor-
mones on the periodontium of women from puberty
through postmenopause. Hormone replacement ther-
apy and osteoporosis will not be addressed here and
are covered in other chapters in the current volume.
Physiology and production of female
sex steroid hormones
Although androgens may also play a role in modulat-
ing periodontal tissue responses in women, the main
sex steroid hormones with evidence for such an
effect are the estrogens and progestins. Produced
by the female gonad, the ovary, the principal preme-
nopausal estrogen in plasma is estradiol, whereas the
main postmenopausal estrogen is estrone (204, 212).
Less potent than estradiol, estrone does not demon-
strate cyclic changes (213). Estradiol is additionally
secreted by the placenta and certain peripheral tis-
sues. Estrogens perform many vital activities in
women including the development and maintenance
of secondary sex characteristics, uterine growth, pul-
satile release of luteinizing hormone from the ante-
rior pituitary gland, thickening and sustainment of
the vaginal mucosa, and ductal development of the
breast. (22, 109).
The principal female progestin is progesterone,
secreted by the corpus luteum, placenta, and the
adrenal cortex. Progesterone is important for many
vital activities in the female including endometrial
development and sustainment, mammary gland
development, maintenance of the pregnant state,
as well as decreasing very low density lipoprotein
and high density lipoprotein secretion, decreasing
insulin action, and enhancing sodium excretion by
the kidneys (22, 109). Estrogens may regulate proges-
terone activities by inducing the production of
Table 5. 1999 International Workshop Classication
for Periodontal Diseases: Section specic to endogen-
ous female sex steroid hormones (Armitage (9))
1. Gingival diseases
A. Dental plaque-induced gingival diseases
2. Gingival diseases modified by systemic factors
a. Associated with the endocrine system
1) Puberty-associated gingivitis
2) Menstrual cycle-associated gingivitis
3) Pregnancy-associated
a) Gingivitis
b) Pyogenic granuloma
68
Mealey & Moritz
progesterone receptors, thus promoting increased
responsiveness of tissues to progesterone (72).
Estrogen formation in the ovary begins between 8
and 10 weeks of gestation, and oogonia in the ovaries
begin developing into primary oocytes by 1011
weeks. A nite number of these germ cells are con-
tained in the ovary with a peak of about 7 million
oogonia present by the 5th to 6th month of gestation.
Thereafter, through the process of atresia, the germ
cells decrease in number until only 1 million primor-
dial follicles containing a single ovum each remain at
birth. The majority of these follicles fail to develop
and after further atresia, approximately 400,000
remain at puberty, and only a few are left at meno-
pause (22). It is during puberty that the nal matura-
tion of the ovarian follicle commences.
The onset of increased production and secretion of
estrogen and progesterone in a cyclic pattern accom-
panies the onset of puberty and is referred to as the
reproductive or menstrual cycle (Fig. 1). The mean
age of the onset of menses (menarche) in the United
States has decreased about 34 months per decade
over the last 100 years and is now about 12 years (22).
The duration of a normal reproductive cycle is 28
days, with the rst half of the cycle referred to as
the follicular or proliferative phase. This phase is
initiated with the release by the hypothalamus of
gonadotropin releasing factor (GnRH). Subsequently,
two major gonadotropic hormones regulating folli-
cular development are produced in the anterior
pituitary follicle stimulating hormone and luteiniz-
ing hormone. Follicle stimulating hormone promotes
the growth of a Graaan follicle that typically con-
tains a single, maturing ovum. The mature follicle
then produces a uid rich in estrogens. Together with
follicle stimulating hormone, estrogen promotes
further maturation of the ovum and thickening of
the uterine lining tissues. The rise in circulating
estrogens initiates a burst of luteinizing hormone
secretion which results in release of the mature ovum
into the fallopian tube. After ovulation at approxi-
mately day 14 of the cycle, the secretory or luteal
phase begins, and is characterized by the synthesis
and release of both estrogen and progesterone by the
follicular cells, which have now become the corpus
luteum. If fertilization does not occur, the corpus
luteum will degenerate, plasma levels of estradiol
and progesterone will decline, and a large portion
of the endometrium will be released as menstrual
ow.
If fertilization and pregnancy do occur, the corpus
luteum will continue to synthesize estrogen and pro-
gesterone in ever increasing amounts. The placenta
develops as an endocrine organ which contributes
additionally to estrogen and progesterone produc-
tion. Plasma progesterone levels during pregnancy
may reach 100 ng/mL, approximately 10 times that
seen during the luteal phase of the reproductive
cycle. Estradiol levels in plasma may similarly be
increased up to 30 times (140).
In stark contrast to pregnancy, menopause is char-
acterized by the irregular and declining production
and secretion of estrogen and progesterone. This
correlates signicantly with the reduced number of
ovarian follicles having the potential for maturation,
along with the progressive loss of ovarian function.
During the reproductive cycle, estradiol, which con-
stitutes 60% of circulating estrogens, is formed pri-
marily by the ovaries, and the remainder is estrone,
formed mainly in extraglandular tissues. After meno-
pause, extraglandular formation becomes the major
route of estrogen formation, with the predominant
plasma estrogen being estrone (22). The permanent
cessation of menstrual ow (amenorrhea) during
menopause occurs in women at an average age of
51 years (22). Considering current life expectancy, it
is apparent that approximately one third of a
womans life will be lived after reproductive function
ceases.
Periodontal manifestations related to
female sex steroid hormones
Puberty
Along with the dramatic rise in female sex steroid
levels during the circumpubertal period, several
Fig. 1. Hormonal variations accompanying the female
reproductive cycle. FSH follicle stimulating hormone;
LH luteinizing hormone.
69
cross-sectional and longitudinal studies have
demonstrated an increase in gingival inammation
without an accompanying increase in plaque levels
(32, 67, 85, 109, 112, 130, 143, 178). This gingivitis
manifests as marginal and interdental gingival enlar-
gement found primarily on the facial surfaces, with
the lingual surfaces remaining relatively unaltered
(55). An increased gingival bleeding tendency has
also been reported (64). Although other factors such
as caries, mouth breathing, tooth eruption status,
and crowding of teeth may inuence the increase
in gingival inammation seen, current disease clas-
sication distinguishes this type of gingivitis as hav-
ing the propensity toward developing clinical signs of
inammation with relatively small amounts of pla-
que present during this period (110). Nevertheless,
evidence for this phenomenon is not absolute and for
the most part may be considered circumstantial at
the present time (80). The reason for this is that most
studies cited as evidence for this phenomenon have
relied mainly on the chronological age of the cohorts
examined, although age in and of itself is not the best
predictor of the onset of puberty (109). Additionally,
few of the studies have evaluated the hormonal levels
of the subjects, which would lend itself to a more
exacting analysis of any association. Nakagawa et al.
(130) did evaluate serum female sex hormone levels
during puberty and found a slight but statistically
signicant increase in gingival index (99) over pre-
pubertal conditions, which was positively correlated
with an increase in serum estradiol and progesterone
levels. In this study as in others, the increased gingi-
val inammation was not accompanied by a signi-
cant change in mean plaque index (171). Studies also
exist that have not found any correlation between
pubertal onset and the gingival condition in females
(193, 211). Further examination to determine a de-
nitive correlation between increased sex steroid
levels is required; however, the data available to date
give some indication that a transient period of
enhanced gingival inammatory response to dental
plaque does indeed exist during this period of female
development.
Several studies have noted a concomitant increase
in subgingival black-pigmented anaerobic bacteria
that parallels the onset of puberty and the increase
in gingival inammation observed during this time
(34, 64, 124, 129, 130, 210). The microbial changes
noted have been postulated to result from a selective
enhancement of the growth of black-pigmented
anaerobic rods, primarily Prevotella intermedia, by
female sex hormones. It has been suggested that
these gram-negative bacteria are able to substitute
estradiol and progesterone for menadione, an essen-
tial vitamin K growth factor for this bacterium (85). A
study evaluating increased serum estradiol and pro-
gesterone levels during puberty noted a positive cor-
relation with signicant increases in proportion,
number, and serum IgG antibody levels to Prevotella
intermedia (130). Capnocytophaga species have also
been noted to increase in number as well as propor-
tion in the subgingival milieu during puberty, and
have been shown to correlate with an increased gin-
gival bleeding tendency (64). However, other studies
have not shown these relationships (63, 211). In a
longitudinal study, Yanover & Ellen (211) were
unable to detect any changes in the oral microbiota
during puberty, and found no correlation between
plasma estradiol levels and levels of black-pigmented
anaerobic bacteria. Thus, more denitive research
may be needed to shed light on the exact mechan-
isms of gingival involvement seen accompanying the
circumpubertal period.
Menstruation
Signicant and observable gingival inammatory
changes have been documented in association with
the menstrual cycle (69, 93, 110, 178). Muhlemann
(128) over 50 years ago described a case of gingivitis
intermenstrualis, which he observed as consisting
of bright red hemorrhagic lesions of the interproxi-
mal papillae identied prior to menstruation. Gingi-
val manifestations of bleeding and swollen gingiva
(45, 69, 93, 178), an increase in gingival exudate most
pronounced in the presence of preexisting gingivitis
(69, 70, 93), and a minor increase in tooth mobility
(93) have been reported in the literature. In a long-
itudinal evaluation, Hugoson (71) reported gingival
exudate increases of at least 20% during ovulation in
more than 75% of the females participating in his
study. The level of exudate appears to peak just
before ovulation, coinciding with the highest levels
of estradiol and progesterone in the circulation (70,
93). Nevertheless, most women with a clinically
healthy periodontium experience few signicant per-
iodontal changes as a result of menstruation (3).
Lindhe & Attstrom (93) noted that during their men-
strual cycles, women without clinical gingivitis
showed no increase in gingival uid, whereas those
with gingivitis showed increases in gingival uid. In
addition to gingival inammation, intraoral recur-
rent aphthous lesions (44), herpes labialis lesions,
and infections with Candida albicans (140) have
been documented in some women and seem to be
associated with increased progesterone levels during
the reproductive cycle.
70
Mealey & Moritz
Pregnancy
The signicant increases in plasma hormone levels
accompanying pregnancy manifest as some of the
most remarkable endocrine-related oral alterations
seen in women. Pregnancy gingivitis is extremely
common and affects 30100% of all pregnant women
(35, 73, 92, 100, 102) (Figs 2 and 3). Its occurrence has
been acknowledged for a substantial period of time,
as pregnancy gingivitis was rst described in 1877
(146), although descriptions had been given even
earlier (37). Gingival inammatory changes in preg-
nancy usually begin during the 2nd month and
increase in severity through the 8th month, after
which there is an abrupt decrease related to a con-
comitant reduction in sex steroid hormone secretion
(100). The greatest involvement appears to be in
anterior areas of the oral cavity, with interproximal
sites most commonly affected (99). Longitudinal and
cross-sectional studies have conrmed that the pre-
valence and severity of gingival inammation is
signicantly higher in pregnant women compared
to postpartum, and appears unrelated to the amount
of plaque present (7, 71, 99). Hugoson (71) addition-
ally conrmed that the severity of gingival inamma-
tion correlated to elevations of sex steroid hormones
during pregnancy, and was reduced following par-
turition and the concomitant drop-off in hormone
production. Other clinical periodontal changes that
have been described during pregnancy include
increased gingival probing depths (71, 99, 119, 150),
increased bleeding upon probing or mechanical sti-
mulation (7, 119), increased gingival crevicular uid
ow (71), and increased tooth mobility (27, 99, 150).
Preexisting gingivitis or periodontitis in pregnant
women has been noted to worsen dramatically (73,
100). However, Tilakaratne et al. (194) studied preg-
nant women and found that pregnancy increased the
clinical measures of gingival inammation, but
attachment levels were not affected. Although signif-
icant adverse gingival conditions may develop during
pregnancy, there are no studies presently available
indicating that periodontitis development is a nat-
ural sequela of steroid sex hormone-induced gingi-
vitis (76, 79). There is, however, current evidence that
periodontal inammation and destruction may be
increased in pregnant diabetic patients compared
to non-diabetic pregnant patients (65). Nevertheless,
in otherwise systemically healthy females, this has
not been demonstrated.
In addition to the gingival changes seen due to an
enhanced inammatory response during pregnancy,
0.59.6% of women who are pregnant also experience
localized gingival enlargement consistent with pyo-
genic granulomas (8, 88, 104, 217) (Figs 4 and 5). First
described in 1874 (28), the pregnancy-associated
pyogenic granuloma, or pregnancy tumor, is not
a neoplasm at all, and clinically and histologically
cannot be distinguished from pyogenic granulomas
occurring in women who are not pregnant. These
lesions have been described as a painless, exophytic
mass that has either a sessile or pedunculated base
extending from the gingival margin or, in most
instances, from the interproximal tissues in the max-
illary anterior (170). The pregnancy tumor develops
as the result of an exaggerated inammatory
response to an irritation (often calculus), enlarges
rapidly, bleeds easily, and may range in color from
purplish red to deep blue, although most commonly
is red in color with small brin spots (3). It rarely
reaches more than 2 cm in size and has a tendency
to recur if not completely removed. The gingiva is
involved in 70% of cases, followed by the tongue,
lips, and buccal mucosa (17). Following parturition,
Fig. 2. Clinical appearance of pregnancy gingivitis. Signif-
icant generalized gingival enlargement and inammation
are evident.
Fig. 3. Clinical view of pregnancy gingivitis in the right
posterior.
71
the lesion may regress or completely disappear
(217).
The etiologic means by which female sex steroid
hormones may inuence the periodontium of
women, especially during pregnancy, are varied
and differ from those ordinarily associated with pla-
que-induced gingivitis. Human gingiva contains
receptors for estrogen and progesterone, and
increased plasma levels result in an increase in accu-
mulation of these hormones in gingival tissues (11,
144, 198, 199). In addition to the observed effects of
increased sex steroid hormone levels on subgingival
microbial parameters described earlier for circumpu-
bertal gingival changes, estrogens, and progestins
may inuence the periodontal tissues by affecting
gingival vasculature, the local immune system, and
the specic cells of the periodontium. Which etiolo-
gic factor plays the greatest role in fostering the exag-
gerated inammatory response to bacterial plaque,
regardless of plaque quantity, has not been deter-
mined, and appears to most likely be a multifactorial
phenomenon (109). Although estrogens play a sig-
nicant role in collagen maintenance and metabo-
lism, a review of the impact of hormones on bone
maintenance and osteoporosis is not given here and
is specically addressed elsewhere in this volume.
Hormonal influences on the microbiota
The effects of sex steroid hormones on the subgingi-
val microbiota during pregnancy have been well
documented. Kornman & Loesche (86) reported that
during the second trimester, plaque levels remained
constant, yet gingivitis and gingival bleeding were
shown to increase in severity. At the same time, the
ratio of subgingival bacterial anaerobes-to-aerobes
increased, as well as proportions of Bacteroides mel-
aninogenicus and P. intermedia (2.210.1%). Subgin-
gival plaque samples from these patients during the
second trimester demonstrated a signicantly higher
accumulation of estradiol and progesterone than pla-
que samples at other time periods. Subsequently,
both estradiol and progesterone were shown to be
selectively accumulated by P. intermedia as a sub-
stitute for vitamin K (87), and thus postulated to be
acting as a growth factor for this microorganism.
Jensen et al. (73) studied the effects of hormone
levels on gingival status in pregnant patients and
those using oral contraceptives versus non-pregnant,
non-contraceptive controls. There was a 55-fold
increase over the control group in the population
of Bacteroides species in the pregnant women and
a 16-fold increase in those taking contraceptives. The
concomitant increases in P. intermedia were most
pronounced in the second trimester and were corre-
lated with increased gingivitis scores.
Using an experimental gingivitis model in patients
during pregnancy and again 6 months postpartum,
Raber-Durlacher (150) demonstrated increased
levels of P. intermedia during pregnancy coinciding
with increased gingival inammation at this time.
This was signicantly different from postpartum
clinical parameters which showed less P. intermedia
and gingival swelling, and reduced probing depths
compared to pregnancy. Plaque scores were similar
at all time points. Not all studies have corroborated
these ndings, and Jonsson et al. (75) found no dif-
ference in levels of P. intermedia at any time during
pregnancy or between pregnant and nonpregnant
controls in a cross-sectional assessment. This has
led to speculation that the increase in P. intermedia
seen during the second trimester of pregnancy may
actually be independent of estrogens or progesterone
and may occur for other reasons. Mariotti (109) has
Fig. 4. Clinical presentation of a pregnancy-associated
pyogenic granuloma (pregnancy tumor) with typical
presentation on the gingiva.
Fig. 5. Pregnancy-associated pyogenic granuloma with
exuberant tissue response.
72
Mealey & Moritz
made observations in this regard. First, P. intermedia
is seen to increase during the second trimester of
pregnancy followed by a decline to postpartum
values during the third trimester, despite highly ele-
vated hormone levels still present during the third
trimester. Additionally, there was no analysis of com-
petitive inhibition with other steroid-like molecules
performed in the heretofore cited studies; therefore,
it is open to question whether the accumulation of
estradiol or progesterone in second trimester plaque
samples or pure cultures of P. intermedia was sex
steroid hormone specic or merely dependent on
the lipophilic nature of the plaque sample.
As discussed for circumpubertal gingival res-
ponses, it has still not been absolutely established
that increases in plasma estrogen or progesterone
during pregnancy stimulate specic bacterial spe-
cies. At the present time, data indicate that absolute
numbers of P. intermedia may be elevated during
pregnancy. However, the relationship of this bacter-
ial species to the development of exaggerated gingi-
val inammatory responses during pregnancy or
whether its growth is enhanced as a direct result of
increasing sex steroid hormone concentrations has
still to be denitively established.
Hormonal influences on the gingival
vasculature
The effects of estrogens and progestins on the gingi-
val vasculature could potentially explain the
increased edema, erythema, gingival crevicular exu-
date, and hemorrhagic gingival tissues noted during
pregnancy as well as other stages of the reproductive
cycle. An increase in gingival crevicular uid ow has
been correlated to elevated sex steroid levels (71),
which indicates that these hormones may affect vas-
cular permeability in the gingival sulcus. Estrogen is
the main sex steroid hormone responsible for altera-
tions in blood vessels in target tissues in females (52,
77), stimulating endometrial blood ow during the
rise in plasma estrogen seen during the follicular
phase. Subsequently, endometrial blood ow
decreases during the luteal phase of the cycle with
waning estrogen levels (147). Progesterone, in con-
trast, has been shown to have little effect on the
vasculature of systemic target tissues (103). The
effects of these hormones on the local vasculature
of the gingiva may be similar; however, the roles of
estrogen and progesterone may be reversed.
In gingiva and other non-periodontal intraoral tis-
sues, more evidence has accumulated for progester-
one affecting the local vasculature than for estrogen.
In addition, progesterone has been shown to reduce
corpuscular ow rate, allowing for accumulation of
inammatory cells (94), increased vascular perme-
ability (70, 95, 120), and increased vascular prolifera-
tion (96, 97). Lindhe et al. (97) administered
progesterone systemically in beagle dogs and
observed that the hormone may induce increased
leakage of leukocytes and plasma proteins from
post-capillary venules by affecting the endothelial
lining. Estrogens in these studies were found to have
minimal effect on the vasculature. Many of these
studies used signicantly higher than pharmacologic
doses of sex hormones in animal models and should
be interpreted with caution. Nevertheless, each of the
effects elaborated have the propensity to contribute
to enhanced inammation in the gingival tissues. It is
evident that further research is needed to fully under-
stand the contribution of sex hormone induced
alterations of the gingival vasculature to the expres-
sion of enhanced gingival inammation.
Hormonal influences on the local
immune system
Several studies have focused on the alteration of local
immune system components to explain the impact
that sex hormones may have on the periodontium
during pregnancy. Given similar levels of bacterial
plaque, local immunologic reactions in the gingiva
enhanced by sex steroid hormones might alter the
pathogenesis of the inammatory lesion, and allow
for exaggerated gingival tissue responses during
pregnancy. This idea is furthered by the observation
that immune system components have been identi-
ed as possessing sex steroid receptors (2). Proges-
terone in particular has been shown to stimulate the
production of the inammatory mediator prosta-
glandin E
2
and to enhance the accumulation of poly-
morphonuclear leukocytes in the gingival sulcus (38,
45). Progesterone has also been found to enhance the
chemotaxis of polymorphonuclear leukocytes, while
low concentrations of estradiol have been shown to
reduce polymorphonuclear leukocyte chemotaxis
(118). In this same study, sex hormones were shown
to have no effect on the chemotaxis of monocytes.
Other studies suggest an increased susceptibility
brought on by immune system alterations. For ins-
tance, sex steroid hormones may modulate the pro-
duction of cytokines (179), and progesterone has
been shown to downregulate IL-6 production by
human gingival broblasts to 50% of that of control
values (90). Kinane et al. (80) have suggested that it
73
may be through IL-6 that estrogen and progesterone
exert their effects on the gingiva.
Kinnby et al. (81) reported that the balance of the
brinolytic system may be disturbed as high proges-
terone levels during pregnancy resulted in lower
levels of plasminogen activator inhibitor type 2
(PAI-2), an important inhibitor of tissue proteolysis.
In addition, the cellular and humoral immune sys-
tem may be affected by sex steroid hormones. Aboul-
Dahab et al. (1) studied pregnant women and non-
pregnant controls and found the percentages of CD3,
CD4, and B lymphocytes decreased in gingival tis-
sues during pregnancy, and greater gingival inam-
mation was seen compared to controls. Other studies
(148, 174) demonstrate a decreased number of CD4
cells in peripheral blood during pregnancy when
compared to postpartum, and Sridama et al. (174)
showed a tendency for the CD4/CD8 ratio to
decrease at the same time, suggesting an immuno-
decient state. Elevated levels of sex hormones, espe-
cially progesterone, have also been linked to
depressed cell-mediated immunity and phagocytosis
(149). Additionally, peripheral blood lymphocytes
showed a decreased response to bacterial antigens
in vitro, including extracts from P. intermedia (101,
139). Although much work remains to be done to
elucidate the exact mechanisms by which female
sex steroid hormones may impact the local immune
system in the periodontium, enough information is
available to suggest that hormonally related immu-
nologic changes during pregnancy may increase sus-
ceptibility to gingival inammation.
Hormonal influences on cells of the
periodontium
The effects of sex steroid hormones on individual
cells of the periodontium may also play a signicant
role in the exaggerated gingival responses seen dur-
ing the female reproductive cycle and pregnancy. Sex
steroid hormones have been shown to directly and
indirectly exert inuence on cellular proliferation,
differentiation, and growth in target tissues, includ-
ing keratinocytes and broblasts in the gingiva (109).
Two theories for the actions of the hormones on
these cells involve the role hormones may play in
altering the effectiveness of the epithelial barrier to
bacterial insult, and in affecting collagen mainte-
nance and repair. Estrogens stimulate epithelial pro-
liferation and increase keratinization of the vaginal
mucosa (3). Some evidence also exists that sex hor-
mones may have a similar effect on the oral mucosal
and gingival epithelia (154, 155, 215), and a reduction
in the keratinization of gingival epithelium of post-
menopausal women has been shown to accompany
declining plasma estrogen levels (195).
These hormones also act in a dynamic fashion on
the extracellular matrix in the gingiva, and these
effects may be exaggerated during times of signi-
cant hormonal uctuations. Fibroblast proliferation
and collagen maturation in gingival connective tis-
sues may be affected by both estrogen and proges-
terone. By altering collagen turnover, estrogens may
stimulate the proliferation of gingival broblasts, and
the synthesis and maturation of gingival connective
tissues (16, 54, 108). In contrast, Lundgren et al. (102)
demonstrated that progesterone may alter the rate
and pattern of collagen production in the gingiva,
resulting in reduced repair and maintenance poten-
tial. This inhibition of human gingival broblast pro-
liferation by progesterone has been demonstrated by
others (54, 207). Sex steroid hormones have also been
shown to increase the rate of folate metabolism in
oral mucosa (141, 190). Since folate is required for
tissue maintenance, increased metabolism could
deplete folate stores and inhibit tissue repair. Addi-
tionally, progesterone in concentrations correspond-
ing to the third trimester of pregnancy has been
shown to lower the synthesis of glycosaminoglycans,
a major constituent of the connective tissue matrix of
gingiva (208). Although little information exists at the
present time, estrogen and progesterone have been
shown to exert inuence on the metabolism of per-
iodontal ligament-derived broblasts in vitro (131).
In particular, estradiol and progesterone inhibited
collagen synthesis by these cells. Taken together,
the evidence suggests that female sex steroid hor-
mones may contribute to gingival tissue mainte-
nance and repair and that these interactions have
the potential to signicantly contribute to enhanced
gingival inammation. As in other areas previously
discussed, further research is needed to fully eluci-
date the exact mechanisms underlying these pro-
cesses.
Menopause
Unlike the rhythmic patterns of the reproductive
cycle, the onset of menopause is accompanied by
irregular hormonal uctuations, as estradiol ceases
to be the major circulating estrogen and estrone,
which lacks cyclic inuence, predominates. With
the overall reduction in estrogen output after meno-
pause, a signicantly different presentation of the
gingiva predominates. Essentially, the sex steroid-
induced gingival inammatory changes witnessed
74
Mealey & Moritz
during the reproductive years no longer occur to any
degree. The effect of reduced estrogen levels on
epithelial keratinization (195), along with decreased
salivary gland ow independent of medications
(177), may have other signicant effects on the per-
iodontium. Friedlander (53) described an atrophic
gingivitis in some post-menopausal women in which
the gingival tissues develop an abnormal paleness. In
other women, a menopausal gingivostomatitis may
develop, characterized by gingival tissues that are
shiny and dry, bleed readily, and may range from
pale to erythematous in color. More commonly
reported gingival lesions seen in women during this
phase of life tend to be desquamative in nature (109).
However, the role that sex steroid hormones play in
the development and progression of these lesions
remains obscure, and only circumstantial data exist
to tie hormones to these lesions. For example, most
of these lesions occur in females of middle age (133),
and exogenous estrogens have been used to treat
these lesions, (197, 216). Oral discomfort is also com-
monly reported by post-menopausal females, with
2090% of women reporting a burning sensation,
xerostomia, or bad taste (15, 43), while only 6% of
pre-menopausal women report the same (203). As
with desquamative lesions, estrogens have been suc-
cessfully used to gain pain relief under these circum-
stances (15, 155, 203), although direct correlation
with these symptoms has yet to be established. In
addition to gingival changes, reduced estrogen levels
certainly may impact overall collagen metabolism,
including bone maintenance, and result in a ten-
dency toward development of osteoporosis.
Conclusion
It is evident from this review that multifactorial
mechanisms involving the endocrine system are
involved to a signicant degree in the homeostasis
of the periodontium during each of the life stages of
the human female. The notable periodontal changes
related specically to female sex steroid levels,
primarily those manifested as pathologic alterations
in the gingiva, attest to the dramatic potential these
hormones possess to impact the oral tissues beyond
normal homeostatic functions. Nevertheless, it is
also apparent that female sex steroid hormones
are neither necessary, nor sufcient to produce
pathologic gingival alterations by themselves, and
attests to the need for further elucidation of the bio-
molecular mechanisms at work to fully understand
how these hormones may exert their signicant
effects.
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81
Osteoporosis and periodontal
disease progression
Nico C. Geurs, Cora Elizabeth Lewis & Marjorie K. Jeffcoat
Osteopenia and osteoporosis are systemic skeletal
diseases characterized by low bone mass and
micro-architectural deterioration with a consequent
increase in bone fragility and susceptibility to frac-
ture. According to the World Health Organization,
osteoporosis is considered to be present when bone
mineral density (BMD) is 2.5 standard deviations
(SD) below the young normal. Osteopenia is dened
as bone density levels between 1 SD and 2.5 SD
below normal BMD (29).
In the third National Health and Nutrition Exam-
ination Survey (NHANES III) the prevalence of osteo-
porosis when assessed at the femoral neck was 20%
of postmenopausal white women (16). An alternative
approach is to use morphological deformities in the
vertebrae to dene osteoporosis. The prevalence of
the dened vertebral deformities was found to be
12% in both men and women. The increase in fre-
quency with age was greater in women, from 5% at
age 5054 to 24% at age 7579. For men this was 10%
at age 5054 years, rising to 18% at age 7579 years
(18). The prevalence of this relatively silent disease is
very high and on the rise. Future projections indicate
a threefold increase in osteoporosis-related hip frac-
tures (9).
The risk factors for osteoporosis can be divided
into non-modiable and modiable risk factors.
The non-modiable include sex, age, early meno-
pause, thin or small body frame, race, and heredity.
Lack of calcium intake, lack of exercise, smoking, and
alcohol are modiable risk factors. Low bone mass,
certain medications, propensity to fall, and systemic
diseases such as hyperparathyroidism are modiable
to some extent. These risk factors have been dis-
cussed previously (5, 6).
The risk factors for osteoporosis include many risk
factors associated with advanced periodontal dis-
ease. Since both osteoporosis and periodontal dis-
eases are bone resorptive diseases, it has been
hypothesized that osteoporosis could be a risk factor
for the progression of periodontal disease.
Relation between systemic bone
mineral density and oral bone
mineral density
Studies discussing the relationship between systemic
BMD and oral BMD are summarized in Table 1. Most
studies reported to date concerning this relationship
are cross-sectional studies using different popula-
tions and different methods to assess BMD.
Kribbs et al. (15) was the rst to address the rela-
tionship in osteoporotic women in a study assessing
total body calcium by neutron activation analysis. An
association was found with mandibular density when
measured by quantitative analysis on intraoral radio-
graphs. In a comparison of 85 osteoporotic women
and 27 normal women, the osteoporotic group had
less mandibular bone mass and density and a thinner
cortex at the gonion than the normal group. The
osteoporotic group also had a greater percentage of
subjects who were edentulous. In dentate subjects a
greater amount of tooth loss was reported for the
osteoporotic group. No differences in clinical period-
ontal measurements were found between osteoporo-
tic and normal groups (1214).
In a study of 12 osteoporotic subjects with a history
of fractures, von Wowern et al. (27) found less man-
dibular bone mineral content as measured by dual
photon absorptiometry than in 14 normal women.
In a longitudinal study of 69 women receiving
hormone replacement therapy, lumbar spine BMD
was assessed by dual photon absorptiometry. When
compared to quantitative measurements of standar-
dized radiographs of the posterior region, a signi-
cant but moderate correlation was found only at the
second visit. During the observation period of an
105
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PERIODONTOLOGY 2000
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average of 5 years, a positive effect of estrogen repla-
cement therapy on the bone mass of the mandible
and the lumbar spine was observed. Different estro-
gen regimens resulted in different increases in bone
mass (4).
Streckfus et al. (24) used quantitative measure-
ments of vertical bitewing and hand radiographs in
patients with active periodontitis. The results of the
study showed that postmenopausal women on estro-
gen therapy had more alveolar bone loss (ABL), more
missing teeth, and reduced alveolar and second met-
acarpal bone density than premenopausal women.
Alveolar bone densities were also strongly correlated
to second metacarpal densities.
Most studies relate systemic BMD with mandibu-
lar mineral density. In a study of both maxilla and
mandible, 41 dentate Caucasian women aged 2078
were evaluated using quantitative intraoral radiogra-
phy and systemic bone densities determined by dual-
energy X-ray absorptiometry (DXA) (23). The density
of maxillary alveolar process bone was signicantly
related to the density of the mandibular alveolar
process, lumbar spine, hip, and radius in healthy
women and maxillary alveolar process bone density
declined with age (23).
Shrout et al. (22) used morphologic measurements
from digitized images of bitewing radiographs to
correlate with lumbar and femoral BMD in 45 post-
menopausal women who had no or only mild period-
ontal disease (no probing depths > 5 mm). The
complexity of the trabecular pattern weakly corre-
lated with lumbar spine and femoral BMD.
In a preliminary report of the oral ancillary study of
the Womens Health Initiative, 158 patients with a
mean age of 62.2 7.6 years were evaluated (6).
Hipbone mineral density was conrmed by DXA
and mandibular bone density was measured by
quantitative digital intraoral radiography. A signi-
cant correlation was found between mandibular
basal bone and hipbone mineral density (6). The
authors posed the question whether intraoral radio-
graphy could serve as a screening tool for osteopenia.
Table 1. Relation between systemic bone mineral density (BMD) and oral bone mineral density
Authors Population Major Result Type of study
Jeffcoat et al. (6) 158 postmenopausal
women
Age 62.2 7.6 years
Significant correlation between
hip BMD and mandibular
basal BMD
Cross-sectional study
Shrout et al. (22) 45 postmenopausal
women with no or
mild periodontitis
Mean age 57.4 5.8
Complexity of the trabecular
pattern weakly correlated with
lumbar spine and femoral BMD
Southard et al. (23) 41 dentate Caucasian
women aged
20 to 78 years
Significant correlation between the
density of maxillary and mandibular
alveolar process, lumbar spine,
hip, and radius in healthy women.
Streckfus et al. (23) 28 healthy women
aged 2378
Strong correlation between alveolar bone
and the second metacarpal densities.
Both reduced in postmenopausal women
Jacobs et al. (4) 69 women receiving HRT
aged 3264 at entry
Correlation between spinal density and
mandibular bone mass at the second
examination (average follow-up 5.1 years)
longitudinal study
von Wowern
et al. (27)
12 women with
osteoporotic fractures
Osteoporotic subjects had less bone
mineral content
Kribbs et al. (14) 50 normal women
aged 2090
Mandibular bone mass correlated with
bone mass at spine and wrist
Kribbs (12) 85 osteoporotic women
and 27 normal
women aged 5085
Osteoporotic group had less mandibular
bone mass and density
Kribbs et al. (13) 85 osteoporotic women Total body calcium, bone mass at radius,
and bone density at spine correlated
with mandibular mass
Kribbs et al. (15) 30 postmenopausal
women
Total body calcium associated with
mandibular bone density
Geurs et al.
106
The usefulness of the alveolar trabecular pattern
analysis and mandibular alveolar bone mass for pre-
diction of the skeletal BMD was further evaluated by
Jonasson et al. (8). They used an index to assess the
alveolar trabecular patterns and found a signicant
correlation with skeletal BMD. The evaluation of the
coarseness of trabeculation of the alveolar bone as
seen on intraoral radiographs could be a helpful clin-
ical indicator of skeletal BMD and better than densi-
tometric measurements of the alveolar bone. Dense
trabeculation is a strong indicator of high BMD,
whereas sparse trabeculation may be used to predict
low BMD.
The data gathered on the mostly cross-sectional
studies appears to indicate a relationship between
systemic BMD and oral BMD. Additional data from
ongoing longitudinal studies will further elaborate
this relationship.
Periodontal disease and
osteoporosis
The relationship of tooth loss and BMD has been
studied. Several reports nd a correlation between
tooth loss and diminished systemic BMD (5, 11, 24,
25). Other reports fail to nd this correlation (1, 10,
19). The use of tooth loss as a surrogate for period-
ontal disease extent has several limitations. The
underlying reason for the loss of the teeth is often
unknown. The extent of the disease around the
remaining teeth is not taken into account in these
analyses. Therefore, an accurate measurement of the
extent of periodontal destruction can not be made by
using tooth loss as a variable in the analysis of the
relationship between osteoporosis and periodontitis.
Several mostly cross-sectional reports have used a
variety of parameters to evaluate the periodontal dis-
ease severity in subjects with decreased BMD. These
reports are summarized in Table 2.
In a report by Elders et al. (1), lumbar BMD and
metacarpal cortical thickness (MCT) were compared
to alveolar bone height measured on bitewing radio-
graphs and clinical parameters of periodontitis. No
signicant relation was observed between the bone
mass measurements and alveolar bone height or per-
iodontal parameters. The mean age in this group was
relatively young, between 46 and 55 years of age,
which could have contributed to the lack of cor-
relation.
Similar ndings were reported in a study of tooth
loss and attachment loss when related to vertebral
and proximal femoral BMD. In that study, 135
women with at least 10 teeth and no evidence of
moderate or severe periodontal disease were exam-
ined. Attachment loss was correlated with tooth loss
but not with vertebral or proximal femur bone den-
sity (3).
When comparing the number of sites with loss of
attachment with BMD in 292 dentate women (aver-
age age 75.5 years) no statistically signicant associa-
tion was found (28).
In an age cohort of 70-year-old women, 15 subjects
with osteoporosis were compared to 21 subjects with
normal BMD (17). No statistically signicant differ-
ences were found in gingival bleeding, probing
pocket depths, gingival recession, or marginal bone
level between the women with osteoporosis and the
women with normal BMD (17).
In contrast to these reports, other authors have
reported a signicant relation between systemic
osteopenia and periodontal bone loss. Von Wowern
et al. (27) found greater amounts of loss of attach-
ment in osteoporotic women in a small population
with a mean age of 68. Osteoporosis was assessed
using bone mineral content of the mandible and
forearm determined by dual photon scanning.
In a study population of 70 postmenopausal
Caucasian women aged 5178, skeletal systemic
BMD was assessed by DXA (26). Clinical attachment
loss and interproximal ABL represented periodontal
disease severity. Mean ABL signicantly correlated
with systemic BMD. A trend for a correlation
between clinical attachment levels and BMD was
found (26).
The cross-sectional studies have limitations. No
information about the diseases studied prior to the
exam is available. Although both osteopenia and per-
iodontal disease are chronic diseases and can be
assumed to have been present prior to the observa-
tions, it is incorrect to conclude that both diseases
have been present. To better evaluate this relation-
ship, prospective longitudinal studies are needed. To
date, few longitudinal studies have been performed.
In a 2-year longitudinal clinical study, the alveolar
bone height and density changes in 21 osteoporotic/
osteopenic women compared with 17 women with
normal lumbar spine BMD were studied. The sub-
jects were postmenopausal women enrolled in a
periodontal maintenance program. Osteoporotic/
osteopenic women exhibited a higher frequency of
alveolar bone height loss and crestal and subcrestal
density loss relative to women with normal BMD.
Estrogen deciency was associated with increased
frequency of alveolar bone crestal density loss in
107
Osteoporosis and periodontal disease progression
the osteoporotic/osteopenic women. The authors
concluded that osteoporosis/osteopenia and estro-
gen deciency are risk factors for alveolar bone den-
sity loss in postmenopausal women with a history of
periodontitis.
Fifty-nine moderate/advanced adult periodontitis
patients and 16 non-periodontitis subjects, all within
5 years after menopause at baseline, were stratied
based on serum estradiol levels. Attachment loss was
assessed over a 2-year period and correlated to BMD
and serum estradiol levels. Serum estradiol levels did
not inuence the percentage of sites losing attach-
ment for either periodontitis or non-periodontitis
groups. The estradiol-decient group had a trend
toward a higher frequency of sites with attachment
loss 2 mm.
Larger prospective longitudinal studies are needed
to further evaluate osteoporosis as a risk factor for
periodontal disease progression. The oral ancillary
study of the Womens Health Initiative at the
University of Alabama at Birmingham was designed
to determine if there is an association between sys-
temic osteoporosis and oral bone loss. In this report,
preliminary prospective longitudinal data will be
presented. The Womens Health Initiative is a study
of womens health after menopause in the United
States. Risk factors for diseases in this population
are being studied nationwide and include heart dis-
ease and osteoporosis. Utilizing the unique opportu-
nity for collaboration with the Womens Health
Initiative at the University of Alabama at Birming-
ham, an oral ancillary study was established.
All subjects enrolled in the study were post-meno-
pausal females. Hipbone mineral density was con-
rmed with DXA. Comprehensive medical histories
and examinations were linked with the results of oral
Table 2. Relationship of periodontal destruction and bone mineral density (BMD)
Authors Population Major result Type of study
Lundstrom et al. (17) 15 women with
osteoporosis, 21 women
with normal BMD
No statistically significant
differences in gingival bleeding,
probing pocket depths, gingival
recession and marginal bone level
Cross-sectional
study
Tezal et al. (26) 70 postmenopausal Caucasian
women aged 5178
Mean ABL was significantly
correlated with BMD.
Cross-sectional
study
Weyant et al. (28) 292 dentate women (average
age 75.5 years)
No statistically significant association
between periodontal disease and
systemic BMD.
Cross-sectional
study
Payne (20) Female periodontal maintenance
patients within 5 years of
menopause; 21 with normal
BMD, 17 osteoporotic women
Greater ABL, crestal and
subcrestal density loss in the
osteoporotic and estrogen-deficient
women.
2-year longitudinal
clinical study
Reinhardt et al. (21) Women within 5 years of
menopause, 59 with adult
periodontitis and 16
non-periodontitis. Stratified
by serum estradiol levels
In non-smoking osteopenic/
osteoporotic periodontitis patients
with estrogen deficiency had
more bleeding on probing and
clinical attachment levels
2-year prospective
longitudinal study
Hildebolt et al. (3) 135 postmenopausal women
aged 4170 years, no moderate,
severe periodontitis
Attachment loss was correlated with
tooth loss but not with BMD.
Cross-sectional
study
Streckfus et al. (24) 28 healthy women
aged 2378
More ABL, more missing teeth, in
postmenopausal women on
estrogen therapy than
premenopausal women.
Cross-sectional
study
von Wowern
et al. (27)
12 women with osteoporotic
fractures
Osteoporotic subjects had more
loss of attachment than normal
subjects
Cross-sectional
study
Elders et al. (1) 216 females between 46
and 55 years
No significant correlation was
observed between probing depth,
bleeding on probing, missing
teeth, alveolar bone height and
bone mass
Cross-sectional
study
108
Geurs et al.
examinations and quantitative digital intraoral radio-
graphy. The intraoral techniques used in this study
have been validated and are over 90% sensitive and
specic in detecting small changes in bone mass and
density (2, 7). Standardized vertical bitewing radio-
graphs were taken at baseline and the 3-year follow-
up visit. The radiographs were digitized and cor-
rected for small angulation errors and contrast. Sub-
traction radiography was used for the enhancement
of the standardized radiographs. Alveolar bone
height was measured using Periovision software.
Measurements were made on the mesial and distal
aspects of posterior teeth. Alveolar bone height was
dened as the measurement from the cemento-
enamel junction to the point of bony attachment to
the root of teeth. The patients were recalled and a
similar examination including the radiographic sur-
veys was performed every 3 years.
The amount of ABL along the root surface over the
3-year period was calculated for 58 subjects using
digital subtraction radiography. The subjects were
divided into two groups based on BMD at the hip
measured at baseline. The osteoporosis group was
dened as hipbone mineral density 2.5 SD below
the normal as conrmed by DXA. Subjects with
BMD above this level were considered the non-
osteoporosis group. The subjects were also stratied
based on ABL as a measure of the periodontal disease
status at baseline. A subject was considered to have
periodontitis when 3 mm or greater of alveolar bone
height was measured at baseline.
Figure 1 and Table 3 represent the 3-year long-
itudinal ABL data for the subjects based on perio-
dontal and osteoporosis status. Subjects with
osteoporosis presented with greater progression of
ABL than subjects with no osteoporosis over the 3-
year period. The subjects with periodontal disease at
baseline exhibited greater amounts of ABL than sub-
jects with periodontal disease. The greatest amount
of ABL was found in the group of subjects with
periodontal disease and osteoporosis. A general lin-
ear model was constructed for the outcome variable
change in logarithmic alveolar bone height. Indepen-
dent variables included smoking, age, current hor-
mone replacement therapy, calcium intake, and
ethnicity (P < 0.0008). In the post hoc comparison
of subjects without periodontitis at baseline, the sub-
jects with osteoporosis had greater mean ABL (0.18
0.21 mm versus 0.66 62 mm; P < 0.02). This was
statistically signicant. When periodontitis was pre-
sent at baseline, the difference in mean ABL between
the osteoporosis and non-osteoporosis groups was
even greater. The mean ABL for patients with period-
ontitis and osteoporosis was 1.08 0.46 mm com-
pared with 0.31 0.20 mm in the non-osteoporosis
group. This was statistically signicant (P < 0.01).
These data present a preliminary report of this
ongoing study and indicate a greater propensity to
lose alveolar bone in subjects with osteoporosis espe-
cially in subjects with preexisting periodontitis. This
would indicate that osteoporosis or low systemic
BMD should be considered a risk factor for period-
ontal disease progression. In the future we will report
the data of the ongoing study for the complete study
population and duration.
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Geurs et al.
Occlusal forces as a risk factor for
periodontal disease
Stephen K. Harrel
The relationship between occlusal forces and the
initiation or progression of periodontal disease has
been controversial for over a century. Early in the
20th century, excessive occlusal forces were consid-
ered to be a major causative factor of periodontal
destruction. In 1901, Karolyi (28) indicated that there
appeared to be a correlation between excessive
occlusal forces and periodontal destruction. In
1917 and 1926, Stillman (56, 57) stated that excessive
occlusal forces were the primary cause of periodontal
disease and that occlusal therapy was mandatory for
the control of periodontal disease. Stillman further
advocated the use of occlusal adjustment to prevent
periodontal destruction. This set the stage for a con-
troversy that continues to this day: What roles, if any,
do excessive occlusal forces play in the initiation and
progression of periodontal disease?
Historical perspective
As noted above, several early authors indicated that
occlusal forces played a signicant role in the initia-
tion and progression of periodontal destruction.
However, these statements were based on clinical
observations rather that scientic evaluation. In the
20 years following Stillman's statement that exces-
sive occlusal forces were the primary cause of period-
ontal destruction, several animal studies on sheep
and monkeys were carried out in an attempt to elu-
cidate the response of the periodontium to occlusal
forces (1, 58). The results of these studies were inter-
preted to show that excessive occlusal forces were at
least a contributing factor in the progression of per-
iodontal disease. At the end of the 1930s it was still
felt that excessive occlusal forces were a major cause
of periodontal disease, that occlusal adjustment
should be part of periodontal treatment, and that
occlusal discrepancies should be prophylactically
treated to prevent periodontal disease (2, 3, 35).
Orban & Weinman, in 1933, used the histologic
observation of human autopsy material as a metho-
dology for the evaluation of the effect of excessive
occlusal forces on the periodontium (40, 66). They
concluded that occlusal forces did not have a major
effect on periodontal destruction and felt that there
was no indication that occlusal forces played a part
in periodontal destruction. Instead, they interpreted
this material as demonstrating that gingival inam-
mation extending into the supporting bone was the
cause of periodontal destruction.
During the 1950s and 1960s, further animal
research was performed. These studies used rats,
monkeys, and dogs to evaluate the effect of occlusal
forces on the periodontium (5, 9, 34, 43, 67). These
studies, by their use of untreated controls, controlled
occlusal forces, experimentally induced periodonti-
tis, and larger number of subjects, introduced a new
level of sophistication in the evaluation of the effect
of occlusal forces. None of these studies gave support
to the concept that excessive occlusal forces were a
primary causative agent of periodontal destruction
and many of the studies indicated that there was
little or no correlation between occlusal forces and
periodontal destruction.
During this period Glickman and co-workers per-
formed a series of animal model and human autopsy
studies which strongly inuenced the perception of
the relationship between excessive occlusal forces
and periodontal destruction. Animal studies where
a ``high'' occlusal contact was created by overcon-
touring a restoration were performed utilizing dogs
and monkeys (16, 22). These studies showed no evi-
dence that occlusal contacts initiated periodontal
destruction. However, in a study on Rhesus monkeys,
a phenomena that was described as an `àltered path-
way of destruction'' was noted on teeth where exces-
sive occlusal forces were present (19). This altered
pathway of destruction was described as a change in
the orientation of the periodontal and gingival bers
111
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PERIODONTOLOGY 2000
ISSN 0906-6713
which occurred in the presence of excessive occlusal
forces that allowed gingival inammation to extend
along the periodontal ligament. This altered pathway
of destruction was felt to result in vertical bony
defects where bone loss followed the periodontal
ligament. Another animal study (21) showed that
bifurcation areas were most prone to stress from
occlusal forces and it was postulated that bone loss
in the furcation area could be related to excessive
occlusal forces. From these studies and later works
by Glickman came the concept that vertical bony
defects and furcation defects were often associated
with excessive occlusal forces.
Glickman and coworkers (4) also performed a ser-
ies of studies using human autopsy material. One of
these studies looked at the histomorphology of
infrabony pockets. The authors felt that the histolo-
gic material showed evidence that occlusal forces
affected the attachment apparatus apical to the bony
defect. Later studies (17, 18) using several human
block sections were interpreted to show that occlusal
forces and gingival inammation were two separate
processes and that occlusal forces altered the envir-
onment in which gingival inammation affected the
bone. They felt that the occlusal forces changed the
alignment of the periodontal ligaments, allowing for
an altered pathway of destruction with the end result
being vertical bony defects. Because two separate
pathologic processes worked together to cause bone
loss, the process was termed a ``co-destructive'' effect.
Glickman and coworkers summarized their work
in a series of review articles (1215, 20). These articles
indicated that excessive occlusal forces (trauma from
occlusion) were a co-destructive force which in the
presence of gingival inammation could lead to ver-
tical osseous defects. Based on this, the use of occlu-
sal adjustment was advocated as part of the
treatment for existing periodontal disease. Because
no evidence existed that excessive occlusal forces
initiated periodontal disease, occlusal adjustment
to prevent periodontitis was not advocated.
Waerhaug (6264) performed studies that utilized a
large number of human autopsy specimens in which
he evaluated the relationship of the morphology of
the bone pocket with the plaque level and presence
or absence of excessive occlusal forces. He found that
the ``plaque front'' (i.e. the border of the subgingival
plaque) was always in very close approximation to
the epithelial attachment level, always resembled the
morphology of the bony defect, and that the relation-
ship of the plaque level between adjacent teeth
(either at the same of different apico-coronal levels)
would yield either horizontal or vertical interproxi-
mal bone loss. He also observed that excessive occlu-
sal forces bore no relationship to the underlying bony
defect and that vertical defects were found equally
around traumatized and non-traumatized teeth.
Waerhaug's conclusion was that bone loss was
always associated with the downgrowth of plaque
and there was no relationship between excessive
occlusal forces and vertical bone loss.
It should be pointed out that a common difculty
encountered in almost all of the occlusal studies that
used human autopsy material is a lack of knowledge
of the patient's occlusal relationship in life. While
certain inferences can be derived from wear patterns,
there is no assurance that the teeth actually occluded
in the assumed manner. Therefore, any statement,
based solely on autopsy material, that a particular
tooth was undergoing excessive occlusal forces at the
time of death can be questioned and any conclusions
about the effects of excessive occlusal forces on the
periodontal supporting structures can not be deni-
tive. A single study (55) evaluated the patient's occlu-
sal relationship prior to the removal of the jaws for
cancer therapy. Only a single bony defect was found
in the block sections and no obvious relationship
between occlusion and periodontal pocketing could
be determined from this limited material.
The results of two extensive animal studies were
published beginning in the 1970s. These studies were
aimed at evaluating the effect of plaque and exces-
sive occlusal forces in the animal models utilized.
Stringent scientic controls and design were used
in all of these studies. These studies were performed
separately by Polson and coworkers (27, 41, 42, 44,
45, 4751) and Lindhe and coworkers (68, 3032, 38,
39, 5961). Polson used squirrel monkeys and mesial-
distal compression forces comparable to orthodontic
forces, while Lindhe used beagle dogs and applied
buccal-lingual forces using a high occlusal contact
and a nger spring. Both groups looked at excessive
occlusal forces with and without plaque as well as
histologic healing which occurs when excessive
occlusal forces are applied for a period of time and
then removed. In the beagle dog study, surgical
defects were created and the effect of excessive
occlusal forces and plaque were evaluated.
The results of these studies were very similar
despite the differences in the animal model and
the manner of applying excessive occlusal forces.
Excessive occlusal forces not in the presence of pla-
que were found to cause loss of bone density and
mobility of the tooth but no evidence was found that
excessive occlusal forces alone would cause attach-
ment loss. When the excessive occlusal forces were
Harrel
112
removed it was found that the loss of bone density
was reversible. In the presence of plaque, inamma-
tion of the gingiva and periodontal supporting struc-
tures were noted and in the presence of excessive
occlusal forces and plaque together there was an
indication that more bone density was lost in both
animal models. In the beagle dog model there was
evidence of attachment loss when plaque and exces-
sive occlusal forces were both present. This was not
found in the squirrel monkey model.
These two series of studies were exhaustive in their
evaluation of the relationship of occlusal forces and
plaque in an animal model. Both studies concluded
that there was no evidence that excessive occlusal
forces alone caused loss of attachment. The studies
by Lindhe showed that in special circumstances that
there may be attachment loss when plaque and
excessive occlusal forces were both present. Both
studies agreed that the control of plaque and gingival
inammation would stop the progress of periodontal
disease in the presence or absence of excessive
occlusal forces. These studies helped to establish that
bacterial plaque is the initiating factor and the main
cause for progression of periodontal disease.
Human studies
There is a paucity of studies evaluating the effect of
occlusion in humans. This is due in part to ethical
difculties related to the non-treatment of diagnosed
periodontal disease. In order to perform a controlled
clinical trial, the gold standard of clinical research, it
is necessary to compare treatment methods. How-
ever, in evaluating the combined effects of excessive
occlusal forces and periodontal disease, it would be
necessary to treat one group of patients and leave the
other group untreated. This approach would be ethi-
cally unacceptable due to the known deleterious
effects of the non-treatment of periodontal disease.
The World Workshop in Periodontics stated ``Pro-
spective studies on the effect of occlusal forces on
the progression of periodontitis are not ethically
acceptable in humans'' (11). As a result, most occlu-
sal studies in humans have been descriptive and/or
retrospective in nature.
It has been reported that patients who have occlu-
sal discrepancies have no more severe periodontal
destruction than do patients without occlusal discre-
pancies (26, 29, 42, 5254). However, it has also been
reported that molars with furcation invasion and
mobility have greater pocket depths than molars that
are not mobile (65). The increased mobility noted in
this study may have been due to occlusal factors or to
greater loss of bony support due to the furcation
involvement. Due to the inability to determine
whether occlusal factors or bone loss was rst pre-
sent, from this study, it is impossible to draw a clear
relationship between occlusion discrepancies, mobi-
lity, and pocket depths. Other studies reported that
patients who received occlusal adjustment as part of
their periodontal therapy had greater attachment
gain than patients who did not receive occlusal
adjustment (3, 10). This study may be an indication
that occlusal adjustment should be performed,
where indicated, as a part of periodontal treatment.
A series of reports on risk factors for periodontal
destruction indicated that mobility and parafunc-
tional habits that are not treated with a biteguard
are associated with increased attachment loss, wor-
sening prognosis, and tooth loss (36). These studies
seems to indicate that untreated (i.e. no biteguard)
parafunctional habits may contribute to increased
periodontal breakdown.
A recent retrospective study evaluated patients
from a private practice who were diagnosed with
advanced periodontal disease and had a comprehen-
sive treatment plan recommended that included
occlusal adjustment if occlusal discrepancies were
detected. Some of these patients self-selected to
not have any periodontal treatment performed
(untreated group). Other patients had only non-sur-
gical periodontal treatment performed (partially
treated group). Others followed through with all
recommended periodontal treatment including sur-
gery (fully treated group). The effect of occlusal dis-
crepancies was studied in each of these groups using
the individual tooth as the experimental unit (24, 25,
37). This means that the progression of periodontal
destruction or the improvement of the periodontium
for each tooth was followed over time. This study
design allowed for the evaluation of teeth with occlu-
sal discrepancies versus teeth without occlusal dis-
crepancies rather than comparing patients with
occlusal discrepancies vs. patients without occlusal
discrepancies. This experimental approach differs
from most past studies where the patient was the
experimental unit and the changes in pocket depth
or attachment levels were expressed as the ``patient
mean''. Using the patient mean may tend to mask
changes that are occurring at the more active sites
and, thereby, may give results that do not reect
what is actually occurring during localized disease
progression.
These studies found that teeth with occlusal dis-
crepancies had deeper presenting pocket depths and
113
Occlusal forces and periodontal disease
a worse prognosis than those teeth that did not have
occlusal discrepancies. Further, when teeth with
occlusal discrepancies were followed over time, a
signicant increase in pocket depth and a worsening
of prognosis was noted when compared to teeth
without occlusal discrepancies. Additionally, teeth
in the partially treated group that had received occlu-
sal adjustment showed a slowing of the progression
of periodontal destruction when compared to teeth
with occlusal discrepancies that had not had occlusal
adjustment. It was concluded that occlusal discre-
pancies appear to be a signicant risk factor that
contributes to more rapid periodontal destruction
and that treatment of occlusal discrepancies seemed
to slow periodontal destruction. The authors postu-
lated that the reason for the difference in their nd-
ings and those of previous studies was the use of
the individual tooth as the experimental unit, which
they felt yielded a more accurate assessment of
the effect of occlusal discrepancies on the perio-
dontium.
Summary of literature review
Most early research on the effects of occlusion on the
progression of periodontal disease focused on a
cause and effect relationship. Stillman clearly felt
that excessive occlusal forces were the cause of per-
iodontal disease and that treatment of the occlusion
was the primary method of effective periodontal
treatment. As it became evident that bacterial plaque
was an integral part of the periodontal disease pro-
cess, the role of occlusal forces became less clear.
Eventually this led to viewing occlusion as a cause
of specic types of periodontal destruction. This was
described by Glickman as the co-destructive roles of
occlusion and bacterial plaque in the formation of
vertical osseous defects and furcation bone loss.
Glickman's theory of Co-Destruction continued to
hold to the thesis that occlusion was, in concert with
bacterial plaque, a causative factor in periodontal
attachment loss and bony destruction. Glickman
described an altered pathway of destruction in an
attempt to articulate a functional mechanism for
the formation of the specic morphology of attach-
ment and bone loss thought to be caused by the co-
destructive action of occlusal forces and bacterial
plaque. The altered pathway of destruction still held
to the concept that occlusion directly changed the
disease process and was thereby, in the presence of
bacterial plaque, a causative agent for periodontal
destruction.
The animal studies on squirrel monkeys and bea-
gle dogs began to shed light on the effect of occlusal
forces on the periodontal attachment structures at a
cellular level. From these studies it was clear that
within these animal models, occlusion had an effect
on the periodontium in the form of bone rarefaction,
which resulted in the clinical manifestation of mobi-
lity. However, it was equally clear that, within the
animal models, loss of attachment and thereby per-
iodontal destruction did not occur in the presence of
excessive occlusal forces only. Loss of attachment
occurred only in the beagle dog model and then only
in the presence of excessive occlusal forces and bac-
terial plaque. While these animal studies gave us an
exhaustive insight into the effect of excessive occlusal
forces on the periodontal supporting structures of
the studied animals, it must be remembered that
these studies were performed using animal models
that show little or no tendency toward periodontal
destruction under natural conditions. The applica-
tion of the information obtained from these animal
models to the periodontal destruction that occurs in
humans must be approached with caution. It is prob-
able that these animal studies give us a picture only
of the physiologic response of the periodontium to
excessive occlusal forces with and without bacterial
plaque. It is unlikely that these animal studies give us
signicant information about the pathophysiology
that may occur when excessive occlusal forces are
present in humans who may be genetically prone to
periodontal destruction and who may also have addi-
tional risk factors for periodontal disease beyond
occlusal forces and bacterial plaque.
Human studies begin to give us some indication of
the effect of excessive occlusal forces on the progres-
sion of periodontal disease in those patients who
show a tendency toward periodontal destruction.
While there are many apparently contradictory nd-
ings from human studies, there appears to be a trend
toward evidence that excessive occlusal forces may
play a role in periodontal destruction and the
response of the periodontium to periodontal treat-
ment. While the available information suggests a
relationship between excessive occlusal forces and
progression of periodontal disease, the 1999 Interna-
tional Workshop for Classication of Diseases and
Conditions indicated that there was no clear evi-
dence that occlusal forces were a factor in plaque-
induced gingivial disease or connective tissue loss
(23). Since the 1999 Workshop, studies have shown
that occlusal interferences have a negative effect on
the periodontium and tend to cause more rapid
pocket formation and poorer prognosis when
114
Harrel
compared to teeth that do not have occlusal inter-
ference.
Occlusal interferences as a risk
factor
Recent human research seems to show a relationship
between occlusal discrepancies and periodontal
destruction (24, 25, 37). If the relationship found in
these studies does exist, then excessive occlusal
forces may need to be classied as a risk factor for
periodontal destruction. Much of the confusion that
exists about the role of occlusal forces in periodontal
disease is based on over 100 years of research and
controversy that has focused, in one way or another,
on a causative role for occlusal forces in periodontal
disease. The concept of risk factors for periodontal
disease, as opposed to causative factors, is a rela-
tively recent occurrence. When the body of informa-
tion on occlusal forces in periodontal disease is
reviewed in terms of occlusal forces as a potential
risk factor instead of a causative or co-destructive
agent, many of the contradictory results of past
research may be more reconcilable. A large body of
research exists which indicates that smoking is a
signicant risk factor for periodontal destruction,
that there is a much higher incidence of periodontal
destruction in smokers vs. non-smokers, and that
smokers do not respond as well to periodontal treat-
ment when compared to non-smokers. Despite the
overwhelmingly strong link between smoking and
periodontal destruction, smoking is not viewed as a
causative or co-destructive agent for periodontal dis-
ease. Instead, smoking is viewed as a cumulative risk
factor for periodontal destruction. Existing research
makes a case for viewing occlusion from a similar
prospective.
It is universally believed that bacterial plaque is the
causative agent for periodontal destruction (33). A
risk factor for periodontal disease appears to be an
environmental or host factor that predisposes a
patient to periodontal breakdown from bacterial pla-
que. The exact mechanism by which each individual
risk factor affects this predisposition is unknown.
The effect of the risk factor may be to alter the host
response or change the bacterial environment or
effect. It may be that some form of localized altera-
tion in the host environment might be the mechan-
ism by which excessive occlusal forces interact with
bacterial plaque. It is also possible that excessive
occlusal forces create an environment whereby the
deleterious effects of bacterial plaque are enhanced,
or there may be a completely different mechanism
that has not been noted in the past. Under any cir-
cumstances, recent human research indicates that
treatment of the occlusion to minimize occlusal
interferences may, in concert with other forms of
periodontal treatment, positively affect the progress
and treatment of periodontal destruction. The risk
factor of excessive occlusal forces is one that can
be minimized with existing clinical armamentarium,
and, as is the case for all risk factors that can be
ameliorated by treatment, treatment of excessive
occlusal forces to minimize the effect of this risk
factor may need to be a part of routine periodontal
treatment.
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117
Understanding the etiology of
periodontitis: an overview of
periodontal risk factors
Martha E. Nunn
Periodontitis is a multi-factorial disease with micro-
bial dental plaque as the initiator of periodontal dis-
ease (50). However, the manifestation and progression
of periodontitis is inuenced by a wide variety of
determinants and factors, including subject charac-
teristics, social and behavioral factors, systemic fac-
tors, genetic factors, tooth-level factors, microbial
composition of dental plaque, and other emerging risk
factors. With the large array of factors that inuence
the development and progression of periodontitis,
understanding what the relationships of these various
factors and determinants are to the initiation and
progression of periodontal disease can be daunting.
The purpose of this paper is to explain some basic
concepts related to understanding periodontal risk
factors and the strength of the association of those
risk factors to the initiation and progression of period-
ontal disease and to present a brief overview of risk
factors for chronic periodontitis. Terminology, study
design, outcome measures, and measures of associa-
tion are presented as basic concepts necessary for
understanding periodontal risk factors. The overview
of risk factors is then systematically divided into sub-
ject determinants, social and behavioral factors, sys-
temic factors, genetic factors, tooth-level factors,
microbial factors, and emerging risk factors.
Terminology
In most epidemiological studies, the researcher seeks
to establish some sort of causal relationship between
a characteristic, behavior, or exposure and the man-
ifestation of a particular disease. Three types of cau-
sation are generally identied: 1) a sufcient cause,
2) a necessary cause, and 3) a risk factor. A sufcient
cause refers to any condition, characteristic, or expo-
sure in the presence of which, the disease will always
occur. This is the strongest type of causal relation-
ship and is relatively rare. Examples of this include
genetic anomalies or conditions. A necessary cause is
any condition, characteristic, or exposure that must
be present for a disease to manifest itself. It differs
from a sufcient cause because its presence does not
necessarily result in manifestation of the disease. A
good example of this is the organism Mycobacterium
tuberculosis, which is a prerequisite for a person to
develop tuberculosis. However, many people can
carry this organism in their bodies without any
symptoms of disease whatsoever. A risk factor is
any characteristic, behavior, or exposure with an
association to a particular disease. The relationship
is not necessarily causal in nature (16). Risk indicator
is a term used to describe a potential risk factor
identied to be associated with disease from case-
control or cross-sectional studies. Risk factor is more
appropriately reserved for those factors that have
been veried to be associated with disease through
longitudinal studies. A risk factor that can be used to
predict the future course of disease, such as an
increased probability of disease, is known as a risk
marker. Some risk factors can be modied to reduce
one's risk of initiation or progression of disease, such
as smoking cessation or improved oral hygiene to
reduce the risk of periodontal destruction, while
other factors cannot be modied, such as genetic
factors. A risk factor that cannot be modied is often
referred to as a determinant (30).
The terminology associated with risk indicators is
indicative of the level of evidence for its association
to periodontal disease. The distinctions among these
terms are not always clear from the literature, nor is it
11
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PERIODONTOLOGY 2000
ISSN 0906-6713
clear how the clinician makes use of such informa-
tion. Whatever terms are used to designate factors
associated with the initiation and progression of per-
iodontal disease, it is the hierarchy of study design
and the relevance of the outcome measure that gov-
ern the strength of the evidence for each risk indi-
cator and its use in clinical decision-making.
In addition to the concept of risk indicators, there
are also terms that refer to the proportion of disease
noted in a particular population. Prevalence is dened
as the number of cases of a disease or condition per
population at risk at a particular point in time. Cross-
sectional studies are used to assess the prevalence of
a particular disease at some point in time for a spe-
cied population. Another measure of disease fre-
quency is incidence. Incidence is dened as the
number of new cases of a disease or condition over
a specied period of time. Longitudinal studies are
used to assess the incidence of a particular disease for
a specied population over a given time period (16).
Study design
The strength of evidence of an association of a risk
factor to any particular disease is based in large part
on the study design utilized. The hierarchy of study
design forms a basis for the relative strength of evi-
dence for any risk factor. The weakest evidence for an
association between a particular factor and the dis-
ease under investigation is the case report or case
history. Although such information may form the
basis for future hypotheses and further study, infor-
mation gained from a case report without further
verication should be given the least weight when
formulating a treatment plan for a patient. The next
type of study in the hierarchy of design is the case
series. This type of study provides only slightly stron-
ger evidence for an association between a risk factor
and a disease than a case report. How much value
one should place upon a case series is related to the
way in which the cases were ascertained and the
number of cases involved. For instance, information
obtained from a series of 10 cases that were hand-
picked by the investigator out of a much larger avail-
able group is far less reliable than a series of 100
cases chosen at random or constituting a conveni-
ence sample. Even in the latter example of a large
case series, the strength of the evidence for an asso-
ciation between a risk factor to a particular disease is
considered quite weak.
The next strongest evidence is provided by case-
control studies. These studies may be retrospective
or prospective in nature. A case-control study is an
outcome-based study. Subjects are classied with
respect to disease status. The investigator takes a
sample of individuals who have the disease in ques-
tion (cases) and a sample of individuals who are also
at risk for the disease but are disease-free (controls).
Subject characteristics and/or the subjects' prior
exposure history to potential risk factors are then
investigated as a function of disease status. Compar-
isons are then made of the relative distributions of
these characteristics or history of exposures among
cases and controls. A subject characteristic or history
of exposure would then be considered to be a risk
indicator if a signicantly greater proportion of cases
possessed that characteristic or had a history of that
exposure in question compared to healthy controls.
The major disadvantages to this type of study include
the inability to completely control for confounding
and the lack of control over the exposures under
investigation. Confounding is the confusion of two
supposedly causal factors so that part or all of the
purported effect of one of these factors is actually
due to the other (16). In addition to these disadvan-
tages, the vast majority of case-control studies are
retrospective in nature with the inherent biases asso-
ciated with a retrospective study:
hypothesis is formulated after events have occurr-
ed.
incomplete and/or poor record keeping, espe-
cially when incomplete records comprise missing
data that are not missing totally at random.
The next strongest form of evidence is the result of
a population-based cross-sectional study. These stu-
dies generally are more powerful than case-control
studies because they involve large populations with
data collected on a multitude of variables so that a
more rigorous assessment of potential risk factors is
possible, with the ability to more completely adjust
for confounding as well as assessing possible effect
modication. Effect modication occurs when a third
variable affects the direction of the association or the
strength of the association between a risk factor and
the disease under investigation (16). Another advan-
tage to a large population-based cross-sectional
study is that data are often collected to study a wide
variety of phenomena so that the bias of formulating
an hypothesis after events have occurred is mini-
mized.
The strongest form of evidence provided by an
epidemiologic study comes from a cohort study,
which is longitudinal in nature. A cohort study is
an exposure-based study design. In this type of study,
subjects are classied with respect to exposure and
Nunn
12
followed longitudinally. The rate of occurrence of the
disease under investigation is studied as a function of
the exposure. The bias of formulating a hypothesis
after events have occurred is eliminated, and poor
record keeping can generally be minimized with
cohort studies. However, subjects may be lost to
follow-up in a longitudinal study, and if the level of
attrition becomes too high, the inference drawn from
the study is seriously compromised.
At the pinnacle of the hierarchy of study design is
the randomized controlled clinical trial. In the case of
risk factors, such a trial is referred to as an interven-
tion trial. In a randomized controlled clinical trial,
subjects are randomized to either a treatment group
or a control group. In the context of an intervention
trial, the hypothesized risk factor is eliminated from
the treatment group while the control group is fol-
lowed without any intervention to eliminate the
hypothesized risk factor. This type of study offers
the strongest evidence of a potential causal relation-
ship between a risk factor and the disease under
investigation.
Outcome measures
The relevance of the outcome measure is also a factor
in assessing the strength of evidence for an associa-
tion between a risk factor and disease. With respect
to periodontal disease, the goal of prevention and
treatment is to maintain teeth in a state of comfort
and function. Hence, the most accurate measure of
the inuence of a risk factor in the progression of
periodontal disease is tooth loss as a direct or indir-
ect result of periodontal disease. Unfortunately, the
number of subjects and the time involved to accu-
rately assess the relationship of potential risk factors
to actual tooth loss from periodontal disease makes it
extremely difcult, if not impossible, to dene all risk
factors for periodontal disease in terms of tooth loss.
In addition, in large longitudinal studies where tooth
loss is known, such as the VA Dental Longitudinal
Study, the actual reason for tooth loss is often not
known. Because of this problem, other clinical mea-
sures are often used to assess the periodontal status
of a tooth at any given point in time.
Two outcome measures that are often used in
place of tooth loss from periodontal disease are
alveolar bone level and clinical attachment level.
Both of these measures are strongly associated with
tooth loss from periodontal disease. Both alveolar
bone level and clinical attachment level are quanti-
tative measures, although these outcome measures
are often reclassied into categorical variables for
evaluating the relationship of a potential risk factor
to these outcomes. Alveolar bone levels can also be
quantied in terms of percentage of alveolar bone
loss based on the ratio of the distance from the height
of the alveolar crest to the cemento-enamel junction
to the length of the root. The percentage alveolar
bone loss determined from these measurements
can then be reclassied into categories of periodon-
tal destruction (mild, moderate, severe). Percentage
bone loss is also often dichotomized into ``signicant
bone loss'', for bone loss above a specied percen-
tage of alveolar bone loss, and ``not signicant bone
loss'', where bone loss falls below this percentage of
alveolar bone loss. Clinical attachment level can
similarly be reclassied categories of periodontal
destruction, or dichotomized into teeth with ``signi-
cant attachment loss'' and teeth with ``no signicant
attachment loss.
In addition to the measures of alveolar bone level
and clinical attachment level described above,
changes in these measures can also be used as out-
come measures when a longitudinal study is con-
ducted. In the case of alveolar bone loss, digitized
subtraction radiography (35) is often employed. With
respect to clinical attachment level in a longitudinal
study, clinical attachment loss over the period of the
study can be found by taking the difference in clinical
attachment levels from the initial measurement to
the nal measurement. In longitudinal studies, these
outcome measures can also be reclassied into cate-
gories. Bothclinical attachment loss and alveolar bone
loss are subject to measurement error. In addition,
alveolar bone loss is not always possible to assess for
each tooth because malposed, overlapping teeth,
cervical caries, and extensive dental restorations
can either obscure the crest of alveolar bone or make
it difcult to locate the cemento-enamel junction.
Other outcome measures gathered on a tooth level
that are used to assess periodontal disease status
include periodontal probing depth, gingival reces-
sion, gingival inammation index, bleeding index,
and furcation involvement. Periodontal probing
depth and gingival recession are quantitative mea-
sures. Gingival inammation index, bleeding index,
and furcation involvement are ordinal measures.
Since periodontal probing depth and gingival reces-
sion are the component measures that constitute
clinical attachment level, these are the next best
tooth-level clinical measures of periodontal disease
destruction. Since gingival inammation index
and bleeding index are both indicators of inamma-
tion but are less strongly associated with actual
13
Understanding the etiology of periodontitis: an overview of periodontal risk factors
periodontal breakdown, these two indices are less
compelling measures of periodontal destruction.
Furcation involvement clearly reects loss of clinical
attachment and alveolar bone loss, but this outcome
measure is limited to molar teeth, so that a risk factor
identied with an increased risk of furcation involve-
ment is limited to inferences of molar teeth.
Tooth-level clinical outcome measures can also be
summarized for each subject to obtain other out-
come measures for assessing periodontal destruc-
tion. For instance, tooth-level alveolar bone loss
can be summarized as the percent of teeth (or sites)
with ``signicant alveolar bone loss'' as dened by
some dichotomized measure of alveolar bone loss.
Similarly, tooth-level clinical attachment loss can be
summarized as the percent of teeth (or sites) with
clinical attachment loss exceeding a certain value,
such as the percent of teeth with clinical attachment
loss greater than 4 mm. The percentage of teeth with
signicant alveolar bone loss can also be used to
classify each subject's level of periodontal disease
(i.e. mild, moderate, severe). Similarly, percentage
of teeth with signicant clinical attachment loss
can also be used to categorize subjects into different
levels of periodontal destruction (i.e. mild, moderate,
severe). When subjects are classied into those with
periodontitis and those without periodontitis, vary-
ing denitions may be employed to dene the sub-
ject's status, but all denitions of periodontitis will
include
a minimum number of teeth with signicant per-
iodontal destruction as measured by alveolar
bone loss or clinical attachment loss,
a percentage of teeth with signicant periodontal
destruction or
some combination of missing teeth and number
of remaining teeth with signicant periodontal
breakdown.
In general, the strongest outcome measure of per-
iodontal disease is that measure that most closely
reects the eventual negative outcome of tooth loss
from periodontal disease and that can be assessed
objectively with the least measurement error.
Measures of association
The strongest association for identifying a parameter
as a risk factor for disease is relative risk. Relative risk
is generally dened as the ratio of the risk of disease
in the `èxposed'' group to the risk of disease in the
`ùnexposed'' group (16). Relative risk is a measure of
association that is only obtained from a longitudinal
study. In the context of periodontitis, the strongest
association for identifying a parameter as a period-
ontal risk factor is the relative risk of tooth loss. Time
until tooth loss (or time until last observation, if tooth
is not lost) is the outcome measure used to estimate
relative risk. Because relative risk is a ratio, a relative
risk of ``1'' means that the presence of the factor
under investigation (exposed) is no more likely to
result in tooth loss from periodontal disease than
when the factor is not present (not exposed). When
the relative risk is less than 1, this means that the
exposure actually reduces the likelihood of tooth loss
from periodontal disease. For instance, a relative risk
of 0.8 for an exposure would mean that a tooth with
that exposure was only 80% as likely to be lost
because of periodontal disease compared to a tooth
without the exposure. When the relative risk is
greater than 1, an exposed tooth is more likely to
be lost from periodontal disease compared to an
unexposed tooth. In other words, for a relative risk
of 2.2, an exposed tooth is 2.2 times as likely to be lost
because of periodontal disease compared to an unex-
posed tooth. The estimate for relative risk alone does
not completely explain any potential association of a
particular exposure to tooth loss. The 95% con-
dence interval for the relative risk provides the sta-
tistical evidence for an increased or decreased risk for
tooth loss as a result of exposure to a particular
factor. If the 95% condence interval for the relative
risk for any factor contains ``1'', there is a lack of
statistical evidence that exposure to the factor under
investigation will either increase or decrease the like-
lihood of tooth loss from periodontal disease. When
the value of ``1'' is not within the 95% condence
interval for the relative risk for a factor, this is equiva-
lent to rejecting the null hypothesis and concluding
that the factor under investigation will either
increase the likelihood of tooth loss from periodontal
disease (when the estimated relative risk is greater
than 1) or decrease the likelihood of tooth loss from
periodontal disease (when the estimated relative risk
is less than 1). For a factor with a statistically signi-
cant relative risk, the greater the value of the relative
risk, the greater is the negative impact by the risk
factor on the periodontium as measured by tooth
loss.
A measure of association obtained from case-con-
trol studies is the odds ratio. The odds ratio is the
ratio of the odds of exposure in the diseased group to
the odds of exposure in the non-diseased group (16).
Because information about exposure is gathered
after the disease status has been ascertained, the
odds ratio is the only way to quantify the relative
14
Nunn
likelihood of disease based on exposure. This odds
ratio is mathematically equivalent to the ratio of the
odds of disease in the exposed group to the odds of
disease in the unexposed group. We make use of this
mathematical equivalence in interpreting an odds
ratio obtained from a case-control study. By making
use of this mathematical equivalence, the interpreta-
tion of the odds ratio is similar to that explained
above for relative risk. An odds ratio of ``1'' indicates
that the odds of disease in the exposed group is equal
to the odds of disease in the unexposed group. An
odds ratio greater than 1 implies that the odds of
disease in the exposed group is greater than the odds
of disease in the unexposed group. For instance, if
cases are dened as subjects with periodontitis and
controls are dened as subjects without periodonti-
tis, then an odds ratio of 3 for a particular exposure
would mean that the odds of periodontitis among
exposed subjects was three times that of the odds
of periodontitis among unexposed subjects. In other
words, an exposed subject is three times as likely to
have periodontitis compared to an unexposed sub-
ject. Similarly, if the odds ratio is less than 1, an
exposure reduces the subject's likelihood of period-
ontitis compared to unexposed subjects. As in rela-
tive risks, the 95% condence interval explains
whether the difference in likelihood of disease
between the exposed group and the unexposed
group has statistical support. If the value of ``1'' is
in the 95% condence interval, then the exposure has
no effect on the likelihood of experiencing disease.
When disease prevalence is relatively rare (<5%) , the
odds ratio can be used to approximate relative risk.
Hence, the interpretation that is often presented for
odds ratios obtained from a case-control study may
be identical to the interpretation for relative risks.
With the advent of methods for analyzing correlated
outcomes, such as the method of generalized esti-
mating equations, the concept of the odds ratio can
be extended to explaining the relationship of a risk
factor to the periodontal status of a tooth.
For continuous outcome measures, such as the
percentage of teeth with signicant clinical attach-
ment loss, correlation can be used to describe the
association. A correlation of zero implies no associa-
tion between the risk factor and the outcome mea-
sure used to describe periodontal disease. A
correlation greater than zero would mean that an
exposure is associated with an increased level of
periodontal disease as measured by percentage of
teeth with signicant periodontal destruction. A cor-
relation less than zero implies that an exposure is
associated with decreased evidence of periodontal
destruction. Further quantication of the relation-
ship between a risk factor and a continuous outcome
measure can be obtained from linear regression
modeling. An increase of one unit of exposure in a
linear model would have the effect of changing the
outcome measure by the value of the coefcient for
the risk factor under investigation. For instance, a
coefcient of 2 for age in years to describe the per-
cent of teeth with signicant periodontal destruction
would mean that for each additional year of age, the
percent of teeth with signicant periodontal destruc-
tion will increase by 2%. A statistically signicant
association between a risk factor and a continuous
outcome measure in a regression model will be
reected by a 95% condence interval for the corres-
ponding coefcient that does not include zero.
Subject determinants
Aging is commonly associated with periodontal dis-
ease, although this relationship is thought to be more
related to the cumulative periodontal breakdown
over time than to an age-related, intrinsic deciency
that contributes to susceptibility to periodontal dis-
ease (30). Epidemiological studies have shown more
periodontal disease, both in terms of attachment loss
as well as bone loss, among older age groups com-
pared to younger age groups (36, 37, 104). In addi-
tion, research has shown that certain physiological
changes in the periodontium occur with age (48,
113), although these changes alone are not respon-
sible for periodontal breakdown. In fact, in a study of
the rst National Health and Nutrition Examination
Survey, the effect of aging on periodontal destruction
appears to be negligible compared to the role of
plaque as represented by oral hygiene practices (1).
These results appear to also hold for the old elderly
(85 years old) as well. In a 5-year follow-up of
elderly patients in Finland, very little change in the
periodontal status was noted, with the authors con-
cluding that periodontal disease in the elderly who
are relatively healthy is not caused by the aging pro-
cess alone (3).
The role of race as a risk factor for periodontal
disease is a more complicated issue. For instance,
in a study of older community-dwelling blacks and
whites (65 years of age and above) , blacks were three
times more likely to exhibit advanced periodontal
destruction compared to whites in the same age
cohort. In addition, Prevotella intermedia was found
to be a risk factor for blacks but not for whites (8).
However, in this study, blacks had more indicators
15
related to socioeconomic status than whites. When
limited to subjects in the same socioeconomic status,
differences in periodontal status between blacks and
whites often disappeared (36, 37). Racial differences
in the distribution of certain genetic risk factors may
also contribute to differences in the prevalence and
severity of periodontal disease among difference
races. For instance, the prevalence of polymorphisms
of the composite interleukin (IL) -1a/IL-1b genotype
among Chinese is signicantly lower than among
Europeans (6).
Associations between gender and periodontal dis-
ease have been mixed. In one study after adjustment
for oral hygiene, socioeconomic status, and age,
males were found to have signicantly more clinical
attachment loss and bone loss (36, 37). Based on
studies of postmenopausal women, estrogen may
be part of the reason for gender differences noted
in periodontal disease. In a study comparing a group
of postmenopausal women receiving estrogen sup-
plements to postmenopausal women without estro-
gen supplementation, women receiving estrogen
exhibited signicantly less gingival bleeding com-
pared to women not on estrogen-replacement ther-
apy (85). Another study suggested that estrogen
supplementation may be associated with reduced
gingival inammation and a reduced frequency of
clinical attachment loss in osteopenic/osteoporotic
women in early menopause (93).
Social and behavioral factors
Cigarette smoking has long been recognized as a risk
factor for periodontal disease. One of the earliest
studies to show a relationship between smoking
and periodontal health was conducted on Swedish
army conscripts where smokers were shown to be at
an increased risk of gingivitis, although no differ-
ences were noted in bone loss or periodontal pock-
eting (89). In another study, the alveolar bone height
of smokers was shown to be signicantly reduced
compared to non-smokers (10). In addition, a case-
control study showed that smokers were 2.7 times as
likely to have moderate to advanced periodontal dis-
ease compared to non-smokers (38). Signicant
associations between cigarette smoking and both
clinical attachment loss and alveolar bone loss were
shown in a study of clinical risk indicators (36, 37). In
addition, cigarette smoking signicantly increases
the risk of tooth loss from periodontal disease (70).
The relationship of cigarette smoking to tooth loss
from periodontal disease also appears to be dose-
related, with heavy cigarette smokers exhibiting a
signicantly greater risk of tooth loss from period-
ontal disease compared to non-smokers and less-
than-heavy smokers (71). Cigar and pipe smoking
have also been shown to be signicantly related to
both tooth loss and alveolar bone loss (4, 49, 55).
Overall, smoking is probably the single most signi-
cant modiable risk factor for periodontal disease.
Socioeconomic status has been proposed as a risk
factor for periodontal disease. On a global level, an
early study suggested that nutritional deciencies in
developing countries could contribute to more
advanced periodontal disease (95). However, a study
comparing the periodontal condition of young men
in India with clinical symptoms of malnutrition to
the periodontal condition of well-nourished men
showed no difference in the periodontal condition
of the malnourished group compared to the well-
nourished group (91). Other similar studies failed
to demonstrate an association between periodontal
disease and nutrition (6, 94, 116).
Related to nutrition, research has been conducted
into possible associations of dietary calcium and
dietary Vitamin C to periodontal disease. In a study
of the Third National Health and Nutrition Examina-
tion Survey (NHANES III) , low calcium intake was
found to be associated with a signicant increased
risk of periodontal disease among young males,
young females, and older males (82). A randomized
trial of supplemental calcium and Vitamin D was
conducted to determine if these supplements would
slow bone loss from various skeletal sites among
subjects 65 years of age and older. Based on this trial,
supplemental calcium and Vitamin D, originally
aimed to reduce osteoporosis, was shown to also
signicantly reduce tooth loss (56). The relationship
of Vitamin C intake and periodontal disease was
explored in the Third National Health and Nutrition
Examination Survey (NHANES III). A weak, but sta-
tistically signicant association was found between
Vitamin C intake and periodontal disease, with that
association being a dose-response relationship. Low
Vitamin C intake among former and current smokers
showed an even higher increased risk of periodontal
disease (83).
Psychological factors have also been proposed as a
risk factor for periodontal disease. In one study,
stress related to nancial strain was found to be sig-
nicantly associated with greater loss of clinical
attachment and greater bone loss (31). In a case-
control study to investigate the relationship of peri-
odontitis to social factors, a signicant association
was found between elevated scores of social strain
16
Nunn
and periodontitis (80). Another case-control study
showed that the negative impact of life events, num-
ber of negative life events, and being unemployed
were all signicantly associated with periodontitis
(21).
Excessive alcohol consumption has been asso-
ciated with an increased risk of clinical attachment
loss as well as an increased risk of gingival bleeding.
However, no association between alcohol consump-
tion and alveolar bone loss has been found (110).
Systemic factors
One of the strongest systemic risk factors for period-
ontal disease is diabetes mellitus. There is strong
evidence of a direct relationship between diabetes
mellitus and periodontitis (51, 92). In addition, dia-
betes mellitus has been shown to be positively asso-
ciated with clinical attachment loss (30). However,
there are also studies that do not support this asso-
ciation of diabetes mellitus with periodontal disease
(102). Insulin and non-insulin dependent diabetics
appear to be at equal risk for periodontal disease. The
severity and extent of periodontitis in the diabetic
patient appears to be related to control of the dia-
betes (109). In addition to the risk progression of
periodontal disease posed by poorly controlled dia-
betes, it has also been suggested that effective peri-
odontal therapy can have a positive effect on the
control of diabetes (111). In a longitudinal study of
non-insulin dependent diabetics, severe periodontal
disease at baseline was found to be a signicant risk
factor for poor glycemic control (107). Similarly, poor
glycemic control in non-insulin dependent diabetics
has been shown to be associated with signicantly
greater alveolar bone loss over time compared to
well-controlled non-insulin-dependent diabetics
(108). A substantial body of evidence has begun to
emerge suggesting a bidirectional relationship
between both types of diabetes and periodontal dis-
ease (106).
Certain systemic conditions require the use of
drugs that may pose a risk for periodontitis from
plaque accumulation as a result of gingival over-
growth. Calcium channel blockers have been asso-
ciated with gingival overgrowth, although the risk for
gingival overgrowth varies according to the specic
drug. Gingival overgrowth is signicantly more pre-
valent among patients taking nifedipine compared to
patients taking either amlodipine or diltiazem,
although the overall prevalence among all patients
taking calcium channel blockers is still fairly low. The
extent and severity of the gingival overgrowth also
appears to be related to local plaque control as well
as genetic factors. Although calcium channel block-
ers have been associated with gingival overgrowth,
no association with periodontal disease has yet been
found (27). Gingival enlargement among patients
taking the anti-epileptic drug phenytoin is well-
documented. The greatest risk factor for phenytoin-
induced gingival overgrowth is plaque (67). The
immunosuppressant drug cyclosporine is a risk fac-
tor for gingival overgrowth which resembles pheny-
toin-induced gingival enlargement. Steroid therapy
has been linked to osteoporosis. However, studies of
the relationship of steroid therapy to alveolar bone
loss are conicting. In one study of long-term pre-
dnisone therapy, no signicant loss of alveolar bone
was noted (18). In another study, hydrocortisone
induced periodontal breakdown in a rat model (60).
Evidence has been mounting for an association
between osteoporosis and periodontal disease (54,
115). In addition, evidence has begun to emerge that
calcium supplements, Vitamin D supplements,
estrogen-replacement therapy, and other drugs used
to treat osteoporosis may also be benecial for redu-
cing alveolar bone loss and tooth loss (54).
Oral lesions manifested in HIV-infected patients
that have previously been noted include:
linear gingival erythema,
necrotizing ulcerative gingivitis (NUG),
severe localized periodontitis, and
severe necrotizing stomatitis which affects both
the gingival and bone (97, 118120).
Associations between periodontal disease and
other systemic diseases have also been reported,
although it is not clear what the role of these relation-
ships play. For instance, several studies have shown
an association between cardiovascular disease and
periodontal disease (9). However, a recent study that
made use of the First National Health and Nutrition
Examination Survey Epidemiologic Follow-up Study
failed to show a signicant association between car-
diovascular disease and periodontal disease (42). An
association between rheumatoid arthritis and peri-
odontitis has also been reported (75). Exactly what
these associations reect is still unknown. With the
discovery of emerging risk factors, inammatory
responses, and genetic regulators of these responses,
it is quite possible that these associations actually
reect common underlying risk factors rather than
any sort of causal relationship. In a recent study, a
relationship between upper body obesity, a risk fac-
tor for other systemic diseases including type 2 dia-
betes and cardiovascular disease, and periodontal
17
disease has been shown in a group of 643 healthy,
dentulous Japanese adults enrolled in the study (96).
This nding reinforces the need to develop a better
understanding of the true relationships of these var-
ious systemic conditions and the manifestation of
periodontal disease.
Genetic factors
In studies of both monozygotic and dizygotic twins
reared together and apart, a signicant genetic com-
ponent for gingivitis, probing depth, attachment loss,
and plaque was found (76, 77). In a recent study
involving both monozygotic and dizygotic twins, it
has been estimated that genetic inuences account
for as much half of the variance in disease in the
population (78).
Specic genotypes have been identied and linked
to periodontal destruction. Polymorphisms of IL-1,
IL-1b, and IL-1RN genotypes have been identied as
potential risk factors for periodontal destruction. In a
recent study that evaluated these polymorphisms
and smoking, it was found that being positive for
the composite IL-1a/IL-1b polymorphism in smokers
resulted in four times the risk of signicant attach-
ment loss (as measured by the percentage of sites
>4 mm attachment loss) compared to genotype-
negative smokers (73). Patients positive for the com-
posite IL-1a/IL-1b polymorphism have also been
shown to have a 2.7 times increased risk of tooth loss
from periodontal disease compared to genotype-
negative patients (71). In addition, interactions bet-
ween a positive IL-1 genotype and age, smoking, and
P. gingivalis have been identied, indicating that the
composite IL-1a/IL-1b polymorphism is a contribu-
tory but non-essential risk factor for periodontal dis-
ease progression (22).
Polymorphism of the tumor necrosis factor- (TNF-
a) gene has been suggested as a possible risk factor
for periodontitis. However, studies have failed to link
polymorphism of the TNF-a gene to either early-
onset periodontitis (98) or adult periodontitis (19).
Recently, a study of a group of middle-aged men
enrolled the VA Dental Longitudinal Study demon-
strated a signicant association between a poly-
morphism of the Vitamin D receptor genotype and
alveolar bone loss, clinical attachment loss, and tooth
loss (43). Studies have also identied an association
between polymorphisms of the Vitamin D receptor
genotype and early-onset periodontitis (40, 122).
Functional polymorphisms of immunoglobulin G
(IgG) Fc receptors (Fc g R) have been shown to be
associated with recurrence of chronic periodontitis
among Japanese patients (52). In another study of
Caucasians, the extent and severity of alveolar bone
loss was found to be signicantly associated with the
genotype of the receptor Fc g RIIIa. The study also
demonstrated an increased risk of severe bone
destruction among subjects carrying the Fc g RIIIa-
VV genotype. This study shows that the Fc g RIIIa
genotype coding for the high afnity receptor and the
Fc g RIIIb genotype coding for the low afnity recep-
tor impose additional risks for alveolar bone loss (72).
Polymorphism of the N-acetyltransferase (NAT 2)
has been shown to be signicantly associated with
more severe bone loss (53). In addition, smoking may
exacerbate the effect of NAT2 on the progression of
periodontal disease (74).
Tooth factors
Various aspects of tooth anatomy have been shown
to be associated with clinical manifestations of per-
iodontal disease. For instance, some studies have
shown an association between enamel projections
and furcation involvement among molars (11, 41,
68). Similarly, enamel pearls have been implicated
in molar furcation involvement (20, 32, 99, 114). It
has been hypothesized that the enamel in these loca-
tions prevents the attachment of connective tissue,
thus making the area more susceptible to periodontal
breakdown.
Tooth position can also present a risk for period-
ontal disease. Malalignment, crowding, and migra-
tion or tipping of a tooth distal to an edentulous area
have all been implicated in loss of periodontal sup-
port (2, 26, 28, 45, 46). In addition, extreme labial or
lingual positioning of a tooth has been correlated
with gingival recession (34, 63). Tooth malposition
poses an even stronger risk among individuals with
suboptimal oral hygiene. Areas with thin or absent
cortical alveolar bone and prominent teeth in the
dental arch are particularly prone to periodontal
breakdown.
Related to tooth position is the issue of occlusion.
A recent cross-sectional study evaluated the relation-
ship of occlusal discrepancies on a tooth level to
periodontal probing depth. Occlusal discrepancies
were found to be an even stronger predictor of per-
iodontal probing depth than smoking (86). In a long-
itudinal study to investigate occlusal treatment of
these occlusal discrepancies, it was shown that teeth
with treated occlusal discrepancies had signicantly
less of an increase in periodontal probing depth than
18
Nunn
teeth with untreated occlusal discrepancies, indicat-
ing that occlusal discrepancies constitute a modi-
able risk factor for periodontal disease (39).
Root proximity has been hypothesized to be a risk
factor for periodontal disease because bone and con-
nective tissue are reduced in these areas so that
inammation and periodontal destruction may be
enhanced. However, there is no scientic evidence
to support root proximity as a risk factor for initiation
and progression of periodontal disease. A long-term
follow-up of subjects treated orthodontically showed
no association between root proximity and perio-
dontal breakdown (7). In another study of periodontal
patients in maintenance therapy following active
treatment, root proximity was not signicantly asso-
ciated with either a worsening in clinical condition as
measured by prognosis (69) or an increased risk of
tooth loss from periodontal disease (70).
Open contacts have been shown to be associated
with increased probing depths and loss of clinical
attachment (47). These results were found in a study
of 104 subjects with unilateral open contacts and
contralateral closed contacts.
Root abnormalities have been shown to be asso-
ciated with periodontal breakdown. In particular,
palato-gingival grooves in maxillary incisors have
been found to be associated with loss of clinical
attachment and bone loss (121). In addition to the
risk posed by palato-gingival grooves among maxil-
lary incisors, proximal root grooves among incisors
and maxillary premolars have also been associated
with attachment loss and bone loss (58). One reason
that these grooves pose such a risk for periodontal
breakdown may be that they impede the removal of
plaque and provide a reservoir for plaque microor-
ganisms in the subgingival area. Cemental tears have
also been associated with attachment loss in an in
vitro study of root surfaces (59). Unsatisfactory root
form has also been linked to the progression of per-
iodontal disease (69) as well as an increased likeli-
hood of tooth loss from periodontal disease (70).
Tooth restorations can also be a risk factor for
periodontal breakdown. This can be the result of
marginal discrepancies of restorations (12, 13, 57,
101) or xed orthodontic appliances (24). Amalgam
overhangs have been linked to signicant alveolar
bone loss (88). Subgingival crown margins as
opposed to supragingival margins have been impli-
cated in increased inammation and greater gingival
recession (81, 87). In addition to the anatomical con-
siderations of restorations, there is also the issue of
tissue response to particular restorative materials.
Allergic reactions to metals used in cast restorations,
such as nickel and palladium, have been reported
(17, 79). However, a study of 16 patients with known
allergies to nickel were treated with crowns or
bridges made of an alloy containing 66% nickel with
porcelain fused to the metal framework and followed
for fteen years without any adverse consequences
reported (103). This latter report does not address
tissue response to cast crowns with alloys containing
nickel without porcelain so that nickel in alloys used
in cast restorations may still pose a risk for some
people with sensitivity to nickel. Acrylics have also
been linked to allergic reactions and may contribute
to gingival inammation (15, 44).
Pulpal involvement may contribute to periodontal
destruction, particularly when there is preexisting
periodontitis. Pulpal necrosis can be associated with
inammatory involvement of the periodontium (29,
90, 100). In addition, when periodontitis is absent, a
sinus tract along the periodontal ligament can be
caused by an endodontic abscess. In some cases,
the defect will persist after endodontic therapy,
necessitating periodontal therapy as well for com-
plete resolution of the problem (14).
Tooth fractures are another factor to consider
among risk factors for periodontal disease. The posi-
tion and extent of a tooth fracture plays a key role in
assessing the risk potential. Thus far, there has been
no evidence that fractures of the crown of a tooth
pose a risk for the development or progression of
periodontal disease, although the fracture itself
may enhance the accumulation of plaque (100). Root
fractures, however, do present a risk for periodontal
disease, especially vertical root fractures that involve
a substantial portion of the root (33, 62, 105).
External root resorption can also present a risk
when there is some communication with the oral
cavity. This provides access to bacteria which can
then destroy the periodontal attachment and alveolar
bone (5).
Microbial risk factors
Microbial dental plaque has long been recognized as
the initiator of periodontal disease. However, not all
bacteria that make up dental plaque have been
shown to be consistently associated with the progres-
sion of periodontal disease. Three specic pathogens
have been repeatedly identied as etiologic agents in
the periodontal destruction associated with chronic
periodontitis: Actinobacillus actinomycetemcomi-
tans, Bacteroides forsythus, and Porphyromonas gin-
givalis (123). However, evaluation of these three
19
pathogens as risk factors for identication of attach-
ment loss over time has resulted in conicting evi-
dence. Three studies indicated that none of these
pathogens were useful in predicting periodontal dis-
ease progression (61, 64, 117). In another study,
B. forsythus was predictive of tooth loss among sub-
jects with little or no periodontitis at baseline (66). In
a recent prospective londitudinal study, B. forsythus
was identied as a risk marker for attachment loss in
a population with low prevalence and severity of
chronic periodontitis (112). B. forsythus and P. gingi-
valis were foundto be associatedwithdisease progres-
sion in established periodontal patients (65) and were
alsofoundtobeassociatedwithalveolar boneloss (36).
Although several studies have evaluated the relation-
ship of A. actinomycetemcomitans to periodontal dis-
ease progression, studies have failed to produce
convincing evidence that A. actinomycetemcomitans
can be used to predict future periodontal destruction.
Emerging risk factors
Increasingly, evidence for a relationship of newly
identied risk factors for systemic diseases, such as
emerging risk factors for cardiovascular disease, to
periodontal disease is starting to emerge. C reactive
protein, which has been implicated in cardiovascular
disease, has also been found in elevated levels in
subjects with adult periodontitis compared to
healthy adults (84). In addition to elevated levels of
C reactive protein, other acute phase reactants and
chemokines, including alpha 1-antitrypsin, hapto-
globin, brinogen, and IL-8, have been signicantly
increased during gingivitis and/or periodontitis in
non-human primates (25). In addition, serum lipid
levels (cholesterol, triglycerides, HDL, LDL) and lipo-
proteins (apoA-I) were also elevated during period-
ontitis (112). Clinical studies have also suggested a
relationship between hyperlipidemia and periodon-
titis (23).
Conclusions
Numerous studies have shown associations between
a myriad of factors and progression of periodontal
disease. In addition, studies of other factors hypothe-
sized to be related to periodontal disease progression
have failed to provide compelling evidence. Under-
standing and evaluation of these risk factors and
determinants demands that the evidence for each
association be weighed according to the study
design, the outcome measures utilized, and the
strength of the association. With further longitudinal
studies evaluating all reported risk factors for perio-
dontal disease progression, it is hoped that we can
one day use these risk factors to more accurately
predict disease progression as well as long-term out-
look for treated teeth.
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23
PERIODONTOLOGY 2000
ISSN 0906-6713
Noninammatory destructive
periodontal disease (NDPD)
Rov C. Pncr & Enwnnn C. S1unnivnN1
Introduction
Prior to the 1970s, the causes and pathobiology of
periodontitis in humans were not understood, and
no widely accepted system of nomenclature and
classication of periodontal diseases existed. The
large number of suspected causes included local irri-
tation from calculus, rough or overhanging restora-
tion margins, or ill-tting oral appliances, systemic
diseases and conditions, diet and nutrition, abnor-
mal occlusal forces, and constitutional conditions.
Various names were used historically to designate
periodontal diseases and boundless theories of the
causation have been espoused. The state of knowl-
edge was summarized by Becks (7) thus:
If we consider the difculties which defy almost all
attempts at classication, we are surprised at the frequency
with which new names and denitions for so-called pyor-
rhea appear in the literature, even before we have made a
clear picture of the single factor which leads to its symp-
toms. The last few years of increased research in this
special subject have resulted in nearly 350 theories of pyor-
rhea and an excessive nomenclature which has confused
the clinical picture for the practitioner more and more.
Over the last half-century, research on the nature of
periodontal diseases has been intensive, and enor-
mous progress has been made on many fronts (51).
Most of the theories alluded to above have been re-
futed and replaced by concepts based on research
ndings. By the 1970s, the central role of bacteria in
causing periodontitis had been documented (12, 15,
32, 63), and concepts about the nature of peri-
odontal disease had begun to shift from those of
classical pathology to the infectious/host defense
paradigm (5). Today the infectious nature of peri-
odontitis is no longer a hypothesis but is a univer-
sally accepted dogma. Experts agree that all forms of
periodontitis are infectious and are characterized by
24
chronic inammation, pocket formation and
deepening, and loss of periodontal attachment and
alveolar bone. Although bacteria are thought to be
essential, bacteria alone are insufcient; a suscep-
tible host is also required, and host susceptibility is
an important determinant of disease status (52).
Currently accepted concepts about the pathobiology,
diagnosis, treatment and prevention of periodontitis
have been built on the essential and dominant role
of bacteria, combined with consideration of the
modulating effects of environmental, acquired, and
hereditary risk factors on host susceptibility. Modern
systems of nomenclature and classication includ-
ing the classication developed by the recent Ameri-
can Academy of Periodontology (AAP) -sponsored
World Workshop are predicated on these character-
istics and assumptions about periodontitis (1, 2, 4,
6, 53). These classications make no allowance for
the possible existence of forms of periodontal dis-
ease that may not full the prescribed character-
istics. When such cases are observed, one has little
choice but to leave the condition unnamed or to
bend the accepted diagnostic criteria to make the
cases t the classication. Consequently, the identity
and classication of diseases of the periodontal
tissues remain problematic. This is especially true of
the Aggressive Periodontitis category.
The present paper questions the validity of the
concept that all forms of destructive periodontal dis-
ease are infectious, and thay they are all character-
ized by chronic inammation, pocket formation and
progressive deepening, and loss of attachment and
alveolar bone. We suggest the existence of at least
one form of severe destructive periodontal disease
that is not recognized in the recent classications. In
this form of periodontal disease, loss of attachment,
resorption of alveolar bone and tooth loss occur, but
gingival inammation and pocket formation and
deepening are not prominent features; antimicrobial
Noninammatory destructive periodontal disease
therapy is not effective in arresting or slowing the
progress of the disease and bacteria may not be the
primary cause. This proposition is, admittedly, out
of the current mainstream of thought in Periodonto-
logy and, to some readers may even seem heretical.
However, making and interpreting observations only
in terms of currently acceptable dogma assures
nothing new will be seen. This volume of Periodon-
tology 2000, which is devoted to periodontal contro-
versies, would seem to be an appropriate place to
describe the disease. In this paper, we trace the his-
torical origins of the disease, present two cases to
illustrate the disease characteristics and discuss the
possible pathobiology and treatment. Our goal is to
call the disease to the attention of practitioners and
periodontal investigators in order to stimulate dis-
cussion and research.
Historical perspective
Much of the ancient and early medical literature re-
ferred to the various maladies of the teeth and their
investing tissues but no specic terminology and no
systematic body of knowledge evolved until the late
eighteenth century. Terms such as spongy gums,
inamed gums and loosening of the teeth were
used. A detailed knowledge and appreciation of the
conception and meaning of dentistry did not take
place until about 1728 (66). The emergence of the
eld of Dentistry was in large part a consequence of
the efforts of Pierre Fauchard and his contemporar-
ies in France. Fauchards book, Le Chirurgien Dentis-
te (The Surgeon Dentist), written in 1723 and pub-
lished in 1728, was the rst compendium of knowl-
edge on diagnosis and treatment of the diseases of
the teeth and associated structures (13). It remained
the predominant textbook for over half a century.
Fauchard was the rst to describe periodontitis ac-
curately and was the rst to refer to the disease as
Scurvy of the Gums.
There is yet another, of which I think no other author has
yet had occasion to speak, and which, without affecting
other parts of the body, attacks the gums, the alveoli and
the teeth. Not only are softened, livid, prolonged and swol-
len gums affected by it but often those which are free from
this vice are not exempt from the disease; it is to be recog-
nized by rather white and sticky pus which can be made to
come out of the gums.
Although Albucasis clearly made the association be-
tween calculus and periodontitis roughly 700years
earlier (66), Fauchard does not speak of this associ-
25
ation, nor did he describe a form of periodontal dis-
ease manifested as a wasting of the alveolar pro-
cesses.
Approximately half a century after the appearance
of Fauchards book, the famous British surgeon, John
Hunter published the Natural History of Human
Teeth, the rst book on Dentistry in the English lan-
guage (28). Between 1771 and 1840, 11 editions of
this book appeared. Hunter described Scurvy of the
Gums in terms very similar to those used by Fauch-
ard. In contrast to Fauchard, Hunter did note that
the accumulation of tartar results in swelling, ulcer-
ation and bleeding of the gums and resorption of
the alveolar processes with exfoliation of the teeth.
Although the text is not very clear, he appears to
have considered Scurvy of the Gums and the tartar-
associated disease as separate entities. Hunter was
the rst to identify and describe a noninammatory,
degenerative form of periodontal disease which he
described as:
... a wasting of the alveolar processes, which are in many
people gradually absorbed, and taken into the system. This
wasting begins rst at the edge of the socket, and gradually
goes on to the root or bottom. The gum, which is supported
by the alveolar processes, loses its connection, and recedes
from the body of the tooth, in proportion as the socket is
lost; in consequence of which, rst the neck, and then more
or less of the fang (root) itself, becomes exposed. The tooth
of course becomes extremely loose, and at last drops out.
In 1806, Joseph Foxs book, entitled Diseases of the
Teeth, the Gums and the Alveolar Processes, appeared
(14). Under the section on Diseases of the Gums, Fox
describes Scurvy of the Gums in much the same
manner as Fauchard and Hunter before him. He also
recognized a form of destructive inammatory dis-
ease associated with the accumulations of tartar, and
he described it in considerable detail. Similar to
Hunter, Fox also acknowledges and further describes
a noninammatory degenerative disease manifested
as absorption of the alveolar processes:
The alveolar processes have certain diseases peculiar to
themselves, independently of afictions arising from the
teeth and gums. The most common diseases of which they
are subject, is a gradual absorption of their substance,
whereby the teeth lose their support, become weak, and at
length are so loosened as to drop out. This disease usually
begins to show itself in persons between 40 and 50years of
age; and, from its frequent occurrence without any evident
cause.
... persons as they approach 50years of age, and others
much sooner, have their teeth become loose from absorp-
tion, or a wasting of the alveolar processes.
... the gums which are supported by the alveolar pro-
cesses, partake also of the disease; thus in the progress of
Page & Sturdivant
the disease, as absorption of the bony matter advances, the
gums lose their attachment to the teeth and recede in pro-
portion to the wasting of the sockets.
... as the disease increases, the teeth become weak, and
at length, by loosing their natural support, are rendered so
exceedingly loose, that, in a short time, they drop out
Sometimes this disease proceeds without the appear-
ance of any assignable cause, the gums retain a very
healthy aspect, are quite free of pain or inammation, and
yet will gradually recede, until the teeth become very loose.
By early in the nineteenth century, inammatory de-
struction of the periodontal tissues, generally but not
always associated with calculus, had become estab-
lished as a common disease, and a noninammatory
form of destructive periodontal disease had been de-
scribed by Hunter and the observation conrmed by
Fox. Very little additional progress was made be-
tween the time of Hunter and Fox and the early
twentieth century. In the United States, Riggs be-
came sufciently known for his methods of treat-
ment that, for a time, periodontitis was known as
Riggs Disease (59), and near the end of the century,
the name Pyorrhea Alveolaris replaced Scurvy of
the Gums and came into common use (55).
Gottlieb had a profound impact upon nomencla-
ture and classication as well as upon the concepts
of pathogenesis of periodontitis. He and his col-
leagues focused on pocket formation as the cardinal
manifestation of inammatory periodontal diseases
and the idea that some forms of periodontal disease
have systemic disease ramications.
Gottlieb recognized four types of periodontal dis-
ease (2022). These included Schmutz-pyorrhoe, Al-
veolar or Diffuse Atrophy, Paradental-pyorrhoe and
Occlusal Trauma. Schmutz-Pyorrhoe was character-
ized by inammation, shallow pocket formation and
alveolar bone resorption associated with deposits on
the teeth. This disease was the equivalent of the
Scurvy of the Gums described by Fauchard, Hunter
and Fox. Alveolar or Diffuse Atrophy was a nonin-
ammatory disease thought to be associated with
systemic causes, resulting in bone loss and loosening
of teeth in the absence of dental deposits, and mani-
festing pockets only in the later stages. This form of
disease appears to be the same as the noninam-
matory wasting of the alveolar bone rst described
by Hunter and by Fox. Paradental-pyorrhoe was
characterized by shallow and extremely deep
pockets, irregularly distributed that could have be-
gun either as Schmutz-Pyorrhoe or as Alveolar or
Diffuse Atrophy. Occlusal Trauma was considered to
result in resorption of the alveolar bone and
loosening of the teeth.
26
The rst system of nomenclature and classi-
cation of periodontal diseases developed by the AAP
was published in 1942 by Orban and further rened
by him in 1949 (44, 45). In this classication, four
types of periodontal disease plus gingival hyper-
trophy were recognized. Clear distinctions were
made between the inammatory diseases, gingivitis
and periodontitis, and degenerative and atrophic
diseases. Periodontitis simplex was considered to be
caused by dental deposits and other local factors,
and was the equivalent of Scurvy of the Gums and
Schmutz-pyorrhoe as described by previous authors.
Periodontitis complex was an inammatory disease
in which systemic diseases and conditions were con-
sidered to play an important role. Degenerative peri-
odontal disease or Periodontosis appears to have
been the equivalent of Gottliebs Paradental-pyor-
rhea. Periodontal Atrophy was accepted as an entity
separate from periodontitis and was characterized
by a noninammatory loss of periodontal tissue
without pocket formation and with gingival re-
cession. Suggested causes of Periodontal Atrophy
were trauma from excessive tooth brushing, disuse,
and aging. In 1949, Orban published radiographs
typical of cases of periodontal disease with extreme
alveolar bone destruction in young individuals (45).
In his gure 1, radiographs of a 17-year-old-girl dem-
onstrate extensive destruction of the alveolar bone
in the complete absence of gingival inammation.
The term Periodontosis was used to designate both
localized and generalized destructive periodontal
disease, especially that which occurs in younger in-
dividuals. Almost all classications of periodontal
diseases published from the time of Gottlieb until
the 1970s included a category of noninammatory
dystrophic diseases including periodontosis (8, 11,
1619, 22, 23, 25, 27, 30, 37, 39, 4446, 65).
Notably, by the 1960s, the terms Periodontitis sim-
plex and complex were no longer in use, in part be-
cause of a lack of evidence supporting their exist-
ence. At the World Workshop in Periodontics in 1966,
the existence of periodontosis, periodontal atrophy
or any form of noninammatory degenerative peri-
odontal disease was called into question (1). In 1977,
participants in the International Conference on Bi-
ology of Periodontal Disease ofcially concluded
that there was no scientic basis for maintaining
that noninammatory degenerative forms of peri-
odontal disease exist (53). Only a single form of peri-
odontitis designated Chronic Marginal Periodontitis
was recognized.
By 1974, the entity previously known as Peri-
odontosis had been redened as a disease affecting
predominantly the permanent incisors and or rst
molars in teenagers, and it reappeared under the
name Localized Juvenile Periodontitis (38). Ad-
vanced generalized forms of periodontitis in young,
otherwise healthy, individuals, described by Orban
(45) as Periodontosis, also reappeared under the
names Rapidly Progressive Periodontitis (RPP),
Early Onset Periodontitis in Young Individuals
(EOP) and Generalized Juvenile Periodontitis (GJP)
(48, 54). Based on these and other observations, a
new classication of periodontitis was suggested
(47). This classication, with minor modication,
was adopted by the AAP in 1986 (1), and rened con-
siderably by the AAP World Workshop on Clinical
Periodontics in 1989 (2). A European classication
was developed in 1994 by the 1st Workshop on Peri-
odontics. In 1999, a World Workshop on the Classi-
cation of Periodontal Diseases was convened by the
AAP to revise and extend the 1989 classication (4).
None of these classications contained a category
for noninammatory destructive periodontal dis-
eases and conditions.
We suggest that noninammatory destructive
periodontal disease does exist and is seen rather
commonly by practicing periodontists, who pre-
viously classied it as RPP, EOP, or GJP and who cur-
rently classify it as Aggressive Periodontitis based on
the 1999 AAP classication, even though it does not
t the diagnostic criteria for any of those diseases.
We also suggest that recognition of the disease and
its correct diagnosis are of considerable importance
as most cases do not respond to the accepted anti-
microbial treatments routinely applied for peri-
odontitis. We propose to designate the disease Non-
inammatory Destructive Periodontal Disease or
NDPD.
The characteristics of NDPD are essentially as de-
scribed by Hunter and Fox (14, 28). They include
generalized loss of attachment and resorption of al-
veolar bone with extensive gingival recession affect-
ing many teeth, with only minor clinical manifes-
tations or history of gingival inammation, without
the formation of deep periodontal pockets, begin-
ning in individuals usually in their 20s and 30s with
excellent daily oral hygiene. The disease may affect
all of the teeth or it may be more severe around the
posterior or the anterior teeth. Affected individuals
are usually in good general health and are receiving
excellent professional dental care. At the time they
are referred to the periodontist, most are exercising
unusually aggressive and frequent daily oral hygiene
manifested by very low levels of plaque, wearing
away of the cemento-enamel junctions, and show a
27
polished appearance of the crowns and exposed
roots of the teeth. Patients may be using multiple
forms of interdental cleaning including dental oss
and interdental brushes and various probes, picks
and sticks. Analysis of the plaque ora fails to reveal
the presence of expected putative periodontal
pathogens such as Porphyromonas gingivalis,
Bacteroides forsythus, Actinobacillus actinomycetem-
comitans, and Treponema denticola or enteric spe-
cies. Furthermore, serum antibody analyses fail to
reveal evidence of prior infection by these organ-
isms, e.g. titers of serum antibody reactive with anti-
gens of these bacteria are not elevated. The evidence
suggests that this form of periodontal disease is non-
infectious. Almost without exception, these individ-
uals do not respond to scaling and root planing or
other currently used antimicrobial periodontal ther-
apies.
Case reports
Case 1
Case 1 is a 45-year-old Caucasian male (W.C.B.) who
was employed in the aerospace industry when he
was rst seen by one of the authors (E.C.S.) in Aug-
ust, 1975. He was in good general health and had
never smoked or consumed alcoholic beverages. All
of the teeth were present except the maxillary left
and mandibular right third molars; the maxillary
right third molar was unerupted. Moderately severe
horizontal alveolar bone loss was apparent around
all of the teeth (Fig. la,b). Clinical examination re-
vealed clean teeth free of visible microbial deposits,
and no clinically apparent gingival inammation.
Gingival recession was apparent on the buccal as-
pect of almost all of the teeth especially the maxillary
premolars and rst molars. Probing depths were
generally 34mm, with occasional 5mm depths, and
early furcation involvement was noted on the buccal
aspect of some of the molars. Several large class V
gold restorations had been placed earlier. The left
premolars were in cross-bite relation and interfering
occlusal contacts were noted (Fig. 1). The diagnosis
made was periodontitis with moderate horizontal al-
veolar bone loss and severe gingival recession affect-
ing the facial surfaces of almost all of the teeth. Fol-
lowing oral hygiene instructions, treatment included
scaling and root planing, occlusal adjustment to re-
move interfering tooth contacts, and placement of
free gingival grafts extending from the canine to the
mesial aspect of the second molars in all four quad-
Page & Sturdivant
Fig. 1. Clinical (a) and radiographic (b) features of W.C.B. and gingival recession especially around the maxillary
at the time he was rst seen in 1975. Note apparent ab- molars and premolars. Moderately severe horizontal al-
sence of microbial deposits on the teeth, the lack of clin- veolar bone loss is apparent in the radiographs (b), espe-
ical manifestations of inammation in the gingival tissues cially around the maxillary molars and premolars.
rants. The maxillary right third molar was extracted.
Upon completion of the planned therapy, the patient
was placed on yearly recall with the periodontist
with interim visits to his general dentist.
It soon became apparent that recession and bone
loss were continuing. The recall interval was
shortened to 3months. The periodontist became in-
creasingly concerned about the continuing and
28
rapid loss of alveolar bone and advancing gingival
recession, more so around the posterior compared
to the anterior teeth. By 1980, advanced horizontal
alveolar bone loss was apparent around all of the
teeth and gingival recession had worsened (Fig.
2a,b). After evaluating the functional occlusion and
observing the patient perform oral hygiene pro-
cedures, it was concluded that continuing trauma
Fig. 2. Clinical (a) and radiographic (b) features of W.C.B. molars and premolars, but the teeth appear to be clean
in 1980. Severe horizontal alveolar bone loss can be ob- and the gingival tissues are free of manifestations of clin-
served around all of the teeth; gingival recession has ical inammation.
worsened, especially on the buccal aspect of the maxillary
from occlusion and aggressive use of the toothbrush
and interdental cleaning devices could be related to
the continuing periodontal deterioration. Additional
selective grinding was performed to remove any re-
maining interfering contacts and to create a cuspid
rise functional occlusion, and a maxillary night
guard was constructed and used. To guard against
the possibility that the patient had been experienc-
ing unrecognized periodontal infections, he was
29
given a prescription for tetracycline 250mg four-
hourly and ask to take the drug for 1week if, in his
opinion, he began to develop gum problems. Less
aggressive oral hygiene procedures were instituted
and the patient was returned to recall every 3
months.
Over the next several years, the patient took numer-
ous courses of tetracycline or minocycline at a stan-
darddaily dose for 7days. Onseveral occasions he was
Page & Sturdivant
30
ask to come to the ofce for observation when he felt
that a problem was developing prior to taking the
antibiotic. On none of these occasions was any in-
ammation, swelling, increasing pocket depth, or
other signs of periodontal infection observed.
In 1983, the patient was diagnosed with rheuma-
toid arthritis and began taking an anti-inammatory
drug (Naproysyn, standard dose) daily for joint pain.
Nevertheless, the recession and bone loss appeared
to be continuing. By 1985, recession and alveolar
bone loss had progressed greatly (data not shown).
In 1988, endodontic therapy was required for one of
the maxillary rst molars. Clinical and radiographic
features of the patient in 1988 are shown in Fig.
3(a,b). Alveolar bone loss was approximately 50% or
greater for all of the teeth except the maxillary in-
cisors. Gingival recession was apparent at the facial
surfaces of all of the maxillary and mandibular teeth
including the incisors, more advanced for the molars
and premolars than for the incisors. Recession was
also advanced on the palatal surfaces of the molars,
somewhat less on the maxillary premolars and man-
dibular incisors and canines. The gingival margins
were located at the cementoenamel junction on the
palatal aspect of the maxillary canines and incisors.
Throughout the mouth, the gingival tissues were free
of clinical manifestations of gingival inammation
and the teeth appeared to be relatively plaque free.
Because of joint pain and limited mobility, bilat-
eral hip replacement was performed for the patient
in 1991 and he was placed on Methotrexate, 6 tab-
lets/week, Plaquenil, 2 tablets/day, Asulfadine, 2 tab-
lets/day, Naprosyn, l tablet/day and prednisone 10
mg/day. This drug regime has been continued to the
present time. Throughout the 1990s the patient also
took several 7-day courses of Keex and 2g Keex
before each dental appointment.
By 1993, approximately one-half to two-thirds of
the alveolar bone had been lost from around all of
the teeth except the maxillary incisors, some of the
teeth had begun to become mobile and gingival re-
cession had continued (data not shown). The clinical
and radiographic features of the disease in 1998 are
Fig. 3. Clinical (a) and radiographic (b) features of W.C.B.
in 1988. The teeth continued to be relatively free of mi-
crobial deposits and the gingival tissues were not clin-
ically inamed. Alveolar bone loss of 50% or more was
apparent around all of the teeth except the maxillary in-
cisors. Gingival recession had increased signicantly on
the labial surfaces of all of the teeth and recession was
apparent on the palatal and lingual surfaces of all of the
teeth except the maxillary incisors.
31
shown in Fig. 4(a,b). Alveolar bone loss had pro-
gressed to approximately two-thirds of the total root
length around most of the teeth. Notably, bone levels
in the furcations of the mandibular molars were pre-
served. Clearly, recession had advanced on all of the
teeth but the gingival tissues were still free of clinical
signs of inammation. In spite of the oral hygiene
challenge presented by extreme gingival recession,
large interdental spaces and open furcations, the
teeth continued to be relatively plaque free, although
minor amounts of plaque were visible on some of
the teeth (Fig. 4a).
Radiographs taken in 2002 revealed continuing
bone loss around all of the teeth, including more
than 50% bone loss around the maxillary incisors
(Fig. 5). One of the maxillary rst molars had become
fractured and the buccal roots had been removed.
The mandibular incisors had required an acrylic-
wire splint because of excessive mobility. Bone loss
had continued in the absence of signicant amounts
of plaque or gingival inammation (data not shown).
As of now, the patient is 66 years old and is ap-
proaching loss of all of his natural teeth.
In 1980, the patient was sent to one of us (R.C.P.)
for consultation. Examination conrmed the obser-
vations that the periodontist had made at that time.
Over a period of several years, beginning in 1981,
plaque samples were harvested and cultured, serum
antibody levels to putative periodontal pathogens
were measured and the functional characteristics of
peripheral blood neutrophils (PMN), monocytes and
lymphocytes were analyzed.
Because an abnormality in PMN chemotaxis had
recently been identied in some patients with pro-
gressing periodontitis (10), in 1981 we harvested pe-
ripheral blood leukocytes and measured PMN and
monocyte chemotaxis in vitro using standard
methods (49). Relative to normal controls, in vitro
PMN and monocyte chemotaxis was within the nor-
mal range, and the patients serum yielded normal
levels of chemoattractant activity following zymosan
activation. In 1990, peripheral blood lymphocytes
were harvested and cell surface markers were meas-
ured using specic monoclonal antibodies and ow
cytometry. All values were within the normal range
except 9% of the patients B lymphocytes were CD5
compared with an expected value of 4% for B

lymphocytes from normal control subjects. CD5
B
cells are thought to produce auto-antibodies, and el-
evated levels have been seen in patients with rheu-
matoid arthritis, scleroderma and Sjgren syndrome.
As noted above, at the time the tests were performed,
the patient had rheumatoid arthritis.
Page & Sturdivant
32
Fig. 5. Radiographic features of W.C.B. in 2002. Although icular bone of the mandibular rst molars remained at
relative to previous radiographs, alveolar bone destruc- normal levels.
tion had continued around all of the teeth, the interrad-
Subgingival plaque was harvested in 1981 from
several sites with the deepest probing depths and
cultured for putative anaerobic periodontal patho-
gens. Bacteroides gingivalis (Porphyromonas gingi-
valis), fusiform Bacteroides (B. forsythus) and A. acti-
nomycetemcomitans were not detected. Additional
plaque samples were harvested and analyzed in
1997, 1999 and 2000. The 1997 sample was analyzed
in the commercial Oral Microbiology Testing Labora-
tory at the School of Dentistry, University of South-
ern California, Los Angeles, CA, and the remaining
Fig. 4. Clinical (a) and radiographic (b) features of W.C.B.
in 1998. Alveolar bone loss had progressed to two-thirds
or more of the root length around most of the teeth, while
bone levels in the furcation areas of the mandibular mo-
lars were preserved. The gingival tissues continued to be
free of clinical manifestations of inammation. The teeth
were relatively clean except for minor amounts of inter-
proximal plaque in some areas (arrows).
33
two samples were analyzed in our anaerobic micro-
biology research laboratories at the School of Den-
tistry, University of Washington, Seattle, WA. The
1997 sample was negative for A. actinomycetem-
comitans, P. gingivalis, B. forsythus, Peptostreptoc-
occus micros, Eubacterium sp., Campylobacter sp.
and for enteric bacteria, while Fusobacterium sp. ac-
counted for 1.25% and Prevotella intermedia for
4.17% of the ora. The 1999 and 2000 samples were
very similar to the 1997 sample and to each other
with both being negative for A. actinomycetemcomit-
ans, P. gingivalis, B. forsythus, and C. rectus, with no
spirochetes observed by dark eld microscopy. Fuso-
bacterium sp. and P. intermedia accounted, respec-
tively, for 2.5% and 7.7% in the 1999 sample and for
2.5% and 3.8% in the year 2000 sample.
In order to obtain information about possible pre-
vious periodontal infections, in 1983 blood was
drawn and serum antibody titers to antigens of puta-
tive periodontal pathogens were measured using
Page & Sturdivant
standard techniques (67). Titers were not elevated to
any of the putative periodontal pathogens tested.
These measurements were not performed in our lab-
oratories and we are uncertain of the accuracy of the
results. Additional blood samples were drawn and
tested in 1990, 1999 and 2000. In addition to the
periodontal pathogens, we also tested for antibodies
reactive with Staphylococcus aureus since elevated
level of specic serum titers for antigens of this or-
ganism have been associated with failing dental im-
plants (34). Results are reported in Table1 relative to
values for sera from periodontally normal controls
set at 100 enzyme-linked immunosorbent assay ELI-
SA Units (EU). Titers were not elevated to antigens
of putative periodontal pathogens tested, except on
one occasion where the titer for antibodies reactive
with antigens of B. forsythus was ll6EU vs. l00EU for
normal controls.
Throughout the 27-year period of observation of
this patient, his teeth were remarkably free of mi-
crobial deposits, the gingiva were relatively free of
inammation and signicant pocket depth did not
occur; yet, extreme loss of periodontal attachment
and alveolar bone did occur and progression of dis-
ease was not slowed by any of the treatments used.
These observations, combined with our studies on
the subgingival ora and serum antibody titers are
consistent with the idea that the periodontal deterio-
ration observed in this patient may not be infectious.
Case 2
Case 2 is a female attorney (J.B.) who was 36years of
age when she began treatment in one of our School
of Dentistry clinics in 1992. She was rst seen by one
of us (R.C.P.) in 2001 because of concern about her
continuing alveolar bone loss and gingival recession,
and a recommendation from her dentist that she
consider full mouth extraction and placement of im-
plants. She was in excellent general health with no
known systemic health problems and was a non-
smoker. She had had excellent routine dental care
Table1. Serum antibody titers
Date blood taken P. gingivalis B. forsythus A. actinomycetemcomitans S. aureus
4/1/90 7 116 1 50
11/17/99 6 36 0 15
3/13/00 4 51 0 22
P. gingivalis, Porphyromonas gingivalis; B. forsythus, Bacteroides forsythus; A. actino., Actinobacilluis actinomycetemcomitans; S. aureus, Staphylococcus aureus.
34
Fig. 6. Radiographic features of J.B. in 1988. Moderately
severe horizontal alveolar bone loss is apparent around
all of the teeth.
and periodontal maintenance. Medical and dental
histories and clinical and radiographic examination
revealed all of the features characteristic of Non-in-
ammatory Destructive Periodontal Disease. A pan-
oramic radiograph from 1988 revealed that all of the
teeth were present except the second premolars (Fig.
6). The maxillary second and third molars and the
mandibular third molars had fused roots. Moder-
ately severe generalized horizontal alveolar bone re-
sorption was apparent at that time. No occlusal ab-
normalities were noted. By 1990 bone loss had pro-
gressed signicantly and by 1998 horizontal alveolar
bone loss of one-half to two-thirds of the total root
length was apparent around all the teeth (Fig. 7). The
mandibular incisors had become so mobile that an
acrylic-wire mesh temporary splint was placed.
When rst seen, the patient was using extremely
aggressive daily oral hygiene. All of the patients oral
hygiene devices were taken and she was placed on a
sonic brush to be used for no longer than 2min,
twice each day. She was seen frequently by dental
Fig. 7. Radiographic features of J.B. in 1990 (uppermost greatly by 1990 and progressed to approximately one-half
set of lms) and in 1998 (lowermost set of lms). Relative to two-thirds of the root length by 1998.
to the 1988 set of lms, alveolar bone loss had advanced
hygienists who helped her develop gentle yet effec-
tive ways to remove and control interproximal
plaque, mostly using interproximal brushes. Stan-
dardized vertical bite wing radiographs were made
after 6 and 12months. These demonstrated that no
perceptible alveolar bone loss had occurred since
less aggressive oral hygiene procedures were insti-
tuted 12months earlier.
Clinical features of the case as seen in March 2002
are shown in Fig. 8. When the photographs were
taken, the patients teeth had not been professionally
cleaned for 6months. Severe gingival recession was
apparent around all of the teeth both on the facial
and lingual/palatal surfaces. Clinical examination
revealed teeth that were relatively free of plaque and
calculus, severe gingival recession affecting all of the
teeth and gingiva free of clinical manifestations of
inammation. Although the free gingival margins
around some of the teeth appeared to be enlarged
(mandibular incisors and right canine and rst pre-
35
molar), clinical examination revealed no bleeding
points upon probing and no probing depths greater
than 3mm. The cementoenamel junction on the fa-
cial aspects of many of the teeth had been worn
away. Note the highly polished appearance of the
crowns and roots of the teeth.
Additional comments
A noninammatory form of destructive periodontal
disease was rst described by Hunter in 1771 and
subsequently conrmed by Fox (14), Gottlieb (20)
and Orban (44, 45). A category of noninammatory
destructive periodontitis was included in the rst
AAP classication of periodontal diseases in 1942
(44), and was an integral part of virtually all sub-
sequent classications published up to about 1970.
The disappearance of NDPD from the literature oc-
Page & Sturdivant
Fig. 8. Clinical features of J.B. in 2002, approximately 1 ammation, and with minor exception (see arrows) the
year after ceasing aggressive oral hygiene procedures. The teeth were free of microbial deposits. Note the polished
gingival tissues were free of clinical manifestations of in- appearance of the crowns and exposed roots.
curred concurrently with increasing documentation
of the important role of bacteria in the etiology of
periodontitis and the shift in concepts about peri-
odontal diseases from the Classic Pathology para-
digm to the Infection/Host Defense paradigm as de-
scribed by Armitage (5). Under the latter paradigm,
all forms of destructive periodontal disease were
considered to be infectious and to be characterized
by inammation, pocket formation and loss of peri-
odontal attachment and alveolar bone. In Armitages
view, the experimental gingivitis studies of Harald
Loe and his colleagues (31, 35, 36, 64) coupled with
the demonstration of bacterial specicity in the
etiology of localized juvenile periodontitis (41, 42)
and the discovery of functional abnormalities in
PMNs from localized juvenile periodontitis patients
(10) rmly established the Infection/Host Defense
paradigm. This development brought to a halt any
further consideration of noninammatory forms of
destructive periodontal disease. We suggest that the
infectious nature of periodontal disease came to
dominate periodontal thought to the point that the
possible existence of a noninfectious form of peri-
odontal destruction that does not manifest inam-
mation and pocket formation became unthinkable.
As demonstrated by the examples we have provided
and more than three decades of clinical experience,
we believe that this disease is still with us even
though it has not been acknowledged in the classi-
cations for about 30years.
NDPD has several distinct diagnostic character-
istics including generalized loss of attachment and
resorption of alveolar bone with extensive gingival
recession, affecting many teeth, without formation
of deep periodontal pockets or signicant clinical
manifestations or history of gingival inammation,
occurring in individuals with excellent daily oral hy-
giene. The disease is recognized most frequently in
individuals in their 30s or 40s, although it may begin
36
in patients in their 20s. It may affect all of the teeth
or it may be more severe around the posterior or
the anterior teeth. Affected individuals are usually in
good general health and are receiving excellent pro-
fessional dental care. At the time they are referred to
the periodontist, most are exercising unusually ag-
gressive and frequent daily oral hygiene manifested
by low levels of plaque, wearing away of the ce-
mentoenamel junctions, and a polished appear-
ance of the tooth crown and root surfaces. In ad-
dition to aggressive tooth brushing, patients may be
using multiple forms of interproximal cleaning, in-
cluding use of various interproximal probes, picks
and sticks, as well as oss and brushes. The oral hy-
giene routine may be performed multiple times each
day or for lengthy time periods of 30min or longer.
NDPD may be a noninfectious form of periodontal
destruction. Analysis of the plaque ora fails to re-
veal the presence of expected putative periodontal
pathogens such as P. gingivalis, B. forsythus, A. actino-
mycetemcomitans, and T. denticola or enteric bac-
terial species. Furthermore, serum antibody analyses
generally fail to reveal evidence for prior periodontal
infection.
There is abundant evidence that periodontitis, as
the disease is traditionally dened, is caused by spe-
cic bacteria that extend apically between the gin-
giva and tooth surface to cause inammation, for-
mation of periodontal pockets with a pocket epithel-
ium and destruction of the periodontal ligament, the
gingival connective tissue and the alveolar bone (51).
Intact bacteria and toxic bacterial components, such
as lipopolysaccharide, gain entry to the connective
tissues and blood. In a susceptible host, bacterial
components initiate and perpetuate an inamma-
tory response in the periodontal tissues. Many pa-
tients produce elevated titers of antibodies specic
for antigens of their infecting periodontal bacteria
(29). Inammatory macrophages and granulocytes
become activated to produce large quantities of
cytokines, especially interleukin-l (IL-1), tumor ne-
crosis factor-a (TNF-a)and prostaglandins, and
members of a large family of enzymes known as the
matrix metalloproteinases (MMP) (24, 40, 43, 50, 56,
58). Resident periodontal broblasts express recep-
tors for IL-l and TNF-a. When these become occu-
pied, the broblasts also become activated and con-
tribute to MMP and prostaglandin production (9, 24,
26, 33, 5658). The evidence demonstrates that
prostaglandins, especially prostaglandin E
2
, mediate
destruction of the alveolar bone and MMP activities
destroy the soft connective tissues (24, 40, 56, 58).
Generally, periodontitis will progress so long as the
driving force, the infecting bacteria above a thresh-
old level, are present.
Although the mechanisms underlying NDPD have
not been directly investigated, we suggest that they
may be the same as for periodontitis except that the
production of prostaglandins and MMP, as described
above, may be initiated and perpetuated by factors
other than bacterial infection, and the mediators may
be produced by cells that are normally resident in the
periodontal tissues rather than inltrating inam-
matory cells. Fibroblasts plus a few macrophages
(histiocytes) comprise the predominant cell popula-
tion present in noninamed gingiva (60). These cells
could become activated to produce, cytokines,
prostaglandins and MMP by application of abnormal
forces as well as by binding of IL-l, TNF-a, or bacterial
substances (3, 62). Such forces could originate from
enduring, frequently applied aggressive oral hygiene.
The resident broblasts could therefore serve as a
source of molecules that mediate resorption of the al-
veolar bone and destruction of gingival and peri-
odontal ligament connective tissues.
Based on the above hypothesis, we have focused
on the idea that soft tissue trauma resulting from
aggressive daily oral hygiene combined with a poss-
ible genetically determined enhanced susceptibility
could account for the pathobiology of NDPD. In two
cases, we have taken away all of the oral hygiene de-
vices being used and replaced them with a sonic
tooth brush to be used for a maximum of 2min twice
each day. The patients were then appointed to a den-
tal hygienist for monitoring and institution of non-
traumatic, gentle interproximal cleaning, generally
using brushes, until plaque control was successful.
For one of these patients, periapical radiographs
taken yearly for period of 3years show no apparent
further alveolar bone loss. In the other patient, case
2 reported here, standardized vertical bite wing
radiographs taken at 6 and 12months following the
37
instituted change in oral hygiene practices revealed
no further alveolar bone loss. Although we appear to
have been successful in arresting alveolar bone loss
in both of these patients, we hasten to add that these
observations are too limited to warrant our recom-
mending changes in oral hygiene as a routine man-
agement method for NDPD and we wish to empha-
size that if such changes are instituted, careful and
frequent patient monitoring for inadequate plaque
control is essential.
In summary, over a period of more than 30years
of practice, we have encountered a large number of
cases of destructive periodontal disease that do not
t the diagnostic criteria of any form of periodontal
disease described in the classications published
since the 1970s. The primary diagnostic features of
the disease include progressive gingival recession
and loss of periodontal attachment and alveolar
bone, the absence of gingival inammation and mi-
crobial deposits and periodontal pocket formation,
and failure of the disease to respond to traditional
antimicrobial periodontal treatments. In addition,
the disease appears to be noninfectious. To distin-
guish the disease from various forms of peri-
odontitis, we suggest the name Non-inammatory
Destructive Periodontal Disease. The disease ap-
pears to have been described accurately by Hunter
and by Fox more than 200years ago, and their obser-
vation has been conrmed by several authors since
that time. Reference to this disease disappeared from
the literature at the time the infectious nature of
periodontitis came to dominate periodontal con-
cepts. Recognition of this disease as a specic entity
is important as it is a signicant cause of tooth mor-
tality at a relatively young age, and it fails to respond
to the usual periodontal therapies. We suggest that
the pathobiology of the disease may be comparable
to that of periodontitis except that the disease may
be noninfectious; the inammatory agents mediat-
ing tissue destruction may be produced by bro-
blasts and macrophages that normally reside in the
periodontal tissues that become activated by trau-
matic forces rather than by inltrating inammatory
cells activated by bacterial substances. Our goal is to
bring the disease to the attention of practitioners
and periodontal investigators in order to develop
better diagnostic criteria and discover successful
means of prevention and treatment.
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2200.
PERIODONTOLOGY 2000
ISSN 0906-6713
Benecial bacteria of the
periodontium
FnnNx A. Rourn1s & Ricnnnn P. Dnnvrnu
Introduction
We propose that bacteria and their products are a
necessary and benecial component of a healthy
periodontium. The primary evidence for our hypoth-
esis is that clinically healthy periodontal tissue con-
tains a highly orchestrated gradient of select in-
ammatory mediators that plays a key role in the
defense of this tissue and the overall health of the
individual. Circumstantial evidence indicates that
these mediators are made in response to a highly
specic microbial consortium residing on the tooth
surface. Therefore it is the dialog that occurs be-
tween the microbial population found in peri-
odontally healthy individuals and the adjacent peri-
odontal tissue that results in a functional periodon-
tium.
Bacteria participate in host tissue
developmental programs
The single most effective therapy for the treatment
of periodontitis is the efcient removal of both supr-
a- and subgingival plaque. This observation alone is
sufcient to warrant the concept that bacteria are
not part of what we normally refer to as healthy peri-
odontal tissue. In addition, the entire medical com-
munity has had similar experiences with micro-
organisms in that their removal is associated with
healing and health. There is no doubt that anti-
biotics represent an important life-prolonging ad-
vance in medical science. These observations, com-
bined with notorious and fatal bacterial diseases
such as the bubonic plague and cholera, have pro-
vided a more than adequate rationale for the theory
in the medical and dental community that bacteria
at the least are expendable and more probably are
usually associated with disease.
40
However, these observations do not accurately
represent the interactions of bacteria with the ani-
mal kingdom (61). Leaving the human system for a
moment there are a plethora of examples of how
bacteria are essential components of normal host
growth and development (see Table1). For example,
at least 11% of all insect species pass their respective
host-associated bacterial community to their pro-
geny (vertical transmission) within or on the egg
(19). This process, termed endosymbiosis, requires
that the microbial community of the parent be trans-
located to the ovary to be present at the time of con-
ception where the bacteria will inuence normal
embryogenesis of the host insect as well as postem-
bryonic development. Furthermore, the larvae of
many invertebrate sessile benthic species, such as
certain oysters and barnacles, will settle normally
and metamorphose only after colonization with spe-
cic bacteria (25, 56). Certain parasitic nematodes
contain a bacterial species that cannot live outside
their specialized nematode tissue and that assist the
worms in killing their insect host (27, 34). These bac-
teria are therefore an integral part of the life cycle of
the nematodes. Termites occupy a unique ecological
niche due to the ability of the highly organized mi-
crobial consortium located within specialized struc-
tures of their hindgut to degrade cellulose so that
it can be utilized as a food source (8). Recent fossil
evidence demonstrates that this hostbacterial as-
sociation dates back 20 million years (96).
Another example of a highly evolved, well-studied
symbiosis is the Vibrio scheri/Euprymna scolopes
pairing (62). Vibrio scheri is a luminous bacterium
that colonizes the light-emitting organ in the mantle
of E. scolopes, a bobtail squid (see Fig. 1). The bac-
terium is the source of the squids characteristic bio-
luminescence. Bioluminescence is believed to pro-
vide a defensive camouage strategy, termed
counterillumination, which the squid employs dur-
ing nocturnal feeding. The light produced appar-
Benecial bacteria of periodontium
Table1. Examples of symbiotic bacterial interactions with host organisms
Host Symbiotic bacteria Benet to host
Nematode Xenorhabdus species Killing of insect larvae
Corals Symbiodinium species Provides nutrients from photosynthesis
Tsetse y Wigglesworthia glossinidia Provides vitamins
Aphid Buchnera aphidicola Provides essential amino acids
Termite Cellulolytic bacteria Digests cellulose
Leguminous plants Rhizobium species Nitrogen xation
Squid Vibrio scheri Bioluminescence
Leeches Aeromonas veronii Digests blood
Cattle Multiple bacteria Rumination
Adapted from Hentschel et al. (34) and Goebel & Gross (27).
ently mimics moonlight passing through the water
so that deep-dwelling predators cannot detect the
squids shadow above them. The association be-
tween the bacteria and the squid results in several
signicant changes in both species. Squid colonized
with V. scheri lose a ciliated epithelium layer, the
epithelial cells swell, and the density of the microvilli
increases, promoting retention of the Vibrio. The as-
sociation also profoundly inuences the bacteria.
Fig. 1. Symbiosis of Vibrio scheri and the Hawaiian sepi-
olid squid Euprymna scolopes. The luminous bacterium V.
scheri colonizes the light-emitting organ in the mantle of
the bobtail squid providing a defense camouage during
nocturnal feeding (courtesy of Margaret McFall-Ngai).
41
They lose their agella, become smaller, reduce their
growth rate and increase cell-specic luminescence.
This system provides additional clear evidence that
animals can require bacterial colonization for nor-
mal growth, development, and function.
Benecial bacterial interactions
occur in human hosts
There is also evidence that similar associations,
perhaps less dramatic, occur in humans. For ex-
ample, the mammalian intestine is occupied by a
variety of different microbes, including up to 400
different bacterial species at any one time (77).
Early studies in germ-free or gnotobiotic mice
demonstrated that bacteria have a direct impact
on the morphology of the intestine (20, 23). Bac-
teria are responsible for degradation of mucus gly-
coproteins, and their absence results in an enlarge-
ment of the cecum. The villi of the small intestine
are longer and the crypts are shorter and contain
fewer cells in germ-free animals. In addition, bac-
teria have effects on intestinal motility. The effects
of bacteria can be seen on the development and
function of the intestinal immune system also. Bac-
teria are required for the complete development of
Peyers patches, the lamina propria, and the intra-
epithelial spaces, the three main immune elements
found in the intestine. The normal vaginal eco-
system also provides a safe habitat for a variety of
different bacterial species and a clear commensal
relationship exists between the host and lactobacilli
species (91). The host vaginal epithelial cells supply
glucose for these bacteria, and in turn the lacto-
Roberts & Darveau
Fig. 2. Innate host defense status in clinically normal peri-
odontal tissue. Clinically healthy tissue expresses low
levels of E-selectin and a gradient of interleukin-8 (repre-
sented in shades of red) that facilitates the transit of neu-
trophils through the tissue and into the gingival crevice
where they protect the host from infection. These me-
diators are made in response to a highly organized bac-
terial biolm termed dental plaque. This representation is
adapted from Whittaker et al. (95) and Tonetti et al. (88).
bacilli produce acid that prevents the growth of
many other species of bacteria that may have del-
eterious effects on the vaginal environment.
However strong the association is between bac-
terial colonization and the proper functioning of hu-
man intestinal and female reproductive tract tissue,
the role of bacteria in their function has been largely
underestimated. There are several possible expla-
nations for this. One is that classic training in micro-
biology has taught that commensal bacteria are tol-
erated by the host, not causing harm, yet enjoying
the benets of a nutrient-enriched environment.
This concept is a long way from appreciating the ac-
tive role these bacteria may contribute to our health.
Some commensal bacterial interactions with the
host have been viewed as benecial for both part-
ners, such as intestinal bacteria providing a source
of essential vitamins for host nutrition, or skin and
mucous membrane bacteria preventing disease by
occupying all of the available host niches precluding
pathogens from establishing an infection. However,
these processes have generally been viewed as pass-
ive, and the bacteria have not been regarded as ac-
tively participating in either nutrition or host de-
42
fense. Another explanation for the underestimation
of the active role that commensal bacteria contrib-
ute to normal tissue structure and function is that
these processes have been historically difcult to
study. This stands in stark contrast to the denition
of the role of bacterial pathogens which has taken
place over the last 100 years, commencing with
Kochs postulates. However, evidence that human
host-associated microbial communities may alter
host tissue development is now accumulating. The
recent developments in mammalian genomics-
based technologies, combined with the molecular
characterization of microbial recognition and re-
sponse components for the innate host defense sys-
tem, now enable the study of more subtle molecular
effects of commensal bacteria on mammalian devel-
opment and functional tissue maturation. For ex-
ample, it was recently demonstrated that coloniza-
tion of the intestines of germ-free mice with specic
commensal bacteria altered the expression of the
host genes involved in postnatal intestinal matu-
ration, including the fucosylation of epithelial glyco-
lipids (36). These sugar residues may serve as recep-
tors or nutrition for further specic bacterial colon-
ization.
The immune system may promote
benecial bacterial interactions
with host tissues
Two conceptual advances in the eld of immunology
have also provided a framework to understand the
potential role of commensal bacteria in host tissue
structure and function. One is that the idea that the
human immune system evolved to recognize nonself
(i.e. bacteria and other nonself entities) is being seri-
ously challenged with an alternative hypothesis (59)
which contends that the human immune system
evolved to recognize threats (i.e. danger from
nondanger). This idea, although originally proposed
to explain autoimmune disorders, has a profound ef-
fect on how we view commensal bacteria immuno-
logically. No longer do we need to view our com-
mensal bacteria as nonself entities which our tissues
have learned to tolerate, but rather we can entertain
the idea that under certain circumstances host
tissues may promote associations with those bac-
teria which benet the host (i.e. present no danger).
The immune system may recognize certain bacterial
types as nonthreatening and choose not to activate
a clearance response. Another conceptual advance is
the pattern recognition hypothesis originally pro-
posed by Janeway (38) to explain how the innate host
defense system evolved a mechanism to recognize
microbes immediately and mount a protective re-
sponse. In contrast to adaptive immunity, where
over a period of days to weeks highly specic anti-
bodies against bacteria are made through a process
of clonal selection from an abundant assortment of
randomly generated T- and B-cell repertoires, innate
responses, such as inammation, occur within min-
utes after bacterial challenge. The pattern recog-
nition hypothesis proposes that the innate host de-
fense system recognizes common conserved struc-
tures, such as lipopolysaccharide, found in a variety
of different microbes rather than the specic bac-
teria. In this way a limited number of host proteins
can detect the wide variety of different microbes
present in the environment (63). These proteins,
designated pattern recognition receptors, continu-
ally monitor the state of host microbial colonization
and elicit appropriate host responses depending
upon the microbial structures detected. Pattern rec-
ognition receptors therefore provide a link facilitat-
ing a molecular dialogue between our commensal
bacteria and the appropriate host response.
The innate host response is
specic
The identication and characterization of several
key host pattern recognition receptors such as lipo-
polysaccharide binding protein (LBP), CD14, and the
Toll-like receptor family (TLR) of membrane recep-
tors have revealed that the innate host defense sys-
tem demonstrates an exquisite microbial specicity.
In contrast to the highly characterized complement
system, where innate defense microbial specicity is
determined by host-directed down-regulation of a
nonspecic highly reactive internal thioester bond in
complement protein C3, pattern recognition recep-
tor microbial specicity results from a dialog be-
tween host and microbe. Initially microbial compo-
nents such as lipopolysaccharide from gram-nega-
tive bacteria or lipoteichoic acid from gram-positive
bacteria bind host pattern recognition proteins such
as LBP or CD14 and are then transferred to other
host proteins such as the TLRs. Lipopolysaccharide
or lipoteichoic acid binding to LBP or CD14 is not
sufcient to elicit host cell activation because of the
lack of cell signaling domains in these molecules. In-
stead, under the appropriate conditions the transfer
43
to the TLRs that are capable of transmitting a signal
into the cell via their interleukin-1 receptor-like sig-
naling domains (24,76) will result in responses such
as the secretion of cytokines or the expression of cell
adhesion molecules. Alternatively, these molecules
may transfer either lipopolysaccharide or lipotei-
choic acid to other nonactivating serum proteins,
such as serum lipoproteins, for eventual removal of
the offending bacterial components from the system
without local activation of the innate host response
(29). Both the type of microbial component detected
by the innate host response system and the availabil-
ity of different host proteins capable of interacting
with the microbial ligand create the dialog that de-
termines what type of host response is elicited.
Several different types of microbial components
have been shown to elicit innate host responses (93).
This has mainly been demonstrated through the
identication of the member of the TLR family that
interacts with the particular microbial component
(see Table2). For example, Toll-like receptor 2 (TLR2)
has been implicated in response to lipoteichoic acid,
lipoproteins, mbriae and peptidoglycan; TLR3 in
response to double-stranded RNA; TLR4 in response
to lipopolysaccharide; TLR5 in response to agella;
and TLR9 in response to DNA. Evidence is accumu-
lating that each TLR yields a different type of host
response by activation of different host cell acti-
vation pathways (2). This represents one mechanism
that the host employs to differentiate between differ-
ent microbial components. Each of the microbial
components recognized by the TLRs are highly con-
served and found in a wide variety of different bac-
teria, consistent with the pattern recognition hy-
pothesis, however, there are ne structural differ-
ences between species in some of these components
that can inuence the host response. For example,
lipopolysaccharide species isolated from diverse
bacteria have different numbers of fatty acid side
chains and phosphate groups that result in different
binding afnities to LBP and transfer rates to soluble
CD14 (sCD14), affecting their entry into the host ac-
tivation pathway (13). Indeed, it has been shown that
Salmonella species can modify the type of fatty acid
side chains on their lipopolysaccharide to alter host
responses (28). Therefore, another mechanism by
which the host is able to discern host-associated mi-
crobial populations is based upon how tightly the
different microbial components are bound to their
appropriate pattern recognition receptors.
The quantity of the different host proteins avail-
able to interact with the microbial ligands also in-
uences the type of innate host response. For ex-
Roberts & Darveau
ample, the levels of both LBP and sCD14 are inu-
enced by bacterial infection, and in this way the host
can augment its response. LBP is an acute-phase
protein made in the liver, and its level in serum can
increase up to eight-fold during infection (71).
Studies have shown that increased levels of LBP can
attenuate the inammatory response to lipopolysac-
charide presumably by directing more lipopolysac-
charide to serum lipoproteins for removal as op-
posed to funneling them to TLRs for host activation
(29). Similarly, the levels of sCD14 in gingival crevic-
ular uid have been shown to be signicantly higher
than those present in serum, indicating that this pat-
tern recognition receptor is made locally in response
to plaque bacteria (39). Furthermore, higher levels of
sCD14 in gingival crevicular uid were associated
with fewer and shallower pockets in a survey of peri-
odontitis patients. The high levels of sCD14 may
serve a protective role by enhancing phagocytosis of
pathogenic plaque bacteria.
Another mechanism by which the host is able to
regulate its innate host response to bacteria is by
varying the TLR expression repertoire on different
cell types. The expression of TLRs has been shown
to be inuenced by bacterial components as well as
by host signaling molecules (2). The expression of
different TLRs can profoundly inuence the ability
of a cell to respond to a microbial component. For
example, it has been shown that human endothelial
cells express more TLR4 than TLR2, accounting for
their ability to respond to lipopolysaccharide (a
TLR4 ligand) but not to Mycobacterium lipoprotein
(a TLR2 ligand). Endothelial cells were activated by
the lipoprotein, however, when the level of TLR2 was
increased through transfection (24). Likewise, hu-
man intestinal epithelial cells do not normally re-
spond to lipopolysaccharide, however, if they are
Table2. Toll-like receptors and their associated microbial ligands
TLR Ligand Source organisms References
2 lipoteichoic acid Gram-positive (72, 79, 85)
lipoproteins Gram-positive, gram-negative, mycoplasma (9, 26, 35, 87)
mbriae Gram-negative (5)
peptidoglycan Gram-positive (33, 64, 86)
3 double-stranded RNA Viruses (3)
4 lipopolysaccharide Gram-negative (74, 75, 78, 81)
5 agella Gram-negative (31)
9 CpG DNA Bacterial DNA (32)
Adapted from Vasselon & Detmers (93).
44
transfected with TLR4 (the ligand for lipopolysac-
charide), a response is elicited (1).
The innate host responses elicited by pattern rec-
ognition receptor binding to microbial components
form a highly coordinated and dynamic process. It
is not surprising therefore that the well-known mi-
crobial composition shift from mostly gram-positive
to gram-negative anaerobic species found during the
development of gingivitis and periodontitis (see be-
low) signicantly alters inammatory mediator ex-
pression patterns in the periodontium. In this review
we propose that this dynamic process is also respon-
sible for the highly orchestrated gradient of select in-
ammatory mediators that keep the periodontal
tissues healthy.
The oral commensal microbial
community.
The oral commensal community has a long history
of characterization initiated in 1683 by van
Leeuwenhoeks discovery of animalcules in gingival
tooth scrapings (18). Although studies from the be-
ginning of the twentieth century were descriptive in
character, they progressed with the use of electron
microscopy and advanced culturing techniques, and
now involve analyses of biolm interactions and
molecular ribotyping. As Socransky & Haffajee de-
scribe in their excellent historical perspective on the
microbiology of periodontal disease, work from the
early 1900s examined the role of amebae, spiro-
chetes, fusiforms, and streptococci in the etiology of
periodontitis, but little was published about those
species present in healthy subjects (83). Appropri-
ately, the emphasis in these studies was to determine
potential microbial etiological agents of peri-
odontitis. More recent studies (49, 51, 82, 98), how-
ever, have speciated many of the organisms thought
to be associated with periodontal health and gingi-
vitis, including several streptococcal species as well
as Actinomyces, Veillonella, and many others (see
Table3). Extensive culture studies by Moore et al.
found certain species, including Actinomyces, Strep-
tococcus, and Veillonella to be associated with health
while more gram-negative species, treponemes, and
higher numbers of Fusobacterium nucleatum were
associated with disease (6669).
The advent of DNA probe analysis methods have
allowed the detection of multiple bacteria concur-
rently and have conrmed and further dened the
shift from mostly gram-positive to gram-negative
species in the transition from health to disease. Ap-
plying a combination of multiple cluster and com-
munity ordination statistical methods to the evalu-
ation of the DNA probe results for 40 taxa of bacteria
from subgingival plaque samples of periodontally
healthy and disease-affected patients, Socransky et
al. found that many of the bacterial taxa appeared
to cluster together including those associated with
gingival health (84). His green cluster included Cap-
nocytophaga species, Campylobacter concisus, Eub-
acteria nodatum and Streptococcus constellatus. The
yellow cluster was formed by a group of streptococci,
and the purple cluster included Actinomyces odonto-
lyticus and Veillonella parvula. These species tended
to occur together in the periodontal crevice and did
not associate with increasing pocket depth or gingi-
val bleeding. Interestingly, in other studies the spe-
cies from those clusters associated with periodontal
health were found to be unaffected or even in-
creased following scaling and root planing (30) and
periodontal maintenance procedures (92). Recently,
Ximenez-Fyvie et al. examined supra- and subgingi-
val plaque in clinically healthy subjects and in peri-
Table3. Microbiology of the periodontal region
Health Gingivitis
Streptococcus sanguis Actinomyces species
Streptococcus mitis Streptococcus species
Veillonella parvula Veillonella species
Actinomyeces naeslundii Fusobacterium species
Actinomyces viscosus
Rothia dentocariosa Prevotella intermedia
Adapted from Listgarten (51) and Darveau et al. (17).
45
odontitis patients for the same 40 bacterial taxa in a
DNA checkerboard analysis and found similar spe-
cies in both supra- and subgingival plaque samples
from healthy and diseased sites (97). However, they
observed a higher mean prevalence of the Acti-
nomyces species in health, with the diseased
(deeper) sites tending to have higher counts of bac-
teria overall as well as greater proportions of the
more pathogenic orange and red complexes of
bacteria including Bacteroides forsythus, Porphyro-
monas gingivalis, Treponema denticola, and Prevotel-
la intermedia (84).
The magnitude of the complexity of the microbial
communities present in the oral cavity has been
further elucidated through ribotyping studies. Using
assay systems with exquisite sensitivity (polymerase
chain reaction-based), hundreds more species of bac-
teria have been detected in the gingival crevice, over
half of which have not been previously identied (47).
As we gain more knowledge of the ora of the peri-
odontium, further denition of the microbial species
associated with health and disease will occur.
Dental plaque is a highly
organized consortium of
co-operating bacterial species that
maintains a long-term
relationship with the host
Recognition of the highly complex nature of dental
plaque came from several sources, including the
electron microscopic studies of Listgarten, the inter-
species coaggregation studies of Kolenbrander and
others (43, 45, 48, 51), and 16S rDNA sequence
analysis (47). That dental plaque exists as a biolm
of interacting microbial colonies has become gener-
ally accepted and supported by several studies (7,
12, 44, 58). The highly specic nature of the biolm
community associated with health is demonstrated
by the ordered microbial succession observed after
cleaning the tooth surface. Following oral hygiene
measures, the tooth is immediately coated by a sali-
vary pellicle. This pellicle provides ligands and re-
ceptors for the attachment of many of the gram-
positive, but not gram-negative, species that com-
prise the initial colonizers of the tooth and are also
associated with periodontal health. The commensal
bacteria that reside on the tooth surface also employ
highly specic adhesion methods to facilitate each
others attachment (95). Experiments involving eluci-
dation of particular proteins used in the aggregation
Roberts & Darveau
and physical co-operation of oral microorganisms in
the biolm have begun, but there is a need for
studies using growing bacteria to augment our
understanding of this area (21, 22, 94). The biolm
on the teeth and other oral structures demonstrates
communities of organisms with multiple nutritional
needs interacting to benet each other.
The molecular consequences that are associated
with the highly specic succession of microbial spe-
cies that occurs as dental plaque develops leave little
doubt that these microbial communities are not a
haphazard collection of bacteria encountered in the
environment but rather have evolved to act as a
community capable of maintaining a long-term re-
lationship with the healthy periodontium. In fact it
is possible that in a similar manner to the vectoral
translocation of symbiotic insect ora, humans show
vertical transmission of oral commensal organisms
to their progeny. Studies of both gram-positive and
gram-negative oral species have demonstrated the
parentchild transmission of streptococci, P. gingi-
valis, and Actinobacillus actinomycetemcomitans (6,
10, 90), but further work examining the transmission
of protective species is needed.
The oral innate host defense status
of healthy periodontal tissue
Histological examination of periodontal tissues has
demonstrated the absence of tight junctions be-
tween the cells of the junctional epithelium, provid-
ing a tissue permeable to bacterial products (50, 60,
65, 80). This structure provides a means for the cells
of the periodontium to sample the crevicular en-
vironment and respond accordingly. The pioneering
work of Page & Schroeder demonstrated the pres-
ence of a mild inammatory inltrate in clinically
healthy tissues that increased with the diagnosis of
gingivitis (73). The cellular inltrate included neu-
trophils, lymphocytes, and monocytes, all of which
are capable of quickly responding to alterations in
the epithelial barrier or to changes in the local ora
(46). This is particularly important since the dental
complex is unique in that a calcied tissue pen-
etrates the epithelium in a particularly nonsterile en-
vironment. Therefore the protective mechanisms
present at this junction may be unique for limiting
pathogen entry into the deeper tissues. These mech-
anisms include the protective effects of the host in
response to the activities of the normal ora.
The innate host response in clinically healthy
46
tissue is highly orchestrated. Studies of cellular acti-
vation in the healthy periodontal crevice show a dis-
tinct gradient of the pro-inammatory cytokine in-
terleukin-8 and the adhesion molecule intercellular
adhesion molecule-1 from the plaque margin to the
deeper connective tissue (see Fig. 2). These mol-
ecules attract neutrophils to the junctional epithel-
ium and ready the tissue for potential pathogenic in-
sult (88, 89). Endothelial cells are also activated for
the production of E-selectin which serves as a
handle to recruit inammatory cells continuously
from the circulation to the periodontal tissues (17).
It is our contention that those bacteria associated
with health, including members of the green, yellow,
and purple complexes, induce this gradient.
Periodontal pathogens may
disrupt the benecial microbial
host dialog
Certain periodontal pathogens, such as P. gingivalis,
are adapted to survival in the pocket, and their outer
membrane lipopolysaccharide is capable of down-
regulating E-selectin and interleukin-8 (15, 16, 37,
55), and thus potentially interrupting the local
supercial inammatory response. This helps the
bacteria to avoid elimination while maintaining
deeper inammation that provides the organism
with needed nutrients in the form of increased cre-
vicular uid ow and blood products from the ulcer-
ated pocket epithelium. Cutler et al. have studied the
role of the dendritic cell and CD4
T helper cell (T
H
)
in control of the immune response, and its regula-
tion by lipopolysaccharide from various bacteria (14,
40). Although classical Escherichia coli lipopolysac-
charide actively induced T
H
1-type responses (gener-
ally pro-inammatory) including high levels of
gamma interferon (IFN-g), P. gingivalis lipopolysac-
charide induced no IFN-g in T
H
cells. Similarly, E.
coli lipopolysaccharide induced production of in-
terleukin-12, a pro-inammatory cytokine, by den-
dritic cells while P. gingivalis lipopolysaccharide did
not. The ability of P. gingivalis to affect these cellular
reactions suggests that this periopathogen can alter
benecial innate and adaptive host responses.
Summary
The interaction between bacteria and host in the
gingival pocket results in relative homeostasis with
occasional aggravated inammation and attachment
loss. When the average lifespan for a 20-year-old-
person (ignoring the effects of childhood illnesses on
mortality) was 42 additional years, or fewer, in the
United States pre-1900 (4), the slow progression of
chronic periodontal disease could allow most per-
sons to retain adequate function for the majority of
their lives. This is seen in modern studies of rural
populations in the developing countries who are
without access to dental care, as well as in studies of
eighteenth century England and of prehistoric popu-
lations where the majority of the people retained
most of their dentition in a functional state to age
65 and older (11, 41, 42, 57). Studies examining the
natural history of periodontal disease in the Sri Lan-
kan tea-worker population (5254, 70) have now re-
ported 20-year data for subjects with a mean age of
42.14.3years, which included subjects up to age
50. Although 100% of the 50-year-olds exhibited gin-
gival recession, the average attachment loss was 5.0
2.0mm. Certainly severe forms of periodontitis
can have a rapidly debilitating effect on oral func-
tion, but these forms are more rare in all popula-
tions. The co-evolution of bacterial species and the
mammalian oral environment have allowed a co-op-
erative system that has usually provided adequate
functioning of the periodontal apparatus throughout
life for the last several million years independent of
adequate professional and home care. Human
beings generally do well with regards to their peri-
odontal status because we have co-evolved with the
commensal bacteria that serve to protect us through
promotion of a benecial host response. However,
this host-bacterial balance is dependent on the spe-
cic genetic markers of each individual (major histo-
compatibility complex type, gene polymorphisms,
etc.), environmental factors (smoking, stress, etc.),
and the continually evolving microbial community.
A more thorough understanding of these factors
should lead to improved periodontal health in the
twenty-rst century.
Acknowledgements
The authors gratefully acknowledge the assistance of
Brian Bainbridge for manuscript content and Laura
Houston for rendering of gure 2. They also thank
the participants of the Benecial Microbial Work-
shop, which was sponsored by the National Institute
for Dental and Craniofacial Research and held dur-
ing November 2001 in Seattle. Discussions that oc-
47
curred at the workshop helped to formulate and re-
ne many of the concepts presented in this review.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Trends in periodontal care
PnuI B. Rourn1soN, MicnnrI A. nrI AcuiIn & MnxwrII H. ANnrnsoN
The purpose of this review is to assess future trends
in periodontal care provided in the private ofces
of general dentists, periodontists and other dental
specialists. The issue is important to developing oral
health care policies that are consistent with the
changing dental needs and demands of the popula-
tion. Such estimates can help provide an indication
of future requirements for the magnitude and train-
ing of the dental workforce.
A number of approaches have been used to pre-
dict future patterns of periodontal care. Foremost is
the use of data reported by periodic epidemiological
studies to determine changes in the prevalence and
severity of the periodontal diseases in various seg-
ments of the population (13, 1012, 15, 17, 19, 23,
26, 27, 28, 30). Interpretations of these epidemio-
logical results have led some to predict declines in
periodontal services, particularly surgical therapy,
based on a decrease in the prevalence and severity of
periodontitis (29, 31, 32). Others suggest escalating
trends in periodontal care as a result of a relatively
intact dentition retained by the population for an in-
creasingly long lifetime (18, 25). For purposes of
forecasting trends in periodontal treatment, this dis-
ease prevalence approach has been controversial. A
substantial literature raises concerns about a wide
range of methodological problems associated with
epidemiological studies of periodontal diseases, in-
cluding differences in study population demo-
graphics, case denitions for the various forms of
periodontal disease, numbers of teeth or sites re-
corded, and methods of expressing clinical ndings
(58, 35, 35, 37). Recent reviews of periodontal epi-
demiology suggest that conclusions about changes
in the prevalence of chronic periodontitis are not
possible at present (13, 33). Also, studies of disease
prevalence would not address changes in dental
practice patterns that might accrue from patients
demands for treatment to improve esthetics or qual-
ity of life.
Dental benet carriers have the potential to pro-
104
vide highly current and reliable information about
changing patterns of oral care (4, 9, 22, 24). Cur-
rently, dental benets companies manage the ma-
jority of dentist ofce revenues in the U.S.A. (36).
Recent retrospective studies using treatment data
from dental carriers have documented major im-
provements in oral health among insured patients
and have suggested a number of factors that may
affect patterns of oral health care for the entire
population (20, 21). We recently reported patterns of
oral health care in an insured Washington state
population for 1993 and 1999 to assess changes in
patient populations, practice characteristics, relative
proportion of procedures, and treatment costs (16).
For both years, general dental ofces were respon-
sible for more than 80% of patient care. Broad cate-
gories of service were similar in 1993 and 1999. The
mix of services varied considerably by patient age,
and between generalists and all specialists in both
years. We used the same database to assess specic
changes in periodontal services.
Washington Dental Service Studies
More than 70% of the Washington state population
has some form of dental benets. The Washington
Dental Service (WDS), a founding member of the na-
tional Delta Dental Plans Association, is the leading
dental benets company in the State of Washington.
WDS provides dental benets to about 30% of the
state population through employer-sponsored pro-
grams. More than 90% of dental ofces in the State
of Washington report claims for reimbursement to
WDS for some portion of patients in their practice.
About 62% of WDS patients participated in fee-for-
service plans, 36% in preferred-provider plans, and
3% in managed-care plans.
Since 1993, WDS has maintained a claims-based
data warehouse that captures all claims data as well
as other major components pertaining to patients,
dentists, purchasers, services rendered, and dental
plan design. The relational nature of the data ware-
house allows longitudinal tracking of individual vari-
ables with granularity down to the individual tooth
surface. The WDS warehouse database is validated
at both treatment planning and completion phases
by random or indicated patient record audit, includ-
ing radiographic conrmation of procedures com-
pleted. In addition, a random chart audit of 200
claims for variables included in this investigation
was conducted. The percentage agreement between
paid claims and patient records was about 97%.
For this assessment of practice trends among gen-
eralists, periodontists and other specialist ofces, we
queried all procedures provided by dental ofces to
all unique patients covered by WDS in 1993 and
1999. About 30% of patients and more than 70% of
dentists in 1999 were also associated with WDS in
1993. None of the analyses involved the identities of
either individual patients or dental professionals.
Data values reported are for persons who had at least
one claim in 1993, 1999, or both years. Patients and
dental team members were limited to those re-
ceiving services or practicing in the State of Wash-
ington. Treatment values in each year reect both
those performed by the dentist as well as those pro-
vided by other members of the dental ofce team.
American Dental Association (ADA) numeric pro-
Table1. Unique patients, and dentists, procedures, expenditures, and patient visits associated with generalists,
periodontists and other specialists in 1993 and 1997
1993 1999 Change
n % n % %
Unique patients 620,293 880,317 41.9
Dentists 2,956 3,402 15.1
Generalist 2,499 84.6 2,801 82.3 12.1
Periodontist 88 3.0 112 3.3 27.3
Other specialist 369 12.4 489 14.4 32.5
Procedures 4,321,256 5,782,729 33.8
Generalist 3,843,191 88.9 5,072,856 87.7 32.0
Periodontist 84,218 1.9 108,177 1.9 28.4
Other specialist 393,847 9.1 601,696 10.4 52.8
Expenditures $306,520,490 $511,084,696 66.7
Generalist $253,478,685 82.7 $414,672,671 81.1 63.6
Periodontist $11,105,036 3.6 $16,859,385 3.3 51.8
Other specialist $41,936,769 13.7 $79,552,640 15.6 89.7
Patient visits 1,852,516 2,738,821 47.8
Generalist 1,651,301 89.1 2,398,781 87.6 45.3
Periodontist 39,823 2.1 60,972 2.2 53.1
Other specialist 161,392 8.7 279,068 10.2 72.9
105
cedure codes were used to group procedures. Null
and irregularly coded records in the database were
infrequent and loss of data for any multiple of vari-
ables assessed never exceeded 2%.
Practice characteristics
An overview of unique patients represented by WDS,
and dentists, procedures, expenditures and patient
visits associated with generalists, periodontists and
other specialists in 1993 and 1997 is shown in Table1.
The number of unique patients withat least one claim
was greater in 1999 than in 1993 by 42%. During the
same 6-year period, the total population in the State
increased by 9.1%, from 5.24 million to 5.75 million
persons (14). Of the 880,317 unique patients in 1999,
309,339 patients (35.1%) also had a reported claim in
1993. For both years, a slight majority of patients were
female and were either a spouse/partner or depend-
ent of the primary subscriber. Of 3,402 unique den-
tists who submitted claims to WDS in 1999, 2,452
(72.1%) also submitted claims in 1993. Generalists,
periodontists and other specialists comprised 84.6%,
3.0%and12.4%of all dentists in1993 and82.3%, 3.3%,
and 14.4% of all dentists in 1999, respectively. Other
specialists for this review included dentists with ad-
vanced training in endodontics, pediatric dentistry,
Robertson et al.
Table2. Dental ofce practice characteristics in
Washington State in 1993 and 1999
1993 1999 Change
Visits per ofce 626.7 805.1 28.5%
Generalist 660.8 856.4 29.6%
Periodontist 452.5 544.4 20.3%
Other specialist 437.4 570.7 30.5%
Procedures per ofce 1,462 1,700 16.3%
Generalist 1,538 1,811 17.8%
Periodontist 957 966 0.9%
Other specialist 1,067 1,230 15.3%
Procedure per patient 2.3 2.1 9.5%
Generalist 2.3 2.1 9.1%
Periodontist 2.1 1.8 16.1%
Other specialist 2.4 2.2 11.6%
Costs per patient $165.46 $186.61 12.8%
Generalist $153.50 $172.87 12.6%
Periodontist $278.86 $276.51 0.8%
Other specialist $259.84 $285.07 9.7%
prosthodontics, orthodontics, oral and maxillofacial
surgery and oral pathology/oral medicine. The num-
ber of procedures reported by all WDS dental ofces
increased by about 34% from 1993 to 1999. This
growth in the number of procedures was lower for
periodontists than generalists and other specialists.
Expenditures for bothyears represent combinedcosts
paid to dental ofces by WDS and by the patient.
These combinedpatient andWDS expenditures for all
procedures in 1993 were adjusted by the Western
United States Consumer Price Index (CPI-U) to reect
1999 dollars. The percentage of total costs paid by the
patient was 29%in 1993, and 28%in 1999. While com-
bined expenditures for all dental services increased
from 1993 to 1999, the percentage distribution of ex-
penditures increased for other specialists with a con-
current decrease among generalists and peri-
odontists. The change in distribution of patient visits
among the three provider groups was similar to that
of expenditures.
In general, ratios between the two years for all pa-
tients and dentists showed an increase in procedures
per dental ofce, expenditures per procedure, and
expenditures per patient and a decline in number
of dentists per patient and procedures per patient.
However, the percentage change in practice trends
from 1993 to 1999 varied considerably among the
three provider groups (Table2). The increase in pa-
tient visits per dental ofce was less for periodontists
(20.3%) than generalists (29.6%) and other specialists
(30.5%). Procedures per dental ofce increased for
106
generalists (17.8%) and other specialists (15.3%) but
rose only slightly for periodontists (0.9%). The de-
crease in procedures per patient was greater for peri-
odontists (16.1%) than for generalists (9.1%) and
other specialists (11.7%). Expenditures per patient
showed an increase for generalists (12.6%) and other
specialists (9.7%) but were decreased for peri-
odontists (0.8%).
Dental expenditures and
procedures
The percentage of total expenditures by WDS and
patients for dental care in 1999 is shown in Fig. 1.
About two-thirds of all dental costs were for exami-
nations, radiographs, cleaning, uoride/sealants,
restorations, and single crowns. Periodontal pro-
cedures (ADA procedures codes 40004999) totaled
$32.7 million or about 6.4% of all dental expendi-
tures for care supported through WDS in 1999. Re-
maining dental costs were divided primarily among
endodontic, orthodontic, and oral surgical services.
Table3 shows the proportion of procedures and
expenditures that were reported by generalists, peri-
odontists and other specialists for ADA service cate-
gories in 1999. General dental ofces were respon-
sible for more than 90% of both total procedures and
total expenditures for diagnostic, preventive, restora-
tive, xed prosthodontic and removable prosthodon-
tic services, and provided a varying proportion of
care in the remaining categories. For periodontal
services, periodontists completed about 21% of all
Fig. 1. Percentage distribution of total expenditures by
Washington Dental Service and patients for 1999.
procedures, and received about 36% of all expendi-
tures. The proportion of procedures and expendi-
tures for implants was divided among generalists
(27%, 13%), periodontists (35%, 48%), and other
specialists (38%, 40%), the majority of whom were
oral and maxillofacial surgeons.
Changes in expenditure rates
Table4 shows the number of periodontics services
(ADA codes 40004999) per 1000 patients served by
WDS in 1993 and 1999 for general practitioners and
periodontists, and the percentage change in those
rates between the two years studied. Since scaling/
root planing and periodontal maintenance pro-
Table3. Percentage of procedures and expenditures reported by generalists, periodontists and other specialists
for the American Dental Association service categories in 1999
Procedures Expenditures
General Periodontist Other General Periodontist Other
Diagnosis and prevention 90.9% 1.0% 8.1% 90.8% 1.3% 7.9%
Restorations and prosthetics 95.3% 0.2% 4.5% 96.4% 0.2% 3.4%
Periodontics 78.3% 20.5% 1.2% 63.5% 35.6% 0.9%
Implants 27.2% 34.5% 38.3% 12.9% 47.5% 39.6%
Endodontics 63.0% 0.5% 36.5% 47.6% 0.4% 52.0%
Orthodontics 49.4% 0.2% 50.4% 49.2% 0.2% 50.6%
Oral surgery and adjunctive 48.2% 2.6% 49.2% 27.3% 2.0% 70.7%
general services
Table4. Rates of periodontics procedures per 1,000 patients served by the Washington Dental Service in 1993
and 1999 for general practitioners and periodontists
General practitioners Periodontists
1993 1999 % Change 1993 1999 % Change
Scaling/root planing and 195.98 231.44 18.10% 48.22 45.74 5.15%
periodontal maintenance
Gingivectomy 2.45 2.30 5.87% 1.21 0.84 30.99%
Periodontal surgery 0.94 0.65 31.62% 10.02 6.10 39.11%
Periodontal re-evaluation 3.56 1.92 46.05% 2.00 1.63 18.58%
Soft tissue grafts 0.68 0.43 36.07% 5.95 5.34 10.28%
Crown lengthening 0.38 0.68 80.44% 0.38 2.55 572.96%
Other 1.34 0.56 58.16% 0.15 0.29 92.02%
Total 205.32 237.99 15.91% 67.93 62.48 8.02%
107
cedures are often reported interchangeably and
since reimbursement policies for these procedures
changed slightly during the study, we are more con-
dent in a combined subtotal for both nonsurgical
procedures. Compared to 1993, the number of scal-
ing/root planing and periodontal maintenance pro-
cedures per 1000 patients completed in 1999 in-
creased about 18% for general dental ofces and de-
creased by 5% for periodontist ofces. The majority
of clinical activity for nonsurgical periodontal ther-
apy in both years occurred in general dental ofces.
Considered collectively, procedure rates for surgi-
cal periodontal services showed substantial de-
creases for both generalists and periodontists. Gingi-
vectomy/gingivoplasty was performed more fre-
quently by generalists than periodontists, but
decreased in both practice groups. The majority of
Robertson et al.
all gingivectomy procedures were reported by tooth
rather than by quadrant. Periodontal surgery in
Table4 includes gingival ap procedures, apically
positioned ap, and osseous surgery that was per-
formed with or without bone replacement grafts or
guided tissue regeneration. The grouping also in-
cluded distal or proximal wedge procedures and sur-
gical revisions of previously provided surgery. These
surgical approaches for the management of peri-
odontitis were combined to minimize reimburse-
ment-related shifts in claims among surgical pro-
cedures and to account for new techniques de-
veloped since 1993. In general, periodontal surgical
services were essentially within the province of peri-
odontists and showed an almost 40% decrease in
rate from 1993 to 1999. Periodontal re-evaluation
visits subsequent to treatment of periodontitis also
declined for both generalists and periodontists.
Soft tissue grafts, comprising pedicle grafts, free
gingival grafts and subepithelial connective tissue
grafts, were performed primarily by periodontists
and the procedure rate in the WDS patient popula-
tion decreased more than 10% during the 6-year
period. The rate of crown lengthening procedures in-
creased about two-fold for generalists and about
seven-fold for periodontists. Other services encom-
passed all remaining procedures in the ADA peri-
odontics category. The increased rate of other ser-
vices among periodontists stemmed largely from ex-
panded clinical activity in localized delivery of
chemotherapeutic agents, provision of periodontal
appliances, minor tooth movement, and special pro-
cedures by report. While WDS provided reimburse-
ment for gingival curettage and periodontal splinting
throughout the study period, the rates of these pro-
cedures were minimal in both years for both general-
ists and periodontists. The net result of these rate
changes in all periodontics procedures reported to
WDS in 1993 and 1999 was a 16% increase in clinical
activity by generalists and an 8% decrease in activity
by periodontists.
Trends in the practice of
periodontics
Figures2(a) and (b) compare the distribution of ex-
penditures that were paid to the ofces of peri-
odontists by WDS and patients for groups of services
in 1993 and 1999. The relative proportion of com-
bined dollars expended for services is a useful out-
come measure of dental practice activity because it
108
reects both the number of procedures completed as
well as the time required and complexity associated
with the procedure. The clinical activity and related
revenue of periodontists were characterized by
marked decreases in periodontal surgical procedures
and increases in implant services, crown lengthening,
and nonsurgical therapy in 1999 compared to 1993.
Conclusions
In order to assess projected changes in periodontal
care, we reviewed the WDS benet claims for 1993
Fig. 2. Distribution of Washington Dental Service and pa-
tient costs paid to the ofces of periodontists in (a) 1993
and (b) 1999.
and 1999 for treatment delivered in the state of
Washington. For both years, the survey represented
about 1.25 million patients, 3,400 general, peri-
odontist and other specialty ofces, 10.1 million pro-
cedures and $817.6 million combined WDS and pa-
tient costs. For this patient population, periodontists
comprised about 3% of all dentists and were respon-
sible for about 2% of all procedures, and 3% of all
expenditures by WDS and patients. The number of
WDS patients and related procedures, as well as
combined expenditures for dental care increased
substantially during the 6-year period. This expan-
sion of dental care was borne primarily by general
dental and other specialty ofces.
The majority of clinical activity in periodontics oc-
curred in general dental ofces. This activity was
dominated by periodontal maintenance, which in-
creased markedly from 1993 to 1999. Implant ser-
vices also grew, the relative proportion of which was
shared by generalists, periodontists and oral and
maxillofacial surgeons. Most periodontal surgical
procedures were provided in periodontics ofces.
Periodontal surgery declined by 40% and soft tissue
grafts declined by 10%, while crown lengthening and
other nonsurgical clinical activity increased substan-
tially.
Observations in these Washington State patients
with WDS dental benets are consistent with projec-
tions suggesting a decreased need for periodontal
surgery concurrent with a decline in the prevalence
of periodontitis. The availability of these data in a
more constant fashion could augment the denition
of prevalence by using treatment as a proxy for dis-
ease. The overall trends here also document an in-
crease in nonsurgical therapy and maintenance care
predicted for a population that retains the dentition
later in life. A surprising feature of these results was
the magnitude of changes in periodontal care pat-
terns during a relatively short period of time.
These changes in periodontal care disproportion-
ately affected the specialty practice of periodontics.
At the same time, the rate of increase in number of
practitioners between 1993 and 2000 was greater for
periodontists than general practitioners, but less
than for other specialists. Moreover, the average cost
per patient (including WDS payment and patient co-
payment) actually decreased for periodontists dur-
ing this period, while it increased for generalists and
other specialists taken as a group.
These trends among an insured U.S. patient popu-
lation highlight the need to monitor closely the en-
vironment in which future generalists, periodontists
and other specialists will practice. For those patients
109
with dental benets, there has been a clear shift in
periodontal care from surgical treatment associated
with periodontitis to crown length and cosmetic
treatment, as well as to implant and preventive ser-
vices. Trends in this and other populations suggest
that future oral care will increasingly focus on pre-
vention in younger patients, and repair and main-
tenance of a relatively intact dentition in adults. For
the practice of periodontics, changes in patterns of
care are occurring rapidly.
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Robertson et al.
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Lindhe J, Mitsis F. Comparative estimation of periodontal
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PERIODONTOLOGY 2000
ISSN 0906-6713
Classifying periodontal
diseases a long-standing
dilemma
G C. A
A long-standing dilemma
Any attempt to group the entire constellation of peri-
odontal diseases into an orderly and widely accepted
classication system is fraught with difculty, and
inevitably considerable controversy. No matter how
carefully the classication is developed, and how
much thought and time are invested in the process,
choices need to be made between equally unsatis-
factory alternatives. Despite this dilemma, in the
past hundred years, experts have periodically as-
sembled to develop a new classication system for
periodontal diseases, or to rene an existing one (1,
2, 4, 5, 11, 19, 58, 80, 81, 86, 91, 106, 122, 139).
Dominant paradigms in the
historical development of
classication systems
The development and evolution of classication sys-
tems for periodontal diseases have been largely in-
uenced by paradigms that reect the understand-
ing of the nature of periodontal diseases during a
given historical period. Over time, thoughts that
guided the classication of periodontal diseases can
be placed into three dominant paradigms primarily
based on the clinical features of the diseases
(18701920), the concepts of classical pathology
(19201970), and the infectious etiology of the dis-
eases (1970present). Classication systems in the
modern era represent a blend of all three paradigms
since there is a certain amount of validity to some of
the earliest thoughts about the nature of periodontal
diseases (2, 4, 5). As classication systems have
evolved, newer thoughts about periodontal diseases
9
have been superimposed on a matrix of older ideas
that are still considered to be valid. Only those ideas
that are believed to be clearly outmoded or incorrect
have been discarded. In a sense, the newest or domi-
nant paradigm rests on a foundation of the still valid
components of the older or previous paradigms.
One of the interesting historical features of classi-
cation systems is the often intense resistance to
their modication. Many people appear to believe
that classication systems are rigid and xed entities
that should not be changed. In fact, classication
systems should be viewed as dynamic works-in-pro-
gress that need to be periodically modied based on
current thinking and new knowledge. Unfortunately,
it seems that once people learn and accept a given
classication, no matter how awed it may be, they
are extremely reluctant to accept revisions to their
favorite system of nomenclature. One group of ex-
perts on the 1949 Nomenclature Committee of the
American Academy of Periodontology (AAP) ex-
pressed their frustration with this subject by stating,
The 1949 Nomenclature Committee is somewhat
pessimistic regarding the possibility of this or any
similar report receiving immediate and enthusiastic
acceptance. Most periodontists have used their own
terms for so long that any suggested change is re-
sisted and resented. (81)
Clinical characteristics paradigm
For the period from approximately 1870 to 1920 very
little was known about the etiology and pathogenesis
of periodontal diseases. Accordingly, the diseases
were classied almost entirely on the basis of their
clinical characteristics supplemented by unsubstan-
tiated theories about their cause. At the time, one
of the main debates about the nature of periodontal
Armitage
diseases was whether they were caused by local or
systemic factors. Most authors considered these dis-
eases to be primarily caused by local factors (16, 53,
93, 111, 112, 125, 127, 136, 149), whereas some be-
lieved that systemic disturbances played a dominant
etiological role (32, 97, 114, 115). Many of the advo-
cates for the etiological role of local factors also ac-
knowledged that in some cases both local and sys-
temic factors were important (93, 112, 113, 136). In
the late 1800s and early 1900s clinicians used case
descriptions and their personal interpretation of
what they saw clinically as the primary basis for
classifying periodontal diseases (15, 17, 28, 53, 111
113, 125, 127, 136, 137, 149). They expressed their
opinions, often with great fervor and conviction, in
oral presentations before local and national meet-
ings of dental or medical societies. Their opinions
survive in the literature in the form of written ab-
stracts or summaries of the proceedings of these
meetings. In many cases the summaries were written
not by the presenter of the paper, but by the editor
of the proceedings (125, 127). Indeed, John M.
Riggs(18111875), an American dentist who lectured
so widely on the treatment of periodontal diseases
that periodontitis was called Riggs disease by many
of his colleagues, rarely published any papers on the
subject (89). Riggs thoughts and opinions were most
often summarized by others (9496, 127).
Formal papers on the classication of periodontal
diseases were rare in the late 1800s and early 1900s.
Typical publications on the subject usually repre-
sented the opinion of a single person who almost
always based the classication on clinical obser-
vations and theoretical explanations of causation. A
good example is a paper published by C.G. Davis in
1879 (28) who believed that there were three distinct
forms of destructive periodontal disease:
O Gingival recession with minimal or no inam-
mation. This was due to ... feeble vascular action
... and trauma from tooth brushing or other
sources.
O Periodontal destruction secondary to lime de-
posits. The gum retires slowly ... and the alveolar
border, deprived of nutrition at the point of press-
ure, is consentaneously absorbed. Davis appar-
ently believed that calculus exerted mechanical
pressure on the gingiva causing the alveolar bone
to resorb because of lack of nutrition.
O Riggs Disease the hallmark of which was, ... loss
of alveolus without loss of gum. The perceived
problem was a necrosed alveolus or death of the
periodontal membrane. ... we get a disease that
10
is initiated and continued without any visible
mechanical irritant in many cases; and I believe
the death of the peridental membrane, depriving
the alveolus of nutrition, accounts for the death
and disintegration of the bone; or, as is believed
by some, among them Dr Waters, of Boston, the
alveolus is destroyed by vegetable parasites.
Similarly, in 1886 G.V. Black (15) published his
thoughts on the classication of periodontal diseases
based on their clinical characteristics and his under-
standing of their cause into ve separate groups.
O constitutional gingivitis; including mercurial gingi-
vitis, potassium iodide gingivitis and scurvy.
O a painful form of gingivitis. Black described a clin-
ical condition that resembled what is now termed
necrotizing ulcerative gingivitis (NUG), but he
never used the term.
O simple gingivitis. This was associated with the ac-
cumulation of debris that eventually led to calcic
inammation of the peridental membrane.
O calcic inammation of the peridental membrane.
This was associated with salivary and/or serumal
calculus. Usually there was an even or generalized
pattern of destruction of alveolar bone. The de-
struction usually occurred slowly. Blacks descrip-
tion best ts the periodontal disease that is now
known as chronic periodontitis.
O phagedenic pericementitis (phagedenic spread-
ing ulcer or necrosis). This condition shared many
features with calcic inammation of the peridental
membrane but there was an irregular pattern of
destruction and not much dental calculus. De-
struction of the alveolar bone can occur slowly or
rapidly. In a later publication Black replaced the
term phagedenic pericementitis with chronic
suppurative pericementitis (17).
The point of these historical examples is to emphasize
that little or no scientic evidence was used to sup-
port the opinions of the clinicians of the time. As one
might expect, the number of theories about what
caused periodontal diseases, how they should be
classied, andthe terminology usedtodescribe them,
seem to have approached the number of clinicians
who treated patients with these diseases. By 1929 one
author estimated that there were ... over 350 theories
of pyorrhea and much confusing terminology (10). It
is not surprising then, that no generally accepted ter-
minology or classication system for periodontal dis-
eases was adopted during this era. As a result, in the
latter part of the 19th century periodontitis went
Classifying periodontal diseases
under numerous names including: pyorrhea al-
veolaris (19, 53, 93, 111, 112, 125, 136, 137, 149),
Riggs disease (28, 9496), calcic inammationof the
peridental membrane (15), phagedenic pericemen-
titis (15, 19), andchronic suppurative pericementitis
(17). During this period, the dominant term used for
destructive periodontal disease was pyorrhea al-
veolaris.
Classical pathology paradigm
(19201970)
As the eld of periodontology began to mature
scientically in the rst half of the 20th century,
many clinical scholars in both Europe and North
America began to develop, and argue about, no-
menclature and classication systems for peri-
odontal diseases (34, 38, 43, 45, 46, 55, 58, 86, 91,
103, 128, 129, 139). What emerged from this debate
was the concept that there were at least two forms
of destructive periodontal disease inammatory and
noninammatory (degenerative or dystrophic). It
had, of course, been known for a very long time that
many periodontal diseases were inammatory con-
ditions. However, the conclusion that some peri-
odontal diseases were caused by noninammatory
or degenerative processes was a somewhat novel
suggestion. This conclusion was primarily based on
the over-interpretation of histopathological studies
from a group of Viennese investigators led by Gottli-
eb and Orban. Gottlieb, in particular, had a signi-
cant inuence on the eld when he postulated that
certain forms of destructive periodontal disease were
due to degenerative changes in the periodontium
(4247). He believed that he had discovered histo-
logical evidence of an impairment in the continuous
deposition of cementum (i.e. cementopathia). This
cemental defect was presumably initiated by the de-
generation of the principal bers of the periodontal
ligament that eventually resulted in detachment of
connective tissue from the tooth followed by resorp-
tion of adjacent bone (4547). Other authors postu-
lated that in some periodontal diseases there was a
degenerative transformation of alveolar bone into
brous connective tissue (139).
Gottliebs ideas were probably widely accepted be-
cause they appeared to explain the long-standing
and perplexing clinical observation that some young
patients with relatively clean mouths had massive
and localized bone loss with only minimal or no
overt signs of gingival inammation (92, 102, 140,
146). The profession was ready to embrace a plaus-
ible etiological explanation for what would eventu-
11
ally be called localized aggressive periodontitis (4).
The impact of Gottliebs work on classication sys-
tems was profound since it suggested that some
periodontal diseases were degenerative. As a result,
almost all classication systems used from approxi-
mately 19201970 included disease categories
labeled as dystrophic, atrophic, or degenerative
(Fig. 1). Classication systems of the period were
dominated by the Classical Pathology paradigm
which is based on the principles of general pathol-
ogy as articulated by Orban et al. (104):
Periodontal diseases follow the same pattern as do dis-
eases of other organs. There are minor differences which
have to be recognized and labeled properly. The basic
pathologic tissue changes, however, are the same as those
of other organs. ... According to principles of general path-
ology, there are three major tissue reactions: inammatory;
dystrophic; neoplastic. Neoplastic changes are not in the
therapeutic realm of periodontics.
Environmental factors, however, dictate the inclusion of
a third and different category of pathologic reaction in Peri-
odontology ... ... pathologic reactions ... produced by oc-
clusal trauma.
Although most classication systems published from
approximately 1920 to 1970 included a degenerative
disease category (24, 34, 37, 39, 40, 48, 58, 80, 128, 129,
139, 144), at the 1966 World Workshop in Periodontics
serious questions were raised about the existence of
periodontosis as a distinct disease entity (1). Many in
attendance at that meeting recommended that the
term be discarded. It was not until the next World
Workshop, held in 1977, that convincing arguments
were provided that there was no scientic basis for re-
taining the concept that there were noninammatory
or degenerative forms of destructive periodontal dis-
ease (122). Information summarized at that meeting
supported the conclusion that periodontosis was ac-
tually an infection and juvenile periodontitis should
become the preferred term for this group of diseases.
Indeed, around 1970 a different paradigm(i.e. the In-
fection/Host Response Paradigm) had begun to
dominate thoughts about the nature of periodontal
diseases.
In retrospect it is puzzling that Gottliebs concept
of cementopathia was so readily accepted, and for
such a long time. Although there has been an oc-
casional report that cemental abnormalities might
be associated with some forms of periodontal dis-
ease (73, 109), there was never any convincing evi-
dence that Gottliebs hypothesis was right. It is worth
noting, however, that there is a rare condition (i.e.
hypophosphatasia) where hypoplasia or absence of
cementum is associated with the early loss of de-
Armitage
*Orban (103) based this classication on a combination of his perceptions of the etiol-
ogic, clinical, and pathologic features of the diseases. He grouped them according to
the pathologic categories of Inammation, Degeneration, Atrophy, Hypertrophy, and
Traumatism. Similar classications were published by other authors (Coolidge & Hine
1951 (24), Fish 1944 (34), Goldman et al. 1956 (39), Goldman & Cohen 1968 (40), Grant
et al. 1968 (48), Hine & Hine 1944 (58), Lyons 1946, (80), Wade 1960 (144)).
Fig. 1. Classication of Periodontal
Diseases Following the Classical
Pathology Paradigm (Orban 1942)*
[103]
12
ciduous teeth (7, 12, 18, 78). Hypophosphatasia is a
hereditary disease characterized by low serum levels
of tissue-nonspecic alkaline phosphatase, elevated
levels of urinary phosphoethanolamine, skeletal ab-
normalities resembling rickets, and premature loss
of anterior deciduous teeth (12, 18, 20, 21, 26, 116,
117, 131). In mild forms of the disease the patients
only complaint may be an unexplained premature
loosening of anterior primary teeth. Extensive bone
loss can be observed around the affected teeth with
no evidence of root resorption (7, 12). On rare oc-
casions, posterior deciduous teeth may be affected;
permanent teeth do not usually become involved
(70, 116). However, there are a few case reports sug-
gesting that cementum may be absent or thin on the
permanent incisors of patients with hypophosphas-
tasia (33, 82, 101). There is also a case report in
which the permanent incisors had severe peri-
odontitis (147, 148). However, the patient in this re-
port harbored Porphyromonas gingivalis in his sub-
gingival ora, suggesting that something other than
hypoplasia of cementum might have contributed to
the periodontal destruction.
Infection/host response paradigm
(1970 to present)
Soon after the 1876 publication of Robert Koch (64)
in which he provided experimental proof of the germ
theory of disease, some dentists began to suggest
that periodontal diseases might be caused by bac-
teria (53, 93, 136). W.D. Miller (93), in particular, was
an early proponent of the infectious nature of peri-
odontal diseases:
In my opinion three factors are to be taken into consider-
ation in every case of pyorrhea alveolaris: (1) predisposing
circumstances, (2) local irritation, (3) bacteria.
... pyorrhea alveolaris is not caused by any specic bac-
terium, which occurs in every case ..., but various bacteria
may participate in it ...
Miller also recognized that certain systemic con-
ditions (e.g. diabetes, pregnancy) could modify the
course of the disease. Although he spent most of his
life studying the oral microora associated with
caries and periodontal disease, his work had very
little impact on convincing his contemporaries that
periodontal diseases were infections (77). He was,
however, an early advocate of the Infection/Host Re-
sponse Paradigm that would come to dominate the
eld nearly a hundred years later.
Despite an extensive amount of work on the
microbiology of periodontal diseases from approxi-
13
mately 1880 to 1965 very little headway was made
in establishing bacterial infections as the foundation
upon which periodontal diseases should be classi-
ed (133). Part of the reluctance of the profession to
accept the idea that most periodontal diseases were
infections was an unfortunate preoccupation with
the notion that some forms of destructive peri-
odontal diseases were degenerative in nature (i.e.
domination of the Classical Pathology paradigm).
In addition, microbiological studies revealed that the
periodontal microora was exceedingly complex and
no clear group of microorganisms could be causally
linked to the diseases. It was not until the classical
experimental gingivitis studies published by Harald
Le and his colleagues from 1965 to 1968 that the
Infection/Host Response Paradigm began to move in
the direction of becoming the dominant paradigm
(62, 75, 76, 138). These studies were signicant be-
cause they provided convincing data that relatively
specic changes occurred in the dental plaque ora
during the development of gingivitis. The next major
discovery in periodontal microbiology was the pre-
liminary demonstration in 19761977 of microbial
specicity at sites with periodontosis (99, 100). This
nding, coupled with the demonstration in 1977
1979 that neutrophils from patients with juvenile
periodontitis (periodontosis) had defective chemo-
tactic and phagocytic activities (23, 68), marked the
beginning of the dominance of the Infection/Host
Response paradigm. Indeed, these seminal ndings
challenged the validity of the 50-year assumption
that degenerative forms of destructive periodontal
disease existed. What followed was over two decades
of hard work that rmly established that juvenile
periodontitis, the new name for periodontosis, was
an infection.
The next major landmark in the classication of
periodontal diseases emerged from the 1989 World
Workshop in Clinical Periodontics where a new
classication of periodontitis based on the Infec-
tion/Host Response paradigm was suggested (2) (Fig.
2). The classication was a renement of one that
had been proposed by Page & Schroeder in 1982
(106) and a similar one that had been adopted by the
AAP in 1986 (2). Five types of destructive periodontal
disease were listed: I, Adult Periodontitis; II, Early
Onset Periodontitis; III, Periodontitis Associated with
Systemic Disease; IV, Necrotizing Ulcerative Peri-
odontitis; and V, Refractory Periodontitis (Fig. 2).
This classication, although soundly based in the In-
fection/Host Response paradigm, depended heavily
on the age of the affected patients (6, 107, 108) and
the rates of progression (107). Other important fea-
Armitage
tures included the acknowledgment that some forms
of periodontitis could be signicantly modied by
host factors (i.e. the category of Periodontitis Associ-
ated with Systemic Disease) and still other forms did
not appear to respond well to conventional therapy
(i.e. the Refractory Periodontitis category). At the
time the classication was proposed it was recog-
nized that, Overlap exists among categories and
cases exist that do not clearly t into any single cat-
egory (2). In addition, it was acknowledged that
considerable heterogeneity existed within the Re-
fractory Periodontitis category since, ... it includes
patients who are unresponsive to any treatment pro-
vided whatever the thoroughness or frequency as
well as patients with recurrent disease at few or
many sites. Assignment of refractory cases to other
categories may be expected to occur as more infor-
mation is acquired. (2) Finally, different forms of
periodontitis proposed in the classication shared
many microbiologic and host response features,
which suggested extensive overlap and heteroge-
neity among the categories (3).
As a consequence of these problems, the 1989
classication was criticized shortly after it was pub-
lished and a different system was proposed by Ran-
ney (123, 124). He suggested elimination of the Re-
Fig. 2. Classication of Various Forms of Periodontitis
Based on the Infection/Host Response Paradigm (World
Workshop in Clinical Periodontics 1989) [2]
14
fractory Periodontitis category since it was a hetero-
geneous group and it was impossible to standardize
the treatment that necessarily would have to be
given prior to making the diagnosis. In addition, he
recommended elimination of the Periodontitis As-
sociated with Systemic Disease category since the,
... expression of all forms of periodontitis can be
modied by some systemic diseases or abnormali-
ties, it is probably better to consider them in that
specic context, rather than treating them as a
unique category. (124) Nevertheless, despite its
problems, the classication was adopted by the
world community as reected by its widespread use
in the periodontal literature. Its acceptance was fa-
cilitated by the ease with which patients could be
placed into age-based categories (i.e. adult vs. early
onset disease). For example, it was logical to assume
that children, adolescents and young adults with ex-
tensive periodontal destruction had a different
group of diseases (i.e. Early Onset Periodontitis)
compared to adults (dened as people 35years of
age) who had a similar amount of periodontal de-
struction. This particularly seemed to make sense in
the three early onset subcategories of prepubertal,
juvenile and rapidly progressive periodontitis. How-
ever, it soon became apparent that there were prob-
lems with some of the assumptions that had been
made.
The disease category of Prepubertal Periodontitis
was the rst to be seriously questioned. In retro-
spect, many of the patients in the original publi-
cation on this disease category (108) turned out to
have either hypophosphatasia (6, 109) or leukocyte
adherence deciency (LAD) (6). Indeed, it is likely
that most prepubertal children with severe peri-
odontal destruction affecting the deciduous teeth
probably have a systemic disease that increases their
susceptibility to bacterial infections such as: LAD
(90, 145), congenital primary immunodeciency (8),
chronic neutrophil defects (31, 63) and cyclic neu-
tropenia (118). Such patients should probably have
been properly placed under the general category of
Periodontitis Associated with Systemic Disease.
Among the other problems with the 1989 classi-
cation were rstly, the uncertainty about the pro-
posal that Rapidly Progressive Periodontitis was a
single entity, and secondly, the questionable criteria
used to determine its presence. For example, do cli-
nicians have to actually document that rapid pro-
gression has occurred prior to giving a patient this
diagnosis? To be designated as rapid, how much
progression has to occur and over what time period?
Can it be assumed from a single examination that
an adolescent or young adult with massive attach-
ment loss has this disease? How can a clinician dis-
tinguish between Generalized Juvenile Periodontitis
and Rapidly Progressive Periodontitis? Since there
are no denitive answers to these, and other similar
questions, the classication lost some of its clinical
utility.
The concept that the rate of progression might be
a useful criterion upon which to base a disease cat-
egory may in itself be awed. The rate at which peri-
odontitis progresses is highly variable and depends
on such factors as
O innate and acquired host susceptibility (30, 36,
60).
O composition and quantity of the subgingival ora
(27).
O the nature of genetically determined hostbac-
terial interactions (54, 66).
Almost any form of periodontitis can progress rapid-
ly or slowly depending on the set of circumstances
governing the nature of the hostbacterial interac-
tions during a given time period. Indeed, longitudi-
nal studies of patients with untreated Chronic
(Adult) Periodontitis, in which disease progression
is usually considered to be slow, can undergo bursts
of progression during which extensive amounts of
attachment loss can occur at localized sites within a
short period of time (e.g. 23mm within 3months)
(41, 49, 50, 61, 132). Did such sites suddenly develop
a different form of periodontal disease (i.e. shift from
Chronic Periodontitis to Rapidly Progressive Peri-
odontitis)? This explanation is possible, but unlikely.
The existence of a group of periodontal diseases
that would eventually be termed Refractory Peri-
odontitis came from a series of studies of private
practice patients who unexpectedly did not respond
to treatment (9, 59, 79, 87, 88, 98). The reasons for
the unresponsiveness to conventional therapy are
not clear, but it is probably due to the emergence of
resistant or super-infecting microorganisms, tissue
invasion by periodontal pathogens, and innate or ac-
quired alterations or defects in host responses (65,
85).
Whatever the reasons, it has been demonstrated
that some patients with periodontitis refractory to
treatment harbor enteric rods, staphylococci and
Candida at unresponsive sites (25, 56, 74, 119121).
Whereas other patients who responded poorly to
treatment, or who developed recurrent disease, con-
tinued to harbor in the subgingival ora at nonre-
sponding sites elevated levels of Porphyromonas gin-
15
givalis (22), Prevotella intermedia (69), Eikenella
corrodens (69), Streptococcus intermedius (84), or mi-
crobial complexes consisting of various combi-
nations of P. gingivalis, S. intermedius, Treponema
denticola, Campylobacter rectus, Bacteroides for-
sythus, Peptostreptococcus micros and Fusobacterium
nucleatum (51, 52). In addition, perturbations in
host responses, such as altered neutrophil chemo-
taxis (83, 105), over-production of certain proin-
ammatory cytokines (57, 69, 126), and elevated
serum (51, 84) or gingival crevicular uid (GCF) anti-
body (21) against putative periodontal pathogens,
have been reported. The striking feature of the
microbiological and host response results in refrac-
tory patients is their extensive variability and hetero-
geneity. The work that has been done on Refractory
Periodontitis does not challenge its existence, since
there are some people who are clearly unresponsive
to conventional treatment. However, studies of such
patients appear to indicate that Refractory Peri-
odontitis is not a single entity. As mentioned in the
proceedings of the 1989 World Workshop in Clinical
Periodontics, it is a heterogeneous grouping (2). In
addition, except in the most unusual of circum-
stances it is very difcult to distinguish between re-
fractory and recurrent periodontal disease (124).
The nal major problem with the 1989 classi-
cation was its arbitrary and heavy reliance on age of
the affected patients or age of onset of the disease. In-
deed, the Adult Periodontitis and Early Onset Peri-
odontitis categories were rmly based on age as a cri-
terion for placing patients into one category or an-
other. In this classication the dividing line between
adult and early onset categories was arbitrarily set at
35years of age (2). Certainly clinical features of a pa-
tients periodontitis were important (e.g. the incisor/
rst molar involvement in Localized Juvenile Peri-
odontitis), but decisions regarding the nal diagnosis
depended greatly on the age of the patient. One of the
advantages of using the age criterionis that it formally
acknowledges the existence of different types of peri-
odontitis in children and adolescents.
There is no question that the patients age is an
important variable in evaluating the nature of an in-
dividuals periodontal disease. For example, a 15-
year-old patient with multiple sites with 3mm of
clinical attachment loss (CAL) has a different kind of
periodontal problem compared to a 90-year-old with
the same amount of damage. However, when age is
used as the single most important determinant in
classifying various forms of periodontitis, difcult
questions arise. Is it necessary to establish the age of
onset of periodontitis before a patient can be cor-
Armitage
rectly classied or diagnosed? As a child or ado-
lescent with periodontitis gets older, should the peri-
odontal diagnosis change (i.e. with time does Prepu-
bertal Periodontitis become Juvenile Periodontitis
which then becomes Rapidly Progressive Peri-
odontitis)? Some might argue that the answer to this
question should be yes since it has been reported
that two or three of these forms of periodontitis have
been observed within highly susceptible families
(134, 142). Authors of one of these reports suggested
that all three forms of Early Onset Periodontitis
might have a common underlying mechanism (134).
It can, however, be argued that it is incorrect or
simply wrong to use age as the main criterion for
assignment of different names to the periodontitis
affecting various family members. Indeed, it is just
as likely that the subcategories of Early Onset Peri-
odontitis are the same disease rather than three sep-
arate forms of periodontitis.
Of all the reasons for questioning the use of age as
a criterion for classication, the most compelling are
data from epidemiological studies indicating that
children and adolescents develop attachment loss
from a type of periodontitis that clinically resembles
that seen in adults (110). In other words, certain
children and adolescents develop what is, for all in-
tents and purposes, identical to Adult Periodontitis,
except that those affected are not adults. Some
authors have avoided this obvious nomenclature
problem by using the term Childhood Periodontitis
(29).
1999 Classication of Periodontal
Diseases and Conditions
Problems, inconsistencies, and deciencies associ-
ated with the 1989 classication led many clinicians
and investigators to call for a revision of the cur-
rently used system. This resulted in a 1999 interna-
tional workshop on the classication of periodontal
diseases (4). One of the goals of this workshop was
to correct the problems associated with the 1989 sys-
tem. There were six major problems with the 1989
classication that needed to be addressed:
O it did not include a gingivitis or gingival disease
category.
O the periodontitis categories had nonvalidated age-
dependent criteria.
O there was extensive crossover in rates of pro-
gression of the different categories of peri-
16
odontitis. Rapidly Progressive Periodontitis was a
heterogeneous category.
O there was extensive overlap in the clinical charac-
teristics of the different categories of periodontitis.
O Refractory Periodontitis was a heterogeneous
category.
O Prepubertal Periodontitis was a heterogeneous
category.
What emerged was a classication that was even
more rmly based on the Infection/Host Response
paradigm, but without some of the inherent prob-
lems of the 1989 classication (Fig. 3) (4). In reality,
the changes could be characterized as a course cor-
rection or ne-tuning of the 1989 classication
since no massive alterations were made. A badly
needed gingivitis or gingival disease category was
added. In addition, the heterogeneous disease cate-
gories of prepubertal, refractory and rapidly pro-
gressive periodontitis were eliminated as distinct or
stand-alone entities. The refractory designation re-
mains in the new classication, but not as a single
entity. Conceptually, all forms of periodontitis can be
unresponsive to treatment. Furthermore, the
troublesome criteria of age and rate of progression
were removed as a basis for classifying different
forms of periodontitis. The reasons for these changes
were not arbitrary, but were based on available data
and the current understanding of the nature of peri-
odontal infections (4, 35, 67, 71, 72).
As might have been predicted, some clinicians and
investigators think that the new classication is non-
sense and will not, ... help much in the discussion
as to how the various forms of periodontitis should
be classied. (141). Remarkably, one critic has fer-
vently recommended that the classication of peri-
odontitis be based on, ... a combination of a number
of clinical symptoms of the disease and the age of
the patient. (141). Indeed, it was suggested that the
classication be based on extent and severity of the
disease, age, and rate of progression (141). Clearly
this would be a return to the domination of the Clin-
ical Characteristics paradigm that reigned from ap-
proximately 1870 to 1920 when we knew little about
the nature of periodontal diseases!
A quick comparison of the 1989 and the 1999
classications could lead to the misconception that
all that was done was to arbitrarily change the
names of Adult Periodontitis to Chronic Peri-
odontitis and Juvenile Periodontitis to Aggressive
Periodontitis. These changes were specically made
to eliminate the nonvalidated age-dependent desig-
nations. They were, however, not the most important
Fig. 3. Classication of Periodontal Diseases and Conditions Based on the Infection/Host Response Paradigm (1999
International Workshop for a Classication of Periodontal Diseases and Conditions) [3]
17
Armitage
changes. Elimination of the categories of Refractory
Periodontitis and Rapidly Progressive Periodontitis
was badly needed because of their extraordinary
heterogeneity. In addition, elimination of the Prepu-
bertal Periodontitis category was important since
existing data do not support the notion that it is a
single entity. Some cases of severe periodontitis in
children are attributable to the presence of a sys-
temic disease, whereas many cases occur without
any modifying systemic conditions (13, 14, 130, 135).
Indeed, the data suggest that chronic periodontitis
has its beginnings in childhood.
To some clinicians, selection of the term chronic
as a replacement for adult to describe the most
common form of periodontitis may seem inappro-
priate since it might be interpreted to mean that the
disease is permanent or incurable. These clinicians
might argue that since most patients with chronic
periodontitis respond favorably to therapy, they
should be considered as cured. However, there are
no data to support the contention that most patients
who have been treated for periodontitis are cured
in the sense that the disease and its underlying
causes are gone. Although treatment may result in
dramatic clinical improvements and reduce the sub-
gingival levels of periodontal pathogens to nonde-
tectable levels (based on cultural data), there are no
convincing data to show that the pathogens have
been permanently eliminated. Indeed, periodontal
infections tend to recur if a rigorous post-treatment
maintenance program is not followed.
Future challenges in the
classication of periodontal
diseases
As we enter the postgenomic era with our increased
understanding of the bacteria associated with peri-
odontal infections and the genetic factors control-
ling host responses to these infections, it would
seem that a more mechanistic or etiological classi-
cation could be devised. Why could modern classi-
cations of periodontal diseases not be based on the
microbiological features of these infections, or on
the genetic factors that seem to control the clinical
expression of these diseases? The answer is simple.
We do not know enough about periodontal infec-
tions to take this next step.
It is very likely that Chronic Periodontitis is a
constellation of diseases (i.e. it is not a single entity).
One of the main problems associated with any
18
attempt at subclassifying this or other forms of peri-
odontitis, is that these infections are polymicrobial
and polygenic. In addition, the clinical expression of
these diseases is altered by important environmental
and host-modifying conditions (e.g. oral hygiene,
smoking, emotional stress, diabetes). It is conceiv-
able that with much more information and the ap-
plication of sophisticated multivariate analyses, it
may eventually be possible to subclassify the
multiple forms of Chronic Periodontitis into dis-
crete microorganism/host genetic polymorphism
groups such as:
O group A Set .1 of microorganisms Set .1 of
genetic polymorphisms.
O group B Set .2 of microorganisms Set .2 of
O group C Set .3 of microorganisms Set .3 of
O group D Set .4 of microorganisms Set .4 of
In addition, it will be necessary to superimpose on
these microorganism/host genetic polymorphism
groupings the effect of environmental or host-modi-
fying factors. It will be necessary to address head on
the nagging question, When are host-modifying fac-
tors (e.g. smoking, diabetes) so important that they
should be a principal part of the disease classi-
cation? That is, in an evidence-based classication
should there be a smoking-induced periodontitis or
a diabetic periodontitis? When do modifying factors
become an essential classication characteristic of
the disease? The same problems also occur when
one addresses the so-called Localized Aggressive
Periodontitis and Generalized Aggressive cate-
gories. It is likely that these diseases also have
multiple forms and are at least as complex as the
Chronic Periodontitis group.
There is a tendency for clinicians and investigators
to jump the gun and to use etiology- or patho-
genesis-based classications or terms prematurely.
For example, it is exceedingly difcult to prove in a
given subset of patients that the presence of a known
periodontal pathogen in the subgingival ora is ac-
tually the cause of the periodontal disease in that
group of individuals. Indeed, it has been amply
shown that known periodontal pathogens, such as
Actinobacillus actinomycetemcomitans, can be found
in the supra- and subgingival ora of patients with-
out periodontitis (3). Nevertheless, some authors
have referred to the existence of an A. actinomyce-
temcomitans-associated periodontitis (143). Al-
though tempting, terms based on assumed etiolog-
ical or pathogenic associations should be discour-
aged until there is a body of data to support their
use.
Summary and conclusions
In the past 130years classication systems for peri-
odontal diseases have evolved based on the under-
standing of the nature of these diseases at the time the
classications were proposed. One consistent feature
of the development of classication systems is the
guaranteed controversy surrounding any suggested
revisions to the previously accepted system of no-
menclature. Revisions to existing systems have been
largely inuenced by three dominant paradigms that
reect thinking at the time the classications were
proposed: the Clinical Characteristics paradigm
(18701920), the Classical Pathology paradigm
(192070), and the Infection/Host Response para-
digm (1970present). Although classication sys-
tems for periodontal diseases currently in use are
rmly based on, and dominated by, the Infection/
Host Response paradigm, some features of the older
paradigms are still valid and have been retained.
The classication system proposed by the 1999
International Workshop for a Classication of Peri-
odontal Diseases and Conditions (4) has corrected
some of the problems associated with the previous
system that had been in use since 1989 (2). Never-
theless, the new system is far from perfect and will
need to be modied once there are sufcient new
data to justify revisions. Since it is probable that
essentially all dentists and periodontists in the world
are convinced that most periodontal diseases are in-
fections, it is unlikely that the Infection/Host Re-
sponse paradigm will be replaced in the near future.
It is highly likely that current disease designations,
such as Chronic Periodontitis, are constellations of
polymicrobial and polygenic infections whose clin-
ical expression is profoundly altered by important
environmental and host-modifying conditions. Be-
fore a classication rmly based on the etiological
and pathogenic characteristics of periodontal infec-
tions can be devised, numerous fundamental break-
throughs will have to occur in our understanding of
hostmicrobial interactions and the environmental
factors that affect them. Until this happens, all
classication systems will continue to create a di-
lemma in that choices will need to be made between
equally unsatisfactory alternatives.
19
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Abnormal pocket depth and
gingival recession as distinct
phenotypes
PHI LI PPE P. HUJ OEL, JOANA CUNHA-CRUZ, HERBERT SELI PSKY &
BARRY G. SAVER
Abnormal pocket depth and pocket-free gingival re-
cession have been recognized as two separate peri-
odontal phenotypes (albeit under different names) at
least since the 18th century. With abnormal pocket
depth referred to here as destructive periodontal
disease the alveolar bone loss is associated with
abnormally deep periodontal pocketing, which can be
associated with signs of clinical inammation and
periodontal abscesses. With pocket-free gingival
recession which we will refer to as periodontal atro-
phy the alveolar bone loss is associated with gingival
recession and often presents without signs of clinical
inammation. In the 1970s, it was decided to label
these two clinically distinct periodontal phenotypes as
one and the same disease, namely chronic periodon-
titis, under the hypothesis that both are caused by
plaque. After 30 years, this speculation, and therefore
the rationale of the diagnostic classication, has re-
mained unsubstantiated. The evidence for plaque
contributing to either has remained surprisingly
sparse and contradictory; indeed, available evidence
suggests that distinctive etiologies are responsible for
eachphenotype. Furthermore, destructive periodontal
disease and periodontal atrophy have different treat-
ments and therefore economic implications, different
anthropological and comparative medicine features,
and possibly different outcomes in terms of quality of
life and tooth loss.
The continued failure to distinguish destructive
periodontal disease from periodontal atrophy may be
a rate-limiting step in understanding the incidence,
etiology, prognosis, and treatment of the different
periodontal phenotypes. We will suggest that both
phenotypes should once again, after a 30-year hiatus,
be recognized as distinct entities and we will also
explore criteria to dene when pockets are abnormal.
Destructive periodontal disease
and periodontal atrophy: two
distinct phenotypes
The recognition of destructive periodontal disease
and periodontal atrophy as two distinct phenotypes
prior to 1970 was recently summarized by Page &
Sturdivant (36). Briey, since the publication of the
rst dental textbooks in the English language in the
18th century (26), two distinct clinical conditions of
alveolar bone loss were recognized:
periodontal atrophy, where the gums retain a very
healthy aspect and are quite free of pain and
inammation, and yet will gradually recede (18);
destructive periodontal disease with the presence
of deepened periodontal pockets and underlying
bone loss (17).
The distinct clinical signs and symptoms of these
two periodontal phenotypes have been described in
detail in older clinical textbooks (see (19)), and con-
tinue to be described as diseases with distinctive
etiologies in at least one oral pathology textbook (9).
In addition to striking phenotypic differences,
destructive periodontal disease and periodontal
atrophy differ with respect to treatments and there-
fore economic implications, suspected causes, and
anthropologic and comparative medicine features.
The treatments and the economics of
periodontal atrophy and destructive
periodontal disease are different
According to one estimate, 90% of the periodontal
procedures performed today would be eliminated
if periodontal pocketing, the cardinal sign of
22
PERIODONTOLOGY 2000
destructive periodontal disease, disappeared (39).
The reasons are twofold. Firstly, treatment guidance
plans established by insurance companies typically
require a certain number of teeth with pockets dee-
per than 4 mm prior to approval of the most widely
used periodontal procedures such as scaling and root
planing. Secondly, the raison detre of most perio-
dontal treatments (other than mucogingival or
clinical crown lengthening) is the presence of perio-
dontal pockets. Periodontal pocket reduction or
elimination surgery can only be performed if pockets
are present in the rst place. Local antimicrobial
therapies are only approved for use if periodontal
pockets are present in which to put the medication.
For instance, the US Food and Drug Administration
(FDA) approved local drugs for the reduction of
pocket depth in patients with adult periodontitis; no
pockets, no FDA-approved local treatments. Simi-
larly, the presence of periodontal pockets remains,
despite the rapid decline in the smoking-associated
epidemic of destructive periodontal disease (24), an
important economic driver of the periodontal speci-
alty. If nothing more, the economic implications of
abnormal pocket depth dictate that its incidence
should be tracked as a distinct clinical entity (see
Fig. 1).
The recent US nationwide drop in the number of
periodontal treatment procedures (5, 39) aimed at
periodontal pockets suggests that the incidence of
destructive periodontal disease among the high
socioeconomic status group has been dropping dra-
matically. In contrast, the incidence of periodontal
atrophy may be increasing due to an increasingly
aging population and increased tooth retention. Yet,
both entities have been grouped together as a loss of
attachment disease and called chronic periodontitis.
Not having the ability to track these two opposing
trends increased incidence of periodontal atrophy
and decreased incidence of destructive periodontal
disease led to an inability to track the pocket-driven
Fig. 1. A 65-year-old dental professional with good plaque
control and no periodontal pocketing present but with
3 mm + palatal and lower buccal recessions. Why diagnose
this personwithchronic periodontitis whentreatment plan
guidelines and the Food and Drug Administration indicate
that this patient is not eligible for chronic periodontitis
procedures (e.g. scaling and root planing, local antibiotics,
pocket reduction surgery). To understand the incidence,
etiology, and prognosis of the deepened pocket cases that
drive 90%of periodontal treatment utilization, we need to
distinguish abnormal periodontal pocketing (not pictured)
frompocket-free gingival recession (pictured here) and not
group them together as chronic periodontitis merely be-
cause they both exhibit attachment loss.
23
Abnormal pocket depth and gingival recession
economics and manpower needs for periodontal
needs.
The etiologies of destructive periodontal
disease and periodontal atrophy may be
different
Emerging epidemiologic evidence suggests that
destructive periodontal disease and periodontal
atrophy differ with respect to their etiologies. Osteo-
porosis (27, 28), aging (14), continuous eruption (4,
14), aggressive oral hygiene procedures (36), and
anatomic periotypes have been suggested as poten-
tial causes of periodontal atrophy. In contrast, studies
in private periodontal practices that focus on the
treatment of periodontal pockets (destructive perio-
dontal disease) indicate that smoking is a primary
driver of destructive periodontal disease (20, 21).
Interestingly, smoking-induced destructive perio-
dontal disease can present with an absence of
bleeding and gingival tissues with an anemic
appearance, bringing into question whether it is
appropriate to label destructive periodontal diseases
as an inammatory disease (i.e. is the term perio-
dontitis appropriate for describing cases of abnormal
periodontal pocketing when no clinical signs of in-
ammation are present?). Another possible driver of
destructive periodontal disease that should be men-
tioned is diabetes (8).
The biological basis for claiming that both
destructive periodontal disease and periodontal atro-
phy have plaque as the common etiologic factor hin-
ges on identifying epidemiologic evidence that plaque
causes both destructive periodontal disease and per-
iodontal atrophy, and refuting existing evidence that
distinct etiologies are responsible for distinct perio-
dontal phenotypes. Since neither type of evidence has
been procured over the past 30 years, the current
plaque-driven diagnostic classication of periodontal
diseases remains mostly supported by a biological
assumption, largely modeled on experimental gingi-
vitis, and not by epidemiologic studies that controlled
for essential factors such as cigarette smoking, dia-
betes, socioeconomic status, and even age.
The anthropologic and comparative
medicine features of destructive
periodontal disease and periodontal
atrophy are different
Clarke & Hirsch (11) have long suggested, based on
anthropologic and comparative medicine evidence,
that destructive periodontal disease and periodontal
atrophy are two distinct periodontal phenotypes and
that the failure to distinguish between these two
phenotypes lies at the root of fundamental misun-
derstandings of the etiology and the historical disease
prevalence estimates of destructive periodontal dis-
ease. Studies of skulls from 23 different population
groups around the world suggest that age-related
alveolar bone loss is a normal physiological process
(12), an observation which is at odds with current
thinking that any attachment loss is pathologic and
the result of an inammatory process caused by
plaque. Similar ndings regarding an age-related
wasting of the alveolar process have been reported by
other investigators (4) and this nding has been
extended to great apes (15). Studies on prehistoric
skeletal remains distinguished alveolar resorption or
alveolar recession from infrabony pockets because of
the distinct phenotypes and suspected etiologies (13).
These different reports indicate that anthropologic
studies, comparative medicine studies, and studies
on prehistoric skulls distinguish between destructive
periodontal disease and periodontal atrophy.
Discriminating between periodontal atrophy and
destructive periodontal disease may have a signi-
cant impact on the study of the epidemiology of
periodontal diseases. Periodontal atrophy is a com-
mon clinical condition; the majority of individuals
have some gingival recession after the age of 30 years
(for a pronounced example, see Fig. 2). If this pocket-
free recession, which can be labeled as periodontal
atrophy, is referred to as destructive periodontal
disease, we end up with the anomalous situation
where close to 100% of the individuals in national
surveys are regarded as having signs of chronic
periodontitis.
Is periodontal atrophy a disease?
Currently, periodontal atrophy is labeled as chronic
periodontitis (dened by attachment loss), and is
therefore considered a disease. But is this type of
pocket-free attachment loss really a disease? Do the
individuals shown in Fig. 1 and 2 have a disease, or
do they simply represent two examples of periodon-
tal changes equivalent to hair loss and wrinkles?
These questions are important, since the prevalence
of periodontal atrophy (a nondisease?) currently may
drive to a large extent the considered prevalence of
so-called chronic periodontitis.
Dening disease is a complex issue. Are meno-
pause and baldness diseases as the FDA suggests, or
are they a reection of normal aging? Can mountain
24
Hujoel et al.
climbing in this genomic era be considered a disease,
or is it normal risk-taking behavior (6)? Are crooked
teeth a disease, or do they reect normal human
variability (38)? Answers to these questions are cul-
ture- and era-specic and depend on whose deni-
tion of disease is used. Disease can be dened as the
sum of abnormal phenomena displayed by a group of
living organisms in association with a specied
common characteristic or set of characteristics by
which they differ from the norm of their species in
such a way as to place them at a biological disad-
vantage (40). In addition, practical factors may come
into play in deciding what disease is, including
whether there is a laboratory test for the condition,
whether there is a treatment available, whether the
diagnosis is billable, and whether there is a major
lobby advocating disease status. For instance, normal
consequences of aging, such as hair loss and wrinkled
skin, can become treatable diseases once protable
treatments appear.
Within this context, is periodontal atrophy a
disease? If a young individual is at a reproductive
disadvantage because of periodontal atrophy, an
argument for disease status could be made. Some
textbooks have considered that recession of the gin-
giva is a normal physiological age-related process (19,
34, 41). Given that national representative samples of
the US population indicate that attachment loss is
almost universal after the age of 30, that attachment
loss increases with aging (1), and that the wear-and-
tear of aging affects every organ system in the human
body (30), it appears logical to consider the possi-
bility that periodontal atrophy is a normal age-related
process. Further investigation needs to determine
whether, if certain conditions do exist, periodontal
atrophy should be considered a disease.
Diagnosing abnormal pocket depth
in destructive periodontal disease
Clinical probing depth is the most obvious marker for
diagnosing destructive periodontal disease; it is sim-
ple to determine, it is a clinical measure that is in use
worldwide, it is predictive of tooth loss, and dee-
pened pocket depths been considered the cardinal
measure of destructive periodontal disease for cen-
turies in humans and more recently in mammals
(35). Abnormal pockets can de dened using nor-
mative or arbitrary values, risk-based reference val-
ues, or treatment-based reference values.
Normative or arbitrary values to
diagnose abnormal pockets
Diseases are sometimes dened based on normative
reference values. Fever can be dened as an oral
temperature greater than 37.7 C, which is the 99th
percentile of the maximum oral temperatures in
healthy persons (32). The normal heart rate is dened
as ranging between 55 and 95 beats per minute,
which corresponds to the range found for 95% of
healthy individuals (42). Children who are in the
bottom rst percentile or the fth percentile of the
Fig. 2. A 20-year follow-up (A: age 23 years, B: age
43 years) in a dental professional with progressive gingival
recession including furcation exposures, but no perio-
dontal pockets and a history of good plaque control.
Diagnostic classication systems for the last 30 years have
been based on the premise that this individual with
pocket-free gingival recession has a disease by the name
of chronic periodontitis, and that both abnormally deep
periodontal pocketing and pronounced gingival recession
have one and the same etiology plaque. This assumption
remains up to this day unsupported by epidemiologic
evidence. We raise the question whether the above con-
dition can be called a disease, let alone an inammatory
disease, and suggest that epidemiologic and clinical re-
search should determine the etiology, prognosis, and
possible treatments for this distinct clinical phenotype
(which we label periodontal atrophy).
25
growth chart may be labeled as diseased with short
stature and in need of growth hormone therapy (7).
If the normal periodontium is postulated to have
no pocket depths deeper than 3 mm, then arbitrary
values could be used to dene destructive perio-
dontal disease. For instance, any individual with
three pockets 5 mm or deeper could be classied as
having destructive periodontal disease. Currently, all
denitions of periodontal diseases are arbitrary,
which should be cause for alarm. Normative values
may be superior to arbitrary values. Normative values
could be based on parametric or nonparametric
percent cut-off values. For instance, the 97.5th per-
centile of the age-specic number of pockets deeper
than 5 mm could be used to dene destructive
periodontal disease. Based on the NHANES III data, a
28-year-old individual with two pockets deeper than
5 mm could be diagnosed as having destructive
periodontal disease, whereas ve periodontal pockets
deeper than 5 mm would be required for that diag-
nosis in a 58-year-old individual (Table 1).
Diagnoses based on normative or arbitrary cut-
offs result in normative or arbitrary disease pre-
valence levels, regardless of the distribution of
underlying risk factors. It would not matter whether
1% or 95% of the population smoked three packs of
cigarettes a day for 30 years the prevalence of
destructive periodontal disease would remain equal
to the selected cut-off value. If all human diseases
were dened based on a 5th percentile cut-off value,
the prevalence of all diseases would be equal to 5%:
for example, 5% of the population would be too
short, 5% would have diabetes, and 5% would have
a fever.
Diagnoses based on percentile distributions can
become disconnected from clinical realities. In such
cases, the number of persons classied as nondis-
eased has potentially no relationship to the number
of patients with adverse outcomes such as tooth loss,
periodontal abscesses, or difculty in chewing.
Complex chronic diseases such as diabetes, coronary
heart disease, and destructive periodontal disease
have too much natural variability to allow a suc-
cessful denition of disease based on arbitrary or
normative values.
Risk-based reference values to diagnose
abnormal pockets
Disease can be dened based on the presence of an
attribute that substantially increases the risk for an
adverse health outcome. A body-mass index above 28
is reective of a diagnosis of obesity, as it carries an
increased risk of morbidity and mortality (43). A
fasting plasma glucose level greater than 126 mg dl
(7.0 mmol l) was selected as a diagnostic criterion for
diabetes because it was associated with a steep in-
creased risk of retinopathy (2). A blood pressure
above 140 90 can be used as a diagnosis of cardio-
vascular disease since it carries an increased risk for
stroke and myocardial infarction (3). The choice of
the reference value for the diagnosis of disease is
typically based on the level of the surrogate marker
where a steep increased risk for adverse health out-
comes is present. The cut-off is still somewhat arbi-
trary, but is connected to clinical realities in terms of
the risk of adverse health outcomes.
A risk-based diagnosis of destructive periodontal
disease requires the conduct of longitudinal perio-
dontal studies, where pocket depth is related to the
risk of adverse outcomes such as tooth loss. The risk
for all-cause tooth loss associated with the maximum
probing depth per tooth was plotted for a cohort of
patients under periodontal specialist care (Fig. 3).
The gure suggests that a pocket depth of 6 mm
could be a diagnostic marker for destructive perio-
dontal disease because a distinct increased risk for
tooth loss is associated with pocket depth values
6 mm or deeper (25).
Risk-based diagnosis of chronic diseases may,
however, do more harm than good. A diagnosis of
obesity based on a BMI index of 28 may, for example,
be counterproductive, since weight loss treatments
may increase the risk of mortality (29). The diagnosis
and treatment of ventricular ectopy following
Table 1. The 97.5 percentiles of the number of peri-
odontal pockets deeper than 5 mm in a representa-
tive sample of the US civilian, noninstitutionalized
population (NHANES III, 198894)*
Age Sample size Normal number
of pockets
2029 years 3311 1
3039 years 3047 3
4049 years 2241 3
5059 years 1410 4
6069 years 1530 4
70+ years 1435 3
All ages 12974 3
*Upper 95% condence limit of the 97.5% percentile; the presence of
pockets deeper than 5 mm on lost teeth cannot be determined in this
sample.
26
Hujoel et al.
myocardial infarction with type I antiarrhythmic
agents resulted in higher mortality (16, 44). A diag-
nosis of high blood pressure (37) or diabetes (33) may
be harmful if the prescribed treatment further
increases the mortality risk.
Similarly, a diagnosis of destructive periodontal
disease based on the presence of periodontal pockets
6 mm or deeper may cause more harm than good if
the suggested periodontal treatments increase dental
or periodontal morbidity. Since destructive perio-
dontal disease is by and large a silent disease, and
since the recent publicity about the potential
association between periodontal pockets and sys-
temic diseases may have increased the psychological
damage of labeling somebody as having destructive
periodontal disease, there is an additional onus on
the profession to ensure that a diagnosis of this dis-
ease and its treatment are associated with tangible
benets (23).
Therapeutic reference values to diagnose
destructive periodontal disease
The most attractive denition of disease is the
therapeutic diagnosis. Under this denition, a person
is screened for a disease only if the diagnosis of dis-
ease leads to better outcomes. The Joint National
Committee on the Detection and Treatment of High
Blood Pressure recommends lower target blood
pressures for persons with diabetes and kidney dis-
ease based on evidence that these lower pressures
improve clinical outcomes (10). A therapeutic
diagnosis denition of disease implies that, for
example, no cancer should be screened for in
asymptomatic individuals unless evidence exists that
cancer treatment actually improves survival or qual-
ity of life. For instance, screening for prostate cancer
is controversial, since it is unclear whether life
expectancy is increased by screening, and treatment
frequently has adverse effects on the quality of life. As
a result, the US Preventive Services Task Force con-
cluded that the evidence is insufcient to recom-
mend for or against routine screening for prostate
cancer using prostate specic antigen testing or
digital rectal examination (22).
The use of therapeutic reference values in clinical
periodontics was rst suggested in one study that
established how deep periodontal pockets needed to
be before the benets of scaling outweighed its
damages (31). Scaling in periodontal pockets less
than 3 mm was reported to result in attachment loss,
whereas the same treatment in pockets deeper than
4 mm resulted in attachment gain. The shortcoming
of this therapeutic denition of destructive perio-
dontal disease is that no evidence exists that short-
term changes in attachment level relate to clinically
relevant outcomes such as tooth loss (23). Nonethe-
less, it does provide an indication as to how clinical
trials could establish pocket depth levels at which a
given treatment will likely result in more tangible
clinical benets than harm.
Conclusion
Destructive periodontal disease and periodontal
atrophy are two phenotypes with distinct clinical
features. Not only are they treated quite differently,
but different lines of evidence suggest that the two
phenotypes have distinct etiologies and different
prognoses. The current custom of labeling both
phenotypes as one and the same disease chronic
periodontitis merely because both exhibit attach-
ment loss, needs to be re-evaluated. Part of this
re-evaluation will need to involve a discussion of
whether periodontal atrophy should be labeled as a
disease, and what constitutes an abnormal perio-
dontal pocket.
Acknowledgments
This study is supported by NIH: NIDCR R-01
DE13912 and a grant from Capes Coordenacao de
Aperfeicoamento de Pessoal de Nivel Superior.
0
3 4 5 6 7 8 9 10 11 12
10
20
30
40
50
60
70
80
90
100
Pocket depth (mm)
I
n
c
i
d
e
n
c
e

r
a
t
e

o
f

t
o
o
t
h

l
o
s
s
Fig. 3. Rate of tooth loss (per 1000 teeth year) as a func-
tion of maximum probing depth per tooth in a cohort of
1021 patients (aged 4065 years) under periodontal spe-
cialist care for destructive periodontal disease.
27
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29
Clinical parameters: biological
validity and clinical utility
ANDREA MOMBELLI
Periodontal diagnosis and monitoring rely upon
clinical parameters to a large extent. Clinical diag-
nosis directly affects decisions to initiate therapy, to
select methods, and to outline the topographical area
of application. Dentists also evaluate the outcome of
therapy, and attempt long-term prognosis based on
clinical parameters.
This paper will specically focus on the biologic
validity and utility of clinical parameters assessed
with the periodontal probe.
Biological validity
Pocket formation and loss of attachment are pathog-
nomonic for periodontal disease. The reduction of
periodontal pocket depth and gain of attachment are
thus obvious clinical goals of periodontal therapy, and
pocket probing appears as the evident method for
diagnosing the disease and evaluating therapy. The
primary parameters assessed by periodontal probing
are probing pocket depth (the distance between the
gingival margin and the bottom of the sulcus pocket),
gingival recession (the distance between the cemento-
enamel junction and the gingival margin), and clinical
attachment level (the distance between the cemento-
enamel junctionandthe bottomof the sulcus pocket).
Measuring probing depth, recession, and attachment
level at the same time is redundant, since with any two
of these parameters the third is also established
(Fig. 1). Periodontal probing also generates informa-
tion regarding bleeding on probing and suppuration
from the periodontal pocket. In addition, the perio-
dontal probe is commonly used to determine the
roughness of the tooth surface and to detect subgin-
gival calculus. These two aspects are not expressed
numerically. Periodontal probing is site specic;
secondary parameters onthe level of the patient canbe
generated using data from several sites (e.g. mean
probing depth and attachment level, the percentage
of deep periodontal pockets or sites bleeding upon
probing).
What do we measure?
Finding the bottom of the pocket by inserting a
blunt instrument between the tooth and the gums
until it meets resistance seems to be a straight-
forward procedure. Various technical and biological
factors, however, determine how a probe tip advan-
ces and where it nally stops. Up close, this proce-
dure is surprisingly complex, and the data it
generates need some interpretation. Firstly, the shape
and the diameter of the probe matter, as they deter-
mine the pressure of the instrument on in the peri-
odontal tissues at any given probing force (47). Probe
design was a discussion topic for a long time (95).
The periodontal probes mostly used today in practice
and clinical research are slightly tapered metal cyl-
inders with horizontal marks, with a rounded tip
0.40.5 mm in diameter. These types of probes have
been used in the majority of clinical trials to record
pocket probing depth and clinical attachment level
on a millimeter scale.
To discuss the biological signicance of perio-
dontal probing, we need to briey review the
anatomic structure of the periodontal pocket and its
histologic components interfering with probe inser-
tion. Under ideal conditions of periodontal health,
the lateral wall of the gingival unit is lined by the
sulcular epithelium in the coronal part and the
junctional epithelium in the apical part. The junc-
tional epithelium tapers apically, and its free surface
lines the bottom of the sulcus in the region of the
cemento-enamel junction. An attachment apparatus
consisting of a basement lamina and hemides-
mosomal junctions, termed epithelial attachment,
provides a union between the epithelium and the
30
PERIODONTOLOGY 2000
tooth surface (87). With the development of perio-
dontal disease, the epithelium progresses apically
from the cemento-enamel junction to follow the
receding connective ber attachment. An inam-
matory inltrate extends apically and laterally into
the connective tissues. Periodontal therapy reduces
inammation and gingival swelling and increases the
rmness of the tissues (10). Histologically, a reduc-
tion of the inammatory inltrate and the formation
of a new epithelial attachment can be demonstrated
(89).
Several studies have indicated that periodontal
probes easily fail to identify the apical termination of
the junctional epithelium, or the coronal level of the
connective tissue attachment (2, 4, 23, 27, 34, 56, 61,
77, 83, 86, 88). The error by which the probe misses
these histological landmarks is variable. Probe tips
penetrate differently in diseased and healthy pockets
(4, 20, 26, 42, 61, 83, 91). In untreated periodontal
disease, probe tips inserted with a force of 0.5 N
penetrated through the junctional epithelium, and
stopped in the connective tissue, approximately
0.5 mm away (23). In smokers, due to reduced
inammation, the probe tips seemed to penetrate
less easily and approached nearer to the actual
attachment than in nonsmokers (13). After treatment,
probes tended to stop between the junctional epi-
thelium and the tooth, at a distance of approximately
0.7 mm coronal to its apical termination (23). Thus,
gain of clinical attachment, observed by probing after
treatment, cannot be solely explained by the forma-
tion of new connective tissue attachment to the root
surface. If the aim of periodontal probing were to
locate the apical termination of the junctional epi-
thelium, one would actually have to apply higher
forces for probing after therapy than at an initial
examination (quite the opposite is probably done by
many clinicians).
Apart from biological properties of the periodontal
tissues, the insertion of a periodontal probe may be
inuenced by factors which change due to treatment,
but are not the object of the measurement: the
roughness of the root surface, or patient comfort
(tendency to lighter probing if a patient signals dis-
comfort to mechanical irritation).
Reproducibility and longitudinal
monitoring of clinical attachment level
and pocket probing depth
Information about dynamic phenomena may be
gained by combining data from repeated assess-
ments. The potential to record loss or gain of clinical
attachment is limited by the resolution of probing
and depends on the reproducibility of a single
measurement. Probing with a manual probe has a
resolution of 1 mm. Electronic probes have been
proposed that may have a resolution up to 0.2 mm,
making it theoretically possible to detect smaller
changes in probing depth or clinical attachment level
over time (43, 69). Thirty to forty percent of perio-
dontal pockets that are re-probed with a manual
probe after 13 weeks may show a positive or neg-
ative deviation in clinical attachment level or probing
depth of 1 mm (41, 67). The standard deviation of a
single measurement of an average periodontal pocket
has been reported to be in the range of 0.81 mm (1,
7, 33, 62, 73, 74). Consequently, in an existing peri-
odontal defect, true changes in probing depth or
clinical attachment level can hardly be discriminated
from probing error in practice unless they exceed
1.3
2.2
2.0
1.6
7.4
4.8
4.4
4.8
1.7
2.3 2.3
0
1
2
3
4
5
6
7
8
9
Baseline Month 2 Month 6 Month 12
m
m
Gingival Recession
Probing Pocket Depth
Clinical Attachment Gain
Fig. 1. Gingival recession, probing
depth, and clinical attachment level
gain in deep periodontal pockets of
subjects treated with scaling and
root planing and adjunctive sys-
temic antibiotics. 0 mm corresponds
to the cemento-enamel junction
(data from 64).
31
Clinical parameters
2 mm. Shallow periodontal sites have a narrower
range in probing depths and therefore show better
reproducibility (69). Analytical procedures have been
designed to test for signicant changes in clinical
attachment level on the basis of pairs of attachment
level measurements, taken 1 week apart, and repea-
ted at 2-month intervals over 1 year (33). These
procedures have been helpful in clinical research to
test the prognostic capability of simpler means to
detect active disease. One-time assessments of pro-
bing depth, clinical attachment level, bleeding on
probing or suppuration, used alone or in combina-
tion, were unable to indicate a state of activity as
determined by these procedures (32, 33). To improve
reproducibility of probing, especially in untreated
patients where the presence of subgingival calculus
may interfere with probe insertion, it has been sug-
gested to measure each site twice using a double-
pass method (73, 74).
Probing force and probing depth
Early studies showed that forces used by clinicians for
periodontal probing varied considerably. Forces
differed between examiners, and when different
regions of the mouth were probed (24, 25, 36). Force
controlled probes have been proposed to reduce these
possible sources of error (12, 14, 15, 28, 60, 77, 93, 96).
By recording probe penetration into a periodontal
pocket as a function of probing force (Fig. 2) it can be
demonstrated that probing depth depends upon the
force applied to the instrument (65, 69). Since depth-
force curves have the characteristics of saturation
curves, which atten with increasing probing force,
small changes of probing force have a greater impact
on the reproducibility of depth readings in the low
force range. In other words, deviations in probing
depth are generally more likely to occur if one uses
light forces (e.g. 0.25 N) for probing than heavier
ones. Thus, if high reproducibility is the primary goal,
one should use a high probing force level.
Comparing depth-force curves recorded before and
after periodontal therapy, it has been found that the
force range chosen for repeated probing inuences
the amount of attachment level change determined
(68, 70). As depth-force curves may have different
shapes before and after treatment, the measurable
outcome of a treatment, expressed as the difference
in probing depth and attachment level, depends on
the force chosen for probing. In theory, if the inser-
tion pressure is smaller than the initial resistance of
the marginal tissues, the probe tip will not penetrate
into the sulcus. In this extreme case, the clinically
determined attachment level would seem to be
identical with the location of the gingival margin. If
therapy produces shrinkage of the gingival tissues,
the use of such a slight probing force would lead to
the false conclusion that treatment has produced
attachment loss. On the other hand, since scaling and
root planing induce a signicant reduction of probing
depth as well, there is a crossover of the depth-force
plots obtained before and after treatment when they
are superimposed. This is illustrated in Fig. 3, where
the mean depth values obtained at 0.25, 0.50, 0.75,
1.00, and 1.25 N are used together with the mean
levels of the gingival margin (intercept with y-axis)
before and after treatment to project depth-force
curves. Crossover is located at about 0.1 N in this
graph (70). Thus, a probing force of 0.1 N would
indicate no change in clinical attachment level.
Even lower forces would indicate mean attachment
loss, and higher forces would indicate attachment
gain. Beyond the crossover area, lighter forces may
yield more attachment gain than higher forces,
because the newly formed long junctional epithelium
may not resist probe penetration at higher probing
forces.
The phenomenon described above appears quite
clearly in the results of a study by Proye et al. (78).
Using standardized probing forces of 0.15, 0.25, and
0.50 N, these authors evaluated the effect of a single
episode of root planing. They reported a mean apical
shift of 0.84 mm of the gingival margin, and found no
attachment level alterations with a probing force of
0.15 N but a signicant attachment gain when the
effects of treatment were evaluated at higher force
levels.
Although not all studies have demonstrated signi-
cant improvements in probing reproducibility
when using force controlled periodontal probes (77,
90, 97), standardization of probing force has been
advocated because it reduces the possibility of
operator bias. A more important source of error may
be probe positioning, which is difcult to standard-
ize under practical conditions (36, 99). Splints have
been used in some trials (41, 98) to secure probe
insertion pathways and to provide vertical reference
points for depth readings. There may be some jus-
tication for the use of these tools in clinical re-
search, but they are too complicated to be useful in
clinical practice.
Bleeding on probing
The characteristics of the gingival tissues associated
with bleeding after probing were investigated histo-
32
Mombelli
logically (30). Specimens from sites bleeding after
probing with 0.25 N showed a signicantly increased
percentage of cell-rich and collagen-poor connective
tissue, but no increase of blood vessel lumens.
Experiments in periodontally healthy subjects dem-
onstrate that occasional bleeding upon probing can
occur even in the absence of disease (51). In subjects
with a reduced but healthy periodontium, a nearly
linear relationship has been found between the per-
centage of sites bleeding on probing and probing
force (46). These studies pointed to tissue trauma due
to probing with high force as a possible reason for
bleeding in the absence of disease. Probing with
controlled forces not exceeding 0.25 N was thus
recommended. Controlled forces were recommended
already in earlier studies to increase the reproduci-
bility of bleeding on probing, but at a much higher
force of 0.75 N (90). It is quite obvious that the
reproducibility of bleeding on probing can be
improved by either increasing or lowering the
probing force level (eventually, either all, or no, sites
will bleed reproducibly).
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0.00 0.25 0.50 0.75 1.00 1.25
Probing force (N)
P
r
o
b
i
n
g

d
e
p
t
h

(
m
m
)
Fig. 2. Mean depth force curves
obtained from probing measure-
ments with a probing device
recording depth and force simulta-
neously (data from 69).
0
1
2
3
4
5
6
0.00 0.25 0.50 0.75 1.00 1.25
Probing force (N)
D
e
p
t
h

(
m
m
)
Fig. 3. Mean depth values obtained
at 0.25, 0.50, 0.75, 1.00, and 1.25 N
are used together with the mean
levels of the gingival margin (inter-
cept with y-axis) to project mean
depth-force curves. Crossover of the
before and after treatment curves is
located at about 0.1 N (data from
70).
33
Clinical parameters
Clinical utility
The utility of a diagnostic parameter depends on its
ability to answer a concrete diagnostic question and
on the clinical context in which this question is
asked. Diagnostic tasks in periodontics may include
the identication of people and dental sites at risk of
developing periodontitis, the detection of early stage
disease in apparently asymptomatic individuals, the
classication of disease categories, the delineation
and local assessment of the disease in affected sub-
jects, the prediction of the likely response to a spe-
cic therapy, monitoring of treatment efcacy, and
nding recurrent disease. The utility of a diagnostic
parameter may not be the same in every one of these
situations it therefore needs to be determined
separately each time. For example, if a parameter has
been conrmed to indicate a risk for further attach-
ment loss in previously treated subjects, it is not
automatically a proven useful diagnostic tool to
detect early stage disease in a large population.
Risk assessment and screening
The issue of periodontal risk factors has been dis-
cussed in detail in recent years. Risk factors may
inuence a subject in general, or may affect perio-
dontal tissues locally (for review, see 81). Individual
variability in periodontal tissue destruction, docu-
mented longitudinally in untreated populations (59),
calls for diagnostic procedures for the early identi-
cation of subjects at high risk for severe periodontal
disease. While the paramount roles of smoking and
systemic diseases, notably diabetes, have been clearly
established on the subject level, much less is known
about specic local factors.
Monitoring untreated disease by recording pocket
probing depth, clinical attachment level, or bleeding
on probing has a limited value for indicating present
activity or predict future attachment loss (32, 33, 35).
However, in a population of elderly subjects, teeth
with reduced attachment levels had an increased
probability of being lost during the next 5 years. In
addition, teeth that actively lost attachment during
an observation period were more likely to be lost
during the following period than were teeth with a
stable clinical attachment level in the rst period
(11). As it is characteristic for periodontal disease that
not all parts of the dentition are affected with equal
severity, is it thus indispensable to monitor all sites
regularly? The answer to this question would be
afrmative if the distribution and temporal occur-
rence of periodontal lesions were entirely random. If
the distribution and occurrence is, however, struc-
tured, a limited assessment in certain areas, and at
specic time points, may be sufcient to obtain
diagnostically useful information. Using data from
comprehensive assessments in the entire dentition,
we estimated the inuence of symmetry on the
variance of clinical and microbiological parameters
in 56 patients with chronic periodontitis (66). The
impact of contralateral conditions was determined
on the level of the site, the tooth (Fig. 4), and the
quadrant. Signicant correlations were detected in
probing depth, recession, clinical attachment level,
total cultivable bacterial counts, and the plaque
index, recorded on the right and left side on all levels
of analysis. Given this amphichiral nature, the diag-
nostic advantage of full mouth recordings over partial
0 2 4 6 8 10 2 4 6 8 10
27
26
25
24
23
22
21
31
32
33
34
35
36
37
17
16
15
14
13
12
11
41
42
43
44
45
46
47
Probing pocket depth (mm)
Fig. 4. Symmetrical behavior of tooth-specic mean probing depths in one subject (data from [66]).
34
Mombelli
assessments should be evaluated carefully in various
clinical situations. Correlations other than symmetry
should also be explored for their potential to improve
the utility of clinical (and other) parameters.
Attention has been focused in the past on the
possibility that periodontal disease may not be a
continuous process, but may be characterized by
episodes of activity, followed by periods of relative
quiescence. As has been discussed above, the
potential of detecting an active episode of perio-
dontitis by simply probing a previously measured site
after a few weeks or months is limited. On the other
hand, the true impact of short bursts of activity on
the accumulated loss of periodontal tissues over time
also remains to be determined, and may have been
overestimated (31, 80). Slow continuous attachment
loss may have considerable consequences in the
long run, although it is undetectable in studies lim-
ited to a few months duration. Given the inaccuracy
of periodontal probing, the detection of a continuous
disease process leading to 6 mm attachment loss
over 60 years would require a minimal study period
of 20 years (subsequent measurements must yield a
difference of at least 2 mm in order to distinguish
tissue destruction from measurement error with
sufcient condence).
Classication and treatment planning
The historical perspective and potential problems
with the current classication are discussed in a
broader context by van der Velden in this volume
(92). The present chapter connes itself to a discus-
sion of the impact of clinical parameters on the
classication process. Eight classes of periodontal
diseases and conditions are currently distinguished.
Among them are chronic periodontitis, aggressive
periodontitis, and periodontitis as a manifestation of
systemic diseases (3). Although these three forms are
all associated with an increased probing depth and
clinical attachment level, their differentiation is not
based on criteria derived from the periodontal pocket
chart. The diagnostic key elements include the
patients age, systemic health, and the occurrence of
similar problems in the family information essen-
tially obtained by assessing the medical and dental
history.
The clinical periodontal examination thus does not
classify causes, it classies destruction patterns. A
major limitation of periodontal probing is its inability
to distinguish previous tissue loss from current
disease activity. Clinical attachment level and pocket
probing depth reect the extent of prior, but not
necessarily current, disease. Since periodontal tissue
damage accumulates over time, the disease may
appear more severe in elderly patients than in young
ones, although in terms of disease progression, the
contrary may be the case. For a further discussion of
this, the reader is referred to the article of Hujoel in
this volume (40).
Decades of clinical experience and well documen-
ted longitudinal studies have shown that all clinically
distinguishable forms of periodontal disease respond
favorably to a nonspecic reduction of the subgin-
gival bacterial mass (52, 53, 71, 72, 75, 84, 85). How-
ever, studies have also indicated that certain
circumstances, identiable by periodontal probing,
may justify specic forms of therapy.
Calculus removal is accomplished less often in
deeper periodontal pockets (19, 79). Calculus
removal in deep pockets has been found to be
more efcient if a surgical access is provided for
root instrumentation (16, 18, 19).
Deep periodontal pockets showed more pocket
reduction following surgical procedures (54, 55, 75,
76).
Sites with shallow initial probing depth have a
tendency to lose attachment (8, 54).
In patients with deep periodontal pockets, sys-
temic antibiotics in conjunction with scaling and
root planing can be of additional benet compared
with scaling and root planing alone (38).
Monitoring
The paramount question when evaluating the success
of periodontal therapy is whether further interven-
tions are necessary or whether the therapeutic phase
can be concluded. In general, this decision is based on
a comparison of clinical attachment level, probing
depth, and radiographs taken before and after treat-
ment. This means that the prognosis for future tissue
changes is based on clinically observed reactions
appearing as a result of treatment. Monitoring of 1688
sites in 49 patients over 24 months indicated that the
outcome of nonsurgical therapy in proximal surfaces
of nonmolar teeth was not compromised by the
severity of the initial soft tissue or bony lesion (9). The
value of clinical and microbiological data assessed
shortly after the completion of nonsurgical perio-
dontal therapy to predict the outcome several months
later is limited (17, 22). The key parameters of probing
depth and clinical attachment level may continue to
improve over a period of 6 months (45). It would be
desirable to have a set of parameters for an early
evaluation of treatment success in order to be able to
35
Clinical parameters
decide rapidly whether additional or alternative
therapy is necessary.
Longitudinal studies on successfully treated
patients show that the results of conventional peri-
odontal treatment can be maintained over many
years if an efcient recall system is provided (5, 6,
48, 53, 57, 85). In well-maintained populations,
recurrent disease seems to be limited to a few
individuals. In these subjects, however, several sites
are often affected at the same time (29, 39, 63).
Clearly, these individuals need to be identied early
on. Very tight recall systems require an expensive
work force and are a nuisance to the patients, not to
mention the tissue damage caused by repeated
instrumentation. This indicates a need for diagnostic
tools to select the optimal recall interval for each
patient individually and to decide which sites need
retreatment in excess of what regular prophylaxis
implies. Clinical indices assessed during the main-
tenance phase are poor prognosticators of attach-
ment loss (44, 50, 94). In one study (49), 41 patients
were monitored after periodontal therapy for
bleeding on probing for 2 1 2 years in a mainten-
ance program. The sensitivity of frequent bleeding
to predict clinical attachment loss >1 mm was only
29%, and the specicity 88%. On the other hand,
continuous absence of bleeding on probing had a
predictive value of 98% for stability. Using the
methodology of the systematic review an attempt
was made to assess the predictive value of residual
probing depth, bleeding on probing, and furcation
involvement in determining further loss of attach-
ment following initial cause related therapy (82).
After a detailed review of 47 publications, only one
study fullled all inclusion criteria. That study (21)
included 16 subjects reviewed over 42 months and
demonstrated that residual probing depth was pre-
dictive of further disease progression, whereas per-
sisting bleeding on probing was not.
Conclusions
Reduction of pocket probing depth and clinical
attachment level gain are the obvious clinical goals of
periodontal therapy. Gain of clinical attachment is
largely due to increased tissue rmness and epithelial
attachment, and cannot be explained solely by the
formation of new connective tissue attachment. If the
aim of periodontal probing were to locate the apical
termination of the junctional epithelium, one would
actually have to apply higher forces for probing after
therapy than at an initial examination.
Certain circumstances, identiable by periodontal
probing, may justify specic forms of therapy. Teeth
with reduced attachment or with evidence of having
actively lost attachment recently are at greater risk of
being lost. In deep pockets, calculus removal is more
efcient after surgical access and systemic antibiotics
can offer an additional benet. Furthermore, residual
probing depth is predictive of future disease pro-
gression.
Since the distribution and temporal occurrence of
periodontal disease is not entirely random, a limited
assessment of clinical parameters in certain areas,
and at specic time points, may be sufcient to obtain
diagnostically useful information in many situations.
Correlations between parameters (37, 58) should be
explored to reduce redundancy and improve the
utility of multiple or repeated measurements.
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39
Clinical parameters
Radiographic parameters:
biological signicance and
clinical use
URS BRA
GGER
This review is based on a literature search in PubMed
using the words radiographic parameter and perio-
dontology. The paper includes the topics of image
acquisition, radiographic parameters obtainable in
periodontal practice, image processing for the per-
ception of biologic processes, the clinical use and
impact of radiographic parameters, and ends with a
proposal for the next generation of diagnostic tools,
which may include repeated electronic probings and
superimposed serial digital radiographs.
According to a recent position paper of the
American Academy of Periodontology (7), clinicians
must still rely on traditional approaches to arrive at a
periodontal diagnosis. Relevant diagnostic factors
listed were the presence or absence of clinical signs
of inammation such as bleeding on probing, pro-
bing pocket depths, the extent and pattern of loss of
clinical attachment and bone, medical and dental
history, the presence of miscellaneous signs and
symptoms including pain, gingival ulceration, as well
as the amount of dental plaque and calculus. Key
ndings to differentiate periodontitis from gingivitis
include presence of inammation at sites with
pathological detachment of collagen bers from
cementum, apical migration of the junctional epi-
thelium, loss of connective tissue attachment, and
resorption of the coronal portion of tooth supporting
alveolar bone (9).
Assessing the degree of gingival inammation, a
detachment of collagen bers, and an early resorp-
tion of alveolar bone can be difcult, even histologi-
cally. Clinically and radiographically, there is no
diagnostic method available to indicate the transition
from gingivitis to periodontitis. The position paper by
the American Academy of Periodontology (7) also
pointed out that patients who already had experi-
enced periodontitis may demonstrate sites with
progressive tissue loss even in shallow sites. When
the next episode of periodontal tissue destruction
may occur is not predictable by any presently avail-
able diagnostic technique. An early detection of
periodontal tissue destruction is, however, crucial in
order to intervene to prevent major tissue damage.
Acquisition of images
Conventional vs. digital imaging
methods
The position paper of the American Academy of
Periodontology (7) recognized that intraoral radio-
graphs obtained with a digital sensor or a fast lm
can provide important detailed information on the
periodontal structures and are an essential compo-
nent of a complete periodontal examination. Current
imaging methods in periodontology have been
thoroughly reviewed by Mol (99). The search for more
sensitive imaging techniques and the search for a
3-dimensional depiction of periodontal sites has
attracted great efforts and investments into the
development of new methodologies such as direct
digital imaging. The traditional method of obtaining
an image has, however, not changed dramatically.
According to Mol (99), there seems to be no evidence
that digital readings, i.e. linear radiographic meas-
urements, are more accurate than analog readings.
The diagnostic efcacy of digital imaging and lm-
based radiographs seems to be similar.
The standard recommendation for the initial
examination of a periodontitis patient was for many
years a full-mouth periodontal probing complemen-
ted by a full-mouth set of intraoral radiographs (29,
44). To decrease the radiation dose, Molander et al.
73
PERIODONTOLOGY 2000
(100) proposed the use of an orthopantomogram
after the initial clinical examination, supplemented
by a limited number of periapical radiographs,
depending on the severity and distribution of
increased probing pocket depths, furcation involve-
ments or various nonperiodontal ndings.
Periapical radiographs, as well as orthopantomo-
grams, may over- or underestimate the actual outline
of the alveolar bone (3, 4, 58). The alveolar bone level
may be obscured, especially in vertical defects (121).
However, if the available diagnostic methods only
detect 1%(orthopantomogram) or 4%(periapical) of
initial vertical lesions, no radiographic method can
really be preferred over the other one, despite
the existence of a statistically signicant difference
between the methods (121). In deeper vertical
lesions conrmed by later ap reection ortho-
pantomogram readings were less accurate compared
to periapical radiographs. Interexaminer variation is,
however, reported to be considerable by both radio-
graphic methods (4).
Mol (99) has pointed out that modern panoramic
X-ray units are able to produce image layers that
resemble the outline of the jaw. However, errors in
patient positioning often result in suboptimal pro-
jections and limit comparisons with follow-up ima-
ges. Less image detail, blurred or over-projected
structures may compromise the picture of the crestal
alveolar bone, especially in the frontal region of the
mouth. Nevertheless, the general convention of not
using orthopantomograms in the initial periodontal
examination may be questioned. Very good agree-
ment has been obtained in assessing alveolar bone
level in half-mouth intraoral radiographs and
in orthopantomograms (126). Apparently, ortho-
pantomograms obtained by latest technology may at
least in part replace periapical radiographs in the
initial periodontal examination as well as in the
maintenance phase of periodontal therapy.
The discussion about the best radiographic tech-
nique for periodontal diagnosis is also driven by a
concern to offer diagnostic information with the least
amount of radiation dose exposure (100). According
to Kiefer et al. (85), the dose of a digital ortho-
pantomogram corresponds to that of 14 intraoral
direct digital radiographs. However, the radiation
dose of an orthopantomogram was considerably
lower than that of a lm-based system. Other factors,
such as comfort for patient, ease of image handling,
costs, diagnostic information of nonperiodontal
ndings, may favor digital orthopantomogram. With
further improvement of digital panoramic radiogra-
phy and digital sensors for periapical images, issues
related to the radiation dose will become increasingly
less critical (101). However, the reality in a daily
practice environment will ultimately determine the
use of improved radiographic techniques, due to
better patient comfort, more detailed diagnosis, less
radiation dose, and or reduced cost. By increasing
the focusskin distance, using a fast lm, and a col-
limation down to the shape of the lm or sensor, the
radiation dose to the patient can be considerably
reduced.
The use of lm holders can improve the repro-
ducibility and accuracy of dental radiographs (132,
133). The paralleling technique has a better per-
formance compared to the bisecting technique (13,
105). However, a survey of the radiographic equip-
ment and techniques used in dental practices in
England and Wales revealed that only 18% of the
responding dentists, always or often, used a low-
dosage technique combining E-speed lm and rect-
angular collimation (148). A similar low number had
been reported for Swedish dentists: 37% of the gen-
eral practitioners routinely applied periapical lm
holders (143). The use of D-lm and round collima-
tion rather than E-lm and rectangular collimation
has been listed as one example of negligent cus-
tomary practice that violates the standard of care
(160). These rather strict recommendations for taking
radiographs are contrasted by the fact that only
an estimated 1% of the Swiss populations total
annual X-ray dose stems from dental diagnostic
procedures (10).
In conclusion, performing conventional radio-
graphy with a low radiation exposure is possible by
using reliable low dose procedures; these radio-
graphic techniques, however, have not yet been fully
adopted by dental professionals. Digital imaging per
se is not superior to lm-based radiographs in its
ability to detect detailed periodontal structures.
Radiographic parameters
obtainable in daily dental practice
Measurements of mesial and distal linear
distance between the cementoenamel
junction and the alveolar crest
An accurate depiction of the distance between the
cementoenamel junction and the mesial and distal
alveolar crest is important in epidemiologic studies as
well as for patient management (115, 116). In 30
adolescents 1618 years of age with clinically healthy
gingiva, Kallestal & Matsson (81) showed in vertical
74
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bitewings a range of 02 mm in the radiographic
distance between the cementoenamel junction and
the alveolar crest. In 1314-year-old adolescents with
no clinical attachment loss during the preceding
18 months, the distance from the cementoenamel
junction to the alveolar crest at rst molars ranged
from 0.4 to 1.9 mm (61). In young adults, a wide
range in the prevalence of sites with a distance
> 2 mm from the cementoenamel junction to the
alveolar crest has been reported (189%) (80). The
large variations in radiographic results among studies
may be due to methodologic problems, but also to
differences in the race of study subjects, their disease
susceptibility and access and use of preventive care
(1, 2, 5, 11, 17, 40, 52, 59, 66, 67, 81, 82, 155). Ado-
lescents not performing oral preventive measures
showed more loss of crestal alveolar bone than ado-
lescents enrolled in a preventive care program (80).
Studies evaluating initial crestal alveolar bone loss
have proposed different thresholds to distinguish
between disease and no disease: > 1 mm (71, 90,
95), > 1.5 mm (34), > 2 mm (66, 67, 86) or > 3 mm
(18, 88). The choice of cutoff values affects the diag-
nostic sensitivity and specicity in detecting alveolar
bone loss. By employing a 1-mm cutoff point and
0.3-mm standard deviation, due to methodologic
error for the diagnosis disease, an individual
experiencing a true crestal alveolar bone loss of
0.1 mm annually would rst be identied as having
bone loss after 713 years of breakdown (14). This
range would increase drastically to an interval from 1
to 19 years if the methodologic error for reading the
distance from the cementoenamel junction to the
alveolar crest reached 0.9 mm. A small error of
0.1 mm in locating the cementoenamel junction
and the alveolar crest produces an error of 0.14 mm
and 0.3 mm at a 95% condence level in assessing
the distance between the cementoenamel junction
and the alveolar crest. These limitations in radio-
graphic accuracy may lead to an overestimation of
disease prevalence and also of the gain in alveolar
bone level following successful treatment (14). If
higher threshold values are used, such as 3 mm, for
the distance between the cementoenamel junction
and the alveolar crest, the true prevalence of early
tissue loss may be completely obscured. By using a
cutoff point of 3 mm, Blankenstein et al. (18) found
alveolar bone loss only in one out of 1645 sites in 13
15-year-old schoolchildren.
Serial radiographs often show considerable pro-
jection divergence. Warping of a nonstandardized
image using known reference points can reduce
projection errors. When similar, moderately similar,
moderately dissimilar and dissimilar pairs of serial
radiographs from an animal study were analyzed, the
mean difference in the reading of interproximal bone
level in uncorrected pairs of images increased
from 0.09 0.06 mm to 0.39 0.06 mm, to 0.66
0.13 mm, and to 1.31 0.23 mm, respectively. After
correcting the second image with a warping algo-
rithm, errors were reduced to 0.06 0.06 mm for
similar pairs radiographs, to 0.13 0.06 mm for
moderately similar, to 0.22 0.19 mm for moderately
dissimilar, and to 0.23 0.13 mm for dissimilar pairs
of radiographs (75). According to an in vitro study
by Hausmann et al. (60), the distance from the
cementoenamel junction to the alveolar crest on
radiographs in the molar region may vary up to
2.35 mm if the rst radiograph is taken at a 90 and
the second radiograph at a 70 angle. In the anterior
and the bicuspid regions, a deviation in angulation of
up to 20 was less critical. Hausmann et al. (62) also
calculated the intra- and interexaminer variability for
two scorers in assessing the distance between the
cementoenamel junction and the alveolar crest in
digital images, employing 20 periodontal sites and
well-dened reference points (62). One scorer pro-
duced readings with a mean accuracy of 0.34 mm.
For longitudinal comparisons, the two scorers would
be able to detect a true change of 0.710.83 mm in
alveolar bone level.
Using a computerized system to measure inter-
proximal alveolar bone level in radiographs has
revealed considerable limitations in intra- and inter-
examiner variation (156). The variation differed for
specic tooth groups and for different levels of peri-
odontal bone loss. A total of 3248% tooth surfaces
could not be assessed by two examiners due to
problems in determining bone root length and
bone tooth interrelationships (156). Other studies
have reported a considerable percentage of non-
readable periodontal sites as well: 25% (138), 34%
(89), and 22% (15).
There is obviously a great difference between the
limited value of linear crestal bone level measure-
ments in daily practice and attempts to optimize
and control conditions affecting methodologic
errors in clinical studies (77). If several radiographs
are to be compared, the total methodologic
error will increase with increasing numbers of
diagnostic outcomes, including variation in projec-
tion geometry, lm development or electronic
noise, locating and structural changes of reference
points, movement of reference points, perception
and interpretation (97). To minimize variation in
longitudinal studies, some researchers have applied
75
Biological signicance and clinical use of radiographs
broader scales to identify loss or gain of alveolar
bone.
Location of the alveolar crest in relation
to root or tooth length
Hugoson & Jordan (68) proposed a classication of
the severity of periodontal disease based on the
location of the alveolar crest: within < 1 3, between
1 3 and 2 3, and >2 3 of the total root length.
Bjorn (16) suggested measuring proximal bone level
as a percentage of the total length of a tooth, using a
ruler with 10 equidistant divisions. By dividing the
distance from the cementoenamel junction or crown
edge to the root apex into equidistant parts, such
rulers help correct methodologic errors stemming
from differences in projection geometry. Some rulers
only accept 1 mm of discrepancy between the
cementoenamel junction and the alveolar bone as a
physiologic condition (138). Other rulers use a bone
loss of as much as > 20%as the threshold of disease.
Broader scales have been used to identify risk factors
for periodontal disease progression and genetic
variance in alveolar bone height (71, 98, 107, 108).
A simple recording of the distance from the
cementoenamel junction to the alveolar crest does
not document the pattern of bone loss. Most often,
horizontal and vertical patterns of alveolar bone loss
are described separately due to perceived differences
in prognosis, treatment needs, and treatment
options. A gradual reduction of periodontal bone
height with increasing age was found by Hugoson &
Laurell (69). Many dentists assume that periodontitis
patients with a horizontal pattern of bone loss are
relatively easy to treat, respond to treatment with a
rapid reduction in probing depth and gingival
bleeding, and exhibit less need for surgical interven-
tion (42).
Vertical defects
The presence of a vertical (angular) periodontal
defect indicates advanced infrabony destruction and
is often a radiographic diagnostic sign of severe per-
iodontitis (53, 106, 124, 157). A recent study
investigated the prevalence and severity of vertical
bony defects in a population of dentally aware
individuals at two points in time, year 1982 and year
1992 (12). Intraoral radiographs of 251 individuals in
1982 and 247 individuals in 1992 in the ages of
2170 years were assessed for the presence or ab-
sence of vertical alveolar bony defects. A vertical
bone defect was dened as a one-sided bone
resorption of the interdental marginal bone 2 mm
that had a typical angulation towards either the
mesial or distal aspect of the root. In subjects initially
2130 years of age, the prevalence of individuals with
vertical defects increased from 11% to 38% from
1982 to 1992. In subjects aged 5170 years in 1982,
the prevalence of individuals with vertical defects
increased from 27% to 64% during the following
decade. The majority of affected individuals had one
to two vertical defects and only about 5%of the study
subjects showed many vertical defects. Vertical
defects were more common in the posterior than in
the anterior region of the dentition and the distri-
bution of vertical defects within the maxilla as well as
the mandible typically revealed a right-hand to left-
hand side symmetry. Vertical defects seemed to be a
rare phenomenon in dentally aware individuals. Both
the prevalence and the severity of vertical defects
increased with the age of the patient (12). ). Un-
treated angular bony defects demonstrated an in-
creased risk for further alveolar bone loss compared
with teeth with a horizontal pattern of bone
destruction (114).
Many treatment plans for periodontitis patients
with vertical defects include surgical interventions in
order to obtain access for better removal of plaque
and calculus (42) and to change an unwarranted
anatomy of the alveolar bone by either resective or
regenerative means. Sites with horizontal bone loss
adjacent to neighboring teeth with a vertical defect
did not respond as well to initial periodontal therapy
as interproximal sites with a horizontal component of
bone loss (41).
Defect angle
If A
1
represents the cementoenamel junction at a
tooth with a vertical defect, D
1
the most apical
extension of the intrabony lesion (the bottom of the
defect), and B
1
the most coronal position of the crest
adjacent to the vertical defect, the angle between the
two lines A
1
D
1
B
1
D
1
can be used as a diagnostic
radiographic parameter.
One year after regenerative periodontal surgery
using a papillary preservation ap technique and an
enamel matrix derivative, Tsitoura et al. (147) found a
signicant association between the characteristics of
the defect angle and clinical attachment gain. The
methodologic error was about 1 using image-pro-
cessing equipment. Narrowperiodontal defects 6 22
healed better than defects P 36. A similar outcome
was previously observed after regenerative procedures
with (145, 146) and without (142) barrier membranes.
76
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Steffensen & Weber (142) expressed alveolar bone
ll as a change in bone height in a given defect and as
a percent change in relationship to total root length,
and assessed the change in alveolar bone level at
baseline and at 1518 months following periodontal
surgery. More favorable healing was noted in defects
with angles <45 than larger angles. The reproduci-
bility of the angle measurements was 0.20 (standard
deviation) when a tracer system was used. Period-
ontal regenerative approaches seem to be most suc-
cessful in deep and narrow defects (145147). The
wider coronally a periodontal defect is and the fewer
bony walls present, the less likely a defect is to im-
prove following therapy.
Area measurements: outline of the
defect
Ehnevid et al. (41) evaluated periodontal healing
1 year after periodontal treatment by comparing one-
and two-dimensional bony changes in superimposed
tracings of radiographs from sites with angular
defects. The relative area changes were larger than
the relative linear changes of the same defects. Some
of the morphologic variables, such as the length of
the bone surface, the original defect area and height
of the defect, correlated with the area gain. Digital
subtraction radiography can determine changes in
area size by calculating the number of pixels dem-
onstrating change in gray-level above a certain
threshold. In standardized radiographs, the area size
can be used as a means to assess treatment success,
complementing parameters such as the number of
defect walls, the determination of which requires a
surgical intervention.
Area measurements: outline of the
remaining bone
Instead of measuring the outline of a defect area,
Heins et al. (64) measured the area of interradicular
bone remaining between mandibular molars, as
dened by the two cementoenamel junctions, the
two apices, and the alveolar crest. The area was
expressed as a percentage of the total area between
the cementoenamel junctions and the tooth apices.
In addition, the portion of the root still embedded in
bone was expressed as a percentage of total root
length at the deepest and the shallowest aspects of a
vertical interproximal lesion. In patients treated by
scaling and root planing with or without the elevation
of a surgical ap, the mean area of remaining inter-
proximal bone decreased signicantly over a period
of 230 years (mean 11.8 years). Progressive bone
loss was mainly observed at the bottom of shallow
vertical lesions, but no additional bone loss was
detected in the deep periodontitis lesions, maybe in
part because of difculty in detecting further bone
loss in deep periodontitis lesions. Bony changes at
the coronal part of an interproximal site seemed to be
independent of changes observed in the adjacent
vertical part of a periodontitis lesion (64).
Density of the crestal alveolar lamina
dura
The presence or absence of a radiographically visible
crestal alveolar lamina dura may be used as an
indicator of either a stable or a deteriorating perio-
dontal situation. Rams et al. (129) studied 1809
interproximal sites for the occurrence of crestal
alveolar lamina dura in periapical and bitewing
radiographs. Changes in probing pocket depth and
clinical attachment level were also assessed every
5 months. Disease recurrence was dened as a site
showing an increase in probing depth P 3 mm or a
clinical attachment loss P 2 mm. Over 36 months,
only 3% of the sites in 45% of the patients exhibited
progressive disease. In predicting periodontal
attachment loss, the absence of crestal alveolar lam-
ina dura had a high sensitivity but a low specicity
and positive predictive value. Sites with a radio-
graphically visible lamina dura at baseline did not
show disease progression for at least the next 2 years,
as assessed by periodontal probings and the thresh-
olds mentioned above. Bitewing radiographs revealed
more sites with crestal alveolar lamina dura than
periapical radiographs (129). These results suggest
the clinical utility of determining the radiographic
status of the crestal alveolar lamina dura, especially
in patients in a maintenance care program. These
ndings are in contrast to a study by Greenstein et al.
(54) a decade earlier, which found no association
between clinical periodontal parameters and the
status of radiographic crestal alveolar lamina dura
(54). The difference in study results may be explained
by technical advances in dentomaxillofacial radiol-
ogy, and by improvement in the ability to distinguish
stable periodontal conditions not requiring thera-
peutic interventions.
Furcation involvement
If the location of the cementoenamel junction and
the root apices is difcult to identify in radiographs,
the radiographic assessment of furcation areas is
77
even more inuenced by the anatomic complexity
and radiographic overprojection of anatomic struc-
tures. Radiographically, the location of the bifurca-
tion in mandibular molars may deviate by an average
of 0.26 mm 0.5 mm in the apical direction from the
true location at rst molars and 0.65 mm 1.15 mm
in the coronal direction at second molars (57). The
study also found a considerable interobserver vari-
ation of 0.5 mm at the rst and 1.06 mm at the sec-
ond mandibular molars. The intraobserver variation
reached 0.47 mm and 1.14 mm, respectively. Of four
morphologic characteristics examined, the mesiodi-
stal width of the bifurcation area had the strongest
effect on accuracy (57). Using receiver operating
characteristic (ROC) curve analysis to determine the
ability of the observers to identify articial study
lesions, 12 observers detected furcation degree I
defects with a mean A
Z
value of 68% (area under the
curve) and furcation degree II defects with a mean A
Z
values of 86% (area under the curve) (56). Thus,
clinical trials may obtain increased sensitivity by
supplementing standardized radiographics with an
image processing technique (8, 27, 33, 38, 102, 113).
Contradictory radiographic results in the literature,
especially in investigations of furcation areas, may be
due to insufcient standardization of radiographic
techniques and inadequate statistical power (33).
In conclusion, the methodologic error of each
radiographic parameter inuences the amount of
alveolar bone change that can be detected at a certain
condence level.
Biological signicance of
radiographic parameters
Image processing for the perception of
biological processes
Digital subtraction radiography can enhance the
possibility of detecting minor bony changes if serial
radiographs are taken with optimal control of
projection geometry. Using image processing, the
radiographic diagnosis based upon standardized
radiographs can be improved by means of a single
scan densitometry (32), a histogram evaluation
of gray-level distributions in regions of interest,
corrected mean gray-level (31) and a computer-
assisted densitometric image analysis (CADIA) (26).
To quantify bone density, references in the form of
aluminum wedges can be included in the images (6,
76, 78, 134, 152, 153). Density changes in dened
regions, such as treated periodontal sites, can then be
compared to density changes in untreated sites (19,
25, 26, 32, 48, 49). Image processing includes contrast
enhancement (22) and computer-aided pattern
recognition (150). A strict chain of exclusion criteria
must be applied to avoid false-positive readings (21,
25, 26, 48, 49, 55, 73, 140). The number of nonread-
able pairs of images may greatly inuence the accu-
racy of the radiographic conclusions.
Several attempts to control the projection geometry
in serial images have been published, including bite-
blocks that are attached to the lm sensor holder,
which then is mechanically coupled to the X-ray
source (25, 26, 38, 39). Jeffcoat et al. (79) proposed an
extraoral method for controlling the projection
geometry in which the position of the patients head
was positioned into a cephalostat with an increased
distance between the X-ray source and the lm. The
head positioning may be guided using reected light,
and the mechanical coupling may be replaced by
sophisticated systems to steer and control the posi-
tion of the tube to the lm holder (28, 63, 91, 104, 136,
159). These new radiographic techniques offer a
noninvasive quantitative assessment of small tissue
change for the comparison of therapeutic procedures
and for detecting disease progression (23, 24, 50, 76,
93, 111, 112, 119, 120, 127, 128). However, the acqui-
sition costs of computer-assisted radiography will
need to be lower for it to become a chair-side proce-
dure. Until then, image processing systems remain
primarily a research tool for clinical trials (118).
Examples of computer-assisted radiography are
shown in Figs. 13. Figure 1 shows a color-coded
digital subtraction image which, as indicated in blue,
depicts an increase in bone density at 6 months after
periodontal treatment with tetracycline bers. The
interproximal site in Fig. 2 showed a loss in bone
density, as indicated in red, at a site that did not
receive special anti-infective treatment. In Fig. 3a,
seven regions of interest were drawn with the cursor
on top of the baseline image of a site treated with
tetracycline bers, scaling and root planing, chlorh-
exidine mouthrinse and improved home care proce-
dures. In Fig. 3b, the increase in bone density
following this treatment, especially in the four crestal
bone regions, is indicative of a successful outcome.
Image processing applying color conversion clearly
can increase the diagnostic performance of inter-
preters compared to an evaluation of pairs of con-
ventional radiographs (22, 27). Bone remodeling,
resorption or apposition can be evaluated densito-
metrically or can be detected visually by assigning
78
Bragger
different colors to bone loss and bone gain using
threshold values to minimize methodologic errors,
i.e. deleting pixels with a change in gray-level within
the methodologic error. Figure 4 depicts a color-co-
ded digital subtraction image representing the
remodeling activity of a vertical periodontal defect
treated according to the principles of guided tissue
regeneration. At 12 months post-treatment, a
considerable portion of the lesion along the root
surface seems to be lled with bone (in blue). A
portion of the crestal demarcation line of the
former defect seems to have undergone resorption
(in red).
In general, the aim of radiographic analysis is to
detect changes in hard tissues, such as bone, dentin
or enamel. Until recently, a radiographic assessment
of periodontal soft tissue conditions was not done.
However, using underexposed serial radiographs,
computer-assisted densitometric image analysis may
reveal progression of disease or healing events in the
supracrestal soft tissue after periodontal treatment.
Use of this method has also provided evidence that
anti-infective topical therapy applied to the entire
dentition results in better periodontal healing, as
assessed by an increase in soft tissue density, than
does topical therapy of discrete periodontal sites (50).
Figure 5 shows a digital subtraction image based on
standardized radiographs obtained with a short
exposure time. Over 6 months, the density of the
supracrestal portion of the interproximal tissue had
increased (in blue). If the antimicrobial regimen is
only applied locally, some periodontal test sites
might gain density and other sites lose density.
Control periodontal sites that were not exposed to
disinfective therapy continued to lose density over
the 6-month study period. In Fig. 6a, regions of
interest in the baseline image included supracrestal
soft tissue. The control site, which was not exposed to
anti-infective therapy, lost soft tissue density inter-
proximally over a 6-month observation period
(Fig. 6b). The regions of interest, drawn in the
Fig. 2. An untreated periodontal
site that lost interproximal bone
density over a period of 6 months,
indicated in red.
Fig. 1. Increase in interproximal crestal bone den-
sity 10 gray-level marked in blue. A pair of standardized
radiographs was taken 6 months apart. The periodontal
site and the entire dentition were treated using tetracyc-
line bers, scaling and root planning, and disinfection.
79
baseline image, missed the region with loss of den-
sity and had to be redrawn to quantify the intensity of
the density change. Interactive steps during image
analysis optimize the detection of remodeling pro-
cesses but may also lead to bias. Only a strict repro-
ducible protocol and preferably blinding the reader
to the images, guarantee objective data.
Numerous in vitro experiments have shown
superior diagnostic performance when interpreters
use the newer radiographic techniques rather than
conventional radiography (20). The biological signi-
cance, however, lies in the possibility of gaining
noninvasive information of tissue conditions reec-
ting physiologic tissue turnover, a decrease in density
as a sign of ongoing disease, or an increase in density
as a sign of healing. Increased interproximal crestal
alveolar bone density, which paralleled a reduction in
probing pocket depth and a gain in clinical attach-
ment, has been observed following subgingival
debridement (37).
By using radiographic techniques, other studies
have documented a phase of bone resorption imme-
diately after periodontal ap procedures, followed
later by bone apposition in patients undergoing crown
lengthening and after surgical access ap procedures
in patients with periodontitis (25). Untreated perio-
dontal sites or sites with unsuccessful healing after
therapy tend to lose bone density (35, 50, 74).
Geurs et al. (51) recently published preliminary
data linking low systemic bone mineral density a
sign of osteoporosis to an elevated risk of losing
crestal alveolar bone. However, it should be cau-
tioned that the parameter change in density does
not reect an well-dened anatomic correlate (135),
i.e. gain in density within a vertical periodontal defect
may not equal new attachment (30). Especially when
placing bony graft particles into regions of interest,
the study design, as well as the interpretation of the
data, has to accommodate the effect of the radio-
opaque material itself on the gray-level of images (43,
Fig. 3. a) Seven regions of interest corresponding to
supracrestal soft tissue, vertical bony defect, crestal
alveolar bone, and the control region, all drawn on top of
the baseline image. b) The same region of interest as in
Fig. 3a projected on top of the color-converted digital
subtraction image, showing an increase in bone density.
80
Bragger
45, 113, 158). A similar precaution applies when using
a resorbable radioopaque grafting material, which
may mistakenly lead to an interpretation of bone loss
as the material resorbs (130).
In conclusion:
Strict standardization of the projection geometry
with serial radiographs combined with image pro-
cessing can result in a more sensitive detection and
quantication of slight tissue changes.
Image processing may reduce both inter- and in-
traexaminator variability.
The histologic correlates to the changes in density
are unknown.
Digital subtraction radiography and computer-
assisted densitometry image analysis are indis-
pensable research tools but will probably not gain
daily chair-side application until the equipment for
navigating and touchless repositioning of the X-ray
source to sensor holders is available at a reasonable
cost.
Clinical use
Tugnait et al. (149) have screened the literature
to assess the role of radiographs in periodontal
diagnosis and management at the time of initial,
corrective and supportive periodontal therapy. Pub-
lished papers were critically reviewed for evidence of
the clinical utility of conventional radiographs. The
authors concluded that, combined with periodontal
probing, radiographs can assist in arriving at a
periodontal diagnosis and in developing a compre-
hensive treatment plan. Radiographs may help in
identifying teeth with a poor prognosis, and may help
locate plaque-retentive structures, dental caries and
periapical pathosis not detected by the clinical eval-
uation. Radiographic features of periodontal lesions
may also have an inuence on the choice of surgical
technique, even though the denitive bony contour
of a periodontal lesion is visible only after ap
reection and removal of granulation tissue. These
careful conclusions by Tugnait et al. were followed by
a series of critical statements. There is not much
evidence on which to decide the question of the
surgical technique preferable for any given type of
periodontitis lesion. During the maintenance phase,
there is a lack of clear evidence supporting recom-
mendations to take radiographs at certain intervals
(46, 47, 65). Tugnait et al. (149) and the Faculty of
General Dental Practitioners (110) recommended
taking radiographs only when they can offer the
possibility of changing either the prognosis or the
management of a patient.
Weems et al. (154) compared treatment plans that
were based either on a clinical examination alone or
on a clinical examination together with intraoral
periapical radiographs. Changes in the two treatment
plans tended to be related to nonperiodontal rather
than periodontal procedures. A periodontal benet of
radiographs was noted in 15% of the patients. The
authors suggested that, had an exact plan for perio-
dontal surgical intervention been contemplated,
more patients would have beneted from an radio-
graphic examination.
Fig. 4. Color-converted digital sub-
traction image of a vertical defect
treated by a guided tissue regener-
ation procedure. After 1 year, a
considerable portion of the vertical
defect along the root surface
appears in blue. Part of the former
demarcation line of the defect
underwent resorption (red).
81
Lundgren et al. (92) calculated the success rates
of periodontal therapy at a 3-year follow-up in 36
patients with moderate to advanced periodontitis. A
series of evaluation steps comprised ve different
levels of various clinical and radiographic criteria.
To fall into success category 1, absolute clinical
health had to be reached, meeting the following
criteria:
a no probing pocket depth > 4 mm;
b no clinical signs of gingival inammation;
c no bleeding on pocket probing;
d no further loss of clinical attachment;
e no further loss of alveolar bone at the 3-year
examination.
To reach success category 2, the clinical conditions
had to fulll criteria b, c, d, and e. Level 5 would just
Fig. 5. Increase in interproximal
soft tissue density above the base-
line bone level as noted in a pair of
standardized radiographs taken
using half the exposure time. Same
treatment as in Fig. 1.
Fig. 6. a). In the series of underexposed radiographs, 2
regions of interest were drawn for potential quantication
of soft tissue changes. b). The loss in density at the papilla
of the teeth was obvious in the digital subtraction image
but could only be quantied after redrawing the regions
of interest. The site was untreated for 6 months and
demonstrated crestal alveolar bone loss.
82
Bragger
use repeated radiographs to verify stable crestal bone
levels using a 2-mm loss as the threshold for true
disease progression. After 30 months of supportive
periodontal care, only 52.1%of the sites and 15.8%of
the individuals exhibited perfect health, reaching
level 1. Successful treatment according to level 2 was
reached in 65.5% of sites and in 34.2% of patients.
Using criteria level 3, 73.2% of sites and 39.5% of
patients were successfully treated. Using level 4,
83.1% of sites and 55.3% of patients reached the
successful category. By applying the criteria no fur-
ther bone loss (level 5), as assessed from bitewing
radiographs, 95.1% of sites and 86.8% of individuals
were categorized as successfully treated and main-
tained. The authors pointed out that the difference in
success rates between sites and individuals is crucial
when considering allocation of resources to treat and
maintain diseased, unsuccessfully treated sites dis-
tributed among many individuals (92). Omitting loss
of clinical attachment (increasing probing pocket
depth) and radiographic loss of interproximal alveo-
lar bone <2 mm in standardized radiographs to reach
level 5, an indicator of true and denitive progressive
loss of periodontal tissue support was dened. The
authors concluded that level 5 would represent a
useful indicator for disease progression with a
favorable costbenet ratio.
A treatment plan considers an estimation of risk, i.e.
grouping the patient, teeth or even single roots into a
secure, doubtful or hopeless category. For teaching
purposes, various institutions have proposed rules for
periodontal therapy based on a certain percentage of
bone loss, a certain crown to root length ratio, the
presence or absence of vertical lesions, furcation
involvement as well as nonperiodontal ndings that
would be graded as favorable or unfavorable for
treatment success. There is a lack of sound evidence
for many of the clinical recommendations for
choosing these risk categories but they may none-
theless have crucial consequences for the patient. The
tooth depicted in Fig. 7ac would be characterized by
most dentists as having a hopeless prognosis. Never-
theless, the tooth has survived for 17 years, and is still
functioning. Expert opinions on appropriate perio-
dontal treatment categories are faced with minimal
agreement and low predictive value (123, 126).
In order to plan treatment and dene the level of
intervention, clinicians attempt to estimate the risk
of deteriorating periodontal conditions in a denti-
tion. Persson et al. (122) presented 23 periodontists
and graduate students with the medical history, data
from an oral examination, full-mouth intraoral
radiographs, and clinical slides of 51 individuals.
Ten subjects were diagnosed with gingivitis and 22
with advanced periodontal breakdown. The inter-
preters were asked to give a score for the risk of
disease progression, if these patients did not
undergo periodontal preventive treatment for the
next 24 years. The study showed that periodontal
experts perceived risk mainly based on the overall
horizontal alveolar bone loss, an age-adjusted pro-
portional radiographic bone height score for the
worst site, and the proportion of pocket probing
depths P 6 mm. Thus, the assignment of risk scores
was mainly based on parameters reecting past
history of disease such as bone loss and the number
of deep pockets, whereas risk factors such as
smoking, diabetes, and poor oral hygiene were not
identied as determining variables.
Since the late 1980s, a number of reports have
evaluated the potential association between poor oral
health, especially signs of chronic periodontitis, and
cardiovascular events (36, 72, 83, 84, 94, 96, 103, 117,
137, 139, 151). Since cardiovascular disease repre-
sents a major public health issue, and since the
control of oral infections may have a benecial effect
on surrogate or even tangible endpoints for patients
suffering from, for example, acute myocardial
infarction, effective prevention of periodontal infec-
tions is receiving increased attention. Persson et al.
(122) examined 80 patients with clinically conrmed
acute myocardial infarction and 80 control subjects
with no evidence of cardiovascular disease recruited
from among friends of the cardiac patients, and
considered potential confounding factors such as
gender, smoking habits, and socioeconomic status.
Each study subject received a periodontal examina-
tion including full-mouth probing, an assessment of
gingival inammation, and a radiographic evaluation
(122). The cut-off point for periodontal disease was
set at P 4 mm, measured from the cementoenamel
junction to the crestal alveolar bone (125). Cardiolo-
gists also examined both subject groups. Study sub-
jects were grouped based on the presence of an
interproximal bone loss 4 mm in 10%, 20%, 30%,
40%, 50% or 60% of periodontal sites. The Odds
ratios of belonging to the patient group with known
acute myocardial infarction ranged between 9 : 1 and
14 : 1, with the highest association observed in sub-
jects demonstrating 50% of periodontal sites with
bone loss P 4 mm. Individual risk proles based on
a ve-vector periodontal risk diagram analysis were
also performed using patients periodontal ndings
(87, 131). Putative risk factors included the proportion
of sites showing bleeding on probing, the number of
pockets >6 mmdeep, the number of teeth lost due to
83
periodontal disease, and the degree of bone loss rela-
tedtoage andsmoking status. The strongest individual
factor associated with acute myocardial infarction
was radiographic evidence of alveolar bone loss in50%
of periodontal sites, which was even enhanced with
increasing number of sites showing bleeding on a
probing and pocket depth P 6 mm. The authors
suggested that subjects with periodontal bone loss in
several teeth and chronic gingival inammation
should be referred for a medical check-up.
Conclusions: Radiographic parameters can be used
to make a periodontal diagnosis, to create a treatment
plan, to estimate disease risk, to document tissue
stability, remodeling or breakdown, and perhaps to
detect periodontal risk factors for cardiovascular
events.
The next generation of periodontal
disease scoring methods
Coming back to the report of the American Academy
of Periodontology on the clinical reality of todays use
of conventional radiographs, it is striking that the
simplest radiographic parameters (linear measure-
ments of mesial and distal bone levels) are not even
systematically included in traditional clinical chart-
ing. Also, the standard is a mere review of images
per se, and is rarely supported by a magnifying glass
or any type of ruler. In contrast, gingival recession,
probing pocket depth, and clinical attachment level
are noted at 46 sites per tooth. The tedious drawing
of periodontal charts is now increasingly being
Fig. 7. a) Periapical radiograph of
the region 14, 13, 12 taken in 1987.
b) Impressive amount of periodon-
tal tissue loss at time of periodontal
surgery. c) Periapical radiograph
from the same region taken in 2004.
The patient received regular main-
tenance therapy for 17 years.
84
Bragger
replaced by a printout using electronic probes. The
common perception that the soft tissue outline
reects the extent and pattern of tissue loss is
frequently used for patient information and motiva-
tion. Changes in mm of probing readings may indeed
represent disease progression or reversion of disease,
but they may also simply represent shrinkage of the
periodontal pocket due to gingival recession or an
increased resistance to the applied probing force.
Viewing of radiographs comprises the usual method
of estimating the appearance of the crestal alveolar
bone or special regions of interest. Combining and
superimposing readings of probing and radiographs
using, for example, the cementoenamel junction as a
reference point can provide valuable information
about the level of periodontal hard and soft tissues.
Figure 8ac describes a proposal for a combined
depiction of radiographic information and clinical
probing. It is assumed that electronic pocket probing
can be combined with a digital image, and that read-
ings are related to the cementoenamel junction, which
must be visible. Any change in clinical attachment
level could be marked, e.g. green for gain, red for loss.
Assuming, furthermore, that two sequential
orthopantomograms are obtained in a standardized
way, bone changes exceeding a certainthreshold value
can be color-marked (green for gain, red for loss in
density). In addition, distances from the contact point
of the teeth to the gingival papilla and the interproxi-
mal alveolar crest could be assessed by marking the
papilla with an opaque radiographic material or by
simply measuring the distance to the contact point
with a probe (144). The combined radiographic-
probing chart could depict a buccal or a lingual oral
viewseparately. A similar chart could be developed for
smaller intraoral, periapical radiographs.
Conclusions
Conventional radiography offers reliable, low dose
procedures which are, however, still not fully adopted
by the dental profession. Digital imaging per se is not
superior to lm-based radiographs in the detailed
depiction of periodontal structures. The magnitude
of the methodologic error of each radiographic
Fig. 8. a) Initial orthopantomogram obtained from a
patient with chronic periodontitis. The gingival recession
and the depth of the pockets are marked in relation to the
cementoenamel junction. b) The orthopantomogram is
opped over to show the lingual view. Probing measure-
ments are marked. c), d) The superimposed change after
treatment is depicted using image processing tools and
threshold values to exclude changes due to methodologic
limitations. Note that the given example is a pure proposal
rather than representing an already available tool.
85
method limits the amount of real change in disease
status that can be detected with a certain level of
condence. Image processing at present is a pure
research tool for the identication of subtle changes
in tissue density. Radiographic parameters can be
used to make a periodontal diagnosis, to create a
treatment plan, to estimate disease risk, to document
tissue stability, remodeling or breakdown, and
perhaps to detect periodontal risk factors for cardio-
vascular events. In the future, a diagnostic system
should be developed that combined and superim-
poses the radiographic image with probing data or
biologic information, thereby providing a compre-
hensive periodontal chart.
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Purpose and problems of
periodontal disease classication
UBELE VAN DER VELDEN
There has been a debate on the diagnosis and
classication of periodontal diseases since dentists
rst became interested in periodontology. In this
respect, periodontology is not unique; comparable
discussions can be encountered in many elds of
medicine, especially in complex diseases. Diagnosis
is dened as the act of identifying a disease from
its signs and symptoms, whereas classication is
dened as the act or method of distribution into
groups. The present article deals with the perio-
dontal condition which is clinically characterized by
three symptoms: loss of connective tissue attach-
ment, loss of alveolar bone support, and inamed
pathological pockets. On the basis of these three
symptoms one diagnostic name for this condition
would be appropriate, e.g. destructive periodontal
disease. However, if age, distribution of lesions,
degree of gingival inammation, putative rate of
breakdown, response to therapy, etc., are also taken
into account, numerous diagnostic names are nee-
ded. In order to be able to communicate about
patients, clinicians have always felt the need for
diagnostic names and classications for these dis-
eases, preferably on the basis of putative etiologic
factors. At present, controversies about denitions
of diseases continues, not only in the periodontal
eld but also in medicine.
An interesting contribution to the discussion on
disease terminology is a paper by Scadding (31)
entitled: Essentialism and nominalism in medicine:
logic of diagnosis in disease terminology. In this
paper the clear distinction between these two types
of denitions is highlighted. The essentialistic idea
implies the real existence of a disease. Essentialist
denitions typically start X is , implying a priori
the existence of something that can be identied as X.
Thus the doctors skills consist in identifying the
causal disease and then prescribing the appropriate
treatment. In relation to this, Scadding states:
The essentialists hankering after a unied con-
cept of diseases as a class of agents causing ill-
ness, is mistaken and misleading for several good
reasons: many diseases remain of unknown
cause; known causes are of diverse types; caus-
ation may be complex, with interplay of several
factors, intrinsic; and, more generally, an effect
the disease should not be confused with its
own cause.
The counterpart of essentialism is nominalism,
which implies that a disease name is just a name given
to a group of subjects who share a group of well-
dened signs and symptoms. Scadding supports the
nominalistic concept and states: The names of dis-
eases are a convenient way of stating briey the
endpoint of a diagnostic process that progresses from
assessment of symptoms and signs towards know-
ledge of causation. Ideally, a nominalistic disease
denition describes a set of criteria that are ful-
lled by all persons said to have the disease, but not
fullled by persons that are considered free from the
disease (40). This set of criteria is dependent on the
level of knowledge of a given disease. For example, if
the etiology is known, e.g. cholera, then the key cri-
terion for the disease is the presence of Vibrio chol-
erae. However, for many diseases the etiology is
complex or not known, and consequently a large
number of diseases are dened as syndromes. A
syndrome constitutes a distinct group of symptoms
and signs which together form a characteristic clinical
picture or entity. In this respect periodontitis is a good
example of a syndromically dened disease (10, 36).
Need for classication
Syndromic classication(s) are needed to cluster
similar disease phenotypes in more homogeneous
13
PERIODONTOLOGY 2000
syndromes. This is the prerequisite to establish
etiology and susceptibility traits, and thus separate
truly different forms of disease or conversely link
different different phenotypic variations to the same
underlying disease (35).
As mentioned above, the names of a disease are a
convenient way of stating briey the endpoint of a
diagnostic process that progresses from the assess-
ment of symptoms and signs towards knowledge of
causation (31). In other words, in order to obtain
more knowledge about the causation of periodontal
diseases the various forms of the disease have to be
classied. The term periodontal disease has referred
for some time to all diseases which affect one or more
tissues from the periodontium (2). However, in 1964
Sherp (32) noted:
Discussions of periodontal disease commonly
begin with the tacit assumption that all partici-
pants are considering the same entity. Since the
variations of periodontal diseases are almost
limitless, depending on ones taste for subclas-
sication, this unqualied usage often leads to
fruitless semantic misunderstandings. What is
usually meant is the most common form of
periodontal disease a chronic, slowly progres-
sive and destructive inammatory process
affecting one or more of the supporting tissues of
the teeth the gingival tissue, the periodontal
membrane, and the alveolar bone.
This statement, made 40 years ago, is still valid
today; it also highlights one of the most frequent
premises in periodontal diagnosis: the assumptions
concerning previous disease progression. In this re-
spect, age has always been an important parameter in
periodontal diagnosis.
Previous classications
Almost all ancient medical works refer to the various
diseases of the teeth and their supporting tissues but
without using any particular terminology. The rst
specic name for periodontal disease was introduced
by Fauchard in 1723 using the term scurvy of the
gums (15). Ever since, researchers have introduced
names for diseases of the periodontium on the basis
of etiologic factors, pathologic changes or clinical
manifestations.
Gottlieb is generally considered to be the rst
author who clearly distinguished various forms of
periodontal disease. In the 1920s he classied perio-
dontal disease into four types (1618): Schmutz-
Pyorrhoe, alveolar atrophy or diffuse atrophy,
Paradental-Pyorrhoe, and occlusal trauma. Schmutz-
Pyorrhoe was thought to be the result of the
accumulation of deposits on the teeth and was
characterized by inammation, shallow pockets, and
resorption of the alveolar crest. Alveolar atrophy or
diffuse atrophy was described as a noninammatory
disease exhibiting loosening of teeth, elongation, and
wandering of teeth in individuals who were generally
free of carious lesions and dental deposits. In this
disease, manifesting pockets are formed only in later
stages. Paradental-Pyorrhoe was characterized by
irregularly distributed pockets varying from shallow
to extremely deep. This form of disease may have
started as Schmutz-Pyorrhoe or as diffuse atrophy.
The fourth type was occlusal trauma, a form of
physical overload which was believed to result in
resorption of the alveolar bone and loosening of
teeth.
More or less at the same time, McCall & Box (24)
introduced the term periodontitis to denote those
inammatory diseases in which all three components
of the periodontium, i.e. the gingiva, bone, and per-
iodontal ligament, were affected. This is in contrast to
the lesions of occlusal traumatism and atrophic
lesions, in which only the bone and periodontal
ligament may be involved. Periodontitis was sub-
classied, on the basis of presumed etiologic factors,
into Simplex periodontitis, considered to be the result
from local bacterial factors, and Complex period-
ontitis, a result of systemic etiologic factors.
Becks (11) made a distinction between parade-
ntitis, a disease which originates from the gum tissue
in the form of gingivitis and genuine paradentosis,
which originates in the bony alveolus, perhaps in the
form of an osteopathy. Orban & Weinmann (25)
adopted this nomenclature using the anglicized term
periodontosis to designate this noninammatory
disease. Periodontosis was considered a separate
disease entity, distinctly different from periodontitis,
which was considered the sequela of gingivitis of the
deeper periodontal structures, and therefore of
inammatory origin. It is remarkable that in relation
to the issue of degenerative disease it is not men-
tioned specically that this was a disease entity par-
ticular to young subjects (23).
During the 1950s and 1960s the importance of
dental plaque as the major etiologic factor for
periodontal diseases became more and more evi-
dent. The ultimate proof of the association between
plaque and gingival inammation was shown by
Loe and coworkers in their experimental gingivitis
14
van der Velden
studies (22, 34). The inuence of this way of
thinking was clearly evident during the 1966
Workshop in Periodontics when the entity perio-
dontosis was revisited (13). In the committee report
it was concluded:
Evidence to support the conventional concept of
periodontosis is unsubstantiated. It was the
consensus of the section that the term perio-
dontosis is ambiguous and that the term should
be eliminated from periodontal nomenclature.
Nevertheless, the committee is aware that some
evidence exists to indicate that a clinical entity
different from adult periodontitis may occur in
adolescents and young adults.
Therefore it is not surprising that soon after the
Workshop a study was published by Butler (12)
introducing the name juvenile periodontitis instead
of periodontosis when describing the periodontal
condition of young individuals with severe perio-
dontal bone loss. According to Butler there was no
proof of any degenerative process, as the sufx osis
would imply.
Numerous classications have since been pub-
lished. Page & Schroeder (28) dened periodontitis
as an inammatory disease of the periodontium
characterized by the presence of periodontal pock-
et(s) and active bone resorption with acute in-
ammation. They suggested at least four distinctly
different forms of periodontitis in humans:
prepubertal, juvenile, rapidly progressive, adult
periodontitis, and acute necrotizing ulcerative gin-
givo-periodontitis (ANUG P). In this classication,
with the exception of ANUG P, the age of onset is
of decisive importance. This item is adopted in
almost all subsequent classications. In 1986 the
American Academy of Periodontology (AAP) adop-
ted the following classication (3):
I Juvenile periodontits
A Prepubertal periodontitis
B Localized juvenile periodontitis
C Generalized juvenile periodontitis
II Adult periodontis
III Necrotizing ulcerative gingivo-periodontitis
IV Refractory periodontitis.
In an attempt to detect groups and individuals at
high risk for periodontal disease, Johnson et al. (20)
presented a more extensive classication:
I Childhood periodontitis including specic syn-
dromes such as Papillon-Leve`fre
II Juvenile periodontitis: localized; generalized
III Post-juvenile periodontitis
IV Adult onset periodontitis: slowly progressive;
rapidly progressive
V Periodontitis associated with systemic diseases
such as diabetes, scurvy, immunodeciencies
(including AIDS), immunosuppressive states,
blood dyscrasias
VI Traumatic periodontitis, e.g. gingival recession
and loss of attachment as a result of abrasion
during oral hygiene practice (toothbrushing,
wood sticks, charcoal, brick dust; trauma from
occlusion)
VII Iatrogenic periodontitis, due to inappropriate
restorations or inappropriate instrumentation of
the gingival crevice.
At the same time a new classication was proposed
by Suzuki (33). Suzuki stated that Additional clinical
observations in our laboratories during investigation
on the mode of inheritance of juvenile and rapidly
progressive periodontitis have suggested that further
qualications can be made. Based on factors such as
age, microbial deposits, and the autologous mixed
lymphocyte reaction, rapidly progressive periodonti-
tis, as introduced by Page & Schroeder (28), can be
subdivided into type A and type B. In addition, the
term postjuvenile periodontitis delineated a slow-
progression-type of juvenile periodontitis.
One year later it was stated in the 1989 World
Workshop in Clinical Periodontics that although the
AAP classication was adopted, legitimizing the idea
that different forms of periodontal diseases exist,
more recently acquired data mandate modication
and revision (4). The following classication was
recommended:
I Adult periodontitis
II Early onset periodontitis
A Prepubertal periodontits
1 Generalized
2 Localized
B Juvenile periodontitis
1 Generalized
2 Localized
C Rapidly progressive periodontitis
III Periodontitis associated with systemic diseases
IV Necrotizing ulcerative periodontitis
V Refractory periodontitis.
Volume 2 of Periodontology 2000, issued in 1993,
was dedicated to the classication and epidemiology
of periodontal diseases. In the contribution of Ran-
ney (30) four major disease categories were proposed,
i.e. adult periodontitis, early onset periodontitis,
necrotizing ulcerative periodontitis, and periodontal
abscess including a large number of subcategories
mainly based on systemic factors. Also in 1993, the
15
Periodontal disease classication
rst European Workshop on Periodontology was
organized. In session I the following position papers
were presented. Papapanou: epidemiology and nat-
ural history of periodontal disease (29), Claffey: Gold
Standard Clinical and radiographical assessment of
disease activity (14), Tonetti: Etiology and patho-
genesis (35) and Johnson: Risk factors and diagnostic
tests for destructive periodontitis (19). On the basis of
these comprehensive reviews a consensus report was
produced (9) that included the following statement
regarding the classication of periodontal diseases:
There is insufcient knowledge to separate truly
different diseases (disease heterogeneity) from
differences in the presentation severity of the
same disease (phenotypic variation). Because of
this, existing classications are unsatisfactory.
Disadvantages of present classications (e.g. AAP
1989) include 1. extensive overlap between the
different diagnostic categories, 2. need for
assumptions concerning previous disease pro-
gression, 3. the necessity for detailed information
on the quality of treatment provided previously
and the patient response to this therapy, and 4.
the apparent lack of a consistent basis for clas-
sication. Ideally classications should be based
on etiologic and host response factors. In order
to deal with the present confusion, a simple
classication distinguishing between 1. Early
onset periodontitis, 2. Adult periodontitis, 3.
Necrotizing periodontitis, might be preferable.
Provided that the relevant information is avail-
able, as many as possible additional secondary
descriptors should be used to further dene the
clinical situation. These include distribution
within the dentition, rate of progression, re-
sponse to treatment, relation to systemic dis-
eases, microbiological characteristics, ethnic
group and other factors.
Although, in my opinion, the conclusion there is
insufcient knowledge to separate truly different
diseases (disease heterogeneity) from differences in
the presentation severity of the same disease
(phenotypic variation) from the European Workshop
on Periodontology in 1993 (9) still holds true today, it
was concluded in the 1996 World Workshop in Peri-
odontics that there was a clear need for a revised
classication system for periodontal diseases (5). This
resulted in a new classication which was agreed
upon at the International Workshop for a Classica-
tion of Periodontal Diseases and Conditions in 1999
(6). This classication included many disease categ-
ories and subcategories and was certainly an
improvement with regard to the category gingival
diseases. However, a number of subcategories pre-
sent in the majority of the previous classications
were eliminated, i.e. prepubertal periodontitis,
juvenile periodontis, postjuvenile periodontitis, rap-
idly progressive periodontitis, early onset periodon-
titis and refractory periodontitis. Amongst others it
was argued that:
in case of early onset periodontitis (prepubertal
periodontitis, juvenile periodontitis, postjuvenile
periodontitis and rapidly progressive periodon-
titis), one must have temporal knowledge of
when the disease started. In addition, there is
considerable uncertainty about arbitrarily setting
an upper age limit for patients with so-called
early onset periodontitis. For example, how does
one classify the type of periodontal disease in a
21-year old patient with the classical incisor-rst
molar pattern of Localized Juvenile Periodontitis
(LJP)? Since the patient is not a juvenile, should
the age of the patient be ignored and the disease
classied as LJP anyway?
On the basis of this and other arguments the
workshop participants decided that it was wise to
discard classication terminologies that were age-
dependent or required knowledge of rates of pro-
gression (6). Therefore it was proposed to re-name
the disease formerly considered under the umbrella
early onset periodontitis and other forms of rapidly
progressive disease by aggressive periodontitis. Al-
though not clearly stated, it can be concluded from
the report that the term aggressive periodontitis is
only applicable for patients with severe periodontal
breakdown. However, it can be argued that this new
classication does not solve the problems because it
is not clear how severe a case must be in order to
be classied as aggressive periodontitis, and know-
ledge about the rate of progression is still needed.
In the same Workshop, adult periodontitis was re-
named chronic periodontitis on the basis of the
assumption that slowly progressive disease can be
present at any age, i.e. in adults as well as in ado-
lescents. But again it can be argued that for this
classication, knowledge about the rate of progres-
sion is still needed.
The problems related to the prediction of the rate
of progression in the future or assumptions on the
rate of progression in the past are clearly illustrated
by the study of Albandar et al. (1). In this longitudinal
study, young individuals, mean age at baseline
16
van der Velden
16 years, were reexamined 6 years later. On the basis
of the baseline measurements the individuals were
classied into localized juvenile periodontitis, gen-
eralized juvenile periodontitis, incidental attachment
loss, and no-periodontitis group. The results showed
low correlations between baseline disease classica-
tions and the classications at the 6-year follow-up
examination. In addition, the cross-sectional classi-
cations were not predictive of the rate of prog-
ression of periodontal disease in these subjects.
Sometimes retrospective documentation of cases
gives interesting information. Figure 1 shows radio-
graphs of a 50-year-old patient when he was referred
to the Department of Periodontology at ACTA. Bite-
wing radiographs could be retrieved from his dentist
when the patient was 45 (Fig. 2) and 49 years old
(Fig. 3). It was obvious that most of the breakdown
had occurred in 1 year. The medical history revealed
no particular problems. This case clearly illustrates
that without documentation, assumptions on the rate
of previous disease progression are made blindly,
although in general, periodontitis is a slowly pro-
gressive disease whose pace may vary between indi-
viduals as well as during life. In a review on classi-
cation of periodontal diseases in 2002, Armitage (7)
stated that if a classication is based on the extent
and severity of the disease, age, and rate of progres-
sion, this would represent a return to the domination
of the Clinical Characteristics paradigm that reigned
from approximately 1870 to 1920, when we knew
little about the nature of periodontal diseases. The
1999 classication is based on the Infection Host
Response paradigm that started to be the dominant
paradigm in the 1970s. According to Armitage, the
1999 classication is even more rmly based on the
Infection Host Response paradigm. However, it
can be argued that, at present, regardless of the
enormous increase in knowledge of periodontal dis-
eases, we still know too little to diagnose and classify
the periodontal disease of a patient on an etiologic
basis.
Fig. 1. Radiographs of a 50-year-old male patient when he
was referred to the Department of Periodontology at
ACTA.
Fig. 2. Bitewing radiographs from the same patient as in
Fig. 1 when he was 45 years old.
Fig. 3. Bitewing radiographs from the same patient as in
Fig. 1 when he was 49 years old.
17
Essentialistic or nominalistic
disease classication
As Sherp (32) noted in 1964:
Discussions of periodontal disease commonly
begin with the tacit assumption that all partici-
pants are considering the same entity. In order to
be able to discuss cases between colleagues it is
for clinicians of paramount importance to be
able to give a diagnostic name to a patient with
periodontitis. One obvious problem is that one of
the most important components of periodontitis
is expressed in all patients in the same way, i.e.
the amount of loss of attachment. This can be
illustrated by the example that 2 mm loss of
attachment mesial of all rst molars in an 8-year
child is a severe problem suggestive for an indi-
vidual that is highly susceptible to periodontal
disease, whereas the same condition in a
60 years old subject may suggest that the indi-
vidual is rather resistant to periodontal disease.
Figure 4 illustrates this problem. The essentialistic
idea implies the real existence of a disease caused by
a class of agents. However, to date, all indications
have been that the causal web for periodontitis is so
complex and involves so many factors in so many
different constellations that a classication of perio-
dontitis based on etiology is effectively precluded
(10). Since periodontitis has to be regarded as a
syndrome, present and future classications of
periodontitis have to be based on the nominalistic
concept.
Classications based on this concept should be
simple to apply and not susceptible to multiple
interpretations. Ideally, such a classication should
be determined on the basis of documented differ-
ences regarding the consequences of the diagnosis
(10). Unfortunately, to date there is insufcient
knowledge to make a classication based on this
principle. However, it is most convenient if the ter-
minology used describes the patient in such a way
that all clinicians immediately have a clear image of a
case. The recent classication into aggressive and
chronic periodontitis (6) does not fulll this criterion
since the criteria are too indenite. However, in a
recent review Armitage (8) again discussed perio-
dontal diagnoses and classication. In this review he
accepted, in a way, the nominalistic concept by sta-
ting that a diagnosis can be phrased many different
ways depending on how precise or detailed one
wants to be. With regard to the distinction between
aggressive and chronic periodontitis it can be argued
that all forms of periodontitis are chronic in nature,
with the exception of acute necrotizing periodontitis
and a periodontal abscess. This would imply that
there is no place for the diagnosis aggressive perio-
dontitis, leaving the diagnosis chronic periodontitis
for all cases of periodontitis, a situation which is not
feasible in practice. Especially in relation to research
into the etiology of the various manifestations of
periodontitis, it is of utmost importance to include
clear phenotypes in the study groups. For clinicians
the most important characteristic of a patient is the
extent and severity of the periodontal destruction in
relation to age.
Classication according to the
nominalistic concept
At present, the best option is to classify the perio-
dontitis syndrome in an exhaustive but also exclusive
way and use a terminology for the various classes of
10 20 30 40 50 60
Age
PD 4 mm + AL 1 mm
PD 4 mm + AL 2 mm
PD 4 mm + AL 4 mm
Severe
Moderate
Minor
S
e
v
e
r
i
t
y

o
f

t
h
e

p
e
r
i
o
d
o
n
t
a
l

p
r
o
b
l
e
m

Fig. 4. Estimation of the severity
of the periodontal problem in rela-
tion to age. PD pocket depth.
AL attachment level.
18
van der Velden
the disease which makes it easy to understand the
case. A classication which comes closest to these
principles was recently published by Van der Velden
(38). This classication was based on four dimen-
sions, i.e. extent, severity, age, and clinical charac-
teristics. The following is a presentation of the
original classication with a few additions.
Dening when periodontitis is considered to be
present. It is suggested to dene periodontitis as
the presence of inamed pathological pockets
4 mm deep in conjunction with attachment loss.
If present, then the next steps can be taken.
Classication based on extent of the disease, i.e.
number of affected teeth (Table 1).
Classication based on severity of disease per
tooth (Table 2). The fact that either attachment loss
or bone loss can be used for the classication of
severity implies that although it may be important
to know the actual root length in a given patient,
radiographs are not a prerequisite for the classi-
cation of severity.
Classication based on age (Table 3).
Classication based on clinical characteristics
(Table 4).
The classication is ascertained in the following
way:
rst, the severity category is determined for each
tooth;
next, the extent category is determined by counting
the number of teeth with the most severe condi-
tion;
diagnosis on the basis of clinical characteristics is
added if applicable;
diagnosis on the basis of age.
In the nomenclature, the parameters for the clas-
sication are set in the following order: extent,
severity, clinical characteristics and age. Thus exam-
ples for diagnoses are: localized minor prepubertal
periodontitis, localized severe juvenile periodontitis,
semi-generalized minor juvenile periodontitis, gen-
eralized severe refractory post adolescent periodon-
titis, localized severe adult periodontitis. One could
make the diagnosis even more detailed by including
two levels of extent and severity when appropriate,
e.g. localized severe, semi-generalized moderate
adult periodontitis.
Traditionally in periodontology, a specic diagno-
sis has been introduced on the basis of severe cases,
Table 1. Classication based on the extent of the disease. If teeth are missing, the class description should still
reect the clinical image of the patient. Therefore it was decided for cases with 14 teeth to omit the class semi-
generalized and to change the number of teeth for the generalized class to 814
Permanent mixed dentition
No. of teeth present
Primary dentition
n 14 n 14
Incidental 1 tooth 1 tooth 1 tooth
Localized 27 teeth 27 teeth 24 teeth
Semi-generalized 813 teeth 59 teeth
Generalized 14 teeth 814 teeth 10 teeth
Table 2. Classication based on the severity of dis-
ease per tooth. The mean estimated root length based
on the literature is approximately 12 mm (21); in the
case of incidental disease, the severity category at
that particular tooth is mentioned
Minor bone loss 1 3 of the root length
or attachment loss 3 mm
Moderate bone loss > 1 3 and 1 2 of the root
length or attachment loss 45 mm
Severe bone loss >1 2 of the root length or
attachment loss 6 mm
Table 3. Classication based on age. If in patients
classied as adult periodontitis it can be demon-
strated on the basis of documentation that they
already had moderate or severe periodontitis before
the age of 36 years, the disease is classied as early
onset periodontitis
Early onset periodontitis
Prepubertal periodontitis 12 years
Juvenile periodontitis 1320 years
Postadolescent periodontitis 2135 years
Adult periodontitis 36 years
19
e.g. paradontosis (11), juvenile periodontitis (12),
rapidly progressive periodontitis (26), and prepuber-
tal periodontitis (27). However, in all patients the
disease started initially with minor breakdown and
progressed over time. It is only a matter of when the
patient is rst diagnosed. For example, in an epi-
demiologic survey carried out in Amsterdam in 15
16-year-old adolescents (39), 230 out of the 4565
subjects were diagnosed as having periodontitis.
These subjects showed a pocket depth 5 mm in
conjunction with attachment loss ranging from 1 to
8 mm. However, the majority (74%) had minor loss
of attachment ( 3 mm). Therefore it is important
that it is possible with a classication system of
periodontitis to make a clinical diagnosis for any
patient with periodontitis. This will also help in epi-
demiologic studies to obtain a better insight of the
periodontal problem in a given population. In addi-
tion, the use of the presented classication based on
the nominalistic principle will help the clinician to
get a better image of the patient population he is
treating. Furthermore, the new classication may
help research into the etiology of periodontitis by
including the same type of patients in the study
protocols. At present, in my opinion response to
treatment is still our chief diagnostic method (37).
Studying the response to treatment in well described
patient populations according to the new classica-
tion may help in the search for a better understand-
ing of the disease.
Conclusion
In order to obtain more knowledge about the caus-
ation of periodontitis and to be able to discuss cases
between colleagues, the various forms of the disease
have to be classied. Since periodontitis must be
regarded as a syndrome with a complex etiology,
classications of periodontitis should be based on the
nominalistic concept. Classications based on this
concept should be simple to apply and not suscept-
ible to multiple interpretations. In this paper an
example of such a classication has been presented.
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interdental gingival necrosis,
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gressive periodontitis. A distinct clinical condition.
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21
PERIODONTOLOGY 2000
ISSN 0906-6713
Inuences of systemic diseases
on periodontitis in children
and adolescents
JOERG MEYLE & JOSE
R. GONZA
LES
Periodontal disease in children and adolescents
comprises mainly gingivitis. There are few cases
where marked periodontal destruction has been ob-
served (111). These patients, if perfectly healthy, may
be described as early-onset periodontitis cases and
prepubertal periodontitis cases, depending on their
age at presentation.
Most prepubertal periodontitis patients have re-
lated systemic diseases that compromise the host re-
sponse to the microbial plaque. These, whether they
are caused by primary immunodeciency, by human
immunodeciency virus (HIV) infection, or by im-
munosuppressive therapy, may have profound ef-
fects on the oral tissues. In the severely immuno-
compromised host, bacteria from the oral cavity can
lead to septicemia and life-threatening infections
(96).
This chapter covers the systemic conditions that
may reduce the host response in children and ado-
lescents, thus increasing their susceptibility to peri-
odontal bone loss and ultimately loss of teeth. Many
of these cases are rare, and thus the evidence com-
prising this review is mainly based on case reports
rather than large-scale epidemiological studies.
Nevertheless, it is important to consider systemic
disease backgrounds to these severe periodontitis
cases, as this may eventually be relevant to treat-
ment and to understanding the pathogenesis of peri-
odontitis (129).
Leukocyte disorders
As far as neutrophils are concerned, inborn (genetic)
defects leading to a depressed or to a complete loss
of cellular chemotaxis are always accompanied by a
severe prepubertal periodontitis (173, 174). In a simi-
lar way, the reduction in the total number of cells
has a comparable effect.
92
Neutropenia
These diseases have periodontal manifestations, and
the group includes agranulocytosis, cyclic neutro-
penia, chronic benign neutropenia, chronic idio-
pathic neutropenia and familial benign chronic neu-
tropenia (178). The cyclic variety, which is inherited
as an autosomal recessive disorder, appears to be the
type most frequently encountered in the literature,
whereas familial benign chronic neutropenia is rela-
tively rare. A case of familial chronic benign neutro-
penia was reported by Deasy et al. (46). This patient,
a 14-year-old Caucasian boy, presented with severe
gingival inammation that had persisted for several
years. The medical history revealed that the patient
had familial benign chronic neutropenia that was
rst diagnosed at the age of 5 years. Subsequent
blood analyses indicated that the neutropenic state
was not of the cyclic variety. Prior to age 5 years, the
patient suffered numerous upper respiratory infec-
tions and experienced several episodes of otitis me-
dia that ceased to reoccur shortly thereafter. Oral ul-
ceration persisted throughout life. Histological
evaluations showed a chronic inammatory cell in-
ltrate with a large plasma cell component. The pa-
tient was advised about the importance of very strict
plaque control and the need for regular recall. The
pedigree of this patient indicated that the type of
familial benign chronic neutropenia segregating in
his family was of the autosomal dominant type.
Since father and son were both affected, the possi-
bility of this being a sex-linked trait was ruled out
(46). This case was similar to those in previous re-
ports in which families were described as exhibiting
an autosomal dominant form of inheritance for the
disorder (100).
Chronic benign neutropenia starts in infancy, al-
though a self-limiting course of 1018 months is
usually characteristic (94). It has been demonstrated
Inuences of systemic diseases on periodontitis in children and adolescents
that neutrophils present in the junctional epithelium
are engaged in phagocytosis of bacteria (150). In the
neutropenic state, either this defensive function is
absent or it is assumed by other cells such as mono-
cytes. Andrews (1965) reported a case of a 4.5-year-
old Caucasian boy presenting with ulceration,
chronic gingivitis and periodontitis. Laboratory
ndings demonstrated the importance of monocytes
in this disease, as monocytes took over the phago-
cytic function usually provided by polymorpho-
nuclear leukocytes. The same ndings were reported
by Wintrobe et al. (180). Ulceration, chronic gingi-
vitis and chronic periodontitis have been reported in
relation to the different forms of neutropenia (23, 40,
47, 65, 176). Ulceration may be episodic in cyclic
neutropenia but tends to be more persistent in
chronic neutropenia (137). The degree of peri-
odontal destruction and susceptibility to systemic
infection depends on the severity of the neutropenia
(90, 137).
More recently, Pernu et al. (133) reported two
cases of adolescents with cyclic neutropenia. Diag-
nostic salivary samples were collected from both pa-
tients. One of them had poor buffer capacity, but the
Streptococcus mutans count was not high because of
intensive caries prevention during the past few
years. One tooth presented with apical periodontitis,
despite appearing clinically intact and without
trauma or occlusal precontact. The question arises
as to whether cyclic neutropenia also affects the oral
defense mechanisms against dental caries and apical
periodontitis. These two patients retained their
teeth, and the authors emphasized the role and im-
portance of prevention. Appointments once or twice
a year were not consider sufcient to maintain peri-
odontal health without progressive attachment loss.
Regular monthly professional removal of dental
plaque and calculus has been recommended. Treat-
ment includes daily rinsing with 0.2% chlorhexidine
gluconate during the neutropenic episodes to
supplement the mechanical cleaning of the den-
tition.
One of the patients received human granulocyte
colony-stimulating factor which has been used re-
cently to raise neutrophil counts 10- to 12-fold in
patients with neutropenia (69, 90, 133). Granulocyte
colony-stimulating factor is a hematopoietic growth
factor that promotes the proliferation and differen-
tiation of neutrophils (38). In patients with malig-
nancies of the blood and blood-forming organs and
other cancers, recombinant human granulocyte col-
ony-stimulating factor is effective at correcting
chemotherapy-induced neutropenia and is useful in
93
the management of infections that complicate neu-
tropenia (64). In HIV patients, granulocyte colony-
stimulating factor has been used successfully to re-
verse neutropenia related to zidovudine therapy and
other causes and thereby reduce the risk of bacterial
and fungal infections (109, 113). This therapy pro-
duces a dramatic reduction in infections, oral ulcer-
ation and gingival inammation.
Chediak-Higashi syndrome
Chediak-Higashi syndrome has frequently been
linked with severe periodontitis (66, 71, 112, 168).
Chediak-Higashi syndrome is a rare autosomal re-
cessive immunodeciency disorder characterized by
large lysosomal granules in granulocytes, partial
oculocutaneous infections and intermittent febrile
episodes (5, 35, 76). Studies of host resistance to in-
fection in this syndrome have centered primarily on
leukocyte regulation and function. It is known that
Chediak-Higashi syndrome neutrophils and mono-
cytes are defective in chemotaxis, bacterial killing
and degranulation and are hyperactive in their
phagocytic capacity (5). Histories of recurrent infec-
tions since early childhood are usually complained
symptoms in these patients. Two boys and a girl be-
tween 9 and 20 years of age were analyzed for defec-
tive granulocyte chemotaxis. Leukocyte migration in
vivo was signicantly less than normal in the three
patients studied. The in vitro studies of granulocyte
chemotaxis utilizing the Boyden chamber technique
clearly demonstrated defective Chediak-Higashi syn-
drome cell migration (37).
There is a lack of information available in the den-
tal literature about Chediak-Higashi syndrome. A re-
view of the literature and a case report were pub-
lished in 1974 (68). They reported a 10-year-old black
boy who presented with marked mobility of the re-
maining dentition, gingivitis and pus exudation. A
roentgenogram revealed severe bone loss. Among
other samples, the bone marrow preparation showed
many granulocytes containing inclusions that were
varyingly azurophilic. Together with the presence in
blood smears of atypical granules in neutrophils,
lymphocytes and rarely in monocytes, these ndings
were considered to be diagnostic of the Chediak-Hi-
gashi syndrome. In the literature reviewed by the
same authors, severe gingivitis was found to be the
most common nding, as previously reported by
others (24). Ulcerations on the buccal mucosa, the
tongue and hard palate are also common ndings.
Severe periodontal destruction has been reported in
Meyle & Gonzales
these young patients, some of them undergoing full-
mouth extractions (24). Some patients with Chediak-
Higashi syndrome develop pancytopenia, high fever
not always associated with infection and lymphohis-
tiocytic inltration of liver, spleen and lymph nodes.
This condition is called the accelerated phase of
Chediak-Higashi syndrome. The etiology of the ac-
celerated phase is not known, but Rubin et al. (146)
suggested that there was a viral cause (virus-associ-
ated hemophagocytic syndrome) with a low blood
CD4/CD8 ratio in a patient with Chediak-Higashi
syndrome in the accelerated phase.
Aslan et al. (8) described a case of a 6-year-old
girl with the accelerated phase of Chediak-Higashi
syndrome. She complained of weakness, paleness,
recurrent febrile episodes, pyogenic infections, ab-
dominal distention and motor-mental retardation
for 5 years. The patient had decreased neutrophil ad-
hesion, chemotaxis and phagocytosis, and monocyte
adhesion, but viral causation (Epstein-Barr and cyto-
megalovirus) could not be substantiated.
Treatment in this phase is still difcult. In the past,
it was reported that functional defects in Chediak-
Higashi syndrome leukocytes were corrected by as-
corbic acid (26, 145, 147). Other treatments con-
sisted of management regimens such as vincristine
corticosteroids, etoposidecorticosteroidsintrathe-
cal methotrexate and high doses of intravenous g-
globulin, inducing a transient remission (17, 24, 89).
In the presented case the patient was successfully
treated with high-dose methylprednisolone and
splenectomy. This regimen should be considered in
treating cases in the accelerated phase of Chediak-
Higashi syndrome associated with hypersplenism
and/or cases nonresponsive to the medical therapy
regimens.
Recently, Delcourt-Debruyne (48) reported a case
of a 14-year-old male with premature exfoliation of
deciduous teeth and who presented extensive peri-
odontal lesions. Clinical, laboratory and histological
ndings revealed most characteristic features of
Chediak-Higashi syndrome, i.e., hematological ab-
normalities such as the presence of huge granules in
leukocytes, oculocutaneous albinism, photophobia,
neurological disorders and frequent oral and remote
infections as well as intestinal hemorrhage syn-
drome. Interesting ndings in gingival biopsies were
also described, with an epithelium showing exten-
sive desquamation, microulceration and inltration
by large numbers of polymorphonuclear leukocytes.
In some areas the epithelium was destroyed, expos-
ing the underlying connective tissue, which was also
inltrated by large numbers of polymorphonuclear
94
leukocytes. Surprisingly, none of the patients sib-
lings nor his parents were found to be affected by
the syndrome, excluding consanguinity as a criterion
of diagnosis in this particular case. Due to the severe
periodontal destruction, tooth extractions were the
only possible treatment in this patient.
Leukocyte adhesion deciency
syndrome
In 1979 a group of patients was identied with delay-
ed separation of the umbilical cord and defective
neutrophil motility, suffering from widespread bac-
terial infections including otitis media, septicemia,
impaired pus formation and delayed wound healing
(6, 170). The blood of these patients showed persist-
ent leukocytosis of 20,000 to 80,000 cells per micro-
liter.
A missing surface adhesion glycoprotein was iden-
tied as a part of a surface antigen rst described in
macrophages (Mac-1) neutrophils and large granular
lymphocytes. Later defects of LFA-1 and p150,95
were also detected. Today it is well known that
leukocyte adhesion deciency is characterized by
defects in the integrin receptors of white blood cells
leading to impaired adhesion and chemotaxis, which
is accompanied by an increased susceptibility for se-
vere infections and early-onset (prepubertal) peri-
odontitis. Both subunits of this receptor-family can
be decient, and Springer suggested, based on
further experiments, that the primary defect is in the
beta-subunit and that this subunit is necessary for
surface expression of the alpha-subunits of the re-
ceptors (161).
Two subtypes of the syndrome are differentiated
(leukocyte adhesion deciency syndrome I and II).
Leukocyte adhesion deciency syndrome I is due to
the absence of the beta subunit of the integrin mol-
ecule, and leukocyte adhesion deciency syndrome
II is caused by a deciency of sialyl 1 Lewis X, the
neutrophil ligand for selectins. In leukocyte ad-
hesion deciency syndrome II, therefore, the initial
steps of leukocyte adhesion to the vessel wall are
affected including the rolling of the cells on the sur-
face. In leukocyte adhesion deciency syndrome I
the binding to intercellular adhesion molecule is de-
fective. Both types are characterized by recurrent
bacterial infections, due to the impaired cell func-
tions with higher severity in leukocyte adhesion de-
ciency syndrome I (154).
Several mutations have been identied as the
source of the defect (43, 50, 91, 104, 161). Periodontal
pocket formation and bone loss around several or all
primary teeth starts shortly after eruption with a
rapid destruction and loss of periodontal attach-
ment. Exfoliation of the deciduous teeth begins prior
to the eruption of the permanent dentition, leading
to transient edentulism (49, 111).
The rst patients suffering from prepubertal peri-
odontitis and leukocyte adhesion deciency syn-
drome were described by Bowen et al. (25). In gingi-
val biopsies no leukocytes were detected within the
affected tissue, although an abundance of cells was
present in the local vessels. A transient improvement
of the local situation was reported after transfusion
of functionally normal neutrophils (127).
Local therapy alone was not successful in most of
the cases. Administration of high doses of ascorbic
acid did not improve the clinical situation (128).
Only bone marrow transplantation could resolve the
problem. It is possible that gene therapy may be of
similar effectiveness (57).
Recently the same defect was discovered in calves
and so an animal model exists to study this genetic
defect meticulously (61, 118120).
Papillon-Lefe`vre syndrome
The Papillon-Lefe`vre-syndrome was rst detected in
1924 in two siblings and described as a diffuse trans-
gradient palmoplantar keratosis with premature loss
of both the deciduous and permanent dentitions
(131). Papillon-Lefe`vre-syndrome belongs to a het-
erogeneous group of skin diseases that are all char-
acterized by hyperkeratosis of palms and soles (163).
Recently 19 different forms of palmoplantar kerato-
derma have been differentiated and classied (Table
1). Papillon-Lefe`vre-syndrome differs from other
types of palmoplantar keratoderma in its severe
periodontopathy and premature loss of the primary
and permanent dentition (Fig. 14).
Papillon-Lefe`vre-syndrome is a rare disease, trans-
mitted in an autosomal recessive manner with a pre-
velance in the general population of 1 to 3 per mil-
lion. At least one third of the families are con-
sanguineous. There is no sex or race preference.
About 200 cases have been reported in the literature.
Another form of the disease, also with palmoplan-
tar keratosis and severe early-onset periodontitis,
has been named Haim-Munk syndrome and differs
from Papillon-Lefe`vre-syndrome in several symp-
toms such as arachnodactyly, acroosterolysis and
onychogryphosis (73). During the last decade some
95
excellent reviews appeared where all the different
forms and cases have been described (72, 163).
The clinical symptoms of Papillon-Lefe`vre syn-
drome may vary. Hyperkeratotic skin lesions may
show a dramatic clinical picture, but in some other
reports hyperkeratosis was hardly visible or even
completely missing (31). Soskolne et al. (159) re-
ported two cases in which the presence of these
symptoms was only conrmed by a dermatological
examination (72) (Fig. 4).
Several authors reported that the involved palmar
and plantar surfaces are initially affected between
the rst and the fourth year of age, although skin
lesions may occur shortly after birth. In the family
reported by Soskolne et al., the mother, who herself
had not suffered from premature loss of teeth, had
already been recognized at the time of her birth as
having the skin lesions, because of their specic clin-
ical appearence.
Actinobacillus actinomycetemcomitans as well as
Haemophilus aphrophilus and Prevotella intermedia
have been isolated from the periodontitis sites in Pa-
pillon-Lefe`vre syndrome patients (22, 81, 92). High
serum antibody titers against the various microbial
species were also reported (138). Other authors,
however, have failed to nd A. actinomycetemcomit-
ans in Papillon-Lefe`vre syndrome patients, nor did
they see differences in antileukotoxin titers between
affected and unaffected patients (161).
Impaired neutrophil chemotaxis has also been re-
ported, which in some cases was accompanied by
decreased phagocytosis and reduced intracellular
killing of Staphylococcus aureus (175). In other re-
ports the chemotactic behavior of the neutrophils
was not affected (151). Already in 1988 Preus had
speculated that the hereditary defect in Papillon-Le-
fe`vre syndrome is located in the epithelial barrier
(138). According to Paghdiwala (130) in Papillon-Le-
fe`vre syndrome and other diseases exhibiting palmo-
plantar keratoderma, the clinical manifestation
could result from the same causative factor but it is
also possible, that different factors or combinations
of factors are leading to similar clinical symptoms.
Aso et al. (9) reported an abnormal keratin molecule
in Papillon-Lefe`vre syndrome as well as other struc-
tural defects as in the cementum and a functional
imbalance of collagenolytic activity in the peri-
odontal ligaments.
Genetic linkage studies of Hart et al. (73) did not
reveal a close relationship between keratin genes on
chromosomes 12 and 17 in the Haim-Munk syn-
drome. In two recent publications it was demon-
strated that the gene for Papillon-Lefe`vre syndrome
Meyle & Gonzales
Table 1. Palmoplantar keratoderma group
Type Name Inheritance Mutation Chromosome
I Jadassohn-Lewandowsky syndrome Autosomaldominant Keratin K16 and K6a 17q12-q21
II Jackson-Sertoli syndrome Autosomaldominant Keratin K17 17q12-q21
III Tylosis Autosomaldominant Unknown Distal locus of gene
cluster on 17q
IV
a
Papillon-Lefe`vre syndrome Autosomalrecessive Unknown 11q14-11q21
V Richner-Hanhart syndrome Autosomalrecessive Tyrosine aminotransferase 16q22.1-q22.3
deciency
VI Olmsted syndrome Autosomaldominant? Unknown Unknown
VII Vohwinkel syndrome Autosomaldominant Involucrin/Lorickrin 1q21
VIII Gamborg-Nielsen syndrome Autosomalrecessive Unknown Unknown
IX Huriez syndrome Autosomaldominant Unknown Unknown
X Fischer-Jacobsen-Clouston syndrome Autosomaldominant Unknown 13q
XI
b
Naegeli-Franceschetti-Jadassohn syndrome Autosomaldominant Unknown Unknown
XII Hyperkeratosis-hyperpigmentation Autosomaldominant Unknown Unknown
syndrome
XIII Dermatopathia pigmentosa reticularis Autosomaldominant Unknown Unknown
XIV Palmoplantar keratoderma, wooly hair, Autosomalrecessive Unknown Unknown
endomyocardial brodysplasia
XV Bart-Pumphrey syndrome Autosomaldominant Unknown Unknown
XVI Keratitis-ichthyosis-deafness syndrome Autosomalrecessive Epidermal glycogen Unknown
deposition
XVII Corneodermatosseous syndrome Autosomaldominant Unknown Unknown
XVIII Charcot-Marie-Tooth disease Autosomaldominant Unknown Unknown
XIX
a
Schpf-Schulz-Passarge syndrome Autosomalrecessive Unknown Unknown
a
Early tooth loss.
b
Enamel defects of teeth.
syndrome was localized on chromosome 11q14-q21
(58, 95). Although a considerable number of genes
have been mapped to chromosome 11, no obvious
candidate gene could be identied in the Papillon-
Lefe`vre syndrome gene region, but a cluster of ma-
trix metalloproteinases was mapped to 11q22 to q23
in a region about 8cM distal to the Papillon-Lefe`vre
syndrome locus (59). Some data suggest, that matri-
lysin (matrix metalloproteinase-7) was assigned to
the telomeric border of the Papillon-Lefe`vre syn-
drome region. As previously reported in one of the
Papillon-Lefe`vre syndrome patients, an altered elec-
trophoretic pattern of gingival collagen was detected
(155). Interestingly, the increased expression of hu-
man tissue collagenase (matrix metalloproteinase-1)
in the skin of a transgenic mouse resulted in hyper-
keratosis, acanthosis and basal cell proliferation (44).
Local therapy of the affected teeth in Papillon-Le-
fe`vre syndrome patients was generally unsuccessful,
and tooth loss was believed to be an inevitable se-
quela of the syndrome. Treatments mostly consisted
of early tooth extraction, and most of the patients
were edentulous before the permanent teeth
96
erupted. Antibiotic treatment alone did not result in
an improvement of the local situation and the teeth
had to be extracted. Successful therapy consisted of
biweekly oral hygiene measures and systemic anti-
biotics in combination with etretinate or acitretin
(27, 60, 70, 86, 108).
The systemic administration of synthetic retinoids
is not only useful to improve and correct the mol-
ecular defect in cytokeratins but also to treat any im-
balance in collagenolytic activity, since it is known
that retinoids also affect production and acitvity of
matrix metalloproteinases. Currently, one case re-
port exists where etretinate medication caused pyo-
genic abscess formation. According to the authors, it
is a possible side effect in all cases with impaired
neutrophil function (171).
A combined approach including meticulous
plaque control, administration of chlorhexidine in
combination with a systemic antibiotic therapy for
the eradication of known periodontal pathogens in
conjunction with retinoids seems to be most promis-
ing as long as the precise nature of the underlying
genetic defect is still not known (86).
Fig. 1. Three-year-old German boy with premature loss of
deciduous tooth 51 due to Papillon-Lefe`vre syndrome and
prepubertal periodontitis
Downs syndrome
Downs syndrome is an autosomal chromosomal
anomaly resulting from trisomy of the chromosome
21 (97). Downs syndrome is one of the most com-
mon causes of mental handicap in children. A high
prevalence of chronic inammatory periodontal dis-
ease in children with Downs syndrome has pre-
viously been described by Cohen et al. (41) and
Johnson & Young (80), but it was rst recognized by
Brousseau (29). In 1976 the prevalence and severity
of periodontal disease in 212 individuals with Downs
syndrome and 124 of their unaffected siblings was
evaluated (126). Both prevalence and severity of the
disease were greatest in the individuals with Downs
syndrome, whereas the siblings demonstrated an ex-
pression not unlike that of the United States popula-
97
tion of children in those years. An extensive review
of these cases has been previously published (141,
142). The authors analyzed such topics as the preva-
lence and severity, progression, distribution, causa-
tion and compared bone loss between Downs syn-
drome patients and other mentally retarded sub-
jects. Local factors were investigated as well as the
bacteriology, saliva, systemic factors and immune
factors related to these patients.
Their conclusions agreed with other investigators
that the prevalence of periodontal disease is almost
100% in children with Downs syndrome under the
age of 30 years. The onset of the disease process is
apparent even in the deciduous dentition. Peri-
odontal disease is often severe, especially in the re-
Fig. 2. a. Radiograph at the time of rst examination with
bone loss around deciduous teeth. b. Three years later
with complete loss of all deciduous teeth during eruption
of permanent dentition. c. At age of 8 years with severe
periodontal destruction around rst molars and incisors.
Meyle & Gonzales
Fig. 3. a. Same patient as in Fig. 1 at the age of 18.
b. Healthy 32-year-old brother at the same time.
gion of the lower anterior teeth. Its progression is
rapid primarily in the younger age groups. Most of
investigators agree that the oral hygiene is poor but
not commensurate with the severity of the peri-
odontal disease. Severity of periodontitis was higher
in subjects living in institutions than those living at
home. Reuland Bosma et al. (141) also concluded
that endogenous factors might contribute to the
rapid progression of periodontal breakdown. The
main immune defect occurs in the thymus-depend-
ent system, which may result in a reduced amount
of mature T cells together with a relatively large pro-
portion of immature ones. This, together with the
possibility of differences in collagen biosynthesis
and an abnormal capillary morphology, may explain
the higher susceptibility to periodontal disease ob-
served in Downs syndrome, the authors suggested.
The same authors performed a clinical study a
year later, where 9 Downs syndrome children and 14
healthy control children were followed in an experi-
mental gingivitis study around their deciduous teeth.
In addition to Plaque Index and Gingival Index (106,
157), gingival exudate measurements were carried
out in one quadrant of the upper jaw, using the
98
methods described by Le & Holm-Pedersen (105)
and the number of crevicular leukocytes recorded in
one lower jaw (10). Their results showed a very low
volume of gingival exudate in both groups, but in
the Downs syndrome group it increased signicantly
from day 0 to day 21. Also, a signicantly larger num-
ber of crevicular leukocytes was found in the Downs
syndrome children than in the controls. The ndings
of this study suggest that Downs syndrome children
have a different leukocyte response together with a
more extensive gingival inammation than normal
children.
Barnett et al. (15) compared the prevalence rates
of periodontitis and dental caries in 30 Downs syn-
drome patients and 30 matched, otherwise retarded,
controls. The study revealed a high prevalence of
periodontitis in patients with Downs syndrome and
thereby conrmed previously reported data. Because
the control population consisted of age- and sex-
matched patients with a similar degree of mental re-
tardation and comparable living arrangements, the
authors concluded that the marked differences in
prevalence and severity of periodontitis between the
Fig. 4. a. Right palm of the patient in Fig. 1 with discrete
dermatological symptoms of hyperkeratosis. b. Right
palm of the healthy brother also with discrete dermate
symptoms of hyperkeratosis.
two groups support the hypothesis that Downs syn-
drome, per se, confers increased susceptibility to
periodontitis. Another study conducted by Modeer
et al. (114) in 80 patients with Downs syndrome be-
tween 10 and 19 years of age revealed a signicantly
(P0.001) higher frequency of sites with alveolar
bone loss compared with a matched control group.
They also reported about a higher frequency of bone
loss in Downs syndrome children around the man-
dibular incisors compared with rst molars.
In an immunohistochemical study looking for
neuronal markers in inamed gingiva obtained from
children with Downs syndrome, the primary objec-
tive was to describe the inammatory involvement
as well as the innervation of the gingiva in these
children. After histopathological and immunohisto-
chemical evaluation, the authors found that patients
with Downs syndrome had profound inammatory
lesions of the gingiva and exhibited a dense inl-
tration of inammatory cells in the lamina propria
and close to the junctional epithelium (16).
A study investigated matrix metalloproteinases in
the saliva and gingival crevicular uid of nine Downs
syndrome children. The results suggested an inap-
propriate regulation of enzymes and matrix metallo-
proteinases in Downs syndrome patients. These
ndings support the novel concept that active matrix
metalloproteinase-8 derived from triggered poly-
morphonuclear leukocytes and/or cytokine-induced
periodontal broblasts may affect the periodontal
tissues and probably alveolar bone destruction seen
in gingivitis and periodontitis associated with
Downs syndrome (67).
More recently, Agholme et al. (1) reported clinical
and microbiological ndings in Swedish patients
with Downs syndrome who were followed up for a
period of 7 years. They found an increase in alveolar
bone loss of approximately 40% during this period,
although clinical signs of inammation were found
to be reduced. No signicant differences in micro-
biological ndings were found between baseline and
follow-up investigations. The authors compared the
development of periodontitis observed in these pa-
tients with that of adult periodontitis and early-onset
periodontitis patients. They concluded that the
mean individual annual bone height reduction was
of the same magnitude as that seen in patients with
adult periodontitis, and much lower than in patients
with early-onset periodontitis.
In another recent study, periodontopathic bac-
teria in children with Downs syndrome were ana-
lyzed. This was the rst study to describe the preva-
lence of periodontopathic bacteria in such a popula-
99
tion using the polymerase chain reaction method.
The subjects were 60 Japanese children with Downs
syndrome who were compared with another 60
healthy children serving as controls. After assessing
clinical parameters, subgingival plaque samples
were taken and investigated for the prevalence of 10
periodontal pathogens. With regard to the clinical
parameters, no signicant differences were found
between Downs syndrome patients and controls.
However, all of the selected children had good oral
hygiene habits maintained either by themselves or
by their guardians. With regard to the periodontal
pathogens, most of the microorganisms were found
with increased frequency in the group of children
with Downs syndrome, except for A. actinomycetem-
comitans, for which no signicant difference was ob-
served between the groups (2).
In conclusion, all these ndings suggest that
Downs syndrome patients may have inappropriate
regulation of enzymes and T-cell immunodeciency
together with functional defects of polymorpho-
nuclear leukocytes and monocytes. This, together
with the possibility of differences in collagen biosyn-
thesis and abnormal capillary morphology and
hyperinnervation of the gingiva, may contribute to
the rapid periodontal destruction observed in these
patients.
Diabetes mellitus
Diabetes mellitus is a syndrome of disturbed glucose
homeostasis caused by a deciency of insulin or of
its action resulting in abnormal metabolism of
carbohydrate, protein and fat. It is the most common
endocrine-metabolic disorder of childhood and ado-
lescence with important consequences on physical
and emotional development.
A total of 40 countries have collected and pub-
lished incidence data of childhood diabetes mellitus
up to the end of the 1980s. The majority of incidence
data comes from regions of high incidence: from
Europe and North America. A clear difference in in-
cidence appeared between the Northern and South-
ern Hemispheres, with no countries below the
equator having an incidence greater than 15 per
100,000. In contrast, above the equator the disease
is common. Between continents the variation in in-
cidence showed that the lowest incidences were
found in Asia, followed by Oceania (Australia and
New Zealand), South and North America, and the
highest rates were in Europe. The incidence varied
from 0.6 per 100,000 in Korea and Mexico to 35.3 per
Meyle & Gonzales
100,000 in Finland, showing prominent worldwide
variation in incidence of insulin-dependent dia-
betes. The largest intracontinental variation in inci-
dence appeared in Europe, varying from the highest
in Finland to the lowest (4.6 per 100,000) in northern
Greece. The highest incidence in the world was in
northern Europe, but within the continental scale
there were some striking exceptions from the overall
level of incidence (85).
Several investigators reported about a higher inci-
dence and severity of periodontal disease in type I
diabetic adults (18, 36, 39, 167). In diabetic children,
however, studies of gingival inammation are rare.
Bernick et al. (19) and Ringelberg et al. (143) found
a higher degree of gingivitis in children with diabetes
than in healthy children. In these studies, the dia-
betics were not grouped by the degree of metabolic
control of the disease. Such an analysis is highly rel-
evant, as there are indications that resistance to in-
fection is lowered in diabetics with poor metabolic
control compared with well-controlled diabetics (14,
123). A clinical study carried out by Gislen et al. (62),
in a group of 43 children with insulin-deciency dia-
betes, aged 7 to 17 years, showed no statistically sig-
nicant differences between those children with
good metabolic control, as assessed by the hemo-
globin A1c level, compared with non-diabetic con-
trols. The diabetic children with poor metabolic con-
trol had numerically higher Gingival Index scores
than the non-diabetics, and the same results were
obtained for Plaque Index. This difference was statis-
tically signicant, so that they concluded that dia-
betic children with good metabolic control show a
similar gingival status to healthy children, whereas
diabetic children with poor metabolic control
tended to be more susceptible to gingivitis than
healthy controls. There are conicting reports in the
literature as regards controlled studies in diabetic
children.
Another study compared 50 children and adoles-
cents aged 7 to 18 years (26 presenting insulin-de-
pendent diabetes and 24 controls), but all of them
were matched by age and gender. Oral examinations
were performed using the Silness & Le index for
assessing plaque, the Le & Silness Gingival Index,
gingival uid ow, plus clinical parameters such as
probing depth, clinical attachment level, recession
and bleeding on probing, which were recorded with
a standardized probe (107, 157). Laboratory meas-
urements of glycosylated hemoglobin were done by
high pressure liquid chromatography. The results
showed that the mean values for attachment loss,
probing depth, Gingival Index, Plaque Index and gin-
100
gival crevicular uid were slightly higher in the dia-
betic group. When using measurements averaged
over all sites, analysis of data did not demonstrate
any statistically signicant differences in periodontal
status between diabetics and controls. They only de-
tected a difference when individual tooth surfaces
were analyzed, demonstrating more gingivitis at the
buccal and lingual sites of the diabetic patients
(136).
Hypophosphatasia
Hypophosphatasia (Rathbun-syndrome) is a con-
genital disease with an autosomal-mode of trans-
mission and characterized by deciency of serum al-
kaline phosphatase, increased urinary excretion of
phosphoethanolamine and defective bone and tooth
mineralization, resulting in cementum hypoplasia or
aplasia and premature exfoliation of the primary
teeth (34, 177). Hypophosphatasia is now recognized
to be an inborn error of metabolism in which there
is decient activity of the tissue-nonspecic (liver,
bone and kidney) alkaline phosphatase. The con-
dition was rst reported in 1948 by Rathbun (140). It
may appear in a lethal neonatal or perinatal form
(congenital lethal hypophosphatasia), a severe infan-
tile form, or a milder form occurring in childhood or
late adolescence (hypophosphatasia tarda). In fam-
ilies, hypophosphatasia has been previously de-
scribed by several authors (13, 20, 28, 30, 33, 45, 83,
135).
In 1985, Baab et al. (11) reported a family where
three of the children manifested premature exfoli-
ation of deciduous teeth. Laboratory studies such as
monocyte and neutrophil chemotaxis count, anti-
body measurements (enzyme-linked immunosorb-
ent assay) for 18 periodontal bacteria, as well as sub-
gingival plaque samples were performed. The results
showed no manifested suppressed neutrophil
chemotaxis in the children, but a signicantly sup-
pressed monocyte chemotaxis was observed in all
three of them.
More recently, Watanabe et al. (177) reported a
case of hypophosphatasia from a 15-year-old patient
who had premature exfoliation of deciduous teeth in
infancy and exhibited alveolar bone resorption simi-
lar to that observed in localized juvenile peri-
odontitis. Biochemical, hematological, immunolog-
ical and bacteriological examinations were con-
ducted. The results showed a reduced alkaline
phosphatase activity and slightly elevated creatine
phosphokinase. Urine analysis revealed a remark-
able elevation of phosphoethanolamine level (310
times above normal). The serum antibody titers
against Porphyromonas gingivalis and Fusobacteri-
um nucleatum were very high. No reduction in the
chemotaxis of neutrophils or monocytes was de-
tected. The bacteriological results indicate that in-
fection with P. gingivalis is probably associated with
the destruction of the periodontal tissue in hypo-
phosphatasia.
While periodontal treatment in the primary den-
tition usually involves extraction of mobile teeth to
prevent discomfort, in the permanent dentition,
more conventional therapy may be attempted. In the
case described above, the patient underwent se-
lected extraction, oral hygiene instruction, scaling
and root planing and periodontal surgical therapy in
several regions. Most areas of the mouth responded
well to therapy and remained stable over the next 4
years; however, two molars were subsequently ex-
tracted due to progressive bone loss. This highlights
the need for close monitoring and recall of these pa-
tients and the need for biochemical tests for accu-
rate disease diagnosis, since clinical features in the
permanent dentition were similar to those of local-
ized juvenile periodontitis.
Lepe et al. (98) described the dental status of three
young adults who where diagnosed as having hypo-
phosphatasia as children, and were the same
children repeatedly reported by Baab et al. (11).
These three individuals have been monitored for 15
years. All of them maintained complete dentitions
with varying degrees of dental restoration. The
authors showed that the actual dental conditions of
these patients were more consistent with poor oral
hygiene or some other causes rather than conse-
quences of familial hypophosphatasia. This allowed
them to conclude that the manifestations of hypo-
phosphatasia are exhibited primarily in the decidu-
ous dentition and not evidenced in adults.
In contrast to this, Hu et al. (80) recently investi-
gated a family with hypophosphatasia. A pair of
twins, a 6-year-old boy and girl, their mother and
maternal grandfather were also affected. The investi-
gators detected a mutation in a single tissue, non-
specic alkaline phosphatase allele that resulted in
the substitution of alanine by threonine at amino
acid position number 99. The exact mechanisms of
identication and characterization of this mutation
were not described in this publication. Some of the
conclusions drawn by this study were that both
males and females are equally affected and that they
had only one affected parent. Affected males did not
transmit the disease to all daughters.
101
Histiocytosis syndromes
A case of hystiocytosis X with periodontal manifes-
tations was described in 1971 by Schoeld et al.
(149). The affected 21-month-old Caucasian girl pre-
sented with recurrent stomatitis, otitis externa and
media, vulvovaginitis and seborrhea. Clinical exami-
nation revealed a pale, thin child with fetid breath.
The primary teeth were completely erupted and well
formed. The anterior gingiva was inamed but
otherwise normal. In contrast, the gingiva around
the molars was markedly tender and gray in color
due to the necrosis, and the surrounding tissue was
erythematous. The gingival tissue in this area could
be reected to reveal deep periodontal pockets,
which exposed the furcations of the highly mobile
molars and contained more gray granulomatous
tissue. Other authors have reported similar clinical
observations, but also radiological ndings such as
several areas of radiolucency in the skull, particularly
in the frontal and parietal regions (139). Large de-
fects of the ramus and body of the mandible and
teeth missing were noted. Four other cases were re-
ported, all of them children between 7 and 8 years.
They all presented with pain in the temporomandib-
ular joint and loss of lower anterior teeth.
Shaw & Glenwright (153) reported on a 6.5-year-
old boy, who after clinical observations was initially
diagnosed as having prepubertal periodontitis. The
biopsy reported that the stratied squamous epithel-
ium covered brous tissue in which there was a
Table 2. Signs and symptoms in cases of oral in-
volvement of histiocytosis X
Eosinophilic granuloma
Localized periodontitis in an otherwise healthy
dentition
Loss of alveolar bone and replaced by soft tissue
Delayed healing after extraction of teeth
Premature loss of teeth
Foul breath
Solitary soft tissue involvement may affect the tongue;
it may also be confused with traumatic granuloma
Hand-Schller-Christian
Generalized stomatitis, soreness
Hemorrhage from the gums
Ulceration and necrosis of the oral mucosa
Progressive bone destruction of the alveolar process
Loosening and premature loss of teeth
Facial asymmetry
Letterer-Siwe
Ulceration of oral mucosa
Diffuse destruction of bone
Premature loss of teeth
Hemorrhage
Foul breath
Suppuration
Meyle & Gonzales
dense, almost conuent inltrate of inammatory
cells. Numerous eosinophils were present, but the
majority of the inammatory cells were histiocytes.
Immunoperoxidase studies on parafn-embedded
tissues showed the majority of inammatory cells
were positive for lysozyme and therefore most likely
to be of the histiocyte lineage. A diagnosis of histio-
cytosis X of the gingiva was made with a recommen-
dation that a skeletal survey should be carried out in
order to determine the extent of the disease. It was
then found that there was evidence of a long stand-
ing intermittent rash characterized by scattered
crusty lesions appearing periodically in the patients
scalp, behind his ears and in his axilla. A skeletal sur-
vey was normal but the chest radiograph revealed
very mild interstitial changes consistent with histi-
ocyte inltration. Histiocytosis X is not uncommon
in the lower jaw. In a series of 50 patients, 36% had
oral involvement and the dentist was the rst to see
them in 16% of the cases (156). In another report,
there were 114 cases with oral involvement among
Table 3. Langerhans cell histiocytoses
Class I Class II Class III
Diseases
Langerhans cell histiocytosis Infection-associated hemophagocytic syndrome Malignant histiocytosis
Acute monocytic leukemia
True histiocytic lymphoma
Familial erythrophagocytic lymphohistiocytosis
Cellular characteristics of the lesions
Langerhans cells with Morphologically normal reactive macrophages Neoplastic cellular proliferation with
Birbeck granules with prominent erythrophagocytosis macrophages or their precursors
Table 4. Types of Ehlers-Danlos syndrome
Type Clinical features Inheritance Biochemical features
I. Gravis Classical features, severe Autosomaldominant Unknown
II. Mitis Classical features, mild Autosomaldominant Unknown
III. Hypermobile Marked joint hypermobility; minimal skin ndings Autosomaldominant Unknown
IV. Sacks ecchymotic Visceral and large vessel rupture Autosomaldominant/ Absence of type III
Autosomalrecessive collagen
V. X-linked Similar to type II; skin highly extensible X-linked Unknown
VI. Ocular Ocular rupture, kyphoscoliosis, hyperelastic skin, Autosomalrecessive Lysyl hydroxylase
joint laxity deciency
VII. Arthrochalasis Congenital hip dislocation, hypermobile joints, Autosomaldominant Structural mutation of
multiplex congenita stretchable, velvety skin type I collagen
VIII. Periodontitis type Cutaneous fragility, pretibial scarring, periodontitis Autosomaldominant Unknown
IX. X-linked recessive Occipital exostoses, widening and bowing of long X-linked Defective collagen
skeletal type bones at tendinous and ligamentous insertion cross-linking
sites, deformed clavicles, mild skin hyperelasticity,
diminished lysyl oxidase activity
X. Dysbronectinemic Striae, moderate skin extensibilitys, Autosomalrecessive Dysfunctional plasma
type joint hypermobility, platelet aggregation defect bronectin
102
1120 patients with histiocytosis X; in 73%, the man-
dible was involved. Indeed, the oral manifestations
may be among the earliest or even the only signs of
the disease but are not pathognomonic (74).
The childhood histiocytoses constitute a rare and
diverse group of disorders. In the past, these dis-
orders were grouped as histiocytosis X. The X re-
ferred to the unknown pathogenesis. The term,
histiocytosis X, was introduced in 1953 by Licht-
enstein (101) to encompass the following group of
related disorders: rst, the localized hystiocytosis
form or eosinophilic granuloma; second, the acute
disseminated histiocytosis or Letterer-Siwe disease;
and third, the chronic disseminated histiocytosis in-
volving multiple sites or Hand-Schller-Christian
disease. The oral manifestations of these diseases are
shown in Table 2.
Combining the three disorders under the term
histiocytosis X solved the problem of trying to differ-
entiate between overlapping syndromes and of nam-
ing incompletely developed cases. However, not all
authors accepted this unitarian view, particularly
those whose studies are mainly conned to younger
age groups (102). Therefore, an international group,
the Histiocyte Society, proposed a system for the
classication of the childhood histiocytoses that in-
cludes the three syndromes previously described.
These disorders are now called Langerhans cell histi-
ocytoses (Table 3). Although the diagnosis and treat-
ment of these disorders are frequently the province
of the pediatric oncologist, the majority of the Lang-
erhans cell histiocytoses are not thought to be malig-
nant. In classes I and II histiocytosis, the cellular in-
ltrate is now thought to be the result of an uncon-
trolled reaction of a normal antigen-processing cell.
The rarity of the histiocytoses has prevented epide-
miological studies. The only form that appears to
have a genetic component is familial erythrophago-
cytic lymphohistiocytosis.
Numerous therapeutic schemes have been evalu-
ated. Radiotherapy and chemotherapy are reserved
for lesions not accessible to surgery (117, 165). Treat-
ment of the disseminated forms of the disease by
combination of surgery, radiotherapy, cortico-
steroids and antimitotic drugs has reduced the mor-
bidity and mortality. In all cases, very thorough root
planing dramatically improved the periodontal con-
dition, although a 16% recurrence rate has been re-
ported (74). Recommendations are that not all teeth
involved need to be removed: only the loose teeth
and the pathological tissue.
The diagnostic management of such children
should include hematological and immunological
investigations at an early stage. If these prove to be
essentially normal, a biopsy should be performed
and the opportunity taken to carry out root planing
in the affected area at the same time (153).
Ehlers-Danlos syndrome
The Ehlers-Danlos syndrome, a disorder mainly af-
fecting the joints and skin, has been classied into
ten types on the basis of clinical symptoms and in-
heritance pattern (Table 4). Of the recognized types
of Ehlers-Danlos syndrome, some have been found
to result from an abnormality of type I or III collagen
(32). Ehlers-Danlos syndrome type VIII was rst rec-
ognized by McKusick (110) in a family with skin fra-
gility, abnormal scarring, early tooth loss and severe
periodontitis. Another family with Ehlers-Danlos
syndrome type VIII was described in 1977. Associ-
ation of joint laxity, skin fragility without bruising or
hyperextensibility and extensive periodontal de-
103
struction was detected (164). In another case a 10-
year-old black girl lost central incisors and extensive
alveolar bone. The young girl was treated with peri-
odontal surgery under general anesthesia, and the
authors reported a profuse gingival bleeding at the
time of surgery and that healing was extremely slow
(103). Although relatively few cases of Ehlers-Danlos
syndrome type VIII have been reported, it appears
from various reports that there exists considerable
interfamilial variability of this form, with phenotypic
heterogeneity (21, 103, 110, 122, 164). Nelson & King
(122) reported two cases where the affected girls, 18
and 19 years old, were both granddaughters of a 70-
year-old white woman, who was also affected. The
distinguishing nding in Ehlers-Danlos syndrome
VIII is early-onset periodontitis, leading to prema-
ture loss of permanent teeth (Fig. 5, 6). Inheritance
is autosomal dominant. However, other clinical
manifestations vary, with different degrees of skin
hyperextensibility, fragility and scarring, minimal to
moderate joint hypermobility (usually limited to di-
gits), and normal to slightly increased tendency to
bruising on mild trauma (53). There also appear to
be overlapping phenotypes (21, 75, 77).
More recently, a case of a 9-year-old girl with clin-
ical signs of Ehlers-Danlos syndrome and general-
ized periodontitis who came from a family with his-
tory of premature loss of teeth was reported. The pa-
tient presented all clinical signs attributable to
Ehlers-Danlos syndrome type IV phenotype, that is,
thinner than normal skin with visible venous pat-
tern, moderate joint laxity, increased tendency to
bruising and unusual skin lesions.
Investigations revealed a normal collagen met-
abolism and dermal broblasts were apparently nor-
mal. The authors concluded that the complex inter-
play between extracellular matrix components may
explain why a defect in another non-collagenous
component of connective tissue could be respon-
sible for the phenotype (53).
When diagnosing Ehlers-Danlos syndrome VIII,
it is important to consider the possibility of Ehlers-
Danlos syndrome type IV, for which there is con-
siderable clinical overlap of symptoms, with the
exception of precocious tooth loss. For this reason,
type III collagen metabolism was investigated in
this case, because mutations affecting the struc-
ture, synthesis or secretion of type III procollagen
are responsible for the Ehlers-Danlos syndrome IV
phenotype. Collagen analysis can readily distin-
guish most forms of Ehlers-Danlos syndrome IV
from Ehlers-Danlos syndrome VIII and is crucial in
the evaluation of these two disorders, as type IV
Meyle & Gonzales
Fig. 5. a. Clinical view of a 14-year-old girl with Ehlers-
Danlos syndrome type VIII and generalized attachment
loss after 2 years of orthodontic treatment. b. Orthopanto-
mogram of the same patient wih bone loss around rst
molars and incisivi (photo and radiograph kindly pro-
vided by Dr. Glicher, Magdeburg).
has life-threatening complications of vascular, uter-
ine and bowel rupture. Interestingly, duodenal rup-
ture has been reported once previously in an adult
male diagnosed with Ehlers-Danlos syndrome VIII,
but this is not believed to be a common compli-
cation (43, 78).
There is no specic treatment for these disorders
and, although death may occur secondary to the
internal manifestations of the disease, life expect-
ancy is usually normal. Because of the history of pro-
longed bleeding in these patients, it seems prudent
to obtain a bleeding history and laboratory evalu-
ation. Complete blood count and bleeding time
should be assessed and consideration should be
given to more in-depth studies, including platelet
aggregometry. A skin biopsy to assess structure and
biosynthesis of collagen is also warranted, as Ehlers-
Danlos syndrome type IV may have similar pheno-
typic features but carries a much graver prognosis
because of the risk of arterial and organ rupture. For
those in whom a bleeding abnormality is identied,
desmopressin may correct this abnormality and
104
allow the patient to undergo procedures with little
or no risk of hemorrhage (43).
Juvenile hyaline bromatosis of
gingiva
Hyaline bromatosis is often familial and until now
of unknown causation. The few reports about the
disease, describe typical multiple gingival and cu-
taneous nodules, papules or tumor masses from 1
mm to about 5 cm, progressively painful exion con-
traction of the major joints that usually appears in
the rst year of life (56, 124). In recent years, one
case of this extremely rare disease has been pub-
lished (134). The case was a 10-year-old girl that
came from Albania to Italy, presenting with cu-
taneous lesions typical of the disease on the head,
back, limbs, nose, ears, scalp and knees. There is no
known cause for the joint contracture, but an inl-
tration of the capsules of the joints by tumor tissues
has been discussed (166). Multiple gingival nodules
Fig. 6. a. Hyperexible and arachnodactyl phalanges of
the patient in Fig. 5. b. Pergament-like skin and hemosid-
erotic staining of lower legs in the same patient (photos
kindly provided by Dr. Glicher, Magdeburg).
were observed completely covering the teeth in both
archs. Generalized osteoporosis, scoliosis, reduced
height and weight as well as other skeletal manifes-
tations have been described (124). The girl also pre-
sented with lesions in the fronto-parietal, temporal
bones and in the right tibia. The hyalin brous tu-
mors appear during the rst few years of life and
slowly enlarge, although some regress over a period
of years. The progressive enlargement of the lesions
is due to an increase in the amount of the intercellu-
lar hyalin produced by the cells (99). The nature of
this hyaline material is not known, but appears to
be a disorder in type VI collagen (63). Differential
diagnosis is necessary, due to the similarity with
other conditions such as gingival bromatosis, a
condition were the lesions are limited to the gingiva
with no other affected body parts.
Acquired immunodeciency
syndrome
The rst case of pediatric acquired immune de-
ciency syndrome (AIDS) was recognized in Novem-
ber 1982, 18 months after the detection of the syn-
drome in adults. Human immunodeciency virus in-
fection has been identied in increasing numbers of
children with otherwise unexplained immune de-
ciency and opportunistic infections of the type
found in adults with AIDS (3, 125).
The risk factors for pediatric HIV infection vary
depending on the age group. Most children with
AIDS are under 5 years of age. The primary risk fac-
tors are perinatal. Infants born by women who are
intravenous drug users or who have bisexual part-
ners comprise the largest group (4). Clinical abnor-
malities are often present by age 3 months but may
also be a late manifestation of the syndrome. About
one third of the infants weigh less than 2500 g at
birth and are small for gestational age.
The rst clinical signs of HIV infection in infants
and children may include one or more of the follow-
ing: failure to thrive, hepatosplenomegaly, diarrhea,
interstitial pneumonitis, lymphadenopathy, oral
candidiasis and recurrent infections.
In addition to candidiasis, recurrent aphthous ul-
ceration and herpes simplex virus, oral lesions seen
in HIV patients or in members of the risk groups
may include severe gingivitis and salivary gland en-
largement, also with xerostomia (12). Salivary gland
enlargement is a condition more common in
children than adults, the cause of which is unknown
105
(82). Treatment with topical uoride to prevent
caries is indicated in these children with xerostomia.
In general, periodontal diseases among younger
patients have been associated with defects in neu-
trophil function (172). There are few reports describ-
ing the periodontal status of children with HIV infec-
tion. An unusual gingivitis with diffuse erythema has
been observed, similar to an atypical form of necrot-
izing ulcerative gingivitis (158, 179).
A recent clinical study carried out by Howell et al.
(79), investigated 60 HIV-infected children and the
relationship of CD4 lymphocyte levels with the
prevalence of selected common oral lesions. A sim-
plied Gingival Index (a modied version of the
Le & Silness Gingival Index) was performed with a
0 to 3 scale. Plaque Index scores were also deter-
mined for the maxillary central and lateral incisors.
Periodontal disease was diagnosed based upon the
advance stage of the lesion. Clinically, this was mani-
fested by attachment loss, spontaneous bleeding, se-
vere inammation and radiographic evidence of
moderate to severe bone loss. Pain and fever were
also universal observations. At the laboratory, CD4
lymphocyte counts were obtained within 3 months
of oral examination. The results showed that almost
50% of the subjects exhibited some soft tissue oral
manifestations of the HIV infection, indicating that
oral ndings may suggest the presence of the dis-
ease. Oral candidiasis occurred in one third of the
subjects, which was similar to the prevalence pre-
viously reported (87). In this study, there was an as-
sociation between CD4 counts and the severity of
periodontal disease, which suggests that periodontal
disease in children may be associated with immune
dysfunction rather than local factors (79).
Recent advances in therapeutics include new
classes of drugs, such as protease inhibitors, that ap-
pear to have persistent dramatic antiretroviral ef-
fects, and a new study indicates that a short course
of antiretroviral therapy administered during pri-
mary infection in adults may improve the sub-
sequent clinical course and increase the CD4
lymphocyte count (88). These ndings raise the

possibility that very early intervention with highly ef-
fective antiretroviral agents in infected neonates may
be able to modify signicantly the natural history of
HIV disease in children. An understanding of the
pathogenesis of perinatal transmission is crucial for
the design of new preventive and therapeutic inter-
ventions (115). The role that vitamin A has on the
transmission of HIV from infected women to the
child has also been investigated. Vitamin A is an es-
sential micronutrient for normal immune function.
Meyle & Gonzales
Vitamin A deciency is common among HIV-in-
fected pregnant women and is associated with
higher mother-to-child transmission of HIV and in-
creased infant mortality. The biological mechanisms
by which vitamin A deciency could inuence
mother-to-child transmission of HIV include impair-
ment of immune responses in both mother and in-
fant, abnormal placental and vaginal pathology and
increased HIV viral burden in breast milk and blood.
Clinical trials are currently in progress to determine
whether daily micronutrient supplementation, in-
cluding vitamin A, during pregnancy can reduce
mother-to-child transmission of HIV (152).
Virus-associated hemophagocytic
syndrome
With the exception of HIV, the literature associating
viral infections with periodontal disease is still rare.
Some manifestations in oral mucosa have been
found in patients with cytomegalovirus infection,
usually in combination with adult HIV infection. Hu-
man viruses, such as cytomegalovirus and Epstein-
Barr virus, have been detected in periodontal
pockets of adults manifesting various forms of peri-
odontitis (132). However, very little information
exists about viral infections and periodontitis of
children and adolescents. Recently, an unusual case
of early-onset periodontitis with concomitant virus-
associated hemophagocytic syndrome was reported
in Japan. Virus-associated hemophagocytic syn-
drome was originally reported by Risdall et al. (144)
as a systemic disorder characterized by benign gen-
eralized histiocytic proliferation. The patient was a
16-year-old male, manifesting all immune disorders
usually related to virus-associated hemophagocytic
syndrome: extremely low number of white blood
cells and platelets, elevated CD4/CD8 ratio and
much higher interferon-g levels than normal. The
patient referred spontaneous gingival bleeding, and
at clinical examination he revealed slight gingival
redness and swelling around the marginal gingiva.
Radiographic examination showed moderate hori-
zontal bone loss in almost all regions, with slight ver-
tical bone resorption in the molar regions. A clinical
diagnosis was made of generalized early-onset peri-
odontitis in combination with the systemic disease.
The patient received treatment that included exten-
sive toothbrushing instruction and scaling and root
planing. P. gingivalis and A. actinomycetemcomitans
were identied by microbiological analysis. After
106
therapy, his serum showed elevated antibody titers
against P. gingivalis and A. actinomycetemcomitans,
and the authors concluded that the patients hu-
moral immunity had recovered following the initial
phase of periodontal therapy, which is consistent
with other studies (93, 116).
Malnutrition
It is almost impossible to nd current literature
about oral ndings and malnutrition in children.
One of the reasons could be that malnutrition in
childhood and in adolescents does not now occur in
industrialized countries per se, and even in most of
the developing countries it would be very rare. Ac-
tual publications describing this condition are also
extremely rare. Some recent crises where mal-
nourished children are seen, such as those in Africa,
could reveal more on this terrible condition, but no
reports have appeared. In addition, there is little
supportive evidence of a statistical relationship be-
tween the energy intake and the presence or absence
of dental caries and the degree of periodontal dis-
ease (51). A paper written in 1985 by Sawyer &
Nwoku (148) published the results of an investiga-
tion done in 52 Nigerian children from 1 to 5 years,
all severely malnourished. Two presented with
cancrum oris and died shortly after the beginning of
the study. Blood samples were collected and com-
pared with samples of seven well-nourished
children. As expected, total serum protein and albu-
min levels were highest in the well-nourished group.
The malnourished children presented with several
oral manifestations such as caries, gingivitis, angular
cheilitis, candidiasis, and some of them manifested
a marked delay in tooth eruption. The authors ob-
served 11 patients from the malnourished group
who were maintained on a high protein and bal-
anced diet for 6 months. Gingivitis improved from
severe to mild in most of them.
In 1973, 872 Nigerian children were examined to
ascertain the inuence of socioeconomic conditions
on enamel hypoplasia. The relationship between
socioeconomic factors and the prevalence of necrot-
izing ulcerative gingivitis was also studied (54, 55).
Prevalence rates of 15.3% among malnourished
children living in a village and 27.2% among children
hospitalized with kwashiorkor were detected.
There is considerable evidence of nonspecic
manifestations in the oral cavity caused by a de-
ciency of vital nutrients such as protein, vitamins
and minerals (160). One of the principal conse-
quences of severe protein deciency is an increased
susceptibility to infection (52). Except for iron and
zinc, no other oral manifestations are known in min-
eral deciency conditions. Iron deciency produces
atrophy of lament-like papillae on the tip of the
tongue. In zinc deciency there is an impaired or
altered taste sensation. However, deciencies of
these nutrients are seen more in elderly people.
In the child, periodontal damage can appear if
there is a deciency of vitamins. The most reliable
sign of a deciency of vitamin C (scurvy) is gingivitis
which starts in the interdental papillae and spreads
to marginal and attached gingivae. In severe de-
ciency, the regeneration of collagen in the peri-
odontal ligament and alveolar bone can fail, with the
shedding of teeth and bone resorption being the ulti-
mate results (169).
Epithelial tissue differentiation is altered in vit-
amin A deciency. The normally nonkeratinized mu-
cosal cells are progressively replaced by keratinizing
cells. Lack of this vitamin can also cause decreased
salivary ow, hyperkeratosis and hyperplasia of the
gingival epithelium.
Food allergies (such as chocolate) or food addi-
tives (such as cinnamon and benzoates) sometimes
have oral manifestations. Ingestion can result in oro-
facial granulomatosis, erythema multiforme, lichen-
oid eruptions, recurrent oral ulceration and burning
mouth syndrome (160).
In recent years, a new perspective for the science
of nutrition has been proposed, but questions such
as the importance of the role that antioxidant nutri-
ents (such as vitamin E and carotene) have on pro-
tective mechanisms against periodontal damage,
and on the immune response in general and the in-
tegrity of periodontal tissues, have still not been
answered (121).
Summary
Systemic diseases affecting the host response as pri-
mary immunodeciencies or secondary defects
caused by lack of nutrients or changes in the local
tissues are very often accompanied by early-onset
prepubertal periodontitis. Local treatment in combi-
nation with systemic antibiotics may in milder forms
improve the situation, but in many cases the success
is questionable and premature loss of teeth occurs.
Since the genetic basis of many of the diseases has
been identied, future developments permit the cor-
rection of at least some of these defects by gene
therapy.
107
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PERIODONTOLOGY 2000
ISSN 0906-6713
Diagnosis and management of
periodontal diseases in children
and adolescents
VALERIE CLEREHUGH & ARADHNA TUGNAIT
A number of different forms of periodontal disease
canpresent inchildrenand adolescents, ranging from
reversible conditions limited to the gingival tissues to
those characterized by destruction of the periodontal
connective tissue attachment and alveolar bone,
which may jeopardize the longevity of the deciduous
or permanent dentition. The prevalence, extent, se-
verity and prognosis of periodontal disease in the
younger age groups vary according to the disease in
question. The diagnostic options are determined by
an up-to-date classication of the periodontal dis-
eases, and this has been an area of ongoing debate
and review (7, 13, 16, 29, 93, 152, 156). Fundamental
principles need to be applied to identify and manage
periodontal problems in these patients together with
an understanding of the causation and contributory
risk factors (Fig. 1) andanappreciationof the different
strategies inherent in working with a younger age
group compared with the adult patient. The aim of
this chapter is to critically review the diagnosis and
management procedures in the treatment of peri-
odontal diseases in children and adolescents.
Diagnosing periodontal disease in
children and adolescents the
patients history
The patients history, in conjunction with the exami-
nation, forms the basis for the diagnosis of the peri-
odontal condition and should involve both the child
or adolescent and the parents or guardians of minors
(Fig. 1). The key aspects to be covered are summar-
ized in Fig. 2 and should include, as for the adult
patient, the presenting complaint or the reason for
attendance, history of any presenting complaint or
problem, past dental history, past medical history
146
and social history. In eliciting this information, the
child or adolescents guardian may be a more re-
liable source of information. A full medical history is
important in identifying children with cardiac
lesions or congenital heart disease, which may put
them at risk of infective endocarditis from bacterae-
mia created by periodontal probing, scaling, polish-
ing, subgingival instrumentation and extractions.
Consultation with the childs pediatrician or consul-
tant physician is essential. Furthermore, the medical
history may elicit certain systemic risk factors for
periodontal diseases (see the chapter by Meyle &
Gonzalez in this volume (108)).
Systemic risk factors
Although periodontal diseases are plaque-induced
infections, not all patients are equally prone to de-
veloping these diseases and, as such, the concept of
individual risk has arisen. A risk factor for peri-
odontal disease is a characteristic, aspect of behavior
or an environmental exposure associated with peri-
odontal disease (69, 96, 124). The association may or
may not be causal (69, 96, 124). Some risk factors are
modiable, whereas others (background factors or
determinants) are not (96). A number of systemic
conditions should alert the clinician to the likelihood
of current periodontal diseases or an increased fu-
ture susceptibility. Furthermore, oral signs may be
the rst presentation of a signicant systemic con-
dition. The management of periodontal diseases in
association with serious systemic conditions re-
quires appropriate medical input, and specialist
periodontal care may be required. Potential systemic
risk factors may be elicited when carrying out a thor-
ough patient medical history (108).
Table 1 provides a list of conditions with gingival
Diagnosis and management of periodontal diseases in children and adolescents
Fig. 1. Key stages of management of the young periodontal patient
manifestations, and Tables 24 provide a checklist of
conditions with destructive periodontal effects. For
detail of the clinical manifestations and pathology,
see the chapter by Meyle & Gonzalez in this volume
(108).
Examining children
Examination of the periodontal tissues in the
younger patient should be a routine part of the den-
tal examination as depicted in Fig. 3. It should in-
clude an initial periodontal screening using a basic
periodontal examination (Fig. 4) to provide a quick,
simple assessment of the periodontal tissues (47, 96).
Any general descriptors of the periodontal condition
should be noted. Special tests such as radiographs
and pulp vitality tests may be required before reach-
ing a diagnosis (47).
147
The need for full periodontal assessments using
periodontal indices is determined after reaching a
diagnosis and should be included in the formulation
of the treatment plan in order to quantify disease
and enable monitoring of the periodontal condition.
Monitoring is typically required for plaque and gin-
givitis. Where screening has detected pockets and
other features of periodontal signicance, then
monitoring (96) should be undertaken for these, as
discussed in the section on initial therapy later.
Periodontal screening
Periodontal screening in children and adolescents
provides a simple and quick method of identifying
periodontal problems which is comfortably tolerated
and gives the dental practitioner an indication of the
need for treatment or further assessment. Following
Clerehugh & Tugnait
Fig. 2. Key stages in history taking for the young periodontal patient
the inception of the Community Periodontal Index
of Treatment Needs (1, 48) as an epidemiological
screening tool, its use in individuals was advocated
(50). In 1986, the British Society of Periodontology
recommended its introduction into general dental
practice (26) and the International Dental Federation
produced guidelines for its use as a simplied peri-
odontal examination for dental practices (65). Since
then it has been adopted as a basic periodontal ex-
amination to provide a quick, simple means of as-
sessing a patients periodontal condition and giving
a general indication of treatment needs (49, 131).
The basic periodontal examination was never in-
tended as a replacement for periodontal indices de-
signed to measure periodontal status such as the de-
tailed six point per tooth measurement of attach-
ment levels, probing depths or bleeding on probing
and the recording of recession, furcation or mobility,
but rather was intended as a basis to determine the
148
patients who would benet from a more detailed
periodontal examination and who may require more
complex periodontal therapy. In 1992, the American
Dental Association and the American Academy of
Periodontology introduced periodontal screening
and recording into general dental practice and rec-
ommended it be conducted for all patients as an
integral part of oral examination (9, 90, 91). Peri-
odontal screening and reporting is a modication of
Community Periodontal Index of Treatment Needs
that dentists in practice in the United States have
found to be a sensitive, simple, rapid and cost-effec-
tive method of screening patients for periodontal
diseases that summarized necessary information
with minimal documentation (67). In children and
adolescents, its use took less time and was better ac-
cepted than full probing with a graduated peri-
odontal probe, and no differences were found in di-
agnosis and clinical management (120).
Table 1. Risk factors associated with gingival manifestations in children and adolescents
Risk factor Nature of risk factor Gingival effects
Hormones Hormones can moderate the response of the tissues An increase in gingivitis has been associated with:
to plaque in teenagers in response to puberty, the O onset of puberty in both sexes (109);
contraceptive pill and pregnancy. Estrogen and O oral contraceptives (117), with a tendency for more
progesterone receptors have been recognized in inammation with longer use; and
the gingiva (159). During pregnancy, increased O pregnancy (114): tooth hypermobility up to the
progesterone levels cause dilation of the gingival eighth month reects the hyperlaxity of the
capillaries, increased permeability and gingival periodontal ligament during pregnancy, and
exudate (144) and altered plaque composition (94) epulides may present in association with local
plaque-retention factors
Drugs Three groups of drugs have been associated with Drug-induced gingival enlargement:
gingival enlargement: anticonvulsants, O may commence within 3 months of starting to take
immunosuppressants and calcium channel- the drug;
blocking agents (107, 144146). Drugs that may be O has a predilection for anterior gingiva; and
used in children and adolescents include phenytoin O has an increased propensity in children and
to control epilepsy or the immunosuppressant adolescents. It has been speculated that this could
cyclosporin to prevent organ rejection following be due to a drug-related increase in gingival
transplantation, possibly with the calcium broblast androgen metabolism that may target
channel-blocker nifedipine. A multifactorial model subpopulations of broblasts and cause either
has been proposed to explain the changes seen in increase in collagen synthesis or decrease in
which genetic factors that give rise to broblast collagenase activity (144146)
heterogeneity, gingival inammation and
pharmacokinetic variables appear to be signicant
in the pathogenesis (146)
HIV/AIDS Acquired immune deciency syndrome (AIDS) is Three forms of periodontal disease associated with
caused by the human immunodeciency virus HIV were agreed at a European Union workshop on
(HIV), which is a retrovirus with a special afnity oral problems associated with HIV infection (56), two
for CD4 receptor molecules on the surface of T- of which have gingival effects:
helper lymphocytes and other cells, such as O linear gingival erythema is seen in HIV-positive
monocytes, macrophages, Langerhans cells and B individuals. This presents as a distinct band of
lymphocytes. The condition causes a reduction in intense marginal gingival erythema, which is
CD4
T-helper cells and an increase in T-suppressor disproportionate to the quantity of plaque

cells, adversely affecting immune functioning (82). present and characteristically fails to respond to
The nature of the condition predisposes the simple plaque removal. There may be diffuse red
individual to candidal infection. or petechia-like lesions more apically. Pain or
Most of the periodontal literature relates to young ulceration are rare (107); and
adults or adults (107). If children are affected, they O necrotizing ulcerative gingivitis includes
are likely to be under 5 years old (108). There are ulceration, sloughing and necrosis of the
few documented case reports of periodontal interdental papilla in this painful gingival
problems in HIV-affected children (108) condition (83, 107). Spirochetes and fusiforms
predominate in the subgingival ora (83)
Leukemia Acute myeloblastic or lymphoblastic leukemia has Generalized gingival swelling may be associated with
been reported to account for 50% of all malignant leukemic inltration, particularly in acute
disease in children and to be the most common myeloblastic leukemia. There may be gingival
cause of non-accidental death in children (143). bleeding related to thrombocyte and coagulation
Oral manifestations may be a presenting feature, abnormalities. Gingival ulceration is a common
therefore prompt referral and hematological feature (143)
investigation are essential
Vitamin C Vitamin C contributes to the formation of collagen, Gingival swelling, sponginess, bleeding and
deciency bone matrix and endothelial components of the ulceration may manifest in vitamin C deciency.
vascular tree. Deciency leads to impaired Formed collagen is unaffected by deciency. True loss
production of collagen and intercellular ground of attachment and pocket formation do not result
substance and weak capillary walls (144) from deciency of the vitamin alone (144)
Special considerations when using
the basic periodontal examination
in children
In children, following the eruption of the incisors and
rst permanent molars, screening using the basic
periodontal examination can be undertaken on the
index teeth 16, 11, 26, 36, 31 and 46 (International
Dental Federation notation) using the WHO 621
probe with the 0.5-mm spherical ball on the tip and
shaded band at 3.55.5 mm to delineate normal sulci
149
and periodontal pockets (63) (Fig. 4). It is rec-
ommended that only codes 0, 1, 2 be determined up
to the age of 11 years because of the likelihood of
pseudopockets associated with newly erupting teeth
(65). However, if the 3.5- to 5.5-mmblack band on the
WHO 621 probe disappears into any unusually deep
pockets, then further periodontal investigation is re-
quired. The 0.5-mmball end on this probe is very use-
ful for detecting subgingival calculus deposits, and
this procedure can be comfortably undertaken on
young individuals, providing the recommendedprob-
Clerehugh & Tugnait
Table 2. Systemic risk factors associated with periodontal destruction in children and adolescents
Risk factor Nature of risk factor Periodontal effects
Diabetes Deciency/lack of insulin production due to Diabetes mellitus is a risk factor for periodontal
mellitus destruction of insulin-producing beta cells of disease. Attachment loss and bone loss are more
(insulin- pancreas. Trigger may be a virus, which induces an prevalent and severe in poorly controlled diabetes
dependent) autoimmune response. Presenting features include and the incidence increases after puberty and with
polyuria, polydipsia and polyphagia associated with age (108, 123, 127).
hyperglycemia. Onset of this form is typically before Factors associated with periodontal effects include
20 years of age. Age of clinical onset has decreased (123):
(70, 154). Treatment involves injection(s) of insulin O decreased functioning of polymorphonuclear
and balancing dietary carbohydrate to control leukocytes (chemotaxis, adherence and
blood glucose; multiple insulin formulations and phagocytosis);
regimes available. Long-term complications include O collagen affected by glucose level (decreased
retinopathy, nephropathy, neuropathy, synthesis, increased collagenase activity, formation
macrovascular disease and altered wound healing. of advanced glycation end-products;
Conclusive evidence of role of good glycemic O increased susceptibility to infections (due to
control in preventing complications in insulin- polymorphonuclear leukocyte defects, vascular
dependent diabetes (54). Good control can be changes and effects of increased insulin resistance
difcult in children and adolescents (106, 138) during acute infection); and
O reduced wound healing
HIV/AIDS Most of the literature relates to young adults/adults Differentiation between necrotizing ulcerative
(107). gingivitis and the initial stages of necrotizing
Of the three forms of periodontal disease ulcerative periodontitis can be difcult, and it is
specically related to HIV (56), two have a gingival unclear whether necrotizing ulcerative gingivitis
manifestation (linear gingival erythema and represents an initial stage of necrotizing ulcerative
necrotizing ulcerative gingivitis see Table 1), periodontitis in HIV-positive individuals. Necrotizing
while necrotizing ulcerative periodontitis (NUP) is ulcerative periodontitis may present at localized sites
associated with periodontal destruction. or generalized with (107):
LGE and the initial stages of NUP may be the rst O necrosis and loss of papillary and marginal
sign of HIV infection, therefore early diagnosis is gingiva;
critical. The presence of LGE and NUP has been O necrosis and loss of alveolar bone, which may be
correlated with decreased CD4 cell counts, and the very rapid;
presence of NUP has been shown to be highly O pocket formation not a feature due to rapid
predictive of CD4 cell counts 200/mm
3
and AIDS necrosis and recession;
diagnosis (107) O exposure of alveolar bone;
O spontaneous gingival bleeding;
O deep, aching pain;
O extension of destruction beyond mucogingival
junction leading to tooth mobility; and
O spread of necrotizing ulcerative periodontitis to
become necrotizing stomatitis involving other
structures, with potentially fatal consequences.
While there are few documented case reports of
periodontal manifestations in HIV-infected children
(108), it is important to note that the conventional
non-HIV-related forms of plaque-associated
gingivitis and periodontitis can also occur in HIV-
infected individuals (107)
ing force of 2025 g is not exceeded. In 12- to 19-year-
old subjects, the full range of scores can be used on
the index teeth so that periodontal pockets can be de-
tectedas early as possible, althoughcare shouldbe ex-
ercised in distinguishing true pockets with attach-
ment loss from pseudopockets (65). If pockets are de-
tected, then full-mouth monitoring should be
undertaken as described later.
While guidelines on the frequency of undertaking
periodontal screening have been proposed for adults
(26), these are generally lacking in younger age
groups. Since screening of six index teeth is very
quick, simple and acceptable to young patients, it
would seem prudent to recommend periodontal
screening of new patients and at the 4 or 6-monthly
recalls in children and adolescents so that any peri-
150
odontal problems are detected early and treated ap-
propriately. This basic periodontal examination
screening system typically takes less than 1 or 2 min-
utes in children and adolescents.
Local risk factors in children
Local factors can increase the risk of periodontal dis-
ease development and progression principally by
acting as plaque retention factors and should be
looked for during the thorough dental examination
of the patient (Fig. 1, 3).
Supragingival and subgingival calculus act as local
plaque retention factors (105, 133). An epidemio-
logical survey on 300 London teenagers found that
Table 3. Smoking as a risk factor for periodontal disease in children/adolescents
Risk factor Nature of risk factor Periodontal effects
Smoking Smoking is a recognized risk Smoking is associated with an increased severity of attachment loss and
factor for periodontal diseases in bone loss at a relatively earlier age than non-smoking peers with
young adults (72, 73, 125, 141), similar plaque levels (72, 73, 125). These effects have been documented
and the habit may develop in in young adults in their twenties and thirties (125). Smoking in children
adolescence (151). A 1992 study of and teenagers therefore has important future implications for
11- to 15-year-olds in secondary periodontal consequences. One study (76) showed that smokers aged
schools in England found 10% of 1930 years were 3.8 times more likely to have periodontitis compared
girls and 9% of boys were regular with nonsmokers, and 51% of their periodontitis could be attributable
smokers, with another 7% of girls to smoking.
and 6% of boys occasional Clinical features (77) include:
smokers (151). O brotic gingiva, rolled margins, tight gingival cuff;
According to the Royal College of O less gingival bleeding and redness;
Physicians, the stage has been O anterior, maxillary, palatal sites worst affected typically;
reached in Great Britain where O anterior recession, open embrasures; and
smoking is considered a O nicotine staining, supragingival calculus.
childrens habit and very few Clinical response to treatment (103, 104, 125) includes:
people take up smoking after age O smokers respond less well to both non-surgical and surgical therapy;
18 years (134). Charlton (32) refers and
to six national surveys and WHO O over 90% of refractory patients have been found to be smokers.
international data documenting Pathogenesis (125) includes:
smoking habits in 15-year-olds O inhibition of phagocytosis and chemotaxis of oral and peripheral
which revealed that, on average, neutrophils exposed to nicotine;
14.4% of both boys and girls O nicotine inhibits defensive functions of neutrophils by inhibiting
smoke daily. production of superoxide and hydrogen peroxide;
The effect of smoking relates to O reduced antibody production, serum IgG
2
;
pack-years smoked: the number of O altered peripheral blood immunoregulatory T-cell subset ratios;
packs of cigarettes smoked per O reduced bone mineralization;
day times the number of years O cytotoxic, vasoactive constituents;
smoked (72, 73). Starting to smoke O nicotine can adversely affect broblast function;
early, even if smoking low numbers O adverse effect on microcirculation, gingival circulation, blood ow,
of cigarettes, will increase the risk chronic vasoconstriction of gingival capillaries, hypoxia of periodontal
of developing periodontal disease tissues, but some ndings are contradictory (125); and
in later life O impairment of vascularization in soft tissues.
Microbiology (167) includes:
O more smokers infected by Actinobacillus actinomycetemcomitans,
Porphyromonas gingivalis and Bacteroides forsythus and higher levels
found in subgingival plaque
subgingival calculus was more prevalent than supra-
gingival calculus (28), and other work has shown that
subgingival calculus is associated with ethnicity
(102), gingival inammation and the development of
attachment loss in adolescents (8, 23, 37, 38, 61).
Subgingival restoration margins, margin discre-
pancies and overhanging restorations have been as-
sociated with gingivitis and attachment loss (21). A
trend of improving child dental health, implemen-
tation of caries prevention measures and newer con-
cepts of cavity design should lead to fewer tooth res-
torations to act on the gingival tissues in the years
to come. However, dental caries still remains a com-
mon condition, with 44% of 12-year-old children in
Great Britain in 1996/97 having caries experience at
the dentinal level of detection (121). Careful restora-
tive techniques must be employed to minimize the
potential deleterious effects of dental restorations on
the periodontal tissues.
Fixed and removable orthodontic appliances are
commonly worn in children and adolescents and re-
quire considerable maintenance by the patient to
151
combat the propensity for increased plaque build-
up (41). The development of gingivitis subsequent
to the placement of orthodontic xed appliances is
common (24, 25). The presence of orthodontic
bands can cause gingival enlargement within 1 to 2
months of placement (25), and decalcication can
occur around bonded attachments (15). However
most studies conclude that gingival changes pro-
duced by appliances are transient and there is no
long-term damage to the periodontal tissues.
An increase in periodontal disease associated with
malocclusion has often been used as an argument
for instituting orthodontic therapy. An association
between the irregularity of teeth and gingivitis has
been demonstrated (14); however, lack of orthodon-
tic treatment does not seem to have much effect on
the periodontal diseases later in life (122).
Mouth breathing, lack of lip coverage at rest and
lip seal (incompetent lips) are associated with higher
levels of plaque and gingivitis (160). However, multi-
variate analysis suggests that lip seal is not indepen-
dently related to plaque and gingivitis and that
Clerehugh & Tugnait
Table 4. Genetic conditions associated with periodontal destruction in children and adolescents
(see also Meyle & Gonzales (108))
Condition Nature of condition Periodontal effects
Leukocyte disorders
O Neutropenia Reduction in number of granulocytes. Various Ulceration, gingivitis, periodontitis (108).
types (108).
O Chediak-Higashi syndrome Rare autosomal recessive immunodeciency Severe gingivitis, periodontitis. Tooth loss
disorder. due to periodontal destruction.
Large lysosomal granules in granulocytes. Ulceration mucosa, tongue, hard palate
Neutrophil and monocyte defects. (150).
Recurrent infections, may be severe (150).
O Leukocyte adhesion Defects in integrin receptors of leukocytes. Early-onset prepubertal periodontitis (55,
deciency syndrome Impaired adhesion and chemotaxis. 115, 127, 152). Rapid attachment loss and
Increased susceptibility to infection, including bone loss shortly after eruption of
otitis media, septicaemia, impaired pus deciduous dentition. Early exfoliation
formation, delayed wound healing (55, 127,
152)
Papillon-Lefe`vre syndrome Autosomal recessive inheritance. Rare 1:3 or 4 Early-onset prepubertal periodontitis.
million. Often history of consanguinous Rapid attachment loss and bone loss
families. Palmoplantar hyperkeratosis. affecting deciduous dentition. Early
Impaired neutrophil chemotactic, phagocytic exfoliation or need for extraction. Therapy
and bactericidal activities and decreased difcult. Permanent dentition may be
migration may play a role in the disease affected resulting in tooth loss. Bacteria
pathogenesis and defects in immune function associated include Porphyromonas
have also been cited (79, 142) gingivalis, Fusobacterium nucleatum, and
Eikenella corrodens, but the etiological
role of Actinobacillus
actinomycetemcomitans seems pivotal
(39, 51, 64, 85, 130). High antibody titers
to A. actinomycetemcomitans have been
reported in some cases (39, 85)
Downs syndrome Autosomal chromosomal anomaly associated Periodontal disease very prevalent and
with trisomy of chromosome 21. Affects 1 of more severe than in age-matched controls
700 live births. Mental handicap. T-cell especially in lower anteriors. Differences
immunodeciency and inappropriate not explained by plaque levels. Rapid
enzyme regulation. Functional defects in progression. Onset apparent in
neutrophils and monocytes. Abnormal deciduous dentition (132)
capillary morphology. Connective tissue
disorders. Hyperinnervation of gingivae (132)
Hypophosphatasia Autosomal inherited trait. Inborn error of Cementum hypoplasia or aplasia.
metabolism. Deciency serum alkaline Periodontal destruction may affect
phosphatase, increased urinary excretion deciduous dentition, resulting in
phosphoethanolamine, defective bone/tooth premature exfoliation, tooth loss. Variable
mineralization. Three forms: lethal neonatal/ effects on permanent dentition, not
perinatal; severe infantile; milder form in necessarily as severe (31)
childhood/late adolescence (31)
Ehlers-Danlos syndrome Collagen disorder affecting joints (loose- Type VIII: aggressive early-onset
jointedness) and skin (fragile and periodontitis leading to premature loss of
hyperextensible). The mucosa is easily permanent teeth (22)
traumatized. Prolonged bleeding may be a
feature, and therefore hematological
investigations are warranted. Ten types; type
VIII has periodontal implications: autosomal
dominant inheritance. Distinguish by skin
biopsy from type IV (autosomal dominant/
recessive) which has life threatening potential
complications. See Meyle & Gonzales (108)
mouth breathing is pertinent to gingivitis of the
palatal surfaces of maxillary anterior teeth, whereas
lip coverage inuences gingivitis at both labial and
palatal surfaces.
Xerostomia is a side effect of radiotherapy where
the radiation elds include the salivary glands. In
children the management of nasopharyngeal carci-
nomas and rhabdomyosarcomas of the orbit may
produce xerostomia. Some types of chemotherapy
152
can produce xerostomia, but this is usually transient.
Xerostomia can be associated with increased gingi-
vitis due to the lack of the innate defensive function
of saliva (164). It may also be a feature in HIV-posi-
tive children (see chapter by Meyle & Gonzales in
this volume (108)).
Enamel projections, enamel pearls, proximal and
palatogingival grooves are all developmental dental
features and have been associated with gingivitis
Fig. 3. Periodontal examination for the child/adolescent
and attachment loss. Cemental tears are described
as complete separation of the cement from the den-
tine or partial separation within the cement and may
be associated with attachment loss (97). However,
the prevalence of these tears in the young has not
been documented.
Radiographs
Due to the cumulative effects of radiation over a pa-
tients lifetime, the decision to take radiographs in
the younger ages groups has to be both conservative
and justiable. Radiographs should only be taken
when they can be clinically justied by changing
either the management or prognosis of a patient
(135) (Fig. 1, 3). The radiation dosage will vary ac-
cording to the choice of radiographic view: 0.002
millisieverts (mSv) for two small intraoral bitewing
lms taken with a 70-kV set, 200 mm focal spot to
skin distance, with rectangular collimation and E
153
speed lm; 0.007 mSv for a panoramic radiograph
with rare earth intensifying screen (135). Dental radi-
ography is considered a low-risk procedure (135): the
International Commission on Radiological Protec-
tion derived a fatal cancer risk of 1 in 20,000 per mSv
for a population of all ages (84).
The use of intraoral radiographs for periodontal
bone levels, primarily bitewings, has recently been
recommended by an Expert Panel of the Faculty of
General Dental Practitioners (United Kingdom) to
supplement the clinical examination (66). In children
and adolescents, these provide a simple means of as-
sessing alveolar crestal bone levels as well as caries di-
agnosis and a consistent geometry is attainable for
longitudinal monitoring of bone changes (66).
In the younger patient or adolescent, panoramic
radiographs can be of value for determining the
presence or absence of permanent teeth, and the
location of unerupted teeth which may inuence fu-
ture orthodontic management. The opportunity to
assess bone levels should always be taken on intra-
Clerehugh & Tugnait
Fig. 4. Screening using the basic periodontal examination for the child/adolescent
oral or panoramic lms, even if these were not taken
primarily for periodontal purposes.
Periodontal diagnosis of periodontal
diseases in children and adolescents
The diagnosis of periodontal problems is reached
after consideration of the ndings of the history and
examination undertaken, and should be based on
current classication systems (13, 93, 152, 156) (Fig.
1).
Gingivitis
Plaque-induced gingivitis is prevalent in children
and adolescents (127, 148). Typical features of
154
plaque-induced gingivitis include gingival redness,
swelling, loss of contour, marginal bleeding and
pseudopockets in the absence of bone loss, which
are reversible following appropriate therapy. There
are various risk factors for other forms of gingival
problem, which can present in a variety of ways
(Table 1). Some of these forms of gingival disease
require additional therapeutic strategies to those ap-
plicable to plaque-induced gingivitis (see later sec-
tions on therapy).
Necrotizing ulcerative gingivitis
Necrotizing ulcerative gingivitis is characterised by:
painful, inamed, bleeding gingivae; necrotic ulcers
affecting the interdental papillae, which have a
punched out appearance and may spread along the
gingival margin; greyish-whitish slough; a character-
istic halitosis foetor ex ore; lymph nodes may be in-
volved (83, 107). Risk factors associated with necrot-
izing ulcerative gingivitis include poor oral hygiene,
stress, smoking, age and HIV infection. It is a mixed
bacterial infection involving anaerobes and spiro-
chaetes: Fusobacterium spp., Selenomonas spp., Pre-
votella intermedia and Treponema spp. (83). If treat-
ment is not sought (see section on therapy below),
the risk factors remain or the patient is immuno-
compromised, (as in HIV-positive individuals) then
necrotizing ulcerative gingivitis may become recur-
rent or progress to necrotizing ulcerative peri-
odontitis.
The differential diagnosis for necrotizing ulcer-
ative gingivitis includes primary herpetic gingivosto-
matitis, caused by primary contact to herpes simplex
virus type 1. This may present acutely with: fever;
malaise; lymphadenopathy; acutely inamed, swol-
len, painful gingiva; herpetic vesicles, especially on
the hard palate, dorsum of tongue and gingiva,
which rupture, leaving shallow ulcers with a red halo
and a yellow-gray oor; and smears show ballooning
degeneration of viral damaged cells (83).
Periodontitis in children and
adolescents
Key features of periodontitis irrespective of the spe-
cic classication are irreversible loss of periodontal
connective tissue attachment and apical migration
of the junctional epithelium with formation of a true
pocket lined by pocket epithelium and alveolar bone
loss. It is of fundamental importance to distinguish
and correctly diagnose the different types of peri-
odontitis, as the management depends on a correct
diagnosis and an appreciation of the causative pro-
cess and contributory risk factors. Kinane (93) has
considered the classication of periodontitis in
children and adolescents and presented three broad
categories: incipient adult periodontitis, early-onset
periodontitis and necrotizing ulcerative peri-
odontitis. Early-onset periodontitis has been further
subdivided into:
O prepubertal periodontitis, localised or general-
ized;
O localized early-onset periodontitis, which en-
compasses the condition previously referred to as
localized juvenile periodontitis;
O generalized early-onset periodontitis, which en-
155
compasses the categories previously referred to as
generalized juvenile periodontitis and rapidly pro-
gressive periodontitis; and
O incidental attachment loss, representing an initial
stage of early-onset periodontitis.
Since the terms incipient attachment loss (7, 93) and
incidental attachment loss (7, 93, 101) have both
been used to describe loss of support in adolescents
but each in a different context, clarication is re-
quired. Dictionary denitions of incipient include
in an initial stage and beginning (44). This seems
to be an appropriate term to describe the early or
initial stage of adult type periodontitis occurring in
teenagers of the type referred to as early peri-
odontitis in several studies (2, 3, 35, 37, 38, 52, 86
89, 99), which has different characteristics from
early-onset periodontitis (Table 5). Le & Brown
(101) have used the term incidental in respect of
early-onset periodontitis in adolescents, which they
suggested may represent an initial stage of a frank
early-onset juvenile periodontitis or even be inci-
dental to other factors.
The most recent developments in the above classi-
cation based on the 1999 International Workshop
for a Classication of Periodontal Diseases and Con-
ditions (13) are discussed in Kinane in this volume
(93).
Determining pocket depths
True periodontal pockets are a feature of incipient
periodontitis, and pockets of 4 to 5 mm or more
can be found at affected sites. In one study, a
group of 15- to 16-year olds from a non-grammar
school with a high proportion of Indo-Pakistanis
(40) were examined on the basis of high suscepti-
bility to periodontal destruction (based on the
ndings of earlier work by that group (37)). The
prevalence of pockets was high (77% overall); 82%
of Indo-Pakistani adolescents had pockets of 4 mm
or more on the mesial or distal surfaces of rst
molars and incisors, compared with 50% of white
Caucasians; on average, 13.3% of sites were
affected (40). However, pockets can also be
found in the absence of detectable attachment loss
in adolescents (23, 40, 61, 62). Such increased
probing depth in the absence of detectable apical
migration of the junctional epithelium beyond the
cementoenamel junction may either be due to the
tissue destruction being too small to detect and
Clerehugh & Tugnait
measure at that stage or, alternatively, it may be
due to coronal swelling of the gingival margin in
the absence of periodontal connective tissue de-
struction (pseudopocket). Therefore in determining
a diagnosis of periodontitis, it is necessary to as-
sess whether or not the base of the pocket is
apical to the cementoenamel junction and to look
for evidence of alveolar bone loss.
Bone loss
Serial bitewing radiographs have been used to
measure small changes in crestal bone level over 18
months in teenage subjects, and it has been sug-
gested that clinical attachment loss of 1 mm or more
precedes these changes (36). The question of what
constitutes a normal distance from the cemento-
enamel junction to crestal alveolar bone was ad-
dressed by Hausmann et al. (80) after perusal of con-
temporary literature revealed a lack of consensus.
They demonstrated that a no bone loss distance
ranging from 0.4 mm to 1.9 mm is consistent with
no clinical attachment loss in 13- to 14-year-old ado-
lescents. Based on these data, Armitage (13) has
commented that the radiographic measurement of
the cementoenamel junction to bone crest of 2 mm
or more is an appropriate cut-off point for bone loss.
The same group reported on a computerized
method for the detection of alveolar crestal bone loss
using serial bitewing radiographs in adolescents and
adults and concluded that changes of less than 1
mm could be measured (81). Albandar et al. (3) used
bitewing radiographs for the assessment of crestal
bone levels in a 3-year longitudinal study of Brazilian
adolescents and concluded that they provided a use-
ful method for monitoring disease progression. In
incipient adult periodontitis, bone loss is typically
horizontal crestal bone loss but will usually affect
only a few sites in those adolescents; individuals
with angular, vertical bone loss affecting at least one
molar have been considered as periodontal risk sub-
jects (3).
Periodontal treatment in children
and adolescents
This is usually undertaken in three phases: i) initial
cause-related therapy to eliminate or control plaque
infections; ii) corrective therapy to provide thera-
peutic measures and restore function and aesthetics;
156
and iii) supportive (maintenance) therapy to prevent
disease recurrence and progression with follow-up
recalls arranged at a time interval appropriate to the
diagnosis (74, 100). The interrelationships between
the three phases of therapy are shown in Fig. 5.
Initial cause-related therapy
This phase of therapy is critical to the successful out-
come of treatment irrespective of the patients age
and the specic diagnosis since it is aimed at con-
trolling the primary causative factor in the peri-
odontal diseases microbial plaque. The various
steps that constitute the initial phase of therapy are
depicted in Fig. 5.
Baseline periodontal indices and monitoring
Monitoring involves measuring the periodontal con-
dition using a chosen index and comparing it after a
dened interval of time to determine change (96).
At the beginning of treatment, periodontal indices
appropriate to the diagnosis (Fig. 3) should be
undertaken (i) to provide a baseline against which
change (whether improvements or deteriorations)
can be measured and (ii) to motivate the patient.
Plaque and gingival inammation (Fig. 3) can be
simply recorded in children and adolescents. The
presence or absence of marginal gingival bleeding
elicited by gently running a blunt periodontal probe
around the gingival margin can be recorded prior to
disclosing and measuring plaque. Plaque can be
measured using indices such as the plaque control
record (113), which gives a score based on the per-
cent of surfaces (buccal, mesial, distal, lingual) with
plaque as a proportion of surfaces examined. The
same system can be used to calculate the percentage
of sites with gingival bleeding. A simple, effective
motivational modication to any plaque or gingivitis
index is to give the child a score based on the
plaque-free surfaces or marginal gingival bleeding-
free surfaces so that, as they improve their tooth
cleaning, the score gets higher. Partial recording can
be undertaken if a very quick index is indicated for
a less cooperative child (such as using only the teeth
designated for the basic periodontal examination).
Where any pockets of 4 mm or more or furcations
have been detected during the initial basic peri-
odontal examination, that is basic periodontal ex-
amination scores of 3 or 4 or *, then full periodontal
charting needs to be undertaken in the affected area.
Other periodontal problems such as tooth mobility
or suppuration noted during the general dental ex-
Fig. 5. Periodontal therapy in children and adolescents: initial, corrective and supportive therapy
amination and screening also pre-empt further in-
vestigations (Fig. 3). Periodontal indices include:
O probing depths/clinical attachment levels in mm
at six sites per tooth;
O bleeding on probing from the base of the pocket
at six sites per tooth (can calculate the percentage
of sites with bleeding on probing);
O recession of the gingival margin in mm apical to
the cementoenamel junction;
O mobility (Iup to 1 mm movement horizontally;
IImore than 1 mm movement horizontally; III
movement of tooth both horizontally and verti-
cally);
O furcation involvement (horizontal probe penetra-
tion into furcation: F1up to 3 mm; F2over 3
mm; F3through and through between two
roots); and
O suppuration.
Attachment levels are indicative of periodontal sta-
tus in subjects with incipient periodontitis (34) and
157
they differentiate true pockets where the attachment
level is apical to the cementoenamel junction, from
pseudopockets. While the indices described here are
applicable to the young as well as the adult patient,
children and adolescents appreciate a tellshow
do management approach (30).
Plaque control instruction
The responsibility for plaque control can be shared
by both the patient and the professional using mech-
anical and pharmaceutical methods. Under the age
of 7 years, parents should take responsibility for
plaque control, as the child does not have enough
manual dexterity to effectively brush his or her own
teeth. Over the age of 7 years, the child can take in-
creasing responsibility for toothbrushing but parents
should be encouraged to supervise this procedure
until the child is old enough to take full responsi-
bility this will vary for different children and fam-
ilies and individual judgment needs to be used. Dis-
closing plaque and showing this to the patient and
Clerehugh & Tugnait
parent with a simple explanation is useful as an
educational and motivational tool. A simple scrub
toothbrushing technique has been found to be effec-
tive in children (137) and adolescents (136), but the
popular Bass technique can equally well be taught
to the older age groups (19).
Periodontal lesions are predominantly interdental
(92), and therefore interdental cleaning under the
contact point is important (59). Research ndings
support the recommendation that interdental plaque
removal every 1248 hours is sufcient (92). Use of
interdental aids such as oss should be reserved for
the adolescent with sufcient manual dexterity to
cope, and individual advice needs to be given (92).
Electric toothbrushes are well liked by younger
patients and are effective (41, 155, 161). Because of
the improved interdental cleaning achieved by vari-
ous electric toothbrushes, it is worth considering
recommending these for the younger age groups (92,
155) and reductions in price make many brands
more affordable than in the past. Based on the litera-
ture (161), the use of a brush with a rotary head
movement has been shown to be more effective at
plaque removal than one with a side-to-side motion.
Furthermore, there is some evidence that electric
brushes may be less abrasive than manual brushes
(161) and useful for children wearing xed ortho-
dontic appliances (41).
Mouthwashes are not indicated in very young
children due to their inability to spit out. Although
certain mouthrinses containing various pharmaceut-
ical agents have been shown to have some adjunctive
antiplaque effect in adults (60), there is little justi-
cation in the literature for their use in adolescents.
However, recent data from a 3-year randomized con-
trolled clinical trial of 641 adolescents demonstrated
that a dentifrice containing 0.3% triclosan with 2.0%
copolymer and 0.243% sodium uoride (62) had a
clinically small but statistically signicant effect in re-
ducing the development of attachment loss in the
subset of adolescents aged 1113 years with the
highest mean periodontal pockets.
Smoking cessation counselling
Smoking cessation counselling is an unwelcome task
to have to consider in a teenage patient, but evi-
dence of nicotine staining or the characteristic odor
pervading the oral cavity from a recent cigarette
should provide the impetus to tackle the problem
sensitively and with clear factual information of the
risks associated with continuing the habit (Table 3).
Smoking counselling delivered by a health pro-
158
fessional may inuence up to 5% of patients to stop
smoking (68).
Scaling, root planing and prophylaxis
The basis of mechanical plaque control by the den-
tal professional is scaling, root planing and prophy-
laxis to remove supragingival and subgingival
plaque and the calculus deposits that constitute lo-
cal plaque retention factors (42, 71). Following the
eruption of the permanent teeth, the need to under-
take both supragingival and subgingival scaling in
children and adolescents should not be underesti-
mated, and persistent gingival inammation in any
young patient who appears to be practicing reason-
able supragingival plaque control is all too fre-
quently related to previously undetected or residual
subgingival calculus deposits. A number of studies
have highlighted the surprisingly high prevalence of
subgingival calculus in young subjects, and its
chronic nature and relationship with subsequent
attachment loss (133) provide a rationale for its re-
moval using nonsurgical scaling techniques. There
is evidence on the efcacy of thorough scaling in
conjunction with oral hygiene programs in adults
(42, 57, 58), adolescents and children (17, 33). When
Chawla et al. (33) monitored different treatment re-
gimes in 12- to 15-year-olds over 2 years, they dem-
onstrated that the most intensive regimes incorpor-
ating professional prophylaxis and oral hygiene in-
struction produced less calculus and better gingival
and periodontal health than in the controls. It is im-
portant to appreciate that the outcome of therapy
may be compromised by absence of either the oral
hygiene component (98, 149) or the professional
scaling component of therapy in young subjects (4).
Suomi et al. (149) showed that there were no sig-
nicant differences in attachment levels in a 3-year
longitudinal study in 17- to 22-year-olds who re-
ceived varying numbers of professional prophylaxes
each year without oral hygiene instruction. More re-
cently, a study of 136 subjects aged 14 to 18 years
from a developing country (98) showed that a single
scaling and oral hygiene instruction without rep-
etition and reinforcement had minimal effects on
the gingival condition over 2 years. Two different
oral hygiene training programs were shown to have
no signicant effects on bone loss in 227 Brazilian
school children aged 13 years who were monitored
over 3 years without professional prophylaxis (4).
On average, the annual bone loss was 0.037 mm.
The authors concluded that impeding the pro-
gression of periodontitis could not be achieved by
self-performed supragingival plaque control and
that secondary prevention of early periodontitis in
adolescents is more appropriate than primary pre-
vention.
'nbTherefore, although relatively few randomized
controlled clinical trials of initial nonsurgical ther-
apy have been undertaken in younger age groups,
reduction in probing depths initially 4 mm or more
and gain in attachment has been consistently dem-
onstrated in many studies over all age ranges (42,
57, 58, 71), and bleeding on probing can be reduced
to 27% or less (71). Critical factors for successful
outcome to therapy are compliance with personal
and professional plaque control measures and
meticulous root debridement.
Restorations, endodontics, extractions, dentures
and elimination of local factors
Any carious lesions, poorly contoured restorations
and pulpally involved teeth requiring endodontics
need to be treated (100). Preventive advice for un-
controlled caries would include appropriate dietary
advice to reduce the frequency and amount of re-
ned carbohydrates and sugars; uoride supple-
ments may also be considered. Extraction of teeth
with a hopeless prognosis should be arranged and
immediate partial denture(s) made where aesthetics
or function are affected. Any other modiable local
factor should be eliminated.
Response to treatment
The response to the initial cause-related periodontal
therapy is crucial in deciding the next phase of
treatment. This is determined by repeating the base-
line periodontal indices around 812 weeks after
completion of the initial therapy (57). Probing
should be avoided in the 34 weeks following root
planing to avoid disruption of the developing long
junctional epithelium and measurement errors re-
lated to soft tissue changes during healing (71). Spe-
cic questions to be posed include:
O Are the oral hygiene and gingival health satisfac-
tory has the child achieved his or her maximum
potential for plaque-free and marginal gingival
bleeding-free surfaces?
O Are probing depths 4 mm and free from
bleeding on probing from the base of the pocket?
If yes, supportive therapy is required. If no, correc-
tive therapy is required (Fig. 5).
159
Corrective therapy
Gingivitis
If gingivitis persists following initial therapy, plaque
control advice needs repeating and reinforcing, and
every effort has to be made to improve the motiva-
tion of the patient and parents (100). For patients
with gingivitis, checks should be made for the pres-
ence of local plaque-retention factors, in particular
residual subgingival calculus deposits requiring
further scaling. Management of hormone-related
gingivitis is the same as for chronic gingivitis, al-
though on occasion in teenagers, pregnancy epulis
removal is needed if the lesion persists postpartum.
Gingivectomy may be indicated for management
of drug-induced gingival enlargement to improve
aesthetics or enhance plaque control, once the ini-
tial therapy has been completed (144, 163). The
medical history should include details of the medi-
cal condition for which the drug has been pre-
scribed as well as the type, dosage, frequency and
duration of therapy. The frequency of seizures, ts
or convulsions is relevant for the youngster with
epilepsy, and it is helpful to be aware if the child or
adolescent has any warning signs of an impending
attack. The medical practitioner, consultant or
specialist with overall responsibility for the young
patient should be contacted for clarication of de-
tails if required. In the most severe cases where the
gingival enlargement compromises function (eating,
chewing) and aesthetics, it may be necessary to li-
aise with the medical consultant about the possi-
bility of adjusting or changing the drug regime. Ad-
junctive use of 0.2% chlorhexidine mouthrinse can
be useful prior to surgery and during the healing
phase post-surgery.
Necrotizing ulcerative gingivitis
Necrotizing ulcerative gingivitis responds to tra-
ditional initial therapy involving mechanical de-
bridement by ultrasonic scaler, oral hygiene instruc-
tion, plus the use of an oxidizing mouthrinse (3%
hydrogen peroxide and equal volume warm water)
and corrective antibiotic therapy (83). Metronidazo-
le 200 mg or 250 mg three times daily may be re-
quired until the ulcers begin to heal, possibly within
3 days. Review should be undertaken every 23 days
until symptoms subside. Adjunctive 0.2% chlorhex-
idine mouthwash aids oral hygiene. Recurrence may
be a feature of this condition unless the risk factors
are eliminated. Therefore, plaque control advice and
smoking cessation counselling are important as-
Clerehugh & Tugnait
pects of periodontal care, but consideration should
also be given to appropriate direction for stress
management in affected teenagers.
Linear gingival erythema in HIV-positive
individuals
Linear gingival erythema is characteristically refrac-
tory to the mechanical therapy and plaque control
measures inherent in the initial phase of treatment.
Daily chlorhexidine rinses may be helpful, and fre-
quent recalls are indicated. If the patient does not
respond, the possibility of fungal infection such as
candidiasis should be considered; this can be
treated with antifungal agents in consultation with
the physician (107, 111).
Incipient adult periodontitis
In patients with incipient adult periodontitis, residual
pockets that bleed on probing should be re-treated,
usually nonsurgically, in conjunction with plaque
control advice and remotivation. Periodontal ap
surgery can be considered for the older adolescent in
selected cases (46). Lang et al. (95) showed that lack of
bleeding onprobing is a goodpredictor of periodontal
stability, whereas 30%of sites that display bleeding on
probing have been shown to lose further attachment.
Since it is not predictable which bleeding sites will
progress, the rationale is to root plane affected sites.
Monitoring after treatment is required to assess re-
sponse to treatment and whether supportive therapy
is indicated.
Early-onset periodontitis
For patients with early-onset periodontitis, monitor-
ing is undertaken a little sooner after initial therapy
around 68 weeks. Use of adjunctive systemic anti-
microbial agents can be considered at this stage
when the cause related therapy will have non-
specically reduced the mass of microbial plaque
(45). Corrective therapy may be effectively under-
taken by nonsurgical or surgical means (45, 46, 75,
116). Ideally, antibiotic therapy should be based on
the presence and relative proportions of the subgin-
gival microora sampled from the deepest residual
pocket in each quadrant; antibiotic sensitivity is not
routinely done as the prole of most of the putative
pathogens is known. There is no consensus regard-
ing the use of antibiotics, but three options that have
been investigated in the management of localized
early-onset periodontitis include (110, 158):
160
O tetracycline 250 mg four times per day for 1214
days;
O metronidazole 200 mg three times per day for 10
days; and
O metronidazole 250 mg and amoxicillin 375 mg
three times per day for 7 days (157).
Tetracycline is a broad-spectrum, bacteriostatic pro-
tein synthesis inhibitor agent that concentrates in
gingival crevicular uid, binds to tooth surfaces and
has anti-collagenase properties (110, 158). Metronid-
azole inhibits DNA synthesis and is a bactericidal
agent that is effective against gram-negative an-
aerobes; emergence of resistant strains is reported to
be uncommon with this drug (158). Saxen et al. (140)
showed that metronidazole was more effective than
tetracycline in eliminating Actinobacillus actino-
mycetemcomitans in localized early-onset peri-
odontitis. However, the combination of metronida-
zole with amoxicillin has been shown to be the most
effective regime due to the synergistic effect of the
two antibiotics and their hydroxymetabolites (118).
Systemic antimicrobial therapy should not be ad-
ministered without prior mechanical therapy (110).
Reduction of the subgingival deposits and disruption
of the subgingival biolm are prerequisites to a
favorable outcome, since the structure of the undis-
turbed biolm prevents the antibiotic from reaching
the target organisms (110).
These principles are equally applicable to general-
ized early-onset periodontitis, but the microora
may be more diverse than the localized form (Table
5). Surgery has been successful in reducing A. actino-
mycetemcomitans, whereas Porphyromonas gingi-
valis was almost eradicated by nonsurgical mechan-
ical therapy (75).
Generalized prepubertal periodontitis may be un-
responsive to mechanical and antibiotic therapy,
whereas localized prepubertal periodontitis is milder
and may respond to mechanical therapy and a
childs dose of penicillin V (115).
Papillon-Lefe`vre syndrome
Management of Papillon-Lefe`vre syndrome is often
difcult, and extraction of all permanent teeth has
in the past been an inevitable consequence of failure
to respond to therapy (Table 4). However, some more
recent reports have provided a successful rationale
for management involving extraction of the affected
deciduous teeth to eradicate the suspected putative
periodontal pathogens and use of systemic anti-
microbial therapy during eruption of the permanent
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161
Clerehugh & Tugnait
dentition to eliminate any re-emergence of these
pathogens (18, 85, 129). The elimination of A. actino-
mycetemcomitans seems critical to successful out-
come (85, 129). Treatment during the mixed den-
tition phase has been more difcult, and extraction
of the deciduous dentition alone has not proved suf-
cient to control the disease without additional sys-
temic antimicrobial agents specically targeted at
the subgingival pathogens identied in the pockets
of the already erupted permanent teeth (85). Having
done bacterial cultures and antibiotic sensitivity
tests for a patient at age 9 years, eradication of A.
actinomycetemcomitans using a succession of differ-
ent antibiotics (minocycline 200 mg twice per day
for 2 weeks, erythromycin and tetracycline com-
bined, then ooxacin 300 mg per day for 1 month)
and ap surgery in one area eventually stopped pro-
gression of the bone loss over the subsequent 3-year
monitoring period (85).
Necrotizing ulcerative periodontitis in
HIV-positive individuals
Following conventional initial therapy to remove
plaque and calculus and prevent progression of dis-
ease, it may be necessary to remove necrotic soft
tissue and bone. This will decrease the microbial
load and facilitate antibiotics reaching the affected
sites. Pain can be controlled by topical povidone iod-
ine. Daily 0.2% chlorhexidine mouthrinses help to
control plaque and inammation. Metronidazole is
the antibiotic of choice since it is specic to an-
aerobes, does not predispose the patient to superin-
fection and reduces pain (107, 111).
Other genetic and systemic conditions associated
with periodontitis
For some genetic or systemic conditions with peri-
odontal manifestations (Table 4), little has been
published on specic therapeutic measures (107).
Therefore, residual periodontal problems after initial
therapy should be identied and managed according
to the therapeutic principles described in previous
sections.
Mucogingival problems
Measurements of recession, clinical photographs
and study models provide a useful means of moni-
toring recession following initial periodontal therapy.
In particular, plaque control advice using an atraum-
atic brushing technique should be monitored. Any
162
traumatic factor identied during the rst phase of
treatment (such as damaging gum-picking habits)
should be reviewed. During a childs normal growth
and development, mucogingival defects may be
eliminated spontaneously provided that an adequate
level of oral hygiene is established and maintained
(10, 119, 128). Therefore, where possible, corrective
therapy should be delayed until the child is beyond
his or her active growth phase and the gingival di-
mensions have reached their maximal potential (11,
12, 20). If gingival recession resolves, decreases or
remains stable and there are no concerns over aes-
thetics or sensitivity, then periodontal supportive
therapy and monitoring are in order.
However, corrective therapy may be indicated on
occasion. Orthodontic treatment may be required
to correct a labially displaced tooth or malocclu-
sion causing direct gingival trauma, and the pa-
tients management should be planned in conjunc-
tion with the orthodontist. There is evidence to
support undertaking a frenectomy to remove a
high frenal attachment that is impeding effective
plaque control (162). Once growth of the ado-
lescent is complete, various root coverage pro-
cedures may be predictable for recession defects
where there is no loss of interdental bone or soft
tissue, including pedicle soft tissue grafts, free soft
tissue grafts (epithelial or subepithelial connective
tissue grafts), combinations of the two or guided
tissue regeneration (162).
Orthodontic therapy
Orthodontic therapy may be needed for alignment
of teeth which have pathologically migrated (drifted)
following severe bone loss associated with early-on-
set periodontitis. Relatively little research has been
undertaken into the causes of drifting, but it has
been shown that affected teeth have greater attach-
ment loss, while other risk factors include inam-
mation of tissues and pressure, occlusal disharmony,
habits (lip habits, bruxism and tongue thrust) and
instrument playing (153); many of these can affect
young individuals. Migration may present on an-
terior teeth as aring (fanning out labially), diaste-
ma, rotation, extrusion below the line of the arch,
tipping and midline shift. The potentially detrimen-
tal affects to the periodontium from moving teeth
with existing active disease must not be outweighed
by the desire to improve aesthetics (165). Initial and
corrective periodontal therapy should be completed
before initiating orthodontic therapy, and there
should be evidence from the periodontal monitoring
that a good tissue response has been achieved in
terms of reduction of pockets to less than 4 mm and
elimination of bleeding on probing. Plaque should
be well controlled, and there should be minimal
marginal gingival bleeding, as wearing of orthodon-
tic appliances is a local risk factor for periodontal
disease (see section on local risk factors). Single tuft-
ed interspace toothbrushes are a useful adjunct to
tooth cleaning around xed appliances (92). Like-
wise, electric toothbrushes with an orthodontic head
have been shown to be of value in controlling gingi-
val health in adolescents with xed appliances (41).
Fluoride mouthrinse supplements are also indicated
to prevent demineralization around brackets and
attachments, which create plaque-retention sites.
There has to be sufcent bone for orthodontic treat-
ment to be undertaken, and permanent retention of
the aligned tooth/teeth may be required. Treatment
planning should be undertaken jointly between the
orthodontist, periodontist, adolescent and his or her
family. Supportive periodontal maintenance is es-
sential during orthodontic treatment.
Supportive therapy and recall
The aims of supportive therapy, formally called
maintenance therapy, are i) to prevent the recur-
rence and progression of disease in patients who
have previously been treated for periodontal disease,
ii) to prevent or reduce the incidence of tooth loss
and iii) to increase the probability of locating and
treating other diseases found within the oral cavity
(126) (Fig. 5). The interval between supportive ther-
apy visits depends on the patients response to treat-
ment, plaque control and the initial diagnosis.
Therefore, evaluation of plaque control is needed in
conjunction with monitoring of the periodontal sta-
tus to determine any treatment needs. The decision
to re-treat is based on these clinical ndings. Recall
intervals of 46 months may be appropriate for most
young patients who have been successfully treated
for gingivitis or incipient adult periodontitis, but this
should be determined on an individual basis, taking
into account the diagnosis, risk factors, patient moti-
vation and compliance. Patients with a history of un-
stable or progressing periodontal disease should be
recalled more frequently. The early-onset forms of
periodontitis need particular vigilance, and since the
return to pre-treatment pathogen levels may take 9
11 weeks, recall intervals could not normally exceed
3 months until there is evidence of periodontal sta-
bility (71). Many studies have reported the efcacy
of supportive therapy in reducing the progression of
163
gingivitis to periodontitis and reducing attachment
loss and tooth loss (126).
Conclusions
Several conclusions can be drawn based on existing
evidence:
O Plaque is the key causative agent in periodontal
diseases affecting younger individuals, but the
balance between bacterial challenge and host re-
sponse is important, as in the older age groups.
O Local and systemic periodontal risk factors can be
identied during the history and examination of
the child or adolescent.
O Periodontal screening should be an integral part
of the dental examination of children and adoles-
cents. The basic periodontal examination takes
less than 12 minutes to perform on the desig-
nated index teeth and indicates the presence of
bleeding following probing, calculus, plaque re-
tention factors and pockets. The basic periodontal
examination provides a basis to determine pa-
tients who would benet from a more detailed
periodontal examination and more complex ther-
apy. The equivalent system in the United States is
the periodontal screening and recording system.
O Some periodontal conditions, such as gingivitis,
are prevalent and respond to simple initial non-
surgical periodontal therapy. Corrective surgical
therapy may be indicated for some young individ-
uals with drug induced gingival enlargement.
O Gingivitis can progress to incipient adult peri-
odontitis in a sizable proportion of adolescents.
Although the prevalence, extent and severity in-
crease with age, this is generally not a severe form
of periodontitis, and progression may be rather
slow if untreated. Nevertheless, this represents the
transition from a reversible to an irreversible peri-
odontal condition, and as the effects of peri-
odontal breakdown will become cumulative over
the lifetime of the patient, therapy should be
aimed at preventing or limiting progression.
O Early-onset forms of periodontitis have a low
prevalence but can be severe and rapidly destruc-
tive, and therefore early detection by routine peri-
odontal screening and examination is essential so
that treatment can be initiated as soon as poss-
ible. Localized and generalized forms have been
identied affecting the deciduous dentition (pre-
pubertal periodontitis) and/or the permanent
dentition (localized early-onset periodontitis or
Clerehugh & Tugnait
generalized early-onset periodontitis). A further
form of early-onset periodontitis designated inci-
dental attachment loss has been described in ado-
lescents.
O A minority of young periodontally involved indi-
viduals may present with rare syndromes, medical
conditions, genetic conditions, systemic problems
or necrotizing periodontal diseases requiring col-
laboration between the physician or pediatrician
and specialist periodontist.
O Mucogingival problems may be a presenting com-
plaint in the younger age groups. These should be
clearly differentiated from plaque-induced peri-
odontal diseases, and understanding the predis-
posing factors will aid diagnosis and manage-
ment.
O Periodontal management should follow the basic
principles of initial cause related, corrective and
supportive therapy. It should take into account the
age, cooperation, motivation and family support
of the child or adolescent and must reect the
periodontal diagnosis.
It is clear that periodontal diseases can present in
children and adolescents in a variety of guises, most
of which are amenable to appropriate periodontal
therapy. The system of classifying periodontitis in
children and adolescents is continuing to evolve, re-
ecting deeper understanding of the nature of the
diseases under study. The end-point has not been
reached in this dynamic area of research, and further
work is still needed before the issues are resolved.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Etiopathogenesis of periodontitis
in children and adolescents
DENIS F. KINANE, MICHELLE PODMORE & JEFFREY EBERSOLE
This chapter provides an account of knowledge on
the etiopathogenesis of the various periodontal dis-
eases that affect children and adolescents. This is
currently an area of active research, and many chal-
lenging questions remain. In many cases we have in-
sufcient knowledge to be denitive about the pro-
cesses involved. This chapter describes the processes
pertinent to the periodontal diseases, with particular
emphasis on the inammatory and immune re-
sponses occurring in the periodontal region during
gingivitis and periodontitis.
This chapter will be structured in the following
way: rstly the basic elements of the host response
processes relevant to periodontal disease will be re-
lated; then follows a description of how the pro-
cesses interact in the pathogenesis of the diseases;
and lastly the specics of the host response which
may be relevant to the causation and diagnosis of
the various forms of periodontal disease will be con-
sidered.
As described in the rst chapter in this volume,
the periodontal diseases affecting children and ado-
lescents are numerous. These diseases can be con-
veniently grouped into the following entities: gingi-
vitis; early-onset forms of periodontitis; necrotizing
gingivitis and periodontitis; incipient adult peri-
odontitis; and periodontitis associated with systemic
diseases. Further categories exist within these broad
denitions, in particular, gingivitis has many forms,
but in children the puberty-related form is common
and most signicant. Similarly, early-onset peri-
odontitis has several categories namely: prepubertal,
which can be localized or generalized; early-onset
periodontitis, which again can be localized or gener-
alized; and incidental early-onset periodontitis (not
extensive enough to t any other category). All of
the early-onset periodontal diseases are initiated by
dental plaque and result in destructive disease pro-
gression in susceptible individuals. The susceptibil-
ity criteria for these diseases remain elusive; the im-
mune causation could be due to variations or defects
54
in the host response to the plaque, however, the im-
mune and inammatory responses during the devel-
opment and progression of periodontitis are highly
complex, making inferences more difcult to draw.
For example, reduced neutrophil chemotaxis has
been reported in localized early-onset periodontitis
and generalized early-onset periodontitis (92, 93,
318, 319, 322324) but is not a consistent nding
across all populations (151, 152). If the abnormalities
actually exist, they do not appear to predispose the
patient to other diseases, suggesting that these are
either mild abnormalities or only relevant in the
periodontal environment (151, 152).
In the many cases where periodontal disease is
predisposed to by systemic disease, identiable
components of the immune or inammatory pro-
cess may be impaired or absent. In leukocyte ad-
hesion deciency or neutropenia, the systemic de-
fect is obvious. The effect of the absence of this com-
ponent in the host response can be deduced from
the description of the normal immune and inam-
matory responses in periodontal disease. The patho-
genesis of the periodontal destruction in the forms
related to systemic diseases is covered in the chapter
by Meyle & Gonzales (202) in this volume.
A pertinent aspect when considering periodontal
disease in children is the hormonal inuences on the
tissues and the host response, which are particularly
relevant in pubertal gingivitis. In addition, the
known variations in the host response in early-onset
periodontitis are described, but the genetics of this
and other periodontal diseases are dealt with fully in
a further chapter in this volume. Briey, early-onset
periodontitis is clearly a familial disease with an
autosomal dominant mode of inheritance. Genes
coding for several aspects of the host immune re-
sponse have been suggested as candidate markers
to be investigated in early-onset periodontitis. These
have included allelic variations in the Fc receptor for
immunoglobulin G
2
(263, 342). The interleukin-1
polymorphisms associated with adult periodontitis
Etiopathogenesis of periodontitis in children and adolescents
have not, however, been found in the early-onset
periodontitis patients studied and further may sug-
gest intrinsic differences between these disease enti-
ties.
The early-onset forms of periodontal disease most
probably share a common pathogenic pathway,
which may, in fact, be similar to that of adult peri-
odontitis, although the causal pathways may differ
considerably. The predisposing aspects responsible
for the much earlier onset and more rapid destruc-
tion in early-onset forms of periodontitis are prob-
ably genetically related but are still not fully under-
stood. Thus, the pathogenesis of adult periodontal
disease is covered in some detail and points relevant
to children and adolescents are highlighted. The eti-
opathogenesis of necrotizing forms of periodontitis
are quite unique and are also discussed.
The host defenses in periodontal
diseases
The host defense system comprises a collection of
tissues, cells and molecules whose function is to
protect the host against infectious agents (274).
The immune response may be subdivided into two
broad divisions, the innate (nonspecic) and the
adaptive (specic) responses. Innate reactions in-
clude the inammatory response and do not in-
volve immune mechanisms. Adaptive or immune
responses tend to be more effective, as the host re-
sponse is a specic immune response to the of-
fending pathogens.
Innate immunity represents an important rst line
of defence against infectious agents. This type of im-
munity is present from birth, is not enhanced by
prior exposure and lacks memory. The innate re-
sponse has the advantage of speed, but lacks speci-
city and may cause host tissue damage. Innate im-
munity entails a number of elements, both cellular
and noncellular. Physical barriers such as the skin
and mucous membranes represent a component
that infectious agents must breach to gain access to
the host. The washing action of uids such as tears,
saliva, urine and gingival crevicular uid keeps mu-
cosal surfaces clear of invading organisms and also
contain bactericidal agents.
The intact epithelial barrier of the gingiva, sulcular
and junctional epithelium normally prevents bac-
terial invasion of the periodontal tissues. It is nor-
mally an effective physical barrier against bacterial
products and components. The epithelial cell wall,
55
secreted proteins and fatty acids are toxic to many
microbes. Salivary secretions provide a continuous
ushing of the oral cavity as well as providing a con-
tinuing supply of agglutinins and specic antibodies.
Furthermore, the gingival crevicular uid ushes the
gingival sulcus and delivers all the components of
serum, including complement and specic anti-
bodies.
The microbial biolm that colonizes the tooth sur-
face releases large quantities of metabolites that may
diffuse through the junctional epithelium. These
metabolites include fatty acids, peptides and lipo-
polysaccharides of gram-negative bacteria. By re-
leasing proteolytic and noxious waste products,
plaque microorganisms may damage cellular and
structural components of the periodontium. As well
as releasing these waste products, microorganisms
could also invade the soft tissues.
Microorganisms produce a large variety of soluble
enzymes in order to digest extracellularly host pro-
teins and other molecules, thereby producing nutri-
ents for growth. They also release numerous meta-
bolic products, such as ammonia, indole, hydrogen
sulde and butyric acid. Among the enzymes re-
leased by bacteria are proteases capable of digesting
collagen, elastin, bronectin, brin, and other com-
ponents of the intercellular matrix of epithelial and
connective tissues. The released waste products
stimulate junctional epithelial cells to release various
inammatory mediators including interleukin-1,
prostaglandin E
2
and matrix metalloproteinases, all
of which can transverse the junctional epithelium
and enter the crevicular uid (1).
The normal ora of the body can also act as an
effective buffer against infection, by inhibiting the
growth of pathogenic organisms by competition for
nutrients or production of inhibitors. Phagocytic
cells in the blood stream and tissues can destroy in-
vading agents. These include polymorphonuclear
leukocytes (or neutrophils), monocyte/macro-
phages, and natural killer cells. Finally, there are the
soluble components. These consist of a wide range
of molecules that are normally protective, but, how-
ever, can also damage microbial cell walls, aid
phagocytosis and cell recruitment or prevent cellular
infection. These soluble components include lyso-
zymes, antimicrobial peptides, cytokines, acute-
phase proteins, complement components and inter-
ferons.
The persistence of an infection in spite of the ac-
tions of the innate immune response leads to the
induction of an adaptive immune response. Adaptive
immune responses are characterized by 1) specicity
Kinane et al.
for the offending antigen(s), 2) memory, which
allows a more rapid and heightened response upon
reinfection by the same or closely related antigen, 3)
diversity, the ability to respond to a wide range of
different antigens, and 4) self versus non-self recog-
nition. The adaptive immune response can be subdi-
vided into humoral and cell-mediated immunity.
Humoral immunity is mediated by antibodies,
whereas cell-mediated immunity involves the direct
action of immune cells.
The lipopolysaccharides of gram-negative micro-
organisms are capable of invoking both the in-
ammatory and immune responses as it interacts
with host cells. The immune response will result in
further release of cytokines and proinammatory
mediators, which in turn will increase the inam-
mation and thus be more harmful to the host. The
quality of the host inammatory response is critical
to the disease process. Although its purpose is pro-
tection and prevention of bacterial invasion into the
tissues, it is also detrimental, as it can be an ineffec-
tive, chronic and frustrated response that actually
causes much of the tissue damage that occurs in
periodontal disease. Inammation, acute and
chronic, nonspecic and immune-mediated, all play
a role in periodontal disease. The components of
these responses are now considered.
Inammatory mediators
Inammatory mediators play a major role in acute
and chronic inammation, and there is a strong evi-
dence for participation of these mediators in peri-
odontitis. These comprise a range of interacting
molecules that include the cytokine system, protein-
ases, proteinase activators, thrombin, histamine,
prostaglandins, leukotrienes, tissue and blood fac-
tors such as Hagemans factor, complement and clot-
ting factors. The purpose of these and many other
mediators is to initiate and perpetuate and eventu-
ally terminate an inammatory response to insult.
In the periodontium they are produced by activated
resident gingival cells and inltrating leukocytes. In
the blood plasma they are produced by the comple-
ment cascade and kinin system. Monocytes from in-
dividuals susceptible to or suffering from severe
periodontitis produce elevated amounts of me-
diators (86). Mediators are present in inamed gin-
giva and gingival crevicular uid from diseased sites
in high concentrations (220). Concentrations of in-
ammatory mediators typically decrease following
successful periodontal therapy. There now follows a
56
discussion of the various components capable of
promoting, sustaining and terminating inamma-
tory and immune reactions.
Cytokines
The term cytokine is derived from the Greek
words kytos meaning cell, and kinesis, meaning
movement. Cytokines are low-molecular-weight
polypeptides of (570 kDa) (17). They function as
soluble mediators produced by cells, to regulate or
modify the activity of other cells. The cytokines
can be broadly split into two groups, those in-
volved in inammatory and immune reactions and
those involved in tissue growth and repair (growth-
and colony-stimulating factors).
The rst group is further subdivided into:
O the interleukins, which transmit information be-
tween leukocytes;
O the chemokines (chemotactic cytokines) involved
in cell recruitment; and
O the interferons that inuence lymphocyte activity.
Various cells secrete cytokines in response to injury
or stimulation. These proteins are biologically active
in even femtomolar concentrations. Most cytokines
are multifunctional molecules that act locally and
have a variety of target or effector cells (340). Cyto-
kines act by binding to specic receptors on the sur-
face of effector cells, which then stimulate intracellu-
lar activation of the cell. In certain situations recep-
tors become saturated and cytokines such as tumor
necrosis factor, interleukin-1 and interleukin-6 may
spill over into the systemic circulation and stimulate
distant tissues and organs (215).
Collectively, the cytokines and other related mol-
ecules form a complex network that controls the in-
ammatory and immune responses (355) and sub-
sequent tissue healing. Many cytokines have over-
lapping functions, and some may inhibit the action
of others (155). A cytokine may have different forms
of receptors that may produce opposing actions, and
paradoxically, elevated levels of proinammatory
cytokines may be evident during periods of healing
as well as during destructive phases. These are im-
portant considerations when interpreting peri-
odontal studies of single or even combinations of
cytokines. These studies may not reect the true
situation in the living organism (355).
Cytokine regulation is achieved through several
mechanisms. Control of cytokines can take place at
the gene activation level, during secretion and circu-
lation and at the cell receptor level (17). Cytokine
production is usually short-lived and self-limiting
(136). In addition, many cytokine receptors exist in
soluble forms, which may be cleaved from the target
cells, allowing them to bind and neutralize cytokines
extracellularly (17). Thus, the local tissue environ-
ment inuences cytokine and cytokine receptor ac-
tivity. Proinammatory cytokines are also regulated
by high-afnity autoantibodies and anti-inamma-
tory cytokines such as interleukin-1 receptor antag-
onist, interleukin-4 and interleukin-10 (17).
Interleukin-1
In humans, two distinct interleukin-1 gene products
have been cloned interleukin-1a and interleukin-1b.
Sources of interleukin-1 production includes mono-
nuclear phagocytes, keratinocytes (185), broblasts
(125), endothelial cells (203) and osteoblasts (119).
Interleukin-1 is a major mediator in periodontitis
(234, 288). Production is induced by lipopolysac-
charide and other bacterial components and by in-
terleukin-1 which is autostimulatory (215). Interleu-
kin-1 is mainly secreted by monocytes or macro-
phages but can also be secreted by most nucleated
cells (143, 196).
All three members of the interleukin-1 group (in-
terleukin-1a, interleukin-1b and interleukin-1 recep-
tor antagonist) bind to interleukin-1 receptors I and
interleukin-1 receptors II. Corticosteroids induce in-
terleukin-1 receptors II messenger RNA synthesis
and the release of secreted interleukin-1 receptors II,
mostly from neutrophils, which may partly explain
their anti-inammatory and immunosuppressive ac-
tions (17).
Tumor necrosis factor
Tumor necrosis factor-a is a multipotential cytokine,
produced mainly by macrophages, with a wide var-
iety of biological effects similar to interleukin-1 (42,
169). Tumor necrosis factor-a and interleukin-1 both
act on endothelial cells to increase recruitment of
polymorphonuclear leukocytes and monocytes to
sites of inammation (20). Tumor necrosis factor-a
and interleukin-1 are key mediators of chronic in-
ammatory diseases and have the potential to in-
itiate tissue destruction and bone loss in periodontal
disease (22). Tumor necrosis factor also mediates
connective tissue destruction through its action on
the matrix metalloproteinase system (50, 199). Tu-
mor necrosis factor receptors exist in membrane-
57
bound and soluble forms similar to the interleukin-
1 receptors (17). These receptors (secreted tumor ne-
crosis factor receptors I and secreted tumor necrosis
factor receptors II) regulate the activity of both tu-
mor necrosis factor-a and the less potent form, tu-
mor necrosis factor-b.
In addition to interleukin-1 and tumor necrosis
factor-a, we must consider additional proinamma-
tory cytokines such as interleukin-6 and related mol-
ecules such as the prostaglandins and leukotrienes
and the inhibitors of inammatory cytokines such as
interleukin-10.
Interleukin-6
Interleukin-6 is produced by macrophages, bro-
blasts, lymphocytes and endothelial cells. Produc-
tion is induced by interleukin-1 and lipopolysac-
charide and suppressed by estrogen and progester-
one. It may be through interleukin-6 that these
hormones exert their effects on gingiva. Interleukin-
6 causes fusion of monocytes to form multinuclear
cells that resorb bone.
Interleukin-8
Interleukin-8 is produced by a wide variety of cell
types, including polymorphonuclear leukocytes,
monocytes, broblasts and keratinocytes in re-
sponse to microorganisms, mitogens and endoge-
nous mediators such as interleukin-1 and tumor ne-
crosis factor. One of the major functions of interleu-
kin-8 is its ability to induce the directional migration
of cells, including polymorphonuclear leukocytes,
monocytes and T cells (229), thus playing a key role
in the accumulation of leukocytes at sites of in-
ammation.
Interleukin-10
Interleukin-10 plays a major role in suppressing im-
mune and inammatory responses. It is produced by
T cells, including human Th0, Th1 and Th2 cells, B
cells and monocytes and macrophages after acti-
vation (54). Interleukin-10 inhibits the antigen-pre-
senting capacity of macrophages by downregulating
class II major histocompatability complex express-
ion. It has also been reported to inhibit antigen pres-
entation by Langerhans cells (76). Human interleu-
kin-10 reduces signicantly the proliferation and
production of cytokines by both Th1 and Th2 clones
exposed to specic antigen and phytohemagglutin
(54). It also has direct inhibitory effects on inter-
Kinane et al.
feron-g production (53, 55). Interleukin-10 has been
found to act as a specic chemotactic factor towards
CD8
T cells while suppressing the ability of CD4
T cells to migrate in response to interleukin-8 (144).

Interleukin-10 is a potent growth and differentiation
factor for activated human B cells and thus plays an
important role in amplifying the humoral immune
response (252). Interleukin-10 inhibits the synthesis
of interleukin-1a, interleukin-6 and interleukin-8
but enhances the production of the interleukin-1 re-
ceptor antagonist, so that this cytokine dampens im-
mune proliferation and the inammatory response
(53, 88). Interleukin-10 abrogates antigen presen-
tation by macrophages and indirectly inhibits T-cell
activation (17).
The lymphocyte cytokines
The lymphocyte-signaling cytokines interleukin-2,
interleukin-3, interleukin-4 and interleukin-5 are all
involved in lymphocyte clonal expansion, differen-
tiation of B cells into antibody-producing plasma
cells and immunoglobulin isotype switching. In-
terleukin-2 is produced by both CD4
(helper) and
CD8
(suppresser) (141), activated T lymphocytes

and induces expression of clones of specic T cells
and secretion of interleukin-3 and interleukin-4. In-
terleukin-4 regulates immunoglobulin production
and suppresses activated macrophages and causes
their apoptosis.
Transforming growth factor-b
Transforming growth factor-b is produced by acti-
vated T cells. It is a chemoattractant for monocytes
and it suppresses their activation. It also induces
production of immunoglobulin A and immuno-
globulin G2b. Transforming growth factor-a is pro-
duced by macrophages and serves as a mitogen for
broblasts, epithelial and endothelial cells. Inter-
feron-g is produced by CD8
T cells and is respon-

sible for recruiting and activating macrophages
and also upregulates the major histocompatibility
complex on virally infected cells and targets them
for killing.
In summary the cytokine network has a crucial
role in immune homeostasis. Complex summative,
overlapping, synergistic and inhibitory interactions
exist between the various members of this family of
molecules. This allows for redundancy within the
system and a high level of ne control which pre-
vents devastating effects to the host when dysfunc-
tion or dysregulation occurs.
58
Role of matrix metalloproteinases in
periodontal disease
Loss of attachment, as occurs in periodontal disease,
is associated with extensive breakdown of collagen
bers in the periodontal tissues. Since the major
structural protein of the periodontium is collagen,
the assessment of matrix metalloproteinase levels
during inammation will reect the association of
matrix metalloproteinases in collagen destruction in
periodontitis (Fig. 1). The involvement of matrix
metalloproteinases in pathological tissue destruc-
tion is well documented, and the evidence for their
role in periodontal destruction has increased over
the years. In earlier studies, collagenases had been
identied in explanted gingival tissues and their cul-
ture uids (22, 82, 248) and in homogenates of gingi-
val biopsies (315). Collagenase activity has also been
demonstrated in gingival crevicular uid, and this
activity increased with the severity of inammation
(100). Further evidence that matrix metalloprotein-
ases are involved in tissue destruction in human
periodontal disease were summarized by Birkedal-
Hansen (22), which includes:
O cells isolated from normal and inamed gingiva
express matrix metalloproteinases in culture;
O several metalloproteinases can be detected in hu-
man gingiva cells in vivo;
O polymorphonuclear leukocytes collagenase and
gelatinases can be detected in gingival crevicular
uid from gingivitis and periodontitis patients.
Each of the major cell types of human periodontal
tissues is capable of expressing a unique comple-
ment of matrix metalloproteinases when properly
stimulated. The cells include polymorphonuclear
leukocyte, broblast, keratinocyte, macrophage and
endothelial cells.
Collagenases in tissues may exist in different
forms, that is, in active form, in inactive form, as an
enzyme inhibitor complex, or in latent form (proen-
zyme). Total collagenase activity has been shown to
be high in gingival crevicular uid of inamed dogs
gingiva (160), in gingival crevicular uid of localized
early-onset periodontitis and chronic adult peri-
odontitis patients compared with gingivitis and con-
trol patients (328, 329), in gingival crevicular uid of
untreated localized early-onset periodontitis pa-
tients and also that the activity decreased after treat-
ment (168). Gingival crevicular uid collagenase ac-
tivity has been noted to increase with disease sever-
ity, that is, adult periodontitis and localized early-
Fig. 1. Macrophage-produced cytokines (such as interleu- teases, which result in bone and tissue breakdown
kin-1) induce broblasts and osteoblasts to produce pro-
onset periodontitis gingivitis healthy gingiva
(328, 329). Ingman et al. (138) demonstrated that sal-
iva samples did not reect periodontal tissue de-
struction clearly as they found only low amount of
total collagenase activity in saliva of untreated local-
ized early-onset periodontitis patient and the level
was comparable to normal patients. Ingman et al.
(139) also demonstrated that in saliva no difference
in collagenolytic activity could be seen in adult peri-
odontitis, localized early-onset periodontitis and
control patients.
Latent collagenase levels were found to be higher
in gingival crevicular uid of gingivitis sites in
humans and dogs compared to healthy sites (160,
161, 170), and also in normal sites (160). It was also
detected in the explant of clinically normal tissue
(124). Higher latent collagenase levels seem to reect
either the normal healthy state or the gingivitis state.
Little or no active collagenase activity was demon-
strated in the gingival crevicular uid of healthy and
gingivitis sites in dogs (166), but higher levels were
found in gingival crevicular uid of localized early-
onset periodontitis compared with normal patients
(168). Higher levels were also detected in gingival
crevicular uid of progressive periodontitis patients
compared with stable and gingivitis patients (170).
In whole saliva, high active collagenase activity was
59
demonstrated in untreated adult periodontitis (84,
138) and localized early-onset periodontitis, but as
treatment progressed more collagenase is present in
the latent form (84). Higher levels of active collagen-
ase correlates with active destructive phases of peri-
odontal disease (170).
Although there is no direct evidence for a causal re-
lationship between matrix metalloproteinases and
periodontal tissue destruction, the evidence for the
involvement of collagenase in collagen breakdown
during chronic inammatory periodontal disease is
strong. Matrix metalloproteinase-8 is detected at high
levels in gingival crevicular uid and saliva during
gingivitis or periodontitis, whereas it is undetectable
in healthy individuals (138, 284). In extracts or homo-
genates of diseased periodontal tissues, matrix
metalloproteinase-1 is abundantly present incontrast
to healthy specimens and, as with matrix metallopro-
teinase-8, a positive correlation was found between
its presence and the severity of inammation (230). In
addition, matrix metalloproteinase-1 has been im-
munolocalized in inamed but not in healthy peri-
odontal tissue (347). These data may suggest that, fol-
lowing production during inammation, most matrix
metalloproteinase-1 remains in the gingival tissue,
whereas most released matrix metalloproteinase-8
nds its way to the pocket.
Kinane et al.
Adhesion molecules
It has been recognized that blood vessels play a
central role in orchestrating the inammatory and
immune processes. Blood vessels are lined by en-
dothelial cells that are activated by cytokines and
bacterial lipopolysaccharide to become more ad-
hesive to circulating polymorphonuclear leuko-
cytes, monocytes, eosinophils, basophils and T and
B lymphocytes. Thus, the endothelial lining is
transformed from being anti-coagulant to becom-
ing pro-coagulant. The changes in the vessels con-
tribute to the inammatory changes in periodontal
disease, but the most important features is the mi-
gration of leukocytes from the circulation. When
the endothelial cells become activated they express
adhesion molecules, such as the intercellular ad-
hesion molecule-1, that enable specic subpopula-
tions of leukocytes to bind and subsequently mi-
grate into the tissues under the inuence of
chemotactic gradients (Fig. 2). Activators include
the proinammatory cytokines, lipopolysaccharide,
interleukin-4 and interferons. In vivo, interleukin-1,
tumor necrosis factor and lipopolysaccharide will
result in rapid accumulation of large numbers of
polymorphonuclear leukocytes. In contrast, in-
terleukin-4 and interferon are likely to be more
important in the recruitment of a mononuclear
cell dominated inltrate.
Intercellular adhesion molecule-1 is expressed on
endothelial cells and shows an increase in express-
ion after stimulation with interleukin-1, tumor ne-
crosis factor and interferon (Fig. 2, 3). It plays an im-
portant part in the adhesion of all leukocytes to en-
dothelial cells and migration. Evidence for this is
leukocyte adhesion deciency, a congenital disease
with low levels of adhesion molecules. Patients sub-
sequently suffer from recurrent infections due to in-
ability of their leukocytes to extravasate into dam-
aged or injured tissue. A constant and characteristic
feature of these individuals is a very rapid severe
generalized prepubertal periodontitis affecting both
the primary and permanent dentition (334).
The endothelial cell leukocyte adhesion molecule-
1 has been found to play an important role in poly-
morphonuclear leukocytes binding to activated en-
dothelial cells. It is rapidly induced by interleukin-1,
tumor necrosis factor or lipopolysaccharide.
In clinically healthy gingiva, endothelial cell
leukocyte adhesion molecule-1 and intercellular ad-
hesion molecule-1 are found in postcapillary venules
in the vicinity of the gingival crevice, with more dis-
tant vessels being negative. A further nding in the
60
study of Moughal et al. (210) was the observation
that junctional epithelium expresses intercellular ad-
hesion molecule-1. This specialized epithelium may
be adapted to express intercellular adhesion mol-
ecule-1 constitutively even in the absence of inter-
feron-g stimulation. This may reect the fact that
junctional epithelium is the site of extensive poly-
morphonuclear leukocytes migration both in health
and in disease (12, 265, 268).
Polymorphonuclear leukocytes
The polymorphonuclear leukocyte is a phagocytic
cell and is the predominant leukocyte in blood and
in the gingival crevice (12). It forms about 62% of
peripheral white blood cells and about 91% of gingi-
val crevicular cells recovered by aspiration (277).
Polymorphonuclear leukocytes are found at sites of
both gingival health and disease (12). The polymor-
phonuclear leukocyte is an important cell because,
along with the integrity of the physical surface bar-
rier, it forms the rst-line defense against invading
pathogens. Evidence for its crucial role in the de-
fense of the periodontal tissues comes from experi-
ments of nature where neutrophils are depleted or
malfunctioning, such as in neutropenia or Che`diak-
Higashi syndrome, and there is severe and rapid loss
of alveolar bone and teeth.
Polymorphonuclear leukocytes develop from plur-
ipotential bone marrow stem cells, and the cells go
through several stages prior to maturity. The lifespan
of a polymorphonuclear leukocyte outside the bone
marrow is short, an average of 7 to 58 hours. As poly-
morphonuclear leukocytes mature they become
more deformable, alter their surface charge and ac-
quire new cell surface receptors (336). Following
these changes mature polymorphonuclear leuko-
cytes can cross endothelial barriers in the bone mar-
row and enter the bloodstream.
In the circulation polymorphonuclear leukocytes
form two pools; circulating and marginated. Mar-
ginated polymorphonuclear leukocytes are stored
in the small vessels, particularly of the lung and
spleen. They can be mobilized at short notice in
times of acute need. The average range of poly-
morphonuclear leukocyte numbers in the circula-
tion is 2.5 to 7.510
9
cells/liter. This number can
increase 10 fold within 28 hours in times of acute
inammation. This rapid and substantial increase
in polymorphonuclear leukocyte number occurs as
a result of several processes; transfer of cells from
the marginated to the circulating pool, increased
Fig. 2. The leukocyte contacts, rolls, sticks and eventually journey to the site of inammation
extravasates out of the blood vessel prior to beginning its
Fig. 3. Molecules, cells and process inuencing the in- they can extravasate and begin to chemotact towards the
creased adherence of leukocytes to blood vessels so that microbes
61
Kinane et al.
release of mature cells from the bone marrow and
an increase in the rate of maturation of cells
within the bone marrow.
Numerous surface receptors are present on poly-
morphonuclear leukocytes (250) of which there are
four major classes:
O receptors for inammatory mediators and bac-
terial products;
O receptors for lymphokines and monokines;
O receptors for opsonization (Fc); and
O receptors for endothelium and proteins of the
tissue matrix.
These receptors are used for all stages of polymor-
phonuclear leukocyte emigration from blood vessels,
chemotactic movement, recognition of foreign ma-
terials, its binding and phagocytosis and for the con-
trol of these processes via lymphokines and monok-
ines.
The polymorphonuclear leukocyte has a charac-
teristic light microscopic appearance with a multi-
lobed nucleus and numerous cytoplasmic granules.
The granular component of the cell consists of many
cytoplasmic granules with an outer membrane en-
closing the densely packed protein in a mucopolys-
accharide matrix.
Three types of granules exist:
O primary or azurophil granules;
O secondary or specic granules; and
O tertiary or C particles.
Primary granules are identied by their peroxidase
content. Positive staining for peroxidase is seen in
the granules, at all stages, and in the secretory ap-
paratus of polymorphonuclear leukocytes during
their formation. The contents of the primary gran-
ules are largely responsible for the non-oxidative kill-
ing mechanisms of the neutrophil (204), and these
granules contain:
O antimicrobial agents or defensins, which include
myeloperoxidase, lysozyme and cationic proteins;
and
O proteinases, which include elastase and cathepsin
G.
Secondary granules are peroxidase negative, of low
density and are twice as numerous as primary gran-
ules in the mature polymorphonuclear leukocyte.
Their size is variable and they are typically spheres
or rods containing:
62
O antimicrobial agents lysozyme
O proteinases collagenase
O other molecules
lactoferrin
B
12
binding protein
laminin receptor (67 kDa)
CD11b/CD18 (C3bi)
bronectin receptor alpha
vitronectin receptor alpha.
The release characteristics and kinetics of primary
and secondary granules are different. Secondary
granules are released chiey extracellularly, with the
granules being found close to the advancing edge of
cells responding to a chemotactic stimulus (348).
Evidence of secondary granule release extracellularly
is more prominent than that of primary granule re-
lease and occurs at an earlier stage. These phenom-
ena have been interpreted in the past as indicating
a secondary and extracellular function of secondary
granules and an intracellular function of primary
granules related to phagocytosis (348).
The process of degranulation is calcium depend-
ent and is not related to cell death (300, 348) with
lysozymal enzymes released without a rise in lactate
dehydrogenase, an indicator of cell death. In vitro,
granule release can be stimulated by dental plaque
(300), binding of immune complexes and comple-
ment, binding to nonphagocytosable surfaces and
passage through membranes (348).
Polymorphonuclear leukocytes in
inammation
When functioning efciently against infectious
agents, polymorphonuclear leukocytes leave blood
vessels and move across tissues towards a chemo-
tactic source where they attempt to kill the organ-
ism. Adhesion molecules relevant to the movement
of polymorphonuclear leukocytes from vessels are
expressed on endothelial cells and polymorpho-
nuclear leukocytes (250). During inammation the
molecules appear before those for other leukocytes,
thus polymorphonuclear leukocytes are the rst cells
to cross the vessel wall. The immediate adhesion of
polymorphonuclear leukocytes is mediated via up-
regulation of P-selectin on endothelial cells by
thrombin or histamine. Stimulation of post-capillary
venules by inammatory mediators triggers the up-
regulation of expression of the endothelial cell ad-
hesion molecules (290). This functions over 24
Fig. 4. Interleukin-8 is one of the major host-produced chemotactic agents to attract neutrophils to the gingival crevice
hours, increasing the interaction of leukocytes with
the cells of the vessel walls. Various endothelial
markers including intercellular adhesion molecule-
1, intercellular adhesion molecule-2 and vascular
cell adhesion molecule-1 bind to the LeuCAM family
of integrins, which are expressed on polymorpho-
nuclear leukocytes. Leukocyte functionassociated
antigen-1 binds specically to intercellular adhesion
molecule-1 and intercellular adhesion molecule-2.
In response to the expression of surface markers,
the cells change shape and roll along the blood ves-
sel wall (Fig. 1). They nally stop moving, atten and
their membranes develope a rufed border. They
then migrate through the endothelial wall. Endo-
thelial cell leukocyte adhesion molecule-1, which is
necessary for this migration, is expressed in response
to interleukin-1 and tumor necrosis factor. Interleu-
63
kin-1 and tumor necrosis factor-a thus have a crucial
role in the control of inammatory mechanisms fol-
lowing the initial stimulus.
Polymorphonuclear leukocytes are capable of re-
sponding to many different chemoattractants in-
cluding N-formyl peptides, complement-derived
C5a, leukotriene B
4
, interleukin-8 and platelet-activ-
ating factor (123). Chemoattractant receptors span
the cell membrane and are GTPase-coupled recep-
tors. Once outside the vessel, cells migrate through
the connective tissue towards the chemotactic
stimulus (Fig. 4). Directed movement of polymor-
phonuclear leukocytes comes about by the interac-
tion of specic chemoattractants with surface recep-
tors on its plasma membrane. This binding activates
a cascade of secondary intercellular events that lead
to either chemotaxis or the respiratory burst, de-
Kinane et al.
pendent on the chemotactic agent (198). Neutrophils
stimulated by chemoattractants undergo morpho-
logical changes, becoming elongated cells with a
rufed border. During movement, the anterior edge
extends forwards and the posterior edge retracts to-
wards the body of the cell. Adherence of the cells to
a substratum occurs via the glycoproteins and
proteoglycans of the extracellular matrix. The con-
centration of extracellular matrix glycoproteins and
proteoglycans changes at sites of gingival inam-
mation (242), and these molecules can become
chemotactic for inammatory cells. Actual cell
movement occurs by the polymerization of globular
actin to brillar actin, and current models propose
that the brillar actin is involved in the generation
of the force required for cell movement (228).
Chemoattractant receptors exist in two intercon-
vertible forms: high- and low-afnity states (278).
Chemotaxis is initiated at receptors by concen-
trations of chemoattractants that are much lower
than those required to produce the respiratory burst.
Snyderman & Pike (283) proposed that binding of
chemoattractant to high-afnity sites results in
chemotaxis, whereas binding to low-afnity sites re-
sults in degranulation. Chemoattractants appear to
be most effective in producing chemotaxis in the fol-
lowing order of potency: interleukin-8, leukotriene
B
4
, C5a, N-formyl peptides and platelet-activating
factor (198).
Once at the site of the chemotactic stimulus and
prior to attachment of the polymorphonuclear
leukocyte to the invading organism, opsonization
takes place either in the presence or absence of com-
plement and antibody. In the presence of comple-
ment and antibody, activated C3 and immunoglob-
ulin G bind their respective polymorphonuclear
leukocyte receptors, CR1 and CR3 for complement
and Fcg receptor II and Fcg receptor III for antibody.
Capsular organisms are able to evade this opsoniz-
ation and phagocytosis due to the masking of the
antigenic portion of their cell wall by the capsule and
by avoiding the deposition of complement on their
surface (279).
If antibody or complement are not available, then
alternative systems exist for the opsonization of bac-
teria. Generally, cell wall antigens determine the ease
with which an organism is phagocytosed. Lipopoly-
saccharide-binding protein is an acute-phase reac-
tant which binds lipopolysaccharide, the cell wall
component of gram-negative bacteria. The binding
of lipopolysaccharide-binding protein to bacteria
enables binding to the CD14 receptor on polymor-
phonuclear leukocytes and macrophages, thus en-
64
hancing phagocytosis (349). Since gram-negative
bacteria are abundant in the gingival crevice and
complement and antibody can be locally destroyed
(91), it is likely that this system is in operation.
Following opsonization, phagocytosis can occur.
Phagocytosis is the process by which the polymor-
phonuclear leukocyte takes up a particle. It involves
attachment of the phagocyte to the particle and its
subsequent engulfment. It starts with the receptor-
ligand binding between polymorphonuclear leuko-
cyte and the microbe. This interaction activates the
ingestion phases which involve actin, myosin and
actin-binding proteins. The membrane surrounds
the particle, pseudopodia are produced and a
phagocytic vacuole is formed. Simultaneously cyto-
plasmic granules fuse with the phagosome mem-
brane to form a phagolysosome. This nal fusion of
the membranes and release of granule contents gen-
erally results in destruction of the engulfed particle
(327).
Once phagocytosed and engulfed by the polymor-
phonuclear leukocyte, two mechanisms exist to kill
the organism; oxygen-dependent and oxygen-inde-
pendent mechanisms (322). Oxygen-dependent kill-
ing is activated when the polymorphonuclear leuko-
cyte undergoes a respiratory burst coincident with
phagocytosis. NADPH in the phagolysosome mem-
brane is activated and reduces O
2
to superoxide
(339). Following the generation of hydrogen peroxide
and OH in this reaction, a second enzyme system
involving myeloperoxidase, a primary granule com-
ponent, becomes active. Myeloperoxidase is released
in large quantities but on its own has little effect. In
combination with H
2
O
2
, myeloperoxidase is capable
of catalyzing the oxidation of plasma halides, the
most important of which is chloride because of its
concentration. It is at this stage that the interaction
between chlorinated oxidants, protease inhibitors
and proteases can occur such that the proteases can
act efciently on their substrate (Fig. 5).
OH is considered the most toxic of all the free rad-
icals. If this reaction occurs in vivo then the rate-
limiting step is the availability of iron. Little free iron
is available due to its binding to transferrin and lac-
toferrin extracellularly and apoferritin intracellularly
(258). In the phagocytic vacuole the pH is reduced
and some iron may be lost from transferrin; however,
lactoferrin retains its ability to bind iron at low pH,
thus protecting the cell and limiting the production
of OH (117).
Oxygen-independent killing is based on the poly-
morphonuclear leukocyte granule network. Anti-
microbial agents that are independent of O
2
are re-
Fig. 5. The four main processes whereby antibodies can ation of microbial toxins (cf. leukotoxin); and 4) opsoniz-
detrimentally affect bacteria: 1) inhibition of adhesion or ation of microbes prior to phagocytosis and enhancement
invasion; 2) complement-mediated killing; 3) neutraliz- of phagocytosis
leased into the phagolysosome during phagocytosis
(310). These granule components, including bacteri-
cidal and permeability-increasing protein, chymo-
trypsin-like cationic protein and defensins (327, 339)
and those outlined above, are strongly bacteriocidal
and in most cases effectively kill bacteria (204). In ad-
dition to killing bacteria, the proteolytic enzymes, in-
cluding elastase and the metalloproteinases, have the
potential to damage extracellular tissue and have
been suggested to operate in periodontal tissues (22).
It has been demonstrated that sera from localized
early-onset periodontitis patients with decreased
neutrophil function can modify the activity of
healthy neutrophils (2). The serum factors respon-
sible for these effects have been identied as being
inammatory mediators such as prostaglandins and
cytokines. Small quantities of bacterial lipopolysac-
charide (in the picogram range) can induce the se-
cretion of tumor necrosis factor-a, interleukin-1 and
prostaglandin E
2
from monocytes. The production of
65
interleukin-1tumor necrosis factor-a and tumor ne-
crosis factor-aprostaglandin E
2
are strongly corre-
lated (207). Very small increases in serum cytokine
levels can alter neutrophil function. Prostaglandin E
2
and cytokines also stimulate broblasts to release
matrix metalloproteinases, which break down the
extracellular matrix. In addition, interleukin-1, tu-
mor necrosis factor-a and prostaglandin E
2
are po-
tent stimulators of bone resorption. Adherent mono-
nuclear cells from localized early-onset periodontitis
patients have been found to secrete higher levels of
prostaglandin E
2
and tumor necrosis factor-a than
generalized early-onset periodontitis patients or
healthy controls (273). More recently, insulin-de-
pendent diabetic patients with periodontitis have
been reported to manifest excessive levels of in-
ammatory mediators (255257). It has been sug-
gested that these observations indicate an underly-
ing immune defect of monocytes in certain diagnos-
tic groups (215).
Kinane et al.
The concept of excessive inammatory mediator
production by monocytes has been proposed as a
possible causative pathway for periodontitis, and in-
deed one of the mechanisms whereby periodontitis
can inuence other systemic conditions such as car-
diovascular disease (215). Early investigations of
genetic polymorphisms of cytokines have attempted
to identify a possible link between different peri-
odontal categories and specic alleles related to an
ability to overproduce cytokines (56, 83, 101, 158). In
addition, a family linkage study of early-onset peri-
odontitis suggested that the COX-1 enzyme system,
which acts as a catalyst for the breakdown of arachi-
donic acid, one of the products of which is prosta-
glandin E
2
, may be associated with susceptibility to
early-onset periodontitis (88).
A further host defense defect that may increase
susceptibility to early-onset periodontitis is neutro-
phil chemotactic defects. A high percentage of mem-
bers of families with a background of localized early-
onset periodontitis have been reported to show ab-
normal neutrophil chemotaxis (322). Analysis of 22
families with localized early-onset periodontitis
demonstrated that, in 19 of these families, the pre-
senting localized early-onset periodontitis patient
and their family members all demonstrated neutro-
phil chemotaxis disorders. In these 22 families the
chemotactic defect was found in almost 50% of the
siblings, indicating a dominant mode of inheritance
(322). Whether the trait is due to an intrinsic defect
of neutrophils or is caused by extrinsic factors in the
sera and thus is environmentally inuenced remains
controversial (2). The neutrophil chemotactic defect
is not noted in all populations exhibiting localized
early-onset periodontitis, and a signicant number
of patients and families with localized early-onset
periodontitis do not show evidence of the defect
(151, 152, 231).
Decreased neutrophil chemotaxis in localized
early-onset periodontitis has been linked to a re-
duced receptors density for chemoattractants such
as N-formyl-methyl-leucyl-phenylalanine, comple-
ment fragment C5a, leukotriene B
4
and interleukin-
8 (48, 51). A cell surface glycoprotein (gp110), which
is thought to be involved in neutrophil movement
and secretion, is reduced in localized early-onset
periodontitis patients (323). In addition, several
other post-receptor defects have been identied
(48). De Nardin (52) found that decreased N-formyl-
methyl-leucyl-phenylalanine binding was related to
variations in N-formyl-methyl-leucyl-phenylalanine
receptor DNA in localized early-onset periodontitis
patients and healthy controls. These preliminary re-
66
sults indicate a possible role for a N-formyl-methyl-
leucyl-phenylalanine receptor alteration in neutro-
phil chemotaxis. However, many molecules related
to neutrophil dysfunction in localized early-onset
periodontitis patients belong to the same family of
surface receptors and have similar morphogenic
characteristics. Similarities in signal transduction
mechanism between cell surface components also
exist. De Nardin (52) hypothesized that a common
underlying defect, at either the cell surface receptor
or post-receptor level, may contribute to the alter-
ation in neutrophil function seen in patients with lo-
calized early-onset periodontitis.
Fc receptors
Fc receptors are the receptors found on leukocytes
that can bind immunoglobulin and are thus relevant
to the microbial opsonization properties of phago-
cytes. Some investigators have shown that patients
with generalized early-onset periodontitis have high
antibody titers against Actinobacillus actinomyce-
temcomitans serotype b outer membrane antigens
(106, 309, 341). It has also been suggested that the
subclass IgG2 is a poor opsonin because it xes com-
plement less effectively than IgG3 and IgG1 and
binds weakly to Fc receptors on the surface of
phagocytes (249), which tends to complicate theor-
ies of protective effects afforded by high antiA. acti-
nomycetemcomitans titers of IgG2 in black localized
early-onset periodontitis patients.
However, a further aspect of the immune response
may yet explain this phenomenon. Fc receptors are
the crucial link between the humoral and inam-
matory components of the host defense system. Fcg
receptors on phagocytes recognize the Fc region of
IgG and facilitate opsonization of microbes. It has
been shown that neutrophils express low-afnity
variants of Fcg receptors (Fcg receptors II and Fcg
receptors III) under normal conditions. Fcg recep-
tors II binds all subclasses of IgG, but Fcg receptors
III only binds IgG3 and IgG1. Many more Fcg recep-
tors III than Fcg receptors II are expressed on the
surface of neutrophils. However, while Fcg receptors
III can evoke lysosomal degranulation, they cannot
promote the necessary respiratory burst activity and
phagocytosis needed for microbial killing of opsonis-
ed bacteria. Fcg receptors II, on the other hand, can
precipitate all three activities. It is possible that Fcg
receptors III may prepare polymorphonuclear leuko-
cytes for phagocytosis mediated by Fcg receptors II
(342).
It has been reported that the numbers of Fcg recep-
tors II and Fcg receptors III on peripheral blood neu-
trophils of localized early-onset periodontitis patients
are withinthe normal range (171). Miyazaki et al. (204)
found that both Fcg receptors II and Fcg receptors III
expression and neutrophil phagocytosis were signi-
cantly depressedingingival crevicular uidcompared
with peripheral blood in adult periodontitis patients.
The reduction in Fcg receptors was signicantly cor-
relatedwithphagocytic activity. Inaddition, the mess-
enger RNA level of Fcg receptors III was also signi-
cantly lower in gingival crevicular uid neutrophils
than in peripheral blood neutrophils but not the
messenger RNA level of Fcg receptors II. These nd-
ings indicate local suppression of Fcg receptor ex-
pression and/or the production of Fcg-binding pro-
teins by periodontal pathogens (129).
Adaptive aspects of the immune
response in early-onset periodontal
disease
In contrast to the innate immune mechanisms dis-
cussed previously, adaptive immune responses are
not initiated at the site where a pathogen rst estab-
lishes a focus of infection.
The humoral immune response
The main function of the humoral immune response
is the destruction of extracellular microorganisms
and prevention of the spread of intracellular infec-
tions. This is achieved by antibodies secreted by B
lymphocytes. Antibodies induced have many func-
tions ranging from neutralization, to leading to op-
sonization. The activation of B cells and their differ-
entiation into antibody-secreting cells is triggered by
antigen and usually requires helper T cells.
Many studies over the years have demonstrated
that antibody responses to various periodontal
pathogens are increased in patients with periodontal
disease compared with subjects without the disease.
Therefore, antibody titers, isotypes and the re-
sponses of the humoral immune system have been
investigated over the years.
Mouton et al. (211) established that antibody to
Porphyromonas gingivalis is found in a signicant
proportion of healthy adults as well as being detect-
able in children as young as 6 months. In both adults
and children there was a positive correlation between
antibody levels andage. Nevertheless, serumIgGanti-
67
body increased by 500800% in adult periodontitis
and generalized early-onset periodontitis patients.
Multiple subsequent studies conrmedthese ndings
and showed a signicant increases in levels and fre-
quency of antibody to P. gingivalis in adult peri-
odontitis and a subset of generalized early-onset peri-
odontitis patients (10, 11). Chen et al. (38) noted that
only some generalized early-onset periodontitis pa-
tients displayed an elevated humoral immune re-
sponse to P. gingivalis, albeit these antibodies were of
low avidity and increased following successful peri-
odontal therapy. C elenligil & Ebersole (31) demon-
strated a signicantly higher level of antibody to the P.
gingivalis strains (that is, strains generally derived
from United States populations) in United States pa-
tients compared with similar disease groups derived
from a Turkish population. However, the Turkish
early-onset periodontitis patients exhibited a signi-
cantly higher frequency of elevated antibody to the P.
gingivalis compared with a geographically matched
normal group. The four concepts originally put forth
by Mouton et al. (211) regarding this pathogen were:
(i) based on the humoral immune response, P. gingi-
valis is probably an causative agent in periodontal
disease; (ii) the humoral immune response is prob-
ably protective; (iii) diseased and healthy individuals
can be distinguished in terms of their antibody re-
sponse to this organism; and (iv) differences in the
antibody response characteristics may denote differ-
ent periodontal disease classications. To these can
now be added that the humoral immune response to
P. gingivalis may contribute a better understanding of
patient susceptibility, diagnostic categories, treat-
ment effects and immune protection and that this
pathogen may have the capacity to express antigenic
diversity between subjects in a population and across
patient populations.
A wealth of literature supports the existence of lo-
cal specic antibody production by plasma cells
present in inamed tissues of the periodontal pocket
(67, 71, 166, 282, 294). Likewise, a proportion of gin-
gival crevicular uid samples within a given subject
have local antibody levels signicantly greater than
can be accounted for by a serum contribution (67,
70, 145). In addition to these inammatory me-
diators, evidence has been provided that indicates
that both C3 and C4 components of the complement
system are cleaved in gingival crevicular uid from
early-onset periodontitis (236, 264), indicating the
potential of antigen-antibody complexes to contrib-
ute to local immune modulation. Cross-sectional
studies have suggested that the gingival crevicular
uid samples with elevated antibody frequently
Kinane et al.
harbor the homologous bacteria and suggest that a
combination of the antigen and the host-response is
frequently associated with progressing disease (71);
these local antibody levels decreased with pocket
depth, attachment level stabilized and inammation
resolved following therapy (71).
Lu et al. (184) have shown that, in certain popula-
tions, serum IgG
2
levels are increased in localized
early-onset periodontitis patients, but not general-
ized early-onset periodontitis, adult periodontitis or
periodontally healthy individuals. IgG
2
is the pre-
dominant immunoglobulin subclass that reacts with
carbohydrate antigens preferentially and is particu-
larly reactive with A. actinomycetemcomitans sero-
type b outer membrane antigens. A. actinomycetem-
comitans is a microbe commonly associated with
early-onset periodontitis (see Darby & Curtis in this
volume). Serum IgG
2
levels increase with age and a
rise can be detected at puberty (184). An interesting
association is that black subjects in general have
higher levels of IgG
2
than whites, and the incidence of
localized early-onset periodontitis among black indi-
viduals has been shown to be up to 15 times greater
than among whites (181, 200, 262). Furthermore, the
increase in serum IgG
2
levels in localized early-onset
periodontitis patients was due to high antiA. actino-
mycetemcomitans serotype b-antibody levels, which
actually only accounted for around 10% of the IgG
2
excess in localized early-onset periodontitis patients.
It thus appears that localized early-onset peri-
odontitis patients, particularly black individuals, have
a tendency to produce increased levels of IgG
2
(309).
A family study of early-onset periodontitis indi-
cated the possibility of genetic transmission of IgG
2
levels (192). Tew et al. (309) suggest a protective role
for high levels of IgG
2
in patients with localiazed
early-onset periodontitis. Smoking has been found
to suppress the IgG
2
response in adult periodontitis
and generalized early-onset periodontitis patients
and is associated with more severe destruction, but
this does not appear to occur in localized early-onset
periodontitis patients (309). Furthermore, the para-
doxically lower IgG
2
titers seen in black generalized
early-onset periodontitis smokers may be specic to
A. actinomycetemcomitans, and the IgG
2
response
against antigens of other bacteria may not be lower
in generalized early-onset periodontitis subjects who
smoke (305).
The cell-mediated immune response
Through nonspecic interactions of the T lympho-
cyte with other cells, controlled by a varying array of
68
adhesion molecules, the naive T-cell migrates
through lymph nodes, makes initial interactions with
antigen-presenting cells and, after specic acti-
vation, eventually moves to peripheral tissues, where
it can interact with target cells. This is thought to be
the case during the maturation of T cells in peri-
odontal disease, culminating in the homing of speci-
c T cells to the gingival tissues where they can effect
their protective function.
Effector T cells fall into 3 functional classes that
detect antigens derived from different types of
pathogens, presented by the 2 different classes of
major histocompatibility complex molecule. Anti-
gens derived from pathogens that multiply in the
cytosol are carried to the cell surface by major histo-
compatibility complex class I molecules and pre-
sented to CD8
cytotoxic T cells, and those derived

from extracellular bacteria and toxins are carried to
the cell surface by major histocompatibility complex
class II molecules and presented to CD4
T cells.
These cells, often referred to as helper cells, can dif-
ferentiate into two types of effector T cell; Th1 and
Th2 cells. Th1 cells secrete both interleukin-2 and
interferon-g when activated by certain types of T-de-
pendent antigens so that they can enhance cell-me-
diated responses. The Th2 subset of T helper cells
produce interleukin-4, interleukin-5, interleukin-10
and interleukin-13 and thereby promote the hu-
moral immune response.
Although much of the literature discusses the im-
portance of the humoral aspect of the immune re-
sponse in periodontal disease, models used in earlier
years suggested that the initial periodontal lesion is
composed mainly of T lymphocytes, with B cells and
plasma cells predominating at a later stage (187, 271,
272). Studies have been carried out reporting differ-
ences in CD4 : CD8 ratios in lesions and peripheral
blood of periodontitis patients (225, 307) compared
with healthy controls, further indicating that the
cell-mediated immune system is potentially as im-
portant in a discussion of this disease as that of the
humoral arm.
It appears that, in terms of periodontal disease,
the antigen is picked up by an antigen-presenting
cell, such as a Langerhans cell, at the site of infec-
tion, whereupon it is carried to a primary lymphoid
tissue where the presentation to a circulating naive T
cell takes place. This leads to antigen-specic clonal
activation, where some activated cells become effec-
tor cells and others remain in the circulation as
memory cells, naive T cells expressing CD45RA and
memory cells CD45RO.
CD45 is a transmembrane tyrosine phosphatase
with three variable exons that encode part of its exter-
nal domain. In naive T cells, high-molecular-weight
isoforms CD45RA are found. In memory T cells, the
variable exons are removed by alternative splicing of
CD45 RNA and this isoformis known as CD45RO. The
general dogma indicates that memory T cells
(CD45RO
) greatly outnumber naive (CD45RA
) T
cells in periodontitis lesions (87, 154, 167, 353). How-
ever, it has also been shown that a proportion of these
cells are CD45RA
, which suggests reactivation of the

memory cell population by specic but as yet iden-
tied antigens in the tissues. It has been reported that
memory T cells adhere to vascular endothelial cells
and augment their permeability to macromolecules
(47). This functional ability of memory T cells to con-
trol endothelial permeability and to adhere to endo-
thelial cells may be a dominant factor that contributes
to the preferential migration of memory T cells into
sites of chronic inammation (239), such as the dis-
eased gingival tissues.
The realization that T lymphocytes are present in
large numbers in the diseased tissues of early-onset
periodontitis patients and not in health has led to in-
vestigations of the numbers, ratios and functionality
of T cells in both the peripheral blood and diseased
tissues of patients withperiodontal disease. Studies of
peripheral blood T-lymphocyte subsets in early-onset
forms of periodontal disease have revealeda wide var-
iety of contradictory results with either depressed
CD4
: CD8
ratios (153) or mixed (147), or even not

correlated to the disease (75). A number of studies
have also looked at the distribution of the T-cell sub-
sets in the tissues. One study reports that the CD4
:
CD8
ratio in the tissues of early-onset periodontitis

patients and even their families tend to be increased
(299). A study by Stou et al. (294) and one by C elenli-
gil et al. (35), however, clearly states that the ratio is
decreased in patients with this disease. What is obvi-
ous and further indicated by a study looking at juven-
ile periodontitis (201) is that the ratios are highly vari-
able betweendifferent patients withthe same disease,
making generalizations and conclusions extremely
difcult. What the above study did suggest however,
was that the high CD4
: CD8
ratios were seen in

those patients that had a greater degree of inamma-
tory cell inltration, while the low CD4
: CD8
ratio
tissues were less inamed.
When diseased gingival tissue is removed and the
cells extracted, both CD4
and CD8
lymphocytes
are present in large numbers (295, 307). Many
studies have indicated raised levels of CD8
cells in
diseased tissues; however, CD4
cells, although
prominent, were found at lower levels than in the
69
peripheral blood. Overall ratios of CD4
: CD8
were
not found to be altered in early-onset periodontitis
and chronic periodontitis patients (195); however,
these ratios were found to be depressed in patients
with localized early-onset periodontitis and general-
ized early-onset periodontitis (153).
The role of CD4
T cells in periodontal
disease
T-cell functions in periodontal granulation and gin-
gival tissues can be elucidated by their cytokine syn-
thesis prole (352). There are two main subsets of T-
helper cells, Th1 and Th2, which are usually deter-
mined by the cytokine prole that is characteristic of
them (see Table 1 for characteristics of cytokines that
play an important role in the progression of peri-
odontal disease). ThO cells have also been identied,
and these cells are thought to secrete interleukin-4
and interferon-g. Their actual role is yet to be con-
rmed; however, it is thought that they may play a
role as a precursor cell to T-helper cells yet to have
differentiated to become either Th1 or Th2 cells
(206). Although the cytokine prole is a good tool for
T-cell subset investigations, the results reported are
often conicting, causing confusion. Often the re-
sults cannot assess the relative importance of the
Th1 and Th2 subsets.
Fujihashi et al. (80) investigated Th1 and Th2 cyto-
kine messenger RNA expression by CD4
T cells
from diseased gingival tissues. They detected inter-
feron-g, a Th1 cytokine, but failed to detect the Th2
cytokine interleukin-4, thus indicating a more domi-
nant role for Th1 cells. Other groups, however, indi-
cate a more dominant role for Th2 cells after detec-
tion of interleukin-4, interleukin-5, interleukin-6 (10,
191, 354) and interleukin-10 (89). One of the current
theories on the Th1/Th2 paradigm is that both cells
have an important but different role. Gemmell et al.
(88), have suggested that Th1 cells remain tightly lo-
calized at sites undergoing an active disease process,
whereas Th2 cells are more widely distributed
throughout the tissue and typify a more quiescent
stage of the disease; that is, the immune response in
periodontal disease is predominantly Th2 driven,
with active phases of the disease leading to a foci of
Th1 activity.
The role of these cell types is that of help for the
immune response. Th1 cells, characterized often by
their production of interferon-g, are important in
microbial killing and are known to augment cyto-
toxic T-cell functions through their production of in-
terleukin-2 and interferon-g. Th2 cells, however, in-
Kinane et al.
Table 1. The role of cytokines in the pathogenesis of periodontal disease
Cytokine Produced by Role
Interleukin-1 Large quantities produced by Proinammatory properties
macrophages Mediator of tissue destruction in peridontal disease (6)
Interleukin-4 Activated T cells Inhibits production of interleukin-1, tumor necrosis factor and
interleukin-6
An absence of interleukin-4 in the periodontal tissues has been
suggested to trigger disease progression (79, 131)
Interleukin-6 Lymphoid and non-lymphoid Mediates inammatory tissue destruction
cell types Thought to be an important cytokine in B-cell differentiation and
Production is triggered by hence to play a role in the induction of the elevated B-cell response
interleukin-1, tumor necrosis in patients with periodontal disease (79)
factor and interferon-g
Interleukin-8 Mononuclear monocytes and Attracts and activates neutrophils into the tissues of the
many of the tissue cells periodontium, particularly as it is produced by gingival broblasts
(303)
Interleukin-12 Monocytes, macrophages, Pleiotrophic effects on natural killer cells and T cells in the tissues
B-cells and accessory cells Induces interferon-g production and is necessary for Th1 induction
Tumor necrosis Macrophages and lymphocytes Similar effects to interleukin-1 and interleukin-6
factor a and b respectively
Interferon g Activated T cells Potent inhibitor of interleukin-1, tumor necrosis factor-a and tumor
necrosis factor-b
hibit much of the work done by Th1 cells. The pro-
duction of interleukin-4 and interleukin-10 by these
cells inhibits the actions of interferon-g, hence has
anti-inammatory characteristics. The production of
interleukin-4, interleukin-5 and interleukin-6 elicits
and helps to maintain a humoral immune response.
Natural killer cells
Natural killer cells are also said to constitute a large
part of the cell-mediated immune system, although
they are classed as a component of the innate im-
mune system. These cells are large granular lympho-
cytes that are often detected by the marker CD16,
thus making the actual numbers detected very dif-
cult to report, since this marker is also found on
other peripheral blood lymphocytes and monocytes.
In contrast to CD8
cytotoxic T cells and killer cells,

which kill infected targets cells in an antigen-specic
manner, natural killer cells kill infected target cells or
tumor cells in an antigen-nonspecic manner (195).
The role of natural killer cells in periodontal dis-
ease is unknown at present. However, there are re-
ports of an increase in the numbers of natural killer
cells in the peripheral blood of patients with early-
onset periodontitis (34, 157, 338).
Other studies have reported no increase in the
numbers of natural killer cells found in the periph-
eral blood of diseased patients compared with health
(357). In the tissues of diseased patients an increase
of natural killer cells is seen, which is actually not
70
difcult or surprising considering the fact that, in
health, very few natural killer cells are present in the
gingival tissues. The numbers of natural killer cells
in the tissues are seen to increase from health to gin-
givitis to periodontitis (43, 81, 350), although the
proportion of these cells relative to total lymphocyte
numbers actually decreases (43).
The activity of natural killer cells is highly in-
creased by soluble mediators such as interleukin-2,
interferon-a, interferon-b and interferon-g. Inter-
ferons a and b are antiviral proteins synthesized and
released by leukocytes, broblasts and virally in-
fected cells; interleukin-2 and interferon-g are re-
leased by activated T cells (18). Surface lipopolysac-
charide from gram-negative bacteria, however, ap-
pear to provide the major activation signal for
natural killer cellmediated cytotoxicity in peri-
odontal disease (173), which leads to natural killer
cell cytotoxic activity against host cells.
Cytokines in periodontal disease
Interleukin-1
In the periodontal tissues, interleukin-1b predomi-
nates over interleukin-1a and is present at higher
levels in active diseased sites compared with healthy
or stable diseased sites (143, 288). The variation in
interleukin-1 concentrations between sites in the
same individual, and the lack of variation in serum
interleukin-1b levels between adult periodontitis pa-
tients and healthy controls, suggests local rather
than systemic production of interleukin-1 (37). Acti-
vated keratinocytes and Langerhans cells produce
interleukin-1a and interleukin-1b (130). Macro-
phages, polymorphonuclear leukocytes, B cells and
broblasts secrete interleukin-1b (89, 143, 301). Not
surprisingly, interleukin-1 receptor antagonist has
been detected in periodontal macrophages and
found in the periodontal crevicular uid of patients
with periodontitis (136, 146).
Numerous periodontal bacteria can stimulate host
cells to produce interleukin-1 (87, 89). Levels of in-
terleukin-1b correlate with the periodontal status
(241). Interleukin-1 stimulates broblasts, endo-
thelial cells, monocytes and granulocytes resident in
the gingival connective tissue to express adhesion
molecules (302). These adhesion molecules aid the
passage of immune response cells from the capil-
laries into the inamed tissues.
Interleukin-1 can induce the gingival broblast
production of plasminogen activator, interleukin-6
and prostaglandin E
2
as well as the matrix metallo-
proteinases, which are involved in tissue destruction,
turnover and remodeling (245). Both interleukin-1
and tumor necrosis factor-a strongly induce matrix
metalloproteinase expression in resident and immi-
grant cells, but interleukin-1 is more potent than tu-
mor necrosis factor-a (22, 317). Interleukin-1 stimu-
lates osteoclasts to resorb bone (135) while inhibit-
ing the formation of bone (287). More specically,
interleukin-1-mediated resorption of bone can be
induced by periodontal pathogens (140).
The proinammatory cytokines tumor necrosis fac-
tor-a, interleukin-1a and interleukin-1b have been
identied in periodontitis lesions from patients with
adult periodontitis at higher levels than in healthy
sites (196, 289). However, cells containing interleu-
kin-1b were found in much greater numbers than
those producing tumor necrosis factor-a and in-
terleukin-1a (289). In contrast to interleukin-1, only
very low concentrations of tumor necrosis factor-a
have been demonstrated in gingival crevicular uid
from both adult and early-onset periodontitis pa-
tients (251, 356). However, high levels of tumor ne-
crosis factor-a in periodontal lesions appear to rep-
resent an established inammatory and immune re-
sponse. Salvi et al. (255) examined gingival crevicular
uid interleukin-1b, tumor necrosis factor-a and
prostaglandin E
2
levels in insulin-dependent dia-
betes mellitus patients with periodontal disease.
71
They found that diabetics had signicantly increased
2
and interleukin-1b but not
tumor necrosis factor-a, compared with non-dia-
betic controls with similar periodontal status. In ad-
dition, diabetics with moderate to severe disease had
almost two-fold higher levels of prostaglandin E
2
and interleukin-1b compared with diabetics with
gingivitis or mild periodontitis.
Prostaglandin E
2
causes increased vascular di-
lation and permeability. It also stimulates macro-
phages to secrete matrix metalloproteinases and has
been shown to trigger bone resorption in vitro (22),
thus acting synergistically with interleukin-1 and tu-
mor necrosis factor-a. Furthermore, it upregulates
receptors for complement and immunoglobulin on
monocytes and neutrophils (215). The major source
of prostaglandins and leukotrienes in inamed peri-
odontal tissue is the activated macrophages, al-
though they can also be produced by broblasts.
Prostaglandins, especially prostaglandin E
2
, com-
prise the primary pathway of alveolar bone destruc-
tion in periodontitis. Leukotrienes, especially leuko-
triene B
4
, are potent chemoattractants for polymor-
phonuclear leukocytes.
Interleukin-10
Interleukin-10 may have a role in the progression of
adult periodontitis. This was suggested by Gemmell
et al. (89), who found that T-cell lines from both the
peripheral blood of these patients and their gingival
tissue secreted interleukin-10, but clones derived
from the peripheral blood of an individual with gin-
givitis did not. It has also been suggested by Stein et
al. (291, 292) that interleukin-10 may contribute to
autoimmune reactions against gingival tissues. In-
terleukin-10 is able to favor the development of a
particular clone of B cells (CD5), which can secrete
high levels of autoantibody and is found in increased
numbers in inamed gingival sections (296). It was
found that a subset of type I diabetics may be more
susceptible to developing periodontitis through an
autoimmune type of reaction directed against gingi-
val connective tissue when exposed to periodontal
pathogens (291, 292).
Gemmell & Seymour (89) have demonstrated a
signicantly reduced number of interleukin-10
CD8
cells from periodontitis lesions (adult peri-

odontitis) than from healthy or gingivitis sections.
They suggested that in gingivitis interleukin-10
might suppress inammation by decreasing macro-
phage activity, thereby preventing progression to
periodontitis. No differences were found in the per-
Kinane et al.
centages of interleukin-10
CD4
cells between the

two disease categories. However, some individuals
were found to have higher numbers of interleukin-
10
T cells regardless of disease category. It appears

that the overall variation between the two diseases
entities in interleukin-10
CD8
cells may have been

due to individual variation in interleukin-10 secre-
tion patterns rather than differences in pathogenesis
between the two disease categories. Further studies
are needed to elucidate the role of interleukin-10 in
periodontal disease.
In summary, interleukin-1, interleukin-6 and tu-
mor necrosis factor are proinammatory cytokines
which are detected and may be modulated within
the periodontium. They are important and ubiqui-
tous aspects of the host defense system, operating in
the periodontal tissues during health, destruction
and healing phases. Similar claims may in future be
made for other members of the cytokine network,
in particular interleukin-10 and various soluble and
cellbound receptors and inhibitors of cytokine func-
tion. These molecules are inter-related and are part
of the immune, inammatory, breakdown and repair
homeostasis ongoing within the periodontium.
The pathology of the
periodontal diseases affecting
children and adolescents
Gingivitis
Various hypotheses have been made over the years
linking the severity of gingivitis in childhood with
various factors including puberty.
An increase in the sex steroid hormones during
the period of puberty is believed to have a transient
effect on the inammatory status of the gingiva
(193). Enlargement of the gingivae occurs in both
males and females in areas of local irritation. The
inammation has been described as marginal in dis-
tribution, characterized by prominent bulbous
proximal papillae. Enlargement is often only found
on the facial gingiva, whereas the lingual surfaces
remain unaltered (97). Although this view is widely
accepted, the evidence obtained from cross-sec-
tional and longitudinal studies for this hypothesis is
mainly circumstantial and often fragmented, al-
though still of importance.
Sutcliffe (298) reported a 6-year longitudinal study
that had been carried out to observe the changes in
gingivitis in 127 children 1117 years old, and the
relationship of these changes with oral hygiene. He
72
reported an increase in gingivitis associated with pu-
berty. It was noted that girls tended to experience
their maximum gingivitis before boys, girls at a mean
age of 12 years and 10 months and boys at 13 years
and 7 months. Hence, he noted that the distributions
of age were consistent with the hypothesis that there
is an increase in gingivitis associated with puberty.
The problems with this and further studies is that
they only provide circumstantial evidence linking
puberty and gingivitis. Thus, the association be-
tween the increase in sex hormone levels and gingi-
vitis needs conrmation. Chronological age is a poor
indicator of puberty, the measurement of the maxi-
mum gingivitis experience is extremely subjective
and, further, none of the studies reported have
measured the hormone levels of the subjects. An
alternative explanation for increased gingivitis dur-
ing puberty is that this is a period of mixed dentition
where erupting and exfoliating teeth present many
sites for plaque retention. The fact that gingivitis de-
creases after puberty may reect the general im-
provement seen in children as they improve their
dexterity and become more aware of oral hygiene.
Other clinical indications of a hormonal effect in-
clude uctuations in gingivitis with phases of the
menstrual cycle (132, 175), increased incidence and
severity of gingivitis during pregnancy (26, 137), gin-
gival enlargement induced by oral contraceptives
(148, 186, 286) and circumstantial evidence sug-
gesting that there is an increased number of de-
squamative gingival lesions in menopausal females
(215). The mechanisms by which the hormones may
inuence the gingiva have so far not been fully eluci-
dated.
Steroid hormone receptors are located in hor-
mone-sensitive tissues, where the hormones tend to
be retained and allowed to accumulate. In a number
of species accumulation of androgens, estrogens and
progestins have been observed in the periodontium
(193), and particularly pertinent are autoradiographic
studies that have demonstrated nuclear localization
of estradiol inhumangingival epitheliumandgingival
broblast cells (331333). The actual accumulation
and effect of sex steroid hormone onthe gingiva is un-
known; however, a number of theories have arisen.
Microbial effects of hormones
Microbial plaque and its presence is a very import-
ant factor on the onset, progression and severity of
gingivitis. It has been reported in other studies of a
hormonal nature that microorganisms are affected.
Kornman & Loesche (137, 148) reported higher levels
of Prevotella intermedia in pregnant females with el-
evated levels of sex hormones and also in women
taking oral contraceptives. Therefore, it seems logical
that if levels of sex hormones can affect the micro-
ora at one time they must be affected during other
periods of hormonal uctuation. Many studies have
been carried out often leading to reports of speculat-
ive results. Several reports of a transient increase of
black pigmented gram-negative anaerobic rods in
children during puberty have arisen (45, 46, 346).
However, none of these studies have used hormone
levels as a measure of puberty, and therefore it is
difcult to draw any inference in this situation.
Vascular effects of hormones
Investigations in animals suggest that the sex hor-
mones, particularly progesterone, may produce in-
creased inammation by generating changes in the
gingival vasculature (137, 148). Local or systemic ad-
ministration of progesterone produces an increase in
vascularity in ear chamber wounds in rabbits (177).
Following systemic administration of progesterone,
certain observations suggested that the hormone
might induce alterations in the plasma-endothelial
lining of the post-capillary venules (177), resulting in
an increased leakage of plasma proteins and leuco-
cytes. Gingival exudate also increased following ad-
ministration of sex hormones to dogs (176), and pro-
gesterone enhanced acute inammation during
wound healing in rabbits (219).
Immune effects of hormones
The importance of sex hormones is emphasized by
the ndings related to gender-related disease sus-
ceptibility. For example, a number of diseases can be
modulated by hormones, mainly estrogens, such as
rheumatoid arthritis (127) and Graves disease (8),
which are both affected during pregnancy. Therefore,
some immune responses and reactions are affected
in the gingiva leading to a possible role for sex hor-
mones in this disease. The degree of inuence of this
is unknown, however.
Cellular effects of hormones
Sex hormones are known to affect the cells of the
body. Histological studies have shown that estrogens
increase epithelial keratinization, stimulate prolifer-
ation (246, 247, 361) and increase the downgrowth
of epithelial attachment (218). Androgens have also
been shown to have effects.
73
Human gingiva is capable of metabolizing sex
hormones (333), and receptors for estrogen have
been found in gingival tissues (16). Furthermore, in-
amed gingiva has been found to metabolize pro-
gesterone more rapidly, and these metabolites differ
from those produced by healthy gingiva (120). Vittek
et al. (333) have shown a positive correlation be-
tween progesterone and gingival inammation. It is
known that the gingival levels of progesterone and
its metabolites increase during pregnancy and that
these metabolites increase the inammation in pre-
existing gingivitis (224). These effects, produced by
progesterone alone or in combination with estrogen,
could account for many of the changes seen in pu-
berty and other conditions in which there are in-
creased circulating sex hormones. In the treatment
of children undergoing puberty with gingivitis, the
increased susceptibility to plaque-induced inam-
matory changes should be reduced by the use of op-
timal levels of plaque control.
The pathogenesis and host responses of
gingivitis and periodontitis
The normal healthy gingiva is characterized by its
pink color and its rm consistency. Interdentally, the
healthy gingival tissues are rm and do not bleed on
gentle probing and ll the space below the contact
areas between the teeth. Healthy gingiva often ex-
hibits a stippled appearance and there is a knife-
edge margin between the soft tissue and the tooth.
In theory, normal gingiva are free from histological
evidence of inammation, but this ideal condition is
very rarely seen due to the ever present microbial
plaque. Even in the very healthy state, the gingiva
has a leukocyte inltrate that is predominantly com-
prised of neutrophils or polymorphonuclear leuko-
cytes. The primary purpose of these phagocytes is to
kill bacteria, which they do by emigrating through
and out of the tissues into the gingival crevicular
area.
Clinical changes in the early stages of inam-
mation of the gingival tissues following plaque ac-
cumulation are subtle, but quite rapidly the gingiva
appears red, swollen and the soft tissue has an in-
creased tendency to bleed on gentle probing. Histo-
logically, this stage is described by increased vascular
dilation and permeability, which leads to an inux
of exudative uid, proteins and inammatory cells
into the tissues giving them their swollen appear-
ance. At this stage there is intense recruitment of
polymorphonuclear leukocytes, macrophages and
their progenitor cells monocytes into the tissues.
Kinane et al.
These leukocytes migrate up a chemoattractant
gradient to the crevice and are aided by adhesion
molecules such as intercellular adhesion molecule-1
and endothelial cell leukocyte adhesion moelcule-1,
which assist leukocyte binding to the post-capillary
venules and help cells to leave the vessels (150). In
addition, as the microbes damage the epithelial cells,
the epithelial cells release cytokines, which further
encourage recruitment of leukocytes (predominantly
neutrophils) into the crevice. The neutrophils within
the crevice can phagocytose and digest bacteria and
so remove these bacteria from the pocket. In ad-
dition to this, if the neutrophil is overloaded with
bacteria, it degranulates or explodes and, by doing
so, causes tissue damage due to the toxic enzymes it
releases. The neutrophil can thus be viewed as being
both helpful and potentially harmful. This neutro-
phil defense may in some instances operate well and
reduce the bacterial load and can be considered im-
portant in preventing the gingivitis lesion from being
established. If, however, there is an overload of mi-
crobial plaque, then the neutrophils and the barrier
that is comprised of epithelial cells will not be suf-
cient.
After approximately 7 days of plaque accumu-
lation, the initial lesion is said to progress to the
early lesion (25, 237, 266, 267). Lymphocytes and
macrophages predominate, and the inltrate occu-
pies 1015% of the gingival connective tissue. Both
clinically and histologically the changes are very vis-
ible. The established lesion as dened by Page &
Schroeder (232) is one dominated by plasma cells.
Plasma cells have been shown to be predominantly
IgG-producing cells, with a lower proportion being
IgA (187, 188, 222, 335). In early-onset periodontitis,
local IgG4-producing cells in particular seem to be
elevated. Clinically in the established lesion, colla-
gen loss continues both laterally and apically and the
dentogingival epithelium continues to proliferate.
Two types of established lesion, however, appear to
exist. One remains stable not progressing for months
or years (174, 233), and the second becomes more
active and converts to progressive destructive
lesions. The very nature of early-onset periodontitis
and the knowledge that it is a rapidly progressing
destructive disease make it clear that the established
lesion here will progress speedily to the destructive
advanced lesion. The advanced lesion is character-
ized by bone loss, periodontal ligament loss, apical
migration of the junctional epithelium to form a true
pocket and histopathologically by an increase to over
50% of plasma cells in the inltrated connective
tissues (150).
74
Gingivitis is primarily a response to the bacteria
in plaque. It includes a vascular response with in-
creased uid accumulation and inammatory cell
inltration. Serum IgG antibody levels to Actinomy-
ces spp. were found to be higher in gingivitis (72),
and a similar study showed that antibodies to at least
three bacteria (Actinomyces spp., Bacterionema ma-
truchotti and Leptotrichia buccalis) were detected in
gingivitis patients with a wide variation in levels (72).
Multiple reports have suggested that gingivitis pa-
tients can present with antibody to suspected oral
pathogens (49, 227), including P. gingivalis and A.
actinomycetemcomitans. Bimstein & Ebersole (21)
noted that IgM antibody levels were signicantly dif-
ferent to many bacteria when comparing children
versus adults with gingivitis. In contrast, the IgG
levels were unrelated to disease in the adult groups
but did help to distinguish between the children
with and without gingivitis. Thus, generally, the gin-
givitis patients exhibit antibody to a wide array of
these bacteria; however, the levels to suspected peri-
odontal pathogens are uniformly signicantly lower
than those seen in periodontitis patients (61, 62). Fi-
nally, we observed lower IgA levels in gingivitis sites
compared to healthy sites of normal subjects (64),
and Grbic et al. (102) demonstrated that IgA levels
were signicantly increased in gingival crevicular
uid from gingivitis sites compared with peri-
odontitis sites, suggesting the potential for IgA to
function as a protective factor in this local environ-
ment. Generally low levels of antibody to bacteria
associated with periodontitis are found in gingival
crevicular uid from gingivitis sites.
The progression from gingivitis to
periodontitis
Brecx et al. (25) suggests that more than 6 months
may be needed in the normal situation for gingivitis
to change to a periodontitis lesion. Furthermore, this
move from gingivitis to periodontitis probably only
occurs in 10% to 15% of the population. The reason
why some patients develop periodontitis more read-
ily than others is highly elusive and thought to be
multifactorial. It should be borne in mind that the
changes during gingivitis, even in prolonged estab-
lished gingivitis lesion, are to a large extent revers-
ible, whereas the changes during periodontitis, that
is, the bone loss and apical migration of the epi-
thelial attachment, are irreversible. Periodontitis is a
cumulative condition whereby once there is bone
loss, it is almost impossible to have it re-established
and therefore, patients lose bone incrementally over
the years. The worst effects of periodontitis are
therefore, commonly seen in older patients rather
than younger patients, who have still to develop
these deep pockets or this extensive bone loss or the
associated gingival recession that occurs in peri-
odontitis. The early-onset forms of periodontitis are,
however, situations where the normal slow destruc-
tive process is greatly accelerated.
Differences between chronic gingivitis
and periodontitis
The similarities in the inammatory inltrate be-
tween the stable established chronic gingivitis lesion
and advanced periodontitis lesions have encouraged
many investigators to look for qualitative and quan-
titative differences that may be involved in deter-
mining the progression of gingivitis to destructive
periodontitis.
Seymour et al. (270) hypothesized that a change
from T-cell to B-cell dominance causes periodontitis.
However, Page (235) has disagreed with this view, as
have Gillett et al. (96), who showed a predominantly
B-cell inltrate to be associated with stable, non-
progressive lesions in childhood gingivitis. Liljenberg
et al. (172) compared plasma cell densities in sites
with active progressive periodontitis and in sites
with deep pockets and gingivitis but no signicant
attachment loss over a 2-year period. The density of
plasma cells (51.3%) was very much increased in ac-
tive sites as compared with inactive sites (31.0%). It
is now generally accepted that plasma cells are the
dominant cell type in the advanced lesion (85).
Necrotizing ulcerative gingivitis and
periodontitis
Necrotizing ulcerative gingivitis is an acute inam-
matory condition associated with a fusospirochaetal
microbial complex. Necrotizing ulcerative gingivitis
begins as linear erythema of the marginal gingiva
and extends to form ulcerated, punched-out, painful
and necrotic papillae and later may extend to ulcer-
ation of the gingival margins (133). The ulcers are
covered by a grayish slough, which if removed causes
bleeding from the exposed ulcers. The condition is
painful and is associated with marked halitosis often
termed fetor oris. As the necrosis progresses, deep
bone cratering appears in the interproximal regions
in the areas where the papillae have been lost. If left
untreated, necrotizing ulcerative gingivitis will pro-
gress to necrotizing ulcerative periodontitis, which
involves destruction of the interproximal alveolar
75
bone and the periodontal ligament (133, 197).
Marked gingival recession and sequestra formation
is a sequel of the rapid necrosis. In industrialized
countries, adolescents and young adults are most
predisposed to this condition, and a prevalence of
approximately 0.5% has been reported (134).
In a classic electron microscopic study by Listgart-
en in 1965 (178) four zones were identied. These
zones are the: 1) bacteria-rich zone or plaque zone,
2) neutrophil-rich zone in which neutrophils pre-
dominate; 3) necrotic zone, which has dead cell,
spirochetes and other bacteria; and 4) the spirochet-
al inltration zone, in which tissue structures is pre-
served but many invading spirochetes are noted.
Necrotizing ulcerative gingivitis has been char-
acterized by the emergence of selected members of
the oral microbiota: Treponema spp. and P. interme-
dia, which develop as a response to altered resis-
tance to infection of the host tissue (immunosup-
pression). These oral spirochetes have been shown
to be capable of invading the infected host tissue
(78, 180, 254). IgG antibody to oral spirochetes (Tre-
ponema denticola and Treponema vincentii) has
been reported; the data have shown below normal
to selected elevations in disease subjects (14, 40, 142,
165, 212, 269, 345). Therefore, while treponemal spe-
cies are frequent in subgingival plaque and comprise
a major proportion of some plaques in periodontitis
and necrotizing ulcerative gingivitis (280, 281), there
does not appear to be an elevation in systemic anti-
body responses, indicative of an active host re-
sponse, to the cultured treponemal species. Serum
antibody to Actinomyces viscosus, Actinomyces israe-
lii, Prevotella melaninogenica and P. gingivalis were
within normal limits in necrotizing ulcerative gingi-
vitis during acute and convalescence; however, anti-
body to P. intermedia was signicantly elevated in
convalescent sera (72), suggesting that acute disease
may exhibit a specic pathogenic microbiota to
which the host responds. The contribution of this
response to resolution of the disease remains un-
known.
Cancrum oris or noma is a devastating orofacial
gangrenous disease that is considered a very severe
form of necrotizing ulcerative periodontitis and is
commonly reported in underprivileged African
children (77). The children often suffer from chronic
malnutrition, numerous endemic communicable
diseases, including viral diseases, and severe adverse
physical conditions. These severe debilitating factors
may deplete the subjects immune and inammatory
system. Measles is the most common infection pre-
ceding the development of noma in Nigerian
Kinane et al.
children (77). Acquired immunodeciency in combi-
nation with the impaired endocrine balance in
malnourished children results in widespread infec-
tion with the measles virus. Anergy resulting from
the combination of malnutrition and measles virus
infection may promote overgrowth and invasion of
anaerobic organisms, including gram-negative bac-
cilli and spirochetes. Due to the severe debilitation
of the malnourished child, the infection is not con-
ned locally as necrotizing ulcerative gingivitis but
spreads rapidly across normal anatomical barriers.
Severe necrosis and possible sequestration as exem-
plied by noma are then seen. Minimal data are
available on host responses in this disease.
Murray et al. (213) also demonstrated that the
microbiota of periodontitis in human immunodec-
iency virus (HIV) patients was similar to that seen in
systemically healthy periodontitis individuals; how-
ever, there were signicant increases in the numbers
of Campylobacter spp. and Treponema spp. Serum
IgG antibody to P. intermedia, P. gingivalis and Fuso-
bacterium nucleatum have been found to be elev-
ated in HIV-positive homosexuals compared with
control normals and seronegative homosexuals
(104). Both necrotizing ulcerative gingivitis and nec-
rotizing ulcerative periodontitis (73) occur in a pro-
portion of HIV-infected patients. Rams et al. (243)
compared the distribution of several bacterial mor-
photypes in necrotizing ulcerative periodontitis and
clinically normal periodontitis subjects and found
that the morphotypes were very similar from areas of
necrotizing ulcerative gingivitislike tissue necrotic
lesions and necrotizing ulcerative periodontitis sites.
Importantly, all the patients with the necrotizing ul-
cerative gingivitislike lesions had signicantly high
levels of spirochetes in their pockets. However, the
levels of serum antibody to oral microorganisms in
HIV subjects has had minimal investigation. Grieve
et al. (103) suggested that HIV periodontitis (necrot-
izing ulcerative periodontitis) patients exhibited el-
evations in serum antibody to P. gingivalis, P. inter-
media, F. nucleatum, A. actinomycetemcomitans and
Eikenella corrodens. We recently examined serum
IgG antibody to a battery of oral pathogens, includ-
ing T. denticola, in HIV patients. We found that,
while some of the patients with necrotizing ulcer-
ative periodontitis exhibited elevated antibody to
suspected periodontal pathogens, the antibody level
to T. denticola isolates was low, and selected HIV pa-
tients had minimal antibody to all of the oral micro-
organisms. Additional studies will be required to de-
lineate the capability of the host response in manag-
ing periodontal infections in these patients.
76
Pathogenic features of adult and
early-onset periodontitis
Experimental short-term clinical studies have shown
that microorganisms quickly colonize clean tooth
surfaces when an individual stops toothbrushing.
Within a few days, microscopical and clinical signs
of gingivitis are then apparent. These inammatory
changes can be resolved when adequate tooth-
cleaning methods are resumed (182, 316). Thus,
microorganisms that form dental plaque and cause
gingivitis probably do so by the release of compo-
nents from the bacterium that induce inammation
in the tissues. Short- and long-term clinical trials
have underlined the importance of supragingival
plaque and subgingival microbial plaque in both the
treatment of gingivitis and periodontitis. Further-
more, animal experiments have indicated that gingi-
vitis only develops in animals that accumulate bac-
terial plaque deposits. Gingivitis is necessary prior to
the tissue developing periodontitis, and this implies
that prevention of gingivitis is also a primary preven-
tive measure for periodontitis.
The pathogenic processes are largely the response
to microbe-induced destructive processes. These
processes are initiated by the microbes but are
undertaken by the host cells, and thus it is the host
tissue itself that results in the destruction seen. The
host produces enzymes that break down host tissue.
This is a necessary process that the host initiates and
controls in order to allow the tissues to retreat from
the microbial destructive lesions.
Microbial plaque accumulation occurs on the sur-
face of the teeth adjacent to gingival tissues, bringing
the oral sulcular and junctional epithelium into con-
tact with colonizing bacteria and their waste prod-
ucts such as proteases, enzymes, lipopolysaccharide
and toxins. As these products build up, the irritation
to the tissues increases, until production of chemical
mediators of inammation commences and a host
inammatory response is induced.
Elements of the host response that could
differentiate adult and early-onset forms of
periodontitis
The various aspects of the host response that may
contribute to peridontal disease have been covered
generally, but several aspects supported within the
literature may be specically relevant to early-onset
periodontitis. These include aspects of the innate,
inammatory and immune defense systems.
The innate defense system includes epithelial,
connective tissue elements and soluble products
that are antimicrobial and in the normal healthy
state may protect against periodontal disease. There
are, however, defects reported in epithelial, connec-
tive tissue (broblasts and cementum), body uids
(saliva) and enzymes (alkaline phosphatase) that
may be relevant to early-onset periodontitis (5, 9, 15,
44). Structural defects such as the absence of endo-
thelial adressins for leukocyte adherence and sub-
sequent migration from the bloodstream into lesions
(seen in leukocyte adhesion deciency with marked
periodontal destruction) and defects of epithelium
and connective tissue (as seen in Papillon-Lefe`vre
syndrome) as well as more obvious periodontal de-
fects such as that of hypophosphatasia (where defec-
tive cementum results in early tooth loss) can all
have major effects on the periodontal tissues and
lead to early loss of teeth.
Pathogens
The microbes involved in adult periodontitis are
largely gram-negative anaerobic bacilli with some
anaerobic cocci and a large quantity of anaerobic
spirochaetes. The main organisms linked with deep
destructive periodontal lesions are P. gingivalis, P. in-
termedia, Bacteroides forsythus, A. actinomycetem-
comitans and T. denticola (360).
P. gingivalis is more frequently detected in severe
adult periodontitis, in destructive forms of disease
and in active lesions than in health or gingivitis or
edentulous subjects. P. gingivalis is reduced in num-
bers in successfully treated sites but is seen in sites
with recurrence of disease after therapy (16, 111,
325). It has been shown that P. gingivalis induces el-
evated systemic and local antibody responses in
subjects with various forms of periodontitis (60). P.
intermedia levels have been shown to be elevated
in certain forms of periodontitis (9). Elevated serum
antibodies to this species have been observed in
some but not all subjects with refractory peri-
odontitis (30). B. forsythus has been found in higher
numbers in sites exhibiting destructive periodontal
disease than in gingivitis or healthy sites (46). In ad-
dition, B. forsythus was detected more frequently in
active periodontal lesions (13). Although spirochetes
such as T. denticola are notoriously difcult to cul-
ture in the laboratory, these strict anaerobes may
comprise more than 30% of the periodontal subgin-
gival microora. The above mentioned putative
pathogens are always part of a large and varied mi-
croora found in the subgingival plaque. Some are
not detected in certain sites with periodontitis and
77
can even be completely absent in cultures from
multiple sites within an untreated periodontitis pa-
tient.
The dominant microorganisms involved in early-
onset periodontitis include A. actinomycetemcomit-
ans, Capnocytophaga spp., Eikenella spp., P. interme-
dia and anaerobic rods such as Campylobacter rectus
(311). Much of the literature regarding the bacterial
effects in early-onset periodontitis have centred
around studies on A. actinomycetemcomitans. A.
actinomycetemcomitans is a short, facultatively an-
aerobic gram-negative rod that is nonmotile and re-
garded as a key microorganism in early-onset peri-
odontitis. Studies of association have shown in-
creased levels of A. actinomycetemcomitans and
increased frequency of antibody to A. actinomyce-
temcomitans in localized early-onset periodontitis
(24). These researchers (24) showed a signicantly
increased level of IgG antibody to A. actinomycetem-
comitans serotype b in 90% of localized early-onset
periodontitis patients, 40% of generalized early-on-
set periodontitis and only 25% of adult periodontitis
patients. Furthermore, clinical studies carried out to
observe the effect of treatment on A. actinomycetem-
comitans load have related poor therapeutic out-
come with an inability to reduce the subgingival load
of A. actinomycetemcomitans (112, 113, 213).
In localized early-onset periodontitis when pres-
ent, A. actinomycetemcomitans, is predominant.
Genco et al. (90) indicated that organisms of the nor-
mal ora play a key role in gingivitis, while exoge-
nous organisms or more unusual anaerobic organ-
isms seem to be implicated in periodontitis. Im-
munological studies involving patients with localised
early-onset periodontitis indicate signicantly elev-
ated titers of serum antibodies to A. actinomycetem-
comitans in localized early-onset periodontitis pa-
tients (66, 68, 72, 94, 179, 190, 259, 314, 330). These
patients have also been seen to produce antibodies
locally to A. actinomycetemcomitans (259).
Accompanying the documentation of specic in-
fections in periodontitis patients has been the de-
nition of active host systemic immune responses to
these bacteria (72). In particular, it appears clear
that, in most populations, patients with localized
juvenile periodontitis exhibit elevated serum IgG
antibody to A. actinomycetemcomitans (29, 72).
These results have been derived from cross-sectional
studies and demonstrated some correlation with the
ability to identify the homologous microorganism in
the subgingival plaque (65, 308). Accumulating evi-
dence has supported the concept that the predomi-
nant serotype of A. actinomycetemcomitans appears
Kinane et al.
to differ throughout the world (11, 39, 59, 74, 98, 99,
108, 209, 223, 261, 313, 351, 358) and may represent
some more general genotypic, phenotypic, and
pathogenetic heterogeneity in this species. Ebersole
et al. (29) reported a study that examined the fre-
quency of oral disease in an adolescent population
and assessed the relationship to A. actinomycetem-
comitans. Heavy accumulations of plaque and calcu-
lus were frequently observed and were associated
with gingival inammation and, signicantly, 25.7%
of the students exhibited early-onset periodontitis
with 1.7% diagnosed as localized early-onset peri-
odontitis. The ndings supported the hypothesis
that A. actinomycetemcomitans may represent a
pathogen in periodontitis and, while oral health care
may be poor, contact with the microorganism ap-
pears to be required to initiate disease in this popu-
lation (29). Ebersole et al. have reported humoral re-
sponses in periodontitis patients in various Turkish
populations (31). All localized early-onset peri-
odontitis patients from Turkey exhibited elevated
antibody levels to A. actinomycetemcomitans, par-
ticularly serotypes c and a, while antibody levels to
A. actinomycetemcomitans serotype b were highest
in United States localized early-onset periodontitis
patients. Fifty percent of the Turkish generalized
early-onset periodontitis patients also showed elev-
ated anti-A. actinomycetemcomitans antibody. These
data suggested that considerable variation exists in
the systemic antibody levels to periodontal patho-
gens and support potential differences in subgingi-
val colonization or antigenic composition of these
pathogens between patient populations from differ-
ent geographical regions. Additional studies by this
group have also noted elevated serum antibody re-
sponses to A. actinomycetemcomitans and selected
other oral periodontopathogens in Sjgrens syn-
drome patients with periodontitis (33) and in pa-
tients with Behcets disease and periodontitis (32).
Thus, numerous investigations of many ethnic and
geographically disparate groups tend to conrm that
A. actinomycetemcomitans is frequently associated
with localized early-onset periodontitis, and prob-
ably an causative agent in the vast majority of cases
and in a substantial proportion of other early-onset
periodontitis patients.
Our ndings and those of others (23, 27, 234, 276,
337, 343, 344) suggest that human serum antibody
reactivities to A. actinomycetemcomitans are ob-
served to a wide variety of antigens derived from the
outer membrane of this pathogen. The results indi-
cated a response to certain antigens that were dis-
tinctive in active disease patients and may: (i) indi-
78
cate changes in antigen expression by A. actinomyce-
temcomitans (114); (ii) reect the overall increase in
serum antibody levels noted in active disease pa-
tients; or (iii) relate to the increase in the A. actino-
mycetemcomitans burden, resulting in greater anti-
genic load, (iv) reect the extent of existing disease
(58) or (v) be associated with more severe disease.
The previously mentioned studies concentrated
their efforts on analysis of serum IgG antibody to A.
actinomycetemcomitans or antigens isolated from
the bacteria. The IgG isotype of immunoglobulin
consists of four subclasses, IgG14, which have been
shown to be elicited selectively by certain types of
antigens and to have diverse functions. Thus, the
isotype and subclass of antibodies in the patient
serum specic to A. actinomycetemcomitans may re-
ect restrictions on the capacity of host antibody to
bind to particular antigens (60, 62, 63, 95, 156). The
repertoire of immunoglobulin isotypes and IgG sub-
class responses to particular antigens may account
for variations in host resistance to infection and dis-
ease.
A wealth of literature supports the existence of lo-
cal specic antibody production by plasma cells
present in inamed tissues of the periodontal pocket
(67, 71, 166, 282, 293). Likewise, a proportion of gin-
gival crevicular uid samples within a given subject
have local antibody levels signicantly greater than
can be accounted for by a serum contribution (67,
70, 71, 145). In addition to these inammatory me-
diators, evidence has been provided that indicates
that both C3 and C4 components of the complement
system are cleaved in gingival crevicular uid from
early-onset periodontitis (236, 264), indicating the
potential of antigen-antibody complexes to contrib-
ute to local immune modulation. Cross-sectional
studies have suggested that the gingival crevicular
uid samples with elevated antibody frequently
harbor the homologous bacteria and suggest that a
combination of the antigen and the host-response is
frequently associated with progressing disease (71).
These local antibody levels decreased with pocket
depth, attachment level stabilized and inammation
resolved following therapy (71).
Studies on early-onset periodontitis patients dem-
onstrating infection with A. actinomycetemcomitans
have demonstrated elevated gingival crevicular uid
antibodies to A. actinomycetemcomitans in the ma-
jority of the patients (115, 116, 259). The presence of
all subclasses of IgG have been identied in gingival
crevicular uid, with IgG1, and/or IgG4 levels in gin-
gival crevicular uid were elevated relative to serum
concentrations (240, 244). The results support a
unique local response in individual sites within cer-
tain patients and are consistent with a progression
of subclass responses at sites of infection and dis-
ease. An intriguing facet of host responses in peri-
odontitis has been the relationship between local
and systemic antibodies. The general paradigm
exists that gingival crevicular uid is comprised pri-
marily of a serum transudate at the site of inam-
mation (36, 41). However, other ndings are consist-
ent with systemic antibody being a manifestation of
the local antibody responses in the gingival tissues
and that a portion of serum antibody may be derived
from local gingival responses to this infection. The
studies described above document some character-
istics of the local immune response and their re-
lationship to infection and clinical presentation in
early-onset periodontitis patients, particularly within
A. actinomycetemcomitans infected periodontitis pa-
tients. The ndings are consistent with the potential
to utilize antibodies or local mediators in gingival
crevicular uid as adjuncts in the diagnosis of peri-
odontitis and in dening the mechanisms of disease.
A. actinomycetemcomitans is not always found in
localized early-onset periodontitis patients (107, 118,
149, 183, 226, 326). Furthermore, A. actinomycetem-
comitans has been found in patients with no clinical
attachment loss (46, 74, 208). Leukotoxin produc-
tion, a factor considered crucial in the virulence
attributes of A. actinomycetemcomitans, varies
among strains and has been found in A. actinomyce-
temcomitans isolated from localized early-onset
periodontitis rather than adult periodontitis or
healthy patients (360). A study in Denmark, however,
found no occurrence of the high leukotoxin-produc-
ing strains of A. actinomycetemcomitans in their
population of localized early-onset periodontitis pa-
tients (122). In summary A. actinomycetemcomitans
does not seem to be found in all cases of localized
early-onset periodontitis. Nevertheless, clinical
studies carried out observing effect of treatment with
A. actinomycetemcomitans load have linked failure of
treatment with inability to reduce the subgingival
load of A. actinomycetemcomitans (113, 213).
Inammatory mediators
It has been demonstrated that sera from localized
early-onset periodontitis patients with decreased
neutrophil function can modify the activity of
healthy neutrophils (2). The serum factors respon-
sible for these effects have been identied as being
inammatory mediators such as prostaglandins and
cytokines, which have correlated with periodontal
79
disease (207). Adherent mononuclear cells from lo-
calized early-onset periodontitis patients have been
found to secrete higher levels of prostaglandin E
2
and tumor necrosis factor-a than generalized early-
onset periodontitis patients or healthy controls
(273). More recently, insulin-dependent diabetic pa-
tients with periodontitis have been reported to
manifest excessive levels of inammatory mediators
(255257). It has been suggested that these obser-
vations indicate an underlying immune defect of
monocytes in certain diagnostic groups (221).
Early investigations of genetic polymorphisms of
cytokines have attempted to identify a possible link
between different periodontal categories and speci-
c alleles related to an ability to overproduce cyto-
kines (56, 158). In addition, a family linkage study of
early-onset periodontitis suggested that the COX-1
enzyme system, which acts as a catalyst for the
breakdown of arachidonic acid, one of the products
of which is prostaglandin E
2
, may be associated with
susceptibility to early-onset periodontitis (88).
Neutrophil chemotactic defects
A further host defense defect that may increase sus-
ceptibility to early-onset periodontitis is neutrophil
chemotactic defects. A high percentage of members
of families with a background of localized early-on-
set periodontitis have been reported to show abnor-
mal neutrophil chemotaxis (322). Whether the trait
is due to an intrinsic defect of neutrophils or is
caused by extrinsic factors in the sera and thus is
environmentally inuenced remains controversial
(2). The neutrophil chemotactic defect is not noted
in all populations exhibiting localized early-onset
periodontitis, and many families with localized
early-onset periodontitis do not show evidence of
the defect (151, 152). Decreased neutrophil chemo-
taxis in localized early-onset periodontitis has been
linked to a reduced receptors density for chemo-
attractants such as N-formyl-methyl-leucyl-phenyl-
alanine, complement fragment C5a, leukotriene B
4
and interleukin-8 (48, 51). De Nardin (52) found that
decreased N-formyl-methyl-leucyl-phenylalanine
binding which was related to variations in N-formyl-
methyl-leucyl-phenylalanine receptor DNA in local-
ized early-onset periodontitis patients and healthy
controls. These preliminary results indicate a poss-
ible role for a N-formyl-methyl-leucyl-phenylalanine
receptor alteration in neutrophil chemotaxis. How-
ever, many molecules related to neutrophil dysfunc-
tion in localized early-onset periodontitis patients
belong to the same family of surface receptors and
Kinane et al.
have similar morphogenic characteristics. De Nardin
(52) hypothesized that a common underlying defect,
at either the cell surface receptor or post-receptor
level may contribute to the alteration in neutrophil
function seen in patients with localized early-onset
periodontitis.
Humoral immune aspects
Lu et al. (184) have shown that in certain popula-
tions serum IgG2 levels are increased in localized
early-onset periodontitis patients, but not general-
ized early-onset periodontitis, adult periodontitis or
periodontally healthy individuals. An interesting as-
sociation is that black subjects in general have
higher levels of IgG2 than whites and the incidence
of localized early-onset periodontitis among black
individuals has been shown to be up to 15 times
greater than among whites (181). It thus appears that
localized early-onset periodontitis patients, particu-
larly black individuals, have a tendency to produce
increased levels of IgG2 (309).
A family study of early-onset periodontitis indi-
cated the possibility of genetic transmission of IgG2
levels (192). Smoking has been found to suppress the
IgG2 response in adult periodontitis and generalized
early-onset periodontitis patients and is associated
with more severe destruction but this does not ap-
pear to occur in localized early-onset periodontitis
patients (309). Furthermore, the paradoxically lower
IgG2 titers seen in black generalized early-onset
periodontitis smokers may be specic to A. actino-
mycetemcomitans and the IgG2 response against
antigens of other bacteria may not be lower in gener-
alized early-onset periodontitis subjects who smoke
(305).
Fc receptors
It has been suggested that the subclass IgG2 is a poor
opsonin because it xes complement less effectively
than IgG3 and IgG1 and binds weakly to Fc receptors
on the surface of phagocytes, which tends to compli-
cate theories of protective effects afforded by high
anti-A. actinomycetemcomtians titers of IgG2 in
black localized early-onset periodontitis patients
(107, 309). However, a further aspect of the immune
response may yet explain this phenomenon. Fc re-
ceptors are the crucial link between the humoral and
inammatory components of the host defense sys-
tem. It is possible that Fcg receptors III may prepare
polymorphonuclear leukocytes for phagocytosis me-
diated by Fcg receptors II (342).
80
It has been reported that the numbers of Fcg re-
ceptors II and Fcg receptors III on peripheral blood
neutrophils of localized early-onset periodontitis pa-
tients are within the normal range (171). There may
be local suppression of Fcg receptor expression and/
or the increased production of Fcg-binding proteins
by periodontal pathogens (129).
Conclusion
The various aspects of the host response that may
contribute to periodontal disease have been covered
in this chapter as have several aspects that may be
specically relevant to early-onset periodontitis, in-
cluding aspects of the innate, inammatory and im-
mune defense systems. In summary, the differences
in the causation and pathogenesis of adult and early-
onset forms of periodontitis are not yet sufciently
elucidated (321). However, multiple specic host de-
fense features are emerging in the early-onset forms
of periodontitis that may have a genetic basis and
that may serve to differentiate the different forms of
periodontitis and may have utility in terms of
screening, diagnosis and therapy.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Post-menopausal bone loss and
its relationship to oral bone loss
MARJORIE K. JEFFCOAT, CORA ELIZABETH LEWIS, MICHAEL S. REDDY,
CHING-YUN WANG & MARYANN REDFORD
Osteoporosis and osteopenia are characterized by
reductions in bone mass and may lead to skeletal
fragility and fracture. In fact, the exact denition of
osteoporosis differs around the world. In much of
Europe, osteoporosis implies a reduced bone mass
that results in a predisposition to fracture. Such de-
creases in bone mass are measured clinically using
new techniques described in this paper. Until the ad-
vent and widespread use of such methods, such as
dual energy X-ray absorption, the denition of os-
teoporosis was usually made using the clinical signs
of a fracture. In 1994 the World Health Organization
dened osteoporosis as a bone mineral density level
more than 2.5 standard deviations below the mean
of young, normal women (52).
The effect of osteoporosis on bone is clearly
shown in Fig. 1 and 2. Fig. 1 is a scanning electron
micrograph of normal trabecular bone. This bone
was obtained by iliac crest biopsy. Note the wide tra-
becular struts, bone mass, and the absence of micro-
fractures. In striking contrast, Fig. 2 is a scanning
Fig. 1. Scanning electron micrograph of normal bone. The
wide trabecular struts are evident.
94
electron micrograph of an iliac crest biopsy from os-
teoporotic trabecular bone. There is a relative lack of
bone mass, and narrow trabecular struts. A perfor-
ated strut is also evident. Such perforations may be-
come microfractures.
The morbidity that may be associated with osteo-
porosis should not be underestimated. While loss of
bone mass, per se, does not cause symptoms, once a
fracture does occur, pain, loss of function, and in
some cases deformity, may result. For these reasons
osteoporosis before fracture is termed a silent dis-
ease. Clearly, fractures of the hip, spine and radius
have signicant effects of the patients quality of life,
but serious fracture can also lead to death. The over-
all reduction in survival for the most serious frac-
tures is illustrated in Fig. 3 and has been estimated
to be 1020% (3, 33, 41).
Risk factors for osteoporosis have been extensively
studied and are summarized in Table 1 (1, 8, 20, 47).
Some of these risk factors are modiable, and others
are not. Women are at a greater risk for osteoporosis
Fig. 2. Scanning electron micrograph of osteoporotic
bone. Note the relative paucity of trabeculation and the
perforation apparent in the center of the micrograph.
Post-menopausal bone loss and its relationship to oral bone loss
Table 1. Risk factors for osteoporosis
Risk factor Modiable?
Sex No
Age No
Early menopause No
Low bone mass To some extent
Thin, small-framed body No
Race No
Lack of calcium intake Yes
Lack of exercise Yes
Smoking Yes
Alcohol Yes
Heredity No
Diseases such as hyperparathyroidism To some extent
Certain medications (such as steroids) To some extent
Propensity to fall To some extent
after menopause. Estrogen levels present prior to
menopause are protective against loss of bone min-
eral, as is hormone replacement therapy after meno-
pause (11, 12, 24, 31, 36). Early menopause, either
naturally occurring or drug or surgically induced,
without hormone replacement therapy predisposes
to osteoporosis (47). The decision of whether or not
to use hormone replacement therapy is dependent
on the risk-benet ratio for the individual woman.
Age is a major non-modiable risk factor for os-
teoporosis (23). In most women, bone mass reaches
its peak in the third decade of life (twenties or thir-
ties) and declines thereafter. This decline in bone
mass is accelerated with the onset of menopause (1,
20, 47). While estimates of the rate of post-meno-
pausal bone loss may differ by population and meas-
urement technology, a rate on the order of 0.51.0%
per year has been reported.
Other non-modiable factors include a thin-
framed body (1, 8), and the fact that Caucasian and
Asian women are at higher risk than African-Ameri-
can women (23), as are women with a history of os-
teoporosis in the family. Modiable contributors to
low bone mass include lack of sufcient calcium in-
take (18), lack of exercise, smoking and alcohol (17).
Certain medications, such as steroids, will alter the
balance between bone formation and resorption re-
sulting in a net loss of bone mass. Finally, a propen-
sity to fall, for any reason, will make fractures in the
patient with low bone mass more likely (1, 8).
Periodontitis is an inammatory disease char-
acterized by loss of connective tissue and alveolar
bone (2). Like osteoporosis, it is a silent disease, not
95
causing symptoms until late in the disease process
when mobile teeth, abscesses and tooth loss may oc-
cur. While the causative agent in periodontitis is a
pathogenic bacterial plaque in a susceptible patient,
periodontitis and osteoporosis have several risk fac-
tors in common.
Fig. 4 shows the prevalence of the proportion of
women with osteoporosis by age (34). Similarly, the
U.S. survey of oral health in adults has shown an
increased prevalence of periodontitis with increasing
age (37). In addition to the increased prevalence with
increasing age, risk factors in common for both dis-
eases include smoking, and inuence of disease or
medications that may interfere with healing (8, 20,
46). Whether the rate of progression of periodontitis
in women sharply increases immediately after
menopause is presently unknown.
Fig. 3. Relative survival following hip fractures. Note rela-
tive survival decreases with increasing years from the ini-
tial diagnosis.
Fig. 4. Percent of women with hip bone mineral density
indicative of osteoporosis. The prevalence increases with
age. Data from a population in Rochester, Minnesota.
Source: Melton (34).
Jeffcoat et al.
The measurement of bone mass
and density
The measurement of loss of bone associated with
osteopenia and osteoporosis has had many similar
problems to those experienced by clinical re-
searchers in periodontics. Both diseases progress
slowly, and the usual measures, such as radiographs,
are fraught with error. Thus, only large changes
could be detected with assurance. It is not surprising
with such measures the original denition of osteo-
porosis was made clinically on the basis of fracture.
Today, early diagnosis of both osteoporosis and
periodontal disease is desirable so that the clinician
can intervene before signicant morbidity such as
fracture, tooth mobility or tooth loss has occurred.
These methods are summarized in Table 2 (1, 50).
Absorptiometry (1, 50) utilizes a gamma source to
measure bone mass in grams (approximate ash
weight) per cm along the axis of the bone. Single
photon absorptiometry requires a means to assure
that the soft tissue equivalent material overlying the
bone is of constant thickness. The original technique
involved submerging the forearm or leg in a water
bath limiting its use to peripheral sites, such as the
distal radius.
The major technology used today to measure
bone mineral density is dual energy X-ray absorption
(1, 50). Dual energy X-ray absorption uses an X-ray
source for measurements of bone mass. This tech-
nology was introduced by Hologic Inc., a major
manufacturer of this equipment in 1987. Dual en-
ergy X-ray absorption measures bone density as
areal density in units of grams/cm
2
(51). Dual en-
ergy X-ray absorption may be used to measure bone
mineral density in central sites such as the spine or
hip or peripheral sites, such as the radius. Fig. 5
Table 2. Clinical measurement methods to assess bone mineral density
Method Sites Units
Single photon absorptiometry Peripheral due to need for water bath Bone mineral content
g/cm
g/cm
2
Dual energy X-ray absorption Central or peripheral Bone mineral density
Areal density
g/cm
2
Bone mineral content
in grams refers
to scanned area
Bone mineral apparent density
g per approximation of volume
Quantitative computed tomography Central or peripheral Apparent bone density
mg/cm
3
96
shows a representative bone mineral scan of the hip.
At rst glance the quality of the image appears to
be poor. In fact, these images are not intended for
diagnosis, but simply to indicate the location of the
regions of interest selected by the operator for the
assessment of bone mineral density. A typical report
may also list demographic and identifying infor-
mation, as well as the computed information con-
cerning each area of interest. Such information in-
cludes area of the region of interest (in cm
2
), bone
mineral content (in grams), and bone mineral den-
sity (in grams per cm
2
).
In order to facilitate interpretation of the examina-
tion, results are often shown as illustrated in Fig. 5
(upper panel). The normal range for women (usually
aged 2080 years) is shown, and is the actual meas-
ured bone mineral density (marked by a cross). Two
standard deviations below the fracture threshold the
mean maximal bone mass is usually shown as the
fracture threshold, although this may be an over-
simplication of the relationship between bone min-
eral density and fracture. The Z score is often used
to further interpret the results. Z represents differ-
ence of the measured bone mineral density from the
appropriate normal population in units of standard
deviations. The Z score is related to a race, age, and
sex matched population. Thus a patient who has the
mean bone mineral density will have a Z score of
zero, and an osteoporotic patient will have a Z score
at or below 2.5. The T score also represents the
difference (expressed in terms of standard deviation)
from the peak bone mass for the population.
Quantitative computed tomography (1, 50) is a
relatively new method that permits direct measure-
ment of either trabecular or total bone density by
further analysis of the information obtained from
special computed tomography protocols. Quantitat-
Fig. 5. Print-out from a dual energy X-ray absorption ex-
amination. The lower panel shows the region of interest
in the hip selected for study. The upper panel shows the
age-matched and population-matched normal values by
age. The cross hair represents the individual patients
bone mineral density and the age of the patient.
ive computed tomography provides measures of
bone apparent density in units of grams/cm
3
.
Radiographs also can provide the basis of meas-
urements such as the thickness of cortical bone,
radiographic densitometry or the use of indices de-
veloped for research protocols. The errors inherent
to non-standardized radiographs have limited the
usefulness of these older methods for the sensitive
measurement of osteoporosis (1, 50).
The mathematics of fractals has also been applied
to the detection of osteoporosis. The fractal dimen-
sion describes the similarity of an object such as tra-
becular bones surface roughness, over a range of
magnication. The underlying concept is that osteo-
porotic bone has a different surface roughness than
normal bone (as can be seen in Fig. 1 and 2). Fractal
dimensions have been used in research, and differ-
ences in fractal dimension have been reported for
97
subjects exhibiting high and low bone mineral den-
sity (4, 5, 50).
A review of biochemical markers of bone forma-
tion and resorption (9, 10) is beyond the scope of
this chapter. Markers of formation in serum include
osteocalcin (bone-gla-protein), alkaline phosphatase
and procollagen I carboxy-terminal extension pep-
tide. Markers of bone resorption are found in plasma
and urine. Plasma markers include tartrate-resistant
acid phosphatase, pyridinoline and possibly pyridin-
oline containing peptides. Urinary markers of re-
sorption include urinary pyridinoline and deoxypyri-
dinoline (collagen cross-link) containing peptides,
fasting urinary calcium and hydroxyproline and uri-
nary hydroxylysine glycosides. Interpretation of
these markers as diagnostic tests for active osteopor-
osis is an emerging eld and remains controversial.
Ongoing studies are addressing their usefulness in
detecting progressive periodontitis (Reddy, M.S.,
personal communication).
For assessment of intraoral sites, research tools
have been and are being utilized. Both absorptiome-
try and dual energy X-ray absorption have been
adapted for intraoral use (14, 16). Most studies have,
however, used radiographs to assess anatomy and
bone density. Methods using both panoramic and
intraoral, periapical or bitewing lms have been de-
scribed (14, 15, 25, 4245). Measures of cortical
thickness, other image features, and indices de-
signed for specic studies have been reported.
Relationship between systemic and
mandibular bone density
It has long been postulated that mandibular bone
density may be indicative of systemic bone mineral
density. In a classic series of studies, Kribbs et al.
addressed this relationship in both normal and oste-
oporotic women. In an early study (30), total body
calcium as assessed by neutron activation analysis
was found to be associated with mandibular density
as measured by quantitative analysis of intraoral
radiographs. A later study (29) in normal, non-osteo-
porotic women revealed that bone mass was not
affected by age but was signicantly associated with
skeletal bone mass at the spine and wrist. A com-
parison of 85 osteoporotic women with 27 normal
women showed less mandibular bone mass and
density and a thinner cortex at the gonion in osteo-
porotic compared with non-osteoporotic women
(27, 28). Similarly, von Wowern et al. reported that
12 osteoporotic subjects with a history of fractures
Jeffcoat et al.
had less mandibular bone mineral content as meas-
ured by dual photon absorptiometry than 14 normal
women (48). It is noteworthy that both Kribbs et al.
and von Wowern et al. described cross-sectional
studies. More recent studies in postmenopausal
women have indicated that a relationship may exist
between osteoporosis and recession (35).
70 post-menopausal women with clinical evi-
dence of periodontitis have been studied by Wactaw-
ski-Wende et al. (49) in order to test the hypothesis
that systemic bone mineral density is related to peri-
odontitis. Positive and signicant correlations were
seen between alveolar bone loss and bone mineral
density at the spine, trochanter, Wards triangle and
total femur (49).
Preliminary data from the
Womens Health Initiative Oral
Ancillary Study
The Womens Health Initiative is an unprecedented
study in the United States of womens health after
menopause. Specic risk factors for diseases includ-
ing heart disease and osteoporosis are being ad-
dressed nationwide. Building on a unique oppor-
tunity for collaboration with the Womens Health
Initiative at the University of Alabama at Birming-
ham, this oral ancillary study was established to de-
termine if an association between systemic osteo-
porosis and oral bone loss exists. In this report, pre-
liminary cross-sectional data on the rst 158
subjects enrolled in the oral ancillary study at the
University of Alabama at Birmingham are presented.
One goal was to determine whether or not image
analysis of intraoral radiographs could be used to
determine if basal bone mineral density of the man-
dible is correlated with hip bone mineral density de-
termined by dual energy X-ray absorption. All sub-
jects enrolled in the study were post-menopausal fe-
males, with a hip bone mineral density conrmed by
dual energy X-ray absorption to be within or below
one standard deviation of young normal subjects.
Subjects with a hip bone mineral density conrmed
by dual energy X-ray absorption one standard devi-
ation or more above that of young normal subjects
were excluded.
Comprehensive medical histories and examina-
tions were linked with results of oral examinations
and quantitative digital intraoral radiography. The
intraoral radiographic techniques used in this study
have been validated and are over 90% sensitive and
98
specic in detecting small changes in bone mass and
density (15, 21, 22). In brief, radiographs are digitized
and corrected for contrast and angulation errors,
and areal bone density is calculated relative to a ref-
erence wedge in the lm holder. The reference
wedge is used so that each patients radiographic im-
age can be displayed with the same brightness and
contrast and that the bone mineral density relative
to the wedge may be determined. A region of interest
in the area of the rst mandibular basal bone was
selected for measurements of basal bone mineral
density. Areal densities for the basal bone density
were calculated in the similar units as dual energy
X-ray absorption measurements of systemic bone
mineral density (gm/cm
2
).
This population had a mean age of 62.27.6 years;
66% of subjects reported taking hormone replace-
ment therapy, and 57.1% of subjects classied them-
selves as Caucasian, 42.5% were African-American,
and 0.4% were American Indian.
General linear models of basal bone mineral den-
sity (of the mandible), hip bone mineral density,
mid-root density, age, race, hormone replacement
therapy and calcium supplements were created. The
result of the generalized linear model for these rst
158 subjects are shown in Table 3. Fig. 6 plots the hip
bone mineral density as measured by dual energy X-
ray absorption versus the basal bone mineral density
measured from the intraoral radiographs. Signicant
correlations were found between mandibular basal
bone mineral density and hip bone mineral density
(r0.74, P0.001). It is important to note that these
data represent a subset of the subjects to be evalu-
ated in this study. Further multivariate analyses will
be performed after the baseline data set is locked.
These preliminary results may lead the clinician
to pose an additional question. That is, can high-
quality intraoral radiographs be used as the basis of
a screening test for osteopenia? Since radiographs
are often taken as part of the periodontal examina-
tion, such analyses would pose no additional risk to
the patient. These analyses would not be used as a
diagnostic, but rather to refer patients for appropri-
ate evaluation and treatment as necessary.
Common strategies for
treatment of osteoporosis and
periodontal disease
Avoidance of the morbidity of osteoporosis begins
with prevention. Adequate calcium intake during
Table 3. General linear model relating mandibular basal bone mineral density to covariate variables
a
Coefcient Standard Test Condence
Variable estimation error statistic P interval
Hip bone mineral density 5.068 0.985 5.145 0.0001 1.1227.014
Bone mineral density at the mid-root of 0.401 0.051 7.922 0.0001 0.3010.501
rst molar of the mandible
Age 0.022 0.017 1.286 0.201 0.0550.012
Race 0.099 0.302 0.327 0.744 0.4970.695
History of hormone replacement therapy 0.005 0.307 0.015 0.988 0.6110.602
Use of calcium supplements 0.120 0.264 0.454 0.651 0.6420.402
Smoking 0.533 0.418 1.276 0.204 1.3580.292
a
Basal bone measurements taken at the mandibular rst molar.
Race, history of hormone replacement therapy, calcium supplement use and smoking status are indicator variables.
adolescence and early adulthood is critical to form-
ing the peak bone mass. The 1994 US National Insti-
tutes of Health Consensus Development Conference
recommended 1000 mg of calcium per day for pre-
menopausal women and 1500 mg per day for post-
menopausal women. In order to maximize the likeli-
hood that bone mass is maintained over a lifetime,
load-bearing exercise is necessary. Like periodontal
disease, smoking is a major risk factor for osteopor-
osis and avoidance of smoking or smoking cessation
contributes to osseous health.
Bone loss in women occurs most rapidly in the
years immediately following menopause when
natural levels of estrogen are greatly reduced. Hor-
mone replacement therapy is designed to replace
estrogen after menopause since this immediate
post-menopausal period is a time of rapid loss of
bone mineral density (6, 11, 12, 31, 36). For
Fig. 6. Hip versus basal bone mineral density (BMD). The
scatterplot demonstrates that hip bone mineral density
and basal bone mineral density are correlated (r0.74,
P0.01).
99
women with a uterus, a combination of estrogen
and progesterone are used; while in women with-
out a uterus estrogen replacement therapy alone is
utilized. Many studies have reported that hormone
replacement therapy and estrogen replacement
therapy is efcacious in sparing bone mineral and
reducing fractures (11, 12, 31).
Therapy for osteoporosis is a rapidly changing
eld. The use of sodium uoride, as well as vit-
amin D metabolites to correct malabsorption of
calcium (1, 8), has been shown to be of some
value in established osteoporosis. Recent advances
have, however, added to the armamentarium for
the treatment of osteoporosis. Calcitonin, which
may be administered by injection or nasal spray,
inhibits osteoclastic activity and over time de-
creases bone turnover (7).
The latest generation of bisphosphonate drugs,
such as alendronate, chemisorb onto bone decreas-
ing osteoclast number and activity, thereby decreas-
ing bone resorption. Alendronate has been shown to
inhibit loss of bone density and decrease the risk of
fracture without disturbance of bone healing ob-
served in earlier drugs (40).
Few studies have directly assessed the relationship
between periodontal disease and its sequelae in
women receiving hormone replacement therapy.
Most of these studies have involved hormone re-
placement therapy, either estrogen or estrogen plus
progesterone, and assessed tooth loss, alveolar bone
loss, or other measures of periodontal health. In a
longitudinal, unblinded study of 69 women receiving
hormone replacement therapy, Jacobs et al. com-
pared lumbar spine bone mineral density, measured
by dual photon absorptiometry, with mandibular
bone mass assessed by quantitative measures of
standardized intraoral radiographs (19). The average
length of study was 5.1 years. A signicant but mod-
Jeffcoat et al.
erate correlation was observed at the second exami-
nation. In contrast, in a cross-sectional study of 228
women, Nordeyd reported no difference in clinical
attachment level or alveolar bone loss (38). Estrogen
replacement therapy was associated with less gingi-
val bleeding after correcting for age.
Two major cohorts of women have been studied
in an attempt to determine if hormone replacement
therapy has reduced the number of lost teeth in
post-menopausal women. Both studies were longi-
tudinal. These include the three year study of 42,171
postmenopausal women in the Nurses Health Co-
hort (13), and the 10-year study of 3,921 women liv-
ing in a retirement community in the Leisure World
Cohort (39). The Leisure World Cohort taking estro-
gen experienced a 36% reduction in tooth loss, and
the Nurses Health Cohort showed an inverse re-
lationship between hormone replacement therapy
and loss of teeth after correcting for smoking and
age. One potential source of bias in these important
studies (and, in fact, addressed in the reports) is the
fact that the same patients who seek to prevent os-
teoporosis may seek preventive dental care as well.
Both of these populations are large, but composed
of relatively well educated, higher socioeconomic
groups. Ongoing longitudinal studies are collecting
the data to address such potential sources of bias
and performing examinations so that assessments of
tooth loss, loss of bone height and bone density can
be made.
The effect of calcium supplementation on tooth
loss has also been assessed, and there is limited evi-
dence that calcium supplementation may be bene-
cial. In a 7-year study of 189 postmenopausal
women not taking hormone replacement therapy,
the subjects were assigned to receive either placebo,
calcium supplementation, or vitamin D plus calcium
supplementation (26). Twelve percent of the pla-
cebo-treated women lost teeth during the study
period compared with 3% of the calcium supple-
mentation group. No effect was observed with vit-
amin D.
In a pilot clinical trial the efcacy of the bisphos-
phonate drug alendronate in slowing alveolar bone
loss due to periodontitis has been investigated (21).
This double-blind placebo-controlled randomized
clinical trial measured loss of bone height and den-
sity using digital subtraction radiography over a 9-
month period. Alendronate reduced the risk of pro-
gressive loss of alveolar bone. The relative risk of
progressive loss of bone height and density was 0.45
for the alendronate-treated patients compared with
placebo-treated patients.
100
Conclusions
While a possible relationship between osteoporosis
and oral bone loss has long been postulated, the
existing studies have been preliminary in nature.
Most studies have used a small number of subjects,
and have been cross-sectional in design. These
studies utilized different outcomes and many did not
have the power or diagnostic techniques to ad-
equately address the questions at hand. The 1992 US
National Institutes of Health Workshop on Osteopor-
osis and Oral Bone Loss recommended population-
based prospective studies of the association between
oral bone loss and systemic bone with particular em-
phasis on cohorts of postmenopausal women both
with and without hormone replacement (32). Longi-
tudinal studies will make it possible to determine if
the progression of periodontal disease is more rapid
in patients with osteopenia than in patients with
normal bone density, as it is impossible to determine
if such a relationship exists from cross-sectional
studies alone. Several centers are currently perform-
ing studies to better elucidate the inter-relationship
between oral and systemic bone loss.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Acute-phase reactants in
infections and inammatory
diseases
JEFFREY L. EBERSOLE & DAVID CAPPELLI
General overviewof the acute-phase
reaction
The acute-phase reaction represents an early and
highly complex reaction of the organism to a variety
of injuries such as bacterial, viral or parasitic infec-
tion, mechanical or thermal trauma, ischaemic ne-
crosis, or malignant growth (78). The acute phase re-
fers to physiological and metabolic alterations that
ensue immediately after onset of infection or tissue
injury. A variety of changes in the organism act in
concert to neutralize the inammatory agent and
foster healing of damaged tissues. In contrast with
the specicity of cellular and humoral immunity, the
acute-phase changes are nonspecic and occur in
response to many conditions (65). The purpose of
these responses is to restore homeostasis and to re-
move the cause of its disturbance. Characteristic fea-
tures of the systemic acute-phase response include
(i) fever, (ii) neutrophilia, (iii) changes in lipid met-
abolism, (iv) hypoferremia, (v) increased gluconeog-
enesis, (vi) increased (muscle) protein catabolism
and transfer of amino acids from muscle to liver, (vii)
activation of the complement and coagulation path-
ways, (viii) hormonal changes, and (ix) induction of
acute-phase proteins (108). The tissue macrophage
is the cell most commonly regarded as initiating the
acute-phase response through direct stimulation
and secretion of various cell communication factors
(78) (Fig. 1).
An additional acute-phase response is an increase
in the polymorphonuclear leukocyte and platelet
count in the blood. This initially reects release from
the storage pool and later reects increased produc-
tion by the bone marrow; accompanying a decrease
in red blood cells contributing to decreased iron
availability to pathogens (Fig. 2). A prominent aspect
of the acute-phase response is also the modication
19
of the vasculature with dilation and leakage of blood
vessels, particularly at the post-capillary venule. This
results in tissue edema, red blood cells extravasation
and associated redness. These alterations are likely
mediated through the release of various inamma-
tory mediators in the inamed tissues, including re-
active oxygen species, nitrous oxide and arachidon-
Fig. 1. Schematic of the cascade of stimulation and re-
sponse activities during the acute-phase response. Source:
adapted from Koj (78). TNFa: tumor necrosis factor a.
IFNg: interferon g.
Ebersole & Cappelli
Fig. 2. Acute-phase response effects on various aspects of
the hematological system. CFU-E, G, GM and Meg denote
colony-forming units for erythrocytes, granulocytes, gran-
ulocytes/monocytes, and megakaryocytes, respectively;
EPO denotes erythropoietin; G- and GM-CSF denote col-
ony-stimulating factors for granulocytes and granulocyte/
monocytes, respectively. Source: adapted from Trey &
Cushner (145).
Fig. 3. Modulation of the hepatic acute-phase reactant
(APR) synthesis by various inammatory mediators. IL-1,
tumor necrosis factor a (TNFa), leukemia inhibitory fac-
tor (LIF), interferon g (IFNg), oncostatin M (OSM), ciliary
neurotrophic factor (CNTF) and transforming growth fac-
tor b (TGFb) represent cytokine stimulators of acute-
phase responses by the liver. Source: adapted from Steel &
Whitehead (136).
20
ate metabolites (Table 1) (6). The results include ac-
tivation of macrophages, aggregation of platelets,
vessel permeability, transudation of biological uids
into the tissues and migration of circulating leuko-
cytes.
The early response to infection or tissue injury is
crucial to the integrity of host defense as well as to
regulation of the ensuing inammatory responses.
The acute-phase response is a primary defense reac-
tion and therefore protects against bacterial prod-
ucts such as endotoxin (78). The systemic acute-
phase response helps to ensure survival during the
period immediately following injury. This systemic
response helps to accomplish the same outcomes as
the localized inammatory response, which is de-
signed to contain or destroy infections or noxious
agents, to remove damaged tissue and to repair the
affected tissue or organ. Acute-phase responses are
thus elicited following infections, surgical wounds or
other traumas such as burns and myocardial infarc-
tions (Fig. 1). The body also responds uniquely by
increasing the hepatic synthesis and the rapid induc-
tion of intravascular secretion of a number of plasma
proteins with a wide diversity of actions. Evidence
indicates that increases in the plasma concentration
of these acute-phase proteins at the site of injury
play a major role in wound healing. The acute-phase
proteins have been found to have an essential role
in the inhibition of extracellular proteases, blood
clotting, brinolysis, modulation of immune cell
function and the neutralization and clearance of
harmful components from the circulation.
The synthesis of these acute-phase proteins has
been shown to be regulated by cytokines and to a
lesser extent by glucocorticoid hormones (Fig. 3). Ar-
bitrarily, cytokines related to the acute phase re-
sponse can be divided into three groups: (i) proin-
ammatory cytokines initiating or enhancing the
cascade of events (tumor necrosis factor a, interleu-
kin 1 (IL-1), interferon-g and IL-8); (ii) interleukin-6-
type cytokines (IL-6, leukemia inhibitory factor, IL-
11, oncostatin M, ciliary neurotrophic factor and
Table 1. Metabolites of arachidonate metabolism
in the acute-phase reaction
Mediator Function
Thromboxane A
2
Vasoconstriction
Prostaglandins I
2
, E
2
, D
2
and F
2a
Vasodilation
Leukotriene B
4
Phagocyte chemotaxis
Leukotrienes C
4
, D
4
and E
4
Smooth-muscle
contraction
Acute-phase reactants in infections and inammatory diseases
cardiotrophin-1), which are held responsible for the
main systemic features of acute-phase response in a
variety of tissues; and (iii) anti-inammatory cyto-
kines downregulating the acute-phase response (IL-
10, IL-4, IL-13 and transforming growth factor b)
(78). None of these individual cytokines induces the
full spectrum of acute-phase protein expression. The
majority of the acute-phase proteins are glyco-
proteins, which play a variety of roles in the homeos-
tatic response to injury. The acute-phase proteins in
humans differ substantially in the magnitude of their
rise after onset of injury. The serum concentration of
a number of these proteins increases rapidly during
infection and concentrations can increase 2- to 100-
fold and remain elevated throughout infection.
Acute phase proteins can be divided into two
groups: type I and type II. Type I proteins include
serum amyloid A, C-reactive protein, complement
C3 and a
1
-acid glycoprotein, which are induced by
the proinammatory, IL-1-like cytokines (IL-1 and
tumor necrosis factor). Type II acute-phase proteins
include brinogen, haptoglobin, a
1
-antichymotryp-
Table 2. Regulation of acute-phase reactant production
Effectors Response of acute-phase proteins
IL-1-type cytokines Stimulation of type I acute-phase proteins
C-reactive protein
a
1
-acid glycoprotein
Ceruloplasmin
Factor B
Serum amyloid A
Serum amyloid P protein
Haptoglobin (rat)
Hemopexin (rat)
Inhibition of type II acute-phase proteins
IL-6-type cytokines Stimulation of most acute-phase proteins
Cysteine proteinase inhibitor
a
2
-macroglobulin
Fibrinogen
a
1
-proteinase inhibitor
Haptoglobin (human)
a
1
-antichymotrypsin
Ceruloplasmin
C1 esterase inhibitor
a
1
-antitrypsin
Synergism with IL-1-type cytokines on type I acute-phase proteins
Glucocorticoids Minor stimulation of most acute-phase proteins,
strong stimulation of rat a
1
-acid glycoprotein
Strong synergistic enhancement of cytokine effects on most
acute-phase proteins
Insulin Reductions of IL-1- and IL-6-type cytokine effect on most
acute-phase proteins
Hepatocyte growth factor, broblast growth factor Minor reduction of IL-1- and IL-6-type cytokine effects on most
and transforming growth factor b acute-phase proteins
Enhancement of IL-1-type cytokine effects on rat a
1
-acid glycoprotein
and C3
Hormones acting via cyclic nucleotides or No detectable regulatory effect on acute-phase proteins
calcium mobilization
Source: adapted from Baumann & Gauldie (6).
21
sin, a
1
-antitrypsin, a
2
-macroglobulin and are in-
duced by the IL-6-like cytokines. The a-helical cyto-
kines, IL-6 and oncostatin M, are the most potent
recognized inducers of acute-phase proteins (150).
The effect of IL-6 on the production of hepatic pro-
teins can also be inuenced signicantly by other
cytokines and by insulin and the counterregulatory
hormones (dexamethasone, glucagon, and epine-
phrine) (Table 2). In general, IL-6-like cytokines sy-
nergize with IL-1-like cytokines to induce type I
acute-phase proteins (108). This phenomenon is
thought to be principally controlled by IL-6 acting
on the hepatocyte and inducing transcriptional acti-
vation of the acute-phase protein genes. As such,
type I acute-phase protein genes contain nuclear
factor-kB, nuclear factor-IL-6 and AP-1 response ele-
ments in their promoter regions (108), whereas type
II acute-phase protein genes contain a hexanucleo-
tide motif (CTGGGA), which is an IL-6 response ele-
ment.
The reciprocal interaction between IL-6 and func-
tionally related other inammatory cytokines, as
Ebersole & Cappelli
well as the hypothalamo-pituitary-adrenal axis, rep-
resents a separate facet of the complex web of regu-
latory neuroendocrino-immunological interactions
(Fig. 4). Glucocorticosteroids decrease the level of IL-
1, tumor necrosis factor, and IL-6 in the peripheral
blood via transcriptional and posttranscriptional
routes and prolong their impact on the target cells
through the elevation of the expression of their re-
ceptors (43). While endogenous pyrogen (IL-1) is
well known to cause fever, IL-6 can also induce fever
(25). Finally, there exist apparent feedback mechan-
isms involving both liver synthesized acute-phase
proteins and neuroendocrine factors from the cen-
tral nervous system, which contribute to regulation
of the acute-phase response to inammation.
Characteristics of acute-phase
cytokines in the acute-phase reaction
IL-1
IL-1 is a broadly reactive cytokine of innate immun-
ity produced by perturbations of the cell membrane
stimulating the macrophage. This is especially not-
Fig. 4. Schematic of various feedback loops within the
acute-phase response which operate to regulate inam-
mation. Cytokines from macrophages activated by in-
ammatory stimuli stimulate the brain to release cortico-
tropin (ACTH), resulting in actions on the adrenal glands
and gluocorticoid production. The glucocorticoids and IL-
6 can suppress both IL-1 and tumor necrosis factor (TNF)
and thus downregulate these proinammatory cytokines.
Additionally, cytokines from the activated macrophages
and other stimulated cells can induce acute-phase protein
(APP) synthesis by the liver. The acute-phase proteins and
IL-6 can induce IL-1Ra and stimulate shedding of soluble
tumor necrosis factor receptors, which both can regulate
the activity of IL-1 and tumor necrosis factor. Source:
adapted from Hogarth et al. (58). PBMCs: peripheral
blood mononuclear cells.
22
able following incorporation of antigenic material.
IL-1 has both local and systemic effects on cell met-
abolism, immune and inammatory reactions. The
IL-1 family contains three ligands (IL-1a, IL-1b, IL-
1Ra) and two receptors (type I and type II). IL1-a
and -b are powerful agonists, whereas IL-1Ra is a po-
tent naturally occurring antagonist of cell activation
by either IL-1a or IL-1b. IL-1a, IL-1b and IL-1Ra are
structurally related and bind with similar afnities to
IL-1 receptors on cells. IL-1a is biologically active
and appears to remain associated with the intact
cell. IL-1a is released when cell integrity is compro-
mised such as in necrosis, apoptosis or cell per-
meation. IL-1b is synthesized as an inactive precur-
sor protein that is processed to its active form upon
release from the cell by the specic proteinase IL-1-
converting enzyme. IL-1Ra can be synthesized as a
secreted protein that contains a classical hydro-
phobic leader sequence and is exported via the en-
doplasmic reticulum and Golgi apparatus, or two in-
tracellular forms that lack the secretory leaders
(141). The balance between agonists and antagonists
in the IL-1 system is likely to have profound effects
on the pathogenesis of inammatory diseases (141).
Separate genes encode IL-1a, IL-1b and IL-1Ra. All
three genes are clustered on the long arm of human
chromosome 2. The three IL-1 gene family members
are regulated transcriptionally by a variety of en-
vironmental signals, which include bacterial cell wall
components, cytokines, immune stimuli and in-
ammatory mediators. The IL-1 family members are
designated as immediate early-response genes (141).
Microbial products such as endotoxins and ex-
otoxins induce IL-1 production by monocytes. The
most common stimulus for secretion of IL-1 is endo-
toxin, particularly as related to lipopolysaccharide
interaction with lipopolysaccharide-binding protein
and CD14 for macrophage stimulation. Endogenous
agents that stimulate monocyte IL-1 production in-
clude complement component 5a (C5a), colony-
stimulating factors, tumor necrosis factor a, trans-
forming growth factor b and IL-1 itself. Cortico-
steroids, as anti-inammatory agents, and prosta-
glandins appear to inhibit the release of IL-1 from
macrophages. Keratinocytes and dermal broblasts
produce IL-1 (primarily IL-1a), often in response to
environmental stress (141). However, many ad-
ditional cell types have also been shown in vitro to
produce IL-1 family members following stimulation
(115).
Like all cytokine hormones, IL-1 acts as a switch
turning specic genes on or off, thereby enabling the
cell to respond appropriately to environmental stress
(141). IL-1 mediates its action on target cells via
high-afnity receptors on the plasma membrane.
Low numbers of IL-1RI are found on many cell types
and are the predominant type of IL-1R on T cells and
broblasts. IL-1RII is most common on neutrophils,
monocytes and B cells (29). The numbers of such
receptors vary from a few hundred on T cells to a
few thousand on broblasts. Expression of IL-1 re-
ceptors is downregulated by IL-1. In contrast, prosta-
glandins and glucocorticoids upregulate the express-
ion of functional IL-1 receptors on some cell types
such as broblasts and B lymphocytes. IL-1R genes
are members of the much larger immunoglobulin
supergene family. The IL-1R genes are located in the
same region of human chromosome 2 as their
ligands (141).
IL-1 is considered an important mediator of in-
ammation, based on its presence at inammatory
sites and its ability to induce many of the hallmarks
of the inammatory response (141). In vivo, IL-1 eli-
cits systemic inammatory reactions, such as fever
and the acute-phase response of the liver. IL-1 elicits
immediate local inammatory reactions that are
characterized by binding of blood neutrophils to the
vessel walls, which results from the induction of ex-
pression of surface structures on endothelial cells
(such as intracellular adhesion molecule 1), and thus
IL-1 is a potent stimulus for neutrophil accumu-
lation in vivo (29). Antigen-presenting cells, T cells,
pre-B cells, and B cells are directly or indirectly
stimulated by IL-1 to participate in innate and adap-
tive immune responses. In vitro IL-1 induces prosta-
glandin E
2
release from many cell types, production
of proteases, catabolism of cartilage and bone, and
growth of broblasts, all of which could contribute
to the pathogenesis of chronic inammation. IL-1 is
also an important macromolecule in the linkage of
the neuroendocrine and inammatory and immune
response systems. It stimulates the hypothalamic-
pituitary-adrenal axis to release bioactive neuropep-
tides into the hypothalamic portal and systemic cir-
culation. Increased levels of corticotrophin-releasing
factor, adrenocorticotropin-releasing hormone, en-
dorphins, vasopressin and somatostatin are detected
in response to IL-1. Products of the cyclooxygenase
pathway appear to mediate IL-1-induced cortico-
tropin release. Under stressful conditions, IL-1 ap-
pears to act directly on neurons. IL-1 is an extremely
potent pyrogen by signaling the hypothalamus
across the blood-brain barrier (141).
IL-1 regulates acute-phase protein synthesis by
hepatocyes (158). Induction of IL-6 by IL-1 partly ex-
plains its role in acute-phase protein production;
23
however, it has been established that IL-1 alone in-
creases the transcription of some acute-phase pro-
teins and decreases transcription of other hepatic
proteins. IL-1 induces serum amyloid A, brinogen,
factor B, metallothioneins, serum clotting factors,
complement components and IL-6. IL-1 with other
cytokines also induces liver production of type 2 ni-
tric oxide synthase. In contrast, IL-1 decreases albu-
min, cytochromes, transferrin and lipoprotein lipase
transcription (141).
Similar to acute-phase proteins, serum levels of
IL-1Ra can rise dramatically during different in-
ammatory and noninammatory conditions such
as sepsis, chronic rheumatic diseases, and nonin-
ammatory tissue injury. Additionally, both IL-1 and
IL-6 increase circulating levels of IL-1Ra, suggesting
that it is regulated similar to classic acute-phase pro-
teins. Thus, serum IL-1Ra is produced by hepato-
cytes and is regulated by proinammatory cytokines
as an acute-phase protein (46).
Tumor necrosis factor is considered a major in-
ammatory mediator. The family of tumor necrosis
factor has two ligands: tumor necrosis factor a,
which is produced by both mononuclear phago-
cytes and certain populations of activated T cells,
and tumor necrosis factor b, which is a product of
cytotoxic T cells. The tumor necrosis factor gene
family also includes the related cytokines,
lymphotoxin-a and lymphotoxin-b. While they
share some overlapping biological activities, they
have distinct receptors and differential regulation
and gene transcription and translation (143). The
other family members include ligands for CD27,
CD30, CD40, OX40, 4-1BB and Fas. There are also
two surface receptors, p55 and p75, both of which
bind tumor necrosis factor a and tumor necrosis
factor b. Both p55 and p75 tumor necrosis factor
receptors can be shed from activated cells and
neutralize the biological activities of tumor ne-
crosis factor a (29). Mature tumor necrosis factor a
undergoes proteolytic cleavage from a membrane-
bound precursor, and recent evidence suggests
that the secreted monomer folds onto itself into
two b-pleated sheets. Three such sandwiches form
a bell-shaped trimer, such that biologically active
tumor necrosis factor a circulates as a homotri-
meric complex (115). Receptor activation presum-
ably occurs by the clustering of two or three recep-
tors around each tumor necrosis factor trimer
(143). Receptors (1000 to 10,000 per cell) with high
Ebersole & Cappelli
afnity for tumor necrosis factor are present on
membranes of target cells that can respond to tu-
mor necrosis factor.
Cells of the macrophage and monocyte lineage are
the predominate source of tumor necrosis factor in
vivo. Although tumor necrosis factor production has
been studied most extensively in monocytes and
macrophages, it is clear that the local production of
tumor necrosis factor by nonhematopoietic cells can
inuence the pathogenesis of a particular inam-
matory state or disease. Tumor necrosis factor is pro-
duced by cardiac myocytes, where its local effects
can profoundly alter systemic physiology through al-
tered myocardial cell function and its local paracrine
activity (143). A variety of cytokines, including IL-1,
induce tumor necrosis factor production by macro-
phages. Local cellular effects of tumor necrosis fac-
tor include the capacity to induce polymorpho-
nuclear leukocytes to bind to vascular endothelium,
activate phagocytosis, elaborate superoxide bursts
and promote degranulation (115). The tissue-injur-
ing effects of tumor necrosis factor are now impli-
cated in a spectrum of pathogenic inammatory
states, including cachexia, autoimmune disease, re-
jection of organ transplants and infections (143).
Tissue alterations induced by tumor necrosis factor
include: (i) an IL-1-like bone-resorptive activity, (ii)
induced collagenase effects on cartilage and en-
hance resorption of proteoglycan with inhibition of
proteoglycan synthesis and (iii) induction of prolifer-
ation of broblasts for wound repair or with poten-
tial pathological effects as noted in brosis induced
by chronic inammation.
Most unstimulated cells contain low levels of tu-
mor necrosis factor a messenger RNA and no detect-
able levels of protein; however, multitudes of endog-
enous and exogenous factors are now known to be
capable of stimulating tumor necrosis factor a secre-
tion (115). Tumor necrosis factor a is made in re-
sponse to a variety of bacteria and bacterial prod-
ucts, especially lipopolysaccharide. A cytokine net-
work controls tumor necrosis factor induction and
action. Tumor necrosis factor, in turn, induces IL-
1 and prostaglandin E
2
production by macrophages,
interferon-g, and interferon-b production by bro-
blasts, and granulocyte-macrophage colony-stimu-
lating factor production by several cell types. Both
tumor necrosis factor a and IL-1 induce intracellular
adhesion molecule 1, a cell adhesion molecule in-
volved in binding and subsequent activation of T
cells by antigen-presenting cells. The effects of tu-
mor necrosis factor a are enhanced in the presence
of IL-1 or interferon-g (29). Although a variety of me-
24
diators are released in response to sepsis, evidence
suggests that tumor necrosis factor a may be a key
player in the pathogenesis of septic shock. The in-
ammatory effects of tumor necrosis factor have
vaulted this cytokine to the forefront of shock and
trauma pathophysiology.
Tumor necrosis factor is an important inamma-
tory mediator and accounts for the activity of
cachectin, a lethally toxic cytokine mediated wasting
(cachexia) during infections or following lipopoly-
saccharide injection. An additional systemic in-
ammatory property of tumor necrosis factor a in-
cludes the induction of fever and several of the acute
phase reactants. Thus, while IL-1 is widely recog-
nized as an endogenous pyrogen, tumor necrosis
factor a has been implicated as both an endogenous
pyrogen and an endogenous cryogen or antipyretic
(89). Tumor necrosis factor has the direct ability to
stimulate hypothalamic prostaglandin E
2
synthesis,
thus stimulating fever. Tumor necrosis factor a also
appears to be capable of contributing to the induc-
tion of several of the acute-phase reactants. The ef-
fects of tumor necrosis factor on acute-phase induc-
tion in human hepatoma cell lines include increased
biosynthesis of complement proteins factor B and
C3, as well as a
1
-anti-chymotrypsin. Tumor necrosis
factor also decreases biosynthesis of albumin and
transferrin (115).
Tumor necrosis factor production occurs as part
of the conserved response to a wide range of cell
stressors, presumably because it participates as a
central mediator of the complex cytokine, hormonal
and cellular signaling cascade underlying the coordi-
nated response to invasive or threatening stimuli.
Peak tumor necrosis factor levels stimulate the pro-
duction of a cascade of other cytokines and hor-
mones, including the glucose counterregulatory hor-
mones epinephrine, glucagon and glucocorticoids,
and these hormones have been implicated in shut-
ting off tumor necrosis factor production (143). After
a rapid distribution phase, circulating tumor ne-
crosis factor is cleared by binding to specic recep-
tors in lungs, spleen, liver, skin, kidneys and other
organs. Very high tumor necrosis factor levels ap-
pearing in the bloodstream represent an unusual
event in nature, associated only with extreme threat
and or pathophysiological crisis (143).
High or low human tumor necrosis factor pro-
ducers have been identied in studies using periph-
eral blood mononuclear cells activated in response
to lipopolysaccharide. The tumor necrosis factor
gene contains a biallelic polymorphism within the 5
DNA region that has been implicated in controlling
transcription. The tumor necrosis factor 2 allele is
strongly associated with human leukocyte antigen
haplotypes correlated to relatively high rates of tu-
mor necrosis factor production. The polymorphism
in the tumor necrosis factor locus might therefore
contribute to susceptibility or severity of disease in
autoimunity, infection, inammation or malignancy
(143).
IL-6
IL-6 is a pleiotropic cytokine involved in the regula-
tion of immune responses, the acute-phase response
and hematopoiesis. Although initially thought to be
a proinammatory cytokine, recent ndings suggest
that IL-6 has many anti-inammatory and immuno-
suppressive activities. IL-6 is related to IL-1 and tu-
mor necrosis factor a in that all three of these cyto-
kines are coordinately released from activated
monocytes and furthermore one can induce produc-
tion of another. Several experimental models suggest
a protective role for IL-6 and specic acute-phase
proteins against inammation, since acute-phase
proteins exert specic functions that are part of a
complex mechanism to control homeostasis, includ-
ing the inhibition of serine proteinases, the binding
of hemoglobin by haptoglobin and the clotting reac-
tion by brinogen and other factors (58). All three
cytokines (IL-1, IL-6 and tumor necrosis factor) can
also be carried via the blood to distant sites, in-
ducing an acute-phase reaction.
IL-6 is one of a group of cytokines that are pre-
dicted to share a common tertiary folding structure
(25), including IL-11, leukemia inhibitory factor, on-
costatin M, ciliary neurotrophic factor, and cardi-
otrophin-1. In several inammatory conditions and
during an acute-phase response induced by endo-
toxin administration or septic shock, levels of leuke-
mia inhibitory factor are increased in plasma and in-
ammatory body uids (108) and induce type II
acute-phase proteins. Oncostatin M is produced by
activated T cells and macrophages and has the ca-
pacity to increase hepatocyte low density lipopro-
tein-receptor expression, stimulates plasminogen
activator activity in endothelial cells and stimulates
hepatocytes to produce type II acute-phase proteins
(108, 124). IL-11 is produced by bone marrow
stromal cells and is involved in hematopoiesis and
B-cell development, as well as inducing acute-phase
proteins (47, 108). Non-neuronal cells of the central
and peripheral nervous tissue normally express cil-
iary neurotrophic factor. This cytokine may induce
acute-phase proteins in extrahepatic cells, or act loc-
25
ally in the liver (108). Cardiotrophin-1 can induce
hypertrophy in cardiac myocytes and has several ac-
tions in common with the other members of this
family, including its actions being mediated by the
leukemia inhibitor factor receptor.
Among the various cytokines, IL-6 is involved as
an autocrine, paracrine and exocrine inammatory
hormone (25). The regulation of IL-6 gene express-
ion occurs through at least three different pathways
of signal transduction, including cyclic adenosine
monophosphate, calcium activation and diacylgly-
cerol (25). A wide range of mediators, including a
number of cytokines such as IL-1 and tumor ne-
crosis factor a and growth factors, lipopolysacchar-
ide, other bacterial products and neuropeptides
stimulates expression of the IL-6 gene. Most, if not
all, nucleated cells have been shown to express and
synthesize IL-6 in vitro. The most prominent source
appears to be stimulated monocyte/macrophages,
and cytokine stimulated stromal cells (such as
broblasts, epithelial cells and endothelial cells) (25).
Furthermore, IL-6 is clearly an interleukin that me-
diates communication between a large number of
cell types by playing a role in the proliferation and
differentiation of B lymphocytes, plasmocytomas
and hybridomas, hematopoietic progenitors,
hepatocytes and T lymphocytes. Other activities
overlap those of IL-1 and tumor necrosis factor, and
thus, IL-6 is considered a major immune and in-
ammatory mediator (Fig. 5). While IL-6 is the pre-
dominant mediator of the local acute-phase re-
Fig. 5. Schematic of the interrelationships of IL-1, IL-6 and
tumor necrosis factor (TNF) in inammatory responses,
acute-phase reactions, and linkages of the neuroendocri-
ne, immune and inammatory systems. Source: adapted
from ONeill (115). NK: natural killer cell. CRF: cortico-
tropin-releasing factor. ACTH: corticotropin. CSF: colony-
stimulating factor.
Ebersole & Cappelli
sponse, in the systemic acute-phase response, at
least with regard to weight loss, hypoglycemia and
induction of acute-phase proteins, IL-6 may not be
essential (79, 108). Nevertheless, plasma levels of IL-
6 rise rapidly in response to bacterial infection and
sepsis and can remain elevated depending on the
severity and duration of the infection (29). Trauma
or infection causes plasma levels of IL-6 to rise. As an
early alarm cytokine (with IL-1 and tumor necrosis
factor), they recruit adjacent stromal cells and endo-
thelial cells to release high levels of IL-6 and other
mediators. The sequential activation and cytokine
cascade leads to raised IL-6 levels in inammation.
Since IL-6 can induce cortisol levels and cortisol is
required for the hepatic acute-phase response, IL-6
plays an inductive role as a second signal to the liver
(25). Biological activities of IL-6 have been closely
associated with that of tumor necrosis factor and IL-
1 (Table 3). IL-6 has been shown to suppress endo-
toxin and IL-1 production induced by tumor ne-
crosis factor. IL-6 exerts a broad spectrum of effects
on various target cells (115). IL-6 stimulates cortico-
tropin release via the activity of corticotrophin-re-
leasing hormone and subsequently corticosteroid
production and thus may indirectly lead to neutro-
philia via corticosteroid-induced demargination and
increased survival of neutrophils (25).
The IL-6 receptor is a member of the hematopoie-
tin receptor family. The binding peptide of the recep-
tor is a member of the immunoglobulin supergene
family, is specic for IL-6, and is present on mono-
cytes, hepatocytes, and T and B cells at approximate-
ly 1700 sites/cell (25).
A potential consequence of excess locally pro-
duced IL-6 is the generation of amyloid proteins,
similar to those present in the cerebral cortex of
Alzheimers disease. These are thought to derive
Table 3. Key properties of cytokines linked to the acute-phase reaction
Mediator Activity
IL-1-type cytokines (IL-1a, IL-1b, tumor necrosis Prototype proinammatory cytokines; stimulation of acute-phase
factor a) protein synthesis
IL-1Ra Member of the IL-1 family; blocks binding of IL-1 to cell surface
receptors; prototype anti-inammatory cytokine
Soluble tumor necrosis factor receptor p55/p75 Naturally occurring tumor necrosis factor inhibitors; comprise
extracellular domains fo the two known tumor necrosis factor
receptors, p55 and p75; block tumor necrosis factorregulated
inammatory processes
gp130 signaling cytokines (IL-6, IL-11, Pro- and anti-inammatory activities, stimulation of most acute-phase
leukemia inhibitor factor, oncostatin M, protein; induction of IL-1ra and soluble tumor necrosis factor receptor
ciliary neurotrophic factor, cardiotrophin) p55 in vivo; several anti-inammatory effects in vivo and in vitro;
evidence for anti-inammatory activities in IL-6-knockout models
Source: adapted from Hogarth et al. (58).
26
from a
1
-antichymotrypsin and amyloid protein pre-
cursor. Elevated levels of IL-6 have been detected in
the body uids of patients with a variety of systemic
autoimmune diseases (such as rheumatoid arthritis
and system lupus erythematosus). In ankylosing
spondylitis, a close correlation has also been found
between serum IL-6 levels and disease activity and
severity. In progressive systemic sclerosis, complexes
of IL-6 antibody with IL-6 that retain activity have
been found in the serum. IL-6 is also involved in
regulating various elements of the endocrine system.
Towards the end of pregnancy, detection of in-
creased levels of IL-6 in cervical secretions has been
shown to correlate with increased incidence of pre-
term labor. The detection of high levels of IL-6 in the
amniotic uid may be a strong indicator of intra-
uterine infection. IL-6 may also play a role in islet
function for insulin production and thus be related
to the development of diabetes. In the systemic im-
mune response syndrome that is frequently triggered
by sepsis and also involves the lung through the de-
velopment of adult respiratory distress syndrome,
high levels of circulating IL-6 have been found dur-
ing the prodrome phase of the illness (25).
IL-8
IL-8 is a proinammatory cytokine whose principal
role in infection and inammation appears to be the
recruitment and activation of circulating and tissue
neutrophils to the site of tissue damage. IL-8 is pro-
duced by a wide variety of cell types including vascu-
lar and lung endothelium, monocytes, eosinophils,
kidney mesangial cells, astrocytes, broblasts and
keratinocytes. Both IL-1b and tumor necrosis factor
a induce transcriptional activation of the IL-8 gene
and synthesis of IL-8 protein. IL-8 has recently been
evaluated for its ability to induce acute-phase pro-
tein production. IL-8 treatment of hepatocytes re-
sulted in a dose-dependent increase in C-reactive
protein, a
1
-acid glycoprotein, and a
1
-antichymo-
trypsin production and a decrease in the production
of both transferrin and albumin. Thus, IL-8 may
have the capacity to modulate the acute-phase re-
sponse (159).
Characteristics of the acute-phase
reaction response molecules
Acute-phase proteins serve important functions in
restoring homeostasis after infection or inam-
mation. These include hemostatic functions (such as
brinogen), microbicidal and phagocytic functions
(such as complement components or C-reactive pro-
tein), antithrombotic properties (such as a
1
-acid gly-
coprotein) and antiproteolytic properties which are
important to contain protease activity at sites of in-
ammation (such as a
2
-macroglobulin, a
1
-antitryp-
sin, a
1
-antichymotrypsin) (108). Although most
acute-phase reactants are synthesized by hepato-
cytes, some are synthesized by other cell types, in-
cluding monocytes, endothelial cells, broblasts and
adipocytes (136). The strong acute-phase proteins
include C-reactive protein, a
2
-macroglobulin, and
serum amyloid A, which responds rapidly to in-
ammatory stimuli and serum levels may increase
several hundred-fold (64, 82, 123, 151). Moderate
acute-phase proteins include haptoglobin, brino-
gen, a
1
-antitrypsin which can increase 2 to 10 fold
(17), while complement component C3 and cerulo-
plasmin are considered weak acute-phase proteins
that may increase up to 2 fold (123) (Table 4).
C-reactive protein
C-reactive protein, when bound to bacteria, pro-
motes the binding of complement, which facilitates
their uptake by phagocytes. This process of protein
coating to enhance phagocytosis is similar to opson-
ization by antibodies. The discovery of C-reactive
protein focused attention on the acute-phase re-
sponse and the role of these proteins during an in-
jury to the body. C-reactive protein was recognized
because of its ability to precipitate with the C-poly-
saccharide extract of Streptococcus pneumoniae. C-
reactive protein did not appear to be a typical anti-
body to pneumococcus for several reasons: (i) C-re-
active protein is present in the sera from patients
27
with other bacterial illness; (ii) C-reactive protein is
not detectable in the sera from normal individuals;
and (iii) the concentration of C-reactive protein
rapidly decreases in patients who recovered from
pneumonia, whereas typical antibody responses are
generally elevated in convalescence. It has since
been shown that C-reactive protein is normally pres-
ent in ng/ml quantities but may increase dramati-
cally to hundreds of mg/ml within 72 h following
tissue injury. This represents a 100- to 1000-fold in-
crease within hours of tissue damage. Similar to anti-
body, C-reactive protein reacts with some specicity
for its substrate to cause lattice formation and pre-
cipitation, causes passive hemagglutination of
erythrocytes coated with substrate, and can elicit
capsular swelling of pneumococcal microorganisms.
The most striking similarities between C-reactive
protein and antibodies are represented by the acti-
vation of the complement cascade through C1 acti-
vation by complexed C-reactive protein analogous to
antibody-antigen complexes, and the capacity of C-
reactive protein to opsonize erythrocytes coated
with C-polysaccharide for enhanced phagocytosis.
While functional similarities between C-reactive pro-
tein and antibody exist, C-reactive protein is pro-
duced by liver hepatocytes while antibody is syn-
thesized by lymphoid tissue plasma cells. Further-
more, there are minimal physical or biochemical
similarties between C-reactive protein and antibody.
Thus, C-reactive protein may be considered a primi-
tive form of antibody specically interacting with
cell membrane components of microorganisms such
as bacteria and fungi as well as for damaged mam-
malian cell membranes. When complexed, C-reac-
tive protein can activate complement to enhance
opsonization and clearance of the bacteria prior to
the production of specic IgM or IgG. C-reactive
protein bound to bacteria or cells can interact with
natural killer cells and with monocytes and may in-
crease the tumoricidal activity of these cells.
Serum C-reactive protein concentrations closely
follow the course of the acute-phase response to in-
ammation or tissue necrosis; measurement can
therefore theoretically provide a valuable and timely
barometer for many disease processes, including in-
fectious, as well as noninfectious conditions. C-reac-
tive protein is a pentamer consisting of ve non-cova-
lently bound, identical subunits, each containing 187
amino acids, with one intrachain disulde bond and
no carbohydrate modications (65). Acute-phase
proteins may provide important mechanisms to
modulate macrophage function, since macrophages
possess C-reactive protein receptors and C-reactive
Ebersole & Cappelli
Table 4. Acute-phase proteins
Protein Species Function
Major changes
C-reactive protein Human, rabbit Opsonin; anti-inammatory effects in vivo and in vitro;
induction of IL-1ra
Serum amyloid A protein Human, mouse, rat Apolipoprotein
Cysteine proteinase inhibitor Rat Antiproteinase
a2-Macroglobulin Rat Antiproteinase
a1-Antitrypsin Most species Antiproteinase; anti-inammatory activities in vivo and in vitro;
induction of IL-1ra
a1-Acid glycoprotein Most species Transport; anti-inammatory activities in vivo and in vitro;
induction of IL-1ra
Moderate changes
Fibronogen Most species Coagulation
a1-Proteinase inhibitor Human, rat, mouse Antiproteinase
a1-Antichymotrypsin Human, rat, mouse Antiproteinase
Haptoglobin Most species Binds and removes hemoglobin
Hemopexin Most species Binds heme
Ceruloplasmin Human, mouse O
2
scavenger, transport
Complement C3 Most species Opsonin
Serum amyloid P Most species
Negative changes
Albumin Most species (?)
Transferrin Most species Transport
protein can potently upregulate proinammatory
cytokine production (158). Thus, C-reactive protein
has been shown to induce the synthesis of IL-1a,
IL-1b, tumor necrosis factor a and IL-6 in human
peripheral blood mononuclear cells and alveolar
macrophages, suggesting that one of its physiological
roles may be the amplication of inammatory re-
sponses; although C-reactive protein probably plays
more of an anti-inammatory role (58).
C-reactive protein reacts with cell surface recep-
tors, resulting in opsonization, enhanced phago-
cytosis and passive protection; activation of the
classical complement pathway; scavenger for
chromatin fragments; inhibition of growth and/or
metastases of tumor cells; and modulation of poly-
morphonuclear leukocyte function.
Serum amyloid A
Serum amyloid A protein was originally described as
an acute-phase protein, with a precursor relation-
ship to the major constituent of amyloid A brils in
reactive amyloidosis. Serum amyloid A protein is
also elevated during inammation and may increase
by as much as 1000-fold, particularly during chronic
inammatory responses. Serum amyloid A is a pre-
cursor of protein amyloid A in secondary amyloid-
28
osis and may be deposited in the interstitium of
tissues, which can interfere with normal tissue func-
tion. Serum amyloid A comprises multiple isoforms
designated SAA
1
, SAA
2
and SAA
4
. The acute-phase
serum amyloid A isoforms, A-SAA
1
and A-SAA
2
, are
upregulated by inammatory cytokines by as much
as 1000-fold during inammation and, like C-reac-
tive protein, are monitored as a surrogate marker of
inammation. Acute-phase serum amyloid A is a
chemoattractant for neutrophils, monocytes, and T
cells. The SAA
4
isoform has been characterized as
constitutive serum amyloid A and appears to be pro-
duced by the liver irrespective of inammation. Both
acute-phase serum amyloid A and constitutive
serum amyloid A circulate as part of high-density
lipoprotein (81).
a
2
-Macroglobulin
a
2
-Macroglobulin is one of two principal protease
inhibitors in human plasma, the other being a
1
-anti-
trypsin. Proteolytic enzymes released from damaged
tissues as well as from phagocytic cells are partially
inhibited after binding to a
2
-macroglobulin. Macro-
phages and broblasts rapidly phagocytose the com-
plexes of proteases and a
2
-macroglobulin. a
2
-
Macroglobulin appears to scavenge proteases by
binding excess molecules that are not eliminated by
more specic inhibitors. Thus, this molecule func-
tions in hemostasis, coagulation, brinolysis, and
complement pathways.
Both hepatocytes and hepatic stellate cells can syn-
thesize a
2
-macroglobulin. In hepatocytes, a
2
-macro-
globulin is regulated as an IL-6-inducible type II
acute-phase protein (108). Interaction with the serum
antiprotease a
2
-macroglobulin represents a mechan-
ism that has been evolutionarily preserved for more
than 550 million years. Macrophages interact with
cytokines via specic receptors, as well as indirectly
with a
2
-macroglobulin. These cells express a 420-kDa
cell surface glycoprotein, which binds the activated
conformation of a
2
-macroglobulin. The activated
conformation is the protease-complexed form of a
2
-
macroglobulin, which is rapidly cleared from the cir-
culation (158). a
2
-Macroglobulin interferes in the -
brotic process by inhibiting plasmin, protease-acti-
vated a
2
-macro binds to transforming growth factor
b, which can be endocytosed, and degraded by
hepatocytes and other inammatory cells; a
2
-macro-
globulin binds and modulates the activity of IL-1, IL-
6, tumor necrosis factor a, transforming growth factor
b, and platelet-derived growth factor (108).
a
2
-Macroglobulin can also be a carrier protein for
IL-6 and thus may modulate the IL-6 activity as part
of the acute-phase response (15).
a
1
-Acid glycoprotein
The acute-phase glycoprotein, a
1
-acid glycoprotein,
can be increased by approximately 2- to 4-fold dur-
ing inammation. The glycosylation of a
1
-acid gly-
coprotein in human sera is subject to marked
changes during acute inammation as a result of the
cytokine-induced hepatic acute-phase response.
Acute inammation induces a strong increase in sia-
lyl Lewis Xsubstituted a
1
-acid glycoprotein mol-
ecules that persist at a high level throughout the in-
ammatory period. The changes may reect physio-
logical feedback response on the interaction
between leukocytes and inamed endothelium since
the sialyl Lewis X structures are associated with en-
dothelial selectin binding to leukocytes (32). a
1
-Acid
glycoprotein may also play an immunoregulatory
role and binds to a number of diverse drugs (130).
a
1
-Antitrypsin
a
1
-Antitrypsin is a serine protease inhibitor in human
plasma that targets proteases released from leuko-
cytes (suchas elastase). Elastase is anendogenous en-
29
zyme capable of degrading elastin and collagen. Once
bound to a
1
-antitrypsin, the activity of the proteases
is completely inhibited, being later removedandcata-
bolized. a
1
-Antitrypsin is synthesized by the liver and
increases 4-fold when stimulated by an inammatory
process. In contrast to complexes of proteases with
a
2
-macroglobulin, a
1
-antitrypsin-protease complex-
es are not taken up efciently by macrophages. a
1
-
Antitrypsin binds to the serpin-enzyme complex re-
ceptors on hepatocytes, neutrophils and macro-
phages. Thus, the acute-phase proteins may function
in a dual role: amplifying inammatory responses
when the inciting pathogen is present within the host
and down-modulating the response when the patho-
gen has been eradicated. a
1
-Antitrypsin-deciency
heterozygous individuals are at more risk thannormal
individuals of developing liver disease, connective
tissue disease (rheumatoid arthritis) and other in-
ammatory diseases. a
1
-Antitrypsin has a role in sev-
eral mediator pathways involved in the inammatory
response and, in the absence of this protein, the pro-
teases degrade tissue surrounding an inammatory
process and cause damage leading to chronic in-
ammation.
Haptoglobin
Haptoglobin binds to and removes free hemoglobin
released by intravascular hemolysis by forming a
complex that is rapidly cleared by hepatocytes. Hap-
toglobin plays a role in the clearance of free hemo-
globin through its ability to form stable complexes
with extra-corpuscular hemoglobin and prevent iron
loss through urinary excretion (31). Following injury,
haptoglobin increases 2- to 4-fold, is found in in-
ammatory exudates and reects the de novo syn-
thesis of the protein by the liver rather than of pre-
viously formed haptoglobin from other tissues. In-
fections or inammation may lead to a 2- to 10-fold
increase in haptoglobin and the biologic signicance
of the elevation in haptoglobin levels should be
interpreted with respect to other acute-phase pro-
teins (that is C-reactive protein).
Acute-phase induction by bacterial lipopolysac-
charide demonstrated that haptoglobin was induced
not only in the liver but also in other tissues, includ-
ing lung, skin, spleen and kidney. Haptoglobin is not
an adult liver-specic gene, and its role as an acute-
phase reactant may well be more diverse than pre-
viously expected (31). Haptoglobin is elevated in
many diseases; however, its glycosylation also
changes, and the type of change observed can vary
with disease. Increased fucosylation is a common
Ebersole & Cappelli
nding and has been suggested to be able to differ-
entiate among diseases (such as inammatory con-
ditions, liver disease), and to monitor disease activity
in cancer. In addition, N-acetyl-neuraminic acid and
N-acetylglucosamine residues suggest that carbo-
hydrate structures may predominate in certain dis-
eases. This glycosylation denotes ongoing intracellu-
lar events that could describe pathological processes
(148).
Fibrinogen
Fibrinogen accumulates at the site of injury and in
the presence of enzymes released from polymorpho-
nuclear leukocytes and platelets, brin is formed. Fi-
brin increases the tensile strength of the wound and
stimulates broblast proliferation and growth. Fi-
brinogen synthesis by hepatocytes can be increased
by approximately 2- to 4-fold and can be stimulated
by brinogen or brin degradation products, indi-
cating a feedback amplication loop that requires
macrophages. This is accomplished since the brin
degradation products do not directly stimulate he-
patocyte synthesis of brinogen but promote IL-1
synthesis by peripheral blood monocytes or Kupffer
cells, which stimulate brinogen synthesis.
Complement components (C)
The complement system is a group of serum proteins
whose general function is to regulate the inamma-
tory response. Several of the components are acute-
phase reactants, as they increase during infection.
These components interact with each other and with
other elements of the innate and adaptive immune
systems. For example, a number of microorganisms
spontaneously activate the complement system via
the alternative pathway. Activation by either the
classical or alternative pathway generates various
peptides that can attract phagocytes to sites of infec-
tion by chemoattractants; increase blood ow to the
site and increase vascular permeability for plasma
molecules; and damage plasma membranes of cells,
viruses or microorganisms that have activated the
systemand can produce lysis of the cell. Complement
has also been linked to macrophage cytokine release.
C5a may act upon macrophages to induce cytokines
such as IL-1. It also synergizes with lipopolysacchar-
ide or interferon-g and appears to function via the
macrophage receptor for C5a (158).
Mannose-binding protein is a plasma protein syn-
thesized by hepatocytes. Mannose-binding protein is
a structural analogue of C1q and can activate com-
30
plement via the classical pathway and plays an im-
portant role in host defense. Mannose-binding pro-
tein messenger RNA expression is increased by IL-6,
dexamethasone and heat shock, suggesting its role
as an acute-phase protein (4).
Ra reactive factor is a lectin present in the sera of
a wide variety of vertebrates. Ra reactive factor binds
specically to the RA and R2 core polysaccharides
shared by certain strains of gram-negative bacteria
and destroys bacteria by triggering the complement
system through activation of C2, C3 and C4. Ra reac-
tive factor is a large (300-kDa) protein composed
of multiple 30-kDa mannose-binding proteins and a
100-kDa serine protease (P100). P100 is related to the
C1r and C1s subcomponents of complement factor
1. P100 activates complement in a manner similar
to that of C1, however, the activation is triggered by
binding to bacterial polysaccharides instead of im-
mune complexes. The liver is the primary site for
P100 expression, and both hepatocytes and hepatic
stellate cells are the primary cellular sources. P100 is
upreguated by IL-6 and may represent a positive
acute-phase gene (76).
Finally, the C4b-binding protein (C4BP) is in-
volved in the uid-phase regulation of the classical
pathway of complement. During an acute phase re-
sponse hepatic levels of messenger RNA for C4b-
binding protein increase by 2.5- to 4-fold. The acute-
phase production of C4b-binding protein is regu-
lated at the transcriptional level by tumor necrosis
factor and IL-6 (105).
Ceruloplasmin
Ceruloplasmin is a glycoprotein that is the major
copper-transporting protein in human plasma (80
95% of the total circulating copper). Ceruloplasmin
appears as the primary copper transport protein that
transfers copper to cytochrome C oxidase, a critical
component to aerobic energy production and gly-
colysis, which necessarily increase during wound
healing. Ceruloplasmin and bound copper are es-
sential to collagen formation and the extracellular
cross-linking and maturation of collagen and elastin.
Ceruloplasmin and copper may also protect the ma-
trix of healing tissue against superoxide ions, gener-
ated by phagocytes in the course of clearing tissue
debris or microorganisms.
Albumin and transferrin
Albumin and transferrin, the serum iron transport
protein, are decreased during inammation, poten-
tially to starve the microorganisms of iron required
for growth and virulence expression. It appears that
cytokines, including IL-1, IL-6 and tumor necrosis
factor a, are important downregulators of the syn-
thesis of these acute-phase reactants.
Other proposed acute-phase reactants
Lipoprotein A is a low-density lipoproteinlike par-
ticle that consists of one apoprotein(a) [apo(a)] mol-
ecule covalently bound to an apo B molecule, sur-
rounding a chlolesterol-rich lipid core. Estrogen
lowers lipoprotein A levels. a
1
-Acid glycoprotein and
haptoglobin are decreased during estrogen treat-
ments. The lack of correlation between the changes
in the two acute-phase proteins and lipoprotein A
suggests different underlying mechanisms for the ef-
fects of estrogen on these liver-derived proteins. El-
evated plasma levels of lipoprotein A have been
shown to be an important independent risk factor
for atherosclerosis in many retrospective case-con-
trol studies (147).
Altered hepatic expression of apolipoproteins oc-
curs during the acute-phase response. Alterations in
liver synthesis of these proteins are believed to be
responsible for the changes in their serum levels. Al-
though the liver is thought to be the source for most
of the acute-phase proteins in the vascular compart-
ment, it has recently been recognized that several of
these proteins are synthesized in extra hepatic
tissues. Serum amyloid A, apo J, apo E, apo A-I and
apo D are produced in response to lipopolysacchar-
ide, tumor necrosis factor or IL-1 in kidney, heart,
stomach, intestine and muscle. The results demon-
strated that widespread extra hepatic regulation of
the apolipoproteins during the acute-phase re-
sponse may be important for the alterations in lipid
metabolism that occur during infection and in-
ammation (54).
The early response to inammation is character-
ized by the synthesis of a variety of proteins under
cytokine and glucocorticoid control. During epi-
sodes of infection or inammation, a secretory phos-
pholipase A
2
appears in the circulation along with a
variety of acute-phase proteins, suggesting possible
common regulatory elements among the secretory
phospholipase A
2
and acute-phase proteins (150).
Secretory group II phospholipase A
2
is a 14-kDa en-
zyme, a calcium-dependent neutral-active and
highly cationic protein whose expression is upregu-
lated by IL-1 and tumor necrosis factor. High con-
centrations of secretory phospholipase A
2
are at-
tained in serum during systemic inammatory re-
31
sposnes, such as septic shock and malaria. In septic
shock, serum secretory phospholipase A
2
concen-
trations may incrase by several hundred-fold (150).
Pancreatic secretory trypsin inhibitor in serum is
an acute-phase reactant and increases remarkably in
respones to surgical stress. Pancreatic secretory tryp-
sin inhibitor exists in various tissues other than the
pancreas, and serum pancreatic secretory trypsin in-
hibitor increases in patients with various malig-
nancies. Following pancreatectomy, the median
plasma level of pancreatic secretory trypsin inhibitor
increased by nearly 10-fold, consistent with its role
as an acute-phase reactant, which appears to be pro-
duced by the liver (68). The physiological role of
serum pancreatic secretory trypsin inhibitor, which
increases in response to tissue destruction or ad-
vanced cancers, is unknown. Several trypsin inhibi-
tors exist in much higher concentration in serum;
thus, its function in inhibition of this enzyme is
questionable. However, some data have suggested
that pancreatic secretory trypsin inhibitor could be
a growth-stimulating factor for endothelial cells. Tu-
mor-associated trypsin inhibitor is closely related or
identical to pancreatic secretory trypsin inhibitor
(112).
Some heparin-binding proteins are also acute-
phase proteins, since induction of the acute-phase
response can dramatically increase the levels of hep-
arin-binding proteins, and vitronectin may play an
important role in nonspecic binding of heparin in
plasma (162).
Acute-phase reactants in infection
Bacterial infections frequently provide a strong
stimulus for a systemic acute-phase response mani-
fested by the increased production of some 25
plasma proteins (50, 136, 144). Studies of neonates
with streptococcal infections and bacteremia suggest
that C-reactive protein is a marker for the acute
period of infection and a
1
-acid-glycoprotein a
marker for recovery (13, 44). Fever and sustained el-
evation of levels of C-reactive protein, erythrocyte
sedimentation rate and other inammatory markers
are common problems during treatment of infective
endocarditis. Elevations in C-reactive protein levels
were signicantly prolonged in the episodes with
complicated courses compared with the episodes
with uncomplicated courses (113). Initial C-reactive
protein levels were increased 90-fold, whereas
bronectin levels were reduced following bacterio-
logically conrmed community-acquired pneu-
Ebersole & Cappelli
monia. IL-1b and IL-6 were elevated 15-fold and tu-
mor necrosis factor a was elevated 3-fold (80). In
both HIV-seropositive and -seronegative patients,
acute-phase proteins were elevated when tubercu-
losis was initially diagnosed and fell during treat-
ment (61). Different lipoproteins, serum amyloid A
and C-reactive protein and activities of lipoprotein
lipase and hepatic lipase during acute and conva-
lescence phases of viral and bacterial diseases have
been evaluated. C-reactive protein binds to low- and
very-low-density lipoproteins and serum amyloid A
protein is associated with high-density lipoproteins.
This suggests that these acute-phase proteins may
interfere with the metabolism of serum lipoproteins
during the acute-phase of infection (127). Recently,
C-reaction protein was shown to increase in Plas-
modium falciparum infection, was higher in more
severe cases, and decreased with treatment (49). The
concentration of mannose-binding protein in-
creased 1.5- to 3-fold following surgery and associ-
ated tissue injury. Increased mannose-binding pro-
tein was observed in malaria patients and was main-
tained for 30 days of treatment (140). Similar results
were noted in animal models, where circulating IL-6
levels in mice tended to delay mortality to Trypano-
soma cruzi infection and appeared to correlate with
increases in serum amyloid P (146).
Certain bacterial compounds, probably deriving
from the gastrointestinal tract, trigger the postopera-
tive acute-phase response and are responsible for
the activation of monocytes or macrophages and
granulocytes. C-reactive protein levels begin to in-
crease in the rst postoperative day after major
surgery. IL-6 levels peaked at the end of the oper-
ation, remaining elevated for 6 h following major
surgery (9). Surgical infections and the inammatory
response to infection have a major role in morbidity
and outcome in surgical patients. Identication of
patients at risk of postoperative complications could
have an impact on the indications for a procedure
as well as permitting modications of treatment to
reduce the surgical risk. Recently, data on preopera-
tive antibacterial host defense suggested that prim-
ing of the immune system may result in an inappro-
priate response to injury or infection. Preoperative
priming of the host response, as detected by an in-
creased acute-phase response, is associated with an
aggressive inammatory response to minor stimuli
(such as a small degree of bacterial contamination)
that otherwise would have been cleared. These data
suggested that, if there are signs of an acute-phase
response preoperatively, the patients response to an
operation and infection during the postoperative
32
period may be adversely affected (55). Several patho-
physiological conditions may contribute to a higher
frequency rate of systemic inammatory response
syndrome in patients undergoing cardiac surgery
with cardiopulmonary bypass compared with pa-
tients undergoing other major surgical procedures.
In response to endotoxemia and cytokine secretion,
the coordinated sequence of systemic and metabolic
changes of the acute-phase response occurs. Serum
amyloid A, an apolipoprotein, has been dened as
the precursor of the secondary amyloid bril pro-
tein. Serum amyloid A has been found to be an ad-
ditional and sensitive marker of the acute-phase re-
sponse following cardiopulmonary bypass; the in-
crease in serum amyloid A concentration parallels
the temporary increase in body core temperature
and is preceded by endotoxemia and IL-6 secretion
(8).
It is established that bacteria evoke a host re-
sponse, including cytokines and other molecules,
which mediate the cellular effects that eventually
lead to the characteristic manifestation of septic
shock. Tumor necrosis factor, IL-1, IL-6, IL-8, IL-10,
interferon-g, the two soluble tumor necrosis factor
receptors and IL-1Ra comprise this list of cytokines.
Septic infections and related syndromes are hetero-
geneous with respect to causation and clinical mani-
festations and have been identied as: (i) systemic
inammatory response syndrome the systemic in-
ammatory response syndrome, which denotes the
systemic inammatory response to a variety of se-
vere clinical insults, (ii) sepsis denotes systemic in-
ammatory response syndrome in response to infec-
tion, (iii) severe sepsis denotes sepsis associated with
organ dysfunction, hypoperfusion or hypotension,
(iv) septic shock describes sepsis induced with hypo-
tension despite adequate uid resuscitation, and (v)
multiple organ dysfunction is the presence of altered
organ function in an acutely ill patient (155). Tumor
necrosis factor, IL-1 and interferon-g have been
identied as important mediator molecules in the
development of septic shock (Table 5). IL-10, soluble
tumor necrosis factor receptors and IL-1Ra appear
to have a couterregulatory effect. IL-6 and IL-8 prob-
ably contribute to manifestations like the acute-
phase response and leukocytosis. Tumor necrosis
factor induced a 40-fold increase in IL-6 concen-
tration, whereas IL-1 appears unaffected (155). Tu-
mor necrosis factor is the rst cytokine to be re-
leased with a peak concentration 11.5 h after con-
tact with lipopolysaccharide. Within 23 h, the peak
concentration of IL-1, IL-6 and IL-8 is attained. In-
terferon-g is released into the circulation later than
Table 5. Cytokine effects in the pathogenesis of septic shock
Mediator Contributory effect Inhibitory effect Other effects
IL-1 Synergism with tumor necrosis factor
Interferon g Synergism with tumor necrosis factor
IL-6 Acute phase response
IL-8 Leukocytosis
IL-10
Soluble tumor necrosis factor receptor p55
Soluble tumor necrosis factor receptor p75 Carrier for tumor necrosis factor
IL-1Ra
tumor necrosis factor. IL-1, IL-6 and IL-8 are induced
directly by lipopolysaccharide as well as indirectly by
tumor necrosis factor. Lipopolysaccharide induces
soluble tumor necrosis factor receptor p55 with a
peak level after 30 min and a gradual decrease, and
soluble tumor necrosis factor receptor p75 with a
peak after 48 h. C-reactive protein levels have been
proposed as a marker to detect and monitor sepsis
in burn patients (88), as well as being useful in moni-
toring septic patients during treatment (35). When
these changes are induced by live gram-negative
bacteria, they can be completely inhibited by speci-
c inhibition of tumor necrosis factor or IL-1. The
cytokine cascade is not triggered by lipopolysacchar-
ide alone, as corresponding observations have been
made with gram-positive bacteria (155). For ex-
ample, the toxin produced by the strain of Strepto-
coccus aureus responsible for toxic shock syndrome
is an inducer of IL-1 from monocytes. Viral particles
from inuenza and respiratory syncytial virus both
can induce IL-1, although respiratory syncytial virus
induces a high-molecular-weight IL-1 inhibitor con-
comittantly (158). The cellular basis for the develop-
ment of shock occurs in part from a depression of
myocardial function associated with decreased con-
tractility, decreased left ventricular ejection fraction
and diminished cardiac lling pressure. These can
all be negatively affected by tumor necrosis factor
(143). Recently, synthesis of acute-phase response
plasma proteins following administration of lipo-
polysaccharide has demonstrated genes for C-reac-
tive protein, serum amyloid A, a
1
-acid glycoprotein,
and haptoglobin with unique extrahepatic tissue
specic patterns of expression in kidney, spleen, thy-
mus, heart, brain, lung, testis and epididymis. How-
ever, other acute-phase proteins (a
1
-antitrypsin,
transferrin, albumin, ceruloplasmin and a
2
-HS-gly-
coprotein) did not exhibit this nonhepatic activity
(70).
33
Marked elevations in IL-6 levels have been ob-
served in acutely ill patients with septic shock and
have been associated with mortality rate. Increased
IL-6 levels were correlated with increases in body
temperature, heart rate, plasma lactate, a prognostic
scale of injury severity and decreases in mean ar-
terial pressure and platelet count (15). Traumatic in-
jury caused by acute thermal injury or multiple
trauma is associated with organ failure and profound
impairment of immune responsiveness. In clinical
studies, IL-6 levels were signicantly elevated within
hours of thermal injury (15). A rise in IL-6 levels may
also provide a warning of impending complications
of organ transplantation (15).
Chronic exposure to elevated tumor necrosis fac-
tor levels mediates loss of skeletal muscle protein,
anorexia, weight loss, anemia and kidney damage.
Mortality from cerebral meningitis correlates with
the levels of tumor necrosis factor detected in the
serum and cerebrospinal uid. Tumor necroris fac-
tor levels correlate with the severity of adult respir-
atory distress syndrome in clinical samples from the
circulation and bronchial washings. Adult respir-
atory distress syndrome appears to be the result of
uncontrolled release of several cytokines and the ef-
fect they have on the neutrophil as an effector cell
and its interactions with endothelium (115). Proin-
ammatory mediators that give rise to neutrophil
and endothelial cell activation and enhanced ad-
hesion-receptor expression are pivotal to the septic
response. It is this complex network of cytokines,
complement degradation products, eicosanoids,
platelet activators and various other chemical me-
diators that exacerbate the normally controlled in-
ammatory response. Tumor necrosis factor has
been implicated in the pathogenesis of acquired im-
munodeciency syndrome (AIDS) cachexia, the acti-
vation of latent human immunodeciency virus
(HIV) infection and the manifestations of organ
Ebersole & Cappelli
toxicity in the HIV-infected patient (143). Cachexia
and the acute-phase response are common manifes-
tations of inammation and are presumed to be the
product of increased synthesis and release of tumor
necrosis factor, IL-1 and IL-6. The inuence of IL-1
on cachexia and the acute-phase response is me-
diated in part by IL-6, and thus, IL-6 may play a piv-
otal role in the cachexia and acute-phase response
of inammation (114). Tumor necrosis factor and IL-
1 play critical roles in severe systemic inammation.
Infusion of IL-1 mimics the systemic inammation
seen in septic shock including hypotension, acidosis,
decreased blood ow and organ failure (141). Leuke-
mia inhibitory factor has been demonstrated to be
active in inammation and cachexia. Leukemia in-
hibitory factor has also been observed to provide
some protection against oxygen-induced lung toxic-
ity. This effect appears to be mediated by increased
manganese superoxide dismutase (as an acute-
phase reactant) and is enhanced by tumor necrosis
factor (47). Serum pancreatic secretory trypsin in-
hibitor is remarkably elevated in association with
serious inammatory and septic complications. Pan-
creatic secretory trypsin inhibitor and C-reactive
protein are positively correlated in patients with va-
rius inammatory diseases. IL-1Ra has shown pre-
liminary results suggesting an ability to provide
therapeutic efcacy in sepsis (5).
Specic signs and symptoms of disease are com-
monly absent in elderly patients, and standard lab-
oratory tests are often unhelpful or misleading. In-
fection, a common cause of admission to hospital of
old people, can be particularly difcult to diagnose.
Major elevation of the serum concentrations of C-
reactive protein and serum amyloid A indicated seri-
ous disease and predicted a poor outcome in elderly
inpatients with infections (58). Additionally, various
cytokines, including IL-1, are produced by uterine
epithelium, inltrating leukocytes and placental
tissues and aid in the communication bewteen mat-
ernal and embryonic cells. Elevated IL-1 levels have
been detected prior to normal parturition and in
premature labor due to intrauterine infections (72).
High IL-1Ra levels have been detected in amniotic
uid of women during normal pregnancy, suggesting
a possible protective role in pregnancy (141). The di-
agnostic relevance of C-reactive protein as a screen-
ing parameter for infections was determined. Due to
a rapid rise in C-reactive protein, all patients with
bacterial pneumonia showed increased C-reactive
protein at the time of hospitalization. In patients
with chronic obstructive pulmonary disease or
asthma and clinical evidence of infection, or in pa-
34
tients with bacterial gastroenteritis, C-reactive pro-
tein was a sensitive measure of infection. Thus C-
reactive protein may be a valuable screening test in
acutely ill patients. In addition, the short half-life of
C-reactive protein makes it a useful parameter for
the follow-up of patients with infections under anti-
biotic therapy (18).
inammation
Several studies have demonstrated a correlation be-
tween elevated serum acute-phase proteins and the
magnitude of joint destruction in rheumatoid ar-
thritis (91, 109). Evidence is also available suggesting
that C-reactive protein levels provide a sensitive and
objective indicator of disease activity and clearly re-
ect the response to therapy of rheumatoid arthritis
(151). In rheumatoid arthritis patients plasma, IL-10
increased, and IL-6 and C-reactive protein were sig-
nicantly elevated versus controls. A negative corre-
lation between IL-10 and IL-6 in serum was also
noted and suggested that IL-10 may cause a decrease
in IL-6 production and by that indirectly affects
acute phase response by decreasing C-reactive pro-
tein, a
1
-acid glycoprotein and a
1
-antichymotrypsin
(85). Serum levels of a
1
-acid glycoprotein, a
1
-anti-
trypsin and a
1
-antichymotrypsin are higher at the
onset of early rheumatoid arthritis compared with
healthy controls. Decreases were noted after 3 years
in patients without anatomical radiological pro-
gression of rheumatoid arthritis (86). Constitutive-
serum amyloid A concentrations vary in serum and
synovial uid independently of acute-phase serum
amyloid A and C-reactive protein levels. In general,
low levels of acute-phase serum amyloid A were
noted in inamed joint uid (81). Synovial uid and
plasma IL-6 levels were signicantly higher in pa-
tients with inammatory arthritis than those with
osteoarthritis. C-reactive protein levels were highly
correlated with plasma IL-6 in patients with other
inammatory arthritides, particularly psoriatic and
HLA B27-positive spondyloarthritis (60). Disease ac-
tivity scores reect the inammatory process either
directly (joint swelling or tenderness) or indirectly
(erythrocyte sedimentation rate or C-reactive pro-
tein level). Suppression of elevated C-reactive pro-
tein in patients with active rheumatoid arthritis is
associated with improvement in functional score,
whereas persistent elevation in C-reactive protein is
associated with functional deterioration (34). The re-
cent trend to start aggressive therapy early in rheu-
matoid arthritis is based on increasing evidence that
irreversible joint destruction may start during the
rst months of the disease and that early changes in
the small joints are predictive of future disability. C-
reactive protein values correlated closely with radio-
logical progression of joint disease in rheumatoid ar-
thritis. The C-reactive protein accurately predicted
outcome from 6 months after presentation and may
be used in a decision support system (152). IL-1 has
also been suggested as a major inammatory me-
diator in a variety of pathological situations such as
rheumatoid arthritis (141). Local production of tu-
mor necrosis factor in the inamed joint has been
implicated in the pathogenesis of rheumatoid ar-
thritis (141). Enhanced IL-1Ra production likely oc-
curs in active rheumatoid arthritis, as evidenced by
very high levels of this protien in synovial uids of
over 80% of patients with this disease. Clinical
studies suggest that IL-1Ra may be of benet to pa-
tients with active arthritis. Acute synovitis secondary
to Lyme disease, high levels of synovial IL-1Ra with
respect to IL-1 correlated with a more rapid recovery
from the episode of arthritis. Also, after a single sub-
cutaneous injection of IL-1Ra or after 7 daily treat-
ments, decreases in tender joint counts, erythrocyte
sedimentation rate and C-reactive protein levels
were noted (5). Supporting evidence for the linkage
of acute phase response with rheumatoid arthritis
has been shown by the effectivness of tenidap in
rheumatoid arthritis. Tenidap combines nonster-
oidal anti-inammatory drug-like cyclooxygenase
inhibition with suppression of the acute-phase re-
sponse (14).
A cross-sectional study of Crohns disease showed
acute-phase reactants (including C-reactive protein
and haptoglobin) changed in parallel with disease
activity; however, the increases did not precede the
disease activity (12). In both Crohns disease and ul-
cerative colitis, activated cells produce various in-
ammatory mediators that may be measured at in-
creased concentrations, in particular when the in-
ammatory bowel disease is active. The prolonged
and increased inammatory reaction seen in the in-
ammatory bowel disease may be caused by an im-
balance of pro- and antiinammatory mediators.
Many inammatory mediators are, however, not
only involved in the immune regulation of intestinal
inammation but contribute to or cause many sys-
temic reactions in inammatory bowel disease [such
as fever, hyperalbuminemia and increased produc-
tion of acute-phase response (C-reactive protein and
serum amyloid A)]. Measurements of circulating IL-6
35
and serum amyloid A proved most useful for clinical
monitoring of the activity of Crohns disease and ul-
cerative colitis. IL-6 was tightly linked with C-reac-
tive protein and serum amyloid A, and IL-6 is prob-
ably the main cytokine factor responsible for hepatic
induction of acute-phase proteins in Crohns disease.
However, measurements of circulating levels of all
the inammatory mediators studied were not useful
at all for differentiating between Crohns disease and
ulcerative colitis (110). Gastrointestinal epithelia
contain signicant levels of pancreatic secretory
trypsin inhbitor and patients with imammatory
bowel disease have changes in mucus structure sug-
gestive of increased proteolysis. Pancreatic secretory
trypsin inhibitor is reduced by 5080% in tissue from
ulceritive colitis and Crohns disease patients. Since
the mucous layer is important in preserving mucosal
integrity, prolonged reduction in mucosal pancreatic
secretory trypsin inhibitor might represent a long-
term reduction in mucosal defense mechanisms and
increased susceptibility to inammation (121). IL-1
has been implicated as a proinammatory mediator
in inammatory bowel disease and Crohns disease
(141) and may be involved in the colonic lesions in
inammatory bowel disease. Finally, IL-1Ra has been
shown to have anti-inammatory effects in an ani-
mal model of ulceritive colitis (5).
Chronic obstructive pulmonary disease affects
about 3% of the population, and in about 20% of
the cases there is a strong familial component that
is predominantly genetic in origin. Genetic de-
ciency of a
1
-antitrypsin results in a 20-fold in-
creased risk of developing chronic progressive lung
damage in cigarette smokers. a
1
-Antitrypsin plays a
major role in protecting the lower respriatory tract
from damage mediated by neutrophil proteinases, in
particular, elastase. This appears to result from a
mutation in the 3 enhancer region of the a
1
-anti-
trypsin gene. During the acute-phase response, the
plasma concentration of a
1
-antitrypsin increases by
3-fold in normal subjects mediated by IL-6. In con-
trast, a
1
-antitrypsin production in response to IL-6
is decient in these patients (107).
Insulin-dependent diabetes mellitus is an autoim-
mune disorder affecting the pancreas. In insulin-de-
pendent diabetes the insulin-producing b cells of the
islets of Langerhans are gradually destroyed, result-
ing in insulin insufciency. IL-1 has been implicated
in pancreatic b-cell damage in patients with insulin-
dependent diabetes. Prolonged incubation with IL-1
is cytotoxic to b cells and intact islets. The toxicity
appears to be mediated by induction of nitric oxide
synthase 2 (141). Along with IL-1 and nitric oxide,
Ebersole & Cappelli
tumor necrosis factor has been implicated in the de-
velopment of macrophage-dependent cytotoxicity of
islets in insulinitis and diabetes (143). IL-1Ra was
found to prevent the IL-1-induced decrease in insu-
lin content and release in cultured islet cells in vitro
(5).
In addition, IL-1 has also been suggested as a
major inammatory mediator in Alzheimers disease
(141). Likewise, tumor necrosis factor has been im-
plicated as a mediator of brain injury during acute
exacerbations of multiple sclerosis (143).
cardiovascular diseases
The primary signals eliciting synthesis of acute-
phase cytokines in aseptic tissue necrosis, such as
myocardial infarction, remain poorly understood, al-
though it has been found that hypoxic stress directly
induces IL-6 from myocytes and vascular cells (78).
Myocardial infarction and other types of tissue injury
generate changes in plasma protein as a part of the
acute-phase response. Numerous sequential
changes in lipid and lipoprotein levels take place fol-
lowing acute myocardial infarction. Characteristics
of the changes in acute-phase lipids and lipoproteins
can be an aid in providing appropriate therapy (126).
Epidemiological evidence suggests that brinogen
represents a major cardiovascular risk factor. As -
brinogen represents an acute-phase protein, its as-
sociation with atherosclerosis could be linked to in-
fection or inammation of other tissues. Moreover,
it could be connected to the inammatory activity
in atherosclerosis more directly (42). Hepatic syn-
thesis of brinogen as well as of other acute phase
proteins, including C-reactive protein, serum amyl-
oid A, haptoglobin and ceruloplasmin, is regulated
by IL-6, while other acute-phase proteins are en-
hanced by IL-1. The results of various studies have
suggested that different inammatory mediators
may act in unstable atherosclerotic plaque and myo-
cardial necrosis. It appears that IL-1 plays an initial
role in activating the acute inammatory response
and that elevations in the acute-phase protein, -
brinogen, during the late phases of myocardial in-
farction may be due to vanishing IL-1 or late induc-
tion of IL-6 by tissue necrosis (41). In addition to
increased plasma levels of brinogen, the acute-
phase response is usually accompanied by increased
production of von Willebrand factor and clotting fac-
tor VIII. Inammation also appears to lead to an in-
36
crease of plasma antithrombin III, while the protein
C system is downregulated. These acute phase re-
sponse related changes in hemostatic variables
would favor the local deposition of brin and plate-
lets while attempting to prevent an intravascular dis-
semination of brin formation (26). A number of
studies have implicated raised plasminogen acti-
vator inhibitor 1 activity as a risk factor of ischemic
heart disease. Since plasminogen activator inhibitor
1, like brinogen, is an acute-phase reactant, plas-
minogen activator inhibitor 1 activity was compared
to infection and vitamin C as an antioxidant. The
results suggested that acute-phase response caused
by infection increases plasminogen activator inhibi-
tor 1 activity and could increase the risk of coronary
artery thrombosis. Moreover, vitamin C appears to
attenuate this response (161).
High-density lipoprotein levels decrease during
the acute-phase response. Hypertriglyceridemia, tri-
glyceride-enrichment of high-density lipoprotein
and dissociation of apolipoprotein A-I from the par-
ticles, possibly by displacement of apo A-I by serum
amyloid A, have been suggested to be important fac-
tors in the decline of high-density lipoprotein during
acute-phase response. IL-6, C-reactive protein and
a
1
-antitrypsin reach maximal levels at 12 days, 3
days and 45 days after episodes of acute myocardial
infarction (111). Lipoprotein(a), an atherogenic par-
ticle that structurally resembles an low-density lipo-
protein, contains a second apolipoprotein, apo(a),
which is attached to the apolipoprotein B-100 in
low-density lipoprotein by a disulde bond. High
concentrations of liproprotein(a) are associated with
an increased risk for coronary heart disease. If lipo-
protein(a) is an acute-phase reactant, then serum
lipoprotein(a) concentratons in acute-phase re-
sponse patients would be expected to be increased
and may confound risk analysis. Lipoprotein(a) was
signicantly higher among acute-phase response pa-
tients, including those with infections, postoperative
patients, tumors, and other systemic diseases (104).
The peak time of synthesis of the acute-phase reac-
tant, lipoprotein(a), after acute myocardial infarction
was delayed. Immunohistochemical ndings sug-
gested that lipoprotein(a) may play an important
role as an acute-phase reactant in the repair of tissue
injury, especially in the process of angiogenesis
(111).
Finally, a dysfunction of the immune system has
been implicated in the cause of essential hyperten-
sion. IL-1b has been strongly associated with the
pathogenesis of atheromatosis and IL-lb is elevated
in hypertensive patients, as well as those with essen-
tial hypertension versus familial hypercholester-
olemia (30).
Acute-phase reactants and stress
Successful adaptationto stress is a prerequisite for the
survival of all organisms living in an environment in
which noxious stimuli are constantly present. Higher
organisms, including humans, have developed com-
plex mechanisms to tolerate the myriad of insults that
occur to cellular constituents and organ systems after
trauma, with its resultant blood loss and tissue injury.
This includes an integrated stress-response axis
linking the neuroendocrine, inammatory and im-
mune response systems (1, 149). IL-1a and IL-1b were
administeredtocancer patients toevaluate endocrine
function changes. C-reactive protein was elevated
within 6 days and resolved after treatment. These
cytokines did cause numerous changes in endocrine
parameters and appeared to inuence the activity of
multiple homeostatic endocrine functions including
increases in cortisol, growth hormone, prolactin and
thyroid-stimulating hormone and decreased levels of
follicle-stimulating hormone and luteinizing hor-
mone (27). In addition, neural cells respond to the
acute-phase response stimulatory cytokines leukem-
ia inhibitory factor and ciliary neurotrophic factor in
a similar manner andupregulate the same set of cellu-
lar proteins (47). Functionally, leukemia inhibitory
factor and ciliary neurotrophic factor promote the
noradrenergic-to-cholinergic switch in cultured sym-
pathetic neurons and affect the survival or differen-
tiation of motor and sensory neurons (47). There is
now some evidence that mental stress may affect
many aspects of the integrative network between the
immune, central nervous and endocrine systems in
bothanimals andhumans. For example, academic ex-
amination stress was found to induce signicant in-
creases in serum immunoglobulin A (IgA), IgG, IgM
and a
2
-macroglobulin in students with high stress
perception. Additionally, stress-induced changes in
IgA, C3 and a
1
-acid glycoprotein were signicantly
higher in individuals with high stress perception ver-
sus those with low stress perception (95).
Stress related to mental disorders has been associ-
ated with decreases in immune function. In ad-
dition, reports have associated alterations in acute-
phase proteins with major depression. Such studies
have demonstrated an increase in a
1
-acid glyco-
protein, haptoglobin, hemopexin and a
1
-antitrypsin
together with elevated levels of IL-6, but only slight
alterations in C3 and C4. Such evidence suggests that
37
depression places individuals at an increased risk of
infection. Extension of this evidence suggests that al-
tered immune status would predispose depressed in-
dividuals to a variety of disorders that include infec-
tious diseases (10). The status of the immune system
in major depression has also demonstrated that the
acute episode of depression may be accompanied by
a systemic immune response that is expressed by in-
creased plasma levels of positive acute-phase pro-
teins such as C-reactive protein, haptoglobin, a
1
-
acid glycoprotein, a
1
-antichymotrypsin, a
1
-antitryp-
sin and hemopexin, as well as complement factors
and immunoglobulins, and by decreased plasma lev-
es of negative acute-phase proteins such as albumin
and transferrin. It is thought that the acute-phase
response in depression is related to increased pro-
duction of IL-1 and IL-6 (134). Thus, major de-
pression may be accompanied by higher concen-
trations of positive and low concentrations of nega-
tive acute-phase proteins. Increased haptoglobin is
the most prominent change and is positively corre-
lated with IL-6 production. Increased production of
IL-6 and IL-1 in major depression may underlie both
immune activation and the acute-phase response in
this illness (93). Major depression in men resulted in
signicantly elevated levels of haptoglobin and a
1
-
antichymotrypsin and were correlated with the se-
verity of the depression, suggesting that an inam-
matory response occurs during depression (69). In
addition to the acute-phase protein responses that
have been reported in major depression, schizo-
phrenic patients had signicantly higher plasma
haptoglobin, brinogen, C3, C4, a
1
-acid glycoprotein
and hemopexin; manic subjects showed signicantly
higher plasma haptoglobin, brinogen, a
1
-acid gly-
coprotein and hemopexin; depressed subjects had
signicantly higher plasma haptoglobin, brinogen,
C3, C4 and a
1
-acid glycoprotein than normal indi-
viduals. Antidepressants, antipsychotics and lithium
all caused decreases in the acute-phase proteins
(94). Additionally, lithium potentiation of antide-
pressant treatment resulted in decreases in acute-
phase proteins (C-reactive protein, a
1
-acid glyco-
protein and a
1
-antitrypsin), while no effect was
noted in patients who did not respond to the lithium
(134).
Acute-phase reactants in cancer
The approach to measurement of acute-phase re-
sponse in cancer generally reects two strategies: (i)
determination of the levels of individual acute-phase
Ebersole & Cappelli
proteins, as predictive variables or biomarkers of
therapeutic efcacy, and (ii) utilization of molecules
that impact upon the immune and acute-phase re-
sponse systems as therapeutic modalities in cancer
treatment.
Serum levels of several acute-phase proteins were
evaluated in patients with various malignancies.
Levels of haptoglobin, a
1
-acid glycoprotein and a
1
-
antitrypsin can be utilized to monitor the course of
disease activity in patients undergoing antilympho-
cyte therapy. Haptoglobin showed the most signi-
cant change in response to therapy. Patients with
progressive tumors or who died showed signicantly
increased a
1
-acid glycoprotein levels. Thus, a de-
creased haptoglobin could indicate a positive re-
sponse to antilymphocyte therapy, while increasing
levels of a
1
-acid glycoprotein are an indicator of a
negative response to therapy. a
1
-Antitrypsin levels
showed a tendency to increase in all patients under-
going antilymphocyte therapy. Additionally, C-reac-
tive protein levels greater than 10-fold over normal
values are better related to microbial infection than
tumor load (77). During a post-injury period, circu-
lating concentrations of trace elements are reduced.
These reductions appear to be related to the magni-
tude of increase in the acute-phase protein response.
There is also evidence that plasma trace element
concentrations are altered in cancer patients. Recent
data have shown increased serum C-reactive protein
levels at the time of diagnosis of the majority of pa-
tients with nonsmall cell lung cancer. The presence
of an acute-phase response has implications for the
interpretation of circulating trace element concen-
trations and the status of patients with nonsmall
cell lung cancer (128). A signicant positive corre-
lation between urinary zinc and C-reactive protein,
a1-acid glycoprotein and haptoglobin and a negative
correlation with transferrin and albumin was ob-
served in patients with solid tumors. Hyperzincuria
in cancer patients appears to be linked to acute-
phase response (100). The incidence and the magni-
tude of the elevation of serum pancreatic secretory
trypsin inhibitor have been reported to be greater in
patients with advanced or metastasized malig-
nancies than in those in early stages (121). Tumor-
associated trypsin inhibitor has also been evaluated
for its relationship to renal cell carcinoma. The tu-
mor-associated trypsin inhibitor level in serum was
correlated with the stage of the disease and showed
a better sensitivity than other markers for renal cell
carcinoma (102). In normal adult tissue, tenascin-C
is usually expressed at low levels. It is strongly in-
duced in many tumors as well as in other patholog-
38
ical conditions often associated with inammation.
Tenascin-C levels are elevated in sera of cancer pa-
tients; however the increase was more pronounced
in persons with high levels of C-reactive protein.
There was a positive correlation between tenascin-
C and C-reactive protein. Thus, elevated tenascin-C
levels appeared to be more reective of infection and
inammation than directly related to tumor burden
(129). The pathogenesis of fever in solid tumors (ma-
lignant lymphoma, rhabdomyosarcoma, neuroblas-
toma) has also been studied. No difference in C-re-
active protein or IL-1 was noted with or without fe-
ver. Tumor necrosis factor was elevated with fever.
Thus, febrile episodes in children with solid tumors
may be primarily related to increases in tumor ne-
crosis factor production (63). Neutropenic children
with cancer and fever were examined and serum
concentrations of tumor necrosis factor a and IL-1b
were elevated, while IL-6 was detected in 68%, and
only 15% of the patients showed elevated IL-6.
Serum amyloid A was more sensitve than C-reactive
protein for the early detection of bacteremia in these
patients. The cytokines and acute-phase proteins
were related to the febrile response, but did not cor-
relate well with documented bacterial causation
(125). Cancer patients in whom infections were sus-
pected showed high levels of C-reactive protein,
haptoglobin, and brinogen, reecting the inam-
matory status of each patients. C-reactive protein
levels were higher in the non-neutropenic patients
and correlated with IL-6 levels (57). Serum IL-1Ra
levels are increased in patients with Hodgkins dis-
ease, particularly in those lacking systemic symp-
toms. Thus, IL-1Ra may have some regulatory con-
trol on the macromolecules contributing to the
symptoms of this disease (5). Raised levels of acute-
phase proteins in patients with cancer at presen-
tation are generally an unfavorable factor for the pa-
tientss survival. The acute-phase proteins can be
prognostic indicators with other clinical evaluations.
The pre-treatment acute-phase protein levels tend to
reect shorter term survival risks up to 18 months
and are often elevated during the last 6 months of
life with disseminated cancer, as well as consistent
with acute events such as sepsis (112).
IL-6 is a pleiotropic cytokine with stimulatory ac-
tions on the hematopoietic system, the immune sys-
tem and hepatocytes and may be a pivotal mediator
in the pathogenesis of shock and sepsis, in modulat-
ing megakaryocytopoiesis and in inhibiting tumor
growth. Clinical interest in the use of this cytokine
exists due to its thrombopoietic properties and its
potential anti-tumor activity. Preliminary studies as
an anti-tumor agent in renal cell carcinoma and
melanoma showed low response rates; however, its
effects in ameliorating chemotherapy-induced bone-
marrow depression and especially thrombocyto-
penia in concert with the stimulaion of acute-phase
proteins appears promising (154). Subcutaneous re-
combinant IL-6 caused signicant increases in C-re-
active protein and brinogen in children with solid
tumors on relapse. Stimulatory effects on thrombo-
cytopoiesis were also observed, although there was
no tumor response to the IL-6 administration (16).
Advanced cancer patients receiving subcutaneous
injections of recombinant human IL-6 showed in-
creased acute-phase responses throughout the treat-
ment and increased tumor necrosis factor a. IL-6 ap-
peared to have systemic activity on the immune sys-
tem, including expansion of monocytes and
activation of Th2-like cells, as well as induction of
acute phase response (71). IL-6 given intravenously
to adult cancer patients caused platelet counts,
white blood cell counts and acute-phase protein
levels to be substantially elevated, although no anti-
tumor responses were noted (156). IL-6 was ad-
ministered subcutaneously for 7 days to patients
with refractory advanced malignancies and did
cause major organ toxicity in some patients. All pa-
tients exhibited fever, chills and minor fatigue. In-
creases in C-reactive protein, brinogen, platelet
counts and IL-2 receptor levels were noted. De-
creases in albumin and hemoglobin were also ob-
served; however, no antitumor response was seen
(157). IL-6 is a part of a network of cytokines control-
ling hematopoiesis. IL-6 has an antineoplastic effect
on myeloid tumor cell lines by inducing terminal dif-
ferentiation. IL-6 also inhibits colony formation and
growth of ovarian carcinoma cells. In addition, dose-
dependent growth inhibitory effects on human
breast and renal carcinoma cells lines have been
found. IL-6 has also shown tumor growth inhibition
in some in vivo models (15). The related cytokines,
oncostatin M and leukemia inhibitory factor, induce
differentiation of leukemic cells, inhibit aortic endo-
thelial cell growth, inhibit embryonic stem cell dif-
ferentiation and induce hepatocytes to form acute-
phase proteins. However, oncostatin M more
specically inhibits the growth of several tumor and
non-tumor cells including melanoma cells, induces
IL-6 production by endothelial cells and stimulates
Kaposis sarcoma cells (47). Cancer patients were
treated with tumor necrosis factor a, and within 30
min to 6 h, signicant increases in serum IL-6 were
noted. C-reactive protein was detectable in serum
and peaked by 48 h. a
1
-Acid glycoprotein, a
1
-anti-
39
trypsin, C3 and C4 rose more slowly and peaked by
day 3. These results showed the relationship be-
tween tumor necrosis factor a and IL-6 in the syn-
thesis of acute-phase proteins (137). In cancer pa-
tients treated with lipopolysaccharide, serum tumor
necrosis factor a increased by nearly 2000-fold and
peaked by 1.5 h, while IL-6 increased by 1000-fold
by approximately 2 h. Parallel to the release of the
cytokines, a marked increased in granulocyte counts
was observed. Also, the lipopolysaccharide caused
the induction of C-reactive protein in the serum (92).
Recombinant human IL-3 was administered to can-
cer patients and caused endogenous IL-6 levels to
increase in a dose-dependent fashion. Increased IL-
6 levels were associated with enhanced production
of C-reactive protein. These results suggested that
IL-3 can augment the acute-phase response (90). In-
travenous injection of recombinant human IL-2 in-
duced dramatic changes in serum lipoproteins, in-
cluding apo B, apo A-I, and apo A-II, which showed
a mean reduction of 2655%. An action on hepatic
synthesis of acute-phase proteins was also noted by
the increase in C-reactive protein and apo S, al-
though there was a decline in brinogen (96).
periodontitis
As described in the preceding sections, the ability
to use acute-phase reactant levels as a measure of
infectious processes or inammatory diseases has
substantial support (19, 20). Acute-phase proteins
(including C-reactive protein and haptoglobin) have
been studied in healthy individuals, and while some
biological variability was noted, changes from nor-
mal can be detected (22). In this regard, circadian
variation of temperatures, both normal and febrile,
is a well-known fact. Mediators of fever are also regu-
lators of the acute-phase response and are associ-
ated with stimulation of the neuroendocrine system,
and thus, components of the acute-phase response
have circadian rhythmicity (120).
Pro-inammatory cytokines and mediators are
signicantly elevated, with gingival inammation
during the destructive phase of periodontitis (11, 39,
73, 87, 116, 122, 142). The clinical ndings in peri-
odontitis have emphasized the local (periodontium)
nature of the inammation and tissue destruction
within the oral cavity. One consequence of these lo-
calized gingival inammatory reactions has been the
identication of elevated levels of various acute-
Ebersole & Cappelli
phase proteins in the gingival crevicular uid (2, 3,
73, 133). These have included a
2
-macroglobulin, a
1
-
antitrypsin and C-reactive protein, which are altered
in the crevicular environment, presumably as a re-
sult of numerous host-bacterial interactions in the
sulcus, and may contribute to the defense of the host
in this milieu. Cytokines appear to play a major role
in the clinical symptoms and tissue destruction as-
sociated with progressing periodontitis (116). There
is also strong evidence for cytokines eliciting the sys-
temic acute-phase response in various chronic in-
ammatory diseases (83, 153). Many of these cyto-
kines are derived from activated macrophages and
can act both locally and distally to amplify cytokine
production from other cell types (such as broblasts
and endothelial cells), which then emerge from the
local tissues and can initiate systemic acute-phase
responses (6). Since multiple cytokines have been
detected in both gingival tissues and gingival crevic-
ular uid (45, 75, 116), changes in local acute-phase
reactants might be expected. Increased levels of
acute-phase proteins have been noted with gingival
inammation, including during experimental gingi-
viitis and periodontitis, reecting the locally stressed
environment (2, 3, 73, 133). The total amount of IL-
1a and IL-1b, but not total IL-1Ra were found to be
correlated with alveolar bone loss score (62). Direct
and indirect immunodot techniques were evaluated
for quantifying acute-phase proteins within gingival
crevicular uid from diseased and healthy sites.
Relative amounts of C-reactive protein and a
2
-
macroglobulin were determined. Periodontitis sites
exhibited lower levels of a
2
-macroglobulin, and C-
reactive protein levels were no different in health
and disease (133). In response to periodontal patho-
gens, neutrophils release oxidants, proteinases and
other tissue-destructive factors. The balance be-
tween these factors, the antioxidants, and endo-
genously synthesized antiproteinases (such as acute-
phase proteins) may determine the extent of peri-
odontal damage. Since the acute-phase response
plays a central role in promoting healing, peri-
odontitis as a wound-healing problem would be di-
rectly affected (40).
While the majority of studies of periodontitis have
emphasized the local nature of this host-bacterial in-
teraction in the periodontium and gingival sulcus
(87, 116, 122, 132), it also appears that systemic
manifestations of this disease also are detected. Due
to the chronic bacterial colonization of the supragin-
gival and subgingival aspects of the teeth, the juxta-
posed gingival tissue often demonstrates some level
of localized inammation (119). As periodontitis en-
40
sues, there are alterations in local host inammatory
mediators (87, 116), the initiation of a localized spe-
cic host response (37, 74, 139), and nally, a serum
antibody response to the bacteria is observed (36,
99). Consequently, these ndings would support
some ability of the localized inammation and/or
infection to be manifest systemically within the
affected host. Serum antibody levels are detected to
many oral bacteria and appeared to be increased
with more extensive dental caries (52) or peri-
odontitis (36, 99) potentially resulting from transient
access of oral bacteria to the circulation (59). Finally,
reports of individual patients and small cohorts have
suggested that patients with more severe disease
may reect systemic changes associated with stress
(131) and symptoms potentially attributable to more
severe bacterial infections (118). The results of this
study showed increased acute-phase proteins in a
group of patients identied as adult periodontitis.
These levels presumably reect both the infection
aspect of periodontitis (48), as well as the manifes-
tations of acute and chronic inammation that exist
in the periodontium (87, 116, 122, 132). Moreover, it
was also clear that patients exhibiting the most se-
vere disease exhibited the greatest levels of each of
the acute-phase reactants. While the severely dis-
eased patients must be considered an extensively
diseased group when compared to the general adult
periodontitis population, this type of patient may
represent the subset of adult individuals who are at
highest risk for extensive and rapid disease and can
provide a model for evaluating the contribution of
the acute-phase response to disease susceptibility or
resistance (117). This nding suggests that an in-
creased burden of infection or elevated inamma-
tory responses exist in this subset of adult peri-
odontitis patients, resulting in the elevated acute-
phase proteins. However, evaluation of the patients
did not support the hypothesis that the quantity of
clinical inammation was greatest in this subset, and
previous studies would support the theory that the
microbial burden is not routinely increased in pa-
tients with more severe periodontitis (135). Alterna-
tive explanations for these ndings would be that the
quality of the microbial ecology confers a higher
probability of systemic manifestations of this local-
ized infection (53) or that there exists a subset of
high-risk patients with increased susceptibility attri-
buted to altered local (gingival) and/or systemic in-
ammatory response characteristics (56). These
alternatives could have important implications for
host-based variations that contribute to increased
susceptibility or resistance to periodontitis pro-
gression. Thus, measurement of serum acute-phase
proteins may help to identify a subset of patients
who are at higher risk for destructive disease or dis-
close the patients who are undergoing a process of
periodontal breakdown (67).
Our study suggested that differences in C-reactive
protein and haptoglobin levels may distinguish a
group of adult periodontitis subjects with more se-
vere disease (38). Previous reports examining pa-
tients with aggressive disease have generally de-
scribed these patients as a portion of a younger
population representing an early-onset type of peri-
odontal disease (56, 67, 117). In this study, the subset
of patients exhibiting the most signicant elevations
in these acute-phase serum proteins represented in-
dividuals exhibiting a very aggressive, rapid disease
in adults. Thus, measurement of acute-phase pro-
teins could provide a valuable tool to identify
changes in the periodontal health of the patients,
particularly in the high-risk subset of periodontitis
patients. Due to the existing variability in serum,
acute-phase reactants within this adult periodontitis
population, conclusions on the effect of treatment
on these levels remains somewhat equivocal. Never-
theless, the study posed some interesting concepts
concerning the identication and monitoring of bio-
logical factors associated with progressing peri-
odontitis, when these factors are not homogeneous
within the population clinically categorized as
having adult periodontitis. The results emphasized
the heterogeneity in the adult periodontitis popula-
tion and may be an important consideration in
evaluating the effects of various treatment modalit-
ies administered to the patients. Both mechanical
oral debridement and treatment with an nonster-
oidal anti-inammatory drug appeared to effect
these serum glycoprotein markers of infection and
inammation. Additional longitudinal studies of
larger, better stratied populations will be required
to validate the usefulness of examining acute phase
reactants in monitoring periodontal disease. We
measured various acute phase proteins in the serum
of early-onset periodontitis smokers compared with
healthy nonsmokers and smokers with only gingi-
vitis and found extensive variation in both the dis-
eased and healthy subjects, although C-reactive pro-
tein appeared to be increased in the early-onset peri-
odontitis group (97).
While prevention of periodontal destruction is of
primary importance, it is clear that strategies for early
intervention, more specic treatment modalities and
more effective assessment of treatment success are
high prole areas of interest in periodontology. Sev-
41
eral studies have been performed to evaluate the ef-
fects of nonsteroidal anti-inammatory drugs on
periodontitis (66, 160), since these pharmaceuticals
have been used as palliative agents in treatment of
rheumatoid arthritis. Recently, tenidap sodiumwhich
is a cytokine reducing anti-rheumatic was found to
decrease C-reactive protein in rheumatoid arthritis
patients (91). Both steroids and disease-modifying
anti-rheumatic drugs also were found to decrease C-
reactive protein by 3070% (91). In both cases these
decreases were associated with a positive effect onthe
inammatory symptoms of the disease. Numerous
studies showed that nonsteroidal anti-inammatory
drugs had minimal effects on C-reactive protein, al-
though two studies indicated that urbiprofen de-
creased C-reactive protein in a subset (80%) of pa-
tients (28, 103). Clark & Fraser (22) suggested that a
change of 150% (C-reactive protein) or 70% (haptog-
lobin) in the levels of these acute-phase reactants are
signicantly different from the individual subject
variabilities. We notedthat mechanical hygiene or low
doses of the non-steroidal anti-inammatory drug
(Flurbiprofen
A
) had a negligible effect on C-reactive
protein or haptoglobin levels in the serum. However,
as was noted with rheumatoid arthritis, patients
higher doses of the non-steroidal anti-inammatory
drug appeared to cause a decrease in C-reactive pro-
tein and haptoglobin, with the haptoglobin changes
being statistically signicant.
The formation of highly reactive and toxic free
radicals, which are described as any species capable
of independent existence that contains one or more
unpaired electrons, in a cell is considered to be a
normal part of the cells metabolic processes. Since
all eukaryotic cells living in an oxygen environment
must be capable of neutralizing these free radical ef-
fects, they have developed metabolic systems that
protect them against the activity of these molecules.
The most notable of these protective mechanisms,
the formation of antioxidants (such as substances
which when present at low concentrations, com-
pared with those of an oxidizable substrate, will sig-
nicantly delay or inhibit oxidation of that sub-
strate), including superoxide dismutase, glutathione
peroxidase, glutathione, glutathione reductase, ceru-
loplasmin, transferrin, a-tocopherol (vitamin E), b-
carotene (vitamin A), ascorbic acid (vitamin C), cata-
lase, uric acid and peroxidase. The vitamins and pro-
vitamins are excellent sources of antioxidants, and
their inclusion in a standard diet routinely provides
some protection from oxidative effects. Growing
literature supports the hypothesis that free radicals
contribute to the formation of several major chronic
Ebersole & Cappelli
diseases, including cancer, cataracts, rheumatoid ar-
thritis and coronary heart disease. Recently, several
reports have described a positive relationship be-
tween the formation of free radicals in the formation
of inammatory lesions elicited by specic peri-
odontal pathogens and the inuence of antioxidants
in saliva and periodontal disease activity (106). The
bacterial species reported to be important to peri-
odontal disease progression and the formation of an
inammatory lesion stimulate the host to respond to
these infecting bacteria by both specic and non-
specic immune responses, as well as the release of
reactive free radicals for phagocytes and a host of
metabolic changes under the control of various cyto-
kines. Chapple et al. (21) determined that the total
antioxidant activity in the serum of the healthy and
periodontitis subjects were similar. However, the
periodontitis subjects were found to have a reduced
level of total salivary antioxidants compared with the
healthy subjects, possibly exposing them to the
damaging effects of free radical species in the oral
and periodontal environment. Therefore, alterations
in the local environment may be essential to peri-
odontal disease activity and may reect increased
levels of local radical production by local polymor-
phonuclear leukocytes and macrophages during dis-
ease progression. Interference with the formation of
these soluble mediators results in the impairment of
the acute-phase response to the infecting bacterial
species. Impairment with the acute-phase response
results in the decreased hepatic synthesis of these
acute-phase proteins and a concomitant inter-
ference with tissue repair. Ebersole et al. (38) have
shown that acute-phase proteins (such as C-reactive
protein and haptoglobin) were signicantly in-
creased in the serum from periodontitis subjects
compared with control individuals. Therefore, it is
more than likely that the increase in these two acute-
phase response proteins in the systemic circulation
response to the localized periodontitis infection has
a signicant impact on the ability of the host to pro-
tect itself from other systemic infections (such as
coronary heart disease). Thus, the potential exists
that the utilization of antioxidants as an intervention
agent in maintaining the acute phase response as a
homeostatic mechanism to control the chronic in-
ammation of periodontitis could have profound lo-
cal and systemic implications.
Recent studies have also indicated a closer linkage
of periodontitis with systemic manifestations of this
chronic infection and inammation. Thus, the
acute-phase response might be useful as biomarkers
of periodontitis contribution to systemic disease, as
42
well as providing a potential mechanistic link be-
tween the local and systemic manifestations of peri-
odontitis. A number of studies within the past dec-
ade have correlated periodontitis with the risk of
coronary artery and cerebral vascular disease (98,
138). The study of Destefano et al. (33) evaluating
data from the NHANES study indicated that peri-
odontal disease associated with poor oral hygiene
was a strong indicator of risk of total mortality and
that in men between the ages of 25 to 49 years, peri-
odontitis signicantly increased the susceptibility to
coronary heart disease and death. Beck et al. (7) have
also analyzed the data from the Normative Aging
Study and the Dental Longitudinal Study to assess
the relationship between oral disease and cardio-
vascular disease. Of the numerous periodontal vari-
ables analyzed in this retrospective analysis, bone
loss appeared to be the most consistent variable as-
sociated with coronary heart disease and thus, peri-
odontal disease appeared to be associated with ex-
cess risk of cardiovascular disease and stroke. More-
over, periodontitis patients presented with increased
levels of serum brinogen (an acute-phase protein)
and elevated white blood cells, signicant risk fac-
tors for coronary heart disease (84). Genco et al. have
recently provided preliminary data associating speci-
c periodontal pathogens, immune responses and
inammation with the formation of atheromatous
plaques and associated with cardiovascular disease
(51). Specically, the host responses to periodontal
disease and cardiovascular diseases were reected by
an increase in the acute-phase proteins (serum
amyloid A and C-reactive protein). Plausible mech-
anisms for this relationship revolve around the abil-
ity of chronic inammation (such as periodontitis)
to initiate and perpetuate systemic elevations in
various cytokines related to the acute-phase re-
sponse. Results from animal studies have demon-
strated the capacity of various acute-phase re-
sponserelated cytokines (such as tumor necrosis
factor, IL-1, IL-6, etc.) to signicantly alter serum tri-
glyceride (Table 6) and cholesterol metabolism
(Table 7).
Additional studies have also supported the con-
cept that circulating non-self materials (such as oral
bacteria and/or their products) may not only con-
tribute to cardiovasular disease but also affect fetal
development if they are not successfully removed by
the reticuloendothelial system. The detection of
acute-phase reactants in the serum of periodontitis
patients suggests that noxious materials from the
oral cavity may have the capacity to challenge vari-
ous tissue and organ systems, in addition to the liver.
Table 6. Effects of cytokines on triglyceride metabolism
Tumor Ciliary Leukemia
necrosis neurotrophic inhibitor
Effect factor IL-1 IL-6 factor factor Interferon a Interferon g
Serum triglycerides
Hepatic fatty acid synthesis
Triglycerides secretion nd nd
Lipoprotein lipase activity nd
Triglycerides clearance nd nd nd
Lipolysis
Source: adapted from Memon et al. (101).
decreased, increased, no change, ndnot determined.
Table 7. Effects of cytokines on cholesterol metabolism
Tumor
necrosis
Effect factor IL-1 IL-6 Interferon a Interferon g
Serum cholesterol
Hepatic cholesterol synthesis nd
Hydroxymethylglutaryl coenzyme A reductase activity nd nd
Hydroxymethylglutaryl coenzyme A reductase messenger RNA nd nd nd
Cholesterol 7a-hydroxylase activity nd nd nd
Cholesterol 7a-hydroxylase messenger RNA nd nd nd
Lecithin cholesterol acyltransferase activity nd nd nd nd
Lecithin cholesterol acyltransferase messenger RNA nd nd nd nd
Low-density lipoprotein receptor protein nd nd nd
Low-density lipoprotein receptor messenger RNA nd nd nd
Cholesterol ester transfer protein nd nd nd
Cholesterol ester transfer protein messenger RNA nd nd nd
Source: adapted from Memon et al. (101).
decreased, increased, no change, ndnot determined.
For example, Collins et al. (23, 24) have employed
a pregnant hamster model and have reported that
subclinical infections with several periodontal
pathogens induced low-birth-weight pups and that
the low birth weight appeared to be associated with
increases in intraamniotic prostaglandin E
2
and tu-
mor necrosis factor a.
Conclusions
The oral disease, periodontitis, has for many years
been considered a disease conned to the oral cav-
ity. It is only in the past several years that substantial
scientic data have emerged that indicate that the
localized infections characteristic of periodontitis
can have a signicant effect on the systemic health
of both humans and animals. Recently data gener-
43
ated from retrospective analysis of existing databases
of human studies has suggested a relationship be-
tween periodontal disease and cardiovascular dis-
eases. The basis of this linkage, and whether this rep-
resents a reection of a noncausal effect (such as
common causation) (Fig. 6) or a causal effect, albeit
potentially via intermediary variables such as acute
phase responses (Fig. 7), will be a challenge to evalu-
ate. Two directions have been the focus of delin-
eating the relationship: (i) bacteria from the oral cav-
ity directly exacerbating the cardiovascular disease
or altering systemic risk factors for cardiovascular
disease; and, (ii) the chronic periodontal inam-
mation at the focus of infection increasing circu-
lating levels of host inammatory macromolecules,
and/or bacteria translocated to the circulation elicit-
ing elevations in systemic host inammatory macro-
molecules that exacerbate cardivascular disease di-
Ebersole & Cappelli
Fig. 6. Model for linkage of chronic infection and in-
ammation of periodontitis with systemic complications
based upon a noncausal relationship (such as common
risk factors) and affected by acute-phase reactants (APR).
For example, genetic predispostion and/or dietary control
of serum lipid proles/levels results in enhanced athero-
sclerotic vascular changes. These negative changes are en-
hanced by the induction of systemic acute-phase reaction
associated with chronic periodontal infection and in-
ammation. The atherosclerotic changes can then present
as increased periodontitis severity through local micro-
vascular changes and decreased wound healing capacity.
In addition, the macrovascular atherosclerotic changes
are reected by coronary and/or cerebral vascular dis-
eases.
Fig. 7. Model for linkage of chronic infection and in-
ammation of periodontitis with systemic complications
based upon a causal relationship and exacerbated by the
acute-phase reactants (APR). The chronic infection and
inammation of periodontitis in susceptible hosts results
in systemic inammatory changes directly (such as de-
rived from local inammation) or indirectly (such as sys-
temic bacterial translocation and stimulation of inam-
mation) which are manifest by altered acute-phase reac-
tion proles. The acute-phase reaction then contribute to
increased risk of atherosclerotic vascular disease and as-
sociated cardiac and/or cerebral complications.
44
rectly or alter other systemic risk factors for cardivas-
cular disease. Thus, oral infections produce
signicant increases in systemic inammatory re-
sponses, manifested by acute-phase cytokines and
acute-phase reactants. Therapeutic oral manipula-
tions or the inappropriate or absence of intervention
of progressing periodontitis could have a signicant
inuence on these systemic diseases. Therefore, an
understanding of the relationship between the pro-
gression of periodontitis and risk factors associated
with cardiovascular disease (such as diet, serum
lipids, acute-phase responses, etc.) and other sys-
temic health complications (such as low-birth-
weight infants, diabetes and systemic inammatory
diseases) would have a profound effect on the strat-
egies for treatment of the periodontal diseases. Fi-
nally, the substantial role of free radicals in peri-
odontitis and their effects on the suppression of
antioxidant defenses and the acute-phase responses
should provide important information relevant to
drawing parallels between periodontitis and sys-
temic health complications.
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PERIODONTOLOGY 2000
ISSN 0906-6713
Periodontal management
of HIV-infected patients
MARK I. RYDER
In the long and broad history of human diseases, it
may be difcult for some to imagine that it has been
20 years since the rst descriptions of the rare
Kaposis sarcomas and Pneumocystis carinii infec-
tions in a small group of patients that heralded the
extensive human immunodeciency virus (HIV) epi-
demic that we know today (11). In addition, with the
extensive body of publications on the unusual oral
manifestations of HIV infection, it is interesting to
note that the very rst reports in this area only began
to appear in the last 18 to 19 years, while the rst
American Academy of Periodontology position paper
on periodontal considerations in the HIV patient ap-
peared a mere six years ago (60). And yet, since these
recent signposts in the history of this critical epi-
demic, there has been dramatic progress in the treat-
ment and diagnosis of the HIV viral infection and in
the myriad of opportunistic diseases that ac-
company HIV infection. In addition, the rapidly
changing epidemiological patterns of HIV infection
have strongly inuenced dental therapeutic ap-
proaches to this infection.
In recent years, the most encouraging news with
regards to the management of the periodontal pa-
tient with HIV infection comes from new treatment
approaches to this disease on a systemic level. For
example, there has been a recent spate of both scien-
tic articles and media publicity on new approaches
to prolonging the lives of HIV-infected patients by
reducing the levels of virus to undetectable levels
through the use of a combination of nucleoside
antagonists such as zivovuodine and didanosine
combined with the newly developed protease inhibi-
tors (5). While the long-term effects of this type of
combined antiviral therapy are still unknown, this
treatment approach may have widespread promise.
For the dental practitioner, these new developments
in therapeutic approaches may alter the incidence,
severity and management of oral manifestations of
HIV infection. In addition, while a single simple vac-
cine approach to preventing HIV infection may not
85
be feasible due to the genetic variation of the differ-
ent HIV viral strains, the use of multiple vaccines to
build up both humoral and cellular immunity may
have future promise (11). Such types of immunity
would also include the development of vaccines to
enhance oral mucosal immunity. Finally, the devel-
opment and renement of treatments to prevent the
occurrence and reduce the severity of some of the
potentially fatal opportunistic infections in HIV such
as P. carinii through antimicrobial agents may in-
directly reduce the incidence and severity of several
HIV-associated intraoral conditions (11, 17, 18). For
a more detailed description of these systemic thera-
peutic approaches and how they may affect the den-
tal management of the HIV patient, the reader is en-
couraged to review the chapter in this volume by
John Phair.
However, amid this encouraging news on the
treatment front, the epidemiological course of the
HIV epidemic has taken several turns that should be
of concern to both the medical and dental prac-
titioner. The initial wave of HIV infection in the
United States and Europe was conned to a few geo-
graphical areas and high-risk groups, particularly
male homosexuals and intravenous drug users.
However, the prevalence and incidence of HIV infec-
tion is increasing, albeit at a much slower rate, in
the heterosexual population in almost all areas of the
Western Hemisphere, Europe, Asia and Africa (11).
This infection trend is not only a concern for those
infected, but also to future newborns of HIV-infected
mothers where the transmission rate can be 25% to
30% (11, 51). For the dentist, this maternal trans-
mission is of concern due to the higher susceptibility
of HIV-infected children to oral opportunistic infec-
tions and other HIV-associated oral manifestations
(6, 29). In addition, although the rate of new infec-
tion in the homosexual population dropped dra-
matically during the late 1980s in the United States,
due in part to better education prevention programs,
the incidence of new infection in this group has
Ryder
risen in the last few years among the younger gener-
ations in this group (4). In this younger generation,
up to 70% of these infected individuals are unaware
that they are harboring the HIV virus (11).
Therefore, the dental practitioner will continue to
play a critical role in recognizing periodontal and
oral manifestations that may herald the HIV infec-
tion at different stages of the disease and to refer
patients for viral and immunological testing. It is im-
portant to keep in mind that about 40% of all HIV-
infected individuals show one or more HIV-associ-
ated oral manifestations, and these rates increase to
over 90% by the time the patient reaches the late
stages of immunosupression of this disease (22, 25).
Despite the recent advances in antiviral therapy and
prevention, the dental practitioner will need to con-
Fig. 1. Oral candidiasis in a patient with HIV infection. In
this photograph we can see both areas of whitish pseudo-
membranous candidiasis as well as erythematous can-
didiasis. Photograph courtesy of Deborah Greenspan.
Fig. 2. Typical appearance of hairy leukoplakia in an HIV-
infected patient. In this photograph we can see the typical
hyperkeratosis along the lateral border of the tongue ex-
tending to the dorsal surface of the tongue. Photograph
courtesy of Deborah Greenspan.
86
tinue to treat both the unique oral lesions and peri-
odontal diseases associated with HIV infection as
well as conventional periodontal diseases in the HIV-
infected patient. This chapter presents what is cur-
rently known about the oral manifestations of HIV
infection, with special emphasis on the periodontal
manifestations of this infection. We then present
newer information on the mechanisms of peri-
odontal disease in HIV-infected patients and how
this information may affect the management of
these diseases. We then discuss the current standard
accepted therapies for the treatment of both the un-
common periodontal lesions seen with greater fre-
quency in HIV infection and the more common peri-
odontal diseases. We conclude with a discussion of
special dental management and infection control
issues with HIV infection.
The course of HIV infection and
the oral cavity
Over the past 15 years, the central role of the dental
practitioner in the management of HIV infection has
essentially remained unchanged. Specically, the
dental practitioner needs to be able to recognize and
to treat both the common and the uncommon
lesions in the oral cavity associated with the HIV in-
fection. It is not the purpose of this chapter to review
each of these changes in detail. Nor do we discuss
in detail the reported variations in the incidence and
prevalence of these oral lesions between different
studies. For a more extensive discussion of these epi-
demiological studies, we refer the reader to some re-
cent excellent reviews by Mealy (42) in the 1996
World Workshop on Periodontal Diseases and by
Murray (46) and by Holmstrup & Westergaard (27).
However, some discussion of the general patho-
genesis of the HIV infection with special focus on
new insights into the relationship of oral and peri-
odontal lesions to HIV immune status is necessary
in order to understand the special role of the dental
practitioner in the management of HIV infection.
The most widely employed classication system
today in monitoring the progression of HIV infection
is the revised US Centers for Disease Control and
Prevention system that employs a matrix of three
ranges of CD4 lymphocyte counts (500, 200499
and 200) combined with a series of three clinical
syndrome classications (3). This current classi-
cation system is particularly important in the study
of oral and periodontal lesions in HIV, since research
Periodontal management of HIV-infected patients
Fig. 3. Early signs of linear gingival erythema in an HIV-
infected patient. At this early stage, this gingival change
resembles the marginal erythema as would be seen in gin-
givitis from patients without HIV infection. A differential
diagnosis for linear gingival erythema is the non respon-
siveness of this gingival erythema to conventional therapy
(scaling and debridement) and possibly the presence of
high populations of C. albicans. Photograph courtesy of
James Winkler.
in this area continues to show a relationship be-
tween the oral manifestation of HIV infection and
decreasing CD4 counts and/or CD4 percentages.
These oral lesions include what are now widely ac-
cepted as indicators of decreasing immunocompet-
ence such as oral candidiasis (Fig. 1), hairy leuko-
plakia (Fig. 2), extensive apthous ulceration and
acute necrotizing ulcerative gingivostomatitis, linear
gingival erythema, and necrotizing ulcerative peri-
odontitis (12, 2325, 32, 35, 56, 63, 70, 71). Perhaps
equally important is the possible relationship of the
levels of immunocompetence in general and CD4
counts in particular with the incidence and severity
of conventional periodontal diseases.
These relationships are borne out by several re-
cent studies which support the notion that the linear
gingival erythema (Fig. 3), characterized by a mar-
ginal band of intense erythema with more apical
focal and/or diffuse areas of erythema that may ex-
tend beyond the mucogingival line (Fig. 4), is associ-
ated with earlier stages of HIV infection and CD4
suppression. On the other hand, necrotizing ulcer-
ative periodontitis, characterized by marginal ne-
crosis of the gingiva and rapid destruction of the al-
veolar bone (Fig. 4, 5), is one of the strongest predic-
tors of a severely depressed CD4 count, which in
turn is the hallmark of progression to overt acquired
immunodeciency syndrome (AIDS) (17, 18).
Equally important are the recent observations that
the more common periodontal diseases may also be
87
associated with the declining immune status in HIV
infection. For example, it was recently reported that
HIV patients with acute necrotizing ulcerative gingi-
vostomatitis had fewer known risk factors than HIV-
negative patients with acute necrotizing ulcerative
gingivostomatitis (28). In addition, in HIV-infected
patients, there was no association between some of
the classic risk factors such as poor oral hygiene and
inadequate sleep with acute necrotizing ulcerative
gingivostomatitis.
Fig. 4. An HIV-infected patient with a mixture of general-
ized severe linear gingival erythema and localized necrot-
izing ulcerative periodontitis on the mandibular anterior
teeth. Possible contributing factors to the necrotizing ul-
cerative periodontitis lesion include the linear gingival
erythema, which may have progressed to this necrotizing
ulcerative periodontitis lesion as well as the generalized
dental neglect in this patient. Photograph courtesy of
James Winkler.
Fig. 5. A more severe form of necrotizing ulcerative peri-
odontitis in an HIV-infected patient. This photograph
shows the typical pattern of soft and hard tissue necrosis
in this condition. In HIV infection staging, this type of
periodontal appearance is closely associated with severe
immunosupression. Photograph courtesy of James
Winkler.
Ryder
Recent publications support the relationship of
HIV immunosuppression to the incidence and sever-
ity of common periodontal diseases such a chronic
adult periodontitis. In the past there were conicting
reports regarding the relationship of HIV infection to
attachment loss and bone loss (2, 10, 13, 34, 40, 64,
67, 73). Some of these studies did not take into ac-
count the CD4 immune status or the periodontal
disease classication. In addition, the use of antiviral
medication may have also confounded these obser-
vations (67). Nevertheless, it appears that in patients
with a preexisting chronic adult periodontal disease,
the degree of attachment loss is greater in the HIV-
infected patient, particularly when CD4 counts drop
below 200 cells/ml (2, 38, 76). Furthermore, in a re-
cent study on HIV-infected patients not receiving
antiviral or antimicrobial therapy, there was a clear
relationship between attachment loss and HIV status
(47).
Recent work on the pathogenesis of periodontal
disease in the HIV-infected patient has also shed
light on the relationship of HIV status and the inci-
dence and progression of a variety of periodontal
diseases. The general observation from earlier
microbiology studies was that the microbiological
prole of linear gingival erythema and necrotizing
ulcerative periodontitis was similar, thereby imply-
ing that they represent a continuum of the same
lesion (4345, 53, 78, 79). These microbiological pro-
les include most of the ora seen in chronic adult
periodontitis (including the newly described patho-
gen-related oral spirochete (57)) as reported in non-
HIV-infected patients and differed from the ora re-
ported in gingivitis in non-HIV-infected patients. In
addition, there were several opportunistic ora ob-
served in these linear gingival erythema and necrot-
izing ulcerative periodontitis lesions. Of particular
importance is the high prevalence of Candida al-
bicans. In the past few years, better understanding
has been gained of the role of C. albicans in the pro-
gression of linear gingival erythema and necrotizing
ulcerative periodontitis in HIV, which may affect the
management of patients with these lesions. Speci-
cally, although C. albicans is detectable in almost all
HIV subjects, its invasion into the soft tissues is
closely associated with periodontal diseases (19, 48).
This invasion not only occurs within the gingival
crevice, but also beneath the oral epithelium. A simi-
lar increase in the local neutrophil inltrate in these
two gingival areas have also been reported and may
be a response to this candida invasion. The candida
invasion may be directly or indirectly related to the
local depression of CD4 cells in the subjacent soft
88
tissues. In addition, this infection may trigger the de-
structive arm of the local host response (17, 19, 58),
which may result in the tissue necrosis as seen in the
necrotizing ulcerative periodontitis lesion and/or fa-
cilitate further loss of attachment and alveolar bone
in more conventional periodontal diseases. This de-
structive arm could include the release of potentially
destructive enzymes to periodontal tissue from ad-
jacent neutrophils which are attempting to neutral-
ize this candida invasion. Such tissue destruction
would occur both within the gingival crevice and
within the oral gingiva. These new studies point to
the increasing need for local and systemic antifungal
therapy in the HIV patient.
The relationship of an HIV-altered host response
to linear gingival erythema, acute necrotizing ulcer-
ative gingivostomatitis, necrotizing ulcerative peri-
odontitis and conventional periodontal diseases has
been investigated over the past 1012 years (49). In
addition to the possible role of altered neutrophil
function, increases in several inammatory me-
diators from the lymphocyte/monocyte arm of the
host response such as interleukin-1b and tumor ne-
crosis factor a have also been reported (31, 39). More
recently, increases in local corticosteroids in the gin-
gival crevicular uid of HIV patients have been re-
ported (9). These increases are often associated with
stress as seen with acute necrotizing ulcerative gingi-
vostomatitis and may lead to a local immunosup-
pression within the periodontal tissues. From this
brief discussion of new developments in our under-
standing of the pathogenesis of oral lesions in the
HIV patient, it is evident that special considerations
must be given in the management of these patients.
Management of periodontal
diseases in the HIV patient
To date, many published reports and reviews discuss
the management strategies for linear gingival ery-
thema and necrotizing ulcerative periodontitis as-
sociated with HIV infection. The majority of these
reports are empirical rather than based on con-
trolled studies. This is fully understandable when
considering the painful and destructive nature of
these diseases, particularly the necrotizing lesions.
Furthermore, these necrotizing periodontal diseases
are harbingers of advanced HIV infection with an as-
sociated reduced lifespan. When reviewing the
literature up to the present time on the management
of these diseases, the therapeutic principles de-
veloped by Winkler and others (21, 72, 74) in the mid
1980s still remain the standard of care for the treat-
ment of these lesions (1, 20, 27, 30, 42, 46, 59, 77).
Since it is probable that the linear gingival erythema
lesion is a precursor for the more destructive necrot-
izing ulcerative periodontitis lesion, similar prin-
ciples should be applied to the management of these
two lesions. These principles involve gross scaling to
remove visible plaque, soft debris and necrotic tissue
when present. Povidine iodine irrigation is rec-
ommended during this debridement procedure due
to its anesthetic and antiseptic effects. Following this
initial debridement, frequent follow-up visits are rec-
ommended to thoroughly remove the remaining
plaque, calculus and other deposits and to provide
plaque control instruction to the patient. In the case
of necrotizing ulcerative periodontitis, this thera-
peutic approach is important to institute as soon as
possible due to the possibility that the bone and soft
tissue necrosis can extend further into the palate and
adjacent tissues leading to a life-threatening NOMA
condition (69).
Antibiotics should be used with caution due to the
risk of overgrowth of C. albicans. In order to prevent
the overgrowth of Candida, the generally accepted
approach is to use a topical antifungal agent such as
clotrimazole troches or nystatin vaginal tablets, and
systemic uconazole in cases of more severe im-
munosuppression. The nonresponse of linear gingi-
val erythema to conventional therapy may be due in
part to a Candida invasion of the gingival tissues. In
these cases, the use of an antifungal agent may be
of benet in reducing the inammatory changes in
this condition. Narrow-spectrum antibiotics that
leave the greater portion of gram-positive ora intact
in order to prevent Candida overgrowth, such as me-
tronidazole, may also be benecial in the control of
the necrotizing ulcerative periodontitis and linear
gingival erythema lesions.
Following this initial debridement, these patients
need to be seen for frequent follow-up visits in order
to remove residual deposits and to receive a thor-
ough plaque control regimen. Chlorhexidine-based
mouthrinses are generally recommended as an ef-
fective therapeutic aid in reducing the acute symp-
toms of linear gingival erythema and necrotizing ul-
cerative periodontitis and in preventing the recur-
rence of these lesions. In the dental literature we
continue to see clinical case reports that employ
these management principles, which demonstrate
the effectiveness of this approach in reducing the
acute symptoms of linear gingival erythema and
necrotizing ulcerative periodontitis (Fig. 6). However,
89
Fig. 6. A. A localized acute ulcerative periodontal lesion in
an HIV-infected patient. Note the diffuse erythema and
the localized tissue necrosis between the central incisors.
Photograph courtesy of James Winkler. B. The same area
2 weeks after local debridement with povidine iodine irri-
gation to remove necrotic tissue, and chlorhexidine
mouthrinses. There is resolution of the soft tissue in-
ammation as well as a partial healing of tissue between
the central incisors. Due to the potential for further
plaque accumulation in this area due to the residual re-
verse architecture, it is important to instruct the patient
with the necessary home care tools to clean these irregu-
lar interproximal areas. Photograph courtesy of James
Winkler.
if there is no resolution of these lesions with this
therapeutic approach, the practitioner should con-
sider other oral lesions associated with HIV infection
that may give a similar clinical picture. These include
lymphomas, neoplastic growths and severe herpetic
or apthous ulcerations of the gingiva (15, 26, 50). In
some cases, particularly where a neoplasm is sus-
pected, a biopsy of the area should be performed to
conrm the diagnosis.
With the development of new strategies for con-
trolling the HIV virus, there may be a signicant de-
lay in the development of overt AIDS in a larger por-
tion of the infected population. This may in turn
lead to a downward trend in the occurrence of acute
Ryder
necrotizing ulcerative gingivostomatitis, linear gingi-
val erythema and necrotizing ulcerative peri-
odontitis in HIV-infected patients. However the den-
tal practitioner will still be faced with the problem
of treatment and maintenance of HIV patients with
more common periodontal conditions such as
chronic adult periodontitis, acute gingivitis etc. The
question facing dental practitioners is how the HIV
infection in general and specically the immune sta-
tus may affect conventional therapeutic approaches
to these diseases. To date there have been only a few
studies on periodontal treatment outcomes in HIV
patients. Much of what can be recommended at this
time is based upon other dental and medical ther-
apies.
The vast majority of HIV-infected periodontal pa-
tients will require a scaling and debridement to re-
move local irritants and hopefully reduce clinical in-
ammation (64). A concern of the practitioner treat-
ing the HIV patient may be the effects of developing
a systemic bacteremia and subsequent systemic in-
fections. To date, the one study that has looked into
this potential problem found a greater percentage of
transient bacteremia cases in HIV patients immedi-
ately after scaling (37). However, these bacteremia
levels returned to normal within 30 minutes. There-
fore, at least from this study, it appears that the scal-
ing and debridement procedure is safe to perform
for HIV-infected patients.
A second major concern of the practitioner is
whether the HIV status of the patient could alter the
treatment plan for periodontal surgical procedures.
To date there have been no studies that compare the
post-periodontal surgery healing response between
HIV infected and non-HIV-infected individuals. One
surgical procedure that has been studied more ex-
tensively and may have similar healing response
concerns is tooth extraction (7, 8, 16, 52, 55). Such
studies on postoperative complications of tooth ex-
traction include observations on delayed hard and
soft tissue healing. While the majority of these
studies a slight increases in these postoperative
complications in HIV-infected patients, these in-
creases were not signicant. However as in all dental
procedures, the general immune status in general
and the CD4 count in particular may alter surgical
decisions. For example, if CD4 counts are below 200,
this may indeed affect the healing response to peri-
odontal treatment. Along these lines, in the recent
American Academy of Periodontology position paper
on management of cancer patients, it was rec-
ommended not to perform extensive elective peri-
odontal surgical procedures if there was severe im-
90
munosuppression (54). Of particular concern in per-
forming periodontal surgery is that a percentage of
HIV-infected subjects will exhibit some degree of
thrombocytopenia during the course of their HIV in-
fection (11). This depressed platelet count may have
an adverse effect on the bleeding and clotting time
during and after periodontal surgical procedures. It
is therefore recommended that the dental prac-
titioner obtain the necessary information of the HIV
patients most current immune and blood status
prior to considering elective surgical procedures.
Transmission risks and
infection control
There are several issues regarding the transmission
of the HIV virus that historically have had an impact
on the management of periodontal patients with
HIV. Perhaps the most important to the dental prac-
titioner is the possibility of acquiring the virus from
the oral uids or oral bleeding from patients (36, 41).
To date there have been several studies which have
cumulatively looked at dental practitioners who
have been exposed to saliva or blood of HIV patients,
with only one possible conrmed case of a trans-
mission to a dentist in a non-risk group (14, 33).
These transmission studies have certainly allayed
many of the concerns of dentists in treating HIV pa-
tients in the dental chair. With the more widespread
use of better infection control techniques as outlined
by the US Occupational Safety and Health Adminis-
tration, and other regulatory agencies, the percen-
tage of dentists who are willing to treat HIV-infected
patients has risen dramatically over the past several
years from 21% in 1986 to 67% in 1993 (61, 68).
A second transmission concern for the dental
practitioner is the possibility of transmission of the
virus orally from an infected subject to an non-in-
fected subject. The initial fears of transmission
through kissing or use of shared utensils have been
allayed in recent years by a lack of clear-cut evidence
(11). It should however be kept in mind that, al-
though very low levels of HIV antigen can be de-
tected in a minority of saliva samples from HIV pa-
tients (75), there is a potential for direct blood con-
tact in subjects with either acute soft tissue
inammation, necrotizing ulcerative periodontitis
and other oral lesions. This problem may be exacer-
bated in patients with HIV-associated thrombocyto-
penia (11). Recent work by several investigators has
demonstrated the presence of the HIV in the
lymphocytes and macrophages in gingival and cre-
vicular uid (65, 66). These potential reservoirs for
HIV transmission through oral bleeding and/or in-
ammatory cells in oral uids point to the need for
the dental practitioner to control the periodontal in-
ammatory process in the HIV patient.
The third transmission concern is the possibility
of transmission from the dentist to the patient. In
the early 1990s there was considerable publicity re-
garding a case of an HIV-infected Florida dentist
who may have transmitted the virus to several pa-
tients. This mode of infection was supported by viral
genotyping. Since this incident, there have been no
other reports of this type of transmission. These pa-
tient concerns can be allayed with the use of ac-
cepted infection control techniques (62).
Summary
Recognition and diagnosis of the general oral mani-
festations and specic periodontal manifestations of
HIV infection will continue to be a major responsi-
bility of the dental practitioner. The dental team
needs to understand the underlying principles of
care for the necrotizing and inammatory peri-
odontal changes associated with HIV. In addition,
the practitioner must take into consideration how
the presence of opportunistic infections in the peri-
odontium such as Candida as well as the patients
level of immunosuppression may affect conven-
tional approaches to the common forms of peri-
odontal diseases. By combining local and systemic
therapy aimed at both preventing and treating oral
lesions and periodontal diseases, combined with
new systemic antiviral and vaccine therapies, dental
and medical practitioners may together help reduce
both the dental morbidity and the overall patient
morbidity in the HIV infected patient.
Acknowledgments
I thank Carol Fiuren for assistance in the preparation
of this manuscript and James Winkler and Deborah
Greenspan for photographic contributions.
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Detection of localized
tooth-related factors that
predispose to periodontal
infections
Debora C. Matthews & Moe Tabesh
Periodontal diseases are bacterial infections that
are present in approximately 70% of the U.S. popu-
lation, with 2030% being severely affected (16).
Over the last century, numerous investigations have
attempted to dene the etiologic agents of these
diseases and it is now clear that specic bacterial
pathogens are the primary etiologic agents. These
bacteria form a biolm at or above the gingival mar-
gin. Supragingival irregularities such as crowding,
calculus and rough restorations enhance the reten-
tion of the supragingival biolm and protect organ-
isms from the action of salivary enzymes and oral
hygiene measures. When the bacteria are left undis-
turbed, the host response is to implement a defense
reaction in the form of gingival inammation, or
gingivitis.
If, for whatever reason, the supragingival biolm is
left undisturbed and the bacteria begin to migrate
subgingivally, the environment changes, allowing
gram-negative anaerobes to ourish. Any irregulari-
ties such as root anatomy, subgingival restorative
margins, overhanging dental restorations and other
aberrations will enhance bacterial adhesion to the
pocket epithelium and the tooth surface, thus allow-
ing the growth of subgingival plaque. While potential
pathogens may colonize a site for decades without
causing disease (105), if the local environment chan-
ges in a manner that upsets the balance between
health and disease, destruction rather than remodel-
ing of the periodontium ensues. ``Local'' factors have
been shown to produce these changes.
Local factors are dened as anything that inu-
ences the periodontal health status at a particular
site or sites, with no known systemic effects. These
may be anomalies in the root anatomy or iatrogenic
features. This paper will review the detection and, to
a limited extent, the role of local factors (66) in the
pathogenesis of periodontal diseases.
Tooth anatomy
Inherent anatomic and morphologic features of teeth
can have a signicant impact on the management
and prognosis of the involved tooth or teeth. To
properly diagnose the severity of attachment loss in
a patient with periodontitis, a thorough understand-
ing of tooth root anatomy is crucial.
Molars
Furcations
Molars, particularly maxillary molars, are the teeth
most frequently lost due to periodontal disease (48,
72, 75). The anatomy of the furcation favors retention
of bacterial deposits and makes periodontal debride-
ment, as well as oral hygiene procedures, difcult.
Teeth that have lost attachment to the level of the
furcation are said to have a furcation invasion, or
involvement. Furcal invasions vary in horizontal
and vertical depth as a result of such features as
cervical enamel pearls, root trunk length, furcation
entrance dimensions, root anatomy and variations in
the anatomy of the roof of the furcation.
Several classication schemes have been devised
to describe the degree of furcation involvement.
Most are based on the amount of horizontal and/or
136
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PERIODONTOLOGY 2000
vertical probe penetration (38, 39, 44, 86, 95, 113),
and are generally divided into three types:
Grade I: Incipient lesion involving up to 3 mm of
horizontal probing depth (Fig. 1).
Grade II: ``Cul-de-sac'' with >3 mm of horizontal
probing depth, without extending through to the
opposite side (Fig. 1).
Grade III: Through-and-through lesion (Fig. 2).
Detection of furcation involvements is not always
straightforward. Good bitewing and periapical radio-
graphs may aid in the diagnosis, but should not be
used as the sole diagnostic tool (3, 61). For example,
less than one quarter of furcation involvements are
picked up on radiographs (98), although the likeli-
hood of detection does increase with the degree of
attachment loss (45).
Clinically, the degree of furcation involvement can
be determined using any of a number of furcation
probes. These probes are curved to allow access into
the furca and have a blunted end for patient comfort.
Clinical assessment, however, may overestimate the
depth of furcation defects. Moriarty et al. (80) demon-
strated that horizontal probing actually measured the
depth of probe penetration into the inamed connec-
tive tissue, rather than the true pocket depth. This is
especially true the deeper the pockets are.
Position of the furcation entrance, particularly in
maxillary molars, is important to remember. The
mesiopalatal entrance of the maxillary rst molar is
located towards the palatal third of the tooth, while
the distopalatal of the furcation is in the middle. In
order to probe maxillary molars, therefore, a palatal
or buccal approach can be used to detect distal fur-
cation involvements (Fig. 3). To detect mesial furca-
tion involvements, it is easiest to detect if probed
from the palatal aspect.
Transgingival sounding under local anesthesia
may be more accurate than regular probing in deter-
mining furcation anatomy. However, this is best
accomplished at the time of initial periodontal ther-
apy when the patient is anesthetized.
Root trunk length
The severity of furcation involvement is highly
dependent on the relationship between the amount
of attachment loss and the distance from the cemen-
toenamel junction to the furcation entrance - the
root trunk length. Hou et al. (53) developed a classi-
cation scheme that takes into account the length of
the root trunk compared to total root length. Type A
has the shortest root trunks, involving a third, or less,
of the cervical area of the root (Fig. 2). Type B root
trunks include up to half of the length of the root,
while in type C, the furcation entrance is in the cer-
vical two-thirds. This system can give a more realistic
prognosis for the tooth, as it takes into consideration
the vertical as well as the horizontal component
of attachment loss. For example, less horizontal
bone loss is required to expose a type A furca than
either the B or C types. On the other hand, a grade I
Fig. 2. Grade III furcation involvement of mandibular rst
molar, with type A root trunk (furcation entrance in the
cervical third of the root) at time of surgery (a). Radio-
graph of area (b).
Fig. 1. Grade I furcation involvement of maxillary second
molar, grade II furcation involvement of rst molar. The
root proximity between these teeth makes access extre-
mely difcult.
Localized tooth-related factors in periodontal infections
137
furcation invasion on a type C root implies a very
poor prognosis, since less than one-third of the root
surface area remains covered by alveolar bone.
In terms of prevalence, Hou et al. (53) reported that
maxillary rst molars are more likely to exhibit either
type B (47.1%) or type A (41.0%), than type C (11.9%).
Mandibular rst molars usually have type A root
trunk lengths (83.5%), while maxillary and mandib-
ular second molars most often have type B (60.8%
and 52.6%, respectively). Others (93, 99) have con-
rmed this in that they found the mean height of the
root trunk is greater in the maxillary molars (3.6
4.8 mm from the cementoenamel junction) than in
mandibular molars (2.43.3 mm). The most apical
furcations are the distal of the maxillary rst molars.
The shortest distance between the cementoenamel
junction and furcation entrance is found in the man-
dibular rst molars, permitting exposure of the fur-
cation at an early stage of periodontitis.
Size of furcation entrance
The diameter of the furcation entrance is extremely
important in predicting the success of periodontal
therapy. Bower et al. (15) reported the majority of
furcation entrances are less than the width of a
new standard Gracey curette. They found that 81%
of furcation entrances were <1.0 mm and 58% of
were <0.75 mm. These ndings are similar to those
found by Chiu et al. (21), who found that 49% of
furcation entrances were <0.75 mm. This anatomic
feature presents additional challenges in the man-
agement of molar furcations. Even with a surgical
approach, narrow furcations are difcult to comple-
tely debride (Fig. 4).
Bifurcation ridges
Using a stereomicroscope and extracted teeth, Svard-
strom & Wennstrom (110) developed complex topo-
graphic contour maps of the furcation areas of
maxillary and mandibular rst molars. Their study
clearly showed the complexity of the anatomy in the
furcation areas. They found various pits and ridges in
the roof of the furcation that would further compli-
cate therapy. These ridges run from one root to
another and, in some maxillary molars, continue
apically. Intermediate ridges connect the mesial
and distal roots and are composed primarily of
cementum. Buccal and lingual ridges are composed
of dentin with overlying thin layers of cementum (3).
In mandibular molars there may be a central bifur-
cation ridge that forms distinct pits in the roof of the
furcation. Hou & Tsai (52) determined that these
ridges are strongly associated with attachment loss
in furcations. These reports emphasize the complex-
ity of the furcation topography of molars that must
be kept in mind when debriding teeth with furcal
attachment loss.
Root concavities
Fluting of roots, or root concavities, are present to
some degree in all molars. Concavities are present in
the roof of furcations, coronal and apical to furca-
tions and on interproximal root surfaces (Fig. 5).
These are often difcult to diagnose unless the
patient is anesthetized during nonsurgical therapy
or the roots surgically exposed. As with any other
anatomic feature, their presence can affect the pro-
gression of attachment loss by harboring bacterial
plaque.
Fig. 3. Note the position of the distal furcation in this
maxillary molar is in the mid-interproximal area. Distal
furcations can be probed from either the buccal or palatal
aspect of the tooth.
Fig. 4. Extracted mandibular molar showing narrow fur-
cation entrance that complicates plaque removal by
patient and clinician.
138
Matthews & Tabesh
Cervical enamel projections
Ectopic deposits of enamel apical to the level of the
normal cementoenamel junction, with a tapering
form and extending towards or into the furcation
areas, are called cervical enamel projections (66).
As with coronal enamel, connective tissue does not
attach to cervical enamel projections, thus they have
been implicated as etiologic factors in furcation
defects (12, 112). Hou & Tsai (50) reported the pre-
valence of cervical enamel projections in molars with
and without furcation involvement. In molars with
cervical enamel projections, 82.5% exhibited furca-
tion involvement, while this was true for 17.5% of
molars that had no cervical enamel projections.
The inuence of a cervical enamel projection is
dependent on the extent of the projection. Masters
& Hoskins (71) introduced a grading system in 1964
that is still in use today:
Grade 1: Short but distinct change in the contour of
the cementoenamel junction extending towards
the furcation.
Grade II: cervical enamel projection approaches
the furcation, without making contact with it.
Grade III: cervical enamel projection extends into
the furcation (Fig. 5).
Roussa (99) found that Grade I and III projections
are more prevalent, and that cervical enamel projec-
tions are more often observed in mandibular second
molars than either the maxillary or mandibular rst
Table 1. Signicant anatomic features of teeth
Tooth Anatomic feature (ref.) Prevalence (ref.)
Maxillary incisors Palatal groove
98% all grooves found in lateral incisors
0.79 (5)21% (51)
Maxillary first bicuspids Root trunk length; averages 414.6 mm (57)
Furcal concavity on palatal aspect of buccal root
Mesial root concavities
Furcation entrance diameter <0.75 mm
62% (57)
100% (14)
57% (15)
Maxillary molars Furcation entrance diameter <0.75 mm
Root trunk length; averages
Mesial: 3.5 mm (99)4.2 mm (15)
Buccal: 4.0 (99)4.8 mm (15, 93)
Distal: 3.3 (12)
63% (15)
32.6% (112)
Mandibular molars Furcation entrance diameter <0.75 mm
Root trunk length; averages
Buccal: 2.4 mm (15)3.14 mm (70)
Lingual: 2.5 mm (15)4.17 mm (70)
First molar
Second molar
Bifurcation ridges
50% (15)
80.4% (52)
48.4% (52)
65.5% (52)76% (18)
Fig. 5. GradeIII cervical enamel projectiononamandibular
rst molar. This projection acts as a lip at the furcation
entrance. Together withthe mesial groove onthe distal root,
these anatomic anomalies compromise plaque removal.
139
molars. These ndings are in line with those of other
studies (see Table 1).
Enamel pearls are less prevalent that cervical
enamel projections. They contribute to the etiology
of furcation involvements nonetheless. Moskow &
Canut (81) reviewed the literature on the prevalence
of enamel pearls and reported a range of 1.19.7%.
Nearly three-quarters of enamel pearls are found on
maxillary third molars. The mandibular third molar
and maxillary second molar are the second most
common sites. Figure 6 is a radiographic example
of an enamel pearl and the resultant periodontal
destruction.
Maxillary bicuspids
Maxillary rst bicuspids often have two roots buccal
and palatal. Joseph et al. (57) examined the furca-
tion anatomy of 100 of these teeth. A furcal concavity
was seen on the palatal aspect of the buccal root
in 62% of bifurcated teeth. This coincides with the
prevalence of 78% reported by Gher & Vernino (35).
The mean furcation width was 0.71 mm; again, less
than the diameter of a new curette. Concavities
were found on the proximal surfaces of all teeth
studied, with a deeper concavity on the mesial than
the distal aspect. Maxillary bicuspids may also dis-
play a V-shaped groove on the proximal surfaces
(Figs 7 and 8). These often persist toward the apical
region, and are associated with a greater loss of
attachment than that found around non-grooved
teeth (67).
Anterior teeth
Palatal grooves
The radicular lingual or palatal groove is a develop-
mental anomaly in which an infolding of the inner
enamel epithelium and Hertwig's epithelial root
sheath create a groove that passes from the cingulum
of maxillary incisors apically onto the root (41).
These grooves usually begin in the central fossa,
Fig. 6. Cervical enamel pearl on the maxillary rst molar
(a). There is alveolar bone loss associated with this anom-
aly that is not present in the contralateral tooth (b).
Fig. 8. Moat-type defect associated with distal and buccal
furcations of maxillary second bicuspid.
Fig. 7. Maxillary rst bicuspid with Grade I buccal furca-
tion involvement. Note the deep mesial groove.
140
Matthews & Tabesh
cross the cingulum and extend apically for various
distances and directions (Figs 9 and 10). Pecora & da
Cruz Filho (90) reported radicular grooves to be pre-
sent in 3.9% of their subjects, primarily on the lingual
surface of the maxillary lateral incisor (3.0%). Less
than 1% of maxillary central incisors showed radicu-
lar grooves on the buccal and/or lingual surfaces.
Others report the prevalence of 0.7921% in both
maxillary incisors (5, 49, 62, 124), and 1.914% in
lateral incisors alone (30, 35, 49, 62, 124).
In contrast to maxillary bicuspids, incisors gener-
ally display a shallow U-shaped groove. Kogon (62)
reported that over half of the grooves extend more
than 5 mm apical to the cementoenamel junction.
These grooves may act as ``funnels'' for the accumu-
lation of microbial plaque in the depth of the groove,
where they are inaccessible to both patient and clin-
ician. The prognosis for teeth with palatal grooves
with apical extension is poor (41).
Dental restorations
The relationship between dental restorations and
periodontal status has been examined for some time.
Research has shown that overhanging dental restora-
tions and subgingival margin placement play an
important role in providing an ecologic niche for
Overhanging dental restorations
An overhanging dental restoration is primarily found
in the Class II restoration, since access for interdental
cleansing is often difcult in these areas, even for
patients with good oral hygiene habits (Fig. 11).
Many studies have shown that there is more period-
ontal attachment loss and inammation associated
with teeth with overhangs than those without. Over-
hangs cause an increase in plaque formation (55, 59,
60, 68, 89, 94, 97), and a shift in the microbial com-
position (64) from healthy ora to one characteristic
of periodontal disease. In addition to their effect on
the inammatory process, overhangs may also cause
damage by impinging on the interdental embrasure
Fig. 9. Periodontal pocket associated with palatal groove
on mesial of central incisor; without probe in place (a),
with probe in place (b).
Fig. 11. Poor quality restorations with overhanging mar-
gins.
Fig. 10. Surgical view of distal palatal groove of maxillary
lateral incisor.
141
and the biologic width. There appears to be no
statistical relationship between the presence of an
amalgam overhang and patient age (89) meaning
an overhang can be equally destructive to young and
old alike.
Restorative matrices may not always adapt well
interproximally, even with careful placement of the
restorative material and tight wedging. Variations in
root anatomy, particularly root concavities, may
make perfect marginal adaptation impossible. It
has been shown that removal of an overhang results
in improved plaque control (97) and restoration of
gingival health (40).
The prevalence of overhangs has been reported in
many patient populations. The reported range on
restored teeth is between 18% (55) and 87% (68).
Criteria used to determine the presence of an over-
hang differ from study to study, which likely
accounts for most of this variation (see Table 2).
Lervick et al. (68) employed bitewing radiographs, a
microscope and magnifying glass. They reported 96%
of overhangs extended less than 0.5 mm from the
tooth. This indicates that studies using the criterion
of 0.5 mm have most likely underestimated overhang
prevalence. Pack et al. (88) found the use of bitewing
radiographs and clinical exploration detected only
35% of interproximal overhangs. Of these, 74% were
found with radiographs alone, while 62% were found
using only clinical inspection.
These studies conrm that removal of overhanging
margins should be part of initial periodontal therapy.
As well, it is obvious that early detection of overhan-
ging dental restorations is an important part of pre-
ventive dental care. A sensitive tactile instrument,
such as a ne explorer, should be used in conjunc-
tion with radiographs to facilitate this detection.
Subgingival restorative margins and
biologic width
The location of the gingival margin of a restoration is
directly related to the health status of the adjacent
periodontium (116). Numerous studies (56, 58, 87,
96, 101, 115) have shown that subgingival margins
are associated with more plaque, more severe gingi-
val inammation and deeper periodontal pockets
than supragingival ones. In a 26-year prospective
cohort, Schatzle et al. (101) followed middle-class
Scandinavian men for a period of 26 years. Gingival
indices and attachment level were compared
between those who did and those who did not have
restorative margins greater than 1 mm from the gin-
gival margin. After 10 years, the cumulative mean
loss of attachment was 0.5 mm more for the group
with subgingival margins. This was statistically sig-
nicant. At each examination during the 26 years of
the study, the degree of inammation in the gingival
tissue adjacent to subgingival restorations was much
greater than in the gingiva adjacent to supragingival
margins. This is the rst study to document a time
sequence between the placement of subgingival mar-
gins and periodontal attachment loss, conrming
that the subgingival placement of margins is detri-
mental to gingival and periodontal health.
Subgingival margins, in addition to inuencing the
progression of periodontitis, can have other effects
on the attachment apparatus. Several authors have
described the area between the depth of a healthy
gingival sulcus and the alveolar crest as the ``biologic
width'' (25, 26, 42, 54, 74, 84). This constitutes the
junctional epithelium and the supracrestal connec-
tive tissue. Using cadaver specimens, Garguilo et al.
(31) calculated the average distance to be 2.96 mm.
Table 2. Prevalence of overhanging dental restorations (adapted from Brunsvold & Lane, 1990 (17))
Reference Diagnostic method for detection % restored surfaces with overhangs
(n number of subjects)
Gilmore & Sheiham, 1971 (37) Bitewing radiographs 25% (n 1976)
Burch et al., 1976 (19) Bitewing radiographs 30% (n 825)
Hakkrainen & Ainamo, 1980 (43) Orthopantograms 50% (n 85)
Than et al., 1982 (114) Calculus probe 60% (n 240)
Lervik & Riordan, 1984 (68) Bitewing radiographs, microscope 25% (n 175)
Keszthelyi & Szabo, 1984 (60) Bitewing radiographs, microscope 86% (n 176)
Coxhead, 1985 (23) Bitewing radiographs, mirror, probe 76% (n 50)
Claman et al., 1986 (22) Bitewing radiographs 27% (n 826)
Jansson et al., 1994 (55) Bitewing radiographs 18% (n 162)
142
Matthews & Tabesh
This is often rounded up to 3.0 mm when referring to
the amount required for the biologic width. It has
since been shown that this is not a magical number,
and the biologic width may vary between teeth and
individuals. The basis for the biologic width is the so-
called ``radius of inammatory effect'' in which there
is a nite distance of approximately 12 mm over
which the tissue-lytic properties of localized inam-
mation operate (117, 118). In other words, plaque at
the apical margin of a subgingival restoration will
cause periodontal inammation that may in turn
destroy connective tissue and bone approximately
12 mm away from the inamed area.
When it is anticipated that placement of a subgin-
gival margin will impinge on the biologic width (i.e.,
be within 12 mm from the alveolar crest), surgical
crown-lengthening procedures should be consid-
ered. Herrero et al. (47) found that this is not always
easy to achieve, even by experienced clinicians. In
the most difcult areas to access, the lingual and
distolingual, clinicians removed an average 0.4 mm
of bone far short of the 3 mm goal. Determination
of the distance between the restorative margin and
the alveolar crest is often done with bitewing radio-
graphs; however, it is important to remember that a
radiograph is a 2-dimensional representation of
3-dimensional structures. Thus, clinical assessment
and judgment are important adjuncts in determining
if, and how much, bone should be removed to main-
tain adequate room for the dentogingival attach-
ment.
Restorative materials
Although surface textures of restorative materials dif-
fer in their capacity to retain plaque (13), all can be
adequately maintained if they are polished and
accessible to patient care (65). This includes the
underside of pontics. While there is little evidence
to substantiate the use of specic materials or
designs for pontics, Wang et al. (119) found that
metal pontics harbor higher proportions of period-
ontal pathogens than porcelain ones. Although there
is no evidence that subpontic plaque leads to attach-
ment loss on abutment teeth, the accumulation of
subpontic plaque for an extended period of time
should be considered a risk factor.
In a trial of porcelain laminate veneers, Kourkouta
et al. (63) found a decrease in the plaque index and
bacterial vitality between teeth with and without
veneers in the same patients. This nding is consis-
tent with other studies that have shown highly glazed
porcelain retains less plaque than enamel (85, 123).
Composite resins are difcult to nish interproxi-
mally and may be more likely to show marginal
defects than other materials (92). As a result, they
are more likely to harbor bacterial plaque (27).
Intra-subject comparisons of unilateral direct com-
posite ``veneers'' showed a statistically signicant
increase in plaque and gingival indices adjacent to
the composites, 56 years after placement (92). In
addition, when a diastema is closed with composite,
the restorations are often overcontoured in the cer-
vical-interproximal area, leading to increased plaque
retention (91). As more plaque is retained, this could
pose a signicant problem for a patient with moder-
ate to poor oral hygiene.
Prostheses
Several investigations have noted that after the inser-
tion of removable partial dentures, the mobility of
the abutment teeth, gingival inammation and per-
iodontal pocket formation increases (10, 11, 20, 102,
126). This may be because partial dentures, especially
those with gingival coverage, have been shown to
favor plaque accumulation (1, 108). Not only is there
an increase in the amount of bacterial plaque, but a
shift to more pathogenic microbiota, as well (100).
In a large case-control study, Zlataric et al. (126)
examined 205 partial denture wearers. Abutment
teeth had higher plaque scores and more severe gin-
gival inammation than non-abutment teeth. Prob-
ing depths of 2 mm were found on 82% of non-
abutment teeth as compared to 54% of abutments.
Not all studies concur with these ndings. Mullaly &
Linden (83) concluded that among regular dental
attenders, those with partial dentures were no more
likely to have periodontal disease than those who did
not wear a partial. It is likely, however, that the sam-
ple size of 14 was too small to detect any potential
difference.
Effect of crowding and tooth
position
Until the 1960s, the dental literature contained few
reports that examined the relationship between mal-
occlusion/malalignment and periodontal disease.
Many studies have concluded that poorly aligned
teeth themselves are not associated with a greater
degree of gingivitis. Rather, crowding can complicate
oral hygiene procedures, leading to an increased
plaque accumulation and subsequent gingival inam-
mation (2, 34, 103). Although Behfelt et al. (9)
143
reported a direct relationship between tooth displa-
cement and gingival inammation, this was only
evident in patients with moderate or poor plaque
control. Similarly, there appears to be no relationship
between crowding and alveolar bone loss in patients
with excellent plaque control (6, 34, 103). Thus, it
appears that malalignment may be more important
in the patient with less than ideal oral hygiene.
However, if interproximal spaces are reduced as a
result of crowding, root proximity may occur (Fig. 1).
This leads to less effective removal of plaque and
subgingival calculus in the inaccessible interproxi-
mal area. Even surgically, these teeth can be difcult
to treat, leading to a compromised prognosis. None-
theless, a well motivated and dexterous patient will
likely prevent plaque accumulation regardless of
tooth shape or position (106).
In some cases of extreme anterior overbite, direct
trauma to the gingiva from the incisal edges of the
mandibular incisors may result in palatal recession
the maxillary incisors. Similarly, in severe Class II
division 2 malocclusions, functional trauma can
cause marginal recession of the facial gingiva of the
mandibular incisors (33).
Vertical root fractures
Vertical root fractures are, by denition, longitudinal
and conned to the root of the tooth. They may be in
a mesiodistal or buccolingual plane, and may occur
at any point along the root (Figs 12 and 13). The
diagnosis of vertical root fracture is difcult because
several of the associated signs and symptoms are
shared with other dental conditions. In some
instances, there may be a halo appearance at the
fracture site of the affected tooth on a radiograph.
However, unless the fractured segment has sepa-
rated, radiographs are often of little use in localizing
the lesion.
The patient may have masticatory pain, with or
without concomitant pulpal pain. Transillumination
may be helpful, or the diagnosis may be made with
the use of a bite test, where a resilient material is
placed between the teeth during gentle occlusion.
Pathognomonic of a vertical fracture is the occur-
rence of a narrow, isolated periodontal pocket. The
affected teeth may exhibit recurrent periodontal
abscesses.
Teeth with cracked roots usually have a poor prog-
nosis, although some success has been reported
using regenerative membranes (82) or resin cement
(109).
Fig. 12. Isolated deep pocket associated with vertical root
fracture and endodontic-periodontal lesion. Radiograph
of tooth prior to extraction (a); extracted tooth (b).
Fig. 13. Not all vertical fractures are visible on a radio-
graph, particularly if they occur in the buccolingual plane.
144
Matthews & Tabesh
Assessment of mucogingival
deformities
Gingival recession
Assessment of the mucogingival status of a patient is
an essential part of an oral examination, particularly
if there is major restorative or orthodontic work
planned. Mucogingival status refers to the quality
and quantity of keratinized gingival tissue, the
amount of gingival recession, the presence of aber-
rant frena and the depth of the vestibules.
Aberrant frenal attachments may be a problem,
especially in shallow vestibules or areas of minimal
attached gingiva (Fig. 14). When the frenum is
stretched, the muscle attachments may pull the mar-
ginal tissue away from the tooth. This then acts as a
``funnel'', allowing accumulation and apical migra-
tion of bacterial plaque. This can lead to an increase
in gingival recession on that particular tooth.
Gingival recession can be a problem for patients
for esthetic reasons, dentinal hypersensitivity or
interference with normal hygiene procedures. A
number of factors have been implicated in the etiol-
ogy of gingival recession. An extreme buccal or lin-
gual positioning of the tooth in the dental arch,
whether natural or due to orthodontic movement,
can lead to thinning of the alveolar plate and asso-
ciated gingival tissues. This makes the area more
susceptible to recession, either from trauma or
inammation (73). Trauma is usually the result of
vigorous toothbrushing, or the use of a medium to
hard brush. Plaque-induced disease can also cause
recession, particularly in patients with a thin period-
ontium (4).
Recession is measured from the midfacial or mid-
lingual/palatal of each tooth to the most coronal
aspect of the marginal gingiva. In multi-rooted teeth,
recession can be measured for each root. Miller's
classication is the standard in categorizing marginal
tissue recession and in predicting the likelihood of
root coverage with periodontal plastic surgery tech-
niques:
Class I: Recession does not extend to the muco-
gingival junction. There is no loss of interdental
bone or soft tissue (Fig. 15).
Class II: Recession extends to or beyond the muco-
gingival junction. There is no loss of interdental
bone or soft tissue (Fig. 16).
Class III: Recession extends to or beyond the
mucogingival junction. There is loss of interdental
bone and/or soft tissue or malpositioning of the
tooth (Fig. 17).
Class IV: There is advanced loss of interdental bone
and/or soft tissue or severe malpositioning of the
tooth (Fig. 18).
Class I and II can be subclassied as narrow or
wide. After gingival grafting procedures, full root
Fig. 15. Grade I recessionhas the best prognosis interms or
root coverage from periodontal plastic surgery procedures.
Fig. 16. Grade II recession on the facial of mandibular
first bicuspid extends beyond the mucogingival junction.
Fig. 14. Frenal pull in an area of no attached gingiva may
accelerate gingival recession.
145
coverage can usually be expected up to the level of
the interdental bone in Classes I and in Class II nar-
row-type defects. For Class III, only partial coverage
can be expected, while little or no coverage occurs in
Class IV lesions treated with current grafting techni-
ques.
Keratinized gingiva
Keratinized gingiva is differentiated from attached
gingiva in that the former includes both attached
and free gingival tissues. Attached gingiva is deter-
mined by subtracting the periodontal probing depth
from the width of keratinized tissue at a given site. It
is measured at the midfacial of mandibular and max-
illary teeth and the midlingual of mandibular teeth.
The clinical impression is that since the attached
gingiva is rmly bound to the underlying periosteum,
it will provide a protective barrier against inamma-
tion and attachment loss. Augmentation of the
attached gingiva is often recommended on this basis.
Several studies have challenged the view that a
wide zone of attached gingiva is a more effective
barrier against recession than a narrow or nonexis-
tent one. It has been demonstrated that in the
absence of inammation, gingival health and attach-
ment levels can be maintained (28, 121, 122). One
long-term study reported that the incidence of reces-
sion was no greater in areas without keratinized tis-
sue than in areas with a wide zone of keratinized
gingiva (120).
Stetler & Bissada (107) studied the relationship
between the width of keratinized gingiva, the pre-
sence of subgingival margins and inammation.
Teeth with subgingival restorative margins and nar-
row (<2.0 mm) keratinized tissue were more likely to
exhibit gingivitis, while those with restorative mar-
gins at or above the coronal aspect of the free gingiva
showed no difference between wide and narrow
bands of keratinized gingiva. These results suggest
that particular attention should be paid to those sub-
gingival restorations associated with <2.0 mm of ker-
atinized tissue. For patients scheduled to receive
subgingival restorations where narrow zones of ker-
atinized tissue exist, and who cannot maintain opti-
mal plaque control levels, it may be desirable to
increase the width of keratinized tissue about those
teeth.
There are other situations in which a wider zone of
attached gingiva may be necessary. Teeth with a thin
periodontium that are planned for orthodontic
movement, particularly if there will be movement
of teeth labially, are likely to have more recession
in areas of minimal attached gingiva (32). Abutment
teeth for removable prostheses are more likely to
exhibit plaque, and therefore be at higher risk of
gingivitis and attachment loss. These areas may be
considered for gingival augmentation, particularly
for patients with inadequate oral hygiene. Sites
where the patient nds oral hygiene difcult because
of the proximity of the mucosa to the cervical area of
the tooth, may also benet from a wider zone of
keratinized tissue.
Effect of periodontally hopeless
teeth on adjacent teeth
The ability to accurately predict the response of the
dentition to periodontal therapy is essential in devel-
oping a denitive periodontal maintenance and
restorative treatment plan. A prognosis is assigned
to an individual tooth based on a number of factors
including type and degree of bone loss, probing
depths, presence and severity of furcations, mobility,
crownroot ratio, root form and pulpal involvement.
Fig. 18. Grade IV recession on mandibular canine that is
further compromised by iatrogenic dentistry.
Fig. 17. Grade III recessionona mandibular central incisor.
146
Matthews & Tabesh
In addition, systemic factors, patient compliance and
type of periodontal disease are key determinants.
Prognostication is a skill not easily acquired and is
highly dependent upon the experience and skill of
the clinician. There are several classication systems
and denitions of prognoses (7, 8, 24, 48, 75). It is
generally agreed that a tooth with a hopeless prog-
nosis is one that, despite the patient's and clinician's
best efforts, is not going to improve. In these situa-
tions, it is difcult, if not impossible, to arrest the
disease process and restore periodontal health. Some
believe that the presence of a hopeless tooth may
also be detrimental to the health of adjacent teeth
(29, 69, 104). Despite this, patients may choose to
retain periodontally hopeless teeth for a variety of
reasons economics, esthetics or an aversion to
extractions.
Several studies have shown that, when appropriate
treatment is rendered and the patient is in a main-
tenance program, teeth previously diagnosed as
hopeless may survive for many years with little or
no effect on proximal teeth (24, 125). Wojcik et al.
(125) examined periodontal maintenance patients,
8 years after the completion of active therapy. There
was no difference in probing depths or alveolar bone
levels between teeth adjacent to the hopeless tooth
and not. Although a small study (n 14), power
calculations showed these results to be robust.
McGuire (76) and Ghiai & Bissada (36) both con-
cluded that in cases where the prognosis is other
than ``good'', it is difcult to predict the long-term
outcome of individual teeth. In cases of hopeless
teeth, prognostication was no more accurate than
ipping a coin (76). In McGuire's study, 25% of teeth
initially diagnosed as hopeless were reclassied as
having a good or fair prognosis 58 years later. These
ndings suggest that periodontally involved teeth
can be maintained for years in health, comfort and
function (8, 24, 36, 46, 48, 7779, 125).
Summary
The primary goal of periodontal therapy is to pro-
duce an environment that is conducive to oral health.
This is achieved by eliminating the subgingival infec-
tion and implementing supragingival plaque control
measures designed to prevent the re-colonization of
the sulcus (111). Local etiologic factors, as described
above, may prevent the removal of subgingival pla-
que, and may even contribute to destruction of the
periodontal tissues. Thus, it is crucial to be able to
recognize and, when possible, eliminate any plaque-
retentive factor that could contribute to disease
progression. Iatrogenic factors such as subgingival
margins, restorative overhangs, overcontoured resto-
rations and unpolished surfaces can be altered.
Similarly, cervical enamel projections, enamel pearls
and, in certain instances, palatal grooves can be
removed or recontoured to enable the patient to
access the area for good plaque control. There are
some things that we cannot alter. Anatomic anoma-
lies, particularly in posterior teeth, cannot be chan-
ged. However, awareness of potential anatomic
variations and early detection of them may be able
to prevent future attachment loss.
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150
Matthews & Tabesh
Occlusal analysis, diagnosis and
management in the practice of
periodontics
William W. Hallmon & Stephen K. Harrel
Occlusal trauma is dened as injury resulting in tissue
changes within the periodontal attachment apparatus
as a result of occlusal forces (1). The controversy that
surrounds this condition including recognition, diag-
nosis, effects, and management, has been widely
debated since the early part of the 20th century (8,
48, 97, 98). Since occlusal trauma can only be con-
rmed histologically, the clinician is challenged to
use clinical and radiographic surrogate indicators in
an attempt to facilitate and assist in its diagnosis.
This chapter will focus on the role of occlusal
analysis, tooth mobility and occlusal therapy in the
clinical practice of periodontics. Diagnostic and ther-
apeutic approaches and effects on treatment out-
comes will be reviewed and discussed. In an
attempt to facilitate this process, the following ques-
tions will be addressed:
What is occlusal trauma?
What is the role of occlusion in the pathogenesis of
periodontitis?
How is occlusal trauma detected clinically?
What is abfraction and are there data to support a
role for occlusion in its development?
What methods are used to detect hypermobility of
teeth? Of what value are assessments of tooth mobi-
lity in the management of periodontitis patients?
Under what clinical circumstances is occlusal
adjustment indicated? Following occlusal adjust-
ment, what clinical outcomes are expected and
how are they evaluated?
What is occlusal trauma?
The International Workshop for a Classication of
Periodontal Diseases and Conditions in 1999 evalu-
ated the available materials relating to the effects of
occlusion on the periodontium and the role that
occlusion may play in periodontal disease. The con-
sensus report of the International Workshop group
evaluating occlusion adopted the following working
denitions for occlusal trauma. These denitions are
critical to the clinical evaluation, diagnosis, and
treatment of occlusion in periodontal disease (1).
Occlusal trauma Injury resulting in tissue
changes within the attachment apparatus as a result
of occlusal force (s).
Primary occlusal trauma Injury resulting in tissue
changes from excessive occlusal forces applied to a
tooth or teeth with normal support (Fig. 1). It occurs
in the presence of: 1) normal bone levels, 2) normal
attachment levels, and 3) excessive occlusal force (s)
(Fig. 1).
Secondary occlusal trauma Injury resulting in
tissue changes from normal or excessive occlusal
forces applied to a tooth or teeth with reduced sup-
port (Fig. 2). It occurs in the presence of: 1) bone loss,
2) attachment loss, and 3) normal/excessive occlusal
force (s) (Fig. 2).
What is the role of occlusion in the
pathogenesis of periodontal
disease?
Occlusal trauma has been associated with periodon-
tal disease for over 100 years. In 1901, Karolyi (48)
reported an apparent association between excessive
occlusal forces and periodontal destruction. In 1917
and 1926, Stillman (96, 97) indicated that excessive
occlusal force was the primary cause of periodontal
disease. Stillman felt that occlusal forces must be
controlled in order to prevent and treat periodontal
151
#
PERIODONTOLOGY 2000
disease. These early reports created a background for
controversy that continues to this day:
Is there an association between excessive occlusal
forces and the progression of periodontal disease?
How are occlusal forces evaluated in a clinical
setting and at what point does an occlusal force
become `èxcessive''?
When an occlusal force is detected and the deter-
mination is made that it is excessive, when should
treatment be initiated and how should this treat-
ment be accomplished?
To effectively evaluate the role of occlusal trauma
in periodontal disease, it is necessary to review stu-
dies that have used human autopsy material or ani-
mal models. As previously stated, several early
authors felt that occlusal forces were the initiating
factor in periodontal disease and led to the ongoing
progression of the periodontal lesion. The associa-
tion between occlusion and periodontal disease was
based on clinical observation as opposed to scientic
evaluation. In an attempt to demonstrate this rela-
tionship, several animal studies on sheep and mon-
keys were conducted. These studies were an attempt
to determine the response of the periodontium to
occlusal forces both clinically and histologically (8,
98). The authors of these studies felt that their nd-
ings showed that excessive occlusal forces were a
contributing factor in the progression of periodontal
disease. By the end of the 1930's, many practitioners
felt that excessive occlusal forces (occlusal trauma)
were a causative factor in periodontal disease, that
occlusal adjustment was a necessary part of period-
ontal treatment, and that occlusal discrepancies
should be prophylactically treated to prevent period-
ontal disease (9, 60).
The role of excessive occlusal forces in the patho-
physiology of periodontal disease has been disputed
by several researchers. Orban & Weinmann in 1933
(70) and Weinmann in 1941 (109), using human
autopsy material, evaluated the effect of excessive
occlusal forces on the periodontium. They concluded
that there was no relationship between occlusal
forces and periodontal destruction and suggested
that occlusal forces played no part in periodontal
destruction. Instead, they indicated that gingival
inammation extending into the supporting bone
was the cause of periodontal destruction.
During the 1950's and 1960's, further animal
research using rats, monkeys, and dogs evaluated
the effect of occlusal forces on the periodontium
(13, 20, 59, 73, 110). The designs of these studies were
more controlled than most of the earlier investiga-
tions. The results from these studies did not support
Fig. 1. Primary occlusal trauma results from excessive
occlusal forces applied to a tooth with normal support.
Note that the center of rotation is near the middle of the
root. (From: T. G. Wilson Jr. et al. Advances in periodontics,
1992. Printed with permission, Quintessence Publishing
Co., Inc.).
Fig. 2. Secondary occlusal trauma results from excessive
occlusal forces applied to a tooth with reduced support.
Note that the center of rotation is in the apical third of the
root. (From: T. G. Wilson Jr. et al. Advances in periodontics,
1992. Printed with permission, Quintessence Publishing
Co., Inc.).
Hallmon & Harrel
152
the concept that excessive occlusal force was a cau-
sative agent of periodontal destruction. Further,
many of the studies showed no obvious association
between occlusal forces and periodontal disease.
In contrast to the above noted studies, at approxi-
mately the same time Glickman and co-workers pub-
lished studies based on animal models and human
autopsy material. Animal studies using a heavy
occlusal contact created by placing a ``high'' restora-
tion were performed utilizing dogs and monkeys (30,
33). While these studies showed no evidence of initia-
tion of periodontal disease by occlusal contacts, the
authors felt that a study using rhesus monkeys
demonstrated a phenomenon described as an
`àltered pathway of destruction'' when excessive
occlusal forces were present (31). This altered path-
way of destruction was described as a change in the
orientation of the periodontal and gingival bers
which occurred in the presence of excessive occlusal
forces, allowing gingival inammation to extend
along the periodontal ligament. The altered pathway
of destruction was postulated to cause vertical bony
defects due to inammation and bony destruction
following the periodontal ligament. Another animal
study (34) showed that bone in bifurcation areas was
stressed by excessive occlusal force and that bone
loss in the furcation area was related to these forces.
Glickman and coworkers also reported evidence of
an altered pathway of destruction in studies utilizing
human autopsy material (30).
From these studies, Glickman and co-workers con-
cluded that excessive occlusal forces in the presence
of plaque-associated inammation caused a change
in the alignment of the periodontal ligaments, allow-
ing an altered pathway of inammation/destruction,
resulting in vertical bony defects. Because there were
two separate pathologic processes working together
to cause bone loss, the process was termed a ``co-
destructive'' effect. Glickman and coworkers sum-
marized their work in a series of review articles (25,
2729, 32). These papers indicated that excessive
occlusal forces (trauma from occlusion) were a co-
destructive force in the presence of gingival inam-
mation and could lead to vertical osseous defects.
Based on these observations, the use of occlusal
adjustment was advocated as part of the treatment
for existing periodontal disease. Because no evidence
existed that excessive occlusal forces initiated peri-
odontal disease, occlusal adjustment to prevent per-
iodontitis was not advocated.
Waerhaug (103105) evaluated a large number of
human autopsy specimens to determine the relation-
ship of subgingival plaque to the morphology of oss-
eous defects and any associations with the presence
or absence of excessive occlusal forces. He found that
the ``plaque front'' (i.e. the apical border of the sub-
gingival plaque) was always in very close approxima-
tion to the epithelial attachment level and always
followed the morphology of the bony defect. In addi-
tion, the relationship of the plaque level between
adjacent teeth (either at the same or different apico-
coronal levels) was associated with either horizontal
or vertical interproximal bone loss. He also observed
that excessive occlusal forces bore no relationship to
the underlying bony defect and that vertical defects
were found equally around traumatized and non-
traumatized teeth. Waerhaug concluded that bone
loss was always associated with the downgrowth of
plaque and there was no relationship between exces-
sive occlusal forces and vertical bone loss.
The use of human autopsy material to study the
effect of occlusal forces has the inherent problem
that rarely if ever is there a true understanding of
the patient's occlusal relationship that existed in life.
Some knowledge can be obtained by studying the
wear patterns on the teeth but there is no assurance
that the teeth actually occluded in the assumed man-
ner or that wear facets represent current and active
occlusal trauma. Therefore, any conclusion or obser-
vations based on autopsy material concerning the
role that occlusal forces may or may not have on
the progression of periodontal disease has to be
questioned. A single histologic study (95) evaluated
the occlusal relationships of four patients prior to the
removal of their jaws for cancer therapy. This study
did not show a relationship between occlusal forces
and periodontal disease. However, it is unclear if
excessive occlusal forces existed in these patients.
Two extensive animal studies were performed in
the 1970s. These studies evaluated the effect of pla-
que and excessive occlusal forces in the animal mod-
els utilized. Unlike most of the earlier investigations,
stringent scientic controls and designs were used.
One series of studies were conducted by Polson and
co-workers (47, 71, 7683) and a different series of
studies was performed by Lindhe and co-workers
(1719, 5557, 67, 68, 100102). Polson's group used
squirrel monkeys and mesialdistal compression
forces comparable to orthodontic forces whereas
Lindhe's group used beagle dogs and applied buc-
callingual forces using a high occlusal contact and a
nger spring. Both groups investigated excessive
occlusal forces in the presence and absence of pla-
que.
These studies yielded similar results despite the
different animal models and the different excessive
153
Occlusal analysis, diagnosis and management in periodontics
occlusal forces used. Excessive occlusal forces in the
absence of plaque were found to cause loss of bone
density and mobility of the affected tooth but no
evidence was found that the occlusal forces alone
could cause attachment loss. When the excessive
occlusal forces were removed, it was noted that the
loss of bone density was reversible. In the presence of
plaque, inammation of the gingiva and periodontal
supporting structures were noted and in the pre-
sence of excessive occlusal forces and plaque
together there was an indication that more bone
density was lost in both animal models. In the beagle
dog model there was evidence of attachment loss
when plaque and excessive occlusal forces were both
present. These results were not observed in the squir-
rel monkey model.
These two series of studies exhaustively evaluated
the relationship of occlusal forces and plaque in an
animal model. They both concluded that there was
no evidence indicating that excessive occlusal force
alone will cause loss of attachment. The studies on
beagle dogs showed that under specic circum-
stances there may be attachment loss when plaque
and excessive occlusal forces are both present. Both
studies agreed that the removal of plaque and the
control of inammation will stop the progression of
periodontal disease whether or not excessive occlusal
forces are present.
Human studies
Only a few studies have evaluated the effects of
excessive occlusal forces in humans. There are many
ethical difculties associated with the non-treatment
of diagnosed periodontal disease that complicate
studying the effect of occlusion on the progression
of periodontal disease. The gold standard of clinical
research is the randomized controlled clinical trial.
These studies require prospective comparisons of
different treatment methods on treatment outcomes.
However, in order to compare the combined effects
of excessive occlusal forces and periodontal disease,
it would be necessary to treat one group of patients
while leaving the other group untreated. This creates
an unacceptable ethical dilemma due to the known
deleterious effects of the non-treatment of period-
ontal disease. The World Workshop in Periodontics
stated, ``Prospective studies on the effect of occlusal
forces on the progression of periodontitis are not
ethically acceptable in humans'' (23). As a result,
human studies are limited to retrospective and
observational research.
It has been reported that patients who have occlu-
sal discrepancies have no more severe periodontal
destruction than do patients without occlusal discre-
pancies (46, 51, 74, 84, 85, 94). However, it has also
been reported that molars with furcation invasion
and mobility have greater probing depths than
molars that are clinically nonmobile (106). The
increased mobility noted in this study may have been
due to occlusal factors or to greater loss of bony
support associated with the furcation involvement.
Due to the inability to determine whether occlusal
factors or bone loss was initially present, it is impos-
sible to draw a clear relationship between occlusal
discrepancies, mobility, and probing depths from
this study. Other studies reported that patients who
received occlusal adjustment as part of their period-
ontal therapy had greater attachment gain than
patients who did not receive occlusal adjustment
(10, 21). These studies suggest that occlusal adjust-
ment should be performed, where indicated, as a
part of periodontal treatment. A report on risk factors
for periodontal destruction indicated that mobility
and parafunctional habits that are not treated with
a biteguard are associated with increased attachment
loss, worsening prognosis, and tooth loss (61). This
study seems to indicate that untreated (i.e. no bite-
guard) parafunctional habits may contribute to
increased periodontal breakdown. Another study
has shown that mobile teeth treated with regenera-
tive surgery did not respond as well as nonmobile
teeth (14). However, no association was drawn
between mobility and occlusal forces.
In a series of retrospective reports, private practice
patients were evaluated who were diagnosed with
advanced periodontal disease and had a comprehen-
sive treatment plan recommended that included sur-
gical treatment. Occlusal adjustment was
recommended if signicant occlusal discrepancies
were detected. Some of these patients self-selected
to not have any periodontal treatment performed
(untreated group). Other patients had only nonsur-
gical periodontal treatment performed (partially
treated group). Others followed through with all
recommended periodontal treatment including sur-
gery (fully treated group). The effect of occlusal dis-
crepancies was studied in each of these groups using
the individual tooth as the experimental unit (40, 41,
66). This means that the progression of periodontal
destruction or the improvement of the periodontium
for each tooth was followed over time. This study
design allowed for the evaluation of teeth with occlu-
sal discrepancies versus teeth without occlusal dis-
crepancies rather than comparing patients with
154
Hallmon & Harrel
occlusal discrepancies vs. patients without occlusal
discrepancies. This experimental approach differs
from most past studies where the patient was the
experimental unit and the changes in probing depth
or attachment levels were expressed as the ``patient
mean.'' Using the patient mean may tend to mask
changes that are occurring at the more active sites
and, thereby, give results that do not reect what is
actually occurring during localized disease progres-
sion.
These studies found that teeth with occlusal dis-
crepancies had deeper presenting probing depths
and worse prognoses than those teeth that did not
have occlusal discrepancies. Further, when teeth
with occlusal discrepancies were followed over time,
a signicant increase in probing depth and a worsen-
ing of prognosis was noted when compared to teeth
without occlusal discrepancies. Additionally, teeth in
the partially treated group that had received occlusal
adjustment showed a slowing of the progression of
periodontal destruction when compared to teeth
with occlusal discrepancies from the same group that
had not had occlusal adjustment. It was concluded
that occlusal discrepancies appear to be a signicant
risk factor that contribute to more rapid periodontal
destruction and that treatment of occlusal discrepan-
cies seemed to slow periodontal destruction. The
authors postulated that the reason for the difference
in their ndings and those of previous studies was
the use of the individual tooth as the experimental
unit, which they felt yielded a more accurate assess-
ment of the effect of occlusal discrepancies on the
periodontium (40, 41, 66).
In summary, animal and human studies have indi-
cated some association between occlusal discrepan-
cies/occlusal trauma and changes in the periodontal
supporting structures. Extensive animal studies have
shown that occlusal trauma does have an effect on
the periodontal supporting structure but does not
initiate breakdown of the attachment apparatus with
resulting measurable attachment loss. The human
studies have indicated that treating occlusal discre-
pancies may lead to better results following period-
ontal treatment. A study using a more contemporary
statistical analysis and utilizing individual sites as the
basis for comparison, has shown a strong association
between occlusal discrepancies and deeper pockets
(66).
Furthermore, existing research does not establish a
cause-and-effect relationship between occlusion and
periodontal disease. However, there are strong data
to indicate that occlusion is a potential risk factor
for periodontal breakdown and that controlling this
risk factor may slow the progression of periodontal
destruction and improve the results of periodontal
treatment outcomes. As is the case with all risk factors
such as smoking, oral hygiene, and systemic factors,
the effect of occlusion on periodontal disease needs
to be minimized by recognizing the risk, diagnosing
the existence of the risk factor, and minimizing the
risk by the use of various treatment modalities such
as selective grinding, orthodontics, and/or occlusal
appliances.
Howis occlusal trauma detected
clinically?
Because trauma from occlusion is dened and diag-
nosed on the basis of histologic changes in the per-
iodontal supporting structure, a diagnosis of occlusal
trauma is impossible without block section biopsy.
Because this is clearly impractical for the clinical
practice of periodontics, the clinician must rely on
the clinical signs of potential occlusal trauma. The
following discussion is based for the most part on
clinical experience due to the extreme paucity of
written material on the subject.
Most periodontal training programs and the Amer-
ican Board of Periodontology require an analysis of
the patient's occlusal relationship as part of a com-
prehensive periodontal examination. Often the Angle
classication is part of this analysis (5). However, the
Angle classication was designed to quantify the ske-
letal relationship between the maxilla and the mand-
ible. While important in determining the growth
pattern of adolescents and recording a starting point
for orthodontic treatment, the Angle classication
has little bearing on the occlusal relationship that
exists between various cusp surfaces. The relation-
ship between cusps is the most important factor in
the transmittal of occlusal forces to the periodontal
supporting structures. Therefore, it is the relation-
ship between opposing cusps that is the most impor-
tant aspect of occlusion and any role it may play in
the progression of periodontal destruction or the
outcomes of periodontal treatment.
The relationship of opposing cusps is usually deter-
mined by using a composite of means that generate a
list of data that must be correlated by the practitioner.
The compiling of data on the relationship of opposing
cusps usually starts with the detection of occlusal
discrepancies. Typically, the initial contact between
the teeth is detected by gently manipulating the
patient's mandible into a ``retruded'' position (15,
26). There is little agreement as to what is meant by
155
a retruded position of the mandible but on a clinical
basis, an attempt is made to guide the mandible into
a position where both right and left condyles are
rmly placed in the fossa of the temporomandibular
joint. This position is one that is felt by the practi-
tioner rather than conrmed by any type of device or
instrument and is therefore subjective in nature.
Once the practitioner feels that a retruded position
has been achieved, the patient is asked to close until
the patient feels the rst contact between the teeth
(Fig. 3a). This contact point is veried by the exam-
iner either by eye, inked marking paper or ribbon, or
both. This initial contact in a retruded position has
been described as contact in ``centric relation''. Fol-
lowing the establishment of the initial contact, the
patient is asked to continue to close the jaws together
until maximum contact between the teeth is
achieved. The jaw position of maximum tooth con-
tact is often termed ``centric occlusion''. The position
of maximum tooth contact is assumed to be the
position that the patient will naturally move to as
the most comfortable or habitual position (Fig. 3b,c).
The distance that the patient moves between the
retruded initial contact and the point of maximal
tooth contact is termed the slide between the posi-
tions of centric relation and centric occlusion. This
slide is often described as the ``centric relation/cen-
tric occlusion slide'' or ``CR/CO shift''. This slide is
usually recorded as the length of the slide in the
anterior, vertical, and lateral planes (26).
No direct correlation with histologic evidence of
trauma from occlusion has been shown between the
presence of a slide between the contacts in centric
relation and the contacts in centric occlusion. How-
ever, indirect clinical evidence of a more rapid pro-
gression of periodontal destruction as evidenced by
increased probing depths has been shown to occur in
patients with untreated periodontal disease (40, 41,
66). While this nding cannot be directly correlated
with animal research showing histologic evidence of
inammation and bone rarefaction in the presence
of experimental occlusal stress and, in beagle dogs,
the loss of attachment when plaque is present in
addition to the experimental occlusal stress, there
is a likelihood that a similar process is occurring in
humans as in the beagle dog model. If this assump-
tion is true, then at least in certain cases the histo-
logic lesion of occlusal trauma is likely to be present
in periodontal patients who have occlusal interfer-
ences.
Other clinical ndings that have been associated
with trauma from occlusion are tooth mobility and
wear patterns on the occlusal surface of the teeth
(Table 1). These clinical ndings are extremely dif-
cult to correlate with occlusal contacts. In the case of
mobility, many other factors such as loss of attach-
ment can affect the presence and severity of the mobi-
lity. In the case of occlusal wear patterns, it is often
impossible to determine whether they are caused by
functional or parafunctional habits that are occur-
ring at present or whether they may be associated
with episodes of bruxism that have occurred in the
past. If bruxism has occurred in the past, what if any
part did it play in the current clinical evidence of
periodontal breakdown? The practitioner must eval-
uate and record all of these ndings so that a picture
of the occlusal stresses being placed on the period-
ontium can be assessed and to help form an assump-
tion of the occurrence of trauma from occlusion.
Table 1. Clinical indicators of occlusal trauma
Clinical indicators of occlusal trauma may include one
or more of the following:
1. Fremitus
2. Mobility (progressive)
3. Occlusal discrepancies
4. Wear facets in presence of other indicators
5. Tooth migration
6. Fractured tooth/teeth
7. Thermal sensitivity
Fig. 3. (a) Initial contact in centric relation. (b) Centric
slide between centric relation and centric (acquired cen-
tric; habitual) occlusion. (c) Centric (acquired centric;
habitual) occlusion, demonstrating maximum intercus-
pation or contact of the teeth of the opposing arches.
(Courtesy Dr. J. Y. Cho).
156
Hallmon & Harrel
What's abfractionandaretheredata
to support a role of occlusion in its
development?
Abfraction has been dened as the ``pathological loss
of hard tooth substance by biomechanical loading
forces'' (37). The lesions have been described as angu-
lar or wedge-shaped defects that occur at the cemen-
toenamel junction of affected teeth as a result of
exure and eventual fatigue of enamel and dentin
(7) (Fig. 4). The prismatic structure of enamel is strong
in compression, but vulnerable in areas of tension,
accounting for the resultant morphology of the pro-
posed lesion (52). It is further noted that occlusal
loads which generate cervical exure may disrupt
hydroxyapatite crystal bonds, and result in micro-
fracture and eventual loss of associated enamel (53).
Noncarious hard-tissue cervical lesions (NCLs)
have been classied as abrasive, erosive, corrosive,
abfractive or combined (38). In contrast to abfrac-
tion, abrasion represents a pathologic loss of tooth
substance resulting from biomechanical wear and is
exemplied by improper or overzealous toothbrush-
ing. This condition is generally accompanied by mar-
ginal tissue recession and may affect one or more
teeth (75) (Fig. 5). Erosion is a chemically induced
loss of tooth substance that occurs primarily through
acid dissolution (44) (Fig. 6). Attrition is dened as
the physiologic wearing away of a substance or struc-
ture, such as the teeth (3). This typically affects the
occlusal and incisal surfaces of the teeth and may
result from functional or parafunctional wear, man-
ifesting as facets (7). These highly polished surfaces
may appear on marginal, transverse and oblique
ridges, and on cusps and restored surfaces (Fig. 7).
Is there evidence for a role of
occlusal loading in the genesis of
noncarious cervical tooth loss?
The presence of lesions consistent with those
described as abfraction have been reported to
increase in size and depth with age of affected indi-
viduals (54). In a study of 913 subjects, 23% pre-
sented with such defects. Sixty-ve percent of the
affected individuals had conrmed parafunction as
compared to 35% who did not (35). Another study
reported that 96% of teeth with noncarious cervical
lesions also presented evidence of occlusal discre-
pancies (86). In comparing canine-guidance with
group-function occlusal relationships, abfraction-
like lesions were observed six times more often in
the latter group, suggesting that increased occlusal
tooth contact during lateral excursion favored the
occurrence of these lesions (87).
Fig. 6. The maxillary right central incisor shows erosion as
a result of chemically induced loss of enamel. (Courtesy
Dr. Terry D. Rees).
Fig. 5. Note the generalized loss of cervical tooth sub-
stance and accompanying marginal tissue recession. The
patient admitted (and demonstrated) aggressive horizon-
tal toothbrushing.
Fig. 4. Maxillary right rst premolar presents a well-deli-
neated noncarious hard-tissue cervical lesion consistent
with abfraction.
157
In addition to clinical observations, evidence sug-
gesting a role of occlusal loading in development of
abfraction-like lesions comes from various stress and
strain studies. These include articulated study mod-
els, strain-gauge studies, nite element stress analy-
sis and photoelastic stress analysis (88). Such studies
have shown that controlled loading (500 N) applied
to inner inclines of the buccal and lingual cusps of
mandibular premolars resulted in stress values
exceeding the failure stress of enamel (89). This load-
ing corresponds to that anticipated during excursive
contacts in group-function occlusal relationships.
These studies are principally in vitro investigations
that fail to take into account the role of the period-
ontal ligament and bone during the course of occlusal
loading. It is important to note that in a strain gauge
study of maxillary incisors in healthy volunteers,
large cervical surface strains were observed (65).
In a clinical investigation of the characteristics and
prevalence of abfraction-like lesions in a U.S. popu-
lation with 103 noncarious cervical lesions, the vast
majority of the lesions were strongly suspected as
being the result of toothbrush abrasion. A small sub-
set (i.e. 15 lesions) was deemed the result of some
other phenomenon. Six (40%) of these teeth mani-
fested premature occlusal contacts. Compared to
control teeth without NCLs, affected teeth did not
signicantly differ in terms of wear facets or occlusal
contacts. No NCLs were reported on lingual surfaces.
However, the authors cautioned that teeth with non-
carious wedge-shaped lesions should be carefully
evaluated for occlusal correction or bite-splint ther-
apy to address interferences that may contribute to
tooth exure (75). In a separate case study of 52
modern human skulls, occlusal ndings failed to
provide evidence for abfraction (43).
There is a strong contention that NCLs represent a
multifactorial phenomenon. It has beenproposedthat
the presence of occlusal stresses in combination with
acidic substances may result in greater damage than
either alone (39). However, one clinical question
remains Why does abfraction occur on the buccal/
facial surfaces, withonlyrare reports of lingual lesions?
In summary, available evidence suggests a plausi-
ble explanation for abfraction. It is equally apparent
that this diagnosis is not absolute. In cases where the
therapist is confronted with NCLs, toothbrushing
habits and occlusal relationships should be thor-
oughly evaluated. Restorative and/or periodontal
plastic surgical intervention should be preceded by
control of any habits and/or untoward occlusal load-
ing which may adversely affect predictable long-term
treatment outcome.
What methods are used to detect
hypermobility of teeth?
Assessment of tooth mobility constitutes a basic part
of the comprehensive periodontal examination (2, 4).
Hypermobility occurs in response to applied force
and is dependent upon its direction, frequency, mag-
nitude, distribution and type (2, 90, 107). The mobi-
lity status of the dentition has traditionally been
evaluated by visual assessment, using two instrument
handles to apply alternating luxating force orofacially
(Fig. 8). In 1950, Miller (63) described a mobility index
basedonthis approach(Table 2). Variations andmodi-
Fig. 7. The mandibular rst molar presents facets on the
mesiobuccal and distobuccal cusps and on the amalgam
restoration at the mesiolingual surface.
Fig. 8. One way to assess tooth mobility is by using two
instrument handles applied to the tooth and applying
alternating luxating force orofacially.
158
Hallmon & Harrel
cations of this index are used extensively throughout
dentistry, and especially periodontics (11, 20).
Clinical assessments of tooth mobility derived by
this means are somewhat arbitrary and dependent
on examiner subjectivity and interpretation. As a
result, they do not usually discriminate well between
small mobility increments (99). Consequently, a
means of more precise and objective measurement
of tooth mobility has been pursued and includes
mechanical, electronic and optical devices, and laser
Doppler vibrometry (12, 64, 69, 72, 92). Despite
objective approaches striving to standardize tooth
mobility evaluation, such devices by and large have
not been well accepted for use in clinical practice.
More recently, the Periotest
1
(Gulden-Medizintech-
nik, Bensheim, Germany), a device resembling a den-
tal handpiece, has gained favor in evaluating and
monitoring tooth mobility and clinical success of
dental implants (6, 93). The instrument is applied
orthoradially to the center of the anatomic crown,
delivering a standardized percussive force. Recorded
values range from 0 to 100, and correspond to time in
milliseconds taken for the supporting structure(s) to
respond to impact deceleration, thus assessing
rebound dynamics and damping characteristics of
the periodontium (42, 58, 91, 108). It is interesting
to note that no signicant change in numeric Peri-
otest1 values has been observed when comparing
baseline values with those recorded after the initial
phase of periodontal therapy (16). This would sug-
gest that Periotest1 mobility evaluation relates pri-
marily to the amount of bone loss (i.e. support) about
teeth being evaluated and may not be directly related
to traditional tooth mobility. Contraindications to
use of this instrument include acute inammation,
traumatic subluxation and dental implants in the
initial phases of healing (91).
One of the diagnostic challenges confronting the
clinician is to determine the associative cause of
the observed tooth mobility. It is widely recognized
that some degree of tooth mobility is always present
in the healthy dentition. This has been termed
physiologic (normal) tooth mobility, in contrast to
pathologic (abnormal) mobility. Factors that have
been associated with tooth mobility include period-
ontal status of the teeth (e.g. inammation, loss of
clinical attachment/bone), periodontal surgery,
occlusal trauma, pregnancy, and pathologic pro-
cesses affecting the jaws/teeth (22, 24, 36, 49, 50).
More recently, it has been proposed that the desig-
nators ``pathologic tooth mobility'' and `àdaptive
tooth mobility'' may be helpful in addressing mobi-
lity status and thus, facilitate management and
maintenance approaches in affected patients (4).
Stability of tooth mobility appears acceptable in
the absence of confounding variables, but progres-
sive mobility is a concern and should be addressed
by controlling inammation, occlusal adjustment
and considering a stabilization appliance or splinting
as indicated. The long-term therapeutic objective is
to maintain the health, stability, comfort and func-
tion of the patient's natural dentition (or implants).
Of what value are assessments of
tooth mobility in the management
of patients with periodontitis?
A limited number of human studies have suggested
that tooth mobility may be associated with greater
attachment loss, probing depth and bone loss when
compared to nonmobile teeth (45, 46, 106). It is dif-
cult to determine from these studies whether tooth
mobility was a result of the associated periodontal
disease process or if, in some way, it contributed
etiologically. Two studies reported that teeth exhibit-
ing a combination of furcation invasion and tooth
mobility were at risk of sustaining greater attachment
loss as compared to nonmobile teeth or teeth with
furcation invasion alone (45, 106). In a longitudinal
study by McGuire & Nunn (62), tooth mobility was
associated with non-improving prognoses of affected
teeth. Other studies have examined the effects of
tooth mobility on periodontal treatment outcomes.
In an 8-year follow-up study in which patients
received scaling, oral hygiene instruction, occlusal
adjustment, periodontal surgery (subgingival curet-
tage, modied Widman ap or pocket elimination)
and 3-month maintenance, baseline and annual
assessments were made of probing depth, attach-
ment level and tooth mobility. Results indicated that
pockets associated with mobile teeth did not respond
as positively (i.e. clinical attachment level gain) to
therapy as rm teeth. This inuence was observed
by the end of the rst year and became more
pronounced by the second year, with only minor
Table 2. Miller Mobility Index (63)
1. The first distinguishable sign of movement
greater than normal (physiologic)
2. Movement of the tooth which allows the
crown to move 1 mm from its normal position
in any direction
3. Teeth which may be rotated or depressed in
their alveoli
159
changes occurring throughout the duration of the 8-
year clinical trial. It should be noted that although
clinically mobile teeth could be effectively treated
and maintained, better responses (i.e. clinical attach-
ment level gain) were generally observed in associa-
tion with rm teeth (21).
In a randomly controlled clinical trial examining
clinical outcomes and postoperative morbidity in
regenerative treatment of deep infrabony defects,
tooth mobility was assessed as a covariate. Among
clinical parameters evaluated in the study were clin-
ical attachment level, probing depth, recession, tooth
mobility, full-mouth plaque scores, and full-mouth
bleeding scores. These were assessed immediately,
prior to surgery and at 1-year post-treatment. Tooth
mobility was evaluated with a purpose-built electro-
nic device (Periotest1). Baseline tooth mobility was
signicantly associated with a reduction in antici-
pated amounts of clinical attachment level gain.
Based on the results of this study, the authors sug-
gested that clinicians may want to consider reducing
tooth mobility prior to attempting periodontal regen-
erative therapy to facilitate therapeutic success (14).
Other studies have examined the effect of splinting
on tooth mobility after initial therapy and after oss-
eous surgery. In the initial therapy study, there was a
reduction in tooth mobility over the 17-week period,
but there was no difference between the splinted and
non-splinted sites. The reduction in mobility
observed in both study groups was attributed to
reduction of inammation and occlusal adjustment
accompanying initial therapy (49). No difference was
observed between splinted and non-splinted teeth
receiving osseous surgery, indicating that splinting
had no lasting effect on tooth mobility. Mobility pre-
dictably increased following surgery, but returned to
baseline presurgical levels after 6 months (22).
In summary, although a signicant role has been
suggested regarding the effect of tooth mobility on
treatment results, it is clear that controlled interven-
tion studies will be necessary in order to clarify the
effect of reducing baseline tooth mobility on period-
ontal treatment outcomes. Until such studies are
conducted, it would appear prudent, based on avail-
able data, to consider reduction/control of tooth
mobility as an integral part of periodontal therapy.
Under what clinical conditions is
occlusal adjustment indicated?
Based on current knowledge, it seems that research
supports the nding that occlusal discrepancies
between centric relation and centric occlusion con-
tribute to the progression of periodontal destruction
and that the presence of mobility will negatively
affect the outcome of periodontal treatment. What
part each of these actually plays in periodontal
destruction, whether these ndings are related to
each other or play separate roles, and whether either
or both of these ndings are associated with the
classic histologic denition of occlusal trauma is
unknown. Furthermore, due to ethical considera-
tions, it is unlikely that these questions will ever be
satisfactorily answered. However, when periodontal
disease and destruction are present, the currently
available evidence appears to support the need for
occlusal treatment that will minimize occlusal inter-
ferences and help decrease tooth mobility.
The treatment of occlusion usually involves either
a reversible approach consisting of some type of bite
appliance (i.e. ``night guard'') and/or the selective
grinding of the occlusal surfaces of the teeth (Fig. 9).
Orthodontic therapy is also an effective method of
changing occlusal relationships and minimizing
Fig. 9. (a) Hard acrylic occlusal nightguard in place. As the
patient's jaw moves into a left lateral excursion, note the
disclusion of the teeth in the anterior and right side. (b)
Note the smooth, highly polished occlusal surface and the
presence of ball clasps between the second premolar and
the rst molar to facilitate retention.
160
Hallmon & Harrel
occlusal forces between opposing teeth. However,
orthodontics is rarely used during the active phase
of periodontal therapy and selective grinding of the
contacting surfaces is usually necessary following
orthodontic therapy. Appliance therapy has the
advantage of causing no permanent changes to the
occlusal surfaces and is therefore fully reversible.
However, appliance therapy is only effective in con-
trolling occlusal forces when the appliance is worn
by the patient. Selective grinding involves the non-
reversible reshaping of occlusal surfaces but has the
advantage of minimizing occlusal forces at all times.
The selection of the treatment modality for treating
occlusal stresses must take into consideration opera-
tor skill and condence in selective grinding, the
presence or absence of parafunctional habits, the
presence or absence of muscle splinting, and the
patient's psychological state.
Measurement of the outcomes from occlusal ther-
apy usually cannot be readily achieved. In cases
where the patient is experiencing discomfort from
occlusal contact, the relief of heavy occlusal pressure
by selective grinding may elicit immediate relief of
the patient's symptoms. In most cases, however, the
changes are subtle and can only be measured in
terms of decreased mobility and long-term favorable
results to periodontal therapy. Due to the fact that
other treatments routinely performed during period-
ontal therapy will also tend to improve mobility and
contribute to long-term favorable outcomes, it is dif-
cult to determine to what extent occlusal treatment
has played a role in any clinical improvements
observed. However, just as periodontal disease
results from a combination of risk factors and con-
tributing causes, long-term favorable outcomes and
decreases in mobility are probably due to the elim-
ination or relief of multiple risk factors. The treat-
ment of periodontal disease consists of attempting to
control the risk factors for the disease such as bac-
terial plaque and the deeper pockets, which harbor
reservoirs of plaque bacteria, ameliorate negative
habits such as smoking, and controllable systemic
factors such as diabetes. The treatment of occlusal
discrepancies/occlusal trauma should also be viewed
in the context of the control of one of the risk factors
contributing to periodontal disease. The successful
treatment of periodontal disease requires the control
of all controllable risk factors. If occlusal discrepan-
cies exist in the presence of periodontal disease, the
occlusal factors should be controlled by the minimi-
zation of the occlusal forces. In other words, occlusal
treatment should be performed, where indicated, as
a routine part of periodontal therapy.
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164
Hallmon & Harrel
Diagnosis, prognosis and
decision-making in the treatment
of combined periodontal-
endodontic lesions
Ilan Rotstein & James H. S. Simon
The pulp and periodontium are intimately related. As
the tooth develops and the root is formed, three main
avenues for communication are created: dentinal
tubules, lateral and accessory canals, and the apical
foramen.
Anatomic considerations
Dentinal tubules
Exposed dentinal tubules in areas of denuded
cementum may serve as communication pathways
between the pulp and periodontal ligament (Fig. 1).
Exposure of dentinal tubules may occur due to devel-
opmental defects, disease, or periodontal proce-
dures. In the root, dentinal tubules extend from the
pulp to the dentinocemental junction (73). They run
a relatively straight course and range in size from 1 to
3 mm in diameter (126). The diameter of the tubules
decreases with age or as a response to a continuous
low grade stimuli by the apposition of highly miner-
alized peritubular dentin. The number of dentinal
tubules varies from approximately 8,000 at the den-
tinocemental junction to 57,000 per square milli-
meter at the pulpal end (126). In the cervical area
of the root there are about 15,000 dentinal tubules
per square millimeter (73). These tubules may be
denuded of their cementum coverage as a result of
periodontal disease, surgical procedures or develop-
mentally when the cementum and enamel do not
meet at the cemento-enamel junction (CEJ) thus
leaving areas of exposed dentin. Patients experien-
cing cervical dentin hypersensitivity are an example
of such a phenomena.
Scanning electron microscopic studies have
demonstrated that dentin exposure at the CEJ
occurrs in 18% of teeth in general and in 25% of
anterior teeth in particular (132). Furthermore, the
same tooth may have different CEJ characteristics
with dentin exposure on one side while the other
sides are covered with cementum (162). This area
becomes important in assessing the progression of
endodontic pathogens (Fig. 2), as well as the effect of
root scaling and planing on cementum integrity, and
bleaching-induced root resorption following the use
of 30% hydrogen peroxide (50, 78, 153, 154).
Other areas of dentinal communication may be
through developmental grooves, both palatogingival
and apical (173).
Lateral and accessory canals
Lateral and accessory canals may be present any-
where along the root (Fig. 3). Their prevalence and
location have been well documented in both animal
and human teeth (26, 44, 69, 101, 115, 141, 155).
It is estimated that 3040% of all teeth have lateral
or accessory canals and the majority of them are
found in the apical third of the root (73). DeDeus
(44) found that 17% of teeth had lateral canals in
the apical third of the root, about 9% in the middle
third, and less than 2% in the coronal third. However,
it seems that the prevalence of periodontal disease
associated with lateral canals is relatively low. Kirk-
ham (101) studied 1,000 human teeth with extensive
periodontal disease and found only 2% had lateral
canals located in a periodontal pocket.
Accessory canals in the furcation of molars may
also be a direct pathway of communication between
165
#
PERIODONTOLOGY 2000
the pulp and the periodontium (69, 115). The pre-
valence of accessory canals may vary from 23% to
76% (26, 64, 101). These accessory canals contain
connective tissue and vessels that connect the circu-
latory system of the pulp with that of the period-
ontium. However, all these canals do not extend
the full length from the pulp chamber to the oor
of the furcation (64). Seltzer et al. (163) reported that
pulpal inammation may cause an inammatory
reaction in the interradicular periodontal tissues.
The presence of patent accessory canals is a potential
pathway for the spread of bacterial and toxic bypro-
ducts, resulting in a direct inammatory process in
the periodontal ligament (Fig. 4).
Apical foramen
The apical foramen is the principal and most direct
route of communication between the pulp and per-
iodontium. Bacterial and inammatory byproducts
may exit readily through the apical foramen to cause
periapical pathosis. The apex is also a portal of entry
of inammatory byproducts from deep periodontal
pockets to the pulp. Pulp inammation or pulp
necrosis extends into the periapical tissues causing
a local inammatory response accompanied with
bone and root resorption (Fig. 5). Endodontic ther-
apy is targeted to eliminate the intraradicular etiolo-
gic factors thus leading to healing of the periapical
tissues.
Fig. 1. (A) Scanning electron micrograph of open dentinal
tubules. (B) Higher magnication. Note absence of
odontoblastic processes.
Fig. 2. Photomicrograph of bacteria in open dentinal
tubules.
Rotstein & Simon
166
Endodontic disease and the
periodontium
When the pulp becomes necrotic, there is a direct
inammatory response by the periodontal ligament
at the apical foramen and/or opening of accessory
canals (164) (Figs 4 and 5). Inammatory byproducts
of pulpal origin may leach out through the apex,
lateral and accessory canals and dentinal tubules to
trigger an inammatory vascular response in the per-
iodontium. Among those are living pathogens such
as bacteria and their toxic byproducts, fungi and
viruses (14, 40, 49, 70, 88, 91, 187), as well as non-
living pathogens (52, 133, 169, 181). Many of these
are similar pathogens encountered in periodontal
infections. In certain cases pulpal disease will stimu-
late epithelial growth that will affect the intergrity of
the periradicular tissues (136, 170).
The effect of periodontal inammation on the pulp
is controversial and conicting studies abound (2, 3,
17, 18, 38, 63, 122, 163, 183, 199). It has been
suggested that periodontal disease has no effect on
the pulp, at least until it involves the apex (38). On
the other hand, several studies suggested that the
effect of periodontal disease on the pulp is degen-
erative in nature including an increase in calcica-
tions, brosis and collagen resorption, as well as a
direct inammatory affect (108, 118).
It seems that the pulp is usually not directly
affected by periodontal disease until recession has
opened an accessory canal to the oral environment.
At this stage, pathogens penetrating from the oral
cavity through the accessory canal into the pulp
may cause a chronic inammatory reaction and pulp
necrosis. However, as long as the accessory canals
are protected by sound cementum, necrosis usually
does not occur. In addition, if the microvasculature
of the apical foramen remains intact, the pulp will
maintain its vitality (108). The effect of periodontal
treatment on the pulp is similar during scaling and
root planing or periodontal surgery if accessory
canals are severed and/or opened to the oral envir-
onment. In such cases microbial invasion and sec-
ondary necrosis of the pulp can occur.
Etiologic factors
Live pathogens
Among the live pathogens encountered in a diseased
pulp and periapical tissues are: bacteria (Fig. 6),
fungi (Fig. 7), and viruses (Fig. 8). These pathogens
and their byproducts may affect the periodontium in
a variety of ways and need to be eliminated during
root canal treatment.
Bacteria
Endodontic disease is caused by bacteria (58, 93,
146). The periapical tissues become involved when
bacteria invade the pulp, causing either partial or
total necrosis. The relationship between the presence
of bacteria and pulpal and periapical diseases was
demonstrated by Kakehashi et al. in a classic work
(93). In that study, pulps of normal (conventional)
rats were exposed and left open to the oral environ-
ment. Consequently, pulp necrosis ensued, followed
by periapical inammation and lesion formation.
However, when the same procedure was performed
on germ-free rats, not only did the pulps remain vital
and relatively non-inamed, but the exposure sites
were repaired by dentin. The study demonstrated
that without bacteria and their products, periapical
lesions of endodontic origin do not occur. Moller et al.
(127) conrmed these ndings in monkeys. They
Fig. 3. (A) Postoperative radiograph showing multiple lat-
eral canals in a mandibular second molar with apical and
furcal radiolucencies. (B) One-year follow-up radiograph
showing bony healing.
167
Combined periodontal-endodontic lesions
reported that non-infected necrotic pulp tissue did
not induce periapical lesions or inammatory reac-
tions. However, once the pulp became infected, peri-
apical lesions and inammation in the apical tissues
ocurred. Korzen et al. (105) reported similar results
and suggested that pulpal infections are by nature
usually mixed infections.
Blomlof et al. (22) created defects on root surfaces
of intentionally extracted monkey teeth with either
open or mature apices. The canals were either
infected or lled with calcium hydroxide and
replanted back in their sockets. After 20 weeks, mar-
ginal epithelial downgrowth was found on the
denuded dentin surface of the infected teeth. Jansson
et al. (86) assessed the effects of endodontic patho-
gens on marginal periodontal wound healing of
denuded dentinal surfaces surrounded by healthy
periodontal ligament. Their results showed that in
infected teeth, the defects were covered by 20% more
epithelium, whereas the non-infected teeth showed
only 10% more connective tissue coverage. Jansson
et al. (87) concluded that pathogens in necrotic root
canals may stimulate epithelial downgrowth along
denuded dentin surfaces with marginal communica-
tion and thus augment periodontal disease. The
same group of investigators (89), in a retrospective
radiographic 3-year study, evaluated 175 endodonti-
cally treated single-rooted teeth of 133 patients.
Patients who were more prone to periodontitis and
exhibited evidence of endodontic treatment failures
showed about a 3-fold increase in marginal bone loss
as compared to patients without endodontic infec-
tion. Jansson & Ehnevid (86) also investigated the
effect of endodontic infection on periodontal prob-
ing depth and the presence of furcation involvement
in mandibular molars. They found that endodontic
infection in mandibular molars was associated with
more attachment loss in the furca. These authors
suggested that endodontic infection in molars asso-
ciated with periodontal disease may enhance period-
ontitis progression by spreading pathogens through
accessory canals and dentinal tubules.
Proteolytic bacteria predominate in the root canal
ora, which changes over time to a more anaerobic
microbiota (55, 179). Rupf et al. (156) studied the
proles of periodontal pathogens in pulpal and per-
iodontal diseases associated with the same tooth.
Specic PCR methods were used to detect Actinoba-
cillus actinomycetemcomitans, Tannerella forsythen-
sis, Eikenella corrodens, Fusobacterium nucleatum,
Porphyromonas gingivalis, Prevotella intermedia,
and Treponema denticola. These pathogens were
found in all endodontic samples and the same
pathogens were found in teeth with chronic apical
Fig. 4. Micrograph stained with Massons Trichrome of a
maxillary lateral incisor with a necrotic pulp associated
with a lateral inammatory process in the periodontal
ligament. (A) Main canal, accessory canal, and the resultant
inammatory response in the periodontal ligament are evi-
dent. (B) Higher magnication of the area shows chronic
inammation with proliferating epithelium.
168
Rotstein & Simon
periodontitis and chronic (adult) periodontitis. They
concluded that periodontal pathogens often accom-
pany endodontic infections and supported the idea
that endodonticperiodontal interrelationships are a
critical pathway for both diseases.
Spirochetes are another type of microorganism
associated with both endodontic and periodontal
diseases. Spirochetes are usually found more fre-
quently in subgingival plaque than in root canals.
Several studies revealed a large diversity of oral tre-
ponemes present in subgingival biolms of period-
ontal pockets (29, 45, 95).
It has been suggested that the presence or absence
of oral spirochetes can be used to differentiate
between endodontic and periodontal abscesses
(187). Today, the presence of spirochetes in the root
canal system is well documented and has been
demonstrated by different identication techniques
such as darkeld and electron microscopy, checker-
board DNADNA hybridization analysis, and 16S
rRNA gene proles (24, 39, 40, 91, 92, 129, 150, 174).
The differences in the prevalence of spirochetes
associated with endodontic disease reported by the
various authors may be attributed to the different
methodologies used. Recent studies demonstrated
that the spirochete species most frequently found
in root canals are T. denticola (150, 174) and Trepo-
nema maltophilum (92). The main virulence factor of
T. denticola includes surface-expressed molecules
with cytotoxic activities such as the major surface
protein and the chymotrypsin-like protease complex,
extracellular or membrane-associated proteolytic
and hydrolytic enzymes, and metabolites (56). This
organism possesses an array of virulence factors
associated with periodontal disease and may also
participate in the pathogenesis of periradicular dis-
ease (150). T. maltophilum is a small, motile trepo-
neme with two periplasmic agella. Although the
virulence factors of this microorganism have not
yet been fully studied, it has been proposed that
the motility of T. maltophilum, caused by the rota-
tion of its periplasmic agella, might contribute to its
pathogenicity (81). T. maltophilum has also been
frequently isolated from patients with rapidly pro-
gressing forms of periodontitis (131).
It has also been suggested that L-form bacteria
may have a possible role in periapical disease
(172). Some bacterial strains can undergo morpho-
logical transition to their L-form after exposure to
certain agents particularly penicillin (96). The L-form
and the bacterium may appear individually or
together and may transform from one variant to
another with numerous intermediate L-form transi-
tional stages. This may occur spontaneously or by
induction in a cyclic manner. Under certain condi-
tions, depending on host resistance factors and bac-
terial virulence, the L-forms revert to their original
pathogenic bacterial form and may be responsible
for acute exacerbation of chronic apical lesions (172).
Fungi (yeasts)
The presence and prevalence of fungi associated with
endodontic disease is well documented (49). Yeast
colonization associated with radicular pathosis has
been demonstrated in untreated root caries (85, 198),
dentinal tubules, (42, 99, 166), failing root canal treat-
ments (128, 134, 142, 180), apices of teeth with
asymptomatic apical periodontitis (114), and in peri-
apical tissues (186). Many studies reported that the
prevalence of fungi in cultured root canal systems
varied from 0.5% to 26% in untreated root canals
Fig. 5. (A) Scanning electron micrograph of the apical
third of a root associated with a periapical inammatory
lesion. Multiple areas of external inammatory root
resorption are evident. (B) Section through the apex of a
maxillary central incisor with pulp necrosis and periapical
lesion. Note opening of accessory canal and dentinal
resorption of the inner surface of the foramen.
169
(15, 65, 85, 98, 110) and from 3.7% to 33% in cases
of previously treated canals (85, 128, 180, 186, 191).
Several studies have demonstrated a higher preva-
lence of 40% to 55% (137, 166). The majority of the
recovered fungi were Candida albicans (191). C. albi-
cans has been detected in 21% of infected root canals
using 18S rRNA directed species-specic primers
(15). C. albicans also showed the ability to colonize
canal walls and penetrate into dentinal tubules (144).
Other species such as Candida glabrata, Candida
guillermondii, and Candida incospicia (191) and
Rodotorula mucilaginosa (49) were also detected.
Factors affecting the colonization of the root canal
by fungi are not fully understood. It appears, how-
ever, that among the predisposing factors of this
process are immunocompromising diseases such as
cancer (42), certain intracanal medicaments (85),
local and systemic antibiotics (121, 198), and pre-
vious unsuccessful endodontic therapy (176, 180).
It has been hypothesized that the reduction of
Fig. 6. Periapical Actinomyces infection. This case graphi-
cally shows the growth of bacteria past the apical foramen
and its invasion of apical cementum and periapical tissues.
(A) Radiograph of a maxillary central incisor with a necrotic
pulp showing a large periapical lesion. (B) Nonsurgical
endodontic therapy was done but the tooth continued to
be symptomatic. (C) Apical surgery was then performed.
Photomicrograph shows part of the root with the attached
lesion. (D) Colonies of Actinomyces in the lumen of the
lesion are evident. (E) Higher magnication shows large
colony of Actinomyces. (F) Foamy macrophages attacking
the bacteria. (G) Edge of the bacterial megacolony showing
the absence of inammatory cells that are unable to pene-
trate the colony. (H) Higher magnication of the bacterial
colony. (I) Center of the colony untouchedby the inamma-
tory cells. (J) Viable bacteria within the apical cementum.
170
Rotstein & Simon
specic strains of bacteria in the root canal during
endodontic treatment may allow fungal overgrowth
in the low nutrient environment (176, 180). Another
possibility is that fungi may gain access from the oral
cavity during treatment as a result of poor asepsis. It
has been found that approximately 20% of chronic
periodontitis patients also harbor subgingival yeasts
(41, 178). As in endodontic infections, C. albicans was
also the most common species of fungi isolated (71).
Recently, it has been demonstrated that the pre-
sence of fungi in root canals is directly associated
with their presence in saliva (49). These ndings
further stress the importance of using aseptic endo-
dontic and periodontal techniques, maintaining the
integrity of dental hard tissues, and covering the
tooth crown as soon as practical with a well-sealed
permanent restoration in order to prevent coronal
leakage.
Viruses
There is increasing evidence to suggest that viruses
play an important role in both endodontic and per-
iodontal diseases. In patients with periodontal dis-
ease, herpes simplex virus is frequently detected in
gingival crevicular uid and in gingival biopsies of
periodontal lesions (32, 34). Human cytomegalovirus
was found in about 65% of periodontal pocket sam-
ples and in about 85% of gingival tissue samples (34).
EpsteinBarr virus type I was detected in more than
40% of pocket samples and in about 80% of the
Fig. 6. continued
171
gingival tissue samples (34). Gingival herpesviruses
were associated with increased occurrence of sub-
gingival P. gingivalis, T. forsythensis, P. intermedia,
Prevotella nigrescens, T. denticola, and A. actinomy-
cetemcomitans, suggesting that they may play a role
in promoting overgrowth of pathogenic periodontal
bacteria (33).
In endodontics, the presence of viruses in the den-
tal pulp was rst reported in a patient with AIDS (62).
DNA of HIV virus has also been detected in perira-
dicular lesions (51). However, it has not been estab-
lished that HIV virus can directly cause pulpal
disease. Herpes simplex virus was also studied in
relation to endodontic disease. However, unlike its
role in periodontal disease, it appears that herpes
simplex virus is not associated with endodontic dis-
ease (79, 148, 158). On the other hand, recent data
suggest that other common types of human viruses
may be involved in pulpal disease and in the devel-
opment of periapical lesions. Sabeti et al. (157) sug-
gested that human cytomegalovirus and Epstein
Barr virus play a role in the pathogenesis of sympto-
matic periapical lesions. It appears that active infec-
tion may give rise to production of an array of
cytokines and chemokines with the potential to
induce local immunosuppression or tissue destruc-
tion (31). Herpesvirus activation in periapical inam-
matory cells may impair the host defense
Fig. 7. Fungi ina persistent periapical lesion. (A) Radiograph
of maxillary lateral incisor with necrotic pulp and periapical
radiolucency. (B) Immediatepostoperativeradiographshow-
inggoodnonsurgical treatment. (C) At the3-monthrecall the
patient is still symptomatic andthe periapical radiolucencyis
larger. (D) Transmissionelectronmicrographshows growing
hyphae of a fungus. (E) Higher magnication of the hyphae
showing the cell wall. (F) Reproductive fungi spores.
172
Rotstein & Simon
mechanisms and give rise to overgrowth of bacteria,
as seen in periodontal lesions. Herpesvirus-mediated
immune suppression may be detrimental in periapi-
cal infections due to already compromised host
responses in the granulomatous tissue (119).
Alterations between prolonged periods of herpes-
virus latency interrupted by periods of activation
may explain some burst-like symptomatic episodes
of periapical disease (158). Frequent reactivation of
periapical herpesvirus may support rapid periapical
breakdown. Absence of herpesvirus infection or viral
reactivation may be the reason that some periapical
lesions remain clinically stable for extended periods
of time (158).
Non-living etiologic agents
Depending on their origin and nature, non-living
etiologic agents can be either extrinsic or intrinsic.
Extrinsic agents
Foreign bodies
Foreign bodies are frequently found to be associated
with inammation of the periradicular tissues (Figs 9
and 10). Although endodontic and periodontal dis-
eases are primarily associated with the presence of
microorganisms, some treatment failures may be
explained by the presence of certain foreign sub-
stances in situ. These include substances such as
dentin and cementum chips (83, 200), amalgam
(61, 104, 200), root canal lling materials (61, 97,
104, 200), cellulose bers from absorbent paper
points (53, 103, 104), gingival retraction cords (57),
leguminous foods (125), and calculus-like deposits
(72). A foreign-body response may occur to any of
these substances and the clinical reaction may be
either acute or chronic. Therefore, such conditions
may be either symptomatic or asymptomatic. Micro-
scopically, these lesions demonstrate the presence of
multinucleated giant cells surrounding the foreign
material in a chronic inammatory inltrate.
Mechanical or surgical removal of the foreign bodies
is usually the treatment of choice.
Intrinsic agents
Cholesterol
The presence of cholesterol crystals in apical period-
ontitis is a common histopathologic nding (20, 25,
135, 167, 189). With time, the cholesterol crystals
would be dissolved and washed away, leaving behind
the spaces they occupied as clefts. The reported pre-
valence of cholesterol clefts in periapical disease
varies from 18% to 44% (25, 167, 189). It has been
suggested that the crystals could be formed from
cholesterol released by disintegrating erythrocytes
of stagnant blood vessels within the periapical lesion
(25), lymphocytes, plasma cells and macrophages,
which die in great numbers and disintegrate in
chronic periapical lesions (189), or by the circulating
plasma lipids (167). However, it is possible that all of
these factors may contribute to the accumulation,
concentration and crystallization of cholesterol in a
periapical lesion (Fig. 11).
Accumulation of cholesterol crystals in inamed
periapical tissues in some cases has been suggested
to be one of the causes of failure of endodontic
therapy (133, 135). It seems that the macrophages
and the multinucleated giant cells that congregate
around cholesterol crystals are not efcient enough
to destroy the crystals completely. In addition, the
accumulation of macrophages and giant cells
around the cholesterol clefts in the absence of other
inammatory cells, such as neutrophils, lympho-
cytes and plasma cells, suggests that the cholesterol
crystals induce a typical foreign-body reaction
(133).
Russell bodies
Russell bodies can be found in most inamed tissues
throughout the body including the periradicular tis-
sues (Fig. 12). These are small, spherical accumula-
tions of an eosinophilic substance found within or
near plasma cells and other lymphoid cells. The
Fig. 8. Transmission electron micrograph of the nucleus
of a macrophage in a periapical lesion showing a possible
viral infection.
173
presence and occurrence of Russell bodies in oral
tissues and periapical lesions is well documented
(60, 113, 120).
Several studies have indicated the presence of Rus-
sell bodies in about 80% of periradicular lesions.
Recently, large intracellular and extracellular Russell
bodies were found also in inammatory pulpal tissue
of carious primary teeth (181). It is hypothesized that
Russell bodies are caused by the synthesis of exces-
sive amounts of normal secretory protein in certain
plasma cells engaged in active synthesis of immuno-
globulins. The endoplasmic reticulum becomes
greatly distended, producing large homogeneous
eosinophilic inclusions (35). However, the preva-
lence of Russell bodies, the mechanisms of their pro-
duction, and their exact role in pulpal inammation
have not yet fully elucidated.
Rushton hyaline bodies
The presence of Rushton hyaline bodies (RHB) is a
feature unique to some odontogenic cysts. Their fre-
quency varies from 2.6% to 9.5% (4). RHB usually
appear within either the epithelial lining or the cyst
lumen (Fig. 13). They have a variety of morphologic
forms, including linear (straight or curved), irregular,
rounded and polycyclic structures, or they may
appear granular (4, 52).
The exact nature of RHB is not fully understood. It
has been variously suggested that they are keratinous
in nature (167), of hematogenous origin (82), a spe-
cialized secretory product of odontogenic epithelium
(130), or degenerated red blood cells (52). Some
authors suggested that RHB are material left behind
from a previous surgical operation (124). It is not
Fig. 9. Foreign-body particles in a periapical lesion. (A)
Radiograph of a symptomatic maxillary central incisor
with a large periapical lesion. Endodontic treatment was
done 27 years ago. (B) Apical surgery was done and apical
tissue submitted for histologic analysis. Photomicrograph
shows foreign-body particles in the presence of giant cells.
(C) Higher magnication of the foreign-body particles and
giant cells. (D) Part of the foreign body. When put under
polarized light, the presence of vegetable matter was
apparent. The diagnosis was conrmed when parts of a
paper point penetrated past the apical foramen.
174
Rotstein & Simon
clear why the RHB form mostly within the epithe-
lium.
Charcot-Leyden crystals
Charcot-Leyden crystals (CLC) are naturally occur-
ring haexagonal bipyramidal crystals derived
from the intracellular granules of eosinophils and
basophils (1, 182, 195). Their presence is most
often associated with increased numbers of peri-
pheral blood or tissue eosinophils in parasitic, aller-
gic, neoplastic, and inammatory diseases (1, 109,
195).
Fig. 10. Multiple etiologic reasons for non-healing of area
past the apical foramen. (A) Radiograph showing treat-
ment failure in a maxillary second premolar. The tooth
was treated by intentional replantation during which the
apical lesion was removed. (B) Photomicrograph of the
lesion showing presence of foreign material. (C) Higher
magnification shows purple unidentified foreign material
and necrotic muscle tissue (dead meat granuloma). (D)
A different area of the lesion showing necrotic muscle with
bacterial colonies. (E) Necrotic muscle tissue infected by
bacteria and presence of lentil beans (pulse granuloma).
(F) One-year follow-up radiograph. The tooth is asympto-
matic, firm and bony healing is evident.
175
Activated macrophages were reported to have an
important role in the formation of CLC in several
disease processes (48, 109). CLC and damaged eosi-
nophils, along with their granules, have been
observed within macrophages (27, 48, 109). It has
been proposed that after the degranulation of eosi-
nophils, CLC protein could be phagocytized into
acidied membrane-bound lysosomes (109). At
some point, CLC protein would begin to crystallize,
forming discrete particles increasing in volume and
density over time. Ultimately, these crystals would be
released via phagosomal exocytosis or by piercing
through the membrane of the phagosome and
macrophage cytoplasm, becoming free in the stro-
mal tissue.
Recent ndings support the theory that activated
macrophages have a role in the formation of CLC
(169). In addition, the presence of CLC can be
detected within a periapical lesion that failed to
resolve after conventional endodontic treatment
(Fig. 14). Although the biological and pathologic role
of CLC in endodontic and periodontal disease is still
unknown, they may be associated with some cases of
treatment failures.
Epithelium
Among the normal components of the lateral and
apical periodontal ligament are the epithelial rests
of Malassez. The term rests, is misleading in that
it evokes a vision of discrete islands of epithelial cells.
It has been shown that these rests are actually a
shnet-like, three-dimensional, interconnected net-
work of epithelial cells. In many periapical lesions,
epithelium is not present and therefore is presumed
to have been destroyed (165). If the rests remain, they
may respond to a stimulus by proliferating to wall off
the irritants coming through the apical foramen. The
epithelium is surrounded by chronic inammation
and is termed an epitheliated granuloma. If this
lesion is not treated, the epithelium continues to
Fig. 11. Cholesterol clefts in a periapical lesion. (A)
Photomicrograph stained with Massons Trichrome of a
cyst with a thick fibrous wall. Embedded in the wall is a
large collection of cholesterol clefts. (B) Higher magnifica-
tion showing empty clefts where cholesterol was dissolved
during the histologic preparation.
Fig. 12. (A) Photomicrograph of a periapical lesion show-
ing presence of Russell bodies. (B) Transmission electron
micrograph demonstrates the round amorphous shape of
these bodies.
176
Rotstein & Simon
proliferate in response to the bacteria and inamma-
tory products from the apical foramen.
The term bay cyst has been introduced for the
microcopic representation of this situation (170).
This is a chronic inammatory lesion that has epithe-
lium lining surrounding the lumen, but the lumen
has a direct communication with the root canal
system through the foramen (Fig. 15). On the other
hand, a true cyst is the completion of the epithelial
proliferative lesion. It is a three-dimensional, epithe-
lium-lined cavity with no communication between
the lumen and the canal system (Fig. 16). When peri-
apical lesions are studied in relation to the root canal
a clear distinction between these two entities should
be made (136, 170).
There has been some confusion in the diagnosis
when lesions are studied only on curetted biopsy
material. Since the tooth is not attached to the lesion,
orientation to the apex is lost. Therefore the criterion
used for diagnosis of a cyst is a strip of epithelium
that appears to be lining a cavity. It is apparent that
curetting both a bay cyst and a true cyst could lead to
the same microscopic diagnosis. A bay cyst could be
sectioned in such a way that it could resemble or give
the appearance of a true cyst. This distinction
between a bay and a true cyst is important from
the standpoint of healing (37). It may be that true
cysts must be surgically removed, but bay cysts that
communicated with the root canal may heal with
nonsurgical root canal therapy. Since root canal ther-
apy can directly affect the lumen of the bay cyst, the
environmental change may bring about resolution of
the lesion. The true cyst is independent of the root
canal system; therefore conventional therapy may
have no effect on the lesion.
The formation of a cyst and its progression from a
bay cyst to a true cyst occurs over time. Valderhaug
(190), in a study done in monkeys, showed no cyst
formation until at least 6 months after the canal
contents became necrotic. Thus the longer a lesion
is present, the greater the chance of becoming a true
cyst. However, the incidence of true cysts is probably
less than 10% (170).
Contributing factors
Poor endodontic treatment
Correct endodontic procedures and techniques are
key factors for treatment success. It is imperative to
completely clean, shape and obturate the canal sys-
tem in order to enhance successful outcomes. Unfor-
tunately, poor endodontic treatments are often
found associated with periradicular inammation.
Poor endodontic treatment allows canal reinfection,
which may often lead to treatment failure (143). Clin-
ical signs and symptoms as well as radiographic evi-
dence of periradicular lesions are usually associated
with endodontic failure.
Fig. 13. (A) Photomicrograph showing Rushton hyaline
bodies in the epithelial lining of a periapical cyst. (B, C)
Higher magnifications demonstrating pleomorphism of
these bodies.
177
Endodontic failures can be treated by either ortho-
grade or retrograde retreatment with good success
rates (Figs 17 and 18). It seems that the success rate
is similar to that of initial conventional endodontic
treatment if the cause of failure is properly diagnosed
and corrected (19). In recent years, retreatment tech-
niques have improved dramatically due to use of
the operating microscope and development of new
armamentarium.
Poor restorations
Coronal leakage is an important cause of failure of
endodontic treatment. Root canals may become
recontaminated by microorganisms due to delay in
placement of a coronal restoration and fracture of
the coronal restoration and/or the tooth (160). Madi-
son & Wilcox (116) found that exposure of root canals
to the oral environment allowed coronal leakage to
occur, and in some cases along the whole length of
the root canal. Ray & Trope (147) reported that defec-
tive restorations and adequate root llings had a
higher incidence of failures than teeth with inade-
quate root llings and adequate restorations. Teeth
in which both the root llings and restorations were
adequate had only 9% failure, while teeth in which
both root llings and restorations were defective had
about 82% failure (147). Saunders & Saunders (159)
showed that coronal leakage was a signicant clinical
problem in root-lled molars. In an in vitro study,
they found that packing excess gutta-percha and
sealer over the oor of the pulp chamber, after com-
pletion of root canal lling, did not seal the root
canals. It was therefore recommended that excess
Fig. 14. Charcot-Leyden crystals in a periapical lesion. (A)
Maxillary lateral incisor with necrotic pulp and periapical
lesion. (B) Nine months after endodontic treatment the
tooth is symptomatic and the lesion is larger. (C) Apical
surgery was done and the lesion submitted for microscopic
analysis. Photomicrograph stained with hematoxylin &
eosin shows only acute and chronic inflammatory infil-
trate. (D, F, H) May-Grunwald-Giemsa stain reveals the
presence of Charcot-Leyden crystals. (E, G) Polarized light
demonstrates refraction of the Charcot-Leyden crystals.
178
Rotstein & Simon
of gutta-percha lling should be removed to the level
of the canal orices and that the oor of the pulp
chamber be protected with a well-sealed restorative
material (159).
Coronal restoration is the primary barrier against
coronal leakage and bacterial contamination of endo-
dontic treatment. Therefore it is essential that the
root canal system be protected by good endodontic
Fig. 14. continued
Fig. 16. Photomicrograph of a true inflammatory cyst
stained with Massons Trichrome showing a 3-dimen-
sional epithelial-lined lesion with no connection to the
root canal system and apical foramen in serial sections.
Fig. 15. Photomicrographshowingabaycyst associatedwith
a root canal that opens directly into the lumen of the lesion.
179
obturation and a well-sealed coronal restoration
(Fig. 19). However, even popular permanent restora-
tive materials may not always prevent coronal leak-
age (197). Cemented full crowns (68, 196) as well as
dentin-bonded crowns (140) also showed leakage.
Heling et al. (80) performed an extensive review of
the literature to determine the factors associated
with long-term prognosis of endodontically treated
teeth and drew the following conclusions:
Post space preparation and cementation should be
performed with rubber-dam isolation.
The post space should be prepared with a heated
plugger.
A minimum of 3 mm of root canal lling should
remain in the preparation.
Fig. 18. Non-healing due to insufficient root canal pre-
paration and obturation in a mandibular second molar.
(A) Radiograph showing a large periapical and furcal
radiolucency. (B) Radiograph taken immediately follow-
ing endodontic non-surgical retreatment. (C) Three-year
follow-up radiograph showing evidence of bony healing.
Fig. 17. Non-healing due to insufficient root canal pre-
paration and obturation in a maxillary second premolar.
(A) Radiograph showing periapical radiolucency asso-
ciated with the tooth involved. (B) Postoperative radio-
graph immediately following endodontic retreatment. (C)
Two-year follow-up radiograph showing evidence of bony
healing. The tooth was restored with post and crown.
180
Rotstein & Simon
The post space should be irrigated and dressed as
during root canal treatment.
Leak-proof restorations should be placed as soon
as possible after endodontic treatment.
Endodontic retreatment should be considered for
teeth with a coronal seal compromised for longer
than 3 months.
Trauma
Trauma to teeth and alveolar bone may involve the
pulp and the periodontal ligament. Both tissues can
be affected either directly or indirectly. Dental inju-
ries may take many shapes but generally can be
classied as enamel fractures, crown fractures with-
out pulp involvement, crown fractures with pulp
involvement, crownroot fracture, root fracture,
luxation, and avulsion (11). Treatment of traumatic
dental injuries varies depending on the type of injury
and it will determine pulpal and periodontal liga-
ment healing prognosis (10).
Enamel fracture involves the enamel only and
includes chipping and incomplete fractures or
cracks. Treatment usually includes grinding and
smoothing the rough edges or restoration of the
missing enamel structure. In cases where only the
enamel is involved, the pulp usually maintains its
vitality and the prognosis is good.
Crown fracture without pulp involvement is an
uncomplicated fracture that involves enamel and
dentin without pulp exposure. Treatment may
include conservative restoration with composite
resin or reattachment of the separated fragments. It
has been reported that reattachment of dentin
enamel crown fragments is a conservative possibility
for crown restoration (9).
Crown fracture with pulp involvement is a compli-
cated fracture involving enamel and dentin and
exposure of the pulp. The extent of the fracture helps
to determine the necessary pulpal and restorative
treatments (11). A small fracture may indicate vital
pulp therapy followed by acid-etched composite
restoration. A more extensive fracture may require
root canal treatment as well. The stage of tooth
maturation is an important factor in choosing
between pulpotomy and pulpectomy (11). The
amount of time elapsed from the injury often affects
pulpal prognosis. The sooner the tooth is treated, the
better the prognosis.
Crownroot fractures are usually oblique and
involve both crown and root. They include enamel,
dentin, and cementum and may or may not include
the pulp. Crownroot fractures often include molars
and premolars, but anterior teeth can also be
affected. A cusp fracture that extends subgingivally
is a common nding and often presents a diagnostic
and clinical challenge (11). Treatment depends on
the severity of the fracture and may vary from only
removing of the fractured tooth fragment and
restoration to endodontic treatment, periodontal
treatment and/or surgical procedures. Sometimes
Fig. 19. Poor coronal seal inamaxillarysecondpremolar. (A)
Radiograph showing inadequate coronal restoration and
root canal treatment. Notethe lateral apical lesionassociated
with the tooth. (B) Radiograph taken upon completion of
endodontic retreatment. The old restoration was removed
and the canal system properly prepared and obturated. (C)
Five-year follow-up radiograph showing bony repair. The
tooth was adequately restored with post and crown.
181
the prognosis is poor and the tooth needs to be
extracted. Due to the complexity of this injury, a
team approach involving endodontists, periodon-
tists, orthodontists, and prosthodontists is highly
recommended (11).
Root fractures involve cementum, dentin, and
pulp. They may be horizontal or transverse. Clini-
cally, root fractures may often present mobility of
the involved teeth as well as pain on biting. Often,
a periodontal defect or a sinus tract is associated with
the fractured root. Radiographically, a root fracture
can only be visualized if the X-ray beam passes
through the fracture line. Horizontal and oblique
root fractures are easier to detect radiographically
while the diagnosis of vertical root fractures is more
challenging.
Treatment, when feasible, usually includes reposi-
tioning of the coronal segment and stabilization by
splinting (11). A exible splint using orthodontic or
nylon wire and acid-etched resin for periods of up to
12 weeks will enhance pulpal and periodontal repair
(8). Teeth with fractured roots do not necessarily
require root canal treatment if healing takes place
with no evidence of pulp disease (201).
Luxations include several different types of tooth
displacement injuries such as concussion, subluxa-
tion, extrusive luxation, lateral luxation, and intrusive
luxation. Generally, the more severe the luxation
injury, the greater the damage to the periodontium
and to the dental pulp (11).
In concussion injuries the tooth is only sensitive to
percussion. There is no increase in mobility, and no
radiographic changes are found. The pulp may
respond normal to vitality tests and no immediate
treatment is usually necessary (11).
In subluxation injuries the teeth are sensitive to
percussion and also have increased mobility (11).
Often sulcular bleeding is present, indicating damage
to the periodontal ligament. Radiographic ndings
are unremarkable and the pulp may respond nor-
mally to vitality tests (11). No treatment is usually
required for minor subluxations. If mobility is severe,
stabilization of the tooth is necessary.
In extrusive luxations the teeth have been partially
displaced from the socket and increased mobility is
found. Radiographs also show displacement. The
pulp usually does not respond to vitality tests and
requires root canal treatment once irreversible pul-
pitis is diagnosed (11). The tooth requires reposition-
ing and splinting usually for a 23-week period.
In lateral luxations the tooth has been displaced
away from its long axis. Percussion sensitivity may or
may not be present. A metallic sound upon percus-
sion indicates that the root has been forced into the
alveolar bone (11). Treatment includes repositioning
and splinting. Lateral luxations that involve bony
fractures usually require up to 8-week splinting per-
iods. Endodontic therapy should be performed only
when a denite diagnosis of irreversible pulpitis or
pulp necrosis is established.
During intrusive luxations the teeth are forced into
their sockets in an axial direction. They have
decreased mobility and resemble ankylosis (11).
Treatment depends on root maturity. If the root is
not completely formed and have an open apex the
tooth may re-erupt. In such cases root canal therapy
is not necessary as the pulp may revascularize (6). If
the tooth is fully developed, active extrusion is indi-
cated. In such cases root canal treatment is indicated
since pulp necrosis develops in almost all cases (6).
Avulsion is when the tooth is totally displaced from
its alveolar socket. If the tooth is replanted soon after
avulsion, the periodontal ligament has a good chance
of healing (11). Extra-alveolar time and the storage
media used to transport the tooth are critical factors
for successful replantation. The degree of recovery of
the periodontal ligament cells will determine long-
term success.
Resorptions
Root resorption is a condition associated with either
a physiologic or a pathologic process resulting in a
loss of dentin, cementum and/or bone (5). It may be
initiated in the periodontium and affect initially the
external surfaces of the tooth (external resorption) or
it may start within the pulp space affecting primarily
the internal dentin surfaces (internal resorption). If
not diagnosed and treated, external root resorption
may invade cementum, dentin and ultimately the
pulp space. In cases of untreated internal resorptions
the process may advance and perforate to the exter-
nal root surface.
External root resorption may be divided into three
main categories (185):
1) progressive inammatory resorption
2) invasive resorption (non inammatory)
3) replacement resorption (non inammatory).
Progressive inammatory root resorption is caused
by stimuli such as pulpal infection and sulcular
infection. It may occur following traumatic displace-
ment injuries, tumors, cysts, certain systemic dis-
eases, periodontal disease, or as a result of pulp
inammation and necrosis. Practically all teeth with
apical periodontitis will exhibit a certain degree of
inammatory root resorption (Fig. 20). This can be
182
Rotstein & Simon
located on either the apical or lateral aspects of the
root but is more frequent at the apex. During the
initial stages the resorption cannot be detected
radiographically; however, it is evident in histologic
sections. If allowed to progress, the resorptive pro-
cess may destroy the entire root. If detected and
treated early, the prognosis is good. Removal of the
inamed pulpal tissue and obturation of the root
canal system is the treatment of choice (36, 177).
Invasive root resorption, also known as invasive
cervical resorption, is a relatively uncommon form
of external root resorption (7476). It is characterized
by its cervical location and invasive nature (Fig. 21).
Invasion of the cervical region of the root is predo-
minated by brovascular tissue derived from the per-
iodontal ligament. The process progressively resorbs
cementum, enamel, and dentin and later may
involve the pulp space. There may be no signs or
symptoms unless it is associated with pulpal or per-
iodontal infection. Secondary bacterial invasion into
the pulp or periodontal ligament space will cause an
inammation of the tissues accompanied with pain.
Frequently, however, the resorptive defect is only
detected by routine radiographic examination.
Where the lesion is visible, the clinical features vary
from a small defect at the gingival margin to a pink
coronal discoloration of the tooth crown (74). Radio-
graphically, the lesion varies from well delineated to
irregularly bordered radiolucencies. A characteristic
radiopaque line generally separates the image of the
lesion from that of the root canal, because the pulp
remains protected by a thin layer of predentin until
late in the process (74).
The etiology of invasive cervical resorption is not
fully understood. It seems, however, that potential
predisposing factors are trauma, orthodontic treat-
ment and intracoronal bleaching with 30% hydrogen
peroxide (75, 153). Treatment of the condition pre-
sents clinical problems because the resorptive tissue
is highly vascular and the resulting hemorrhage may
impede visualization and compromise placement of
a restoration (76). Successful treatment relies upon
the complete removal or inactivation of the resorp-
tive tissue. This is difcult to obtain in more
advanced lesions characterized by a series of small
channels often interconnecting with the periodontal
ligament apical to the main lesion. In most cases,
surgery is necessary to gain access to the resorptive
defect and often may cause loss of bone and period-
ontal attachment. Topical application of a 90% aqu-
eous solution of trichloracetic acid, curettage and
sealing of the defect has proved successful in most
cases (76). Large defects associated with advanced
stages of this condition have a poor prognosis.
Replacement resorption or ankylosis occurs fol-
lowing extensive necrosis of the periodontal ligament
with formation of bone onto a denuded area of the
root surface (185). This condition is most often seen
as a complication of luxation injuries, especially in
avulsed teeth that have been out of their sockets in
dry conditions for several hours (Fig. 22).
Certain periodontal procedures have been
reported to induce replacement root resorption
(117). The potential for replacement resorption was
also associated with periodontal wound healing (94).
Granulation tissue derived from bone or gingival
connective tissue may induce root resorption and
ankylosis (23). It seems that the culprit is the lack
of ability to form connective tissue attachment on a
denuded root surface. The only cells within the per-
iodontium that seem to have this ability are the per-
iodontal ligament cells (23). In general, if less than
20% of the root surface is involved, reversal of the
ankylosis may occur (7). If not, ankylosed teeth are
incorporated in the alveolar bone and will become
part of the normal remodeling process of bone. This
is a gradual process and the speed by which the teeth
are replaced by bone varies depending mainly on the
metabolic rate of the patient. In most cases, it may
take years before the root is completely resorbed.
Clinically, replacement root resorption is diag-
nosed when lack of mobility of the ankylosed teeth
is determined (7). The teeth will also have a metallic
sound upon percussion, and after a period of time
will be in infraocclusion. Radiographically, absence
of a periodontal ligament space is evident and the
ingrowth of bone into the root will present a char-
acteristic moth-eaten appearance (185).
Fig. 20. Photomicrograph of a tooth with a periapical
lesion showing multiple resorptive areas, inflammatory
infiltrate, and osteoclasts.
183
Internal root resorption occurs as a result of multi-
nucleated giant cell activity in an inamed pulp
(Fig. 23). The origin of this condition is not fully
understood but it appears to be related to chronic
pulpal inammation associated with an infected cor-
onal pulp space (193). Internal resorption will only
take place in the presence of granulation tissue and if
the odontoblastic layer and predentin are affected or
lost (185, 194).
Causes for internal resorption are usually trauma,
but bacteria may play a role in the process (193).
Traumatic factors can be either mechanical, chemi-
cal, or thermal. Extreme heat has been suggested as a
possible cause for this type of resorption (188).
Therefore, the clinician must use sufcient irrigating
solutions when performing root scaling with ultra-
sonic devices as well as when using cauterization
during surgical procedures.
Internal root resorption is usually asymptomatic
and diagnosed during a routine radiographic exam-
ination. Early diagnosis is critical for the prognosis
(Fig. 23). When diagnosed at an early stage endodon-
tic treatment of such lesions is usually uneventful
(Fig. 24). The radiographic appearance of the resorp-
tive defect discloses a distorted outline of the root
canal. A round or an oval-shaped enlargement of the
root canal space is usually found. In most cases,
resorption of the adjacent bone does not occur
unless large parts of the pulp become infected. His-
tologically, pulpal granulation tissue with multinu-
cleated giant cells and coronal pulp necrosis are
commonly found.
Fig. 21. Invasive root resorption in a maxillary lateral
incisor. (A) Radiograph shows a large diffuse resorptive
defect in the cervical region. The tooth was extracted and
submitted for microscopic analysis. (BD) Photomicro-
graphs of a horizontal cross-section of the resorptive area.
Note the multiple resorption bays as well as bone-like
material deposited directly on dentin (ankylosis). Also
note the absence of inflammation.
184
Rotstein & Simon
Perforations
Root perforations are undesirable clinical complica-
tions that may lead to treatment failure (184). When
root perforation occurs, communications between
the root canal system and either periradicular tissues
or the oral cavity may often reduce the prognosis of
treatment. Root perforations may result from exten-
sive carious lesions, resorption, or from operator
error occurring during root canal instrumentation
or post preparation (106, 184).
Treatment prognosis of root perforations depends
on the size, location, time of diagnosis and treat-
ment, degree of periodontal damage as well as the
sealing ability and biocompatibility of the repair
material (59). It has been recognized that treatment
success depends mainly on immediate sealing of the
perforation and appropriate infection control.
Among the materials that have been recommended
to seal root perforations are mineral trioxide aggre-
gate, Super EBA, intermediate restorative material,
Cavit
1
, glass-ionomer cements, composites, and
amalgam (12, 43, 90, 112, 139, 149).
Developmental malformations
Teeth with developmental malformations tend to fail
to respond to treatment when they are directly asso-
ciated with an invagination or a vertical developmen-
tal radicular groove. Such conditions can lead to an
untreatable periodontal condition. These grooves
usually begin in the central fossa of maxillary central
and lateral incisors crossing over the cingulum, and
continuing apically down the root for varying dis-
tances. Such a groove is probably due to the failure
of the tooth germ to form another root. As long as the
epithelial attachment remains intact, the periodon-
tium remains healthy. However, once this attach-
ment is breached and the groove becomes
contaminated by bacteria, a self-sustaining infrab-
ony pocket can be formed along its entire lengh. This
ssure-like channel provides a nidus for accumula-
tion of bacterial biolm and an avenue for the pro-
gression of periodontal disease. Radiographically, the
area of bone destruction follows the course of the
groove.
From a diagnostic standpoint, the patient may
present symptoms of a periodontal abscess or a vari-
ety of asymptomatic endodontic conditions. If the
condition is purely periodontal, it can be diagnosed
by visually following the groove to the gingival mar-
gin and by probing the depth of the pocket, which is
usually tubular in form and localized to this one area,
as opposed to a more generalized periodontal pro-
blem. The tooth will respond to pulp-testing proce-
dures. Bone destruction that vertically follows the
groove may be apparent radiographically. If this
entity is also associated with an endodontic disease,
the patient may present clinically with any of the
spectrum of endodontic symptoms.
The prognosis of root canal treatment in such
cases is guarded, depending upon the apical extent
of the groove. The clinician must look for the groove
since it may have been altered by a previous access
opening or restoration placed in the access cavity.
The appearance of a teardrop-shaped area on the
radiograph should immediately arouse suspicion.
The developmental groove may actually be visible
on the radiograph. If so, it will appear as a dark
vertical line. This condition must be differentiated
from a vertical fracture, which may give a similar
radiographic appearance.
Treatment consists of buring out the groove, pla-
cing bone substitutes, and surgical management of
the soft tissues and underlying bone. Radicular
grooves can result in self-sustaining infrabony pock-
ets and therefore scaling and root planing will not
Fig. 22. Radiograph showing replacement root resorption
in a maxillary central incisor that was avulsed and
remained 2 h out of its socket.
185
sufce. Although the acute nature of the problem
may be alleviated initially, the source of the chronic
or acute inammation must be eradicated by a sur-
gical approach. Occasionally, the tooth needs to be
extracted due to a poor prognosis.
Differential diagnosis
For differential diagnostic purposes the endo-
perio lesions are best classied as endodontic,
Fig. 23. Internal root resorption in a maxillary central
incisor. The patient reported that a small lesion was
diagnosed 2 years previously and was left untreated. (A)
Clinical view. Note a large pink spot defect in the crown.
(B) Radiograph showing a large internal resorptive defect in
the crown and cervical area. Note that the defect has
perforated into the surrounding periodontal ligament.
(CE) Histologic section of the internal resorptive area
showing chronically inflamed connective tissue and
dentin resorption by multinucleated giant cells.
186
Rotstein & Simon
periodontal or combined diseases (171). They can
also be classied by treatment depending on whether
endodontic, periodontal or combined treatment
modalities are necessary. They include: primary
endodontic disease, primary periodontal disease, and
combined diseases. The combined diseases include:
primary endodontic disease with secondary period-
ontal involvement, primary periodontal disease with
secondary endodontic involvement, and true com-
bined diseases.
Primary endodontic disease
An acute exacerbation of a chronic apical lesion on a
tooth with a necrotic pulp may drain coronally
through the periodontal ligament into the gingival
sulcus. This condition may mimic clinically the pre-
sence of a periodontal abscess. In reality, it is a sinus
tract from pulpal origin that opens through the per-
iodontal ligament area. For diagnosis purposes, it is
imperative for the clinician to insert a gutta-percha
cone into the sinus tract and to take one or more
radiographs to determine the origin of the lesion.
When the pocket is probed, it is narrow and lacks
width. A similar situation occurs where drainage
from the apex of a molar tooth extends coronally into
the furcation area. This may also occur in the pre-
sence of lateral canals extending from a necrotic pulp
into the furcation area.
Primary endodontic diseases usually heal follow-
ing root canal treatment (Fig. 25). The sinus tract
extending into the gingival sulcus or furcation area
disappears at an early stage once the necrotic pulp
has been removed and the root canals are well sealed
(Fig. 26). It is important to recognize that failure of
any periodontal treatment will occur when the pre-
sence of a necrotic pulp has not been diagnosed, and
endodontic treatment has not followed.
Primary periodontal disease
These lesions are caused primarily by periodontal
pathogens. In this process, chronic periodontitis pro-
gresses apically along the root surface. In most cases,
pulp tests indicate a clinically normal pulpal reaction
(Fig. 27). There is frequently an accumulation of pla-
que and calculus and the pockets are wider.
The prognosis depends upon the stage of period-
ontal diseaseandtheefcacyof periodontal treatment.
The clinician must also be aware of the radiographic
appearance of periodontal disease associated with
developmental radicular anomalies (Fig. 28).
Combined diseases
Primary endodontic disease with secondary
periodontal involvement
If after a period of time a suppurating primary endo-
dontic disease remains untreated, it may become
secondarily involved with periodontal breakdown
(Fig. 29). Plaque forms at the gingival margin of the
sinus tract and leads to plaque-induced periodontitis
in the area. When plaque or calculus is detected, the
treatment and prognosis of the tooth are different
that those of teeth involved with only primary endo-
dodntic disease. The tooth now requires both endo-
dontic and periodontal treatments. If the endodontic
treatment is adequate, the prognosis depends on the
severity of the plaque-induced periodontitis and the
Fig. 24. (A) Radiograph showing an
internal inflammatory resorptive de-
fect in the coronal third of the root
canal of a maxillary central incisor.
The tooth tested positive to pulp
sensitivity tests. (B) Postoperative
radiograph showing obturation of
root canal and resorptive defect.
187
efcacy of periodontal treatment. With endodontic
treatment alone, only part of the lesion will heal to
the level of the secondary periodontal lesion. In gen-
eral, healing of the tissues damaged by suppuration
from the pulp space can be anticipated.
Primary endodontic lesions with secondary peri-
odontal involvement may also occur as a result of root
perforation during root canal treatment, or where pins
or posts have been misplaced during coronal resto-
ration. Symptoms may be acute, with periodontal
abscess formation associated with pain, swelling,
pus or exudate, pocket formation, and tooth mobility.
A more chronic response may sometimes occur with-
out pain, and involves the sudden appearance of a
pocket with bleeding on probing or exudation of
pus. When the root perforation is situated close to
the alveolar crest, it may be possible to raise a ap
and repair the defect with an appropriate lling
material. In deeper perforations, or in the roof of the
fucation, immediate repair of the perforation has a
better prognosis than management of an infected
one. Use of mineral trioxide aggregate has resulted in
cemental healing following immediate repair (145).
Root fractures may also present as primary endo-
dontic lesions with secondary periodontal involve-
ment. These typically occur on root-treated teeth,
often with post and crowns. The signs may range
from a local deepening of a periodontal pocket to
more acute periodontal abscess formation. Root frac-
tures have also become an increasing problem with
molar teeth that have been treated by root resection
(107, 151).
Primary periodontal disease with secondary
endodontic involvement
The apical progression of a periodontal pocket may
continue until the apical tissues are involved. In this
case the pulp may become necrotic as a result of
infection entering via lateral canals or the apical fora-
men. In single-rooted teeth the prognosis is usually
poor. In molar teeth the prognosis may be better.
Since not all the roots may suffer the same loss of
supporting tissues, root resection can be considered
as a treatment alternative.
The effect of the progression of chronic perio-
dontitis on the vitality of the pulp is controversial
Fig. 25. Primary endodontic disease
in a mandibular first molar with a
necrotic pulp. (A) Preoperative
radiograph showing periapical and
interradicular radiolucencies. (B)
Radiograph taken upon completion
of root canal treatment. (C) Two-
year follow-up radiograph showing
evidence of bony healing.
188
Rotstein & Simon
(2, 3, 108). If the blood supply circulating through the
apex is intact, the pulp has good prospects for survi-
val. It has been reported that pulpal changes result-
ing from periodontal disease are more likely to occur
when the apical foramen is involved (108). In these
cases, bacteria originating from the periodontal
pocket are the most likely source of root canal infec-
tion. A strong correlation between the presence of
microorganisms in root canals and their presence in
periodontal pockets of advanced periodontitis has
been demonstrated (100, 102). Support for this con-
cept has come from research in which cultured sam-
ples obtained from the pulp tissue and radicular
dentin of periodontally involved human teeth
showed bacterial growth in 87% of the teeth (2, 3).
The treatment of periodontal disease can also lead
to secondary endodontic involvement. Lateral canals
and dentinal tubules may be opened to the oral
environment by scaling and root planing or surgical
ap procedures. It is possible for a blood vessel
within a lateral canal to be severed by a curette
and for microorganisms to be pushed into the area
during treatment, resulting in pulp inammation
and necrosis (Fig. 30).
True combined disease
True combined endodonticperiodontal disease
occurs less frequently than other endodonticperiod-
ontal problems. It is formed when an endodontic
disease progressing coronally joins with an infected
periodontal pocket progressing apically (163, 171).
The degree of attachment loss in this type of lesion
is invariably large and the prognosis guarded
(Fig. 31). This is particularly true in single-rooted
Fig. 26. Primary endodontic disease in a mandibular first
molar with a necrotic pulp. (A) Preoperative radiograph
showing large periradicular radiolucency associated with
the distal root and furcal lucency. (B) Clinically, a deep
narrow buccal periodontal defect can be probed. Note
gingival swelling. (C) One year following root canal
therapy, resolution of the periradicular bony radiolucency
is evident. (D) Clinically, the buccal defect healed and
probing is normal.
189
teeth (Fig. 32). In molar teeth, root resection can be
considered as a treatment alternative if not all roots
are severely involved. Sometimes, supplementary sur-
gical procedures are required (Fig. 33). In most cases
periapical healing may be anticipated following suc-
cessful endodontic treatment. The periodontal tissues,
however, may not respond well to treatment and will
depend on the severity of the combined disease.
The radiographic appearance of combined endo-
dontic-periodontal disease may be similar to that of a
vertically fractured tooth. A fracture that has invaded
the pulp space, with resultant necrosis, may also be
labeled a true combined lesion and yet not be amen-
able to successful treatment. If a sinus tract is pre-
sent, it may be necessary to raise a ap to determine
the etiology of the lesion.
Clinical diagnostic procedures
Clinical tests are imperative for obtaining correct
diagnosis and differentiating between endodontic
Fig. 27. Primary periodontal disease in a mandibular
second molar. Patient was referred for endodontic therapy.
(A) Preoperative radiograph showing periradicular radio-
lucency; however, the response of the tooth to pulp
sensitivity tests was normal. The referring dentist insisted
that endodontic therapy be done. (B) Photomicrograph of
the pulp tissue removed during treatment. Note normal
appearance of the pulp. (C) Higher magnification shows
normal cellular components as well as blood microvascu-
lature. (D) Postoperative radiograph. The tooth was
subsequently lost to periodontal disease. A periapical
lesion of endodontic origin will not occur in the presence
of a normal vital pulp.
190
Rotstein & Simon
and periodontal disease. The extraoral and intraoral
tissues are examined for the presence of any
abnormality or disease. One test is usually not suf-
cient to obtain a conclusive diagnosis.
Visual examination
A thorough visual examination of the lips, cheeks,
oral mucosa, tongue, palate and muscles should be
Fig. 28. Primary periodontal disease in a maxillary second
premolar. (A) Radiograph showing alveolar bone loss and
a periapical lesion. Clinically, a deep narrow pocket was
found on the mesial aspect of the root. There was no
evidence of caries and the tooth responded normally to
pulp sensitivity tests. (B) Radiograph showing pocket
tracking with gutta-percha cone to the apical area. It
was decided to extract the tooth. (C) Clinical view of the
extracted tooth with the attached lesion. Note a deep
mesial radicular developmental groove. (D) Photomicro-
graph of the apex of the tooth with the attached lesion. (E,
F) Higher magnification shows the inflammatory lesion,
cementum and dentin resorption, and osteoclasts. (G, H)
Histologic sections of the pulp chamber shows unin-
flamed pulp, odontoblastic layer, and intact predentin.
191
done routinely. Digital examination of the same tis-
sues is performed simultaneously. The alveolar
mucosa and attached gingiva are examined for the
presence of inammation, ulcerations, or sinus
tracts. Frequently, the presence of a sinus tract is
associated with a necrotic pulp (See below in section
on stula tracking).
The teeth are examined for abnormalities such as
caries, defective restorations, erosions, abrasions,
cracks, fractures, and discolorations. A discolored per-
manent tooth may often be associated with a necro-
tic pulp. A pink spot detected in the tooth crown
may indicate an active internal resorption process. A
conclusive diagnosis for pulpal disease cannot be
achieved by visual examination alone. It therefore
must always be accompanied by additional tests.
Visual examination is dramatically improved by
the use of enhanced magnication and illumination.
Fig. 28. continued
Fig. 29. Primary endodontic disease
with secondary periodontal involve-
ment in a mandibular first molar.
(A) Preoperative radiograph demon-
strating interradicular defect ex-
tending to the apical region of the
mesial root. (B) Radiograph taken
upon completion of root canal ther-
apy. (C) One year follow-up radio-
graph showing resolution of most of
the periradicular lesion, however, a
bony defect at the furcal area re-
mained. Note that endodontic treat-
ment alone did not yield complete
healing of the defect. Periodontal
treatment is necessary for further
healing of the furcal area and in-
flamed gingival tissues.
192
Rotstein & Simon
Magnifying loops and the operating microscope are
currently widely used among dental professionals
(Fig. 34). These accessories facilitate the location of
calculus, caries, coronal and radicular fractures,
developmental defects, and areas of denuded dentin
mainly at the cementumenamel junction.
Palpation
Palpation is performed by applying rm digital pres-
sure to the mucosa covering the roots and apices.
With the index nger the mucosa is pressed against
the underlying cortical bone. This will detect the
presence of periradicular abnormalities or hot
zones that produce painful response to digital pres-
sure. A positive response to palpation may indicate
active periradicular inammatory process. However,
this test does not indicate whether the inammatory
process is of endodontic or periodontal origin. Also,
as with any other clinical test, the response should be
compared to control teeth.
Percussion
Percussion is performed by tapping on the incisal or
occlusal surfaces of the teeth either with the nger or
with a blunt instrument such as the back end of a
mirror handle. The tooth crown is tapped vertically
and horizontally. Although this test does not disclose
the condition of the pulp, it indicates the presence of
a periradicular inammation. An abnormal positive
response indicates inammation of the periodontal
ligament that may be either from pulpal or period-
ontal origin. The sensitivity of the proprioceptive
bers in an inamed periodontal ligament will help
identify the location of the pain. This test should be
done gently, especially in highly sensitive teeth. It
should be repeated several times and compared to
control teeth.
Mobility
Mobility testing can be performed using two mirror
handles on each side of the crown. Pressure is
applied in a faciallingual direction as well as in a
vertical direction and the tooth mobility is scored.
Tooth mobility is directly proportional to the integ-
rity of the attachment apparatus or to the extent
of inammation in the periodontal ligament (30).
Teeth with extreme mobility generally have little
Fig. 30. Primary periodontal disease with secondary en-
dodontic involvement in a maxillary premolar. (A) Radio-
graph showing a bone loss in one-third of the root and a
separate periapical radiolucency. The crown was intact
but pulp sensitivity tests were negative. (B) Radiograph
taken immediately following root canal therapy showing
lateral canal that was exposed to the oral environment
due to bone loss. Exposed lateral canal is one of the
possible pathways of infection of the root canal.
Fig. 31. True combined endodonticperiodontal disease
in a mandibular first molar. Radiograph showing separate
progression of endodontic disease and periodontal dis-
ease. The tooth remained untreated and consequently the
two lesions joined together.
193
periodontal support, indicating that the primary
cause may be periodontal disease.
Fractured roots and recently traumatized teeth
often present high mobility. Frequently, however,
a periradicular abscess of pulpal origin may cause
similar mobility. This can only be veried if other
tests indicate pulp necrosis or if mobility improves
a short time after completion of endodontic ther-
apy. Pressure exerted by an acute apical abscess
may cause transient tooth mobility (84). This may
also occur as a result of orthodontic movement
and pulp necrosis of previously traumatized teeth
(152).
Radiographs
Radiographs are essential for detection of anatomic
landmarks and a variety of pathological conditions.
In addition, radiographs are of utmost importance
for documentation and legal purposes. Radiographic
examination will aid in detection of carious lesions,
extensive or defective restorations, pulp caps, pulpo-
tomies, previous root canal treatment and possible
mishaps, stages of root formation, canal obliteration,
root resorption, root fractures, periradicular radio-
lucencies, thickened periodontal ligament, and alveo-
lar bone loss.
The integrity of the dental pulp cannot be deter-
mined by radiographic images alone. Radiographic
changes will only be detected once the inammation
or bacterial byproducts originating from the dental
pulp cause sufcient demineralization of the cortical
bone. Often, the initial phases of periradicular bone
resorption from endodontic origin is conned only to
cancellous bone. Therefore it cannot be detected
unless the cortical bone is also affected (16, 111).
Also, certain radiographic features are susceptible
to multiple interpretations (66, 67). On the other
hand, periodontal disease causing alveolar bone loss
can be effectively detected by radiographs. For pur-
poses of differential diagnosis, periapical and bitew-
ing radiographs should be taken from several angles.
Sometimes, other types of radiographs are also
required.
A number of radioloucent and radiopaque lesions
of non-endodontic and non-periodontal origin may
simulate the radiographic appearance of endodontic
or periodontal lesions. Therefore, clinical signs and
symptoms as well as ndings from the other clinical
tests should always be considered at the time of
radiographic evaluation.
Pulp vitality testing
These tests are designed to assess the response of the
pulp to different stimuli. An abnormal response may
indicate degenerative changes in the pulp. In general,
no response indicates pulp necrosis, and moderate
transient response indicates normal vital pulp. A
quick painful response may often indicate reversible
pulpitis and lingering painful response indicate irre-
versible pulpitis. Since some of these tests may pro-
voke a painful reaction they should be carefully
performed and their nature and importance
explained to the patient. When correctly performed
and adequately interpreted these tests are reliable in
differentiating between pulpal disease and period-
ontal disease. The most commonly used pulp vitality
tests are: cold test, electric test, blood ow tests, and
cavity test (192).
Fig. 32. True combined endodontic-
periodontal disease. (A) Radiograph
showing bone loss in two-thirds of
the root with calculus present and a
separate periapical radiolucency. (B)
Clinical examination revealed coro-
nal color change of the tooth in-
volved and pus exuding from the
gingival crevice. Pulp sensitivity
tests were negative indicating pulp
necrosis.
194
Rotstein & Simon
Cold test
This test is performed by applying a cold substance,
or agent, to a well-isolated tooth surface. Tooth iso-
lation can be achieved by drying the crown surfaces
with cotton rolls, gauze and a very gentle air blast.
Several cold methods are used: ice sticks, ethyl chlor-
ide, carbon dioxide (dry ice), and refrigerants such as
dichlorodiuoromethane (DDM). Carbon dioxide
(78 8C) and DDM (50 8C) are extremely cold and
are only used when the pulp does not respond to less
cold agents. Extremely cold agents may cause crazing
and infraction lines on the enamel.
Teeth with vital pulps will react to cold with sharp
brief pain response that usually does not last more
than a few seconds. An intense and prolonged pain
Fig. 33. True combined endodontic-periodontal diseases
in a mandibular first molar. (A) Preoperative radiograph
showing periradicular radiolucencies. Pulp sensitivity
tests were negative. (B) Immediate postoperative radio-
graph of nonsurgical endodontic treatment. (C)
Six-month follow-up radiograph showing no healing.
Gutta-percha cone is inserted in the buccal gingival
sulcus. (D) Clinical photograph showing treatment of the
root surfaces and removal of the periradicular lesion. (E)
One-year follow-up radiograph demonstrating healing.
195
response often indicates abnormal pulpal changes
and irreversible pulpitis. Lack of response may indi-
cate pulp necrosis. When adequately performed, this
test is reliable in determining whether the pulp has
undergone irreversible damage. However, false-posi-
tive and false-negative responses may occur, espe-
cially in multiradicular teeth where not all roots are
affected or in teeth with calcied root canals.
Electric test
This test is performed by applying an electric stimu-
lus to the tooth using a special pulp tester device. The
tooth is rst cleaned, dried and isolated. A small
amount of toothpaste is placed on the electrode of
the pulp tester, which is then put into contact with
the clean tooth surface. Only sound tooth structure
should be contacted. Electric current is gradually
applied until the patient reports sensation. Many
devices are currently available; all are effective and
used in a similar manner (Fig. 35). The purpose of
the test is to stimulate the sensory nerve bers of the
pulp to produce a response. No response frequently
indicates pulp necrosis. A positive response may be
interpreted as either intact vital pulp or partially
necrotic pulp. However, the electric test does not
provide any information about the condition of the
vascular supply of the pulp.
While interpreting the results the clinician must
take into consideration the various false-positives
and false-negatives of this test (144). The most com-
mon causes for false-positive responses are: partial
pulp necrosis, patient anxiety, ineffective isolation,
and inadvertent contact with metallic restorations.
The most commoncauses for false-negative responses
are: obliteratedroot canals, recentlytraumatizedteeth,
teeth with immature apices, patient taking drugs that
elevate the pain threshold, and poor electrodetooth
contact. In general, however, the electric pulp test is
easy to perform and provides accurate determination
of pulp necrosis in adult teeth.
Blood flow test
This test is designed to determine the vitality of the
pulp by measuring its blood ow rather than the
response of its sensory nerve bers. Different sys-
tems such as dual wavelength spectrophotometry,
pulse oximetry, and laser Doppler have been devel-
oped to measure either oxyhemoglobin, low concen-
tration of blood, or pulsation of the pulp (46, 54, 138,
161). Sensors are applied to the external surfaces of
the crown and the pulp blood ow is recorded and
compared to controls. The procedure is non-invasive
and painless. These tests are relatively new and are
not used routinely.
Cavity test
This test is highly reliable in determining the vitality
of the pulp. It basically consists of creating a cavity in
the tooth without anesthesia. A high-speed hand-
piece with a new sharp bur is generally used. A posi-
tive response indicates presence of vital pulp tissue,
while a negative response accurately indicates pulp
necrosis. If no response is obtained, the cavity is
extended into the pulp chamber and endodontic
treatment is initiated.
This test is not routinely performed since it may
produce pain in cases where the pulp is vital. It
should only be limited to cases where all other tests
proved inconclusive and a denitive diagnosis of the
pulp condition could not be established.
Fig. 34. The operating microscope provides enhanced
magnification and illumination of the working field. It is
used for both diagnosis and treatment purposes.
Fig. 35. An example of a popular pulp tester device. It
produces a low electric current to stimulate the sensory
nerve fibers of the pulp.
196
Rotstein & Simon
Restored teeth testing
Testing teeth with extensive coronal restorations is
somewhat more challenging. Whenever possible, the
restoration should be removed to facilitate pulp test-
ing. In cases where restoration removal is not feasi-
ble, a small access opening is made through the
restoration until sound tooth structure is reached.
Cold test and cavity test will give the most reliable
results. In most instances electric pulp testing will
not prove benecial.
Access through full gold crowns can usually be
done without affecting the strength and stability of
the restoration. Access repair is done with amalgam,
or another permanent lling material (123). Access
for pulp testing can be done through porcelain
restorations as well. In such cases, access is done
slowly and with copious water irrigation.
Pocket probing
Periodontal probing is an important test that should
always be performed when attempting to differenti-
ate between endodontic and periodontal disease. A
blunt calibrated periodontal probe is used to deter-
mine the probing depth and clinical attachment
level. It may also be used to track a sinus resulting
from an inammatory periapical lesion that extends
cervically through the periodontal ligament space. A
deep solitary pocket in the absence of periodontal
disease may indicate the presence of a lesion of
endodontic origin or a vertical root fracture.
Periodontal probing can be used as a diagnostic
and prognostic aid (192). For example, the prognosis
for a tooth with a necrotic pulp that has developed a
sinus track is excellent following adequate root canal
therapy. However, the prognosis of root canal treat-
ment in a tooth with severe periodontal disease is
dependent on the success of the periodontal therapy.
Therefore, correct identication of the etiology of the
disease, whether endodontic, periodontal or com-
bined, will determine the course of treatment and
long-term prognosis.
Fistula tracking
Endodontic or periodontal disease may sometimes
develop a stulous sinus track. Inammatory exu-
dates may often travel through tissues and structures
of minor resistance and open anywhere on the oral
mucosa or facial skin. Intraorally, the opening is
usually visible on the attached buccal gingiva or
in the vestibule. Extraorally, the stula may open
anywhere on the face and neck. However, it is most
commonly found on the cheek, chin, and angle of the
mandibule, and occasionally also on the oor of the
nose (78). If the etiology is pulpal, it usually responds
well to endodontic therapy.
The identication of the sinus tract by simple
visual examination does not necessarily indicate
the origin of the inammatory exudate or the tooth
involved. Occasionally, the exudate exists through
the periodontal ligament, thus mimicking a pocket
of periodontal origin. Identifying the source of
inammation by tracking the stula will help the
clinician to differentiate between diseases of endo-
dontic and periodontal origin.
Fistula tracking is done by inserting a semi-rigid
radiopaque material into the sinus track until resis-
tance is met. Commonly used materials include gutta-
percha cones or presoftenedsilver cones. Aradiograph
is then taken that will reveal the course of the sinus
tract and the origin of the inammatory process.
Cracked tooth testing
Transillumination
This test is designed to aid in the identication of
cracks andfractures inthe crown. Aberoptic connec-
ted to a high-power light source is used to illuminate
the crown and gingival sulcus. The contrast between
the dark shadowof the fracture andthe light shadowof
the surrounding tissue will clearly reveal the size and
orientation of the fracture line. An existing restoration
may need to be removed to enhance visibility.
Wedging
This technique aids in the identication of vertical
crown fractures or crownroot fractures. Such frac-
tures cause a painful response to the patient at the
time of chewing. During the test, wedging forces are
created as the patient is instructed to chew on a
cottonwood stick or other rm material. This test is
fairly reliable in identifying a single tooth causing
pain during mastication. Many of these fractures
involve only the tooth crown and terminate in the
pulp chamber. Such cases are treated successfully
with endodontic therapy.
Staining
Staining identies lines of fracture in the crown and
root and is often used in conjunction with the wed-
ging test. The tooth crown is dried and a cotton pellet
soaked with methylene blue dye is swabbed on the
occlusal surface of the tooth. The patient is asked to
197
bite on a stick and perform lateral jaw movements.
This way the dye penetrates well into the zone of the
fracture. The dye is then rinsed from the tooth sur-
faces and visual examination with magnifying loops
or the microscope will reveal a distinctive fracture
line darkened with dye.
Selective anesthesia test
This test is useful in cases where the source of pain
cannot be attributed to a specic arch. Disappear-
ance of pain following a mandibular block will con-
rm the source of pain originating from a
mandibular tooth. The periodontal ligament injec-
tion is often used to narrow down the zone in ques-
tion, however, it cannot anesthetize a single tooth
without affecting adjacent teeth (47). In the maxillary
arch the test may be more focused to a specic tooth
by injecting a small amount of anesthetic solution in
an anteriorposterior direction at the root apex level.
No conclusive diagnosis differentiating between
endodontic and periodontal disease can be made
using this type of test.
Treatment decision-making and
prognosis
Treatment decision-making and prognosis depend
primarily on the diagnosis of the specic endodontic
and/or periodontal disease. The main factors to con-
sider are pulp vitality and type and extent of the
periodontal defect. Diagnosis of primary endodontic
disease and primary periodontal disease usually pre-
sent no clinical difculty. In primary endodontic dis-
ease the pulp is infected and nonvital. In primary
periodontal disease the pulp is vital and responsive
to testing. However, primary endodontic disease with
secondary periodontal involvement, primary period-
ontal disease with secondary endodontic involve-
ment, or true combined diseases are clinically and
radiographically very similar. If a lesion is diagnosed
and treated as primarily endodontic disease due to
lack of evidence of plaque-induced periodontitis, and
there is soft-tissue healing on clinical probing and
bony healing on a recall radiogragh, a valid retro-
spective diagnosis can then be made. The degree of
healing that has taken place following root canal
treatment will determine the retrospective classica-
tion. In the absence of adequate healing, further
periodontal treatment is indicated.
The prognosis and treatment of each endodontic
periodontal disease type varies. Primary endodontic
disease should only be treated by endodontic therapy
and has a good prognosis. Primary periodontal dis-
ease should only be treated by periodontal therapy.
In this case, the prognosis depends on severity of the
periodontal disease and patient response. Primary
endodontic disease with secondary periodontal
involvement should rst be treated with endodontic
therapy. Treatment results should be evaluated in 2
3 months and only then should periodontal treat-
ment be considered. This sequence of treatment
allows sufcient time for initial tissue healing and
better assessment of the periodontal condition (28,
141). It also reduces the potential risk of introducing
bacteria and their byproducts during the initial heal-
ing phase. In this regard, it was suggested that the
periodontal healing was adversely affected by aggres-
sive removal of the periodontal ligament and under-
lying cementum during interim endodontic therapy
(21). Areas of the roots that were not aggressively
treated showed unremarkable healing (21). Prognosis
of primary endodontic disease with secondary
periodontal involvement depends primarily on the
severity of periodontal involvement, periodontal
treatment and patient response.
Primary periodontal disease with secondary endo-
dontic involvement and true combined endodontic
periodontal diseases require both endodontic and
periodontal therapies. It has been demonstrated that
intrapulpal infection tends to promote epithelial
downgrowth along a denuded dentin surface (22).
Additionally, experimentally induced periodontal
defects around infected teeth were associated with
20% more epithelium than non-infected teeth (88).
Non-infected teeth showed 10% more connective
tissue coverage than infected teeth (88). The prog-
nosis of primary periodontal disease with secondary
endodontic involvement and true combined diseases
depends primarily upon the severity of the period-
ontal disease and the response to periodontal treat-
ment. Cases of true combined disease usually have
a more guarded prognosis than the other types of
endodonticperiodontal problems. In general, assum-
ing the endodontic therapy is adequate, what is of
endodontic origin will heal. Thus the prognosis of
combined diseases rests with the efcacy of period-
ontal therapy.
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203
Diagnosis of acute periodontal
lesions
Esmonde F. Corbet
A thorough understanding of the interpretation of
the ndings from a clinical and a radiographic exam-
ination of dental and periodontal tissues still forms
the basis for the initial diagnosis and often for estab-
lishing the denitive diagnosis of most acute period-
ontal lesions. Components of the complete clinical
periodontal examination are covered in detail by
Armitage (5). Imaging methods in periodontics are
covered by Mol (65). Only those aspects particular to
the clinical and radiographic examinations in the
diagnosis of acute periodontal lesions are covered
in this chapter. Microbial testing, as outlined by
Loomer (56), is principally conned to testing in
chronic periodontal disease conditions, and micro-
bial testing plays little part in the diagnosis of most
acute periodontal lesions, although they are infective
in origin. The diagnosis of the common acute period-
ontal lesions can still be established, for the most
part, on the basis of the clinical and radiographic
examination.
Acute periodontal lesions and gingival ulcera-
tions may of course occur in patients with systemic
diseases, and the testing of patients for systemic
disease is covered by Mariotti (59). Localized tooth-
related factors that predispose to periodontal infec-
tions, including acute purulent infections, are
covered by Matthews & Tabesh (60). Combined per-
iodontalendodontic lesions may present as acute
purulent lesions. The diagnosis of these combined
periodontal-endodontic lesions is covered by Rotstein
& Simon (75). The diagnosis of periodontal manifes-
tations of systemic disease, which may include both
acute periodontal lesions and more evidently, gingi-
val ulcerations, are covered by Jordan (46).
This chapter aims to cover those aspects of the
diagnostic aspects applied in the diagnosis of acute
periodontal lesions not covered in other chapters
of this volume, to which readers are referred for
a more complete background to comprehensive
periodontal diagnosis. This chapter aims to cover
establishing a diagnosis of acute periodontal
lesions. Àcute' when used to describe a periodontal
condition is taken to mean rising to its crisis (i.e. its
worst point) sharply and having a short and relatively
dramatic course in the acute phase before the crisis is
reached.
The two acute periodontal conditions are necrotiz-
ing periodontal disease and acute periodontal
abscess, and the diagnosis of these is considered.
Whether differentiating acute necrotizing ulcerative
gingivitis from acute necrotizing ulcerative period-
ontitis in the healthy patient has any practical impli-
cations is discussed, as is necrotizing periodontal
disease and HIV infection. A common differential
diagnosis for acute necrotizing gingivitis, often given
in periodontal textbooks, is acute herpetic gingivos-
tomtatis. The question is asked if it is reasonable to
misdiagnose necrotizing ulcerative gingivitis for
acute herpetic gingivostomatitis. Furthermore, other
conditions that a practitioner should consider when
faced with a patient presenting with acute ulcera-
tions of the gingiva are given attention.
Acute abscesses of the periodontium, which are
not endodontic in origin, must be differentiated from
those that are endodontic in origin for practitioners
to be able with reasonable certainty to establish a
diagnosis and institute appropriate therapy. As the
American Academy of Periodontology's Parameter
on Acute Periodontal Diseases (3) argues, failure to
treat acute periodontal diseases appropriately is
what may result in progressive loss of periodontal
attachment possibly affecting an adverse change in
prognosis and possible tooth loss. For the period-
ontal practitioner the rst step towards appropriate
therapy of acute periodontal lesions is a sound diag-
nosis. A very useful guiding principle for practi-
tioners is that acute periodontal lesions usually
respond dramatically to appropriate èmergency'
periodontal therapy; if the emergency therapy has
been optimal, yet the response has not been rapid,
then a diagnosis other than an acute periodontal
lesion should be sought.
204
#
PERIODONTOLOGY 2000
Necrotizing periodontal diseases
These have recently been classied (4) as either
necrotizing ulcerative gingivitis or necrotizing
ulcerative periodontitis.
Necrotizing ulcerative gingivitis (NUG)
This condition has been exclusively reviewed (44, 76).
It is a distinct, painful, bacterial infection of the
interdental and marginal gingival tissues. While
opportunistic bacteria are the primary etiologic
agents (55), microbiological testing is not part of
the diagnostic process for NUG. Good evidence for
the role of bacteria in NUG comes from the rapid
clinical response to mechanical disruption of the
bacterial mass on the teeth adjacent to the gingival
ulcerations (28), to antibiotic therapy (84) and to
both debridement and antibiotic therapy (30). This
rapid clinical response to appropriate antimicrobial
therapy can be used diagnostically indirectly if there
is doubt over the etiology of necrotizing ulcerations
of the gingiva. The histopathologic features of NUG
have been described (41), but procuring biopsy spe-
cimens for histologic examination of suspected NUG
lesions is not a diagnostic procedure. While bacteria
are the primary causative agents, a variety of predis-
posing factors for NUG are known. These include
psychological stress, malnutrition, tobacco smoking,
preexisting gingivitis, trauma and, of greater signi-
cance since the 1980s, immunosuppression and
immunodeciency (44). While history taking which
seeks responses about the known predisposing fac-
tors is an important component of the diagnostic
process for a patient with necrotizing ulcerative gin-
gival lesions, NUG may occur in individuals without
any obvious predisposing factors. Therefore, the
absence of detectable predisposing factors does not
rule out the diagnosis of NUG, which is made almost
exclusively on the basis of its clinical features.
Diagnostic clinical features of NUG
A patient presenting with NUG almost invariably has
a complaint of pain or discomfort. This may have had
a rapid onset (91). Manipulations of the affected
areas may be very painful and the patient may guard
against physical disturbance of affected gingiva, and
may resist investigations such as periodontal probing
of affected sites. Invariably, there is ulceration of the
interdental papillae. The ulcerations, which may
spread from the interdental papillae to the marginal
gingiva, may result in a reduction in width of
attached gingiva due to the marginal necrosis. Char-
acteristically the ulcers are crater-like; ``punched-
out'' is the term used to describe the crater-like
ulcers of the interdental papillae. If undisturbed,
the ulcerated surface is usually covered by a slough
of necrotic debris. These cardinal diagnostic features
are given in Table 1. These are evident in the young
Chinese male photographed in Fig. 1, who at a time
of heavy work stress and heavy tobacco smoking
developed exquisitely painful interdental and then
marginal gingiva. Further clinical features that can
contribute to the establishment of the diagnosis of
NUG are not invariably present (8) and these are
given in Table 2. The ulcerated lesions are often cov-
ered by a slough, which has been usually called a
``pseudomembrane''. This slough is composed of
brin, necrotic tissue, inammatory cells and masses
of dead, dying and viable bacteria. This slough is
readily dispersed, revealing an underlying bleeding
Fig. 1. NUG in a heavy smoking, Chinese male in his 20s,
enduring a period of work-related stress.
Table 2. Non-essential clinical features of NUG, the
absence of which does not preclude the diagnosis of
NUG
1. ``Pseudomembrane'' of sloughed necrotic debris and
bacteria covering the ulcerated area
2. Foetor ex ore (foetor oris, fetid breath)
3. Fever, malaise
4. Lymphadenopathy submandibular (and cervical)
Table 1. Diagnostic essentials for NUG
1. Lesions are painful
2. Lesions are gingival ulcers, punched-out crater-like,
of interdental papilla and may involve marginal
gingiva
3. Gingival ulcers bleed spontaneously or readily
Acute lesions
205
gingival ulceration, diagnostically conrmatory of
this condition. Thus a name for this slough that
implies a covering imitating or resembling a `mem-
brane' is not really accurate. The foetor ex ore (foetor
oris, fetid breath) is a variable clinical nding and,
while not diagnostic, when present and characteristic
is helpful in establishing the diagnosis. The patient
photographed in Fig. 1 displayed a pseudomem-
brane on some ulcerated lesions and a characteristic
fetid breath. Fever and malaise are not consistent
ndings in NUG and fever when present is usually
not marked (28, 83, 91). Lymphadenopathy, if pre-
sent, is found more commonly in submandibular
lymph nodes and less commonly in cervical lymph
nodes, and may be less common in adults, without
other systemic medical conditions, than has been
reported in children with NUG (43).
Necrotizing ulcerative periodontitis
(NUP)
While NUG has recently been dened as an infection
characterizing by gingival necrosis presenting as
``punched-out'' papillae, with gingival bleeding and
pain, necrotizing ulcerative periodontitis (NUP) has
been dened as `àn infection characterized by
necrosis of gingival tissues, periodontal ligament,
and alveolar bone'' (18). The same consensus report
noted that the group speculated that NUG and NUP
may be different stages of the same infection and
that the only difference between the two named con-
ditions is that NUG is an infection limited to gingiva
whereas NUP involves the attachment apparatus.
Periodontal attachment loss may be associated
with NUG, either when NUG occurs at a site with
preexisting attachment loss, or as a result of the NUG
itself (58) or recurrence(s) of NUG (80). Treatment for
NUG has been reported to allow for regeneration of
the lost interdental gingival height (30), but none-
theless NUG may result in attachment loss. Figure 2
shows periodontal attachment loss around mandib-
ular incisors of an HIV-1 and HIV-2 seronegative
Caucasian healthy adult male resulting from NUG.
Therefore the nding of attachment loss in associa-
tion with, or as a consequence of, NUG does not
confer a need for a diagnosis of NUP, especially in
patients known to be HIV seronegative who are sys-
temically healthy and not immunosuppressed. It
would appear that necrotizing stomatitis could be a
late stage of the NUGNUP continuum (42), and this
understanding has not been altered by the classica-
tion of NUG and NUP as necrotizing periodontal
diseases (18).
Diagnostic clinical features of NUP
In NUP, the necrosis and ulceration, commencing at
the interdental papilla, results in deep interproximal
crater-like defects. These result from and denote
necrosis of the interdental periodontal ligament
and alveolar bone. Denudation of the interdental
alveolar bone may occur, which may lead to seques-
tration of pieces of bone. Upon removal of the
sequestrum, involvement of buccal and/or lingual
alveolar bone may be found (40).
The consensus report on necrotizing periodontal
diseases (18) concluded that the lesions of NUP are
most commonly observed in individuals with sys-
temic conditions including, but not limited to, HIV
infection, severe malnutrition and immunosuppres-
sion. Accordingly, the nding of lesions consistent
with NUP in a patient for whom HIV infection or
other immunosuppression is not known and who is
not malnourished, might call for investigation of the
patient's systemic health. For the healthy patient
without systemic diseases, the treatment for NUG of
NUP would probably be the same, which may render
precision in the differentiation of NUG and NUP for
the systemically healthy of little practical value.
Necrotizing periodontal diseases
and HIV infection
NUG may be the rst clinical sign of HIV infection
(77). Figure 3 shows one such example of this in a
young Chinese male who complained of exquisite
tenderness of the interdental area between the max-
illary left third and second molars, which showed the
characteristic NUG appearance, albeit very localized,
and who on testing was HIV seropositive. NUG/NUP
are considered to be among the HIV-associated
Fig. 2. Periodontal attachment loss in a healthy, HIV ser-
onegative Caucasian male around mandibular incisors
resulting from NUG.
206
Corbet
periodontal conditions (52). Yet, as has been recently
pointed out, while it can occur, NUG/NUP has not
commonly been reported in Asians infected with HIV
(19). The reasons for this apparent difference in HIV-
associated oral conditions in different populations
are unclear.
Gingival ulcerations in HIV-infected individuals
may not all be due to NUG/NUP. While most
NUG-like ulcerations in HIV seropositive patients
respond to treatment for NUG, a minority do not,
and other causes such as neutropenia (74) must be
investigated. Also in the HIV-infected, gingival ulcers
may result frominfectious agents suchas viruses, cyto-
megalovirus alone or in combination with herpes
virus (25), and herpesvirus and fungi such as Penicil-
liosis marneffei (68). Thus, HIV-seropositive patients,
withor without AIDS, whopresent withgingival ulcera-
tions that either are unlike the lesions of NUG or are
supercially similar to NUGbut which do not respond
to usual anti-NUG therapy, should be tested for their
blood state. Additionally, the gingival ulcers may
require microbiological testing before a diagnosis can
be established and appropriate treatment delivered.
Knowledge of HIV serostatus is useful for persons
for whom this knowledge can guide behaviors; for
those HIV-seropositive, if anti-retroviral therapy is
available, this knowledge can be life prolonging.
However, as is beginning to be recognized in the
dental literature in many developing countries such as
Cambodia (10), anti-retroviral therapy is not available.
The value of having patients presenting withNUG, and
especially NUP, who do not know their HIV serosta-
tus, tested for their HIV serostatus depends upon the
health care delivery system and the economic deve-
lopment of the country in which the patient lives.
Necrotizing ulcerative gingivitis and
acute herpetic gingivostomatitis
In 1973, Klotz published a differentiation between
necrotic ulcerative gingivitis (NUG) and primary
(acute) herpetic gingivostomatitis (AHG) (51). It is
often assumed in standard periodontal textbooks
that these two conditions are not infrequently
Fig. 3. NUG as rst presenting symptom (exquisite pain
between the maxillary left third molar and the maxillary
left secondmolar) andsigns - bleeding, punched-out, crater-
likeulcer of theinterdental gingival tissuebetweentheaffec-
ted teeth in a 25-year-old Chinese male with HIV/AIDS.
Table 3. Diagnostic clinical features of NUP
1. Deep interproximal craters with denudation of
interdental alveolar bone
2. Sequestration of interdental, and possibly buccal and/
or lingual, alveolar bone
Table 4. Differences in clinical features of necrotizing ulcerative gingivitis (NUG) and acute herpetic gingivosto-
matitis (AHG)
NUG AHG
Site of ulcers Interdental papilla
Marginal gingival
Gingiva, no predilection for interdental
papilla
Entire oral mucosa
Character of ulcers Punched-out, crater-like
Covered by yellow/white/gray slough
Bleed readily or spontaneously
Painful on stimulation
Multiple vesicles that coalesce and
form shallow, fibrin-covered,
regular-shaped ulcers
No marked tendency to bleed
Not specially tender
Fever Doubtful or slight only 38 8C (or more)
Symptoms Painful gums, `dead-feeling teeth' Sore mouth
Duration of ulcers
and discomfort
Short-lived (13 days), with appropriate therapy More than 1 week, even with therapy
207
Acute lesions
mistaken one for the other. This should not be so.
The features of the two are summarized in Table 4. It
should not be difcult to differentiate one bacterial
(NUG), one viral (AHG), one contagious (AHG), one
not (NUG), and one capable of causing periodontal
attachment loss (NUG) and one not (AHG), on the
basis of the clinical features alone, without the need
for further diagnostic tests. Even the character of the
ulcers is different and the ulcers of NUG (and NUP),
unlike those of AGH, do not occur on the nonkerati-
nized mucosa.
Both acute necrotizing ulcerative gingivitis and
oral herpetic ulcers are considered to be the precur-
sor lesions of orofacial gangrene (noma/cancrum
oris) which is a devastating disease affecting children
and teenagers in sub-Saharan Africa whose immune
systems are impaired from infections and malnutri-
tion (24). So it seems as though differentiation
between NUG and AHG could be helpful if prompt
diagnosis of NUG followed by effective therapy could
prevent the onset of such a devastating disease. How-
ever, in the circumstances pertaining in many of the
countries in which orofacial gangrene occurs, it
seems unlikely that for the majority of affected chil-
dren prompt diagnosis and effective treatment of
NUG would be a regular occurrence. Indeed, differ-
entiation between NUG and AHG, both of which
when left untreated in malnourished children and
adolescents with repeated infections can result in
orofacial mutilation or death, seems unlikely to be
commonplace.
Other differential diagnoses for
NUG
Many standard periodontal textbooks list potential
differential diagnoses for NUG. For example one
from America (16) lists desquamative gingivitis and
benign mucous membrane pemphigoid and one
from Europe (40) lists desquamative gingivitis,
benign mucous membrane pemphigoid, erytherma
multiforme exudativum, streptococcal gingivitis and
gonococcal gingivitis. Both textbooks note, however,
that none of these conditions are clinically like NUG
in that none have punched-out ulceration of the
interdental papilla as a clinical feature. In addition
to acute herpetic gingivostomatitis and recurrent
intraoral herpes caused by herpes simplex type 1 or
type 2, other viral infections may have gingival
ulcerations as a clinical feature (73, 82). The vari-
cella-zoster virus causes both varicella (chickenpox)
and herpes zoster (shingles), both of which can have
gingival ulcerations as a feature (38, 82). EpsteinBarr
virus causing infectious mononucleosis may give rise
to gingival ulcerations as part of the intraoral features
of this condition (64).
In addition to NUG, streptococcal and gonococcal
gingivitis, other bacterial infections may also feature
gingival ulcers (e.g. infection by Treponema pallidum)
(85). The chronic painless ulcer of tuberculosis may
affect the gingiva (73) and the lesions of lepromatous
types of leprosy may present as gingival ulcers (72,
73). Fungi and parasites very seldom cause gingival
ulcerations in immunocompetent patients (90).
Other mucocutaneous conditions, in addition to
desquamative gingivitis, which is really just a mani-
festation of a range of vesiculobullous disorders (81),
benign mucous membrane, pemphigoid (26) and
erythema multiforme (9), oral lichen planus of the
ulcerative (66, 92) or erosive (38) types, pemphiqus
vulgaris (63, 98) and oral discoid and systemic lupus
erythematosus (79) may all give rise to gingival
ulcerations. However, these ulcerations due to infec-
tions or mucocutaneous conditions should be cap-
able of being differentiated from the gingival ulcers
of NUG/NUP on clinical grounds alone, as they
do not have the characteristic clinical features
and will not show a rapid favorable response to
anti-NUG therapy. The diagnosis of infections caus-
ing gingival ulceration may require microbiological
Table 5. Conditions that may include gingival ulcera-
tion as a clinical feature. The gingival ulceration, how-
ever, is not characteristic of NUG/NUP
Viral infections Acute herpetic gingivostomatitis
Recurrent intraoral herpes
Varicella
Herpes zoster
Infectious mononucleosis
Bacterial
infections
Streptococcal gingivitis
Gonococcal gingivitis
Syphilis
Tuberculosis
Leprosy
Mucocutaneous
conditions
Desquamative gingivitis
Benign mucous membrane
pemphigoid
Erythema multiforme
Oral lichen planus
Pemphigus vulgaris
Lupus erythematosus
Traumatic
conditions
Traumatic ulcerative gingival lesions
Toothbrushing
Flossing
Toothpicks/woodsticks
Factitious gingival ulceration
208
Corbet
and immunologic testing and the mucocutaneous
conditions may require biopsy and histologic, histo-
chemical, and immunohistochemical investigation
to establish the diagnosis.
Gingival ulceration may also result from allergy to
toothpaste constituents (23). The gingival ulcerative
conditions that may most closely mimic the clinical
features of NUG/NUP are induced by trauma. Gingi-
val lesions induced by improper toothbrushing have
been cited as important in the differential diagnosis
of NUG (11). These lesions have been further
described and the morphology of the lesions in rela-
tion to the vigor, duration, frequency and direction of
the toothbrush abuse has been detailed (88). From
this description of the clinical features, it should be
possible to differentiate the (predominantly) buccal
elongated supercial ulcerative lesions of toothbrush
trauma from the punched-out ulcers of NUG with
the predilection of NUG for affecting the interdental
papilla. This type of toothbrush lesion when chronic
has been termed the ``traumatic ulcerative gingival
lesion'' (6). However, the toothbrush is not the only
oral hygiene device that can cause traumatic injuries
to the gingiva. Improper ossing may also cause
gingival ulceration (27), restricted largely to the inter-
dental areas, often associated with a linear cleft on
the buccal or more commonly the lingual marginal
gingiva usually on the mesial and/or the distal aspect
of the interdental papilla. Toothpick use in some
parts of the world is common (19). The toothpick
is, however, as a dangerous instrument, especially
for children (7, 12). Toothpicks also have a potential
for damage outside the mouth including the gastro-
intestinal tract, liver, and bladder. They can cause
osteomyelitis and even necrotizing fasciitis resulting
in death, all of which is documented in the medical
literature. However, the damage often encountered
from improper use, especially after vigorous inter-
dental use of a large-sized or inappropriately shaped
toothpick, can clinically mimic the ulcerative lesions
of NUG conned to the interdental papilla (49), and
this is not well reported in the dental literature. The
top of the papilla can be abraded and blunted and
can be very tender, simulating the supercial clinical
appearance of NUG. Interdental brushes, when
newly introduced to a patient, may produce the same
type of lesion, but the history makes this diagnosis
easier. The diagnostic test for suspected traumatic
ulcerative gingival lesions is cessation of mechanical
plaque control and its substitution by chemical pla-
que control for a period of 2 weeks. Under the
current classication (4) these would be classied
as traumatic lesions (accidental) physical, and
removal of the physical trauma should result in heal-
ing within 10 days. Forceful food packing into an
open contact can leave a punched-out appearance
to the injured, painful interdental papilla, but this
lesion should be easy to differentiate from NUG even
when the papilla is very painful.
Another type of physical traumatic lesion is the
factitious lesion (4), previously known as self-
aficted gingival injury (70) or gingivitis artefacta
(89). This type of lesion may indeed produce a clin-
ical picture usually on accessible buccal gingival sur-
faces, of a ``punched-out'' type of ulceration on the
gingival margin, which if long-standing is often asso-
ciated with localized recession. Initially this lesion
can present as an ulcerative lesion similar to NUG
on the marginal gingival except that the interdental
papilla is usually spared, which it would not normally
be in NUG, and there should be no supercial slough,
as the surface is constantly being disrupted by the
self-induced trauma. This lesion is usually encoun-
tered in children and young patients, but may be a
presenting complaint in those with a psychiatric pro-
blem (32). When found in those whose mental age is
impaired (45, 47) its diagnosis is easier, but when
occurring in the apparently emotionally stable teen-
ager or adult, its diagnosis can be challenging and
factitious lesions can be mistaken for NUG/NUP, but
lack of a rapid response to anti-NUG therapy should
rule out NUG as the diagnosis.
Acute periodontal abscesses
Abscesses of the periodontium have been recently
classied as gingival abscess, periodontal abscess and
pericoronal abscess(17). Agingival abscesswasdened
as a localized purulent infection that involves the
marginal gingiva or interdental papilla. A periodontal
abscess was dened as a localized purulent infection
within the tissues adjacent to the periodontal pocket
that may lead to the destruction of periodontal liga-
ment and alveolar bone. A pericoronal abscess was
dened as a localized purulent infection within the
tissues surrounding a partially erupted tooth.
Gingival abscess
The diagnosis of a gingival abscess is uncomplicated,
as a gingival abscess is conned to marginal gingival
tissues, often at previously non-diseased sites. A
gingival abscess is often an acute inammatory res-
ponse to the impaction of a foreign body or material
into the gingiva from the oral surface or from the
gingival sulcus. The nding and retrieval of the
209
Acute lesions
offending foreign material is, thus, often diagnostic.
The diagnosis of a gingival abscess can be made on
the basis of a history of 12 days of pain and very
localized gingival swelling and the clinical nding of
a red, shiny swelling conned to marginal gingival
tissues. Figure 4 shows one such gingival abscess due
to a husk of a nut in a young Chinese female with
minimal periodontal disease experience.
Pericoronal abscess
A pericoronal abscess is also an uncomplicated diag-
nosis if a partially erupted tooth can be detected
clinically, and if an abscess is present in the absence
of any periodontal pocket on the vital tooth or vital
teeth adjacent to the partially erupted tooth.
Periodontal abscess
The periodontal abscess has been recently reviewed
(36, 62). A periodontal abscess may be acute in pre-
sentation or may be chronic in nature, with possible
acute exacerbations. The microbiology of periodon-
tal abscesses has been studied. Putative periodontal
pathogens are more frequently isolated from period-
ontal abscesses than from periapical abscesses (35).
Porphyromonas gingivalis has been reported to form
a sizeable proportion of microorganisms from peri-
odontal abscesses (29, 35, 67, 93). It has been recently
suggested that these studies may have inadvertently
mixed the ora of the abscesses with the ora fromthe
periodontal pockets (20). The percentage of spiro-
chetes in the exudate from abscesses, as visualized
by darkeld microscopy, has been suggested to be a
diagnostic aid in differentiating between periapical
abscesses and periodontal abscesses, in which the
percentage of spirochetes is higher (94). There has
been one study reported where this was one of the
microbiological investigations undertakenonexudates
from draining abscesses (78), but this diagnostic aid
does not appear to be used routinely, so the diagnosis
of periodontal abscesses relies usually on the history,
clinical examination and radiographic ndings.
Diagnosis of acute periodontal abscesses
A patient presenting with an abscess may have a
history known to the oral health care provider, or
this may be elicited through a history taking. Many
standard periodontal textbooks deal with differences
in the pain history elicited from patients with a per-
iodontal abscesses compared to that from patients
with a periapical abscess, and while the pain severity
varies (2), most but not all patients presenting with
an acute periodontal abscess complain of pain (35).
Other aspects worth investigating in the patient-cen-
tered interview include the history of previous peri-
odontal therapy, as some case series have shown a
reasonably high proportion of periodontal abscesses
presenting in patients undergoing periodontal treat-
ment (87). An explanation for this based on incom-
plete removal of calculus has been proposed (21),
though the possibility of allowing microbial penetra-
tion of the soft tissue pocket wall during subgingival
instrumentation (21), shown to occur in periodontal
abscesses (22), also cannot be ruled out. A history of
previous active periodontal therapy and current
maintenance or supportive periodontal therapy
may also be consistent with the presentation of an
acute periodontal abscess (14, 61) as may be the
history of a previous abscess at the site. Recent pre-
vious antibiotic therapy, often for non-oral reasons,
may be a trigger for periodontal abscess formation
(33, 34, 93). These should all be asked about in the
patient-centered interview. More controversial is the
predisposition of those suffering from diabetes mel-
litus to periodontal abscess formation (62), but the
history taking should always inquire about all current
medical conditions and medications. Patients may
also recall traumatic events, because the same force-
ful impaction of foreign bodies or materials which
can give rise to a gingival abscess in healthy sites,
may predispose toward periodontal abscess forma-
tion at previously periodontally involved sites (69).
The clinical ndings associated with periodontal
abscesses are swelling, edema and redness of the
affected periodontal sites (35, 36). Bleeding on prob-
ing the affected site will invariably occur (35), and
suppuration on pressure or on probing occurs in the
majority (35). Figure 5 shows the release of pus from
Fig. 4. Gingival abscess buccal to the mandibular right
lateral incisor, due to the husk of a nut, in a Chinese female
with minimal periodontal disease experience.
210
Corbet
an acute painful abscess on a tooth with a positive
response to pulp testing. That suppuration does not
always occur in response to periodontal probing is
due to the tortuous nature of some pockets. How-
ever, a ready release of pus on periodontal probing,
with alleviation of the symptoms of pain, pressure
and discomfort, can be helpful towards establishing
the diagnosis of a periodontal abscess. A tooth with
an associated periodontal abscess often displays
hypermobility (35), which can be helpful in deter-
mining the tooth a periodontal abscess arises from,
as periodontal abscesses may migrate from the
involved tooth and present at a site some distance
from the origin (35, 87). Tenderness of the tooth to
palpation may be present (2, 35, 36, 62). Lymphade-
nopathy occurs only in a minority of patients (35),
and fever and malaise probably are rare. Periodontal
pocket probing depths, while increased, are variable
(35). While some investigators have proposed a cut-
off of a pocket of 6 mm to be associated with a
periodontal abscess for investigative purposes (95),
a periodontal abscess may occur in association with
a shallower pocket. Although the majority of period-
ontal abscesses have been reported to occur inter-
dentally in one series (87), they may occur on any
tooth surface (35). A radiograph or radiographs may
show bone loss consistent with previous periodontal
disease experience and may reveal bone loss asso-
ciated with the periodontal abscess per se, but this is
not invariably so as a periodontal abscess in its initial
stage may precede any radiographic indication of
demineralization. Radiographs may reveal an apical
radiolucency, previous endodontic therapy with an
associated endodontic lesion, endodontic or intraca-
nal post preparation perforations or root fractures.
Any or all of these might suggest that the abscess is
not a periodontal abscess (2, 36, 62). In addition, an
indication that the abscess may not originate from a
periodontal pocket is when the abscess does not
resolve in response to usually effective emergency
periodontal treatment (i.e. drainage of pus and
pocket debridement). Standard periodontal text-
books describe the radiographic appearance of a per-
iodontal abscess (13). A tooth with an apical lesion
may also have a periodontal abscess or what is now
classied as a combined (periodontalendodontic)
lesion (4). A tooth with previous endodontic therapy
or a post-retained restoration may also experience a
periodontal abscess, so the radiographic picture
must be carefully correlated with the clinical ndings
in establishing a diagnosis. For instance, a narrow
probing depth, short of or to the tooth apex, com-
bined with a radiographic appearance of an apical
lesion in an abscessed tooth, would usually indicate a
periapical lesion as being the likely cause of the
abscess. `Èxceptions to the rule'' of the clinical nd-
ing of a narrow probing depth being more likely
associated with apical lesions, endodontic in origin,
have recently been detailed (31). The use of a radio-
paque marker, such as a guttapercha point inserted
into a sinus, if present, or fully into the narrow prob-
ing depth, can be helpful prior to exposing an
intraoral radiograph as this traces the tract of a defect
and can indicate the origin of the lesion by where the
guttapercha point terminates.
Table 6 presents other possible causes of the clin-
ical features of an acute periodontal abscess, namely
a red, painful swelling of the periodontal tissues, and
Table 6. Differential diagnoses for periodontal absces-
ses, when the clinical presentation is of a painful, red
swelling of periodontal tissues
Condition Differentiation
Gingival abscess Location
Periodontal health
Periapical abscess Pulpal response
Apical lesion on radiograph
No, or only narrow,
increased probing depth
Pericoronal abscess Partially erupted tooth
Adjacent vital teeth with no
increased probing pocket
depths
Incomplete root
fracture
Clinical finding of fracture
Radiographic finding of
fracture
Endodontic perforations
Post perforations
Radiographic features (more
than one exposure at
different angulations)
Fig. 5. Periodontal abscess, with pus on probing on the
maxillary left central incisor that responded as positive
to pulp testing in a Chinese adult.
211
Acute lesions
some ideas about how to differentiate between these.
Some standard texts also include infected periodontal
cysts in the differential diagnoses for periodontal
abscesses (50). The lateral periodontal cyst has been
reviewed, and while relatively common with a predi-
lection for the caninepremolar regions (48), there is
little evidence of these frequently becoming infected.
For red painful swellings in the periodontal tissues
around a tooth, Table 7 gives some indications as to
whether a periodontal abscess or a periapical abscess
or an abscess endodontic in origin is the likely cause.
Actually, in practice, the ``classic'' periodontal abscess
on a vital tooth is easy to differentiate from other
acute lesions which arise in the pulp or from within
the tooth. However, tests of pulpal responsiveness
(sensitivity) do not always represent the pulpal status
(71) and this may have particular relevance when the
challenge to the pulp has arisen from periodontal
disease. For instance, when periodontal destruction
reaches the apex of one root of a multi-rooted tooth
in this circumstance, partial pulpal necrosis may
result (53). This further complicates the diagnosis.
A periodontal abscess is dened as a purulent
infection (17). Many reactive and neoplastic lesions
may share the clinical appearance of a red swelling of
periodontal tissues, but these lesions, unless super-
infected, are not purulent, and often are not painful,
and so should not be misdiagnosed as periodontal
abscesses.
Tooth fractures leading to acute
abscesses
A fracture of a tooth extending from the supragingival
oral environment in an apical direction subgingivally
can give rise to a periodontal infection which will not
respond to conventional periodontal treatment.
Hence such fractures must be excluded in sites with
acute periodontal purulent infections. Figure 6(a,b)
shows the presentation of an acute abscess associated
with a deep probing depth at the abscessed site on
a tooth which was so tender that the patient would
not permit tenderness to percussion to be elicited.
Table 7. Differences between periodontal and periapical abscesses, indicating which may be more likely
Periodontal (more likely)
periodontal in origin
Periapical (more likely)
endodontic in origin
History Periodontal disease
Periodontal treatment
Previous antibiotic therapy
Caries, Fracture, Tooth wear,
Restorative treatment
Endodontic treatment
Clinical findings Vital pulp responses
Periodontal probing releases pus
Periodontal disease experience evident
Questionable or non-responsive to pulp tests
Narrow probing defect (may be isolated lesion)
Advanced caries, advanced toothwear, large
restorations, discolored tooth
Radiographic findings Alveolar crestal bone loss, angular bone
defect(s)
Furcation involvement(s)
Apical radiolucency,
(/ endodontic therapy)
Endodontic filling
Endodontic or post perforation(s)
Response to treatment Responds dramatically to release of pus,
subgingival debridement
Responds poorly, or not at all, to periodontal
therapeutic interventions
Fig. 6. (a) Abscess presenting in association with a loca-
lized probing depth on the maxillary rst molar, which was
extremely tender. (b) Crown-root fracture apparent on
investigating tooth under local anesthesia.
212
Corbet
Under local anesthesia, the fracture became appar-
ent (Fig. 6b). The patient had no recollection of a
specic traumatic event during chewing.
Crownroot fractures (37) leading to acute period-
ontal infections should be easy to diagnose, if no
complete extracoronal coverage restoration is in place
on the affected tooth or if no restoration obscures or
covers the fracture line. However often the presence
of restorations makes the diagnosis more difcult.
Viewing the affected tooth with magnication, using
either loupes or operating microscopes, it is believed
should assist in the visualization of cracks/fractures
extending from the supragingival environment to the
subgingival environment. The bite test, transillumi-
nation with a ber-optic light, and the use of dyes
applied to the tooth in the area of a suspected frac-
ture, even at the precursor stage of a crack, may all be
useful if the crown of the tooth is available for exam-
ination and is not obscured by restorations (1, 54).
Vertical crownroot fractures, both incomplete and
complete, may often be associated with an increased
periodontal probing depth adjacent to the fracture
(57). A ne caries probe or a ne root surface explorer
may be useful in attempting to conrm and trace the
course of a crownroot fracture within the connes
of the increased periodontal probing depth. Radio-
graphs, which should be a component of the com-
prehensive examination of a tooth presenting with an
acute periodontal infection, are not always helpful in
diagnosing vertical splits and fractures (86). It is
coming to be more widely recognized that Chinese
adults can experience vertical root fractures of non-
endodontically treated, non-carious, non-restored
teeth, especially molars (15, 62, 96, 97). Why this
Fig. 8. A periapical radiograph of
the mandibular left rst molar in a
57-year-old Chinese man with a his-
tory of painonchewing using the left-
hand side, which had not responded
to periodontal debridement of the
furcation involvement between the
distal and the distolingual roots of
the three-rooted tooth. The arrow
shows the loss of continuity of the
pulp canal space in the mesial root,
conrmed on exploratory open ap
surgery to be a vertical root fracture.
Fig. 7. Panoramic oral radiograph
showing a root-surrounding radiolu-
cency in the alveolar bone of the
mesial root of the mandibular left
first molar, and the suggestion of a
straightening of the pulp canal space
of the mesial root, in a 60-year-old
Chinese man presenting with an
acute abscess on the buccal surface
of the affected tooth, in whom a root
fracture was confirmed.
213
Acute lesions
happens so commonly is probably a combination of
the tooth anatomy in Chinese people and the dietary
habits of many Chinese that exert occasional great
pressures on the posterior teeth when used for open-
ing crab and lobster claws and in chewing bones.
Abrasive characteristics of the diet have usually
resulted in fairly extensive occlusal tooth wear prior
to the fracture (96). The diagnosis of vertical root
fractures, when the tooth crown is intact, is based
largely on the history taking, which characteristically
yields a history of sharp pain on chewing. With luck,
this pain can be elicited using a instrument, such as
the tooth-sleuth, or any other rubber stop on which
to close, and having the patient bite on this rubber
stop at various angles. The characteristic radio-
graphic nding, if the projection geometry is favor-
able and if the fractured part of the root has not
separated, is of a widening (and perhaps straighten-
ing) of the pulp canal space in a root and a surround-
ing radiolucency of the alveolar bone of the root
concerned. Figure 7 shows such an appearance
around the mandibular left rst molar, which pre-
sented with an acute abscess.
The vertical root fracture, with its attendant pain
history, can occur on a periodontally involved tooth.
Effective periodontal treatment does nothing to alle-
viate the symptoms, and often this can be diagnostic.
Figure 8 shows such a case. The patient complained
bitterly of pain on chewing using the left-hand side of
the mouth. Periodontal treatment aimed at debriding
the furcation involvement between the distal and
distolingual roots of this three-rooted mandibular rst
molar hadnot relievedthe symptoms, whichremained
unabated. The loss of continuity of the radiographic
pulp canal space in the mesial root (arrowed) indica-
tes the vertical fracture which was conrmed during
exploratory open ap surgery, which is a diagnostic
procedure occasionally found to be very useful, and
on the subsequent tooth extraction.
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216
Corbet
Diagnosis of periodontal
manifestations of systemic
diseases
Richard C. K. Jordan
Many systemic diseases may present or are mani-
fested in the gingiva, producing signs and symptoms
that resemble chronic gingivitis or periodontitis.
Although, by comparison, most of these conditions
are uncommon, their management is markedly dif-
ferent, hence the need for prompt and effective diag-
nosis. Moreover, the mechanism to establish the
diagnosis may differ from that used for chronic per-
iodontitis, relying more on histologic, immunohisto-
chemical and in some cases serologic testing.
Therefore, a high index of suspicion is often neces-
sary to identify specic signs and symptoms of these
systemic diseases and prompt the clinician to pursue
further investigations. This chapter will review a
number of systemic conditions that may mimic clini-
cally both gingivitis and chronic periodontitis, focus-
ing on the features that will assist in making the
diagnosis. The conditions are presented in order of
frequency from most to least common.
Systemic diseases that mimic
gingivitis
Lichen planus
The hallmark of lichen planus is reticulated, hyper-
keratotic lesions (60). However, when lichen planus
occurs on the gingiva it is frequently erythematous
with little evidence of reticulation and can be mis-
diagnosed as plaque-induced gingivitis (5). Figure 1
illustrates the gingival lesions arising in an otherwise
well patient. There are patchy erythematous lesions
but little evidence of hyperkeratosis. Biopsy of one
area of the gingiva showed features of lichen planus.
Lichenplanus is acommonchronicmucocutaneous
condition of unknown etiology. It affects between
0.22.0% of the population, with women slightly more
oftenaffectedthanmen(60). Withinthe mouth, lichen
planus presentsasbilateral, reticulated, hyperkeratotic
lesions that may be associated with erythema and
ulceration. On the gingiva, lichen planus is an impor-
tant cause of gingival inammation and pain (34).
The cause of lichen planus is not known; however,
many of the features suggest an immune-mediated
hypersensitivity reaction. The epithelium and under-
lying connective tissues are inltrated by T lympho-
cytes that express CD4 and CD8 antigens. In
addition, there is an increase in the number of intrae-
pithelial Langerhans cells, the antigen-presenting
cells of skin and mucosa (66). Inammation med-
iates basal epithelial cell damage that ultimately
results in hyperkeratinization. More marked inam-
mation can lead to more extensive epithelial break-
down and resultant oral ulceration (56).
Clinically, lichen planus affects middle-aged men
and women. It is rare in children. There is an asso-
ciation between lichen planus and hepatitis C infec-
tion but the proposed relationship between lichen
planus and diabetes mellitus or hypertension is more
likely due to a drug reaction than to the conditions
themselves (27, 67). The classical form of lichen pla-
nus has ne, white reticulations on the buccal
mucosa, tongue and gingiva with or without inam-
mation (Fig. 2). Often on the gingiva there is marked
inammation but with little discernible hyperkerato-
sis. The inammation is frequently patchy and dis-
tributed in all four quadrants, although focal disease
can also occur. The key to clinical diagnosis is the
identication of reticulated hyperkeratotic areas
either on the gingiva or at other intraoral sites. On
the gingiva, superimposed inammation and abun-
dant plaque will make this diagnosis more challen-
ging (34).
217
#
PERIODONTOLOGY 2000
The diagnosis of lichen planus is made by biopsy
showing a band-like inltrate of lymphocytes in the
supercial connective tissue and into the epithelium
that exhibits hyperkeratosis and degeneration of
basal keratinocytes (Fig. 3)(56). For equivocal cases,
direct immunouorescence can be helpful, showing
a band of brinogen deposited at the epithelial
mesenchymal interface (Fig. 4)(35).
Topical corticosteroids are the mainstay of treat-
ment for lichen planus. Occasionally systemic ster-
oids can be used for patients with severe disease,
prior to initiation of topical therapy (69). Other treat-
ments have included vitamin A analogues and anti-
fungal medications but these have proven less
effective than corticosteroid therapy (43). Topical
cyclosporin has also been used with promising
results although the medication costs are high in
comparison with corticosteroids (25).
Primary herpes simplex infection
Figure 5 shows the clinical presentation of an
11-year-old slightly febrile female patient with a 2-day
history of generalized, erythematous, painful gingiva.
Previously she had good oral hygiene and there was
minimal plaque. Questioning of the parent revealed
that she had been exposed to a relative with recurrent
Fig. 2. The hallmark of oral lichen planus is reticulated
hyperkeratotic lesions on the buccal mucosa. In this case
there is very little evidence of inammation and no ulcera-
tion.
Fig. 1. There is patchy inammation affecting the man-
dibular and maxillary marginal gingiva with little evidence
of hyperkeratosis. Biopsy showed features of oral lichen
planus.
Fig. 5. Primary herpetic gingivostomatitis typically has an
acute presentation with painful, diffusely erythematous
gingiva with or without areas of ulceration.
Fig. 3. Histology of oral lichenplanus shows hyperkeratosis
of the epithelium covering connective tissue containing a
band-like inltration of lymphocytes in the lamina propria.
Fig. 4. Immunouorescence studies of oral lichen planus
show deposits of brinogen (green) at the epithelial-
mesenchymal interface (courtesy of Dr. T. Daniels, UCSF).
Jordan
218
herpes labialis several days earlier. Based on the his-
tory and clinical ndings, a diagnosis of primary
herpetic gingivostomatitis was made.
Herpes simplex virus infections are common in the
mouth. Herpes simplex virus is a DNA virus and
member of the herpes family. There are two forms
of the disease: primary (systemic) infection and sec-
ondary (localized) disease. In immunocompetent
patients, both forms are self-limiting. Primary herpes
simplex virus produces generalized gingivitis termed
primary herpetic gingivostomatitis. Infection is
transmitted in aerosolized droplets or by direct con-
tact (65). Following an incubation period of several
days to 2 weeks a primary vesiculoulcerative eruption
develops on any oral mucosal surface including the
gingiva, which are red, slightly hyperplastic and ten-
der (Fig. 6). Primary herpes simplex virus most com-
monly affects children but adults who have not been
previously exposed or infected may also develop the
disease. Resolution of the primary infection can be
expected in 710 days, during which the virus
becomes latent in basal ganglia (64).
The diagnosis is made based on history and clin-
ical ndings. In equivocal cases, serology demon-
strating a rising antibody titer can be useful in
establishing the diagnosis. In the immunocompe-
tent, treatment is palliative until resolution occurs
(8). Acyclovir can also be used either topically or
systemically early in the course of the disease.
Mucous membrane pemphigoid
Mucous membrane pemphigoid occurs commonly
on the gingiva and can frequently mimic plaque-
induced gingivitis (75). Indeed, when mucous mem-
brane pemphigoid occurs on the gingiva it is often
described under the clinical rubric desquamative gin-
givitis or gingivosis. Figure 7 illustrates a 56-year-old
female patient with persistent erythematous gingivi-
tis that was unresponsive to local plaque control
procedures. A biopsy was taken from the gingiva that
showed subepithelial separation consistent with
mucous membrane pemphigoid and the diagnosis
was conrmed by direct immunouorescence exam-
ination, showing deposits of IgG and complement at
the epithelialmesenchymal interface.
Mucous membrane pemphigoidis a chronic blister-
ing disorder that affects the oral and ocular mucosae.
Other sites can be affected include the oropharyngeal
mucosa and the skin. The condition is also known as
cicatricial pemphigoid, benign mucous membrane
pemphigoid, and ocular pemphigoid.
The etiology of mucous membrane pemphigoid is
unknown, although it is believed to be an autoim-
mune disease (75). Immunoglobulins and comple-
ment are frequently identied along the basement
membrane of oral epithelium and their presence, as
demonstrated using immunouorescence study, is
diagnostic. The target antigens are laminin 5 and a
180 kDa protein bullous pemphigoid antigen (6).
The clinical features of mucous membrane pem-
phigoid are variable. It typically affects adults and the
elderly, with women more often affected than men.
The condition is rare in children. Erythema is fol-
lowed by vesicle and bulla formation that rupture
to leave ulcerations (Fig. 8). On the gingiva, there is
band-like or patchy erythema that affects all four
quadrants. Usually there is supercial gingival
Fig. 7. Mucous membrane pemphigoid can present as dif-
fusely erythematous gingiva. Collapsed vesicles and bulla
may also be seen with careful inspection.
Fig. 6. Extensive involvement of the palatal gingiva is also
a common nding in primary herpetic gingivostomatitis.
219
Periodontal manifestations of systemic diseases
ulceration. Patient discomfort frequently results in
discontinuation of routine oral hygiene, leading to
a build-up of plaque and consequent worsening of
the condition (70). A key point in the diagnosis is the
identication of collapsed vesicles or bullae,
although it is rare to see these intact since trauma
commonly induces rupture.
Mucous membrane pemphigoid can also affect
other mucosal surfaces suchas the conjunctiva produ-
cing erythema and ulceration (Fig. 9). Conjunctival
scarring leads toinversionof the eyelashes (entropion)
that abrade the cornea, thereby compromising vision.
Other mucosal sites affected include the pharynx and
genital region. About 10% of patients with mucous
membrane pemphigoid also have skin lesions.
The diagnosis of mucous membrane pemphigoid
is established by biopsy showing separation of the
epithelium and connective tissue at the level of the
basement membrane (Fig. 10). Areas of ulceration
are not diagnostic and are not suitable for histologic
examination. Therefore, intact or collapsed vesicles
or an area adjacent to ulceration are preferable sites
to biopsy. Direct immunouorescence, showing lin-
ear deposits of IgG and complement along the base-
ment membrane region, is often necessary to
establish or conrm the diagnosis (Fig. 11). Indirect
immunouorescence studies of serum are usually
negative and are not often performed (19).
Mucous membrane pemphigoid is treated using
topical and systemic steroids often in combination.
Other medications that have been used to control the
disease include sulfapyridine, sulfones, antibiotics,
gold injections and nutritional supplements (70).
Linear IgA disease
Figure 12 shows a female patient presenting for
assessment of erythematous lesions affecting the
Fig. 8. A collapsed bulla is present on the attached gingiva
of this patient with mucous membrane pemphigoid.
Trauma frequently leads to rupture of the bulla contents.
Fig. 10. Biopsy of mucous membrane pemphigoid shows
characteristic separation of the epithelium from the
underlying connective tissue at the level of the basement
membrane.
Fig. 11. Immunofluorescence study of an oral lesion from
a patient with mucous membrane pemphigoid shows
deposits of IgG at the epithelial-mesenchymal interface
(yellow line) (courtesy of Dr. T. Daniels, UCSF).
Fig. 9. Patients with ocular involvement in mucous mem-
brane pemphigoid may show marked conjunctivitis. If left
untreated, there may be scarring of leading to inversion of
the eyelash and abrasion of the cornea.
220
Jordan
mucogingival junction of the maxillary tissues. Her
oral hygiene was good and she was otherwise well. A
biopsy was performed that showed separation of the
epithelium from the underlying connective tissue at
the level of the basement membrane and, by immu-
nouorescence examination, linear deposits of IgA at
the epithelialmesenchymal interface.
Linear IgA disease is a chronic mucocutaneous
disease of the skin that also affects the oral mucosa
and gingiva. Similar to dermatitis herpetiformis this
condition is also characterized by deposits of IgA in
the tissues but is not associated with gluten-sensitive
enteropathy (33). Linear IgA disease resembles
mucous membrane pemphigoid both clinically and
histologically. The oral lesions are erythematous and
ulcerative; when involving the gingiva, lesions are
seen in all four quadrants (14). Microscopically, there
is separation of the epithelium from the connective
tissue at the level of the basement membrane
(Fig. 13). By direct immunouorescence, linear
deposits of IgA are seen along the basement mem-
brane with the target a 120 kDa protein (11). Linear
IgA disease is treated in a manner similar to mucous
membrane pemphigoid using topical and systemic
steroids. Cyclosporine has also proven effective in
the management of the disease but, unlike dermatitis
herpetiformis, linear IgA disease usually shows a
poor response to dapsone (44, 76).
Pemphigus vulgaris
Pemphigus comprises a group of autoimmune, vesi-
culobullous disorders characterized by involvement
of the skin, mouth and other mucous membrane
sites. Four main types are recognized: pemphigus
vulgaris, pemphigus foliaceous, pemphigus erythe-
matosus and pemphigus vegetans. They differ in
the level of intraepithelial involvement. Pemphigus
vulgaris and pemphigus vegetans affect the supraba-
sal and prickle layers while pemphigus foliaceous
and pemphigus erythematosus affect the upper
prickle cell and keratin layers. Only pemphigus vul-
garis and pemphigus vegetans involve the oral
mucosa (74). Pemphigus vegetans is very rare and
is generally considered a variant of pemphigus vul-
garis. All forms of pemphigus arise because patients
have circulating immunoglobulin directed against
desmosomes of skin and mucous membranes. The
target antigen is desmoglein 3, a protein in the des-
mosome-tonolament complex (29). The antibody
binding to these sites activates complement and
plasminogen activator, resulting in acantholysis,
vesicle formation, erosion and ulceration.
The clinical features of pemphigus vulgaris are
nonspecic with areas of erosion at any mucosal site,
although nonkeratinized sites appear to be affected
most often. When the lesions involve the gingiva,
there is erythema, vesiculation and ulceration
(Fig. 14)(20). Skin lesions may or may not be present.
Pemphigus vulgaris is usually a disease of older peo-
ple, with women being more affected than men. The
oral mucosa is involved initially in about 50% of
cases of pemphigus vulgaris and, indeed, oral invol-
vement often precedes involvement at other sites.
Because the condition is life threatening without
treatment, it is important to conrm clinical suspi-
cion of the disease by histologic and immunologic
investigations at an early stage (74).
The diagnosis is made by biopsy of an intact or
recently ruptured vesicle or bulla (Fig. 15). Tissue
Fig. 12. Linear IgA disease often has a clinical presenta-
tion similar to mucous membrane pemphigoid. Here there
is bilateral, band-like inflammation of the maxillary
gingiva and alveolar mucosa.
Fig. 13. Biopsy of linear IgA disease shows separation of
the epithelium from the underlying connective tissue at
the level of the basement membrane. There is inflamma-
tion in the connective tissues with inflammatory cells and
extravasated red blood cells present in the cleft.
221
should be sent for both routine histopathology and
direct immunouorescence. A blood sample should
be sent for indirect immunouorescence, which will
demonstrate circulating autoantibody (IgG) (19). The
titer of circulating antibody is important since it
reects the degree of disease activity and can be used
to monitor the effectiveness of immunosuppression
therapy.
Pemphigus vulgaris is managed with systemic cor-
ticosteroids, often taken for prolonged periods. Topi-
cal corticosteroids are also used for oral lesions in
conjunction with systemic therapy since they often
show poor response to the systemic route (58). Once
control is achieved and no new lesions develop, then
the dose of steroids is reduced to a maintenance
level. Drugs such as azathioprine or cyclophospha-
mide have an important role in management as they
allow the dose of steroid to be reduced (61). Because
pemphigus is a lifelong disease, corticosteroid ther-
apy can be reduced but usually never discontinued.
Occasionally, complications of long-term steroid
therapy, such as cataracts, diabetes and duodenal
ulcers, can develop and these need appropriate
investigation and treatment.
Leukemia
Figure 16 shows the clinical presentation of a patient
with diffusely hyperplastic and erythematous gingiva
that bled easily. She had been unwell prior to pre-
sentation suffering lethargy and fatigue. Biopsy of the
gingiva showed sheets of atypical myeloid cells con-
sistent with a diagnosis of chronic myeloid leukemia.
This diagnosis was conrmed by bone marrow
biopsy showing similar features.
Leukemias encompass a group of disorders char-
acterized by neoplastic proliferation of bone marrow
lymphocyte or myeloid precursors that replace the
marrow and can be identied in the peripheral
blood. Neoplastic cells can also inltrate other
organs such as the liver, spleen, lymph nodes and
other tissues (50). A number of different causes have
been attributed to the development of specic forms
of leukemia, including genetic factors, specic chro-
mosome translocations in chronic myeloid leukemia
and environmental agents such as benzene, ionizing
radiation and viruses such as HTLV1 in adult T-cell
leukemia (12, 15, 46).
Leukemias are classied based on the type of pro-
genitor cell (myeloid or lymphoid lineage) and the
clinical presentation (acute or chronic). Acute leuke-
mias are characterized by the presence of immature
cells and a fulminant clinical course and chronic
Fig. 14. Pemphigus vulgaris often presents first in the
mouth. Here there is a collapsed bulla affecting the upper
labial mucosa. It is uncommon to see intact vesicles or
bullae in pemphigus vulgaris.
Fig. 16. Markedly and diffusely hyperplastic gingiva in a
patient who was shown to have chronic myelogenous
leukemia.
Fig. 15. Biopsy of a gingival lesion from a patient with
pemphigus vulgaris shows separation within the epithe-
lium (acantholysis) with free-floating epithelial cells in the
cleft.
222
Jordan
leukemias are characterized by the presence of better
differentiated, mature cells and a more indolent clin-
ical course. Chronic leukemias of the myeloid lineage
are the most common form to inltrate the gingiva
causing edema and erythema. The gingivae are red,
boggy, edematous, and bleed easily. Sometimes this
may be the initial presenting feature of chronic mye-
loid leukemia. The gingival appearance is due to
inltration by neoplastic myeloid cells and is initially
out of proportion to the amount plaque that is pre-
sent.
The diagnosis of leukemia is made by biopsy show-
ing sheets of neoplastic, immature myeloid cells
(Fig. 17). Histochemistry showing chloroacetate
esterase expression and immunohistochemistry to
demonstrate myeloid lineage will assist making the
diagnosis. Bone marrow biopsy will conrm the diag-
nosis and is used to type the disease (28, 37).
Chemotherapy is used to manage all forms of leu-
kemia (30). Some acute leukemias show good
response to treatment and cures can be expected
while others such as chronic lymphocytic leukemia
are essentially incurable but characterized by a pro-
tracted clinical course (23).
Wegener's granulomatosis
Wegener's granulomatosis is an inammatory con-
dition of unknown etiology. The classical clinical
triad consists of necrotizing granulomatous vasculitis
in the upper respiratory tract, lung and kidney; how-
ever, most often patients present with only two of the
three sites of involvement. Patients with Wegener's
granulomatosis present with sinusitis, rhinitis, nasal
stufness, epistaxis, hemoptysis, and hematuria (10).
Ulceration of the maxilla is common, as is perfora-
tion of the hard palate in the midline (36). The
gingivae appear hyperplastic, red and granular with
a generalized, uniform distribution (Fig. 18). This
appearance has been likened to a ``strawberry stuck
on the gums'' (49).
The diagnosis of Wegener's granulomatosis is
made by biopsy showing necrotizing vasculitis and
granulomatous inammation. Serologic demonstra-
tion of antineutrophil cytoplasmic antibodies is posi-
tive in over 90% of patients with active disease and
helpful to establish the diagnosis. This assay is also
used to monitor disease response to therapy (9). Prior
to the introduction of chemotherapy Wegener's
granulomatosis had a uniformly poor prognosis.
Wegener's granulomatosis is now managed with
the cytotoxic agent cyclophosphamide supplemen-
ted with prednisone. Remission can be expected in
about 75% of cases (1, 51).
Systemic diseases that mimic
periodontitis
Tuberculosis
Tuberculosis is caused by the aerobic bacillus Myco-
bacterium tuberculosis. Tuberculosis is one of the
most common infectious diseases and is particularly
common in the developing world. Spread of the
infection is favored by poor living conditions, low
socioeconomic status, low native resistance, and
compromised immunity.
Tuberculosis is spread by infected aerosol droplets
to the lungs where the organism is engulfed by
macrophages, leading to a characteristic granuloma-
tous reaction. The infection may lay dormant in the
lungs or spread to regional lymph nodes. Clinically,
the lesions occur mainly in the lung, usually in the
Fig. 17. Gingival biopsy of a patient with chronic myelo-
genous leukemia shows a diffuse infiltrate of immature
myeloid cells.
Fig. 18. The oral presentation of Wegener's granulomatosis
shows hyperplastic, erythematous gingivaethat bleedeasily.
223
upper lobes. Lesions can occur in other sites by
implantation of infected sputum (47). Within the
mouth, any mucosal site may be involved, including
the gingiva. Here, the appearance is nonspecic,
showing ulceration and bone destruction (3, 42).
The diagnosis of tuberculosis is established by
biopsy since the clinical features are nonspecic,
resemblingother ulcerativeconditions suchas neopla-
sia. On histology, there are well-formed granulomata,
necrosis and Langhans-type giant cells (Fig. 19). His-
tochemical staining will demonstrate acid-fast bacilli
contained within the granulomata (13).
Tuberculosis is managed with an antibiotic regi-
men consisting of combinations of isoniazid, rifam-
pin, streptomycin and ethambutol with treatment
times typically prolonged amounting to several
months or a year (2).
Deep fungal infections
Figure 20 shows a male patient who presented with a
painless gingival ulceration of several weeks' dura-
tion. It had been unresponsive to local plaque control
procedures. He also had a cough and recent weight
loss. Gingival biopsy showed granulomatous inam-
mation and giant cells containing organisms consis-
tent with histoplasmosis.
Deep fungal infections are caused by a group of
organisms that typically infect the lungs but may also
produce secondary lesions elsewhere including the
gingiva. This group includes the diseases histoplas-
mosis, coccidiodomycosis, blastomycosis and cryp-
tococcosis. Histoplasmosis is endemic in the Eastern
United States and acquired by inhalation of dried
pigeon droppings (22). Coccidiodomycosis is also
endemic in the Western United States, particularly
the San Joaquin Valley of California, where it
is known locally as ``valley fever''. Blastomycosis is
also seen in North America particularly in the Ohio-
Mississippi river basin (63). Cryptococcosis has a
widespread distribution and is acquired by inhala-
tion of avian excrement.
The initial lesions of these deep fungal infections
occur in the lungs. Symptoms include cough, fever,
night sweats, weight loss, and chest pain. Oral infec-
tions typically follow implantation of infected spu-
tum from the lung. On the gingiva there are
ulcerative, indurated, and frequently painful bone-
destroying lesions that can be single or multiple.
Diagnosis is established by biopsy showing granulo-
matous inammation and demonstration of the
organism using histochemical stains (Fig. 21). Cul-
ture and serologic testing do not play a signicant
role in the diagnosis of these diseases (22, 63).
Treatment consists of the antifungal medication
uconazole, ketoconazole or amphotericin B. The
Fig. 20. A solitary ulcer causing bone loss around a
mandibular tooth in this patient with biopsy-proven
histoplasmosis.
Fig. 19. Biopsy of a patient with oral tuberculosis lesions
shows caseating granulomas with Langhans-type giant
cells.
Fig. 21. A silver stained (GMS) biopsy of histoplasmosis
show small round organisms (black) surrounded by
inflammatory cells (green).
224
Jordan
choice depends on the clinical setting, severity of
infection and prior antifungal use.
Metastatic carcinoma
Figure 22 shows a gingival mass from a patient
known to have esophageal carcinoma. The mass
was not painful and had been growing for several
weeks. Biopsy showed carcinoma consistent with
an esophageal primary.
In terms of frequency, the most common malig-
nancy of the skeleton is metastatic carcinoma.
Approximately 1% of all malignancies metastasize
to the jaws with 80% of these to the mandible, about
15% to the maxilla and 5% to both jaws (77). Meta-
static tumors can also metastasize to the gingiva,
producing a gingival mass, or it may mimic period-
ontal disease. In adults the most common metastatic
malignancies to the jaws are breast carcinoma in
women and lung carcinoma in men (53). In children,
neuroblastoma and osteosarcoma are the most com-
mon tumors to metastasize to the jaws (21). In about
30% of cases a jaw metastasis will be the rst sign of
the primary malignancy.
The most common sites in the jaws for metastatic
tumors are the premolarmolar areas and the angle
and body of the mandible. Bone pain, loosening of
the teeth, paresthesia, swelling and gingival mass are
the most common presenting features (26). Radio-
graphically, there is a poorly demarcated, irregular,
expansile radiolucency with a moth-eaten periphery.
However, some tumors such as prostatic and thyroid
carcinomas are often osteoblastic.
The diagnosis is established by biopsy showing
metastatic tumor. If the primary is not recogniz-
able by histology alone, immunohistochemistry is
often necessary to establish the diagnosis. Solitary
metastatic deposits are managed with surgery and
or chemoradiotherapy. Jaw deposits that represent
generalized metastases are managed with palliation.
Overall, metastasis is a poor prognostic sign asso-
ciated with a 10% 5-year survival rate (53).
Langerhans cell disease
Figure 23 shows a female patient who presented with
hyperplastic gingiva and periodontal bone loss of
several weeks' duration. The condition was unre-
sponsive to local plaque control procedures. A
panoramic radiograph showed extensive bone loss
around the roots of the teeth, prompting biopsy,
which showed sheets of Langerhans cells mixed with
eosinophils consistent with a diagnosis of Langer-
hans cell disease. A skeletal survey failed to identify
any other lesions and she was then treated with local
radiotherapy.
Langerhans cell disease, formerly known as histio-
cytosis X, represents a proliferation of antigen- pre-
senting Langerhans cells. There is spectrum of
behavior from localized, relatively indolent, intra-
osseous disease to widespread, fulminant disease of
theskinandorgans resemblingneoplasia. The etiology
of Langerhans cell disease is unknown. The acute dis-
seminated form may represent overt neoplasia but
less disseminated forms may represent an immune
response to an unknown antigenic challenge (40).
Classically, there are three distinct types of Langer-
hans cell disease. Chronic localized Langerhans cell
disease, or eosinophilic granuloma, produces single
or multiple bone lesions only. Chronic disseminated,
or HandSchullerChristian, disease consists of a clas-
sical triad of lytic bone lesions, exophthalmos, and
diabetes insipidus. Acute disseminated Langerhans
cell disease, or LettererSiwe disease, is characterized
Fig. 22. Esophageal carcinoma metastatic to the mandib-
ular gingiva.
Fig. 23. The clinical presentation of a patient with
Langerhans cell disease showing hyperplastic and erythe-
matous gingiva.
225
by a fulminant, rapidly progressive usually fatal
course with widespread inltration of the skin, bones
and organs. Typically, neonates and infants are
aficted (38).
In general, Langerhans cell disease is a disease of
children and young adults. Males are slightly more
often affected than females. Any bone can be
involved including the skull, mandible, maxilla, ribs,
vertebrae and the long bones (55). Involvement of the
jaws consists of single or multiple radiolucencies
with sharply dened borders producing a punched-
out appearance. Most often lesions are located at the
apices of teeth, producing the appearance of teeth
`òating in air'' (Fig. 24)(16). Teeth become loose in
the affected area and there may be pain, tenderness
and swelling. The gingival tissue is frequently
inamed, hyperplastic and ulcerated.
Diagnosis is made by biopsy showing sheets of
Langerhans cells intermixed with eosinophils. Imm-
unohistochemical staining will show expression of
CD1a and S100 antigens by the Langerhans cells (32).
Treatment varies depending on the extent of the
disease and site. For small, well localized chronic
Langerhans cell disease surgical excision may be an
option but, more commonly, low-dose radiation
therapy is used. Chemotherapy is reserved for more
extensive disease or for those with acute dissemi-
nated forms. Cure rates vary and depend on the
extent and type of Langerhans cell disease (39).
Papillon-Lefevre syndrome
Papillon-Lefevre syndrome is a hereditary disorder
transmitted in an autosomal recessive manner. The
predominant clinical manifestations are palmar and
plantar keratosis coupled with rapid periodontal dis-
ease (57). Patients show an immunologic decit
related to altered or impaired neutrophil function
that is the result of mutation in the cathepsin C gene
located on chromosome 11q14 (68). The prevalence
of Papillon-Lefevre syndrome is about 14 per mil-
lion in the general population.
The dermatologic manifestations consisting of pal-
mar and plantar keratosis appear in the rst 3 years
of life. Other sites that can be involved include the
legs, thigh, toes, and ngers. Dramatic, advanced
periodontitis affecting both the primary and perma-
nent dentition is also characteristic. The gingiva
appear hemorrhagic and hyperplastic with loss of
bone and tooth exfoliation (7). The diagnosis of
Papillon-Lefevre syndrome is made by history and
clinical ndings. Biopsy of gingival tissues is nonspe-
cic, showing acute and chronic inammation with
granulation tissue.
Retinoids are used to treat the skin lesions (45).
Treatment of the periodontal disease is often dif-
cult, with poor success. Most patients with Papillon-
Lefevre syndrome show rapidly progressing period-
ontal disease and eventually lose their teeth (24).
Scrupulous oral hygiene coupled with topical anti-
septic mouthrinses and antibiotic prophylaxis for
Actinobacillus actinomycetemcomitans can slow the
pace of periodontal destruction but it may not be a
cure (73).
Hypophosphastasia
Hypophosphastasia is a rare, hereditary condition
transmitted in an autosomal recessive manner that
produces a deciency of tissue-nonspecic alkaline
phosphatase. Over 60 different mutations of the alka-
line phosphatase gene have been described in associ-
ation with this disease (31, 48). Alkaline phosphatase
is thought to play a role in the production of bone but
its mechanism of action is unknown (71).
Four types of hypophosphatasia are recognized,
depending on the time of onset and severity of symp-
toms (62). The perinatal form is congenital and
invariably fatal due to respiratory failure. The infan-
tile form appears within 6 months of life and is asso-
ciated with 50% morbidity. Infants so affected show
vomiting and hypotonia. Skeletal manifestations that
resemble rickets are common, as are nephrocalcino-
sis and nephrolithiasis. If these infants survive, there
is premature shedding of the primary teeth. The
childhood form is detected within 624 months of
age and has a range of severity. The most consistent
feature is premature loss of the primary teeth, which
have enlarged dental pulps. The cementum is hypo-
plastic or aplastic and there is frequently hypoplasia
Fig. 24. Radiographic changes in a patient with Langer-
hans cell disease showing punched-out radiolucencies
surrounding the roots of the mandibular teeth. Biopsy of
one of these lesions confirmed the diagnosis.
226
Jordan
of the enamel. There is alveolar bone loss, especially
in the anterior mandible and maxilla. Long bones
also show inadequate mineralization and suffer
stress fractures. The adult form is rare, occurring in
late adolescence and adulthood, and has a mild pre-
sentation. There is premature loss of the primary or
permanent teeth. Stress fractures are also common,
particularly following relatively minor trauma (54).
The diagnosis of hypophosphatasia is made based
on the clinical ndings coupled with the demonstra-
tion of reduced levels of serum alkaline phosphatase
and increased levels of phosphoethanolamine in the
urine and blood (72). Since there is no therapeutic
means to replace the alkaline phosphatase, treat-
ment is essentially symptomatic. The prognosis var-
ies by the onset of symptoms and type of the disease.
Patients with both childhood and adult forms have a
normal life span.
Cyclic neutropenia
Neutropenia is dened as an absolute reduction in
circulating neutrophils. Prolonged or persistent neu-
tropenia is associated with leukemia, certain blood
dyscrasias, many drugs and radiation or chemother-
apy. Cyclic neutropenia is a rare disorder character-
ized by a severe, cyclical depression of neutrophils
from the blood and bone marrow. Mean periodicity
is about 21 days (17). Recent genetic, molecular, and
cellular studies have shown that autosomal-dominant
cyclic neutropenia and sporadic cases are due to a
mutation in the gene for neutrophil elastase (ELA2),
located at chromosome 19p13.3. This enzyme is
synthesized in neutrophil precursors early in the pro-
cess of primary granule formation (4, 18).
During episodes of cyclic neutropenia there is
fever, malaise, cervical lymphadenopathy, infections,
and oral ulcers. Mouth ulcerations are common on
nonkeratinized surfaces and may appear as single or
multiple discrete lesions. Patients are often prone to
severe periodontal disease (52).
The diagnosis is established by examination of the
peripheral blood differential showing a reduction in
circulating neutrophils during episodes of oral
ulceration. Usually, it is necessary to sample the
blood sequentially over several days to document a
drop in circulating neutrophils. The neutrophil count
should be less than 500/mm
3
for 35 days during
each of three successive cycles to establish the diag-
nosis (17).
There is no specic management for the condition.
Medical investigations may be needed to rule out
other causes of neutropenia (41). During episodes
of neutropenia, antibiotics may be given to prevent
oral infection. Scrupulous oral hygiene is needed to
minimize periodontal disease (59).
Summary
Many systemic diseases can mimic periodontitis or
gingivitis. These include many immunologic, infec-
tious, neoplastic and metabolic disorders. In com-
parison with periodontitis and gingivitis, these
disorders are considerably less common. However,
since their diagnosis and management differ, a high
index of suspicion is frequently needed when con-
fronted a patient presenting with gingival or period-
ontal disease. This chapter has reviewed a number
of systemic conditions that may mimic clinically
both gingivitis and chronic periodontitis and has
focused on the features that may assist the clinician
in making the diagnosis and providing specic thera-
pies.
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229
Clinical evaluation of dental
implant treatment
Christoph H. F. Hammerle & Roland Glauser
During the past decades, implant therapy has devel-
oped into a successful treatment for partial and com-
plete edentulism. Based on the healing response of
bone leading to osseointegration, a desired type of
anchorage of the endosseous part of dental implants
can predictably be obtained. The nature of this bone-
to-implant relationship is obviously capable of sup-
porting the loads resulting from forces exerted onto
the implant-borne suprastructures.
In addition, under standard clinical conditions a
stable and competent soft tissue seal is established
during the phase of tissue integration at the part of
the implant penetrating the tissues and extending
into the oral cavity. This soft tissue seal comprises
an epithelial attachment and a connective tissue
adaptation to the neck portion of the implant. A
stable mechanical seal is thus provided by these tis-
sues along with their capacity to mount a competent
immunologic reaction to the microbial challenge
occurring at the marginal area of the implant.
An understanding of the factors crucial for obtain-
ing stable and lasting tissue integration of functional
implants has led to a therapeutic approach rendering
excellent long-term success. Many factors play
important roles in obtaining long-lasting implant
stability. These include factors related to the beha-
vior and systemic health of patients, to the health of
the implant recipient site, to the amount and quality
of the tissues at the recipient site, to the forces
exerted onto the implant and surrounding tissues,
to the implant system and type of implant chosen,
and to the skill of the therapists providing care
including surgical, prosthetic as well as maintenance
procedures.
During treatment planning, execution of therapy,
and maintenance of patients, the clinician is faced
with the challenge of making the right decisions for
rendering implant success to an individual patient.
In order to do so, the clinician has to be able to
obtain all pertinent information regarding the
patient history and to perform the necessary exam-
inations prior to implant therapy, during implant
therapy and also during the maintenance phase.
The aim of this chapter is to review the critical
parameters available to evaluate patients in need of
implant therapy and monitor implants on a long-
term basis.
Diagnostic procedures prior to
implant placement
Competent treatment planning is a key to long-term
success of implant therapy. Indications and contra-
indications must carefully be balanced and optional
treatments must be taken into the decision-making
process in each individual patient eligible for implant
therapy.
Systemic factors
Information derived from a thorough medical history
is mandatory as an initial step of treatment planning.
The aim of this history is to identify current and past
diseases, medications taken by the patient, and pre-
sent or past therapeutic interventions relevant to the
treatment with oral implants. In the case of systemic
conditions that may inuence implant therapy, con-
tacting the physician treating the patient for further
clarication is recommended.
Prerequisites for implant therapy include an
undisturbed wound healing capacity, the possibility
of mounting a competent host response to microbial
challenges, and the absence of bleeding disorders. In
addition, jaw growth should have been completed.
An exception to this rule is the placement of perma-
nent or temporary implants as elements of ancho-
rage for orthodontic therapy.
230
#
PERIODONTOLOGY 2000
Systemic diseases including rheumatoid arthritis,
osteomalacia, or developmental disorders like osteo-
genesis imperfecta are considered high risk factors in
implant therapy. Whether or not and to what degree
osteoporosis is to be considered a risk factor is still
under debate (9, 22).
Patients severely immunocompromised as a result
of medications, systemic diseases, or irradiation
therapy are considered at risk for implant therapy
failure (50). However, when approved protocols are
followed in irradiated patients, e.g. regarding the
time span between termination of irradiation and
implant placement, survival rates similar to those
reported for implants placed in native, non-irra-
diated bone can be obtained (38).
Non-controlled severe diabetes, in particular insu-
lin-dependent diabetes (type I), is considered a risk
factor due to an impaired healing response and an
increased rate of implant loss. Once the diabetes is
under control, implants can successfully be used in
such patients.
Bleeding disorders pose a risk for oral surgical
interventions in general and implant placement in
particular. Necessary precautions need to be taken in
coordination with the responsible physician prior to
incorporation of implants.
The importance of genetic factors known to be
associated with the development and progression
periodontal disease in predicting higher risks for
implant failures remains to be elucidated.
Patient compliance
Certain aspects of patient behavior have been associa-
ted with increased risk for complications relative to
implant therapy. Lack of proper daily oral hygiene
measures has been associated with an increased
rate of peri-implant disease including the destruction
of bone supporting the implant (50). In addition,
heavy and long-standing cigarette smoking has
been demonstrated to lead to higher rates of early
failures and to adversely affect long-term prognosis
of implants (8, 23, 51).
Aspects of the stomatognathic system
A thorough intra- and extraoral examination is per-
formed to identify general and local factors that
might adversely affect long-term success of implants.
In this context, the soft tissues and the bone are
inspected for diseases by means of clinical, radio-
graphic and possibly histologic methods.
An association has been found in a number of
studies between loss of osseointegration of dental
implants and parafunctional activities such as brux-
ism (67, 70, 74). Although more data are needed, it
seems at present that bruxism is a risk factor for
maintaining osseointegration over time.
In cases of reduced salivary ow rate the protective
function of the components of the immune system in
saliva and the rinsing effect of the saliva are ham-
pered. This may pose a threat to the wound healing
after implant placement and to the long-term main-
tenance of the implants.
Untreated periodontal disease is also a risk factor
for loss of implants resulting from an increased
chance for contamination of the implant surfaces
with periodontopathogenic bacteria and subsequent
infection of the peri-implant tissues. Various studies
have shown an association between the microbial
ora developing on the implants and the ora on
the remaining dentition (62).
Pathologic changes of the soft tissues like erosive,
bullous or hyperkeratotic lesions are potential risk
factors and should be thoroughly examined and ade-
quately treated before implant therapy in the region
is conducted.
In addition, the prospective host bone has to be
examined to detect ongoing disease processes or
lesions resulting from previous diseases. Based on
the ndings, a decision can be taken as to whether
implant therapy is possible.
Finally, insufcient bone volume in orofacial and/
or mesiodistal directions at the prospective implant
site is considered to be a factor associated with com-
plications related to the peri-implant tissues or with
early implant loss. These conditions are evaluated
using periapical radiographs, orthopantomograms,
computer tomograms or combinations thereof. In
more simple cases involving only anterior sites, peri-
apical radiographs are sufcient. In more complex
cases, especially in the area of the inferior alveolar
nerve, mental foramen, and the maxillary sinus,
orthopantomograms or computer tomograms are
chosen.
Clinical inspection combined with measurements
of the thickness of the soft tissues further help to
estimate the available bone volume. The amount of
bone necessary for implant placement may vary
depending on the requirements of the specic
implant system recommended by the manufacturer.
Recommendations based on data from studies in the
literature are also important.
Subsequently, treatment planning is performed by
taking into consideration all of the factors mentioned
above. In addition, the planning phase for determin-
ing the best sites for implant placement is governed
Evaluation parameters for implants
231
by the prosthetic needs of the patient. Ideally, a sur-
gical stent based on a wax-up of the planned nal
reconstruction is used for guiding the placement of
the implants in the best location. This stent is applied
to simplify placing the implant in the correct location
regarding the implant axis, the implant position in
the orofacial as well as mesiodistal dimension, and
the implant sink depth.
Diagnostic procedures between
implant placement and initiation of
prosthesis fabrication
Evaluation during the phase of tissue
integration
During implant placement and immediately there-
after, various methods are available to judge the
quality of the clinical procedures. Measurements of
insertion torque as well as assessments of bone-
implant damping reactions (e.g. tapping, resonance
frequency analysis) are used to determine the initial
stability (i.e. primary stability) of the newly placed
implants. Radiographic analysis will make it possible
to judge the location of the implant within the bone
and with respect to the neighboring structures like
roots of adjacent teeth, sinuses, and nerve structures.
In the early period of clinical application of
endosseous osseointegrated implants, it was recom-
mended that primary stability following placement
could be assessed by tapping the implant with a
metallic instrument (1). Such a test is more of a
raw, qualitative character since a clear determination
of the resonance and damping characteristics of an
implant-bone-interface is simply not possible. The
availability of a clinically applicable, simple and
non-invasive test to assess implant stability is con-
sidered to be of major interest. Therefore, different
non-destructive techniques mainly based on vibra-
tion methods in the sonic or ultrasonic range have
been investigated to study implant integrity (25, 40,
42, 55). These methods can basically be divided into
transient or impact methods and steady-state or
swept-frequency techniques (54). The Periotest
1
is
an example of an impact technique, whereas the
resonance frequency analysis is a typical steady-
state, swept-frequency technique.
Periotest
1
The Periotest
1
(Gulden, Bensheim, Germany) is an
electronic device designed to perform quantitative
measurements of the damping characteristics of
the periodontium, thereby establishing a value for
tooth mobility (82). The mobility of a tooth is eval-
uated as a function of the contact time of a small
metal plug containing an accelerometer, which is
used to strike a tooth. In the search for a quantitative
method of assessing implant stability, a number of
studies have been performed using the Periotest
1
to
measure dental implants (21, 57, 60, 61, 69, 84, 85, 90,
93, 94). It has been shown experimentally that the
device also measures contact time when applied on
dental implants or abutments (43, 58). In an early
review of the literature, Periotest
1
values were
described that had been obtained for a number of
implant systems (69). For example, typical values
reported for ITI implants were 5 to 5, which repre-
sents a narrow range over the possible scale from 8
to 50. In a clinical assessment on the use of the
Periotest
1
device for a baseline mobility measure-
ment of craniofacial implants, a good interexaminer
reliability has been reported (24). But the authors
also listed a number of variables that inuence Peri-
otest
1
values: the vertical measuring point on the
abutment, the angulation of the handpiece relative
to the abutment, and the horizontal distance of the
handpiece to the abutment. The inuence of opera-
tor variability in the application was also reported in
other investigations (53, 58). Moreover, Periotest
1
measurements on a group of 2,212 osseointegrated
implants revealed that the range of the mean Peri-
otest
1
values was less than 2 units for implants
placed in dense or in soft bone (95). In conclusion,
lack of resolution, poor sensitivity, and susceptibility
to operator variables limit the use of the Periotest
1
device as a clinical diagnostic aid to measure implant
stability.
Resonance frequency analysis
A method based on a steady-state, swept-frequency
technique termed Resonance Frequency Analysis
(RFA) has been developed by Meredith and co-work-
ers. The method utilizes a small transducer, which is
screwed onto an implant or abutment (55). The
transducer is excited by a steady-state signal, and
its response is measured. The resonance frequency
value of an implant is a function of its stiffness in the
surrounding bone and the level of the marginal bone.
The overall stiffness of an implant placed in the reci-
pient bone is inuenced by the stiffness of 1) the
implant itself, 2) the implant-tissue interface, and
3) the surrounding bone. In vitro and in vivo inves-
tigations have revealed that the RFA technique is
232
Hammerle & Glauser
non-invasive, easy to use, and capable of eliciting
quantitative information on implant stability and
stiffness. Therefore, this technique has been widely
used to assess implant stability during healing and
bone formation (30, 31, 3335, 39, 56, 68, 7579, 83,
86, 87). Experimental and clinical studies have
demonstrated that a slight increase or decrease in
resonance frequency is related to mechanical stress
relaxation and bone remodeling following implant
placement (30, 31, 57). An increase in resonance
frequency values following implant placement has
been documented for implants placed in soft bone
conditions, where bone formation during healing is
obviously more pronounced as compared to dense
bone conditions (31). A pronounced decrease in
resonance frequency value is related to a major
decrease in stiffness at the bone-implant-interface,
which may be indicative of potential failure (56).
Soft tissue integration
In the case of transmucosal location of the implant or
the healing cap, additional evaluations are used to
assess the congruency between the margins of the
soft tissue aps and the implant structures pene-
trating these tissues. A tight adaptation of the soft
tissues is expected to enhance soft and hard tissue
integration of the implant during the early phases of
healing.
During healing, the health of the mucosal tissues
adjacent to the implant are regularly checked with
respect to adequate plaque control by chemical or
mechanical means and to the absence of loading
forces exerted onto the implant (e.g. via temporary
prosthesis). In addition, the patient is examined for
adverse reactions resulting from the surgical inter-
vention or appearing during the phase of healing.
In situations of a submerged mode of healing, fol-
low-up visits are only necessary for removal of the
sutures and for the examination regarding adverse
reactions.
Numerous animal and clinical studies in humans
have demonstrated that neither the transmucosal
nor the submerged mode of healing show signicant
advantages regarding tissue integration of implants
and their long-term prognosis (12, 28, 29, 37, 96).
Radiographs
Immediately following implant placement or shortly
thereafter, a periapical radiograph is taken to control
for correct placement of the implant regarding posi-
tion, sink depth, and angulation (14). In addition, this
radiograph is used as baseline documentation for the
analysis of subsequent bone level changes. Depend-
ing on the circumstances, this radiograph may also
have forensic meaning.
Healing times
Recommended healing times with a documented
scientic background sufcient for recommendation
for clinical practice range from 6 weeks (20, 80) to
12 months (32). The choice within this range is deter-
mined by local and by patient factors. Hence, in bone
quality of types IIII, implants with a sand-blasted
and acid-etched surface (SLA) that were loaded after
6 weeks have been shown to be highly successful (20)
and this clinical protocol can be as predictable as
traditional protocols with loading initiated only
3 months after implant placement (80). In contrast,
implants placed in bone of low density in osteoporo-
tic patients have been demonstrated to still be suc-
cessful provided that extended healing periods are
allowed before prosthetic loading is initiated (32).
To date, parameters are lacking that permit the
determination of the minimal healing time necessary
for the occurrence of sufcient bone integration to
allow functional implant loading. Measurements of
implant stability using RFA are presently being ana-
lyzed for their value in providing this information.
Ideally, an assessment obtained immediately after
implant placement should yield this kind of data.
In this case, further treatment planning could be
done at the end of the surgical session for implant
placement.
The possibilities for immediate loading and the
short- as well as the long-term outcomes of this
approach are being extensively investigated. Loading
of implants immediately following their placement
has been demonstrated to be possible while still
obtaining excellent long-term results when four or
more implants placed in the mandible are splinted in
one single reconstruction (6, 41, 48).
Diagnostic procedures following
incorporation of prosthetic
reconstructions
Peri-implant soft tissues
Various parameters are available to the clinician to
determine the state of the peri-implant tissues and
from which to draw the appropriate conclusions in
order to initiate the correct interceptive therapy.
233
Microbiota at implant sites
Similar to the situation with other hard, non-shed-
ding surfaces in uid systems, a biolm develops on
implant surfaces once they penetrate the mucosal
tissues and are exposed to the oral environment.
These microorganisms may lead to inammation of
the peri-implant tissues, eventually causing destruc-
tion of the implant-supporting structures.
It has been demonstrated in a large number of
studies that the microbiota associated with healthy
peri-implant soft tissues is similar to the microbiota
detected in sulci of healthy sites at teeth (62).
Furthermore, implant sites exhibiting diseased tis-
sues have been demonstrated to harbor a periodon-
topathogenic ora similar to the one found in
periodontal pockets of sites showing advanced peri-
odontitis.
Hence, the information derived from analysis of
the microbiota in conjunction with the interpretation
of the clinical ndings is used to make a decision as
to the need and the mode of therapy in cases of peri-
implant infections.
Mucosal inflammation
Various studies in animals and humans have demon-
strated that the development of increased amounts
of plaque will lead to peri-implant mucositis (11, 71,
98), much like the gingival inammation observed
around teeth under similar conditions (52).
It may be concluded that the occurrence of peri-
implant mucositis can in general be interpreted as
the result of increased amounts of plaque and as a
sign of an inammatory reaction within these tis-
sues. Therefore, the assessment of the status of the
mucosa around implants is of value when trying to
discern a healthy from a diseased site.
This peri-implant mucositis has been studied with
respect to the cellular and humoral defense mechan-
isms. Basically, the mechanisms leading from
healthy to inamed tissues and the sequence of
events occurring during this process are similar
within the peri-implant soft tissues and the period-
ontal tissues (49, 91, 92).
The Gingival Index originally developed for the
assessment of the degree of inammation of the
marginal periodontal tissues (52) has been adapted
for use at implants (63). This modied index
describes the same degrees of severity of the inam-
mation as the original Gingival Index and can be
used to monitor peri-implant mucosal inammation.
It has to be taken into consideration, however, that
it has not been demonstrated so far that peri-implant
mucositis, if left untreated, will develop into peri-
implantitis.
Peri-implant probing
Originally, the value of peri-implant probing in deter-
mining the status of the peri-implant tissues was
questioned. However, in recent years the usefulness
of the information derived from it has generally been
accepted (3, 13). Probing the peri-implant soft tissue
zone renders information regarding the:
level of the mucosal margin;
peri-implant probing depth;
level of the tissues in the peri-implant zone provid-
ing resistance to probing;
effects of probing regarding bleeding, exudation
and suppuration.
Various factors affect the reliability of probing at
teeth and implants. Probing at implants is affected by
the size of the probe tip, the probing force, the direc-
tion of probe insertion, the health of the peri-implant
tissues, the macroscopic form and surface structure
of the implant, and the presence and the design of
the suprastructure.
Probing around teeth is a very useful tool to gather
information regarding the health of the tissues and
various anatomic landmarks. However, one should
be careful in expecting the same information from
probing around implants (13). The anchorage of
teeth and implants in the jawbone are distinctly dif-
ferent. With respect to probing the peri-implant and
the periodontal tissues the most important differ-
ence relates to the supracrestal soft tissue zone.
Whereas teeth exhibit connective tissue bers insert-
ing into supracrestal root cementum, the connective
tissue bers at implants generally show an orienta-
tion parallel to the implant surface without evidence
of insertion. This is particularly important when dis-
cussing the meaning of probing, since the bers
around teeth represent a primary source of resis-
tance to probe penetration (5).
Probe penetration at implant sites is heavily
dependent upon the conditions of the peri-implant
tissues (26, 47). At healthy sites the probe tip stopped
at around the level of the most coronal aspect of the
connective tissue adhesion to the implant neck. At
inamed sites the probe consistently reached close
to or was in contact with the bone level.
The optimal force for probing at teeth has been
suggested to be 0.25 N (64, 65). Results from clinical
studies suggest that a change in probing force
level has a higher impact at implants compared
to teeth (66). Hence, the use of a standardized force
is of higher importance when interpreting the
234
Hammerle & Glauser
measurements obtained at implants compared to
teeth. Acceptable levels of reproducibility regarding
probing measurements in patients using three differ-
ent pressure-calibrated probes have been reported
(19).
Various studies have described the probing depths
at healthy implant sites to be around 3 mm (2, 16, 27,
60, 63). Microbiological studies have shown a clear
difference between the microbiota in shallow and in
deep peri-implant pockets (63, 73, 81). Deeper pock-
ets harbored signicantly higher proportions of per-
iodontopathogenic microorganisms.
Regarding probing at implants, clinical studies
have shown a correlation between the probing depth
and the level of the marginal peri-implant bone as
determined on radiographs (17, 72). These data sup-
port the conclusion that probing represents a non-
invasive method free from irradiation to estimate the
marginal bone level. It has further been described in
one of these clinical studies that the mean distance
from the probe tip to the bone level amounted to
1.7 mm (17).
Despite the important information derived from
peri-implant sulcus probing, various shortcomings
decrease its value compared to the one obtained
from probing at teeth:
the conguration of the implant or the suprastruc-
ture may hinder proper probing;
increased probing depths, in particular in esthetic
sites, can be difcult to interpret.
Bleeding on probing
It has been established that bleeding on probing is a
valuable parameter in assessing the health status of
periodontal tissues (4, 7). In particular, the absence
of bleeding on probing has been shown to be a pre-
dictor of periodontal stability (45). The size of the tip
of the probe as well as the probing force should be
standardized to obtain meaningful data (44, 46).
Studies comparing bleeding scores at teeth and
implants in the same mouth have reported that the
bleeding on probing frequencies are higher at
implants compared to teeth (13). However, the value
in assessing healthy or diseased sites using bleeding
on probing registered at the peri-implant sulcus or
the peri-implant pockets has not yet been deter-
mined.
In this context, studies including the health status
of the peri-implant tissue in their success criteria also
assessed the presence or absence of suppuration
from the peri-implant sulcus or pocket (17, 18). His-
tologic studies have shown an inltration with large
numbers of polymorphonuclear leukocytes in
acutely inamed peri-implant soft tissues indicating
the clinical diagnostic value of suppuration.
Level of the mucosal margin
It has been reported that recession of the marginal
soft tissues occurs following incorporation of recon-
structions (10). Whereas this recession primarily tak-
ing place during the rst 6 months has never been
shown to adversely affect long-term survival of
affected implants, such recession may cause esthetic
problems in anterior areas of the mouth.
Keratinized mucosa
The question of whether or not keratinized mucosa is
required for the long-term maintenance of peri-
implant tissue health is still under debate. Neverthe-
less, several clinical studies in humans revealed that
the absence of marginal keratinized tissue is compa-
tible with soft tissue health provided adequate levels
of plaque control are maintained (10, 59, 61, 97).
In one of these studies, a retrospective analysis of
171 implants in 39 patients was conducted in order
to analyze the inuence of the presence or absence of
masticatory mucosa at the implant neck on the pre-
sence of plaque and the health status of the marginal
tissues (97). The results failed to reveal an inuence
of the condition of masticatory mucosa or marginal
soft tissue mobility on the outcome variables of pla-
que and bleeding on probing.
In another study, a prospective analysis of 163
implants in 41 patients evaluated the occurrence
and degree of recession during the rst two years
of implant function (10). Lack of masticatory mucosa
and mobility of the peri-implant soft tissues were
poor predictors of soft tissue recession. The investi-
gators concluded that the recession, which primarily
occurred during the rst 6 months of function, was
due to a physiological soft tissue remodeling aimed
at establishing appropriate biological dimensions of
the soft tissues.
Assessment of implant stability
During loading
Following successful osseointegration, implant load-
ing leads to an effective load distribution at the bone-
implant interface. Therefore, it has been proposed
that osseointegration of screw-shaped titanium
implants may be tested clinically by the application
of a counterclockwise torque up to 20 Ncm (reverse
torque test) at second-stage surgery (88). While
osseointegrated implants will resist a reverse torque
235
at this level, osseointegration failure with brous
encapsulation will lead to an unscrewing. Based on
this all-or-none result, the reverse torque test is not
able to discriminate the degree of healing or bone
formation around an implant. Moreover, the method
has some destructive potential since the test relies on
the direct application of shear stresses at the bone
implant interface, which have been reported to
induce irreversible bone deformations (15).
Radiographic evaluation is the most widely used
chairside technique to assess the boneimplant
interface during healing and implant function. The
objective is to determine marginal bone levels as well
as peri-implant radiolucencies. In general, the radio-
graphic evaluation has inherent shortcomings due to
the two-dimensional projection and the limitation to
the interproximal interfaces, as well as due to dif-
culties with standardization. In a clinical study on the
accuracy and precision of diagnosing implant stabi-
lity using radiographs, it was concluded that the
probability of predicting clinical implant instability
from radiographic examination was low in popula-
tions with a low prevalence of implant instability
(89).
Resonance frequency analysis as a rapid, non-
invasive method has also being used as a means of
monitoring the changes in stability related to bone
formation and the dynamics of osseointegration (i.e.
establishing secondary stability) over time. Clinically,
the technique has been used to follow implants
placed in combination with a non-submerged heal-
ing, to document implants subjected to immediate
loading, or to monitor implants placed in demanding
bone conditions (33, 35, 79).
Conclusions
A thorough history coupled with an evaluation of
relevant systemic factors is mandatory prior to the
initiation of implant therapy in order to recognize
conditions that may adversely affect treatment
results.
In addition to evaluating the site of the planned
implantation, a comprehensive examination of the
stomatognathic system is highly recommended
depending on the complexity of the clinical case.
Between implant placement and the initiation of
prosthetic therapy the peri-implant mucosa should
be examined regarding proper soft tissue integration.
Clinical visual examination aided by the use of a
periodontal probe allows the relevant information
to be gathered.
The most valuable clinical parameters for the
assessment of the health status of the peri-implant
tissues are the presence or absence of mucosal
inammation, signs of infection, and probing depth.
Standardized radiographs taken at regular intervals
are presently the best means to assess the bone-
implant relationship over time.
Resonance frequency analysis appears to be a use-
ful tool for assessing implant stability during the
phase of tissue integration and during implant func-
tion. More data, however, are needed to determine
discriminative levels of stability regarding measure-
ments taken at single as well as at repeated time
points.
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239
Periodontal diagnoses and
classication of periodontal
diseases
Gary C. Armitage
What is a periodontal diagnosis?
A periodontal diagnosis is an important label that
clinicians place on a patient's periodontal condition
or disease. In the current practice of periodontics it is
primarily derived from information obtained from
the patient's medical and dental histories combined
with ndings from a thorough oral examination. The
entire constellation of signs and symptoms asso-
ciated with the disease or condition is taken into
account before arriving at a diagnosis. In some
instances additional information provided by labora-
tory tests is useful in the overall decision-making
process. Under the best of circumstances a period-
ontal diagnosis is a clinician's best guess as to what
condition or disease the patient has. Assignment of a
diagnosis carries with it the implication that the clin-
ician has ruled out other possible diseases that the
patient might have had. Since diagnostic deductions
(i.e. reasonable guesses) are made on the basis of
incomplete knowledge of a patient's actual condi-
tion, it is important to realize that the assigned diag-
nosis might be wrong. Because there is always some
uncertainty, experienced clinicians routinely develop
a differential diagnosis that is a listing of the possible
diagnoses of a patient's condition ranked from most
likely to least likely. A differential diagnosis is impor-
tant because it provides the clinician with other diag-
nostic options if the initial diagnosis subsequently
proves to be wrong.
A carefully considered periodontal diagnosis is of
major importance in the subsequent management of
a patient's periodontal disease. An accurate diagno-
sis is often a rst step toward development of a well-
designed and appropriate treatment plan that when
implemented leads to resolution of the patient's per-
iodontal infection. An incorrect diagnosis often leads
to an ill-conceived treatment approach that ulti-
mately fails to resolve the patient's periodontal pro-
blem.
The diagnostic label captures, in a few words, a
clinician's entire past experience with a disease or
condition. It is a summary term that helps guide the
clinician toward answering questions that are impor-
tant to both the dentist and patient:
What is the cause of the disease or condition?
Is referral to another, more experienced, clinician
appropriate?
What will happen if the disease or condition is not
treated?
What are the treatment options?
What is the best treatment?
What is the expected outcome of treatment (i.e.
prognosis)?
What are the anticipated side effects of treatment?
Will the treatment be painful?
Will the treatment result in esthetic problems?
How long will treatment take?
How much will the treatment cost?
Importantly, most patients will want these
questions addressed before treatment has started. It
should be emphasized that from the perspective of
many patients a ``healthy'' periodontium is one that
is comfortable and free from functional and esthetic
problems. Therefore, it is always a good practice to
establish a diagnosis and discuss its implications
with the patient prior to starting any therapeutic
procedures.
Current classificationsystem what
are possible periodontal diagnoses?
Plaque-induced periodontal diseases have tradition-
ally been divided into three general categories:
health, gingivitis, or periodontitis. In this context,
9
#
PERIODONTOLOGY 2000
the diagnosis of health implies that there is an
absence of plaque-induced periodontal disease. Pla-
que-induced gingivitis is the presence of gingival
inammation without loss of connective tissue
attachment (7). Plaque-induced periodontitis is the
presence of gingival inammation at sites where
there has been apical migration of the epithelial
attachment onto the root surfaces accompanied by
loss of connective tissue and alveolar bone (7). In
most patients, increased probing depths or the for-
mation of periodontal pockets accompany the devel-
opment of periodontitis. Plaque-induced gingivitis
and periodontitis are, by far, the most frequent of
all forms of periodontal disease. They are not, how-
ever, the only diagnostic possibilities.
In the 1999 classication system for periodontal
diseases and conditions, over 40 different gingival
diseases were listed (Table 1)(10). In some of the
non-plaque-induced gingival lesions, loss of attach-
ment and destruction of alveolar bone may occur (28,
51, 57, 74, 83, 94). In addition, seven major categories
of destructive periodontal diseases were listed:
Chronic periodontitis (Fig. 1),
Localized aggressive periodontitis (Fig. 2),
Generalized aggressive periodontitis (Fig. 3),
Periodontitis as a manifestation of systemic dis-
ease (Fig. 4),
Necrotizing ulcerative gingivitis/periodontitis
(Figs 5 and 6),
Abscesses of the periodontium (Fig. 7),
Combined periodonticendodontic lesions (10).
Periodontitis can be a manifestation of at least 16
systemic diseases. In most of these systemic diseases
there is either decreased host resistance to infections
or perturbations in gingival connective tissue that
increases its susceptibility to inammation-induced
degradation (Table 2).
Three basic diagnostic questions
components of a periodontal
diagnosis
Before arriving at a periodontal diagnosis the clini-
cian must answer three basic questions:
1 What periodontal disease or condition does the
patient have?
2 How severe is the problem?
3 Is the disease or condition localized or generalized?
The rst of these questions is the most difcult
since it requires an assimilation and understanding
of all of the information collected during the history-
taking process and the clinical examination. In unu-
sual cases where something other than plaque-
induced periodontal disease is suspected, laboratory
tests, histopathologic examination of biopsy speci-
mens, and a medical consultation may be required
Table 1. Partial list of possible diagnoses for gingival
diseases (1999 Classication) (10)
Dental plaque-induced gingival diseases
Plaque-induced gingivitis (no other
local contributing factors)
(60)
Plaque-induced gingivitis with local
contributing factors
(60)
Necrotizing ulcerative gingivitis (NUG) (23, 80)
Puberty-associated gingivitis (60, 93)
Menstrual cycle-associated gingivitis (60)
Pregnancy-associated gingivitis (54, 60)
Pregnancy-associated pyogenic
granuloma
(60, 87)
Diabetes mellitus-associated gingivitis (34, 60)
Leukemia-associated gingivitis (60, 99)
Drug-influenced gingival enlargement (60, 85)
Oral contraceptive-associated gingivitis (60)
Ascorbic acid-deficiency gingivitis (19, 60)
Non-plaque-induced gingival lesions
Neisseria gonorrhea-associated lesions (41, 83, 86)
Treponema pallidum-associated
lesions
(41, 59, 82, 86)
Streptococcal species-associated
lesions
(41, 53)
Mycobacterium tuberculosis-
associated lesions
(27, 83)
Bacillary angiomatosis (57)
Primary herpetic gingivostomatitis (40, 83)
Recurrent oral herpes (41, 83)
Varicella-zoster infections (41, 83)
Generalized gingival candidosis (41, 83)
Linear gingival erythema (32)
Histoplasmosis (28, 83)
Hereditary gingival fibromatosis (33, 42)
Gingival manifestations of
Lichen planus (41, 46, 82)
Mucous membrane pemphigoid (24, 41, 82)
Pemphigus vulgaris (41, 66, 82)
Erythema multiforme (14, 41, 58, 82)
Lupus erythematosus (41, 82)
Linear IgA disease (20, 46, 82)
Wegener's granulomatosis (44, 46)
Psoriasis (100)
Allergic reactions of the gingiva to
Restorative materials (e.g. mercury,
nickel, acrylic)
(41)
Toothpastes (25)
Mouthrinses (41)
Chewing gum additives (49)
Foods and food additives (41, 84)
Traumatic lesions of the gingiva
Chemical injury (4, 48, 67)
Physical injury (51, 74, 94)
Thermal injury (41)
Armitage
10
Fig. 2. Advanced localized aggressive periodontitis in a
medically healthy 19-year-old Caucasian male. Note that
there are minimal amounts of plaque and gingival inam-
mation (A). Radiographs of the mandibular anterior teeth
showing the massive amount of bone loss (B). The period-
ontal damage was conned to the permanent incisors and
rst molars.
Fig. 1. Chronic periodontitis associated with poor oral
hygiene in a medically healthy 45-year-old Japanese male.
Inammation, gingival recession, and attachment loss are
most obvious in the mandibular anterior area.
Fig. 3. Generalized aggressive periodontitis in a medically
healthy 15-year-old Caucasian male. Note the intense
inammation and heavy deposits of plaque and calculus.
Massive bone loss was present around all teeth.
Fig. 4. Periodontitis as a manifestation of systemic dis-
ease. Plaque-induced periodontitis aggravated by
increased susceptibility to infection in a 24-year-old male
with Down's syndrome. Note the intense gingival inam-
mation.
Fig. 5. Necrotizing ulcerative gingivitis in a 25-year-old
Caucasian male. Note the cratering of the interproximal
gingiva in most areas. The gingival lesions were painful.
11
Diagnoses and classification of periodontal diseases
(46, 61). If there is uncertainty, clinicians often use
what is sometimes called a working diagnosis to
guide the information-gathering process. The rele-
vant information is identied and then analyzed
within the context of the entire patient. A decision
is then made as to what label or diagnosis should be
placed on the patient's disease or condition. It
should be emphasized that patients may have more
than one disease or condition simultaneously affect-
ing the periodontium. For example, Fig. 8 shows
a 50-year-old white female who has both chronic
periodontitis and benign mucous membrane pem-
phigoid.
Table 2. Partial list of possible diagnoses for destructive types of periodontal diseases (1999 Classication) (10)
Chronic periodontitis (Localized/Generalized) (30)
Localized aggressive periodontitis (52, 95)
Generalized aggressive periodontitis (52, 95)
Periodontitis as a manifestation of systemic diseases
Associated with hematologic disorders:
Acquired neutropenia (43)
Leukemias (6, 91)
Associated with genetic disorders
Familial and cyclic neutropenia (76, 90)
Down's syndrome (78)
Leukocyte adhesion deficiency syndromes (65, 97)
Papillon-Lefevre syndrome (38, 50)
Chediak-Higashi syndrome (37)
Langerhans cell disease (histiocytosis syndromes) (68, 96)
Glycogen storage disease (50)
Chronic granulomatous disease (17)
Infantile genetic agranulocytosis (81)
Cohen syndrome (1, 50)
Ehlers-Danlos syndrome (Types IV and VIII) (50, 75)
Hypophosphatasia (18)
Crohn disease (inflammatory bowel disease) (30, 47)
Marfan syndrome (92)
Necrotizing ulcerative periodontitis (NUP) (23, 69)
Abscesses of the periodontium (23, 39, 63)
Combined periodonticendodontic lesions (64, 79)
Fig. 7. Periodontal abscess that rapidly developed over a
2-day period between the maxillary second and third
molars in a 43-year-old African American male with
untreated chronic periodontitis. Both molars were free
of pulpal disease.
Fig. 6. Necrotizing ulcerative periodontitis in a medically
healthy 37-year-old Caucasian male. The severity and pat-
tern of attachment loss in the mandibular anterior region
is similar to that often observed in HIV-positive patients.
However, the photograph was taken in 1968, well before
the AIDS epidemic. The patient complained of pain from
the affected areas.
12
Armitage
The second question is a bit easier to answer since
it only requires one to decide on a way to character-
ize the severity of the patient's plaque-induced per-
iodontal disease. Most clinicians designate severity
by using a 3-tiered system: 1 slight, 2 moderate,
or 3 severe. Sometimes `ìnitial'' and ``mild'' are
used interchangeably with ``slight''; `àdvanced''
may be used instead of ``severe.'' It makes no differ-
ence what words are chosen as long as the terms are
understood by other clinicians. In cases of period-
ontitis, it has been recommended that severity be
categorized on the basis of the amount of clinical
attachment loss as follows: Slight 12 mm, Mod-
erate 34 mm, and Severe 5 mm clinical attach-
ment loss (10). Clinical attachment loss is measured
with a periodontal probe and is the distance from the
cementoenamel junction to the base of the probe-
able crevice (7). In cases of gingivitis, a similar
3-tiered system is frequently used: 1 slight, mild,
2 moderate, and 3 severe. However, these desig-
nations are based on subjective clinical assessments
of the intensity of the gingival inammation (i.e.
degree of redness, swelling, bleeding).
In answering the third question a decision needs to
be made as to whether a patient's periodontal dis-
ease is localized or generalized. This decision is arbi-
trary since there are no hard-and-fast rules that can
be applied for determining the extent or intraoral
distribution of a patient's periodontal disease. Never-
theless, it has been recommended that the disease be
called localized if up to 30% of the teeth are affected
and generalized if >30% of the teeth are involved
(10). Since during a routine periodontal examination,
probing depths and clinical attachment loss mea-
surements are taken from six sites around each tooth,
it is theoretically possible to assign a diagnosis to a
single surface on a given tooth, a single tooth, a
quadrant, or the entire patient. However, for practi-
cal reasons, clinicians usually combine all of the
clinical ndings in a summary or global statement
by assigning a diagnosis for the entire patient. Excep-
tions to this practice are common. The diagnosis can
be phrased many different ways depending on how
precise or detailed one wants to be. For example,
Table 3 shows multiple ways to correctly phrase
the periodontal diagnosis for a middle-aged patient
with slight gingivitis around most teeth, who also has
severe chronic periodontitis (clinical attachment
loss 6 mm) localized to the mesial surfaces of
three teeth on the mandibular left where there are
8 mm probing depths and bleeding on probing at the
periodontitis-affected sites.
Table 3. Some of the multiple ways to correctly phrase a periodontal diagnosis for a middle-aged patient with slight
gingivitis around most teeth, who also has severe chronic periodontitis (clinical attachment loss 6 mm) localized
to the mesial surfaces of three teeth on the mandibular left where there are 8 mm probing depths and bleeding on
probing at the periodontitis-affected sites
Diagnostic field and emphasis Phrasing of the diagnosis
Focus on one disease (periodontitis)
Single surfaces of affected teeth Severe chronic periodontitis on the mesial surfaces of teeth #18, 19, and 20
Single teeth Severe chronic periodontitis localized to teeth #18, 19, and 20
Quadrant Severe chronic periodontitis localized to the mandibular left quadrant
Patient Localized severe chronic periodontitis
Focus on both diseases (gingivitis and periodontitis)
Patient and single surfaces of
affected teeth
Generalized slight gingivitis with severe chronic periodontitis on the
mesial surfaces of teeth #18, 19, and 20
Patient and single teeth Generalized slight gingivitis with severe chronic periodontitis localized to
teeth #18, 19, and 20
Patient and quadrant Generalized slight gingivitis with severe chronic periodontitis localized to
the mandibular left quadrant
Patient Generalized slight gingivitis with localized severe chronic periodontitis
Fig. 8. A 50-year-old female with chronic periodontitis
and benign mucous membrane pemphigoid.
13
It should be noted that probing depth measure-
ments, while not used as the primary criterion for
establishing the severity of periodontitis, are a useful
piece of information. Probing depth is measured with
a periodontal probe and is the distance from the
gingival margin to the base of the probeable crevice.
It is not the main criterion for severity because the
gingival margin is not a xed reference point from
which to measure. For example, in cases where there
is gingival enlargement and swelling, the gingival
margin can be considerably coronal to the cemento-
enamel junction (Fig. 9). In other instances, where
there has been recession, the gingival margin is apical
to the cementoenamel junction (Fig. 10). Most impor-
tantly, the gingival margin can move coronal or api-
cal to the cementoenamel junction over time and
therefore is not a good reference point from which
to assess longitudinal changes in clinical attachment.
Nevertheless, probing depth measurements are of
considerable importance since they provide a useful
assessment of location and size of the principal habi-
tats (i.e. periodontal pockets) of subgingival bacteria.
Deep pockets are of concern because they are dif-
cult for both the patient and therapist to clean (7). It
is because of this that one of the goals of periodontal
therapy is probing depth reduction.
Establishing a diagnosis in treated
patients
Important steps in the management of patients with
plaque-induced periodontal diseases are evaluations
of post-treatment results. Such evaluations can occur
at two critical points in monitoring the outcomes of
therapy. The rst is performed at the end of active
therapy when the clinician must decide if treatment
has beensuccessful. Has thedesiredshort-termclinical
outcome been attained (9)? The second set of evalua-
tions are repeatedly performed at multiple stages
during the maintenance phase of therapy. Has the
patient remained free of recurrent disease (9)? At each
of these post-treatment evaluations a periodontal
examination is performed and a diagnosis is made.
Treatment of plaque-induced periodontal diseases
often results in the resolution of the patient's period-
ontal infection. It is important to note that period-
ontal therapy can change the pretreatment diagnosis
to a more favorable post-treatment diagnosis. For
example, effective treatment routinely converts pla-
que-induced gingivitis into a state of periodontal
health (i.e. a Gingivitis to Periodontal Health shift).
Successful treatment of plaque-induced periodonti-
tis is often converted to a state of periodontal health
with a reduced periodontium. In such cases, damage
persists from the previous periodontitis in the form
of gingival recession. An interesting diagnostic pro-
blem arises when successfully treated patients who
once had periodontitis subsequently develop gingival
inammation during the maintenance phase of ther-
apy. Do such patients have gingivitis superimposed
on a reduced periodontium or do they have a recur-
rence of periodontitis? There is no simple answer to
this question. However, depending on a variety of
circumstances, most clinicians err on the side of
caution and entertain the notion that the periodon-
titis may be recurring.
At a single evaluation visit one cannot determine if
previously treated periodontitis is recurring. Data
collected during multiple maintenance visits are
required to make this determination. Perhaps the
Fig. 9. Gingival enlargement in a patient taking phenytoin
for cerebral seizures. The gingival margins on most teeth
are coronal to the cementoenamel junction (CEJ).
Fig. 10. Gingival recession in a 40-year-old female with
chronic periodontitis. The gingival margin is apical to the
CEJ on the facial surfaces of the two mandibular central
incisors.
14
Armitage
earliest indication that the disease might be return-
ing is the presence of bleeding on probing at multiple
maintenance visits. This notion is supported by the
results of a meta-analysis of treated populations of
patients receiving periodontal maintenance care. In
this analysis sites that exhibited the repeated presence
of bleeding on probing were at a threefold higher risk
of losing additional attachment compared to sites that
did not bleed at most of the maintenance visits (9).
Higher clinical attachment loss with time is the best
single indication that periodontitis has probably
recurred. One can be certain that the disease has
recurred if there is bleeding on probing, increased
probing depth, and higher clinical attachment loss
measurements.
Although treatment of plaque-induced periodonti-
tis usually results in resolution of the patient's peri-
odontal infection, in some cases therapy is
unsuccessful. Usually the causes for the unsuccessful
treatment are unknown. One might reasonably sus-
pect that the infection did not resolve because of
poor plaque control by the patient or incomplete
removal of subgingival calculus and plaque by the
therapist. Additional plaque control instructions and
further scaling and root planing are often successful
in controlling the periodontal infections in these
`ìncompletely treated'' patients. However, there are
some cases where the patient has beencompliant with
all recommendations and performs excellent plaque
control. In addition, the therapist has provided a
course of conventional periodontal therapy that suc-
ceeds in most patients. Nevertheless, the treatment
fails toprevent thefurther progressionof periodontitis.
Such patients are sometimes assigned the diagnosis
of ``refractory'' (21, 22, 98) or ``therapy-resistant'' (12,
13, 26) periodontitis. The diagnostic category of
refractory periodontitis is a heterogeneous grouping
since there are probably multiple forms of periodon-
titis that are nonresponsive to conventional therapy.
However, most of the patients who have been studied
started out with an initial diagnosis of chronic (adult)
periodontitis. In retrospect, an appropriate diagnosis
for this group of patients is refractory or treatment-
resistant chronic periodontitis. Diagnostic approaches
for this group of patients are discussed elsewhere in
this volume (56).
Diagnosis and classification of
developmental or acquired deformities
and conditions of the periodontium
There are a large number of developmental or
acquired deformities and conditions that affect the
periodontium (Table 4). Some of them are simply
departures from normal periodontal anatomy that
may cause functional or esthetic problems for
patients. Others can create an environment that pro-
motes the development of plaque-induced periodon-
tal diseases. Methods for the detection and diagnosis
of these conditions are discussed elsewhere in this
volume (62). Diagnostic methods for identifying the
clinical effects of an injury-producing occlusion are
also discussed (36).
Distinguishing between chronic and
aggressive forms of periodontitis
Most patients with plaque-induced periodontitis will
have the chronic form (2, 72). The main clinical fea-
tures and characteristics of chronic periodontitis are
listed in Table 5. The typical patient is over 30 years of
age with substantial deposits of plaque and calculus
associatedwiththe presence of gingival inammation,
periodontal pockets, and attachment loss. In most
cases the disease is slowly progressing (16, 55, 73),
but short periods of rapid attachment loss can occur
(45, 89). Chronic periodontitis was once called `àdult
periodontitis'' since it was believed that only adults
developed the disease (5). However, epidemiologic
Table 4. List of developmental or acquired deformi-
ties and conditions affecting the periodontium (1999
Classication) (10)
Tooth-related factors that modify or predispose to
plaque-induced gingival diseases/periodontitis
(15, 62)
Tooth anatomic factors
Dental restorations/appliances
Root fractures
Cervical root resorption and cemental tears
Mucogingival deformities and conditions (62, 77)
Gingival/soft tissue recession
Facial or lingual surfaces
Interproximal (papillary)
Lack of keratinized gingiva
Decreased vestibular depth
Aberrant frenum/muscle position
Gingival excess
Pseudopocket
Inconsistent gingival margin
Excessive gingival display
Gingival enlargement
Abnormal color
Occlusal trauma (35, 36)
Primary occlusal trauma
Secondary occlusal trauma
15
data clearly show that the disease can also be found
in children and adolescents (3, 72). Although chronic
periodontitis can occur in localized or generalized
patterns, the two forms appear to be identical with
regards to their etiology and pathogenesis.
Aggressive periodontitis is less common than
chronic periodontitis and principally affects young
patients (3, 72). It occurs in localized and generalized
forms that differ in many respects with regard to their
etiology and pathogenesis (52). Localized aggressive
periodontitis (LAP) and generalized aggressive peri-
odontitis (GAP) were previously called ``localized and
generalized juvenile periodontitis,'', respectively (5).
Features of aggressive periodontitis that are common
to both the localized and generalized forms of the
disease are shown in Table 6.
The thought process followed in distinguishing
between chronic and aggressive forms of periodon-
titis initially focuses on 1) the amount and pattern of
periodontal destruction and 2) the patient's age and
medical status. One begins to suspect that a patient
might have a form of aggressive periodontitis if they
are young, medically healthy and present with exten-
sive periodontal destruction. If the periodontal
destruction is localized to interproximal areas of
permanent rst molars and incisors, the diagnosis
of LAP is usually made. In addition, in most cases
of LAP the traditional view is that the, `àmounts of
microbial deposits are inconsistent with the severity
of periodontal tissue destruction'' (52). If the
destruction is found around at least three permanent
teeth other than rst molars and incisors, the diag-
nosis of GAP is usually made (52).
As in the case of chronic periodontitis, both forms
of aggressive periodontitis are plaque-induced infec-
tions and host responses to plaque bacteria are
responsible for most of the tissue destruction. The
plaque biolms are, however, often clinically thinner
than in cases of chronic periodontitis. As mentioned
above, this is particularly true in cases of LAP.
Based on several specic clinical and host-
response differences between LAP and GAP, it is clear
that LAP is not merely a localized form of GAP. Fea-
tures that are said to be specic for each of these
forms of aggressive periodontitis are shown in
Table 7. Importantly, LAP generally has a circumpu-
bertal onset or is rst detected and diagnosed during
puberty, whereas GAP is usually detected and diag-
nosed in people under 30 years of age. However,
some patients with GAP may be older than 30 years
of age. It has been suggested that patients with LAP
usually mount a robust serum antibody response to
Table 6. Features of aggressive periodontitis that are common to both the localized and generalized forms of the
disease (1999 Classication) (52)
Primary features
Except for the presence of periodontitis, patients are otherwise clinically healthy
Rapid attachment loss and bone destruction
Familial aggregation
Secondary features (often present)
Amounts of microbial deposits are inconsistent with the severity of periodontal tissue destruction
Elevated proportions of Actinobacillus actinomycetemcomitans and, in some populations, Porphyromonas gingivalis
may be elevated
Phagocyte abnormalities
Hyperresponsive macrophage phenotype, including elevated levels of prostaglandin E
2
(PGE
2
) and interleukin-1b
(IL-1b)
Progression of attachment loss and bone loss may be self-arresting
Table 5. Main clinical features and characteristics of chronic periodontitis (1999 Classication) (10)
Most prevalent in adults, but can occur in children and adolescents
Amount of destruction is consistent with the presence of local factors
Subgingival calculus is a frequent finding
Associated with a variable microbial pattern
Slow to moderate rate of progression, but may have periods of rapid progression
Can be associated with local predisposing factors (e.g., tooth-related or iatrogenic factors)
May be modified by and/or associated with systemic diseases (e.g., diabetes mellitus)
Can be modified by factors other than systemic disease such as cigarette smoking and emotional stress
16
Armitage
periodontal pathogens, whereas patients with GAP
exhibit a poor antibody response to the infecting
agents (52). Comparisions of the main clinical charac-
teristics of chronic, localized aggressive, and general-
ized aggressive periodontitis are shown in Table 8.
Noninflammatory destructive
periodontal disease (NDPD) does it
exist?
Most of the destructive forms of periodontal disease
discussed in this chapter are primarily caused by
dental plaque biolms. Classication of these afic-
tions follows an infection/host response paradigm in
which it is held that noxious materials from dental
plaque bacteria induce an inammatory response in
the adjacent periodontal tissues (11). The lytic activ-
ities associated with inammation are the primary
way in which periodontal tissues are destroyed. Cen-
tral to this paradigm is the notion that destruction of
periodontal tissues is accompanied by an inamma-
tory response.
However, for the past 250 years some clinicians
have described what they believed were ``noninam-
matory destructive periodontal diseases'' in which
periodontal tissues were gradually lost without the
presence of inammation (71). Indeed, from
approximately 1920 to 1970, most classication sys-
tems for periodontal diseases included noninam-
matory (i.e. degenerative, atrophic) categories. The
primary reasons for including these categories were
the clinical impressions (i.e. opinions) of clinicians
and the long-held assumption that periodontal dis-
eases followed the ``principles of general pathology''
in which ``there are three major tissues reactions:
inammatory, dystrophic, neoplastic'' (11, 70).
Despite the lack of supporting scientic data, the
existence of such diseases was never seriously chal-
lenged until it was convincingly shown in the 1970's
that ``periodontosis,'' a name given to a destructive
periodontal disease that was presumably a degenera-
tive noninammatory condition, was actually an
infection with a denite inammatory component
(11). As a consequence of this discovery, most clas-
sication systems for periodontal diseases published
after 1977 do not include degenerative or noninam-
matory categories.
Page & Sturdivant have recently proposed the exis-
tence of a form of ``noninammatory destructive
periodontal disease (NDPD)'' and that it ``. . .is seen
rather commonly by practicing periodontists'' (71).
These authors supported their suggestion by present-
ing two case reports of patients who progressively
experienced gingival recession and loss of attach-
ment over a period of several years. Importantly,
both patients practiced intense and frequent tooth-
brushing and use of ``. . .multiple forms of interdental
cleaning including dental oss and interdental
brushes and various probes, picks and sticks'' (71).
In addition, the authors reviewed the early literature
in which references have been made to the
``. . .absorption, or wasting of the alveolar processes.''
They quote an early book by Joseph Fox (1823) who
stated, ``Sometimes this disease proceeds without
the appearance of any assignable cause, the gums
retain a very healthy aspect, are quite free of pain
or inammation, and yet will gradually recede, until
the teeth become very loose'' (31). Finally, Page &
Sturdivant state that the 1999 classication of period-
ontal diseases and conditions adopted by the Amer-
ican Academy of Periodontology (AAP) makes ``. . .no
allowance for the possible existence of forms of per-
iodontal disease that may not full [sic] the pre-
scribed characteristics'' (71).
Although their paper is interesting and thought
provoking, the arguments presented by Page & Stur-
divant for the existence of a specic noninamma-
tory destructive periodontal disease are weak and
unconvincing. Examination of the two case reports
strongly suggests that the progressive gingival reces-
sion and loss of attachment were secondary to self-
inicted injury caused by longstanding abusive oral
Table 7. Specic features of localized and generalized aggressive periodontitis (1999 Classication) (52)
Localized aggressive periodontitis
Circumpubertal onset
Robust serum antibody to infecting agents
Localized first molar/incisor presentation with interproximal attachment loss on at least two permanent teeth, one
of which is a first molar, and involving no more than two teeth other than first molars and incisors
Generalized aggressive periodontitis
Usually affecting persons under 30 years of age, but patients may be older
Poor serum antibody response to infecting agents
Pronounced episodic nature of the destruction of attachment and alveolar bone
Generalized interproximal attachment loss affecting at least three permanent teeth other than first molars and incisors
17
hygiene procedures. Similar cases appear in Hirsch-
feld's classic book, The Toothbrush: its Use and Abuse
(40). It should be pointed out that the 1999 AAP
classication includes a category of ``Non-plaque-
induced gingival lesions'' with a subgroup of ``Trau-
matic lesions'' under which ``physical injury'' is spe-
cically listed (10). It is unclear why the gingival
recession and loss of attachment observed in the
two cases was not attributed to the cumulative
effects of repeated physical injury to the periodon-
tium. Certainly, repetitive physical injury to the gin-
giva and periodontium can result in gingival
recession and loss of attachment (51, 74, 94). Con-
trary to the statements of Page & Sturdivant, there is
nothing in the AAP classication to indicate that
``. . .all forms of destructive periodontal disease are
infectious, and thay [ sic] they are all characterized by
chronic inammation, pocket formation and pro-
gressive deepening, and loss of attachment and
alveolar bone'' (71). Indeed, periodontal destruction
can be a feature of some non-plaque-induced gingi-
val lesions (28, 51, 57, 74, 83, 94).
The most troublesome suggestion regarding the
existence of NDPD is the notion that it is ``nonin-
ammatory.'' If the observed periodontal destruction
is due to repetitive physical injury to the periodon-
tium (which is likely), it is difcult to imagine the
existence of small and short-lived traumatic wounds
without the development of transient inammation
during healing. Therefore, use of the word ``nonin-
ammatory'' is ill-advised since it suggests that
inammation is not part of the process associated
with tissue destruction and remodeling. Although it
is possible that inammation may not be a central
component of the tissue-destructive processes
involved in the two cases described by Page & Stur-
divant, it is highly unlikely that the injured tissues
were free of inammation at all times during the
development of attachment loss and gingival reces-
sion. Alternatively, no data are presented to rule out
the possibility that injury-induced inammation is
involved in the process leading up to the observed
periodontal damage. It is unlikely that intermittent
clinical observations on two patients would permit
the detection of transient episodes of periodontal
inammation. Without a sound scientic basis, it
is unwise to resurrect the old concept that some
forms of destructive periodontal disease are non-
inammatory.
Finally, Page & Sturdivant appear to have uncriti-
cally interpreted some very old literature and
assumed that what was written by John Hunter (circa
1771) and Joseph Fox (circa 1806) must be true.
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18
Armitage
Although Hunter and Fox made signicant and
insightful contributions to the eld of their day,
over-interpretation of what they wrote should be
avoided. Misinterpretation of clinical observations
was common among many early authors (11). In
retrospect, with the limited technology and informa-
tion base available to them, the failure of the pio-
neers of periodontics to recognize the infectious and
inammatory nature of such diseases as localized
aggressive periodontitis is understandable. Indeed,
early failures to recognize the presence of periodon-
tal inammation is not surprising since in many
cases of chronic and aggressive periodontitis the tis-
sues supercially look healthy. It is only upon closer
inspection with calibrated periodontal probes, which
were not invented until 1925 (88), that bleeding on
probing (now a universally accepted sign of period-
ontal inammation) permitted the detection of hid-
den-from-view periodontal inammation.
Nevertheless, the two cases presented by Page &
Sturdivant clearly demonstrate that not all patients
who experience progressive periodontal destruction
must necessarily have clinical features commonly
associated with chronic periodontitis (i.e. formation
of periodontal pockets, overt and persistent signs
of inammation, abundant accumulation of dental
plaque biolms) (71). The important question that
might have been raised by these authors is: Does
progressive periodontal destruction presumably
caused by self-inicted vigorous oral hygiene pro-
cedures t under the 1999 AAP category of ``Non-
plaque-induced gingival lesions'' (subcategory
``Traumatic lesions'')? Should the classication be
modied to include a category of ``Non-plaque-
induced periodontal lesions'' (subcategory ``Trauma-
tic lesions'')? If one chooses to make inexible or
rigid interpretations of the 1999 AAP classication,
it could be argued that gingival lesions and period-
ontal lesions should be separate items.
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21
The complete periodontal
examination
Gary C. Armitage
Components of a complete
periodontal examination
A thorough periodontal examination is a critically
important data-collection activity that is necessary
to arrive at a diagnosis and develop a treatment plan.
In medically healthy patients with simple and rather
straightforward periodontal conditions, the examina-
tion can usually be completed in one visit. For medi-
cally compromised patients with complex periodontal
and dental problems, multiple visits may be needed
to complete the data-gathering process.
The purpose of this chapter is to itemize and
describe the basic components of a complete period-
ontal examination and briey review their impor-
tance in the overall care of the patient.
History and chief complaint
Prior to conducting the hands-on examination, the
information-gathering process begins with taking
medical and dental histories from the patient. Many
practitioners prefer to initiate this process by having
the patient ll out a questionnaire. However, ques-
tionnaires are only a starting point, since a discus-
sion with the patient about past and present
medical/dental problems nearly always provides
additional important information. A valuable aspect
of this history-taking discussion is that it begins to
develop the doctorpatient relationship. Indeed, the
critically important patient-periodontal therapist
partnership starts during these initial conversations.
In addition, the discussion helps clarify important
variables that may affect the periodontal health of
the patient. For example, sometimes the only way to
obtain a reasonably accurate smoking history is by
one-on-one questioning. Procurement of an accurate
list of medications that the patient may be taking is
facilitated by talking with the patient.
A key component of the history-taking session is
determination of the patient's chief complaint. Prior
to the examination it is important to know why the
patient is seeking a periodontal evaluation. With
advance knowledge of the chief complaint, the exam-
iner can, during the course of the examination, spe-
cically look for possible explanations or causes of
the patient's problems and concerns.
The initial periodontal examination
Prior to conducting a periodontal examination it is
customary to routinely inspect the extraoral tissues
of the head and neck. In addition, examination of all
non-periodontal tissues in the mouth should be per-
formed. In other words, a detailed periodontal eva-
luation is the last component of a thorough oral
examination.
Performance of a periodontal examination is a
multi-task activity. While keeping in mind all of the
information gathered during the history-taking ses-
sion, the examiner looks for any signs of periodontal
disease or other abnormalities. It is, of course, neces-
sary to have a good idea what healthy periodontal
tissues look like (Fig. 1). In general, an overall
inspection is made during which changes in color,
shape, and texture of the gingival tissues are
assessed. An appraisal of potential etiologic and
predisposing factors is continuously being made
during the examination process. Detailed measure-
ments of probing depths and clinical attachment
loss are taken and recorded. Finally, the teeth are
inspected for occlusal relationships and restorative
needs.
Recognition of gingival inflammation
One of the very rst things to note during a period-
ontal examination is the presence or absence of
22
#
PERIODONTOLOGY 2000
disease. This often can be determined in seconds by
looking for signs of gingival inammation. The four
most common signs of gingival inammation that
are routinely observed during a periodontal exami-
nation are redness, swelling, bleeding on probing,
and purulent exudate (pus).
Gingival redness and swelling usually are seen
together andoccur rst at the gingival margin. Without
treatment the inammation can eventually involve
the entire interproximal area (Fig. 2a) and in some
cases extend into portions of the attached gingiva
(Fig. 3). Sometimes the redness associated with gin-
gival inammation can be quite subtle. If one is
uncertain about the presence of inammation-asso-
ciated gingival redness, it is useful to compare the
color of the site in question with that of a conrmed
healthy site (e.g. such a site is often the adjacent
attached gingiva) (Fig. 3).
Recognition of gingival swelling or edema requires
that the clinician have a very clear mental picture of
the shape and texture of healthy gingiva (Fig. 1).
Healthy gingiva is rm and resilient, whereas edema-
tous tissue is often enlarged and puffy (Figs 2, 4 and
5). If there is some uncertainty about the presence or
absence of gingival edema, it is sometimes useful to
gently press the side of a periodontal probe against
the tissue for a few seconds and then remove it. At
edematous sites the imprint of the periodontal probe
can often be seen (Fig. 5), whereas at sites without
marked edema no imprint will be observed. Recogni-
tion of the presence or absence of gingival edema
helps the clinician determine if the tissues are
Fig. 1. Clinical appearance of healthy gingival tissues with
no abnormalities in color, form, contour, or texture.
(a) 14-year-oldCaucasianfemale. (b) 36-year-oldCaucasian
female. (c) 40-year-old African-American female with nor-
mal gingival pigmentation. (d) 62-year-old Caucasian
female.
Fig. 2. Mandibular anterior region of a 55-year-old female
with chronic periodontitis. (a) Note that the gingival
papilla between the canine and lateral incisor is red and
swollen. (b) Bleeding on gentle probing at the same site.
Note: the 7 mm probing depth at the site. The clinical
attachment loss is somewhat less than 7 mm since the
gingival margin is coronal to the cementoenamel junction
(not visible). The combination of gingival inammation
plus a considerable amount of clinical attachment loss
indicates that the site has periodontitis (2).
The complete periodontal examination
23
healthy or diseased. In addition, it also serves another
very important purpose anticipating the response
to treatment. Gingival edema and the accompanying
redness often disappears shortly after scaling and
root planing. Therefore, by noting that the tissues
are edematous during the examination, the clinician
can predict the likely response to therapy.
It should be remembered that not all areas of gin-
gival redness and swelling are due to periodontal
diseases. Endodontic infections sometimes drain
through the orice of a periodontal pocket thereby
mimicking a periodontal abscess (Fig. 6). Elsewhere
this volume discusses in detail the diagnosis of endo-
dontic-periodontal lesions (10).
Bleeding on probing is a somewhat objective sign
of gingival inammation; it is either present or
absent (Fig. 2b). Inamed gingival tissues bleed
when gently probed because of minute ulcerations
in the pocket epithelium and the fragility of the
underlying vasculature. At the initial examination
the percentage of sites that exhibit bleeding on prob-
ing prior to treatment is a clinically useful piece of
information since it provides a full-mouth pretreat-
ment assessment of the extent of gingival inamma-
tion. For example, if 70% of the sites exhibit bleeding
on probing prior to treatment, a decrease to 20% of
the sites after initial scaling and root planing and oral
hygiene instructions is encouraging to both the
patient and periodontist by indicating that progress
has been made. In other words, knowledge of this
improvement reassures the patient and periodontist
Fig. 3. Gingival swelling and color change (redness)
between the mandibular canine and rst premolar affect-
ing the attached gingiva in a 45-year-old female with
chronic periodontitis. The color change can be rapidly
determined by comparing the affected site with the
appearance of the healthy attached gingiva in an adjacent
area such as the second premolar and rst molar.
Fig. 4. Swollen gingival papilla in a 17-year-old male. In
this case the center of the swollen papilla has developed a
``dimple'' due to loss of tissue tone.
Fig. 5. Pitting edema of an edematous interproximal area
in a 65-year-old male with chronic periodontitis. The side
of the periodontal probe was gently pressed against the
edematous area for a few seconds and then removed. The
resulting ``pit'' is indicative of the presence of gingival
edema. The tissue is no longer rm and resilient.
Fig. 6. Gingival swelling of endodontic origin on the inter-
proximal gingiva between the molar and second bicuspid.
The molar was undergoing endodontic treatment for a
necrotic pulp.
24
Armitage
that their joint efforts to control the periodontal
infection are working.
Although purulent exudate (pus) can occasionally
be found at sites with gingivitis, it is most often
detected at sites with chronic periodontitis. Pus is a
neutrophil-rich exudate that is found in about 35%
of sites withuntreated periodontitis (1). Without ques-
tion, its presence signies that the site is inamed
and infected. The best way to detect the presence of
pus is to gently apply digital pressure to the overlying
gingiva in a coronal direction (Fig. 7). Conventional
wisdom suggests that the presence of pus is an unfa-
vorable sign. However, available data suggest that
suppuration is not a good stand-alone predictor of
the progressionof chronic periodontitis (1). This state-
ment only applies to the relatively small amounts of
pus produced at sites with chronic periodontitis. The
importance of copious amounts of pus often seen at
sites with periodontal abscesses is a different situa-
tion (Fig. 8). Highly purulent periodontal abscesses
are associated with rapid and extensive destruction
of bone and surrounding tissues. The diagnosis of
acute periodontal lesions is discussed in detail else-
where in this volume (3).
Detection of departures from normal
anatomy, shape, and form
During the examination, notations should be made
of any deviations from normal periodontal anatomy
such as alterations in contour, aberrant frenal attach-
ments, and minimal amounts or lack of keratinized
gingiva. These items are of particular importance if
they interfere with the patient's ability to perform
oral hygiene procedures.
Altered gingival contours can be the result of a
wide range of factors. They become clinically impor-
tant if they create esthetic problems, make plaque
control difcult, or interfere with function. For exam-
ple, gingival enlargement is a well-known side effect
Fig. 7. Mandibular anterior region of a 55-year-old female
withchronic periodontitis (same patient as showninFig. 2).
(a) Note the inamed area between the lateral and central
incisor. Color change including all of the attached gingiva
overlying the central incisor can be seen. (b) Same area
after digital pressure has been applied to the gingiva of the
central incisor. Note the purulent exudate at the distal
gingival margin of the central incisor.
Fig. 8. Highly purulent periodontal abscess on a mandib-
ular central incisor in a 38-year-old female. (a) The entire
vestibule in the lower anterior region was markedly swollen
from a large accumulation of pus. (b) A massive amount of
pus was released immediately after an incision was made
to drain the abscess. (Courtesy of Dr. Gilbert V. Oliver.)
25
of certain medications (e.g. phenytoin, nifedipine,
cyclosporine) (Fig. 9). Sometimes the enlargement
is due to unusual anatomic variations (Fig. 10). Occa-
sionally, mandibular tori can be come so large that
they interfere with chewing or impede access for
plaque control procedures (Fig. 11). Little needs to
be said about these alterations in contour because
they are clinically obvious and are often associated
with the patient's chief complaint. However, men-
tion should be made of subtle changes in gingival
contour that are sometimes overlooked, but have
clinical importance. In some patients with long-
standing chronic periodontitis, the gingiva becomes
rm and enlarged in reaction to the chronic inam-
mation (Fig. 1214). Sometimes such tissues are
referred to as ``brotic.'' In contrast to gingival enlar-
gement due to tissue edema, brotic enlargements
will not disappear after scaling and root planing. This
knowledge is important since it helps the clinician
anticipate what tissue changes will occur after non-
surgical therapy. The best way to conrm that the
tissue is brotic is to gently press on the gingiva with
Fig. 9. A 35-year-old female with gingival enlargement
associated with the ingestion of phenytoin to help control
cerebral seizures. The gingival enlargement created
esthetic problems for the patient and was her chief com-
plaint.
Fig. 10. Localized enlargement of the palatal gingiva in
the molar region in a medically healthy 32-year-old
female. The enlargement was bilateral and was considered
to be an unusual anatomic variation. The enlarged gingiva
was the focus of the patient's chief complaint.
Fig. 11. Bilateral large mandibular tori in a 45-year-old
female that interfered with oral hygiene procedures.
Fig. 12. A 49-year-old male with chronic periodontitis. (a)
The altered contours and enlargement of the interproximal
gingival papillae were due to the longstanding inflamma-
tion. The papillae were firmand ``fibrotic'' and the contours
did not appreciably change after nonsurgical therapy. (b)
Lingual view of the same teeth shown in A. The
interproximal gingiva with increased redness was highly
edematous, thereby increasing the likelihood of consider-
able shrinkage after nonsurgical therapy.
26
Armitage
the side of the periodontal probe. Unlike the reaction
of edematous tissue to this procedure, no imprint of
the probe will be left behind.
During the examination, notations should be made
about narrow bands, or the complete absence, of
keratinized gingiva. This item is discussed in detail
elsewhere in this volume (6). However, the main
clinical importance of an adequate zone of kerati-
nized gingiva is that it is often necessary for the
patient to comfortably perform oral hygiene proce-
dures. The gingiva overlying teeth with narrow zones
of keratinized gingiva is often thin and is thereby
prone to toothbrush-induced damage followed by
recession (Fig. 15).
Aberrant frenal attachments are anatomic features
that should be noted if they contribute to a clinical
problem. This item is discussed in detail elsewhere in
this volume (6). However, the most common situa-
tion in which they become a problem is when they
interfere with oral hygiene or other self-care proce-
dures. For most patients it is uncomfortable to brush
non-keratinized tissues such as frena and alveolar
mucosa. Therefore, if the frenal attachments are
located close to the gingival margin, patients tend
to avoid cleaning these areas and plaque-induced
periodontal disease develops (Fig. 16). In some
instances a frenum is located near the gingival mar-
gin of a tooth in an area with little or no keratinized
gingiva (Fig. 17). Such a combination should be con-
sidered to increase the risk for the future develop-
ment of periodontal problems at the site and should
most certainly be noted at the time of the initial
examination. In other cases a very prominent frenum
may be attached at the mucogingival junction where
there is an adequate band of keratinized healthy
gingiva coronal to the attachment (Fig. 18). Under
these circumstances the risk of developing a period-
ontal problem is unlikely because the patient can
adequately clean the site.
Assessment of etiologic and predisposing
factors
During the course of a periodontal examination the
clinician should begin to develop an idea of what
Fig. 13. Fibrotic gingiva in the maxillary anterior region in
a 52-year-old male. The gingival contours will not change
after nonsurgical therapy.
Fig. 14. Gingiva with altered contours in the same patient
shown in Fig. 13 Some of the enlargement was due to
edematous changes and some had already become
fibrotic. In such cases, nonsurgical therapy will only
result in partial resolution of the gingival enlargement.
Fig. 15. An unusual case of a 24-year-old female who
lacked keratinized gingiva in most areas of her mouth.
The atypically thin gingiva was at high risk for developing
recession from both plaque-induced inflammation and
damage during toothbrushing. Both causes of recession
were etiologic factors in this patient. Cases similar to this
have been reported by Moskow & Baden (8).
27
etiologic and predisposing factors are present. As the
examination is being performed the clinician should
develop an impression of what modiable factors
might be responsible for, or increase the risk of, per-
iodontal infections. Where are the heaviest deposits
of plaque and calculus? Are there local contributing
factors that might increase the risk for periodontal
infections? Conceptually, one is looking for etiologic
items or predisposing factors that can be modied by
therapeutic interventions. Tooth-related factors such
as close roots, palatal-gingival grooves, furcation
anatomy, cervical enamel projections, overhangs
on dental restorations, and other local contributing
factors are all discussed elsewhere in this volume (6)
and will not be repeated here. However, when pre-
sent they should be noticed and recorded. As the
information is being collected it is important to keep
in mind what is known about potential risk factors
for chronic periodontitis such as cigarette smoking,
poor compliance, aging, psychological stress, and
genetic susceptibility. These and other risk factors
are discussed in detail elsewhere in this volume (9).
Assessment of periodontal damage
Assessments of periodontal damage are a mandatory
component of a complete periodontal examination.
Measurements taken with calibrated periodontal
probes are the main way in which damage to
the periodontium is assessed. Such measurements
include probing depth, clinical attachment loss, and
gingival recession. Probing depth and clinical attach-
ment loss measurements are routinely recorded at
six sites around each tooth (i.e. mesiobuccal, buccal,
distobuccal, mesiolingual, lingual, and distolingual).
During the course of periodontal probing the instru-
ment is stepped around the entire circumference of
the tooth and the deepest reading nearest each of the
six sites listed above is recorded. In other words, an
attempt is made to probe every portion of the gingi-
val crevice or pocket around each tooth. This routine
of thoroughly probing all locations is usually fol-
lowed since it is often impossible to tell from the
supercial appearance of the gingiva if sites will have
deepened probing depths and loss of attachment
(Fig. 19). In addition to the above assessments,
radiographs are a necessary adjunct to a thorough
periodontal examination. A detailed discussion of
imaging methods (including radiography) can be
found elsewhere in this volume and will not be
repeated here (7).
Fig. 17. Attachment of a frenum between two mandibular
central incisors near the gingival margins of both teeth.
Note the lack of keratinized gingiva in the area. Both teeth
have an increased risk of developing plaque-induced
periodontal problems if oral hygiene cannot comfortably
be performed at the sites. The situation should be
recorded at the initial periodontal examination.
Fig. 18. Attachment of a prominent frenum at the muco-
gingival junction in an area where there is enough
keratinized gingiva to permit good oral hygiene.
Fig. 16. Multiple frena attached to positions on the gingiva
that make oral hygiene difficult and uncomfortable. Note
the heavy calculus deposits, an indication that the patient
does not (or cannot) effectively clean the areas.
28
Armitage
Probing depth is the distance from the gingival
margin to the base of the probeable crevice. Probing
depth measurements are important because they
give a good approximation of the principal habitat
of periodontal pathogens (i.e. periodontal pockets).
Knowledge of the depth, extent, and location of
pockets gives the clinician a good idea where therapy
might be directed. Indeed, probing depth reduction
is often one of the important goals of many forms of
periodontal therapy. However, probing depth mea-
surements do not necessarily give the best approx-
imation of the amount of periodontal damage since
the reference point from which the measurements
are taken (i.e. the gingival margin) may uctuate in
apical or coronal directions. For example, at one
examination the probing depth at a given site might
be 4 mm, but at a later date gingival inammation
can cause gingival swelling that results in migration
of the gingival margin 2 mm coronally. The probing
depth at this later date would be 6 mm (i.e. 4 mm
2 mm) even though no additional periodontal
damage had occurred. Conversely, if at a later date
2 mm of additional attachment loss occurred and the
gingival margin receded 2 mm apically, the probing
depth would still be 4 mm. In other words, the gin-
gival margin is not a xed landmark from which valid
assessments of additional damage can be made.
Clinical attachment loss is the distance from the
cementoenamel junction (CEJ) to the base of the
probeable crevice. If the CEJ landmark is missing
because it has been destroyed by dental caries or has
been removed by placement of a dental restoration,
another xed reference point can be used to measure
attachment loss. Such landmarks might include the
apical margin of a restoration or the incisal edge of a
tooth. When attachment loss measurements are taken
from a xed landmark other than the CEJ they are
calledrelative attachment loss measurements. Clinical
attachment loss or relative attachment loss measure-
ments are the best way to assess the presence or
absence of additional periodontal damage.
Some clinicians elect not to take clinical attachment
loss measurements at the initial examination but wait
until active treatment has been completed. The main
reasons for this are that many changes occur in clin-
ical attachment loss as a result of therapy and the
measurements are easier to obtain once supragingi-
val and subgingival calculus has been removed.
Nevertheless, prior to placing patients in the main-
tenance phase of therapy, clinical attachment loss
readings should be taken since these measurements
serve as a baseline from which future determinations
of additional attachment loss are judged.
Gingival recession is the distance from the CEJ to
the gingival margin (GM). Recession is often of major
concern to patients since it is a readily visible man-
ifestation of periodontal damage and can cause
esthetic problems when in occurs around anterior
teeth. Indeed, many patients have a chief complaint
of ``receding gums.'' Therefore, at the initial exam-
ination it is important to record the amount and
location of gingival recession.
Damage fromperiodontal disease often involves the
furcation areas of multirooted teeth. The severity of
furcation involvement is an important factor in deve-
loping a treatment plan for affected sites. Therefore,
during a complete periodontal examination the loca-
tion and severity of furcation involvements should be
recorded. Onecommonclassicationsystemfor furca-
tion involvement includes: Class I (beginning), Class II
(cul-de-sac), and Class III (through-and-through). A
detailed discussion of the classication, diagnosis,
and importance of furcation involvements can be
found elsewhere in this volume (6).
The nal assessment of periodontal damage that
should be recorded during a complete periodontal
examination is abnormal tooth mobility. Although
this symptom may have several causes other than
periodontal infections (5), loss of alveolar bone from
periodontitis is a major cause of abnormal tooth
mobility. In addition, it is sometimes part of the
patient's chief complaint (e.g. ``My teeth are loose.'').
Fig. 19. Gingiva in a 38-year-old female that superficially
looks healthy (a). Insertion of a periodontal probe on the
mesiobuccal side of the first molar reveals a probing depth
of 8 mm (b). The patient had received scaling and root
planing by the referring dentist approximately 6 weeks
prior to taking these photographs.
29
The diagnosis and overall importance of tooth mobi-
lity are discussed in elsewhere in this volume (4).
Inspection of the teeth
Although the primary focus of a periodontal exam-
ination is the periodontium, the teeth also need to be
carefully inspected for dental caries, restorative pro-
blems (6), and occlusal discrepancies (4). Tooth-
related problems have considerable importance in
the overall periodontal treatment plan.
Recording the findings
There are many types and styles of periodontal charts
to choose from. Selection of one charting system over
another is entirely up to the preferences of the indi-
vidual practitioner. Most acceptable charting sys-
tems are simple, easy to ll out and read, and
contain all of the relevant information collected dur-
ing the periodontal examination. An example of such
a chart is shown in Fig. 20. The periodontal chart is a
permanent record that can be used in assisting the
practitioner to arrive at a diagnosis and prognosis,
develop a treatment plan, and longitudinally evalu-
ate the response to therapy.
To efciently ll out a periodontal chart requires
the help of a dental assistant who serves as a recorder
of the examination ndings. As the clinician calls out
the measurements or assessments they are recorded
in the chart. The chart shown in Fig. 20 has places for
assessments of probing depth, the presence or
absence of plaque, clinical attachment loss, the pre-
sence or absence of bleeding on probing, and the
distance from the CEJ to the gingival margin
(CEJ GM). As mentioned above, in the section on
`Àssessments of periodontal damage'' measure-
ments or assessments at six sites around each tooth
are usually recorded.
Some examiners prefer to record, as the very rst
step, the presence or absence of plaque on each
tooth and surface. In the chart shown in Fig. 20, if
supragingival plaque is present a ``dot'' ( ) is placed
in the box where the probing depth measurements
will be inserted. The second step is to measure the
probing depth, CEJ to GM distance, and the presence
or absence of BOP. These three pieces of information
are collected virtually at the same time. In this step
the examiner calls out a probing depth reading. Then
a second number is called out that represents the CEJ
to GM distance. Finally, if the site exhibits bleeding
on probing, the examiner says ``bleeding,'' and the
recorder places a ``dot'' ( ) in the box where the
clinical attachment loss measurement will eventually
be inserted. As will be seen in a moment, the clinical
attachment loss reading is a derived number obtained
by adding the CEJ to GM distance to the probing
depth measurement.
For beginners the CEJ toGMreading canbe a source
of confusion. There is usually no problem in under-
standing how to measure gingival recession (i.e. the
CEJ to GMdistance when the gingival margin is apical
to the CEJ). The CEJ to GMmeasurement can be easily
obtained since both of the reference points (i.e. CEJ
and GM) are in full view. In addition, there is usually
not a problem in understanding that the clinical
attachment loss can be obtained by adding the prob-
ing depth to the amount of gingival recession. For
example, if there is a 4 mm probing depth and 2 mm
of gingival recession, the clinical attachment loss at
the site is 6 mm (i.e. 4 mm 2 mm). The problem
occurs when the gingival margin is coronal to the CEJ
(i.e. when there is no gingival recession). In this case,
only one of the reference points (i.e. GM) is in full
view of the examiner. To determine the CEJ to GM
measurement the examiner must feel for the CEJ
with the tip of the periodontal probe and estimate
how far coronally the GM is from the CEJ. If the GM is
at the CEJ, the number called out by the examiner
would be ``zero.'' If the GM is 1 mm coronal to the
CEJ, the number called out by the examiner would be
``minus one.'' If the GM is 2 mm coronal to the CEJ,
the number called out by the examiner would be
``minus two.'' In other words, when the GM is cor-
onal to the GM, the CEJ to GM measurement is
recorded as a negative number. For example, if there
is a 4 mm probing depth and the CEJ to GM distance
is 2 mm, the calculated clinical attachment loss at
the site would be 2 mm (i.e. 4 mm 2 mm).
The chart in Fig. 20 has been lled out using the
examination ndings from a patient with generalized
severe chronic periodontitis. In addition to the
probing depth, clinical attachment loss, and other
assessments discussed above, commonly used
symbols have been placed to reect the extent of
Fig. 20. Example of a periodontal chart showing some of the clinical information collected during examination of a 36-
year-old male with generalized chronic periodontitis. CAL clinical attachment loss; BOP bleeding on probing; PD
probing depth; Plaque visible plaque (plaque index score 2 using Silness & Loe system (11)); CEJ GM distance
from cementoenamel junction to gingival margin.
30
Armitage
31
furcationinvolvement (L incipient; D cul-de-sac;
~ through-and-through) and mobility (I slight,
II moderate, III severe). Many other symbols to
signify a variety of clinical ndings are used by prac-
titioners. There is no standard set of symbols that are
universally accepted by the majority of clinicians.
The important thing is that the symbols be consis-
tently used and are understood by others who might
be called upon to read the chart (e.g. a referring
dentist or colleague).
Although the chart shown in Fig. 20 provides site-
specic details of the collected assessments, it also
provides the raw data from which some valuable
full-mouth information can be calculated. In this
patient, full-mouth summary data include: sites with
probing depth 5 mm 53.7%, clinical attachment
loss 5 mm 64.2%, bleeding on probing 71.6%,
and visible supragingival plaque 66.7%. This infor-
mation is useful in a number of ways and it has
immediate applications in communicating with the
patient. For example, it is very easy to point out to the
patient that one of the reasons they have periodontal
disease is that two-thirds of their tooth surfaces have
visible plaque. Over 70% of the sites bleed, which is a
sign of the infection and well over half of the sites
have deep pockets with signicant damage. After
successful treatment, marked reductions in the per-
centages of sites with plaque and bleeding on prob-
ing will always occur. Simply showing the patient the
improved percentages can be gratifying and
encouraging to both patient and therapist. The
important point being raised here is that data col-
lected during a periodontal examination are not just
for the therapist's eyes. If presented in an under-
standable way, patients can also benet from seeing
the clinical data.
In addition to the data traditionally recorded on
the periodontal chart, the initial examination usually
generates other important information that may be
valuable in the subsequent development and execu-
tion of a treatment plan. Such items are usually
entered, in detail, in the ``Progress Notes'' section
of the patient's chart.
Follow-up (maintenance-phase)
examinations
After completion of active periodontal therapy, prior
to placing the patient on a maintenance program, the
examination should be repeated. The information
to be collected is the same as that obtained during
the initial examination. A key purpose of this post-
treatment evaluation examination is to determine if
the administered therapy was successful in arresting
the patient's disease. The examination also provides
baseline or benchmark data to which all clinical data
collected at subsequent examinations can be com-
pared.
In a busy practice, as the number of patients on
recall/maintenance programs increases, it is often
difcult to rapidly assimilate, review, and compare all
of the information collected from multiple examina-
tions. Indeed, the sheer amount of clinical data
becomes overwhelming after the patient has been
on a maintenance program for several years. If one
patient has 28 remaining teeth, each examination will
generate 168 data points (i.e. six per tooth) for each
assessment made or measurement taken. Since the
periodontal chart shown in Fig. 20 has places for ve
clinical variables (i.e. clinical attachment loss, prob-
ing depth, bleeding onprobing, plaque, CEJ GM dis-
tance), each exam could generate 840 pieces of data
(i.e. 5 168). If the patient is placed on a 3-month
recall program and the examination is repeated at
every visit, 3,360 data points will be generated in
1 year, 16,800 in 5 years and 33,600 in 10 years. Ways
toovercomeor deal withthis potential burdeninclude:
utilizing computers during data entry and subse-
quent analysis, emphasizing or focusing on clinical
attachment loss measurements, and reducing the
number of examinations from 4 per year to 1 per year.
Computerized systems including software are
needed to cope with the large amount of data col-
lected during multiple examinations. Computers can
automatically track changes in each of the assess-
ments and bring them to the attention of the clin-
ician. Since clinical attachment loss measurements
are the best way to track the progression of period-
ontal disease, it makes sense to emphasize this
assessment in the longitudinal evaluation of period-
ontal status. Finally, for most patients it is probably
sufcient to conduct full examinations or data col-
lection procedures once per year instead of quarterly.
If this last option is chosen, the clinician should
selectively and closely monitor high-risk or fragile
sites at frequent intervals. The best option, of course,
is to examine as frequently as is practical and enlist
the aid of computer technology to assist in tracking
any changes in periodontal status.
References
1. Armitage GC. Periodontal diseases: diagnosis. Ann Period-
ontol 1996: 1: 37215
32
Armitage
2. Armitage GC. Clinical evaluation of periodontal diseases.
Periodontol 2000 1995: 7: 3953
3. Corbet EF. Diagnosis of acute periodontal lesions. Period-
ontol 2000 2004: 34: 204216.
4. Hallmon WW, Harrel SK. Occlusal analysis, diagnosis and
management in the practice of periodontics. Periodontol
2000 2004: 34: 151164.
5. Jordan RCK. Diagnosis of periodontal manifestations of sys-
temic diseases. Periodontol 2000 2004: 34: 217229
6. Matthews DC, Tabesh M. Detection of localized tooth-
Periodontol 2000 2004: 34: 136150.
2000 2004: 34: 3448.
8. MoskowBS, BadenE. Unusual gingival characteristics havinga
familial tendency: a case report. Periodontics 1967: 5: 259264
9. Ronderos M, Ryder MI. Risk assessment in clinical practice.
Periodontol 2000 2004: 34: 120135.
10. Rotstein I, Simon JHS. Diagnosis, prognosis and decision-
making in the treatment of combined periodontal-endo-
11. Silness J, Loe H. Periodontal disease in pregnancy. Part II.
Correlation between oral hygiene and periodontal condi-
tion. Acta Odontol Scand 1964: 22: 121135
33
Imaging methods in
periodontology
Andre Mol
Advances in basic periodontal research have trans-
formed our understanding of almost all aspects of
the periodontal disease process. These developments
have translated into meaningful clinical applications
improving the way we prevent, diagnose and treat
periodontal disease. In comparison, the impact of
radiographic imaging on the management of the per-
iodontal patient has essentially remained unchanged
for decades. Substantial advances in X-ray generator
and X-ray detector technology have resulted in sig-
nicant dose reductions and improved image qual-
ity. However, the basic information content of oral
radiographic images has changed very little.
This does not imply that the imaging needs in
periodontology are met or that there is little room
to improve patient outcomes by using better imaging
methods. For many years a number of limitations in
current radiographic methods have been recognized,
such as the two-dimensional representation of the
alveolar bone and uncertainty regarding the validity,
accuracy and precision of quantitative measure-
ments (22).
Relatively few new technologies have emerged to
address the critical needs in periodontal diagnosis.
Digital imaging has been hailed as a panacea for many
of the limitations associated with traditional lm-
basedradiography. However, most of these limitations
are associated with X-ray transmission and image
interpretationand not with the choice of image recep-
tor. Although scientic studies have provided a better
understanding of the strengths andlimitations of digi-
tal technology in dentistry, a number of misconcep-
tions prevail. The added value of digital radiography
in clinical practice is mainly at a practical level and
its diagnostic efcacy has in most cases been shown
to be equivalent to lm radiography. Digital imaging
does, however, offer a new starting point to readdress
some of the periodontal imaging needs, and signi-
cant scientic progress has been made.
In this chapter the current status of conventional
and digital radiographic methods used in periodonto-
logy and implantology will be examined. A number of
excellent review articles have been published on this
topic in the recent past (4, 22, 33, 34, 55, 63). The
purpose of this paper is not to present a comprehen-
sive re-reviewof similar concepts. Rather, an overview
will provided of the most relevant periodontal imaging
issues focusing on the relationship between basic
diagnostic principles and clinical applications. Spe-
cically, issues will be addressed related to why and
when we use radiographic imaging and what imaging
modalities are best suited for a given task. A section
on new frontiers covers developments that may soon
change the role of imaging in periodontology.
Why imaging?
The use of radiographic imaging as an aid in the
diagnosis and treatment of periodontal disease is
widely accepted. Its main purpose is to assess the
level of the alveolar bone including the pattern and
extent of bone resorption. Linear measurements
from the cementoenamel junction to the alveolar
crest and from the cementoenamel junction to the
base of the osseous defect are commonly used to
quantify crestal bone levels and osseous defects.
Radiographs also show the periodontal ligament
space, lamina dura and periapical region and are
useful in identifying related factors, such as calculus
and defective restorations. Radiographs can provide
critical information for diagnosis and treatment
planning, which can also serve as baseline informa-
tion for the assessment of treatment outcomes.
Few people will question the value of radiographs
for the above stated purposes. Yet good clinical prac-
tice requires that the use of radiographs be justied
on the basis of sound selection criteria tting the
34
#
PERIODONTOLOGY 2000
needs of each individual patient. A decision to pre-
scribe any radiograph should only be made if addi-
tional information is required and when that
information cannot be obtained through methods
carrying a lower risk to the patient. Such a decision,
like many others, is based on a costbenet analysis.
Consequently, the clinician needs to understand
both the costs and benets of a radiographic exam-
ination.
Cost is usually associated with the absorbed dose
from exposure to ionizing radiation, although eco-
nomic and patient-related factors need to be consid-
ered as well. The effective dose to the patient in oral
radiography is generally low (71). However, it con-
tinues to be assumed that each exposure to ionizing
radiation carries a risk and that less is better.
On the other side of the equation, the practitioner
needs to understand the benets of radiography.
This is usually less obvious than it appears, because
radiography, even when used appropriately and opti-
mally, is far from perfect. As with any diagnostic test
lacking perfect accuracy, there is a chance that false-
positive or false-negative results will emerge. For
instance, it is well-known that it remains fairly uncer-
tain whether or not a patient has furcation involve-
ment when the radiograph shows no bone loss in the
furcation area. In other words, the negative predictive
value of radiography for furcationinvolvement is fairly
low. On the other hand, if the radiograph demon-
strates bone loss in the furcation area it is virtually
certainthat thepatient hasfurcationinvolvement. This
means that the positive predictive value of radio-
graphs for furcation defects is high. The positive and
negative predictive values are important measures of
the clinical benet of a diagnostic test. The factors
that determine the predictive values are the diagnos-
tic accuracy of the test and the probability that the
disease is actually present. Since the absence, pre-
sence or extent of the disease is unknown before the
patient has been examined, a clinical examination
needs to be performed to obtain a realistic estimate
of the probability or risk that the disease is present.
This estimate of risk is important in judging the out-
come of the radiographic interpretation (28).
Decisions to use radiographs are sometimes made
solely for the purpose of collecting diagnostic infor-
mation. However, the imaging examination only
benets the patient when it inuences treatment
decisions (27, 41). Therefore, radiographs should
only be prescribed if the information they provide
can make a difference in patient management and if
this difference has an impact on outcomes that are
important to the patient.
When to use imaging?
Radiographs are generally considered to provide
essential information for the assessment, diagnosis
and management of periodontal disease. Yet very
little is known about the impact of radiography on
the outcome of care for the periodontal patient (63).
Much of the information required to diagnose peri-
odontal disease can be obtained through clinical
examination alone. The hypothesis that the informa-
tion on bone levels and bone resorption provided by
radiographs has a signicant impact on treatment
and patient outcome has hardly been tested.
It is quite common for practitioners to seek general
guidelines that can be applied to decide when and
how often to perform a radiographic examination.
However, the timing of a radiographic examination
is dictated by its purpose, which depends on indivi-
dual patient factors. Practitioners must use profes-
sional judgment to decide on the type and frequency
of each radiographic examination. The routine use of
any type of imaging based on a preset timetable does
not serve the needs of the individual patient (72). It
follows that a decision to take radiographs must be
preceded by a clinical examination and review of
existing radiographs.
Radiographs used for diagnosis and treatment
planning generally provide adequate information
for the entire patient-management phase. If the
initial phase of therapy indicates a need for correc-
tive surgery, additional periapical radiographs may
be required if they were not part of the initial diag-
nostic workup. During the maintenance phase of
treatment, healing or recurrence of the disease is
mainly detected by clinical examination. Clinical
signs usually precede radiographic signs and provide
sufcient diagnostic information during this phase
(63). The sensitivity of radiography for the detection
of osseous change signicantly improves when high
quality images are produced, radiographic para-
meters are standardized and digital image processing
techniques are applied (26, 34). High sensitivity is
particularly useful for outcomes assessment of
regenerative therapy, where early detection of small
changes may affect patient management.
The same criteria for justication of a radiographic
examination apply during the short-term and long-
term maintenance phase. When clinical signs pro-
vide sufcient information regarding the periodontal
health of the patient, there is no need for further
radiographic documentation. If the clinical examina-
tion raises doubt regarding the progress of the
Imaging methods in periodontology
35
disease, radiographic examination may be indicated.
The timing of such examinations is highly dependent
on individual patient needs and can not be dictated
by general guidelines.
What type of imaging?
A number of intraoral and extraoral imaging modal-
ities are available to assist in the management of the
periodontal patient (27). Commonly used modalities
include bitewing, periapical and panoramic radio-
graphy. All of these modalities can provide important
diagnostic information, but none of them are without
limitations. One of the main limitations is the two-
dimensional representation of three-dimensional
structures. Important morphologic or pathologic
aspects of the alveolar bone may go undetected as
a result of superimposition of teeth and other ana-
tomic structures. Only the interproximal alveolar
bone levels can be assessed with some level of cer-
tainty. The detection and quantitative assessment of
2-wall and 3-wall defects remains a challenge even in
these areas. There also needs to be a substantial
amount of mineral loss (3050%) before bone resorp-
tion can be detected. These limitations reduce the
sensitivity of conventional radiography and generally
result in underestimating actual bone loss even when
high quality images are produced. Misdirection of
the central ray of the X-ray beam as well as exposure
and processing errors further limit accuracy. There-
fore, proper use of radiographs requires that high
quality images are produced and that the limitations
imposed by the image formation model are recog-
nized in the interpretation process.
Intraoral radiography
Intraoral radiographs are simple to acquire, are rela-
tively low-cost and provide a level of image detail
that is unprecedented in medical imaging. Patient
dose is also very low, especially when recommended
parameters of radiologic care are followed (72). The
two most effective ways to reduce patient dose are to
use a fast image receptor and rectangular collima-
tion. Fast receptors include E- or F-speed lm as well
as digital detectors. These receptors have repeatedly
been shown to be diagnostically equivalent to D-
speed lm, which requires at least double the expo-
sure of fast receptors. Therefore, D-speed lm has no
place in a contemporary dental practice. Rectangular
collimation can further reduce the dose to the patient
by as much as 5080%. It is a simple method to
signicantly cut the dose to the patient and improve
image contrast by reducing scatter radiation. Unfor-
tunately, the extra effort and skill required for the
successful implementation of rectangular collima-
tion are often considered prohibitive and higher
patient doses are accepted.
The most accurate radiographic projection of the
alveolar bone level is obtained when the receptor is
placed parallel to the tooth and the central ray of the
X-ray beam is aimed at a right angle relative to the
tooth and the receptor (73). The technique that ful-
lls these requirements most often is bitewing radio-
graphy (Fig. 1a) (11). The ideal imaging conditions are
usually not compromised for bitewing radiographs
and both maxillary and mandibular structures are
shown in a single image. It is therefore the modality
of choice to assess the level of the alveolar bone (21,
24, 34, 73). Horizontal bitewing radiographs are
appropriate in most cases (Fig. 1b). When moderate
or severe bone loss is suspected, vertical bite-wing
radiographs should be taken to ensure that the cres-
tal bone of both the maxilla and mandible is shown
(Fig. 1c). Correct positioning of vertically oriented
image receptors is slightly more critical and may be
less comfortable for some patients.
In the posterior mandible, periapical radiographs
could be a substitute for bitewing radiographs when
the paralleling technique can be applied successfully.
This requires that the oor of the mouth provide suf-
cient space to position the receptor parallel to the
long axis of the tooth. Very rarely can this be achieved
Fig. 1. The ideal projection geometry
creates an image without distorting
the relationship between the buccal
alveolar crest, the lingual alveolar
crest and the cementoenamel junc-
tion (a). (b) Example of a horizontal
bitewing radiograph. (c) Example of a
vertical bitewing radiograph.
36
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in the maxilla because of the limited space provided
by the hard palate. Therefore, posterior maxillary
periapical radiographs are expected to produce a
more distorted view of the buccal and lingual period-
ontal bone levels.
Optimal projection geometry in the anterior
regions is achieved with periapical radiographs using
the paralleling technique (11). The receptor is placed
parallel to the long axis of the tooth and the central
ray of the X-ray beam is at a right angle to both. Small
deviations from the optimal projection geometry are
usually better tolerated in the anterior region
because of the smaller buccolingual dimension of
the teeth and the alveolar bone.
When the entire tooth and its surrounding tissues
need to be visualized, periapical radiography is the
technique of choice. A full-mouth series of periapical
and bitewing radiographs usually provides overlap-
ping coverage of posterior areas. Whereas too much
overlap is undesirable from a radiation safety point of
view, some overlap provides images of the same area
from different projection angles, thus increasing the
opportunity to detect bone defects and allowing the
clinician to build a mental three-dimensional picture
of the dentoalveolar structures.
The selection of the appropriate imaging techni-
que cannot overcome the fundamental limitations of
intraoral radiography, even when the images are of
high quality. Several studies have demonstrated that
intraoral radiographs tend to underestimate the
amount of bone loss (1, 9, 49, 62), whereas radio-
graphic assessment of severe osseous destruction
was shown to overestimate actual bone loss (49).
The use of radiopaque or transparent millimeter
grids, such as the Fixot-Everett grid or any other
analog or digital grid superimposed on the image,
does not improve the measurement accuracy. Grids
may facilitate the measurement process, but they do
not account for magnication or distortion.
Indirect methods have been used to account for
angulation errors, expressing the amount of bone
loss as a percentage of root length. However, mea-
suring the ratio between bone height and root length
does not provide a reliable measure of the alveolar
bone level, since this ratio varies with the length of
the root (55). The indirect method is also likely to
produce more observer variability since it relies on
the identication of three points (cementoenamel
junction, alveolar crest, apex), whereas the direct
method relies on the identication of only two points
(cementoenamel junction and alveolar crest).
One of the key components of reliable assessment
of osseous changes in serial radiographs is to
standardize the image acquisition geometry. It has
been shown that the reproducibility of horizontal
and vertical angulation is better when positioning
instruments are used (51). The skill and diligence
of the radiographer using the positioning instru-
ments remain critical in achieving high quality
images (57). When properly used, positioning instru-
ments have been shown to reduce the minimally
detectable amount of bone loss to less than 1 mm.
Further standardization allows reliable bone loss
measurements as little as 0.5 mm (23).
Extraoral radiography
Panoramic radiography is a useful imaging modality
for a number of oral and maxillofacial applications.
Image acquisition is relatively fast and simple with-
out the need for any intraoral manipulation. It shows
all dentoalveolar structures in a single image at a
dose that is considerably lower than for an intraoral
full-mouth series (71).
However, panoramic radiography has a number of
drawbacks which limit its usefulness as a diagnostic
tool in periodontology. One of the main limitations is
the potential for image distortion. The fan beam is
directed upwards and a sharp image layer is formed
by the coordinated movement of the beam and the
receptor. Horizontal and vertical magnication are
determined by different mechanisms and vary inde-
pendently as a function of the location of a structure
within the image layer. Even when the patient is
placed in an ideal position, lingual structures will
be projected higher on the lm than buccal struc-
tures. Although modern panoramic units use sophis-
ticated engineering to create an image layer that
closely resembles the shape of the jaws, a right-angle
horizontal orientation of the beam relative to the
arch cannot always be guaranteed. These effects,
along with the limited width of the image layer,
makes patient positioning a critical factor. Although
most units are equipped with positioning guides,
patient-positioning errors represent one of the main
sources of error in panoramic radiography. This
technique sensitivity also makes it difcult to repro-
duce the imaging geometry at a later date.
The tomographic nature of the technique and the
use of a screen-lm combination results in images
showing less image detail than intraoral images.
Structures outside the image layer are not completely
removed and dense structures on the opposite side of
the rotation center cause so-called ghost images. For
example, the shadowof the cervical spine often makes
interpretation of the anterior region challenging. For
37
assessment of the alveolar bone level, panoramic
radiography is not recommended as the primary
imaging modality (11). A panoramic radiograph can
still be valuable for the assessment of other dentoal-
veolar properties. It can be used as an alternative for
an intraoral full-mouth series when combined with
bitewing radiographs and selected periapical radio-
graphs when indicated.
Digital radiography
The use of lm as the X-ray detector in dental radio-
graphy is no longer self-evident. Digital detectors have
been on the market for about 15 years and the evolu-
tion of this technology has made digital imaging in
dentistry a viable alternative to lm-based imaging.
Digital imaging offers a number of advantages com-
pared to lm. The elimination of chemical processing
is considered one of the main benets. Other advan-
tages include a shorter exposure-to-display time and
integration with existing electronic ofce and patient-
management systems. Image processing can be used
to enhance the perceived quality, either to restore the
subjective quality of the image as a whole or to
enhance a selected region in the image for a specic
diagnostic task. Furthermore, the software offers a
variety of measurement tools, most of whichare digital
versions of existing analog tools. The benets of digi-
tal imaging are obtained along with a reduction in
radiation exposure, although the amount of exposure
reduction is dependent on the type of receptor.
There are currently two competing technologies
available for the implementation of digital imaging.
One uses solid-state detectors, the other photosti-
mulable phosphor (Fig. 2). Both technologies are
available for intraoral as well as extraoral applica-
tions. Solid-state detectors are based either on
charge-coupled device technology (CCD) or on com-
plimentary metal oxide semiconductor technology
(CMOS). Although quite different from a technical
perspective, their clinical application is very similar.
Both are rigid sensors that are directly linked to the
computer.
The dimensions of solid-state sensors have
improved dramatically since their rst introduction.
Active image areas are currently very similar to those
of intraoral lm and the sensors have become much
thinner. They are still considerably thicker than lm,
which, together with their rigidity and cable attach-
ment, can make sensor placement more challenging
and patient discomfort more likely. One of its key
features, however, is the immediate availability of
the image. Not only does this provide immediate
feedback to the radiographer, it also results in a con-
siderable time savings.
Digital imaging systems based on photostimulable
phosphor (PSP), also called storage phosphor (SP),
offer an indirect approach to digital image acquisi-
tion. The PSP plates are dimensionally comparable to
lm and handle quite similarly. Exposed plates are
scanned in an external laser scanner, which gener-
ates the digital image data for storage and display on
the computer. The plates are then erased and can be
reused. Advocates of this technology point out the
advantages of digital imaging and the lm-like hand-
ling properties of the PSP plates. This benet comes
at the cost of increased time and effort for scanning
the plates and preparing them for reuse.
Contrary to some manufacturers' claims, digital
images do not improve the diagnostic efcacy of
dental radiography. Current evidence suggests that
there is neither an improvement nor a reduction in
diagnostic efcacy. This reects the use of lm
according to manufacturer's recommendations for
storage and processing. If current clinical procedures
for lm processing are substandard, digital imaging
may indeed improve image quality and diagnostic
performance, albeit for the wrong reason. Film re-
mains technically equivalent, if not superior, to digi-
tal receptors. The fact that differences in technical
Fig. 2. Examples of current size-2 intraoral image recep-
tors. (a) Kodak Insight F-speed lm. (b) Wrapped Gendex
photostimulable phosphor plate. (c) Gendex Gs-X CCD
sensor. (d) Schick CMOS sensor.
38
Mol
performance among various image receptors have
very little effect on diagnostic efcacy supports the
notion that the receptor is not the limiting factor in
the diagnostic imaging chain. Digital imaging in its
current form does not fundamentally change the tra-
ditional image formation model. The limitations of
transmission radiography as discussed in previous
sections still exist. Therefore, a decision to implement
digital imaging should mainly be based on functional
grounds as well as on individual preference.
It might be argued that digital measurement tools
are better than analog instruments. For example, bone
height measurements are easily made with a digital
length measurement tool. Since the starting point and
the end point are both pixels, the limit of resolution
of the measurement can theoretically be expressed in
terms of pixel size. However, the accuracy of measure-
ment is not as high as the resolution suggests. The
accuracy remains limited by the image formation
model andthe interpretive skills of the operator. There
is no evidence suggesting that digital measurements
are more accurate than analog measurements.
Digital imaging remains a relatively new technol-
ogy in dentistry. Most clinicians lack the basic train-
ing and knowledge to judge the quality and reliability
of digital imaging systems. Moreover, a wide variety
of manufacturers offer digital imaging systems and
their long-term commitment to the quality and ser-
vice of their digital products remains to be evaluated.
At the same time, technologic developments con-
tinue to be rapid and seem to follow those in the
electronics and computer industry. This, along with
the relatively high initial costs, provides legitimate
reasons to reconsider a decision to implement digital
imaging at this time.
The true added value of digital imaging in terms of
diagnostic outcomes needs to be sought in new
image acquisition schemes and image processing
applications. Such applications can vary in complex-
ity between simply altering the display format of
serial images to increase the reliability of linear
measurements (3, 23), and developing automatic
classication schemes (25). Most digital image pro-
cessing applications in dentistry have been developed
for digital subtraction radiography and other specia-
lized techniques.
Specialized techniques
The introduction of digital imaging in dentistry gen-
erated many new research initiatives aimed at
unlocking the diagnostic potential of radiography
through image processing. Some of these initiatives
have resulted in meaningful applications that have
been shown to increase the diagnostic utility. While
these applications are not yet easily implemented in
a clinical setting, they begin to address some of the
diagnostic imaging needs in periodontology in terms
of early detection, quantitative assessment and
three-dimensional (3-D) imaging.
Digital subtraction radiography (DSR)
The application of subtraction imaging predates the
invention of computers and was rst demonstrated
by Ziedses des Plantes in 1935, who used a photo-
graphic technique. The application of subtraction
radiography in dentistry was facilitated by the deve-
lopment of the microcomputer, allowing conventional
radiographs to be digitized and subtracted (20). The
concept of DSR is relatively simple. When two images
of the same object are registered and the image inten-
sities of corresponding pixels are subtracted, a uni-
formdifference image will be produced. If a change in
the follow-up image has occurred, this change will
show up as a brighter area when the change repre-
sents gain and as a darker area when the change
represents loss. The strength of DSR is that it cancels
out the complex anatomic background against which
this change occurs. As a result, the conspicuousness
Fig. 3. Application of digital subtraction radiography for
detection and quantification of periodontal bone healing
in a clinical research setting. (a) Baseline image, (b) 1-year
follow-up image, and (c) subtraction image showing bone
gain (arrow).
39
of the change is greatly increased (Fig. 3). Numerous
studies have shown the high diagnostic utility of DSR
relative to conventional radiography, as reviewed
previously (54, 55, 66).
In order for DSR to be diagnostically useful it is
imperative that the baseline projection geometry and
image density be reproduced. Standardization of
these image acquisition parameters is usually con-
sidered a serious limitation, but it is precisely this
requirement that makes the comparison of serial
radiographs more meaningful.
The projection geometry is dened by the position
and orientation of the X-ray source, the patient, and
the detector relative to each other. If the projection
geometry used for the follow-up image is different
from the projection geometry used for the baseline
image, the subtraction image will show these differ-
ences. They can be difcult to distinguish from
actual change within the patient or may hide actual
change. Perfect reproduction of the projection geo-
metry would be ideal, but is virtually impossible to
achieve. However, not all changes in projection geo-
metry are equally detrimental. Most changes can be
reversed through image processing. There are only
two changes in the projection geometry that cannot
be reversed: differences in horizontal beam angula-
tion and differences in vertical beam angulation.
Several image-processing techniques have been
developed to adjust for reversible projection errors
prior to subtraction (6, 12, 13, 60, 67). A few investi-
gators have used standardization software to mini-
mize the effects of irreversible projection errors (8,
14, 36, 37, 46, 70). Results suggest that a limited
amount of change in beam angulation, approxi-
mately 68, is permissible (42). Although the actual
tolerance of these types of differences in the projec-
tion geometry depends on how much osseous
change needs to be detected, the level of standardi-
zation required for successful subtraction is attain-
able under clinical conditions. While exact
reproduction of the projection geometry is not
strictly necessary, some form of mechanical standar-
dization will reduce the reliance on image processing
and generally produces better results.
Differences in image contrast and density between
the baseline image and the follow-up image can
hamper the detection of actual change and make
quantitative measurements unreliable. Fluctuations
in the exposure factors and changes in projection
geometry account for most of the discrepancies.
When lm is used as the image receptor, lm proces-
sing variations are also a main concern. There are a
number of different approaches to dealing with con-
trast and density differences. One method uses a
reference device, such as an aluminum wedge, which
allows the image intensities of the follow-up image to
be adjusted according to the image intensities of the
baseline image (35, 42, 53). Another method com-
pares the image intensity distributions of both
images and adjusts the follow-up image accordingly
(58). The computer-aided densitometric image ana-
lysis method (CADIA) designates image intensities as
true change only after comparison with the image
intensities in an unchanged reference area (5, 15). All
of these methods are quite successful in accounting
for contrast and density differences under reasonably
controlled clinical conditions.
Subtraction images allow detection of mineral
changes of as little as 5%. In addition to early detec-
tion, a number of quantitative measurements can be
made, such as linear, area, perimeter and density
measurements. All can be helpful in quantifying
bone mineral changes within a specic site, provid-
ing an opportunity for early assessment of the course
of the disease. Various approaches for detection and
quantication have been reported in the literature.
While some rely upon visual interpretation and man-
ual measurement, others use some form of auto-
mated image analysis. Image analysis algorithms
designed to automatically distinguish between true
osseous changes and background noise are funda-
mentally dependent on threshold selections and on
assumptions regarding the characteristics of radio-
graphic signs of osseous change and the character-
istics of the background noise. Trade-offs between
the level of automation and the generalizibility of
image processing applications are not uncommon.
Fully automated image analysis techniques can only
attain high accuracy if the radiographic signs of oss-
eous change can be uniquely characterized. This
characterization is complicated by the unpredictable
behavior of noise introduced by suboptimal image
standardization. Therefore, better image standardi-
zation usually results in more accurate estimates of
actual osseous change.
It is often stated that the time and effort involved in
producing subtraction images with a high diagnostic
quality is prohibitive in clinical practice. True as this
may be, the expectation that an increase in the qual-
ity and quantity of diagnostic information should
come at no extra cost or effort is unrealistic. At one
level or another, a price has to be paid for increased
diagnostic utility, either by the clinician or staff in
terms of time and effort, or by the patient in terms of
absorbed dose, time and money, to name a few. In
the case of DSR, the detection and quantication of
40
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actual changes within the patient requires that other
factors impacting such measures need to be con-
trolled. There is no free lunch.
Current DSR research focuses on developing image
processing techniques to facilitate image standardi-
zation and rening image analysis techniques to
detect and quantify osseous changes. The clinical
application of DSR is not yet a routine procedure.
The balance between the ability to generate standar-
dized radiographs, advances in technology and the
need for objective and quantitative information will
determine its future clinical use.
Tuned aperture computed tomography
(TACT
1
)
The need for assessment of dentoalveolar tissues in
three dimensions has long been recognized. The
motivation behind the development of TACT
1
was
to be able to achieve this with existing dental equip-
ment and without the high cost and dose associated
with computed tomography.
TACT
1
is built on the basic principles of tomo-
synthesis: by shifting and combining a set of basis
projections, arbitrary slices through the object can be
brought into focus (19, 59). The basis projections are
conventional transmission radiographs. Each radio-
graph is taken from a different angle relative to the
object and the receptor. Only a limited number of
basis projections are required to generate a stack of
tomographic slices. Each slice is a two-dimensional
representation of the object at a different location in
the third dimension (69).
The diagnostic efcacy of TACT
1
for imaging the
alveolar bone has been tested in a number of studies
(Fig. 4). TACT
1
was shown to improve the ability of
observers to detect osseous defects around implants
(30, 68). The feasibility of using TACT
1
as an alter-
native for preoperative imaging of the implant site
has also been investigated (38). Results of studies
testing TACT
1
and TACT
1
subtraction for the detec-
tion and localization of osseous changes in the cres-
tal bone are encouraging (7, 44, 52).
Ongoing research seeks to further improve this
technology and determine optimal parameters for
various clinical applications. The development of
specialized equipment to acquire basis images in a
rapid and convenient manner may be expected in the
future.
Computed tomography (CT)
The pursuit of three-dimensional (3-D) information
has led to exploring the value of CT for the assess-
ment of alveolar bone height. CT machines use a
rotating fan beam to image one thin slice of the
patient at the time, generally in an axial orientation.
Modern CT machines use a continuous table motion
during image acquisition, resulting in a spiral or
helical image formation pattern. Once the image
volume has been generated, image slices can be
reconstructed in various orientations through a pro-
cess called multi-planar reformatting (MPR). In addi-
tion, most software applications are capable of
rendering 3-D surfaces and volumes, allowing the
clinician to study the various tissues in a more intui-
tive manner. A number of companies have developed
reformatting software specically for dental applica-
tions. CT with dental MPR is particularly useful for
planning complex implant and surgical cases.
While CT provides exquisite 3-D views, its ability to
show very small details remains limited, usually not
more than 12 mm. Currently, thin multi-slice spiral
CT is capable of rendering uniform sub-millimeter
resolution in all three dimensions (isotropic pixels).
Although the level of image detail remains consider-
ably lower than with conventional intraoral imaging,
these advancements in CT technology satisfy almost
all periodontal imaging needs from a pure technical
and possibly diagnostic perspective. Studies have
shown that CT assessment of alveolar bone height
and intrabony pockets is reasonably accurate and
precise (17, 18, 45, 50).
However, despite its attractive features and the
optimism of the investigators, the application of
CT imaging for periodontal diagnosis appears to have
an unfavorable costbenet ratio. Studies have
shown that the effective dose of CT for imaging the
mandible and the maxilla is much higher than with
Fig. 4. Application of Tuned Aperture Computed Tomo-
graphy (TACT). (a) Image slice through the buccolingual
center of the alveolar bone. (b) Image slice through the
buccal aspect of the alveolar bone. The presence of a bone
defect in the buccal aspect of the interproximal alveolar
bone is evident (arrow).
41
conventional radiography (10, 61). While develop-
ments in CT scanner technology continue to reduce
patient dose, the acquisition of high-resolution CT
images remains a high-dose procedure. Other draw-
backs include the limited availability of medical CT
imaging to dental health care providers and the cost
of obtaining and reformatting a scan for this purpose,
which is often prohibitive (61).
The development of a new generation of compact
CT scanners specically designed for imaging the
head and neck region is likely to reduce the use of
medical CT scanners for dental purposes. These new
scanners offer many advantages over conventional
CT scanners and are likely to become a major asset
of the oral and maxillofacial radiologist (see section
on New Frontiers below).
Implant site imaging
The placement of a dental implant requires thorough
planning to optimize its success and to minimize
morbidity. Once the optimal location and orientation
of the implant has been established based on restora-
tive criteria, the practitioner needs to determine
whether the recipient site can accommodate and
support the implant in terms of length, width and
angulation. One of the key requirements before pre-
paring the site is to know with a high level of cer-
tainty how much bone can be removed without
violating critical anatomic structures. How much
and what type of radiographic information needs
to be gathered depends on site-specic factors and
on the amount of information that can be gained
from clinical examination.
It is generally accepted that some form of preo-
perative imaging is necessary. The choice of the ima-
ging modality depends on many factors and can
range from a single intraoral radiograph to a com-
plete 3-D workup using computed tomography. A
number of excellent review papers have been pub-
lished on imaging of the dental implant site (34, 74).
The American Academy of Oral and Maxillofacial
Radiology (AAOMR) published a position paper on
this topic in 2000 (64). In that publication, basic
radiographic principles are discussed as well as avail-
able modalities, dosimetry, costs and selection cri-
teria. The AAOMR recommends that some form of
cross-sectional imaging be used for implant cases
and that conventional cross-sectional tomography
is the method of choice for most patients (Fig. 5).
This recommendation is qualied by acknowledging
that there is no published evidence to support that
the use of cross-sectional imaging changes the over-
all success rate for dental implants. It is also recog-
nized that the experience of the practitioner can be a
determining factor in the selection of the imaging
modality.
The radiographic imaging modalities available for
implant site assessment are similar to those for per-
iodontal diagnosis and have been discussed else-
where in this paper. The invasive nature of implant
surgery makes it even more important that the
strengths and limitations of the selected imaging
modality are fully understood. Treatment decisions
and implant selection are essentially based on quan-
titative assessments and small errors may have ser-
ious consequences. The inherent accuracy and
precision of conventional radiographic modalities
such as intraoral and panoramic radiography are
limited. They also do not provide cross-sectional
information. When the reliance on radiographic ima-
ging becomes critical, it needs to be recognized that
an increase in diagnostic efcacy will come at some
cost. A choice needs to be made either to improve the
reliability of conventional modalities or to switch to a
more costly and dose-intense modality with higher
inherent reliability.
The fabrication of a reference marker to account for
horizontal and vertical magnication is an example
of improving the reliability of panoramic radiography.
When conventional or computed tomography is the
method of choice, the fabricationof a stent withradio-
paque markers provides a simple but effective means
Fig. 5. Example of parasagittal (a) and paracoronal (b)
cross-sectional images of the right mandibular body taken
with the CommCAT tomographic unit (Imaging Sciences
International, Hateld, PA). The lead foil is placed in a
stent and identies the region of interest. The vertical
center lines of the images are horizontally correlated.
42
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to identify the location of the recipient sites. The
most reliable method of identifying the recipient site
and correlating the image with the surgical site is the
use of guiding tubes as radiographic markers.
The use of CT has become more prevalent with the
advent of MPR software specically for dental
implant treatment planning (Fig. 6). The latest gen-
eration of CT units is capable of producing thin
image slices with isotropic pixels. This means that
high quality images can be generated in any two-
dimensional (2-D) plane through the patient. Despite
its accuracy and versatility, the use of CT should be
limited to complex cases for which the benets of CT
images outweigh the higher dose and the higher fee.
A new type of CT is being developed that combines
the 3-D versatility of conventional CT with a lower
patient dose and lower cost. This type of CT is based
on cone-beam technology and holds great promise
as an imaging modality for preoperative implant site
assessment (see section on New Frontiers below).
Postoperative imaging is usually performed for
evaluation of the peri-implant bone. A radiograph
taken immediately after placement of the implant
can serve as a baseline for longitudinal assessments.
While some bone loss is expected at the crestal bone
around the implant, a follow-up radiograph can
assist the practitioner in determining whether this
loss is within reasonable limits. Whether or not the
implant shows osseointegration can not be deter-
mined radiographically, although an increase in
bone density near the implant-bone interface is
usually a favorable sign (Fig. 7). The diagnostic ef-
cacy of postoperative radiographs will be higher if
high quality radiographs are obtained in a standar-
dized manner. The application of digital subtraction
radiography can be particularly useful to detect oss-
eous changes around the implant at an early stage.
As stated in the AAOMR position paper (64), there
is no evidence suggesting that the success rate of
implants is affected by the imaging modality
employed during the treatment planning stage. How-
ever, it is important to consider how success is being
dened. Immobile retention of the implant after
5 years is a rather narrow denition of success. The
end point of any implant procedure should be the
functional and esthetic value of the nal prosthesis.
Other factors, such as length of surgery, intraopera-
tive complications and surgical morbidity, also need
to be included in dening success. These factors are
not always easily quantied and the incidence of their
occurrence is most likely underreported. However,
they can be important factors in the practitioner's
and the patient's perception of success. Until scien-
Fig. 6. CT study for dental implant
treatment planning using SimPlant
software (Materialise - Columbia
Scientic, Inc.). This example shows
correlated cross-sectional, axial and
panoramic images for assessment of
the maxillary right premolarmolar
region.
Fig. 7. (a) Original baseline image taken immediately
post-surgery. (b) Follow-up image taken after placement
of the final restoration. The density and the projection
geometry have been standardized. (c) Subtraction image
showing bone loss at the alveolar crest and bone gain at
the bone-implant interface.
43
tic studies can elucidate the impact of the preopera-
tive imaging modality on implant success in this
broader context, each individual practitioner needs
to rely upon best available evidence and sound clin-
ical judgment. While personal expertise and experi-
ence of the practitioner are good predictors of
success, an overly self-condent approach may
result in underutilization of preoperative imaging
and put the patient at unnecessary risk.
Newfrontiers
The future of oral and maxillofacial radiology is being
shaped by digital technology. Both solid-state and
photostimulable phosphor detectors will continue
to evolve. The acceptance of solid-state detectors will
increase when they become even thinner than the
current generation of detectors and when wireless
image transfer is feasible. Improvements in PSP
technology need to be sought in simplifying plate
handling by automating plate scanning, erasing, and
repackaging.
No matter how revolutionary the developments
and improvements in detector technology, the repla-
cement of lm by digital detectors does not represent
a fundamentally different approach to dental diag-
nostic imaging. The impact of digital receptors on the
clinical practice of periodontology will therefore not
be signicant if the innovation ends there.
Fortunately, the ability to capture dental radio-
graphic images in a digital format has inspired scien-
tists to take a fresh look at existing diagnostic needs
and to study novel approaches to address these needs.
One approach is the development of image-proces-
sing techniques to characterize radiographic features
in ways previously not feasible. Examples include
characterization of the structure and complexity of
the radiographic trabecular pattern and computer-
aideddetectionandquanticationof osseous changes.
Another approach is to take advantage of the abil-
ity to digitally combine multiple conventional images
in order to synthesize new ones. When two images
with identical projection angles are combined, a sub-
traction or addition image can be synthesized. When
more than two images with different projection
angles are combined, a set of tomosynthetic or
TACT
1
views can be synthesized providing some
3-D information. As the disparity of projection angles
increases, a more complete 3-D model can be gen-
erated. The maximum disparity of 3608 is used by CT
and allows a complete 3-D image representation of
the tissues.
The prospect of specialized techniques like DSR
and TACT
1
being developed for clinical applications
is quite good. Advanced image registration algo-
rithms are being developed to facilitate automated
digital subtraction in a clinical environment. This
does not diminish the requirement to acquire high
quality standardized images, but this requirement is
not much different when serial radiographs are used
for longitudinal assessment in a conventional man-
ner. The clinical application of TACT
1
may be
expected when user-friendly image acquisition and
image reconstruction modules become commer-
cially available.
Despite the advancements in medical CT technol-
ogy, the use of conventional CT in dentistry will
remain restricted to a limited number of applica-
tions. Alternative approaches to 3-D image acquisi-
tion are being investigated using cone-beam
technology rather than fan-beam technology.
Cone-beam CT
The ability to synthesize 3-D views from multiple
projections is not limited to fan-beam geometry as
used in medical CT units. Cone-beam geometry, like
in conventional dental X-ray units, can also be used
to generate 3-D CT images. With cone-beam geome-
try a patient volume can be scanned in a single rota-
tion. Together with fast area image receptors, such as
image intensiers or at panel detectors, a much
lower patient dose is achieved than with conven-
tional CT for a similar size volume. The simplied
design of cone-beam CT units also allows for a con-
siderable cost savings relative to medical CT units.
One of the main drawbacks of cone-beam CT (CBCT)
is the increased effect of scatter radiation on image
quality. Scatter radiation reduces contrast and limits
the imaging of soft tissues. Therefore, CBCT is mainly
indicated for imaging hard tissues.
Already, CBCT units have been developed speci-
cally for oral and maxillofacial imaging (2, 29, 31, 32,
43). For example, the NewTom QR-DVT-9000 (QR-
NIM s.r.l., Verona, Italy) is a large area CBCT unit and
is employed by several institutions and practices
throughout Europe and the United States. Initial
dose estimates for this unit indicate that a full scan
of the mandible and the maxilla generates an effec-
tive dose approximately 36 times that of a single
panoramic radiograph (40). Main applications of this
unit include implant site evaluation, orthodontics,
oral surgery and temporomandibular joint imaging
(Fig. 8). Investigations regarding the usefulness of
CBCT for periodontal applications are in progress.
44
Mol
Local CT
Local CT (LCT) is a form of CBCT. LCT distinguishes
itself by using a small-eld high-resolution detector
to generate a limited high-resolution 3-D volume.
The eld or volume size varies, but is generally com-
parable to the dimensions of conventional intraoral
radiographs. LCT generates exquisite image detail in
three dimensions while retaining the advantages of
reduced patient dose and reduced cost (Fig. 9). This
makes LCT particularly suited for dental applications
(65). This technology is still relatively new and com-
mercial availability is limited. The characteristics of
LCT make it a very promising modality for imaging
the alveolar bone, both for the assessment of bone
destruction and for implant site assessment.
Optical coherence tomography
Optical coherence tomography (OCT) generates
cross-sectional images of biological tissues using a
near-infrared light source. The light is able to pene-
trate into the tissue without biologically harmful
effects. Differences in the reection of the light are
used to generate a signal that corresponds to the
morphology and composition of the underlying tis-
sues. A prototype OCT system was developed and
tested in vitro as well as in vivo (47, 48). The feasi-
bility of its clinical use was demonstrated by captur-
ing high-resolution images of oral structures,
including soft tissue and hard tissue boundaries of
the periodontium. While it is yet too early to judge
the potential success of OCT as a routine clinical tool,
the initial results warrant keeping an eye on further
developments of this technology.
Where do we go fromhere?
The digital era is only in its infancy and there is
already a lot of new imaging technology on the mar-
ket. Some of this technology will address part of the
diagnostic dilemmas of the periodontist. Some of it
will not. Science and common sense are required to
distinguish between hype and truth and between pro-
mise and performance. Howmuch information do we
need to best serve the interests of our patients? Some
argue that 3-D information is better than 2-D infor-
mation simply because the patient is 3-Dand alveolar
bone destructionoccurs in 3-D. They assume that 3-D
images will provide critical information for diagnosis
and treatment planning, thus improving patient out-
come. Unfortunately, thisisnot alwaysthecase. Even if
the diagnostic efcacy increases, this does not auto-
matically translate into better treatment or improved
patient outcomes (16). Gathering more information
simply because we have the capability to do so does
not make sense. Periodontists need to understand
the strengths and weaknesses of diagnostic imaging
and weigh the costs and benets of the test before
prescribing it. This is the best assurance that new
technology will be utilized at its greatest potential.
Improvements in the efcacy of diagnostic imaging
do not all have to be based on high-tech solutions.
Fig. 9. Examples of image slices createdwith LocalCTusing
an intraoral sensor. Two horizontal cross-sections (a and
b) and two vertical cross-sections (c and d). Courtesy A.N.
van Daatselaar et al., Amsterdam, The Netherlands.
Fig. 8. Examples of NewTom cone-beam CT images. Axial
images through the maxilla and the mandible with para-
sagittal and paracoronal cross-sections.
45
Simple, low-cost changes in current radiographic
procedures can have a major impact on the value
of radiography. Prescribing the appropriate type
and number of radiographs is critical for optimizing
the impact of radiographs on treatment and patient
outcomes. The use of standardized procedures for
selecting exposure parameters, applying projection
geometry principles and controlling lm processing
appear self-evident, but in reality rarely are. Most
clinicians are procient in recognizing radiographic
features of anatomy and pathology, but the informa-
tion embedded in the relative location of image fea-
tures under different projection conditions is often
underutilized (39, 56). Reducing ambient light and
blocking extraneous light from the view box are cri-
tical for the detection of small differences in image
density. The fact that this is not commonly done
implies that bone lesions presenting as subtle radi-
olucencies are going undetected. The prevalence of
technical errors in general and periodontal practices
has led to a call for improved education in radiology
and better quality-assurance procedures (4).
With the exception of eliminating chemical pro-
cessing errors, digital imaging will not contribute to
solving any of these issues. The fact that new digital
receptors do not improve diagnostic performance
implicitly conrms that other factors are currently
limiting the diagnostic potential of radiography.
The development of digital imaging has helped con-
siderably to identify and study these factors, but the
results of these studies will only slowly translate into
improved clinical applications.
The adoption of digital imaging as the radiographic
modality of the future, when based on sound scien-
tic evidence, has the potential to transform the way
we visualize the periodontal tissues. Digital image
standardization, subtraction radiography, 3-D ima-
ging and quantitative image analysis are already a
reality. There is little doubt that periodontists of
the future will be using more advanced imaging
modalities, either directly as a chairside procedure,
or indirectly through the services of an oral and max-
illofacial radiologist.
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48
Mol
How effective is surgical therapy
compared with nonsurgical
debridement?
LI SA J. A. HEI TZ-MAYFI ELD
Chronic periodontitis is dened as an inammatory
disease of the supporting tissues of teeth caused by
groups of specic microorganisms, resulting in pro-
gressive destruction of the periodontal ligament and
alveolar bone with either pocket formation or
recession, or both. The aim of effective treatment of
periodontal diseases is to arrest the inammatory
disease process by removal of the subgingival biolm
and establish a local environment and microora
compatible with periodontal health. Reduction of
probing pocket depths, maintenance or improvement
of clinical attachment levels, and reduction in
bleeding on probing are the most common outcome
measures used to determine whether treatment is
successful. The treatment offered to the periodontal
patient by the clinician may be nonsurgical or sur-
gical mechanical debridement.
Aim
The aim of this chapter is to address the important
clinical issue of effectiveness of surgical therapy
compared with nonsurgical therapy by evaluating
published systematic reviews, which are considered
the highest level of evidence in the literature (Center
for Evidence-Based Medicine: http://www.cebm.net/).
A systematic review follows a well-dened protocol
whereby a clearly formulated question is addressed
using systematic and specic methods to identify,
select, critically appraise, and summarize relevant
research. In a systematic review, unlike a traditional
or narrative review, the aim of the review process is to
minimize bias and be transparent, allowing the rea-
der to assess the quality of the review. A systematic
review may include a meta-analysis evaluation where
the results from several studies are statistically
combined to provide increased power for the iden-
tication and quantication of differences between
therapies. However, this is only a small part of the
systematic review (22).
The three papers reviewed in this chapter Heitz-
Mayeld et al. (8), Antczak-Bouckoms et al. (2) and
Hung & Douglass (10) have reviewed all combined
randomized controlled trials comparing scaling and
root planing with open ap debridement in meta-
analysis evaluations. While only one paper (8) was
titled a systematic review by the authors, all papers
have used a systematic approach and are therefore
evaluated as systematic reviews in this chapter.
The aim of this chapter was to present the results of
the three papers, and to evaluate the methods and
quality of the systematic reviews in order to facilitate
clinical decision-making in the choice of surgical vs.
nonsurgical therapy for the treatment of chronic
periodontitis.
A systematic review of the effect of
surgical debridement vs.
nonsurgical debridement for the
treatment of chronic periodontitis
(Heitz-Mayeld et al. (8))
Focused question
The focused question addressed in this systematic
review was clearly stated: What is the effect of sur-
gical debridement vs. nonsurgical debridement in
terms of changes in clinical attachment level, probing
pocket depth and bleeding on probing for patients
with chronic periodontitis?
Literature search
A literature search was conducted of two databases
(MEDLINE and the Cochrane Oral Health Group
72
PERIODONTOLOGY 2000
specialist trials register) from 1965 to April 2000 in
addition to searching reference lists of original and
review articles. The search strategy including MeSH
terms and text words was published. Hand searching
of journals for missed trials was not carried out. Only
English language articles were considered and
unpublished data were not sought.
Study selection
The specic inclusion criteria were studies of at least
12 months duration comparing surgical (open ap
debridement) with nonsurgical (scaling and root
planing) treatment of patients with a clinical diag-
nosis consistent with the current classication of
chronic periodontitis. A patient-based analysis was
also required for inclusion.
Validity assessment
Two reviewers independently screened 589 titles and
abstracts of the search results for possible relevance
against the stated inclusion criteria.
Two reviewers, focusing on specic criteria known
to affect study outcomes, assessed the methodologi-
cal quality of the papers independently. Two
reviewers also performed data abstraction inde-
pendently for further analyses.
Outcome variables
Although the intention was to evaluate a number of
outcome variables, there were only sufcient data to
evaluate clinical attachment level change (DCAL) and
probing pocket depth change (DPPD).
Quantitative data analysis
Data from studies were combined in meta-analyses
where possible in order to evaluate the treatment
effect of surgical vs. nonsurgical therapy. The results
of the meta-analyses were expressed as weighted
mean differences and 95% condence intervals (CI).
The heterogeneity of studies was assessed to deter-
mine whether the studies were similar enough for the
results of meta-analyses to be valid. A random effects
model was used when studies showed statistically
signicant heterogeneity, otherwise a xed effects
model was used where appropriate.
Variance imputation methods were used to esti-
mate appropriate variance in some studies, where the
appropriate standard deviation of the differences was
not included in the study reports (6).
Due to the limited number of studies in the ana-
lyses, formal testing for publication bias was not
possible.
Study characteristics
The search provided 589 potentially relevant publi-
cations, 14 of which were retrieved for detailed eval-
uation. Of these, eight studies did not meet the
inclusion criteria, leaving six randomized controlled
trials for evaluation (11, 14, 18, 20, 23, 26), in ve of
which the data presentation was appropriate for
meta-analyses. In four of the randomized controlled
trials, two or more publications per study population
were available for data abstraction (12, 16, 19, 20, 23
25). Reasons for the exclusion of studies and the
characteristics of the included studies were discussed.
Methodologic quality of included studies
The review reported on the quality of the individual
trials and found a lack of reporting on the method
used for generation of the random sequence for test
and control procedures. Only two studies described
the randomization method adequately (12, 14).
Regarding allocation concealment, none of the
studies indicated that the random sequence was
hidden from those recruiting subjects for the study.
A number of studies were based on a decreasing
number of subjects compared with baseline (14, 20,
23, 26). Data from these subjects were included until
the time of exit from the study.
Study design
The six randomized controlled trials were all of split
mouth design with a nonsurgical and a surgical
procedure performed within each patient. There was,
however, considerable variation in the study design
and data presentation among the studies.
Initial therapy
Initial therapy varied among studies with regard to
presurgical scaling. In two studies initial therapy
consisted of oral hygiene instruction alone (18, 20).
Three studies incorporated scaling and root planing
during initial therapy with repeated scaling and root
planing in both control and test groups (12, 14, 26). In
one study the control group received a single session
of scaling and root planing, whereas the test group
received scaling and root planing at initial therapy
and again during the ap procedure (23).
73
Surgical therapy vs. nonsurgical debridement
Professionally supervised maintenance
care
All included studies incorporated regular supervised
maintenance care in their protocols. This ranged
from professional prophylaxis at 2-week intervals for
a period of up to 1 year following treatment proce-
dures (11, 18, 19), to supra- and subgingival plaque
removal at 34-month intervals (14, 23, 26).
Data presentation
Some studies did not report the standard deviation or
standard error of results. Furthermore, data for the
meta-analyses were frequently obtained by estima-
tion from gures in the papers. Five trials presented
data in a form suitable for possible inclusion in meta-
analyses (14, 18, 20, 23, 26).
Data presentation also varied in relation to initial
probing depth severity. One study presented data for
outcome variables for all initial probing depths
combined (20), while four studies presented data
using the initial probing depth categories 13 mm,
46 mm, and >6 mm (18, 20, 23, 26). An alternative
initial probing depth classication of 14 mm, 5
6 mm, and > 6 mm was used by Kaldahl et al. (14).
Thus for the initial probing depth categories 13 mm
and 46 mm there were four studies available for
meta-analysis evaluation, and for deep pockets
(> 6 mm), ve studies were available.
Change in clinical attachment level and
probing pocket depth
Differences in clinical outcomes (change in clinical
attachment level and probing pocket depth) between
surgical and nonsurgical therapies were combined
where appropriate in meta-analyses and presented
for respective initial probing depth categories. The
results were clearly displayed in a meta-analysis
summary table (Table 1) and forest plots (Fig. 1af).
Summary at 12 months
The meta-analyses for difference in clinical attach-
ment level change and probing pocket depth change
in shallow pockets (13 mm) showed statistically
Table 1. Summary of meta-analyses for clinical outcomes Heitz-Mayeld et al. (8)
Outcome
12 months
Initial
PPD
category
No. of
studies
Studies
included
(ref. nos.)
WMD
(mm)
95% CI P-value
for WMD
Heterogeneity
Method P-value
CAL gain 13 mm 4 18, 20, 23, 26 ) 0.513 [) 0.737, ) 0.290] 0.000 random 0.005
PPD reduction 13 mm 2 23, 26 0.101 [) 0.036, 0.239] 0.147 random 0.008
CAL gain 46 mm 4 18, 20, 23, 26 ) 0.373 [) 0.485, ) 0.261] 0.000 xed 0.331
PPD reduction 46 mm 2 23, 26 0.351 [0.234,0.467] 0.000 xed 0.108
CAL gain > 6 mm 5 14, 18, 20, 23, 26 0.191 [0.035,0.347] 0.017 xed 0.897
PPD reduction > 6 mm 3 14, 23, 26 0.584 [0.383,0.785] 0.000 xed 0.687
CAL, clinical attachment level. CI, condence interval. PPD, probing pocket depth. WMD, weighted mean differences.
Fig. 1. (a) Forest plot of studies investigating the difference
in probing depth change between open ap debridement
(OFD) andscalingandroot planing(SRP) at sites withinitial
PPD > 6 mm. The rectangles represent the individual re-
sults for each study; in this case the weighted mean differ-
ence (WMD) in mm between open ap debridement vs.
scaling and root planing. The size of the rectangle repre-
sents the weighting given to the study in the meta-analysis
and is directly related to the precision of the study. The
horizontal line extending from each square represents
the 95%condence interval (95% CI). The diamond at the
bottom is the pooled value from the meta-analysis. The
centre of the diamond is the summary value and the hori-
zontal points represent the 95% CI. Whenthe diamondis to
the left of the zero line the outcome is in favour of scaling
and root planing. When the diamond is to the right of the
zero line the outcome is in favour of open ap surgery. This
analysis shows a 0.58 mm greater pocket depth reduction
for surgery than scaling and root planing (95% CI: 0.38,
0.79, P < 0.001). (b) Difference in the PPD change between
OFDandSRPat sites withInitial PPD: 4- 6mm. Fixedeffects
forest plot. (c) Difference in the PPD change between OFD
and SRP at sites with initial PPD 1 3 mm. Random effects
forest plot. (d) Difference in the CAL change between OFD
and SRP at sites with initial PPD: > 6 mm. Fixed effects
forest plot. (e) Difference in the CAL change between OFD
and SRP at sites with initial PPD 4 6 mm. Fixed effects
forest plot. (f) Difference in the CAL change between OFD
and SRP at sites with initial PPD 1 3 mm. Random effects
forest plot.
74
Heitz-Mayeld
signicant heterogeneity. However, graphically dis-
played in a random effects forest plot (Fig. 1f) the
results of the four individual studies all indicated a
greater attachment loss following surgery compared
to scaling and root planing (weighted mean differ-
ences )0.51 mm; 95% CI [) 0.74, ) 0.29]; hetero-
geneity Q 12.863, 3 df, P 0.005). This consistency
indicates that in shallow pockets, scaling and root
planing is less harmful to clinical attachment; how-
ever, the size of the difference compared with surgery
is unclear. The random effects forest plot (Fig. 1c)
showed no signicant difference in probing
pocket depth reduction between procedures for both
studies.
diff
2 1 0 1 2
Combined
Pihlstrom 1983
Ramfjord 1987
Kaldahl 1996
Favours SRP

Favours OFD
Weighted mean difference (mm)
diff
2 1 0 1 2
Combined
Pihlstrom 1983
Ramfjord 1987
diff
2 1 0 1 2
Combined
Ramfjord 1987
Pihlstrom 1983
diff
2 1 0 1 2
Combined
Lindhe 1982
Lindhe &
Nyman 1985
Kaldahl 1996
Ramfjord 1987
Pihlstrom 1983
diff
2 1 0 1 2
Combined
Lindhe &
Nyman 1985
Ramfjord 1987
Pihlstrom 1983
Lindhe 1982
diff
2 1 0 1 2
Combined
Ramfjord 1987
Lindhe &
Nyman 1985
Pihlstrom 1983
Lindhe 1982
Favours SRP

Favours SRP

Favours SRP

Favours SRP

Favours SRP

(a) (b)
(c) (d)
(e) (f)
Favours OFD
Favours OFD
Favours OFD Favours OFD
Favours OFD
75
In 46 mm pockets, scaling and root planing
resulted in 0.4 mm more clinical attachment gain
(weighted mean differences )0.37 mm; 95% CI
[) 0.49, ) 0.26]) and 0.4 mm less probing depth
reduction (weighted mean differences 0.35 mm;
95% CI [0.23,0.47]) than surgical therapy.
In deep pockets (> 6 mm), surgical therapy resul-
ted in 0.6 mm more probing pocket depth reduc-
tion (weighted mean differences 0.58 mm; 95% CI
[0.38,0.79]) and 0.2 mm more clinical attachment
gain (weighted mean differences 0.19 mm; 95%CI
[0.04,0.35]) than nonsurgical therapy.
Incidence of bleeding on probing
The incidence of bleeding on probing was not
reported consistently and a meta-analysis was not
possible for this outcome variable. A similar reduc-
tion in the percentage of sites with presence of
bleeding on probing following nonsurgical and sur-
gical treatment modalities was reported in the two
studies (16, 18) that addressed this issue.
Overall, the studies showed a substantial reduction
in the percentage of sites with bleeding on probing
following both treatment modalities, reecting the
systematic approach to plaque control incorporated
in the included studies.
Molar and nonmolar teeth
(Tables 2a and 2b)
Two studies evaluated the treatment outcome in
molar and nonmolar teeth (20, 24). A meta-analysis
was not performed as it was not possible to derive the
standard error in the paper by Pihlstrom et al. (24).
Both studies reported that, 12 months following
surgery, initially deep sites (probing pocket
depth > 6 mm) showed greater probing pocket
depth reduction for nonmolar teeth than for molar
teeth (P 0.001 in (25); the P-value was not provided
in the paper by Lindhe et al. (20)).
Mean attachment level changes, however, showed
little variation between tooth-types and treatment
modalities.
Angular defects nonmolar teeth
(Table 2a)
One study evaluated nonmolar teeth over a period of
5 years (11). Similar results in probing depth reduc-
tion and clinical attachment level gain were achieved
following surgical and nonsurgical procedures. An
analysis of angular defects was also included and
indicated no difference in outcome between treat-
ment modalities. Data on the initial severity of pro-
bing depth were not presented in this paper, and it
therefore could not be combined in a meta-analysis
with the data from the paper by Lindhe et al. (20).
Molar furcations
Kalkwarf et al. (15) evaluated molar furcations and
reported similar treatment outcomes 12 months after
surgical and nonsurgical periodontal therapy (Table 2c).
Long-term treatment outcomes
While the focused question of this systematic review
addressed treatment outcomes at 12 months, a num-
ber of studies presented long-term treatment out-
comes after 5 years (11, 19, 26), 6.5 years (23), and
7 years (14). Ramfjord et al. (26) reported a total of 101
teeth in 24 patients requiring re-treatment due to
persistent inammation(bleedingandsuppurationon
probing); 44 teeth in the nonsurgical treatment group
and 21 teeth in the surgical group were re-treated.
While this suggests that there may be some differences
inoutcome betweentherapies, the long-termresults of
scaling and root planing in this study were equivalent
to those of the surgical procedure with regard to
maintenance of mean attachment levels and preven-
tion of loss of teeth. However, this lack of a difference
might have been achieved though greater treatment
need in the nonsurgical group. Such a hypothesis
could be evaluated in future research.
Similarly, Kaldahl et al. (14) reported that break-
down sites (attachment loss 3 mm year) were more
frequently found in deep sites treated by scaling and
root planing. Despite this, they reported that similar
numbers of teeth were extracted due to progressive
periodontal disease following nonsurgical treatment
(21 teeth) and open ap debridement (21 teeth).
Pihlstrom et al. (23) reported a similar incidence of
tooth loss following surgical (5 teeth) and nonsurgical
(6 teeth) therapy over a 5-year observation period.
Conclusions of the systematic
review by Heitz-Mayeld et al. (8)
When sites with an initial probing pocket depth of
13 mm were treated by open ap debridement,
there was more clinical attachment level loss than
after scaling and root planing (weighted mean dif-
ferences )0.51 mm; 95% CI [ 0.74, ) 0.29]).
46 mm were treated by open ap debridement,
76
Heitz-Mayeld
therewas less clinical attachment level gainthanafter
scaling and root planing (weighted mean differences
)0.37 mm; 95%CI [)0.49, 0.26]). There was a greater
reduction in probing pocket depth reduction after
the open ap debridement procedure (weighted
mean differences 0.35 mm; 95%CI [0.23,0.47]).
Table 2a. Change in clinical attachment level and probing pocket depth at nonmolars treated with scaling and root
planing or open ap debridement
Reference Non-molar teeth Scaling and root
planing
Open ap
debridement
12-month results Initial PPD n Mean SD SE* Mean SD SE*
Lindhe et al. (20) D CAL mm 13 mm 15 ) 0.9 0.4* ) 0.2 0.2*
46 mm 15 0.7 0.4* 0.3 0.2*
> 6 mm 15 0.9 0.9* 1.5 0.6*
Pihlstrom et al. (24) 46 mm 14 0.34 0.06
> 6 mm 10 0.41 1.19
Isidor & Karring (11) all PPD 16 0.6 0.2* 0.2 0.3*
Angular defects 16 1.6 0.3* 0.9 0.5*
Lindhe et al. (20) D PPD mm 13 mm 15 0.4 0.2* 0.6 0.2*
46 mm 15 2.0 0.4* 2.3 0.4*
> 6 mm 15 2.6 1.0* 3.4 0.8*
> 6 mm 10 1.71 3.14
Isidor & Karring (11) all PPD 16 2.3 0.3* 2.5 0.4*
Angular defects 16 3.7 0.3* 3.5 0.3*
CAL, clinical attachment level. PPD, probing pocket depth.
Table 2b. Change in clinical attachment level and probing pocket depth at molars treated with scaling and root
planing or open ap debridement
Reference Molars Scaling and root
planning
Open ap
debridement
12-month results Initial PPD n mean SD SE* mean SD SE*
Lindhe et al. (20) D CAL in mm 13 mm 15 ) 0.3 0.2* ) 1.0 0.6*
46 mm 15 0.3 0.4* ) 0.1 0.2*
> 6 mm 15 0.9 0.6* 0.7 0.6*
> 6 mm 10 0.6 1.2
Lindhe et al. (20) D PPD in mm 13 mm 15 0.2 0.2* 0.5 0.4*
46 mm 15 1.2 0.4* 1.4 0.4*
> 6 mm 15 2.0 1.0* 2.0 1.2*
> 6 mm 10 0.9 2.3
SD SE *denotes standard deviation standard error.
77
>6 mm were treated by open ap debridement,
there was a greater increase in the clinical attach-
ment level than after scaling and root planing
(weighted mean differences 0.19 mm; 95% CI
[0.04,0.35]). Open ap debridement resulted in a
greater reduction in probing pocket depth than
scaling and root planing in these deep pockets
[0.38,0.79]).
There is limited evidence (no meta-analysis) sug-
gesting little difference between open ap debri-
dement and scaling and root planing when treating
furcations or angular bony defects.
There are no data available to address the
important issue of patient-centered treatment
outcomes and the evaluation of adverse effects.
Quality assessment of the
systematic review by Heitz-
Mayeld et al. (8)
An assessment of the quality of the systematic review
was made using a specic checklist (7) and showed
that most requirements were met (Table 3). A
focused question and search strategy was clearly
dened and an independent assessment of the
inclusion criteria and quality assessment of studies
was performed by two reviewers. An assessment of
heterogeneity was carried out and reasons for vari-
ation were discussed. Results of meta-analyses were
clearly displayed in tables and forest plot diagrams,
enabling correct interpretation of the results.
The following checklist requirements were not met:
inclusion of unpublished literature; inclusion of lit-
erature in languages other than English; and hand
searching of journals for missed trials (Table 3). The
lack of reporting of study quality in the original
publications hindered the incorporation of this
parameter into the meta-analyses. One strategy to
overcome this limitation is to seek clarication from
the authors of the original research.
It should be emphasized that the assessment of
study quality should not be viewed as judgmental
comments about the original studies (or systematic
reviews). Research methodology continues to evolve
and appreciation or application of these issues has
not been clear in the past.
Meta-analysis of the effect of
scaling and root planing, surgical
treatment and antibiotic therapies
on periodontal probing depth and
attachment loss (Hung & Douglass
(10))
Aim
The objective of this systematic review was to
evaluate the effect of scaling and root planing on
periodontal probing depth and clinical attachment
level. Scaling and root planing was rst examined
independently and then in comparison with surgical
treatment and selected local antibiotic therapies
incorporating meta-analysis evaluations.
Focused question
A number of questions were addressed in this review.
Inpart II of the paper, evidence relatedtothe reduction
of periodontal probing depth and gain of attachment
level measurements after scaling and root planing as
compared to surgical treatment was evaluated.
Literature search
A MEDLINE search was performed to identify
studies related to periodontal treatment. In
Table 2c. Change in clinical attachment level and probing pocket depth at molar furcation sites treated with
scaling and root planing or open ap debridement
Reference Molar furcation sites Scaling and root planing Open ap debridement
12-month results n mean SD SE* n mean SD SE*
Kalkwarf et al. (15) D CAL in mm vertical 78 0.8 74 0.6
D CAL in mm horizontal 78 0.2 74 ) 0.4
D PPD in mm 78 1.2 74 1.5
SD SE *denotes standard deviation standard error.
78
Heitz-Mayeld
addition, searching from the bibliographies of ori-
ginal and review articles was performed. The exact
search strategy and search terms used were not
described.
Study selection
Specic inclusion criteria were dened: scaling and
root planing as one of the primary treatment arms;
random assignment to study groups; 80%of enrolled
patients available for the 12-month evaluation; pro-
bing pocket depths and clinical attachment levels
reported in mm; reporting of the sample size; pres-
entation of data by stratication of initial probing
depth.
Outcome variables
The outcome variables evaluated were clinical
attachment level change (DCAL) and probing pocket
depth change (DPPD).
Data from studies were combined in meta-analyses
to evaluate the treatment effect of surgical vs. non-
surgical therapy. The results of the meta-analyses
were expressed as weighted mean differences and
95% condence intervals (CI). Standard errors were
not reported in all studies and the sample size of each
study was used to weight the relative contribution of
Table 3. Assessment checklist for systematic reviews (Glenny et al. (7))
Question (possible categories) Heitz-Mayeld
et al. (8)
Antczak-Bouckoms
et al. (2)
Hung & Douglass
(10)
A Did review address a focused question?
(yes, no, cant tell)
yes yes yes
B Did authors look for appropriate papers?
yes yes yes
C Do you think authors attempted to identify
all relevant studies? (yes, no, cant tell)
yes cant tell cant tell
D Search for published and unpublished literature?
no cant tell cant tell
E Were all languages considered?
no no cant tell
F Was any hand searching carried out?
no no cant tell
G Was it stated that the inclusion criteria were carried
out by at least two reviewers? (yes, no, cant tell)
yes no no
H Did reviewers attempt to assess the quality
of the included studies? (yes, no, cant tell)
yes yes no
I If so did they include this in the analysis?
(yes, no, cant tell, not applicable)
no no not applicable
J Was it stated that the quality assessment was carried
out by at least two reviewers? (yes, no, cant tell)
yes yes no
K Are the results given in a narrative or pooled
statistical analysis? (narrative, pooled, not applicable)
pooled pooled pooled
L If the results have been combined was it reasonable
to do so? (yes, no, cant tell, not applicable)
M Are the results clearly displayed?
(yes, no, not applicable)
yes no no
N Was an assessment of heterogeneity made and reasons
for variation discussed? (yes, no, not applicable)
yes no no
O Were results of review interpreted appropriately?
(yes, no, cant tell, not applicable)
79
each study. The authors stated that they were unable
to test for heterogeneity as they did not have the
standard errors of the results from most of the
included studies.
Eight papers were identied (3, 9, 13, 14, 20, 21,
23, 26), reporting on ve study populations com-
paring changes in periodontal probing depth and
clinical attachment levels between scaling and root
planing and open ap debridement. One study
included only posterior teeth and systemic anti-
biotics were prescribed for 4 days following treat-
ment (2).
Data presentation
The majority of studies presented data using the
initial probing depth categories 13 mm, 46 mm,
and >6 mm. One study population used an alter-
native initial probing depth classication: 14 mm,
56 mm, and >6 mm (13, 14). In the meta-analysis
evaluations it is unclear how the analyses dealt with
the difference in initial probing depth stratication
among studies. The categories used were shallow,
medium and deep without providing a range in
millimeters, which may have resulted in inappropri-
ate pooling of data.
At the 12-month evaluation, ve studies were inclu-
ded in each meta-analysis. Mean differences in pro-
bing pocket depth reduction and clinical attachment
level change between open ap debridement and
scaling and root planing were combined in meta-
analyses and presented for the respective initial
probing depth categories (Table 4).
Summary at 12 months
In initially shallow sites, nonsurgical therapy resulted
in 0.3 mm less clinical attachment loss (weighted
mean differences 0.33 mm; 95% CI [) 0.44,
) 0.22 mm]) and 0.1 mm less probing pocket depth
reduction than surgical therapy (weighted mean
differences 0.14 mm; 95% CI [0.11,0.17]).
In initially medium sites, nonsurgical therapy
[) 0.36, ) 0.22]) and 0.3 mm less probing pocket
depth reduction (weighted mean differences
0.30 mm; 95% CI [0.22,0.39]) than surgical therapy.
In initially deep sites, surgical therapy resulted in
0.6 mm more probing pocket depth reduction
[0.50,0.77]) than nonsurgical therapy. There was no
statistically signicant difference in clinical attach-
ment level gain between procedures.
Four studies were combined in meta-analyses at
2 years and two studies at 3, 4, 5 and 6 years fol-
lowing treatment. This review showed that surgical
treatment initially resulted in greater probing pocket
depth reduction than nonsurgical therapy. However,
with increasing follow-up time the mean differences
in probing pocket depth reduction became smaller.
After 6 years, all differences in probing pocket depth
reduction were nonsignicant.
The 6-year follow-up results of this review showed
an advantage for nonsurgical therapy in terms of
attachment level gain for all initial probing depth
categories.
Table 4. Summary of meta-analyses for clinical outcomes (10)
Outcome
12 months
Initial PPD
category
No. of
studies
Studies included
(ref. nos.)
Mean
difference (mm)
95% CI P-value for
mean difference
CAL gain shallow 5 3, 14, 20, 23, 26 ) 0.33 [) 0.44, ) 0.22] 0.0001
PPD reduction shallow 5 3, 14, 20, 23, 26 0.14 [0.11,0.17] 0.0001
CAL gain medium 5 3, 14, 20, 23, 26 ) 0.29 [) 0.36, ) 0.22] 0.0001
PPD reduction medium 5 3, 14, 20, 23, 26 0.30 [0.22,0.39] 0.0001
CAL gain deep 5 3, 14, 20, 23, 26 0.09 [) 0.07,0.24] 0.24
PPD reduction deep 5 3, 14, 20, 23, 26 0.64 [0.50,0.77] 0.0001
80
Heitz-Mayeld
Conclusions
The authors concluded:
Surgical treatment is better for reduction of per-
iodontal probing depth and these benets be-
come greater with the increase of initial probing
depth.
The gain of attachment level differences indicates
an advantage for nonsurgical treatment in shal-
low and medium initial periodontal probing
depths.
For deep initial periodontal probing depths,
surgical therapy showed similar attachment
gains when compared with scaling and root
planing.
systematic review (Hung &
Douglass (10))
An assessment of the quality of the systematic
review was made using a specic checklist (7)
(Table 3). This review addressed a number of
focused questions and specic inclusion criteria
were dened. While this review met some of the
criteria on the checklist, search terms were not
clearly dened and it was unclear whether the
authors had considered inclusion of unpublished
literature, or literature in languages other than
English. Hand searching of journals for missed trials
was not reported. Assessment of study quality
and an assessment of inclusion criteria by two
independent reviewers were not performed. It was
difcult to tell whether the pooling of data was
appropriate due to the ambiguous description of
initial probing depth categories. An assessment of
heterogeneity to determine whether the studies
were similar enough for the results of meta-analyses
to be valid was not made.
Meta-analysis of surgical vs.
nonsurgical methods of treatment
for periodontal disease (Antczak-
Bouckoms et al. (2))
Aim
The objective of this review was to perform a meta-
analysis on randomized controlled trials comparing
surgical with nonsurgical treatment for periodontal
disease.
Focused question
This review addressed the difference in probing
pocket depth and attachment level changes when
comparing open ap debridement with nonsurgical
treatment of periodontal disease.
Literature search
A literature search was conducted of MEDLINE
(198090) by reviewing the appropriate MeSH key-
words. In addition, searching from the bibliographies
of original and review articles was performed. The
exact search strategy and search terms used were not
described.
Study selection
Studies were selected according to specic inclusion
criteria: comparison of scaling and root planing or
subgingival curettage (with anesthesia) with open
ap debridement for the treatment of chronic adult
periodontitis; random assignment to study groups;
articles published in English.
Validity assessment
Two reviewers independently assessed the quality of
the methods of the study and of the data derived
from it using a point scoring system adapted from
Chalmers et al. (4). In an effort to minimize bias,
information including the title, authors, journal,
institution, and study results was removed from
photocopies of the original papers during the quality
assessment process.
Outcome variables
The outcome variables evaluated were clinical
attachment level change (DCAL) and probing pocket
depth change (DPPD).
Data from ve studies were combined in meta-
analyses to evaluate the treatment effect of surgical
vs. nonsurgical therapy. The results of the meta-
analyses were expressed as weighted mean differ-
ences and 95% condence intervals. Standard
errors were not reported in all studies and the
sample size of each study was used to weight the
relative contribution of each study. An assessment
of heterogeneity of results was not performed,
81
making it difcult to assess whether the studies
were similar enough for the summary values from
the meta-analyses to be valid.
Twenty-two papers were selected for initial review. Of
these, ve randomized controlled trials (2, 14, 15, 19,
21) met the inclusion criteria and were combined in
meta-analysis evaluations. Reasons for exclusion of
studies and the characteristics of included studies
were discussed.
In this review, nonsurgical therapy was dened as
scaling and root planing or subgingival curettage with
anesthesia. The authors combined the results repor-
ted by Ramfjord et al. (26) for subgingival curettage
with those of scaling and root planing. The study by
Knowles et al. (17) was also included, in which sub-
gingival curettage was considered as a nonsurgical
procedure. One study included only posterior teeth
and systemic antibiotics were administered for
4 days following treatment (3).
Methodologic quality of included studies
Quality assessment revealed low scores related to the
lack of blinding for the randomization process of
patients and observers, lack of consideration of
power, and unknown handling of withdrawals.
Study design
Similarities and differences among studies were
described. All studies were of split mouth design with
a nonsurgical and a surgical procedure performed
within each patient. The data were analyzed accord-
ing to initial probing depth categories dened as level
1 (13 mm), level 2 (46 mm), and level 3 (> 6 mm).
Considerable variation in study design and data
presentation among the studies was observed,
including variation in sample sizes, tooth type
included, time at which baseline measurements were
recorded, statistical tests used in analyses, and the
duration of the studies.
Differences in changes in clinical attachment level
and probing pocket depth between surgical and
nonsurgical therapies were combined in meta-ana-
lyses and presented for the respective initial probing
depth categories.
At 12 months, the meta-analysis evaluation
showed a signicantly greater reduction in probing
depth for surgical treatment for all three initial levels
of probing pocket depth. Surgical therapy resulted in
signicantly less attachment gain than nonsurgical
therapy for shallow and medium initial probing
depths (Table 5).
Summary
In shallow pockets (13 mm), compared with sur-
gical therapy, nonsurgical therapy resulted in
0.3 mm less clinical attachment loss (weighted
mean differences )0.27 mm; 95% CI [) 0.49, 0.48])
(note: the CI is incorrect as there was a printing
error in the original paper; an attempt was made to
contact the authors without success). There was
also 0.1 mm less probing pocket depth reduction
[0.05,0.17]).
In 46 mm pockets, scaling and root planing
(weighted mean differences )0.27 mm; 95% CI
[) 0.44, ) 0.10]) and 0.3 mm less probing depth
reduction (weighted mean differences 0.34 mm; 95%
CI [0.20,0.49]) than surgical therapy.
In deep pockets (> 6 mm), surgical therapy
resulted in 0.5 mm more probing pocket depth
reduction (weighted mean differences 0.48 mm; 95%
CI [0.12,0.84]) than nonsurgical therapy. There was
no statistically signicant difference in clinical
attachment level gain between procedures.
Long-term treatment outcomes showed no signi-
cant difference between treatment modalities, with
the exception of a greater reduction in probing
pocket depth following surgery at 2 years for initial
probing pocket depth 46 mm (four studies) and at
5 years for initially deep pockets (probing pocket
depth > 6 mm).
Conclusions
The authors concluded:
When the objective is reduction of probing depth,
surgical therapy provides a greater benet than
nonsurgical therapy for all levels of initial disease
severity.
When the objective is to increase attachment level,
nonsurgical therapy provides a greater benet for
initial disease severity levels 13 mm and 46 mm,
and surgical therapy for > 6 mm.
82
Heitz-Mayeld
the interpretation of the results of the meta-ana-
lysis must take into account potential sources of
bias related to lack of blinding, subjective assess-
ment of outcomes, and the inuence of publication
bias.
systematic review (Antczak-
Bouckoms et al. (2))
An assessment of the quality of the systematic review
was made using the checklist published by Glenny
et al. (7) (Table 3). The assessment showed that the
review addressed a focused question and that an
independent assessment of the quality of included
studies was made by two reviewers.
Although this review met some of the checklist
criteria, search terms were not clearly dened and it
was unclear whether the authors had considered
inclusion of unpublished literature. Papers in lan-
guages other than English were not considered. Hand
searching of journals for missed trials and assessment
of inclusion criteria by two independent reviewers
were not stated. Quality assessment of studies was
performed; however, the assessment was not used in
the analysis. The inclusion of subgingival curettage as
a nonsurgical procedure may have resulted in inad-
equate pooling of results. Furthermore, an assess-
ment of heterogeneity to determine whether the
studies were similar enough for the results of meta-
analyses to be valid was not made.
Discussion
Differences in results of the three
systematic reviews
Although the results of the three systematic reviews
showed similar patterns in differences in attachment
level and probing depth changes between therapies,
the outcomes of the meta-analysis evaluations dem-
onstrated variations in the magnitude and signi-
cance of these differences (Tables 1, 4 and 5, Figs 2
and 3).
Two of the reviews (2, 10) concluded that open ap
debridement resulted in greater probing depth
reduction than scaling and root planing in all initial
probing depth categories. In contrast, the review by
Heitz-Mayeld et al. (8) showed greater probing
pocket depth reduction following surgery in initial
probing depth categories 46 mm and >6 mm. The
results of this review showed that in initially deep
pockets (probing pocket depth > 6 mm) there was a
greater attachment level gain following open ap
debridement. However, the other two reviews (2, 10)
showed no signicant difference in attachment level
change between the two therapies in deep pockets.
A number of important factors may explain the
variation in the results of the systematic reviews
(Table 6).
Inclusion criteria study populations
Due to differences in inclusion criteria the study
populations in the three systematic reviews were not
identical (Table 6). The review by Antczak-Bouckoms
et al. (2) included one study (17) where subgingival
curettage was considered as a nonsurgical procedure.
The reviews by Antczak-Bouckoms et al. (2) and
Hung & Douglass (10) included one study (3) where
posterior teeth were excluded and systemic anti-
biotics were administered for 4 days after treatment.
Pooling of results
The methods used for pooling results also varied
among the reviews. The study sample size was used
for the reviews by Antczak-Bouckoms et al. (2) and
Hung & Douglass (10), but the review by Heitz-
Mayeld et al. (8) used the preferred method,
whereby the inverse of the standard error was used
for pooling of the results (5).
In the review by Hung & Douglass (10) it was un-
clear how initial probing depth categories were de-
termined, which may have resulted in inappropriate
pooling of results.
Number of studies combined
in meta-analyses
In the review by Heitz-Mayeld et al. (8) there
were varying numbers of studies combined in the
meta-analyses, depending on the initial probing
depth category and the availability of standard errors
(Table 6). In contrast, the reviews by Hung &
Douglass (10) and Antczak-Bouckoms et al. (2)
combined ve studies in each meta-analysis for each
probing depth category.
Assessment of heterogeneity
In the systematic review by Heitz-Mayeld et al. (8),
heterogeneity was tested and an appropriate statis-
tical model was used for the pooling of results
83
where signicant heterogeneity between studies was
found.
As stated earlier, testing for heterogeneity and, if
present, investigating its causes is an essential ele-
ment for meta-analysis. Without such testing, it is not
clear whether the statistical combining of data is
valid, even if inclusion criteria for studies to be
incorporated into the review are stated. Furthermore,
if heterogeneity is present, such analysis provides
opportunities to examine possible causes of the ob-
served variation. These could include prognostic
factors such as plaque control and smoking, meth-
odologic issues, especially protection from bias and
other characteristics such as the study population
and nature of the intervention.
Quality assessment of the three
systematic reviews (2, 8, 10)
While all of the reviews presented followed a struc-
tured protocol there was considerable variation in the
quality of the reviews as assessed using a checklist for
systematic reviews by Glenny et al. (7) (Table 3). This
is perhaps not surprising considering that the reviews
were published in a period spanning more than
10 years (19932004). As evidence-based dentistry
has evolved, research methodology has developed
and improved, and specic guidelines have been
formulated for a rigorous systematic review process
(NHS Center for Reviews & Dissemination: http://
www.york.ac.uk/Institute/crd/report4.htm). Thus
quality assessment should not be considered to be
judgmental comments about the reviews, as some
quality criteria listed in the checklist of Glenny et al.
(7) may not have been recognized as being important
in the past.
The value of a good systematic review to clinical
decision-making is that it should minimize bias, pro-
vide a comprehensive and contemporary overview, be
objective in its appraisal of quality and, above all, be
transparent to allow others to appraise the methods
and quality of the review itself. If such conditions are
met, the reader should have greater condence in the
Table 5. Summary of meta-analyses for clinical outcomes (2)
Outcome
12 months
Initial PPD
category
No. of
studies
Studies included
(ref. nos.)
Mean
difference (mm)
95% CI P-value for
mean difference
CAL gain 13 mm 5 3, 17, 20, 23, 26 ) 0.27 [) 0.49, 0.48]
a
0.03
PPD reduction 13 mm 5 3, 17, 20, 23, 26 0.11 [0.05,0.17] 0.01
CAL gain 46 mm 5 3, 17, 20, 23, 26 ) 0.27 [) 0.44, ) 0.10] 0.01
PPD reduction 46 mm 5 3, 17, 20, 23, 26 0.34 [0.20,0.49] 0.00
CAL gain > 6 mm 5 3, 17, 20, 23, 26 0.18 [) 0.18,0.54] 0.22
PPD reduction > 6 mm 5 3, 17, 20, 23, 26 0.48 [0.12,0.84] 0.02
a
Error in original publication.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
2 8 10
w
e
i
g
h
t
e
d

m
e
a
n

d
i
f
f
e
r
e
n
c
e

(
m
m
)
1 3 mm 4 6 mm/medium > 6 mm
s n
Favours OFD
Favours SRP
Fig. 2. Results of the 3 systematic reviews (2, 8, 10). Dif-
ference in PPD change (WMD, 95%CI) between OFD and
SRP at sites with initial PPD 1 3 mm, 4 6mm medium,
>6 mm).
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
8 2 10
w
e
i
g
h
t
e
d

m
e
a
n

d
i
f
f
e
r
e
n
c
e

(
m
m
)
1 3 mm 4 6 mm/medium > 6 mm
Favours OFD
Favours SRP
Fig. 3. Results of the 3 systematic reviews (2, 8, 10). Dif-
ference in CAL change (WMD, 95% CI) between OFD and
SRP at sites with initial PPD 1 3 mm, 4 6mm medium,
>6 mm).
84
Heitz-Mayeld
conclusions of the systematic review than in other
summaries of clinical evidence (22).
Strengths of the systematic
review (8)
Of the three systematic reviews, the review by Heitz-
Mayeld et al. (8) had the highest quality as assessed
by the Glenny et al. (7) checklist, thereby providing
the most reliable data of the three reviews. A well-
dened search strategy was carried out, and an
objective appraisal of the quality of evidence was
made. The reader can be condent that the conclu-
sions of this systematic review addressed the focused
question what is the effect of surgical debridement
vs. nonsurgical debridement in terms of changes in
clinical attachment level and probing pocket depth
and bleeding on probing for patients with chronic
periodontitis?, while minimizing bias.
Limitations of the systematic
review (8)
The main limitations of the review by Heitz-Mayeld
et al. (8) were the lack of incorporation of quality
assessment into meta-analysis and the possibility
that trials were missed due to restriction to the
English language articles only. Furthermore, unpub-
lished literature was not included and hand searching
of journals was not performed.
A further limitation to this review was the inability
to formally test for publication bias due to the limited
number of studies in the analyses. This bias is due to
the tendency for studies with positive outcomes to be
more prevalent in the literature than negative studies,
as studies that showed no effect (or a negative effect)
are not published. This may result in an overesti-
mation of the effect of an intervention.
It should be emphasized that at the time the
studies included in these papers were performed, the
1999 Classication of Periodontal Diseases (1) did not
exist and a range of terminology was used to describe
study populations and disease severity. Furthermore,
when considering the studies included in this sys-
tematic review, the age range was 2268 years, sug-
gesting that participants may have varied in their
susceptibility to periodontitis.
It is also important to recognize that when most of
the studies were conducted, subject characteristics
such as smoking and their possible effect on treat-
ment outcome were not considered. The lack of
patient-centered data available to the systematic
review is a major limitation. Clinical differences, in
terms of probing changes, could be less important to
patients than other outcomes such as aesthetics, pain
and overall experience of treatment. Without these
Table 6. Differences observed between the three papers (2, 8, 10) accounting for differences in the results of the
meta-analyses
Heitz-Mayeld et al. (8) Hung & Douglass (10) Antczak-Bouckoms et al. (2)
Inclusion criteria Chronic periodontitis
Nonsurgical procedure
scaling and root planing
No classication stated
PPD stratication
Chronic adult periodontitis
subgingival curettage or
Study populations references 14, 18, 20, 23, 26 3, 14, 20, 23, 26 3, 17, 20, 23, 26
Test for heterogeneity yes no no
Method for pooling of results standard error sample size sample size
Initial PPD categories 13 mm, 46 mm, > 6 mm shallow, medium, deep 13 mm, 46 mm, > 6 mm
No. of studies in meta-analysis
CAL gain Initial PPD 13 mm: 4 Initial PPD shallow: 5 Initial PPD 13 mm: 5
Initial PPD 46 mm: 4 Initial PPD medium: 5 Initial PPD 46 mm: 5
Initial PPD > 6 mm: 5 Initial PPD deep: 5 Initial PPD > 6 mm: 5
PPD reduction Initial PPD 13 mm: 2 Initial PPD shallow: 5 Initial PPD 13 mm: 5
Initial PPD 46 mm: 2 Initial PPD medium: 5 Initial PPD 46 mm: 5
Initial PPD > 6 mm: 3 Initial PPD deep: 5 Initial PPD > 6 mm: 5
85
data, informed consent must be based on a limited
range of outcomes.
Limited data ontoothsurvival was found (23). Tooth
survival is a long-term outcome, and is therefore very
difcult to test in a randomized controlled trial.
Interpretation of the results of the
systematic review (8)
All studies included in the review by Heitz-Mayeld
et al. (8) incorporated regular supervised mainten-
ance care in their protocols and the results should be
interpreted with this in mind. The importance of a
high standard of oral hygiene was emphasized by
Lindhe et al. (19) in a 5-year study where patients
followed a strict program of professional supragin-
gival plaque removal at 2-week intervals for 6 months
which was then extended to 3-month visits including
subgingival scaling as required. The reduction of
probing depth and gain of clinical attachment oc-
curred predominantly in the group of patients with
excellent plaque control (plaque index 10%).
All studies included in the systematic review were
university-based. This may have inuenced the
comparison of treatment modalities in that condi-
tions were ideal for research, with no time con-
straints during treatment and maintenance.
Finally, operator experience was inconsistently
reported and this may also have had an inuence on
the outcome of treatment modalities.
Implications for future research
The systematic review (8) identied a number of
important areas which need to be addressed in future
research, including the evaluation of adverse effects,
and patient-based outcomes, such as aesthetics and
patient preference, which were not reported in any of
the included studies. This systematic review also
acknowledged that there is insufcient evidence to
evaluate treatment effects at specic sites such as
molar furcations and angular defects.
Clinical implications
If the choice of therapy is made solely on the basis of
the outcome measures of probing pocket depth
reduction and gain in clinical attachment level, then
the decision is relatively straightforward for a given
pocket. If probing pocket depth reduction is the main
aim, surgical therapy will be the treatment of choice
for moderate and deep pockets. If the objective is to
increase the clinical attachment level, nonsurgical
therapy is of greater benet for shallow (13 mm)
and moderate (46 mm) pockets, and open ap de-
bridement for deep pockets (> 6 mm). However, in
many cases the area to be treated will include a
combination of shallow, moderate, and deep pockets.
In addition, the predictability of treatment outcome
at sites with furcation involvement or angular defects
is unclear.
The choice of therapy may depend not only on the
outcome measures of probing pocket depth reduc-
tion and clinical attachment level gain, but also on
the inuence of other variables, including the evalu-
ation of adverse effects and patient-centered out-
comes, such as patient discomfort and apprehension,
root sensitivity and aesthetic considerations, which
were not addressed.
The selection of surgical or nonsurgical periodontal
therapy should be based on a careful consideration
and discussion of the benets and harm for that
particular patient and site.
References
1. American Academy of Periodontology. Consensus Report:
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2. Antczak-Bouckoms A, Joshipura K, Burdick E, Tulloch JFC.
Meta-analysis of surgical versus non-surgical methods of
treatment for periodontal disease. J Clin Periodontol 1993:
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3. Becker W, Becker BE, Ochsenbein C, Kerry G, Caffesse R,
Morrison EC, Prichard J. A longitudinal study comparing
scaling, osseous surgery and modied Widman procedures.
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4. Chalmers TC, Smith H Jr, Blackburn D, Ambroz A. A
method for assessing the quality of a randomized control
trial. Control Clin Trials 1981: 2: 3149.
5. Deeks J, Altman D, Bradburn M. Statistical methods for
examining heterogeneity and combining results from sev-
eral studies in meta-analysis. In: Egger M, Davey Smith GD,
Altman D, eds. Systematic Reviews in Health Care. Meta-
Analysis in Context. London: BMJ Publishing Group, 2001.
6. Follmann D, Elliot P, Suh I, Cutler J. Variance imputation
for overviews of clinical trials with continuous response.
J Clin Epidemiol 1992: 45: 768773.
7. Glenny AM, Esposito M, Coulthard P, Worthington HV. The
assessment of systematic reviews in dentistry. Eur J Oral
Sci 2003: 2: 8592.
8. Heitz-Mayeld L, Trombelli L, Heitz F, Needleman I, Moles
D. A systematic review of the effect of surgical debridement
vs. non-surgical debridement for the treatment of chronic
periodontitis. J Clin Periodontol 2002: 29 (Suppl. 3): 92102.
9. Hill RW, Ramfjord SP, Morrison EC, Appleberry EA,
Caffesse RG, Kerry GJ, Nissle RR. Four types of periodontal
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655662.
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10. Hung H-C, Douglass CW. Meta-analysis of the effect of
scaling, root planing, surgical treatment and antibiotic
therapies on periodontal probing depth and attachment
loss. J Clin Periodontol 2002: 29: 975986.
11. Isidor F, Karring T. Long-term effect of surgical and non-
surgical periodontal treatment. A 5-year clinical study.
12. Isidor F, Karring T, Attstrom R. The effect of root planing as
compared to that of surgical treatment. J Clin Periodontol
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13. Kaldahl WB, Kalkwarf KL, Patil KD, Dyer JK, Bates RE Jr.
Evaluation of four modalities of periodontal therapy. Mean
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changes. J Periodontol 1988: 59: 783793.
Long-term evaluation of periodontal therapy. I. Response
to 4 therapeutic modalities. J Periodontol 1996: 67: 93102.
15. Kalkwarf KL, Kaldahl WB, Patil KD. Evaluation of furcation
region response to periodontal therapy. J Periodontol 1988:
59: 794804.
16. Kalkwarf KL, Kaldahl WB, Patil KD. Evaluation of gingival
bleeding following 4 types of periodontal therapy. J Clin
Periodontol 1989: 16: 601608.
17. Knowles JW, Burgett FG, Nissle RR, Shick RA, Morrison EC,
Ramfjord SP. Results of periodontal treatment related to
pocket depth and attachment level. Eight years. J Period-
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18. Lindhe J, Nyman S. Scaling and granulation tissue removal
in periodontal therapy. J Clin Periodontol 1985: 12: 374
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19. Lindhe J, Westfelt E, Nyman S, Socransky SS, Haffajee
AD. Long-term effect of surgical non-surgical treatment
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Bratthall G. Healing following surgical non-surgical treat-
ment of periodontal disease. A clinical study. J Clin Peri-
odontol 1982: 9: 115128.
21. Morrison EC, Ramfjord SP, Hill W. Short-term effects of
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22. Needleman IG. A guide to systematic reviews. J Clin
Periodontol 2002: 29 (Suppl 3): 69.
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periodontal disease. A review of current studies and addi-
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24. Pihlstrom BL, Oliphant TH, McHugh RB. Molar and
nonmolar teeth compared over 6 years following two
methods of periodontal therapy. J Periodontol 1984; 55:
499504.
25. Pihlstrom BL, Ortiz-Campos C, McHugh RB. Molar and
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87
Which reconstructive procedures
are effective for treating the
periodontal intraosseous defect?
LEONARDO TROMBELLI
Deep intraosseous defects represent a major chal-
lenge for the clinician. Sites with intraosseous lesions
have been shown to be at higher risk of disease
progression in subjects who had not received
systematic periodontal therapy (78). Treatments of
intrabony defects include scaling and root planing
with or without surgical access ap. When the
periodontal debridement is surgically performed,
additional osseous resective therapy and or recon-
structive therapy by means of the application of
membranes, biological agents or grafting biomate-
rials can be used to correct the bone deformities
induced by destructive periodontal disease.
In this respect, reconstructive procedures have
been used with varying success during the past dec-
ades to accomplish the restitutio ad integrum of lost
attachment apparatus in deep intraosseous defects.
Although observations from histologic studies in
humans (6, 19, 41, 105, 125) and data from controlled
clinical trials have demonstrated that some of the
available grafting procedures may result in healing
that can be termed periodontal regeneration, com-
plete and predictable reconstruction of periodontal
tissues is still difcult to obtain. Despite the widely
accepted effectiveness of the reconstructive proce-
dures previously documented (28, 36, 89), there is a
substantial variation in clinical response that can
only in part be attributed to the biomaterials biolo-
gical agents adopted (54).
Several traditional reviews have previously repor-
ted the additional effect of the use of different
reconstructive procedures in intraosseous defects
(9, 28, 32, 42, 46, 48, 50, 64, 65, 72, 87, 90, 101, 117).
Unfortunately, it has been shown that many tradi-
tional medical reviews are haphazard and biased,
often reecting the opinion of the reviews authors.
In contrast, systematic reviews follow explicit,
well-documented, scientic methodology in order to
reduce both systematic errors (biases) and random
errors (those occurring by chance) and provide a
more objective, comprehensive view of the research
literature (30). Since all reconstructive procedures
are time-consuming, technically demanding and
nancially costly, in view of the great expense of
the reconstructive device biomaterials and surgi-
cal time, systematic reviews are of paramount
importance to assess the efcacy of reconstructive
procedures in the treatment of periodontal intra-
osseous defects measured against the current
standard of surgical periodontal treatment, access
ap surgery.
The aim of this article is to determine the effect of
the use of guided tissue regeneration, grafting pro-
cedures or the application of enamel matrix proteins
in addition to conventional open ap debridement
(OFD) in the treatment of deep intraosseous defects.
Specically, the additional efcacy of each recon-
structive procedure in comparison to the OFD-only
procedure will be evaluated by means of clinical and
patient-centered outcome parameters on a short-
and long-term basis.
Guided tissue regeneration
Background
One of the most frequently documented reconstruc-
tive procedures is guided tissue regeneration (GTR)
(28). In this procedure, a biocompatible barrier
membrane (either resorbable or nonresorbable) is
surgically implanted to isolate and protect the bone
defect. If non-resorbable, the barrier is surgically
removed 46 weeks after implantation. Connective
tissue and bone regeneration may then occur within
the bony lesion protected by the barrier.
88
PERIODONTOLOGY 2000
The biological rationale of the procedure is
based on prevention of migration of the epithelial
periodontal tissues into the osseous defect, allowing
time for bone and other attachment tissues to heal.
During normal healing, it appears that the epithelial
tissues migrate rapidly into the wound, preventing
regeneration (47). The placement of a barrier
membrane would thus ensure that the detached
root surface becomes repopulated with cells from
periodontal ligaments capable of forming bone,
periodontal ligament and cementum. An alternative
explanation is that the membrane provides suf-
cient space for optimal wound stability, an essential
prerequisite for periodontal regeneration to occur
(37, 119).
Results from systematic reviews
Recently, two systematic reviews have been pub-
lished aimed at assessing the additional efcacy of
GTR in the treatment of periodontal intraosseous
defects with respect to OFD (71, 73). The reviews
were based on randomized clinical trials of at least 6
(71) or 12 months (73) duration. In one systematic
review (71) case-control studies as well as cohort
studies were included. The literature search was
extended up to and including October 2000 in one
systematic review (73), and up to January 2002 in the
other (71). Quality assessment of GTR systematic
reviews is summarized in Table 1.
The study population was limited to patients with a
clinical diagnosis of chronic adult periodontitis or
periodontitis in subjects aged 21 years or older. The
lower age limit was selected to include as many
studies as possible, but studies specically treating
early onset forms of disease were excluded.
Short-term clinical outcome was dened as chan-
ges in clinical attachment levels, probing depths and
gingival recession. Short-term changes in the mor-
phology of the bony lesion were evaluated by means
of radiographic assessment and hard tissue probing
at surgical re-entry. Long-term clinical outcomes
included: disease recurrence (as percentage of sites
with 2 mm loss of probing attachment measured
from 12 months after treatment), and tooth loss. Ease
of maintenance (as percentage of sites with <4 mm
probing depth), change in aesthetic appearance,
postoperative complications, cost benet and
patient well-being were considered as patient-
centered outcomes.
A weighted treatment effect was calculated and the
results were expressed as the weighted mean differ-
ence and 95% condence interval for continuous
outcomes and relative risk and 95% CI for dichot-
omous outcomes. The primary outcome measure was
gain in attachment. The signicance of discrepancies
in the estimates of the treatment effects from the
different trials (or heterogeneity) were assessed by
means of Cochrans test for heterogeneity. If any
signicant heterogeneity (P < 0.1) was detected, the
signicance of the treatment effects was re-assessed
by using a random effects model and any hetero-
geneity was investigated. Subgroup analyses were
planned to investigate the effects of factors thought
to have the most inuence on periodontal outcomes,
including smoking status and initial disease severity.
In the two systematic reviews, four types of inter-
vention were considered:
GTR vs. open ap debridement;
GTR and bone substitutes combined vs. OFD;
GTR plus some form of augmentation, usually a
particulate bone graft, vs. GTR alone;
GTR using an expanded polytetrauoroethylene
(ePTFE) barrier membrane vs. GTR using a bioab-
sorbable barrier device.
GTR vs. OFD
The evaluation of clinical attachment level change
for GTR alone vs. OFD was based on 10 studies (4, 12,
14, 15, 17, 59, 68, 82, 85, 111) in Needleman et al.s
systematic review (73). The other review (71), using
broader inclusion criteria for study selection, found
19 studies comparing GTR vs. OFD (1, 4, 7, 14, 15, 17,
18, 45, 51, 52, 59, 74, 81, 83, 85, 98, 110, 111, 129).
The results from both systematic reviews showed a
limited but statistically signicantly greater attach-
ment gain for test groups compared with OFD. For
GTR, the weighted mean difference between test and
control was 1.11 mm (95% CI: 0.631.59) in one
systematic review (73), and 1.15 mm (range: 0.20
2.90) in the other (71, data not shown) (Table 2). In
both systematic reviews, meta-analysis showed a
high level of heterogeneity among the results from
different studies.
The nding of heterogeneity is important as it
suggests that the studies within the meta-analysis are
too dissimilar to combine meaningfully, i.e. there is a
systematic difference in one or more aspects of the
studies that is not due to chance. This could be
related to the types of patients defects, the type of
treatment or the internal validity of the studies. A
more detailed discussion on heterogeneity will be
addressed in a separate section. Despite these
differences, the most likely conclusion is that GTR
provides a greater gain in attachment than the
89
Reconstructive procedures for intraosseous defects
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90
Trombelli
Table 2. Mean differences (in mm) in clinical outcomes between reconstructive procedure and control (open ap
debridement) procedures as assessed in systematic reviews
Reconstructive
procedure
Systematic
reviews
Outcome No. of
studies
Weighted
mean
difference
(mm)
95% CI
(SD) [SE]
P-value
for
difference
P-value for
heterogeneity
GTR Murphy &
Gunsolley
(71)
CAL change 18 1.15 N A <0.0001 N A
PD change 15 1.04 N A <0.0001 <0.01
Needleman
et al. (73)
CAL change 10 1.11 0.63, 1.59 * <0.001
PD change 5 0.80 0.14, 1.46 * 0.04
Bone gain at
re-entry
3 1.39 1.08, 1.71 * 0.65
GTR + bone
substitutes
Needleman
et al. (73)
CAL change 2 1.25 0.89, 1.61 * 0.91
PD change 2 1.24 0.89, 1.59 * 0.85
Bone gain at
re-entry
1 3.37 3.14, 3.61 *
Autogenous
bone graft
Trombelli
et al. (115)
CAL change 1 1.20 [0.39] >0.20
Reynolds
et al. (88)
CAL change 3 0.72 (1.82) 0.030 NS
PD change 1 0.60 (1.35) 0.062
Bone ll 2 1.62 (1.53) 0.058 0.004
Bone allograft Trombelli
et al. (115)
CAL change 6 0.36 )0.16, 0.87 0.174 0.013
PD change 6 0.41 0.16, 0.66 0.001 0.067
Reynolds
et al. (88)
CAL change 11 0.44 (2.25) 0.008 NS
PD change 9 0.43 (2.25) 0.032 NS
Bone ll 12 1.06 (1.97) <0.0001 NS
Dentin
allograft
Trombelli
et al. (115)
CAL change 1 0.80 [0.38] >0.50
Coralline
calcium
carbonate
Trombelli
et al. (115)
CAL change 4 0.90 0.53, 1.27 <0.001 0.104
PD change 4 0.04 )1.78, 1.87 0.962 <0.001
Reynolds
et al. (88)
CAL change 4 0.91 (1.94) 0.004 NS
PD change 4 0.09 (2.16) 0.886 NS
Bone ll 3 2.21 (1.82) <0.0001 NS
Bioactive glass Trombelli
et al. (115)
CAL 4 1.04 0.31, 1.76 0.005 0.024
PD change 4 0.60 0.20, 1.00 0.003 0.684
Reynolds
et al. (88)
CAL change 4 1.05 (1.89) 0.022 NS
PD change 4 0.71 (2.22) 0.018 NS
Bone ll 4 1.61 (1.47) 0.086 0.006
Porous
nonporous
hydroxyapatite
Trombelli
et al. (115)
CAL change 4 1.40 0.64, 2.16 <0.001 0.013
PD change 5 0.98 0.67, 1.29 <0.000 0.070
Reynolds
et al. (88)
CAL change 4 1.20 (2.22) 0.003 NS
PD change 6 0.74 (2.12) 0.030 NS
Bone ll 5 1.58 (1.77) <0.000 0.04
91
control treatment, although the size of this superi-
ority is unclear.
When considering the number of sites gaining less
than 2 mm of attachment (73), again a signicant
benet was shown for GTR [relative risk 0.58 (95%CI:
0.380.88, chi-square for heterogeneity 5.72 (3 df),
P 0.13)] (20, 21, 80). The number needed to treat
with GTR to achieve one extra site gaining 2 mm or
more attachment over OFD was therefore 8 (95% CI:
433), based on an incidence of 32% of sites in the
control group failing to gain 2 mm or more of
attachment.
For change in probing depth, ve studies were
found for GTR alone including change in probing
depth as an outcome (4, 17, 59, 68, 85) in one sys-
tematic review (73), and 18 studies (1, 4, 7, 14, 15, 17,
18, 45, 51, 52, 59, 74, 81, 83, 99, 110, 111, 129) in the
other (71). The results demonstrated a small but
signicantly greater probing depth reduction for
GTR. Weighted mean difference ranged from
1.04 mm (range: 0.052.66 (71, data not shown) to
0.80 mm (95% CI: 0.141.46) (73) (Table 2). Again, a
signicant heterogeneity among studies was ob-
served in both systematic reviews.
The only study (59) containing the radiographic
data in one systematic review (73) showed a 0.6 mm
gain in bone from the base of the defect in both test
and control groups. The other systematic review (71)
included only one study (51) that demonstrated a
difference in gain of bone height of 0.90 mm, favor-
ing GTR over OFD.
A statistically signicantly greater gain in hard
tissue probing was found for GTR compared with
open ap debridement. This amounted to a weighted
mean difference of 1.39 mm [95% CI: 1.081.71,
chi-square for heterogeneity 0.85 (2 df), P 0.65]
(Table 2) (73). In Murphy & Gunsolleys systematic
review (71), the additional bone gain provided by
GTR at re-entry varied from 0.20 mm to 1.16 mm,
with one of the selected studies favoring OFD.
GTR + bone substitutes vs. OFD
In one systematic review (73) two studies were
selected to evaluate the adjunctive benet of GTR +
bone substitutes vs. OFD (4, 53). GTR + bone
substitutes resulted in an additional clinical
attachment level gain of 1.25 mm [95% CI: 0.89
1.61, chi-square for heterogeneity 0.01 (1 df),
P 0.91] (Table 2). For change in probing depth,
the results demonstrated a small but signicantly
greater probing depth reduction for GTR + bone
Table 2. Continued
Reconstructive
procedure
Systematic
reviews
Outcome No. of
studies
Weighted mean
difference
(mm)
95% CI
(SD) [SE]
P-value
for
difference
P-value for
heterogeneity
PMMA-PHEMA Trombelli
et al. (115)
CAL change 1 0.90 [0.22] 0.001
PD change 1 0.90 N A 0.003
Reynolds
et al. (88)
Bone ll 1 1.26 N A 0.001
Polylactic acid
granules
Trombelli
et al. (115)
CAL change 1 )1.45 N A N A
PD change 1 )1.60 (0.55) N A
Reynolds
et al. (88)
Bone ll 1 )0.28 N A 0.519
Enamel
matrix
proteins
Trombelli
et al. (115)
CAL change 5 1.33 0.78, 1.88 <0.000 <0.001
PD change 5 1.60 0.59, 2.62 0.002 <0.001
Esposito
et al. (21)
CAL change 8 1.31 0.84, 1.78 <0.001 <0.001
PD change 8 0.96 0.50, 1.41 <0.001 0.002
Radiographic
bone level
1 2.0 0.88, 3.12 <0.001
CAL, clinical attachment level. CI, condence interval. GTR, guided tissue regeneration. PD, probing depth. PMMA-PHEMA, polymethylmethacrylate and
polyhydroxylethylmethacrylate. SD, standard deviation, SE, standard error. NS, not signicant.
*P-values are not given for Needleman et al. (73) because the 95% condence interval was reported.
92
Trombelli
substitutes [weighted mean difference 1.24 mm
(95% CI: 0.891.59, chi-square for heterogeneity
0.03 (1 df), P 0.85] (Table 2). Data stemming from
one study (4) showed a difference in hard tissue
probing of 3.37 mm (95% CI: 3.143.61).
GTR with ePTFE membrane vs. GTR with
bioabsorbable membrane
In one systematic review (71) three studies were
included which directly compared the efcacy of a
bioabsorbable barrier (polylactic acid or polylactic
acid 910) with that of an ePTFE membrane (11, 20,
118). Meta-analysis failed to demonstrate a signi-
cant difference in clinical attachment level gain and
probing depth reduction between bioabsorbable and
non-resorbable membranes.
GTR + bone substitutes vs. GTR alone
The efcacy of the addition of a bone substitute
(mainly demineralized freeze-dried bone allograft)
under a membrane device was investigated in one
systematic review (71). Meta-analysis of the seven
selected studies (3, 10, 31, 33, 62, 77, 112) did not
reveal any adjunctive effect on either clinical
attachment level gain or probing depth reduction.
There was signicant heterogeneity among the
studies when clinical attachment level gain was
considered (P < 0.03).
Long-term and patient-centered
outcomes
Interestingly, no data were found on either long-term
clinical outcomes or patient-centered outcomes.
Therefore, no statements can be made regarding the
efcacy of periodontal therapy using various GTR
procedures to enhance tooth retention.
Heterogeneity
When considering the additional effect of GTR with
or without bone substitutes with respect to OFD,
meta-analysis showed a statistically signicant het-
erogeneity of results from different studies, implying
that the differences between the outcomes of inclu-
ded studies are greater than would occur by chance.
The studies appear too dissimilar (some studies favor
GTR, some studies show no difference between
treatments) in certain respects to be sensibly com-
bined and overall summary values should be inter-
preted with caution.
How condent can we be that GTR is of greater
benet than OFD? The most likely threat to this
conclusion is bias. Several possible sources of bias
have been investigated in one systematic review,
including publication bias, concealment of group
allocation and blinding of examiner and surgeon (73).
Publication bias is the preferential publication of
studies with positive outcomes over studies with
negative results. Investigation of this showed that it
was unlikely to contribute to the results of the meta-
analysis (73). However, tests for publication bias
have a low power of detection. Since study quality
has been shown to have a direct impact on the size
of the effect of treatment (67, 93), Needleman et al.
(73) explored this effect with sensitivity analyses
including a compound quality scale (44) and the
individual parameters of allocation concealment,
examiner and therapist blinding. The results of these
analyses did not help to explain the differences
between studies, as heterogeneity was not elimin-
ated despite the reduced number of included
studies. In view of the limitations in study design,
we should be aware that bias could be contributing
to these results.
A possible explanation for the heterogeneity might
be variability between studies in prognostic factors
that have been documented to affect the outcome of
regenerative surgery. Factors affecting GTR outcome
have been extensively explored in the past 15 years. It
has been suggested that the primary factors that
inuence the successful management of intraosseous
defects when treated by barrier membranes include,
but are not limited to, bacterial contamination,
innate wound healing potential, local site charac-
teristics, and surgical procedures (54). Evaluation of
the relative impact exerted by these factors may
help to explain the variability in outcome variables
in studies selected for meta-analysis (high level of
heterogeneity). Variability of results is clearly an
important issue when considering the relevance of
a treatment to clinical practice. In their systematic
review, Needleman and coworkers (73) have attemp-
ted to explore some of the possible causes of the
heterogeneity that emerged from the results across
studies (sensitivity analysis).
A longitudinal study on the GTR procedure for
the treatment of intraosseous defects has clearly
demonstrated that the stability of the regenerative
outcome is strictly dependent on a regular recall
program including supra- and subgingival plaque
control (13). To investigate the robustness of the
results with respect to the very frequent mainten-
ance, which may be impractical to provide in many
93
clinics (more often than every 3 months), a sensitivity
analysis was conducted from which studies with very
frequent follow-up were excluded. This eliminated
heterogeneity and showed a small reduction in the
weighted mean difference in clinical attachment level
gain favoring GTR vs. OFD (0.9 mm, 95%CI: 0.61.1).
The benets of plaque control on the short- and
long-term response to periodontal therapy are well
established. In contrast, smoking habit is associated
with adverse healing after therapy and, particularly,
less favorable regeneration (15, 109, 113). When
approaching a sensitivity analysis regarding plaque
and smoking, it was apparent that substantial dif-
ferences in the way that both factors are reported
between studies prevented sensible comparisons
(73). The effect of smoking on reducing the gain in
attachment following surgery is reported in only
one subgroup analysis of a randomized clinical trial
(59). This highlights the need for more research
into prognostic factors to help explain heterogen-
eity.
One factor of paramount importance in explaining
the study differences is the surgical technique used,
with particular emphasis on preservation of the
interdental tissues and or coronal positioning to
ensure primary closure and prevent limit membrane
exposure. In Needleman et al.s review (73), two
studies from the same group (14, 15) reported an
attachment gain approximately twice that of the total
group estimate. The authors also observed that when
this same treatment approach was examined in a
multicenter trial, although with less frequent main-
tenance (11 different clinicians), the clinical attach-
ment level gain was highly variable between centers,
showing a smaller overall benet of GTR compared to
OFD (111). They conclude that although the efcacy
of GTR has been demonstrated in some studies, the
effectiveness and external validity of such a tech-
nique may be questioned.
Concluding remarks
Data stemming from the two available systematic
reviews on the effect of GTR for intraosseous defects
indicate that:
GTR most likely provides an additional benet, in
terms of clinical attachment level gain, probing
depth reduction and defect ll, when compared to
OFD in the treatment of deep intraosseous defects.
A limited number of studies showed that the
additional effect of the combination treatment (i.e.
GTR + bone substitutes) is similar to GTR alone
when compared to OFD with respect to attach-
ment gain, but results in slightly more probing
depth reduction and greater gain in hard tissue
probing at re-entry surgery.
There is no evidence for a difference between
bioabsorbable and non-resorbable (ePTFE) mem-
branes in producing clinical attachment level gain
and probing depth reduction.
The addition of a bone substitute to a membrane
device does not seem to produce any adjunctive
effect compared to the use of a membrane device
alone.
Long-term clinical outcomes or patient-centered
outcomes could not be determined due to the lack
of available data.
A substantial variation was observed in the results
from the included studies. In both systematic
reviews, the attachment gain varied greatly
following both GTR and OFD. The mean difference
between GTR and OFD surgery varied from 0.02 to
2.60 mm between studies in one systematic review
(73), and from 0.20 mm and 2.90 in the other
(71). The heterogeneity was large and unexplained.
The role of bias could not be eliminated.
Grafting procedures
Background
One of the most investigated methods used to
achieve the reconstruction of intraosseous defects is
to combine access surgery with placement of bone
grafts or implant biomaterials into the debrided bony
lesion in order to regenerate the lost periodontal
tissues. Grafting biomaterials include autogenous
grafts, allogenic grafts, xenogenic grafts and allo-
plastic materials. The assumption behind the clinical
use of grafting procedures is that the complete
regeneration of the attachment apparatus (including
new bone formation and new connective tissue
attachment) would be enhanced by the various bio-
materials due to their osteogenetic potential (if the
graft contained viable bone-forming cells), osteoin-
ductive capacities (exerted by the release of bone-
inducing substances), or osteoconductive properties
(i.e. the possibility to create a scaffold to support
bone formation). Observational and controlled trials
have generally reported an additional benet in terms
of soft and hard tissue improvements following the
use of grafting procedures. However, due to the large
variety of graft biomaterials on the market, the
magnitude of such improvements as well as the
consistency of the advantage achieved when grafting
94
Trombelli
procedures are compared to an access ap procedure
remains to be determined.
Data to assess the effect of the use of grafting proce-
dures in the treatment of intraosseous defects were
derived from two recent systematic reviews (88, 115).
Our estimation was limited to assessing the additional
efcacy of different bone substitutes, either alone or in
combination, when compared to OFD. In both sys-
tematic reviews, all studies grouped for the meta-
analysis were identied as randomized clinical trials.
The literature searchwas extendeduptoandincluding
June 2001 in one systematic review (115), and up to
October 2002 in the other (88). The characteristics of
the two systematic reviews are summarized inTable 1.
The therapeutic endpoints examined included
short-term (as evaluated 12 months after interven-
tion) and long-term (as evaluated 13 months or more
after intervention) changes in clinical attachment
level, probing depth, and bone level (defect ll). The
incidence of disease recurrence and incidence of
tooth loss were also considered outcome variables.
Data synthesis and analysis were similar to those
reported for GTR. In one systematic review (115),
only studies where the patient, not the defect site,
was regarded as the statistical unit were selected. In
Reynolds et al.s systematic review (88), multiple
defect sites from the same subject were averaged to
provide a pooled estimate of the true outcome
variable for the individual. Moreover, the primary
outcome measures were clinical attachment level
gain in our systematic review (115), and change in
bone level in the other (88). Our review (115) was
based on 21 studies (2, 4, 5, 22, 26, 27, 49, 52, 55, 58,
60, 69, 70, 76, 80, 94, 100, 122124, 126), while the
other (88) was based on 27 studies (2, 4, 5, 8, 22, 25,
26, 49, 51, 52, 55, 57, 58, 60, 61, 63, 69, 76, 80, 84, 86,
91, 92, 120, 122124). Differences in the inclusion
criteria for study eligibility may partly explain the
discrepancy in the number of selected studies
between the two systematic reviews.
In one systematic review (115), grafting procedures
agents were separately analyzed as the following:
autogenous bone, bone allograft, dentin allograft,
coralline calcium carbonate, bioactive glass,
hydroxyapatite, calcium-layered composite of
polymethylmethacrylate and polyhydroxylethyl-
methacrylate (PMMA-PHEMA), and polylactic acid
granules. In the other systematic review (88), graft
materials were categorized on an a priori basis into
one of the following categories: autogenous bone,
bone allograft, porous nonporous hydroxyapatite
(calcium phosphate ceramic), bioactive glass, and
other (including coralline calcium carbonate; poly-
lactic acid; PMMA, PHEMA and calcium hydroxide
polymer; hydroxyapatite cement; hydroxyapatite-
glycosaminoglycan).
Autogenous bone
Only one parallel-arm trial (70) comparing the auto-
genous bone to the OFD procedure was selected in
our systematic review (115). The results indicated a
greater clinical attachment level gain for the grafted
group (clinical attachment level gain: 3.2 mm, SD 0.5)
than for controls (clinical attachment level gain:
2.0 mm, SD 0.8). However, the difference in clinical
attachment level gain between groups (1.20 mm, SE
0.39) was not statistically signicant (Table 2). In the
systematic review by Reynolds et al. (88) autogenous
bone treatment resulted in signicantly greater clin-
ical attachment level gain (weighted mean difference:
0.72 mm, SD 1.82) and bone ll (weighted mean
difference: 1.62 mm, SD 1.53) for autogenous bone
compared to OFD (Table 2).
Bone allograft
Six studies (2, 4, 5, 22, 58, 124) met the inclusioncriteria
for comparing bone allograft to OFDin one systematic
review (115) and 12 studies (2, 4, 5, 8, 22, 57, 58, 60, 63,
69, 92, 124) met the criteria in the other (88). Meta-
analysis showed a greater clinical attachment level
gain for the grafted group with respect to OFD (Ta-
ble 2). Weighted mean difference between graft and
control ranged from 0.36 mm (95% CI: )0.160.87;
P 0.174) (115), to0.44 mm(SD2.25) (88). Signicant
heterogeneity [Q-test for heterogeneity: 14.40 (5 df),
P 0.01] was foundbetweenstudies inone systematic
review (115), but not in the other (88).
Adjunctive defect ll amounted to 1.06 mm (SD
1.97) with the use of bone allograft (88). In both
systematic reviews, a signicantly greater probing
depth reduction was reported for bone allograft
treatment [0.41 mm, 95% CI: 0.160.66 (115);
0.43 mm, SD 2.25 (88)] compared to OFD (Table 2).
These results were consistent among the different
studies (heterogeneity not signicant).
Dentin allograft
Clinical attachment level gain following treatment
with dentin allograft was evaluated in one trial (70).
Clinical attachment level gain amounted to 2.8 mm
95
(SD 0.7) for grafted patients and 2.0 mm (SD 0.8) for
controls. The difference in clinical attachment level
gain between groups (0.80 mm, SE 0.38) was not
statistically signicant (Table 2).
Coralline calcium carbonate
Meta-analysis of four selected studies (52, 69, 94, 123)
resulted in a consistent and statistically signicant
difference in clinical attachment level gain between
groups [weighted mean difference 0.90 mm (95%CI:
0.531.27; Q-test for heterogeneity: 6.16 (3 df),
P 0.10] (115) (Table 2). Reynolds et al. (88) con-
sistently reported a weighted mean difference in
clinical attachment level gain of 0.91 mm (SD 1.94)
(Table 2). When changes in bone level were consid-
ered, the analysis from three studies (52, 69, 123)
suggested that coralline calcium carbonate produces
a signicant (P 0.001) and consistent (heterogen-
eity not signicant) treatment effect (gain in bone ll:
2.21 mm, SD 1.82) (88). In contrast, treatment with
coralline calcium carbonate did not produce any
signicant improvement in probing depth reduction
[weighted mean difference: 0.04 mm (115), and
0.09 mm (88)] (Table 2).
Bioactive glass
Treatment of intraosseous defects by means of bio-
active glass resulted in an improvement of the bony
lesion when compared to the OFD procedure.
Weighted mean difference in clinical attachment
level gain between bioactive glass and OFD was
1.04 mm [95%CI: 0.311.76; Q-test for heterogeneity:
9.44 (3 df), P 0.02] in one systematic review (115),
and 1.05 mm (SD 1.89) in the other (88) (Table 2).
Meta-analysis of change in bone ll revealed a
greater, although not signicant, increase (1.61 mm,
SD 1.47) for bioactive glass than for OFD (Table 2).
Signicant heterogeneity was found across the studies
(P 0.006), the inconsistency being attributable to
one study (76) that reported a more favorable change
in bone ll following an OFD procedure (88). Meta-
analysis also showed that bioactive glass resulted in
signicantly greater probing depth reduction than the
OFD procedure [weighted mean difference: 0.60 mm,
95% CI: 0.201.00 (115); 0.71 mm, SD 2.22 (88)]
(Table 2).
Porous nonporous hydroxyapatite
Various forms of hydroxyapatite resulted in signi-
cantly greater clinical attachment level gain
compared with the OFD [weighted mean difference:
1.40 mm, 95% CI: 0.642.16 (115); 1.20 mm, SD 2.22
(88)] (Table 2). However, meta-analysis from one
systematic review (115) revealed that there was highly
signicant evidence of heterogeneity among studies
[Q-test for heterogeneity: 10.72 (3 df), P 0.01].
Weighted mean difference in bone ll between
hydroxyapatite and OFD was 1.58 mm (SD 1.77), the
difference being statistically signicant (88) (Table 2).
Again, signicant heterogeneity was found in the
studies (P 0.04). Meta-analysis showed that hy-
droxyapatite resulted in signicantly greater probing
depth reduction than the OFD procedure (Table 2).
Weighted mean difference ranged from 0.98 mm
(95%CI: 0.671.29) in one systematic review (115) to
0.74 mm (SD 2.12) in the other (88). Signicant het-
erogeneity in studies was found in one systematic
review (115), but not in the other (88).
Polymethylmethacrylate and
polyhydroxylethylmethacrylate
Meta-analysis was not possible for this group since the
SE of the difference could not be imputed in one (100)
of the two selected studies (100, 123). One study
showed a clinical attachment level gain of 3.43 mm in
the test group, and 2.73 mm in the control group, dif-
ference 0.70 mm (100). In the other systematic review
(115) a clinical attachment level gain of 1.9 mm (SD
1.1) in the test group, and 1.0 mm (SD 0.9) in the
control group was found. The difference between
treatments (0.90 mm, SE 0.22) was statistically signi-
cant (P < 0.001) (Table 2). Polymethylmethacrylate
and polyhydroxylethylmethacrylate was also found to
improve bone levels and result in a statistically signi-
cant probing depth reduction relative to OFD (122).
Polylactic acid granules
One trial (60) evaluated the adjunctive effect of
polylactic acid plus OFD compared to OFD only.
Clinical attachment level gain was 0.50 mm for
polylactic acid treatment and 1.95 mm for the OFD
procedure (difference )1.45 mm) (Table 2).
Long-term outcomes
Most of the studies did not provide information
about long-term outcomes of the grafting procedure,
in terms of either disease recurrence or incidence
of tooth loss during follow-up. In one study (22)
6-month vs. 36-month clinical attachment level
recordings were compared. A 0.12 mm gain in
96
Trombelli
clinical attachment level was observed for the test
procedure and a 0.43 mm decrease for the control
procedure from 6 to 36 months postsurgery. In
another study (27) the 48-month clinical attachment
level was assessed. When compared to the 12-month
clinical attachment level, a 0.27 mm decrease for the
grafted group and a 0.14 mm gain for the OFD group
were observed. A long-term evaluation of a hydroxy-
apatite graft compared to OFDshowed that 40%of the
OFD defects lost attachment over the 5-year follow-
up, whereas two thirds of the hydroxyapatite defects
gained attachment over the same interval (121).
Patient-centered outcomes
There are insufcient and inconsistent data to enable
analytic comparisons among different grafting pro-
cedures to be made with respect to OFD related to
patient-centered outcomes. In most of the studies, no
systemic or local adverse effects in grafted patients
were observed. Adverse effects included pebbled
surface texture of grafted site (76), transient slight
gingival inammation (5), delayed soft tissue healing
(70), and exfoliation shedding of implanted bioma-
terial (27, 55, 122). The information available revealed
the absence of patient-related differences in fre-
quency of complaints, level of comfort, and need for
analgesia (122, 124, 125). Patient-centered outcomes
where no data were found were ease of maintenance,
change in aesthetic appearance, estimation of patient
well-being derived from additional use of grafting
biomaterials biologicals, and cost benet ratio.
Heterogeneity
In both systematic reviews (88, 115), different grafting
procedures have been crudely grouped and separately
analyzed with respect to their nature or physico-
chemical characteristics. This may have resulted in
pooling graft materials with different biologic poten-
tial in enhancing periodontal regeneration and, con-
sequently, clinically assessed outcomes. However, the
heterogeneity in clinical attachment level gain or bone
ll reported between different studies dealing with the
same bone substitute seems to suggest that other
factors may signicantly inuence the variability in
clinical response. It should be also pointed out that for
some grafting procedures (such as bone allograft and
bioactive glass) the heterogeneity was due to studies
which reported a more favorable change in bone ll
following an OFD procedure along with studies fav-
oring the grafting procedure, thus calling into ques-
tion the clinical validity of the graft biomaterial. In
contrast, for hydroxyapatite-derived biomaterials,
heterogeneity was associated with a generally positive
treatment effect across studies. In this case only the
magnitude of benet for grafting procedures over
OFD needs to be determined.
Due to limited sample sizes for each treatment
modality, sensitivity analysis including individual
components of quality assessment could not be
performed. The limited number of studies in the
analyses also prevented formal testing for publication
bias. It was therefore not possible to determine the
effect of publication bias on the results. Nevertheless,
the possibility that publication bias may have exag-
gerated the treatment effects should be considered
when interpreting the results of the review.
Among patient characteristics, supragingival pla-
que accumulation and smoking status have been
reported to be negatively correlated with treatment
outcome associated with the use of reconstructive
procedures (54). In the studies considered in our
systematic review, differences in methods for plaque
assessment between studies (full-mouth plaque
scores, full-mouth plaque index, plaque index at the
experimental sites only) prevented exploration of the
relevance of this potential prognostic factor on out-
come variability. It should, however, be pointed out
that in all selected studies, the surgical phase was
always preceded by multiple sessions of cause-
related therapy and followed by a stringent main-
tenance program. This may have limited the
detrimental impact of plaque on healing response.
Regarding the effect of smoking status, no studies
reported a separate analysis of outcome variables in
smokers and nonsmokers, thus impeding any evalu-
ation of this predictor.
Previous studies reported that variability in clinical
outcome may reect variability in defect characteris-
tics, including preoperative attachment level and
probing depth, intrabony wall components, anddefect
depth and angle (54, 56). In particular, the magnitude
of the differences in outcome parameters, such as
clinical attachment level gain or bone ll, seems to
parallel initial differences in average pretreatment
depth of the intrabony component of the defect (56). It
was not possible with the data available to perform a
sensitivity analysis to explore the relevance of these
factors in determining the heterogeneity.
Periodontal reconstructive surgery for intraosseous
defects is a technique-sensitive procedure. Selection
of a specic ap design, in relation to anatomic
characteristics of interdental space and loca-
tion morphology of bony lesion, and proper suturing
technique may signicantly contribute to soft and
97
hard tissue changes following surgery (115). This is
partly conrmed by a signicant center-related effect
on treatment outcome observed when a specic bone
substitute has been evaluated in a multicenter trial
(124). Further studies are therefore needed to deter-
mine how and to what extent surgical variables may
inuence treatment outcome when a regenerative
procedure is performed in association with grafting
procedures.
Study quality has been shown to have a direct
impact on the size of the effect of treatment (67, 93),
thus potentially contributing to heterogeneity
between studies. However, due to limited sample
sizes for each treatment modality, sensitivity analysis
including individual components of quality assess-
ment could not be performed. The limited number of
studies in the analyses also prevented formal testing
for publication bias. It was therefore not possible
to determine the effect of publication bias on the
results. Again, this effect should be considered when
interpreting the results of these systematic reviews.
Concluding remarks
In conclusion, the results from the two available
systematic reviews on the effect of grafting proce-
dures for the treatment of intraosseous defects indi-
cate the following:
Apart from polylactic acid, the use of grafting
procedures produces a greater clinical attachment
level gain and bone ll when compared to the OFD
procedure. A greater probing depth reduction is
also generally observed with graft biomaterials.
However, differences in clinical attachment level
gain and bone ll between grafting procedures and
OFD procedures varied greatly with respect to
different graft biomaterials.
A marked variability in hard and soft tissue
improvements among studies dealing with the
same bone substitute (heterogeneity) was also
observed. This variability prevents a formal esti-
mation of how great a difference will result from
treatment.
Due to limited information on long-term outcome,
it is unclear whether stability of periodontal sup-
port and tooth survival are affected by application
of grafting procedures.
Variations in grafting biomaterials and procedures
as well as lack of objective standardized data did
not allow for a meaningful summary of treatment-
related adverse effects as well as evaluation of
cost benet ratio.
Enamel matrix proteins
Background
Enamel matrix proteins (Emdogain
, Biora AB,
Malmo, Sweden) constitute a commercially available
compound consisting mainly of amelogenin and
related proteins derived from porcine tooth buds.
During fetal life, these enamel matrix proteins are
secreted and temporarily deposited on the root sur-
face by the cells of Hertwigs epithelial root sheath,
being essential for the formation of acellular
cementum and development of associated perio-
dontal ligament and alveolar bone (34, 43, 106, 107).
It is believed that enamel matrix proteins used in
periodontal lesions mimic the development of the
tooth-supporting apparatus during tooth formation
(34). Recently, enamel matrix proteins have been
shown to be effective in regenerating the periodontal
attachment apparatus both in animals (35, 97) and
in humans (39, 40, 66, 95, 96, 116, 125), and in
improving the clinical attachment level in deep
intrabony defects (3840, 79, 81, 95, 96, 103).
Three systematic reviews have been recently pub-
lished with the purpose to determine the additional
efcacy of enamel matrix proteins in the treatment of
periodontal intraosseous defects with respect to
either OFD (21, 29, 115) or GTR (21). Two systematic
reviews were based on randomized clinical trials
(115) or quasi-randomized clinical trials (29) of at
least 68 months duration, and one systematic
review included only randomized clinical trials with a
1-year follow-up (21). The literature search was
extended up to and including June 2001 for Trombelli
et al.s systematic review (115), up to April 2002 for
Giannobile & Somerman systematic review (29), and
up to January 2003 for Esposito et al.s systematic
review (21). Search strategy and inclusion criteria
resulted in the selection of ve studies (24, 75, 81,
103, 108) for Trombelli et al.s (115), eight studies for
Giannobile & Somerman (24, 40, 75, 81, 99, 103, 108,
127), and 10 studies (23, 40, 75, 81, 98, 99, 103, 104,
108, 128) for Esposito et al. (21). Quality assessment
of systematic reviews of enamel matrix proteins is
summarized in Table 1.
The study population was extended to patients
affected by periodontitis with intraosseous defects to
be treated. The clinical attachment level change was
regarded as the primary outcome measure. Changes
98
Trombelli
in probing depth, gingival recession and radiographic
bone level were also considered. Evaluation of long-
term benets included ease of maintenance based on
residual probing depth, incidence of relapsing or
recurrent disease, and tooth loss. Changes in aes-
thetic appearance, postoperative complications
(infection, soft tissue dehiscences), pain, tooth
hypersensitivity, cost benet and patient well-being
were considered as patient-centered outcomes.
In data pooling, a weighted treatment effect was
calculated and the results were expressed as weighted
mean differences (and 95% CI) for continuous out-
come variables using both xed and random models
(115) or a random model only (21). In one systematic
review (21) the signicance of any discrepancies in
the estimates of the treatment effects from the dif-
ferent trials was assessed by means of Cochrans test
for heterogeneity and any heterogeneity was investi-
gated. In Trombelli et al. (115) if any signicant het-
erogeneity (P < 0.05) was detected, meta regression
was performed to explore heterogeneity.
Enamel matrix proteins vs. OFD
Overall, the results derived from the systematic
reviews indicate that there were signicant differences
between enamel matrix proteins and the OFD for
the three outcomes measured as change from the
baseline values: clinical attachment level, probing
depth, and radiographic marginal bone levels. There
was a signicant gain in mean clinical attachment
level for enamel matrix proteins compared with OFD
defects, with weighted mean difference of 1.33 mm
[95% CI: 1.011.42; Q-test for heterogeneity: 24.27
(4 df), P < 0.001] in one systematic review (115), and
1.31 mm (95% CI: 0.841.78, chi-square 32.9, 7 df,
P < 0.001) in another (21) (Table 2). However, in both
systematic reviews the analysis contained statistically
signicant heterogeneity in the results among studies.
In one systematic review (29), the additional clinical
attachment level gain achieved with enamel matrix
proteins with respect to OFD was not explicitly
reported.
A signicant reduction in probing depth was also
observed, with a weighted mean difference ranging
from 0.96 mm (95% CI: 0.501.41; P 0.002) (21) to
1.60 mm (95% CI: 0.592.62; P < 0.001) (115)
(Table 2). No data regarding the adjunctive probing
depth reduction with enamel matrix proteins was
reported in one systematic review(29). The signicant
increase in marginal bone levels favoring enamel
matrix proteins was only based on one trial in one
systematic review (21) with weighted mean difference
2.0 mm (95% CI: 0.883.12) (Table 2). There was no
statistically signicant difference in gingival recession
between Emdogain and OFD (21). No postoperative
infections or other adverse events were reported.
Enamel matrix proteins vs. GTR
Six trials from one systematic review (21) provided
data for this comparison between enamel matrix
proteins and GTR. A signicantly greater reduction in
probing depth was found for GTR compared to
enamel matrix proteins (weighted mean difference:
0.58 mm, 95% CI: 0.081.07; chi-square 8.9, 5 df,
P 0.11). There was also a signicant increase in
change in gingival recession for GTR with a weighted
mean difference of 0.47 mm (95% CI: 0.170.76;
chi-square 1.5, 4 df, P 0.82). No statistically sig-
nicant differences for clinical attachment level and
postoperative infections were observed between
treatments.
Heterogeneity
Systematic reviews have shown that the use of
enamel matrix proteins resulted in a statistically sig-
nicant improvement in average clinical attachment
level and probing depth over control ap surgery
when used in intrabony defects. Although all studies
generally showed an additional benet with the use
of enamel matrix proteins, a high degree of hetero-
geneity was found in the included trials.
In one systematic review (21) random effects meta
regression analysis was used to investigate which
factors might, at least in part, explain the hetero-
geneity in the comparisons between enamel matrix
proteins and OFD. Factors included antibiotics given,
surgical technique used in control group, funding by
manufacturer, risk of bias, baseline depth of intra-
bony defects and trial location. Only one of the ran-
dom effects meta regressions was signicant, where
manufacturer-funded studies found less recession
than the unfunded studies. Apart from this, Esposito
et al. (21) were unable to explain the heterogeneity
found between the studies. In their systematic
review, a planned subgroup for maintenance was not
possible as all studies were categorized as providing
very high levels of maintenance.
In our systematic review (115) we have attempted
to explore the effect of preoperative defect depth on
difference in clinical attachment level and probing
depth change as recorded in patients treated with
enamel matrix proteins compared to OFD. Meta
regression failed to detect an effect of initial defect
99
depth on the difference in clinical attachment level
gain and probing depth reduction between test and
control procedures. The lack of signicant correlation
may be partly explained by limited variability in
mean defect depth, as reported in different studies,
since a threshold for defect severity represented a
consistent inclusion criterion for most of the studies.
In all systematic reviews the relevance of publica-
tion bias on heterogeneity was not tested, therefore
preventing the exploration of the effect of publication
bias on the results. Again, the possible impact of
publication bias in exaggerating the size of the
treatment effect should be considered when inter-
preting the results of the review.
One of the potential drawbacks inherent in enamel
matrix protein treatment is its gel-like consistency
after reconstitution. This limits the space-making
potential of the preparation when used in intrabony
defects (66). Moreover, if primary closure is not
properly ensured over the interdental space, dis-
placement or contamination of the material may take
place, thus jeopardizing the regenerative potential. As
a consequence, provision of a secluded space by
means of adequate interdental tissue management
may allow enamel matrix protein-induced healing
process to occur undisturbed, maximizing the clinical
outcome. Adequate preservation of interdental soft
tissues may also limit the collapse of the ap into the
bone defect, thus optimizing available space for
regeneration (102, 114). These observations suggest
that the surgical application of enamel matrix pro-
teins is a technique-sensitive procedure. Although
the exploratory sensitivity analysis was not able to
detect any signicant difference with respect to the
surgical procedures used (traditional modied Wid-
man ap or new papilla preservation aps) (21), dif-
ferences in ap management with respect to the
physical properties of enamel matrix proteins may in
part explain the great variability in clinical attach-
ment level gain reported in the systematic reviews.
In this respect, results from a large multicenter
randomized clinical trial comparing enamel matrix
proteins to OFD (108), which involved eight expert
clinicians, showed that the difference in clinical
attachment level gain between the center performing
best and the center performing worst was more than
4-fold higher than the additional effect achieved by
the application of enamel matrix proteins.
Concluding remarks
Data from the three systematic reviews seem to
suggest the following:
Application of enamel matrix proteins resulted in
statistically signicant improvements in attach-
ment levels (additional clinical attachment level
gain of 1.3 mm) and probing depth reduction
(ranging from 1.0 mm to 1.6 mm) in comparison
with OFD.
General conclusions about the clinical relevance
(i.e. magnitude of the additional effect) of enamel
matrix proteins are limited by the high level of
heterogeneity found across the studies.
No evidence of major differences between enamel
matric derivative and guided tissue regeneration
could be found with the exception of slightly
more probing depth reduction (0.6 mm) due to
increased gingival recession (0.5 mm) in GTR-
treated sites.
Because of insufcient information on long-term
outcome, it was not possible to conrm the efc-
acy of enamel matrix proteins on the stability of
periodontal support and tooth survival.
Conclusions
The aim of this article was to determine the effect of
GTR, grafting procedures or the application of
enamel matrix proteins in addition to OFD in the
treatment of deep intraosseous defects. Overall, data
resulting from systematic reviews indicate that all
reconstructive treatment modalities produce com-
parable and more favorable clinical improvements in
hard and soft tissue parameters of healing response
(i.e. clinical attachment gain, pocket reduction
and bone ll) compared to conventional OFD
procedures. Although the biomaterial-supplemented
reconstructive procedures are associated with a
generally positive treatment effects with respect to
OFD, a signicant heterogeneity was found among
studies in the different reconstructive procedures.
This limits the possibility of drawing general con-
clusions about the clinical relevance (in particular,
the magnitude of the adjunctive effect) of the addi-
tional use of GTR, grafting procedures or enamel
matrix proteins for the treatment of intraosseous
defects. Some of the possible causes of heterogeneity
have been explored; however, the limited number of
studies currently available did not permit denite
conclusions about which factors account for the
variability in treatment outcome. More research is
therefore needed to identify patient, site, choice of
material and technique factors associated with the
successful outcome of treatment of intraosseous
defects.
100
Trombelli
This review indicates that different reconstructive
procedures support comparable clinical outcomes. It
should, however, be considered that similar
improvements in clinical parameters do not neces-
sarily imply similar wound healing processes on a
histologic level. Whereas the use of some recon-
structive procedures, such as GTR and enamel matrix
proteins, has been demonstrated to result in a true
and complete periodontal regeneration, for some of
the graft biomaterials the effect on the formation
of a new attachment apparatus, including bone,
cementum and periodontal ligament, rather than
periodontal repair, is still a matter of debate.
Due to limited information on long-term outcomes,
it is unclear whether the stability of periodontal sup-
port and tooth survival are affected by the additional
application of reconstructive devices biomaterials.
While the improvements inprobing recordings may be
reasonably considered surrogate measurements rela-
ted to a better long-term tooth prognosis, we recom-
mend that more clinical studies should examine
whether and to what extent more compromised teeth
could be saved using a reconstructive procedure.
There are at present insufcient data to permit
analytic comparisons among different reconstructive
procedures with OFD with respect to patient-cen-
tered outcomes. When considering the adjunctive
effect of reconstructive procedures, evaluation of
adverse effects related to the additional use of bio-
materials biological agents, postoperative complica-
tions, ease of maintenance, change in aesthetic
appearance, estimation of patient well-being, and
cost benet ratio (including estimation of additional
treatment time and costs for implant placement of
biomaterials biological agents) should be carried
out. Studies including patient-centered outcomes will
be critical, as well as long-term follow-up cohorts to
examine the effect of a reconstructive biomateri-
al device on true therapeutic endpoints.
Acknowledgment
This study was supported by the Research Centre for
the Study of Periodontal Diseases, University of Fer-
rara, Italy. I wish to thank Dr. Roberto Farina for his
help in preparing the manuscript.
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105
Guided tissue regeneration for
periodontal intrabony defects a
Cochrane Systematic Review*
IAN NEEDLEMAN, RI CHARD TUCKER, ELAI NE GI EDRYS-LEEPER & HELEN
WORTHI NGTON
The key concept for periodontal therapy is to im-
prove periodontal health and by these means to sat-
isfy a patients aesthetic and functional needs or
demands (52, 55). Mostly, this is achieved though
repair of the periodontium; however, the rst reports
of guided tissue regeneration (GTR) as a clinical
periodontal surgical procedure appeared more than
20 years ago (21, 39). The rationale behind this
therapy is to achieve regeneration of the tooth sup-
porting structures, i.e. cementum, periodontal
ligament and alveolar bone. This is different from
conventional periodontal surgery, which achieves
healing by repair (20). Indeed, human histology has
demonstrated that such regeneration can occur fol-
lowing this procedure (9, 39, 49). However, histolog-
ical differences are not sufcient to recommend one
form of therapy over another. To consider a change in
clinical practice a new therapy must offer superior
properties; these may include clinical, patient-
centred or economic outcomes. In view of the num-
ber of research reports investigating GTR, pooling the
data is essential in order to understand the charac-
teristics of the intervention and to investigate poss-
ible causes of differences in outcomes.
Systematic reviews are widely recognized as the
least biased and most rational way to summarize
research evidence that evaluates heath care inter-
ventions (35). They are themselves research and are
rigorous and exhaustive syntheses of the literature
attempting to nd and appraise all available evi-
dence. As a result, such reviews are now considered
the highest level of evidence for medical interven-
tions (44). We rst reported the results of such a
systematic review of GTR in 2001 (37) and this is the
version of the review which has been considered in
a chapter of this volume of Periodontology 2000 (51).
Since then, additional studies have appeared and
this paper extends the review to include signicant
new data as well incorporating changes in review
methodology. The objective of this research was
therefore to assess systematically the efcacy of GTR
in the treatment of periodontal infrabony defects
measured against the current standard of surgical
periodontal treatment, open ap debridement, with
respect to clinical and patient-centred and econo-
mic outcomes.
Selection of included studies
To be considered for this review, studies needed to
be randomised controlled clinical trials of at least
12 months duration. Other criteria for inclusion
were patients with a clinical diagnosis of chronic
periodontitis or periodontitis in subjects aged
21 years or older. Studies that addressed GTR
exclusively around furcation-involved teeth were
excluded. The lower age limit was selected to in-
clude as many studies as possible but studies spe-
cically treating aggressive forms of disease were
excluded. Quality assessment issues are addressed in
Methods of review. The interventions investigated in
this study were GTR vs. open ap debridement and
*The results of a Cochrane Review can be interpreted
differently, depending on peoples perspectives and circum-
stances. Please consider the conclusions presented carefully.
They are the opinions of review authors, and are not necessarily
shared by the Cochrane Collaboration. A version of this review
has been published in The Cochrane Library. Cochrane
systematic reviews are regularly updated to include new
research, and in response to comments and criticisms from
readers. If you wish to comment on this, or other Cochrane
reviews of interventions for oral health, please send it to
emma.tavender@manchester.ac.uk.
106
PERIODONTOLOGY 2000
GTR with bone substitutes combined vs. open ap
debridement.
To determine the effect of GTR, the following
outcome measures were to be investigated:
Primary outcomes
Tooth loss.
Change in attachment levels.
Patient well-being or quality of life.
Secondary outcomes
Change in probing depths.
Change in gingival recession.
Changes in bone/hard tissue:
i radiographic;
ii hard tissue probing at surgical re-entry;
Disease recurrence (% sites with 2 mm loss of
probing attachment measured from 12 months
after treatment).
Percentage of sites with 4 mm probing depth at
completion of study.
Aesthetics (change: better or worse in patients
opinion).
Post-operative complications (including pain,
infection).
Economic outcomes.
Search strategy
A search of the Cochrane Oral Health Group trials
register, EMBASE and MEDLINE was conducted up
to and including April 2004. We used the strategy:
guided tissue regeneration OR guided-tissue-
regeneration OR GTR OR periodontal regeneration
OR periodontal-regeneration OR intra bony de-
fect* OR intrabony defect* OR intrabony defect*
OR infra bony defect* OR infrabony defect* OR
infra-bony defect* OR intra osseous OR intraos-
seous OR intra-osseous. For MEDLINE this also
included the rst two phases of the sensitive search
strategy for randomised controlled trials of the
Cochrane Oral Health group and was adapted for
other databases.
Hand searching included a complete search of
Journal of Periodontology, Journal of Clinical Peri-
odontology and Journal of Periodontal Research up to
April 2004 and bibliographies of all relevant papers
and review articles. In addition, we contac-
ted experts/groups/companies involved in surgical
research to nd other trials or unpublished material
or to clarify ambiguous or missing data.
Methods of the review
Titles and abstracts of the search results were
screened by two independent reviewers (R.T., I.N.).
The full text of all studies of possible relevance was
obtained for independent assessment by three
reviewers (R.T., I.N., E.G.L.) against the stated inclu-
sion criteria. Any disagreement was resolved by dis-
cussion amongst the reviewers and authors of the
trials were contacted to provide missing data where
possible. Data entry on to computer was performed
by two reviewers (R.T., H.W.).
Methodological quality
The methodological quality of included studies was
assessed using both components shown to affect
study outcomes (typically by exaggeration of treat-
ment effect) such as randomization, allocation con-
cealment (concealment of the randomization code
from those recruiting patients to avoid selection bias)
and blinding of examiners and therapists (24, 33, 45).
The quality assessment of included trials was under-
taken following the guidelines in the Cochrane
Reviewers Handbook (1) (Table 1). All quality assess-
ments were conducted independently by two review-
ers (R.T., I.N.) and agreement determined by Kappa
scores. Methodological quality was used in sensitivity
analyses to test the robustness of the conclusions but
was not used to exclude studies qualifying for the
review on the basis of their inclusion criteria.
Data synthesis
A weighted treatment effect was calculated and the
results were expressed as mean differences (MD and
95%CI) for continuous outcomes and relative risk
(RR and 95%CI) for dichotomous outcomes using
the REVMAN ANALYSES software (available free from the
Cochrane website, http://www.cochrane.org). The
analysis for the continuous outcome variables was
conducted using the generic inverse variance statis-
tical method where the mean differences and stand-
ard errors are entered for all studies so that the
parallel group studies and intra-individual (split-
mouth) studies could be combined (see Results).
Variance imputation methods were used to esti-
mate appropriate variance estimates in some split-
mouth studies, where the appropriate standard
deviation of the differences was not included in study
reports (19). The primary outcome measure was gain
107
in attachment. Random effects models were used for
analyses and the signicance of discrepancies in the
estimates of the treatment effects from the different
trials were assessed by means of Cochrans test
for heterogeneity. If any signicant heterogeneity
(P < 0.1) was detected it was planned to investigate
this. Publication bias was examined using both the
Begg and Mazumdar rank correlation test and the
Egger regression asymmetry test (3, 16).
A subgroup analysis was conducted for the eight
parallel group and eight split-mouth studies for
attachment change to see whether it is appropriate to
combine these study designs in the meta-analysis.
Only one of the ve split-mouth studies (8) stated the
standard deviation of the mean difference for two of
the outcomes (change in attachment and change in
probing depth). It was possible to calculate this for
these two outcomes fromthe rawdata inanother split-
mouthstudy (7) andto estimate this fromP-values ina
further study (34). The intra-patient correlations from
these studies rangedfrom0.14 to 0.43. Anintra-patient
correlation of 0.25 was used throughout this review to
estimate the standard errors for the other ve split-
mouth studies (4, 30, 39, 40, 42). Sensitivity analyses
were conducted imputing standard errors for the in-
trapatient correlation of zero, which would provide a
conservative estimate of the standard error.
Subgroup analyses were planned to investigate the
effects of factors thought to be most inuential on
periodontal outcomes including smoking status,
barrier membrane type (resorbable vs. nonresorba-
ble) and surgical technique (conventional ap vs.
papilla preservation ap). A further sensitivity ana-
lysis examined periodontal outcomes with exclusion
of studies providing more frequent postoperative
maintenance than is practicable in clinical practice
(dened as maintenance visits more frequent than
every 3 months, commencing 3 months postsurgery).
Main ndings
Search
The search produced 626 titles with or without ab-
stracts. Of these, 596 were clearly not relevant to the
review due to addressing completely different re-
search questions, or involving animal research only
or because they were review articles. The full-text of
32 studies of possible relevance was obtained and 15
studies were excluded (see below for reasons).
Therefore 17 randomised controlled trials were
included in this review.
Characteristics of studies
Characteristics of excluded studies
There were 15 excluded studies, seven of which
reported 6 months data only (6, 17, 25, 27, 28, 38,
Table 1. Criteria for methodological assessment
Criterion Key
Randomization Adequate would include any one of the following methods of randomization:
computer generated or table of random numbers, drawing of lots,
coin-toss, shufing cards or throw of a dice.
Inadequate method of randomization utilizing any of the following:
case record number, date of birth or alternate numbers will be judged inadequate.
Unclear where the method cannot be determined.
Concealment of allocation: Adequate methods of allocation concealment would include either central
randomization or sequentially numbered sealed opaque envelopes.
This criterion will be considered inadequate if there is an open allocation
sequence and the participants and those carrying out the trial can foresee
the upcoming assignment.
Unclear where the method cannot be determined.
Blinding of outcomes
assessment
Whether persons assessing the outcome of care were aware of which
treatment the participant received, will be graded as yes, no and unclear.
Blinding of operator This could only be achieved if defect preparation was complete prior to
informing the operator of treatment allocation. For split-mouth studies,
this would demand preparation of both test and control sites simultaneously.
Handling of withdrawals
and losses
Was there a clear description given of the difference between the two groups
of losses to follow up? Graded as yes, no and unclear.
108
Needleman et al.
42). Of the other eight studies, six were written in
English, one in German and the other in Japanese.
The latter two were translated, revealing that one (15)
did not have access ap control and the other (53)
was not a randomised controlled trial. Of the
remaining studies, three were either not fully rand-
omised controlled trials (2, 18, 56), one was quasi-
randomised (5) and one did not employ GTR (47).
One used a unique radiographic assessment for
which the outcomes could not be compared with
other radiographic techniques (23).
Characteristics of included studies
Of the 17 included trials (Table 2), three were multi-
centre studies with the following numbers of indi-
viduals completing each study; 136 patients in a
parallel group design (50), 23 patients in an intrain-
dividual design (8) and 109 patients in a parallel
group design (14). Of the single-centre trials, there
were ve intraindividual (split-mouth) trials ranging
from 10 to 40 participants (4, 7, 34, 40, 41), seven
parallel group trials ranging from 36 to 90 partici-
pants (10, 11, 27, 32, 46, 48, 55) and two studies
containing both split-mouth and parallel group ele-
ments (30, 42) contributing 16 and 25 individuals,
respectively. Six trials were based in private practice
(8, 10, 11, 14, 46, 50), nine trials were university-based
(4, 7, 27, 31, 32, 34, 40, 41, 43) and for two trials, the
setting was not clear (48, 55). Three studies (7, 32, 50)
were supported, in part, by companies whose prod-
ucts were being used as interventions in the trials.
The age range of all the included studies was from
21 to 81 years. However, one study (25) did not give
details of their age range and another (31) described
their mean age as 48.7 years (SD 11.6). All studies had
both male and female participants, but with differing
proportions. One study had more participants who
were smokers than nonsmokers, with 21 out of 38
smokers (32).
Methodological quality of included
studies
Randomization was reported in all studies included
in this review. An adequate method of randomization
was present in the following studies: (references 4, 8,
10, 11, 14, 31, 34, 41, 46, 50, 55). It was unclear as to
how the following studies randomised their treat-
ment groups (7, 26, 32, 40, 43, 48).
Concealment of the randomization code during
patient selection was adequate in nine studies (8, 10,
11, 14, 34, 41, 46, 50, 55) and was not clear in seven
studies (4, 7, 26, 31, 32, 40, 48). Examiner blinding
was described in eight of the studies (10, 11, 26, 31,
32, 40, 46, 55). Operator blinding (such as by reveal-
ing the treatment code only after defect preparation
was complete) was present in the following eight
studies: (references 8, 10, 11, 31, 40, 41, 46, 50).
In all studies, all participants entering a study
could be accounted for at study completion.
However, a high number of withdrawals or drop-outs
were reported for two studies with 33%drop-outs (7),
35% losses (28) and much smaller numbers for the
multicentre studies (8, 14, 50).
Attachment level change
Sixteen studies presented attachment level data for
GTR alone: eight parallel group trials (10, 11, 14, 32,
46, 48, 50, 55) and eight split-mouth studies (4, 7, 8,
31, 34, 40, 41, 43) (Fig. 1). The results for this analysis
show a statistically signicantly greater attachment
gain for test groups compared with open ap debri-
dement. For GTR the mean difference between test
and control was 1.22 mm (95%CI Random Effects
[0.80,1.64], chi-square for heterogeneity 69.1
(df 15), P < 0.001, I
2
78%). There appeared to be
a difference between the two subgroups based on the
study design, with the subgroup of split-mouth
studies having a lower treatment effect (0.79 mm;
95%CI Random Effects [0.37,1.21]) than the parallel
group studies (1.71 mm; 95%CI Random Effects
[1.02,2.40]). This difference was statistically signi-
cant (meta-regression slope coefcient 0.91, 95%CI
[) 1.71, 0.11], P 0.026). The sensitivity analysis
imputing an intrapatient correlation of zero only
made a small difference to the estimate for the split-
mouth studies, with a mean difference of 0.82 mm
(95%CI Random Effects [0.40,1.24]).
There were two studies for GTR + bone substitutes
(4, 26) and the mean difference for these two studies
was 1.25 mm (95%CI [0.89,1.61], chi-square for het-
erogeneity 0.01 (df 1), P 0.91), similar to the
overall result for GTR alone.
Proportion of sites gaining less
than 2 mm in attachment level
From an additional (posthoc) analysis for GTR, in six
parallel group studies (10, 11, 14, 32, 50, 55)) it was
possible to extract data for number of sites gaining
less than 2 mm of attachment. This again showed a
signicant benet for GTR, a relative risk of 0.62
109
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t
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l
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2
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l
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r
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y
112
Needleman et al.
R
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(
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3
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2
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r
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t
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)
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t
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2
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o
l
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5
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t
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m
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2
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o
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l
:
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b
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113
(95%CI Random Effects [0.41, 0.95], chi-square for
heterogeneity 5.9 (df 5), P 0.31) (Fig. 2). The
number needed to treat for GTR to achieve one extra
site gaining 2 mm or more attachment over open ap
debridement was therefore 11 (95%CI [7,33]), based
on an incidence of 28% of sites in the control group
failing to gain 2 mm or more of attachment. For
baseline incidences in the range of the control groups
of 3% and 55% the number needed to treat is 104
and 6. It should be noted that the calculation of
Study
or sub-category
01 Parallel group studies
mean difference (SE)
mean difference (random) Weight
% 95% Cl
mean difference (random)
95% Cl
Cortellini et al. (10)
Blumenthal & Steinberg (4)
02 Split-mouth studies
Mayfield et al. (32)
Sculean et al. (46)
Silvestri et al. (48)
Tonetti et al. (50)
Chung et al. (7)
Loos et al. (31)
Mora et al. (34)
Pontoriero et al. (40)
Pritlove-Carson et al. (41)
Ratka-Kruger et al. (43)
Zuccheli et al. (55)
Subtotal (95% Cl)
Subtotal (95% Cl)
Total (95% Cl)
2.2000 (0.6480)
2.6000 (0.4110)
0.9000 (0.3800)
0.2000 (0.6380)
1.4000 (0.5700)
3.6000 (0.7400)
0.8600 (0.2700)
2.3000 (0.3300)
0.4200 (0.2400)
1.2700 (0.2700)
1.4000 (0.4800)
0.1100 (0.3400)
1.3000 (0.3600)
1.3000 (0.3800)
0.0200 (0.4400)
0.1800 (0.9200)
Test for heterogeneity: Chi
2
= 34.37, df = 7 (P < 0.0001), I
2
= 79.6%

Test for overall effect: Z = 4.87 (P < 0.00001)
2
= 18.46, df = 7 (P = 0.01), I
2
= 62.1%

2
= 69.07, df = 15 (P < 0.00001), I
2
= 78.3%

Test for overall effect: Z = 3.69 (P = 0.0002)
Test for overall effect: Z = 5.66 (P < 0.00001)
4.87
6.60
6.83
4.93
5.40
4.29
7.63
7.21
47.77
7.83
7.63
6.07
7.14
6.99
6.83
6.37
3.37
52.23
100.00
2.20
2.60
0.90
0.20
1.40
3.60
0.86
2.30
1.71
0.42
1.27
1.40
0.11
1.30
1.30
0.02
0.18
0.79
5.05]
2.52]
1.45]
1.64]
3.41]
3.47]
[2.15,
1.39] [0.33,
2.95] [1.65,
2.40] [1.02,
[0.28,
[1.05,
[0.16,
[1.79,
[0.93,
2.04]
2.01]
0.78]
2.34]
1.80]
0.89]
[0.56,
0.88] [0.84,
1.98] [1.62,
1.21] [0.37,
1.22 1.64] [0.80,
[0.59,
[0.56,
[0.46,
[0.74,
[0.05,
Favours GTR Favours access flap
0 2 4 4 2
Fig. 1. Forest plot of guided tissue regeneration surgery vs. access ap surgery for attachment change.
Study
Tonetti et al. (50)
Total (95% CI)
Total events: 32 (Treatment), 55 (Control)
2
= 5.92, df = 5 (P = 0.31), I
2
= 15.6%

Treatment
n/N
Control
n/N
1/30 2/15
2/12
17/54
11/20
22/67
1/30
0/24
10/55
10/18
11/69
0/30
226 198
0.1
100.00 0.62
0.33 7.87]
0.95]
0.92]
1.79]
1.15]
2.01]
2.54]
0.49
0.58
0.10
0.25
1.01
[0.41,
[0.01,
[0.26,
[0.57,
[0.29,
[0.01,
[0.02,
1.72
30.29
35.37
27.51
1.96
3.15
0.2 0.5 1 2 5 10
RR (random)
95% CI
RR (random)
95% CI
Weight
% or sub-category
Fig. 2. Forest plot of guided tissue regeneration surgery vs. access ap surgery for sites with less than 2 mm loss of
attachment.
114
Needleman et al.
number needed to treat values based on arbitrary
cut-thresholds derived from continuous data may not
be appropriate. This is currently being examined.
In order to investigate the subgroup analysis of very
frequent maintenance, that may be impractical to
provide in many clinics (< 3 monthly after the rst
3 months post surgery) vs. maintenance after
3 months, we conducted a random effects meta
regression to compare these two subgroups. This was
not found to be signicant (meta-regression slope
coefcient 0.62 (SE 0.43), P 0.15) (Table 3). A
subgroup analysis was also conducted comparing the
six studies with papilla preservation with the 10
studies where the papilla was not preserved. Although
a higher attachment change was found for papilla
preservation, this subgroup analysis was also not
signicant (P 0.09, Table 3). Comparison of studies
using an absorbable vs. nonabsorbable membrane
type also gave rise to a nonsignicant difference be-
tween the subgroups (P 0.11; Table 3).
Change in probing depth (Fig. 3)
There were 11 studies for GTR alone including
change in probing depth as an outcome: ve parallel
group studies (14, 32, 46, 48, 55) and six split-mouth
studies (4, 8, 31, 34, 40, 43). The standard errors for
one split-mouth study were given in the report (8)
and could be estimated for a further study (34). The
intra-patient correlations were 0.11 and 0.1224 and so
a value of 0.1 was used to calculate the standard
errors for the other split-mouth studies. A sensitivity
analysis was conducted imputing standard errors for
the intrapatient correlation of zero, which would
provide a conservative estimate of the standard error.
The results demonstrated a signicantly greater
probing depth reduction for GTR, weighted mean
difference 1.21 mm (95%CI [0.53,1.88], chi-square for
heterogeneity 62.9 (df 10), P < 0.001, I
2
84%).
Although the treatment effect for split mouth studies
was lower, this was not statistically signicant (meta
regression slope coefcient 0.72, 95%CI [) 2.21,
0.78], P 0.35).
There were also two studies for GTR + bone sub-
stitutes (4, 26) with a weighted mean difference of
1.24 mm (95%CI: [0.89,1.59], chi-square for hetero-
geneity 0.03 (df 1), P 0.85) similar to that for the
GTR alone.
Gingival recession (Fig. 4)
Nine studies for GTR had gingival recession as an
outcome: four with a parallel group design (14, 32, 46,
55) and ve with a split-mouth design (4, 31, 34, 40,
43). There was one study for GTR + bone substitutes
(4). We were unable to estimate any of the intra-
patient correlations and we decided to use a value of
0.25 for the split-mouth studies for this outcome. A
statistically signicant difference between GTR and
open ap debridement controls was evident (mean
difference 0.26 mm, 95%CI Random Effects
[0.08,0.3], chi-square for heterogeneity 2.7 (df 8),
P 0.95), with a greater change in recession from
baseline for the control group.
The single study of GTR + bone substitutes showed
slightly greater recession for test than controls, with a
mean difference of 0.33 mm (95%CI [ 0.43, 0.23]).
Bone/hard tissue change
Radiographic data were given in only one study (31).
This showed a 0.6 mm gain in bone from the base of
the defect in both test and control groups. Regarding
surgical re-entry, three studies (4, 7, 32) reported data
Table 3. Results from the random effects meta-regression analysis of attachment change
Characteristic No. of studies Slope estimate 95%CI Slope interpretation P-value
Frequency of
maintenance
16 ) 0.62 [) 1.46,0.22] Higher attachment
change for visits
< 3 months
0.15
Type of GTR barrier 18 comparisons ) 0.70 [) 1.55,0.15] Higher attachment
change for nonabsorbable
membrane
0.11
Surgical technique 16 0.75 [) 0.10,1.59] Higher attachment
change for papilla
preservation
0.09
GTR, guided tissue regeneration.
115
for GTR alone and one study for the combination of
GTR + bone substitute (4). For GTR, a statistically
signicant greater gain in hard tissue probing was
found for GTR compared with open ap debride-
ment. This amounted to a weighted mean difference
of 1.39 mm (95%CI [1.08,1.71], chi-square for het-
erogeneity 0.85 (df 2), P 0.65). For GTR + bone
substitutes the difference was greater, with a mean
difference of 3.37 mm (95%CI [3.14, 3.61]).
Impact of methodological quality
Heterogeneity
Sensitivity analyses for attachment level change
which excluded poorer quality studies resulted in re-
duced numbers of incorporated studies, although
heterogeneity remained statistically signicant
(Table 4). Subgroup analysis of parallel group and
split-mouth studies reduced the heterogeneity,
although there was still a great deal of unexplained
heterogeneity. However, as all the ndings for each
outcomes were in the same direction we felt it was
appropriate to undertake the meta-analyses as shown.
Prognostic factors
The variability in reporting data on prognostic factors
such as initial defect depth, plaque levels and smo-
king prevented a meaningful comparison. One study
(32) presented a subgroup analysis comparing clin-
ical changes in smokers and nonsmokers. This
showed reduced benets in smokers for attachment
gain (GTR group: nonsmokers 1.9 mm SD 1.5,
smokers 0.8 mm SD 0.8), although with little effect on
probing depth change. However, this result should be
viewed with some caution as the groups were not
balanced with respect to disease levels.
Publication bias
We tested publication bias using the Begg and Maz-
umdar rank correlation test (3, 16), which found no
evidence of a correlation between the effect estimates
0 2 4 4 2
Sculean et al. (46)
Silvestri et al. (48)
Study
or sub-category mean difference (SE)
% 95% Cl
95% Cl
2
= 43.60, df = 4 (P < 0.00001), I
2
= 90.8%

Subtotal (95% Cl)
2
= 9.06, df = 5 (P = 0.11), I
2
= 44.8%

2
= 62.90, df = 10 (P < 0.00001), I
2
= 84.1%

Subtotal (95% Cl)
Total (95% Cl)
0.8000 (0.4400)
0.1000 (0.6200)
0.5000 (0.5300)
4.5000 (0.5400)
2.0000 (0.3400)
0.4800 (0.2600)
1.3000 (0.5400)
0.1400 (0.5000)
1.8000 (0.5200)
1.1300 (0.3400)
0.2300 (1.0300)
9.63
8.34
8.99
8.92
10.28
46.16
10.73
8.92
9.21
9.06
10.28
5.65
53.84
0.80
0.10
0.50
4.50
2.00 2.67]
5.56]
1.54]
1.32]
1.66]
[1.33,
1.59
0.48
1.30
0.14
1.80
1.13
0.23
0.87
2.97] [0.21,
[3.44,
[0.54,
[1.12,
[0.06,
1.80]
2.82]
1.12]
2.36]
0.99]
[0.46
2.25] [1.79,
1.36] [0.38,
100.00 1.21 1.88] [0.53,
[0.78,
[0.84,
[0.24,
[0.03, Blumenthal & Steinberg (4)
Loos et al. (31)
Mora et al. (34)
Fig. 3. Forest plot of guided tissue regeneration surgery vs. access ap surgery for probing pocket depth change.
116
Needleman et al.
and their variances, indicating no evidence of publi-
cation bias (P 0.53). The results of the Egger
regression asymmetry test also suggested no publica-
tion bias (P 0.43). There is no strong evidence of
publication bias and 16 studies were included in this
analysis.
Other outcomes
No data were found for the following outcomes:
disease recurrence (% sites with 2 mm loss of pro-
bing attachment measured from 12 months after
treatment), tooth loss, % of sites with 4 mm probing
depth, aesthetics (change: better or worse in patients
opinion), postoperative complications (including
pain, infection), economic outcomes, patient well-
being or quality of life.
Implication of ndings
GTR alone
This systematic review has shown an overall mean
increase in attachment gain for GTR over open ap
debridement (mean difference 1.22 mm, 95%CI
[0.8,1.64]). However, this value is not a valid estimate
of effect because the heterogeneity is substantial and
statistically signicant. Therefore, the value should
not be quoted to demonstrate the magnitude of dif-
ference between the two therapies. It should be noted
that 11/16 trials produced a statistically signicant
greater gain in attachment with GTR than OFD and
with no statistically signicant publication bias (see
below). Therefore, we suggest that the data indicate
that GTR can produce a statistically signicant
greater gain in attachment. The magnitude of this
superiority, however, is not clear.
The results of the 16 randomised controlled trials
for GTR alone included in this review show a sub-
stantial variation in their results. The mean additional
attachment gain fromGTR over that achieved by open
ap debridement surgery ranged between studies
from 0.02 to 3.60 mm. This range is large and the
differences are difcult to reconcile. We have
attempted to explore some of the possible causes of
this heterogeneity and these analyses should be
viewed as exploratory observations. These investiga-
tions included analyses of protection from bias as well
as factors affecting clinical heterogeneity.
Sculean et al. (46)
Study
or sub-category
2
= 0.97, df = 3 (P = 0.81), I
2
= 0%

Subtotal (95% Cl)
2
= 0.64, df = 4 (P = 0.96), I
2
= 0%

2
= 2.69, df = 8 (P = 0.95), I
2
= 0%

Test for overall effect: Z = 2.84(P = 0.005)
Subtotal (95% Cl)
Total (95% Cl)
Blumenthal & Steinberg (4)
Loos et al. (31)
Mora et al. (34)
mean difference (SE)
% 95% Cl
95% Cl
0 0.5 1 1 0.5
0.0000 (0.2300)
0.2000 (0.2920)
0.0000 (0.4910)
0.3000 (0.2320)
0.2800 (0.2000)
0.2700 (0.5600)
0.3500 (0.2400)
0.4700 (0.2300)
0.1400 (0.4300)
15.34
9.52
3.37
15.08
43.30
0.00
0.20
0.00
0.30 0.75]
0.96]
0.77]
0.45]
[0.15,
0.15 0.42] [1.12,
[0.96,
[0.37,
[0.45,
2.59
14.09
15.34
4.39
56.70
0.27
0.35
0.47
0.14 0.98]
0.92]
0.82]
1.37]
[0.70,
0.34 0.57] [0.10,
100.00 0.26 0.43] [0.08,
[0.02,
[0.12,
[0.83,
20.29 0.28 0.67] [0.11,
Fig. 4. Forest plot of guided tissue regeneration surgery vs. access ap surgery for recession change from baseline.
117
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n
.
118
Needleman et al.
Protection from bias
Since study quality has been shown to have a direct
impact on the size of the effect of treatment we
explored this effect with sensitivity analyses including
allocation concealment, examiner and therapist
blinding. The results of these analyses were not
consistent. For attachment gain, excluding studies
without adequate concealment of the randomization
code or examiner blinding did not substantially affect
the estimate or reduce heterogeneity (Table 4).
However, when studies without surgeon blinding
were excluded, the summary estimate was reduced
and heterogeneity became non-statistically signi-
cant (four studies: mean difference 0.67 mm 95%CI
[0.06,1.39], heterogeneity P 0.027). When studies
without both surgeon blinding and examiner blind-
ing were excluded, the difference between GTR and
OFD became non-statistically signicant and with no
signicant heterogeneity (three studies: mean differ-
ence 0.41 mm, 95%CI [) 0.33,1.08], heterogeneity
P 0.41). Caution should be exercised here as the
number of studies in these comparisons was also
much lower, which in itself could reduce heterogen-
eity. However, the trend to a reduction of the
magnitude of effect with greater protection from bias
is in line with previous studies of the impact of bias
(24, 33, 45). If further studies are to be conducted on
GTR, it is critical that they employ greater meth-
odological rigour, in particular in their protection
against these biases.
Publication bias was investigated and found not to
be statistically signicant. Whilst these tests are
conservative in their ability to demonstrate such bias,
the number of studies included (16) should be ade-
quate to identify publication bias if it was present.
Therefore, we can conclude that publication bias did
not appear to affect the summary values.
Clinical heterogeneity
Available data allowed the investigation of several
clinical aspects that we hypothesized, a priori, could
affect heterogeneity. These included frequency of
supportive maintenance care (more frequently vs.
after the rst 3 months of healing), type of GTR
barrier membrane (resorbable vs. nonresorbable) and
surgical technique (papilla preservation technique vs.
conventional approach). Random effects meta-
regression analyses were conducted to compare each
of these subgroups.
Membrane type might be important. The differ-
ence between GTR and OFD for attachment gain was
greater for non-absorbable barriers than for absorb-
able barriers; however, this difference was not sta-
tistically signicant by meta-regression (P 0.11).
The hypothesis for excluding studies with frequent
maintenance was based on the nding that frequency
of maintenance care can affect periodontal surgical
results (52). However, the analysis for this review
showed neither a statistically signicant effect on the
summary estimate nor an elimination of heterogen-
eity (P 0.15).
Surgical techniques aiming to retain the interden-
tal soft tissue have been proposed with the potential
to achieve and maintain primary closure during
wound healing. It has been suggested that such
methods could therefore produce greater clinical
improvements (12). The meta-analysis for the six
studies with papilla preservation did not show a
statistically signicant difference compared with the
overall estimate despite the apparent greater attach-
ment gain (0.75 mm, 95%CI [) 0.10,1.59], P 0.09)
and therefore the heterogeneity remained highly
statistically signicant. However, the 95%condence
interval only just includes a value of no difference,
suggesting that papilla preservation might be
important and should be examined in future studies.
Another explanation for the heterogeneity might be
variability between studies in prognostic factors that
have been documented to affect the outcome of
regenerative surgery. These include plaque levels,
cigarette smoking and defect severity as expressed by
baseline probing depth, attachment level and bone
defect depth at baseline. Regarding plaque and
smoking, it is apparent that differences in the way
that both factors are reported between studies pre-
vent sensible comparison. For instance, some studies
present full mouth plaque scores, others measure
plaque at the experimental sites only, and other
studies present plaque index values or no plaque
data. The effect of smoking on reducing the gain in
attachment following surgery is reported in only one
subgroup analysis of a randomised controlled trial
(32). This result highlights the need for more research
into prognostic factors to help explain heterogeneity.
Therefore, the extent to which we have been
successful in explaining the troubling extent of the
heterogeneity has been limited. It should be noted,
however, that heterogeneity exists not only between
studies. In two large multicentre trials (14, 50) the
results between centres within each study showed a
substantial variability (from best to worst) in attach-
ment gain of between 1.73 mm and 2.1 mm,
respectively. Therefore, although the efcacy of GTR
has been demonstrated in some studies, the effect-
119
iveness and external validity of such a technique may
be questioned.
Variability of results is clearly an important issue
when considering the relevance of a treatment to
clinical practice. Although we have explored some of
the possible causes of heterogeneity in this system-
atic review, we have been unable to determine
denitively which factors account for this. The
number and characteristics of studies currently
available is insufcient to answer this clinically rele-
vant problem.
In terms of clinical signicance, mean difference
values are difcult to interpret. The relative risk for
sites gaining less than 2 mm attachment demon-
strated that sites treated with GTR were 38% less
likely to fail to attain a gain of 2 mm of attachment or
more than those treated by open ap debridement
(Relative risk 0.62, 95%CI [0.41,0.95]). There was no
signicant heterogeneity between the studies. The
number needed to treat for GTR to achieve one extra
site gaining 2 mm or more attachment over open ap
debridement was 11 (95%CI [4,33]), i.e. 11 patients
need to be treated for one to achieve this benet over
OFD. This is a slight increase from the number nee-
ded to treat of 8 in the original review and includes an
additional two studies (14, 55). The increase seems
related to an improvement in performance of OFD-
treated sites as the proportion achieving at least
2 mm attachment gain has increased from 77/114
(67.5%) to 143/198 (72.2%), whereas the proportion
of GTR-treated sites achieving this improvement has
changed little; 119/141 (84.4%) vs. 194/226 (85.5%).
This observation still suggests two comments. Firstly,
that OFD should remain the control procedure for
the evaluation of GTR and, secondly, that improve-
ments in conventional surgical methods should be
explored further.
Other clinical outcomes indicate statistically
greater improvements with GTR than with OFD. As
with attachment level gain, heterogeneity with pro-
bing depth hampers a conclusion about the size of
this improvement. The analysis of recession indica-
ted greater change for OFD than GTR and with no
heterogeneity (0.26 mm, 95%CI [0.08,0.43], hetero-
geneity P 0.96, n 9). The result for gingival
recession is interesting because although 7/9 studies
produced more recession with OFD, this difference
was statistically signicant in only one study (40).
However, the greater precision that is achievable
when multiple studies are combined with meta-
analysis means that overall, statistically signicantly
more recession can be shown to occur following
OFD.
Effect of study design
This analysis has demonstrated a statistically
signicant difference between parallel group and
split-mouth studies with respect to attachment level
change. Split-mouth studies produced a more con-
servative estimate of attachment level gain. Whilst
there remained statistically signicant heterogeneity
in both subgroups, the difference between split-
mouth and parallel groups was statistically signi-
cant by meta-regression. To our knowledge, this is
the rst time that such a difference has been dem-
onstrated and underlines the importance of analysing
by study design. The reasons for the smaller differ-
ences between two interventions can only be specu-
lated upon. They might be a chance nding as a re-
sult of producing two subgroups of studies. Possibly,
protection from bias could be more straightforward
in split-mouth studies. For instance, selection bias
might be less of a risk as the patient provides both
experimental groups. Furthermore, it might be more
successful to maintain masking for patient, examiner
and therapist if the patient provides both groups.
Alternatively it may be possible that there is some
cross-over benet, either local or systemic, as has
been previously suggested (22).
GTR plus bone grafts
Only two studies could be located examining the
combination of GTR and bone grafts vs. open ap
debridement in a randomised, 12-month design (4,
26). The effects of the combination treatment were
similar to GTR alone for attachment gain, but with
slightly more probing depth reduction (1.2 mm) and
greater gain in hard tissue probing at re-entry surgery
(3.4 mm) in one study and a gain in gingival recess-
ion in another study.
Reporting quality
We found that many study reports were incomplete in
their presentation of methods or results. We contacted
19 authors to clarify missing or ambiguous data.
However, we would recommend that authors of
randomised controlled trials follow the CON-
SORTguidelines (http://www.consort-statement.org),
which provide clear guidance on presentation of trial
reports and would help systematic reviewing of the
literature.
Statistical methods
This review considered parallel group and split-
mouth studies. In theory the combining of the
120
Needleman et al.
treatment effects from these studies should be
straightforward; however, due to the poor reporting
of the data from the split-mouth studies, the standard
deviation of the difference had to be estimated. This
was achieved by using the results of the split-mouth
studies that presented the necessary data to calculate
the intrapatient correlation for the other split-mouth
studies. Sensitivity analyses were carried out impu-
ting different values for the intrapatient correlation
and the results of these were very similar to the
results presented in this review.
Comparison with previous thorough reviews and
meta-analyses
Two recent meta-analyses have reported greater
benets to GTR than found in the present systematic
review. One review indicated a difference in attach-
ment gain between GTR and open ap debridement
of 2.7 mm (29) and a second review reported 1.6 mm
difference (13). Differences in the methods of these
reviews that may help to explain the results include
the incorporation of uncontrolled and nonblinded
studies in one (29) and unclear selection criteria for
randomised controlled trials in the second, including
the inclusion of studies of shorter duration (13). A
recent systematic review (36) on GTR produced
broadly similar ndings to our present review. In
terms of gain in clinical attachment, GTR produced
0.81 mm greater gain than OFD (P < 0.001) (no
condence interval presented for the difference). The
result contained highly statistically signicant het-
erogeneity. This group also did not nd a statistically
signicant difference between different types of
barrier material. Differences in search strategy,
inclusion of both randomised and nonrandomised
studies, and of studies of shorter duration of follow-
up may have accounted for these differences.
Trombelli, in this volume of Periodontology 2000
(51), considered the previously published version of
this review (37, 51). The results of this update are
similar to those of the original review, even though
six more trials have been included. The major dif-
ference was found for gingival recession, which is
now signicant, with a greater increase in gingival
recession from baseline found in the control group
access ap group. In addition, further exploration of
heterogeneity is now possible.
Reviewers conclusions
Eleven out of 16 studies showed greater attachment
gain for guided tissue regeneration than for open
ap debridement. However, this systematic review
has shown that the outcomes following GTR are
highly variable both between and within studies.
A meta-analysis comparing GTR with OFD indi-
cates overall an increase in clinical attachment
gain of 1.22 mm (95%CI [0.80,1.64]) and a probing
depth reduction of 1.21 mm (95%CI [0.53,1.88]) of
GTR over OFD. However, the highly statistically
signicant heterogeneity between studies indicates
that these summary values should not be used to
indicate the magnitude of the greater probing
changes.
Statistically signicantly greater gingival recession
occurs following access ap surgery than following
the use of GTR (mean difference 0.26 mm, 95%CI
[0.08,0.43]) with no statistically signicant hetero-
geneity.
Few data are available from controlled studies of
12 months duration on the combination of bone
substitutes with GTR but the results suggest little
added benet beyond a gain in hard tissue probing
at surgical re-entry.
No data exist on important questions such as
patient evaluation of outcomes or effect of
therapyon denitive outcomes such as tooth loss.
Choice of papilla preservation aps vs. conven-
tional designs may be important.
Control of bias (especially randomization, con-
cealment of allocation and blinding of examiner
and operator where possible) needs to be more
rigorously employed in study designs and this
might be an important factor in explaining
heterogeneity. More research is needed to identify
the most important patient, site and technique
factors associated with successful outcomes. This
should then be followed by independent trials
showing a more consistent benet of GTR over
OFD, before acceptance into wider practice.
Until consistent benets from GTR can be shown,
open ap debridement should remain the control
comparison.
Greater attention in designing and reporting stud-
ies should be given to study quality issues as set
out in the CONSORT guidelines. This will help to
facilitate the evaluation of these studies once
published.
Greater consideration in study design should be
given to outcomes that capture the boarder patient
experience including patient centred and econo-
mic outcomes and evaluation of adverse effects.
Two treatments that produce similar clinical
changes may be quite different from the patient or
economic perspective.
121
Declaration of interests
All authors are funded by UCL or the NHS. None of
the review authors have a nancial interest in any
regenerative product or therapy.
Acknowledgments
We would like to thank Mrs Sylvia Bickley at the
Cochrane Oral Health Group in Manchester, UK, for
her tremendous help in searching the literature and
Ms Emma Tavender, also at the Cochrane OHG, for
her administrative support.
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123
Systematic review of the effect of
smoking on nonsurgical
periodontal therapy
ANTONELLA LABRI OLA, IAN NEEDLEMAN* & DAVI D R. MOLES
Periodontitis is the result of complex interrelation-
ships between infectious agents and host factors.
Environmental, acquired, and genetic risk factors
modify the expression of disease and may therefore
affect the onset or progression of periodontitis (27).
Among the environmental risk factors, tobacco
smoking has been found to be associated with an
increased prevalence and severity of periodontal
disease (13, 30). It is also apparent that a dispropor-
tionately high number of people with severe perio-
dontal disease are smokers (2, 7) and that a strong
association exists between smoking and an unusual
form of periodontitis that is resistant to treatment
(20).
Smoking has also been implicated as a factor that
reduces the effectiveness of treatment (17). It appears
that smokers may respond to nonsurgical periodontal
therapy less favorably than nonsmokers, especially in
terms of probing depth and bone level (1, 18, 21).
When the effect of the level of cigarette consumption
is considered, it seems that the response to perio-
dontal therapy is related to the amount of cigarettes
smoked (16), and that previous smokers (quit-smok-
ers) have a similar response to treatment compared
to nonsmokers (4, 12, 16). However, the size of the
effect on treatment response in these studies is not
consistent, making it difcult to draw conclusions
about the clinical signicance of smoking and the
effect of quitting smoking on treatment.
The mechanisms by which smoking could affect
the response to periodontal treatment might be
related to the altered inammatory and immune
response that has been observed in smokers (17, 19,
22) or to the persistence of pathogenic ora in
smokers after treatment (11, 12).
Periodontitis represents an important health issue
because it may lead to changes in appearance,
impairment in function, signicant pain and, nally,
tooth loss, all of which may affect the quality of life
(25). In addition to the impact on the individual,
there is a signicant impact on healthcare resources
needed to manage the condition. In the USA in 1999,
the expenditure on periodontal and preventive care
amounted to over $14 billion (5). In England and
Wales, 174 million was spent on treatment of peri-
odontal disease by the NHS (National Health Service)
in the year 20012002 (9).
Therefore, as a public health measure, it is critical
to establish the effect of smoking on periodontal
therapy. To date there has been no reliable estimate
of the impact of smoking on periodontal treatment
response. The aim of this systematic review was
therefore to investigate the effect of smoking on
nonsurgical periodontal therapy in patients with
chronic periodontitis. The null hypothesis was that
there is no difference between smokers and non-
smokers in their response to nonsurgical periodontal
therapy. The focused question was: In patients with
chronic periodontitis, what is the effect of smoking
and smoking cessation on the response to nonsurgi-
cal periodontal therapy in terms of clinical and
patient-centered outcomes?.
Methods
Protocol development
We developed the protocol specifying all aspects of
the review methods before commencing the review.
These included the following: inclusion criteria for
studies, search strategy, screening method, data *Corresponding author.
124
PERIODONTOLOGY 2000
abstraction, quality assessment, and data analysis.
This aspect of the design was planned to minimize
the effect of our possible bias on the review and, in
particular, on the potential to alter the methods or
analytical techniques based on study ndings.
Search strategy
The search strategy involved the use of the following
electronic databases: MEDLINE, EMBASE and the
Cochrane Central Register of Controlled Clinical
Trials (CENTRAL), as well as hand searching of bib-
liographies of found references, review articles and
consensus statements. All databases were searched
from their earliest records until March 2003; only
English language publications were searched. The full
search strategy is included as Appendix 1.
Study selection
The primary study design selected was controlled
clinical trials as smoking habit cannot be random-
ized. In addition, arms of randomized controlled tri-
als investigating the effectiveness of nonsurgical
periodontal treatment that reported results sepa-
rately for smokers and nonsmokers were included.
Other inclusion criteria were studies that assessed
systemically healthy patients who had been diag-
nosed with chronic or adult periodontitis and where
the patient was the unit of analysis (rather than a
tooth- or site-based analysis). Studies were not
excluded on the basis of quality, only on whether
they fullled the inclusion criteria for entry. We
planned to investigate the impact of quality on study
outcome if there was heterogeneity between studies.
Types of intervention
The intervention of interest was nonsurgical perio-
dontal treatment, including oral hygiene instructions
and scaling and root-planing or root debridement.
Studies considering nonsurgical periodontal therapy
as oral hygiene alone or deliberate curettage were
excluded.
Types of outcomes measured
The following outcome measures were reported:
primary outcomes: tooth loss, changes in probing
pocket depth and clinical attachment level (clinical
attachment level);
secondary outcomes: changes in bleeding on pro-
bing and complications post-treatment; patient-
centered outcomes, such as quality of life, changes
in appearance, and patient experience.
Validity assessment
The lead investigator (A.L.) was initially calibrated for
screening against another investigator with experi-
ence of several systematic reviews (I.N.). Sixty records
in batches of 20 were screened in this manner until a
kappa (K) score of >0.80 was achieved. Titles and
abstracts were then screened for possible relevance
by one investigator (A.L.). For all studies of possible
relevance, the full text was retrieved. This was
examined independently and in duplicate with a
second investigator. Disagreement was resolved in all
cases by discussion; the K-score for agreement was
0.75. Evaluation of studies was not masked in relation
to study authors or afliations as this has not been
shown to signicantly alter outcomes (23).
Study quality was assessed for the similarity
between groups at baseline, the report of adjustment
for confounding factors, blindness of examiner to
smoking status, proportion of cohort followed up,
and presence of specied eligibility criteria. The
criteria were modied from a guideline for quality
assessment of follow-up studies (26).
Data abstraction
The data abstraction form was piloted over 20 studies
and used to abstract general information about the
paper, study characteristics, outcome measures,
treatment characteristics and quality assessment
data. Abstraction was performed in duplicate inde-
pendently. Where disagreement arose, this was
resolved by discussion.
Study characteristics and quantitative
data synthesis
From evidence tables, studies were analyzed for
similarity in key components and suitability for
meta-analysis. For the studies that could be included
in the meta-analysis, the weighted mean difference
was used for continuous outcomes comparing non-
smokers, smokers and quit smokers (STATA version 7).
Where heterogeneity between studies existed, it was
investigated using a limited number of factors
thought most likely to generate differences in out-
comes, including clinical and methodologic varia-
bles. These were dened a priori. Some studies
reported mean values calculated from all sites in the
mouth (full-mouth studies) including both diseased
125
Smoking and mechanical periodontal therapy
and nondiseased sites. Other studies calculated mean
values only for sites above a dened disease thresh-
old (threshold studies); for instance, initial pocket
depth 5 mm. Data from these two sets of studies
were analyzed separately.
Results
Search (Fig. 1)
From the 330 studies initially obtained from the
search, 80 full text articles were independently
screened by two reviewers and the level of agreement
was determined by Kappa score (K-score for full
text screening: 0.57). Of the 80 full text articles
screened, 67 were not relevant and were excluded
and 13 were considered eligible for inclusion in the
review. The most common reason for study exclusion
was the lack of a control group receiving the same
treatment but not exposed to smoking (58 studies).
Other reasons were site-based analysis (2 studies),
data for nonsurgical and surgical therapy combined
(1 study), selected patients for subgroup not repre-
sentative of initial sample (1 study), duplicate data
(1 study) and not a clinical trail (1 paper). The
characteristics of the included studies are shown in
Tables 1 and 2.
The quality assessment revealed that of the 13 eli-
gible studies, seven showed a clear similarity between
groups at baseline. In most other cases, baseline
values were not reported. The proportion of the
patients followed up was unclear in seven articles
and examiner blinding to smoking status was unclear
in most studies (11 articles). Potential confounding
factors were listed in 11 studies, but only ve studies
reported making an adjustment for these factors.
Three of the studies evaluated all sites in the mouth,
seven studies only sites above a certain threshold of
baseline probing depth, and three studies both full-
mouth and deeper sites.
The heterogeneity between studies was investi-
gated using meta-analysis regression. No statistically
signicant difference between studies was found
after adjustment for baseline values and the duration
of follow-up. Similarly, no statistically signicant
difference was found when considering whether or
not the studies were adjusted for baseline values,
suggesting a reasonable similarity between groups.
Primary outcomes (Table 3)
Tooth loss
No studies reported data on tooth loss.
Probing depth reduction in smokers compared to
nonsmokers
All sites. The difference in full-mouth probing depth
reduction after nonsurgical therapy between smokers
and nonsmokers was assessed in six studies, of
which ve showed a better response in nonsmokers,
although the difference was small (Fig. 2). The results
showed a mean difference in probing depth reduc-
tion of 0.133 mm (95%CI [0.038,0.227], P 0.006)
with a chi-squared value for heterogeneity of 7.69
(5 df, P 0.18), i.e. the reduction in probing depth
was 0.133 mm greater in nonsmokers than in smok-
ers and there was no evidence to suggest that the
studies were dissimilar in their estimates of this result
(no evidence of heterogeneity, P > 0.05).
Only sites with an initial probing depth of 5 mm. A
separate analysis was undertaken for the threshold
studies, evaluating only sites with an initial probing
depth of 5 mm. Eight out of nine available studies
were included. One study (31) could not be included
in the meta-analysis because it was not comparable
with the others, due to an upper limit for probing
depth of experimental sites (i.e. only sites 46 mm
were evaluated). A random effects meta-analysis
indicated a weighted mean difference in probing
Initial search
Screening of
titles and
abstracts: n=330
Screening of
full-text articles:
n=80
Included
full-text articles:
n=13
Excluded
articles: n=250
Excluded
articles: n=67
Articles included
in meta-
analysis: n=12
Fig. 1. Flow of articles through the review.
126
Labriola et al.
Table 1. Characteristics of included studies
1st Author year
(ref. no.)
No. of
smokers
No. of
nonsmokers
No. of
treatment
sessions
Duration of
treatment
sessions (h)
Follow-up
(months)
Experimental
sites
Preber &
Bergstrom (31)
40 35 mean: 7.8 1 h session 1 Initial probing depth: 46 mm
Palmer et al. (28) n.r.
a
n.r. 2 3 h 6 Initial probing depth: 5 mm
Grossi et al. (12) 60 28 46 n.r.
a
3 Full-mouth and initial probing
depth: 5 mm
Machtei et al. (21) n.r. n.r. 4 n.r. 15 Full-mouth
Williams et al. (38) 91 159 1 n.r. 9 Initial probing depth: 5 mm
Haffajee et al. (14) n.r. n.r. 4 34 h 9 Full-mouth
Pucher et al. (34) 38 59 1 1 h 9 Initial probing depth: 5 mm
Preshaw et al. (33) 15 12 up to 4 up to 4 h 6 Full-mouth and test sites
(8 subject)
Preber et al. (32) 17 15 68 n.r. 2 Full-mouth and 1 site with initial
probing depth: 5 mm
Winkel et al. (39) 32 17 36 1 h session 6 Full-mouth
Ryder et al. (36) 61 48 2 n.r. 9 Initial probing depth: 5 mm
Renvert et al. (35) 13 15 n.r. n.r. 6 Initial probing depth: 6 mm
Mongardini et al. (24) 5 7 4 n.r. 8 Initial probing depth: 7 mm
a
n.r., not recorded.
Table 2. Quality of included studies
1st Author year
(ref. no.)
Similarity between
groups at baseline
Confounding factors Examiner blind to
smoking status
Proportion
followed-up
Listed Adjusted
Preber & Bergstrom (31) Yes Yes Unclear Unclear 100%
Palmer et al. (28) Yes Yes Yes Unclear Unclear
Grossi et al. (12) Yes Yes Yes Unclear Unclear
Machtei et al. (21) Unclear Yes No Unclear Unclear
Williams et al. (38) Unclear Yes Unclear Unclear Unclear
Haffajee et al. (14) No Yes No Unclear 100%
Pucher et al. (34) Yes Yes No Yes 8791%
Preshaw et al. (33) Unclear No No Unclear Unclear
Preber et al. (32) Unclear Yes Yes No 100%
Winkel et al. (39) Yes Yes Yes Unclear Unclear
Ryder et al. (36) Unclear No No Unclear 8594%
Renvert et al. (35) Yes Yes Unclear Unclear 100%
Mongardini et al. (24) Yes Yes Yes Unclear Unclear
127
depth reduction of 0.433 mm, favoring nonsmokers
(95%CI: [0.235,0.631], P < 0.001; chi-squared test for
heterogeneity 18.666, 8 df, P 0.009) (Fig. 3). The
highly signicant heterogeneity suggests that these
studies are not similar in their estimate of the result.
Because of limitations in reporting within the original
Table 3. Meta-analysis of differences in treatment effect in the smoking groups
Smoking
groups
Variable Probing
depth
category
Pooled
estimate
95% CI P-value
for
estimate
P-value
or hetero-
geneity
Effects Studies
(ref. nos.)
Lower Upper
S vs. NS Probing depth reduction Full mouth 0.133 0.038 0.227 0.006 0.180 Fixed (12, 14, 21,
32, 33, 39)
S vs. NS Probing depth reduction 5 mm 0.433 0.235 0.631 < 0.001 0.009 Random (12, 24,
28, 32,
3436, 38)
QS vs. NS Probing depth reduction Full mouth )0.016 )0.117 0.085 0.753 0.728 Fixed (12, 14, 33)
QS vs. NS Probing depth reduction 5 mm 0.130 )0.340 0.600 0.588 0.005 Random (12, 36)
S vs. NS Clinical attachment level
gain
Full mouth 0.114 )0.021 0.249 0.097 0.996 Fixed (12, 14,
21, 39)
S vs. NS Clinical attachment level
gain
5 mm 0.116 )0.047 0.278 0.164 0.337 Fixed (12, 24, 28,
3436)
QS vs. NS Clinical attachment level
gain
Full mouth )1.059. )4.027 1.910 0.485 < 0.001 Random (12, 14)
QS vs. NS Clinical attachment level
gain
5 mm 1.340 0.654 2.025 < 0.001 0.006 Random (12, 36)
Probing pocket depth mean improvement (mm)
0.5 0 0.5
Combined
Winkel et al. 2001 (39)
Preshaw et al. 1999 (33)
Machtei et al. 1998 (21)
Grossi et al. 1997 (12)
Haffajee et al. 1997 (14)
Preber et al. 1995 (32)
Study (ref)
Results in this direction indicate a
better result in non-smokers
better result in smokers
Note: Each study is represented by a blue box with a black horizontal line. The centres of the
boxes represent each studys mean estimate of the difference in PD change between smokers
and non-smokers. The size of each box is proportional to the weight given to each study when
calculating the combined (pooled) estimate. The lengths of the horizontal bars represent the 95%
confidence intervals for each studys mean estimate. The orange diamond represents the pooled
estimate of the mean difference from all the studies combined. The centre of the diamond is the
point estimate and the left and right tips are the 95% confidence interval for the pooled estimate.
The green vertical line represents the position at which there would be no difference in improvement
between smokers and non-smokers. Thus any confidence intervals that cross the green line
indicate no statistically significant difference between smokers and non-smokers.
Fig. 2. Forest plot of mean differ-
ence in probing pocket depth
reduction between smokers and
non-smokers (all sites).
128
Labriola et al.
publications, it was not possible to account for the
differences. As a cautious observation, it is clear that,
with one exception (34), all studies produced a
summary estimate favoring nonsmokers, although
the difference in Mongardini et al. (24) was not sta-
tistically signicant. It is therefore possible that
unreported differences in characteristics between
these studies might account for these differences in
outcomes.
Probing depth reduction in quit-smokers compared
with nonsmokers
All sites (Fig. 4). Only ve of the included studies
assessed the response of quit-smokers to nonsurgical
therapy (12, 14, 21, 33, 36). Among these, three could
be included in the meta-analysis for full-mouth
evaluation, as one (21) did not report results for quit-
smokers and another (36) considered only sites ini-
tially 5 mm. Fixed effects meta-analysis showed no
statistically signicant difference between quit-
smokers and nonsmokers ()0.016 mm, 95%CI
[0.117,0.085], P 0.753) and no signicant hetero-
geneity (chi-squared test for heterogeneity 0.636,
2 df, P 0.728).
Only sites with an initial probing depth of 5 mm
(Fig. 5). Three threshold studies comparing quit-
smokers and nonsmokers were found (12, 21, 36).
However, one (21) was not eligible to be included in
the meta-analysis because results were not reported
for quit-smokers. The difference was not statistically
signicant by random effects meta-analysis
(0.130 mm, 95%CI [)0.340,0.600], P 0.588; chi-
1 0 1 2
Combined
Williams et al. 2001 (38)
Ryder et al. 1999 (36)
Palmer et al. 1999 (28)
Renvert et al. 1998 (35)
Pucher et al. 1997 (34)
Preber et al. 1995 (32)
Study (ref)
Mongardini et al. 1999 (24)
reduction between smokers and
non-smokers (threshold studies).
0.4 0.2 0 0.2
Combined
Study (ref)
better result in quit-smokers
Preshaw et al. 1999 (33)
reduction between quit-smokers
and non-smokers (all sites).
129
squared test for heterogeneity 7.784, 1 df, P 0.005).
However, the heterogeneity between studies indi-
cates that it may not be appropriate to pool the
studies into a single overall estimate.
Clinical attachment level gain in smokers compared
to nonsmokers (Figs 6 and 7)
Four studies could be included in the meta-analysis
of the difference in clinical attachment level gain
between smokers and nonsmokers after nonsurgical
periodontal therapy. No statistically signicant dif-
ference was found between the two study groups
(0.114 mm, 95% CI [)0.021,0.249], P 0.097; chi-
square for heterogeneity 0.063, 3 df, P 0.996). For
sites with an initial probing depth of 5 mm (six
studies), the difference in clinical attachment level
gain between smokers and nonsmokers was not
statistically signicant (0.116 mm, 95%CI [)0.047,
0.278], P 0.164; chi-squared test for heterogeneity
5.699, 5 df, P 0.337).
Clinical attachment level gain in quit-smokers
compared to nonsmokers
All sites (Fig. 8). Two studies were available to
examine the difference in clinical attachment level
change between quit- and nonsmokers. A random
effects meta-analysis showed no statistically signi-
cant difference between quit- and nonsmokers in
terms of full-mouth clinical attachment level gain
(difference in clinical attachment level gain: 1.06 mm
1.0 0 1.0
Combined
Winkel et al. 2001 (39)
Clinical attachment level mean improvement (mm)
Study (ref)
Haffajee et al. 1997(14)
Machtei et al. 1998 (21)
ence in clinical attachment level
gain between smokers and non-
smokers (all sites).
0.5 0 0.5 1.0
Combined
Study (ref)
reduction between quit-smokers and
130
Labriola et al.
in favor of quit-smoking group: 95%CI [)4.027,1.910],
P 0.485; chi-squared test for heterogeneity 52.105,
1 df, P < 0.001) with highly statistically signicant
heterogeneity (P < 0.001).
Only sites with an initial probing depth of 5 mm
(Fig. 9). The meta-analysis of the two studies com-
paring the change in clinical attachment level
between quit-smokers and nonsmokers in sites with
an initial probing depth of 5 mm showed a differ-
ence in clinical attachment level gain of 1.34 mm,
favoring the nonsmokers (95% CI [0.654,2.025],
P < 0.001; chi-squared test for heterogeneity 7.470,
1 df, P 0.006). In both of these analyses, the degree
of heterogeneity suggests that it is not appropriate to
pool the results as the studies appear to be estima-
ting different results.
Investigating the heterogeneity between the
threshold studies
The reports of the studies included sufcient infor-
mation to investigate the effects of two of the a priori
dened potential sources of heterogeneity. Differ-
ences between smokers and nonsmokers in baseline
disease severity were available for seven of the eight
studies (not for [38]). Meta-analysis regression indi-
cated no evidence that this inuenced the pooled
estimate (change in estimate per 1 mm change in
baseline difference 0.523, 95%CI [ 0.099,1.145]).
Seven of eight studies (not [35]) could be utilized to
investigate the effect of the number of sessions of
treatment on the outcome. These also provided no
evidence that differences in this factor between
4.0 2.0 0 2.0
Combined
Study (ref)
gain between quit-smokers and
non-smokers (all sites).
1.0 0.5 0 0.5 1.0
Combined
Palmer et al. 1999 (28)
Pucher et al. 1997 (34)
Study (ref)
Renvert et al. 1998 (35)
Mongardini et al. 1999 (24)
gain between smokers and non-
smokers (threshold studies).
131
studies were a source of heterogeneity in the pooled
estimate (change in pooled estimate per addi-
tional session of treatment 0.079, 95%CI [)0.048,
0.020]).
Secondary outcomes
Bleeding was assessed after therapy in seven stud-
ies. However, due to great heterogeneity in the
methods used to of assess bleeding, it was decided
not to perform a meta-analysis and data are
reported for each study individually in Table 4. Of
these seven studies, one (33) reported bleeding on
probing results only on graphs and it was not
possible to extrapolate the data because of the small
scale. No statistically signicant differences in
bleeding were found between smokers and
nonsmokers either at baseline or after therapy in
most of the studies. However, one study (12) found
signicantly less bleeding in smokers than in non-
smokers at baseline and another (35) found a re-
duced response in terms of bleeding in smokers
compared to nonsmokers. In the two studies eval-
uating the change in bleeding in quit-smokers also
(12, 36) no statistically signicant difference was
found after treatment.
Patient-centered outcomes
No data were reported for any of the included studies
on patient-centered outcomes such as quality of life,
ease of maintenance, changes in aesthetic appear-
ance, or patient experience.
Discussion
This systematic review has shown that smoking can
have a negative effect on mechanical nonsurgical
periodontal therapy as indicated by less probing depth
reduction in smokers than in nonsmokers. Whereas
the analysis for sites with an initial probing depth of at
least 5 mm indicated statistical heterogeneity, a
glance at the forest plot (Fig. 3) demonstrates that six
out of the eight studies had outcomes that statistically
signicantly favored nonsmokers and the other two
studies had outcomes showing no statistically signi-
cant difference between smokers and nonsmokers.
Therefore, although the degree of heterogeneity is
troubling, the most likely conclusion is that smoking
decreases the effect of probing depth reduction.
Data were only available to explore the hetero-
geneity in terms of differences in baseline disease
severity between smokers and nonsmokers and the
number of sessions of treatment that patients
received. Neither of these factors signicantly inu-
enced the outcome of the studies. Nevertheless, this
should not be taken to mean that the impact of
smoking is unclear. Instead, we would interpret this
nding as indicating that smoking very likely affects
the treatment response, but that the size of the effect
remains uncertain.
It is not possible to reject the null hypothesis of no
difference between smokers and nonsmokers for all
outcomes. There were no statistically signicant dif-
ferences in the change in clinical attachment level
between smokers and nonsmokers either when
0 0.5 1.0 1.5 2.0
Combined
Study (ref)
gain between quit-smokers and
132
Labriola et al.
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133
studies considered full mouth sites or when they
considered only initially deeper sites.
We could speculate that the nding of a difference
between smokers and nonsmokers with respect to
probing depth and not clinical attachment level
could be explained, at least in part, by a reduced level
of edema in the periodontal tissues of smokers at
baseline. The increased vasoconstriction of periph-
eral blood vessels observed in smokers has been
related to reduced bleeding and edema in periodon-
tal patients who smoke, compared to nonsmokers (3,
8). If this is generally the case, smokers would have
less potential for resolution of inammation and
edema within the marginal tissues and therefore less
potential for gingival recession. Thus, there could be
a decrease in probing depth reduction, but no dif-
ference in clinical attachment level change.
Possible sources of heterogeneity between studies
in the size of treatment effect were investigated by
subgroup analysis and meta-analysis regression. One
possible source of heterogeneity could be related to
differences in initial disease severity, since previous
studies have shown that clinical outcomes of both
nonsurgical and surgical periodontal therapy are
related to the initial attachment level and probing
depth (6, 15, 29). However, meta-analysis regression
failed to detect an effect of initial defect depth or
duration of follow-up on the difference in probing
depth reduction and clinical attachment level gain
after nonsurgical therapy in smokers, nonsmokers
and quit-smokers. No statistically signicant differ-
ence was found when considering whether or not the
studies were adjusted for baseline values, suggesting
a reasonable similarity between groups. Other factors
that were initially planned to be investigated could
not be assessed due to missing data. These factors
included plaque level and tooth type.
When the difference in response to nonsurgical
treatment between quit-smokers and nonsmokers
was assessed, the data were not consistent. Regarding
probing depth change, the data from studies inclu-
ding all sites regardless of initial probing depth sug-
gest no difference between groups. For studies done
only on pockets initially 5 mm and deeper, one study
showed no statistically signicant difference and one
indicated greater pocket depth reduction in non-
smokers. For clinical attachment level gain, in the
meta-analysis for studies on all sites, one study fav-
ored quit smokers and one study nonsmokers. For
initially diseased sites only, both studies favored
nonsmokers over quit-smokers, although the sizes of
the difference between the two studies was quite
different. Possible causes of the differences between
these two studies are treatment characteristics and
the denition of smoking. Four to six sessions of
scaling and root-planing were performed in one of
the studies (12) and outcomes assessed at 3 months,
whereas only two sessions were performed in the
other study (36) and the patients were reevaluated at
9 months. The denition of smokers was also differ-
ent. Any smoker was included in one study (12),
whereas only subjects smoking 10 cigarettes or more
per day were selected in the other (36).
Clearly, the validity of drawing conclusions about
the early effects of quitting smoking from the avail-
able data is questionable. This is due partly to the
limited number of studies and partly to differences in
their outcomes. Therefore, the data on the effect of
quitting smoking (compared with nonsmokers) is
currently and perhaps surprisingly inconclusive. This
is an area that should be a high priority for future
research.
Secondary clinical outcomes such as tooth loss and
complications post-treatment were never reported.
On the other hand, changes in bleeding after therapy
were reported in about half of the included studies
(six of 13). The great variability in the methods of
assessing bleeding did not allow us to perform the
meta-analysis. However, it is apparent that most
studies did not nd a difference between smokers
and nonsmokers with respect to this. All but one
study (12) reported no statistically signicant differ-
ence in bleeding in smokers, nonsmokers and quit-
smokers at baseline. Similarly, no signicant differ-
ences between groups were found after treatment.
Only one study (35) found a statistically signicant
difference between smokers and nonsmokers in
terms of a change in bleeding after therapy (P < 0.05).
Limitations
One recurring problem in this review was the vari-
ability (or complete absence) of denitions of
smoking status. In addition, no study veried self-
reported smoking status with biochemical measures
such as salivary cotinine or exhaled carbon monox-
ide. Self-reported history may not be a reliable
method to assess smoking exposure; biochemical
tests to measure serum levels of metabolites of
nicotine should be used instead (10). We would
recommend that future studies investigate the utility
of biochemical measures of smoking exposure in
periodontal therapy. We have such a study in pro-
gress and hope to report the results soon.
A further limitation was the lack of data on tooth
loss. This meant that we had to rely on surrogate
134
Labriola et al.
measures such as change in probing depth and
clinical attachment level. Capturing the impact of
smoking on tooth loss would require follow-up peri-
ods lasting several years and such studies are difcult
to conduct. Rigorous observational studies could
provide such data and could also examine the effect
of smoking on additional treatment needs to secure
oral health. Such data would be valuable to estimate
the impact of this impaired treatment response.
Limiting the search to English language studies
could have introduced a selection bias. However, no
non-English studies were identied despite the
search of EMBASE, which has a greater coverage of
non-English journals.
Clinical implications
The reduction in the effectiveness of nonsurgical
periodontal treatment in periodontal patients
indicates that smoking cessation therapy should
be offered to smokers requiring such treatment.
Smoking cessation interventions can be successful in
the dental setting (37) but may require further training
and resources. Although this review has not investi-
gated the impact of smoking on future periodontal
treatment needs, other data also suggest that the
recurrence of disease is a greater problemfor smokers.
Thus, proper consent to treatment for smokers with
periodontal disease should include this information.
The further aim of this review was to assess the
effect of smoking on the response to nonsurgical
treatment in terms of patient-centered outcomes
such as quality of life, ease of maintenance, changes
in aesthetic appearance, and patient satisfaction.
However, no data on these outcomes were found in
any of the included studies.
Studies evaluating the effect of smoking on treatment
response should be based on reliable methods of
assessing smoking exposure, in place of patient-
reported data. These methods include the assessment
of salivary or serum levels of metabolites of nicotine,
such as cotinine, and the measurement of exhaled
carbon monoxide. Such objective measures are nee-
ded to investigate the impact of quitting smoking on
treatment outcomes. More emphasis should also be
given to the difference in the long-term response to
periodontal therapy in smokers, nonsmokers and
quit-smokers. In this respect, a useful outcome
measure could be tooth loss.
Conclusions
Following nonsurgical periodontal therapy, people
who smoke will experience less reduction in pro-
bing depth than nonsmokers. There is no evidence
of a difference in gain in clinical attachment
between smokers and nonsmokers or a reduction
of bleeding on probing between smokers and
nonsmokers. Differences in study design and lack
of data precluded an adequate and complete
pooling of data for a more comprehensive analysis.
In short-term studies, it is unclear whether people
who quit smoking will respond as favorably to
nonsurgical therapy to those who have always
been nonsmokers.
Progress in understanding the effects of smoking
on periodontal therapy will require the evaluation
of objective measures of smoking exposure such
as cotinine and exhaled carbon monoxide in place
of sole reliance on patient reported information.
Declaration of interest
We are grateful to the Eastman Dental Institute for
part funding of this study through an Eastman
Foundation for Oral Research and Training (EFFORT)
grant. Salaries were paid as HEFCE funding for Uni-
versity academics.
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Labriola et al.
Appendix 1
Search strategy
1 exp PERIODONTITIS th [Therapy]
2 periodontal therapy.mp. [mp title, abstract, cas
registry ec number word, mesh subject heading]
3 periodontal treatment.mp. [mp title, abstract,
cas registry ec number word, mesh subject
heading]
4 initial periodontal therapy.mp. [mp title,
abstract, cas registry ec number word, mesh
subject heading]
5 mechanical periodontal therapy.mp. [mp title,
subject heading]
6 non surgical periodontal therapy.mp. [mp title,
subject heading]
7 non surgical periodontal treatment.mp.
[mp title, abstract, cas registry ec number
word, mesh subject heading]
8 dental scaling.mp. [mp title, abstract, cas
9 exp Dental Scaling
10 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9
11 NICOTINE
12 smok$.mp. [mp title, abstract, cas registry ec
number word, mesh subject heading]
13 smoking cessation.mp. [mp title, abstract, cas
14 previous smokers.mp. [mp title, abstract, cas
15 former smokers.mp. [mp title, abstract, cas
16 SMOKING or exp SMOKING CESSATION
17 TOBACCO or exp TOBACCO USE CESSATION
18 cigarette smoking.mp. [mp title, abstract, cas
19 cigarettes.mp. [mp title, abstract, cas regis-
try ec number word, mesh subject heading]
20 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19
21 exp Gingival Pocket th [Therapy]
22 Periodontal Attachment Loss th [Therapy]
23 Periodontal Pocket th [Therapy]
24 Periodontal Diseases th [Therapy]
25 Dental Plaque th [Therapy]
26 prophylaxis.mp. [mp title, abstract, cas regis-
27 planing.mp. [mp title, abstract, cas registry ec
number word, mesh subject heading]
28 Root Planing
29 planing.ab. or planing.in. or planing.ti.
30 debridement.mp. [mp title, abstract, cas regis-
31 DEBRIDEMENT or debridement.mp.
32 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or
30 or 31
33 10 or 32
34 20 and 33
137
Effectiveness of mechanical
nonsurgical pocket therapy
JEAN E. SUVAN
The context
A number of systematic reviews have been published
on the subject of mechanical nonsurgical pocket
therapy. They have provided summaries of evolving
perspectives which continue to inform and direct
further research. This chapter aims to address the set
question what is the effectiveness of mechanical
nonsurgical pocket therapy? through the review of
published systematic reviews. At the same time as
examining the effectiveness, this article will highlight
current knowledge of the effect, efcacy, and ef-
ciency of this therapy, addressing the question in the
context of what is known about the four Es (effect,
efcacy, effectiveness, efciency). The objective is to
appraise and discuss research synthesis results and
conclusions, providing a summary of clinical impli-
cations for the patient and clinician and scientic
implications for the researcher. In this article,
mechanical nonsurgical pocket therapy refers to the
treatment of gingival and periodontal inammation
through mechanical removal of tooth and root sur-
face irritants to the extent that the adjacent soft tis-
sues maintain or return to a healthy, noninamed
state (70, 97). Additional terms often used to refer to
this therapy include mechanical debridement or
scaling and root planing.
Historical perspectives
Evidence-based healthcare and
periodontology
Evidence-based healthcare has evolved as a tool to
help clinicians apply the results of research to the
treatment of the individual patient (25). Its focus
starts and ends with the patient and is about decision
making. It was developed in response to a need to
deal with the rapid rate of technologic development,
the explosion in volume of literature available, and
disparities between research ndings and clinical
practice. In its attempts to get closer to the truth, it
carries with it an underlying principle and goal to
minimize bias in all contexts: clinical care, research,
and setting of health policy (19, 84).
Although suggested as early as 1884 by Lord Ray-
leigh in an address to the British Association for the
Advancement of Science (29), it was only in the mid
1980s that the healthcare community began to accept
that interpretation and clinical application of single
study results in isolation was unethical and imposs-
ible. The mandate was set that critical summaries
were needed to improve patient care (89). This
prompted the development of research synthesis
methodologies and increased efforts by all healthcare
disciplines, including dentistry, to provide systematic
reviews that would facilitate decision making (69).
Further to the benets this development has
brought to patient clinician and health policy decis-
ion making have been the effects for the research
community. In an ideal evidence cycle, new studies
should be designed and implemented in the context
of research synthesis (systematic summary of previ-
ous research) and research synthesis should in turn
provide guidance for further research (Fig. 1) (17).
With the intent to minimize bias at the center of the
research and the development of systematic review
methodology, much has been elucidated about the
quality of research. In this context, quality relates to
the extent to which research design, conduct and
analysis minimizes biases (29, 54). Research synthesis
has provided us with an improved framework for the
clinical and scientic application of evidence.
This framework has highlighted the importance of
precisely dened research questions as the founda-
tion for choosing other elements of study design. The
associated emphasis on patient-centered decision
making has facilitated the focus on patient outcomes;
in particular, research design for questions of thera-
peutic effect based on a clear understanding of the
48
PERIODONTOLOGY 2000
differences between effect, efcacy, effectiveness,
and efciency. Effect is the observed association
between interventions and outcomes or a statistic to
summarize the strength of observed association.
Efcacy, dened as the extent to which an interven-
tion can produce a benecial outcome under ideal
circumstances, provides a specic focus on the
patient in highly controlled conditions. Effectiveness
is the extent to which an intervention (therapy,
prevention, diagnosis, screening, education, social
care, etc.) produces benecial outcomes under
ordinary day-to-day circumstances. Efciency (inclu-
ding cost effectiveness) is the extent to which the
balance between input (effort or costs) and output
(outcomes including benets, side-effects) of inter-
ventions represents value for money (or resources
expended) (6, 18, 53, 54). Effectiveness and efciency
are clearly helpful in relating the results to every day
clinical practice. Each of the four Es has an
important role in the generation, interpretation and
application of evidence. Once effect is established,
the next step should include studies addressing
efcacy, followed in sequence by questions of
effectiveness and efciency, with each aspect
providing the basis for the next or feeding back into
previous aspects for further research (Fig. 2).
Research synthesis
New studies
Single Group
Cross Sectional
Cross
Sectional
Controlled
Case
Control
Cohort
Multi Centre
Randomized
Controlled
Trial
Randomized
Controlled
Trial
Ecological
Observational
Experimental
Descriptive Analytical
Fig. 1. Evidence cycle.
Effectiveness Efficiency Efficacy Effect
Definition: Definition: Definition: Definition:
Observed
association between
interventions and
outcomes
Extent to which an
intervention can
produce a beneficial
outcome under ideal
circumstances
Extent to which an
intervention
produces beneficial
outcomes under
ordinary day-to-day
circumstances
Extent to which the
balance between
input and output of
interventions
represents value for
resources expended
(time, effort, money)
Fig. 2. The four Es.
49
Mechanical nonsurgical pocket therapy
The primary goal of periodontal therapy is to
preserve the natural dentition by achieving and
maintaining a healthy periodontium. Mechanical
nonsurgical pocket therapy has long been docu-
mented as part of periodontal therapy. In the 1950s,
disease progression was understood to be associated
with the amount of plaque or calculus, which were
thought to be a physical irritant to the gingival tis-
sues. Therefore, removal of all tooth deposits was
considered the rst step of therapy directed at
inammation and pocket depth reduction (32, 36, 83,
95). As awareness of the role of plaque in the
inammatory process increased, the enzymes and
endotoxins released by the bacteria were considered
to directly destroy the periodontium (57, 58, 73).
Inthe last three decades, scientic advancement has
extended our understanding of periodontal disease
fromthe cellular to the molecular level and even to the
genetic level (7476, 96). New developments in
microbiology and genetics have added to our
understanding of the infectious nature and patho-
genesis of the disease (24, 33, 71). Current concepts
suggest that most forms of periodontal disease are
caused by a limited number of so-called periodontal
pathogens that accumulate on the tooth surfaces and
in the gingival sulcus, initiating interwoven elements
of host response that result in destruction of the
structures supporting the teeth (57, 75, 76). Simulta-
neously, the accepted understanding of plaque accu-
mulations has also changed. Bacterial plaque is now
known to be present in the form of a complex biolm
and calculus is perceived as a plaque-retentive factor
capable of harboring bacterial biolm (12, 24, 8688).
Despite signicant advancements in the knowledge
of disease pathogenesis and factors affecting the
progression of disease, traditional approaches to
mechanical debridement of the tooth surface to
remove tooth accretions continue to be an integral
part of periodontal therapy. New knowledge deter-
mining the rational for mechanical nonsurgical
pocket therapy continues to validate the importance
of therapies directed at removal or disturbance of the
plaque biolm and removal of factors facilitating
biolm formation (1, 20, 21, 23, 2628, 47, 59, 60, 79).
In 1984, Badersten et al. concluded that nonsurgi-
cal mechanical debridement of the periodontal
pocket will, in the majority of cases, result in
improvement of gingival health, arrest disease pro-
gression, and therefore, reduce the risk of tooth loss
(79). Now 20 years later, how can current best evi-
dence be summarized? What is known of mechanical
nonsurgical pocket therapy in the context of effect,
efcacy, effectiveness and efciency? Where might
the next steps be taken to develop this key aspect of
periodontal therapy?
Methodology
The highest level of evidence for examining a ques-
tion related to treatment effect is a well designed and
conducted systematic review of relevant studies (4,
69). Systematic reviews differ from narrative reviews
in that they employ methodologies directed at min-
imizing biases and random errors (18, 19, 25, 29, 38).
To conrm the possibility of using existing research
synthesis to address the set question posed at the
beginning of this chapter, an initial electronic search
of MEDLINE was performed. This search identied a
number of reviews as potential sources of evidence.
However, these were either narrative or systematic in
nature.
Search strategy
A comprehensive electronic search strategy, based on
evidence of search techniques, was then designed
and carried out by a representative of the Cochrane
Oral Health Group to isolate systematic reviews
published in the eld of periodontology between
1966 and December 2003 (10, 11). The databases
searched included Cochrane Oral Health Group List
of Systematic Reviews in Dentistry, DARE, and
MEDLINE. A total of 106 articles were found, which
were then screened in duplicate to identify reviews
employing elements of systematic review methodo-
logy and providing information related to mechanical
nonsurgical pocket therapy. A further electronic
search was performed by the author as an update
extending to March 2004 and included a search of
EMBASE and SCISEARCH with no date or language
restriction. Search strategy terms are outlined in
Appendix 1. The extended search found 191 articles,
which were then screened in duplicate based on the
previous criteria. Reference lists of located reviews
were checked for additional references. The com-
bined search and screening resulted in 12 reviews to
be considered in the current summary (13, 30, 31, 40
45, 48, 93, 94).
Appraisal of the evidence
As with new studies, systematic reviews may differ in
focus, design and conduct according to the research
50
Suvan
question set by the reviewers. It is also recognized
that not all systematic reviews are of equal quality
and that differences in methodology can result in
varying conclusions (29, 35, 49, 50, 62, 63, 72, 80).
Included reviews were assessed using existing criteria
for the appraisal of systematic reviews previously
published by Glenny et al. (35), Greenhalgh (37) and
Oxman (72). Key methodologic elements of the
included reviews and a summary of the characteristic
components of the evidence, including results, are
found in Tables 1 and 2, respectively. These are fol-
lowed by a narrative summary of the key aspects of
the included reviews with the articles grouped
according to the context of therapy rendered. Where
reviews included additional intervention arms, such
as adjunctive use of antimicrobials, they are dis-
cussed primarily in the context of results relating to
mechanical nonsurgical pocket therapy.
Narrative summary
Mechanical debridement in initial
therapy
On the basis of the question, the population and
the intervention inclusionexclusion criteria, ve
reviews considered mechanical nonsurgical pocket
therapy as a part of initial therapy or initial therapy
combined with maintenance therapy (30, 41, 48, 93,
94).
Hallmon & Rees (41)
The focus of this review question is on the effect of
manual vs. machine-driven instruments, with or
without adjunctive agents. The review incorporates
appropriate methodology with detailed transparent
reporting of methods and results. An extensive search
strategy was employed; however, in spite of broad
inclusionexclusion criteria, minimal evidence was
available, primarily due to a lack of information
reported in the studies obtained. It is unclear whether
searching additional electronic databases would have
yielded additional evidence. The evidence included is
described in tables with summary narrative state-
ments, as pooled analysis was correctly deemed
inappropriate. Inclusion of studies with data from
either site- or patient-based analyses renders pooled
analysis inappropriate as site-based analysis is likely
to signicantly overestimate treatment effect.
Variability in study design including populations,
interventions and outcomes was reported as a
deterrent to research synthesis. Based on the best
evidence available, the authors appropriately con-
clude that hand and machine-driven instruments
appear to be comparably efcacious in the reduction
of probing pocket depth, although this is better stated
as there being no evidence of differences between the
two instrument types. It is also clear that both types
of instruments gave results substantially better than
those in untreated controls, conrming the benet of
mechanical debridement. The authors highlight a
number of additional outcomes that should be
included in future research, including those focused
on effectiveness in day-to-day clinical practice and
efciency.
Van der Weijden & Timmerman (94)
This review addresses the effect of subgingival deb-
ridement on clinical outcomes in chronic period-
ontitis and is a methodologically sound, transparent,
and clearly reported systematic review. A compre-
hensive search strategy was implemented; however,
the authors did not report whether this included
unpublished literature and hand searches. Narrow
inclusion criteria were appropriately set to focus on a
question addressing effect. Subsequent analysis was
suitable for the evidence included, with similar
studies being grouped together, rendering minimal
pooled analysis possible.
The authors reported that a number of factors
varied between selected studies, including research
study design, conduct, analysis, and reporting.
Details of time, thoroughness of debridement, and
instruments used were seldom reported in the
included studies, and therefore were suggested to be
a potential source of variability in the therapy given.
Appropriate inclusion criteria set according to the
research question in this review resulted in limited
available evidence. However, the interpretation of
results within this context suggests that the review
provides evidence of the positive effect of subgingival
debridement on probing pocket depth and clinical
attachment level under controlled conditions (efc-
acy), with the effect being greater than that of
supragingival plaque control alone. While presenting
clear evidence of efcacy, the authors conrm the
need for additional research that would be of
value to clinicians, including data analysis of initial
pocket depth by subgroup to facilitate assessment of
treatment effect in shallow, moderate, and deep
pockets. Another desired requirement for nonsurgi-
cal therapy studies was the inclusion of multiple sites
per patient.
51
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(
9
3
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53
T
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l
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1
.
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t
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.
(
4
5
)
I
n
c
h
r
o
n
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c
o
r
a
g
g
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s
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v
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d
o
n
t
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t
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w
h
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s
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f
f
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t
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m
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j
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c
t
t
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c
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a
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d
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c
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m
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d
w
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R
P
a
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?
D
a
t
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b
a
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s
:
M
E
D
L
I
N
E
-
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u
b
M
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d
,
C
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c
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O
r
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l
H
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h
G
r
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c
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d
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g
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s
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M
B
A
S
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.
Y
e
a
r
s
:
1
9
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a
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l
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R
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s
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.
H
u
n
g
&
D
o
u
g
l
a
s
s
(
4
8
)
A
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m
t
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d
u
c
t
a
m
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t
a
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.
54
Suvan
Tunkel et al. (93)
These authors provide a well outlined, conducted,
and reported systematic review focused on a ques-
tion related to how mechanical debridement is per-
formed, that is, the aim was to compare the efcacy
of hand instruments to machine-driven instruments
in mechanical debridement. Although this initially
targets the efcacy of the two instruments, the results
also provide information about the overall effective-
ness and efciency of mechanical therapy, because
both instruments relate to the specic delivery of the
intervention. The outcomes sought in the reviewed
studies were patient and operator focused, including
but going beyond clinical surrogate outcomes.
The results reported inthis reviewoffer indisputable
evidence of the efcacy of mechanical nonsurgical
pocket therapy, with these results being in agreement
with those cited in the review by Van der Weijden &
Timmerman (94). In addition, the results are highly
applicable to clinical practice, suggesting no evidence
of a difference in the efcacy of the two instruments,
with machine-driven instruments being suggested to
be faster. The authors put the patient at the centre of
the review and highlight the need for additional evi-
dence in the area of side-effects for the patient and
clinician, as well as additional efciency data.
Hung & Douglass (48)
The authors of this paper meet their objective to
conduct a meta-analysis to determine the effect of
scaling and root planing on probing pocket depth
and clinical attachment level. This appears to be the
initial focus; however, the article goes on to describe
two additional meta-analyses associated with two
additional questions. Few details of the search strat-
egy are reported and therefore the extent and scope
of the search including modications necessary to
address the additional questions are not known.
These details are essential for an accurate assessment
of the completeness of the review.
The inclusion and exclusion criteria are broad and
include all periodontal disease population studies
where scaling and root planing was a stand alone
intervention and where outcomes were stratied
according to baseline probing pocket depth. The
inclusion criteria limited the selection of studies to
randomised controlled trials, the most robust study
design for investigating treatment effect. Quality
assessment of trial characteristics might have added
precision to the review. Weighting was applied in the
pooled analysis, based solely on sample size, a
B
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&
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3
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.
61
method considered to be less robust than inverted
standard error weighting. The validity of the results is
slightly unclear as minimal information is presented
on the completeness and appropriateness of the
included studies for the meta-analysis.
The authors state that with the increase of initial
periodontal probing depths, scaling and root planing
results in more positive clinical outcomes. This
statement could imply that the therapy is more pre-
dictable or effective in deeper pockets. It is intuitively
logical that pockets with a greater depth at baseline
have the greatest opportunity for improvement.
Improvement in clinical parameters following
mechanical debridement reported in this meta-
analysis is minimally higher than those reported in
robust systematic reviews which included quality
assessment in the methodology.
Elley et al. (30)
The review by Elley et al. is an example of a highly
systematic detailed report of research literature.
However, it is awed by an inadequate under-
standing and interpretation of the subject area.
There appears to be some misunderstanding of
periodontal disease and therapy as described in the
discussion of the background (which is not refer-
enced or evidence based). The investigation might
have beneted from a single focused question.
Pooled analysis might have produced clearer results
had the study inclusion criteria been limited to
designs appropriate for showing treatment effect
(randomized clinical trials).
There is an insufcient distinction between initial
and maintenance therapy in setting inclusionexclu-
sion criteria and in the interpretation of results in the
review. The author states information was only
available about outcomes from initial scaling after
one year as a proxy for annual scaling. No studies
were found where annual scaling was carried out over
a long period. This alone precludes the possibility to
address the initial research question of the review as
the assumption that initial scaling results after 1 year
act as proxy for annual scaling is inaccurate.
The author concludes that quarterly dental scaling
is not supported in specialist units and that the
effectiveness of quarterly scaling over annual scaling
in primary care is not proven. Interpretations of the
results focus on a particular health system in a
specic context. This limited perspective, together
with the lack of a background understanding of per-
iodontal diseases, renders conclusions inapplicable
to clinical practice.
T
a
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.
(
4
3
)
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1
3
(
9
r
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4
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.
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F
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,
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.
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P
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k
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,
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p
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a
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g
.
62
Suvan
The results are, however, valuable in identifying
issues and challenges of research synthesis in perio-
dontal disease trials. The author comments that the
quality of trials assessed was generally poor as judged
on the criteria of critical appraisal for each study
design. In particular, interventions, patient popula-
tions, randomization procedures, blinding, allocation
concealment and completeness of follow-up were
often not reported.
This review clearly highlights the importance of
and difculties in assessing the effectiveness and
efciency of therapies such as mechanical debride-
ment. The complexity of the multifactorial nature of
the periodontal disease, the factors affecting the
therapy given, in the context of a particular economy
provides an enormous challenge. The economic
analysis performed suggests that research money
invested to conrm effectiveness and efciency could
have tremendous impact on expenditures for perio-
dontal care.
Mechanical debridement in
maintenance therapy
Only one of the included reviews addressed
mechanical nonsurgical therapy solely in the context
of maintenance therapy (44).
Heasman et al. (44)
This carefully conducted and clearly presented
review is based on the well dened aim to investigate
the effectiveness of two regimens of supportive
periodontal care maintenance. Inclusion criteria
excluded patients who had surgery previously (eight
studies being excluded). Inclusion of these studies
with the use of subgroup analysis to cope with
differences between study populations might have
introduced additional evidence.
A detailed consideration of the studies included in
this review highlights numerous varying factors
between studies, making combined analysis contro-
versial. Combined analysis was performed but the
authors reported it to be compromised by variability
between studies, requiring the authors to make a
number of assumptions in pooling data.
The authors indicate that there was little difference
between pooled data estimates and the single study
included; however, the deciencies in study design of
both scenarios were noted. Condence intervals are
considerably wider for supragingival than subgingival
care, suggesting more uncertainty or less precision. It
could be argued that the changes that could be
expected during maintenance therapy might be
small, depending on the population characteristics at
the commencement of supportive therapy. There is
therefore a need for robust studies of sufcient
sample size in this area.
This review is an excellent example of research
providing no evidence of effect (as compared to
evidence of no effect). In clinical practice, main-
tenance therapy effectiveness has been based
partially on extrapolation of effect and efcacy of
debridement in initial therapy and partly on the
role of the biolm in disease pathogenesis. Further
research is needed specically in the area of
supportive therapy with clearly dened population
characteristics at baseline to avoid combining
studies which include diverse populations (e.g. non-
responding cases combined with cases containing
few residual signs of disease).
Mechanical debridement as
control therapy for antimicrobial
therapies
The results following mechanical debridement car-
ried out as part of control therapy in studies investi-
gating the adjunctive use of systemic or local delivery
antimicrobials were considered from six reviews (13,
31, 40, 42, 43, 45).
Hanes & Purvis (42)
This review describes each step in the systematic
process in intricate detail. The authors excluded
studies that did not clearly report the identied
quality assessment criteria (randomization proce-
dures, blinding, denition of study groups). This
highlights the challenge of deciding how to manage
unclear information in systematic reviews. Follow-up
with the respective authors might have increased the
number of studies included, providing the authors
could be contacted and could provide missing
information. In spite of the exclusion criteria, a
cohort trial and case-control study were included in
the combined results for scaling and root planing
(SRP) alone studies. The reason for this is unclear.
The cohort trial was reported to be split mouth,
although it was suggested that only parallel groups
were used for summary of SRP alone results to avoid
carryover effect.
The authors chose to exclude a well conducted
trial because therapy was limited to teeth with
63
furcation involvements (follows the inclusionexclu-
sion criteria). This identies yet another challenge in
systematic review methodology, that is, the inclusion
and meaning of full mouth data vs. selected site data.
It might have been helpful to include such studies in
a narrative evidence table as this information, the
differences in results depending on tooth or site
variation, is precisely what is lacking in many studies.
The summary of scaling and root planing results
alone conrms that mechanical nonsurgical pocket
therapy is successful in improving clinical parame-
ters. The authors very nicely state that more studies
are needed looking at the effectiveness of therapies
in all forms of periodontal diseases and efciency
aspects of the included interventions. Overall, this
review provides solid evidence that mechanical
nonsurgical therapy is efcacious.
Haffajee et al. (40)
This is an extremely detailed report of a thorough
systematic review based on a clearly dened ques-
tion. The question investigates effect, and therefore
inclusion of only RCTs and controlled clinical trials
(CCTs) is most appropriate. Although the inclusion
criteria state that other trial designs would be inclu-
ded, these were later excluded based on quality
assessment criteria and sufcient availability of RCTs
and CCTs. Quality assessment was performed using
numerous criteria. Pooled analysis was undertaken
with consideration of sources of heterogeneity. Study
results in control groups are listed in evidence tables.
No specic results of mechanical nonsurgical pocket
therapy are reported; however, it is important to note
that the authors clearly state that there is insuf-
cient evidence to support the use of systemic anti-
biotics as a mono-therapy in periodontitis patients.
The review endorses mechanical debridement as a
foundation therapy for the adjunctive use of systemic
antimicrobials.
Herrera et al. (45)
This review also meets the criteria outlined as key in
the appraisal checklists. The inclusionexclusion
criteria were set appropriately according to the
focused question. The focus of the review was
systemic antimicrobials; however, it offers some
valuable information applicable to mechanical non-
surgical pocket therapy.
First of all, results in the control groups, that is,
the mechanical debridement alone groups, were
comparable to results in other systematic reviews
showing signicant pocket depth reduction and
clinical attachment gain, therefore conrming the
efcacy of this therapy. In addition, the meta-analysis
undertaken demonstrated varied responses to ther-
apy (in this case, systemic antimicrobials) associated
with initial disease classication. No pooled analysis
was performed for mechanical debridement. These
results may suggest that further synthesis of the
research on scaling and root planing should be
carried out to increase knowledge of the predict-
ability of mechanical nonsurgical pocket therapy,
based for example on disease classication.
Elter et al. (31)
The authors of this review suggested that the aim, set
a priori, was to conduct a meta-analysis rather than
to explore the possibility of meta-analysis. As men-
tioned previously, current systematic review meth-
odology suggests that the decision to undertake
meta-analysis should not be made until the included
studies have been assessed for trial characteristics,
including quality assessment (18, 29, 51, 54, 63).
The search strategy was suited to the aim in the
context of the date of this review; however, current
search methods reect recent progress in this area. In
particular, hand searching of a number of journals
has been conducted by the Cochrane Collaboration
Oral Health Group, giving current review authors
access to more evidence. As well, author and indus-
try contact has been facilitated more recently by
electronic mail.
As in the previous reviews, results showed vast
differences in studies, making analysis and inter-
pretation problematic. Although the focus of the
review was the use of systemic metronidazole, the
analysis shows an effect from scaling and root
planing alone. The magnitude of this effect is in
agreement with results of other reviews performed at
this time. The inclusion of additional outcomes (e.g.
patient outcomes including side-effects) might have
increased the understanding and application of
results.
Bollen & Quirynen (13)
Although this older review includes some aspects of
systematic review methodology, based on current
criteria, it cannot be considered a systematic review.
Search and general inclusion criteria were dened on
the basis of population, intervention and outcomes,
but screening of articles or quality assessment was
not reported. By current standards, the search could
Suvan
64
be biased. All articles found in the search were
included. Due to the general nature of the question,
which includes four possible interventions, the
conducted search provides a large pool of evidence.
Results are presented in subgroups due to diversity
of studies identied by the authors; however, the
conclusions reached are fairly general. The authors
state that scaling and root planing alone had a
positive effect on probing pocket depth reduc-
tion, with results of improvement somewhat higher
than from the evidence available in subsequent
reviews.
The article is an excellent example of early
attempts at systematic review methodology in
periodontology. The author appropriately states the
difculty in summarizing the content of the 85
selected articles. Evidence tables provide trans-
parency of included evidence. Very thorough report-
ing is employed. However, reports of effect could be
biased by the narrative nature of the review. It is
difcult to make more specic conclusions about
mechanical debridement from this review.
Hayes et al. (43)
This systematic review, undertaken early in the 1990s,
was conducted and reported with a high level of
precision and still serves as an example of the issues
faced in research synthesis. The extensive reporting
of quality assessment performed, using a checklist of
items assessed, highlights inadequacies in study
design, conduct, analysis, and reporting that unfor-
tunately are still not being addressed 12 years later (5,
17, 67). Some parts of the methodology have pro-
gressed since this time. For example, the search was
current at that time but would now be considered
outdated. This may be a critical issue in relation to
information provided about antibiotic therapy but
is less of a concern in relation to outcomes follow-
ing scaling and root planing as this therapy has not
seen major technological advancements since that
time. Additional studies of improved quality may
have been conducted in the 12 years following this
review providing additional data, therefore current
reviews should be considered in clinical decision
making.
No narrative description is given of results from
studies that could not be included in pooled analysis
(called crude combining by the authors), which
included only limited studies. The data reported is at
site level, which is known to overestimate effect.
When comparing these results to patient level data,
this would appear to be the case.
Discussion
Evidence-based periodontology and
mechanical nonsurgical pocket therapy
The acceptance, publication and application of sys-
tematic reviews have increased rapidly in dentistry
over the last 10 years, with nine of the included
reviews being published within the last 3 years (30,
4042, 44, 45, 48, 93, 94). Simultaneously, substantial
progress has been made in research synthesis meth-
odologies (61, 68, 90). The 12 included reviews
highlight some of these differences. For example,
search strategy and results have changed signicantly
over the last decade not only due to investigations of
search strategies, but also as a consequence of tech-
nological advancement in databases and the sheer
number of databases now easily accessible (10, 11).
Meta-analysis is currently understood to be a statis-
tical technique for combining the individual effects of
a number of studies incorporated according to suit-
ability of studies included (18, 19, 29, 54, 64, 65).
There are still inconsistencies in approach in this
area, with some researchers setting meta-analysis as
the objective of the review rather than a well for-
mulated research question (78).
The evaluation of reviews to conrm the most
appropriate evidence to answer the clinical question
in a given context is essential in the application of
systematic review results (29, 37). Validity, applica-
bility and clarity of results can be diverse, as was
evident in the included reviews using critical
appraisal based on predened criteria. Each review
focused on a different population, interventions,
outcomes, and was undertaken using different
methodologies, at varying time points with or
without updates. Critical appraisal identied nine
reviews deemed to be the most appropriate to
answer the research question (31, 4042, 44, 45, 48,
93, 94).
In spite of immense variability in study character-
istics, the mechanical nonsurgical pocket therapy
given, patient populations, and the outcomes meas-
ured, mechanical debridement was shown in all nine
reviews to have a positive effect or to be efcacious
in the treatment of periodontal diseases, with the
exception of sites with probing pocket depth of
3 mm, where clinical attachment loss may occur.
Although the reviews reported signicant
improvements in clinical parameters to be expected
from mechanical nonsurgical pocket therapy, results
quoted in narrative and statistical summaries were
mean values, therefore camouaging detailed indi-
65
vidual patient information. Condence intervals were
often wide, suggesting uncertainty in the size of
change. This would be expected based on current
knowledge of disease pathogenesis together with
potential patient and environmental factors thought
to inuence disease progression and response to
periodontal therapy (16, 39, 52, 77, 91). In addition to
patient and environmental elements, operator factors
affecting therapy delivery have been suggested to
inuence results of mechanical debridement (3, 8, 15,
34, 46, 55, 56).
The effect and efcacy of mechanical nonsurgical
pocket therapy are supported by the summarized
evidence; however, the list of possible additional
factors, suggested in the reviews for further investi-
gation, allude to information on effectiveness and
efciency that is still lacking. Figure 3 presents a
synopsis of the numerous factors listed in the inclu-
ded reviews, placed in categories relating to popula-
tion, intervention, and outcome. All are relevant in
the context of time and cost issues faced by each
patient and clinician in everyday practice.
Many of these factors are related to how mechan-
ical pocket therapy is performed, or how it is best
incorporated into treatment regimens (effectiveness
and efciency). Two of the included reviews focused
on instruments to perform mechanical debridement
and conrmed that there is no evidence of a differ-
ence between hand and machine-driven (ultrasonic)
instruments in terms of clinical outcomes in the
treatment of single rooted teeth (41, 93). The time
needed to perform instrumentation was included as
an outcome, with some evidence that machine-dri-
ven instruments are faster than hand instruments.
One review cited a well conducted study investigating
the response of furcated molars to mechanical non-
surgical pocket therapy that was excluded from the
review due to its focus on limited sites (92). Research
synthesis protocols need to be designed to seek
information from these types of investigations.
All of the reviews highlighted the increasing need
to focus on the effectiveness and efciency of this
therapy. The issues to be addressed in future research
could perhaps best be summarized by the question
how can effectiveness be maximized while minim-
izing patient or operator harm and effort (efciency)?
Additional research focused on issues listed in
Fig. 3 would provide information on how mechanical
debridement is best performed or incorporated into
treatment regimens. It could facilitate interpretation
of statistical clinical signicance of surrogate clinical
outcomes through investigation of patient outcomes.
Full mouth disinfection and recent studies investi-
gating the time used for treatment are examples of
efforts in this direction (81, 82). Treatment regimen,
frequency and interval are particularly important in
the context of supportive therapy. This is a complex
area to investigate due to the multiple factors
affecting the long-term stability of sites treated in
periodontal patients (2, 14, 92).
Effectiveness / Efficiency
Cost
(Financial, psychological, physical)
Population Intervention Outcome
Patient Level
Disease classification
Disease extent and severity
Genetic factors
Environmental factors
Systemic conditions
Site and Tooth Level
Pocket depth
Single root vs. multi root
Defect topography
Debris (amount and tenacity)
Mobility
Therapy Delivery
Instrument type
Calculus detection
Time spent
Frequency
Interval
Number of visits
Post -treatment monitoring
Role of plaque control
Operator
Experience
Skill
Training
Patient
Tooth loss
Disease progression
Site vs. patient analysis
Adverse effects (root sensitivity,
recession, root surface
alteration, abscess)
Quality of life
Patient experience/satisfaction
Operator
Adverse effects
Safety
Fig. 3. Effectiveness efciency.
Suvan
66
Adjunctive therapies continue to be explored.
However, there are still no stand alone replacement
therapies for mechanical debridement and it is
therefore the best single therapy option available. It
remains the foundation therapy for many adjunctive
antimicrobial therapy investigations (31, 40, 42, 48,
85).
Continued development of research synthesis
methodologies has the potential to enhance know-
ledge of the effectiveness and efciency through the
synthesis of quantitative efcacy data together with
other study designs, including qualitative research
(22). This would include consideration of various
population groups, interventions and additional
outcomes including patient and operator outcomes
and cost effectiveness to enhance details of the pre-
dictability of the therapy. Combined with informa-
tion on patient status and preference, this informa-
tion could facilitate clinical practice. If periodontal
diseases and responses to therapy continue to be
viewed as inuenced by numerous factors, treatment
will need to be viewed as individualized. Therefore,
information on effectiveness and efciency are
essential to facilitate clinical decision making.
Implications for clinical practice
Initial therapy
In periodontitis patients, mechanical nonsurgical
pocket therapy reduces inammation, pocket
depth, and increases clinical attachment level.
The magnitude of pocket depth reduction cor-
relates with greater pocket depth before treatment.
Nonsurgical mechanical debridement may cause
loss of attachment in shallow pockets ( 3 mm).
There is no evidence of a difference in efcacy of
machine-driven (ultrasonic and sonic) and hand
instruments (insingle rootedteeth). Machine driven
instruments may be faster than hand instruments;
Adjunctive therapies have been developed and
investigated, but, to date, no therapy exists as a
stand alone replacement for mechanical nonsur-
gical pocket therapy.
Maintenance therapy
In periodontal maintenance patients, mechanical
debridement reduces inammation and disturbs
the bacterial biolm understood to be key in
disease control, including prevention of disease
progression.
The effect of mechanical nonsurgical pocket ther-
apy on pocket depth reduction and clinical
attachment gain in maintenance patients is
unclear; however, maintenance or stability of
pocket probing depth and clinical attachment level
has been demonstrated and meets the goal of
maintenance therapy.
Evidence for time, thoroughness, and frequency of
mechanical debridement recommended for perio-
dontal maintenance care is unclear.
High quality research evaluating the efcacy of
mechanical nonsurgical pocket therapy in various
sites defects, teeth and patients is needed to
assess factors potentially inuencing the predict-
ability of the therapy in various population groups
under certain conditions (including time or thorough-
ness, frequency, interval, instrument used, etc.).
To facilitate understanding of therapy effective-
ness, research should include assessment of oper-
ator aspects such as experience, technique, and
skill. Operator well being and safety should be
incorporated in risk benet assessments.
Research focused on psychosocial patient-centered
outcomes such as patient experience comfort,
satisfaction, adverse effects, esthetics, and quality
of life needs to be conducted and synthesized in
combination with clinical outcomes.
Efciency studies need to be performed which take
into account effectiveness data in order to evaluate
the cost effectiveness or the benet vs. harmrisk
for both patient and clinician.
Mechanical nonsurgical pocket therapy in the con-
text of maintenance therapy should be investigated
in further detail as a distinct population group.
Study design based on a clearly stated question
needs to be emphasized as the foundation of high
quality research, whether in new studies or
research synthesis. Study designs may need to be
altered to facilitate inclusion of patient outcomes.
For example, search strategies should be designed
to incorporate databases potentially containing
additional patient outcome (including psychoso-
cial aspects) and efciency studies (including cost-
effectiveness studies).
Additional research is needed to address method-
ologic issues of analysis of new studies and sys-
tematic reviews to further rene standards for
research specic to periodontology. Issues to be
67
addressed might include: site vs. patient level
analysis, split mouth studies, impact of search
strategies, sources of study heterogeneity, issues of
meta-analysis, and research synthesis of nonrand-
omised trials.
Researchers should provide details of essential
elements of study design, conduct and analysis,
adhering to standard reporting guidelines such as
the CONSORT statement (66) to increase the trans-
parency of researchandfacilitate researchsynthesis.
Future studies should be designed with the intent
of being incorporated in future systematic reviews.
Adherence to CONSORT will aid this objective.
Conclusions
Existing evidence in the form of systematic reviews,
the highest level of evidence for evaluating a therapy,
provide conclusive support for the benecial effect
and efcacy of mechanical nonsurgical pocket ther-
apy in the treatment of periodontal diseases. Con-
tained in this diverse evidence are suggestions of
factors related to effectiveness and efciency of this
therapy, facilitating the denition of future research
required to advance knowledge of these two ele-
ments. Additional investigations could be invaluable
in maximizing the predictability of treatment effect in
individual case scenarios (effectiveness) and could be
fundamental in simultaneously illuminating items
essential for minimizing patient and operator harm,
effort, or expenditure (efciency). Evidence-based
decisions related to the performance of nonsurgical
mechanical pocket therapy will continue as elements
of individual clinical judgment, therapy phase,
patient status and preference, and operator preference.
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Appendix 1 : Search strategy and
results
Database: Ovid MEDLINE(R) < 1966 to
April Week 12004 >: Search Strategy
1 exp Periodontal Diseases (44076)
2 mechanical.mp. [mp title, original title, abs-
tract, name of substance, mesh subject heading]
(86263)
Suvan
70
3 (instrument$ or debrid$). mp. [mp title, ori-
ginal title, abstract, name of substance, mesh
subject heading] (89678)
4 exp dental prophylaxis or dental scaling (4436)
5 2 or 3 (173780)
6 (nonsurgical or (nonadj surgical)).mp. [mp title,
original title, abstract, name of substance, mesh
subject heading] (7947)
7 1 and (5 or 6 or 4,3457)
8 limit 7 to (review or review, academic or review
literature or review, multicase or review of
reported cases or review, tutorial) (458)
9 1 and (5 or 6, 1769)
literature or review, multicase or review of
reported cases or review, tutorial) (302)
11 systematic.mp. [mp title, original title, abs-
tract, name of substance, mesh subject heading]
(45873)
12 11 and 10 (15)
13 exp Anti-Infective Agents (791358)
14 9 not 13 (1305)
literature or review, tutorial) (191)
16 from 15 keep 1 to 191 (191)
71
Effects of nonsurgical
periodontal therapy on the
microbiota
MAKOTO UMEDA, YASUO TAKEUCHI , KAZUYUKI NOGUCHI , YI HUANG,
GEENA KOSHY & ISAO ISHI KAWA
The microbiologic etiology of periodontal disease has
been conrmed by numerous studies conducted over
the years. Experimental gingivitis studies initially
proved the role of plaque in the etiology of perio-
dontal infections (88, 186). Although there has been a
shift in the paradigm (122), the role of microbiota in
the initiation and progression of periodontal disease
is conclusive. Periodontal treatment thus focuses on
the thorough removal of plaque, calculus, and plaque
products. Nonsurgical mechanical treatment, which
includes mechanical plaque control, scaling and root
planing, is the rst recommended step and is an
indispensable phase of periodontal therapy (26). In
this chapter, we intend to update the information
about periodontal microbiology and review recent
studies on the effect of nonsurgical periodontal
therapy on microbiota. In addition, we evaluate the
currently available microbial detection methods.
Current concepts in periodontal
microbiology
Early on, it was believed that plaque as a whole was
responsible for periodontal disease, and its removal
was essential for resolution of periodontal disease, a
theory which came to be called the nonspecic pla-
que hypothesis (187). The concept of bacterial spe-
cicity in the etiology of progressive periodontal
disease has now almost replaced that of nonspecif-
icity. One of the signicant advances in periodontal
research is the identication of specic pathogenic
species in periodontitis. Advances in anaerobic cul-
turing techniques, bacterial taxonomy, and molecular
identication have made this possible. A consensus
list of important periodontal pathogens has been
compiled based mainly on the organisms degree of
association with disease (174). Specic periodonto-
pathic bacteria such as Actinobacillus actinomyce-
temcomitans and Porphyromonas gingivalis were
detected in actively progressing periodontal pockets,
and are considered to be major pathogens in
aggressive periodontitis. Dening methods to selec-
tively eradicate these specic pathogens remains a
challenge in periodontal research.
The concept of dental plaque as a biolm has been
reviewed recently (21, 101, 175, 206). According to
this concept, plaque bacteria do not exist as free-
oating single-cell bacteria (planktonic bacteria), but
as attached bacteria in biolms (sessile bacteria), and
have a unique ecologic position in the structure of
biolms. Synergistic relationships exist among bac-
teria present in the biolms (73). Microorganisms
composing biolms cooperate with each other and
form a protective barrier of exopolysaccharide. In
addition, biolm bacteria are relatively inactive
metabolically, and exert more resistance to antibiot-
ics than planktonic bacteria (27, 43, 65, 158). More
than 500 times higher concentration of antibiotics is
required to kill biolm-forming organisms. The host
immune response is insufcient against biolm
infections (27), thus allowing periodontal breakdown
to proceed.
With these new concepts in mind, periodontal
treatment should not be limited to elimination of
specic periodontal pathogens but should also be
aimed at destroying biolms that protect resident
bacteria from antibiotics or the host immune system.
However, the concept of the biolm does not change
the importance of specic pathogenic bacteria in
periodontitis (21). Chemotherapeutic agents affect
certain periodontal pathogens as well as modify the
98
PERIODONTOLOGY 2000
composition of plaque nonspecically. Chen (21)
pointed out that biolm research is still in its infancy
and that periodontal treatment has yet to integrate
this concept. He also suggested that certain key
members of the dental plaque community may be
relatively easy to remove, resulting in a collapse of
the dental plaque community, which subsequently
can no longer support pathogenic species. It is also
possible that removal of exopolysaccharide by
chemical agents may weaken the biolms structural
integrity and sensitize the bacteria to antimicrobial
agents. Clearly, the removal of mature plaque is a
critical step in periodontal therapy.
Effect of supragingival plaque
control on subgingival plaque
bacteria
It is known that a certain level of oral hygiene is
essential for successful periodontal therapy (85, 86,
146). Early plaque-forming bacteria create an envi-
ronment necessary for the establishment of a more
fastidious microora, and development of the sub-
gingival ora depends on the presence of supragin-
gival plaque (169). It appears plausible that removal
of supragingival plaque inuences the development
and composition of subgingival plaque. However, the
effect of supragingival plaque control on subgingival
microora remains unresolved.
Several reports have shown that supragingival
plaque control cannot signicantly affect subgingival
bacteria in deep periodontal pockets. Kho et al. (69)
reported no signicant changes in the subgingival
bacterial composition of pockets 7 mm or deeper
after 18 weeks in patients who received only oral
hygiene instructions and supragingival professional
toothcleaning. Beltrami et al. (12) observed no sig-
nicant changes after 3 weeks in the composition of
subgingival plaque in pockets 6.5 mm or deeper,
following only professional supragingival plaque
control. Loos et al. (92) evaluated the effect of a
12-week period of oral hygiene alone on gingival
conditions and subgingival microora in 15 patients
with severe periodontitis. They reported that no sig-
nicant changes were noted in the subgingival
microora in patients who complied with oral
hygiene instructions and less compliant patients,
although the gingival condition improved moderately
in patients with good oral hygiene. Westfelt et al.
(205) studied the effect of meticulous supragingival
plaque control on subgingival ora in a split-mouth
study. They showed that supragingival plaque control
only fails to prevent periodontal tissue destruction in
subjects with advanced periodontal disease. These
studies show that subgingival bacteria have an eco-
logic system partly independent of supragingival
plaque, at least in deep periodontal pockets.
In contrast, some recent studies have demon-
strated positive changes in subgingival plaque fol-
lowing removal of supragingival deposits (28, 62,
104, 173). McNabb et al. (104) reported that supra-
gingival plaque control by professional toothclean-
ing induced signicant changes in the composition
of subgingival microora, including a decrease of
P. gingivalis and spirochetes. Dahlen et al. (28)
examined the effect of improved self-performed
plaque control after supragingival scaling on the
subgingival microbiota during a 2-year period.
Supervised supragingival plaque control markedly
changed the quantity and composition of subgingi-
val microbiota, in both deep and shallow pockets.
Furthermore, the number of subjects and sites from
which microorganisms such as P. gingivalis,
A. actinomycetemcomitans, and Campylobacter rec-
tus were detected was markedly decreased between
years 2 and 4. These ndings may imply that the
subgingival microbiota is inuenced not only by
local factors in periodontal pockets, but also by the
supragingival environment.
Meticulous supragingival plaque control may cause
shrinkage of the gingiva and a reduced probing depth
at periodontally involved sites. Thus, a reduced pro-
bing pocket depth may partly account for the chan-
ges in subgingival microora. Hellstrom et al. (62)
evaluated the inuence of carefully exercised supra-
gingival plaque control on subgingival microbiota at
periodontal sites with suprabony, infrabony, or fur-
cation pockets, hypothesizing that there would be no
signicant changes in the probing depth in infrabony
or furcation pockets. However, even in sites with no
signicant alterations in the probing depth, meticu-
lous and prolonged supragingival plaque removal
reduced the total number of microorganisms, as well
as the percentage of sites with P. gingivalis. There-
fore, it may be assumed that supragingival plaque
removal affects subgingival microbial composition to
some extent. Ximenez-Fyvie et al. (213) showed that
professional supragingival plaque removal on a
weekly basis signicantly diminished counts of
supra- and subgingival species, creating a microbial
prole comparable to that observed in periodontal
health and that, surprisingly, this prole was main-
tained at the nal monitoring visit 9 months
after completion of therapy. They suggested that
99
Nonsurgical periodontal therapy
professional cleaning may have changed the subjects
attitude towards performance of plaque control pro-
cedures and caused disruption of the supragingival
biolm, as well as a subsequent alteration of the
periodontal ecosystem, which led to the observed
prolonged effect on the subgingival microbiota.
The discrepancy in reports about the effect of su-
pragingival plaque control on subgingival microora
may be attributed to the differences in methodology,
extent of supragingival plaque control and initial
pocket depth. In the studies which failed to show a
signicant effect of supragingival plaque control on
subgingival microora (12, 69, 92), periodontal
pockets with a probing depth of greater than 6 mm
were selected. Dahlen et al. (28) reported that there
was no change of subgingival ora in periodontal
pockets with a probing depth greater than 6 mm,
although there was a signicant effect of supragin-
gival plaque control on subgingival microora in
periodontal pockets with a probing depth of less than
6 mm. In a recent review, Petersilka et al. (128)
mentioned that both patient-performed and profes-
sional supragingival plaque removal affects subgin-
gival microbiota only in the marginal 3 mm of the
periodontal pocket. Hence, it appears that the depth
of the pocket may determine the effect of supragin-
gival plaque control on subgingival bacteria.
Effect of supragingival plaque
control on recolonization of
subgingival bacteria
Several studies have demonstrated that supragingi-
val plaque is partly responsible for recolonization of
subgingival bacteria after treatment. Mousque`s
et al. (110) investigated the effect of a single session
of full-mouth scaling and root planing without oral
hygiene instruction on subgingival periodontal ora
of 14 adult periodontitis patients. The results indi-
cated that debridement was capable of disturbing
the proportions of certain bacterial forms, including
spirochetes in the subgingival ora, and that
approximately 42 days may be required for the
bacterial proportions to return to baseline levels.
Sbordone et al. (157) examined the recolonization
pattern of subgingival microora of adult perio-
dontitis patients who received a single session of
scaling and root planing, but no oral hygiene
instructions. Seven days after scaling and root pla-
ning, the microbial composition of treated sites was
similar to that of periodontally healthy sites, but at
60 days, there was no signicant variation between
the microbial composition prior to and after treat-
ment. Magnusson et al. (98) demonstrated that
supervised oral hygiene measures following sub-
gingival scaling resulted in marked improvement of
periodontal conditions and a pronounced and sus-
tained reduction in subgingival motile rods and
spirochetes, whereas in the presence of supragin-
gival plaque, subgingival microbiota containing
large numbers of motile rods and spirochetes was
reestablished in the pockets within 48 weeks.
MacAlpine et al. (97) and Braatz et al. (15) reported
that the rate of spirochetes in subgingival micro-
ora was maintained at a low level when good
plaque control was practiced after scaling and root
planing. In another split-mouth study by Lavanchy
et al. (81), the bacterial distribution tended to
return to baseline levels within 70 days after treat-
ment, although clinical parameters signicantly
improved.
The differences in ndings may be explained by the
performance of supragingival plaque control or the
extent to which subgingival bacteria was eliminated.
Subgingival bacteria after subgingival instrumenta-
tion may originate from microbiota which were not
completely removed, or microbiota in supragingival
plaque which proliferated, matured, and then spread
subgingivally. From the available data, it seems that
proper supragingival plaque control following sub-
gingival instrumentation may be essential for pre-
venting subgingival recolonization of pathogenic
bacterial species.
Effect of scaling and root planing
on subgingival microora
Supragingival plaque control can partially improve
clinical gingival symptoms, but to produce sub-
stantial improvement, it is necessary to perform
subgingival plaque control (205). Subgingival plaque
in biolm can evade the defense mechanisms of the
host and diminish the effect of chemotherapeutic
agents. Biolms cannot be eliminated by daily oral
hygiene methods. Thus, mechanical debridement,
including scaling and root planing, is required for
successful periodontal treatment. Subgingival sca-
ling and root planing clinically induces a reduction
in periodontal pocket depth, a decrease in the
number of gingival sites with bleeding on probing,
attachment level gain, and a shift from a predom-
inantly gram-negative to a gram-positive subgingival
100
Umeda et al.
microbiota. In addition, it decreases the number
of microorganisms, including black-pigmented
species and spirochetes (81, 110, 170, 185). It has
also been shown that ultrasonic instrumentation
can cause reduction in spirochete and motile rod
counts with a concomitant increase in coccoid cells
(11).
Haffajee et al. (54, 55) examined the levels of 40
bacterial species including A. actinomycetemcomi-
tans, P. gingivalis, Prevotella intermedia and Trepo-
nema denticola using checkerboard DNADNA
hybridization before and after scaling and root pla-
ning in 57 adult periodontitis patients. After scaling
and root planing, a mean gain in attachment level
and a reduction in gingival redness, bleeding on
probing, and mean pocket depth were observed.
Mean prevalence and levels of P. gingivalis, T. den-
ticola and Tannerella forsythia (Bacteroides forsythus)
(152) were signicantly reduced, whereas the level of
Actinomyces viscosus increased. Interestingly, when
the patients were divided into two groups, a poor
response group (18 subjects with mean attachment
loss) and a good response group(39 subjects with
mean attachment level gain), the prevalence of
Actinomyces naeslundii genospecies 2 (A. viscosus),
T. denticola, Campylobacter gracilis and C. rectus was
signicantly higher before treatment in the good
response group.
Recently, Dougudomdacha et al. (32) reported
changes in levels of subgingival microorganisms after
nonsurgical therapy by quantitative polymerase
chain reaction (PCR). According to their results, the
number of P. gingivalis was positively associated with
periodontal pocket depth and attachment loss in
adult periodontitis. Furthermore, they showed that
none of the species was eradicated, and attachment
levels and bleeding on probing were not improved,
although the numbers of P. gingivalis, P. intermedia
and A. actinomycetemcomitans decreased and perio-
dontal pocket depth improved after scaling and root
planing. They suggested that a threshold number of
P. gingivalis associated with early clinical signs of
disease may be approximately 2.6 10
4
cells. How-
ever, Haffajee et al. (57) had previously reported the
threshold to be 6 10
5
.
Darby et al. (30) investigated the effects of sca-
ling and root planing on subgingival microora.
PCR was used to determine the presence of
A. actinomycetemcomitans, P. gingivalis, T. forsythia,
P. intermedia, and T. denticola in four sites from 28
patients before and after scaling and root planing.
The treatment resulted in clinical improve-
ment, and there were signicant reductions in
P. intermedia, T. forsythia, and T. denticola at a site
level. However, on a subject basis, there was little
or no change in the microora. Renvert et al. (140)
also demonstrated that root debridement resulted
in clinical improvement, such as reduction in per-
iodontal pocket depth and bleeding on probing
sites and reduced levels of subgingival bacteria.
While P. gingivalis was eliminated from a majority
of infected subgingival sites, A. actinomycetem-
comitans still remained in a high proportion of
sites after therapy. Other studies have also showed
that it is difcult to eliminate A. actinomycetem-
comitans from periodontal pockets of juvenile per-
iodontitis by scaling and root planing alone (23, 75,
171), probably due to the ability of A. actinomyce-
temcomitans to invade gingival tissues (17, 22, 44,
151). Surgical excision of the gingival tissues har-
boring these bacteria or the use of systemic anti-
biotics might be possible ways to eradicate the
pathogen. It is reported that, although it is possible
to suppress A. actinomycetemcomitans immediately
following surgery, reinfection of subgingival sites
frequently occurs several months later, due to
translocation from other sites in the oral cavity
(139). On the other hand, Ali et al. (3), Rosenberg
et al. (145), and Darby et al. (30) reported that
scaling and root planing eradicated A. actinomyce-
temcomitans from infected sites. This discrepancy
may be dependent on pretreatment levels of
A. actinomycetemcomitans in infected sites.
It has been shown that scaling and root planing can
signicantly affect the quantity of subgingival
P. gingivalis (55, 96, 140, 208). The rate of removal of
P. gingivalis was reported to be 70% according to
Renvert et al. (140), 68% according to Wilkstrom
et al. (208), and 66% according to Takamatsu et al.
(184). While the decrease of P. gingivalis is associated
with improvement of clinical symptoms, high levels
of the bacterium are detected in sites that show poor
response to therapy (140, 208).
There are only a few available reports as to the
effect of scaling and root planing on T. forsythia.
The change in percentage of the sites colonized by
T. forsythia before and after nonsurgical therapy was
43% to 27% according to Haffajee et al. (55), 77.9%
to 35.6% according to Takamatsu et al. (184), and
57.6% to 36.6% according to Darby et al. (30). All
the studies show the difculty in eliminating
T. forsythia by nonsurgical therapy. However, recent
studies utilizing a full-mouth approach to nonsur-
gical treatment (131133) have shown promising
results in terms of eradicating P. gingivalis and
T. forsythia.
101
Effect of nonsurgical therapy on
subgingival microora in furcation
involved sites
It has been shown that moderately deep and deep
molar furcation sites respond less favorably to non-
surgical therapy than do nonmolar sites and molar
at surface sites of similar probing depth (94, 119).
The poor response to therapy in molars with furca-
tion involvement may be due to specic anatomic
conguration, poor accessibility to the fornix, con-
cavities of the furcations, and inadequate plaque
control in furcation sites. In a longitudinal study with
an observation period of 52 weeks, Loos et al. inves-
tigated the clinical and microbiologic effects of pla-
que control and root debridement in grade II molar
furcation sites (93). They reported that sites with
probing attachment loss exhibited higher microbial
counts and higher proportions of spirochetes, black-
pigmented colony-forming units, and P. gingivalis
colony-forming units than sites with probing
attachment gain. Leon & Vogel (82) showed that in
severely involved furcations, ultrasonic instrumenta-
tion succeeded in decreasing the counts of spiro-
chetes and motile rods. Drisko (34) commented that
power-driven instrumentation using newly designed
micro-ultrasonic tips may be more effective for
debridement of furcation areas. Although furcations
are difcult to treat due to limited access, use of new
technology might help to improve the clinical and
microbial outcomes of nonsurgical treatment in fur-
cations.
Antimicrobial agents in
periodontal therapy
Elimination or adequate suppression of periodonto-
pathic bacteria in subgingival microbiota is essential
for periodontal healing. Mechanical root debride-
ment is a prerequisite for controlling periodontal
infections, and clinical improvement following
mechanical root debridement is directly related to
the degree to which pathogenic subgingival microbes
are removed (54, 184). However, conventional
mechanical root debridement does not usually
eradicate all periodontopathic bacteria from the
subgingival ecosystem (3, 20, 23, 45, 55, 106, 115, 137,
139, 157, 161). Sites with deep periodontal pockets,
grooves, furcations, and concavities are difcult to
access with periodontal instruments. Thus, perio-
dontopathic bacteria may remain in those sites. In
addition, periodontal bacteria have been detected on
the mucosa, tongue, tonsils, and gingiva (125, 197,
217) from where they may colonize dental plaque.
Also, P. gingivalis, A. actinomycetemcomitans, and
spirochetes are capable of invading gingival epithelial
cells, subepithelial connective tissues, and dentinal
tubules (1, 24, 35, 100, 149). In such cases, antimi-
crobial agents may help to further suppress perio-
dontal pathogens. Furthermore, most bacterial
species reside in a biolm within subgingival pockets
(31), and microorganisms in biolm are more
resistant to the bactericidal action of antibiotics in
comparison to their planktonic forms (5, 6). There-
fore, treatment of periodontal disease with antimi-
crobial agents alone may not often be sufcient, and
mechanical instrumentation should routinely be
performed to disrupt the biolm and to remove the
bulk of bacterial deposits prior to antimicrobial
therapy (51).
Selection of an antimicrobial agent
Identication of periodontal pathogens and delinea-
tion of the type of periodontal infection is necessary
for selecting a proper strategy for antimicrobial
therapy in periodontitis. The choice of antimicrobial
agent depends on the target microorganisms.
Although more than 500 bacterial taxa have been
identied within periodontal pockets, only a limited
number are associated with periodontal diseases
(216). Identifying the key species considered
responsible for the infection is a diagnostically valid
and economically viable approach in periodontal
treatment. Microbiologic analysis helps determine
the optimal antibiotic therapy and effectiveness of
treatment. Microbial analysis may be repeated after
treatment to verify the suppression of the pathogens.
It is also performed to screen for possible superin-
fecting organisms, as periodontal superinfections
may be caused by previous use of antibiotics (56, 63,
135).
The antimicrobial susceptibility of several perio-
dontal pathogens has been examined in vitro to
determine the antibacterial activity and spectrum of
various agents (72, 129, 194). This information is
useful to select the proper type and dosage of anti-
microbial agents. However, these data must be
interpreted with caution, because the efcacy of an
antibiotic is usually determined using planktonic
bacterial cells in vitro, which does not necessarily
account for the biolm effect (5, 6). In addition, the
percentage of organisms developing resistance to
102
Umeda et al.
commonly used drugs has increased dramatically
during the past decade (200). In general, microor-
ganisms that are not strongly associated with perio-
dontitis, including the Streptococcus or Actinomyces
species, tend to show the most resistance to antibi-
otics (77). However, there have been reports of some
periodontopathic bacteria that exhibit resistance to
antibiotics (72, 130) or possess the resistance gene to
antibiotics (25, 42, 78).
Antimicrobial effects of subgingival
irrigation
Various antimicrobial agents have been used for
subgingival irrigation by dental professionals. Clin-
ical benets of subgingival irrigation with chlorhex-
idine, povidone-iodine, tetracycline, SnF
2
, hydrogen
peroxide, etc. (138), have been compared to those of
conventional mechanical root debridement alone.
Several studies have found that subgingival irriga-
tion with these agents reduced the amount of sub-
gingival pathogens, such as spirochetes (59, 79, 163,
180). These investigations established the biologic
rationale for irrigation therapy, but the long-term
antimicrobial effect of a single course of subgingival
irrigation is limited. Southard et al. (179) reported
that reduction of P. gingivalis was observed only
until 2 months after 2% chlorhexidine irrigation.
Furthermore, the effect of subgingival irrigation with
various antimicrobial agents did not enhance the
effect of bacterial suppression achieved with scaling
and root planing, and many studies revealed little or
no benets from irrigation therapy (97, 141, 159
161, 204). Some researchers suggested the use of
povidone-iodine (PVP-I) for subgingival plaque
control (50, 67, 165). Recently, Slots recommended
subgingival irrigation of 10% povidone-iodine by a
syringe for 5 min to control subgingival colonization
of periodontal pathogens and various types of peri-
odontal disease (166). In addition to its ability to
rapidly kill bacteria and yeasts, povidone-iodine may
exert activity in the presence of organic matter, and
carries no risk of the emergence of resistant bacteria
(50). Rosling et al. (147) reported favorable clinical
and microbiological effects of adjunctive povidone-
iodine irrigation in periodontal treatment, including
furcation lesions in multirooted teeth. However,
additional studies are needed to verify the efcacy of
povidone-iodine against subgingival microorgan-
isms.
Wikesjo et al. (207) reported that A. actinomyce-
temcomitans was eliminated in 46% of periodontitis
sites irrigated with 3% hydrogen peroxide biweekly
over 6 months, despite the lack of adjunctive clinical
benet. Interestingly, MacAlpine et al. (97) also
investigated the effects of biweekly subgingival irri-
gation with chlorhexidine and tetracycline over
6 months. They demonstrated that although the sites
treated with these agents exhibited reduction in
spirochetes, similar improvement was observed in
saline irrigated and nonirrigated control sites. Simi-
larly, Taggart et al. (183) compared the efcacy of
chlorhexidine and water administered via an ultra-
sonic device while scaling, and showed that both
treatments reduced the number of motile rods and
spirochetes. These results suggest that the washing
out effect may be more important than the antimi-
crobial actions of the agents.
Antimicrobial effects of local delivery
agents
The benet of local antimicrobial therapy is the
higher concentration of an antimicrobial agent,
which can be attained in subgingival sites compared
with a systemic drug regimen (138). The disadvantage
of local antimicrobial therapy is the difculty of
placing therapeutic concentrations of the antimicro-
bial agents into deeper parts of periodontal pockets
or furcation lesions. Thus, topical antimicrobials may
not be adequate to eliminate all periodontopathic
bacteria. The efcacy of locally applied antimicrobial
agents in periodontal therapy also depends on whe-
ther there is sufcient contact time between the
antimicrobial agent and the target microorganisms
(48).
Table 1 shows recent clinical studies involving
professional application of local antibiotics with
sustained pocket delivery techniques. These studies
used local antimicrobial agents as an adjunct to
scaling and root planing, and evaluated the micro-
biological effects of subgingivally administered anti-
microbial agents in chronic periodontitis patients.
Tetracycline, minocycline, doxycycline, and met-
ronidazole have been used in sustained delivery
devices. Tetracycline, minocycline, and doxycy-
cline have a similar spectrum of activity, and are
considered effective against gram-positive and
gram-negative bacteria, mycoplasma, rickettsia, and
chlamydia. Tetracyclines are considered to be bac-
teriostatic, but may have a bactericidal effect at high
concentrations (199). In addition, they can inhibit
microbial attachment (80, 127). Metronidazole is
effective against gram-positive and gram-negative
anaerobes, including P. intermedia, P. gingivalis, and
Fusobacterium sp. (129).
103
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Umeda et al.
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106
Umeda et al.
Although most studies showed that antibiotic
therapy combined with mechanical treatment was
equally as effective as conventional scaling/root pla-
ning, there was little or no difference between the two
treatment modalities in the numbers/incidence of
periodontal pathogens (83, 142, 148, 189, 192). The
clinical and microbial effects of local antimicrobial
agents have been well documented (64, 66, 182, 201,
212, 215). However, most of these studies showed
only a transient reduction of periodontal pathogens,
and may not provide a compelling reason for routine
use of antimicrobial agents in conjunction with root
debridement. However, sites and patients unrespon-
sive to conventional therapy frequently respond to
antibiotic treatment (33, 194). Local drug delivery as
an adjunct to conventional care should be reserved
for nonresponding sites or patients with recurrent
disease who need an alternate treatment approach.
However, there is limited data to indicate that local
antibiotic therapy provides a clinically signicant
improvement in these situations. Furthermore, only a
few reports have compared the efcacy of different
local delivery systems (71, 134). Thus, it is difcult to
conclude that one device is superior to another (52).
Antimicrobial effects of systemic
antibiotic therapy
Systemic drug therapy offers several benets over
local drug delivery. Since systemic drugs can be
delivered widely to intraoral tissue via serum, they
may be more practical for a patient with multiple
diseased sites than local antimicrobial therapy. In
addition, systemic drug therapy can affect the peri-
odontal pathogens colonizing oral mucosa or are
present in saliva (29, 107, 125), and the risk for sub-
gingival recolonization of pathogens after periodontal
therapy may be reduced (111).
Systemic antimicrobial therapy has been studied
using several antibiotics in various types of perio-
dontitis (Table 2). Many researchers have reported
elimination or marked suppression of periodontal
pathogens with tetracyclines (103, 136). However, it
has also been reported that tetracycline treatment
together with scaling and root planing has only a
minor effect on subgingival microbiota compared to
scaling and root planing alone (10, 84, 170). In
addition, tetracycline resistance has been observed
in periodontal pathogens, and a failure of suppres-
sion of periodontal pathogens has been reported
(39, 76, 113, 156, 195). Clindamycin may be con-
sidered for periodontal infections comprised of high
levels of peptostreptococcus, b-hemolytic strepto-
cocci, and various oral gram-negative anaerobic
rods (202), such as the species in aggressive and
refractory periodontitis (49, 99). Sigusch et al. (162)
showed that two-step root debridement and
administration of clindamycin almost completely
eradicated P. gingivalis and A. actinomycetemcomi-
tans after 24 months of treatment. Amoxicillin
possesses substantial antibacterial activity for gram-
negative species, and shows high concentrations in
serum and high susceptibility to periodontal
pathogens (47). However, amoxicillin is susceptible
to degradation by b-lactamase and is therefore
generally used together with metronidazole. Aug-
mentin
(GlaxoSmithKline, Brentford, UK), a com-

posite formulation of amoxicillin and clavulanic acid,
has been shown to be effective against A. actin-
omycetemcomitans and P. gingivalis in recurrent/
progressive periodontitis patients (58, 102, 198).
Metronidazole has been the focus of research in many
years due to its antimicrobial activity against gram-
positive and gram-negative anaerobes (68, 108). It can
suppress subgingival pathogens like spirochetes,
motile rods, P. intermedia, and P. gingivalis, and is
effective in various forms of periodontitis (2, 38, 89
91, 120, 162, 210). However, there have been reports of
reappearance with time of pathogenic bacteria after
systemic metronidazole therapy (53, 108). Recently,
the clinical and microbiological effects of a subanti-
microbial dose regimen of doxycycline (SDD) have
been examined. SDD plus scaling and root planing
has resulted in a small, but signicant, clinical
improvement compared to scaling and root planing
alone (18, 19, 46), but there was no difference in the
composition of microbial ora. Although it has been
suggested that SDD has the potential to induce drug-
resistant bacterial strains, several in vivo studies that
addressed this issue failed to detect any resistance
development (18, 46, 188, 203).
Subgingival microbiota in periodontitis often har-
bors more than one periodontopathic species, and
certain combinations of bacterial species have been
implicated in periodontal breakdown (36, 37, 176).
Since the subgingival microbiota in destructive peri-
odontal disease consists of various periodontal
pathogens with different antimicrobial susceptibility,
combination drug therapy using two or more anti-
biotics is sometimes warranted. Combinations of
antibiotics broaden the antimicrobial range beyond
that attained by a single antibiotic, and also help
prevent emergence of bacterial resistance. Combined
therapy may also help to lower the dose of individual
antibiotics by synergistic effect between two antibi-
otic drugs.
107
T
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108
Umeda et al.
W
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109
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110
Umeda et al.
Metronidazole plus amoxicillin has been used
successfully in the treatment of several types of
periodontitis, especially with A. actinomycetemcom-
itans-associated infections (13, 112, 125, 195, 196),
where they have a synergistic effect (124). Berglundh
et al. (13) and Winkel et al. (211) administered
metronidazole and amoxicillin in conjunction with
scaling and root planing to advanced periodontitis
patients, and revealed elimination or marked sup-
pression of periodontal pathogens such as P. gingi-
valis, T. forsythia and P. intermedia. Lopez et al. (95)
applied metronidazole-amoxicillin in advanced
adult periodontitis patients without mechanical de-
bridement and showed that the proportions of
subgingival P. gingivalis and P. intermedia was sig-
nicantly reduced in the antibiotic treated group
compared to the placebo group. Flemmig et al. (41)
reported that antibiotic therapy with metronidazole
plus amoxicillin suppressed A. actinomycetemcomi-
tans in the entire oral cavity to below detectable
levels for over 12 months, but P. gingivalis persisted
or recurred. They suggested that the metronidazole
amoxicillin therapy might not be effective in cases
of mixed infections involving A. actinomycetem-
comitans and P. gingivalis. Metronidazole and ci-
prooxacin is another combination currently used
against anaerobic and facultative bacteria (124, 168).
The combination of antibiotics frequently used for
periodontal therapy is metronidazoleamoxicillin
(250375 mg of each 3 daily for 8 days) and met-
ronidazoleciprooxacin (500 mg of each 2 daily
for 8 days).
A position paper by the American Academy of
Periodontology (164) and a recent review of literature
(172) indicated that systemic antibiotic therapy is not
acceptable for standard use in chronic periodontitis,
though many studies reported clinical and microbi-
ologic benets (13, 38, 40, 95, 120, 162, 211). Above
all, the majority of patients with chronic periodontitis
can be successfully managed with conventional
mechanical therapy alone. Systemic antibiotic ther-
apy should be considered in patients with various
types of aggressive periodontitis.
Limitations of nonsurgical
periodontal therapy
Periodontal pathogens are capable of invading per-
iodontal tissues. Supercial penetration by spiro-
chetes in the region of acute necrotizing ulcerative
lesions was reported by Listgarten (87). Saglie et al.
(150) found bacteria invading the epithelial wall of
deep periodontal pockets in ve of eight cases using
scanning electron microscopy. Gingival invasion by
A. actinomycetemcomitans in localized juvenile per-
iodontitis lesions has been identied by immuno-
peroxidase and immunouorescent studies, and the
number of gram-negative bacteria in connective
tissue was signicantly higher in active sites than in
inactive sites (116). Also, A. actinomycetemcomitans
has the ability to invade human gingival epithelial
cells cultured in vitro (14, 105). Invasive strains of
P. gingivalis from subgingival plaque at periodon-
tally diseased sites tended to spread along tissue
planes rather than grow in colonies, while nonin-
vasive strains in dental plaque from periodontally
healthy sites exhibited localized abscess formation
(116). P. gingivalis can adhere to and enter oral
epithelial cells as well (154). Periodontal pathogens
have also been recovered from oral tissues such as
tongue, oral mucosa, tonsils, and saliva (9, 191, 197).
Mechanical therapy alone may not completely
eliminate the periodontal pathogens residing in
these extradental oral sites. Adriaens et al. (1) have
shown that in spite of meticulous scaling and root
planing and personal oral hygiene, bacteria
remained in radicular cementum and dentinal
tubules. Periodontal pathogens residing in various
domains of the mouth may translocate to perio-
dontal sites. Incomplete elimination of periodontal
pathogens by nonsurgical therapy might result in
relatively rapid recolonization and recurrence of the
disease.
Periodontal pathogens reside in the strictly anaer-
obic environment of the deep periodontal pocket,
which may compromise important antimicrobial
mechanisms of polymorphonuclear leukocytes and
other host cells. Several putative pathogens possess
mechanisms to evade the host defense and cause
tissue breakdown. Kigre et al. (70) reported the dis-
tribution of P. gingivalis and T. denticola in human
subgingival plaque at different periodontal pocket
depths using immunogold-silver staining and im-
muno electron microscopy. They reported that cells
of both P. gingivalis and T. denticola were predom-
inantly found in subgingival plaque located at a
depth of more than 4 mm in the periodontal pockets,
and the coexistence of P. gingivalis and T. denticola
was observed in deep subgingival plaque. Coaggre-
gation between P. gingivalis and T. denticola has also
been observed in vitro (121). Kigure et al. (70) sug-
gested the possibility that T. denticola could carry
attached P. gingivalis cells deeper into periodontal
pockets, and could affect the progress of periodontal
diseases.
111
It is difcult to access a periodontal pocket deeper
than 5 mm adequately while scaling and root pla-
ning. The anatomy of the periodontal pocket also
affects the effectiveness of debridement. Noiri &
Ebisu (117, 118) examined periodontal pathogens in
the plaque-free zone at the bottom of periodontal
pockets using scanning immunoelectron microscopic
techniques, and detected the presence of P. gingiva-
lis, C. rectus, T. denticola, Prevotella nigrescens and
A. viscosus. They also observed that lm-like struc-
tures coated several cells in the plaque-free zone.
They suggested that C. rectus and T. denticola may
invade the most apical border plaque area by use of
their motility and associate with the biolm forma-
tion in the plaque free zone. It is difcult to gain
access and debride the tooth surface at the apical
border of periodontal pockets, and thus mechanical
treatment may be insufcient to completely disrupt
the biolm in deep pockets.
A. actinomycetemcomitans is a well-recognized
periodontal pathogen, and should be completely
eradicated from periodontally diseased sites. Various
antimicrobial regimens seemto be effective in treating
A. actinomycetemcomitans periodontal infections. In
case of periodontal breakdown by mixed infection of
A. actinomycetemcomitans and P. gingivalis, antibi-
otic-resistant biolms may complicate eradication of
the pathogens. Slots (166) recommends periodontal
treatment that includes a battery of professionally and
patient-administered antimicrobial agents, including
appropriate systemic antibiotics, povidone-iodine and
sodium hypochlorite subgingival irrigants, and
chlorhexidine mouthrinse, to control periodontal
infections.
Advances in microbiological
diagnosis
Detection of specic periodontal pathogens at per-
iodontally involved sites seems to be important for
treatment of certain types of periodontal diseases. It
certainly would appear to be important to identify at
least A. actinomycetemcomitans and P. gingivalis in
cases of severe periodontal disease. Among the
microbial detection methods, bacterial culture is
considered the gold standard. However, culture
methods are time consuming, laborious, and tech-
nique sensitive, and may fail to grow some import-
ant organisms. Selective culturing is an easy means
of identication of A. actinomycetemcomitans, while
culture and identication of P. gingivalis requires
skill and expertise. Recent technical advances have
resulted in the use of nucleic acid probes and
amplication techniques for the identication of
DNA of potential periodontal pathogens. DNA
probes were the rst genetic method for the detec-
tion of specic periodontal pathogens (109, 155,
214), but DNA probes lack adequate sensitivity and
specicity for some organisms. PCR, which targets
the gene of a specic region of periodontal patho-
gens, is commonly used these days (8, 143, 190) for
highly sensitive and specic detection of periodontal
pathogens. Among these periodontal pathogens,
PCR can detect highly toxic clones of A. actin-
omycetemcomitans with deletion (16) or insertion
point (61) in the leukotoxin gene, which possess
enhanced (1020 times) leukotoxic activity, and can
also detect P. gingivalis with a variety of mA genes
(4) or abscess-forming strains (114). PCR can detect
organisms with virulent clones by targeting the gene
related to the pathogenesis (60), as well as oral
microorganisms that are difcult or impossible to
culture (153). In addition, recent advances in PCR
technology have made it possible to perform quan-
titative microbial analysis rather than only qualitat-
ive analysis.
Future directions
A successful choice of treatment procedures for
each patient depends on an accurate diagnosis
based on comprehensive data, including clinical
and microbiologic ndings. Integrating a well-
developed and sensitive microbiologic detection
system in our diagnostic armamentarium would
help in the diagnosis and treatment of periodontal
diseases. Recently introduced quantitative PCR
methods are accurate and specic in detecting and
quantifying periodontal pathogens in biolms. Easy
and rapid chairside detection systems of microor-
ganisms should also be developed for microbial
monitoring.
Several bacteria have been currently accepted as
periodontopathic. However, other microorganisms,
as yet unidentied, may also be related to perio-
dontitis. Recent studies have indicated that besides
various gram-negative anaerobic bacteria, several
types of viruses including human cytomegalovirus,
Epstein-Barr type 1 virus and other members of the
Herpes viridae family, are associated with destructive
disease (167). Thus, further investigations for
unknown microorganisms, including bacteria and
viruses, responsible for periodontal destruction need
to be conducted.
112
Umeda et al.
As discussed in this chapter, there is a limit to the
ability of conventional nonsurgical methods to re-
move bacteria, especially in subgingival periodontal
pockets. It has been proposed that full-mouth disin-
fection therapy is more effective than conventional
scaling and root planing (74). Laser application has
also been demonstrated to be effective in the elim-
ination of subgingival bacteria (7). New treatment
protocols, including full-mouth disinfection and laser
application, need to be investigated for effective
disruption of the biolm and proper disinfection.
Furthermore, drugs effective against microorganisms
in biolms should be developed, because biolms
can hamper the effect of antimicrobial drugs.
Development of drugs which can destroy the biolm
or inhibit biolm formation may be helpful in con-
trolling periodontal disease.
Nonsurgical treatment is indispensable for many
periodontitis patients, especially for those with sys-
temic diseases, such as heart disease and diabetes,
who may not be suitable for periodontal surgery.
Recent studies showing the association of periodon-
tal pathogens with various systemic diseases under-
line the importance and necessity of elucidating the
effects of nonsurgical therapy on supra- and sub-
gingival microorganisms.
Conclusions
It is now well-known that dental plaque exists as
a biolm, and specic plaque bacteria such as
A. actinomycetemcomitans and P. gingivalis are
responsible for the initiation and progression of
periodontitis. Plaque bacteria within the biolm are
partially protected from antibiotics and host immune
reactions. The strategy of periodontal treatment
should include a break-up of the biolm and subse-
quent elimination of periodontal pathogens.
From the available data, it appears that supragin-
gival plaque control reduces the subgingival bacterial
load to some extent. A considerable body of evidence
indicates that mechanical therapy is relatively
effective in suppressing periodontal pathogens and in
promoting clinical improvement. Conventional
mechanical therapy is a necessary step in periodontal
therapy, but it does not always succeed in eliminating
periodontal pathogens completely, especially in
furcations, deep periodontal pockets, and other
intraoral niches. In view of the complex ecosystem
within the subgingival pocket, antimicrobial agents
may be required, in conjunction with scaling and root
planing, to eliminate the pathogenic ora in some
cases of periodontitis. Antimicrobial agents might be
effective in removing potential periodontal patho-
gens inhabiting sites inaccessible to instrumentation,
such as furcation involvements, convex part of deep
root surfaces, soft tissues, and dentinal tubules. The
need to mechanically disrupt the biolm before
administering antimicrobials should be emphasized
for maximal efcacy of the drug. Systemic antibiotic
therapy should be restricted to aggressive types of
periodontitis, and routine use of antibiotics for
standard management of chronic periodontitis can-
not be justied, as mechanical therapy has proven to
be adequately effective in controlling this form of
disease.
Acknowledgments
This research was supported by the grant for Center
of Excellence Program for Frontier Research on
Molecular Destruction and Reconstruction of Tooth
and Bone in Tokyo Medical and Dental University.
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Umeda et al.
120
Effects of nonsurgical
periodontal therapy on hard
and soft tissues
PATRI CK A. ADRI AENS & LAURENCE M. ADRI AENS
Introduction
Nonsurgical periodontal therapy is directed toward
removal of the microbial biolm from the root sur-
faces of periodontally diseased teeth. Conceptually,
the aim of this treatment approach is to eliminate
both living bacteria in the microbial biolm and
calcied biolm microorganisms, i.e. dental calculus,
from the root surface and from the subgingival area
without the surgical reection of the soft tissues
surrounding the teeth. From a practical point of view
the result of this treatment is a more or less complete
removal of the calcied accretions covering the root
surfaces, a reduction of the number of biolm
microorganisms, and a disturbance of the ecology of
the microbial biolm (Fig. 1 and 2). As a conse-
quence, the host tissues can better cope with the
remaining microorganisms, reducing the inamma-
tory changes of the soft tissues and producing a
varying degree of closure of the subgingival pocket.
The host should therefore be able to better control
the microbial recolonization of the dentogingival area
by personal oral hygiene measures.
Although there have been tremendous advances in
the understanding of the events leading to peri-
odontal breakdown and of the effects of periodontal
treatment in the past few decades, the concept and
understanding of subgingival plaque elimination and
oral hygiene performed by the patient as being the
essential elements of a successful periodontal treat-
ment approach date back more than a century. In
1886, Black (29) stated that the most important
measure in the treatment of calcic inammation of
the periodontal membrane and gums is the removal
of the concretions from the teeth, and the next most
important, instilling in the mind of the patient an
active determination to keep their teeth clean in the
future. With the demonstration of the etiologic role of
bacterial deposits in the development of experimen-
tal gingivitis (132), the principal aim of periodontal
therapy became the thorough removal of the deposits
from supra- and subgingival tooth surfaces.
This review will focus on the effects of nonsurgical
periodontal therapy on the distinct compartments of
the periodontal lesion, i.e. the root surface, the soft
tissues surrounding the tooth, and the osseous
compartment of the periodontium. Furthermore, this
review will intentionally be limited to the effects
induced by mechanical nonsurgical therapy. The
effects of chemical antimicrobial therapy alone or in
combination with mechanical periodontal therapy
are extensively discussed in another contribution in
this volume (196), as are the effects of laser therapy
(14). Mechanical nonsurgical periodontal therapy in
this review includes all nonsurgical treatment mod-
alities that are performed with the use of hand
instruments, sonic or ultrasonic instruments, motor-
driven instruments, or any combination of these
instruments.
Effects of nonsurgical therapy on
the root surface
Removal of bacterial deposits (microbial
biolm and dental calculus)
Most studies addressing the efcacy of nonsurgical
periodontal therapy in removing the microbial bio-
lm and dental calculus have been performed on
periodontally diseased teeth scheduled for extraction.
Their extraction was indicated due to extensive loss
of periodontal supporting tissues or a prosthetic
121
PERIODONTOLOGY 2000
treatment plan that considered these teeth unreliable
due to their periodontal condition. The mechanical
debridement of the root surface in the subgingival
area was performed immediately before extraction
(Table 1). Immediately following extraction, the teeth
were processed for evaluation by light microscopy,
stereomicroscopy, or scanning electron microscopy.
In most of the studies no time limit was set for the
instrumentation of the root surfaces. However, in
several studies the time used by the operator was
recorded.
As a general observation from all studies in the
literature, it is evident that subgingival scaling and
root planing is an efcient method to reduce the
amount of bacterial plaque and calculus attached to
the subgingival root surface. However, most studies
also indicate that none of the instrumentation tech-
niques is totally effective in eliminating all bacteria
and calculus from the subgingival surface of the tooth
(13, 24, 4244, 46, 58, 67, 94, 100102, 108, 113, 114,
142, 144, 146, 152, 155, 164, 175, 187, 195, 204, 205).
With increasing probing pocket depth, the number of
teeth with subgingival surfaces completely free of
residual plaque or calculus decreases (46). The per-
centage of the treated root surface with residual
plaque or calculus is directly related to the probing
pocket depth present at the time of instrumentation
(Table 1). For probing pocket depth values up to
3 mm, 443% of the root surface was still covered
with remnants of plaque or calculus after thorough
instrumentation (42, 44, 58, 75, 164). In pockets with
a probing pocket depth of 46 mm, 1538% of the
instrumented root surface still demonstrated the
presence of calculus or plaque (42, 44, 47, 58, 75, 108,
164). In pockets deeper than 6 mm, these values
varied between 19 and 66%(13, 42, 44, 47, 58, 75, 108,
118, 164). Complete debridement of furcations was
notably more difcult using nonsurgical techniques
(43, 118, 152, 204). Repeated nonsurgical subgingival
debridement comprising a rst episode of not more
than 10 min, followed 24 h later by two additional
episodes of subgingival instrumentation for a maxi-
mum of 5 min each, was not more effective in
removing subgingival deposits than a single 10-min
episode of subgingival scaling and root planing per
tooth (13). Nonsurgical subgingival instrumentation
was slightly less effective (42, 44, 46, 100) in removing
subgingival plaque and calculus than access ap
therapy combined with root debridement under
direct visual control. Interestingly, these studies
demonstrated that even with direct visual control
during surgical debridement, increasing amounts of
remaining calculus and plaque were found with
increasing values for probing pocket depth (42, 44,
46). This might indicate that the visual control of
these surfaces during access ap surgery is not
optimal due to anatomic factors and constraints.
Fig. 1. Scanning electron microscopic view of the
bacterial biolm covering the subgingival root surface.
Microcolonies of rods are emerging from the complex
mixture of bacteria forming the attached bacterial pla-
que.
Fig. 2. In the subgingival microbial biolm, coccoid
bacteria interact with lamentous microorganisms and
rods (scanning electron microscopy).
122
Adriaens & Adriaens
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s
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d
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l
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r
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M
e
a
n
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i
s
t
a
n
c
e
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e
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o
f
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l
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m
e
a
n
P
P
D
1
6
4
1
1
9
2
m
m
o
r
m
o
r
e
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
S
t
e
r
e
o
-
m
i
c
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o
s
c
o
p
y
N
o
t
m
e
n
t
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o
n
e
d
C
u
r
e
t
t
e
s
(
6
2
)
U
n
t
r
e
a
t
e
d
(
5
7
)
1
7
1
3
%
P
P
D
3
m
m
:
1
0
%
P
P
D
5
m
m
:
2
3
%
P
P
D
7
m
m
:
3
5
%
5
3
2
5
%
P
P
P
3
m
m
:
4
6
%
P
P
P
5
m
m
:
6
5
%
P
P
P
7
m
m
:
8
4
%
9
4
5
0
5
m
m
o
r
m
o
r
e
S
t
e
r
e
o
-
m
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c
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p
y
N
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t
m
e
n
t
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o
n
e
d
C
u
r
e
t
t
e
s
(
2
5
)
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l
t
r
a
s
o
n
i
c
(
2
5
)
5
.
7
8
%
6
.
1
7
%
4
6
1
2
7
1
m
m
o
r
m
o
r
e
(
h
o
p
e
l
e
s
s
t
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e
t
h
)
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t
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c
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p
y
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t
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e
n
t
i
o
n
e
d
S
c
a
l
i
n
g
(
4
3
)
F
l
a
p
+
s
c
a
l
i
n
g
(
4
2
)
U
n
t
r
e
a
t
e
d
c
o
n
t
r
o
l
s
(
4
2
)
P
P
D
<
4
m
m
:
1
4
%
P
P
D
4
6
m
m
:
5
7
%
P
P
D
>
6
m
m
:
6
8
%
P
P
D
<
4
m
m
:
1
4
%
P
P
D
4
6
m
m
:
2
4
%
P
P
D
>
6
m
m
:
5
0
%
P
P
D
<
4
m
m
:
8
2
%
P
P
D
4
6
m
m
:
9
5
%
P
P
D
>
6
m
m
:
1
0
0
%
7
5
2
4
3
1
1
2
m
m
(
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p
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l
e
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n
s
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t
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d
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r
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n
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r
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t
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r
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t
t
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s
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0
0
)
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n
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l
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r
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n
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6
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6
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s
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g
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y
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d
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8
%
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4
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o
s
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r
g
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y
n
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e
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d
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6
%
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7
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3
1
8
n
o
s
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r
g
e
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y
n
e
e
d
e
d
(
3
7
%
)
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8
/
2
5
4
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e
t
r
e
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t
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d
s
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r
f
a
c
e
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2
7
%
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D
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4
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m
:
1
9
%
P
P
D
4
5
m
m
:
3
8
%
P
P
D
>
5
m
m
:
4
3
%
7
5
/
2
3
5
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e
t
r
e
a
t
e
d
s
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r
f
a
c
e
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(
3
2
%
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P
D
<
4
m
m
:
1
4
%
P
P
D
4
5
m
m
:
3
3
%
P
P
D
>
5
m
m
:
5
9
%
3
4
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2
0
1
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e
t
r
e
a
t
e
d
s
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r
f
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c
e
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1
7
%
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P
D
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4
m
m
:
1
1
%
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P
D
4
5
m
m
:
1
5
%
P
P
D
>
5
m
m
:
2
9
%
124
Adriaens & Adriaens
4
4
8
6
5
.
7
2
.
4
m
m
6
.
0
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3
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m
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6
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m
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e
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p
y
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9
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1
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1
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5
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.
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+
(
6
.
6
1
.
9
)
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C
u
r
e
t
t
e
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l
t
r
a
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o
n
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c
(
2
9
)
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e
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3
5
)
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n
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e
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t
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d
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2
2
)
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8
%
6
3
%
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%
2
4
%
P
P
D
<
4
m
m
:
7
%
P
P
D
4
6
m
m
:
2
3
%
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P
D
>
6
m
m
:
3
9
%
1
4
%
P
P
D
<
4
m
m
:
5
%
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P
D
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6
m
m
:
1
5
%
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P
D
>
6
m
m
:
1
7
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8
2
%
1
0
8
4
3
H
o
p
e
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h
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o
n
t
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o
l
s
4
.
6
7
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4
%
9
5
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8
8
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3
%
4
2
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1
4
2
m
m
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r
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r
e
(
h
o
p
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l
e
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s
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n
g
l
e
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t
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h
)
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t
e
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e
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-
m
i
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s
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o
p
y
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o
t
i
m
e
l
i
m
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t
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l
t
r
a
s
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n
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c
+
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r
e
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t
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s
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t
r
a
i
n
e
d
p
e
r
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o
d
o
n
t
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s
t
s
(
2
4
)
t
r
a
i
n
i
n
g
r
e
s
i
d
e
n
t
s
(
2
1
)
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a
m
e
+
a
c
c
e
s
s
a
p
:
t
r
a
i
n
e
d
p
e
r
i
o
d
o
n
t
i
s
t
s
(
2
8
)
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r
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n
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g
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e
s
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d
e
n
t
s
(
2
6
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D
<
4
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m
:
4
%
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P
D
4
6
m
m
:
2
1
%
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P
D
>
6
m
m
:
1
9
%
P
P
D
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4
m
m
:
1
4
%
P
P
D
4
6
m
m
:
3
4
%
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P
D
>
6
m
m
:
6
6
%
P
P
D
<
4
m
m
:
0
%
P
P
D
4
6
m
m
:
4
%
P
P
D
>
6
m
m
:
5
%
P
P
D
<
4
m
m
:
0
%
P
P
D
4
6
m
m
:
1
2
%
P
P
D
>
6
m
m
:
1
7
%
4
3
8
0
4
7
m
m
(
h
o
p
e
l
e
s
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t
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t
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(
4
0
s
i
n
g
l
e
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r
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o
t
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d
t
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e
t
h
)
(
4
0
m
o
l
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r
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)
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t
e
r
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o
-
m
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c
r
o
s
c
o
p
y
3
5
m
i
n
/
t
o
o
t
h
C
u
r
e
t
t
e
s
:
s
i
n
g
l
e
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r
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t
e
d
m
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l
a
r
s
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l
t
r
a
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n
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c
s
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l
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r
:
s
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n
g
l
e
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r
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t
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m
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r
s
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n
t
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l
t
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e
t
h
2
9
.
8
%
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0
.
4
%
3
4
.
5
%
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3
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5
%
1
0
0
%
1
0
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1
0
3
5
.
3
0
.
4
m
m
S
t
e
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-
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y
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l
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P
a
p
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r
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t
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n
+
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e
r
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t
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m
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t
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n
+
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n
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t
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o
n
t
r
o
l
s
6
.
4
%
1
.
3
%
0
.
5
%
125
T
a
b
l
e
1
.
C
o
n
t
i
n
u
e
d
R
e
f
N
o
.
o
f
t
e
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t
h
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n
i
t
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l
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b
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n
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k
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t
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v
a
l
u
a
t
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e
t
h
o
d
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l
o
g
y
T
r
e
a
t
m
e
n
t
t
i
m
e
(
m
i
n
)
/
t
o
o
t
h
I
n
s
t
r
u
m
e
n
t
s
(
n
o
.
o
f
t
e
e
t
h
)
%
o
f
t
e
e
t
h
w
i
t
h
o
u
t
c
a
l
c
u
l
u
s
/
p
l
a
q
u
e
%
o
f
s
u
r
f
a
c
e
s
w
i
t
h
r
e
s
i
d
u
a
l
c
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l
c
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l
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s
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r
s
t
a
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n
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a
t
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i
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l
M
e
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n
d
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s
t
a
n
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o
f
c
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l
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e
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n
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D
1
5
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o
d
e
l
i
n
p
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a
n
t
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m
h
e
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-
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i
c
r
o
s
c
o
p
y
N
o
t
i
m
e
l
i
m
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t
:
8
7
s
9
9
s
5
4
s
6
6
s
8
2
s
7
7
s
C
u
r
e
t
t
e
s
(
4
0
)
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l
t
r
a
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n
i
c
s
t
r
a
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g
h
t
t
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p
(
4
0
)
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l
t
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a
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n
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c
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r
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e
d
t
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p
(
4
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)
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a
x
i
l
l
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r
y
m
o
l
a
r
f
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r
-
c
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t
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o
n
:
6
1
.
1
1
0
.
8
%
M
a
n
d
i
b
u
l
a
r
m
o
l
a
r
f
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r
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c
a
t
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o
n
:
3
9
.
5
1
3
.
1
%
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a
x
i
l
l
a
r
y
m
o
l
a
r
f
u
r
-
c
a
t
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o
n
:
5
0
.
3
1
1
.
6
%
M
a
n
d
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b
u
l
a
r
m
o
l
a
r
f
u
r
-
c
a
t
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o
n
:
4
4
.
1
1
7
.
1
%
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a
x
i
l
l
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o
l
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r
f
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r
-
c
a
t
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o
n
:
1
5
.
1
7
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6
%
M
a
n
d
i
b
u
l
a
r
m
o
l
a
r
f
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r
c
a
t
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o
n
:
1
6
.
7
7
.
9
%
1
4
4
3
5
A
t
l
e
a
s
t
1
s
u
r
f
a
c
e
w
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t
h
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P
D
>
6
m
m
(
m
e
a
n
P
P
D
:
4
8
m
m
)
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t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
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a
x
.
1
5
m
i
n
/
t
o
o
t
h
R
i
g
i
d
G
r
a
c
e
y
c
u
r
e
t
t
e
s
(
1
3
)
R
i
g
i
d
l
o
n
g
s
h
a
n
k
c
u
r
e
t
t
e
s
(
1
3
)
C
o
n
t
r
o
l
t
e
e
t
h
(
9
)
3
8
%
4
2
%
7
5
%
6
7
2
8
>
6
m
m
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
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t
e
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o
-
m
i
c
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s
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o
p
y
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a
x
.
1
0
m
i
n
/
t
o
o
t
h
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d
e
n
t
i
s
t
s
+
5
h
y
g
i
e
n
i
s
t
s
w
i
t
h
>
1
0
y
e
a
r
s
e
x
p
e
r
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e
n
c
e
C
u
r
e
t
t
e
s
(
1
3
)
M
o
d
i
e
d
u
l
t
r
a
s
o
n
i
c
t
i
p
s
(
1
2
)
S
t
a
n
d
a
r
d
u
l
t
r
a
s
o
n
i
c
t
i
p
(
3
)
3
.
5
m
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%
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F
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l
s
(
5
)
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%
2
4
%
7
0
%
126
Adriaens & Adriaens
1
1
3
5
6
0
M
o
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:
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.
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%
127
T
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.
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C
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.
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.
*
:
t
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e
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+
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a
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g
a
p
s
u
r
g
e
r
y
)
.
128
Adriaens & Adriaens
The experience of the operator is an important
factor for the nal result of the subgingival debride-
ment (Table 1). In vivo (42) and in vitro (in phantom
heads) (113) it was demonstrated that trained peri-
odontists succeeded in leaving less residual plaque
and calculus on the subgingival root surfaces than
periodontists in training (42) or inexperienced den-
tists (113). This observation was made for the use of
Gracey curettes (113), Perio-Planer (113), sonic or
ultrasonic scalers (113), and the combined use of
ultrasonic scalers and Gracey curettes (42). The
experienced operators instrumented the root surfaces
approximately 1015% longer, suggesting a higher
degree of quality control and/or an improved ability
to evaluate the end-point of the root surface treat-
ment.
When comparing different instruments used for
the subgingival debridement during nonsurgical
periodontal therapy, no major differences between
these approaches could be demonstrated (Table 1).
In early studies no major differences were found in
the plaque and calculus removing ability of different
types of hand instruments (curettes, les, hoes),
ultrasonic instruments, and rotary diamond instru-
ments (24, 94, 101, 102, 142, 146, 175, 187, 195). These
observations were further conrmed for different
hand instruments such as rigid curettes and long
shank curettes (144), sonic instruments (75), ultra-
sonic scalers (43, 58, 113, 114, 118, 152, 155, 205), EVA
reciprocating instruments (118), and Perio-Planer or
Per-io-tor (113, 114). Curved tips for ultrasonic scal-
ers appear to allow better plaque and calculus re-
moval in furcation areas of mandibular and maxillary
molars (152). Modied ultrasonic inserts have been
developed for improved penetration into the sub-
gingival pocket without loss of adequate cooling (58,
67, 108). The use of diamond coated ultrasonic tips
does not reduce the proportions of surfaces that are
still covered with remnants of plaque or calculus, but
it does cut down the time needed to achieve the
appropriate treatment of the subgingival root surface
(205).
Surface characteristics of the
treated root surface
Smoothness of the root surface
Clinicians have long striven for a smooth root surface
as the desired end-point of the mechanical root sur-
face debridement during both nonsurgical and sur-
gical periodontal therapy. Root planing with different
hand instruments, such as curettes, hoes and les, or
the polishing of the treated root surface with different
motor-driven instruments, such as ne-grain dia-
mond coated rotary instruments, Perio-Polisher and
rubber cups, has been advocated as the nal step in
root preparation. A smooth surface is accepted as
being more likely to also be a clean surface. Fur-
thermore, it was suggested that a smooth root surface
would be less prone to colonization by oral bacteria,
thus delaying the formation of a new biolm on the
treated root surfaces. This was based on an experi-
ment performed by Waerhaug in dogs (194). He
demonstrated that intentional roughening of the
subgingival enamel surfaces enhances plaque for-
mation and its transformation into subgingival cal-
culus. As a corollary, Lindhe et al. have reported that
normal healing of the dentogingival tissues occurs in
contact with a smooth root surface (121).
A direct relationship between supragingival sur-
face roughness and bacterial colonization was
demonstrated in several studies (45, 109, 119, 163).
Teeth with rough surfaces are also more frequently
associated with the presence of gingivitis and peri-
odontitis (161). In contrast to these studies,
Rosenberg & Ash (171) could not detect any
signicant effect of root surface roughness as pro-
duced by instrumentation with curettes or ultra-
sonic scalers on the retention of dental plaque or on
the inammation of the marginal gingival tissues.
Later studies could not demonstrate any relation-
ship between the root surface texture after
instrumentation according to different protocols
and the healing response of the periodontal tissues,
the probing pocket depth reduction and the clinical
attachment level changes (110, 151).
The latter ndings should be interpreted in the
light of the ndings of an in vitro study in which the
average surface roughness was determined for dif-
ferent instruments (176). The average surface
roughness (Ra) value was highest after treatment with
sonic scalers (Ra 2.71 lm 1.12) (mean standard
deviation), whereas the lowest value was observed
after the root surface treatment with a rotating
instrument coated with 15 lm diamond particles
(Ra 1.64 lm 0.81). All other instruments tested
yielded Ra values between these upper and lower
values, i.e. Ra 1.90 lm 0.84 for surfaces treated
with Gracey curettes, Ra 2.10 lm 1.03 for the
Perio-Planer curette treated surfaces, Ra 2.48 lm
0.90 for surfaces following treatment with a piezo-
electric scaler, and Ra 2.60 lm 1.06 for those root
surfaces subjected to treatment with a rotating dia-
mond tip coated with 75 lm diamond particles.
129
In a study with implants receiving titanium abut-
ments with different surface roughness, it was shown
that a surface roughness with values of 0.2 lm or less
had no inuence on plaque accumulation. If, how-
ever, the surface roughness was increased to a value
of 0.8 lm or more, the amount of plaque accumula-
ting on this surface increased 25-fold (162). Taking
into account this threshold Ra value of 0.2 lm, the Ra
values for the root surfaces after treatment with dif-
ferent instruments that are currently available for use
during nonsurgical periodontal therapy were
between 8 and 14 times higher (176). This observa-
tion indicates that with the currently available
instruments for planing or smoothing the subgingival
root surfaces during nonsurgical or surgical peri-
odontal therapy, the surface roughness would still be
far above the threshold Ra value where the surface
roughness no longer inuences the colonization by
subgingival plaque bacteria. Nevertheless, even if
these treatments leave behind a surface that to a
certain extent promotes plaque formation by its
residual roughness, the clinician should still attempt
to obtain a surface with the lowest possible surface
roughness. Thus, it might still make sense to com-
plete the surface treatment with Gracey curettes or, if
possible, with ne grain rotating diamond points,
after the initial use of sonic or ultrasonic instruments.
Removal of diseased root
cementum
During the progression of periodontal disease, root
cementum becomes exposed to the subgingival and/
or oral environment as periodontal attachment loss
occurs and progresses. Thus, the exposed root ce-
mentum constitutes the inner wall of the periodontal
pocket. It is also the surface which is colonized by the
bacteria that form the attached portion of the sub-
gingival bacterial biolm. Exposed radicular cemen-
tum does not display any of the cellular activity or
turnover observed in the adjacent tissues involved in
periodontal disease and inammation, i.e. epithe-
lium, connective tissue, and alveolar bone. Never-
theless, a number of changes affecting the exposed
root cementum have been described, such as the
formation of localized areas of hypermineralization
(Fig. 3) and demineralization (140, 178180), pro-
gressive loss of proteins contained in the root ce-
mentum, as well as loss of collagen matrix (140, 178),
adsorption of endotoxins and other mediators of
inammation (10, 11), formation of localized
cell-mediated resorption lacunae (Fig. 3, 4) (2, 3, 8,
166) and invasion of bacteria in the root cementum
and radicular dentin (Fig. 47) (28, 76).
The presence of these disease-mediating factors
and the morphologic changes have formed the basis
for mechanical treatment of the root surface. This
treatment should therefore not only remove the
bacteria and the calculus from the subgingival root
surface, but should also attempt to remove the con-
taminated part of the root cementum. The achieve-
ment of decontamination of the exposed root surface
is essential for the healing, repair, and regeneration of
the periodontal tissues. However, at present there is
no means by which the clinician can determine
whether all contaminated root cementum has been
removed. Because of this, extensive and aggressive
scaling and root planing has been advocated (1, 10,
11, 41, 53, 61, 62, 107, 134, 202). The therapeutic end-
point of this approach is a completely smooth and
hard root surface, assuming that this represents
complete removal of subgingival plaque and calculus
and maximum removal of contaminated root
cementum.
Fig. 3. Scanning electron micrograph of longitudinally
fractured root with the plaque mass covering the root on
the left and the fractured root on the right. The bottom of
the lacuna lled with subgingival bacteria (b) demon-
strates a higher density of the mineralized tissues, sug-
gesting a hypermineralization of this area.
130
Adriaens & Adriaens
In contrast with this aggressive approach, a gentler
treatment of the root surface has been advocated
based on the observation that endotoxin does not
penetrate into the exposed root cementum, but
rather forms a loosely attached supercial layer on its
surface (38, 54, 92, 139, 149, 150, 154, 184). This
supercial endotoxin layer could be almost
completely removed by using gentle scaling with
hand instruments (53), conservative instrumentation
with ultrasonic scalers (184) or, on surfaces exposed
Fig. 4. Scanning electron microscopic image of the root
surface of an extracted periodontally diseased tooth after
mechanical debridement of the surface. Resorption
lacunae are visible. Plaque bacteria remaining on the
tooth surface suggest that mechanical periodontal ther-
apy did not succeed in removing all bacteria. In the
bottom of the resorption lacunae the openings of the
dentinal tubules are visible.
Fig. 5. Opening of a dentinal tubule emerging to the root
surface in the bottom of a resorption lacuna. Multiple
bacteria are present at the entrance of the dentinal tubule
(scanning electron micrograph).
Fig. 6. Scanning electron microscopic image of dentinal
tubules in radicular dentin. The dentinal tubules contain
invading bacteria in their lumen.
Fig. 7. The presence of invading bacteria in deeper parts
of the dentinal tubules suggest that a purely mechanical
therapy of scaling and root planing is unable to reach and
eliminate these bacteria (scanning electron micrograph).
131
during periodontal surgery, gentle brushing and
washing for 1 min (139). Chemical detoxication of
the root surface with citric acid (61, 62, 73, 154, 172)
or EDTA applications (29, 3036) on the instrumen-
ted root surfaces has been advocated as a procedure
to more adequately remove the endotoxin and smear
layer (Fig. 8) and improve the detoxication of the
root surface.
Conceptually, the aggressive approach in which an
attempt is made to remove all contaminated root ce-
mentum, may be increasingly questioned. The rst
argument against this approach is the presence of
endotoxin as a loosely bound supercial layer on the
exposed root surface. The second is that it is almost
impossible to remove all of the root cementum,
especially in the middle and apical portions of the root
(40, 153). A third argument is found in the observa-
tions that viable bacteria were demonstrated in the
dentinal tubules of mechanically treated roots of
periodontally diseased teeth (28, 76). These bacteria
could not be reached unless substantial amounts of
root cementum and radicular dentin were removed,
signicantly damaging and weakening the root. Fi-
nally, although mechanical root planing achieves
almost complete if not total removal of the cemen-
tum-bound endotoxin from the subgingival root sur-
face, during the weeks following this treatment the
root surface is likely to become recontaminated (135).
These ndings would tend to support a gentler
treatment approach of the root surface, leaving in
place most of the root cementum but at the same
time removing and disturbing as much as possible
the bacterial biolm attached to the surface of the
root cementum. The supportive periodontal care
program, including the personal home care of the
patient, should subsequently aim at reducing the
recontamination of the treated root surfaces to levels
that are compatible with a clinically healthy and
stable periodontal condition.
Amount of root structure removed
during nonsurgical therapy
From what is described above, it is apparent that
aggressive scaling and root planing aiming at the
complete removal of all contaminated root cemen-
tum is no longer a desired and meaningful clinical
end-point of nonsurgical or surgical periodontal
therapy. Nevertheless, the question arises as to how
much of the root cementum is removed during
mechanical root debridement. The relevance of this
question lies in understanding the amount of root
structure being removed during a single episode of
root surface instrumentation and how often this
procedure can be repeated without causing clinically
signicant damage to the hard tissues of the root.
In answering this question it should be realized
that several factors might inuence the amount of
hard tissue being removed, such as the force applied
to the instrument, the number of strokes performed
with the instrument, the degree of mineralization of
the supercial layers of the root cementum, the
degree of sharpness of the curette or scaler at the
start of the treatment and the gradual dulling of these
instruments during their continued use. Most of the
studies addressing this question have been per-
formed in an in vitro setting to allow the control of a
number of parameters such as the pressure applied
to the instrument and the extent and number of
strokes performed with the instruments. It is difcult,
however, to standardize the degree of mineralization
of the root surface of the experimental teeth. More-
over, most of the teeth used in these experiments
were unerupted teeth or nondiseased teeth extracted
for orthodontic or prosthetic reasons. The degree of
mineralization and thus also the hardness of the root
surfaces of these teeth might signicantly differ from
those found in periodontally diseased teeth (179,
180). From these arguments is should be clear that
the few studies that are available in the literature
should be analyzed carefully and that extrapolation of
these in vitro results to the in vivo situation should be
done with great care.
Fig. 8. Scanning electron microscopic image of the root
surface covered by a smear layer following mechanical
instrumentation of the subgingival root surface.
132
Adriaens & Adriaens
In an in vitro study evaluating the amount of root
surface structures removed during the mechanical
treatment of the root with curettes, it was shown that
an increasing number of strokes did remove
increasing amounts of mineralized root structures
(63). After 20 strokes applied with a force of between
700 and 1200 g of force, an average of 60 lm was
removed. The amount of material removed increased
to 65 lm for 30 strokes, 89 lm for 40 strokes, 112 lm
for 50 strokes, 174 lm for 60 strokes and 205 lm for
70 strokes. Based on the average values for the
thickness of the root cementum, that study also
demonstrated that it can be expected that 20 strokes
should be sufcient to remove all root cementum in
the cervical third of the root.
Zappa et al. (206) demonstrated in their in vitro
study that not only an increasing number of strokes
with the curette leads to increased amounts of root
surface material being removed, but also the force
applied inuences the removal of mineralized
material. At a force of 3.04 N, the amount of removed
mineralized material increased gradually with
increasing number of strokes applied: after 5 strokes
the instrument has removed 34 lm, after 10 strokes
65 lm, after 20 strokes 110 lm, and after 40 strokes an
average of 149 lm of external root structures (ce-
mentum and dentin) had been removed. When the
curette was applied with a force of 8.48 N, the values
were 103 lm (5 strokes), 165 lm (10 strokes), 245 lm
(20 strokes) and 343 lm (40 strokes). For both types of
forces a difference in the amount of removed miner-
alized material was found during the rst 5 strokes
with the instrument and during the strokes 2140 with
the same instrument. With a force of 3.04 N the
amount of substance removed with each stroke was
6.8 lm for the rst 5 strokes and 2.3 lm per stroke for
the last 20 strokes. The corresponding values for a
force of 8.48 N were 20.6 lmand 5.6 lm, respectively.
These differences illustrate the effect of gradual dull-
ing of the curettes during prolonged use on the
amount of tooth substance being removed during
instrumentation of the root surface.
The relationship between the amount of material
removed from the root surface and the forces applied
to the instrument were conrmed in an in vitro study
with different types of instruments (170). With
increasing forces (100 p, 200 p, and 400 p) applied to
an ultrasonic scaler used during 12 strokes, the
amounts of material removed from the root surface
were 11.6 lm, 18.2 lm, and 85.9 lm, respectively. For
a manual curette the forces of 250 p, 500 p, and 1000 p
resulted in loss of mineralized material to a depth of
60 lm, 109 lm, and 264 lm, respectively. When the
number of strokes was standardized to 12 and the
amount of pressure applied to the instrument to 100 p,
the amount of mineralized material removed during
the use of a ne grid diamond bur was 119 lm, while
the values for a curette, a sonic scaler, and an ultra-
sonic scaler were 109 lm, 94 lm, and 12 lm,
respectively. The use of different Per-io-tor instru-
ments resulted in the removal of less than 7.0 lm of
the root surface (138). It was therefore suggested that
the Per-io-tor might be an instrument best suited for
use during periodontal maintenance therapy.
Effects of nonsurgical therapy on
the periodontal tissues
Changes in gingival inammation
In many clinical studies on the effects of nonsurgical
or surgical periodontal therapy, bleeding after pro-
bing was used as an indicator of (residual) disease
activity. Several investigators (20, 22, 55, 103, 115)
have demonstrated that sites in which bleeding after
probing occurred repeatedly at successive observa-
tions during the periodontal supportive therapy, had
a higher probability for showing periodontal break-
down as evidenced by loss of clinical periodontal
attachment. The coefcient of correlation between
loss of clinical attachment (2 mm or more) and the
presence of bleeding after probing at repeated
observation times during the supportive periodontal
care period up to 5 years varied between 0.2 and 0.4
(55, 103). In a different type of analysis, it was
reported that 14% of the sites followed during a
5-year periodontal supportive care program demon-
strated 1.5 mm or more of clinical attachment loss
after the completion of the nonsurgical periodontal
therapy (22). However, for those sites that bled on
probing at 75% of the evaluation moments, clinical
attachment loss occurred in 29% of the sites. Lang
et al. have tried to avoid the problem of weak corre-
lation between the repeated occurrence of bleeding
after probing and further clinical attachment loss, by
using the absence of bleeding after probing as an
indicator of a stable periodontal health (115).
Nonsurgical periodontal therapy leads to a reduc-
tionof the periodontal inammationas evidencedby a
reduction in bleeding tendency (Table 2). Table 2
summarizes the percentage reduction in bleeding
after probing for nonmolar sites at different time
points after therapy and in function of the initial pro-
bing pocket depth values of the sites included in the
different studies. In the rst part of Table 2 the studies
133
giving meanvalues for the initial probing pocket depth
are grouped, whereas in the second part those studies
are listed that reported their results in function of a
range of initial probing pocket depth values.
This reduction in inammation in the periodontal
tissues could not be obtained by supragingival plaque
control alone (52, 120, 137, 201). Although the
development of supragingival plaque control led to
some reduction of the periodontal inammation, the
majority of the reduction in inammation was only
obtained following the subgingival instrumentation
(16, 17, 52, 88, 106, 127, 188, 201). Moreover, the long-
term stability of the periodontal situation is improved
when the subgingival debridement is performed in
conjunction with self-performed plaque control. Over
a 3-year period further periodontal breakdown as
evidenced by 2 mm or more loss of clinical attach-
ment, was observed in 9% of the sites that received
only plaque control, whereas this was limited to 2%
of the sites receiving subgingival debridement com-
bined with improved plaque control (201).
Data from studies in which sites with initial pro-
bing depth between 4 and 7 mm were treated by the
nonsurgical approach, demonstrated an average
reduction from baseline in bleeding after probing of
approximately 50% (12, 16, 17, 28, 39, 51, 52, 64, 68,
71, 81, 84, 86, 96, 97, 106, 108, 112, 116, 117, 122, 123,
125, 127, 130, 145, 156160, 167, 169, 173, 183, 185,
186, 189, 198, 199). Data from selected studies are
presented in Table 2 and indicate that the decrease in
bleeding after probing has a tendency to further
stability or even to a slight additional improvement
with an increasing length of the post-operative
observation period. The range of reduction of the
occurrence of bleeding after probing was 664%after
the rst month, 1280%at 3 months post-treatment,
1287% at 6 months and 3787% at 12 months after
completion of the nonsurgical periodontal treatment.
Several studies have compared the reduction in
periodontal inammation, i.e. the reduction in
bleeding after probing or the reduction in gingival
index scores, between sites that received nonsurgical
and surgical periodontal therapy. These studies could
not nd any short-term or long-term differences
between the results of both treatment modalities in
reducing the periodontal inammation (28, 96, 97,
106, 122, 123, 125, 157, 158, 167, 169, 199).
Changes in probing pocket depth and
clinical attachment level
During the weeks following subgingival debridement
and mechanical instrumentation of the root surfaces
combined with an appropriate home care program
aiming at adequate supragingival plaque control, a
reduction in probing pocket depth is observed. This
probing pocket depth reduction is benecial since it
results in an environment that is less favorable for the
establishment of anaerobic periodontopathic micro-
organisms. Moreover, the reduced values for probing
pocket depth also facilitate the access for later
debridement and polishing during the maintenance
phase of the supportive periodontal care and for
plaque removal during self-performed oral hygiene
(85, 124, 168, 182, 193, 200).
Table 2. Percentage reduction in bleeding after pro-
bing (BOP) for different values of initial probing
pocket depth (IPPD) (mean values and ranges of
values) at nonmolar sites
Ref. Month
1 3 6 9 12 60
Mean
IPPD
(mm)
4 16 6 87 87
5 189
39
108
64
17
159
173
68
96
42
53
75
64
20
12 63 67 81
41
47
60
73
6 185
145
156
112
9
82
186
25
43
25
42
40
59
35
40
30
56
26
28
IPPD
range
(mm)
46 106
52
12
86
84
130
71
42
44
55
74
10
17
12
35
21
39 37
76
47 160
51
38
48
>4 116 34 80
134
Adriaens & Adriaens
The reduction in probing pocket depth is the result
of both a gain in clinical attachment level and a
recession of the marginal gingival tissues (93, 160).
The gingival recession results from the reduction in
swelling of the marginal gingival tissue. The inamed
tissue with its inammatory cell inltrate and the
increased numbers of capillaries present in the gin-
gival connective tissue is gradually replaced by a
more collagen-rich tissues (25, 48, 85, 188, 195).
These changes are accompanied by a gradual
shrinkage of the tissue in an apical direction and
towards the root surface. The interface between the
root surface and the former pocket epithelium is
partially transformed into a long junctional epithe-
lium (48, 49). Both the presence of the long junctional
epithelium and the increased content in collagen
bers in the gingival connective tissue result in the
gain in clinical attachment level, i.e. an increased
resistance of the tissues against the penetration of a
periodontal probe. Due to these phenomena the
probe that did penetrate the base of the pocket in an
inamed untreated site, does no longer reach the
base of the junctional epithelium of a site treated
with nonsurgical periodontal therapy (50, 72, 126,
190, 191). Although only few studies have tested the
stability of a long junctional epithelial attachment to
the root surface, no difference could be found in
resistance to disease between a long junctional
epithelial adhesion and a true connective tissue
attachment (26, 133).
Table 3 summarizes the mean reductions in pro-
bing pocket depth and the mean changes in clinical
attachment level for nonmolar sites at different time
points following nonsurgical periodontal therapy and
in function of the initial probing pocket depth values
of the sites included in the different studies. The
studies giving mean values for the initial probing
pocket depth are grouped in the rst part of Table 3,
and the studies that reported their results in function
of a range of initial probing pocket depth values are
listed in the second part. The studies listed have
reported on changes occurring as early as after 1
month and up to 60 months post-therapy.
The magnitude of the probing pocket depth
reduction and the gain in clinical attachment level is
related to the initial measurement of probing pocket
depth (Table 3). This early observation led to the fact
that in most studies a distinction is made between
the treatment changes observed in pockets with an
initial probing pocket depth of 13 mm (shallow
sites), 46 mm (moderate sites) and 7 mm or more
(deep sites). Moreover, it should be understood that
most of the studies in the literature have reported on
the clinical changes occurring at nonfurcation sites
(Table 3) (for review see [59, 60, 192]). Few studies
have specically examined the tissue changes after
nonsurgical treatment of furcation sites (57, 105, 129,
131, 147). Recent data suggest that the clinical
changes occurring following nonsurgical periodontal
therapy are signicantly less favorable in smokers
than in nonsmokers (99). Unfortunately, many pub-
lished studies on the clinical effects of nonsurgical
periodontal therapy did not take into account the
smoking status of their patient population. This
might explain some of the variability observed when
comparing the results of different studies (Table 3).
The evaluation of clinical changes occurring in the
periodontal tissues following nonsurgical therapy
should ideally not be performed earlier than 4 weeks
after the nonsurgical periodontal treatment was
performed (51, 66, 104). Studies that have recorded
the changes in different clinical parameters have
indeed demonstrated that the major changes occur
during the initial 13 months after completion of the
nonsurgical periodontal treatment (16, 17, 52, 65, 104,
141, 159, 160). Subsequently, up to 12 months, some
additional healing and maturation of the periodontal
tissues may occur, as evidenced by some further
minor improvements in the clinical parameters.
Clinical changes at nonmolar sites
Based on a series of studies published between 1979
and 2002 (for review see [59, 60, 192]) it was calcu-
lated that for pockets with an initial probing pocket
depth of 3 mm or less, nonsurgical periodontal
therapy resulted in a negligible reduction of the
probing pocket depth of 0.03 mm (Table 3) (12, 28,
86, 90, 104, 128, 131, 147, 158). The mean values
varied between an increase in probing pocket depth
of 0.19 mm (90) and a reduction in probing pocket
depth of 0.40 mm (104). However, this reduction in
probing pocket depth was accompanied by a mean
loss of clinical attachment of 0.34 mm, with values
ranging from a loss of 0.80 mm (131) to a slight gain
of 0.29 mm in clinical attachment level (104). The
follow-up period in these studies varied between 1
(128) and 68 (86) months. It has been suggested that
the majority of perceived loss of attachment due to
subgingival scaling and root planing in sites of min-
imal probing depth may be due to a statistical phe-
nomenon called regression towards the mean (83).
For nonmolar defects with an initial probing
pocket depth between 4 and 6 mm the nonsurgical
periodontal therapy achieved a mean reduction of
the probing pocket depth of 1.29 mm accompanied
135
Table 3. Mean decreases in probing pocket depth (mm) and mean changes in clinical attachment level for
different values of initial probing pocket depth (IPPD) (mean values and ranges of values) at nonmolar sites
Ref. Month
1 3 6 9 12 24 60
Mean
IPPD
(mm)
4 16 1.25 {0.25} 1.10 {0.10}
5 189
39
64
74
17
159
96
1.10 {n.r.}
1.83 {1.12}
0.70 {n.r.} 0.75 {n.r.}
1.00 {0.45}
1.20 {0.10}
0.9 {0.50}
1.40 {0.20} 1.60 {0.20}
1.56 {0.19}
2.10 {0.40}
6 185
80
112
156
198
143
181
9
82
186
0.56 {n.r.}
0.74 {0.41}
1.30 {n.r.}
1.30 {0.50}
1.30 {0.50}
1.38 {n.r.}
2.70 {1.25}
1.50 {n.r.}
1.00 {0.40}
1.20 {n.r.)
7 167
23
74
129
1.50 {0.80}
2.40 {1.58}
1.70 {0.95} 1.65 {1.05}
2.40 {0.90}
IPPD
range
(mm)
13 128
86
104
12
158
28
90
131
147
0.02 {)0.15}
0.03 {)0.03}
0.40 {0.29}
0.00 {)0.30}
0.02 {)0.73}
)0.04 {)0.27}
)0.19 {)0.50}
0.00 {)0.80}
0.01 {)0.60}
46 128
52
86
104
181
12
158
124
84
28
56
90
131
147
0.81 {0.27}
1.00 {0.10}
1.03 {0.69}
1.23 {0.96}
1.25 {0.20}
1.72 {1.02}
0.40 {0.20}
0.94 {0.56}
1.06 {0.50}
0.45 {0.25} 0.50 {0.25} 0.50 {0.20}
0.86 {0.49}
1.30 {0.40}
0.64 {)0.07}
1.20 {0.00}
1.50 {0.59}
136
Adriaens & Adriaens
by a mean gain of clinical attachment level of
0.55 mm (Table 3) (9, 12, 17, 28, 39, 51, 52, 56, 68, 79,
80, 82, 86, 87, 89, 90, 96, 104, 112, 125, 128, 131, 147,
156, 158, 174, 181, 197). Mean probing pocket depth
reductions ranged from 0.30 mm (190) to 2.1 mm
(96). The mean values for the changes in clinical
attachment level in these sites with initial moderate
probing pocket depth ranged from a loss of 0.07 mm
(90) to a gain of 1.20 mm (197). The follow-up period
in these studies varied between 1 (39, 89, 128) and 68
(86) months.
In deep sites, i.e. sites with initial probing pocket
depth values of 7 mm or more, the nonsurgical peri-
odontal therapy resulted in a probing pocket depth
reduction of 2.16 mm (Table 3) (12, 23, 28, 56, 68, 86,
90, 104, 117, 125, 128, 129, 131, 143, 147, 158, 167,
181). For these sites the mean gain in clinical
attachment level was 1.19 mm. The mean reduction
in probing pocket depth was between 1.38 mm (143)
and 2.85 mm (12), whereas the mean gain in clinical
attachment varied between 0.55 (90, 128) and
2.50 mm (117). The follow-up period in these studies
varied between 1 (128) and 68 (86) months.
Clinical changes at molar sites
The clinical changes occurring at molar furcation
sites following nonsurgical periodontal therapy are
documented in a limited number of clinical studies
(57, 105, 129, 131, 147). A total of 141 patients with
over 500 molar sites with class 2 furcation defects
were included in these studies, with observation
periods ranging between 3 (105) and 42 months (57).
For molar sites with an initial probing pocket depth
of 14 mm the mean probing pocket depth reduction
was 0.4 mm and these sites lost an average of 0.2 mm
Table 3. Continued
Ref. Month
1 3 6 9 12 24 60
47 51 1.39 {0.70}
58 98 0.73 {0.63} 0.73 {0.63} 0.72 {0.70} 0.65 {0.58}
> 3 79 0.54 {0.50}
>4 116 26% {n.r.} 66% {n.r.}
< 6 87 1.78 {n.r.}
< 7 68 1.08 {1.06}
>5 148
89
111
174
197
1.30 {0.59}
2.46 {0.45}
0.71 {n.r.}
0.30 {n.r.}
1.70 {1.20}
>6 117
158
169
124
65
2.87 {2.50}
1.00 {0.50}
1.66 {1.40}
2.30 {0.90}
2.30 {1.00}
1.20 {0.50} 1.70 {0.50} 1.56 {0.90}
>7 128
104
12
28
68
56
90
131
147
86
1.50 {0.54}
2.18 {1.66}
2.85 {1.54}
1.54 {1.54}
2.01 {1.20}
2.20 {0.80}
2.14 {0.57}
2.30 {1.00}
2.60 {1.10}
2.28 {1.51}
n.r.: not reported. IPPD: initial probing pocket depth.
Values not in parentheses are the mean reduction in probing pocket depth (expressed in mm); values between { } are the changes in clinical attachment level
(expressed in mm) (positive values: gain of clinical attachment / negative values: loss of clinical attachment).
137
in clinical attachment level (57, 105). For molar sites
with a moderate initial probing pocket depth
(between 4 and 6 mm) the mean probing pocket
depth reduction varied between 0.00 mm (131) and
1.02 mm (57, 105) and the mean changes in clinical
attachment level varied between a loss of 0.80 mm
(57, 131) and a gain of 0.28 mm (105). For molar sites
with initially deep probing pocket depth values
(7 mm or more) the changes in probing pocket depth
varied between 0.00 mm (131) and 1.52 mm (57,
105). The mean values for the changes in clinical
attachment levels ranged between a loss of 0.50 mm
(57, 147) and a gain of 0.84 mm (105).
From these results it is apparent that the tissue
changes obtained with nonsurgical periodontal
therapy in molar furcation sites are less pronounced
than those obtained in nonmolar sites. Moreover,
periodontal status and healing after nonsurgical
treatment in proximal sites are negatively inu-
enced by the presence of a deep furcation involve-
ment in the adjacent site in the same proximal
space (70). These observations combined with data
from previous studies showing an inferior long-term
prognosis for molars with furcation involvement
(27, 78, 91, 136, 165, 203), have led to the use of
more aggressive treatment protocols being advo-
cated for the treatment of molars with furcation
lesions, including mainly resective procedures or, to
a lesser extent, regenerative techniques for class 2
lesions.
Gingival recession
As a result of nonsurgical periodontal therapy,
changes in probing pocket depth and clinical
attachment level occur. However, the magnitude of
the changes for both parameters is not equal. The
difference between the values of these two parame-
ters results in gingival recession. For nonmolar sites
with initial probing pocket depths of between 1 and
3 mm, the amount of gingival recession was
approximately 1 mm (1521, 57). For sites with
moderately deep (46 mm) or deep (7 mm or more)
probing pocket depth at baseline, the mean values for
the gingival recession were 1.2 mm and 1.9 mm,
respectively. No signicant changes occurred for the
amount of recession when hand instruments or
ultrasonic instruments were used (1517, 19). Neither
was the magnitude of gingival recession inuenced
by the variability of operators (19) or by the number
of sessions of subgingival instrumentation (18).
Although the amount of gingival recession observed
on the molar at surfaces was similar to that
observed around nonmolar teeth, there was signi-
cantly less gingival recession at the molar furcation
sites (57). Both this smaller gingival recession and the
smaller gain, or sometimes even loss, of clinical
attachment level at molar furcation sites explain the
less favorable values for probing pocket depth
reduction at the furcation sites treated with nonsur-
gical therapy.
Changes in alveolar bone structures
While most studies on nonsurgical periodontal ther-
apy have addressed the clinical changes occurring in
the soft tissue compartment of the periodontium,
such as the degree of inammation, the amount of
probing pocket depth reduction and the changes in
clinical attachment levels, only few studies have also
followed the changes occurring in the osseous com-
partment of the periodontium. These studies have
used bone probing levels as determined by bone
sounding under local anesthesia (167, 168) or stan-
dardized radiographic techniques, mostly combined
with subtraction radiography (69, 95, 177).
Comparing the effect of different surgical and
nonsurgical periodontal treatment modalities on
changes in bone height, Isidor et al. observed that
nonsurgical periodontal therapy did not produce any
changes in the bone height for sites with horizontal
bone loss (95). Although a mean gain in bone height
of 0.5 mm was observed for intraosseous defects
following surgical therapy with a classical modied
Widman ap procedure, the changes in bone height
after nonsurgical periodontal treatment of these sites
were not signicant.
In intraosseous defects treated with nonsurgical
periodontal therapy there is an increase in bone
probing levels of 0.2 mm at 6 months (167, 168),
0.3 mm at 12 months and 0.5 mm at 24 months after
therapy (168). These values are 5060% lower than
the increase in bone probing levels obtained fol-
lowing surgical access ap therapy including full
coverage ap procedures combined with root pla-
ning and citric acid conditioning of the roots. Over
the course of the following 3 years, the gain in bone
probing levels after nonsurgical periodontal therapy
was gradually lost, most probably due to the absence
of any additional professional subgingival instru-
mentation during the 5-year follow-up in these
studies.
Using subtraction radiography on standardized
intraoral radiographs it could be determined that
60% of the sites receiving nonsurgical periodontal
therapy demonstrated some amount of bone gain at
138
Adriaens & Adriaens
the 314 months post-treatment observation (177). In
contrast with this nding, in 95% of the sites that
received access ap surgery some amount of bone
loss occurred during the same observation period.
Changes in bone density in interproximal intraos-
seous defects depend on when the observation is
made (69). During the initial 2 months, there is a
decrease in bone density. This decrease is followed by
a signicant increase in bone density in the next
4 months. Only minor changes occur 612 months
after therapy, indicating a further maturation of the
osseous structures.
In an animal study with surgically treated perio-
dontal defects the use of hand instruments and
ultrasonic instruments resulted in similar amounts of
changes in bone density (77).
Conclusions
Although subgingival scaling and root planing as a
nonsurgical treatment approach in periodontal ther-
apy is an efcient method to reduce the amount of
calculus and biolm bacteria attached to the sub-
gingival root surface, none of the currently used
techniques or instruments is totally effective in
completely eliminating all calculus and bacteria.
Initial probing pocket depth, root anatomy, instru-
ment design, and operator skill and experience
inuence the efcacy of the calculus and biolm
removal from the subgingival surfaces. Smoothness
of the treated root surfaces is determined by the
nature of the instruments used during the treatment.
The importance of root surface smoothness as
obtained with the currently available instruments
may have been overestimated. The need for removal
of diseased root cementum has been discussed. The
amount of root substance removed during nonsur-
gical periodontal therapy is determined by the nature
of the instruments used, the force applied during
their use, and the number of strokes performed on a
given part of the root surface.
Nonsurgical periodontal therapy induces bene-
cial changes to the periodontal tissues, as expressed
by a reduction of the gingival inammation, a
reduction of probing pocket depth, and a gain in
clinical attachment level. The magnitude of the
changes is related to the initial defect size as
expressed by initial probing pocket depth, tooth type
(nonmolar sites versus molar sites), and other envi-
ronmental factors such as the quality of oral hygiene
and smoking status of the patient. Changes in pro-
bing pocket depth and clinical attachment are also
accompanied by changes in the position of the
gingival margin and changes to the crestal parts of
the alveolar bone.
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145
Supportive periodontal therapy
STEFAN RENVERT & G. RUTGER PERSSON
Chronic periodontitis is an infectious disease
caused by an opportunistic microora. The infec-
tion triggers host inammatory responses resulting
in the destruction of the tooth supporting tissues.
Neither the infection nor the hosts inammatory
responses are completely understood. Epidemio-
logical studies suggest that in adult populations the
prevalence of chronic periodontitis is common (16,
33, 76).
Untreated chronic periodontitis has been des-
cribed as a slowly progressive disease affecting
individual teeth or tooth sites, showing evidence of
periods of stability and periods of progression (32,
55). Thus, at untreated tooth sites, clinical meas-
ures of oral hygiene, gingivitis, pocket probing
depths, and clinical attachment levels were poor
predictors of disease activity over 18 months (87).
In fact, Reddy et al.s (87) study demonstrated that
a majority of untreated sites (approximately 75%)
experienced no progression of disease or improved
in the absence of care. This suggests that the
evaluation of the efcacy of supportive periodontal
therapy (SPT) must be carried out over an extended
period of time.
Several recent investigations have demonstrated
the signicance of host specic factors that can
either contribute to the exacerbation of periodon-
titis or effectively control the infection. On a
patient-based level it is now evident that several
conditions such as genetic predisposition, cigarette
smoking, and bio-behavioral factors signicantly
alter the host ability to control disease. Previous
exposures to periodontitis and treatments, systemic
diseases, and medications are other factors that
may signicantly inuence patient-based responses
to treatment.
Standard care of patients with previously untreated
chronic periodontitis cases includes oral hygiene
instructions and mechanical nonsurgical debride-
ment, sometimes supplemented with surgical pro-
cedures. One of the treatment objectives has been to
reduce probing pocket depths. This treatment phase
is referred to as Initial Cause Related Therapy (ICRT).
Over the last decade, antimicrobials have been used
as an adjunct to ICRT.
Signicant efforts have been made to develop anti-
microbial treatments and regenerative procedures. At
present there is no denitive periodontal treatment
that can cure the disease. Furthermore, the chronic
nature of periodontitis as well as the inability of
existing clinical parameters to predict disease pro-
gression mean that continuous adjunct monitoring
and treatments are necessary to prevent recurrence of
the disease. The principles of periodontal mainten-
ance care are well established and considered the
standard of care.
It is also common practice to assess the outcome
of ICRT after 3 months as it appears that limited
improvements occur thereafter (10).
The general principles of the post-treatment
phase of periodontal therapy are well established.
However, in the literature different terms such as
periodontal maintenance, supportive periodontal
care, and supportive periodontal therapy have been
used and represent somewhat different entities.
Supportive periodontal care is a broader term and
focuses on patients previously treated for perio-
dontal disease (7).
In a position paper by the American Academy of
Periodontology (25), supportive periodontal ther-
apy, formerly referred to as periodontal mainten-
ance, should include an update of the medical and
dental histories, examination of extra- and intra-
oral soft tissues, dental examination, radiographic
review, evaluation of the patients oral hygiene
performance, periodontal evaluation and risk
assessment, with supra- and subgingival removal
of bacterial plaque and calculus, and retreatment
of disease when so indicated. The therapeutic
goals of SPT are to:
prevent or minimize the recurrence and progres-
sion of periodontal disease in patients who have
been previously treated for gingivitis, periodontitis,
and peri-implantitis.
179
PERIODONTOLOGY 2000
prevent or reduce the incidence of tooth loss by
monitoring the dentition and any prosthetic
replacement of natural teeth.
increase the probability of locating and treating in
a timely manner, other diseases or conditions
found within the oral cavity.
Once ICRT has been successfully completed it
becomes critical that the clinician considers risk
factors for the recurrence of periodontitis, and pre-
scribes adequate treatments andintervals of treatment
in order to fulll the goals of SPT listed above. The
objectives of this review are to assess the efcacy of
SPT and the role of risk factors in order to provide
guidance for practice and suggestions of future
research.
Literature search strategy
The search covered MEDLINE and was limited to
papers published in English from 1980. The search
strategy was: periodontal maintenance, supportive
periodontal therapy, compliance and periodontal,
periodontal recall, supportive and periodontal and
therapy, oral hygiene and periodontitis and pre-
vention, antimicrobials or antibiotics and periodon-
tal maintenance, peri-implantitis and maintenance,
periodontal maintenance and complication, perio-
dontal maintenance and caries/decay, supportive
periodontal therapy and caries/decay, supportive
periodontal therapy and complication, perio-
dontal maintenance and root sensitivity, supportive
periodontal therapy and root sensitivity, periodontal
maintenance and endodontic, as well as supportive
periodontal therapy and endodontic.
The search strategy was designed for high recall
rather than high precision in the rst instance. Case
reports and letters were explicitly excluded, whereas
both prospective and retrospective studies were
allowed. A total of 1,173 papers were retrieved.
Additional hand search of the Journal of Clinical
Periodontology, the Journal of Periodontology and
Clinical Oral Implants Research was performed.
Criteria for considering studies
for this review
To assess the overall efcacy of SPT, only studies with
a minimum duration of 36 months were included. In
specic areas, however, such as the adjunct value of
antimicrobial treatments of SPT and the prevention
of peri-implantitis, studies of shorter durations were
also included.
Efcacy of supportive periodontal
therapy
The present review has identied that the initial
results obtained following ICRT could not be
sustained using standardized SPT (i.e. 36-month
intervals) over 3 years. A slight increase in pocket
probing depth and loss of attachment over time as
well as loss of teeth are reported (Table 1). It is
difcult to draw denitive conclusions about the
efcacy of standardized SPT programs due to differ-
ences in study design, inadequate description of
subject characteristics, small study populations, sig-
nicant patient drop-out rate and where intent to
treat was not accounted for. Routine mechanical
subgingival debridement of shallow bleeding sites at
SPT visits results in attachment loss (6, 23).
Studies vary in SPT procedures and may or may
not include subgingival debridement of sites with
bleeding on probing. In a recent subject-based sys-
tematic review (37) no rm clinical recommenda-
tions regarding the choice of supra- or subgingival
debridement could be made. However, data repor-
ted in this review imply that SPT programs should
be individualized in accordance to the patients risk
prole.
The prerequisite and importance of good to
excellent oral hygiene to obtain a reliable and suc-
cessful outcome of periodontal therapy has been
identied in many studies (Table 1). Professionally
delivered and frequently repeated supragingival
toothcleaning, in combination with self-performed
plaque control, has a signicant effect on the sub-
gingival microbiota of moderate to deep periodontal
pockets (38). Meticulous oral hygiene instruction,
supragingival scaling and professional monitoring
during a 2-year period have demonstrated that such
treatments effectively change the quantity and the
composition of the subgingival microbiota (27).
SPT long-term effects on tooth
mortality
Several retrospective studies have evaluated the
effectiveness of ICRT followed by SPT. Overall, SPT
seems to be effective in preventing recurrence of
periodontitis. However, disease reoccurs in a small
group of individuals who often are identied as high
risk or extreme downhill patients. One of the primary
objectives of therapy is to maintain a well functioning
and esthetically acceptable dentition. Tooth loss may
180
Renvert & Persson
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1
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o
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.
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.
(
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6
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M
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3
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K
o
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g
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t
a
l
.
(
4
9
)
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4
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<
3
0
%
a
t
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n
d
p
o
i
n
t
182
Renvert & Persson
M
a
t
t
h
e
w
s
e
t
a
l
.
(
5
9
)
3
3
5
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)
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.
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e
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.
(
9
9
)
*
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6
4
A
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r
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x
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o
l
(
<
1
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%
)
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2
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f
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s
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D
r
o
p
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f
1
2
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b
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t
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(
1
8
%
)
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P
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r
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e
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m
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t
u
d
y
.
C
A
L
,
c
u
m
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l
a
t
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v
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t
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a
c
h
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t
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s
.
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,
c
o
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o
n
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l
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n
g
.
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C
R
T
,
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n
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t
i
a
l
C
a
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s
e
R
e
l
a
t
e
d
T
h
e
r
a
p
y
.
M
W
,
W
i
d
m
a
n
a
p
.
O
S
,
o
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s
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o
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s
s
u
r
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e
r
y
.
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D
,
p
r
o
b
i
n
g
d
e
p
t
h
.
P
P
D
,
p
r
o
b
i
n
g
p
o
c
k
e
t
d
e
p
t
h
.
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A
L
,
c
l
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n
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c
a
l
a
t
t
a
c
h
m
e
n
t
l
e
v
e
l
.
O
H
I
,
o
r
a
l
h
y
g
i
e
n
e
i
n
s
t
r
u
c
t
i
o
n
.
S
P
T
,
s
u
p
p
o
r
t
i
v
e
p
e
r
i
o
d
o
n
t
a
l
t
r
e
a
t
m
e
n
t
.
183
therefore be considered (25) an indisputable treat-
ment failure. Whether ICRT includes surgical treat-
ment, it does not appear to have an impact on future
tooth loss in patients referred to as downhill (64). In
the absence of SPT there is an increased risk for tooth
loss (9). It has been demonstrated that in patients
with periodontally seriously compromised teeth,
microbial monitoring and the use of systemic anti-
biotics as an adjunct to nonsurgical SPT can effect-
ively reduce the need for tooth extractions (57). This
demonstrates that carefully designed SPT is of the
utmost importance for successful periodontal ther-
apy.
Frequency of supportive
maintenance care
The rationale for 3-month recall intervals for SPT is
most likely based on published studies that used 34-
month intervals as part of study design rather than a
result of studies comparing the efcacy and safety of
different time intervals for SPT (5, 10, 18, 23, 41, 45,
46, 57, 82, 83, 85, 95, 99). Another rationale for short
intervals between clinic visits is the understanding
that frequent maintenance care is necessary to
eliminate/reduce subgingival proportions of patho-
gens associated with periodontitis. Recolonization of
pathogens in previously treated periodontal pockets
occurs quickly if oral hygiene is not properly main-
tained (58, 98, 112). Therefore, 34-month mainten-
ance care intervals have been suggested (84, 116,
117).
However, several other studies have demonstrated
that longer intervals between maintenance care visits
can effectively prevent further disease progression (2,
42, 56, 90, 114). Axelsson et al. (2), in a 15-year follow-
up study of 375 adult individuals, demonstrated a low
incidence of caries and almost no further loss of
periodontal support even though maintenance visits
were performed only once or twice yearly for the
previous 9 years. Lindhe et al. (56), using a main-
tenance program restricted to oral hygiene instruc-
tion and supragingival cleaning every 46 months,
found that patients who consistently had a high fre-
quency of plaque-free surfaces showed little evidence
of additional loss of attachment. Thus rigorous oral
hygiene, frequent recalls do not appear to be as
important as in individuals with inadequate oral
hygiene.
Few studies have compared the impact of different
recall intervals. However, Rosen et al. (94) studied the
effects of 3, 6-, 12-, and 18-month intervals between
supportive recall treatments. With the exception of a
trend of some rebounding sites 6.0 mm and
attachment loss at molar sites with furcation invasion
in the 18-month recall group, no differences were
found between the groups. The results of this study
suggest that recall intervals could be extended to at
least 1 year in subjects with a history of limited sus-
ceptibility to periodontitis.
Compliance with supportive
periodontal recall visits
It is well accepted that regular maintenance care is
essential for the long-term success of periodontal
therapies. It appears that few, if any, studies have
assessed the level of patient compliance considering
both acceptable levels of oral hygiene and attendance
to scheduled regular maintenance care visits. Studies
that have assessed compliance with attendance only
during at least 3 years suggest that the attendance
compliance varies between 26%and 77%(28, 49, 59,
65, 7175) (Table 2). The studies by Demetriou et al.
(28) and Demirel et al. (29) suggested that females are
more compliant than men. Two studies showed that
older patients are more compliant than younger
patients (72, 75), whereas the study by Demetriouet al.
(28) suggested the opposite. It is also unclear whether
patients with extensive surgical procedures are more
compliant that patients treated with SRP only.
Assuming that compliance with maintenance care
visits is important for the successful results of peri-
odontal therapies, these results with a mean of 54%
compliance are discouraging. However, a study by
Johansson et al. (42) demonstrated that it may be
possible to maintain successful results of periodontal
therapy in patients with less personal and profes-
sional effort than traditionally recommended.
In a previous review by Wilson (116), economic
problems and fear of dental treatment were identied
as factors keeping patients from complying with
scheduled recall intervals. In the study by Demetriou
et al. (28), subjects belonging to higher socioeco-
nomic groups appeared to be more compliant. From
our review of the literature, however, it is not possible
to conclude that nancial factors discriminate
between compliant and noncompliant patients. The
role of professional fees for maintenance services
and dental insurance policies differ greatly through-
out the world. Axtelius et al. (3) reported that
nonresponding periodontal patients experienced
184
Renvert & Persson
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185
signicantly more unpleasant feelings towards dental
procedures and a tendency to experience more pain
in connection with dental procedures than respond-
ing patients. However, in a recently published paper
(47) most patients on maintenance care showed a
low pain response to both probing and instrumen-
tation. Only 15% had a painful experience and these
individuals could be identied by responses to an
anxiety questionnaire. The role of dental fear and
pain in maintenance therapy is as yet poorly inves-
tigated and it is therefore difcult to draw any
denite conclusions regarding the unwillingness to
attend maintenance visits due to dental fear.
SPT with adjunct use of
antimicrobials/antibiotics
It appears that many patients are unable to achieve
an oral hygiene consistent with periodontal health.
Therefore antimicrobials have been used to com-
pensate for inadequate mechanical oral hygiene.
Antimicrobials can be administered using different
delivery systems, i.e. dentifrices, solutions for oral
rinses or ushing of the periodontal pockets, and
other local delivery systems. There are few long-term
studies suggesting the efcacy of such antimicrobials
in SPT programs.
Dentifrices as the delivery system for antimicro-
bials have been evaluated. Rosling et al. (93, 96)
demonstrated that a triclosan/copolymer containing
dentifrice reduced the subgingival microbiota both
quantitatively and qualitatively over a 3-year period
without concomitant use of subgingival mechanical
treatment. The frequency of deep periodontal pock-
ets and number of sites exhibiting additional probing
attachment and bone loss was also reduced when
using such a dentifrice over 3 years. Furuichi et al.
(31) reported that patients using a triclosan/copoly-
mer dentifrice demonstrated signicantly more gain
of attachment and decrease in mean pocket probing
depth as compared to a control group.
Chlorhexidine was found to be effective as an
adjunct rinse to inadequate mechanical oral hygiene
used as an alternative to structured mechanical oral
hygiene in nonsurgical treatment of chronic to
advanced periodontitis patients over an observation
period of 1 year (22). Use of chlorhexidine rinse over
3 years at varying intervals may also prevent tooth
loss (79).
Administration of chlorhexidine in a controlled
release delivery system (Periochip) in patients with
residual pockets after ICRT appeared to be effective
in a 6-month study (36). The long-term effects of
such treatments are unknown.
A number of short-term studies (12 months or less)
imply that the use of antibiotics are effective adjuncts
to ICRT and that the effect may be sustained over a
longer period of time (19, 43, 57, 68, 69, 115). How-
ever, the advantage of adjunct antibiotic therapy
during SPT is unknown.
Maintenance care of patients with
dental implants
The use of dental implants has become an integrated
part of periodontal practice. The long-term favorable
results of dental implants have been reported in a
large volume of studies. However, infections (peri-
implantitis) occur in about 419% of implants (53,
100, 110, 111, 113). Different treatment modalities of
peri-implantitis have been evaluated and the use of
mechanical debridement with or without adjunct use
of local or systemic antibiotics, guided tissue regen-
eration, and autogenous bone transplants have been
described in the literature (for review see [92]). Few
studies exist on the long-term efcacy of treatment of
peri-implantitis. A 3-year follow-up study of implants
treated with autogenous bone suggested that initial
improvements can be stabilized (12). In a 5-year case
report study by Leonhardt et al. (54) it was found
that, in spite of antibiotic retreatment for the reoc-
currence of peri-implantitis, four of nine individuals
demonstrated implant loss during the follow-up
period. In the four patients experiencing implant loss,
33% of their implants were lost over a period of
5 years. Proposed strategies for treatment of peri-
implantitis identied in the literature were found to
have many recommendations in common. Due to the
lack of controlled studies these recommendations
must, however, be recognized as empiric. In a recent
Cochrane literature review by Esposito et al. (30)
controlled clinical trials on maintenance were eval-
uated. From this report it can be concluded that there
is little reliable evidence for what methods should be
used for long-term maintenance of dental implants.
Although limited data exist on the long-term effects
of SPT of dental implants, it seems reasonable to
anticipate that the long-term success of dental
implants can be achieved using the same principles
as used for the maintenance of teeth in patients with
a past history of periodontitis. It seems reasonable to
anticipate that the same predisposing factors as those
186
Renvert & Persson
currently considered for the risk of periodontitis also
apply for dental implants. This conclusion is sup-
ported by a recent 5-year SPT study with 3-month
recall intervals in which no signicant differences in
increasing probing depth and clinical attachment
loss around teeth and implants were found (66).
Complications of supportive
periodontal therapy
One of the objectives of periodontal therapy is to
prevent tooth loss. Once ICRT is completed, SPT
should be aimed at preventing further loss of teeth as
a consequence of periodontitis or treatment of peri-
odontitis. Several studies have demonstrated that
tooth loss can not be completely prevented by ICRT
(48, 106). It also appears that tooth loss occurs in
subsets of subjects and that risk-proling subjects on
SPT might allow prevention or reduction in tooth loss
in subjects on SPT (105).
Caries
Few studies have specically addressed root caries as
a complication during a period of SPT. However,
studies suggest that the prevalence of root caries in
periodontally treated patients is very high (86). One
of the consequences of periodontal therapy is the
removal of root cementum. It has been suggested
that intact root cementum prevents dentin caries
(62). Due to the potential of exposed root surfaces
without root cementum as result of initial ICRT, and
further removal of dentin during SPT, subjects sus-
ceptible to caries are at a high risk for root caries. In a
study of patients who had received ICRT and were on
routine SPT, the data suggested an association be-
tween the level of oral hygiene and the number of
root surface lesions and likewise an association with
salivary Streptococcus mutans counts. However, no
relation was found between previous experience of
coronal caries, salivary ow rate, or salivary buffer
capacity and root lesions (88). Studies have also
shown a relationship between root caries and sub-
gingival presence of S. mutans (109). Molars treated
with root resection also carry a higher risk of root
caries, resulting in treatment failure in spite of SPT
(8). Therefore, repeated oral hygiene instructions and
adjunctive preventive measures including diet coun-
seling and uoride rinses, as well as uoride and
chlorhexidine varnishes, should be advocated in
high-risk patients on SPT (88). An extensive review of
the use of uorides in the management of patients
with periodontitis in preventing caries has been
published (77).
Endodontic lesions
Endodontic complications during SPT may result in
tooth extraction. Data suggest that approximately
30%of all extractions of teeth over a 4-year period of
SPT are the consequence of peri-apical lesions (106).
Additional information about the relationship
between periodontitis and endodontic lesions was
recently published (35).
Periodontal abscesses
Periodontal abscesses appear to occur in approxi-
mately 35%of subjects on SPT and predominantly in
subjects who can be identied as rapid downhill
cases (63). It appears that subjects on SPT who only
received nonsurgical therapy during the ICRT may be
at a greater risk of periodontal abscesses during the
SPT phase (46).
Root sensitivity
It is well established that following ICRT, root sensi-
tivity is common, especially if treatment involved
surgical procedures. In most cases such sensitivity
decreases over time. Reports on root sensitivity dur-
ing SPT vary from 15% to 98% and are often asso-
ciated with root surface exposure and gingival
recession (21, 47, 102). The very high prevalence of
root sensitivity reported by Chabanski et al. (21) was
based on patients previously treated for periodontitis.
Data conrm that meticulous plaque control will
diminish root sensitivity (103). Treatment of root
sensitivity is consistent with preventive measures of
root caries (77).
Risk assessment for recurrence
of disease in patients with a
history of periodontitis
Many studies have shown that the predictive value of
routine periodontal parameters is relatively low.
Thus, at what level the prevalence of bleeding on
probing or plaque scores is compatible with perio-
dontal stability of an entire dentition is not well
understood. Bleeding on probing cannot be used
as a predictor of periodontal disease progression.
187
However, bleeding on probing at approximately 25%
or less of sites is a good predictor of stable conditions
(6, 24, 44). On a tooth-site basis the presence of fur-
cation involvements, tooth mobility, and probing
depth are predictive of tooth survival (60). Longitud-
inal clinical data collected from older subjects have
indicated that the presence of deeper periodontal
pockets and irregular dental visits can be positively
associated with progression of periodontitis (11).
It is currently well established that smoking rep-
resents a true risk factor for periodontitis (13, 14, 34,
70). Several SPT studies have conrmed that smoking
is a risk factor for further progression of periodontitis
(Table 3). However, the study with the longest SPT
period and the largest study population failed to
demonstrate the signicance of smoking as risk fac-
tor for tooth loss (59).
Although much recent interest has been focused on
the associations between risk for periodontitis and
systemic disease, it remains unclear to what extent
common systemic diseases have an impact on the
outcome of SPT.
Published data suggest that genetic factors may
explain approximately 50% of all cases of periodon-
titis (67). A genetic marker has recently become
available to determine a polymorphism genotype of
patients who may be more susceptible for chronic
periodontitis (50). Prospective studies have shown
that interleukin (IL)-1 gene positive nonsmoking
subjects over the age of 50 have signicantly deeper
Table 3. Patient-based prognostic factors regarding tooth loss and or attachment loss over time
Authors No. of
subjects
Time period Smoking IL-1 Compliance Microbiology
Kaldahl et al. (45) 74 72 months.
SPT at 3-month
interval
Yes, smokers less
favorably
No
McGuire & Nunn
(60)
100 60 months
or more
Yes, smokers less
favorably
Bostrom et al. (15) 57 60 months.
SPT at
12 months
interval
Yes, trend towards
smokers less
favorably

McGuire & Nunn (61) 42 60 months
or more
Yes, smokers less
favorably
Yes IL-1 positive
individuals have
increased risk
of tooth loss
Buchmann et al. (17) 13 36 months.
SPT at 3-month
intervals
Positive for
A.a had no
impact on
post treatment
conditions
De Sanctis & Zucchelli
(97)
40 48 months.
Monthly recalls
during rst year,
then every
3rd month
Yes. IL-1 positive
individuals have
signicantly more
loss of attachment

Matthews et al. (59) 335 > 120 months.
SPT interval
unknown
No No
Cattabriga et al. (20) 60 120 months.
SPT at 3-month
intervals
No
Cullinan et al. (26) 295 60 months.
SPT interval
unknown
No
A.a., A. actinomycetemcomitans. IL, interleukin. SPT, supportive periodontal therapy.
188
Renvert & Persson
periodontal pocket probing depths than their IL-1
negative gene counterparts (26). Analysis of data from
young adults has also suggested that the IL-1A
(+ 4845) [1,1]/IL-1B (+ 3953) [2,2] genotype is asso-
ciated with periodontitis (104). However, contradict-
ory results have also been reported, the studies being
unable to demonstrate differences in periodontitis
severity between IL-1 gene positive and negative
subjects (78, 108).
Genetic predisposition and the immune host
responses may have an impact on the progression of
periodontitis during SPT. IL-1 genotype positive
nonsmoking patients enrolled in an SPT program for
several years previously had signicantly higher
bleeding on probing percentages at recall visits than
IL-1 gene negative patients (52). In a 4-year study
comprising 224 subjects using individualized SPT
based on a composite risk prole (% bleeding on
probing, tooth loss, and the number of pocket pro-
bing depths 5 mm) IL-1 genotype positive subjects
responded less favorably to SPT than did IL-1 neg-
ative subjects (81).
Recent studies have evaluated the effect of IL-1
gene polymorphism on the outcome of SPT (see
Table 2). McGuire & Nunn (61) in a mixed group of
smokers and nonsmokers reported IL-1 gene positive
patients to be more susceptible to tooth loss. After
the initial year of maintenance, De Sanctis et al. (97)
were unable to nd differences between IL-1 gene
positive and negative subjects, but at the 4-year
examination IL-1 gene positive subjects demonstra-
ted more attachment loss than IL-1 gene negative
subjects. Following SPT for 10 years in a strictly
nonsmoking population, Cattabriga et al. (20) were
unable to detect differences in tooth loss between
IL-1 gene positive and negative subjects. The data are
not congruent and therefore the role of genetic fac-
tors predisposing for recurrence of periodontitis
needs to be further elucidated.
Major efforts attempting to develop and recom-
mend laboratory assays to predict risk of future per-
iodontal disease progression have been made, but
have been poorly received by the profession.
Although useful for the understanding of the etiology
and pathogenesis, site-based assays based on gingival
uid content of enzymes and cytokines have, so far,
failed to attain clinical acceptance as periodontal risk
predictors in spite of data supporting the value of
gingival crevicular uid analysis as a marker of
inammatory response (4, 39, 40, 80).
Microbiological monitoring during SPT has been
explored in many studies with duration of less than
12 months. Subgingival proles have been sugges-
ted to identify patients with refractory periodontal
disease (101). On a patient basis, the presence or
absence of Actinobacillus actinomycetemcomitans
failed to identify subjects at risk for progressive
periodontitis (17). Tran et al. (107) reported that
subjects with persistent presence of Tannerella for-
sythensis (formerly Bacteroides forsythus) over a
24-month period had 5.3 times higher odds of having
at least one site in their mouth losing attachment
compared to subjects with occasional or no presence
of the pathogen. Other studies have also shown that it
might be impossible to eliminate the presence of
A. actinomycetemcomitans from periodontal pockets
by root debridement and periodontal surgery alone
(91). Regular monitoring of the presence of pathogens
associated with periodontitis during SPT appears to
provide useful data to revise scheduled treatments
and procedures and include a prescription of antibi-
otics as found necessary (57).
Using patient-based data as the unit of observa-
tion, a recent systematic review revealed that the
presence of deep pockets ( 6 mm) at reevaluation is
predictive of periodontitis progression. However, no
studies were found demonstrating that bleeding on
probing or the presence of furcations on a patient
basis were predictive of disease progression (89).
Thus it appears that smoking habits, the presence
of deep pockets following ICRT, IL-1 gene poly-
morphism and other genetic factors should be con-
sidered and might be used in a risk assessment
prole of the patient. Systems have been suggested
where risk factors are combined in order to assess an
individual patient-related risk prole (81).
Multifactorial risk diagram
To dene SPT intervals and procedures, the risk for
further periodontitis progression can be assessed
using a combination of risk factors in a multifactorial
risk diagram. This can be facilitated by the EXCEL
Microsoft software program (EXCEL XP for PC, Red-
mond, WA). The number of risk factors can vary in
the presented diagram of six vectors:
proportion of sites with bleeding on probing;
number of sites with a pocket probing depth
>5.0 mm;
number of missing teeth (tooth loss);
proportion of mesial/distal sites showing evidence
of a distance CEJ to bone level 4.0 mm on radi-
ographs;
genetic factors;
smoking status with regard to pack/years.
189
The scoring model used to identify the position on
each vector is presented in Table 4. The surface area
that could be outlined between ve different risk
scores was calculated and could be used as the risk
score in this example (Fig. 1). It would also
be possible to use the number of vectors reaching
the peripheral perimeter of the diagram to assess the
risk. The more vectors reaching the score of 5, the
higher the risk. The surface area between the various
risk scores can provide a numerical score of risk
which can be compared to risk scores identied at
different time points and guide the clinician to
change SPT strategy (81). A hypothetical case
responded with a decrease in the proportion of sites
with bleeding on probing to a score of 12% and to
four sites with a maximum pocket probing depth
>5 mm with a resulting comprehensive risk score
of 6.93 (Fig. 2) instead of a score of 36.64 (Fig. 1). To
further reduce risk, the clinician should focus on the
reduction of sites with probing depths >5.0 mm.
The scale as well as alternative vectors can be used
as deemed necessary. For example, the proportion of
sites with distance of the cementoenamel junction to
bone level could be changed to a bone loss index
representing proportional bone loss in relation to
subject age (51).
Strategies of SPT
It is well recognized that periodontitis is a multifac-
torial disease. Few of the factors contributing to the
onset and progression of periodontal disease can,
however, be altered by the patient or the clinician to
prevent the recurrence of periodontitis following
ICRT. Thus a young adult patient presenting with
periodontitis is most likely a carrier of one or more
genetic factors that can not be altered. Patients may
also have one or more chronic lifelong systemic
Table 4. Coding system used for the multifactorial risk diagram
Axis score* Bleeding on
probing
No. of sites with
probing depth
> 5.0 mm
Tooth loss % sites with bone
4.0 mm on
radiographs
Smoking
Pack/year
0 04% 01 0 09% 0
1 59% 23 12 1019% 139
2 1016% 45 34 2029% 4089
3 1725% 67 56 3039% 90189
4 2535% 89 78 4049% 180364
5 36+ % 10+ 9+ 50+ % 365+
*A score 0 begins at the center with a score 5 in the outer periphery.
Multi-factorial risk diagram
BOP%
PD > 5 mm
Tooth loss Bone loss
Smoking status
Fig. 1. Graphic illustration of the multifactorial risk dia-
gram. The center axis represents a score of 0 and the
peripheral axis a score of 5. The surface area risk score
was 34.6.
Multi-factorial risk diagram
BOP%
PD > 5 mm
Tooth loss Bone loss
Smoking status
Fig. 2. Hypothetical case responding to therapy with a
decrease in the proportion of sites with bleeding on pro-
bing. The surface area risk score was 6.9.
190
Renvert & Persson
diseases associated with an increased risk for perio-
dontal disease. This leaves few factors that actually
can be modulated, i.e. bacterial colonization and
smoking cessation. Good control of supragingival
plaque is an effective way to prevent disease pro-
gression (1). It has been demonstrated that an
effective supragingival oral hygiene even may affect
the subgingival microbiota (27, 38). If an effective oral
hygiene cannot be accomplished by the patient, pro-
fessional care at frequent intervals and/or the use of
supplementary antimicrobials are options available
to the dental team. Socioeconomic factors that
inuence the ability of the patient to attend SPT
programs can to some degree be overcome by
insurance systems focusing on prevention of further
disease.
As a result of this review, a standardized SPT pro-
gram cannot be recommended. However, categor-
izing the patients into risk proles may be a useful
strategy to assess appropriate and individualized SPT
time intervals and procedures.
Clinical recommendations
Based on the information obtained from the pre-
sent critical review on supportive periodontal care,
the following clinical recommendations can be
made:
SPT should be based on assessment of the patient
risk prole for further periodontal disease pro-
gression. Such risk assessment should be per-
formed after the completion of ICRT and be
revisited continuously.
A standardized SPT routine cannot be considered
to be consistent with best practice and an indi-
vidualized approach is needed.
SPT resulting in good oral hygiene is essential to
minimize the risks of periodontal disease progres-
sion. Issues of compliance must be considered.
The use of a triclosan/copolymer dentifrice could
be of value to enhance oral hygiene.
In patients with inadequate oral hygiene, chlorh-
exidine rinses could be advocated.
There does not seem to be scientic evidence of
additional value of routine subgingival debride-
ment of sites presenting with bleeding on probing
at SPT visits without concomitant increase in
probing depth. Such treatment should therefore be
avoided in sites without increasing probing depth.
In the absence of long-term evaluation of SPT
programs for dental implants it seems appropriate
to use the same principles of SPT as listed above.
Recommendations for research
Overall, studies must be conducted over a time per-
iod that reects the rate of natural progression of
periodontitis around teeth and implants (i.e. 3 years
or more).
Studies are needed to evaluate the efcacy of
supragingival treatment alone as compared to
subgingival debridement during SPT.
Studies are needed to assess the value of antibiotics
and antimicrobials as adjunct and stand alone
treatment during SPT.
Patient-based factors must be considered in the
analysis of data.
Prospective and not retrospective studies on the
efcacy of SPT must be performed comparing
different methods of SPT allowing the examiner(s)
to be blinded. Multicenter studies may be required
to obtain statistical power.
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Current concepts and advances
in manual and power-driven
instrumentation
SHI GERU ODA, HI ROSHI NI TTA, TAKASHI SETOGUCHI , YUI CHI IZUMI &
ISAO ISHI KAWA
Mechanical debridement consisting of scaling and
root planing is an important procedure in the
treatment of periodontal diseases. By root
instrumentation, toxic substances can be removed
from periodontally affected root surfaces resulting in
the biologic detoxic condition of the root surface,
which is favorable for periodontal tissue healing. In
addition to supragingival plaque control, subgingival
plaque control by means of mechanical debridement
is essential for elimination of the microbial causative
factors of periodontal disease. Meticulous scaling and
root planing is performed during the surgical and
nonsurgical phases of periodontal treatment, as well
as in the maintenance phase. Until now, mechanical
debridement has been performed with the help of
scalers. Scalers can be divided into manual, power-
driven, and other types. In this chapter, we discuss
the various usages of these scalers and the current
concepts and advances in the eld of mechanical
debridement.
Manual instruments
Manual instruments are generally classied into ve
types: sickle, curette, le, hoe, and chisel types. In
recent years, the sickle and curette types are the most
commonly used. The sickle scaler is used for removal
of supragingival calculus, while the curette type is
usually used for removal of subgingival calculus and
root planing (Fig. 1). The main sickle type scalers are
University of Southern California sickle, Turner
sickle, Jaquette sickle, and Morse sickle. The cross-
section of the blade is triangular. Some sickle type
scalers are designed for use in both anterior and
posterior teeth.
Curette type scalers are more frequently used now
than before. There are two basic types of curettes:
universal and Gracey curette. These two types of cur-
ettes differ in the area-specicity, number of cutting
edges, curve of cutting edge, and the angle of the face
to terminal shank. Columbia curettes and Gothen-
burg curettes are representatives of the universal
curette. Gracey curettes were designed by Dr. Gracey
in the 1930s and are manipulated by a push stroke,
followed by a pull stroke. Initially, Gracey curettes
were available as a set of 14 instruments, but now
mainly double-ended Gracey curettes (7 instruments)
are used (Fig. 2). File type scalers are used to fracture
or to crush calculus. This group includes a set of four
scalers, for buccal, lingual, mesial and distal sites. The
blade is narrow and used for access to deep and
narrow pockets. Hoe scalers are used for removal of
subgingival calculus, and are a set of four scalers
similar to the le type. They can be used for root
planing and to remove calculus from the base of the
pocket. Chisel scalers, designed for proximal surfaces,
are usually used in the anterior part of the mouth and
therefore, the adaptation area is limited.
Power-driven instruments
Ultrasonic and sonic scalers are referred to as power
scalers or power-driven scalers. Ultrasonic scalers are
either piezoelectric or magnetostrictive. High vibra-
tional energy generated in the oscillation generator is
conducted to the scaler tip, causing vibrations with
frequencies in the range of 25,00042,000 Hz (69).
The amplitude ranges from 10 to 100 lm. Microvi-
bration crushes and removes calculus under cooling
water (26, 69).
45
PERIODONTOLOGY 2000
In the piezoelectric scaler, the transducer is com-
pletely contained within the handpiece, and not
connected to the working tip insert. The working tip
is small and easily inserted into the handpiece.
Alternating electrical current applied to reactive
crystals causes a dimensional change, which is then
transmitted to the working tip as ultrasonic vibra-
tions. These vibrations can dislodge tenacious cal-
culus deposits from the tooth surface. The tip
movement of the piezoelectric scaler is primarily
linear in direction. Some popular brands available in
the market include Piezon Master
2730 kHz (EMS,

Nyon, Switzerland); ENAC
30 kHz (Osada Electro-

nics Co. Ltd, Tokyo, Japan); Solphy
2729 kHz
(J-Morita Corp., Tokyo, Japan); Amdent
25 kHz
(Amdent, Nynashamn, Sweden); and Suprasson
30 kHz (Satelec, Merignac, France). In the magneto-

strictive scaler, either a stack of at metal strips
(Cavitron
25 kHz; Dentsply, Des Plaines, IL), or a rod

of ferromagnetic materials (Odontoson 42 kHz;
Odont-wave, Fort Collins, CO) acts as the transducer.
This magnetostrictive transducer comes attached
with the working tip to constitute a handpiece insert.
With passage of electrical current, a coil within the
handpiece insert reacts to the magnetic eld by
expanding and contracting in accordance with the
alternating current. This rapid expansion and con-
traction result in vibrations, which are transmitted to
the working tip. The tip of the magnetostrictive scalers
moves in an elliptical or circular manner. Sonic
instruments utilize mechanical rather than electrical
vibrations of the working tip. The Sonic scaler tip vi-
brates by compressed air from the dental units with
frequencies ranging from 2,000 to 6,000 Hz (17, 57).
Titan
sonic scaler 6.5 kHz (StarDental, Lancaster,

PA), SonicFlex
6.0 kHz (Kavo, Biberach, Germany),

Quixonic
sonic scaler 6.0 kHz (Dentsply, Des Pla-

ines, IL) and Emie
560 6.0 kHz (Micron Co. Ltd,

Tokyo, Japan) come under this group. The handpiece
is composed of a hollow rod, a rotor and several
rubber O-rings. Compressed air is forced through the
hollow rod in the handpiece. The rotor is a 6-mm-
wide thin metal ring, which encircles the hollow rod
above a series of the scientically angled holes. The air
escapes through the 10 holes and causes the rotor to
vibrate, which in turn triggers the entire rod to vibrate.
The working motion of the sonic scaler is generally
elliptical. Although a coolant is not necessary, its use
enhances the effect through acoustic streaming and
lavage. Sonic scalers have been widely used due to
their clinical effectiveness, and as they cause less pain.
Power-driven scalers were originally designed for
supragingival use. The advantages of power-driven
instruments are less operator fatigue, ease in use, and
a simultaneous ushing effect by coolant. Disad-
vantages include difculty in inserting the bulky
working tips into deep periodontal pockets to per-
form root planing, risk of damage to the root surface,
poor tactile sensation, and aerosol contamination. In
an effort to overcome these problems, the design of
the working tip has been modied by several manu-
facturers for use in subgingival or furcation areas.
Fig. 2. Curette type scalers are used for removal of sub-
gingival calculus. A set of double-ended Gracey curettes,
from left to right, Gracey 1-2, 3-4, 5-6, 7-8, 9-10, 11-12,
13-14 curettes (Hu-Friedy Co. Ltd., Chicago, IL).
Fig. 1. Sickle type scalers are used for removal of supra-
gingival calculus. University of South California #3 4
(Nordent Co. Ltd., Chicago, IL).
46
Oda et al.
Other instruments
Apart from the abovementioned types, there are
instruments mounted on air turbines or microen-
gines. Rotosonic scalers are mounted on an air
turbine. A hexagon pyramid-shaped bur on the air
turbine removes calculus with rotational movement.
Diamond points with ne diamond particles are also
used. However, due to excessive removal of tooth
substance the use of Rotosonic scalers and diamond
points is limited. The instruments mounted on
microengines are mainly used for professional
toothcleaning. This group includes the rubber cup,
brush, prophy cup, EVA tip, etc.
Various aspects of manual and
power-driven instrumentation
Effectiveness in plaque and calculus
removal (Table 1)
Manual and ultrasonic scalers have been reported to
be equally effective in subgingival plaque removal
(7, 38, 49, 64, 65). Oosterwaal et al. (49) investigated
the effects of manual and ultrasonic scaling (Cavi-
tron
with TFI-10 tip) on the subgingival microora

in periodontal pockets with a probing depth of
69 mm. They concluded that both treatments
equally reduced the microscopic counts of rods,
spirochetes, and motile organisms, and reduced the
total colony-forming units and number of black-
pigmented anaerobic rods and Capnocytophaga.
Copulos et al. (13) also reported similar bacterio-
logical observations after manual and ultrasonic
scaling (Cavitron
with modied tip 17.5 mm long

and having a shaft width of 1.1 mm). Baehni et al. (5)
compared the effects of ultrasonic (Piezon-Master
with type A tip) and sonic (Titan-S
with Perio tip

type no. 56420) scaling on the subgingival microora,
and reported no difference in microscopic or culture
observations.
Jotikasthira et al. (24) compared the efcacy of
three sonic scalers (Phatelus
with a curette-shaped
universal tip No. T390761; Nakanishi Dental MFG,
Tokyo, Japan; SonicFlex2000
with a curette-shaped
universal tip no. 5715071, Titan-S
with a curette-
shaped universal tip no. 56801), two ultrasonic
scalers (Hygienist
III with a standard probe-like

straight tip; Lysta Trading A S, Farum, Denmark,
Cavitron
with a probe-like TFI-EWPP tip) and a

reciprocating scaling instrument (EVA
instrument
with insert Type III for at areas; Dentatus Inter-
national AB, Stockholm, Sweden) in calculus
removal and extent of loss of tooth substance
in vitro. Their ndings showed that sonic scalers as
a group removed calculus more completely, but
caused signicantly more roughness and loss of
tooth substance than did ultrasonic or reciprocating
instruments. Recently, Busslinger et al. (8) found
that the calculus remaining was similar after
removal by a magnetostrictive ultrasonic scaler
(Cavitron
Jet SPS with Slimline
insert), a piezo-
electric ultrasonic scaler (Sonosoft
with prototype
modied insert from KaVo Innovations-GmbH;
Biberach, Germany) and a hand curette (a new
M23A universal hand curette; Deppeler, Rolle,
Switzerland) in vitro. Therefore, it may be concluded
that manual and power-driven scalers are equally
effective in removal of plaque bacteria and calculus.
Effectiveness in elimination of virulent
substances (endotoxin and others) from
the periodontally involved root
During the development of periodontal disease, the
root surface, especially the cementum, is exposed to
a new pathogenic environment which results in
structural changes. This change affects the wound
healing after treatment.
The changes to an exposed root surface, especially
exposed cementum, have been extensively reported
since 1960. These changes include alterations in the
levels of calcium or phosphate, degradation of col-
lagen bers, decalcication of the surface of cemen-
tum, hypercalcication, and absorption of bacterial
toxins, such as endotoxin. Throughout periodontal
therapy, from plaque control to the maintenance
phase, management of the deleterious effects of
periodontitis on the exposed root surface inuences
the success of the treatment (2). The most important
and noteworthy change in exposed cementum is the
presence of endotoxin. The presence, toxicity, and
management of endotoxins have been investigated in
the last few decades.
In 1971, Hateld & Baumhammers (19) reported
that cells cultured with periodontally involved roots
showed irreversible morphologic changes, which
suggested the presence of toxic factors associated
with the roots. In the following years, Aleo et al. (2, 3)
showed that the endotoxin attached to cementum
was capable of inhibiting cell growth of broblasts,
and the attachment of broblasts to the periodontally
involved root surface was suppressed. Removal of
47
Manual and power-driven instrumentation
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48
Oda et al.
diseased cementum promoted cell attachment by
broblast. These studies suggested that the exposed
root surface contained endotoxin, which could inhi-
bit wound healing.
In 1982, Daly et al. (14) reported the penetration of
microorganisms to the depth of the cementodentinal
junction, and suggested that all periodontally
involved cementum should be removed during root
planing to achieve a root surface free of bacterial
contamination. However, in the same year, Nakib
et al. (44) immersed healthy teeth in an endotoxin
solution and examined the extent of penetration of
endotoxin into cementum by indirect immuno-
uorescence examination. They concluded that
endotoxin adheres to the tooth surface without pen-
etration into cementum of either periodontally
healthy or diseased teeth, and binding of the endo-
toxin to the root surface appears to be weak.
Other investigators have suggested that extensive
root planing is not essential for endotoxin removal
from the root surface (21, 42, 46, 48, 60). Oda (46)
investigated the extent of endotoxin penetration into
57 extracted periodontally involved teeth. Following
plaque and calculus removal, exposed root surfaces
were scaled with a universal curette in 13 strokes.
Root debris after the rst two strokes (the rst layer),
another three strokes (the second layer), the fol-
lowing four strokes (the third layer) and the nal
four strokes (the fourth layer) were collected to
measure the endotoxin contents of each layer.
Results indicated that the rst layer contained 7.424
times more endotoxin than other layers. He con-
cluded that two scaling strokes with a sharp manual
scaler were enough to eliminate endotoxin from
periodontally involved root surfaces. Moore et al.
(42) investigated the distribution of lipopolysaccha-
ride on periodontally involved root surfaces. They
showed that 39% of the lipopolysaccharide could be
removed by gently washing in water for 1 min and
60% by brushing for 1 min with a slowly rotating
brush. These studies suggested that an almost
complete debridement of root surfaces might be
achieved by relatively simple and atraumatic meas-
ures (21, 42, 46, 48, 60). Therefore, intentional
excessive removal of cementum during root planing
in order to eliminate endotoxins from the exposed
root was not justied.
The efcacy of ultrasonic scaler on removal of
endotoxin has been investigated (10, 11, 41, 60). The
cavitational activity is considered effective for
removal of plaque and endotoxin (42, 7072). These
studies suggested that ultrasonic scalers are effective
for periodontal treatment, as they are capable of
removing endotoxin located on the root surface
without excessive removal of cementum or dentin.
Root surface removal by scaling
and root planing
Cementum removal during scaling and root planing
with manual scalers was reported to be 57.8 lm with
40 strokes by Horning et al. (20) and 60 lm with 20
strokes by Coldiron et al. (12). Ishizuka and cowork-
ers (22) reported that the root surface removal by
Gracey curette was 39 lm with 750 g lateral pressure
per stroke, for the rst 50 strokes. The amount of root
substance removal increased with force. When using
a ne curette, the loss of substance per working
stroke with clinically applied forces was reported to
be 9.1 lm. Zappa et al. (74) also reported that signi-
cantly more root substance was removed when the
force applied was strong. Ritz et al. (54) found that
the amount of root substance removed by an ultra-
sonic scaler, sonic scaler, diamond bur, and ne
curette per stroke was 17.2 lm, 4.37.8 lm, 7.9
15.5 lm and 522 lm, respectively. While comparing
manual and ultrasonic scalers, some reports indica-
ted that the manual scaler removes more root
substance (55, 67), whereas others reported that
ultrasonic scalers do so (43, 50). According to these
studies, the root substance removal with one stroke
was 120 lm and it varied depending on the site of
the tooth, the power of the power-driven scaler, the
shape of the tip, and whether the root surface was
exposed or not.
Required time and clinical outcome for
scaling and root planing (Table 2)
Badersten et al. (4) compared the clinical effects of
subgingival debridement using manual and ultra-
sonic instruments, and reported no differences in
terms of probing depth, clinical attachment level, and
gingival recession after 2 years. However, they poin-
ted out that manual instrumentation took longer to
achieve the same clinical outcome.
Copulos et al. (13) used a curette and an ultrasonic
scaler (Cavitron
) with a modied tip in nine patients

for supportive periodontal treatment. After 6 months,
clinical parameters including plaque index, gingival
index, and bleeding on probing were recorded and
microbiologic observations using dark-eld micros-
copy were performed. The results showed that
treatment with the ultrasonic scaler was as effective
as treatment with the Gracey curette in all clinical
parameters measured. However, instrumentation
49
T
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50
Oda et al.
time for ve sites was signicantly less with the
curette (3.9 min vs. 5.9 min). Sherman et al. (58)
evaluated the calculus-removing efcacy of an
ultrasonic scaler (Cavitron
with P-10 universal tip)

and a Gracey curette. The total time spent for
instrumentation per tooth was 3.6 min with the
ultrasonic scaler and 5.8 min with the manual scaler.
Other studies comparing ultrasonic and manual
scalers also reported similar clinical effects (66), but
differences in instrumentation time.
Jotikasthira et al. (24) scaled root surfaces with
plaque and calculus using sonic or ultrasonic
instruments or with a new reciprocating scaling
insert for the EVA PROFIN system in vitro. Although
the reciprocating insert gave results similar to those
of the ultrasonic scaler, the scaling time was signi-
cantly longer for the new cleansing principle.
Busslinger et al. (8) compared the time needed, cal-
culus removal, and root surface roughness after sca-
ling with a magnetostrictive ultrasonic scaler, a
piezoelectric ultrasonic scaler, and a hand curette
in vitro. The time needed for instrumentation was
126.1 38.2 s for the curette, which was longer than
the piezoelectric ultrasonic scaler (74.1 27.6 s;
p < 0.05) and the magnetostrictive ultrasonic scaler
(104.9 25.4 s; p>0.05). The piezoelectric scaler was
more efcient than the magnetostrictive scaler in
removing calculus, but left the instrumented tooth
surface rougher.
Loos et al. (39) compared the effects of a single
episode of supra- and subgingival debridement using
either a sonic or an ultrasonic scaler in 10 patients
with adult periodontitis. There was no difference in
the clinical response between sites treated with the
sonic or ultrasonic instrument. A slightly longer time
was required for instrumentation with the sonic than
ultrasonic instrument. The average time of active
instrumentation was 4.0 min tooth with the sonic
scaler and 3.3 min tooth with the ultrasonic instru-
ment. However, the difference was not signicant. It
may be concluded that manual scalers require more
time in scaling and root planing than power-driven
scalers.
Limitation of scaling and root planing
Waerhaug et al. (68) examined to what extent it was
possible to remove subgingival plaque and calculus
by means of manual scalers. After scaling and root
planing, the rate of success (reformation of junction
epithelium) or failure (reformation of subgingival
plaque) was evaluated on 212 surfaces of 53 teeth. In
pockets measuring less than 3 mm, 83% of the root
surfaces reformed junctional epithelium. In pockets
between 3 and 5 mm the rate of success was 39%,
and pockets deeper than 5 mm showed 11% rate of
success. Similarly, Stambaugh et al. (61) reported the
inability to remove all subgingival plaque and cal-
culus from all tooth surfaces with more than
3.73 mm periodontal pocket.
Matsuo et al. (40) extracted 40 teeth after subgin-
gival scaling and root planing using various manual
instruments, and evaluated residual calculus and
deposits. The amount of residual calculus and
deposits was greatest on the molar teeth, followed by
premolar and anterior teeth. Teeth with pockets less
than 3 mm had the least amount of residual calculus
and deposits, whereas teeth with pockets over 5 mm
showed greater residual calculus and deposits than
teeth with pockets less than 5 mm. They concluded
that scaling and root planing with manual scaler was
effective when the pocket depth was less than 5 mm.
The conditions differed for single and multirooted
teeth.
Izumi et al. (23) compared the effects of scaling
and root planing performed from approximately
1 mm coronal to the base of the pocket or from the
base of the probable pocket to the gingival margin.
For periodontal pockets deeper than 3.5 mm, the
pocket reduction and attachment gain were greater
when scaling and root planing was performed from
the base of the pocket. They concluded that the
trauma caused to the most coronal part of the con-
nective tissue attachment by scaling and root planing
seemed to be of minor importance compared to the
effective removal of subgingival deposits.
Recent developments of manual and
power-driven scalers (Table 3)
In the past few years, researchers have tried to modify
scaling instruments for use in various situations
where operators experienced difculties in perform-
ing mechanical debridement (51). These modica-
tions were aimed to improve the efcacy, as well as
ease of scaling and root planing. Recently, extended
shank curettes for deep subgingival scaling or ap
operation, and mini-bladed curettes for narrow
pockets at the midlingual or midpalatal have become
available (37, 59). In another type, the diameter of the
shank has been modied (Fig. 3). In addition, many
other types of curettes have been developed. Two
double-ended curettes for mesial and distal surfaces
of the posterior teeth have been added to the set of
Gracey curettes (Fig. 4). A comparative study of a
short-blade, long-shank curette newly developed for
51
T
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52
Oda et al.
deep pockets at incisors, and a conventional Gracey
1 2 curette demonstrated that the new curettes were
more effective for anterior narrow and deep pockets
(59), although they made the root surface rougher
(37).
Various modied power-driven scaler tips, such
as tiny, thin, periodontal probe type, rounded top,
diamond coated and contra-angled inserts for use
in deep pockets have been developed (16) (Fig. 5
and 6). Dragoo et al. (15) examined a manual scaler
(universal curette) and ultrasonic scalers (Cavi-
tron
) with modied (contra-angled tips EW-P10R

and EW-P10L which resemble a periodontal probe
Fig. 4. New addition of two double-ended curettes
designed for mesial and distal surfaces of the posterior
teeth to the set of Gracey curettes. From left to right,
Standard 11-12, Fit 11-12, Fit 13-14 (Hu-Friedy Co. Ltd.).
Fig. 3. Rigid type curettes for heavy calculus removal,
extended shank curettes for deep subgingival scaling or
ap operation, mini-bladed curettes for narrow pockets
at the mid-lingual or mid-palatal, Ohta type curettes for
Asian populations have been developed. From left to
right, Standard, Rigid, Mini-ve, After Five, Ohta type
curettes (Hu-Friedy Co. Ltd.).
Fig. 5. Slim periodontal probe type inserts for use in
deep pockets have been developed. From left to right,
Perio slim tip
, Perio tip
, A tip
(EMS).
Fig. 6. Ball-ended ultrasonic inserts for use in furcation
have been developed. From left to right, a set of furcation
tips for Solfy
(Morita), a set of furcation inserts for

ENAC
(Osada), a set of furcation inserts for Piezon

Master
(EMS).
53
in size and shape) and an unmodied (P-10 type)
inserts in debridement of hopeless single or multi-
rooted teeth. They evaluated the pocket depth,
instrument limit, and instrument efciency. The
modied inserts showed added benets. Yukna
et al. (73) developed a diamond-coated ultrasonic
tip, the shape of which resembled a universal one
(Cavitron
) and evaluated its effectiveness in the

removal of subgingival calculus on single-rooted
teeth with probing depths of 512 mm in 15
patients. Results indicated that the diamond-coated
ultrasonic tips were much more efcient than
conventional or the universal tip in removing cal-
culus. But these diamond-coated tips left the
roughest root surface after instrumentation.
Gantes et al. (18) developed a plastic tip, made of a
strong plastic material, for use with a sonic scaler,
and evaluated its efcacy in removing dentin sub-
stance in vitro and mature supragingival plaque
in vivo. Instrumentation with the sonic scaler tted
with the plastic tip removed considerably less dentin
and resulted in a smoother dentin surface than
instrumentation with the curette, or the sonic scaler
with an ordinary metal tip. The plastic tip ef-
ciently removed mature plaque within 5 s. They
concluded that the new plastic tip might be useful
especially for maintenance therapy, with less risk for
iatrogenic effects on root surfaces.
Kocher et al. (27, 29, 30, 35) developed Teon-
coated sonic inserts that were as effective as con-
ventional sonic inserts in removal of plaque. They
compared the clinical effects of subgingival debride-
ment using the new Teon-coated sonic inserts with
manual scaling. As subgingival debridement with
Teon-coated sonic scaler inserts seemed to be
nearly as effective as conventional scaling, the
authors suggested that they may be employed for
maintenance treatment of residual pockets.
A new instrument designed for root debridement,
Periosonic
, which is a modied version of the

endodontic system, has also been introduced (6, 52,
56). The instrument has two types of les inserted in
a sonic handpiece. The Periosonic
1 le resembles a
reamer with a 16-mm working tip, and is used to
remove heavy supra- and subgingival calculus. The
Periosonic
2 le is more exible and less aggressive

than Periosonic
1. Its one-sided working tip is

21 mm long. This le was designed for subgingival
debridement, where the smooth part of the le faces
the soft tissue wall in a periodontal pocket to min-
imize trauma.
A split-mouth clinical study was performed to
evaluate the clinical effectiveness of the Periosonic
instruments compared to hand curettes (6). The

results suggested that Periosonic
instruments are
clinically as effective as curettes in terms of pocket
depth reduction in pockets with an initial pocket
depth <6 mm, and show better clinical attachment
gain with less recession in pockets with an initial
pocket depth >7 mm.
Access to furcation areas (Table 4)
The access to furcations or deep pockets is inu-
enced by the shape of the instrument and the shape
of the pocket or root surface. Some reports showed
that the skill of the operator was highly important (7,
25). The comparative study of manual scaling vs.
ultrasonic debridement by Leon & Vogel (38) con-
cluded that both instruments were equally effective
in Class I furcations. However, ultrasonic debride-
ment was signicantly more effective than manual
scaling in Class II and III furcations.
A newly designed tip, which resembles a furca-
tion probe with a spherical end, was developed to
improve access to the furcation area. Oda & Ishi-
kawa (47) introduced this new ultrasonic scaler
insert specically designed for furcation areas. This
insert is shaped like a short section of a spiral with
a curvature radius of about 9 mm. The tip is a
sphere with a diameter of 0.8 mm. This insert
seemed to have better access to furcation areas
than a straight probe-like insert, and in vitro
experiments have demonstrated that it is signi-
cantly more effective than Gracey curettes. Taskacs
et al. (63) compared the scaling efcacy of four
types of modied tips of Cavitron
(prototype
Cavitron insert with a spherical tip of diameter
0.8 mm (Ball Point Tip), Cavitron insert (EWP-12 L:
Pointed Tip), ENAC
(furcation insert with a

spherical tip of diameter 0.8 mm), and Titan
sonic
scaler (universal type insert: no. 56801) in the fur-
cation area. They reported greater efcacy with the
ball point inserts of Cavitron
and ENAC
or the
universal type insert of Titan
.
Kocher et al. (28, 3134, 36) developed a sonic
scaler set with diamond-coated ellipsoid terminal
tips, and compared it with the instruments used for
furcation treatment, hand instruments, hand instru-
ments in conjunction with diamond burs, a conven-
tional ultrasonic scaler insert, and a conventional
sonic scaler insert in vitro (28, 36). A greater area was
instrumented with the diamond-coated inserts than
with the other instruments. In addition, tooth sub-
stance removal was greater with diamond-coated
inserts than the others. They concluded that
54
Oda et al.
T
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55
diamond-coated inserts could be used during initial
treatment and ap operations, but with considerable
care.
Adjunctive effects of chlorhexidine as
irrigants during ultrasonic scaling
Adriaens et al. (1) examined the bacterial invasion in
root cementum and radicular dentin of periodontally
diseased human teeth by scanning electron micros-
copy. After scaling and root planing, they could still
detect bacteria in the remaining root cementum and
in the dentinal tubules. They concluded that it would
be appropriate to combine mechanical periodontal
therapy with the use of chemotherapeutic agents.
Several ultrasonic scalers with autoclavable uid
reservoirs, such as Piezon
Master 400, ENAC
,
Cavitron
, and Odontoson
, are now capable of

using saline or antimicrobial lavage as an irrigant.
The application of chlorhexidine in ultrasonic scalers
instead of water has been investigated. Nosal et al.
(45) evaluated the penetration depth of the water
coolant for medicament lavage of an ultrasonic
(Cavitron
with EWPP tip) device into the perio-

dontal pocket and concluded that the ultrasonic
scaler may be an effective system to mechanically
remove plaque and calculus at the same time as
delivering a chemotherapeutic agent. Taggart et al.
(62) reported that 0.02% chlorhexidine had a slight
adjunctive effect in terms of reduction of pocket
depth when used as a coolant during ultrasonic root
planing (Cavitron
) for the treatment of chronic

periodontitis. Reynolds et al. (53) studied the clinical
and microbial effects of a single episode of simulta-
neous ultrasonic scaling and subgingival irrigation
with 0.12% chlorhexidine, and reported that sub-
gingival irrigation with chlorhexidine during ultra-
sonic scaling (CaviMed
TM
200; Dentsply) provided
differential clinical benets that were site-dependent.
However, Chapple et al. (9) failed to show any sig-
nicant benets of using 0.2% chlorhexidine as irri-
gant during ultrasonic (CaviMed
TM
200) scaling
and root planing. Statistical analysis at the 3- and
6-month post-treatment stages revealed no signi-
cant differences between the experimental groups in
terms of probing attachment levels (p>0.5), bleeding
index (p>0.1), and plaque index (p>0.05).
Summary
It is clear from the literature that scaling and root
planing play a pivotal role in the elimination of
causative factors of periodontal disease throughout
periodontal therapy, including the nonsurgical, sur-
gical and maintenance phases. In the past, it had
been generally agreed that excessive root surface
removal by hand instruments was necessary to
remove the tenacious calculus deposits. However,
research over the past years has shown that denitive
root surface detoxication can be achieved without
excessive cementum removal or aggressive instru-
mentation. Complete cementum removal is no lon-
ger a requisite. Many studies have demonstrated that
hand and power-driven instruments are equally
effective in reducing the probing depth, attaining
attachment level gains and reducing inammation by
removal of plaque bacteria, calculus, and endotoxin.
Power-driven instruments have many advantages
over the manual scalers; however, further studies are
needed to improve the performance of currently
available instruments. These include the develop-
ment of a more effective tip and ultrasonic generator
unit. Long-term randomized controlled studies are
also required to examine the efcacy of the newly
designed scalers. These studies would help to provide
treatment based on exact information regarding the
instrument and technology.
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Oda et al.
58
Lasers in nonsurgical
periodontal therapy
AKI RA AOKI , KATI A MI YUKI SASAKI , HI SASHI WATANABE & ISAO ISHI KAWA
This article reviews the current and potential appli-
cations of laser technology in nonsurgical therapy for
the treatment of periodontal diseases. Based on its
various characteristics, such as ablation or vaporiza-
tion, hemostasis, and sterilization effect, laser treat-
ment may serve as an adjunct or alternative to
conventional, mechanical periodontal therapy. The
Carbon dioxide (CO
2
) and the Neodymium-
doped:Yttrium-Aluminum-Garnet (Nd:YAG) lasers
were previously approved for soft tissue treatment in
periodontics (1, 2, 4), because of their superior ability
of soft tissue ablation, accompanied by strong he-
mostatic and bactericidal effects (6, 37, 143, 170, 218).
However, when these lasers are applied to dental
hard tissues the result is major thermal damage,
especially at a high-energy output, rendering them
unsuitable for hard tissue treatment (56, 214).
Recently, the Erbium-doped:Yttrium-Aluminum-
Garnet (Er:YAG) laser was developed in dentistry (71,
85, 87). As it is capable of ablation in both soft and
hard tissues, the Er:YAG laser can be used for perio-
dontal hard tissue treatment such as root surface
debridement, as well as soft tissue management (78).
The use of lasers within the periodontal pocket has
become a topic of much interest and is a promising
eld in periodontal therapy. This article deals with
recent advances in nonsurgical laser therapy for
periodontal disease, and will briey describe the
advantages and disadvantages of various laser types.
and lasers
In periodontal pockets, the root surfaces are con-
taminated with an accumulation of plaque and cal-
culus, as well as inltration of bacteria and bacterial
endotoxins into cementum (5). Complete removal of
these harmful substances is essential for the healing
of periodontal tissue. Formation of biolms on the
exposed root surface within periodontal pockets
impedes the inltration of antibiotics, and therefore
mechanical disruption of the biolm is necessary
during periodontal treatment (36).
Basically, the aim of periodontal treatment is to
restore the biological compatibility of periodontally
diseased root surfaces for subsequent attachment of
periodontal tissues to the treated root surface. During
the initial periodontal treatment, debridement of the
diseased root surface is usually performed by
mechanical scaling and root planing using manual or
power-driven instruments. Power-driven instruments
(power scalers) such as ultrasonic or air scalers are
frequently used for root surface treatment as they
render the procedure easy and less stressful for the
operator, while improving the efciency of treatment.
However, conventional mechanical debridement
using curettes is still technically demanding and time
consuming, and power scalers cause uncomfortable
stress to the patients from noise and vibration.
Complete removal of bacterial deposits and their
toxins from the root surface and within the perio-
dontal pockets is not necessarily achieved with con-
ventional, mechanical therapy (5). In addition, access
to areas such as furcations, concavities, grooves, and
distal sites of molars is limited. Although systemic
and local antibiotics are occasionally administered
into periodontal pockets for the purpose of disinfec-
tion, with frequent use of antibiotics there is a
potential risk of producing resistant microorganisms.
Therefore, development of novel systems for scaling
and root planing, as well as further improvement of
currently used mechanical instruments, is required.
As lasers can achieve excellent tissue ablation with
strong bactericidal and detoxication effects, they are
one of the most promising new technical modalities
for nonsurgical periodontal treatment. Another
advantage of lasers is that they can reach sites that
conventional mechanical instrumentation cannot.
The adjunctive or alternative use of lasers with
59
PERIODONTOLOGY 2000
conventional tools may facilitate treatment, and has
the potential to improve healing.
Conventional mechanical treatment usually pro-
duces a smear layer and, sometimes, deep grooves on
the root surface. Asmear layer may adversely affect the
healing of periodontal tissues as it contains bacteria
and inammatory substances such as debris of infec-
ted cementum and calculus (140). Therefore, ways to
eliminate the smear layer have been investigated in
recent years. Many researchers have examined the
effects of root conditioning after mechanical debri-
dement, using chemical agents such as tetracycline,
citric acid, and ethylenediaminetetraacetic acid
(EDTA). Root conditioning has been shown to remove
the smear layer, and to expose collagen bers and
dentinal tubules, enhancing the histocompatibility
and new connective tissue attachment with cement-
ogenesis (25, 120, 154). Laser irradiation has been
reported to exhibit bactericidal and detoxication ef-
fects without producing a smear layer, and the laser-
treated root surface may therefore provide favorable
conditions for the attachment of periodontal tissue.
Gingival curettage after scaling and root planing
using mechanical instruments has been shown to
have no added benet over routine scaling and root
planing (3, 41, 108, 146). Therefore, the root surface
has been the focus of mechanical debridement, and
root surface debridement alone is the main step of
nonsurgical periodontal therapy at present. However,
the poor clinical outcome of gingival curettage may
have been due to the lack of an effective tool for soft
tissue debridement. Contrary to mechanical treat-
ment with conventional instruments, the excellent
ablation of tissue with laser treatment is expected to
promote healing of periodontal tissues, ablating the
inamed lesions and epithelial lining of the soft tissue
wall within periodontal pockets. This procedure
might be more effective for the treatment of residual
pockets after initial therapy and during maintenance.
Part of the laser energy scatters and penetrates
during irradiation into periodontal pockets. The
attenuated laser at a low energy level might then sti-
mulate the cells of surrounding tissue, resulting in
reduction of the inammatory conditions (131, 162,
183), in cell proliferation (8, 103, 135), and in increased
ow of lymph (184), improving the periodontal tissue
attachment and possibly reducing postoperative pain.
Although there is no clear evidence to date that laser
applications improve clinical outcome due to the
action of curettage (3), laser treatment has a potential
advantage of accomplishing soft tissue wall treatment
effectively along with root surface debridement, and
should be further investigated.
Characteristics of laser
LASER is an acronym for Light Amplication by
Stimulated Emission of Radiation. The physical
principle of laser was developed from Einsteins
theories in the early 1900s, and the rst device was
introduced in 1960 by Maiman (114). Since then,
lasers have been used in many different areas in
medicine and surgery. Laser light is a man-made
single photon wavelength. The process of lasing
occurs when an excited atom is stimulated to emit
a photon before the process occurs spontaneously.
Spontaneous emission of a photon by one atom
stimulates the release of a subsequent photon and so
on. This stimulated emission generates a very
coherent (synchronous waves), monochromatic (a
single wavelength), and collimated form (parallel
rays) of light that is found nowhere else in nature
(28). Lasers can concentrate light energy and exert a
strong effect, targeting tissue at an energy level that is
much lower than that of natural light. The photon
emitted has a specic wavelength that depends on
the state of the electrons energy when the photon is
released. Two identical atoms with electrons in
identical states will release photons with identical
wavelengths. The characteristics of a laser depend on
its wavelength (Table 1, Fig. 1).
The termwaveform describes the manner in which
laser power is delivered over time, either as a con-
tinuous or as a pulsed beam emission. A continuous
wave laser beam emits an uninterrupted beam at the
output power set for as long as the switch is turned on.
The pulsed beam may be delivered in two different
modes: free-running pulse, in which pulsation occurs
within the laser tube, and gated (chopped) pulse, in
which the continuous wave beam is interrupted by a
shutter at various rates. The gated pulse has the same
maximumpower as that set on the control panel of the
laser, whereas the free-running pulse is the result of
power storage for given time periods. Release of stored
power within a very short time creates an emission
that exhibits a peak power greater than the power
selected on the control panel (28, 159).
When laser light reaches a tissue, it can reect,
scatter, be absorbed or be transmitted to the sur-
rounding tissues (Fig. 2). In biological tissue,
absorption is mainly due to the presence of free
water molecules, proteins, pigments, and other
macromolecules. The absorption coefcient strongly
depends on the wavelength of the incoming laser
irradiation. In thermal interactions, absorption by
water molecules plays a signicant role (129) (Fig. 3).
60
Aoki et al.
The absorption coefcient (a : cm
)1
) for water is
0.00029 for argon laser (514 nm), 0.020 for diode laser
(800 nm), 0.61 for Nd:YAG laser (1,064 nm), 12,000
for Er:YAG laser (2,940 nm) and 860 for CO
2
laser
(carbon dioxide laser) (10,600 nm) (68, 129) (Fig. 3).
Application of lasers in periodontal
therapy
Lasers were rst employed in dentistry in hard tissue
treatments, such as caries removal and cavity
preparation, as a substitute for mechanical cutting
and drilling. After the discovery of the ruby laser in
1960, Goldman and coworkers (65) attempted caries
removal in vitro using the ruby laser in 1964. Since
then, many researchers have investigated the effects
of various lasers such as the argon, CO
2
, and Nd:YAG
lasers on dental hard tissues and caries (95, 190).
However, previous laser systems were basically not
indicated for hard tissue procedures due to major
thermal damage (55, 214). Thus, these laser systems
showed only limited potential for caries prevention
Table 1. Type and wavelength of lasers
Laser type Wavelength Color
Excimer lasers Argon Fluoride (ArF) 193 nm Ultraviolet
Xenon Chloride (XeCl) 308 nm Ultraviolet
Gas lasers Argon 488 nm Blue
514 nm Blue-green
Helium Neon (HeNe) 637 nm Red
Carbon Dioxide (CO
2
) 10,600 nm Infrared
Diode lasers Indium Gallium Arsenide Phosphorus (InGaAsP)
Gallium Aluminum Arsenide (GaAlAs)
Gallium Arsenide (GaAs)
Indium Gallium Arsenide (InGaAs)
655 nm
670830 nm
840 nm
980 nm
Red
Red-infrared
Infrared
Infrared
Solid state lasers Frequency-doubled Alexandrite 337 nm Ultraviolet
Potassium Titanyl Phosphate (KTP) 532 nm Green
Neodymium:YAG (Nd:YAG)
1,064 nm Infrared
Holmium:YAG (Ho:YAG) 2,100 nm Infrared
Erbium, chromium:YSGG (Er,Cr:YSGG) 2,780 nm Infrared
Erbium:YSGG (Er:YSGG) 2,790 nm Infrared
Erbium:YAG (Er:YAG) 2,940 nm Infrared
Infrared X-ray Ultraviolet
Visible light
(400~700 nm)
CO
2
(10,600)
( nm )
Microwave
Wavelength
Excimer
(193 - 350)
Argon
(458 - 515)
Er:YAG
(2,940)
He-Ne
(637)
Nd:YAG
(1,064)
Diode
(655 - 980)
Er:YSGG
(2,790)
Er,Cr:YSGG
(2,780)
Ho:YAG
(2,100)
Frequency-doubled
Alexandrite
(337)
1 10 10
2
10
3
10
4
10
5
10
6
10
7
Fig. 1. Electromagnetic spectrum and wavelengths of
lasers.
Laser Tissue Effects
Transmission
Scattering
Reflection
Laser Irradiation
Tissue
Absorption
Fig. 2. Effects of laser irradiation on tissue. When laser
light impinges on tissue, it can reect, scatter, be
absorbed or transmitted to the surrounding tissue.
61
Lasers in nonsurgical periodontal therapy
(217) and for the polymerization of light-cured
restorative materials (142) in the eld of preventive
and operative dentistry.
The initial and most important stage of periodontal
therapy is the nonsurgical mechanical debridement
of periodontally diseased root surfaces. In 1965,
Kinersly et al. (95) had already reported the possi-
bility of removing dental calculus by ruby laser.
However, they warned that limiting the vaporization
selectively to calculus without damaging the under-
lying tooth might present clinical problems.
Since the periodontium is composed of gingiva,
periodontal ligament, cementum, and alveolar bone,
both soft and hard tissues are always targeted when
using lasers for the treatment of periodontal lesions.
The commonly used high power lasers CO
2
and
Nd:YAG are capable of excellent soft tissue ablation,
and have an adequate hemostatic effect. As such,
these lasers have been generally approved for soft
tissue management in periodontics and oral surgery
(1, 2, 4, 137, 159). However, these lasers are not useful
for treatment of the root surface or alveolar bone, due
to carbonization of these tissues and major thermal
side-effects on the target and surrounding tissues
(159). Until the beginning of the 1990s, the use of
laser systems in periodontal therapy was limited to
soft tissue procedures, such as gingivectomy and
frenectomy (1, 2, 137, 160), as application to perio-
dontal hard tissues had previously proved to be
clinically unpromising.
In the early and mid 1990s, scientic research was
begun on root surface debridement and pocket
curettage using an Nd:YAG laser. The clinical appli-
cation of the Nd:YAG laser had already been tried by
general practitioners because its convenient, exible
ber optic delivery system made it appropriate for
use in periodontal pockets (124).
Meanwhile, in 1988, Hibst et al. (72) and, in 1989,
Keller & Hibst (71, 87) and Kayano et al. (85) reported
the possibility of dental hard tissue ablation by Er:
YAG laser irradiation, which is highly absorbed by
water. Since then, numerous studies on hard tissue
treatment using the Er:YAG laser have indicated the
ability of this laser to ablate dental hard tissues and
caries lesions without producing major thermal side-
effects. Promising results in basic and clinical appli-
cation have been demonstrated in the eld of caries
therapy (11, 34, 76, 86, 89, 92, 115). Later, in the mid
1990s, Aoki et al. (10) and Keller et al. (90) began to
investigate the application of the Er:YAG laser for
periodontal hard tissue procedures, such as dental
calculus removal anddecontaminationof the diseased
root surface. Anumber of basic studies onEr:YAGlaser
application to root surface treatment followed and,
recently, promising results have been reported in
clinical studies on nonsurgical pocket therapy. Appli-
cation of the Er:YAG laser for bone surgery has been
also studied in vitro and in vivo (15, 94, 141, 165, 166,
204). Development of this laser brought the prospect of
hard tissue treatment in periodontics (78) and end-
odontics (42, 185), as well as in operative dentistry
including pediatric dentistry (38, 83).
The diode lasers as well as Nd:YAG lasers are cur-
rently used for pocket curettage by clinicians because
of their exible ber delivery system, which is suit-
able for pocket insertion. However, to date, there is a
shortage of basic and clinical research providing
scientic support for these procedures.
In the eld of dentistry, Nd:YAG, CO
2
, diodes, Er:
YAG, Er,Cr:YSGG, Argon, excimer and alexandrite
lasers are being studied in vitro or are in clinical use
(Table 2). Their use in the treatment of periodontal
pockets and/or debridement of the periodontally
diseased root surface is presently under investigation.
Advances in research on clinical application of each
laser system for nonsurgical periodontal therapy are
discussed in the next section of this chapter.
Characteristics and current
research on different laser systems
CO
2
laser
Characteristics
The CO
2
laser has a wavelength of 10,600 nm and is
used as both a pulsed and a continuous wave laser.
wavelength [nm]
Er,Cr:YSGG
CO
2
Ar
Diode
Nd:YAG
Er:YAG
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
10
1
10
-2
10
-3
10
-4
10
-1
a
b
s
o
r
b
t
i
o
n

c
o
e
f
f
i
c
i
e
n
t

[
1
/
c
m
]
Fig. 3. Absorption spectrum for water. Data were calcu-
lated from Hale & Querry (68). Ar (488 nm), Diode
(810 nm), Nd:YAG (1,064 nm), Er,Cr:YSGG (2,780 nm),
Er:YAG (2,940 nm), and CO
2
(10, 600 nm) are indicated in
the gure.
62
Aoki et al.
This laser is readily absorbed by water and therefore
is very effective for the surgery of soft tissues, which
have a high water content. The primary advantage
of CO
2
laser surgery over the scalpel is the strong
hemostatic and bactericidal effect. Very little wound
contraction and minimal scarring are other advan-
tages of laser surgery, especially for the CO
2
laser
(113). The CO
2
laser has been used for soft tissue
surgery since the early 1970s (27, 54, 112, 134, 206).
In 1976, it was approved by the US Food and Drug
Administration (FDA) for soft tissue surgery, inclu-
ding the surgery of the oral tissues.
Since the CO
2
laser (10,600 nm) produces severe
thermal damage, such as cracking, melting, and car-
bonization when applied to hard tissues (111, 168), its
use has been limited to soft tissue procedures. Even
though the water absorption coefcient of the CO
2
laser is approximately one-tenth that of the Er:YAG
laser, it still shows a relatively high level (Fig. 3).
Considering only the water absorption coefcient, we
may assume that the CO
2
laser (10,600 nm) to some
extent demonstrates similar hard tissue ablation to
the Er:YAG laser. However, the CO
2
laser is also highly
absorbed by the main, mineral components of hard
tissue, especially phosphate ions ( PO
4
) in the car-
bonated hydroxyapatite (44, 45, 58, 59, 97). The
energy applied is readily absorbed in the hard tissue
but causes instantaneous heat accumulation in
the irradiated inorganic components, resulting in
carbonization of organic components and melting of
the inorganic ones, instead of the water-mediated
physical collapse of the hard tissue observed in
Er:YAG laser irradiation (59, 180).
The CO
2
laser is absorbed at the tissue surface with
very little scatter or penetration. Since ablation is
mainly due to the action of heat generation, car-
bonization easily occurs on the irradiated surface but
the heat produced does not scatter. Therefore, the
CO
2
laser produces a relatively thin layer of thermally
changed tissue (coagulation) around the ablated site.
The width of the coagulation layer was reported to be
100300 lm in an incision of pigskin with the con-
tinuous mode CO
2
laser at 6 W (16). Tissue penetra-
tion from this laser irradiation will be approximately
Table 2. Current and potential applications of lasers in dentistry
Laser type Current/Potential dental application
Excimer lasers Argon Fluoride (ArF)
Xenon Chloride (XeCl)
Hard tissue ablation, Dental calculus removal
Gas lasers Argon (Ar)
Helium Neon (HeNe)
Carbon Dioxide (CO
2
)
Curing of composite materials, Tooth whitening, Intraoral soft tissue surgery,
Sulcular debridement (subgingival curettage in periodontitis
and peri-implantitis)
Analgesia, Treatment of dentin hypersensitivity, Aphthous ulcer treatment
Intraoral and implant soft tissue surgery, Aphthous ulcer treatment, Removal
of gingival melanin pigmentation, Treatment of dentin hypersensitivity,
Analgesia
Diode lasers Indium Gallium Arsenide
Phosphorus (InGaAsP)
Galium Aluminum
Arsenide (GaAlAs) and
Galium Arsenide (GaAs)
Caries and calculus detection
Intraoral general and implant soft tissue surgery, Sulcular debridement
(subgingival curettage in periodontitis and peri-implantitis), Analgesia,
Treatment of dentin hypersensitivity, Pulpotomy, Root canal disinfection,
Aphthous ulcer treatment, Removal of gingival melanin pigmentation
Solid state
lasers
Frequency-doubled
Alexandrite
Neodymium:YAG
(Nd:YAG)
Erbium group
Erbium:YAG (Er:YAG),
Erbium:YSGG (Er:YSGG),
Erbium,chromium:YSGG
(Er,Cr:YSGG)
Selective ablation of dental plaque and calculus
Intraoral soft tissue surgery, Sulcular debridement (subgingival curettage
in periodontitis), Analgesia, Treatment of dentin hypersensitivity, Pulpotomy,
Root canal disinfection, Removal of enamel caries, Aphthous ulcer treatment,
Removal of gingival melanin pigmentation
Caries removal and cavity preparation, Modication of enamel and dentin
surfaces, Intraoral general and implant soft tissue surgery, Sulcular
debridement (subgingival curettage in periodontitis and peri-implantitis),
Scaling of root surfaces, Osseous surgery, Treatment of dentin
hypersensitivity, Analgesia, Pulpotomy, Root canal treatment and
disinfection, Aphthous ulcer treatment, Removal of gingival
melanin/metal-tattoo pigmentation
63
0.5 mm deep, depending on power density (160). In
the eld of periodontics, there have been several
reports on laser application for gingivectomy and
gingivoplasty in the late 1980s (19, 138, 139, 160).
Transmission of the CO
2
laser through optical
bers was very difcult and therefore the CO
2
laser
system previously employed mirror systems using
articulated arms for laser beam delivery. Recently,
new exible ber optic delivery and hollow tube
wave-guiding systems have been developed, along
with the development of contact tips. These advances
may render the use of the CO
2
laser for periodontal
pockets possible in the near future.
Basic and clinical studies on root surface
preparation and calculus removal
Several basic studies have shown the effects of con-
tinuous wave CO
2
laser irradiation on root surfaces.
The continuous wave CO
2
laser readily produces car-
bonization, melting, and cracking of root cementum
and dentin (80, 164, 167, 182, 187) (Fig. 4, Table 3).
Spencer et al. (187) found cyan-derived toxic
products, such as cyanamide and cyanate ions, on
the carbonized layer of the CO
2
lased (continuous
wave, 8 W) root surface by chemical analysis using
Fourier transformed infrared (FTIR) spectroscopy.
Tucker et al. (200) evaluated the effects of the CO
2
laser on calculus in vitro and reported that the pulsed
CO
2
laser at 6 W and 20 Hz (pulse duration: 0.01 s)
was able to remove dental plaque on the root surface,
whereas only melting and carbonization occurred on
the dental calculus of extracted teeth. In an animal
study, Gopin et al. (66) demonstrated that the root
surface treated by pulsed CO
2
laser (pulse duration:
0.01 s) at 6 W and 20 Hz, featuring a residual char
layer, inhibited periodontal soft tissue attachment.
Misra et al. (118) examined the root conditioning
effects of the defocus mode CO
2
laser after scaling
and root planing in vitro. Laser irradiation at 3 W for
1 s completely removed the smear layer with min-
imal change in the diameter of the dentinal tubules;
however, irradiation times of 1.2 and 1.4 s produced
surface charring and carbonization, and were totally
ineffective in exposing the dentinal tubules. Barone
et al. (20) investigated the effects of the pulsed
defocus mode CO
2
laser. The CO
2
laser at 2.0 W and
4 Hz with 4.0 mm spot size did not result in any
extensive damages to the root surface, which was at
and smooth with apparent fusion of the smear layer.
They concluded that the pulsed defocus mode may
present the advantage of decontaminating the root
surface. Crepsi et al. (35) reported that after the
pulsed defocus mode CO
2
laser treatment at 2 W and
1 Hz, the periodontally diseased root surface showed
the highest number of tightly attached broblasts
compared with the nontreated control and scaling
and root planing (SRP) alone. They concluded that
pulsed defocus mode CO
2
laser treatment combined
with mechanical instrumentation constitutes a useful
tool for root conditioning. Coffelt et al. (31) found
that, when used at an energy density between 11 and
41 mJ/cm
2
in the defocused mode, the CO
2
laser
destroyed microbial colonies without inicting
undue damage to the root surfaces.
Thus, the CO
2
laser, when used with high-energy
output, especially in a continuous wave mode, is not
appropriate for calculus removal and root surface
debridement due to major thermal side-effects, such
as carbonization. However, when used with relatively
low energy output in a pulsed and/or defocused
mode, this laser may have root conditioning, detoxi-
cation and bactericidal effects on the contaminated
root surfaces.
Miyazaki et al. (119) applied CO
2
laser irradiation
for pocket treatment on the external surface of the
marginal gingiva. They used a continuous wave mode
CO
2
laser (2.0 W, 120 s) and reported decreased
inammation and probing depth after treatment.
However, so far there have been no reported clinical
studies on application of the CO
2
laser in periodontal
pockets.
Nd:YAG laser
Characteristics
The Nd:YAG laser is a free-running pulsed wave laser
with a wavelength of 1,064 nm. Unlike the CO
2
and
Er:YAG lasers, the Nd:YAG laser has low absorption in
Fig. 4. Scanning electron micrograph of the root surface
after CO
2
laser irradiation (continuous wave, focused
beam, 0.5 W), showing numerous typical microcracks.
Original magnication: 750. Bar = 10 lm.
64
Aoki et al.
water, and the energy scatters or penetrates into the
biological tissues. In water, the Nd:YAG laser will
theoretically penetrate to a depth of 60 mm before it
is attenuated to 10% of its original strength (4).
The photothermal effect of the Nd:YAG laser is
useful for soft tissue surgery. Due to the charac-
teristics of penetration and thermogenesis, the
Nd:YAG laser produces a relatively thick coagula-
tion layer on the lased soft tissue surface, and
thereby shows strong hemostasis. Hence, the
Nd:YAG laser is basically effective for ablation of
potentially hemorrhagic soft tissue. The width of
the coagulation layer was 0.30.8 mm in an incision
of bovine oral soft tissue in vitro at 310 W (136,
208, 212).
In dentistry, soft tissue surgery using the Nd:YAG
laser has been widely accepted (117, 125, 137, 157,
207). In 1990, the FDA approved soft tissue removal
by means of a pulsed Nd:YAG laser (193). White
et al. (210) successfully used the Nd:YAG laser for
intraoral soft tissue application without anesthesia,
and with minimal bleeding compared to scalpel
surgery. It is easy to deliver the Nd:YAG laser by a
exible optical ber with a contact tip of 400 lm
(core diameter: 320 lm) suitable for pocket inser-
tion, and basic research and clinical trials have been
performed on periodontal pocket curettage and root
surface debridement. In 1997, the FDA approved
sulcular debridement by means of a pulsed Nd:YAG
laser (193).
The Nd:YAG laser is not suitable for ablation of
intact hard tissues. However, caries removal using
this laser is possible to some extent (21, 211). White
et al. (211) reported a safe and effective procedure
for selective removal of enamel caries with the
Nd:YAG laser, and the FDA approved removal of
enamel (rst degree) caries using an Nd:YAG laser in
1999 (193). As the Nd:YAG laser is well absorbed by
dark substances, Indian ink or other kinds of black
pigment are often applied to increase the efciency
of ablation (82).
Basic studies
Calculus removal: In an in vitro study, Tseng & Liew
(198, 199) demonstrated that partial removal and
detachment of the calculus from the root surface was
achieved with the Nd:YAG laser at 2.0 or 2.75 W and
20 Hz. However, melting of calculus and thermal
Table 3. CO
2
laser Basic studies on root surface treatment and a clinical study on periodontal pocket treatment
Author and Year References Laser
parameters
Findings
Shariati et al. 1993
Israel et al. 1997
Sasaki et al. 2002
(182)
(80)
(164, 167)
5 or 10 W, CW
511 W, 10 or 20 Hz
0.5 W, CW
Carbonization, melting and cracking of root
cementum and dentin
Spencer et al. 1996
Sasaki et al. 2002
(187)
(167)
8 W, CW
0.5 W, CW
Presence of cyan-derived toxic products on the
carbonized layer
Tucker et al. 1996 (200) 6 W, 20 Hz Dental plaque removal and calculus carbonization
Coffelt et al. 1997 (31) 25 W,
10 or 20 Hz
Destruction of microbial colonies without inicting
undue damage to the root surfaces in defocused mode
Gopin et al. 1997 (66) 6 W, 20 Hz Inhibition of periodontal tissue attachment by residual
char layer in vivo
Misra et al. 1999 (118) 3 W, CW
defocus
Root conditioning effects in defocused irradiation
Barone et al. 2002 (20) 2 W, 4 Hz
defocus
Root decontamination effects on the root surface
in pulsed defocus mode
Crespi et al. 2002 (35) 2 W, 1 Hz
defocus
Increased broblast attachment after root conditioning
in pulsed defocus mode
Miyazaki et al. 2003 (119) 2 W, CW Decreased inammation and probing depth at 12 weeks
after pocket treatment by irradiation on the external
surface of the marginal gingiva (see Table 5)
CW: continuous wave.
65
damage was noted in localized areas of the original
cementum and even dentin after irradiation at high
power (198). They also reported that, after laser
irradiation, removal of remaining calculus by curettes
was facilitated (198, 199). Arcoria & Vitasek-Arcoria
(17) assessed the effects of the Nd:YAG laser on cal-
culus removal from root surfaces in vitro. Nd:YAG
laser irradiation was performed at the calculusce-
mentum interface in contact mode at 1.5 or 3.0 W
(15 Hz) with the tip inclined 45
to the root surface.

The integrity of the calculusroot surface attachment
was not appreciably affected by 1.5 W irradiation,
whereas 3.0 W irradiation detached the calculus
without root surface damage, similarly to conven-
tional hand instrumentation. In an in vitro study,
Radvar et al. (144) reported that Nd:YAG laser irra-
diation, perpendicular to the root surface, at 0.5
2.0 W (50 or 100 mJ/pulse, 10 or 20 Hz) caused
greater ablation of calculus than either cementum or
dentin. However, the specimens with complete eva-
poration of calculus also showed some degree of
damage to the underlying cementum (Table 4).
Judging by the above, limited studies, the ability of
the Nd:YAG laser to remove calculus is insufcient,
and removal of calculus at a level equivalent to
mechanical treatment can not be expected clinically.
As subgingival calculus is originally dark in color, the
Nd:YAG laser has the advantage of being absorbed
Table 4. Nd:YAG laser ) Basic studies on calculus removal and root surface treatment
Author and Year References Laser parameters Findings
Tseng & Liew
1990, 1991
(198, 199) 2.0 or 2.75 W
100 or 137.5 mJ/pulse, 20 Hz*
Partial calculus removal with thermal damage to the
root surface
White et al.
1991
(209) 0.33.0 W
20 s and 2 min
6-log reduction in B. subtilis and E. coli on dentin
section after 2 min of laser exposure greater
than 0.5 W
Arcoria & Vitasek-
Arcoria 1992
(17) 1.5 or 3.0 W
100 or 200 mJ/pulse, 15 Hz*
Detachment of calculus without thermal damage
to the root surface
Morlock et al.
1992
(123) 1.251.50 W
62.575 mJ/pulse, 20 Hz
Surface pitting and crater formation with charring,
carbonization, melting, and crater production
Spencer et al.
1992
(189) 0.8 W*
80 mJ/pulse, 10 Hz
Decreased protein/mineral ratio and potential
surface contamination with protein by-products
Trylovich et al.
1992
(197) 0.8 W*
80 mJ/pulse, 10 Hz
Root surface alteration unfavorable for broblast
attachment
Ito et al.
1993
(81) 20 W
CW
Smear layer removal at high energy level not suitable
for clinical use
Fukuda et al.
1994
(62) 0.30.5 W
3050 mJ/pulse, 10 Hz, 24 s
Inactivation of endotoxin in the supercial layer of
periodontally diseased root surface of extracted teeth
Thomas et al.
1994
(195) 1.5 W
75 mJ/pulse, 20 Hz
Recovery of cell attachment to the lased surface
when followed by conventional SRP
White et al.
1994
(207) 0.33.0 W
30150 mJ/pulse, 10 or 20 Hz
Should not cause devitalizing intrapulpal
temperature rise at 0.33.0 W
Radvar et al.
1995
(144) 0.52 W
50 or 100 mJ/pulse, 10 or 20 Hz
Greater ablation of calculus than either cementum or
dentin with some thermal damage to the root surface
Wilder-Smith et al.
1995
(215) 5 W
1,000 mJ/pulse, 5 Hz*
Smear layer removal with signicant
temperature rise
Spencer et al.
1996
(187) 420 W
80400 mJ/pulse, 50 Hz*
Production of cyan-derived toxic substances such as
cyanamide and cyanate ions
Liu et al.
2002
(110) 0.5 W)3 W
50 mJ/pulse and 10 Hz to
150 mJ/pulse and 20 Hz
2 min
Less effectiveness of Nd:YAG laser noncontact
irradiation with a distance of 1.5 cm to the
moistened, cementum particles in destroying
diseased cementum endotoxin
*Calculated from data presented in paper. CW: continuous wave.
66
Aoki et al.
well into subgingival calculus. Use of higher energy
levels may ablate calculus more efciently but may
be inappropriate for clinical usage due to increased
thermal side-effects.
Root surface treatment: Several basic studies have
shown the effects of Nd:YAG laser irradiation on root
surfaces. Nd:YAG laser produced thermal changes
such as carbonization, melting, and resolidication of
root cementum and dentin (80, 123, 187, 189, 197)
(Fig. 5, Table 4).
Morlock et al. (123) showed that the Nd:YAG laser
at 1.251.50 W (62.575 mJ/pulse, 20 Hz) produced
surface pitting and crater formation with charring,
carbonization, melting, and crater production, even
when irradiation was performed parallel to the sur-
face. Spencer et al. (189) reported a decrease in the
protein/mineral ratio in cementum samples lased
with the Nd:YAG laser at 0.8 W (80 mJ/pulse and
10 Hz) using FTIR analysis. They considered that the
decreased protein/mineral ratio and the potential
surface contamination with protein by-products
might ultimately affect cell reattachment at the
cementum surface. Later, they suggested that the
protein by-products would be cyanamide and cya-
nate ions (39, 40, 187).
Trylovich et al. (197) showed that the root surface
after Nd:YAG laser irradiation at 0.8 W (80 mJ/pulse,
10 Hz) was unfavorable for broblast attachment
in vitro. Thomas et al. (195) also reported that the
nondiseased root surfaces treated at 1.5 W (75 mJ/
pulse, 20 Hz) with water coolant exhibited a signi-
cantly decreased broblast attachment, but the root
planing or polishing with air-powder abrasive that
followed, increased the cell attachment. They con-
cluded that alterations in the laser-irradiated surface
are reversible and additional root treatment following
laser irradiation appears essential to render the root
surface biocompatible. FTIR spectroscopy of the
lased root surface revealed reduction of the Amide II
band, suggesting denaturation of surface protein
upon laser exposure.
Nd:YAG laser treatment has been reported to re-
move the smear layer on the root surface. In Ito
et al.s study (81) Nd:YAG laser was effective in
removing the smear layer after conventional root
planning. However, the extremely high energy level
used (20 W, continuous wave [CW]) could not be
used clinically. Wilder-Smith et al. (215) examined
the effect of smear layer removal using the Nd:YAG
laser at 5 W (pulse durations and intervals of 0.1 s;
5 Hz and calculated energy output 1,000 mJ/pulse)
without coolant. Although the smear layer was
removed without microstructural changes of the hard
tissue, a signicant rise in the intrapulpal and root
surface temperature occurred. They concluded that
the irradiation parameters used may not be appro-
priate for clinical use.
Interestingly, Fukuda et al. (62) performed Nd:YAG
laser irradiation to the periodontally diseased root
surface of extracted teeth, and reported that the
Nd:YAG laser at 0.30.5 W (3050 mJ/pulse, 10 Hz,
24 s) could inactivate the endotoxin in the super-
cial layer of the root surface. Liu et al. (110) explored
the in vitro effectiveness of the Nd:YAG laser for the
elimination of cementum-bound endotoxin by
measuring interleukin (IL)-1b changes in stimulated
monocytes. Nd:YAG laser varying between 50 mJ/
pulse and 10 Hz and 150 mJ/pulse and 20 Hz for
2 min did not seem to be effective in destroying
diseased cementum endotoxin. However, in their
study, the thermal effect of Nd:YAG laser would have
been weakened by noncontact irradiation with a
long distance (1.5 cm) to the moistened cementum
particles.
Regarding the bactericidal effect, in an in vitro
study, White et al. (209) examined the decontam-
ination effects of the Nd:YAG laser on root surface.
They treated dentin sections contaminated with
bacteria using the Nd:YAG laser, and reported that
there was a 6-log reduction in Bacillus subtilis and
Escherichia coli after 2 min of laser exposure greater
than 0.5 W.
Clinical studies
Cobb et al. (29) performed Nd:YAG laser irradiation
into periodontal pockets in vivo in 18 teeth from eight
patients, and examined the effects of root debride-
ment using a power of 1.753.0 W (87.5150 mJ/
after Nd:YAG laser irradiation (1 W: 100 mJ/pulse and 10
Hz), showing typical melting and resolidication with a
porous structure. Original magnication: 750. Bar =
10 lm.
67
pulse, 20 Hz), either alone or in combination with
manual instrumentation. Application of the Nd:YAG
laser resulted in ineffective and patchy removal of
calculus from the root surface. The remaining calcu-
lus had a characteristic porous surface due to
carbonization, melting, and resolidication but the
surface was free of microbial plaque. They suggested
that laser therapy should be followed by mechanical
debridement. They reported the effect of Nd:YAG
laser on both root surface and subgingival microora
in periodontal pockets, employing teeth that required
extraction. DNA probe analysis showed a decreased
number of bacteria in periodontal pockets after laser
treatment. In addition, there were root surface alter-
ations similar to those observed in the in vitro studies.
Horton & Lin (75) compared subgingival applica-
tion of the Nd:YAG laser with conventional scaling
and root planing. Each of three segments in 15
patients received Nd:YAG laser irradiation (2 W:
100 mJ/pulse and 20 Hz, 2 min), scaling and root
planing by curette, or no treatment. The subgingival
application with the Nd:YAG laser was equally or
more effective than scaling and root planing in
reducing or inhibiting recolonization of specic
bacterial species and, though less effective in
removing calculus, was at least equally effective on
measures of probe depths and attachment loss.
Gold & Vilardi (64) performed Nd:YAG laser curet-
tage invivo, andstudiedmicroscopically 24 specimens
of gingival tissue from six patients following the laser
application at 1.25 and 1.75 W (62.5 and 87.5 mJ/
pulse, 20 Hz). The pulsed Nd:YAG laser removed
pocket-lining epithelium in moderately deep perio-
dontal pockets without causing necrosis or carbon-
ization of the underlying connective tissue. Radvar
et al. (145) performed pocket treatment using Nd:YAG
laser at a low power level of 0.5 or 0.8 W (50 or 80 mJ/
pulse, 10 Hz), or scaling and root planing in 80 perio-
dontally affectedsites of teethscheduledfor extraction
in 11 patients. Scanning electron microscope exam-
ination revealed that the low Nd:YAG laser energy
levels did not cause any heat damage to the root sur-
face, but failed to improve the clinical and microbio-
logical parameters of periodontal disease as compared
to scaling and root planing.
Ben Hatit et al. (22) compared the in vivo effects of
conventional scaling plus Nd:YAG laser at 0.81.5 W
(100 mJ/pulse, 815 Hz) treatment with those of
scaling alone, on root cementum and levels of peri-
odontopathic bacteria, in 150 sites from 14 patients.
The post-treatment reduction of levels of periodont-
opathic bacteria in the conventional scaling followed
by laser treatment group was signicantly greater
than the scaling alone control. However, scanning
electron microscope examination of the specimens
treated with the Nd:YAG laser exhibited different root
surface alterations.
Neil & Mellonig (128) performed a double-blind,
randomized, controlled clinical trial for sulcular
debridement on 186 teeth from 10 patients using a
split-mouth design to compare the adjunctive use of
the pulsed Nd:YAG laser at 2 W (80 mJ/pulse, 25 Hz)
to scaling and root planing alone in the nonsurgical
treatment of moderate to advanced adult periodon-
titis. The reduction of probing depth for 6 months
after treatment was similar between the scaling and
root planing plus laser therapy and scaling and root
planing alone, but the scaling and root planing plus
laser therapy showed signicantly greater improve-
ments in gingival index and gingival bleeding index at
specic time points. The mean attachment level of
laser-treated pockets also showed a tendency to
improve steadily for 6 months, whereas the scaling
and root planing group showed a reduction in
attachment level.
Liu et al. (109) performed a randomized, controlled
clinical trial in a split-mouth design, comparing the
effects of Nd:YAG laser treatment with scaling and
root planing treatment on crevicular IL-1b levels,
which is closely associated with periodontal destruc-
tion, in 52 sites from eight patients. Data showed that
laser therapy alone (3 W: 150 mJ/pulse and 20 Hz)
was less effective than traditional SRP for the reduc-
tion of crevicular IL-1b. Laser treatment followed by
SRP after 6 weeks showed greater reduction of IL-1b
and more clinical improvement than scaling and root
planing followed by laser treatment after 6 weeks. No
additional benet was found when laser treatment
was used secondary to traditional scaling and root
planing therapy.
Miyazaki et al. (119) compared the effectiveness of
the Nd:YAG and CO
2
laser treatment to that of
ultrasonic scaling in periodontal pockets of chronic
periodontitis patients. The 41 sites of 18 patients
were randomly assigned for treatment with the
Nd:YAG laser alone (2.0 W: 100 mJ/pulse and 20 Hz,
120 s), the CO
2
laser alone (2.0 W, 120 s), or ultra-
sonic scaling alone (maximum power, 120 s).
Decreased inammation and probing depth were
observed in all three groups after treatment. There
were signicant decreases in both Porphyromonas
gingivalis and the amount of gingival crevicular uid
in the Nd:YAG and scaling groups. The Nd:YAG group
also tended to show a decrease in IL-1 level. Nd:YAG
laser and ultrasonic scaling treatments showed
signicant improvements regarding the clinical
68
Aoki et al.
parameters and subgingival microora compared to
the baseline, but no signicant difference was
observed between the three groups.
Various combinations of irradiation parameters
have been reported for the clinical use of the Nd:YAG
laser for pocket treatment (208). White (208) recom-
mended 1.5 W (100 mJ/pulse, 15 Hz) irradiation for
removal of the sulcular diseased tissue and 2.0 W
(100 mJ/pulse, 20 Hz) irradiation for coagulation of
soft tissue wall after mechanical debridement. Col-
uzzi (33) recommended laser soft tissue curettage at
1.8 W (30 mJ/pulse, 60 Hz) after mechanical debri-
dement, followed by irradiation at 2 W (100 mJ/
pulse, 20 Hz) for hemostasis and bacterial reduction.
Gutknecht et al. (67) suggested the use of the Nd:YAG
laser at 2 W (100 mJ/pulse, 20 Hz) for curettage be-
fore mechanical debridement to reduce the risk of
bacteremia after the scaling and root planing proce-
dures, and to facilitate mechanical debridement.
Summary of the Nd:YAG laser clinical application
In 1997, the US FDA approved the application of the
Nd:YAG laser for sulcular debridement or soft tissue
curettage (193). It was the rst approval of laser
application in periodontal pockets by the FDA.
Pocket curettage with laser as an adjunct to con-
ventional mechanical root surface treatment has
been increasingly performed by general practitioners
because of its ease of use. However, in spite of the
widespread use of the Nd:YAG laser among general
practitioners, there is still insufcient proof from
scientic studies of the positive clinical effects of this
laser (Table 5). Opinions differ as to the use of lasers
in periodontal pockets. The American Academy of
Periodontology does not recommend the use of laser
curettage (3, 4) but the Academy of Laser Dentistry
(ALD) approves the adjunctive use of lasers for
curettage following conventional mechanical root
debridement (32).
Based on the results of previous in vitro and in vivo
studies, the Nd:YAG laser cannot achieve root surface
debridement to a satisfactory degree, due to insuf-
cient ability to remove calculus and distinct root
surface alteration induced by heat generation during
irradiation. If utilized, the Nd:YAG laser should be
employed as an adjunct to conventional mechanical
treatments, rather than as a primary instrument in
the treatment of periodontal pockets. That is, the
Nd:YAG laser should be used for curettage of perio-
dontal pockets to remove infected granulation tissue
and epithelial lining as well as disinfection and
detoxication of periodontal pockets, at a relatively
low energy level, rather than debridement of calcied
deposits and contaminated cementum at a high
energy level, having the potential of producing ther-
mal damages. Some clinical studies that used the
Nd:YAG laser as a primary debriding instrument
could not demonstrate the same or better clinical
outcomes than with conventional SRP. There was
insufcient calculus elimination as well as occasional
inevitable thermal damages of root surface following
Nd:YAG laser irradiation.
Many studies have reported that Nd:YAG laser
irradiation produces unfavorable thermal changes on
the root surface (30). However, variations in the
manner and conditions of irradiation, such as energy
output, use or nonuse of water irrigation, and the
degree of contact tip angulation to the root surface,
may produce different results. The root surface
alterations following Nd:YAG laser irradiation for soft
tissue curettage need to be reevaluated. Also, as soft
tissue attachment to the root surface exposed to the
Nd:YAG laser has not been reported in vivo, further
investigations are necessary to histologically deter-
mine the tissue attachment to the Nd:YAG laser-
treated root surface in vivo.
Regarding thermal inuence, White et al. (207)
examined in vitro the changes of intrapulpal tem-
peratures during Nd:YAG laser irradiation at 0.3
3.0 W (30150 mJ/pulse, 10 or 20 Hz) of root surfa-
ces. They reported that, within the parameters out-
lined in their study, when pulsed Nd:YAG laser energy
is applied to root surfaces with adequate remaining
dentin thickness it should not cause devitalizing
intrapulpal temperature rise.
Nonetheless, it is always necessary to bear in mind
that, at a high energy level on oral tissues, the Nd:YAG
laser will penetrate into deeper tissues of the target
site. In particular, when the irradiation is directed
perpendicular to the target, this deep penetration
may occasionally cause unexpected thermal damage
in the tooth pulp (196), alveolar bone, and other
surrounding tissues. Spencer et al. (188) reported a
mean temperature increase at the bone surface of
4.511.1

C during Nd:YAG laser soft tissue surgery at
high energy levels of 59 W (100180 mJ/pulse,
50 Hz) with and without water coolant. Friesen et al.
(60) reported a delayed healing response in bone
tissue irradiated by Nd:YAG laser. Care must be taken
when using the Nd:YAG laser, especially at a high
energy level, in the treatment of periodontal pockets.
Further studies areneededtoestablishthe useof this
laser as an effective and safe treatment modality in
nonsurgical periodontics, including repeated inter-
ventions of residual pockets after initial periodontal
therapy and in the maintenance stage. Improved
69
treatment outcomes must be demonstrated with the
adjunctive use of the Nd:YAG laser over those of con-
ventional mechanical treatment alone, as well as
suitable irradiation conditions and techniques.
Er:YAG laser
Characteristics
The Er:YAG laser was introduced in 1974 by Zharikov
et al. (219) as a solid-state laser that generates a light
with a wavelength of 2,940 nm. Of all lasers emitting in
the near- and mid-infrared spectral range, the
absorption of the Er:YAG laser in water is the greatest
because its 2,940 nm wavelength coincides with the
large absorption band for water. The absorption
coefcient of water of the Er:YAG laser is theoretically
10,000 and 15,00020,000 times higher than that of the
CO
2
andthe Nd:YAGlasers, respectively (68, 155). Also,
as part of the apatite component, OH groups show a
relatively high absorption at 2,940 nm, although the
maximum absorption is around 2,800 nm (44). Since
the Er:YAG laser is well absorbed by all biological tis-
sues that contain water molecules, this laser is indi-
cated not only for the treatment of soft tissues but also
for ablation of hard tissues (Fig. 6 and 7).
Table 5. Nd:YAG laser Clinical studies on periodontal pocket treatment
Cobb et al. 1992 (29) 1.753 W
87.5150 mJ/pulse, 20 Hz
Ineffective removal of calculus with thermal damage to
the root surface, and decreased number of bacteria
in periodontal pockets after laser treatment (Case series,
8 patients, 18 teeth)
Horton & Lin 1992 (75) 2 W
100 mJ/pulse, 20 Hz*
2 min
Laser was equally or more effective than SRP
in reducing or inhibiting recolonization of specic
bacterial species for pocket treatment (CT, 15 patients,
45 segments).
Gold & Vilardi 1994 (64) 1.25 and 1.75 W
62.5 and 87.5 mJ/pulse,
20 Hz
Laser removed pocket lining epithelium in periodontal
pockets, without causing necrosis or carbonization
of the underlying connective tissue (Case series,
6 patients, 24 specimens).
Ben Hatit
et al. 1996
(22) 0.81.5 W
100 mJ/pulse, 815Hz
SRP plus laser treatment showed more reduction
of periodontopathic bacterial levels compared to SRP
alone, but root surface alterations were observed after
laser treatment (RCT, 14 patients, 150 sites).
Radvar et al. 1996 (145) 0.5 or 0.8 W
50 or 80 mJ/pulse, 10 Hz
No thermal damage of root surface, but no clinical
or microbiological improvement after laser treatment
compared to SRP (RCT, 11 patients, 80 sites)
Neil & Mellonig
1997
(128) 2 W
80 mJ/pulse, 25 Hz
Similar reduction of probing depth was observed after
SRP plus laser therapy and SRP alone, but SRP plus laser
therapy showed signicantly higher improvements in
gingival index and gingival bleeding index (RCT in
a split-mouth design, 10 patients, 186 teeth).
Liu et al. 1999 (109) 3 W
150 mJ/pulse, 20 Hz
Laser was less effective in reduction of crevicular IL-1b
than traditional SRP (RCT in a split-mouth design,
8 patients, 52 sites).
Miyazaki
et al. 2003
(119) 2 W
100 mJ/pulse, 20 Hz
Improvements regarding clinical parameters and
subgingival microora after Nd:YAG, CO
2
and
Ultrasonic treatments (RCT, 18 patients, 41 sites).
Decrease of mean PD (NS between the 3 groups)
Nd:YAG: from 6.50 1.09 mm to 5.07 0.83 mm
(n 14)
CO
2
: from 6.92 1.50 mm to 5.92 1.93 mm (n 13)
Ultrasonic: from 6.86 2.63 mm to 5.50 2.06 mm
(n 14)
*Calculated from data presented in paper. SRP: scaling and root planing. CT: controlled trial. RCT: randomized controlled trial. PD: probing depth. NS: not
signicant.
70
Aoki et al.
Nagasawa (126), one of the pioneers in laser den-
tistry, demonstrated an approximately 3,000 nm
absorption peak of enamel and dentin in the infrared
spectral region. The absorption peak that Nagasawa
found for dried dental hard substances was probably
mainly due to absorption by intrinsic water in apatite
crystals, and minimally due to OH groups of the
mineral apatite (148). Kumazaki (107) speculated
that, in the case of dental hard tissue, the Er:YAG
laser is selectively absorbed by solid water and the
hydration shell of hydroxyapatite, causing a collapse
of apatite bindings.
In dentistry, the free-running pulsed Er:YAG laser
has already been used clinically for caries removal
and cavity preparation (34, 89, 92, 107, 115) and soft
tissue treatment (14, 203). The FDA approved the
pulsed Er:YAG laser for hard tissue treatment such as
caries removal and cavity preparation in 1997 (193),
unchanged for soft tissue surgery and sulcular
debridement in 1999 and for osseous surgery in 2004.
The high absorption of the Er:YAG laser into water
minimizes thermal inuences on the surrounding
tissues during irradiation. When the Er:YAG laser
was used for an incision of pigskin in a noncontact
mode, it showed formation of a thermally changed
layer of only 1050 lm (202). In the case of hard
tissue procedures, some degree of heat generation is
inevitable with the Er:YAG laser, since the Er:YAG
laser emits in the infrared region and hard tissues
have very low water content. However, the use of
water coolant minimizes heat generation by cooling
the irradiated area and absorbing excessive laser
energy (26, 133, 201). In addition, a water spray
facilitates hard tissue ablation by keeping the target
moist (26). Er:YAG laser irradiation using water
irrigation has been reported to produce an altered
layer 515 lm in width on cementum and dentin
surfaces (11, 12, 61).
The recently introduced Erbium,Chromium-doped:
Yttrium-Scandium-Gallium-Garnet (Er,Cr:YSGG) laser
with 2,780 nm wavelength and the Erbium-doped:
Yttrium-Scandium-Gallium-Garnet (Er:YSGG) laser
with 2,790 nm wavelength, which are more highly
absorbed by OH ions than water molecules (44), are
expected to have a performance similar to that of
the Er:YAG laser.
Mechanism of tissue ablation with the Er:YAG laser
A mechanism of biological tissue ablation with the
Er:YAG laser has been proposed, based on the
optical properties of its emission wavelength and
morphologic features of the surface ablated by Er:
YAG laser. During Er:YAG laser irradiation, the laser
3.5
7.1
10.6
17.7
28.2
42.4
Fig. 6. Photograph of the root surface after single pulse
irradiation of the Er:YAG laser at various energy levels.
Single pulse Er:YAG laser irradiation was performed
under water irrigation in a contact mode using a con-
ventional cylindrical tip with a 600 lm diameter on the
intact root surface at an energy density of 3.5, 7.1, 10.6,
17.7, 28.2, and 42.4 J/cm
2
per pulse (energy output of 10,
20, 30, 50, 80, and 120 mJ/pulse, respectively) starting
from the upper left in the gure. The ablation defect
became deeper according to the increase in energy out-
put. The ablation defect shows chalky appearance with-
out carbonization.
after single pulse Er:YAG laser irradiation. The irradiation
was performed in a contact mode at an energy density of
42.4 J/cm
2
(energy output of 120 mJ/pulse) under water
irrigation. The lased site shows crater-like ablation defect
with a well dened edge and microstructured bottom,
and without major crackings and thermal alterations.
Original magnication: 100. Bar = 100 lm.
71
energy is absorbed selectively by water molecules
and hydrous organic components of biological
tissues, causing evaporation of water and organic
components and resulting in thermal effects due to
the heat generated by this process (photothermal
evaporation). Moreover, in hard tissue procedures,
the water vapor production induces an increase of
internal pressure within the tissue, resulting in
explosive expansion called microexplosion (71, 87).
These dynamic effects cause mechanical tissue
collapse, resulting in a thermomechanical or
photomechanical ablation (97). This phenomenon
has also been referred to as water-mediated explo-
sive ablation (59, 180) (Fig. 8).
Unlike CO
2
laser (10,600 nm) irradiation, the Er:
YAG laser is capable of ablating hard tissues. Koort
et al. (97) reported that its effectiveness depends on
the balance of absorption into water and hydroxy-
apatite ( PO
4
). Fried et al. (58, 59) suggested that the
difference between the Er:YAG laser and CO
2
laser
Before irradiation
Er:YAG laser irradiation
After irradiation
Deep layer Intermediate layer Superficial layer
Fig. 8. Schematic view of hard tissue ablation by Er:YAG
laser irradiation. During Er:YAG laser irradiation, the la-
ser energy is absorbed selectively by water molecules and
hydrous organic components of the biological hard tis-
sues and causes evaporation of water (photothermal
evaporation). Further, in the case of hard tissue, the va-
por produces an increase in the internal pressure leading
to micro-explosions and consequent mechanical tissue
collapse (thermomechanical or photomechanical abla-
tion). The Er:YAG laser irradiation produces a very thin
altered layer on the ablated surface, which consists of two
distinct sub-layers: a supercial, signicantly altered
layer and a deeper, less affected (intermediate) layer. In
the directly irradiated, supercial layer, micro-cracking,
disorganization and slight recrystallization of the original
apatites and reduction of surrounding organic matrix are
evident after microexplosion, while the intermediate
layer, which receives less energy, suffers mainly from the
effects of energy accumulation, such as heat and subse-
quent microexplosion. The deep layer under the inter-
mediate layer shows no change. W: water molecules. BA:
biological apatites. PM: protein matrix. (Schema from
Sasaki KM et al. Ultrastructural analysis of bone tissue
irradiated by Er:YAG laser. Lasers Surg Med 2002: 31: 322-
332; with permission. Lasers in Surgery and Medicine
copyright (2002) Wiley-Liss, Inc.).
72
Aoki et al.
(10,600 nm) in the mechanism of enamel ablation is
based primarily on the principal absorber in the hard
tissue. He reported that primary absorption in water
results in water-mediated ablation, and primary
absorption in the bulk of enamel rods results in
melting and vaporization. The absorption of the Er:
YAG laser by inorganic components (hydroxyapatite)
is much lower than that of the CO
2
laser (97). Thus, in
the hard tissue ablation with the Er:YAG laser, the
absorption into water and hydrous organic compo-
nents rapidly occurs before heat accumulation
caused by absorption into inorganic components
takes place, resulting in thermo-mechanical, explo-
sive ablation. The excellent ablation effect of the
Er:YAG laser of both soft and hard tissues has
received a lot of attention in the eld of periodontal
therapy, and has been extensively researched.
Basic studies
Removal of subgingival calculus: Dental calculus
contains water in its structural micropores as well as
in its intrinsic components. Since the Er:YAG laser
has the ability to ablate dental hard tissues such as
enamel and dentin, the laser was expected to be
capable of removing dental calculus at much lower
energy levels. Several researchers have already
reported the promising ability of the Er:YAG laser to
remove subgingival calculus in vitro (10, 12, 51, 55,
90, 91, 172, 191) (Table 6, Fig. 10, 11 and 12).
In 1994, Aoki et al. (10) rst documented the ability
of the Er:YAG laser to remove subgingival calculus
in vitro. They showed that the pulsed Er:YAG laser
used with water irrigation was capable of removing
subgingival calculus from the root surface effectively
at 30 mJ/pulse (energy density of single pulse at the
tip: 10.6 J/cm
2
per pulse) and 10 Hz, in the contact
mode, directed perpendicular to the root surface
using a conventional cylindrical contact tip 600 lm
in diameter. In addition, ablation of the tooth sub-
stance following laser scaling was generally observed
within cementum, with a slight rise in temperature of
pulpal side during scaling. Their study suggested the
potential for clinical application of the Er:YAG laser in
subgingival scaling. Following the study, in 1995
Keller & Hibst (90) recommended that in contact
irradiation perpendicular to the root surface under
water irrigation, an energy level of 50 mJ/pulse (tip
diameter 600 lm, energy density 18 J/cm
2
per pulse)
should be used for effective calculus removal to avoid
damage to the cementum.
Soon after, Stock et al. (191) introduced a newly
developed contact tip (chisel type) suitable for root
surface treatment within periodontal pockets. They
performed Er:YAG laser scaling at 120 mJ/pulse (8 J/
cm
2
per pulse) and 15 Hz with water spray and with
the tip inclined at an angle of 20
to the root surface.

They reported that only smooth ablation traces were
visible in the cementum after Er:YAG laser scaling.
Keller & Hibst (91) tried Er:YAG laser scaling at 120
and 150 mJ/pulse (calculated energy density 15.0 and
18.8 J/cm
2
per pulse) and 10 and 15 Hz under water
irrigation using the rotatable ber tip with a chisel
shaped prole (dimensions of the rectangular end:
0.5 1.6 mm), at 20 or 40
to the root surface

(Fig. 9c). Calculus was effectively removed from the
root surface without thermal alteration of the surface.
Similarly, Folwaczny et al. (51) and Frentzen et al.
(55) reported that the Er:YAG laser scaling with water
spray using a chisel tip resulted in complete or
adequate calculus removal without thermal change
of the root surface.
Aoki et al. (12) evaluated the effectiveness of Er:
YAGlaser scaling comparedtoconventional ultrasonic
scaling. They performed laser scaling at 40 mJ/pulse
(14.2 J/cm
2
per pulse) and 10 Hz with water spray,
using a conventional tip at 30
to the root surface in a

sweeping motion. The level of calculus removal
achieved by laser scaling was similar to that by ultra-
sonic scaling, although the laser scaling was slightly
less efcient. Schwarz et al. (173) comparedthe degree
of calculus removal with in vivo Er:YAG laser irradi-
ation at 160 mJ/pulse (energy output 120 mJ/pulse
and chisel tip size 1.65 0.5 mm; calculated energy
density 14.5 J/cm
2
per pulse) and 10 Hz with removal
after water spray or scaling and root planing with hand
instruments. The study used roots of teeth that had
been planned for extraction due to severe periodontal
destruction. The laser treatment was performed in a
coronal to apical direction in parallel paths, with the
ber tipinclined1520
tothe root surface. The Er:YAG

laser treatment providedselective subgingival calculus
removal to a level equivalent to that provided by sca-
ling and root planing.
Root substance removal during laser scaling: In a
preliminary in vitro study, Aoki et al. (10) reported
that the average depth of cementum ablation was
approximately 40136 lm following Er:YAG laser
scaling in a straight line at 20120 mJ/pulse (7.1
42.4 J/cm
2
per pulse) and 10 Hz in perpendicular
contact irradiation using a conventional tip. They
later demonstrated that the depth of cementum
ablation was generally 1530 lm and occasionally
reached 80 lm in localized areas exposing dentin
surface, after in vitro Er:YAG laser scaling at 40 mJ/
pulse (14.2 J/cm
2
per pulse) and 10 Hz in oblique
contact irradiation at 30
in a sweeping motion (12).

73
Stock et al. (191) reported that the maximum depth
of ablation traces was approximately 100 lm after
Er:YAG laser scaling at 120 mJ/pulse (8 J/cm
2
per
pulse) and 15 Hz with water spray at 20
inclination
of the chisel tip to the root surface. They also repor-
ted that the threshold for both calculus and cemen-
tum ablation was 0.8 J/cm
2
. Folwaczny et al. (51)
examined root substance removal while irradiating
on root surfaces with or without calculus with the
Er:YAG laser at 60150 mJ/pulse (calculated energy
density: 7.318.2 J/cm
2
per pulse) and 15 Hz with
water spray using a chisel type contact tip (tip end
1.65 0.5 mm) in oblique contact irradiation at 30
.
They observed that the root substance removal with
the Er:YAG laser irradiation on the teeth without
calculus was approximately 38, 70, 143, and 484 lm,
respectively at 60 (7.3), 80 (9.7), 100 (12.2) and
150 mJ/pulse (18.2 J/cm
2
per pulse). They concluded
that the root substance removal with the Er:YAG
laser at lower energy densities up to 100 mJ/pulse
(12.2 J/cm
2
per pulse) was comparable to that after
conventional root surface instrumentation with cur-
ettes, and that selective calculus removal may be
feasible using lower radiation energies.
However, Frentzen et al. (55) reported that,
although Er:YAG laser scaling achieved complete
debridement clinically, laser scaling at a panel setting
of 160 mJ/pulse (output energy 100 or 120 mJ/pulse
and calculated energy density 18.8 or 14.5 J/cm
2
per
pulse in the use of chisel tip 1.1 0.5 mm or
1.65 0.5 mm, respectively) and 10 Hz with water
spray resulted in an increased loss of cementum and
dentin in vitro compared to mechanical scaling. They
considered that this loss should be taken into
Table 6. Er:YAG laser ) Basic studies on removal of subgingival calculus
Aoki et al. 1994 (10) 10120 mJ/pulse
(3.542.4 J/cm
2
per pulse)
10 Hz, water irrigation
90
contact (round-end tip)

Effective subgingival calculus removal at 10.6 J/cm
2
per pulse with little temperature increase
and minimum cementum ablation
Keller & Hibst 1995 (90) 50 mJ/pulse
(18 J/cm
2
per pulse),
1.5 or 3 Hz, water irrigation
90

Effective calculus removal at 18 J/cm
2
per pulse
without greater damage to the cementum
Stock et al. 1996 (191) 120 mJ/pulse
(8 J/cm
2
per pulse)
15 Hz, water spray
20
contact (chisel tip)

Smooth ablation traces on the root surface after
scaling with a new chisel-type contact tip
Keller & Hibst 1997 (91) 120 or 150 mJ/pulse
(15.0 or 18.8 J/cm
2
per pulse*)
10 or 15 Hz, water spray
20 or 40

Effective calculus removal with a chisel-type
tip without thermal alteration of the root surface
(14.2 J/cm
2
per pulse)
10 Hz, water spray
30

Calculus removal ability comparable to ultrasonic
scaling
Folwaczny et al. 2000 (51) 60150 mJ/pulse
(7.318.2 J/cm
2
per pulse*)
15 Hz, water spray
30

Complete calculus removal without thermal change
of root surface
Frentzen et al. 2002 (55) 100 or 120 mJ/pulse
(18.8 or 14.5 J/cm
2
per pulse*)
10 Hz, water spray
20

Clinically adequate debridement without
carbonization or other side-effects of clinical
relevance.
Schwarz et al. 2003 (173) 120 mJ/pulse
(14.5 J/cm
2
*)
10 Hz, water spray
1520

In vivo selective subgingival calculus removal to a
level equivalent to that provided by scaling and
root planing
*Calculated from data presented in paper and/or data obtained during personal communication with author.
74
Aoki et al.
account in the clinical situation. The crater depth of
the treated root surface was approximately 40 and
80 lm with the use of tip 1.65 and 1.10, respectively.
Regarding their results, Ishikawa (77) pointed out
that Frentzen et al. employed a relatively high energy
output for Er:YAG laser scaling, and commented that
although the efciency of laser scaling can be easily
improved by using a higher output power, caution
should be used when deciding on the power output,
considering a balance of effectiveness and unneces-
sary tissue removal. Improvement of the effectiveness
of laser scaling should rely on other variables, such as
pulse repetition rate and pulse duration, rather than
only on an increase of energy output.
Thus, the Er:YAG laser does not accomplish select-
ive ablation of dental calculus in vitro, as the tissue
underlying dental calculus is also removed during
laser scaling (Table 7, Figs 11 and 12). For safe and
effective clinical use, a combination of a higher pulse
repetition rate and lower energy output is recom-
mended in order to increase the efciency of calculus
ablation and simultaneously decrease the amount of
cementum loss. With such irradiation conditions, the
efciency is improved without increasing the
uncomfortable vibration stress experienced by
patients. At the same time, selective calculus removal
using the Er:YAGlaser may be more feasible (12). Also,
Folwaczny et al. (53) reported that the angulation of
the application tip to the root surface has a strong
inuence on the amount of root substance removed
during Er:YAG laser irradiation. The angulation of the
application tip is another important factor for
decreasing root substance removal.
Recently, Schwarz et al. (172) reported interesting
ndings. First, they performed in vivo Er:YAG laser
scaling on periodontally diseased roots of teeth con-
sidered for extraction due to severe periodontal
destruction, at panel settings of 120180 mJ/pulse
(energy output 71106 mJ/pulse, calculated energy
density 8.612.8 J/cm
2
per pulse) and 10 Hz using a
Fig. 9. Contact tips used for Er:YAG laser irradiation. a)
Conventional cylindrical contact tip, which has a curva-
ture of 80, a round end with a diameter of 600 lm and
approximately 65% laser transmission (80 Tip 600
Micron, Er:YAG laser DELight
TM
; HOYA ConBio, CA, USA,
and Er:YAG laser Dentlight
TM
; HOYA Photonics Corp.,
Tokyo, Japan). b) Conventional cylindrical contact tip
with a tapered end which enables lateral irradiation,
used for soft tissue incision and periodontal pocket
curettage (S600T, Er:YAG laser Erwin AdvErL
TM
; Morita
Mfg. Corp., Kyoto, Japan). c) Chisel-type tip which has a
rectangular end with dimensions 1.65 0.5 mm, and is
generally used with a contra-angle handpiece for pocket
treatment (Truncated-core end 1.65 0.5 mm and per-
iodontal handpiece, KEY Laser
TM
; KaVo, Biberach,
Germany).
75
chisel type tip under water coolant. This was followed
by in vitro Er:YAG laser scaling to different surfaces of
the same roots after extraction. The Er:YAG laser-
treated root surfaces after in vitro and in vivo laser
scaling were then compared. Clinical use of the Er:
YAG laser resulted in smooth root surface morphol-
ogy, even at higher energy settings such as 180 mJ/
pulse (energy output 106 mJ/pulse, calculated energy
density 12.8 J/cm
2
per pulse) and 10 Hz, which was
not comparable to the marked morphologic changes
that were produced in vitro, and they suggested
calculus removal can be done selectively in vivo,
contrary to the situation in vitro.
Supragingival laser scaling on enamel surface using
the Er:YAG laser is contraindicated, since complete
calculus removal without affecting the underlying
enamel is difcult during Er:YAG laser scaling.
However, in subgingival scaling, not only the removal
of calculus but also removal of contaminated
cementum may be clinically acceptable to some
extent. To avoid excessive removal of sound root
substance during Er:YAG laser subgingival scaling,
further in vitro and in vivo studies are required to
determine a suitable combination of laser irradiation
parameters, such as energy output and pulse rate in
conjunction with the irradiation manner and type of
contact tip used.
Root surface alteration following Er:YAG laser irra-
diation: The Er:YAG laser does not cause carboniza-
tion of the irradiated root surface, but it has been
demonstrated that the ablated surface becomes
chalky after drying due to micro-irregularities on the
lased surface (10, 12, 61, 80) (Fig. 6 and 7). The sur-
face of Er:YAG laser-treated calculus under water
coolant has been reported to exhibit a micro-irregular
appearance without melting and carbonization, likely
due to the effects of the mechanical ablation (12).
Israel et al. (80) reported that the root surface trea-
ted with the Er:YAG laser at 2.515 mJ/pulse and
20 Hz with water spray in the noncontact irradiation
Fig. 10. Photographs of the root surfaces before and after
Er:YAG laser scaling of subgingival calculus. Er:YAG laser
scaling was performed in vitro at an energy density of
10.6 J/cm
2
per pulse (energy output of 60 mJ/pulse) and
30 Hz under water spray. The laser was applied using a
chisel-type tip (1.4 0.45 mm) in contact mode, main-
taining the tip oblique to the root surface at an angle of
approximately 20, and moving the tip in a back-and-
forth motion. The time required for laser scaling was 112
s. Macroscopically, the laser-scaled area appears smooth
and slightly chalky, but without any major thermal
changes such as carbonization. a) Before laser scaling. b)
After laser scaling. Part of the subgingival calculus was
intentionally left on the edge of the treated root surface.
Original magnication: 25.
76
Aoki et al.
Table 7. Er:YAG laser ) Basic studies on root substance removal and root surface alteration
(3.542.4 J/cm
2
per pulse)
90

Chalky and micro-irregular appearance without melting
and carbonization after laser scaling, and cementum
ablation with a depth of 40136 lm on average, fol-
lowing laser scaling in perpendicular contact irradiation
in a straight line
Benthin et al.
1995
(23) Er:YSGG 7.560 J/cm
2
5 Hz,
With and without water
spray Noncontact
Reduction of in vitro broblast attachment after Er:
YSGG laser irradiation on an intact root surface
compared to attachment on a mechanically prepared
surface
Stock et al. 1996 (191) 120 mJ/pulse
(8 J/cm
2
per pulse)
15 Hz, water spray
20

A maximum root ablation of 100 lm after laser scaling
Israel et al. 1997 (80) 2.515 mJ/pulse
20 Hz, water spray
Noncontact
An etched appearance with exposure of numerous tufts
and/or ber bundles of mineralized collagen as well as
sharply dened microfractures of mineralized structure
after irradiation
Fujii et al. 1998 (61) 75 mJ/pulse
(26.5 J/cm
2
per pulse*)
1 pulse, water spray
90

Microstructured root surface with denaturation of col-
lagen bers up to a depth of 15 lm
(14.2 J/cm
2
per pulse)
10 Hz, water spray
30

Micro-irregular appearance and minimal change with
characteristic staining of the lased root surface and 15
30 lm of cementum ablation
Folwaczny
et al. 2000
(51) 60150 mJ/pulse
(7.318.2 J/cm
2
per pulse*)
15 Hz, water spray
30

Root substance removal comparable to conventional
instrumentation and selective calculus ablation at lower
energy level up to 100 mJ/pulse (12.2 J/cm
2
per pulse)
Folwaczny
et al. 2001
(53) 60180 mJ/pulse
(7.321.8 J/cm
2
per pulse*)
10 Hz, water spray
1590

Angulation of application tip to the root surface has a
strong inuence on the amount of root substance re-
moved during laser irradiation.
Gaspirc &
Skaleric 2001
(63) 60100 mJ/pulse
(11.919.9 J/cm
2
per pulse*)
10 Hz, no water
Noncontact
Inuence on the morphology and diffusion process of
root surfaces and no changes in the chemical structure
of the root surface
Schwarz et al.
2001
(172) 71106 mJ/pulse*
(8.612.8 J/cm
2
per pulse*)
10 Hz, water spray
20

Smooth root surface morphology after laser scaling
in vivo not comparable to the marked morphologic
changes in vitro
Frentzen et al.
2002
(55) 100 or 120 mJ/pulse
(18.8 or 14.5 J/cm
2
per pulse*)
10 Hz, water spray
20

Increased loss of cementum and dentin compared to
mechanical debridement
Sasaki et al. 2002 (164) 40 mJ/pulse
(14.2 J/cm
2
per pulse*)
10 Hz, water spray
30

Slight melting with cluster formation of enlarged
microparticles in cementum and dentin
Sasaki et al. 2002 (167) 40 mJ/pulse
(14.2 J/cm
2
per pulse*)
10 Hz, water spray
30

No major compositional or chemically deleterious
changes on the root surface except for reduction of
organic components
77
exhibited an etched appearance with exposure of
numerous tufts and/or ber bundles of mineralized
collagen as well as sharply dened microfractures of
mineralized structure. Fujii et al. (61) showed a
microstructured root surface with denaturation
of collagen bers up to a depth of 15 lmin cementum,
following single-pulse, perpendicular contact Er:YAG
laser irradiation at 75 mJ/pulse (calculated energy
density: 26.5 J/cm
2
per pulse) under water spray.
Aoki et al. (12) reported that numerous rounded
or sharp pointed projections were evident on the
root surface after Er:YAG laser scaling with water
spray, and that the supercial layer of the root sur-
face ablated by Er:YAG laser irradiation presented
minimal change with characteristic staining, and
was subdivided histologically into two distinct lay-
ers: a supercial, signicantly altered layer and an
underlying, less affected (intermediate), layer. The
supercial layer showed a fragile structure with
micro-irregularities and degradation. They specula-
ted that the deeply stained supercial layer is
approaching ablation and is highly damaged,
exhibiting both microstructural degradation and
thermal denaturation, whereas the underlying sub-
surface layer is affected only by thermal denatura-
tion, and is not degraded structurally.
Using FTIR spectroscopy analysis, Sasaki et al.
(167) observed that the Er:YAG laser at 40 mJ/pulse
(14.2 J/cm
2
per pulse) and 10 Hz used with water
coolant did not cause major compositional changes
or chemically deleterious changes in the root
cementum and dentin. However, OH and amide
bands related to organic components were signi-
cantly reduced compared to carbonate and phos-
phate bands related to inorganic components. Laser
irradiation without water coolant produced slightly
cyan-derived toxic substances. Gaspirc & Skaleric
(63) reported that the Er:YAG laser at 60100 mJ/
pulse (calculated energy density 11.919.9 J/cm
2
per
pulse) and 10 Hz inuenced only the morphology
and diffusion process of root surfaces but did not
change the chemical structure of the root surface.
Sasaki et al. (164) also reported that the surface lased
by Er:YAG at 40 mJ/pulse (14.2 J/cm
2
per pulse) and
10 Hz under water irrigation showed slight melting
with cluster formation of enlarged microparticles of
inorganic components in scanning electron micro-
scope observation at ultra-high magnication. The
use of water spray during Er:YAG laser irradiation
resulted in a cleaner and less porous surface. Thus,
Er:YAG laser irradiation produces a characteristic
microstructural change as well as minimal thermal
damage on the treated root surface (Table 7, Fig. 11
and 12). On the other hand, Ishizaka et al. reported
that a changed layer of maximum thickness 600 lm
was detected in the dentin after cavity preparation
with the Er:YAG laser at 80140 mJ/pulse and 4 Hz
using a ne water mist, as observed using polarizing
and light microscopy (79). Folwaczny et al. (48) per-
formed in situ Er:YAG laser irradiation in periodontal
pockets of human corpses, at 60100 mJ/pulse (cal-
culated energy density 7.312.1 J/cm
2
per pulse) and
10 Hz (50 or 100 pulses) with water spray using a
chisel type tip (1.65 0.5 mm) and reported that the
Table 7. Continued
Schoop et al.
2002
(169) 100 mJ/pulse
(5.98 J/cm
2
)
15 Hz, water spray
3045

Better conditions for broblast attachment after laser
debridement
Feist et al. 2003 (46) 35 or 59 mJ/pulse
(4.2 or 7.2 J/cm
2
per pulse*)
10 Hz, water spray
45

Faster adhesion and growth of broblasts on the sur-
faces treated with 35 mJ/pulse irradiation than those
treated with either root planing or 59 mJ/pulse irradi-
ation
Folwaczny
et al. 2003
(48) 60100 mJ/pulse
(7.312.1 J/cm
2
per pulse*)
10 Hz, water spray
30

A deep semicircular area of thermal change with a
depth of 255611 lm in the root dentin close to the
bottom of the pocket after laser irradiation in situ
Schwarz et al.
2003
(171) 120 mJ/pulse
(14.5 J/cm
2
per pulse*)
10 Hz, water spray
1520

Signicantly greater cell attachment in vitro on the
in vivo laser treated root than on the in vivo SRP treated
root
*Calculated from data presented in paper and/or data obtained during personal communication with author. SRP: scaling and root planing.
78
Aoki et al.
root dentin close to the bottom of the pocket showed
a deep semicircular area of thermal change with a
depth of 255611 lm after Er:YAG laser irradiation.
Considering the wide variance described above in the
potential thermal effects of the Er:YAG laser, further
detailed investigations are required to evaluate the
extent of thermal alteration of lased root surface
in vivo.
The microstructurally and thermally changed root
surface produced by Er:YAG laser irradiation may
inuence the attachment of soft periodontal tissues.
Benthin et al. (23) observed a reduction of in vitro
broblast attachment after Er:YSGG laser irradiation
on an intact root surface compared to attachment on
a mechanically prepared surface, although the root
surface irradiated with water cooling exhibited better
broblast attachment than that treated without water
cooling. Similarly, using the intact roots, we observed
decreased in vitro periodontal ligament cell attach-
ment on the root surface treated by Er:YAG laser
under water irrigation, compared to attachment on a
mechanically prepared surface (unpublished data).
Thus, the Er:YAG laser irradiation on intact root
surfaces may result in decreased cell attachment on
the treated surface, compared to mechanical treat-
ment.
Studies that employed periodontally diseased root
surfaces showed better results. Schoop et al. (169)
reported that the surface structure of periodontally
diseased root after Er:YAG laser irradiation at 100 mJ/
pulse (energy density 5.98 J/cm
2
) and 15 Hz with
water spray offered better conditions for the adher-
ence of broblasts in vitro than a root surface after
mechanical scaling only. Schwarz et al. (171) per-
formed in vivo Er:YAG laser irradiation at the panel
setting of 160 mJ/pulse (energy output 120 mJ/pulse
and chisel tip size 1.65 0.5 mm; calculated energy
density 14.5 J/cm
2
per pulse) and 10 Hz with water
spray or scaling and root planing with hand instru-
ments on periodontally diseased roots of teeth con-
sidered for extraction due to severe periodontal
destruction, and cultured broblasts on the treated
teeth following extraction. They observed signi-
cantly greater cell attachment in vitro in the laser
treatment group than in the SRP treatment group.
Feist et al. (46) performed in vitro Er:YAG laser irra-
diation at the panel setting of 60 or 100 mJ/pulse
(energy output 35 or 59 mJ/pulse and chisel tip size
1.65 0.5 mm; calculated energy density 4.2 or 7.2 J/
cm
2
per pulse) and 10 Hz with water spray or scaling
SC
C
CDJ
D
Fig. 11. Photomicrograph of a histologic section of the
root after Er:YAG laser scaling. Root surface was scaled
in vitro using the Er:YAG laser at 14.2 J/cm
2
per pulse (40
mJ/pulse ) and10 Hz under water spray witha conventional
cylindrical tip contacted obliquely to the root surface at an
angle of 30. The treated surface generally shows micro-
irregularity due to the ablationof the supercial part of the
cementum layer. Minimal change with deep staining and
microirregularities are noted on the lased surface. SC:
subgingival calculus that was intentionally left on the edge
of the treated root surface, and the border of the scaled
area clearly indicated. C: cementum. CDJ: cementodenti-
nal junction. D: dentin. Bar = 50 lm. (Photograph from
Aoki A et al. In vitro evaluation of Er:YAG laser scaling of
subgingival calculus in comparison with ultrasonic scaling.
J Periodontal Res 2000: 35: 5: 266-277; with permission.
Journal of Periodontal Research copyright (2000)
Blackwell Munksgaard, Inc.)
scaled by Er:YAG laser, at high magnication. Er:YAG
laser scaling was performed in vitro at 14.2 J/cm
2
per
pulse (40 mJ/pulse) and 10 Hz with water spray using a
conventional cylindrical tip contacted obliquely to the
root surface at an angle of 30 in a sweeping motion. The
laser-treated surface shows a characteristic microstruc-
tured appearance. Numerous rounded projections are
evident on the lased root surface. Original magnication:
1,000. Bar = 10 lm. (Photograph from Aoki A et al. In
vitro evaluation of Er:YAG laser scaling of subgingival
calculus in comparison with ultrasonic scaling. J Perio-
dontal Res 2000: 35: 5: 266-277; with permission. Journal
of Periodontal Research copyright (2000) Blackwell
Munksgaard, Inc.)
79
and root planing with curettes on periodontally dis-
eased roots of teeth. They reported that the surfaces
treated with 35 mJ/pulse (4.2 J/cm
2
per pulse)
Er:YAG laser irradiation promoted faster adhesion
and growth than those treated with either root pla-
ning or 59 mJ/pulse (7.2 J/cm
2
per pulse) Er:YAG
laser irradiation. These better results may be due to
the disinfection and detoxication effects of Er:YAG
laser irradiation on the diseased root surface and
absence of a smear layer on the treated root surface
after irradiation, which are described later in this
chapter.
Periodontal connective tissue attachment to the
Er:YAG-lased root surface has not been fully studied
yet. Further in vitro and animal experiments are
required to clarify the biocompatibility of the root
surface prepared by Er:YAG laser. In addition, whe-
ther supplementary treatment, using chemical or
mechanical methods, is required to remove the
supercially altered layer of the lased root surface
needs to be investigated histologically.
Thermal generation during Er:YAG laser treatment
and its inuence on the pulp tissue: When applying
lasers for hard tissue ablation, thermal side-effects
have been a major problem. Heat generation during
laser irradiation often caused inammation and
necrosis of the pulp (6). Aoki et al. (10) demonstrated
that the use of water coolant effectively prevented
thermal generation during laser scaling without
compromising the efciency of laser scaling. They
examined the temperature rise on the pulpal wall of
proximal surfaces of mandibular incisors during Er:
YAG laser scaling for 20 s with and without water
coolant, at 30 mJ/pulse (10.6 J/cm
2
per pulse) and
10 Hz, in contact mode, directed perpendicular to the
root. They observed that the maximum temperature
rise without water coolant was 39

C in the root
surface and 18.4

C in the pulpal wall on average,
whereas that with water coolant was 2.4

C in the
root surface and 0.8

C in the pulpal wall (the mean
thickness of root: 1.38 mm, experiment at room
temperature). In another study, they observed the
temperature rise on the pulpal wall of proximal sur-
faces of mandibular incisors during Er:YAG laser
scaling with water coolant was 1.4

C in the pulpal
wall (the mean thickness of root: 1.30 mm, experi-
ment at room temperature) (12). Keller et al. (91)
reported that the maximum temperature increase of
the pulpal side was 4

K (4

C) at 10 Hz or 5.5

K
(5.5

C) at 15 Hz on average during Er:YAG laser
scaling at 120 and 150 mJ/pulse (calculated energy
density 15.0 or 18.8 J/cm
2
per pulse) under water
irrigation using a chisel type tip, with a tip inclination
of 20 or 40
to the root surface. Thus, temperature

elevation in the pulpal wall during Er: YAG laser
scaling used with water coolant would be limited
within the physiologically tolerable level of pulp
tissue (Table 8).
There are no histologic studies of pulp tissue
response after Er:YAG laser scaling of subgingival
calculus. However, regarding the pulpal response
after cavity preparation with the Er:YAG laser at high
energy levels, in vivo reports have indicated minimal
thermal effects of the Er:YAG laser on pulp tissue (88,
213). Comparative studies of pulpal reactions after
cavity preparation with the Er:YAG laser and those
with the conventional high-speed bur have shown
that pulpal responses induced by Er:YAG laser treat-
ment with water coolant were acceptable, and that no
histopathologic differences were observed between
Er:YAG laser treatment and high-speed bur cutting
(181, 186).
Thus, in animal studies, no major damage has been
reported to occur in the pulp tissue after cavity pre-
paration with Er:YAG laser using much higher energy
levels than those employed in laser scaling. We ob-
served no thermal effects on the pulp tissue following
Er:YAG laser scaling of supragingival calculus in an
experiment in dogs (unpublished data). Therefore, it
may be presumed that Er:YAG laser subgingival sca-
ling at a low energy level, especially with the contact
tip directed obliquely or parallel to the root surface,
does not produce any major deleterious outcomes in
the pulp tissue.
Disinfection and detoxication: The Er:YAG laser
may offer several antimicrobial advantages over
conventional mechanical scaling, due to its benecial
properties such as bactericidal effect (9, 50), degra-
dation and removal of bacterial endotoxins (192,
216), and ablation effects without producing a smear
layer (194) (Table 8).
Andoet al. (9) reportedthat the Er:YAGlaser exhibits
a high bactericidal potential against periodontopathic
bacteria such as P. gingivalis and Actinobacillus
actinomycetemcomitans at a low energy level of
0.3 J/cm
2
, and signicant reduction of viable bacteria
was observed in a P. gingivalis colony of diameter
0.8 mm after single pulse irradiation at above
7.1 J/cm
2
. Folwaczny et al. (50) reported that Er:YAG
laser irradiation at 60 mJ/pulse (10 J/cm
2
per pulse)
and 15 Hz causes reduction in bacteria on root surfa-
ces in vitro. However, they noted that complete elim-
inationof bacteria couldnot be achievedintheir study.
Yamaguchi et al. (216) showed in vitro that the
infrared spectrum of bacterial lipopolysaccharide had
a peak at 2,940 nm, which also corresponded to the
80
Aoki et al.
wavelength of the Er:YAG laser, and that the Er:YAG
laser at 100 mJ/pulse and 1 Hz (35.4 mJ/cm
2
) could
effectively and rapidly remove most of the lipopoly-
saccharide that had been coated on the extracted root
surfaces. Sugi et al. (192) reported that the amount of
endotoxin on the diseased root surface treated by
Er:YAG laser at 30 mJ/pulse (16.1 J/cm
2
per pulse)
and 10 Hz under water spray was signicantly less
than that on the control diseased root surface from
which calculus was removed by hand scaler, without
damaging the underlying cementum. Also, Sasaki
et al. (167) reported that root cementum and dentin
treated with the Er:YAG laser used with water coolant
was free of toxic substances such as cyanate (NCO
)
and cyanamide (NCN
2
) that were observed on sur-
faces irradiated by CO
2
and Nd:YAG lasers. Thus,
improved disinfection and detoxication may be
expected on the Er:YAG laser-treated root surface.
Clinical studies
Based on the results of in vitro studies, Watanabe
et al. (203) performed clinical application of Er:YAG
laser scaling in 1996. The laser scaling was carried out
under water coolant at a panel setting of approxi-
mately 40 mJ/pulse on average (calculated energy
output: 32 mJ/pulse, calculated energy density:
11.3 J/cm
2
per pulse), 10 Hz, using a straight contact
tip (600 lm diameter), targeting the supra- and sub-
gingival calculus on the root surface of 60 teeth in 60
patients. They reported that the Er:YAG laser could
remove calculus from root surfaces in 95% of cases.
Although scaled sites showed some irregularity, this
was not clinically signicant in 98% of cases, and
reduction of pocket depth was obtained. Only a few
patients complained about the unpleasant sound and
vibration. There were no complications or side-
effects during this clinical trial. Watanabe et al.
suggested that laser scaling was safe and effective,
and clinically useful.
Recently, Schwarz et al. (176) reported interesting
clinical data of nonsurgical periodontal treatment,
comparing Er:YAG laser irradiation with conventional
scaling and root planing in a randomized, controlled
clinical study using a split-mouth design in 20
patients. Periodontal pockets of 110 teeth having
subgingival calculus with moderate to advanced
periodontal destruction were treated under local
anesthesia with either the Er:YAG laser or scaling and
Table 8. Er:YAG laser ) Basic studies on temperature elevation, disinfection, and detoxication
(10.6 J/cm
2
per pulse)
90

Slight temperature rise of 0.8

C in the pulpal
wall during laser scaling under water coolant
Ando et al. 1996 (9) 0.32.6 J/cm
2
1 pulse, no water
Noncontact
High bactericidal potential with periodonto-
pathic bacteria at a low energy level
Keller & Hibst 1997 (91) 120 or 150 mJ/pulse
(15.0 or 18.8 J/cm
2
per pulse*)
10 or 15 Hz, water spray
2040

A temperature increase of 4 K (4

C) at 10 Hz
or 5.5 K (5.5

C) at 15 Hz on the pulpal side
Yamaguchi et al. 1997 (216) 100 mJ/pulse (35.4 mJ/cm
2
)
1 Hz, no water
Near contact
Effective and rapid removal of lipopolysac-
charide from root dentin surface
Sugi et al. 1998 (192) 30 mJ/pulse (16.1 J/cm
2
per pulse)
10 Hz, water spray
30

Reduction of the amount of endotoxin after
Er:YAG laser scaling on the diseased root sur-
face
Aoki et al. 2000 (12) 40 mJ/pulse (14.2 J/cm
2
per pulse)
10 Hz, water spray
30

Slight temperature rise of 1.4

C in the pulpal
wall during laser scaling under water coolant
Folwaczny et al. 2002 (50) 60 mJ/pulse (10 J/cm
2
per pulse)
15 Hz, no water
Noncontact
Reduction in bacteria on root surfaces in vitro
*Calculated from data presented in paper and/or data obtained during personal communication with author.
81
root planing using hand instruments. Er:YAG laser
treatment was performed using chisel type contact
tips (1.10 0.5 mm, or 1.65 0.5 mm) at the panel
setting of 160 mJ/pulse (energy output 100 and
120 mJ/pulse and energy density 18.8 and 14.5 J/cm
2
per pulse for size 1.10 and 1.65 tip, respectively) and
10 Hz with water coolant. The laser treatment was
performed in a coronal to apical direction in parallel
paths, with an inclination of the ber tip at 1520
to
the root surface. The laser treatment required less
time than the scaling and root planing treatment. At a
6-month post-treatment evaluation, the laser treat-
ment showed similar or better results than the scaling
and root planing treatment in terms of reduction of
bleeding on probing, pocket depth, and clinical
attachment level. In particular, the laser group pre-
sented a signicantly higher reduction of bleeding on
probing and improvement of clinical attachment
level compared to the scaling and root planing group.
Furthermore, the difference between laser and hand
instrumentation in treatment outcomes was much
more signicant in deeper pockets. The researchers
concluded that the Er:YAG laser may present a suit-
able alternative to conventional mechanical debri-
dement in nonsurgical periodontal treatment.
Schwarz et al. (174) also reported that the clinical
attachment gain obtained following Er:YAG laser
nonsurgical periodontal treatment was maintained
over a 2-year period.
Schwarz et al. (175) also investigated the neces-
sity of adjunctive scaling and root planing after
Er:YAG laser treatment. They performed a clinical
study similar to the above-mentioned study (176),
and found no additional improvement in clinical
outcomes for the laser treatment followed by sca-
ling and root planing compared to laser treatment
alone. Schwarz et al. (172) previously showed that
the clinical use of the Er:YAG laser resulted in a
smooth root surface morphology without marked
morphologic changes, and suggested that calculus
removal can be selectively done in vivo. Based on
these ndings, it appears that in vivo Er:YAG laser
treatment, as per their method, results in a root
surface favorable for tissue attachment, without
producing a typical microstructured root surface,
and therefore without requiring additional scaling
and root planing.
Most recently, Sculean et al. (179) compared the
effectiveness of an Er:YAG laser to that of ultrasonic
scaler for nonsurgical periodontal treatment. Twenty
patients with moderate to advanced periodontal
destruction were randomly treated in a split-mouth
design with a single episode of subgingival debride-
ment using either an Er:YAG laser device (energy
output 120 mJ/pulse; energy density 14.5 J/cm
2
per
pulse; 10 Hz) combined with a calculus detection
system with uorescence induced by 655 nm
InGaAsP diode laser radiation, or an ultrasonic
instrument. Six months following treatment, there
was a statistically signicant improvement in the
mean values of bleeding on probing, probing pocket
depth, and clinical attachment level in both groups.
However, no statistically or clinically signicant
differences were observed between the treatment
modalities.
Summary of clinical application of the Er:YAG laser
Research conducted so far has indicated the safety
and effectiveness of clinical application of the Er:YAG
laser for periodontal pocket treatment, including root
surface debridement. Er:YAG laser irradiation may be
a promising, useful adjunctive or alternative method
to the conventional technique for root preparation
and pocket curettage (Table 9, Fig. 13 and 14).
However, the effects of the Er:YAG laser need to be
demonstrated in further randomized controlled trials
and a subsequent meta-analysis.
With respect to healing after Er:YAG laser scaling,
no histologic studies have been reported. Although
uneventful clinical wound healing has been reported
after periodontal treatment using the Er:YAG laser,
minimal thermal changes have been reported after
Er:YAG laser irradiation on both hard and soft tissue.
Therefore, further studies are necessary to clarify the
histologic attachment of periodontal tissues to the
irradiated root surface in vivo.
The Er:YAG laser has some shortcomings when
used for subgingival scaling. For clinical application
in periodontal pockets where the operator cannot
visualize the irradiated target, special tips should be
designed to facilitate insertion into the periodontal
pocket and detection of the presence of dental cal-
culus on the surface (91, 205) (Fig. 9). Also, since
Er:YAG laser irradiation causes splashing of water and
blood from pockets as a result of explosive ablation,
adequate high speed evacuation by means of not only
an intraoral suction but also an extraoral evacuation
apparatus is required to prevent contamination by
blood and water splattering.
Recently, as a novel application of laser, the use of
diode laser uorescence spectroscopy for detection
of dental calculus has been suggested by Hibst et al.
(74). Keller et al. (93) reported a novel method of
subgingival root cleaning with the Er:YAG laser
combined with diode laser uorescence spectros-
copy, and this work has already resulted in the
82
Aoki et al.
Table 9. Er:YAG laser ) Clinical studies on periodontal pocket treatment
Author and
Year
Reference Laser parameters Findings
Watanabe
et al. 1996
(203) Approximately 32 mJ/pulse*
(panel setting: approximately
40 mJ/pulse on average)
(11.3 J/cm
2
per pulse*)
10 Hz, water spray
oblique contact
round-end tip
(600 lm in diameter)
Safe and effective calculus removal and
subsequent pocket depth reduction at 4 weeks
Case series, 60 patients, 60 sites of 60 teeth)
Removal of supragingival calculus (n 25):
Decrease of mean RD: from 2.9 1.3 mm
to 2.5 1.4 mm
Removal of subgingival calculus (n 35):
Decrease of mean PD: from 5.6 2.0 mm
to 2.6 0.9 mm
Schwarz
et al. 2001
(176) 100 or 120 mJ/pulse*
(panel setting:
160 mJ/pulse)
(18.8 or 14.5 J/cm
2
per pulse*)
10 Hz, water spray
1520
contact
chisel tip
(rectangular end: 1.10 0.5
or 1.65 0.5 mm)
Similar or better improvement of clinical
parameters at 6 months after pocket treatment
with the laser, compared to SRP treatment
(RCT in a split-mouth design, 20 patients,
660 sites of 110 teeth in total)
Decrease of mean BOP(+) score (P < 0.05
between the 2 groups):
Laser group: from 56% to 13%
SRP group: from 52% to 23%
Decrease of mean PD (NS between the 2 groups):
Laser group: from 4.9 0.7 mm to 2.9 0.6 mm
SRP group: from 5.0 0.6 mm to 3.4 0.7 mm
Decrease of mean CAL (P < 0.001 between the
2 groups):
Schwarz et al.
2003
(174) 100 or 120 mJ/pulse*
(panel setting:
160 mJ/pulse)
(18.8 or 14.5 J/cm
2
per pulse*)
10 Hz, water spray
1520
contact
chisel tip (rectangular end:
1.10 0.5 or 1.65 0.5 mm)
Maintained improved clinical parameters
of periodontal pockets after 2 years for both laser
and SRP treatments (RCT in a split-mouth design,
20 patients, 660 sites of 110 teeth in total)
Decrease of mean CAL (P < 0.001 between
baseline and 1 year/2 years, and NS between
1 and 2 years, for both groups):
at 1 year and to 4.9 0.4 mm at 2 years
at 1 year and to 5.8 0.4 mm at 2 years
Schwarz et al.
2003
(175) 100 or 120 mJ/pulse*
(panel setting:
160 mJ/pulse)
(18.8 or 14.5 J/cm
2
per pulse*)
10 Hz, water spray
1520
contact
chisel tip (rectangular end:
1.10 0.5 or 1.65 0.5 mm)
No additional improvement in the clinical
parameters at 12 months after pocket treatment
with the laser + SRP treatment, compared to laser
treatment alone (RCT in a split-mouth design,
20 patients, 600 sites of 100 teeth in total)
Decrease of mean PD (NS between the 2
groups):
Laser + SRP group: from 5.2 0.8 mm
to 3.2 0.8 mm
Decrease of mean CAL (NS between the
2 groups):
Laser + SRP group: from 6.9 1.0 mm
to 5.3 1.0 mm
*Calculated from data presented in paper and/or data obtained during personal communication with author. PD: probing depth. SRP: scaling and root
planing. RCT: randomized controlled trial. BOP: bleeding on probing. CAL: clinical attachment level. NS: not signicant.
83
development of a commercial device (179). Er:YAG
laser treatment combined with an automatic calcu-
lus-detecting system may be a novel technical
modality for pocket therapy in the near future.
In addition, with the Er:YAG laser, the results of
studies should include a description of the energy
density (uence) per pulse at the end of contact tip,
since the size and shape of the contact tip varies
among the laser apparatuses.
Diode lasers
Characteristics
The diode laser is a solid-state semiconductor laser
that typically uses a combination of Gallium (Ga),
Arsenide (Ar), and other elements such as Aluminum
(Al) and Indium (In) to change electrical energy into
light energy. The wavelength range is about 800
980 nm. The laser is emitted in continuous-wave and
gated-pulsed modes, and is usually operated in a
contact method using a exible ber optic delivery
system. Laser light at 800980 nm is poorly absorbed
in water, but highly absorbed in hemoglobin and
other pigments (7). Since the diode basically does not
interact with dental hard tissues, the laser is an
excellent soft tissue surgical laser (156), indicated for
cutting and coagulating gingiva and oral mucosa, and
for soft tissue curettage or sulcular debridement. The
FDA approved oral soft tissue surgery in 1995 and
sulcular debridement in 1998 by means of a diode
laser (GaAlAs 810 nm).
The diode laser exhibits thermal effects using the
hot-tip effect caused by heat accumulation at the
end of the ber, and produces a relatively thick
coagulation layer on the treated surface (7). The
usage is quite similar to electrocauterization. Tissue
penetration of a diode laser is less than that of the
Nd:YAG laser, while the rate of heat generation is
higher (147), resulting in deeper coagulation and
more charring on the surface compared to the
Nd:YAG laser. The width of the coagulation layer was
reported to be in excess of 1.0 mm in an incision of
bovine oral soft tissue in vitro (208). The advantages
of diode lasers are the smaller size of the units as well
as the lower nancial costs.
Basic studies
Concerning the effects on the root surface, Kreisler
et al. (106) examined the periodontal ligament cell
attachment to the 810 nm diode laser-treated root
surface. After scaling and root planing the peri-
odontally diseased root surface with curettes fol-
lowed by air-powder abrasive treatment, the laser
group received diode laser irradiation at 1 W in the
continuous wave mode for 20 s and the control
group was left unirradiated. There was no signi-
cant difference between the laser and the control
groups in cell attachment. This nding suggested
that the diode laser did not produce any deleterious
effect on the root surface. Thus, it is generally
considered that diode laser surgery can be per-
formed safely in close proximity to dental hard
tissue.
b a c d
Fig. 13. Clinical application of Er:YAG laser for scaling.
Er:YAG laser scaling was performed on the exposed sub-
gingival calculus on the root surface of lower right second
premolar with gingival recession. Laser irradiation was
performed at an energy density of 16.0 J/cm
2
per pulse
(energy output 45 mJ/pulse) and 10 Hz under water spray
in a contact mode, keeping the contact tip oblique to the
root surface in a sweeping motion. Calculus was effect-
ively removed by laser without producing major thermal
changes on the root surface. Patient did not feel any
stress, vibration or pain during irradiation. After treat-
ment, no complications or side effects were observed. a)
Before laser scaling. b) During laser irradiation. Conven-
tional cylindrical tip positioned obliquely to the root
surface. c) Immediately after laser scaling. d) 2 years after
treatment.
84
Aoki et al.
However, Kreisler et al. (99) further evaluated
possible morphologic alterations of the root surfa-
ces with a human blood lm after noncontact Ga-
AlAs-diode laser (809 nm) irradiation (0.52.5 W,
continuous wave, 1030 s). Interestingly, they
reported that lasing dry or saline-moistened root
specimens resulted in no detectable alterations;
however, the blood-coated specimens showed se-
vere damage depending on the irradiation condi-
tions. Irradiation at 1 W and below had barely any
negative effect on the root surface, whereas irradi-
ation at 1.5 W and higher resulted in partial or total
carbonization. Kreisler et al. (101) also examined
intrapulpal temperature elevations during diode
laser (809 nm GaAlAs) irradiation on the root
surface, performing laser irradiation at 0.52.5 W in
the continuous wave mode for 120 s. Temperature
elevations between 0.5 and 32.0

C were registered
in an energy- and time-dependent manner. They
reported the risk of temperature elevation of the
pulpal side during diode laser irradiation on the
root surface. Schwarz et al. (173) performed in vivo
GaAlAs diode laser treatment (810 nm, 1.8 W,
pulsed, pulse/pause relation 1 : 10) on periodontally
diseased roots of teeth considered for extraction
due to severe periodontal destruction and examined
the degree of calculus removal after extraction.
They reported that diode laser was unsuitable for
Fig. 14. Clinical application of Er:YAG laser for the
treatment of periodontal pocket. Subgingival curettage in
combination with gingivectomy was performed using
Er:YAG laser, to treat a periodontal pocket having gingival
enlargement at the buccal side of an maxillary left canine
with a porcelain-fused metal crown. The lesion did not
successfully improve after repeated conventional
mechanical debridements using curettes and ultrasonic
scaler. First, marginal gingiva, approximately 2 mm wide,
was removed with the Er:YAG laser at an energy density
of 10.6 J/cm
2
per pulse (energy output 30 mJ/pulse) and
10 Hz under local anesthesia. The gingiva was easily re-
sected with minimal bleeding. Then, subgingival curet-
tage was performed with the laser in contact mode using
a conventional cylindrical contact tip with a tapered end
(Fig. 9b) which enables lateral irradiation, keeping the tip
parallel to the gingival wall within the periodontal
pocket. Diseased granulation tissues were effectively re-
moved by laser without any major thermal damage and
with minimal bleeding. The patient did not experience
any stress and vibration during irradiation, and did not
complain of any postoperative pain or discomfort. a)
Before laser treatment. The mid-buccal site was 5 mm
deep and positive for bleeding on probing (BOP). b)
Immediately after treatment. c) One week after treat-
ment. The wound healing was uneventful and favorable.
d) Nine months after treatment. The periodontal pocket
improved and pocket depth reduced to 1 mm with no
BOP and without unaesthetic exposure of root surface
due to marked gingival recession.
85
calculus removal and altered the root surface in an
undesirable manner.
Thus, diode lasers at a high energy level, especially
in a continuous mode, can cause root surface alter-
ations in the presence of blood and elevated tem-
peratures, depending on the power employed.
Clinical studies
Some studies have demonstrated that a diode laser
facilitated bacterial elimination from periodontal
pockets, resulting in better healing. Moritz et al.
(121) reported pocket irradiation with a diode laser
(805 nm) following scaling. Irradiation with the
diode laser at a power output of 2.5 W in pulsed
mode (50 Hz, pulse duration 10 ms), produced
considerable bacterial elimination from periodontal
pockets at a much higher level than the scaling
alone group, especially in terms of A. actinomyce-
temcomitans. Moritz et al. (122) also performed a
clinical study using a diode laser (805 nm) as an
adjunctive treatment for periodontal pockets in
order to reduce or eliminate bacteria. The pulsed
irradiation at 2.5 W (50 Hz, pulse duration 10 ms)
was performed three times 1 week, 2 months, and
4 months after scaling, while the control group was
rinsed with H
2
O
2
. After 6 months, the bacterial
reduction in the laser therapy group was signi-
cantly higher than in the control group. The
improvement of bleeding on probing scores and
pocket depths were greater in the laser group. They
concluded that diode laser therapy, in combination
with scaling, supports healing of periodontal pockets
by eliminating bacteria (Table 10).
Regarding clinical use of the diode laser for pocket
treatment, Coluzzi (33) recommended laser soft tissue
curettage at 0.4 W in continuous wave mode after
mechanical debridement of root surface, followed by
irradiation at 0.6 W for hemostasis and bacterial
reduction, while Gutknecht et al. (67) suggested the
use of adiode laser at 2 Wincontinuous wave mode for
curettage before mechanical debridement. Further
detailed studies are required to establish proper irra-
diation conditions, including the use of water coolant,
for periodontal pocket therapy with diode lasers.
Other applications
Hibst et al. (73, 74) developed a novel laser device for
caries detection (Diagnodent
, KaVo, Biberach,
Germany), which uses laser uorescence induced by
the 655 nm InGaAsP diode laser. It was suggested
that caries-associated bacteria or their byproducts
might be the source of reaction to the increasing
uorescence (44, 96). Hibst et al. (74) identied the
source of red excited uorescence present in caries
lesions as porphyrins, especially proto-porphyrin IX,
which are products of oral bacteria, such as Prevotella
intermedia and P. gingivalis. They also suggested
another application of laser uorescence for calculus
detection (74, 163). Recently, the 655 nm diode laser
system has been reported to be useful for detection of
Table 10. Diode laser ) basic and clinical studies on root surface and periodontal pocket treatments
Moritz et al. 1997 (121) 2.5 W, 50 Hz
805 nm
Bacterial elimination from periodontal pockets
at a much higher level than scaling alone group
Moritz et al. 1998 (122) 2.5 W, 50 Hz
805 nm
Diode laser therapy in combination with scaling
supports healing of periodontal pockets by eliminating
bacteria
Kreisler et al. 2001 (106) 1 W, CW
810 nm
No signicant difference in the cell attachment
to the root surface between laser treatment and the
control groups
Kreisler et al. 2002 (99) 0.52.5 W, CW
809 nm
Dry or saline-moistened root specimens resulted in
no detectable alterations; however, blood-coated
specimens showed severe damage depending
on the irradiation conditions
Kreisler et al. 2002 (101) 0.52.5 W, CW
809 nm
Risk of temperature elevation of pulpal side during
diode laser irradiation on the root surface
Schwarz et al. 2003 (173) 1.8 W, Pulsed
810 nm
Diode laser was ineffective for calculus removal,
and caused alteration of root surface such
as grooves and crater-like defects in vivo
CW: continuous wave.
86
Aoki et al.
calculus that includes a signicant amount of bac-
teria or their byproducts.
Keller et al. (93) reported that the degree of root
debridement can be assessed by laser uorescence
and that subgingival root cleaning with the Er:YAG
laser can be optimized with the aid of laser uor-
escence spectroscopy. An apparatus combining the
Er:YAG laser with the diode laser uorescence for
root surface preparation is already being marketed.
Folwaczny et al. (49) also reported that subgingival
calculus can be reliably detected in vitro using laser
uorescence induced by 655 nm InGaAsP diode
laser. Krause et al. (98) observed that laser uores-
cence values decreased signicantly after in vitro
scaling of extracted teeth, and the values were
strongly correlated with the presence of calculus.
Schwarz et al. (173) reported that the Er:YAG laser,
combined with a calculus detection system with
uorescence induced by 655 nm InGaAsP diode
laser, provided selective subgingival calculus
removal to a level equivalent to that provided by
scaling and root planing.
Traditionally, calculus detection has been per-
formed manually by judging the ruggedness of the
root surface using a periodontal probe. The laser
uorescence probe may be a novel, valuable tool for
clinical detection of calculus in the near future.
Argon laser
Characteristics
The argon laser uses argon ion gas as an active
medium and is ber optically delivered in continuous
wave and gated pulsed modes. This laser has two
wavelengths, 488 nm (blue) and 514 nm (blue-green),
in the spectrum of visible light. The argon laser is
poorly absorbed in water and therefore does not
interact with dental hard tissues. However, it is well
absorbed in pigmented tissues, including hemoglobin
and melanin, and in pigmented bacteria. Although
not widely used in periodontal therapy, in operative
dentistry the 488 nm argon laser is commonly used
for curing composite resin (142) and bleaching teeth
in the dental ofce, and the application for caries
prevention has also been studied (127). The argon
laser was approved by the FDA for oral soft tissue
surgery and curing of composite materials in 1991
and for tooth whitening in 1995 (193).
Basic and clinical studies
Henry et al. (69, 70) reported that the argon laser at a
low level has a bactericidal effect on the Prevotella
and Porphyromonas species in the presence of
oxygen. They suggested that low doses of argon laser
radiation may be effective in the treatment of clinical
infections caused by biolm-associated species of
Prevotella and Porphyromonas. In a clinical study,
Finkbeiner (47) treated a total of 1,328 pockets from
30 patients, with argon laser pocket thermolysis in
combination with mechanical root planing. Argon
laser treatment was performed using a 300-lm ber
in contact at 0.4 W, for 2030 s per pocket, with
coaxial irrigation. He reported that the 45 mm
pockets were reduced by a mean of 1.62 mm, the
67 mm pockets by 2.85 mm, and the 89 mm
pockets by 3.30 mm.
Considering the advantages of eradication of pig-
mented bacteria, this laser may be useful for the
treatment of periodontal pockets. Further in vitro
and in vivo studies are required to demonstrate the
clinical benets of this laser.
Alexandrite laser
The Alexandrite laser is a solid-state laser employing
a gemstone called Alexandrite, which is chromium-
doped:Beryllium-Aluminum-Oxide chrysoberyl (Cr
+3
;
BeAl
2
O
4
) and is one of the few trichroic minerals.
In 1995 Rechmann & Henning (149) rst reported
that the frequency-doubled Alexandrite laser (wave-
length 337 nm, pulse duration 100 ns, double spikes,
q-switched) could remove dental calculus in a com-
pletely selective mode without ablating the underly-
ing enamel or cementum. Based on the difference in
spectral region of uorescence emission from dentin
and that from subgingival calculus, they assumed
that the wavelength of the Alexandrite laser may be
favorable for selective calculus ablation (149, 150).
Their studies revealed that the Alexandrite laser at the
uence of 1 J/cm
2
and pulse repetition rate of 55 Hz
under water-cooling could selectively ablate supra-
and subgingival calculus as well as dental plaque.
This laser has a wavelength in the ultraviolet spec-
trum and therefore does not produce any morpho-
logic damage to enamel surface or root cementum
(151), although extremely slight compositional
change such as minimal reduction of amide II band is
detected in the lased cementum by FTIR spectros-
copy analysis (153). Rechmann et al. (152) also
demonstrated that there was no pulpal damage after
removal of calculus with the Alexandrite laser at
1.56 J/cm
2
and 70 Hz (pulse duration 1 ls) under
water-cooling in dogs. However, the mechanism of
selective ablation has not been claried yet. The
development of this laser for clinical use is widely
87
expected due to its excellent ability for selective
calculus removal from the tooth or root surface
without ablating the tooth structure. However, there
is concern regarding use of light in the ultraviolet
spectral region, and further studies are required to
demonstrate the safety and effectiveness of this laser
in clinical usage and to develop a laser apparatus
appropriate for clinical use.
Excimer lasers
Excimer lasers are lasers that use a noble-gas halide,
which is unstable, to generate radiation, usually in
the ultraviolet region of the spectrum. Excimer laser
wavelength depends on the chemical component
serving as the medium of the laser. It has been sug-
gested that tissue ablation occurs in the nonthermal
process of photoablation, likely due to an instanta-
neous increase of the temperature or a straight
combination of chemical elements (57).
In an in vitro experiment, Frentzen et al. (57)
demonstrated that the ArF excimer laser, wavelength
193 nm, could effectively remove dental calculus
without causing any damage to the underlying sur-
face. The cementum surface was clean, and only a
slight roughness could be observed after irradiation,
supporting the use of excimer lasers for laser scaling.
Folwaczny et al. (52) have reported that the 308 nm
wavelength XeCl excimer laser could effectively ab-
late dental calculus without thermal damages or
smear layer production.
Recently, exible quartz glass bers for XeCl-exci-
mer laser delivery systems have become available.
However, apparatus cost and size still constitute an
obstacle for clinical application of these lasers.
Furthermore, ultraviolet rays should be used with
caution, as they may have deleterious effects on
biological tissues.
Application of lasers for the
treatment of peri-implantitis
It is well known that adherent bacterial plaque and
calculus develop on the surface of implant abut-
ments, as in natural teeth. The maintenance treat-
ment is required to keep the peri-implant tissue
healthy in implant therapy. However, mechanical
instruments such as metal curettes and ultrasonic
scalers are prohibited for decontamination of tita-
nium implant surfaces, since they easily damage the
titanium surface.
Recently, lasers have been widely used for soft
tissue incision in exposing submerged implants.
Lasers may be used for decontamination of implant
surface and treatment of peri-implantitis without
damaging the implant surface (18). Regarding the
effect of lasers on titanium, the Nd:YAG laser is not
suitable for implant therapy, since it easily ablates the
titanium irrespective of output energy. However,
diode lasers basically do not interact with titanium or
the coated material (24, 104, 158). As for the Er:YAG
and CO
2
lasers, Kreisler et al. (104) suggested that the
power output must be controlled so as to avoid
damage of implant surfaces. Matsuyama et al. (116)
also observed that the Er:YAG laser causes damage on
the titanium surface at a high energy level, such as
100 mJ/pulse (35.4 J/cm
2
per pulse), but does not
result in any morphologic change or major tem-
perature elevation at a low energy level under 50 mJ/
pulse (17.7 J/cm
2
per pulse) and 30 Hz, with water
coolant, which is suitable for periodontal treatment.
Schwarz et al. (177) observed that the Er:YAG laser at
100 mJ/pulse (energy out put of 85 mJ/pulse, calcu-
lated energy density 10.3 J/cm
2
per pulse) and 10 Hz
under water irrigation does not damage titanium
surfaces and does not affect the attachment of oste-
oblast-like cells. Their preliminary clinical results
(178) have also shown that nonsurgical treatment of
peri-implantitis with an Er:YAG laser at 100 mJ/pulse
and 10 Hz (energy density 12.7 J/cm
2
per pulse)
under water spray led to a statistically signicant
reduction in pocket depth and gain in clinical
attachment level.
Effective decontamination of the implant surface
without excessive temperature elevation and any
morphologic changes by CO
2
(energy density
286 J/cm
2
and 245 J/cm
2
) or Er:YAG (calculated
energy density 26.2 or 52.4 J/cm
2
per pulse) lasers
has been reported in vitro by Kato et al. (84) and
Kreisler et al. (100, 105). However, the risk of
moderate to high temperature elevation has been
noted after CO
2
or diode laser irradiation (102, 132).
Laser treatment of peri-implantitis may also be a
promising eld; however, further studies are
required for application of lasers in implant main-
tenance therapy.
Disadvantages and precautions
in the clinical use of lasers
Lasers may be novel, effective tools for the treatment
of periodontitis. However, lasers have disadvantages
88
Aoki et al.
as well as advantages. Therefore, precautions should
be taken when performing laser surgery (Table 11).
The position paper of the American Academy of
Periodontology suggests several important precau-
tions in the use of lasers (4).
Laser light interacts with target tissues not only in
the contact irradiation mode but also in the non-
contact irradiation mode. Therefore, inadvertent
irradiation to the patients eyes, throat, and delicate
oral tissues outside the target site may occur during
treatment and must be prevented (4). Particular care
must be taken to avoid accidental irradiation to the
eyes. The most important precaution in laser surgery
is the use of glasses for eye protection. Before laser
treatment, protective eyewear, specically blocking
the wavelength of the laser in use, must always be
worn by patients, operator, and assistants (4). The
laser beam may be reected off shiny metal surfaces
of dental instruments, such as retractors or mouth
mirrors, which can cause accidental irradiation to
adjacent tissues (4). Use of wet gauze packs may be
occasionally useful for protection of the oral tissues
surrounding the surgical site from accidental beam
impact (138). Also, adequate high speed evacuation is
necessary to capture the laser plume, which is a
biohazard (4, 43). Contact with tooth enamel during
periodontal treatment should be avoided during CO
2
and Er:YAG laser emission, as they easily cause
melting or ablation.
There are few studies on the safety criteria for
intraoral usage of lasers. With the Er:YAG laser,
Aoki et al. (13) evaluated the effects of inadvertent
irradiation on the tongue of rat. Er:YAG laser irradi-
ation in a noncontact defocused mode caused no
major damage to the tongue, especially when used
with water irrigation. Although contact irradiation
caused a tissue defect, thermal damage was rarely
observed, and the healing process was without clin-
ical problems. They concluded that inadvertent irra-
diation with the Er:YAG laser within the usual power
setting used for dental treatment did not cause severe
damage to surrounding soft tissues in the oral cavity.
There also exists a risk of excessive tissue destruc-
tion by direct ablation and thermal side-effects of
periodontal tissues during irradiation into periodon-
tal pockets. Improper use of lasers could cause fur-
ther destruction of the intact attachment apparatus
at the bottom of pockets as well as excessive ablation
of root surface and gingival walls. Root surface with
major thermal damage could render the tissue
incompatible for normal cell attachment and healing.
Thermal injury to the pulp tissue and underlying
bone tissue would also be a concern with some lasers,
especially with those exhibiting deep penetration.
Therefore, thermal injury must be prevented by
proper irradiation conditions and techniques.
Regarding the laser apparatus, development of a
new laser system as well as improvement of currently
available laser systems, such as miniaturization of
device size and advances of performance, are re-
quired. Also, development of a new contact probe
and handpiece suitable for periodontal treatment is
necessary, as accessibility of contact probes into
periodontal pockets is limited due to complex root
morphology and furcated roots.
The high nancial cost of the laser apparatus is still
somewhat prohibitory, and this has prevented the
spread of laser treatment among general practition-
ers. However, the price is expected to decrease with
developments in laser technology and with increas-
ing demand.
Conclusions
With conventional mechanical instruments, complete
access and disinfection may not be achieved during
the treatment of periodontal pockets. The effective-
ness of instrumentation may vary with the skills
and experience of the practitioner and is therefore
Table 11. Disadvantages and precautions in clinical
use of lasers
1. Caution before and during irradiation
Use of glasses for eye protection (patient, operator, and
assistants)
Inadvertent irradiation (action in noncontact mode)
Protection of patients eyes, throat, and oral tissues
outside the target site
Reection from shiny metal surfaces
Adequate high speed evacuation to capture the laser
plume
2. Risk of excessive tissue destruction by direct abla-
tion and thermal side-effects
Destruction of the attachment apparatus at the bottom
of pockets
Excessive ablation of root surface and gingival tissue
within periodontal pockets
Thermal injury to the root surface, gingival tissue, pulp
and bone tissue
3. Problems of laser systems
Further development of a new laser system as well as
improvement of currently available laser systems
Development and improvement of contact probes suit-
able for periodontal treatment
Reduction of high cost of laser apparatus
89
technique sensitive. Conventional mechanical treat-
ment has various limitations in techniques and
effects, and lasers have been introduced as an
adjunctive or alternative tool for mechanical therapy.
Basically, lasers have the potential advantages of
bactericidal effect, detoxication effect, and removal
of the epithelium lining and granulation tissue, which
are desirable properties for the treatment of perio-
dontal pockets. Some lasers may be capable of
effectively removing not only dental plaque but also
calculus from the root surface with extremely low
mechanical stress and no formation of a smear layer
on the treated root surface. Furthermore, potential
biostimulation effects of scattering and penetrating
lasers on the cells surrounding the target tissue dur-
ing irradiation might be helpful for the reduction of
inammation and healing of periodontal tissues.
Considering the various advantages of laser irradi-
ation, its use in combination with conventional
mechanical treatment or alone has the potential to
improve the condition of the periodontal pockets
more than mechanical therapy alone.
Also, considering the evidence of bacterial invasion
into the soft tissue of periodontal pockets (130, 161),
not only debridement of the root surface but also
removal of the epithelium lining and granulation
tissue of the gingival wall within periodontal pockets
could be important factors in the treatment of mod-
erate to deep pockets in order to promote attachment
of gingival connective tissue to the root surface. This
might be particularly applicable to residual pockets,
after initial therapy and during the maintenance
period, that are not resolved by mechanical therapy
alone. Lasers may be used to accomplish curettage of
the soft tissue wall, and provide favorable conditions
more effectively than the currently available instru-
ments. Comprehensive treatment including prepar-
ation of both hard and soft tissue walls within
pockets should be considered for more effective
nonsurgical periodontal therapy in the future, and
this is what may be accomplished by lasers. Thus,
lasers could play an important role in comprehensive
pocket therapy.
Based on the limited research so far, the Er:YAG
laser holds promise as a useful tool to debride safely
and effectively both the root surface and gingival
tissue of the periodontal pockets, and the Nd:YAG,
diode and Ar lasers have a potential for soft tissue
curettage and disinfection of periodontal pockets.
The Alexandrite laser has also shown highly promis-
ing results for selective calculus removal. Another
promising characteristic is the ability of diode laser
uorescence to detect dental calculus.
The ultimate applicability and benets of a novel
treatment modality must be strictly evaluated based
on scientic evidence and critical review of existing
literature (3). Although the use of lasers for subgin-
gival curettage and calculus removal in the treatment
of periodontal pockets has been increasing among
practitioners, the scientic studies indicating positive
clinical results of lasers are still insufcient. Further
basic and clinical studies, such as randomized con-
trolled studies, are necessary to elucidate the actual
effects and effectiveness of lasers in comparison with
conventional treatment as well as negative side-
effects.
To use lasers safely in a clinic, the practitioner
should have precise knowledge of the characteristics
and effects of each laser system and their applica-
tions as well as a full understanding of the conven-
tional treatment procedures, and nally should
exercise appropriate caution during their use.
A reliable procedure for laser application in non-
surgical periodontal therapy should be established by
further studies, and clinicians should follow the
results of scientic investigations to obtain successful
outcomes. As understanding of the nature of laser
light evolves, lasers will be used more effectively in
the treatment of periodontal diseases. Laser systems
applying the ablation effect of light energy, which is
completely different from conventional mechanical
debridement, may emerge as a new technical
modality for nonsurgical periodontal therapy in the
near future.
Acknowledgements
The authors wish to thank Dr Frank Schwarz, Hein-
rich Heine University, Dusseldorf, Germany;
Dr Geena Koshy, Dr Koji Mizutani and Dr Aristeo
Atsushi Takasaki, Tokyo Medical and Dental
University, Tokyo, Japan; and Dr Yoshinori Ando,
private practice, Tokyo, Japan for their kind advice
and help in manuscript preparation. This review was
supported by the grant for Center of Excellence Pro-
gram for Frontier Research on Molecular Destruction
and Reconstruction of Tooth and Bone in Tokyo
Medical and Dental University.
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97
Treatment of small defects
adjacent to oral implants with
various biomaterials
William Becker
Primary stability of an endosseous implant can be
compromised by bone defects. Placement of bone
grafts or other biomaterials in bony defects adjacent
to oral implants should promote osseointegration
and improve adjacent soft tissue esthetics. Autolo-
gous bone has a long history of use and is considered
the gold standard for graft materials (24). The ratio-
nale for using autologous bone is its osseoinductive
potential. This type of graft may retain the matrix,
with its bone inductive properties and undifferen-
tiated cells. Autologous bone grafts have the poten-
tial to retain vital cells, which are replaced by the
host. It does not induce an immunologic reaction.
Small autologous grafts can be harvested from the
chin, while larger grafts are harvested from the man-
dibular ramus or iliac crest (17, 1921, 54). Osseous
defects adjacent to oral implants have been success-
fully treated with intraoral autologous bone; how-
ever, there is a lack of evidence as to the ultimate
fate of these bone grafts. Such defects can be grafted
with small autologous bone chips (7, 16), which
results in signicant improvement. In the study of
Becker et al. (7), the initial mean vertical defect depth
was 5.7 mm, and the average residual depth at
second stage surgery was 0.3 mm. These changes
were statistically (P < 0.001) signicant and clini-
cally relevant.
The use of DFDBA
In the United States, implantation of demineralized
freeze-dried bone allograft (DFDBA) has a long, safe
history (3739). The rationale for implanting DFDBA
is the possibility of initiating the bone induction cas-
cade. Urist isolated bone morphogenetic proteins
from cortical human bone (57). These proteins have
been shown to initiate the bone formation cascade in
rats and rabbits, and other species (58). When bones
containing morphogenetic proteins are implanted
into ectopic sites in athymic mice, bone ossicles form
within 21 days (56). The highest percentages of bone
morphogenetic proteins (BMPs) in human bone are
present in the rst two decades of life (59). Other
researchers conrmed that the bone inductive capa-
city of demineralized freeze-dried bone is related to
age, but not gender (51).
A study evaluated the bone induction potential of
human DFDBA in large dogs (8). The purpose of this
study was to compare bone promotion around
implants which were augmented with expanded
polytetrauoroethylene (ePTFE) membranes alone
or in combination with cortical DFDBA or the com-
bination of platelet derived growth factor-B (PDGF)
and insulin-like growth factor I (PDGF/IGF-I). Mem-
branes were placed over titanium implants that had
been inserted into fresh extraction sockets with large
buccal dehiscences. Clinically, a signicant gain in
bone levels was present in both the ePTFE mem-
brane alone group (P < 0.005) and in the ePTFE plus
PDGF/IGF-I group (P < 0.01), but not in the ePTFE
plus DFDBA group. Histologic measurements demon-
strated that sites treated with ePTFE membranes plus
PDGF/IGF-I had the highest bone density compared
with sites which received ePTFE membranes alone or
ePTFE membranes and DFDBA. The results of this
study question the use of DFDBA and support the
use of ePTFE membranes alone or with PDGF/IGF-I
as potential methods of promoting bone formation
around endosseous implants. A follow-up study
implanted DFDBA prepared from long bones of a
dog of the same species as used in the study. The
26
#
PERIODONTOLOGY 2000
ISSN 0906-6713
Fig. 1. (a) Implants placed in dehiscences created after
removal of P1,P2,P3. (b) Autologous bone chips were
taken from the site distal to the mandibular canine. (c)
Distal site is barrier membrane only, middle site received
autologous bone chips, anterior site is untreated control.
(d) Three sites received DFDBA grafts together with
ePTFE barrier membranes. (e) Barrier membranes have
been adjusted to cover the DFDBA grafts. (f) Barriers
have been adjusted to cover the barrier and autologous
bone chip sites. The control site is not augmented. (g)
DFDBA augmented site presents a composite of woven
bone (arrows) and incorporated DFDBA particles. There
is no bone ingrowth into the implant threads. (h) Biopsy
of site which received DFDBA. Arrows point to breakup of
DFDBA particles, without evidence of new bone forma-
tion. (i) Autologous bone chips have been incorporated
into the surrounding bone (arrows). (j) Histologic view of
site that received a barrier membrane. Note sparse bone
to implant contacts. (k) Histologic view of untreated
control.
Placement of bone grafts adjacent to compromised implants
27
teeth P2, P3, and P4 were extracted bilaterally, and
buccal defects were created and measured. Twelve
Branemark System
TM
implants were placed into the
extraction sockets. Two implants acted as untreated
controls, two implants received only GoreTex Aug-
mentation Membrane (GTAM), and two implants
received GTAM with autologous bone, while six sites
received GTAM and DFDBA (Fig. 1af). At 12 weeks,
clinical measurements were taken and the dogs
sacriced. The untreated control defects had a mean
clinical bone ll of 1.8 mm (37%). Sites treated with
autologous bone had a mean of 5.0 mm (95%) of
clinical bone ll within the original defects. Sites
treated with DFDBA and barriers had 3.8 mm
(75%) of bone ll, while sites treated with mem-
branes alone had a mean of 4.2 mm (80%) of bone
ll. Histologic evaluation revealed that DFDBA sites
had retained nonviable bone chips in 45.4% of the
bone matrix, and only 8.3% was lamellar bone
(Fig. 1gh). Autologous graft sites had 26.2% retained
bone chips within the bone matrix, and 61% of the
matrix consisted of lamellar bone (Fig. 1i). For
GTAM-only sites, 70.2% of the matrix was lamellar
bone and 29.8% woven bone. Figure 1(j) shows the
site that received an ePTFE barrier only. Bone
appeared to form beneath the barrier; however,
the bone was not in contact with the adjacent
implant threads. The implant in the control site
Fig. 1. continued
28
Becker
was integrated, with four exposed threads (Fig. 1k).
The general histologic description revealed that
retained DFDBA bone chips were nonviable, occa-
sionally surrounded by woven bone, and appeared to
break up and then remineralize without the presence
of osteoclastic or osteoblastic activity. Retained auto-
logous bone chips on the other hand were sur-
rounded and incorporated by the host bone. The
autologous bone grafts and DFDBA implants were
considered to be osteoconductive. For all three treat-
ment groups, there were sparse bone-implant con-
tacts within the defects. The results indicate that
GTAM barriers alone or with autologous bone grafts
produced the best clinical and histologic results.
DFDBA did not appear to induce bone formation
in any of the evaluated specimens.
In an effort to determine the bone induction capa-
city of commercially available DFDBA, bone from
ve different banks was placed in the hindquarters
of athymic mice for a 20-day period, after which the
mice were sacriced (9). The hindquarters were radio-
graphed and histologic sections were prepared. The
primary histologic picture revealed brous encapsula-
tion of dead allograft material (Fig. 2a). Evaluation of
allograft infused with human bone morphogenetic
protein hBMP/ncp and implanted in athymic mice
for 21 days revealed extensive new bone formation
(Fig. 2b). When bone banks were compared, the per-
centage of deadbone variedbetween78.4%and92.5%.
Other researchers have repeated this study and also
reported wide variability between bone induction
capacity from different banks.
Biopsies were obtained from human titanium
microimplant sites augmented with small autotolo-
gous bone chips, DFDBA, and deproteinized bovine
bone (Fig. 3ac) (11). Histologic evaluation demon-
strated encapsulation of the implanted materials
adjacent to the titanium microimplants without
bone to implant contacts (Fig. 3df). When new bone
forms around autologous bone chips, allografts, or
xenografts the process is known as osteoconduction.
In this situation, the graft particles may act as a
scaffold for new bone formation. Bone formation
was occasionally seen adjacent to the allograft parti-
cles. When bone was noted, it was considered to have
formed by osteoconduction. Other researchers placed
DFDBAinto celluloidcapsules andimplantedthe cap-
sules into prepared defects below the apices of man-
dibular molars (43). Histologic analysis of all recovered
Fig. 1. continued
Fig. 2. (a) Section contains DFDBA particles which were
implanted in an athymic mouse for 21 days. Particles are
dead, with no evidence of osteoblastic or osteoclastic
activity. (b) DFDBA particles were infused with hBMP/
ncp (human bone morphogenetic protein and non-col-
lagenous proteins). Note extensive active bone formation.
29
Fig. 3. (a) Periodontally diseased teeth have been removed
and a micro implant has been placed into the lateral wall of
the extraction socket. (b) Micro implants were inserted and
stabilized into the lateral wall of the sockets. Defects adja-
cent to the mini screws were implanted with DFDBA, intra
oral bone chips, or bovine bone. (c) Flaps were coapted with
interrupted 4-0 silk sutures. (d) Healing of implanted sites at
4 months seen in (b). (e) Biopsy of site implanted with
deproteinized bovine bone. Non-vital particles are seen
throughout the section. (f) Site received autologous bone
chips. Bone chips have coalesced, but are not in contact
with the micro implant. (g) Histologic view of site that
received DFDBA graft. Arrows point to DFDBA particles.
Implant is surrounded by connective tissue.
30
Becker
capsules by two independent oral and maxillofacial
pathologists could not conrm the presence of either
osteoblastic or osteoclastic activity associatedwiththe
DFDBA particles, although two longer-term speci-
mens clearly exhibited trace amounts of vital bone
non-contiguous with the implanted material. Evalua-
tionof the biopsies did not support the osteoinduction
potential of DFDBA.
Bovine bone has been used to ll deshiscence
defects adjacent to oral implants in monkeys (25).
Conclusions from clinical and histologic measures
indicated the material was osteoconductive and
could be used to augment defects adjacent to oral
implants. A subsequent study in patients indicated
that bovine bone together with a bioabsorbable bar-
rier can be used to reduce vertical defects adjacent to
immediately placed oral implants (26).
Implants in extraction sockets
Placement of implants at the time of extraction has
become a predictable method (5, 6, 12, 16, 23, 31, 34,
49, 50). Since the implant does not completely oblit-
erate the extraction socket, many clinicians feel the
necessity to ll the gap between the surrounding
bony walls and the oral implant. To appreciate the
healing dynamics of these sites, it is necessary to
understand the healing sequence of extraction sock-
ets (1, 32, 36). Immediately after tooth extraction
there is hemorrhage into the socket with blood clot
formation. By 72 h, granulation tissue begins to
invade the clot, and within 72 h, epithelialization is
initiated. Within a week the socket is lled with
immature connective tissue, primary osteoid is being
formed and there is continued epithelial prolifera-
tion. Twenty days after extraction there is connective
tissue maturation, mineralization of the osteoid and
continued epithelialization. By 6 weeks, there is
ongoing connective tissue maturation, woven bone,
trabeculation and the wound is epithelialized. Over
46 months, there is further maturation of the con-
nective tissue and mineralization of woven and tra-
becular bone. This dynamic sequence of healing
events can be expected to occur whenever teeth
are atraumatically removed and four bony socket
walls are present. Implants placed into extraction
sockets with four bony walls can be expected to heal
uneventfully with bone (Fig. 4a,b) (10). Several case
reports have demonstrated that implants placed at
the time of tooth extraction osseointegrate without
brous tissue interposition between the implant sur-
face and surrounding bone (22, 60). One group of
investigators evaluated implants placed into fresh
extraction sockets with compromising defects in mon-
keys (41). The defects were lledwith small autologous
Fig. 3. continued
Fig. 4. (a) Figure shows implants placed at the time of
tooth extraction. (b) Photograph clinically demonstrates
bone surrounding the implants. Picture taken 6 months
postoperatively.
31
bone chips without barrier membranes. The study
demonstrated that when screw-type oral implants
are placed without the use of barrier membranes or
other regenerative materials into freshextractionsock-
ets with a bone-to-implant gap of 2 mm or less, the
clinical outcome and degree of osteointegration does
not differ from implants placed in healed, mature
bone. A similar study in monkeys reported results after
placement of 36 immediately placed implants (46).
The defects adjacent to the implants were augmented
with autologous bone chips without barrier mem-
brane augmentation. At retrieval, all implants were
covered by compact, mature bone under examination
in light microscopy. A very high bone-to-implant con-
tact percentage (6570%) was observed. No bone loss
was present after the loading period. These results
indicate that implants placedinto freshextractionsites
grafted with autogenous bone chips will heal in a pre-
dictable way.
One of the objectives for placing implants at the
time of extraction is to maintain ridge width by mini-
mizing crestal width resorption. An excellent study
placed implants following the delayed extraction
approach (40). Socket width and depth was mea-
sured at the time of implant placement. At second
stage the distance between the implant cover screw
and adjacent ridge was remeasured. The horizontal
distance decreased from 2.5 mm to 0.36 mm. When
extraction sockets are protected with barriers, it
appears there will be less crestal resorption (35).
Bovine bone has been implanted in small defects
after simultaneous extraction and oral implant pla-
cement. This achieved signicant reduction in the
vertical component of the defects (55). A retrospec-
tive study evaluated changes in ridge crest after pla-
cement of implants at the time of extraction and at
second stage surgery (13). Measurements were made
from 35 mm occlusal views taken at implant place-
ment and at second stage surgery. Measurements
were made for sites augmented with ePTFE barriers
alone, from those receiving small autologous bone
chips from adjacent edentulous ridges (Fig. 5a,b) and
when no augmentation or grafting was performed.
Results indicated bone crest loss for the three treat-
ment groups. The least bone remodeling occurred for
sites receiving no augmentation treatment.
There is evidence that placement of various graft-
ing materials in extraction sockets may interfere
with the normal healing sequence (6, 11). In the
former study small turned titanium implants were
placed into the lateral walls of extraction sockets in
eight patients. The sockets were implanted with allo-
graft, autologous bone chips or deproteinized
bovine bone. Implant biopsies including surround-
ing tissues were taken 3 to 6 months after the initial
surgery. Histologic evaluation was similar for the
three treatment groups. The overriding picture was
brous encapsulation of the grafted materials with
absence of new bone to implant contacts. Periopera-
tive bone traps have been a popular method to har-
vest bone during drilling procedures. This harvested
material has been used to ll small gaps between
implants and surrounding bone. An animal study
indicated that implants receiving bone shavings
from bone traps had less bone to implant contacts
than untreated controls (47). A recent report indi-
cated that in the rat model, bovine bone augmented
with barrier membranes may interfere with bone
formation (53).
Fig. 5. (a) An autologous bone chip graft taken from the
edentulous ridge has been placed into the buccal defect.
(b) Photograph taken 6 months postoperatively. Note loss
of crestal width when compared with (a).
32
Becker
Alveolar ridge augmentation
Augmentation of edentulous alveolar ridge defects can
be classied as either hard or soft tissue in nature.
Hard tissue augmentations primarily facilitate implant
placement into bone-decient ridges. Soft tissue aug-
mentations are utilized to achieve improved esthetics
adjacent to oral implants. Buser et al. have demonst-
rated consistent long-term clinical results after lateral
augmentation of decient edentulous ridges (1721).
The augmentations were made with autologous bone
harvested from either the chin or the lateral ramus.
Other researchers have demonstrated similar results
without barrier membrane augmentation (14, 44, 45).
Augmentation of compromised soft tissue defects
adjacent to implant sites can be successfully accom-
plished in a variety of ways. Subepithelial connective
tissue grafting for esthetic enhancement of soft tissue
deformities has been reported in numerous papers
(2, 27, 28, 30, 33, 52). Biomaterials can be implanted
between the inner ap margins and adjacent alveolar
bone. Deproteinized bovine bone is especially useful
for enhancement of soft tissue inconsistencies, since
it is safe, slowly resorbed and becomes encapsulated
with brous tissue.
Rehabilitationof large, compromiseddefects inmax-
illas or mandibles may require large, corticocancellous
hip grafts. Implants placed using the simultaneous
approach had, after 10 years a 95% cumulative survival
rate (54). Corticocancellous grafts have been success-
fully used to augment large vertical and horizontal
defects (3, 4). Aseries of 12 men and 13 women ranging
in age from 24 to 71 years underwent two- or three-
dimensional reconstruction of type C, D, or E ridges
and placement of anterior implants. The mean hori-
zontal augmentation was 6.4 mm (range 217 mm),
and the mean vertical augmentation was 4.22 mm
(range 015 mm). The 67 implants were all loaded,
with the time averaging 34.4 months for the maxillary
implants and 19 months for the mandibular implants.
None of the implants have been lost to date. Patients
initially presenting with Type C jaw were converted to
Type B after grafting. In the future, distraction osteo-
genesis may provide a predictable method for reducing
or eliminating compromised alveolar defects (15).
Conclusions
Considerable efforts have been made to maintain
ridge width after tooth extraction and oral implant
placement. Various grafting materials with and with-
out barrier membranes have been used for this pur-
pose. It appears possible to increase alveolar bone
width and height with autologous bone chips, demi-
neralized freeze-dried bone or bovine bone, with or
without the use of barrier membranes, but there is
insufcient evidence that these procedures are pre-
dictable (29, 42). Even though these materials are
biologically acceptable bone substitutes, there is lit-
tle evidence these materials signicantly contribute
to osseointegration.
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35
Bone augmentation by means of
barrier membranes
Christoph H. F. Hammerle & Ronald E. Jung
The development of guided bone regeneration (GBR)
has substantially inuenced the possibilities for
using implants. The use of bone augmentation pro-
cedures has extended the use of endosseous implants
to jaw bone areas with insufcient bone volume.
Lack of bone volume may be due to congenital,
post-traumatic, or post-surgical defects or the result
of disease processes. The recently achieved predict-
ability and success of this procedure can change the
treatment of a compromised patient into a nearly
normal challenge.
Based on pioneer experiments investigating heal-
ing of periodontal tissues following surgical therapy,
a principle of tissue healing was discovered by
Nyman & Karring in the early 1980s (60, 62, 87,
88). It was found that cells which have access to
and migrate into a given wound space determine
the type of tissue regenerating in that space. Both
the exclusion of undesired cells from the wound area
and the formation of a wound space into which
desired cells are allowed to migrate were initially
achieved by the placement of barrier membranes
(61). The present understanding of the mechanisms
leading to regeneration of desired tissues is still in
accordance with these initial concepts.
The aim of the present chapter was to review
the techniques and membrane materials applied
for GBR in conjunction with implant based oral reha-
bilitation.
Types of membranes
General aspects
A wide range of membrane materials have been used
in experimental and clinical studies to achieve GBR
including polytetrauoroethylene (PTFE), expanded
PTFE (ePTFE), collagen, freeze-dried fascia lata,
freeze-dried dura mater allografts, polyglactin 910,
polylactic acid, polyglycolic acid, polyorthoester,
polyurethane, polyhydroxybutyrate, calcium sulfate,
micro titanium mesh, and titanium foils. Devices
used for GBR in conjunction with endosseous
implants should be safe and effective. Since no life-
threatening diseases or deciencies are treated, pos-
sible adverse effects emerging from the implanted
devices should be kept to a minimum. At the same
time, documentation of the effectiveness of the pro-
cedures and materials should be available. Certain
critical criteria regarding membranes used for guided
tissue regeneration have been postulated (47): bio-
compatibility, cell occlusiveness, integration by the
host tissues, clinical manageability, and the space
making function. For bioresorbable and biodegrad-
able membranes, additional criteria need to be ful-
lled. Tissue reactions resulting from the resorption
of the membrane should be minimal, these reactions
should be reversible, and they should not negatively
inuence regeneration of the desired tissues (38).
Although GBR is a quite successful procedure, a
better understanding of the factors critical for suc-
cess or failure is mandatory. This understanding
will lead to more rened clinical protocols and to
the manufacture of membrane materials with
improved performance for a given indication. Some
of these factors include the ideal size of membrane
perforations, membrane stability, duration of barrier
function, enhanced access of bone and bone-mar-
row-derived cells to the area for regeneration, ample
blood ll of the space, and prevention of soft tissue
dehiscence.
Non-resorbable membranes
Polytetrafluoroethylene
With the presentation of the rst successful GBR
procedures and the subsequent wide and successful
application of ePTFE membranes, this material
became a standard for bone regeneration. Expanded
PTFE is characterized as a polymer with high stability
36
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PERIODONTOLOGY 2000
ISSN 0906-6713
in biological systems. It resists breakdown by host
tissues and by microbes and does not elicit immu-
nologic reactions.
A frequent complication with membrane applica-
tion in conjunction with implants is membrane
exposure and infection (98). Wound dehiscence
and membrane exposure have been reported to
impair the amount of bone regenerated in a number
of experimental animal (36, 63, 68) and clinical inves-
tigations (13, 34, 44, 106).
Many of the factors critical for successful bone
formation were identied in experimental studies
applying ePTFE membranes. Furthermore, clinical
protocols regarding surgical procedures, postopera-
tive care and the healing times required were estab-
lished using non-resorbable membranes. Today, as
evidence of the effectiveness of bioresorbable mem-
branes increases, non-resorbable membranes are
losing importance in clinical practice and their use
is increasingly limited to specic indications. Since
the use of ePTFE membranes have been documented
to result in successful GBR therapy, results obtained
using new materials should always be compared with
results of ePTFE membranes.
Titanium-reinforced ePTFE
In situations where bone formation is desired in large
defects or in supracrestal areas, conventional ePTFE
membranes do not adequately maintain space unless
supported by grafting materials. The alternative
approach involves the use of membranes with a sta-
ble form, such as titanium-reinforced membranes.
Titanium-reinforced membranes consist of a double
layer of ePTFE with a titanium framework interposed
(57, 110, 114). Recent research has demonstrated the
successful use of these membranes in vertical ridge
augmentation and in the treatment of large defects in
the alveolar process.
Bioresorbable membranes
An obvious disadvantage of ePTFE materials is that
they are non-resorbable and therefore have to be
removed during a second surgical procedure. With
regard to patient morbidity and psychological stress,
risk of tissue damage, and cost versus benets, the
replacement of non-resorbable by bioresorbable
membranes is highly desirable. Hence, recent experi-
mental research in GBR has aimed at developing
bioresorbable barrier membranes for application in
the clinic (Fig. 1).
Apart from the fact that the surgical intervention
for removal of the membrane is omitted, bioresorb-
able membranes offer some additional advantages
(Table 1). Among these are improved soft tissue heal-
ing, the incorporation of the membranes by the host
tissues (depending on material properties), and a
quick resorption in case of exposure, thus eliminat-
ing open microstructures prone to bacterial contam-
ination (72, 130).
In general, soft tissue healing is improved in the
presence of bioresorbable compared to non-resorb-
able membranes (69, 70, 130). However, some inves-
tigators have reported contrasting ndings (72). With
ePTFE membranes, 22% (GTAM) and 47% (TR-
GTAM) of sites showed dehiscences, while 50% of
sites treated with a bioresorbable membrane (self-
reinforced polyglycolic acid) became dehiscent (72).
The average gain of bone was decreased in cases
with dehiscences compared to an uneventful sub-
merged regeneration period. Both in the presence
and in the absence of soft tissue problems the
amount of bone ll of the defects was greater with
the ePTFE membranes.
Several studies have compared bioresorbable
membranes to non-resorbable membranes made of
ePTFE. In situations where no membrane exposures
were noted, the results regarding the relative amount
of bone formation were usually more favorable using
the ePTFE membranes compared to the bioresorb-
able ones (55, 81, 83, 109).
Various reasons for the generally lower defect ll
with bioresorbable membranes as compared to
ePTFE membranes include:
the better space-making capacity of ePTFE;
controlled time of barrier function;
lack of a resorption process and lack of the gen-
eration of resorption products that negatively
affect bone formation;
longer experience with ePTFE membranes resulting
in surgical protocols better tailored for their use.
In summary, it appears that bioresorbable mem-
branes in general allow for more bone regeneration
than non-resorbable ePTFE membranes. However, if
soft tissue dehiscences can be avoided, the ePTFE
membranes allow for slightly more bone regenera-
tion than bioresorbable ones.
Choice of material
Obviously, choice of material is very critical when it
comes to bioresorbable membranes for GBR. A vari-
ety of bioresorbable and non-resorbable devices for
GBR have been evaluated in a recent animal and
clinical experiments. Depending on the material,
inammatory reactions have been documented in
Barrier membranes
37
Fig. 1. a) Radiographic view of a single tooth gap in the
anterior maxilla following extraction of a partially
resorbed central incisor. b) A screw implant (ITI
1
Dental
Implant System, Straumann AG, Waldenburg, Switzer-
land) has been placed at the location of the right central
incisor of the maxilla. A dehiscence defect is visible expos-
ing part of the buccal aspect of the rough surfaced implant
body. c) A bioresorbable collagen membrane (Bio-Gide,
Geistlich AG, Wolhusen, Switzerland) supported by a
deproteinized bovine bone mineral (Bio-Oss, Geistlich)
has been placed in an attempt to augment the bone buc-
cal to the implant, thus integrating the titanium surface.
d).At the reentry surgery 6 months later, the initially
exposed surface of the implant is completely covered by
newly formed bone. The buccal bone plate is of sufcient
width to adequately support the soft tissues. e) Radio-
graphic view of the implant following incorporation of
the nal reconstruction. Note the excellent height of
the crestal bone and the adaptation of the bone to the
implant surface.
38
Hammerle & Jung
the tissues adjacent to bioresorbable membranes,
ranging from mild (1, 93, 97) to severe (37). Another
recent study reported on therapeutic failures using
polylactic membranes for bone regeneration at peri-
implant defects in dogs (98). Soft tissue complica-
tions were frequent, and the results did not reveal
any improvement over control sites without the use
of membranes.
Bioresorbable membranes that are commercially
available at present are not capable of maintaining
adequate space unless the defect morphology is very
favorable (77). Even if the membranes initially seem
able to maintain space, they generally lose their
mechanical strength soon after implantation into
the tissues. Only in situations where the bony bor-
ders of the defects adequately support the membrane
have favorable results been reported. When defects
are not space making by themselves, failure of bone
regeneration results (82, 126). Therefore, they need to
be supported in one way or another.
A different approach was taken in experimental
studies evaluating a form-stable bioresorbable mem-
brane made of polylactic acid in conjunction with a
bone substitute in a rabbit skull model (41, 43, 100).
New bone was demonstrated to form underneath the
membrane beyond the borders of the former calvar-
ium. On the one hand, this experiment demonstrated
that, in principle, stiff, bioresorbable membranes are
conducive to bone regeneration and bone neoforma-
tion. On the other hand, after the observation period
of 4 months, no overt signs of breakdown of the
membrane were reported. In many clinical situations
a resorption time not extending beyond 612 months
is mandatory in order not to lose the advantages of
resorbability.
Bioresorbable materials that may be used for the
fabrication of membranes all belong to the groups of
natural or synthetic polymers. The best-known
groups of polymers used for medical purposes are
collagen and aliphatic polyesters. Currently tested
and used membranes are made of collagen or of
polyglycolide and/or polylactide or copolymers
thereof (for review see (54)).
Convincing results of bone regeneration in animal
experiments have been reported for collagen mem-
branes (53, 125). Furthermore, reports of human
cases or case series (45) as well as controlled clinical
studies (130) have been presented describing the
successful use of collagen membranes for GBR at
exposed implant surfaces.
In two recent experiments, a polylactic acid (PLA)
membrane was tested for its ability to increase the
bone volume in conjunction with an autogenous
bone graft; this was compared to controls that were
grafted only (74, 75). Both experiments showed more
bone formation when the membranes were applied.
These results and results from other investigations
(83, 109) demonstrate that soft PLA or polyglycoid
acid (PGA) membranes are suitable for GBR proce-
dures in conjunction with autogenic grafts.
In contrast, unfavorable results were obtained
using a bioresorbable barrier composed of poly
(d,l-lactic)-co-trimethylene carbonate (52). Control
sites treated with ePTFE showed signicantly better
bone regeneration. Generally, only a mild inamma-
tory reaction was found associated with the mem-
branes. Five months following implantation, some of
the membranes had only just started to lose their
original integrity, while others were completely
resorbed. This high variability in resorption kinetics
complicates clinical use of the material. Similar unfa-
vorable ndings were documented in another study
using the same PLA membrane (98).
In summary, animal experiments, human case
reports and initial controlled clinical studies demon-
strate that bioresorbable collagen, PLA and PGA
membranes can be used successfully for bone regen-
eration at implants with exposed surface areas.
Table 1. Characteristics of bioresorbable membranes
Advantages
No need for membrane removal surgery
Simplified surgical procedure with an implant system with two-stage surgical approach
Wider range of surgical techniques possible at abutment connection (which coincides with membrane removal for non-
resorbable membranes)
Better cost-effectiveness
Decreased patient morbidity
Disadvantages
Uncontrolled duration of barrier function
Resorption process possibly interfering with wound healing and bone regeneration
Need for membrane supporting material
39
Barrier membranes
Types of bone defects
Horizontal bone defects include dehiscence, fenes-
tration and infra bony defects.
A large number of animal experiments have
demonstrated the successful use of bioresorbable
membranes in GBR (7, 24, 43, 64, 74, 75, 76, 97, 99,
100, 104, 125), whereas others have reported failures
(28, 37, 72, 98, 123).
Horizontal bone defects
Since the early 1990s, various studies have been
published describing the successful clinical employ-
ment of bioresorbable PLA and PGA membranes for
GBR at exposed implant surfaces (8, 77, 79, 89, 109).
In contrast, some investigators have reported a
reduced defect ll when applying bioresorbable
(60%) compared to non-resorbable (8184%) mem-
branes (72).
In one of these studies, 18 implants with exposed
surfaces were treated in nine patients (109). In the
test sites, bioresorbable membranes of polylactic and
polyglycolic acid (PLA/PGA) were used, whereas
non-resorbable membranes of ePTFE (GTAM) were
applied in the control sites. In addition, autogenic
bone was placed to cover the exposed implant
threads prior to membrane adaptation. The results
at reentry 67 months later revealed a more favorable
healing using ePTFE (98% defect ll) compared to
the bioresorbable group (89% defect ll). This pattern
of increased bone formation resulting from the use of
ePTFE membranes compared to PLA/PGA mem-
branes has been demonstrated in a histologic study
in humans (108).
Vertical bone defects
The indications for vertical ridge augmentation
include situations where the remaining bone height
is too small for proper anchorage of oral implants;
unfavorable crown to implant ratios and unfavorable
esthetic outcomes will result from the lack of remain-
ing hard and soft tissues.
Data from animal experiments have clearly
demonstrated that growth of bone above the external
borders of the skeleton was possible using GBR (43,
64, 65, 74, 78, 100, 101).
In a recently presented model system, vertical
growth of bone has been studied experimentally in
humans (42). Titanium hollow cylinders were placed
in the retromolar area of healthy subjects. The regen-
erating tissue was harvested at different time points.
The results revealed that up to 12 weeks soft tissue
was lling the cylinder space, while increasing
amounts of mineralized bone was lling the space
at later time points up to 9 months.
In the rst attempt to augment bone above the
borders of the alveolar crest at titanium implants in
dogs, reinforced ePTFE membranes showed a gain of
1.8 mm, standard ePTFE membranes revealed a gain
of 1.9 mm, and in the controls without membranes
the bone height increased by 0.5 mm (58). No graft
materials had been placed. In both membrane
groups, about 1 mm of non-mineralized tissue was
present between the mineralized bone and the mem-
brane at its highest point. Non-mineralized tissue
has been reported in contact with the inner surfaces
of membranes in other studies as well (57, 106).
In the rst clinical study on vertical ridge augmen-
tation in humans, a titanium-reinforced ePTFE
membrane was used to cover implants that were
allowed to protrude up to 7 mm above the crest
(107). The results after 9 months of submerged heal-
ing showed bone formation reaching up to 4 mm
above the previous border of the alveolar crest. The
remainder of the space between the newly formed
bone and the membrane was occupied by non-
mineralized tissue. Within the area of the newly
formed bone, osseointegration of the implants had
occurred as demonstrated by histologic analysis of
experimentally retrieved test implants.
In another study, six patients were treated with
titanium-reinforced membranes and autogenic bone
collected in a suction lter (114). Twelve months
following membrane placement, an average gain of
5 mm of vertical bone height was measured, reach-
ing up to a maximum of 7 mm.
Later studies reported improved amounts of verti-
cal bone gain when using autogenous bone grafts or
bone substitute materials in addition to the titanium-
reinforced ePTFE membranes (110, 115).
The ePTFE/tissue interface has recently been char-
acterized in humans (31). The tissue layer in contact
with the membrane was described as non-minera-
lized, rich in cells and vessels, and containing irre-
gularly distributed collagen bers. According to the
investigators, this tissue layer may have developed
due to micro movements or broblasts penetrating
through the membrane from the covering soft tissue
ap. The phenomenon of non-mineralized tissue in
contact with membrane surfaces has been reported
for bioresorbable materials as well (94, 100). The
reasons for this are not fully understood and need
clarication. Furthermore, it has been postulated
40
Hammerle & Jung
that lack of adequate stability of membranes will
prevent complete bone formation.
Time frame of implantation
In relation to GBR surgery
In principle, attempts at regenerating bone for
improved anchorage of oral implants may be per-
formed in conjunction with the placement of the
implants or during a surgical intervention prior to
implant placement.
The staged approach is primarily chosen in situa-
tions with large bone defects. Such situations often
do not allow obtaining primary stability of the
implants in the prosthetically desired position.
Hence, the alveolar ridge is augmented in a rst
surgical intervention. After the appropriate time for
healing, the implants are then placed into a site of
sufcient bone volume.
Staged approach
Experimental research on ridge augmentation using
GBR was presented in the early 1990s (103). In a dog
model, large defects of the alveolar ridge were surgi-
cally prepared both in the mandible and in the max-
illa to allow for four different treatment modalities.
The defects were treated one of the following ways:
covered with ePTFE membranes;
covered with membranes and grafted with porous
hydroxyapatite or with a tissue growth matrix of
porous PTFE;
grafted with these same materials but not covered
with membranes;
neither grafted nor covered.
Morphological and histologic analysis revealed that
in sites treated with membranes, with or without the
addition of grafts, the entire space between the mem-
brane and the jawbone was lled with bone. In the
absence of membranes, bone formation did not occur.
More recently, studies in humans and animals have
lead to further development and renement of this
method. In a clinical study involving 40 partially eden-
tulous patients lateral ridge augmentations were
performed prior to implant placement (21). Ridge
width at the rst surgical intervention precluded
implant placement in prosthetically proper position.
Autogenous cortico-cancellous block grafts and parti-
culate bone were used in conjunction with ePTFE
membranes for ridge augmentation. At the reentry
surgery 9 months later, the bone volume had
increased to values sufcient for implant placement
in all cases. The investigators concluded that the
combination of autogenous bone grafts and ePTFE
membranes rendered predictable success.
The effect of barrier membranes on the resorption
of bone block grafts has recently been studied in an
animal experiment (6). Resorption of the grafted
bone occurred to various degrees at sites not covered
with ePTFE membranes, whereas at the sites with
membranes no loss of graft volume due to resorption
was recorded.
The necessity for membranes was tested in a pro-
spective randomized clinical study involving 13
patients (3). Patients were either treated with onlay
bone grafts alone or additionally covered by ePTFE
membranes. The width of the ridges was clinically
evaluated immediately following graft placement
and at the time of membrane removal 6 months
later. In the group with membranes signicantly less
resorption had occurred. This controlled clinical
study conrms animal experimental data and is in
accordance with previous case series reporting the
occurrence of pronounced resorption of bone grafts
(4, 121, 27).
Grafts of autogenous bone have traditionally been
used for augmentaion of narrow ridges prior to
implant placement. In a recent clinical study a
deproteinized bovine bone mineral was applied in
conjunction with a bioresorbable collagen mem-
brane (128). Spongiosa granules of 0.251 mm parti-
cle size were mixed with sterile saline and
tetracycline powder and applied to the defect. The
grafting material was subsequently covered with a
collagen membrane, which was xed to the adjacent
bone by use of titanium pins. Flaps were adapted and
sutured for primary healing. After 67 months, reen-
try surgeries were carried out. Biopsies taken from
the augmented sites reveal the grafted particles to be
well integrated into the newly formed bone.
In another study the sites were augmented with
bioactive glass immediately following tooth extrac-
tion. Membranes were not used in all cases. An overall
success rate of 88.6% was reported for the implants
after a mean period of function of 29.2 months. For-
mation of new bone in association with the grafting
particles was histologically documented.
These newer studies indicate that provided the
appropriate protocols are developed, ridge augmen-
tation without the use of autogenous block grafts
could possibly evolve into a standard procedure.
Combined approach
Compared to the staged approach the combined
method offers some advantages:
41
Barrier membranes
decreased patient morbidity due to the fact that
only one surgical intervention is necessary;
decreased treatment time since regeneration and
implantation are performed at the same time;
decreased costs.
Based on these advantages it has been postulated
that the combined approach is to be preferred when-
ever the clinical situation allows doing so (Fig. 2).
One of the key points of discussion regarding
combined implantation and regeneration was the
Fig. 2. Preoperative view of a single tooth gap in the ante-
rior maxilla following extraction of a central incisor. (a:
occlusal view; b: buccal view). Intraoperative view of a
screw implant (TiUnite Mk III Branemark
1
, Nobel Biocare,
Gothenburg, Sweden) showing a buccal and oral dehis-
cence. (c: occlusal view; d: buccal view). The defect site
has been grafted with a deproteinized bovine bone mineral
(Bio-Oss, Geistlich) and autogenous bone harvested from
the surrounding area. A titaniumreinforced non-resorbable
ePTFE membrane (Gore-Tex TR, W.L. Gore & Associates,
Inc., Flagstaff, USA) has been trimmed and shaped to cover
the bone substitute materials and the implant. The
membrane has been secured at the buccal aspect by two
non-resorbable pins. (e: occlusal view; f: buccal view). Six
months after implantation and regeneration, a signicant
gain of the buccal contour is discernable. (g: occlusal view;
h: buccal view). At membrane removal surgery, the surface
of the implant is completely covered with new bone (i). A
pronounced gain of bone also in buccal direction is obser-
ved following uncovering of the closure screw(j). Radiogra-
phic view of the implant with an individualized zirconium
oxide abutment screwed onto the implant. Note the close
adaptation of the bone at the surface of the implant and the
crestal bone level being located coronal to the st thread.
42
Hammerle & Jung
question whether or not implant surfaces initially
not covered by bone can successfully be osseointe-
grated by the regenerating bone under GBR. A
large number of animal experimental studies have
histologically demonstrated that such surfaces are
truly osseointegrated during the process of bone
regeneration (10, 11, 63, 118). In addition, studies
in humans have conrmed the ndings reported
in the animal experiments mentioned above (91,
92, 122).
In relation to tooth extraction
With respect to loss or extraction of teeth, implants
may be placed in an immediate, a delayed or a late
fashion. The time requirements for immediate
Fig. 2. continued
43
Barrier membranes
implant placement are fullled when the implant is
placed into an extraction socket during the same
surgical intervention as the removal of the tooth to
be replaced by the implant. The delayed approach
indicates that several weeks, usually up to a maxi-
mum of 12 weeks, have passed between tooth extrac-
tion and implant placement. In situations where
more time elapses between tooth extraction and
implant placement the term late implant placement
is appropriate.
In a recent retrospective study immediate, delayed
and late implant placements were compared using
survival data as well as clinical and radiographic
parameters (119). The survival rate for all groups
was above 97% 2 years after implantation. In another
investigation, no difference regarding bone ll was
found between immediate and delayed implant pla-
cement at reentry surgery (130). In this study the
defects had been treated by collagen membranes
and a deproteinized bovine bone mineral.
Immediate implant placement
Increasing numbers of clinical studies demonstrate
that the immediate placement of implant into extrac-
tion sockets is a method that renders success rates
similar to the ones reported for conventional implant
placement. Success rates usually well above 90%
have been reported in a large number of studies
(14, 16, 18, 33, 35, 102).
A problem encountered when placing implants
immediately into extraction sockets is a lack of soft
tissues for primary healing. This soft tissue decit is
due to the area of tissue penetration of the tooth
prior to extractions. Hence, in order to obtain pri-
mary closure, the buccal ap is often mobilized by
vertical and horizontal releasing incisions. However,
as a result the soft tissue contours, in particular the
level of the mucogingival line, are altered and the
esthetic outcome may be hampered. Consequently,
techniques have recently been published aimed at
facilitating primary ap closure through the place-
ment of pedicle or free grafts (23, 32, 84, 96). Closure
of the wound over an immediate implant can suc-
cessfully be obtained by connective tissue grafts,
eliminating the need for excessive mobilization of
the buccal ap (23, 32). In addition, this technique
increases the amount of keratinized tissue and thus
provides better conditions for an optimal esthetic
result.
Immediate implants without regeneration
Whereas controlled human studies have demon-
strated enhanced bone regeneration when exposed
implant surfaces are treated with barrier membranes
(29, 90), other investigators have reported adequate
bone formation in extraction sockets without the use
of membranes (12, 92, 102, 112, 122).
In a prospective controlled human clinical trial,
test implants were placed into extraction sockets
immediately after tooth extraction (92). Implants
were located in the premolar and molar region of
maxilla and mandible. The diameter of the experi-
mental implants was chosen with the aim of obtain-
ing as much contact with the socket walls as possible.
Thus, the distance from the bone to the implant sur-
face was consistently kept at values smaller than
2 mm. The neck of the implants was located at the
level of the alveolar crest. No lling materials or
membranes were applied. Control implants were
inserted into sites with sufcient bone volume. Pri-
mary wound closure was performed at both test and
control implants. Six months after implant place-
ment, a second stage surgery was performed and
healing caps were inserted. After another 6 months,
test and control implants were removed with an
appropriate trephine drill. Results of clinical mea-
surements, radiographic assessments of bone levels,
histologic analysis of crestal bone levels and surface
fraction of the implants in direct bone contact
revealed no difference between test and control
implants. In only three out of 48 test implants was a
crestal zone of connective tissue contact discernible
extending 1.52 mm. The investigators concluded that
implant placement immediately into extraction sock-
ets leads to tissue integration similar to the one
obtained with implants placed into intact alveolar
crests provided the gap width is smaller than 2 mm.
In a human study treating more demanding
defects including dehiscence defects with a mean
depth of 7 mm, a deproteinized bovine bone was
used to ll the gaps between the implants and the
bony walls of the defects (112). Again, no membranes
were used to cover the defect areas. Although, defect
ll was not as predictable as reported in other studies
using membranes (108), the majority of the initial
defect area was lled with new bone at the 6-month
reentry surgery. Compromised results were asso-
ciated with exposure and loss of the grafting material.
There seems to be some consistent results that a
gap width of less than 2 mm between the implant
and the socket wall will be lled with bone in the
absence of membranes. However, more research is
needed to characterize the clinical conditions, where
membranes in bone regeneration procedures are not
necessary and where they are necessary for predict-
able success.
44
Hammerle & Jung
Delayed implantation
Since ap dehiscence and resulting membrane expo-
sure are the most frequent complications using the
immediate implantation protocol, differing treatment
concepts have been developed. In contrast to immedi-
ate implantation following tooth extraction, the
delayed approach is aimed at allowing soft tissue
healing prior to the surgical intervention. The in-
creased amount of soft tissue present simplies the
surgical technique for ap closure. Furthermore, it
renders esthetically optimal results more predictable.
Since the resorption processes of bone are rather
small, the loss in bone volume occurring during the
rst 12 weeks will usually not hamper placement of
the implant and can be compensated by the planned
regeneration procedure.
In a recent study a comparison between the fre-
quency of membrane exposures was done between
three treatment groups: immediate implantation,
delayed implantation, and late implantation (130).
The results demonstrated that in particular when
using non-resorbable ePTFE membranes, the inci-
dence of membrane exposure was signicantly ele-
vated when using the immediate (60%) as compared
to the delayed approach (17%). The late approach,
however, was associated with even fewer exposures
than the delayed approach. Although still present,
these differences were less pronounced when a bior-
esorbable collagen membrane was used.
The disadvantages of the delayed approach
include the increased treatment time, the need for
an additional surgical intervention, the fact that the
excellent bone regenerative potential of the fresh
extraction socket can no longer fully be taken advan-
tage of, and the decreased bone volume resulting
from the occurring bone resorption during soft tissue
healing.
Late implantation
Late implant placement is performed in situations
where teeth have been extracted months before. Clin-
ical experience shows that many implantation proce-
dures can be performed in a standard way without the
need for GBR. Such procedures would qualify for the
term late implantation. However, the focus of this
review lies on treatment using GBR for compromised
sites. In situations where the late approach is chosen
and the need for GBR is still present, no signicant
advantages have been described compared to GBR
using the delayed approach.
However, it has been reported that the incidence of
soft tissue dehiscence and subsequent membrane
exposure is somewhat smaller when using the late
approach compared to the delayed one (130). This
difference may have signicant effects since bone
formation is generally less favorable when dehis-
cences occur, compared to situations where the soft
tissues remain intact during the entire regeneration
period (13, 20, 44, 51, 67, 106, 117, 127).
Soft tissue dehiscences represent an important
complication during GBR with ePTFE membranes.
They range from a minor problem affecting only a
few percent of membranes up to a serious complica-
tion leading to a high rate of complete treatment
failures, impaired results, and suffering for the
patient (30, 72, 109, 117, 127, 129). This emphasizes
the importance of carefully balancing benets and
risks when planning GBR therapy. Based on the
reports in the literature, there is general agreement
that GBR is a difcult procedure to perform and that
the skills and experience of the clinician are a critical
factor in the success of treatment. Recently, evidence
of lower rates of dehiscences associated with biore-
sorbable membranes compared to non-resorbable
membranes has been presented, further promoting
the use of bioresorbable membranes.
Clinical mode of healing
Submerged implants
In general, when GBR is performed in conjunction
with implant placement a submerged mode of heal-
ing is attempted. The rationale for attempting a sub-
merged mode of healing includes the prevention of
bacterial contamination of the implant and the
membrane surfaces by bacteria from the oral cavity.
In addition, in esthetically difcult regions an addi-
tional soft tissue volume is present at the initiation of
the prosthetic procedures, which increases the range
of therapeutic possibilities to reach an optimal
esthetic result. Finally, a submerged implant has
been claimed to be optimally protected from adverse
loading forces (2).
In contrast, recent studies have demonstrated that
successful treatment outcomes may also be obtained
when implants are kept in a transmucosal location
during bone regeneration and tissue integration (18,
25, 40, 45, 66). Furthermore, histologic data obtained
in animal experiments as well as from human speci-
mens demonstrate osseointegration of the previously
exposed surface areas (63, 122).
In contrast to the submerged approach, transmu-
cosal healing eliminates the surgical intervention
45
Barrier membranes
necessary to obtain access to the implant head for
performing the prosthetic procedures.
Transmucosal implants
The technique for transmucosal positioning of
implants during GBR procedures was rst described
in a clinical report of a series of cases (25). Subse-
quently, another series of cases using ePTFE mem-
branes for regeneration of bone around implant
immediately placed into extraction sockets was pre-
sented (66). In this study a treatment regimen includ-
ing the directions for meticulous plaque control was
described involving both mechanical means and
chemical agents. The results demonstrated 20 out
of 21 implants with complete defect ll with new
bone upon membrane removal surgery. In a retro-
spective controlled study, transmucosal implants
with GBR were compared with transmucosal
implants that did not need any GBR procedures
(18). No differences were found regarding the pre-
sence of plaque, the prevalence of bleeding on prob-
ing, values for the level of the mucosal margin, or
probing depths around the implants.
Two recent studies presented a defect ll of 94%
when using ePTFE membranes (40) and 86% when
using bioresorbable membranes (45) for GBR at
transucosal implants. Measurements were taken at
the time of implant placement and 6 months later.
In summary, although studies on bone regenera-
tion at transmucosal implants are still not very
numerous, the available data demonstrate that GBR
at transmucosal implants is a successful procedure.
Hence, primary closure of the site of implantation
and regeneration is not a prerequisite for successful
treatment outcomes. However, investigators report-
ing on the transmucosal technique stress the impor-
tance of meticulous plaque control during the
regeneration period.
Long-termresults
Survival rate of implants placed into sites with regen-
erated/augmented bone using barrier membranes
have been reported to vary between 79% and 100%
with the majority of studies indicating more than 90%
after at least 1 year of function. Thus, in a number of
studies a total of 656 implants treated with GBR
yielded survival rates ranging from97.5% to 100% after
a 2-year observation period (19, 29, 73, 80, 86, 73).
Three studies reported 5-year data (15, 22, 131).
Survival rates were 79.4% for 37 implants with dehis-
cence/fenestration defects treated with ePTFE mem-
branes (15), 92.6% for 41 implants treated with
ePTFE membranes (131), 93.9% for 33 implants in
extraction sites treated with ePTFE membranes (15)
95.4% for 112 implants treated with a collagen mem-
brane (131), and 100% for 12 implants treated with
e-PTFE membranes (22).
In a 5-year prospective controlled study, implants
placed into pristine bone were compared with
implants associated with bone regeneration (131).
No signicant differences were found regarding the
cumulative implant survival rates in the three groups:
sites augmented with a collagen membrane and a
deproteinized bovine bone mineral (95.4%), sites
augmented with an e-PTFE membrane and a depro-
teinized bovine bone mineral (92.6%), and sites with-
out augmentation (97.3%).
Another study providing controls reported on 38
implants in seven patients (80). In this study, 21
implants were placed in conjunction with GBR and
17 could be placed into the host bone without the
need for regenerative therapy. A polylactic, polygly-
colic acid membrane was used in all regenerative
procedures. After an average of 25 months following
incorporation of xed reconstructions, a 100% survi-
val rate was found for both test and control implants
and no signicant difference in marginal bone levels
was recorded between the two groups.
A multicenter evaluation of implants inserted at
the time or after vertical ridge augmentation revealed
stable crestal bone levels over a period of 15 years
(111). Initially, 123 implants had been placed. At the
follow-up examination one implant had been lost,
while the remainder could be reexamined. Augmen-
tation had been performed using ePTFE membranes
in conjunction with a blood clot, with demineralized
freeze-dried bone allografts or with autografts. Stable
crestal bone levels were reported in the majority of
the implants. Only two implants demonstrated an
increased crestal bone loss of 3.5 mm and 4 mm,
respectively. The investigators concluded that verti-
cally regenerated bone reacts to implant placement
in a manner similar to non-regenerated bone.
Although the data are derived from a small number
of studies showing an adequate design, survival rates
similar to the ones generally reported for implants
placed conventionally into sites without the need for
bone augmentation have been documented.
Outlook into the future
In recent years, many developments in medicine
have been based on a better understanding of
46
Hammerle & Jung
the development of diseases at the cellular and
the molecular level. This is a key step to the devel-
opment of new strategies and materials in medicine.
The strategies in most of the medical disciplines
are changing in character from a mechanical to a
more biological one. The aim is to come up with
more effective techniques that predictably promote
the bodys natural ability to regenerate lost tissue
instead of using external materials to repair lost
tissue.
The most intriguing method currently being inves-
tigated is the application of polypeptide or natural
proteins that regulate wound and tissue regenera-
tion. With the improvements in cellular and molecu-
lar technologies to isolate, sequence and produce
these molecules, a new eld of therapeutic options
will be opened.
Growth and differentiation factors in oral
surgery
In the past decade a number of basic science experi-
ments and clinical studies have claried biological
mechanisms of several growth and differentiation
factors in the regeneration of oral soft and hard tissue
(49). Growth and differentiation factors currently
believed to contribute to periodontal and alveolar
ridge augmentation include platelet-derived growth
factor (PDGF), insulin-like growth factor (IGF-I and -
II), transforming growth factor beta (TGF-a and -b),
broblast growth factor (a- and b-FGF), and bone
morphogenetic proteins (BMPs 1-15). Of these, bone
morphogenetic protein is the most widely studied in
the dental literature. In 1965, Urist discovered the
inductive bone capacity of a protein extracts
from demineralized bone (116). This protein extract
was termed bone morphogenetic protein (BMP). The
induction process triggered by BMPs involves
chemotaxis, proliferation and differentiation of
mesenchymal progenitor cells (124). Recombinant
biotechnology has made it possible to characterize
at least 15 BMPs and to produce quantities of pur-
ied recombinant protein for therapeutic application
(39). Recombinant human BMP-2 (rhBMP-2) has
been found to exhibit very high osteogenic activity
in experimental (26, 46, 48, 105) and clinical studies
(17, 46).
Local delivery of molecules
The regenerative potential of growth and differentia-
tion factors depends upon the carrier material (50,
105). The effect of such proteins is dependent upon a
carrier material, which serves as a delivery system
and as a scaffold for cellular ingrowth (95).
Collagen has been extensively studied as a carrier
material for growth factors applied to promote bone
formation in different kinds of indications (17, 49,
85). Different studies (9) using rhBMP-2 in an
absorbable collagen sponge (ACS) for alveolar ridge
augmentation gained only small amounts of bone.
The investigators concluded that the compromised
results were due to the failure of the ACS to ade-
quately support the supra-alveolar wound space. In
order to overcome this lack of structural strength, rh-
BMP-2/ACS has been combined with hydroxyapatite
in an experiment attempting lateral ridge augmenta-
tion (9). In contrast to rhBMP-2/ACS alone, a signif-
icant clinical improvement in ridge width was
observed with the addition of HA to rhBMP-2/ACS.
However, the hydroxyapatite particles were largely
encapsulated by brous tissue and they appeared
to partially obstruct bone formation. The investiga-
tors concluded that space maintenance in BMP-
induced bone formation is an important factor (9).
Previous animal studies indicated that a xenogenic
bone substitute mineral performed well as carrier
material for osteoinductive proteins (105, 113). A
recent study demonstrated that the results of GBR
procedures in human could be improved by the addi-
tion of rhBMP-2 to a xenogenic bone substitute
mineral. This improvement was documented by a
higher degree of bone maturation and an increased
graft to bone contact fraction at the BMP-treated
sites (59) (Fig. 3). In addition, the xenogenic bone
substitute mineral has some therapeutic advantages
over HA due to its three-dimensional structure and
its resorbability (56). Synthetic polymers, on the
other hand, offer possibilities for being combined
with biologic factors. Synthetic polymers can repro-
ducibly be fabricated and the growth factors can be
incorporated under controlled manufacturing condi-
tions (120).
However, the ideal carrier material, which is easy
to apply, able to provide space for regeneration, bior-
esorbable, and allows controlled release of the bioac-
tive molecules has not yet been discovered. Further
research is needed to determine the ideal combina-
tion of factors for regeneration, the best delivery sys-
tem, and the optimum doses.
Membranes
The benet of using both barrier membranes and
BMP for bone augmentation remains controversial
(26, 49, 59, 71). In a recent study it was found that
47
Barrier membranes
non-resorbable ePTFE membrane initially inhibited
bone formation with rhBMP-2 (4 weeks) but not at
later time points (12 weeks) (26). It has been shown
that bone induction by rhBMP-2 occurs at early time
points and that rhBMP-2 undergoes rapid clearance.
Hence, the use of a membrane may potentially
reduce the bone-forming effect of rhBMP-2 due to
limited availability of inducible cells. Another animal
study in rats reported no difference in bone healing
with the combination of rhBMP-2 and an ePTFE
membrane compared to rhBMP-2 alone (71). From
a clinical point of view, the use of a membrane sim-
plies the handling and the stabilization of the bone
substitute mineral at the time of bone augmentation,
but from a biological point of view the use of a
membrane may block the recruitment of cells from
the environment.
The question whether or not bioactive molecules
are best used in conjunction with membranes or in
the absence of membranes has not been answered
conclusively yet. On the one hand, it is reasonable to
apply them as adjunctive agents to GBR therapy to
accelerate the membrane-guided bone regeneration.
On the other hand, it may be hypothesized that their
mode of action is best taken advantage of when
membranes are not simultaneously applied, thus
allowing all inducible cells from the wound environ-
ment access to the area to be regenerated.
Developments in bone augmentation
procedures
Further developments in bone augmentation proce-
dures can be related either to simplication of the
clinical handling or to inuencing of biological pro-
cesses. To simplify clinical handling, new materials
should comprise a matrix with optimal cell ingrowth
capacities and good mechanical properties, provid-
ing space for tissue regeneration. No membrane and
no specic procedures for mechanical xation
should be necessary. This would reduce the techni-
que sensitivity and increase the predictability of bone
augmentation.
From a biological point of view the growth and
differentiation factors may induce earlier bone
growth into the area to be regenerated. Thus, the
area of regeneration would be stabilized at an earlier
time point. Furthermore, the use of such materials
would allow treatment of extended bone defect
volumes. At present, large bone defects are regularly
augmented with autogenous block grafts and mem-
branes (5). The use of synthetic materials would
result in lower surgical risks and lower morbidity in
augmentation procedures and would represent an
important step forward in simplifying bone regenera-
tion techniques.
Conclusions
Presently available data demonstrate GBR therapy
to be a predictable and successful procedure to
augment bone in a horizontal direction at sites
exhibiting insufcient bone volume for implant
placement under standard conditions.
Although data are not plentiful, vertical augmenta-
tion using this technique has successfully been
performed by various groups of investigators.
An increasing body of literature demonstrates
bioresorbable membranes to render success rates
similar to the ones obtained with non-resorbable
membranes for the treatment of horizontal defects.
Fig. 3. Histologic section of a site treated with Bio-Oss and
rhBMP-2 (a) showing a signicantly higher surface frac-
tion of the bone substitute particles in direct contact with
newly formed bone compared to a site treated with
Bio-Oss alone (b). Lamellar bone (lb); woven bone (wb);
non-mineralized tissue (nt), bone substitute particles (bo).
(Original magnication 100; toluidine blue stain).
48
Hammerle & Jung
Some studies indicate that implant placement
immediately following tooth extraction without
the use of membranes may lead to complete bone
regeneration of the gap between the implant and
the socket wall, provided this gap does not surpass
a certain distance.
A few long-term studies indicate that the survival
rate of implants partly resting in augmented bone
is similar to implants placed in native bone.
It is expected that bone regeneration using growth
and differentiation factors will play an important
role in the future.
The literature on GBR for implant placement
is mostly limited to compromised sites and studies
evaluating this procedure in compromised
patients are not available.
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53
Barrier membranes
Distraction osteogenesis for
vertical bone augmentation prior
to oral implant reconstruction
Bradley S. McAllister & Thomas E. Gaffaney
Distraction osteogenesis has been employed in the
lengthening of long bones for the last 100 years (8).
During the last 50 years, predictable results having
been developed through scientic studies by the
Russian surgeon, Gavriel Ilizarov (1619). The basic
principle developed by Ilizarov has the following
three distinct phases:
a latency phase of approximately 7 days of initial
post surgical healing;
the distraction phase, consisting of the gradual
incremental separation of two bone pieces at a rate
of approximately 1 mm per day;
consolidation phase, during which new bone forms
in the regenerate zone between the separated bone
pieces.
Over the last 7 years the technique of distraction
osteogenesis has been under development for verti-
cal augmentation of the mandible and maxilla prior
to implant reconstruction (6). With such a short his-
tory of employing alveolar distraction for the specic
application of implant reconstruction, the technique
is clearly still in its infancy. Yet, one must not lose
track of the fact that the general principle of distrac-
tion osteogenesis has been extensively investigated
and successfully applied to a variety of bone pro-
blems over the last century.
While no controlled long-termstudies exist with any
of the commercially available alveolar bone devices
available today, excellent case reports and animal stu-
dies exist for the distraction devices currently avail-
able. These publications demonstrate the potential for
successful results with a variety of intraosseous and
extraosseous distractors. The rst published case
report of alveolar bone distraction was that of a single
mandibular case using an intraosseous distractor with
a threaded rod, a threaded transport plate and a sta-
bilizing unthreaded base plate (6). Additional case
reports have been published over the past few years
with intraosseous, extraosseous and implant distrac-
tors (4, 10, 15, 21, 24, 34). Recently, a case history
review of prosthetically restored distraction cases,
loaded for a minimum of 3 years, revealed a success
rate of 90.4% for the 84 implants placed in distracted
bone utilizing devices that are not commercially avail-
able (20). This high rate of success compares favorably
with other reports evaluating rates of oral implant
success in regenerated bone (14).
Several animal studies have been published demon-
strating successful vertical augmentation. Customized
ossseointegrated implant supported distraction dev-
ices were utilized in dog mandibles to gain 9 mm of
vertical augmentation. The regeneratedbone was eval-
uated histologically (2, 3). The distraction gap, or
regenerate zone, was lled with new bone and both
a lingual and buccal cortex were formed. The crestal
bone levels did not change during a year of implant
loading (3). Additional dog studies by Oda and collea-
gues have evaluated botha two-stage andsingle-phase
approach to implant placement (27, 28). In the single
stage approach, implants were used as the distraction
device and left to integrate (27). While the regenerated
bone in the distraction gap was found to consist of a
comparable percent of bone area, 20% of the implants
failed to integrate, minimizing the clinical validity of
this approach. Their more successful two-stage
approach utilized a simple intraosseous screw for
active distraction. Implants were placed during the
consolidation phase and evaluated at 12 weeks. Less
than 1 mm of crestal loss was found and minimal
differences were noted between the transport segment
and the regenerated bone for both percent bone area
and percent bone-to-implant contact.
54
#
PERIODONTOLOGY 2000
ISSN 0906-6713
Studies using in vitro techniques to gain a better
understanding of the phenomenon of distraction
osteogenesis have evaluated how osteoblasts respond
to mechanical stimulation. Elevations ingrowthfactor
and cytokine gene expression have been demonstra-
ted in response to the mechanical stimulation in vitro
(7). Together with the extensive animal studies in long
bones and the numerous craniofacial applications
(25, 32), the principle of distraction osteogenesis is
now a part of the periodontist's armamentarium for
implant placement.
Surgical technique of distraction
osteogenesis
Proper treatment planning is imperative for distrac-
tion osteogenesis. Typically, for distraction to be
considered, a minimum of 67 mm of bone height
must remain above vital anatomic structures and at
least a 4 mm vertical defect of sufcient length
(edentulous zone of three or more missing teeth)
must exist when measuring from the height of the
adjacent bony walls to the vertical depth of the oss-
eous defect. In the event teeth adjacent to the eden-
tulous region being considered for distraction show
considerable marginal bone loss, it is reasonable to
consider extraction and extension of the edentulous
zone to create a true vertical defect of at least 4 mm
depth. With no evidence existing that the attachment
level on teeth can be improved through distraction, it
may be necessary to sacrice a compromised tooth
to optimize the amount of vertical bone improve-
ment. In fact, in animal studies, attempts to improve
the attachment level on natural teeth with distraction
were unsuccessful (1). Small vertical defects of only
one or two teeth tend to have a higher rate of com-
plication when distracted and should usually be trea-
ted with conventional bone grafting techniques (20).
While it is desirable to perform distraction under
conscious sedation or general anesthesia, it is possi-
ble to perform the surgical procedure using only
regional anesthesia. Either a vestibular mucosal inci-
sion or a mid-crestal incision placed at the buccal
line angle staying in gingival (keratinized) tissue may
be successfully utilized to access the bone (22, 24). A
full thickness ap is elevated on the buccal aspect
only, taking care not to reect the tissues on the
alveolar crest or towards the lingual. The horizontal
and vertical osteotomies are prepared with either a
ssure bur, or a saw, taking great care not to damage
the lingual periosteum. The specic order of distrac-
tor placement, distractor xation and nal osteot-
omy preparation is specic to the system being
used. Once the distractor is placed and the osteo-
tomies are complete, device function is tested to
make sure that there are no interferences. If the ver-
tical osteotomies slightly converge to the coronal,
and to the lingual aspect, there will be little risk for
interference problems. Suturing can be easily accom-
plished by primary closure using a slowly resorbing
suture material such as vicryl.
A 1-week latency healing period should be
employed prior to initiation of distraction. In young
patients with rapid healing, a shorter period may be
utilized. In older patients, or those with slow soft
tissue healing, a slightly longer latency period may
be utilized. With complete soft tissue closure, dis-
traction may be initiated at a rate of up to 1 mm
per day. A slower rate of distraction may be utilized
in older individuals and in cases of dense bone with
minimal vascularity. It is important to optimize the
incremental traction for proper tension stress and
ultimate osseous healing. While continuous distrac-
tion, or incremental movement over multiple daily
advancements, has been shown to improve the bone
regeneration it is not clear what the optimal rate and
frequency is for alveolar distraction (16, 19, 30). A
reasonable approach for alveolar distraction would
be to have the patient turn the device three times
daily for incremental advancements of 0.25 to
0.33 mm. Optimal bone formation was found to
occur at physiologic levels of 2,000 microstrain with
some decreases in hydroxyapatite crystal formation
by 20,000 microstrain/one cycle per day. Only
brous tissue was formed in specimens distracted
with the hyperphysiologic levels of 200,000 and
300,000 microstrain, indicating the importance of
not placing excessive tension on the tissues (26).
Meyer et al. (26) also demonstrated that peak strain
magnitudes rather than frequency inuence the
bone cell differentiation and matrix production, indi-
cating smaller more physiologic advances, even if
frequent, will likely optimize the bone regeneration.
While some slight crestal resorption is often found
during consolidation, it usually is no further apical
than the adjacent bone levels. Therefore, it may be
benecial to overdistract by 23 mm. Any further
overcorrection may minimize the potential for
bone-to-bone contact between the vertical osteo-
tomies and the transport segment resulting in a
higher incidence of non-union or incomplete dis-
traction gap ossication.
Distractor removal and implant placement will be
performed during the consolidation phase. It is pos-
sible to place implants at the time of distractor
Distraction osteogenesis
55
removal, or it may be benecial to delay placement
until further hard and soft tissue consolidation has
occurred. The decision should not only be based on
how the area is healing, as determined clinically and
radiographically, but also on the position of the pro-
posed implant. In partially edentulous cases the
implant treatment plan typically consists of placing
implants in the locations of the vertical osteotomies,
indicating the need to have adequate consolidation
to prevent segment mobilization during implant
osteotomy preparation, or actual implant placement.
In a signicant number of distraction cases supple-
mental bone and soft tissue grafting may be required
to optimize the nal result (20, 23, 24). As a minimum
timeline for consolidation, the long bone literature
has suggested 5 days per 1 mm of distraction (29).
Standard integration periods consistent with newly
regenerated bone should be employed prior to pros-
thetic loading of the dental implants placed in dis-
tracted bone.
ACE surgical distractor
The intraosseous ACE distractor (ACE Surgical Sup-
ply, Brockton, MA) is made of titanium alloy and has
three main components during active distraction
(Fig. 1). The distractor body engages the bony trans-
port segment with external threads that are of the
same pattern as that of a conventional 3.75 mm oral
implant. The distractor body comes in both a 5 mm
thread length (long body) and a 3 mm thread length
(short body). Unless anatomic constraints exist, it is
advisable to utilize the long body distractor for max-
imal xation. The axial distraction screw is threaded
through the distractor body and used for active dis-
traction. The base plug has an internally threaded
hole in which the axial distraction screw sits and
engages for the distraction process. As the axial dis-
tractor screw is turned in a clockwise direction
(2.5 turns/1 mm), the upper distractor body with
the bony transport segment advances in a coronal
direction away from the intact bony bed with the
stationary base plug. This distraction system has a
very simple removal procedure that does not require
mucoperiosteal ap reection unless implants are
placed at the time of the distractor removal surgery.
At distractor removal the base plug is easily removed
by threading the base plug removal tool onto the
internal threads of the base plug. Reports to date with
this system have shown favorable results (21, 24, 34).
The following examples illustrate the capabilities
of this intraosseous distractor. A 42-year-old male
presented for oral implant reconstruction following
a motor vehicle accident. A signicant vertical defect
was present in the area of missing teeth 32 through
41 (Fig. 2). After horizontal osteotomy preparation,
the distractor was placed. Once distractor stability
was conrmed, vertical osteotomies were completed
utilizing a straight ssure bur (Fig. 3). After a 1-week
latency period, distraction was initiated at the rate of
1 mm/day for 8 days utilizing guidance components
Fig. 1. ACE distractor components during activation. The
axial distraction screw is shown during activation with the
0.88 mm hex driver. A long body distractor with an axial
distraction screw and base plug is shown.
56
McAllister & Gaffaney
and an appropriate temporary (Fig. 4). Without
mucoperiosteal ap reection the distractor was
removed after 2 months of consolidation. After a
total of 4.5 months of consolidation two implants
were placed and a biopsy was taken from the regen-
eration zone (Figs 5 and 6). After a standard integra-
tion period the implants were restored and loaded.
Fig. 2. Radiographic view of the vertical bone loss.
Fig. 3. The segment after placement of the distractor and
completion of both the horizontal and vertical osteotomies
to mobilize the segment.
Fig. 4. Radiographic view at the completion of approxi-
mately 8 mm of distraction.
Fig. 5. Two 18-mm implants have been placed and a
biopsy was taken from the regeneration zone.
57
Radiographic and clinical evaluation after 3 years
shows excellent preservation of bone and soft tissues
(Fig. 7). A 36-year-old female presented with teeth 32
through 43 lost from untreated aggressive periodon-
titis. The 6 mm vertical defect that remained (Fig. 8)
was treated with two distractors (Fig. 9). For this
system, one distractor is typically placed for every
three missing teeth up to a maximum of three dis-
tractors. Following 2 months of consolidation, imp-
lants were placed at the time of distractor removal
and allowed to heal for 7 months prior to nal
restoration (Fig. 10a,b).
As with most distraction systems, it is imperative
that the guidance components and a suitable
Fig. 6. Regeneration zone biopsy. (a) The entire histologic
specimen (original magnication 3). (b) Magnied view
of an area containing immature woven bone (original
magnication 40). (c) Magnied view of an area contain-
ing more mature lamellar bone (original magnication
40) (Stevenel's blue Van Gieson's picric fuchsin stain).
Fig. 7. The radiographic view of the distraction case after
3 years.
Fig. 8. Surgical view after the reection of only the buccal
mucoperiosteal ap prior to the placement of two ACE
distractors.
58
temporary be utilized to insure proper transport seg-
ment positioning. Base plug instability may arise
during placement of the ACE distractor, especially
if an insufcient amount of bone remains apical to
the base plug. Due to the dense inferior cortex of
the mandible, planned vertical distraction can be
achieved even with complete disattachment of the
base plug. Currently, the preassembled device con-
tains both the base plug and the distractor body, so
instability is not likely to occur. Radiographic con-
rmation at the completion of surgery and during
distraction is advised to conrm proper positioning
of the distractor components.
The Leibinger Endosseous Alveolar
Distraction (LEAD) system
The intraosseous LEAD system (Stryker Leibinger,
Kalamazoo, MI) consists of a 2 mmdiameter threaded
rod, a threaded transport plate, and a stabilizing
unthreaded base plate (Fig. 11). The threaded distrac-
tionrodcomes in17, 22 and32 mmlengths andcanbe
advanced0.4 mmper turn. The angle of the osteotomy
preparation for the threaded rod should be consistent
with the proposed vector of distraction. The transport
plate and base plate are then bent and xed into place
with xation screws to maintain the proposed vector
(Fig. 12). With the signicant forces from the palatal
tissues and lingual musculature it is advisable to use a
guidance temporary toensure the transport segment is
Fig. 9. Radiographic view at the completion of approxi-
mately 8 mm of distraction with 2 mm of overcorrection.
The guidance axial distraction screwis engaging the ortho-
dontic band retained lingual arch wire xed/removable
temporary.
Fig. 10. The nal restoration after consolidation (a, b).
59
orientated correctly with this system (Figs 13 and 14).
With the narrownature of the threaded rod it is impor-
tant not to apply too much horizontal force as bone
resorption can occur and the rod may become dis-
placed from the transport segment. With the narrow
threaded rod a vestibular incision can be made and
drilling the threaded rod osteotomy can be made with-
out mucoperiosteal ap reection even in cases with
narrow ridges. It is, however, still necessary to later
augment the ridge in the horizontal direction if it has
not been completed prior to distractor placement.
Reports to date with this systemhave shown favorable
results (6, 12, 13).
KLS Martin distractor
The extraosseous Track distractors (KLS Martin, Jack-
sonville, FL) are made of titanium with microplates
that have been welded onto the sliding mechanism of
the actual distraction screw (Fig. 15). Multiple sizes
are available depending on the regenerative needs.
For full arches the Track 1.5 is indicated and for very
small segments the Track 1.0 microdistractor may be
utilized. For most partially edentulous distraction
patients the Track Plus distractor is indicated,
because it has the most rigidity due to the apical
extension and it is still of manageable size.
Fig. 11. The LEAD system showing the threaded rod, sta-
bilizing base plate and threaded transport plate.
Fig. 12. The LEAD system xated in place after the com-
pletion of the horizontal and vertical osteotomies.
Fig. 13. The ridge as seen prior to the start of distraction.
Fig. 14. After the completion of distraction the coronal
advancement of the transport segment can be appreciated.
60
The following case study demonstrates how this
extraosseous distractor functions. A 41-year-old male
presented following untreated trauma that fractured
teeth 11 and 21. Secondarily this resulted in 75%
bone loss on the mesial and facial aspects of tooth
12 (Fig. 16). The patient was a smoker, but had no
other medical issues. All three hopeless teeth were
extracted. A graft of anorganic bone (Bio-Oss, Osteo-
health, Shirley, NY) was placed and the area was
allowed to heal for 5 months. After a vestibular inci-
sion was made, a Track Plus device was modied to
t the bony topography of the area and screwed to
place. The locations for vertical and horizontal osteo-
tomies were marked, the device removed, the osteo-
tomies completed with saws and the device was
replaced with additional xation screws (Fig. 17).
After a 1-week latency healing period, distraction
was initiated at a rate of approximately 1 mm/day
(1 turn for 0.3 mm). Concurrent with completion of
distraction, excellent vertical height was obtained
(Fig. 18). However, some soft tissue dehiscence of
the distraction device was noted at the end of the
consolidation period (Fig. 19). With extraosseous
devices, soft tissue complications may occur more
frequently due to the compromised blood supply, yet
this does not appear to affect the osseous outcome as
long as soft tissue grafting is completed at the time of
distractor removal. After 5 months of consolidation,
the distractor was removed, a soft tissue graft was
added, and implants placed (Fig. 20). After 4 months
of further healing, a provisional implant supported
restoration was placed (Fig. 21).
Fig. 15. The KLS distractors: (a) Track 1.5, (b) Track Plus
and (c) Track 1.0.
Fig. 15. continued
61
With the extraosseous distractors there is no
bone width requirement; however, ultimate implant
reconstruction requires a 57-mm-wide ridge. Thus,
grafting with autogenous bone to achieve the neces-
sary width may be necessary prior to distraction. A
split ridge approach for increasing the ridge width is
the suggested approach prior to distraction (9, 31).
While the potential for nerve injury exists for any
posterior mandible distraction case, the extraosseous
design is the best suited for this region (Fig. 22). An
insufcient number of patients have been treated for
a predictive incidence of nerve damage from the
distraction osteotomies in this area to be deter-
mined. The small number of surgeries performed
in this area is likely due to the limited number of
indications for distraction in this area. A minimum of
56 mm bone is required superior to the nerve to
allow for clearance with the nerve during horizontal
osteotomy preparation and to maintain a transport
segment height of 34 mm. For any amount less
than this 56 mm, nerve repositioning should be
Fig. 16. Initial radiographic appearance showing exten-
sive evidence of bone loss on teeth 12, 11 and 21.
Fig. 17. The KLS Martin Track Plus fully xated with
completed vertical and horizontal osteotomies prior to
suturing.
Fig. 18. Radiographic appearance at the completion of
6 mm of distraction.
Fig. 19. At the end of the consolidation phase, excellent
vertical height can be appreciated.
62
considered, or no implant placement and use of an
alternative prosthetic replacement. Considering that
when 8 mm of bone height is present above the
nerve 10 mm implants can usually be placed with
minimal particulate vertical guided bone regenera-
tion. Therefore only those cases with 57 mm of
bone superior to the nerve should be considered
for distraction, leaving a fairly small number of actual
cases. Reports to date with this extraosseous system
have shown favorable results (4, 15).
Distractor and oral implant
combination devices
The concept of a prosthetically restorable distractor
(Fig. 23) was introduced by SIS Trade Systems (Kla-
genfurt, Austria). A histologic study in sheep has
demonstrated that this distractor becomes osseoin-
tegrated and can therefore function as a loaded oral
implant (11). A study in 35 patients evaluating these
distraction implants found a range of 46 mm
increase in vertical height and no complications in
29 of the 35 patients (10). Conceptually, this
approach is clearly superior because the secondary
surgeries for distractor removal and implant place-
ment are eliminated. There are, however, several
major complications of concern that could arise spe-
cic to this approach, including a lack of device
osseointegration, improper device orientation for
restoration, crestal bone loss during distraction
exposing the rough coronal device threads, and the
inability to initially place the devices in ideal pros-
thetic position due to the interference of the
vertical osteotomies. The Veriplant distraction device
Fig. 20. Radiographic presentation at the time of implant
placement.
Fig. 21. After integration, a provisional implant supported
restoration has been placed.
Fig. 22. Distraction in the posterior mandible with a KLS
Martin Track Plus.
Fig. 23. The SIS implant distractor in the start position
(left) and in full extension (right).
63
(EverFab, East Aurora, NY) is also a combination oral
implant and distractor device (33).
Potential complications with
distraction osteogenesis
While the complication rate for distraction is fairly
low, a variety of complications such as infection,
extensive bleeding, nerve injury, adjacent tooth
damage, and ap dehiscence may occur. If the per-
iodontist is not prepared for the potential complica-
tions the treatment will more likely result in an
unfavorable outcome. With proper treatment plan-
ning and careful surgical manipulation, most of these
complications can be avoided. In addition to general
surgical complications, there are several potential
complications related to the alveolar distraction pro-
cedure itself.
Fracture of the host bone or transport segment
may occur during insertion of the distraction device,
or distraction xation screws. This is most often a
concern in narrow ridges of dense bone quality when
the transport segment dimensions are small. As a
result of fracture, the distractor, or distractor xation
screws, will lose stability and therefore should be
removed. In cases with insufcient distractor stabi-
lity it may be appropriate to remove the distractor,
place a bone graft and delay distraction surgery for 2
3 months. In order to prevent fracture of dense bone,
tapping is recommended during the ACE distractor
insertion. This avoids stress on the buccal plate of the
transport segment. For the distractors using a micro
screw xation system, a larger diameter drill before
placement may minimize damage to a transport seg-
ment with dense bone. Care should also be taken if
completion of the osteotomies is performed with an
osteotome, particularly near the maxillary sinus oor
and the piriform rim. It is recommended that only
the lateral walls be used for leverage of the transport
segment during mobilization.
Distractor instability can develop due to poor bone
quality, soft tissue dehiscence, transport or host seg-
ment fracture, or extensive site preparation for dis-
tractor placement. Placing the ACE distractor apical
enough to engage the wider implant shoulder will
increase distractor stability. Using longer or wider
diameter xation screws for situations of question-
able KLS or LEAD distractor stability can also
improve xation. If adequate distractor stability can-
not be obtained, the distractor must be moved to a
different location, or the site must be closed for later
distraction.
Either with a single distractor or multiple distrac-
tors, undesirable movement of the transport bone
segment may occur, often due to lingual ap or mus-
cle tension. A guidance axial distraction screw may
be utilized to maintain the desired direction of move-
ment or to correct malalignment with the ACE dis-
tractor. Temporary or orthodontic hardware may be
used with the KLS or LEAD distractors if segment
guidance is an issue. Making the lingual aspect of
the vertical cuts slightly convergent will also resist
undesirable segment movement, especially when lar-
ger transport segments are used that involve more
arch curvature.
When both vertical osteotomies are properly com-
pleted and the distraction rate is kept to 1 mm per day
or less, the transport segment should advance without
signicant resistance. If premature consolidation
occurs prior to completion of distraction, it is likely
due to incomplete osteotomy or mineralization at the
vertical osteotomy sites. In these cases, the transport
segment can be freed under local anesthesia by apply-
ing a nger pressure on the bone segment. Alterna-
tively, the transport segment premature consolidation
can be re-osteotomized using a small-size interdental
osteotome through a small incision. Keeping the ver-
tical osteotomies slightly divergent to the crestal
aspect will prevent segment binding and minimize
premature consolidation.
Typically, there is no detectable transport segment
mobility at the end of the consolidation period. In
some cases, however, delayed consolidation may
occur, potentially leading to the development of a
nonunion. If signicant transport segment mobility
exists at the proposed time of distractor removal, the
device may be left in place to allow further consoli-
dation. Sufcient stabilization of the transport seg-
ment is an important aspect in prevention of
nonunion during distraction. During active distrac-
tion and during the consolidation phase, segment
mobility must be controlled. In cases with an imma-
ture regeneration, nger pressure can be applied to
the transport segment at the time of distractor
removal to resist rotational forces, thereby stabilizing
the segment. In addition, immediate placement of
oral implants at this point will also support further
stabilization of the newly regenerated bone. Finally,
if a partial or complete nonunion exists at the nal
uncovering of the implants, debridement must be
performed followed by bone grafting and plate sta-
bilization.
While any of these complications are possible, the
publications to date indicate these complications
occur at a very low rate (4, 5, 12, 13, 20, 22). For
64
distraction cases involving sites that have had multi-
ple prior surgeries the incidence of complications is
higher (5), suggesting that distraction should be the
rst line of treatment rather than a last resort after
other techniques have failed. Considering there is no
established ideal bone augmentation approach for
the treatment of vertical defects, the minimal inci-
dence of complications gives further support to the
technique of distraction osteogenesis for treatment
of vertical defects.
Conclusions
Favorable clinical results have been observed for
alveolar ridge augmentation via distraction osteogen-
esis with the different distractor systems described.
These systems are relatively simple to apply and will
be a valuable adjunct to the contemporary implant
reconstruction armamentarium in periodontology.
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Mommaerts MY, Jensen OT. Alveolar distraction devices.
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66
Management of the posterior
maxilla in the compromised
patient: historical, current,
and future perspectives
Thomas J. Balshi & Glenn J. Wolnger
The posterior maxilla has been described as the most
difcult and problematic intraoral area confronting
the implant practitioner, requiring a maximum of
ingenuity for the achievement of successful results
(39, 59). Both anatomic features and mastication
dynamics contribute to the challenge of placing tita-
nium implants in this region.
Anatomic factors include decreased bone quantity,
especially in older edentulous or partially edentulous
patients who have experienced alveolar resorption in
the wake of tooth loss. The antrum also tends to
enlarge with age, as well as with edentulism, and this
further decreases the amount of available bone. In
addition to the diminished quantity, bone in the
posterior maxilla often is softer and of poorer quality.
Radiographs typically reveal a dearth of trabecula-
tions, and the tactile experience of drilling here often
more closely resembles the penetration of styrofoam
rather than anthracite. Limited access to the ptery-
gomaxillary region constitutes yet another problem.
Mastication dynamics also affect the long-term
stability of implants placed in the posterior maxilla.
Whereas masticatory forces of 155N have been
reported in the incisor region, the premolar and
molar regions have exhibited forces of 288N and
565N, respectively (37). Parafunction can increase
these forces as much as three-fold (16, 12, 21), apply-
ing signicant stress to the bone-implant interface
and the component hardware.
Despite the biomechanical impediments to creating
prostheses in the posterior maxilla, patients who have
lost teeth in this area have sought some means of
restoring both their chewing ability and their appear-
ance. One solution has been the use of posterior can-
tilevers on implant prostheses. When designed to
minimize the occlusal forces applied to the pontic,
short cantilevers can function successfully. One key
is the availability of several long and strong implants
anterior to the cantilever. The author also suggests the
use of implants of 4 mm diameter or greater, if the
intent is to create a cantilevered prosthesis.
If sufciently strong anchors are unavailable or
longer cantilevers are required, problems are likely
to ensue. Complications associated with posterior
cantilevers include screw loosening and fracture,
bone loss around the most distal xtures, and loss
of osseointegration (6) (Fig. 1). As awareness of such
consequences has grown, the alternative of creating
non-cantilevered bone-anchored restorations has
become increasingly desirable.
The following discussion reviews the development
of implant solutions in the posterior maxilla and
examines the feasibility of applying these solutions
to the compromised patient. Future prospects are
also briey assessed.
Standard implant placement
Studies of the long-term success of osseointegrated
implants placed in the posterior maxilla have painted
a mixed picture. Jafn & Berman, reporting speci-
cally on implants used in this region (26), noted a
higher failure rate related to Type IV bone. Schnit-
man (44) showed that only 72% of implants placed in
the posterior maxilla achieved osseointegration.
When Widmark et al. (55) studied the results of
implants placed in the severely resorbed maxillae
67
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PERIODONTOLOGY 2000
ISSN 0906-6713
of 36 patients (16 of whom received bone grafting
and 20 of whom did not), they found that after
35 years, the success rates in the two groups were
74 and 87%, respectively.
Other investigators, however, have found signi-
cantly higher success rates. Bahat (3), analyzing the
experience with 660 Branemark System implants
placed in the posterior maxilla and followed in 202
patients for up to 12 years after loading, found a
cumulative success rate of 94.4% at 56 years and a
93.4% rate after 10 years. Lazzara and coworkers
found a success rate of 93.8% among 529 implants
placed in the posterior maxilla (35). The Kaplan
Meier success rate for 167 IMZ posterior maxillary
implants after 80 months was 96.9%, according to
Haas and colleagues (20). And when Buchs and
associates studied Steri-Oss HA-coated threaded
implants, including 416 placed in the posterior max-
illa, their life-table analysis indicated a 96.6% 5-year
success rate (13).
Fig. 1. (a) Panoramic and periapical
radiographs of maxillary xed deta-
chable prosthesis with cantilever
illustrating advanced bone loss on
posterior xture. (b) Transition from
xed detachable prosthesis to max-
illary implant overdenture on ve
implants after removal of three pos-
terior implants with advanced bone
loss. Note the bone loss on the
remaining implants as well. (c) Addi-
tional xtures placed in the ptery-
goid region for extension of the
overdenture bar for better stability.
Balshi & Wolfinger
68
A number of recommendations for achieving
predictable implant osseointegration in the poster-
ior maxilla have been made. To obtain a greater
surface area for bone contact, Langer et al. suggested
the use of wider diameter implants (33). More
recently, Bahat recommended placement of a
sufcient number of implants to support the
occlusal load in a way that avoids nonaxial loading
(3).
In the author's experience, standard implant pla-
cement in the posterior maxilla is indicated if at
least 8 mm of bone is available below the sinus.
In such cases, a 10 mm implant can be utilized.
The apical threads of the implant will engage the
layer of cortical bone that forms the antral oor,
thereby creating bi-cortical stabilization of the x-
ture and a slight apical tenting of the sinus mem-
brane. This tenting, or mini sinus lift, is similar in
effect to the osteotome technique for xture place-
ment (48).
Another alternative is to utilize longer implants,
tilting them anteriorly between the oor of the sinus
and the apex of the canine or other anterior
teeth. Such off-axis loading of maxillary anterior
implants has been shown (1, 32) to achieve osseoin-
tegration and create a stable support system for the
prosthesis.
Immediate extraction sites also offer opportunities
for standard implant placement in the posterior max-
illa because residual bone usually exists around the
extraction site.
When standard implants are placed in the poster-
ior maxilla of partially edentulous patients, the nal
prosthesis will not enjoy the benet of cross-arch
stabilization. Therefore, placement of more implants
are recommended to prevent the overload bending
moment forces that can cause bone loss around the
implants (Fig. 2).
Hard-tissue grafting in conjunction
with standard implants
When standard implant placement is contraindi-
cated because of inadequate bony volume, one
approach historically has been to augment the ridge.
Breine & Branemark rst described the use of onlay
composite bone grafts for reconstruction of compro-
mised severely atrophic ridges in 1980 (11). Although
the original technique has evolved considerably since
then, unpredictable resorption of the graft material
has been a continuing problem (49). Verhoeven et al.,
assessing various studies of onlay grafts, sandwich
osteotomies, and onlay grafts plus hydroxyapatite
augmentation, found that in the rst year after bone
grafting, resorption is signicant and may continue
for up to 3 years (53).
Even when successful, grafting of the ridge may
reduce the posterior interocclusal space so signi-
cantly as to cause prosthetic restorative problems
(25). Another approach, therefore, has been to aug-
ment the oor of the sinus. Introduced by Dr. Hilt
Tatum in 1975 (46), the sinus lift graft has gradually
gained proponents over the years, and a 1996 con-
sensus conference on sinus grafts organized by the
Academy of Osseointegration found that sinus graft-
ing should be considered a highly predictable and
effective therapeutic modality (27).
Today, two basic sinus grafting strategies exist. In
the rst, elevation of the sinus and placement of
the implants occur simultaneously. This approach
offers the advantage of requiring fewer surgeries,
while at the same time allowing for a shorter treat-
ment time and reduced expense. However, at least
5 mm of bone must be present in order to ensure
rigid xation of the implant at the time of place-
ment (40) (Fig. 3).
Fig. 1. continued
69
Management of the posterior maxilla
When atrophy of the antral oor is more advanced,
the alternative is to stage the surgeries, placing the
implants 610 months after the initial bone graft.
Allowing additional time for the implants to heal in
the grafted bone, the overall procedure may require a
time commitment of close to 2 years, a prospect that
is unattractive to many patients.
Furthermore, all grafting may result in complica-
tions, including infection and loss of grafted bone. As
a result, placement of implants in more distant sup-
port sites in the maxilla has emerged as another
potentially attractive alternative.
Tuberosity and pterygoid implants
There has been a longstanding feeling among clin-
icians that the pterygomaxillary region of the maxilla
was unsuitable for implants because of large fatty
marrow spaces, limited trabecular bone, and the rare
presence of cortical bone covering the alveolus.
However, subsequent clinical trials showed that tita-
nium xtures could successfully osseointegrate in
this area (7). Indeed, the density of some of the pter-
ygomaxillary structures may provide stability that
exceeds that offered by the anchorage in any other
part of the maxilla (51).
Reiser's anatomic investigations using cadaver dis-
section have shown that the specic structures that
may support implants are the tuberosity of the max-
illary bone, the pyramidal process of the palatine,
and the pterygoid process of the sphenoid bone
(41). At times it is possible to place an implant com-
pletely within the rst of these (and avoid angling the
implant apex more distally), depending on the tuber-
osity's dimensions and quality. If the height, length,
and/or width of the tuberosity are not adequate,
however, the implant can be angled and the apex
made to engage either the pterygoid process, the
pyramidal plate of the palatine bone, or both. Recent
observations and measurement of the height, ante-
roposterior distance, and mediolateral distance of
the pyramidal process indicate that placement of
implants in the lower half of the pyramidal process
is advantageous (36).
Such xtures have provided successful support for
a variety of tissue-integrated prosthesis forms,
including multi-xture complete-arch xed pros-
theses (Fig. 4), complete removable overdentures
with xed retention bars (Fig. 1c), multiple xture-
supported restorations independent of the natural
dentition (Fig. 2c), and terminal abutments for par-
tial xed prostheses connected to the natural denti-
tion (Fig. 5).
Treatment planning
Several factors should be weighed by the treatment
team when considering the use of implants in the
tuberosity or pterygomaxillary region. Access to the
oral cavity is often limited. Prior to surgery, therefore,
Fig. 2. (a) Unilateral maxillary posterior implant recon-
struction showing advanced bone loss around distal x-
ture and subsequent implant fracture. (b) Replacement of
xed prosthesis without cantilever after distal implant was
resurfaced. (c) Additional implants placed for better sta-
bility of unilateral maxillary posterior prosthesis.
70
Balshi & Wolfinger
it is critical to measure the vertical opening available
for xture placement and restoration. The amount of
space required for the drilling instrumentation and
the xture mount, as well as the length of the implant
to be placed, must be considered.
Accurate radiographic analysis of the available bone
using computerized tomography and/or panoramic
radiographs also is important in planning implant
placement in this complex region.
Finally, because of the limited access in the pter-
ygomaxillary area, placement of implants here requ-
ires considerable surgical skill. Extensive experience
in xture placement in other areas of the maxilla is
recommended.
Clinical results
Given adequate surgical expertise, the success rate
for implants in the pterygomaxillary regions com-
pares favorably with the results of previous studies
of implants placed in the maxillary arch. In 1999, the
author reported on the results of placing 356 ptery-
gomaxillary implants in edentulous arches and found
a cumulative survival rate of 88.2% after an average
functional period of 4.68 years (10). Five other stu-
dies of pterygomaxillary implants (2, 9, 18, 30, 50)
also have revealed cumulative survival rates that
were consistently above 86.0%.
Zygoma fixtures
The volume of bone in the pterygomaxillary area is
not always sufcient to support placement of
implants. In such cases, when patients have severely
atrophic maxillas and are unwilling or unable to
undergo extensive bone grafting, Zygoma xtures
(Nobel Biocare, Goteborg, Sweden) may provide an
alternative.
Ranging in length from 30 to 52.5 mm, Zygoma
xtures are anchored in two different types of bone.
The head of the xture normally emerges in a slightly
palatal position in the second premolar or rst molar
Fig. 3. (a) Preoperative panoramic view of advanced bone
loss in maxillary posterior region. (b) Caldwel-luc proce-
dure in fracturing buccal plate of bone and placement
of three xtures to support and elevate the bone plate
and sinus membrane. (c) Placement of 50/50 mixture of
autogenous bone and Bio-Oss bovine bone material for
sinus grafting. (d) Placement of a Bio-Gide resorbable
membrane with the use of four titanium tacks. (e) Post-
operative panoramic radiograph showing the placement of
three implants in the grafted area and a pterygoid xture
for posterior support. (f) Clinical view 5 months postop
showing complete bone ll. (g) Panoramic view of nal
prosthesis in place. (h) Periapical views showing nal
prosthesis in place and bone graft 1 year after surgery.
71
area of the maxilla, while the other end of the xture
engages the very dense midfacial zygomatic bone.
The body of the xture thus traverses the posterior
portion of the maxillary sinus, ideally avoiding pene-
tration of the sinus mucosa. Initial sinoscopic studies
of patients treated with Zygoma xtures indicate that
the presence of a titanium foreign material inside the
sinus cavity does not appear to increase the risk of
inammatory reactions in the nasal and maxillary
sinus mucosa.
Fig. 3. continued
72
Balshi & Wolfinger
Preparation of the xture sites is accomplished
with the patient under deep sedation or general
anesthesia. After determining the exact point on
the alveolar crest to start the drilling sequence and
the direction of the long axis of the xture, a series of
long twist drills of increasing diameter is used to
prepare the receptor sites. A Zygoma xture is then
placed and allowed to heal for 56 months before
being loaded.
Because of the greatly increased length of the x-
tures and the limited bone support commonly found
in the alveolar crest, Zygoma xtures have an
increased tendency to bend under horizontal loads.
Since bending forces can jeopardize the long-term
stability of implant-supported restorations, Zygoma
xtures must be placed in combination with at least
two, and preferably more, standard implants in the
anterior maxilla, in order to distribute the functional
load and prevent rotation. The restoration should
ideally include cross-arch stabilization (Figs 6 and
7), decreased buccal lever arms, decreased cantile-
vers, balanced occlusion, and decreased cuspal incli-
nation.
Placement of the Zygoma xture is demanding and
difcult, requiring considerable surgical expertise.
On the other hand, this approach offers patients
and implant practitioners a number of advantages,
including shorter treatment and hospitalization
times than those required by most grafting proce-
dures, as well as reduced pain and risk of morbidity.
The ability to use fewer implants may also result in
lower treatment cost.
One study of the Zygoma xture by Branemark has
indicated an overall success rate of 97% (19), but this
evaluation is only preliminary.
Treatment of the maxillary sinus in
the compromised patient
A variety of medically compromising conditions may
be encountered in patients who lack dentition in the
Fig. 4. (a) Five implants to support a
mandibular implant overdenture in
the anterior region. (b) After loss of
one of the anterior implants, impl-
ants were placed in the pterygoid
maxillary region to support a full
arch maxillary xed detachable por-
celain fused to gold prosthesis.
73
posterior maxilla and seek implant therapy as a
means of restoring form and function in that area.
Unfortunately, the body of clinical studies evaluating
the success of various implant modalities in various
categories of compromised patients is limited. Until
more denitive evidence emerges, the following set
of guidelines may prove useful.
Contraindications
Three conditions are considered by the author to be
absolute contraindications for the placement of any
type of implant in the posterior maxilla: a recent or
imminent course of cancer chemotherapy and radia-
tion, drug or alcoholism addiction, and blood dys-
crasias that directly affect bone metabolism.
Chemotherapy and radiotherapy disturb the bone
metabolism, suppress the immune system, and
diminish healing potential. All three elements must
function well for osseointegration to succeed. A ret-
rospective study by Wolfaardt et al. (57) found that
the implant loss rate for patients who had had che-
motherapy was 21.9%. That study also found one
reported case in which all eight mandibular implants
placed in a patient who had received chemotherapy
1 day prior to surgery were lost. Although more
investigation of the affect of chemotherapy upon
osseointegration is needed, the author currently
recommends delaying implant therapy for several
months after completion of the chemotherapy.
The metabolic and psychologic problems exhib-
ited by patients who are addicted to drugs or alcohol,
coupled with their tendency to be non-compliant,
make them poor candidates for any sort of implants,
let alone those in the challenging and compromised
posterior maxilla. As for patients with blood dyscra-
sias such as hemophilia or leukemia, the author
believes that the risk of an adverse outcome due to
uncontrolled bleeding or compromised healing war-
rants recommending against any posterior maxillary
implant placement.
Indications in compromised
conditions
A number of medical conditions may signicantly
increase the risk of posterior maxillary implant fail-
ure when unaddressed. Coupled with appropriate
corrective action, however, implant placement in
patients with such conditions can enjoy a reasonable
likelihood of success. These conditions include dia-
betes, smoking, severe parafunctional habits, osteo-
porosis, and Crohn's disease.
Diabetes
Diabetes has been associated with numerous com-
plications, including an increased incidence of caries
(38) and periodontitis (31), a higher susceptibility to
Fig. 5. (a) Panoramic view showing advanced bone loss
in maxillary posterior region. (b) Placement of one x-
ture in the pterygomaxillary region and restoration in
connection to the natural tooth. (c) Panoramic view
13 years later showing the response of the restoration
of an implant in the pterygomaxillary region connected
to a natural tooth.
74
Balshi & Wolfinger
infection (17, 34, 42), and slower healing after surgery
(42). However, evidence has accumulated that dia-
betic patients who effectively control their disease
incur a lower risk of various health complications
than their uncontrolled cohorts (31, 34, 19). When
Kapur et al compared 37 diabetic patients who
received conventional removable mandibular over-
dentures with 52 individuals who were tted with
implant-supported ones, the researchers concluded
that implants could be successfully used in diabetic
patients with even low to moderate levels of meta-
bolic control (29). A 1994 study found a 92.7%
implant success rate for Type II diabetic patients
under acceptable glucose control (8). And when the
author conducted a study of 227 implants in 34 dia-
betic patients, a survival rate of 94.3% was found (8).
This study included 73 implants placed in the poster-
ior maxilla, where the success rate was 94.5%.
Implant practitioners should make clear to dia-
betic patients the importance of achieving adequate
Fig. 6. (a) Preoperative panoramic
view of patient with congenitally
missing teeth. (b) Placement of a
total of 10 implants: 6 in the anterior
maxilla, 2 in the zygomatic area and
2 in the pterygomaxillary area. A
total of 10 implants: 2 zygomatic
and 2 in the pterygomaxillary area.
(c) Restoration of the maxillary arch
utilizing implants inboththe zygoma
and pterygoid regions. (d) Anterior/
posterior cephalometric radiograph
showing projection of implants in
the zygoma area. (e) Lateral cepha-
lometric radiograph showing projec-
tion of posterior implants in the
pterygomaxillary andzygomaregions.
75
metabolic control, along with stressing the need
to take all diabetic medications on the day of sur-
gery and maintain them throughout the healing
period. A 10-day regime of broad-spectrum antibio-
tics beginning on the day of surgery is also recom-
mended.
Smoking and parafunctional habits
The deleterious impact of smoking on osseointegra-
tion has been well documented (5, 4). Furthermore,
implants placed in the maxillary posterior of smokers
appear to fare worse than those placed in maxillary
Fig. 6. continued
76
Balshi & Wolfinger
posterior sites in non-smokers (28, 55). Patients
should thus be urged to enroll in a smoking cessation
program before and after undergoing implant place-
ment.
Patients who nd it impossible to stop smoking
should be counseled as to the additional risk of
implant failure that they may be incurring. Further-
more, they should be advised that utilization of
Fig. 7. (a) Preoperative panoramic radiograph with old
implant restoration. (b) Post-surgical radiograph showing
implants in the severely resorbed maxilla; a total of two
implants on each side were placed in the zygoma region.
(c) Lateral cephalometric radiograph illustrating the
implants in the pterygomaxillary region and the four
implants in the zygoma region. (d) Post-treatment panora-
mic radiograph showing reconstruction of severely resor-
bed maxillary utilizing the four implants in the zygoma
region. (e) Anterior/posterior cephalometric radiograph
showing projection of the four implants in the zygoma
region to support the full xed maxillary reconstruction.
77
additional implants might compensate for the failure
of some xtures to osseointegrate and thus increase
their overall chances for prosthesis success. The
author employs a similar strategy when counseling
individuals with severe parafunctional habits. Addi-
tional biomechanical support has proven effective in
counteracting the harmful effects of bruxism and
clenching upon the prosthesis supported by osseoin-
tegrated implants.
Osteoporosis
Osteoporosis currently threatens the health of 25 mil-
lionAmericans. Of those individuals (80%of whomare
women), some 78 million are estimated to have the
disease already, and an additional 17 million have low
bone mass and consequently are at increased risk for
osteoporosis and the fractures it causes.
Screening for osteoporosis is thus a prudent course
whenconsidering placement of implants inthe poster-
ior maxilla of post-menopausal females. This should
include comprehensive reviews of medical history and
family history regarding bone fractures. Whenever
osteoporosis or osteopenia is identied, a program
of supplementation should begin immediately. This
should include 12001500 mg of calcium taken three
times a day withmeals tomaximize absorption, as well
as a multiple vitamin that includes C and E and
between600 and800 mg of vitaminD. Pharmaceutical
preparations such as alendronate sodiumor raloxifene
HCl also should be prescribed.
Osteoporotic patients should be advised about the
importance of continuing this therapeutic regime,
not only throughout the healing period, but on a
continuing basis. Counseling about lifestyle aspects
of avoiding osteoporosis such as engaging in weight-
bearing exercise and avoiding smoking, caffeine,
excessive alcohol, carbonated sodas, and corticoster-
oids is also recommended.
Crohn's disease
Crohn's disease is a serious inammatory disorder
that predominates in the ileum and colon but may
Fig. 7. continued
78
Balshi & Wolfinger
occur in any section of the gastrointestinal tract.
Some 500,000 cases are estimated to exist in the
U.S. alone (22).
Because of the potential for involvement of the oral
mucosa, the author urges patients with Crohn's dis-
ease to achieve effective control of their condition
before undertaking any form of implant therapy. Sev-
eral categories of drugs constitute the mainstay of
treatment for Crohn's disease today, including anti-
biotics, immune modiers, oral and rectal aminosa-
licylates, and oral and rectal corticosteroids (15).
Other considerations
Although pregnancy in itself has no adverse impact
on the osseointegrative process, the stress of surgery
or the use of narcotics for pain relief may potentially
compromise the unborn baby. Delay of implant ther-
apy until after the baby is delivered is thus recom-
mended.
One other compromising condition worth noting is
that of the psychotic patient. When the psychosis
relates to the teeth and/or mouth, as is not uncom-
mon, implant therapy may create complications for
both the implant practitioner and for the patient's
psychiatrist.
Future considerations
Over the next decade, technologic and scientic
advances have the potential to transform the place-
ment of posterior maxillary implants in all patients
both healthy and compromised into a mundane
and predictably successful operation. Likely develop-
ments include the implementation of genetic and
tissue engineering in conjunction with bone surgery
and implant placement, as well as systemic enhance-
ment of bone metabolism.
These developments are already well underway.
More than 35 years ago, Urist coined the term ``bone
morphogenetic protein'' (BMP) to describe the bone-
inducing substance that he hypothesized had caused
the formation of new cartilage and bone after
implantation of decalcied bone matrix in rabbits
and rats (52). By the late 1980's, a group of proteins
from bovine bone had been identied (54) and the
rst recombinant human BMP (rhBMP) had been
cloned and characterized (58). Today more than 20
BMPs have been described (56).
A number of preclinical studies have demonstrated
that rhBMP-2 may be used successfully in animals to
augment alveolar defects (14, 23, 45), and Hanisch
and colleagues found signicantly greater bone
height in augmented subantral space that had been
implanted with rhBMP-2 in the Cynomolgus monkey
(24). Recent human studies have also shown rhBMP
delivered on an absorbable collagen sponge (ACS) to
be safe, predictable, and effective (11).
Other delivery methods seem certain to develop
with the introduction of new implant technologies.
The TiUnite surface (Nobel Biocare), for example,
seems a precursor to the eventual ability to lace
implant surfaces with genetically engineered pro-
teins to stimulate bone growth.
Platelet Rich Plasma (PRP) in conjunction with
autogenous grafting is already in use to biologically
reconstruct and graft the sinus area. In the future, the
growth factors made available through the use of PRP
may very well be available through recombinant
technology, thereby simplifying the entire treatment
process.
Conclusion
Although management of the posterior maxilla pre-
sents many challenges for the implant practitioner,
progress on a number of fronts has made it increas-
ingly possible to create successful bone-anchored
restorations in this region predictably. When at least
8 mm of bone exists below the sinus, standard
implant therapy can be considered, particularly in
immediate extraction sites or when wider-diameter
implants can be employed. A variety of grafting pro-
cedures, including sinus lift grafts, may be consid-
ered when bone loss is more extensive. Alternatively,
the use of implants that obtain support from more
distant bony sites such as the pterygomaxillary
region or the zygomatic bone has also proven suc-
cessful. Future breakthroughs in the areas of tissue
and genetic engineering are likely to enhance these
developments still further. In the meantime, altho-
ugh a few compromising conditions contraindicate
the placement of implants in the posterior maxilla,
the use of such implants in patients who are diabetic,
smokers, osteoporotic, or who have Crohn's disease
or severe parafunctional habits can be a prudent
option, given proper management of each condition.
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81
Use of zygomatic implants
to deal with resorbed posterior
maxillae
Chantal Malevez, Philippe Daelemans, Philippe Adriaenssens &
Francoise Durdu
For some 40 years, osseointegration has been
clinically applied in both the maxilla and the mand-
ible.
With a high predictability for some systems (19),
increased applications have been developed for com-
promised patients.
Unfortunately, restrictions have appeared in the
use of oral implants. One of them is the lack of
sufcient bone volume, especially in the posterior
maxilla. This insufcient bone volume can be due
to bone resorption as well as to pneumatization of
the sinus or to a combination of both. In any case,
insertion of implants in this region remains extre-
mely unpredictable.
According to some clinical reports, the minimal
bone height for a standard implant in the posterior
region should be at least 10 mm (33) to ensure
acceptable success rate. This is conrmed by a retro-
spective study of 660 implants using the Branemark
system in the posterior maxilla followed for 5
12 years showing a cumulative success rate of
94.4% but with a loss of 10 implants on 49 implants
with a length of 7 mm and a diameter of 3.75 mm
(22).
With the introduction of wide implants 5 and
6 mm in diameter (17, 18) the contact surface
between implant and bone is increased and assumes
a cortical anchorage with an initial stability in bone
type IV even if the bone height is no greater than
6 mm. Implants of wide diameter limit the biome-
chanical complications in the treatment of the pos-
terior maxillas.
Pterygomaxillary implants have also been pro-
posed for posterior anchorage in totally edentulous
patients (3).
Although wide implants could be a solution for
crestal heights up to 6 mm, many patients present
a maxillary height of 0.86 mm (39). For these cases,
several solutions have been proposed to augment the
volume of bone in this region, such as onlay/inlay
bone grafting (33).
Although autologous bone grafting remains the
gold standard, different types of grafting materials
have been proposed for these procedures: deminer-
alized bone from human cadavers, bovine bone and
synthetic materials. Although many have claimed
good clinical results, there is insufcient long-term
data from histologic and histomorphometric investi-
gations to provide real guidelines for these proce-
dures.
Contraindications for sinus lifting (33), such as
Caldwell Luc operations, Underwoods septae, severe
sinus oor convolutions and narrow sinuses, limit
the use of this clinical technique. Perforation of the
Schneidarian membrane could also jeopardize the
nal result.
The gold standard for sinus-lifting procedures
uses autogenous bone (25) but bone harvesting is
most often performed under general anesthesia
and, as in a majority of these cases these procedures
necessitate a two-stage since surgery, since the
implants are not inserted at the moment of the bone
grafting.
Complications like sinusitis or loss of grafts
have also been described with the sinus graft
technique. Final results (15) show a success rate of
up to 75% for the sinus-lifting procedure together
with simultaneous insertion of implants and it
is recommended to avoid this one-stage pro-
cedure.
82
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PERIODONTOLOGY 2000
ISSN 0906-6713
Vertical ridge augmentation using membrane has
also been proposed. In short series of prospective
and retrospective clinical studies histologically
documented (34, 35), it was proven that a ridge
augmentation of 4.95 mm could be obtained
around implants inserted and covered by barrier
membrane. In a retrospective multicenter study (29)
on 123 implants with a follow-up of up to 5 years, a
ridge augmentation of a maximum of 3 mm was
found.
However, in a histologic and histomorphometric
analysis of 30 patients (24) with provisional implants
inserted with threads only covered by bone chips,
membranes and soft tissue, there was a lower
(43.5%) bone density around the exposed threads
of the implants than around the threads (60.3 %) in
the nonexposed regions.
Surprisingly, in a recent animal study (26), no bone
growth appeared after 12 weeks of healing around
the noninserted part of the implants inserted in the
mandible of beagle dogs.
Distraction osteogenesis is a quite new procedure
for bone augmentation. However, although this new
procedure is in some areas well documented, no
publication concerning bone lengthening in the pos-
terior severely resorbed maxilla could be found.
From his own experience based on animal
research and human experiments, P. I. Branemark
(8) knowing that the introduction of an implant in
the sinus would not necessarily jeopardize sinus
health considered using the zygoma bone as an
anchorage for prosthetic rehabilitation in hemimax-
illectomy patients as well as for other defects. As
these reconstructions (23) were successful and
long-term stability of these implants was established,
in 1997, Branemark developed a specic implant
called the zygomaticus xture to provide xed solu-
tions even when the conditions for implant insertion
were poor in the posterior maxilla. This new techno-
logic development offers alternatives to bone grafting
or sinus-lifting procedures, which involve rather
invasive surgery.
Description of the zygomatic
implant
The zygomatic implants (Fig. 1) are self-tapping
screws in c.p. titanium with a well-dened machined
surface. They are available in eight different lengths
ranging from 30 to 52.5 mm. They present a unique
458 angulated head to compensate for the angulation
between the zygoma and the maxilla. The portion
that engages the zygoma has a diameter of 4.0 mm,
and the portion that engages the residual maxillary
alveolar process a diameter of 4.5 mm. At the max-
illary level the angulated implant platform extremity
offers the possibility to screw any kind of abutment
from the Branemark system
1
. However, for the new-
est generation of abutments a separate slightly
shorter abutment screw must be utilized for the con-
struction of conventional screwed prosthesis.
Fig. 1. The zygomatic implant.
Fig. 2. Three-dimensional CT image and occlusal view
showing connection of the zygoma with the maxilla.
Zygomatic implants for resorbed posterior maxillae
83
Indications for the use of the
zygomatic implant
Resorption of the maxilla appears in two dimen-
sions height and width. Posterior resorption can
lead to a maxillary bone height of 0.8 mm in the
posterior region. In the anterior region, height and
width can also be dramatically reduced (39).
Zygomatic implants are indicated in cases of
severe resorption of the maxilla: free end situations
in the maxilla where insufcient bone height is
Fig. 4. Radiologic (a) two- dimensional and (b) three-
dimensional view of the virtual insertion and placement
of the zygomatic implant from the maxillary level up to the
zygoma.
Fig. 5. Left (a) and right (b) view of the virtual emergence
of the zygomatic implant as previewed on the 3D recon-
struction before surgery.
Fig. 3. Tridimensional CT image showing the supposed
sites of insertion and end of the zygomatic implant.
84
Malevez et al.
available for standard implant insertion and in total
edentulism, when together with reduced bone height
of the posterior region, pneumatization of the
sinuses decreases the anterior area of the maxilla,
allowing the placement of only 2, 3 or 4 implants.
Because of its insertion in the zygoma region, the
zygomatic implant can be used in all of these situa-
tions.
In cases of very severe resorption of the anterior
maxilla in totally edentulous patients, when bone
grafting cannot be avoided, the use of zygomatic
implants reduces the dimensions of the bone graft
and the surgery is made easier.
The zygoma as an anchorage
The zygoma (Fig. 2) bone can be compared to a
pyramid, offering an interesting anatomy for the
insertion of implants (16, 38). Histologic analysis
(12) of the zygoma shows regular trabeculae and
compact bone with an osseous density of up to
98%. Due to this high bone density, the zygoma bone
has also been used in the treatment of maxillofacial
fractures, for the insertion of miniplates (11) and
during orthodontic treatment, offering a xed ancho-
rage to allow dental arch retractions (21).
In an animal study (30) the zygoma bone provided
an anchorage place for implants to obtain protrac-
tion of the maxilla by means of distraction. In max-
illofacial prosthesis, the zygoma bone is also utilized
as an anchorage for the placement of extraoral
implants sustaining a facial prosthesis (22, 27, 36).
After maxillectomy, zygomatic implants were con-
nected with standard ones to anchor a screwed pros-
thesis (14, 23).
In a recent study on cadavers (31) it could be
established that the mean length of the zygoma
was 14.1 mm, allowing the insertion of zygomatic
implants as described above.
Fig. 7. Reection of the Schneidarian membrane with a
gauze mesh (a) and drilling with the rst bur (b).
Fig. 6. Reection of the soft tissue (a) with identication
of the zygoma and suborbital nerve and (b) achievement of
the sinusal window.
85
For all these reasons, the zygoma should be con-
sidered as an extended anchorage in a region situ-
ated at an important distance from the occlusal level.
This can make a crucial difference to patients with
compromised maxillary anatomy.
Presurgical evaluation of feasibility
Inserting implants (Fig. 3) from the maxillary level
through the sinus and up to the zygoma is a challen-
ging venture. Three levels have to be investigated :
the maxillary level, the sinus and the zygoma. Clin-
ical examination is not sufcient for this evaluation
and radiologic assessment has to be considered. The
OPG can give distorted information and therefore,
the examination of choice is the spiral or helico d
computed tomography (CT) scan, which makes
two- and three-dimensional imaging possible
(Fig. 4a,b) (40).
Oralim
1
(Medicim, Leuven) provides appropriate
software. The implants can be placed via virtual
images and the surgeon only has to convert
these images to the reality of the clinical situation
(Fig. 5).
The CT scan also gives the opportunity to visualize
the health of the maxilla and the sinus. Sinusitis,
polyps or any sinusal pathology can be excluded.
The density, length and volume of the zygoma can
be evaluated and special templates for inserting the
zygomatic implants can be constructed on stereo-
lithographic models to facilitate the orientation of
the zygomatic implants during the surgery with
minimal errors in angulation and position (31).
Surgical procedure
The zygomatic implant surgical procedure should
involve atraumatic surgery, avoiding overheating in
the zygoma bone as well as in the maxilla under
sterile circumstances with what is in reality still a
two-stage approach.
Although the operation can be carried out under
local anesthesia, for the patients comfort, it has been
done up to now under total anesthesia or neuroleptic
deconnection (13). After a palatal 458 incision of the
soft tissue along the entire maxillary crest, the soft
tissue is completely reected from maxillary crest to
zygomatic buttress and the suborbital nerve identi-
ed. A window is then made by drilling at the upper
limit between the zygoma and the sinus (Fig. 6b) to
determine the orientation of the zygoma and to
reect the Schneidarian membrane. This window
will also be helpful during the surgical procedure
for cooling the drills to avoid overheating (Fig. 7a,b).
Fig. 8. Insertion with a low speed motor of the zygomatic
implant. The head of the implant is seen at the top
of the zygoma.
86
Malevez et al.
Different drills are used with increasing dia-
meters, ending with the insertion at low speed of
the self-tapping zygomatic implant (Fig. 8). The
length of this is carefully chosen by means of a
special gauge.
After insertion of the implant, a cover screw is
placed on the top of the zygomatic implant and the
soft tissue closed. There are no evidence-based argu-
ments that advocate the use of a membrane to cover
the hole made in the sinus.
The other implants are placed during the same
surgical procedure. At the second stage surgery 6
months later, the abutments are screwed on the
implants and an immediate (same day) provisional
prosthesis is provided for the patient (Fig. 9).
Prosthetic procedure
The prosthesis is made of gold and acrylic or gold
and porcelain routinely like a standard screwed
reconstruction on standard implants (Figs 10 and
11). Although screwed bridges allow a better adjust-
ment of the occlusion, overdentures retained by rigid
bars are also considered sometimes because of the
important cantilever due to the palatal emergence of
the zygomatic implants and to the distance between
the two maxillas or simply the resorption of the
maxilla.
Considering the biomechanical aspects of the
prosthetic reconstructions on zygomatic implants,
it is well known that when masticatory load is
applied to a rigid semicircular arch connecting
four anterior implants and two zygomatic ones,
the masticatory load in the posterior region is trans-
ferred to the bony support situated in the zygoma
(Fig. 12).
Fig. 11. Occlusal view of a bridge
made of gold and porcelain on top
of two zygomatic implants and four
standard implants.
Fig. 10. Occlusal view of a screwed prosthesis placed on
four standard implants and two zygomatic ones.
Fig. 9. Placement of an acrylic screwed provisional pros-
thesis.
87
Results
Although this new development of a specially
designed implant has been elaborated for more than
10 years in Sweden and has, since 1997, been the
subject of a worldwide multicenter prospective
study, so far, few data are available from the litera-
ture (46, 13, 37). Case reports as well as technical
reports show satisfactory results and a high success
rate.
In our own experience of 55 edentulous patients
involving 103 zygomatic implants, the survival rate is
100% with an observation period up to 48 months
(20).
Conclusions
The zygomatic implant the zygomaticus xture -
appears to be a promising development in implant
technology. It offers an interesting alternative solu-
tion to heavy bone grafting in the severely resorbed
posterior maxilla. It has been in use for more than
10 years and gives a predictable outcome in the reha-
bilitation of totally as well as partially edentulous
patients. More published reports are needed and
more follow-up has to be provided to assess its nal
goal and predictability.
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31. van Steenberghe D, Malevez C, Van Cleynenbreugel J,
Bou Serhal C, Dhoore E, Schutyser F, Suetens P, Jacobs R.
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89
Clinical examples of what can be
achieved with osseointegration
in anatomically severely
compromised patients
Daniel van Steenberghe, Marc Quirynen, Barbro Svensson &
Per-Ingvar Branemark
The present chapter will illustrate by means of as
series of examples of patient treatments how far one
can go in the rehabilitation of patients with extremely
compromised jaw bones using osseointegrated im-
plants. Those happen to be the patients who often
are the most in need of this treatment. This contribu-
tion presents a series of patients with different types
of very compromising situations, ranging from con-
genital defects to major jawbone resection because
of cancer. The clinical examples also show that the
team approach, where high-level skills from different
disciplines join forces, can go beyond what routine
clinical practice may envisage. It is important that
patients who are suffering because of certain anato-
mical or functional defect are being offered this treat-
ment option. Therefore it is important that clinicians
become familiar with the far-reaching possibilities
offered by this approach, even if they do not provide
the treatment themselves.
Extremely resorbed maxillae
A long period of edentulism and/or untreated severe
periodontitis can result in a nearly complete resorp-
tion of the alveolar process in the maxilla, rendering
the retention of an ordinary complete denture nearly
impossible. Such patients can be rehabilitated with
osseointegrated implants after the reconstruction of
the jawbone (via a grafting procedure or guided bone
regeneration).
Onlay graft
The surgical protocol for this procedure has been
outlined in papers by Branemark and co-workers
(2, 8). The reconstruction of the jawbone and implant
insertion is an excellent example of a synchronized
team approach with a patient under general anesthe-
sia, where periodontists can work at the oral site
while another surgical team harvests grafts at the
hip site (14, 15).
After local inltration of analgesics in the vestibular
fold of the mouth, a bevel incision is made about 2 cm
apical of the remaining alveolar crest from maxillary
tuberosity to tuberosity. The mucosa is dissected in a
slightly coronal direction and the periosteum is cut
just coronal of the base of the apertura piriformis.
The mucoperiosteal ap is further dissected taking
special care to maintain the integrity of the perios-
teum. The gross size of the remaining alveolar crest is
evaluated via a series of prefabricated horseshoe-
shaped templates. This information is given to the
second team working at the hip. The nasopalatine
neurovascular bundle is sacriced and the canal
thoroughly curetted to be free of fatty tissue (Fig. 1a)
and to be lled with bone chips. The incisal canal site
often has some remaining bone, which can serve to
harbor an implant. Periosteal remnants on the dis-
sected bone are removed. The cortex of the recipient
jawbone is perforated with a small round bur at sev-
eral sites to allow proper blood supply. Blood con-
tains the necessary ingredients for optimal healing
(cells, growth factors).
In the meantime, the team at the hip surgical site,
separated by a screen of sterile drapes over the
patient's abdomen, has prepared the donor site.
The skin incision is made on top of the palpable crest
and, after multilayered dissection, the periosteum is
cut over a length of 5 cm on top of the crest starting
some 2 cm dorsal of the spina iliaca anterior. Both
90
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PERIODONTOLOGY 2000
ISSN 0906-6713
periosteum and gluteus muscle are reected. A tem-
plate corresponding to that used in the oral cavity is
placed on the lateral surface of the iliac bone and,
under continuous cooling, a series of holes are drilled
around it. These holes are connected with each other
by means of an oscillating saw or by using osteo-
tomes. Finally, the graft is freed in the vertical plane
from the underlying squamous part of the iliac bone
with large thin bone scissors, starting from the top of
the crest. As such, a horseshoe-shaped cortico-cancel-
lous bone graft is progressively retrieved (Fig. 1b,c,d).
At the same time, some additional cancellous bone is
collected from the iliac defect together with some
blood (see later). While the team at the oral site takes
Fig. 1. (a) Uncovered mandible with the curetted nasopa-
latine canal. (b, c, d) The surgical team at the hip side
prepares, while a horseshoe-shaped corticocancellous
graft is progressively mobilised. (e) The cancellous part
is adapted to the jaw bone crest to achieve a stability of
the graft. (f, g) The graft is immediately xed by a series of
self-tapping implants. h) 3-D CT-scan image of the pelvic
bones showing a remaining defect on the left iliac crest
after bone harvesting. (i) Connecting the abutments by a
rigid prosthesis should be done as soon as possible to
spread the occlusal loading. (j) After some time the nal
prosthesis can be installed. (k) During the rst months
after installation of an autologous bone graft a deminer-
alization occurs which can be detected on the radiographs.
This will be reversed later. (l, m) With a team approach and
good cooperation with the restorative dentist it seems pos-
sibletomaintainmarginal bone levels for years. Patient with
a full maxillary graft at year 1 and after 10 years.
Osseointegration in severely compromised patients
91
over the graft, the surgical team at the hip starts the
closure in a routine multilayered manner, often
together with the installation of Redon suction to
deal with any postoperative bleeding.
Once the team at the oral site receives the graft, it
immediately starts to adapt the cancellous side of the
graft to the anatomy of the remaining jaw bone crest
by means of rongeurs and a foil adapted to the crest
(Fig. 1e). The latter is prepared from tin foil. To pre-
vent undue bone damage, no rotating instruments
are used except sometimes for a major reduction of
the cortical plate of the graft at the end of the pro-
cedure before closing the wound. The nasopalatine
canal and other undermining cavities are lled with
cancellous bone. Once an optimal t is obtained (the
graft should already be stable by itself), the graft is
xed to the maxilla via several implants (Fig. 1f).
With this team approach, the graft is outside the
body for less than 10 min.
The installation of the self-tapping implants starts
with one implant in the front area. During this pro-
cedure the surgeon has to pay special attention to
immobilizing the graft. The stability obtained with this
rst implant together with the optimal t renders the
remaining implant insertion a routine intervention.
The remaining space between graft and basal bone
is packed with the earlier retrieved bone chips. The
wound is closed with a one-layered monolament
suture with three mattress sutures and a series of
supercial sutures. However, in order to prevent
Fig. 1. continued
92
van Steenberghe et al.
perforations due to the graft, it is sometimes neces-
sary to dissect the labial mucoperiosteal fold.
After 47 days the patient can leave the hospital,
but complaints of difculties and pain upon walking
are frequently encountered. Indeed, some perma-
nent defects can occur at the iliac crest (Fig. 1h). A
denture cannot be worn during the rst 2 months.
Before using the old denture, a meticulous adapta-
tion with soft liner is essential. The abutments can be
inserted no earlier than 8 months after implant
insertion. Immediately after abutment connection, a
rigid provisional prosthesis is prepared which should
be ready within 2 weeks (Fig. 1i). In the meantime,
and even during the rst months with the provisional
prosthesis, a soft diet is instituted. The nal prosthe-
sis is installed 6 months or 1 year later (Fig. 1j) to
allow a stable, soft tissue adaptation and to compen-
sate for early graft remodeling and any supercial
resorption, thus achieving good esthetics.
Several follow-up studies have reported survival
rates around 85% (3); the above-mentioned proce-
dure led to a cumulative survival rate for individual
xtures of more than 90% after 10 years of function
(15). Sometimes an initial bone resorption/remodel-
ing of 12 mm (1) has been observed during the rst
12 months of loading, followed by a further bone loss
that falls within the 0.1 mm range, which is consid-
ered success using the criteria of Albrektsson and
Sennerby (4). The two-team approach and the
immediate xation of the autologous graft by means
of self-tapping implants led to minimal marginal
bone resorption (Fig. 1k,l,m).
Onlay graft in maxilla with congenital
discontinuities
In cleft palate patients (Fig. 2a) a similar procedure
as described above can be followed, except that the
bone graft adaptation to the complex anatomic situa-
tion requires more time. Instead of a horseshoe-
shaped graft, a full bone plate in installed (Fig. 2b).
If a connection with the nasal cavity is still present,
soft tissue handling by means of a sliding ap should
achieve a hermetic closure (Fig. 2c,d). The inserted
implants can later either be used to support a full
bridge or often to anchor an overdenture (Fig. 2e,f).
Guided bone regeneration using a
occlusive titanium membrane
Some patients badly want a xed prosthetic recon-
struction in the maxilla but are reluctant to undergo
an autologous hip bone graft procedure because of
the postoperative pain and limping (15). These
patients can be helped with a guided bone regenera-
tion procedure via the placement of a subperiosteal,
rigid, commercially pure titanium, barrier mem-
brane. Although it is still in a clinical development
stage, the concept is inspired by guided bone
augmentation techniques under hermetically closed
membranes (13) but this time applied for a larger size.
The approach, previously described in detail by
van Steenberghe and co-workers (17) for a series of
10 patients, is briey summarized by the clinical
achievement in a 31-year-old female patient with
extreme resorption of the maxilla (Fig. 3a,b), who
Fig. 1. continued
93
was in the need of a xed full bridge but who refused
harvesting of a hip graft to obtain the necessary bone
volume. With the use of the data from the spiral CT
scan of the maxilla, a stereolithographic model was
prepared (Fig. 3c) to which the amount of desired
bone was added by a wax-up technique (Fig. 3d).
The blocked out model was used to press a 0.2 mm
custom-t commercial pure titanium membrane
with a perfect adaptation (Fig. 3e). The latter serves
as rigid membrane below which undisturbed bone
regeneration can occur.
The crest of the maxilla was exposed under local
anesthesia via a mucoperiosteal ap with an incision
in the vestibular fold from molar to molar region.
Special attention was paid to maintaining an intact
periosteum. All periosteal remnants were removed
Fig. 2. (a) Patient with a bilateral cleft palate. (b) The
autologous graft should have a large surface to close the
entire oroantral and or oronasal communication. (c, d)
When soft tissue defects are still present, they should be
closed by a multilayered technique. One can see from this
view that the patient could not wear a denture. (e, f) The
clinical result (two pictures 13 years apart) on two
implants only (the ones immediately installed over the
congenital defect did not integrate) which can retain an
overdenture. (g, h) Radiographic outlook before surgery
and some years after. (i, j) In this female patient suffering
from a bilateral palatoschisis, four implants could be
installed in an autologous graft. (k) The same patient after
some 12 years with a stable situation concerning implants
but with gingivitis due to a Candida infection (she did not
remove her denture at night).
94
from the dissected bone. The cortex of the recipient
jawbone was, as for a grafting procedure, perforated
with a small round bur at many sites to allow proper
blood supply. The individualized membrane ts very
well (Fig. 3f) and a few xation screws were installed
(Fig. 3g) to guarantee the immobility when the
patient was allowed to wear his denture again or
preferably 2 months after insertion. The volume con-
tained under the membrane lls itself by blood,
which will allow a progressive ossication as has
been demonstrated from experiments on the rabbit
skull (17). The postoperative pain is limited but swel-
ling can be substantial for 2 weeks.
After 12 months the membrane was removed and
a whitish dense and eventually hard tissue lled the
entire space (Fig. 3h). Implants can be installed
(Fig. 3i) and, 6 months later, abutments. The patient
received a xed prosthesis with very satisfying
result from both a functional and esthetic point of
view (Fig. 3j). Intraoral control radiographs after
3 years show the perfect stability of the marginal
bone level.
A total of 10 patients have been treated in Leuven
via this approach (17). In all but three patients suf-
ciently large bone volume increases, up to 13 mm in
height, were obtained to install implants. The failures
were related to an early perforation of the mem-
brane. The latter can be prevented by the following:
an optimal ap design and possibly dissection of
the labial fold of the vestibulum
optimal postoperative care
2-month healing without denture
perfect adaptation of the denture for the following
6 months
Fig. 2. continued
95
From a total of 39 implants, one failed. The mean
bone loss around the remaining implants remained
within the success criteria formulated by Albrektsson
& Sennerby (4).
Guided bone regeneration but for smaller volumes
can also be obtained with disposable membranes as
illustrated by Becker (5). One can conclude that
membrane techniques are predictable and therefore
Fig. 3. (a) The preoperative view illustrates the knife-edge
jaw anatomy. (b) Panoramic radiograph of the referring
dentist illustrates an extremely radiolucent maxilla with a
relative good height of the crest. The canine and molar
were removed at surgery to allow a guided bone regenera-
tion over the entire maxilla. (c, d) An example of a litho-
graphic model prepared with the data from the spiral CT
scan of the maxilla on which the augmentation will be
mimicked with some wax-up and later transformed to a
plaster model. (e) Such a blocked out model is used to
press a 0.3 mm custom-t commercial pure titanium
membrane with a perfect adaptation. (f) The borders of
the individualized membrane ts perfectly to the basal
bone. Before inserting the membrane it is lled with
patient's blood retrieved after perforating the crest. (g)
Close up picture of optimal t between occlusive mem-
brane and basal bone. To stabilize the membrane, small
xation screws are added. (h) The bony tissue volume at
12 months is impressive when compared to the initial
situation. A whitish dense and underlying hard tissue lls
the entire space below the membrane. The supercial
layers showa good blood supply. (i) The bone regeneration
allows the insertion of six implants even following indica-
tions of the dentist to achieve the optimal implant loca-
tion/angulation. j) The provisional prosthesis is in place
on the bulky crest.
96
should be offered to patients with, at rst sight, too
little bone for an implant-based rehabilitation. This
should be done even if patients have to be referred to
a more skilled surgeon.
Extremely resorbed mandible
An extreme resorption of the mandible often renders
retention of the full prosthesis impossible. Such
patients, who are used to living with a denture, often
only seek more retention of their denture. The inser-
tion of two implants interconnected with a dolder
bar offers a satisfactory solution for most patients
and seems reliable in the long-term (16). For some
patients, however, even such a minor surgical inter-
vention becomes risky because of the extremely
resorbed jawbone volume. In such patients a grafting
procedure can recommended. The procedure is illu-
strated via another clinical case.
In this patient, a 45-year-old man, edentulous
since he was 18, which presented an extreme resorp-
tion of both edentulous jaws, the risk of fracture
during or soon after implant could not be ignored.
Furthermore, the distance to the occlusal plane was
more than 3 cm, which would lead to an unfavorable
relationship between the lengths of the endosseous
implant and of the prosthetic suprastructure. It was
agreed with the patient that an autologous hip graft
would be installed to heighten the mandible,
together with implant installation. The patient asked
only for a sufcient retention of his denture, so just
two implants to retain an overdenture were planned.
A horseshoe-shaped hip graft was taken and imme-
diately xed by means of three screw-shaped
implants (Branemark system) (Fig. 4a). The graft
reached the molar region since this is where mucosal
support for the overdenture is needed. It has also
been observed that the bone resorption of the mand-
ible in this distal region is increased when implant-
retained overdentures are placed (7). Although for an
overdenture retention only two implants are needed
(10, 11, 16), the third implant was installed as a
reserve in case one of the other two did not integrate.
As can be seen from the panoramic radiograph
taken some 6 months after the grafting procedure,
a reduced bone density is evident (Fig. 4b). Only two
abutments were installed on the lateral implants.
They were connected by a bar and to anchor the
overdenture with clips. After more than 12 years
the patient still happily functions with his abut-
ment-retained overdenture. The panoramic radio-
graph (Fig. 4c) shows only a partial resorption of
Fig. 3. continued
97
the graft, especially in the distal areas where no x-
tures had been inserted. The intraoral, long cone
radiographs taken at his last visit (13 years after abut-
ment connection) demonstrate the stable peri-
implant conditions (Fig. 4d).
Anatomic defects/discontinuities
after resection surgery
Oncologic surgery in the head and neck region
often includes the resection of major parts of the
jawbones. Today's maxillofacial surgical approaches
can almost guarantee the re-establishment of tissue
continuity. However, tissue repair is not synony-
mous with functional rehabilitation. Osseointegrated
implants can be essential to re-establish normal oro-
facial function.
Mandibular discontinuity
As seen in this female patient (65 years old) who
underwent a subtotal mandibulectomy (together
with chemotherapy and radiotherapy) for a spinocel-
lular epithelioma of the oor of the mouth, the
resulting intraoral situation did not allow a proper
retention of a full removable denture (Fig. 5a). A
plastic surgeon performed a subtotal reconstruction
of the resected mandible by means of an iliac crest
bone graft (Fig. 5b). This involved an arterial end-to-
end anastomosis between the arteria circumexa
and the facial artery to insure a proper vasculariza-
tion of the graft after the irradiation the patient had
undergone. The clips of arterial anastomosis can be
seen (Fig. 5b). This element encouraged us to try for
a functional rehabilitation by means of endosseous
implants from this orally seriously disabled patient.
Indeed, the concavities of the lower crest and the
intraoral skin graft that had accompanied the
bone graft were unfavorable elements for a prosthe-
tic retention. Skin does not offer the moisture nor-
mally encountered with oral mucosae, which
guarantees the vacuum effect under the custom-t
removable denture. Intraoral hair growth was also
disturbing.
In 1987, six implants were inserted under general
anesthesia, which all uneventfully integrated, as
Fig. 4. (a) An autologous graft immediately xed by three
self-tapping implants leads to an ample jaw bone volume.
(b) The panoramic image, taken immediately prior to
abutment connection, shows a reduced density of the
graft. The three implants seem to be well integrated. (c)
The panoramic radiograph taken after 12 years shows only
a partial resorption of the graft, especially in the distal
areas where no xtures had been inserted. (d) The
intraoral, long cone radiographs taken at the last visit
(13 years after abutment connection), demonstrate an
acceptable marginal bone level.
98
could be checked at the abutment surgery some
6 months later (Fig. 5c,d). On these abutments a
xed prosthesis was installed by the Department of
Prosthetic Dentistry, which, according to the
patient's reactions, gave an enormous improvement
of her quality of life (Fig. 5e).
The maintenance of inammation-free soft tissues
around the abutments demanded a meticulous
Fig. 5. (a) As part of the treatment of a spinocellular
epithelioma of the oor of the mouth, a subtotal mandi-
bulectomy had been performed in this 65-year-old women
by a plastic surgeon. Even though the patient had under-
gone a subtotal reconstruction of the resected mandible by
means of an iliac crest bone graft, the resulting intraoral
situation did not allow a proper retention of a full remo-
vable denture. (b) The intraoral view shows the skin graft
and compromised anatomy, which explain the lack of
retention of the denture. (c) It was decided to install six
Bra nemark system implants under general anesthesia,
which all uneventfully integrated, as shown by this radio-
graph taken just before abutment connection at 8 months.
(d) During the abutment insertion, all implants were
mechanically diagnosed as well integrated. (e) Up to the
molar region, a 10-unit bridge, installed by the prostho-
dontist, gave the patient satisfactory function. (f) The same
patient after 15 years with a stable functional situation,
even though a slight inammation of the gingiva and
mucosa is apparent. (g) Since the patient had other prio-
rities in her life, she did not seek a more esthetic solution
in the maxilla.
99
plaque control, which the patient, although very moti-
vated, was not always able to achieve. Around the
most distal implants a pocket deepening occurred
and after some years this became purulent, though
this did not lead to subjective complaints (Fig. 5f).
This peri-implantitis was treated by means of
repeated rinsing with a chlorhexidine gel at each con-
trol visit. It did not seem to improve whatever was
tried. After some time, the most distal implant in the
3rd quadrant failed. The latter was replaced by two
new implants. All remaining implants remained
functional and even after 15 years the appearance
was good. The patient passed away because of an
unrelated illness.
Bilateral maxillectomy
A male adult patient had undergone a total bilateral
maxillectomy in 1968 (in another department)
because of a carcinoma. Only the palatal bones
and some of the lateral walls of the maxillae were
left (Fig. 6a). The patient experienced great difcul-
ties in chewing and speech functions. He could not
wear a retention prosthesis because of pain due to
the fragility of the sinusal epithelial linings after the
irradiation. Even subtle contacts of those mucosae
many years after the irradiation still led to bleeding.
The patient normally wore tissues in the sinusal cav-
ities to allow a more or less understandable speech.
When he consulted the Department of Periodontol-
ogy in 1988, the option of rehabilitation by means of
endosseous implants was considered, even though
the prognosis at that time before hyperbaric oxy-
gen therapy was routinely used, was reserved (6).
Explorative surgery was performed to search for any
available bone volume to harbor implants. Since no
bone whatsoever could be found in the anterior areas,
it was decided to insert two long implants in the
zygomatic bone. At that time no special design was
available (9), so two long (25 mm) self-tapping im-
plants were inserted in the zygoma through an infra-
zygomatic arch approach. To allow the insertion of
two more implants, a split crestal hip bone graft was
xed on top of each of the palatine bones, and two
implants were screwed in these grafts (Fig. 6b). After
8 months of healing abutment surgery took place and
all four implants seemed rigidly anchored. On top of
that, a complex two-piece frame was constructed by
the Department of Prosthetic Dentistry to retain an
overdenture (Fig. 6c,d). Although there was no frontal
support, this superstructure functioned properly for
6 years. After that time, one of the zygomatic implants
fractured and had to be replaced (Fig. 6e). The pros-
thetic framework was replaced by a shorter framework
(Fig. 6f,g), which also gave a comfortable retention.
The psychologic benets for the patient of re-estab-
lished phonetic and chewing functions are enormous.
After more than 15 years this patient happily enjoys
his implant-supported rehabilitation.
Resection of maxillary and nasal
structures
A male patient had undergone a resection of maxillary
and midfacial structures for carcinoma treatment in
1980. He was given xtures in the frontal and zygo-
matic bones to support a metallic frame which
allowed him to anchor both an oral and a midfacial
prosthesis. All implants functioned well, although the
patient had been fully irradiated, until he passed away
11 years later from an unrelated cause (Fig. 7ae).
Anatomic defects after traumatic
injuries
In a number of instances, traumatic loss of jaw struc-
tures can exclude the insertion of any implant or lead
to unfavorable biomechanical outcomes. If the length
of the abutment plus the prosthetic superstructure to
reach the occlusal plane is larger than the endoss-
eous part, overloading can occur with loss of integra-
tion (12). Therefore it can be mandatory to increase
the available bone volume prior to or together with
the implant insertion.
Fig. 5. continued
100
In a young patient (21 years of age) who suffered a
trafc accident (Fig. 8a), besides dental trauma a
substantial part of the periodontal tissues was lost
in the symphyseal area. To achieve a proper recon-
struction, a small hip graft was taken, inserted on top
of the remaining symphyseal bone and xed with
two screw-shaped xtures (Fig. 8b,c). At abutment
surgery (Fig. 8d,e) an optimal situation was encoun-
tered and a xed prosthesis was installed (Fig. 8f),
which thanks to the increased bone and gingival
Fig. 6. (a) The bilateral maxillectomy to resect the tumor
created severe difculties in chewing and speech for this
patient. Only the palatal bones wer

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Environmental and other

modifying factors of the

The native periodontium includes cementum, a

berg T, Wozney J, Thesleff I. Expression patterns of bone

zkaynak E, Oppermann H, Rueger DC.

Fig. 2. Tumor necrosis factor-a (TNF-a) concentration

bo Akademi University, 2000.

compared with an expected value of 4% for B

kesson L, Hakansson J, Rohlin M. Comparison of pan-

kesson L, Hakansson J, Rohlin M. Comparison of pan-

lymphocyte count (88). These ndings raise the

T-helper cells and an increase in T-suppressor disproportionate to the quantity of plaque

T cells while suppressing the ability of CD4

T cells to migrate in response to interleukin-8 (144).

(suppresser) (141), activated T lymphocytes

T cells and is respon-

cytotoxic T cells, and those derived

) greatly outnumber naive (CD45RA

, which suggests reactivation of the

ratios (153) or mixed (147), or even not

ratio in the tissues of early-onset periodontitis

ratios were seen in

cytotoxic T cells and killer cells,

cells from periodontitis lesions (adult peri-

cells between the

T cells regardless of disease category. It appears

cells may have been

T cell clones and resting T

T lymphocytes and an inhibitor of IL-8 induced

T lymphocyte migration. J Immunol 1993: 151:

ngen Z. The importance of acute phase pro-

. Cytokine networks and cortico-

nerud A, Boysen H, Burgin W, Loe H.

hman AE, Moller A

JR, Fabricius L, Dahlen G, O

hman AE, Heyden G.

, Gardner DG, Bostanci H, Gunham M. Familial

nlu F, Gurses N, Seckin T, U

nal T. Multifocal eosinophilic

kesson L, Hakansson J, Rohlin M. Comparison of panora-

) for periodontal tissue regener-

stgren A, Gestrelius S. Clinical safety of enamel

(GlaxoSmithKline, Brentford, UK), a com-

sman B, Bergstrom K. Granulocyte

sman B, Soder B, Purliene A, Bergs-

strand P. The applicability of osseo-integrated oral

2730 kHz (EMS,

30 kHz (Osada Electro-

30 kHz (Satelec, Merignac, France). In the magneto-

25 kHz; Dentsply, Des Plaines, IL), or a rod

sonic scaler 6.5 kHz (StarDental, Lancaster,

6.0 kHz (Kavo, Biberach, Germany),

sonic scaler 6.0 kHz (Dentsply, Des Pla-

560 6.0 kHz (Micron Co. Ltd,

with TFI-10 tip) on the subgingival microora

with modied tip 17.5 mm long

with type A tip) and sonic (Titan-S

with Perio tip