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Periodontology 2000
Periodontology 2000
Periodontology 2000
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?
Gingival crevice uid 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.
Gingival crevice uid ow
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.
Gingival crevice uid ow
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.
Gingival crevice uid ow
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.
Gingival crevice uid ow
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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Periodontology 2000, Vol. 31, 2003, 167180 Copyright C Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
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
GCF markers of risk for periodontal diseases
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).
GCF markers of risk for periodontal diseases
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
levels of prostaglandin E
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
GCF markers of risk for periodontal diseases
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-
GCF markers of risk for periodontal diseases
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, 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
Toothepithelial interface in health and disease
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.
Toothepithelial interface in health and disease
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.
Toothepithelial interface in health and disease
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-
Toothepithelial interface in health and disease
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
Toothepithelial interface in health and disease
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-
Toothepithelial interface in health and disease
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
Periodontal ligament homeostasis
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
Periodontal ligament homeostasis
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
Periodontal ligament homeostasis
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
Periodontal ligament homeostasis
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
Periodontal ligament homeostasis
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-
Periodontal ligament homeostasis
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, Vol. 24, 2000, 927 Copyright C Munksgaard 2000
Printed in Denmark All rights reserved
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
Development and general structure of the periodontium
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).
Development and general structure of the periodontium
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-
Development and general structure of the periodontium
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
Development and general structure of the periodontium
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
Development and general structure of the periodontium
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.
Development and general structure of the periodontium
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
Periodontology 2000, Vol. 42, 2006, 712
Printed in Singapore. All rights reserved
2006 The Authors.
Journal compilation 2006 Blackwell Munksgaard
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
Haffajee & Socransky
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
Haffajee & Socransky
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
Periodontology 2000, Vol. 42, 2006, 1326
Printed in Singapore. All rights reserved
2006 The Authors.
Journal compilation 2006 Blackwell Munksgaard
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
Molecular genetics analyses of biolm formation in oral isolates
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
Molecular genetics analyses of biolm formation in oral isolates
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
Periodontology 2000, Vol. 42, 2006, 2735
Printed in Singapore. All rights reserved
2006 The Author.
Journal compilation 2006 Blackwell Munksgaard
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
Techniques for the growth of Porphyromonas gingivalis biolms
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
Techniques for the growth of Porphyromonas gingivalis biolms
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
Periodontology 2000, Vol. 42, 2006, 219258
Printed in Singapore. All rights reserved
2006 The Authors.
Journal compilation 2006 Blackwell Munksgaard
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
Effect of periodontal therapy on the subgingival microbiota
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
Effect of periodontal therapy on the subgingival microbiota
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
2 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
a i d e m r e t n i . P
9 . 0
3 . 1
8 . 1
2 . 2
a i h t y s r o f . T
6 . 2
3 . 1
5 . 1
8 . 1
0 . 2
2 . 2
i i t n e c n i v s s m u t a e l c u n . F
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
Effect of periodontal therapy on the subgingival microbiota
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
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
2 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
0 . 0 6 . 1 3 . 3 9 . 4 5 . 6 0 . 0 6 . 1 3 . 3 9 . 4 5 . 6
*
* * *
* * *
*
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* *
* * *
* *
* * *
* * *
*
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* *
* * *
*
* * *
* * *
* *
*
* * *
* * *
* * *
* *
* *
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
* *
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
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
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
Fig. 14. 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, 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
a i h t y s r o f . T
6 . 0
3 . 2
0 . 4
7 . 5
s i l a v i g n i g . P
4 . 0
2 . 1
9 . 1
7 . 2
4 . 3
a l o c i t n e d . T
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
Effect of periodontal therapy on the subgingival microbiota
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
* *
* *
* * *
* *
* *
* * *
*
*
* * *
* * *
* * *
* * *
* *
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
2 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
P O B P O B o N
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
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
* *
* *
*
* * *
*
* * *
* * *
* * *
* *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
*
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
2 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
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
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
0 1 x s t n u o C
5
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
Fig. 17. Proles of mean counts (10
5
) of 40 taxa in sub-
gingival plaque samples from 461 chronic periodontitis
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
Effect of periodontal therapy on the subgingival microbiota
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
) of 40 taxa in sub-
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
Effect of periodontal therapy on the subgingival microbiota
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.
Fig. 22. Plots of mean counts (10
5
) of 40 taxa in sub-
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
Effect of periodontal therapy on the subgingival microbiota
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
1 . 7 9 . 3 7 . 0 5 . 2 7 . 5
m m 2 > e s a e r c n i D P m m 2 > n o i t c u d e r D P < e g n a h c D P m m 2
7 . 5 5 . 2 7 . 0 9 . 3 1 . 7 7 . 5 5 . 2 7 . 0 9 . 3 1 . 7
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
2 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
0 1 x s t n u o C
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
Fig. 24. Plots of mean counts (10
5
) of 40 taxa in sub-
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
Effect of periodontal therapy on the subgingival microbiota
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
g
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
from baseline (pretherapy) to 12 months post-therapy in
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
Effect of periodontal therapy on the subgingival microbiota
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
5 1
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
>
L
A
s
e
x
h
i
b
i
t
i
n
g
e
t
i
s
N
0
0
0
,
0
1
r
e
p
m
m
3
k
s
i
r
t
a
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
Effect of periodontal therapy on the subgingival microbiota
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
Effect of periodontal therapy on the subgingival microbiota
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
*
* *
* * *
*
* *
* * *
* * *
* * *
*
* *
* *
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
2 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
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
s e c y m o n i t c A
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
Fig. 32. Proles of mean counts (10
5
) of 40 taxa in sub-
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
l e v e l t n e m h c a t t A
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
Effect of periodontal therapy on the subgingival microbiota
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
* * *
* * *
* * *
* * *
* * *
*
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* * *
* *
* * *
* * *
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
2 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
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
s e c y m o n i t c A
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
Fig. 34. Proles of mean counts (10
5
) of 40 taxa in sub-
gingival plaque samples from 169 chronic periodontitis
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
a i h t y s r o f . T
9 . 0
7 . 1
5 . 2
3 . 3
1 . 4
s i l a v i g n i g . P
4 . 0
6 . 0
8 . 0
0 . 1
2 . 1
a l o c i t n e d . T
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
Effect of periodontal therapy on the subgingival microbiota
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
a i h t y s r o f . T
8 2 . 5
6 9 . 3
4 6 . 2
2 3 . 1
0 0 . 0
s i l a v i g n i g . P
3 0 . 1
4 7 . 0
6 4 . 0
8 1 . 0
a l o c i t n e d . T
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
Effect of periodontal therapy on the subgingival microbiota
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
h t p e d t e k c o P
l e v e l t n e m h c a t t A
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
h t p e d t e k c o P
l e v e l t n e m h c a t t A
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
Effect of periodontal therapy on the subgingival microbiota
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
a i h t y s r o f . T
7 . 0
5 . 1
3 . 2
0 . 3
s i l a v i g n i g . P
3 . 0
5 . 0
6 . 0
8 . 0
0 . 1
a l o c i t n e d . T
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
Fig. 41. Mean counts ( standard error of the mean) for
Eubacterium nodatum, Tannerella forsythia, Porphyro-
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
a i h t y s r o f . T
6 . 0
4 . 1
2 . 2
0 . 3
s i l a v i g n i g . P
2 . 0
4 . 0
6 . 0
8 . 0
0 . 1
a l o c i t n e d . T
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
Fig. 42. Mean counts ( standard error of the mean) for
Eubacterium nodatum, Tannerella forsythia, Porphyro-
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
Effect of periodontal therapy on the subgingival microbiota
0 4 8
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
2 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
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
s e c y m o n i t c A
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)
from baseline (pretherapy) to 12 months post-therapy in
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
s p u o r g t n e m t a e r T
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
Effect of periodontal therapy on the subgingival microbiota
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
s p u o r g t n e m t a e r T
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
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112
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
Regeneration of vascularized bone
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
Regeneration of vascularized bone
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
Regeneration of vascularized bone
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, Vol. 41, 2006, 136156
Printed in Singapore. All rights reserved
Copyright Blackwell Munksgaard 2006
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
Advances in dental implant materials and tissue regeneration
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)
,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
Regeneration of periodontal tissues: cementogenesis revisited
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s
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n
c
r
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a
s
e
N
D
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(
8
9
)
H
u
(
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r
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r
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)
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s
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c
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b
1
,
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L
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6
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-
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B
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(
1
3
9
)
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(
p
r
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m
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n
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c
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(
2
0
4
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M
o
(
c
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l
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)
*
*
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(
2
7
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7
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s
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l
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u
(
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1
7
/
2
.
8
)
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(
2
2
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1
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(
1
6
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(
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T
2
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f
f
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(
2
6
8
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(
K
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/
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1
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s
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a
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s
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l
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1
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s
-
m
o
r
e
(
2
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(
p
r
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s
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n
c
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a
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e
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D
(
1
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(
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6
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1
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3
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a
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a
n
d
l
e
s
s
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C
N
(
2
5
4
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u
(
2
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9
p
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l
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s
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N
o
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f
f
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t
N
D
(
2
1
5
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u
(
M
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6
3
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l
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a
s
e
A
P
,
O
C
N
,
T
G
B
1
N
D
(
2
1
5
)
204
Zeichner-David
T
a
b
l
e
1
.
C
o
n
t
i
n
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e
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e
l
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A
t
t
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m
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t
P
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f
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f
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t
i
a
t
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o
n
M
i
n
e
r
a
l
i
z
a
t
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o
n
R
e
f
e
r
e
n
c
e
H
u
(
p
r
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m
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)
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s
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n
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e
a
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e
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,
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n
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,
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1
N
D
(
2
1
5
)
H
u
(
M
G
6
3
)
Y
e
s
N
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e
s
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n
c
r
e
a
s
e
N
D
N
D
(
8
9
)
P
(
p
r
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(
2
0
4
)
H
u
(
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s
1
7
/
2
8
)
*
N
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D
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n
c
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a
s
e
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(
2
2
8
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b
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e
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l
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R
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m
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e
(
1
1
5
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u
(
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y
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s
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a
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e
N
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D
(
2
0
3
)
H
u
(
p
r
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N
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n
c
r
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a
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v
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c
a
n
,
b
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y
c
a
n
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d
e
c
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n
,
h
y
a
l
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o
n
a
n
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(
7
3
)
H
u
(
p
r
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m
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r
y
)
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e
f
f
e
c
t
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f
f
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n
d
T
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F
-
b
1
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(
2
5
6
)
H
u
(
p
r
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m
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r
y
)
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e
s
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e
s
i
n
c
r
e
a
s
e
N
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N
D
(
8
9
)
R
a
t
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D
N
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e
s
i
n
c
r
e
a
s
e
M
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r
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E
C
M
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o
(
1
1
5
)
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a
t
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o
d
i
f
f
e
r
e
n
c
e
N
D
N
D
(
7
2
)
P
(
p
r
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m
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)
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(
2
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4
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e
n
t
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l
f
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c
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e
M
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(
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V
4
0
)
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s
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n
c
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h
i
b
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(
7
4
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m
e
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s
M
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(
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4
0
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n
c
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n
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n
h
i
b
i
t
s
(
2
5
4
)
M
o
(
O
C
C
M
-
3
0
)
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N
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N
D
N
D
D
e
c
r
e
a
s
e
B
S
P
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n
h
i
b
i
t
s
(
2
5
8
)
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(
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C
M
-
3
0
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N
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N
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e
f
f
e
c
t
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e
c
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a
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e
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C
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,
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n
c
r
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a
s
e
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n
d
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P
G
I
n
h
i
b
i
t
s
(
1
7
)
F
i
b
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o
b
l
a
s
t
s
M
o
(
L
9
2
9
)
N
D
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D
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o
d
i
f
f
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e
n
c
e
N
D
N
D
(
7
2
)
R
a
b
b
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t
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v
a
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l
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m
.
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r
o
w
t
h
f
a
c
t
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r
s
(
1
6
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)
H
u
m
a
n
(
p
r
i
m
a
r
y
)
Y
e
s
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D
Y
e
s
i
n
c
r
e
a
s
e
N
D
N
D
(
2
0
3
)
M
e
s
e
n
c
h
y
m
a
l
s
t
e
m
c
e
l
l
s
H
u
(
C
2
C
1
2
)
N
D
N
D
N
D
Y
e
s
i
n
c
r
e
a
s
e
A
P
.
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s
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b
l
a
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p
h
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p
e
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(
1
7
7
)
E
p
i
t
h
e
l
i
a
l
c
e
l
l
s
H
u
(
H
E
L
A
)
N
D
N
D
I
n
h
i
b
i
t
e
d
I
n
c
r
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a
s
e
c
A
M
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a
n
d
P
D
G
F
-
A
B
N
D
(
1
1
3
)
H
u
(
S
C
C
2
5
)
N
D
N
D
I
n
h
i
b
i
t
e
d
I
n
c
r
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a
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p
2
1
W
A
F
1
/
c
i
p
1
;
d
e
c
r
e
a
s
e
C
K
-
1
8
N
D
(
1
1
3
,
1
1
2
,
1
1
4
)
E
R
M
P
(
p
r
i
m
a
r
y
)
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s
Y
e
s
i
n
c
r
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a
s
e
I
n
c
r
e
a
s
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O
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(
2
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)
E
n
d
o
t
h
e
l
i
a
l
c
e
l
l
s
H
u
(
H
U
V
E
C
)
Y
e
s
i
n
c
r
e
a
s
e
N
D
N
o
e
f
f
e
c
t
N
D
N
D
(
2
7
1
)
A
P
,
a
l
k
a
l
i
n
e
p
h
o
s
p
h
a
t
a
s
e
;
B
M
P
,
b
o
n
e
m
a
t
r
i
x
p
r
o
t
e
i
n
;
B
S
P
,
b
o
n
e
s
i
a
l
o
p
r
o
t
e
i
n
;
C
o
l
,
c
o
l
l
a
g
e
n
;
H
u
,
h
u
m
a
n
;
I
G
F
-
1
,
i
n
s
u
l
i
n
-
l
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k
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r
o
w
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f
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c
t
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r
;
I
L
-
6
,
i
n
t
e
r
l
e
u
k
i
n
-
6
;
M
M
P
S
,
m
a
t
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x
m
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t
a
l
l
o
p
r
o
t
e
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n
a
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s
;
M
o
,
m
o
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s
e
;
N
D
,
n
o
t
d
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t
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r
m
i
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e
d
;
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C
N
,
o
s
t
e
o
c
a
l
c
i
n
;
O
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N
,
o
s
t
e
o
p
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n
t
i
n
;
O
P
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,
o
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p
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e
r
i
n
;
P
,
p
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g
;
P
D
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-
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,
p
l
a
t
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t
d
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d
g
r
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w
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f
a
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r
A
B
;
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G
H
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-
2
,
p
r
o
s
t
a
g
l
a
n
d
i
n
G
/
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s
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n
t
h
a
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e
2
;
T
G
F
-
b
1
,
t
r
a
n
s
f
o
r
m
i
n
g
g
r
o
w
t
h
f
a
c
t
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r
b
-
1
.
*
M
o
u
s
e
r
e
c
o
m
b
i
n
a
n
t
a
m
e
l
o
g
e
n
i
n
.
M
o
u
s
e
r
e
c
o
m
b
i
n
a
n
t
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m
e
l
o
b
l
a
s
t
i
n
.
M
o
u
s
e
l
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u
c
i
n
e
r
i
c
h
a
m
e
l
o
g
e
n
i
n
p
e
p
t
i
d
e
(
L
R
A
P
)
.
205
Regeneration of periodontal tissues: cementogenesis revisited
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
Regeneration of periodontal tissues: cementogenesis revisited
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
Regeneration of periodontal tissues: cementogenesis revisited
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, Vol. 41, 2006, 915
Printed in Singapore. All rights reserved
Copyright Blackwell Munksgaard 2006
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
Tissue engineering: challenges and potential
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15
Tissue engineering: challenges and potential
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
Printed in Denmark. All rights reserved
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
Smoking and periodontal disease
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.
In summary, the preponderance of the evidence
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).
In summary, the preponderance of the evidence
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
Smoking and periodontal disease
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
Smoking and periodontal disease
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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
Periodontology 2000, Vol. 32, 2003, 5981 Copyright
#
Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
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
Hormonal influences on periodontium
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
Hormonal influences on periodontium
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
Hormonal influences on periodontium
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
Hormonal influences on periodontium
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
Hormonal influences on periodontium
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
Hormonal influences on periodontium
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
Hormonal influences on periodontium
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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#
Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
PERIODONTOLOGY 2000
ISSN 0906-6713
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
Cross-sectional study
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.
Cross-sectional study
Streckfus et al. (23) 28 healthy women
aged 2378
Strong correlation between alveolar bone
and the second metacarpal densities.
Both reduced in postmenopausal women
Cross-sectional study
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
Cross-sectional study
Kribbs et al. (14) 50 normal women
aged 2090
Mandibular bone mass correlated with
bone mass at spine and wrist
Cross-sectional study
Kribbs (12) 85 osteoporotic women
and 27 normal
women aged 5085
Osteoporotic group had less mandibular
bone mass and density
Cross-sectional study
Kribbs et al. (13) 85 osteoporotic women Total body calcium, bone mass at radius,
and bone density at spine correlated
with mandibular mass
Cross-sectional study
Kribbs et al. (15) 30 postmenopausal
women
Total body calcium associated with
mandibular bone density
Cross-sectional study
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 ``altered 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
Occlusal forces and periodontal disease
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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#
Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
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 ``exposed'' group to the risk of disease in the
``unexposed'' 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
Understanding the etiology of periodontitis: an overview of periodontal risk factors
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
Understanding the etiology of periodontitis: an overview of periodontal risk factors
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
Understanding the etiology of periodontitis: an overview of periodontal risk factors
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
Understanding the etiology of periodontitis: an overview of periodontal risk factors
Periodontology 2000, Vol. 30, 2002, 2439 Copyright C Blackwell Munksgaard 2002
Printed in Denmark. All rights reserved
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
Noninammatory destructive periodontal disease
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
Noninammatory destructive periodontal disease
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
Noninammatory destructive periodontal disease
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
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
Noninammatory destructive periodontal disease
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
Noninammatory destructive periodontal disease
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
Noninammatory destructive periodontal disease
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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Periodontology 2000, Vol. 30, 2002, 4050 Copyright C Blackwell Munksgaard 2002
Printed in Denmark. All rights reserved
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
Benecial bacteria of periodontium
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
Benecial bacteria of periodontium
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
Benecial bacteria of periodontium
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, Vol. 30, 2002, 104110 Copyright C Blackwell Munksgaard 2002
Printed in Denmark. All rights reserved
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
Trends in periodontal care
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.
Trends in periodontal care
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.
Trends in periodontal care
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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Periodontology 2000, Vol. 30, 2002, 923 Copyright C Blackwell Munksgaard 2002
Printed in Denmark. All rights reserved
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
Classifying periodontal diseases
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
Classifying periodontal diseases
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
Classifying periodontal diseases
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
genetic polymorphisms.
O group C Set .3 of microorganisms Set .3 of
genetic polymorphisms.
O group D Set .4 of microorganisms Set .4 of
genetic polymorphisms.
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-
Classifying periodontal diseases
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, Vol. 39, 2005, 2229
Printed in the UK. All rights reserved
Copyright Blackwell Munksgaard 2005
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
Abnormal pocket depth and gingival recession
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
Abnormal pocket depth and gingival recession
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29
Abnormal pocket depth and gingival recession
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, Vol. 39, 2005, 3039
Printed in the UK. All rights reserved
Copyright Blackwell Munksgaard 2005
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, Vol. 39, 2005, 7390
Printed in the UK. All rights reserved
Copyright Blackwell Munksgaard 2005
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
Bragger
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
Bragger
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
Biological signicance and clinical use of radiographs
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
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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
Biological signicance and clinical use of radiographs
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
Biological signicance and clinical use of radiographs
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
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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
Biological signicance and clinical use of radiographs
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
Biological signicance and clinical use of radiographs
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.
References
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Variation in radiographic alveolar bone loss in subgroups
of 14-year-old schoolchildren in Oslo. J Clin Periodontol
1988: 15: 130133.
2. Aass AM, Tollefsen T, Gjermo P. A cohort study of radio-
graphic alveolar bone loss during adolescence. J Clin
Periodontol 1994: 21: 133138.
3. A
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Salonen L. A computerized system to measure inter-
proximal alveolar bone levels in epidemiologic, radio-
graphic investigations. II. Intra- and interexaminer
variation study. Acta Odontol Scand 1988: 46: 3339.
157. Wouters FR, Salonen LE, Hellden LB, Frithiof L. Preval-
ence of interproximal periodontal intrabony defects in an
adult population in Sweden. A radiographic study. J Clin
Periodontol 1989: 16: 144149.
158. Zamet JS, Darbar UR, Grifths GS, Bulman JS, Bragger U,
Burgin W, Newman HN. Particulate bioglass as a grafting
material in the treatment of periodontal intrabony de-
fects. J Clin Periodontol 1997: 24: 410418.
159. Zappa U, Simona C, Graf H, van Aken J. In vivo deter-
mination of radiographic projection errors produced by a
novel lmholder and an X-ray beam manipulator. J Peri-
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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, Vol. 39, 2005, 1321
Printed in the UK. All rights reserved
Copyright Blackwell Munksgaard 2005
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
Periodontal disease classication
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
Periodontal disease classication
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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21
Periodontal disease classication
Periodontology 2000, Vol. 26, 2001, 92112 Copyright C Munksgaard 2001
Printed in Denmark All rights reserved
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
Inuences of systemic diseases on periodontitis in children and adolescents
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).
Inuences of systemic diseases on periodontitis in children and adolescents
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.
Inuences of systemic diseases on periodontitis in children and adolescents
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-
Inuences of systemic diseases on periodontitis in children and adolescents
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
Inuences of systemic diseases on periodontitis in children and adolescents
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).
Inuences of systemic diseases on periodontitis in children and adolescents
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
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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
Diagnosis and management of periodontal diseases in children and adolescents
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, Vol. 26, 2001, 5491 Copyright C Munksgaard 2001
Printed in Denmark All rights reserved
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
Etiopathogenesis of periodontitis in children and adolescents
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
(helper) and
CD8
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
Etiopathogenesis of periodontitis in children and adolescents
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
) T
cells in periodontitis lesions (87, 154, 167, 353). How-
ever, it has also been shown that a proportion of these
cells are CD45RA
: CD8
:
CD8
: CD8
: 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
CD8
CD4
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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, Vol. 34, 2004, 165203 Copyright
#
Blackwell Munksgaard 2004
Printed in Denmark. All rights reserved
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
Combined periodontal-endodontic lesions
(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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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
Combined periodontal-endodontic lesions
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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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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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
Reconstructive procedures for intraosseous defects
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
Reconstructive procedures for intraosseous defects
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.
Results from systematic reviews
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
Reconstructive procedures for intraosseous defects
(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
Reconstructive procedures for intraosseous defects
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).
Results from systematic reviews
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
Reconstructive procedures for intraosseous defects
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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113
Guided tissue regeneration
(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
Cortellini et al. (11)
Cortellini et al. (14)
Mayfield et al. (32)
Sculean et al. (46)
Silvestri et al. (48)
Tonetti et al. (50)
Chung et al. (7)
Cortellini et al. (8)
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)
Test for heterogeneity: Chi
2
= 18.46, df = 7 (P = 0.01), I
2
= 62.1%
Test for heterogeneity: Chi
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
Cortellini et al. (10)
Cortellini et al. (11)
Cortellini et al. (14)
Mayfield et al. (32)
Tonetti et al. (50)
Zuccheli et al. (56)
Total (95% CI)
Total events: 32 (Treatment), 55 (Control)
Test for heterogeneity: Chi
2
= 5.92, df = 5 (P = 0.31), I
2
= 15.6%
Test for overall effect: Z = 2.22 (P = 0.03)
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
Favours GTR Favours access flap
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
Guided tissue regeneration
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
Favours GTR Favours access flap
0 2 4 4 2
Cortellini et al. (14)
Mayfield et al. (32)
Sculean et al. (46)
Silvestri et al. (48)
Zuccheli et al. (56)
Study
or sub-category mean difference (SE)
mean difference (random) Weight
% 95% Cl
mean difference (random)
95% Cl
01 Parallel group studies
Test for heterogeneity: Chi
2
= 43.60, df = 4 (P < 0.00001), I
2
= 90.8%
Test for overall effect: Z = 2.26 (P = 0.02)
Subtotal (95% Cl)
Test for heterogeneity: Chi
2
= 9.06, df = 5 (P = 0.11), I
2
= 44.8%
Test for heterogeneity: Chi
2
= 62.90, df = 10 (P < 0.00001), I
2
= 84.1%
Test for overall effect: Z = 3.49 (P = 0.0005)
Test for overall effect: Z = 3.51 (P = 0.0004)
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)
Cortellini et al. (8)
Loos et al. (31)
Mora et al. (34)
Pontoriero et al. (40)
Ratka-Kruger et al. (41)
02 Split-mouth studies
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.
Cortellini et al. (14)
Mayfield et al. (32)
Sculean et al. (46)
Study
or sub-category
01 Parallel group studies
Test for heterogeneity: Chi
2
= 0.97, df = 3 (P = 0.81), I
2
= 0%
Test for overall effect: Z = 1.08 (P = 0.28)
Zuccheli et al. (56)
Subtotal (95% Cl)
Test for heterogeneity: Chi
2
= 0.64, df = 4 (P = 0.96), I
2
= 0%
Test for heterogeneity: Chi
2
= 2.69, df = 8 (P = 0.95), I
2
= 0%
Test for overall effect: Z = 2.82 (P = 0.005)
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)
Pontoriero et al. (40)
Ratka-Kruger et al. (43)
02 Split-mouth studies
mean difference (SE)
mean difference (random) Weight
% 95% Cl
mean difference (random)
95% Cl
Favours GTR Favours access flap
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]
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1.37]
[0.70,
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100.00 0.26 0.43] [0.08,
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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
Guided tissue regeneration
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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
Guided tissue regeneration
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.
Guided tissue regeneration
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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Guided tissue regeneration
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, Vol. 37, 2005, 124137
Printed in Denmark. All rights reserved
Copyright Blackwell Munksgaard 2005
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
Smoking and mechanical periodontal therapy
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
Results in this direction indicate a
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-
Probing pocket depth mean improvement (mm)
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)
Grossi et al. 1997 (12)
Preber et al. 1995 (32)
Study (ref)
Results in this direction indicate a
better result in non-smokers
Results in this direction indicate a
better result in smokers
Mongardini et al. 1999 (24)
Fig. 3. Forest plot of mean differ-
ence in probing pocket depth
reduction between smokers and
non-smokers (threshold studies).
0.4 0.2 0 0.2
Combined
Grossi et al. 1997 (12)
Probing pocket depth mean improvement (mm)
Study (ref)
Results in this direction indicate a
better result in non-smokers
Results in this direction indicate a
better result in quit-smokers
Haffajee et al. 1997 (14)
Preshaw et al. 1999 (33)
Fig. 4. Forest plot of mean differ-
ence in probing pocket depth
reduction between quit-smokers
and non-smokers (all sites).
129
Smoking and mechanical periodontal therapy
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)
Grossi et al. 1997 (12)
Results in this direction indicate a
better result in non-smokers
Results in this direction indicate a
better result in smokers
Haffajee et al. 1997(14)
Machtei et al. 1998 (21)
Fig. 6. Forest plot of mean differ-
ence in clinical attachment level
gain between smokers and non-
smokers (all sites).
0.5 0 0.5 1.0
Probing pocket depth mean improvement (mm)
Combined
Study (ref)
Results in this direction indicate a
better result in non-smokers
Results in this direction indicate a
better result in quit-smokers
Grossi et al. 1997 (12)
Ryder et al. 1999 (36)
Fig. 5. Forest plot of mean differ-
ence in probing pocket depth
reduction between quit-smokers and
non-smokers (threshold studies).
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
Grossi et al. 1997 (12)
Study (ref)
Clinical attachment level mean improvement (mm)
Results in this direction indicate a
better result in non-smokers
Results in this direction indicate a
better result in quit-smokers
Haffajee et al. 1997 (14)
Fig. 8. Forest plot of mean differ-
ence in clinical attachment level
gain between quit-smokers and
non-smokers (all sites).
1.0 0.5 0 0.5 1.0
Combined
Ryder et al. 1999 (36)
Palmer et al. 1999 (28)
Pucher et al. 1997 (34)
Grossi et al. 1997 (12)
Study (ref)
Clinical attachment level mean improvement (mm)
Renvert et al. 1998 (35)
Results in this direction indicate a
better result in non-smokers
Results in this direction indicate a
better result in smokers
Mongardini et al. 1999 (24)
Fig. 7. Forest plot of mean differ-
ence in clinical attachment level
gain between smokers and non-
smokers (threshold studies).
131
Smoking and mechanical periodontal therapy
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)
Clinical attachment level mean improvement (mm)
Results in this direction indicate a
better result in non-smokers
Results in this direction indicate a
better result in quit-smokers
Grossi et al. 1997 (12)
Ryder et al. 1999 (36)
Fig. 9. Forest plot of mean differ-
ence in clinical attachment level
gain between quit-smokers and
non-smokers (threshold studies).
132
Labriola et al.
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133
Smoking and mechanical periodontal therapy
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.
Implications for future research
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,
abstract, cas registry ec number word, mesh
subject heading]
6 non surgical periodontal therapy.mp. [mp title,
abstract, cas registry ec number word, mesh
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
registry ec number word, mesh subject heading]
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
registry ec number word, mesh subject heading]
14 previous smokers.mp. [mp title, abstract, cas
registry ec number word, mesh subject heading]
15 former smokers.mp. [mp title, abstract, cas
registry ec number word, mesh subject heading]
16 SMOKING or exp SMOKING CESSATION
17 TOBACCO or exp TOBACCO USE CESSATION
18 cigarette smoking.mp. [mp title, abstract, cas
registry ec number word, mesh subject heading]
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-
try ec number word, mesh subject heading]
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-
try ec number word, mesh subject heading]
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
Smoking and mechanical periodontal therapy
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, Vol. 37, 2005, 4871
Printed in Denmark. All rights reserved
Copyright Blackwell Munksgaard 2005
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
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
Mechanical nonsurgical pocket therapy
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(
4
4
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A
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D
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1
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T
u
n
k
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t
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l
.
(
9
3
)
A
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m
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C
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1
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p
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l
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f
R
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s
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t
s
:
Y
e
s
.
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t
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:
Y
e
s
.
53
Mechanical nonsurgical pocket therapy
T
a
b
l
e
1
.
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E
D
L
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N
E
-
P
u
b
M
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d
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M
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e
a
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s
:
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6
6
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n
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8
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o
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l
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R
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:
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s
.
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d
:
N
o
.
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a
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:
Y
e
s
,
b
u
t
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a
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.
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l
l
e
y
e
t
a
l
.
(
3
0
)
W
h
a
t
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r
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e
f
f
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?
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s
s
c
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g
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y
3
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o
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?
W
h
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D
a
t
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b
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s
:
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E
D
L
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N
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-
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M
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,
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o
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O
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G
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R
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s
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)
1
9
7
6
9
8
.
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e
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.
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:
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s
.
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e
t
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:
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a
n
d
c
o
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t
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t
.
E
l
t
e
r
e
t
a
l
.
(
3
1
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a
q
u
a
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D
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9
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.
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e
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f
r
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m
a
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t
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f
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u
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t
.
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
o
l
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n
&
Q
u
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(
1
3
)
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r
s
:
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9
6
6
9
4
.
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:
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:
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R
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:
Y
e
s
.
H
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r
o
g
e
n
e
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y
a
s
s
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s
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d
:
R
e
p
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d
a
s
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x
i
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t
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t
a
n
d
c
o
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s
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d
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p
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s
.
I
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t
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r
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t
a
t
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f
r
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s
u
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t
s
a
p
p
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p
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:
D
i
f
c
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t
o
a
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d
u
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t
o
b
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d
a
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a
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n
c
l
u
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d
.
H
a
y
e
s
e
t
a
l
.
(
4
3
)
T
o
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n
v
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s
t
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g
a
t
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t
h
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D
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N
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-
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M
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C
o
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G
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R
e
g
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.
Y
e
a
r
s
:
1
9
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6
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9
.
S
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p
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.
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a
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:
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n
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n
p
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p
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.
H
a
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d
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N
a
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r
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r
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c
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Y
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P
o
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1
6
c
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o
r
c
o
m
b
i
n
a
t
i
o
n
u
s
e
o
f
s
y
s
t
e
m
i
c
a
n
t
i
b
i
o
t
i
c
.
N
o
n
a
n
t
i
b
i
o
t
i
c
t
h
e
r
a
p
y
(
S
R
P
,
p
l
a
c
e
b
o
,
s
u
r
g
e
r
y
)
.
D
C
A
L
D
P
P
D
N
o
.
o
f
s
t
u
d
i
e
s
e
l
i
g
i
b
l
e
:
2
9
.
N
a
r
r
a
t
i
v
e
a
n
a
l
y
s
i
s
:
N
o
s
p
e
c
i
c
d
e
t
a
i
l
s
s
u
m
m
a
r
i
z
e
d
f
o
r
n
o
n
-
s
u
r
g
i
c
a
l
d
e
b
r
i
d
e
m
e
n
t
;
h
o
w
e
v
e
r
,
a
n
t
i
b
i
o
t
i
c
a
l
o
n
e
d
i
d
n
o
t
p
r
o
v
i
d
e
s
i
g
n
i
c
a
n
t
b
e
n
e
t
o
v
e
r
p
l
a
c
e
b
o
.
M
e
c
h
a
n
i
c
a
l
d
e
b
r
i
d
e
m
e
n
t
a
s
a
c
o
n
t
r
o
l
g
r
o
u
p
d
i
d
s
h
o
w
a
n
e
f
f
e
c
t
.
N
o
s
p
e
c
i
c
c
o
n
c
l
u
s
i
o
n
s
o
f
m
e
c
h
a
n
i
c
a
l
t
h
e
r
a
p
y
a
r
e
r
e
p
o
r
t
e
d
b
y
t
h
e
a
u
t
h
o
r
s
,
a
l
t
h
o
u
g
h
a
n
t
i
b
i
o
t
i
c
t
h
e
r
a
p
y
c
l
e
a
r
l
y
s
t
a
t
e
d
a
s
a
t
h
e
r
a
p
y
a
d
j
u
n
c
t
i
v
e
t
o
m
e
c
h
a
n
i
c
a
l
d
e
b
r
i
d
e
m
e
n
t
.
V
a
n
d
e
r
W
e
i
j
d
e
n
&
T
i
m
m
e
r
m
a
n
(
9
4
)
C
l
i
n
i
c
a
l
t
r
i
a
l
o
r
l
o
n
g
i
t
u
d
i
n
a
l
s
t
u
d
i
e
s
w
i
t
h
m
i
n
i
m
u
m
3
m
o
n
t
h
f
o
l
l
o
w
-
u
p
w
i
t
h
p
a
t
i
e
n
t
l
e
v
e
l
a
n
a
l
y
s
i
s
(
p
r
o
v
i
d
i
n
g
m
e
a
n
o
r
i
n
c
r
e
m
e
n
t
a
l
d
a
t
a
)
.
A
d
u
l
t
s
(
m
e
a
n
a
g
e
r
e
p
o
r
t
e
d
)
.
C
h
r
o
n
i
c
p
e
r
i
o
d
o
n
t
i
t
i
s
.
G
o
o
d
g
e
n
e
r
a
l
h
e
a
l
t
h
.
N
o
p
e
r
i
o
d
o
n
t
a
l
t
r
e
a
t
m
e
n
t
w
i
t
h
i
n
p
r
i
o
r
6
m
o
n
t
h
s
.
N
o
a
n
t
i
b
i
o
t
i
c
s
w
i
t
h
i
n
p
r
i
o
r
6
m
o
n
t
h
s
.
M
i
n
i
m
u
m
2
s
i
t
e
s
e
v
a
l
u
a
t
e
d
.
N
o
n
s
u
r
g
i
c
a
l
s
u
b
g
i
n
g
i
v
a
l
d
e
b
r
i
d
e
m
e
n
t
.
N
o
l
o
c
a
l
d
e
l
i
v
e
r
y
a
n
t
i
m
i
c
r
o
b
i
a
l
s
w
i
t
h
i
n
s
a
m
e
p
a
t
i
e
n
t
.
R
e
c
a
l
l
f
r
e
q
u
e
n
c
y
e
v
e
r
y
3
m
o
n
t
h
s
f
o
l
l
o
w
i
n
g
t
h
e
r
a
p
y
.
M
e
a
n
D
C
A
L
M
e
a
n
D
P
P
D
M
e
a
n
D
B
O
P
N
o
.
o
f
s
t
u
d
i
e
s
e
l
i
g
i
b
l
e
:
2
6
N
a
r
r
a
t
i
v
e
a
n
a
l
y
s
i
s
:
P
P
D
r
e
d
u
c
t
i
o
n
:
S
R
P
v
s
.
O
H
d
i
f
f
e
r
e
n
c
e
s
r
a
n
g
e
0
.
4
0
1
.
4
0
m
m
f
a
v
o
r
i
n
g
S
R
P
.
S
R
P
v
s
.
n
o
t
x
d
i
f
f
e
r
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n
c
e
s
r
a
n
g
e
0
.
5
4
0
.
7
0
m
m
f
a
v
o
r
i
n
g
S
R
P
.
C
A
L
g
a
i
n
:
S
R
P
v
s
.
O
H
d
i
f
f
e
r
e
n
-
c
e
s
r
a
n
g
e
0
.
1
4
1
.
0
0
m
m
f
a
v
o
r
i
n
g
S
R
P
v
s
.
n
o
t
x
d
i
f
f
e
r
e
n
c
e
s
r
a
n
g
e
0
.
6
5
1
.
0
0
m
m
f
a
v
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r
i
n
g
S
R
P
.
P
o
o
l
e
d
a
n
a
l
y
s
i
s
:
4
s
t
u
d
i
e
s
c
o
m
p
a
r
i
n
g
S
R
P
t
o
O
H
:
c
h
a
n
g
e
i
n
P
P
D
,
S
R
P
v
s
.
,
O
H
w
e
r
e
+
1
.
1
8
m
m
a
n
d
+
0
.
5
9
m
m
r
e
s
p
e
c
t
i
v
e
l
y
.
C
h
a
n
g
e
i
n
C
A
L
,
S
R
P
v
s
.
O
H
w
e
r
e
+
0
.
6
4
m
m
a
n
d
+
0
.
3
7
m
m
,
r
e
s
p
e
c
t
i
v
e
l
y
(
b
o
t
h
s
t
a
t
i
s
t
i
c
a
l
l
y
s
i
g
n
i
c
a
n
t
)
6
s
t
u
d
i
e
s
c
o
m
p
a
r
i
n
g
p
r
e
a
n
d
p
o
s
t
t
x
,
C
A
L
i
n
p
o
c
k
e
t
s
4
m
m
:
p
r
e
S
R
P
v
s
.
p
o
s
t
S
R
P
+
0
.
7
4
m
m
.
2
s
t
u
d
i
e
s
,
c
o
m
p
a
r
i
n
g
p
r
e
a
n
d
p
o
s
t
t
x
C
A
L
i
n
c
l
u
d
i
n
g
a
l
l
s
i
t
e
s
m
e
a
s
u
r
e
d
:
p
r
e
-
S
R
P
v
s
.
p
o
s
t
S
R
P
+
0
.
2
2
m
m
.
I
n
p
a
t
i
e
n
t
s
w
i
t
h
c
h
r
o
n
i
c
p
e
r
i
o
d
o
n
t
i
t
i
s
,
s
u
b
g
i
n
g
i
v
a
l
d
e
b
r
i
d
e
m
e
n
t
(
i
n
c
o
n
j
u
n
c
t
i
o
n
w
i
t
h
s
u
p
r
a
g
i
n
g
i
v
a
l
p
l
a
q
u
e
c
o
n
t
r
o
l
)
i
s
a
n
e
f
f
e
c
t
i
v
e
t
r
e
a
t
m
e
n
t
i
n
r
e
d
u
c
i
n
g
p
r
o
b
i
n
g
p
o
c
k
e
t
d
e
p
t
h
a
n
d
i
m
p
r
o
v
i
n
g
t
h
e
c
l
i
n
i
c
a
l
a
t
t
a
c
h
m
e
n
t
l
e
v
e
l
.
I
n
f
a
c
t
,
i
t
i
s
m
o
r
e
e
f
f
e
c
t
i
v
e
t
h
a
n
s
u
p
r
a
g
i
n
g
i
v
a
l
p
l
a
q
u
e
c
o
n
t
r
o
l
a
l
o
n
e
.
57
Mechanical nonsurgical pocket therapy
T
a
b
l
e
2
.
C
o
n
t
i
n
u
e
d
C
i
t
a
t
i
o
n
T
y
p
e
s
o
f
s
t
u
d
y
i
n
c
l
u
d
e
d
S
t
u
d
y
p
o
p
u
l
a
t
i
o
n
s
i
n
c
l
u
d
e
d
I
n
t
e
r
v
e
n
t
i
o
n
s
i
n
c
l
u
d
e
d
O
u
t
c
o
m
e
s
i
n
c
l
u
d
e
d
R
e
s
u
l
t
s
(
m
e
c
h
a
n
i
c
a
l
d
e
b
r
i
d
e
m
e
n
t
)
A
u
t
h
o
r
c
o
n
c
l
u
s
i
o
n
s
(
m
e
c
h
a
n
i
c
a
l
d
e
b
r
i
d
e
m
e
n
t
)
H
e
a
s
m
a
n
e
t
a
l
.
(
4
4
)
P
r
o
s
p
e
c
t
i
v
e
c
l
i
n
i
c
a
l
t
r
i
a
l
s
w
i
t
h
1
2
m
o
n
t
h
d
a
t
a
r
e
p
o
r
t
i
n
g
p
a
t
i
e
n
t
l
e
v
e
l
a
n
a
l
y
s
i
s
.
A
d
u
l
t
s
a
g
e
>
2
0
.
C
h
r
o
n
i
c
p
e
r
i
o
d
o
n
t
i
t
i
s
N
o
p
a
t
i
e
n
t
s
w
i
t
h
i
m
p
l
a
n
t
s
P
a
t
i
e
n
t
s
p
r
e
v
i
o
u
s
l
y
t
r
e
a
t
e
d
w
i
t
h
n
o
n
s
u
r
g
i
c
a
l
t
h
e
r
a
p
y
o
n
l
y
.
S
u
p
r
a
g
i
n
g
i
v
a
l
p
r
o
p
h
y
l
a
x
i
s
(
i
n
c
l
u
d
e
s
d
e
p
o
s
i
t
r
e
m
o
v
a
l
)
.
S
u
b
g
i
n
g
i
v
a
l
d
e
b
r
i
d
e
m
e
n
t
.
M
a
i
n
t
e
n
a
n
c
e
r
e
g
i
m
e
n
f
o
r
a
t
l
e
a
s
t
1
2
m
o
n
t
h
s
.
M
e
a
n
D
P
P
D
M
e
a
n
D
C
A
L
N
o
.
o
f
s
t
u
d
i
e
s
e
l
i
g
i
b
l
e
:
1
1
N
a
r
r
a
t
i
v
e
a
n
a
l
y
s
i
s
:
O
n
e
s
t
u
d
y
o
f
d
i
r
e
c
t
c
o
m
p
a
r
i
s
o
n
o
f
i
n
t
e
r
v
e
n
t
i
o
n
s
o
f
i
n
t
e
r
e
s
t
(
1
2
-
m
o
n
t
h
f
o
l
l
o
w
-
u
p
)
.
D
P
P
D
S
u
p
r
a
g
i
n
g
i
v
a
l
0
.
5
9
m
m
[
0
.
1
3
]
S
u
b
g
i
n
g
i
v
a
l
0
.
3
7
m
m
[
0
.
1
5
]
D
A
L
S
u
p
r
a
g
i
n
g
i
v
a
l
)
0
.
1
3
m
m
[
0
.
1
9
]
S
u
b
g
i
n
g
i
v
a
l
)
0
.
1
4
m
m
[
0
.
1
8
]
P
o
o
l
e
d
a
n
a
l
y
s
i
s
:
P
P
D
:
5
s
t
u
d
i
e
s
s
u
p
r
a
g
i
n
g
i
v
a
l
D
P
P
D
1
.
1
5
m
m
[
-
0
.
1
7
,
2
.
3
8
]
4
s
t
u
d
i
e
s
s
u
b
g
i
n
g
i
v
a
l
D
P
P
D
0
.
9
2
m
m
[
0
.
3
7
,
1
.
4
7
]
D
i
f
f
e
r
e
n
c
e
i
n
m
e
a
n
D
P
P
D
0
.
2
3
m
m
[
-
1
.
2
0
,
1
.
2
4
]
A
L
6
s
t
u
d
i
e
s
s
u
p
r
a
g
i
n
g
i
v
a
l
D
A
L
0
.
1
8
m
m
[
-
0
.
3
8
,
0
.
7
4
]
4
s
t
u
d
i
e
s
s
u
b
g
i
n
g
i
v
a
l
D
A
L
0
.
5
0
m
m
[
0
.
1
1
,
0
.
8
9
]
D
i
f
f
e
r
e
n
c
e
i
n
m
e
a
n
D
A
L
0
.
3
2
m
m
[
-
0
.
3
6
,
1
.
0
0
]
.
B
e
s
t
e
v
i
d
e
n
c
e
a
v
a
i
l
a
b
l
e
i
n
d
i
c
a
t
e
s
t
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(
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;
h
o
w
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a
k
.
H
e
r
r
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r
a
e
t
a
l
.
(
4
5
)
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d
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o
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.
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a
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n
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d
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p
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t
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s
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t
h
e
s
i
a
.
59
Mechanical nonsurgical pocket therapy
T
a
b
l
e
2
.
C
o
n
t
i
n
u
e
d
C
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t
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n
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d
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t
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d
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t
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d
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t
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d
R
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60
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&
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u
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.
61
Mechanical nonsurgical pocket therapy
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.
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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
Mechanical nonsurgical pocket therapy
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-
Mechanical nonsurgical pocket therapy
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.
Implications for future research
Mechanical nonsurgical pocket therapy
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
Mechanical nonsurgical pocket therapy
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)
10 limit 9 to (review or review, academic or review
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)
15 limit 14 to (review or review, academic or review
literature or review, tutorial) (191)
16 from 15 keep 1 to 191 (191)
Mechanical nonsurgical pocket therapy
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, Vol. 36, 2004, 98120
Printed in Denmark. All rights reserved
Copyright Blackwell Munksgaard 2004
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
Nonsurgical periodontal therapy
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
Nonsurgical periodontal therapy
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m
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t
R
u
d
h
a
r
t
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t
a
l
.
(
1
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)
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n
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D
5
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m
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(
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T
w
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n
7
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s
W
o
n
g
e
t
a
l
.
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1
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)
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a
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B
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d
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b
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n
g
.
104
Umeda et al.
T
a
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.
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M
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)
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7
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n
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o
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t
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t
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l
.
(
9
6
)
3
1
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Nonsurgical periodontal therapy
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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
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t
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l
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(
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)
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P
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m
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m
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%
M
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N
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6
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a
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m
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F
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c
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c
l
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n
e
108
Umeda et al.
W
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(
2
0
9
)
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1
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F
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t
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.
(
3
8
)
1
7
A
P
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d
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m
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C
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C
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8
,
1
9
)
1
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6
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o
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9
m
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2
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N
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m
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A
P
,
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o
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s
.
E
O
P
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.
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P
P
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R
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d
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n
t
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t
i
s
.
109
Nonsurgical periodontal therapy
T
a
b
l
e
2
b
.
A
n
t
i
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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
Nonsurgical periodontal therapy
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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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, Vol. 36, 2004, 121145
Printed in Denmark. All rights reserved
Copyright Blackwell Munksgaard 2004
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
T
a
b
l
e
1
.
E
f
c
a
c
y
o
f
p
l
a
q
u
e
a
n
d
c
a
l
c
u
l
u
s
r
e
m
o
v
a
l
f
o
r
d
i
f
f
e
r
e
n
t
i
n
s
t
r
u
m
e
n
t
s
d
u
r
i
n
g
r
o
o
t
s
u
r
f
a
c
e
d
e
b
r
i
d
e
m
e
n
t
R
e
f
N
o
.
o
f
t
e
e
t
h
I
n
i
t
i
a
l
p
r
o
b
i
n
g
p
o
c
k
e
t
d
e
p
t
h
E
v
a
l
u
a
t
i
o
n
m
e
t
h
o
d
o
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
a
l
c
u
l
u
s
o
r
s
t
a
i
n
e
d
m
a
t
e
r
i
a
l
M
e
a
n
d
i
s
t
a
n
c
e
f
r
e
e
o
f
c
a
l
c
u
l
u
s
/
m
e
a
n
P
P
D
1
7
5
1
8
2
7
m
m
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
L
i
g
h
t
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
S
c
a
l
i
n
g
(
6
)
R
o
o
t
p
l
a
n
i
n
g
(
1
2
)
0
/
6
(
0
%
)
1
1
/
1
2
(
9
2
%
)
2
4
9
0
5
m
m
o
r
m
o
r
e
L
i
g
h
t
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
H
o
e
s
(
3
0
)
F
i
l
e
s
(
3
0
)
C
u
r
e
t
t
e
s
(
3
0
)
C
u
r
e
t
t
e
s
&
h
o
e
s
w
e
r
e
m
o
s
t
e
f
c
i
e
n
t
1
8
7
1
5
0
5
m
m
o
r
m
o
r
e
L
i
g
h
t
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
U
l
t
r
a
s
o
n
i
c
(
7
5
)
C
u
r
e
t
t
e
s
(
7
5
)
N
o
d
i
f
f
e
r
e
n
c
e
i
n
c
a
l
c
u
l
u
s
r
e
m
o
v
a
l
N
o
p
l
a
n
i
n
g
w
i
t
h
u
l
t
r
a
s
o
n
i
c
1
4
2
9
5
5
m
m
o
r
m
o
r
e
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
U
l
t
r
a
s
o
n
i
c
(
5
4
)
C
u
r
e
t
t
e
s
(
4
2
)
4
3
/
5
3
(
8
1
%
)
3
7
/
4
2
(
8
8
%
)
1
0
1
5
4
5
m
m
o
r
m
o
r
e
E
l
e
c
t
r
o
n
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
U
l
t
r
a
s
o
n
i
c
(
2
7
)
C
u
r
e
t
t
e
s
(
2
7
)
N
o
d
i
f
f
e
r
e
n
c
e
i
n
c
a
l
c
u
l
u
s
r
e
m
o
v
a
l
1
0
2
4
8
5
m
m
o
r
m
o
r
e
L
i
g
h
t
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
C
u
r
e
t
t
e
s
(
4
8
)
3
9
/
4
8
(
8
1
%
)
1
9
5
3
1
2
m
m
o
r
m
o
r
e
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
L
i
g
h
t
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
H
o
e
s
,
c
u
r
e
t
t
e
s
&
r
o
t
a
r
y
d
i
a
m
o
n
d
s
3
/
3
1
(
1
0
%
)
(
a
l
l
4
s
u
r
f
a
c
e
s
f
r
e
e
o
f
p
l
a
q
u
e
)
6
/
3
1
(
2
0
%
)
(
a
l
l
4
s
u
r
f
a
c
e
s
w
i
t
h
r
e
m
n
a
n
t
s
o
f
p
l
a
q
u
e
)
1
4
6
9
2
5
m
m
o
r
m
o
r
e
L
i
g
h
t
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
C
u
r
e
t
t
e
s
(
4
6
)
U
l
t
r
a
s
o
n
i
c
(
4
6
)
3
6
/
4
6
(
7
8
%
)
3
2
/
4
6
(
7
0
%
)
123
Effects on hard and soft tissues
T
a
b
l
e
1
.
C
o
n
t
i
n
u
e
d
R
e
f
N
o
.
o
f
t
e
e
t
h
I
n
i
t
i
a
l
p
r
o
b
i
n
g
p
o
c
k
e
t
d
e
p
t
h
E
v
a
l
u
a
t
i
o
n
m
e
t
h
o
d
o
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
a
l
c
u
l
u
s
o
r
s
t
a
i
n
e
d
m
a
t
e
r
i
a
l
M
e
a
n
d
i
s
t
a
n
c
e
f
r
e
e
o
f
c
a
l
c
u
l
u
s
/
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
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
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
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
C
u
r
e
t
t
e
s
(
2
5
)
U
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
e
e
t
h
)
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
N
o
t
m
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
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
I
n
s
i
t
u
d
u
r
i
n
g
s
u
r
g
e
r
y
a
f
t
e
r
r
e
e
v
a
l
u
a
t
i
o
n
(
i
.
e
.
o
n
l
y
s
i
t
e
s
n
e
e
d
i
n
g
s
u
r
g
e
r
y
w
e
r
e
e
v
a
l
u
a
t
e
d
)
N
o
t
m
e
n
t
i
o
n
e
d
C
u
r
e
t
t
e
s
(
1
0
0
)
S
o
n
i
c
s
c
a
l
e
r
(
9
0
)
S
o
n
i
c
+
c
u
r
e
t
t
e
s
(
5
3
)
3
4
6
/
6
0
0
n
o
s
u
r
g
e
r
y
n
e
e
d
e
d
(
5
8
%
)
3
0
5
/
5
4
0
n
o
s
u
r
g
e
r
y
n
e
e
d
e
d
(
4
6
%
)
1
1
7
/
3
1
8
n
o
s
u
r
g
e
r
y
n
e
e
d
e
d
(
3
7
%
)
6
8
/
2
5
4
r
e
t
r
e
a
t
e
d
s
u
r
f
a
c
e
s
(
2
7
%
)
P
P
D
<
4
m
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
r
e
t
r
e
a
t
e
d
s
u
r
f
a
c
e
s
(
3
2
%
)
P
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
/
2
0
1
r
e
t
r
e
a
t
e
d
s
u
r
f
a
c
e
s
(
1
7
%
)
P
P
D
<
4
m
m
:
1
1
%
P
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
2
.
3
m
m
6
.
0
2
.
6
m
m
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
1
2
.
9
2
.
1
1
1
.
5
2
.
0
+
(
6
.
6
1
.
9
)
*
C
u
r
e
t
t
e
s
+
u
l
t
r
a
s
o
n
i
c
(
2
9
)
S
a
m
e
+
a
p
s
u
r
g
e
r
y
(
3
5
)
U
n
t
r
e
a
t
e
d
(
2
2
)
3
8
%
6
3
%
0
%
2
4
%
P
P
D
<
4
m
m
:
7
%
P
P
D
4
6
m
m
:
2
3
%
P
P
D
>
6
m
m
:
3
9
%
1
4
%
P
P
D
<
4
m
m
:
5
%
P
P
D
4
6
m
m
:
1
5
%
P
P
D
>
6
m
m
:
1
7
%
8
2
%
1
0
8
4
3
H
o
p
e
l
e
s
s
t
e
e
t
h
U
l
t
r
a
s
o
n
i
c
s
c
a
l
e
r
U
n
t
r
e
a
t
e
d
c
o
n
t
r
o
l
s
4
.
6
7
.
4
%
9
5
.
8
8
.
3
%
4
2
1
1
4
2
m
m
o
r
m
o
r
e
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
(
s
i
n
g
l
e
-
r
o
o
t
e
d
t
e
e
t
h
)
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
N
o
t
i
m
e
l
i
m
i
t
U
l
t
r
a
s
o
n
i
c
+
c
u
r
e
t
t
e
s
:
t
r
a
i
n
e
d
p
e
r
i
o
d
o
n
t
i
s
t
s
(
2
4
)
t
r
a
i
n
i
n
g
r
e
s
i
d
e
n
t
s
(
2
1
)
S
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
)
t
r
a
i
n
i
n
g
r
e
s
i
d
e
n
t
s
(
2
6
)
P
P
D
<
4
m
m
:
4
%
P
P
D
4
6
m
m
:
2
1
%
P
P
D
>
6
m
m
:
1
9
%
P
P
D
<
4
m
m
:
1
4
%
P
P
D
4
6
m
m
:
3
4
%
P
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
s
t
e
e
t
h
)
(
4
0
s
i
n
g
l
e
-
r
o
o
t
e
d
t
e
e
t
h
)
(
4
0
m
o
l
a
r
s
)
S
t
e
r
e
o
-
m
i
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
-
r
o
o
t
e
d
m
o
l
a
r
s
U
l
t
r
a
s
o
n
i
c
s
c
a
l
e
r
:
s
i
n
g
l
e
-
r
o
o
t
e
d
m
o
l
a
r
s
C
o
n
t
r
o
l
t
e
e
t
h
2
9
.
8
%
3
0
.
4
%
3
4
.
5
%
2
3
.
5
%
1
0
0
%
1
0
0
1
0
3
5
.
3
0
.
4
m
m
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
N
o
t
m
e
n
t
i
o
n
e
d
S
o
n
i
c
s
c
a
l
i
n
g
P
a
p
i
l
l
a
r
e
e
c
t
i
o
n
+
b
e
r
o
p
t
i
c
i
l
l
u
m
i
n
a
t
i
o
n
+
s
o
n
i
c
s
c
a
l
i
n
g
U
n
t
r
e
a
t
e
d
c
o
n
t
r
o
l
s
6
.
4
%
1
.
3
%
0
.
5
%
125
Effects on hard and soft tissues
T
a
b
l
e
1
.
C
o
n
t
i
n
u
e
d
R
e
f
N
o
.
o
f
t
e
e
t
h
I
n
i
t
i
a
l
p
r
o
b
i
n
g
p
o
c
k
e
t
d
e
p
t
h
E
v
a
l
u
a
t
i
o
n
m
e
t
h
o
d
o
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
a
l
c
u
l
u
s
o
r
s
t
a
i
n
e
d
m
a
t
e
r
i
a
l
M
e
a
n
d
i
s
t
a
n
c
e
f
r
e
e
o
f
c
a
l
c
u
l
u
s
/
m
e
a
n
P
P
D
1
5
2
1
2
0
M
o
d
e
l
i
n
p
h
a
n
t
o
m
h
e
a
d
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
N
o
t
i
m
e
l
i
m
i
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
)
U
l
t
r
a
s
o
n
i
c
s
t
r
a
i
g
h
t
t
i
p
(
4
0
)
U
l
t
r
a
s
o
n
i
c
c
u
r
v
e
d
t
i
p
(
4
0
)
M
a
x
i
l
l
a
r
y
m
o
l
a
r
f
u
r
-
c
a
t
i
o
n
:
6
1
.
1
1
0
.
8
%
M
a
n
d
i
b
u
l
a
r
m
o
l
a
r
f
u
r
-
c
a
t
i
o
n
:
3
9
.
5
1
3
.
1
%
M
a
x
i
l
l
a
r
y
m
o
l
a
r
f
u
r
-
c
a
t
i
o
n
:
5
0
.
3
1
1
.
6
%
M
a
n
d
i
b
u
l
a
r
m
o
l
a
r
f
u
r
-
c
a
t
i
o
n
:
4
4
.
1
1
7
.
1
%
M
a
x
i
l
l
a
r
y
m
o
l
a
r
f
u
r
-
c
a
t
i
o
n
:
1
5
.
1
7
.
6
%
M
a
n
d
i
b
u
l
a
r
m
o
l
a
r
f
u
r
c
a
t
i
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
i
t
h
P
P
D
>
6
m
m
(
m
e
a
n
P
P
D
:
4
8
m
m
)
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
M
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
)
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
M
a
x
.
1
0
m
i
n
/
t
o
o
t
h
5
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
i
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
m
/
6
.
4
m
m
(
5
4
%
)
4
.
7
m
m
/
6
.
4
m
m
(
7
4
%
)
3
.
1
m
m
/
7
.
5
m
m
(
4
2
%
)
2
0
4
6
0
F
u
r
c
a
t
i
o
n
l
e
s
i
o
n
s
I
I
o
r
I
I
I
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
(
m
u
l
t
i
r
o
o
t
e
d
t
e
e
t
h
)
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
N
o
t
i
m
e
l
i
m
i
t
C
u
r
e
t
t
e
s
(
2
0
)
C
u
r
e
t
t
e
s
+
a
c
c
e
s
s
a
p
(
2
0
)
C
o
n
t
r
o
l
t
e
e
t
h
(
2
0
)
5
4
%
3
3
%
9
1
%
1
3
3
5
H
o
p
e
l
e
s
s
t
e
e
t
h
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
M
a
x
.
1
0
m
i
n
/
t
o
o
t
h
M
a
x
.
1
0
+
5
+
5
m
i
n
/
t
o
o
t
h
C
u
r
e
t
t
e
s
1
e
p
i
s
o
d
e
(
1
5
)
C
u
r
e
t
t
e
s
3
e
p
i
s
o
d
e
s
(
1
5
)
U
n
t
r
e
a
t
e
d
c
o
n
t
r
o
l
s
(
5
)
2
5
%
2
4
%
7
0
%
126
Adriaens & Adriaens
1
1
3
5
6
0
M
o
d
e
l
i
n
p
h
a
n
t
o
m
h
e
a
d
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
N
o
t
i
m
e
l
i
m
i
t
:
6
.
5
m
i
n
/
t
o
o
t
h
5
.
8
m
i
n
/
t
o
o
t
h
7
.
0
m
i
n
/
t
o
o
t
h
6
.
2
m
i
n
/
t
o
o
t
h
5
.
5
m
i
n
/
t
o
o
t
h
5
.
0
m
i
n
/
t
o
o
t
h
5
.
6
m
i
n
/
t
o
o
t
h
4
.
8
m
i
n
/
t
o
o
t
h
C
u
r
e
t
t
e
s
:
T
r
a
i
n
e
d
p
e
r
i
o
-
d
o
n
t
i
s
t
s
(
1
0
)
I
n
e
x
p
e
r
i
e
n
c
e
d
d
e
n
t
i
s
t
(
1
0
)
P
e
r
i
o
-
p
l
a
n
e
r
:
T
r
a
i
n
e
d
p
e
r
i
o
-
d
o
n
t
i
s
t
s
(
1
0
)
I
n
e
x
p
e
r
i
e
n
c
e
d
d
e
n
t
i
s
t
(
1
0
)
S
o
n
i
c
s
c
a
l
e
r
(
S
o
n
i
c
e
x
)
:
T
r
a
i
n
e
d
p
e
r
i
o
-
d
o
n
t
i
s
t
s
(
1
0
)
I
n
e
x
p
e
r
i
e
n
c
e
d
d
e
n
t
i
s
t
(
1
0
)
U
l
t
r
a
s
o
n
i
c
:
T
r
a
i
n
e
d
p
e
r
i
o
-
d
o
n
t
i
s
t
s
(
1
0
)
I
n
e
x
p
e
r
i
e
n
c
e
d
d
e
n
t
i
s
t
(
1
0
)
1
3
.
0
9
.
8
%
2
3
.
9
9
.
5
%
1
8
.
6
8
.
6
%
2
6
.
7
7
.
8
%
2
1
.
4
1
4
.
8
%
2
8
.
3
1
2
.
0
%
2
0
.
4
6
.
8
%
2
8
.
1
1
1
.
3
%
2
0
5
8
0
5
1
2
m
m
(
h
o
p
e
l
e
s
s
t
e
e
t
h
)
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
4
.
8
3
.
2
m
i
n
/
t
o
o
t
h
3
.
2
1
.
1
m
i
n
/
t
o
o
t
h
2
.
8
1
.
2
m
i
n
/
t
o
o
t
h
2
.
5
1
.
5
m
i
n
/
t
o
o
t
h
C
u
r
e
t
t
e
s
(
2
0
)
U
l
t
r
a
s
o
n
i
c
(
s
t
a
n
d
a
r
d
t
i
p
)
(
2
0
)
U
l
t
r
a
s
o
n
i
c
(
n
e
d
i
a
m
o
n
d
)
(
2
0
)
U
l
t
r
a
s
o
n
i
c
(
m
e
d
i
u
m
d
i
a
m
o
n
d
)
(
2
0
)
2
/
2
0
(
1
0
%
)
8
/
2
0
(
4
0
%
)
8
/
2
0
(
4
0
%
)
5
/
2
0
(
2
5
%
)
4
.
6
5
.
3
%
4
.
7
6
.
4
%
4
.
3
5
.
2
%
3
.
4
4
.
2
%
1
5
5
9
6
M
o
d
e
l
i
n
p
h
a
n
t
o
m
h
e
a
d
S
t
e
r
e
o
-
m
i
c
r
o
s
c
o
p
y
4
m
i
n
/
t
o
o
t
h
C
u
r
e
t
t
e
s
(
4
8
)
U
l
t
r
a
s
o
n
i
c
s
c
a
l
e
r
(
4
8
)
I
n
s
i
d
e
m
e
s
i
a
l
r
o
o
t
:
4
2
.
2
2
.
6
%
I
n
s
i
d
e
d
i
s
t
a
l
r
o
o
t
:
3
3
.
9
2
.
8
%
I
n
s
i
d
e
m
e
s
i
a
l
r
o
o
t
:
5
3
.
2
3
.
2
%
I
n
s
i
d
e
d
i
s
t
a
l
r
o
o
t
:
7
4
.
3
2
.
1
%
127
Effects on hard and soft tissues
T
a
b
l
e
1
.
C
o
n
t
i
n
u
e
d
R
e
f
N
o
.
o
f
t
e
e
t
h
I
n
i
t
i
a
l
p
r
o
b
i
n
g
p
o
c
k
e
t
d
e
p
t
h
E
v
a
l
u
a
t
i
o
n
m
e
t
h
o
d
o
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
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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
Effects on hard and soft tissues
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
Effects on hard and soft tissues
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
Effects on hard and soft tissues
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
Effects on hard and soft tissues
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
Effects on hard and soft tissues
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
Effects on hard and soft tissues
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, Vol. 36, 2004, 179195
Printed in Denmark. All rights reserved
Copyright Blackwell Munksgaard 2004
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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181
Supportive periodontal therapy
T
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182
Renvert & Persson
M
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l
.
(
5
9
)
3
3
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.
183
Supportive periodontal therapy
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
T
a
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.
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r
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(
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j
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185
Supportive periodontal therapy
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
Supportive periodontal therapy
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
Supportive periodontal therapy
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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effect of supragingival plaque control on the subgingival
microbiota in subjects with periodontal disease. J Clin
Periodontol 1992: 19: 802809.
28. Demetriou N, Tsami-Pandi A, Parashis A. Compliance
with supportive periodontal treatment in private perio-
dontal practice. A 14-year retrospective study. J Period-
ontol 1995: 66: 145149.
29. Demirel K, Efeodly A. Retrospective evaluation of patients
compliance with supportive periodontal treatment. J Ni-
hon Univ Sch Dent 1995: 37: 131137.
30. Esposito M, Worthington HW, Coultard P, Jokstad A.
Intervention for replacing missing teeth: maintaining and
re-establishing healthy tissues around dental implants
(Cochrane review). Cochrane Database Syst Rev 2002: 3:
CD003069.
31. Furuichi Y, Rosling B, Volpe AR, Lindhe J. The effect of a
triclosan/copylymer dentifrice on healing after non-sur-
gical treatment of recurrent periodontitis. J Clin Period-
ontol 1999: 26: 6366.
32. Goodson JM, Tanner ACR, Haffajee AD, Sornberger GC,
Socransky SS. Patterns of progression and regression of
advanced destructive periodontal disease. J Clin Period-
ontol 1982: 9: 472481.
33. Grifths GS, Duffy S, Eaton KA, Gilthorpe MS, Johnson
NW. Prevalence and extent of lifetime cumulative
attachment loss (LCAL) at different thresholds and asso-
ciations with clinical variables: changes in a population of
young male military recruits over 3 years. J Clin Period-
ontol 2001: 28: 961969.
34. Haber J, Wattles J, Crowley M, Mandell R, Joshipura K,
Kent R. Evidence for cigarette smoking as a major
risk factor for periodontitis. J Periodontol 1993: 64: 16
23.
35. HarringtonGW, Steiner DR, Ammons WF. The periodontal-
endodontic controversy. Periodontol 2000 2002: 30: 123
130.
36. Heasman PA, Heasman L, Stacey F, McCracken GI. Local
delivery of chlorhexidine gluconate (PerioChip) in perio-
dontal maintenance patients. J Clin Periodontol 2001: 28:
9095.
37. Heasman PA, McCracken GI, Steen N. Supportive perio-
dontal care: The effect of periodic subgingival debride-
ment compared with supragingival prophylaxis with
respect to clinical outcomes. J Clin Periodontol 2002: 29
(Suppl. 3): 163172.
38. Hellstrom MK, Ramberg P, Krok L, Lindhe J. The effect of
supra-gingival plaque control on the sub-gingival micro-
ora in human periodontitis. J Clin Periodontol 1996: 23:
934940.
39. Jin LJ, Soder P-O, A
2729 kHz
(J-Morita Corp., Tokyo, Japan); Amdent
25 kHz
(Amdent, Nynashamn, Sweden); and Suprasson
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
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
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
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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
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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
, 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
(EMS).
53
Manual and power-driven instrumentation
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
1 le resembles a
reamer with a 16-mm working tip, and is used to
remove heavy supra- and subgingival calculus. The
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
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
a
b
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e
4
.
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55
Manual and power-driven instrumentation
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
,
Cavitron
, and Odontoson
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
Author and Year References Laser parameters Findings
Aoki et al. 1994 (10) 10120 mJ/pulse
(3.542.4 J/cm
2
per pulse)
10 Hz, water irrigation
90
)
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
Author and Year References Laser parameters Findings
Aoki et al. 1994 (10) 30 mJ/pulse
(10.6 J/cm
2
per pulse)
10 Hz, water irrigation
90
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):
Laser group: from 6.3 1.1 mm to 4.4 1.0 mm
SRP group: from 6.5 1.0 mm to 5.5 1.0 mm
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):
Laser group: from 6.3 1.1 mm to 4.5 0.4 mm
at 1 year and to 4.9 0.4 mm at 2 years
SRP group: from 6.5 1.0 mm to 5.6 0.4 mm
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
Laser group: from 5.0 0.7 mm to 3.3 0.7 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
Laser group: from 6.6 1.1 mm to 5.0 0.7 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
Lasers in nonsurgical periodontal therapy
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
Lasers in nonsurgical periodontal therapy
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
Author and Year References Laser parameters Findings
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
Lasers in nonsurgical periodontal therapy
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
Lasers in nonsurgical periodontal therapy
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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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, Vol. 33, 2003, 5466 Copyright
#
Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
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
Distraction osteogenesis
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
McAllister & Gaffaney
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
Distraction osteogenesis
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
McAllister & Gaffaney
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
Distraction osteogenesis
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
McAllister & Gaffaney
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
Distraction osteogenesis
(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
McAllister & Gaffaney
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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66
McAllister & Gaffaney
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
Periodontology 2000, Vol. 33, 2003, 6781 Copyright
#
Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
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
Management of the posterior maxilla
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
Management of the posterior maxilla
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
Management of the posterior maxilla
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
Management of the posterior maxilla
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
Management of the posterior maxilla
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
Periodontology 2000, Vol. 33, 2003, 8289 Copyright
#
Blackwell Munksgaard 2003
Printed in Denmark. All rights reserved
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
Zygomatic implants for resorbed posterior maxillae
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
Zygomatic implants for resorbed posterior maxillae
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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