Periodontology 2000, Voi.
19, 1999, 40-58
Pririted ill Dennzark All rights reserved
Biological mediators for
periodontal regeneration
& J O H N M . WOZNEY
DAVIDL. COCHRAN
Successful periodontal reconstruction comprises re-
generation of multiple tissues including cementum,
periodontal ligament, bone and gingiva. The pro-
duction or regeneration of any tissue type is a com-
plex biological process in itself, requiring intricately
regulated interactions between cells, locally acting
growth factors, systemic hormones and growth fac-
tors, and the extracellular matrix components in
which these entities interact (31-34, 106).In fact, the
identity of a particular tissue is defined by the bio-
chemical nature of the extracellular matrix it con-
tains as well as the phenotype of the cells within it
positioned in a particular spatial relationship to one
another and to neighboring tissue types. Regenera-
tive therapies have generally been directed at pro-
duction of one tissue type of the periodontium. For
example, as bone is required for successful regenera-
tion, a variety of therapies (both biological and
membrane types) have been directed at its regenera-
tion with the assumption that the appropriate pro-
duction of other required tissues will subsequently
occur. The key to tissue regeneration is stimulating
a series of events and cascades at a point, which can
result in the coordination, and completion of inte-
grated tissue formation. Various biological ap-
proaches to the promotion of periodontal regenera-
tion have been used. These can be divided into the
use of growth and differentiation factors, application
of extracellular matrix proteins and attachment fac-
tors and use of mediators of bone metabolism.
Growth and differentiation factors
Growth factors are proteins that may act locally or
systemically to affect the growth and function of
cells in several ways (Table 1). They may act in an
autocrine fashion, where the cells that produce them
are also affected by them; or more commonly, in a
paracrine fashion, such that the production of a
growth factor by one cell type affects the function of
40
C o p y r i g h t 0 M u n k s g a a r d 1999
PERIODONTOLOGY 2000
ISSN 0906-6713
a different cell type. These factors may control the
growth of cells and hence the number of cells avail-
able to produce a tissue. In addition, they may con-
trol the metabolism of a particular cell type: for ex-
ample, the rate of production of an extracellular ma-
trix component such as collagen. Differentiation
factors control the phenotypic state of cells, causing
precursor cells to become fully functional mature
cells of a particular type, such as undifferentiated
mesenchymal cells to become osteoblasts.
Several growth factors, as single agents or in com-
binations, have been examined for their periodontal
regenerative potential in animal models and in the
clinic. Platelet-derived growth factor is a dimeric
molecule; several subtypes exist consisting of homo-
dimers or heterodimers of the platelet-derived
growth factor-A and platelet-derived growth factor-B
gene products (37, 102). While it was originally iden-
tified in platelets, many cell types have subsequently
been determined to synthesize platelet-derived
growth factor. Reciprocally, many different cell types,
particularly those of mesenchymal origin, respond to
platelet-derived growth factor. The primary effect of
platelet-derived growth factor is that of a mitogen,
initiating cell division. In studies using fibroblastic
cell types, platelet-derived growth factor has been
characterized as a competence factor. A competence
factor classically is a growth factor that makes a cell
competent for cell division; a progression factor such
as insulin-like growth factor-I or dexamethasone is
then necessary to induce mitosis. Thus, in some sys-
tems, there is synergy between growth factors of the
two groups. However, some cell types respond to
platelet-derived growth factor by division without
the exogenous addition of other growth factors, per-
haps due to autocrine production and stimulation
by progression factors. For example, osteoblasts pro-
liferate in response to platelet-derived growth factor
without the addition of other growth factors (16, 20,
35). Similar results have been found with isolated
periodontal ligament cells (70).

Table 1. Activities of growth factors
Fibroblast
uroliferation
Platelet-derived growth factor +f
Insulin growth factor + ++
Bone morphogenetic protein - +
Transforming growth factor-B + or -
Fibroblast growth factor ++
++=greatly increased, +=increased, no effect or negative effect.
a=indirect effect
Insulin growth factor-I and insulin growth factor-I1
are peptide growth factors with biochemical and
functional similarities to insulin (25).As noted above,
they are mitogens, and in fibroblastic systems appear
to be progression factors. In bone cell systems, insulin
growth factors stimulate both proliferation of pre-os-
teoblasts as well as the differentiation of osteoblasts,
including type I collagen synthesis (3, 17).Thus, insu-
lin growth factor increases both the number of cells
synthesizing bone and the amount of extracellular
matrix deposited by each cell. The function of insulin
growth factors is regulated at one level by the pres-
ence of a family of insulin growth factor-binding pro-
teins, which may either increase or inhibit insulin
growth factor activity (84). Combinations of platelet-
derived growth factor and insulin growth factor have
been tested in periodontal systems (see below). This
combination could potentiate the growth of the
multiple tissue types including the periodontal liga-
ment and gingival tissue by combining a competence
and progression factor; and, bone through its mito-
genic effects on bone precursor cells.
Bone morphogenetic proteins constitute a large
family of regulatory factors. Originally discovered
based on their presence in bone-inductive extracts
of bone, they are now known to have pivotal roles in
patterning of the embryo as well as functions in the
adult animal (44). While the bone-inductive activity
of bone matrix has been widely recognized (96), it
was not until extensive purification from bovine
bone (92) and subsequent molecular cloning (18, 71,
107), that it became clear what the proteins respon-
sible for this activity were (98). Several bone mor-
phogenetic protein molecules and preparations have
been tested as outlined below in preclinical models
of periodontal regeneration. Preparations derived
from bovine or human bone, which contains a com-
plex mixture of bone morphogenetic protein mol-
ecules and possibly other factors and proteins, have
Biological mediators for periodontal regeneration
Pre-osteoblastl
osteoblast Extracellular Mesenchymal cell
uroliferation matrix svnthesis differentiation Vascularization
++ - - +a
++ - -
- 2 ++ ++a
+ or - ++ ~
+a
++ - - ++
been used in several studies. The other two bone
morphogenetic proteins tested are both manufac-
tured using recombinant DNA methods, yielding
pure individual proteins. Recombinant human bone
morphogenetic protein-2 and recombinant human
bone morphogenetic protein-7 (also called osteo-
genic protein-1) are both synthesized using mam-
malian cell expression systems. The cellular activities
of the recombinant proteins have been determined
in a number of cell systems, and the effects of re-
combinant human bone morphogenetic protein-2
and recombinant human bone morphogenetic pro-
tein-7 appear to be similar. While they are mitogenic
on some cell types such as rat calvarially derived
cells, their primary activity appears to be one of dif-
ferentiation (79, 105). That is, they change the
phenotype of mesenchymal precursor cells into
those of mature osteoblasts and/or chondroblasts. In
addition to these activities, bone morphogenetic
proteins are chemotactic for some cell types of the
osteoblastic lineage (47, 58). In terms of their activity
in vivo, extensive preclinical testing has documented
the ability of these bone morphogenetic proteins to
induce bone in a wide range of anatomic locations
(57, 106). The bone morphogenetic proteins are the
only known factors which are capable of inducing
bone formation at extraskeletal sites, apparently
causing differentiation of cells derived from the soft
tissue into bone-producing cells (74). This activity
makes bone morphogenetic proteins an obvious
candidate for regeneration of alveolar bone. In ad-
dition, the bone morphogenetic proteins are re-
quired for the embryonic development of the skel-
eton and teeth as well as many other organ and
tissue types. Thus they may also have direct effects
in the adult on regeneration of other aspects of peri-
odontal tissue formation. In fact, bone morphogen-
etic proteins have been shown to affect the pheno-
typic expression of periodontal ligament cells.
41
Cocliran & Woznev
Several other growth factors may have potential
for the regeneration of periodontal tissues. One of
these is transforming growth factor-p. Transforming
growth factor-p is a multifunctional growth factor
structurally related to the bone morphogenetic pro-
teins, but functionally quite different (65, 90). It is
synthesized by many cell types, and effects the func-
tion of almost every tissue cell type examined. Gen-
erally, it increases matrix production and leads to an
increase in fibrosis. As one of the major growth fac-
tors present in bone matrix, its effects on bone cells
have been studied in detail (9, 19). Transforming
growth factor-p has been shown to be chemotactic
for bone cells, and may increase or decrease their
proliferation, depending on the differentiation state
of the cells, culture conditions, and concentration of
transforming growth factor- p applied. In most
studies, it increases the differentiated function of os-
teoblasts and osteoblast precursors; for example, in-
creases in extracellular matrix formation such as
type I collagen have been observed. In vivo, it pro-
duces new cartilage and/or bone if injected in prox-
imity to bone; however, it does not induce new bone
formation when implanted away from a bony site (4,
5, 52, 69, 75). In spite of its effects on augmentation
of soft and hard tissue types, no positive data have
been reported on in viuo healing in a periodontal
setting.
Fibroblast growth factors are members of a family
of at least nine related gene products (48). Named
for its general growth-promoting effects on most
fibroblastic cell types, it also stimulates angiogen-
esis, wound healing and cell migration. Studies on
the effects of fibroblast growth factor on individual
cell types have shown that it can stimulate endo-
thelial cell and periodontal ligament cell migration
and proliferation (92, 95). It also stimulates bone cell
replication, but under some conditions can inhibit
matrix synthesis by bone cells. In vivq fibroblast
growth factor has been shown to increase bone for-
mation, and accelerate the rate of fracture repair (2,
51). Some of these effects may be mediated through
increases in transforming growth factor-p produc-
tion. Again, in spite of the presence of a biologic
rationale for the use of this growth factor in peri-
odontal repair, positive in uivo data are not available.
Extracellular matrix proteins and
attachment factors
A variety of proteins and other extracellular matrix
components control where cells migrate, how they
42
adhere and how they function. Amelogenins are a
family of extracellular matrix proteins that regulate
the initiation and growth of hydroxyapatite crystals
during mineralization of the enamel. The expec-
tation is that this material will direct the formation
of cementum, based on the circumstantial evi-
dence that, during embryonic development, these
enamel proteins are involved in the formation of
dentin (88). The formation of dentin is dependent
on a matrix-cell interaction, and in vitro studies
have demonstrated that mesenchymal cells of the
dental follicle develop a hard tissue matrix be-
lieved to be cementum when exposed to enamel
matrix proteins (38). An acidic extract of enamel
(enamel matrix derivative) is being evaluated for
clinical use (27). The expected primary effect of
enamel matrix derivative is generation of ce-
mentum; however, it has also been shown that ex-
posure of periodontal ligament cells to enamel ma-
trix derivative results in increased cell proliferation,
total protein synthesis, and numbers of mineral-
ized nodules formed (28). While amelogenins com-
prise a majority of the protein in enamel matrix
derivative, it is likely that this extract contains ad-
ditional growth regulatory molecules, as other
growth factors including bone morphogenetic pro-
teins, have been found within tooth matrix.
Fibronectin is a large glycoprotein present in
serum and produced by a wide variety of cell types
(14). Its major function is to aid in the attachment
of cells to the extracellular matrix, and it thus
plays a pivotal role in tissue regeneration and
wound healing. Most tissue cell types require the
presence of an extracellular matrix for fully differ-
entiated function, and fibronectin is one molecule
that has been shown to assist in this cell-matrix
interaction. In periodontal healing, the application
of fibronectin has been coupled with surface de-
mineralization of the root. The rationale is that de-
mineralization will expose collagen fibers within
the root surface, and fibronectin will facilitate in-
teraction of the gingival fibroblasts and the tooth.
In this manner, connective tissue attachment may
be increased. In support of this, in vitro studies
have demonstrated the ability of fibronectin to in-
crease the attachment and proliferation of fibro-
blasts to dentin explants, while decreasing epi-
thelial cell attachment (91, 93). Once again, this
therapy is directed at one aspect of periodontal re-
generation; the belief here is that augmentation of
connective tissue attachment will also lead to in-
creases in periodontal ligament and bone forma-
tion.

Mediators of bone metabolism
Several other agents which affect the growth of bone
have been used, either as single agents or in combi-
nation with one of the growth factors described
above, to augment periodontal regeneration. Prosta-
glandins and other eicosanoids have multiple roles
in bone metabolism, and the effects of many growth
factors and cytokines are dependent on the endo-
genous synthesis of prostaglandins (53, 73). For ex-
ample, interleukin-1 and tumor necrosis factor-a
stimulate prostaglandin production by bone cells
and are believed to have a role in the bone loss after
estrogen decrease (54). They are usually thought of
as activators of bone resorption and are important
mediators of bone loss in periodontal disease, as
well as in rheumatoid arthritis. However, they also
have both positive and negative effects on bone for-
mation. At the cellular level, prostaglandins are
mitogenic for osteoblasts and stimulate differen-
tiation, but may decrease matrix synthesis by differ-
entiated osteoblasts. Zn v i m studies have demon-
strated that prostaglandins increase periosteal and
endosteal bone formation, suggesting the possibility
of augmentation of the alveolar bone with this agent
(63, 64, 66, 67).
Glucocorticoids such as dexamethasone similarly
have complex direct and indirect effects on bone
metabolism (59). Chronic glucocorticoid administra-
tion is known to result in bone loss, through a de-
pression of osteoblast function and an increase in
the number of bone remodeling units. However, it
augments the activity of some growth factors and
Table 2. Studies of biological mediators on periodontal regeneration (reference number)
Platelet-derived growth factor + insulin growth factor
Platelet-derived growth factor + dexamethasone
Platelet-derived growth factor
Insulin growth factor + fibroblast growth factor +
transforming growth factor-p
Transforming growth factor-0
Bone morphogenetic proteina
Enamel matrix
aBone-derivedbone morphogenetic protein
bRecombinant bone morphogenetic protein-7 = recombinant OP- 1
Bioloaical mediators for aeriodontal reaeneration
synergistically increases the differentiation of osteo-
blastic cells and hence the formation of bone tissue.
Thus experiments on the effect of glucocorticoids on
bone in vitro reveal both anabolic and catabolic ef-
fects, depending on the concentrations used and the
differentiation state of the cells. Generally, glucocort-
icoids enhance differentiation of osteoblastic precur-
sor cells. For example, bone nodule formation by cal-
varial cells and stromal cells is enhanced in the pres-
ence of glucocorticoids, and can synergize with the
differentiative effects of bone morphogenetic pro-
teins (7, 8). On the other hand, decreases in bone
formation markers are observed in a number of or-
gan culture systems (15, 21, 36).
Studies investigating the use of
biological agents for periodontal
and alveolar bone regeneration
As noted above, much excitement has been centered
on the use of proteins and molecules for regenera-
tion of tissues around teeth, in edentulous areas of
the alveolus, and in the maxillary sinus. Initial
attempts to regenerate oral tissues centered on re-
generation of the periodontium and often included
membrane exclusion techniques or guided tissue re-
generation. In sequence, similar approaches, termed
guided bone regeneration, were used for regenera-
tion of alveolar bone. This work helped focus thera-
peutic approaches to regenerating tissues at the
cellular and molecular levels. In the following, we
Preclinical studies Clinical studies
Periodontal Implant Periodontal Implant
29, 30, 61, 62, 80 6,60 46
81
22, 30, 72, 99
83
104
77 50, 82, 100, 101 10
39 43,109
43
Cochrari & Wozney
examine animal and human studies using proteins
and molecules to regenerate oral tissues to include
the rationale, the data, and the direction of the re-
search (Table 2).
Mitogenic polypeptides
In 1989, Lynch et al. (62) reported that a combi-
nation of purified platelet-derived growth factor and
recombinant insulin growth factor-I stimulated peri-
odontal regeneration. One microgram of each factor
in a methylcellulose gel carrier was applied to the
root surfaces after full thickness flaps had been re-
flected in three dogs with natural periodontitis de-
fects. After a two-week healing interval, control sites
(7 teeth) receiving the carrier alone exhibited a long
junctional epithelial attachment without evidence of
cementum or bone regeneration in the histological
evaluation. In the growth factor-treated sites (5
teeth), new cementum and bone formation was ob-
served with the newly formed bone lined by osteo-
blasts. The results suggest that growth factors that
had stimulated tissue formation in other models also
may stimulate tissue regeneration around teeth. Per-
haps one of the most striking observations was that
tissue formation, including cementum and bone, oc-
curred within a two-week interval.
In a second report, Lynch et al. (61) described
periodontal regeneration in 13 beagle dogs exhibit-
ing natural periodontitis defects treated with a com-
bination of 3 pg of recombinant platelet-derived
growth factor-BB and insulin growth factor-I in a
methylcellulose gel carrier. Radio-labeled growth
factor was evaluated in four additional animals to
determine clearance rate. The results indicate that
both factors were cleared rapidly with 96% removed
by four days and essentially all removed by 14 days.
The half-life approximated four hours for platelet-
derived growth factor and three hours for insulin
growth factor. Increased bone metabolic labeling
was found at two and four weeks post-surgery in
growth factor treated sites compared to control with
five- to ten-fold increases in bone and cementum
regeneration in growth factor treated sites compared
to control at two and five weeks post-surgery. A peri-
odontal ligament of appropriate dimensions was ob-
served. Ankylosis was not observed in the growth
factor-treated sites.
Following these first demonstrations of peri-
odontal regeneration induced by growth factors in
the dog, it was important to determine whether
similar results could be obtained in nonhuman pri-
44
mates. Rutherford et al. (80) evaluated healing fol-
lowing surgical implantation of recombinant plate-
let-derived growth factor combined with insulin
growth factor-I into ligature-induced periodontitis
defects in the cynomolgus monkey. Three animals
were treated with 3% methylcellulose and 5 pg of
each growth factor. Plaque control was not per-
formed during the one-month healing interval. The
histological analysis showed regeneration of ap-
proximately half of the attachment including bone
formation in septa1 areas with horizontal bone loss.
The results support a concept that exogenously ap-
plied growth factors may stimulate residual tissues
to regenerate cementum, periodontal ligament and
bone. Because the half-life for release of the growth
factors from the carrier is rapid (within hours) and
since these factors are rapidly cleared or bound, the
single dose application suggests that the mechanism
for regenerating tissues occurs via stimulation of a
cascade of events that result in multiple tissue for-
mation and integration.
In a subsequent study, Rutherford et al. (81) evalu-
ated platelet-derived growth factor with dexametha-
sone in a collagen matrix carrier for periodontal re-
generation. A single application of the growth factors
was evaluated histologically following an one-month
healing interval in the nonhuman primate model.
Two micrograms of recombinant platelet-derived
growth factor-BB and 2.3 ng of dexamethasone were
combined with 1.5 mg of collagen matrix to treat
each periodontal defect. Fivefold increases in ce-
mentum and periodontal ligament were observed
with seven-fold increases in bone formation.
Giannobile et al. (29) compared regeneration fol-
lowing surgical implantation of recombinant plate-
let-derived growth factor-BB and insulin growth
factor-I into canine natural periodontitis defects
and into nonhuman primate ligature-induced peri-
odontitis defects. Significant regeneration versus
control occurred in both systems. Increased re-
generation of the periodontal attachment occurred
in the nonhuman primate compared to the canine
model. In contrast, increased bone fill occurred in
the canine model compared with that in nonhu-
man primates. After one month of healing, 64%
new attachment versus 34% in the control was
found in the nonhuman primate, and 51% new
attachment versus 9% in the control occurring in
the canine model. Osseous defect fill amounted to
65% in the canine versus 22% in the nonhuman
primate model. Bone maturation appeared similar
in both models. This report confirms earlier
studies suggesting that platelet-derived growth fac-

tor-BB and insulin growth factor-I may stimulate
periodontal regeneration.
In a subsequent study, Giannobile et al. (30)
examined the effect of surgical implantation of 10
pg of recombinant platelet-derived growth factor-
BB and insulin growth factor-I alone, and in com-
bination, on periodontal regeneration in the non-
human primate model. The healing intervals were
one and three months. Control defects (carrier
alone) exhibited a 15% osseous defect fill and 27%
new attachment at both healing intervals. Healing
in insulin growth factor-I-treated defects was simi-
lar to that in the control. Defects treated with
platelet-derived growth factor-BB exhibited 70%
new attachment at the three-month healing inter-
val. The combination of platelet-derived growth
factor-BB/insulin growth factor-I resulted in 43%
osseous fill and 75% new attachment at three
months. It appears that insulin growth factor-I has
no stimulatory effect on periodontal regeneration
while platelet-derived growth factor-BB significant-
ly stimulates regeneration similar to the combi-
nation of platelet-derived growth factor-BB and in-
sulin growth factor-I.
Wang et al. (99) used autoradiography to evaluate
the cellular response to topical platelet-derived
growth factor. Bilateral periodontal fenestration de-
fects were surgically created in the posterior man-
dible in six dogs. The 4 x 4 mm defects received
either a 10 pg/ml solution of platelet-derived growth
factor-BB or saline (control) with or without pro-
visions for guided tissue regeneration using ex-
panded polytetrafluoroethylene membranes. Two
animals each were euthanised after receiving triti-
ated thymidine at one, three, and seven days post-
surgery. The results suggest that fibroblast prolifer-
ation was significantly enhanced in the presence of
exogenous platelet-derived growth factor-BB. This
enhancement also occurred in the presence or ab-
sence of guided tissue regeneration. No differences
were found in proliferation rates in controls with or
without guided tissue regeneration.
Cho et al. (22, 72) also evaluated the effect of im-
plantation of platelet-derived growth factor-BB with
guided tissue regeneration. Induced, chronic,
through-and-through, horizontal, mandibular pre-
molar furcation defects in six beagle dogs were sub-
jected to reconstructive surgery including citric acid
root conditioning, application of recombinant plate-
let-derived growth factor-BB (5 pg) and guided tissue
regeneration using expanded polytetrafluoroethy-
lene membranes. Control defects received the same
protocol without growth factor. Two dogs each were
Biological mediators for periodontal regeneration
killed at 5, 8 and 11 weeks post-surgery. Minima1
amounts of regeneration were observed at five weeks
post-surgery. At eight weeks, increased amounts of
bone and periodontal ligament were found in the
defects receiving platelet-derived growth factor-BB.
Eighty percent of the furcation defect was filled with
new bone compared to 14% in the control. After 11
weeks, there was 87% fill in the growth factor-treated
defects compared with 60% in the control.
A two-center clinical trial (FDA phase 1/11) was
performed to evaluate the safety and efficacy of the
recombinant platelet-derived growth factor-BB/re-
combinant insulin growth factor-I combination for
periodontal regeneration (46). In a split-mouth de-
sign, 38 patients with bilateral intrabony defects
were treated with 50 pg/ml of each factor or with 150
pg/ml of each factor. Controls included sham
surgery or surgery with the carrier alone. Re-entry
surgery for treatment evaluation was performed at
six to nine months post-surgery. Statistically signifi-
cant increases in bone formation were observed with
the high dose of growth factors but not with the low
dose. Vertical bone height increased 2.1 mm versus
0.8 for control with 42% defect fill compared to 19%
fill in control sites. In furcation defects, although
limited in numbers, 2.8 mm of horizontal defect fill
was observed. No safety issues were observed, in-
cluding immunological reactions. In conclusion, sur-
gical implantation of recombinant platelet-derived
growth factor-BB and recombinant insulin growth
factor-I may support significant periodontal re-
generation in humans in a dose-dependent fashion.
In other studies, Lynch et al. (60) evaluated bone
formation at endosseous dental implants receiving
the combination of platelet-derived growth factor
and insulin growth factor-I combination. Forty im-
plants with apical holes containing either platelet-
derived growth factor-BB and insulin growth factor-
I or carrier alone (control) were evaluated following
a 7- and 21-day healing interval in eight beagle dogs.
Significantly enhanced bone fill and bone-to-im-
plant contact was observed in presence of growth
factors compared with control at seven days. At 21
days, significantly increased amounts of new bone
were found around dental implants treated with
growth factors. No differences in bone fill or bone-
to-implant contact between growth factor and con-
trol sites could be established at this observation in-
terval, suggesting that the control sites had time to
form bone in sufficient amounts. Thus, it appears
that the combination of platelet-derived growth fac-
tor and insulin growth factor-I accelerated bone for-
mation around endosseous dental implants.
45
Cocliran & Wozney
A subsequent study evaluated platelet-derived
growth factor and insulin growth factor with guided
bone regeneration in fresh extraction sockets with
buccal dehiscences and endosseous dental implants
in four dogs (6).Histological analysis was performed
following a healing interval of 4.5 months. A two-fold
increase in bone-to-implant contact, area of bone
adjacent to the implant, and in bone-to-implant
contact in the defect area compared to sites treated
with guided bone regeneration alone was observed.
Bone density was enhanced in sites treated with the
growth factor combination compared to sites with-
out growth factors. These results confirm the obser-
vations above regarding bone stimulation at endos-
seous dental implants.
A question arises as to why this approach to peri-
odontal and alveolar regeneration is not being ag-
gressively pursued. Several possibilities exist. One
reason is resources. Many studies were funded with
private investment dollars, and these sources may
have diminished over time. The reason for this is not
known but could be attributable to the fact that mar-
ket approval is a long and costly process for new
therapeutic approaches and/or new directions for
investors to pursue. Another possibility is that, while
periodontal regeneration occurs with exogenous
platelet-derived growth factor, the magnitude of the
response is not overwhelming compared to existing
therapies that the investment in optimizing this ap-
proach with carrier molecules, delivery systems etc.
is not feasible. A further possibility is that although
theoretically platelet-derived growth factor makes
sense at the cellular level and can be shown to be
effective in animal models, when used in humans
with all inherent variables, the efficacy of exogenous
platelet-derived growth factor is either difficult to
discern, or the effect becomes less obvious due to
other factors in the environment. Whatever the rea-
son, this approach to periodontal regeneration is not
actively pursued.
In other studies evaluating growth factors for peri-
odontal regeneration, Selvig et al. (83) screened the
effect of a insulin growth factor-II/basic fibroblast
growth factor/transforming growth factor-pl combi-
nation in four beagle dogs. Standardized fenestration
defects were surgically created through the cortical
bone into the dentin in mandibular and maxillary
teeth. Collagen sponges loaded with 200 ng of insu-
lin growth factor-11, 20 ng of basic fibroblast growth
factor and 6 ng of transforming growth factor-pl
were implanted into randomly assigned defects with
contralateral defects receiving the carrier without
growth factors. Surgeries were scheduled to allow
46
-
histological observations of healing at 3, 7, 10 and
14 days post-surgery. The results demonstrated no
differences in fibroblast and collagen density in de-
fects treated with the growth factor combination
compared with control. Bone regeneration was sig-
nificantly greater and occurred more frequently in
the control compared with growth factor-treated de-
fects, indicating that this growth factor combination
did not stimulate periodontal regeneration and, in
fact, inhibited bone formation.
More recently, Wikesjo et al. (104) reported on the
effect of transforming growth factor-pl combined
with guided tissue regeneration in five beagle dogs.
In this study, 20 pg of recombinant transforming
growth factor-pl in a calcium carbonate/starch car-
rier protected by an expanded polytetrafluoroethy-
lene membrane was compared with carrier plus
guided tissue regeneration alone. Coronally placed
flaps reaching above the cemento-enamel junction
covered the supra-alveolar critical size defects. His-
tometric evaluation was performed following a one-
month healing interval. Bone regeneration was
limited to the most apical area of all defects. Simi-
larly, cementum regeneration was not different in
growth factor compared with control defects and
was not significant in quantity. New bone area in
transforming growth factor-pl treated defects
amounted to 1.8 mm2 versus 1.3 mm2 in controls.
The density of the newly formed bone as measured
by the ratio of bone-to-marrow area was 0.3 and 0.2
for growth factor and control defects, respectively.
The authors concluded based on the observations in
this discriminating preclinical model that this ap-
proach for stimulating regeneration may have
limited clinical potential.
Differentiation polypeptides
In 1991, Toriumi et al. (94) reported the significant
observation that a bone differentiation factor, re-
combinant bone morphogenetic protein-2, may
stimulate regeneration of 3-cm mandibular disconti-
nuity defects. In this canine model, the mandibles in
26 animals were stabilized with stainless steel recon-
struction plates. Three groups of animals received
treatment including recombinant bone morphogen-
etic protein-2 in an inactive allogeneic bone matrix
carrier, inactive bone matrix alone (control), or
served as sham-operated controls. The jaw segments
were evaluated three and six months post-surgery
using radiography, histology and biomechanical
testing. The reconstruction plates could be removed

after 2.5 months in animals receiving recombinant
bone morphogenetic protein-2, and the animals
were returned to a solid diet. Stiff, noncompressible
bone formed across the defects in these animals.
Bone formation did not occur beyond the bound-
aries of the defect and the borders between the re-
constructed segment and the natural bone on either
side of the defect. Control animals exhibited mini-
mal bone formation and the defects remained un-
stable. These animals could not use a solid diet and
could not have the reconstruction plate removed.
Histologically, control defects were occupied by fi-
brous scar tissue. This study demonstrates the po-
tential of bone morphogenetic protein to regenerate
substantial amounts of bone (Fig. 1).
Also in 1991, Bowers et al. (10) reported that an
allogeneic bone morphogenetic protein preparation
in a freeze-dried, allogeneic, demineralized bone
matrix carrier significantly enhanced regeneration in
human periodontal defects. Bone morphogenetic
protein-3 (osteogenin) was combined with either de-
mineralized bone matrix or a bovine tendon-derived
collagen matrix. Controls were either demineralized
bone matrix or the collagen matrix alone. Both sub-
merged and nonsubmerged wound closure tech-
niques were used in the soft tissue management of
the intrabony periodontal defects. Appropriate his-
tological evaluation of block biopsies was performed
following a six-month healing interval. Bone mor-
Fig. 1. Osseous repair of mandibular defects using recom-
binant bone morphogenetic protein-2 in the canine criti-
cal-sized segmental defect model of Toriumi et al. (94).
A. Radiographic appearance of defect treated with buffer
and absorbable collagen sponge. B. Defect treated with
recombinant bone morphogenetic protein-2 and ab-
sorbable collagen sponge (12 weeks post-surgery).
Biological mediators for periodontal regeneration
phogenetic protein-3 and demineralized bone ma-
trix in a submerged environment resulted in signifi-
cant periodontal regeneration. Bone morphogenetic
protein-3 and demineralized bone matrix and de-
mineralized bone matrix alone resulted in signifi-
cantly more new tissue formation than bone mor-
phogenetic protein-3 and collagen matrix or the col-
lagen matrix alone in both submerged and
nonsubmerged environments. No differences in
tissue formation were found between bone morpho-
genetic protein-3 and collagen matrix or the collagen
matrix alone. A lymphocyte blastogenesis test was
performed at six months post-surgery to evaluate
any immune reaction to bone morphogenetic pro-
tein-3. No interference was observed in this assay in
patients treated with bone morphogenetic protein-
3, and no cell-mediated immunity was noted.
Rutherford et al. (82) used a partially purified pro-
tein fraction from bovine bone in combination with
a collagen matrix to stimulate bone growth in mon-
key extraction sites in the presence or absence of en-
dosseous dental implants. No surgical preparations
for the implants were performed and, as such, the
lesions were not standardized between sites. In
seven sites that could be evaluated new bone was
observed within three weeks in close proximity to
the implants in the treated sites compared to the
nontreated sites. These results suggested that pro-
tein fractions containing bone morphogenetic pro-
tein might be useful in stimulating bone formation
around endosseous implants.
Wang et al. (50, 100, 101) confirmed the obser-
vations by Rutherford et al. (82) and suggested that
the rate of dental implant osseointegration may in-
crease in the presence of bone morphogenetic pro-
tein. In this study, purified bovine bone morphogen-
etic protein or bovine serum albumin (control) was
placed in a 2-mm hole at the apical end of 10-mm
endosseous dental implants. The implants were
placed into the edentulous premolar area in 15 dogs.
Histologic and scanning electron microscopy analy-
ses of block biopsies harvested from three animals
each at 1 , 2 , 4 , 8and 12 weeks post-surgery revealed
direct bone apposition after four weeks in sites
treated with bone morphogenetic protein, while fi-
brous connective tissue encapsulated the control
implants with new bone formation only at the mar-
gins of the implant preparation. After eight weeks
more mature bone was found surrounding implants
treated with bone morphogenetic protein, whereas
the controls had a few areas of fibrous tissue remain-
ing around the implants.
Ripamonti et al. (77) reported on periodontal re-
47
Cochran & Wozney
Fig. 2. Periodontal regeneration with recombinant bone
-
momhogenetic ~rotein-2using canine SuDra-dveolar de-
feet mozel (86): Buccal-lingual histological sections fol-
lowing implantation of buffer/carrier (left,A and C ) or re-
combinant bone morphogenetic protein-2/carrier (right,
B and D).
generation following surgical implantation of bone
morphogenetic protein fractions obtained by ex-
tracting bovine demineralized bone matrix. These
preparations consisted primarily of bone morpho-
genetic protein-2 and bone morphogenetic protein-
3. The study included 12 surgically induced furcation
defects (approximately 10-12 mm in a buccal-lingual
direction) in three baboons. The bone morphogen-
etic protein preparations (250 pg) were combined
with insoluble collagenous bone matrix (150 mg),
which also served as control. Histological evaluation
was performed upon a two-month healing interval.
Significant amounts of new bone, periodontal liga-
ment and cementum were observed in defects
treated with bone morphogenetic protein compared
with control. In a review on bone morphogenetic
proteins and periodontal regeneration (78), Ripa-
monti & Reddi pointed out that, although promising
results are emerging using bone morphogenetic pro-
teins to stimulate regeneration, there is a need to
identify the origin of the cells that respond to bone
morphogenetic proteins more precisely, to under-
stand the role of each of the different bone morpho-
genetic proteins and their combinations and to
better understand delivery systems for the bone
morphogenetic proteins.
Ishikawa et al. (49) used recombinant bone mor-
phogenetic protein-2 to stimulate periodontal re-
generation in surgically induced three-wall intra-
bony defects in dogs. Recombinant bone morpho-
genetic protein-2 (0.2 mglml) in poly (D,L-lactide-co-
glycolide) microparticles mixed with autologous
blood was implanted into the intrabony defects.
Sham-operated defects served as control. Following
a one-month healing interval, both recombinant
bone morphogenetic protein-2 defects and control
demonstrated new bone and cementum with Shar-
pey’s fibers. The coronal extent of the newly formed
tissues was significantly greater in defects receiving
recombinant bone morphogenetic protein-2 com-
pared to control.
Periodontal regeneration following surgical im-
plantation of recombinant bone morphogenetic pro-
tein-2 has also been evaluated in the critical size, sup-
ra-alveolar periodontal defect model (86). This study
used the same recombinant bone morphogenetic
protein-2 delivery system as Ishikawa et al. (49).Con-
trols consisted of the carrier alone. Upon implan-
tation of recombinant bone morphogenetic protein-2
(0.2 mg/ml), the gingival flaps were coronally ad-
vanced and sutured to submerge the 5-mm mandibu-
lar premolar defects. The animals were killed at two
months post-surgery, and block biopsies were pro-
cessed for histometric analysis. The height of regener-
ated bone in recombinant bone morphogenetic pro-
tein-2 sites averaged 3.5 mm compared with 0.8 mm
in the control (Fig.2). New bone area amounted to 8.4
and 0.4 mm2, respectively. Cementum regeneration
averaged 1.6 mm for recombinant bone morphogen-
etic protein-2 sites compared with 0.4 mm for the
control. Substantial root resorption was observed in
control sites with small amounts observed in the sites
treated with recombinant bone morphogenetic pro-
tein-2. Ankylosis was found occasionally but was lo-
calized and appeared unrelated to the bone morpho-
genetic protein treatment.
Subsequently, Sigurdsson et al. (87) evaluated re-
combinant bone morphogenetic protein-2 with vari-
ous carriers for periodontal regeneration. Canine de-
mineralized bone matrix, bovine deorganified crys-
talline bone matrix (Bio-Oss), an absorbable type I

bovine collagen sponge, poly (D,L-lactide-co-gly-
colide) microparticles and polylactide acid granules
(Drilac) were examined with a single concentration
of recombinant bone morphogenetic protein-2 (0.2
mglml). The critical size, supra-alveolar periodontal
defect model with transgingival tooth positioning at
wound closure was used. After a two-month healing
interval, new bone regeneration was observed in all
sites implanted with recombinant bone morphogen-
etic protein-2. Cementum regeneration and anky-
losis were observed and varied among carriers. The
authors concluded that the quantity and the quality
of recombinant bone morphogenetic protein-2
stimulated periodontal regeneration, in particular
regeneration of alveolar bone, appeared to be sig-
nificantly affected by the specific carrier system.
Ripamonti et al. (76) evaluated cementogenesis
following application of recombinant bone morpho-
genetic protein-7 to furcation defects in the baboon.
Surgically created furcation defects in the first and
second mandibular molars in three animals received
either 20 or 50 pg of recombinant bone morphogen-
etic protein-7 in a bovine collagen matrix or the col-
lagen matrix alone (control). Histological analysis
following a two-month healing period revealed new
cementum formation with both recombinant bone
morphogenetic protein-7 dosages compared with
control. Areas of a functionally oriented periodontal
ligament without bone formation were observed.
The authors suggested that the presence of exposed
dentin provides a substrate that serves to preferen-
tially direct cementum formation.
Recently, Kinoshita et al. (56) reported on recom-
binant bone morphogenetic protein-2 induced peri-
odontal regeneration to root surfaces with a history
of dental plaque and calculus exposure. Ligature-in-
duced, circumferential periodontal defects approxi-
mating 50% of the root length in the mandibular pre-
molar teeth in six beagle dogs received recombinant
bone morphogenetic protein-2 (0.4 mg/ml) in gela-
tin and polylactic acid polyglycolide acid copolymer
carrier or the carrier alone (control) in contralateral
defects. The histometric evaluation revealed signifi-
cantly more new bone and cementum with Sharpey’s
fibers in defects treated with recombinant bone
morphogenetic protein-2 compared with control fol-
lowing a three-month healing interval. Root resorp-
tion was a rare finding. Ankylosis was not observed
in bone morphogenetic protein or control sites. The
results suggest that periodontal regeneration in-
duced by recombinant bone morphogenetic protein-
2 may also occur at root surfaces with a history of
periodontal disease.
Biological mediators .for periodontal regeneration
King et al. (55) used a rat fenestration defect to
evaluate periodontal wound healing stimulated by
recombinant bone morphogenetic protein-2. An
extraoral approach was used to create and treat the
defects avoiding any communication between the
defect and the oral cavity. Buccal, acid-conditioned
defects on the molar roots were implanted with re-
combinant bone morphogenetic protein-2 in a colla-
gen gel carrier or carrier alone (control). Histometric
evaluation was performed following a 10- and 38-
day healing period. Significant bone formation was
observed at ten days in recombinant bone morpho-
genetic protein-2 treated sites. New osteoblasts and
osteocytes were confirmed by immunostaining of
the histological sections. Twice as much cementum
formation was observed in sites treated with recom-
binant bone morphogenetic protein-2 compared
with control. At 38 days, complete healing was noted
in recombinant bone morphogenetic protein-2 and
control sites without any observable differences be-
tween the sites. The results indicate that recombin-
ant bone morphogenetic protein-2 accelerates bone
formation and selectively enhances cementum for-
mation during early wound healing.
Nevins et al. (68) demonstrated that recombinant
bone morphogenetic protein-2 stimulates bone
growth in the maxillary sinus, a site where bone for-
mation would not occur in absence of bone grafting.
Bilateral maxillary sinus floor elevation procedures
were performed in six adult goats. Each animal re-
ceived recombinant bone morphogenetic protein-2
and absorbable collagen sponge containing 1.7 mg
of recombinant bone morphogenetic protein-2 or
buffer and absorbable collagen sponge (control) in
contralateral sites. Computerized tomography over
three months revealed increasing radiopacity in sites
receiving recombinant bone morphogenetic protein-
2, with controls exhibiting unchanged or reduced
radiopacity. Significant amounts of new bone com-
pared with control were demonstrated histologically,
with normal progression of the bone formation pro-
cess. Bone formation occurred rapidly, with detec-
tion in radiographs and histological specimens by
one month. The implantation procedure and healing
progressed without observable adverse effects or im-
mune response.
The effect of recombinant bone morphogenetic
protein-2 on peri-implant bone formation was re-
ported by Sigurdsson et al. (85). Endosseous 10-mm
dental implants were placed 5 mm into the reduced
edentulous mandibular ridge, leaving 5 mm of the
implant in a supra-alveolar position. Then recom-
binant bone morphogenetic protein-2 and ab-
49
Cochran & Wozney
Fig. 3. Histological analysis of peri-implantitis defect re-
pair in the monkey (41). Defects were treated with buffer
and absorbable collagen sponge (A) or recombinant bone
morphogenetic protein-2 and absorbable collagen sponge
(B).
sorbable collagen sponge (recombinant bone mor-
phogenetic protein-2 at 0.4 mg/ml) or buffer and ab-
sorbable collagen sponge (control) was implanted in
contralateral peri-implant defects. Histological and
radiographic analyses were performed following a
four-month healing interval. Sites treated with re-
combinant bone morphogenetic protein-2 exhibited
swelling within the first week of healing. Contra-
lateral controls exhibited minimal swelling. Large
variability was noted between the sites treated with
recombinant bone morphogenetic protein-2. Inter-
estingly, the authors reported that the extent of bone
regeneration appeared to correlate with the extent
of post-surgery swelling. Density of the regenerated
bone exhibited an inverse relationship with the
amount of bone formation. The height of regener-
ated bone averaged 4.2 mm for sites treated with re-
combinant bone morphogenetic protein-2 com-
pared with 0.5 mm for the control. Average new bone
area amounted to 6.1 mm2 and 0.2 mm2, respec-
tively. Bone-to-implant contact within regenerated
bone averaged 19% and 8% for recombinant bone
morphogenetic protein-2 and control sites, respec-
tively.
Hanisch et al. (41) were the first to demonstrate
dental implant re-osseointegration using a nonhu-
man primate peri-implantitis model and surgical
implantation of recombinant bone morphogenetic
protein-2. Ligature-induced chronic peri-implantitis
lesions were created around hydroxyapatite-coated
endosseous dental implants over 11 months in four
rhesus monkeys (40). The advanced peri-implantitis
50
defects received recombinant bone morphogenetic
protein-2 and absorbable collagen sponge (recom-
binant bone morphogenetic protein-2 at 0.4 mg/ml)
or absorbable collagen sponge with buffer (control)
in contralateral mandibular and maxillary defects.
Histological analysis was performed following a four-
month healing interval. The mean coronal extent of
new bone formation was 2.6 mm in sites treated with
recombinant bone morphogenetic protein-2 com-
pared with 0.8 mm in the control (Fig. 3). Mean
bone-to-implant contact within the defect area was
29% in the recombinant bone morphogenetic pro-
tein-2 sites compared with 4% for controls. Within
the area of new bone formation, bone-to-implant
contact averaged 40% in bone stimulated with re-
combinant bone morphogenetic protein-2 com-
pared with 9% for the control. Thus, recombinant
bone morphogenetic protein-2 appears capable of
stimulating bone formation to support dental im-
plant re-osseointegration in advanced nonhuman
primate peri-implantitis defects exhibiting a micro-
biological profile similar to that of advanced human
periodontal disease and that of advanced human
peri-implantitis.
In a subsequent study, Hanisch et al. (42) showed
substantial bone augmentation of the maxillary si-
nus in the cynomolgus monkey, allowing placement
of endosseous dental implants. Recombinant bone
morphogenetic protein-2 and absorbable collagen
sponge (recombinant bone morphogenetic protein-
2 at 0.4 mg/ml) or buffer and absorbable collagen
sponge was surgically implanted into the subantral
space in four animals. After three months of healing,
dental implants were placed into the augmented su-
bantral space and anteriorly to the sinus (control
placed in native bone). Histological analysis, follow-
ing another three months of healing, revealed sig-
nificantly greater bone height in recombinant bone
morphogenetic protein-2 treated sites compared
with control (6.0 versus 2.6 mm, respectively). Bone
density and bone-to-implant contact were similar in
recombinant bone morphogenetic protein-2 aug-
mented, control sites and native bone surrounding
the augmented sinus.
Cochran et al. (23) examined bone regeneration
around endosseous dental implants following im-
plantation of recombinant bone morphogenetic pro-
tein-2 with and without provisions for guided bone
regeneration in a dog model. Standardized, intra-
bony peri-implant defects were created around 96
endosseous implants with a sandblasted, acid-
etched titanium surface in the edentulous mandible.
Recombinant bone morphogenetic protein-2 and

absorbable collagen sponge or recombinant bone
morphogenetic protein-2 in the poly (w-lactide-co-
glycolide) microparticle carrier were placed into the
defects. Half these sites were additionally prepared
for guided bone regeneration using an expanded PO-
lytetrafluoroethylene membrane. Controls included
defects without recombinant bone morphogenetic
protein-2 with or without guided bone regeneration.
Standardized radiography was used to assess bone
regeneration following one and three months of
healing. Peri-implant bone fill was determined by
linear measurements on the radiographs and by
bone density changes using computer-assisted
densitometric image analysis. At one month, non-
guided bone regeneration sites exhibited signifi-
cantly enhanced bone regeneration compared with
the guided bone regeneration sites. At three months,
sites treated with recombinant bone morphogenetic
protein-2 exhibited significantly enhanced bone re-
generation compared with control. Bone fill was
most enhanced in sites treated with recombinant
bone morphogenetic protein-2 and guided bone re-
generation followed by sites treated with recombin-
ant bone morphogenetic protein-2 alone. When con-
trol sites were analyzed, guided bone regeneration
sites exhibited enhanced bone regeneration over
non-guided bone regeneration sites; however, both
treatments exhibited less regeneration than sites re-
ceiving recombinant bone morphogenetic protein-
2. At three months, guided bone regeneration sites
exhibited greater bone density than non-guided
bone regeneration sites. When the carriers were
compared, greater bone density was observed with
use of the absorbable collagen sponge carrier. The
results suggest that recombinant bone morphogen-
etic protein-2 induced bone within peri-implant de-
fects is dependent on the healing interval, the carrier
system employed and whether guided bone re-
generation was used.
Recently, Cochran et al. (24) reported the histo-
metric analyses from the peri-implant defect study
(23). Induced bone area, bone-to-implant contact in
the defect area, and defect bone fill were deter-
mined. Surgical implantation of recombinant bone
morphogenetic protein-2 resulted in significantly in-
creased induced bone area, bone-to-implant con-
tact, and defect bone fill at one and three months
post-surgery. New bone area was reduced in guided
bone regeneration sites at one month, though there
was no significant difference between guided bone
regeneration and non-guided bone regeneration
sites at three months. In some specimens, new bone
was observed coronal to the expanded polyte-
Biological mediators for periodontal regeneration
trafluoroethylene membranes. In these sites in-
creased bone formation was observed in the pres-
ence of recombinant bone morphogenetic protein-
2 compared with the absence of recombinant bone
morphogenetic protein-2. Furthermore, bone-to-im-
plant contact was significantly enhanced along the
titanium implant surfaces. Guided bone regenera-
tion and non-guided bone regeneration sites ex-
hibited similar amounts of bone formation at three
months, while at one month the guided bone re-
generation sites exhibited less bone formation. The
histometric analysis demonstrates that recombinant
bone morphogenetic protein-2 may be used to
stimulate bone growth both around and onto endos-
seous dental implants in clinically significant peri-
implant osseous defects.
Clinical trials have been initiated in the evaluation
of recombinant bone morphogenetic protein-2.
These trials are required prior to Food and Drug Ad-
ministration approval and practitioner availability.
Once adequate data are accumulated to assure
safety and efficacy, the product is submitted to the
Food and Drug Administration for approval of com-
mercial introduction. Boyne et al. (11) evaluated
maxillary sinus augmentation using recombinant
bone morphogenetic protein-2 and absorbable col-
lagen sponge in 12 patients. Computerized tomo-
graphy was used to assess the effect of the augmen-
tation procedure over a four-month healing interval.
Eleven patients were available for the evaluation. All
11 patients implanted with recombinant bone mor-
phogenetic protein-2 had induced bone formation.
The average height of induced bone amounted to 8.5
mm. Histological analysis of bone cores removed at
dental implant placement revealed that the quality
of bone appeared similar to that of the native bone.
Clinical observations revealed no serious adverse re-
actions to the recombinant bone morphogenetic
protein-2 and absorbable collagen sponge implant
including toxicity and significant immune reactions.
This clinical trial suggests that recombinant bone
morphogenetic protein-2 may be used safely to
stimulate bone formation for maxillary sinus aug-
mentation.
Clinical trials have also demonstrated the safety
of recombinant bone morphogenetic protein-2 and
absorbable collagen sponge when used for alveolar
ridge augmentation or preservation (45). In a two-
center trial, 12 patients were treated with recombin-
ant bone morphogenetic protein-2 and absorbable
collagen sponge and were followed for four months.
Patient safety was evaluated through a battery of oral
and radiographic examinations, and collection of
51
Cockran & Woznev
blood samples to determine serum chemistries,
hematology and potential antibody formation. Re-
combinant bone morphogenetic protein-2 and ab-
sorbable collagen sponge was found to be well toler-
ated locally and systemically without serious adverse
effects. Significant bone formation could not be es-
tablished following the ridge augmentation pro-
cedure. However, extraction sites healed without en-
suing alveolar ridge reduction. These patients have
been followed for three years without any observable
complications from the alveolar ridge augmentation
or preservation procedure. Taken together, the clin-
ical trials demonstrate that recombinant bone mor-
phogenetic protein-2 may be used safely in patients.
Arachidonic acid metabolites
In 1988, Marks & Miller (63) reported on yet another
approach to stimulate oral and maxillofacial bone
formation. Prostaglandin El, at doses of 0.5 to 2.0
mglweek, was infused adjacent to the mandible in
five beagle dogs using cannulated osmotic mini-
pumps. Vehicle alone served as control. Fluor-
escence microscopy and microradiography following
five weeks of treatment indicated localized new bone
formation exhibiting normal lamellar structure and
mineralization.
In other studies, Miller & Marks (66, 67) used os-
motic minipumps and controlled-release pellets to
present prostaglandin E, subperiosteally next to the
mandibular cortex. Dose-response curves were pro-
duced for both delivery systems. The osmotic pumps
released prostaglandin El for three weeks, and the
tissues were collected for examination one week
later. Sustained release polymers were encapsulated
with an absorbable gelatin sponge. Dose-related in-
creases were found in areas of maximal response.
Subperiosteal bone formation was greater with the
use of minipumps. The response to prostaglandin El
was dependent on the method of administration,
dose and the proximity to the delivery device. The
histological appearance of the newly formed bone
formed appeared to be dependent on the local
prostaglandin E, dose. In areas of high doses, pre-
dominantly woven bone was found, whereas in areas
of lower amounts, there were greater quantities of
lamellar bone often surrounding a core of woven
bone. In the areas of the most enhanced periosteal
activity, intracortical bone changes were noted in the
native bone, with the changes varying depending on
proximity to the prostaglandin E,. Most of the ob-
served changes were a result of activated osseous re-
52
modeling. At the highest doses of prostaglandin E l ,
there was an observed increase in the mineral ap-
positional rate. These alterations in bone activity
suggested that the locally delivered prostaglandin El
increased bone production at the cellular level and
also increased recruitment of osteoblast cells.
In 1994, Marks & Miller (64) reported that, as a
result of adding locally delivered prostaglandin on
the lateral mandibular surface 1.5 cm from the al-
veolar crest after gingival flap reflection, periodontal
regeneration was stimulated around the teeth.
Eleven beagle dogs were utilized; doses from 5 to 25
mg of prostaglandin E, were delivered for three
weeks, and the dogs were lulled a week later after
fluorochrome labeling. New bone, cementum and
periodontal ligament were observed that was histo-
logically indistinguishable from native periodontal
ligament. New attachment occurred in all 18 exper-
imentally treated sites and in only one of seven con-
trol sites. This report indicated that, in addition to
increasing alveolar bone width, locally delivered
prostaglandins can increase alveolar bone height
and stimulate cementogenesis and periodontal liga-
ment formation around teeth. The new attachment
that formed was the result of local delivery at a site
remote from the periodontal defect and thus repre-
sents another possible strategy for the use of growth-
promoting molecules in the oral cavity.
The rationale for using prostaglandin E series for
bone stimulation comes from studies that demon-
strate that the systemic administration of these mol-
ecules (26, 108) has measurable effects (stimulates
tissue formation) on the skeleton and can increase
bone mass in humans. Prostaglandins are the result
of cyclo-oxygenation of precursors derived from ara-
chidonic acid. These compounds are ubiquitous and
found in a variety of tissues. The effect of the prosta-
glandins varies considerably from stimulating in-
flammation and bone resorption to the enhance-
ment of bone formation. It appears that the specific
result is dependent on a number of issues, including
whether skeletal or local effects are being examined,
the dose delivered and the model system used for
evaluation. For example, early reports in the peri-
odontal literature focused on a model to evaluate
bone resorption and the prostaglandins became
known for their effects for stimulating bone resorp-
tion, as this was the effect this model system best
evaluated. Reports on the systemic effects of prosta-
glandins vary as well, ranging from effects on bone
resorption to effects on bone formation depending
on the factors noted above. It is not surprising, then,
that, as regards the oral cavity, that bone formation

as well as bone resorption might occur with these
molecules. It appears that the addition of prosta-
glandin stimulates or accelerates the natural pro-
cesses of coupled bone resorption and formation
and that, depending on a number of factors and
evaluation techniques, that one will find effects on
bone resorption or formation. Therefore, a logical
extension of these studies was to evaluate these
compounds for the local stimulation of bone forma-
tion in the oral cavity. It appears at this point, how-
ever, that further work in this area for periodontal
regeneration is limited. A likely reason for this is the
difficulty in controlling the precise response from
the application of the molecules depending again on
the local delivery system, carrier, dose etc. However,
given that so many arachidonic acid metabolites
exist, future investigations could focus on the appli-
cation of these other molecules, and their result may
prove effective and worthy of pursuit.
Extracellular matrix proteins and
attachment factors
Fibronectin, applied in conjunction with citric acid
demineralization of the root surface, has been dem-
onstrated to increase the level of connective tissue
attachment in a canine model (12). In this study
using two beagle dogs with natural periodontal dis-
ease defects, attachment levels were increased by
approximately 2 mm compared with flap surgery
alone. Similar results were obtained in a subsequent
study using six dogs (89). Three different concen-
trations of fibronectin were applied to surgically in-
duced defects; no advantage was found to increasing
the fibronectin concentration over plasma levels.
Contrasting results were observed following root sur-
face demineralization and fibronectin application in
supra-alveolar periodontal defects in a 14-dog study
by Wikesjo et al. (103). Animals receiving root sur-
face application of fibronectin exhibited significantly
less connective tissue attachment compared to con-
trol. These results were confirmed in a human histo-
logical case study by Alger et al. (1). In still another
human study, the clinical effect of placement of
fibronectin in periodontal defects following treat-
ment of the root surfaces with citric acid demin-
eralization was investigated (13). Groups treated
with flap surgery alone, and flap surgery plus
fibronectin and citric acid treatment, showed attach-
ment level gains after one year. While there was a
difference between the two treatments, the differ-
Biological mediators for periodontal regeneration
ences in pocket depth and clinical attachment levels
were slight, less than 1 mm. Thus, this treatment
modality remains of uncertain clinical benefit.
Periodontal regeneration stimulated by enamel
matrix proteins has been suggested in a nonhuman
primate buccal dehiscence model (39). Alveolar
bone, periodontal ligament and cementum were re-
moved from maxillary canines, premolars and first
molars. Different preparations of porcine enamel
matrix with or without carrier were applied prior to
flap repositioning. Histological analysis was per-
formed following a two-month healing interval. Re-
generation including acellular cementum with colla-
genous fibers connecting to new alveolar bone oc-
curred when either homogenized enamel matrix or
an extract containing amelogenins was used. Prep-
arations without amelogenin resulted in minimal ce-
mentum and bone formation and was similar to the
results in the control sites. Of three different carriers
examined, only propylene glycol alginate combined
with fractions containing amelogenin resulted in sig-
nificant periodontal regeneration. The results of
these experiments suggest that a cell-matrix interac-
tion is permissive for tissue formation around teeth.
In this study, amelogenin containing protein prep-
arations as a matrix isolated from developing tooth
enamel was combined with undifferentiated mes-
enchymal cells in the periodontal ligament to allow
for tissue formation.
The clinical use of enamel matrix derivative has
been shown to be safe, also after multiple appli-
cations (109). A total of 107 patients requiring peri-
odontal surgery received the enamel matrix protein
at two separate intrabony defects adjacent to single-
rooted teeth. Within two to six weeks following treat-
ment of the first defect, the second defect received
surgery including application of enamel matrix pro-
teins. Thirty-three patients with similar periodontal
defects served as surgical controls. Serum samples
were analyzed for total and specific antibody levels.
The results suggested that none of the antibody
levels differed from baseline, demonstrating that the
immune potential is low to the enamel matrix pro-
teins. This is not particularly surprising, given the
highly conserved amino acid sequence of enamel
matrix proteins. Clinical and radiographic analysis at
eight months and after three years indicated a sig-
nificant difference between protein-treated and non-
treated teeth. The enamel matrix derivative treat-
ment under these conditions resulted in a 2.5 to 3.0
mm increase in clinical attachment and bone level.
A multicenter randomized placebo-controlled
clinical trial evaluated surgical implantation of en-
53
Cochran & Woznev
amel matrix derivative in 33 patients exhibiting 34
paired intrabony periodontal defects (43). Inter-
proximal defects in the same jaw with a pocket
depth of greater than 6 mm and a radiographic de-
fect of greater than 4 mm in depth and 2 mm in
width were used. The paired defects in each patient
received modified Widman flap surgery, one of the
defects additionally received application of enamel
matrix derivative. Clinical attachment and radio-
graphic changes were evaluated following an 8-, 16-
and 36-month healing interval. Clinical attachment
gain in defects treated with enamel matrix derivative
was significantly greater than in the control aver-
aging 2.2 and 1.7 mm, respectively at 36 months
post-surgery. Radiographic bone gain at 36 months
post-surgery was 2.6 mm for defects treated with en-
amel matrix derivative, whereas the controls mini-
mally differed from baseline values. No adverse ef-
fects of the treatment were noted. The results sug-
gest that topical application of enamel matrix
derivative during periodontal surgery may result in
a significant gain in clinical attachment and radio-
graphic bone fill.
Summary
A review of the literature on the use of growth-regu-
latory molecules in the oral cavity permits a model
in which to consider approaches to oral tissue engin-
eering. These concepts apply to periodontal re-
generation and to regeneration of alveolar bone. In
either case, the formation of tissues is complex but
proceeds in a deliberate and orderly sequence. In
these sequence of events resulting in either bone or
cementum formation, periodontal ligament and
bone can be stimulated at various points. Different
signals can apparently be used to stimulate tissue
formation including mitogenic signals and differen-
tiation factors. Additionally, both hard and soft tissue
stimulatory molecules appear to be permissive. Clas-
sic receptor-mediate'd peptides or extracellular ma-
trix molecules for soft and hard tissues appear to
allow stimulation of tissue formation cascades. Im-
portantly, it also appears that the stimulatory event
is transitory (that is, short-lived) and leads itself to a
sequence of cellular events. These cellular events in
turn stimulate a number of subsequent events (such
as chemotaxis, proliferation, differentiation or angio-
genesis), which lead to further progression of tissue
formation.
While a solid scientific rationale exists for the use
of a variety of growth and attachment factors in re-
54
generation of oral tissues, only a small number are
being pursued clinically. Many therapeutic regimens
have failed in preclinical testing or have resulted in
limited regenerative capacity. The mitogenic poly-
peptides that stimulate soft tissue growth (such as
platelet-derived growth factor) and both hard and
soft tissue growth (such as transforming growth fac-
tor+) appear to have not led to successful enough
outcomes to facilitate further work towards regula-
tory approval. The demonstrated ability of bone
morphogenetic proteins to generate substantial
quantities of bone suggest many applications in the
oral cavity where this is the only tissue desired. An-
other therapeutic candidate is enamel matrix deriva-
tive, a set of matrix proteins. Enamel matrix deriva-
tive appears to stimulate first acellular cementum
formation, which may allow for functional peri-
odontal ligament formation. It will be of interest in
the future to determine whether the protein matrix
contains classic mitogenic or differentiation factors
as well as the amelogenins. It is also evident that the
bone morphogenetic proteins permit periodontal
ligament formation. The conditions for stimulating
predictable periodontal ligament tissues with bone
morphogenetic proteins however are not known. It
is clear that the bone morphogenetic proteins are
excellent molecules for stimulating oral bone forma-
tion. The results of all these studies will determine
the future therapeutic potential for these growth
molecules such that they may be used to optimally
stimulate and direct specific points along tissue for-
mation cascades.
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