Biological mediators for periodontal regeneration articles/LR141/Cochran... · chemical nature of...

Periodontology 2000, Voi. 19, 1999, 40-58 Pririted il l Dennzark All rights reserved

Copyr ight 0 Munksgaard 1999

PERIODONTOLOGY 2000 ISSN 0906-6713

Biological mediators for periodontal regeneration DAVID L. COCHRAN & JOHN M. WOZNEY

Successful periodontal reconstruction comprises regeneration of multiple tissues including cementum, periodontal ligament, bone and gingiva. The production or regeneration of any tissue type is a complex biological process in itself, requiring intricately regulated interactions between cells, locally acting growth factors, systemic hormones and growth factors, 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 biochemical nature of the extracellular matrix it contains 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 production of one tissue type of the periodontium. For example, as bone is required for successful regeneration, a variety of therapies (both biological and membrane types) have been directed at its regeneration with the assumption that the appropriate production 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 approaches to the promotion of periodontal regeneration have been used. These can be divided into the use of growth and differentiation factors, application of extracellular matrix proteins and attachment factors 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

a different cell type. These factors may control the growth of cells and hence the number of cells available to produce a tissue. In addition, they may control the metabolism of a particular cell type: for example, the rate of production of an extracellular matrix 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 combinations, 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 systems, 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).

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Biological mediators for periodontal regeneration

Table 1. Activities of growth factors

Pre-osteoblastl Fibroblast osteoblast Extracellular Mesenchymal cell uroliferation uroliferation matrix svnthesis differentiation Vascularization

+a - - Platelet-derived growth factor + f ++ - Insulin growth factor + ++ ++ -

Bone morphogenetic protein - - + 2 ++ ++a

Transforming growth factor-B + or - + or - ++ + a ~

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-osteoblasts as well as the differentiation of osteoblasts, including type I collagen synthesis (3, 17). Thus, insulin 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 presence of a family of insulin growth factor-binding proteins, 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 ligament and gingival tissue by combining a competence and progression factor; and, bone through its mitogenic 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 morphogenetic 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 complex mixture of bone morphogenetic protein molecules and possibly other factors and proteins, have

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 osteogenic 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 recombinant human bone morphogenetic protein-2 and recombinant human bone morphogenetic protein-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 differentiation (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 addition, the bone morphogenetic proteins are required for the embryonic development of the skeleton 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 periodontal tissue formation. In fact, bone morphogenetic proteins have been shown to affect the phenotypic expression of periodontal ligament cells.

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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 proteins, but functionally quite different (65, 90). It is synthesized by many cell types, and effects the function 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 factors 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 osteoblasts and osteoblast precursors; for example, increases 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 proximity 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 endothelial 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 formation, and accelerate the rate of fracture repair (2, 51). Some of these effects may be mediated through increases in transforming growth factor-p production. Again, in spite of the presence of a biologic rationale for the use of this growth factor in periodontal 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

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 evidence 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 believed 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 cementum; however, it has also been shown that exposure of periodontal ligament cells to enamel matrix derivative results in increased cell proliferation, total protein synthesis, and numbers of mineralized nodules formed (28). While amelogenins com- prise a majority of the protein in enamel matrix derivative, it is likely that this extract contains additional growth regulatory molecules, as other growth factors including bone morphogenetic proteins, 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 differentiated 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 demineralization of the root. The rationale is that demineralization will expose collagen fibers within the root surface, and fibronectin will facilitate interaction 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 increase the attachment and proliferation of fibroblasts to dentin explants, while decreasing epithelial cell attachment (91, 93). Once again, this therapy is directed at one aspect of periodontal regeneration; the belief here is that augmentation of connective tissue attachment will also lead to increases in periodontal ligament and bone formation.

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Bioloaical mediators for aeriodontal reaeneration

Mediators of bone metabolism

Several other agents which affect the growth of bone have been used, either as single agents or in combination 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 example, 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 formation. At the cellular level, prostaglandins are mitogenic for osteoblasts and stimulate differentiation, but may decrease matrix synthesis by differentiated osteoblasts. Zn v i m studies have demonstrated 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 administration 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

synergistically increases the differentiation of osteoblastic cells and hence the formation of bone tissue. Thus experiments on the effect of glucocorticoids on bone in vitro reveal both anabolic and catabolic effects, depending on the concentrations used and the differentiation state of the cells. Generally, glucocorticoids enhance differentiation of osteoblastic precursor cells. For example, bone nodule formation by calvarial cells and stromal cells is enhanced in the presence of glucocorticoids, and can synergize with the differentiative effects of bone morphogenetic proteins (7, 8). On the other hand, decreases in bone formation markers are observed in a number of organ 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 regeneration of tissues around teeth, in edentulous areas of the alveolus, and in the maxillary sinus. Initial attempts to regenerate oral tissues centered on regeneration of the periodontium and often included membrane exclusion techniques or guided tissue regeneration. In sequence, similar approaches, termed guided bone regeneration, were used for regeneration of alveolar bone. This work helped focus therapeutic approaches to regenerating tissues at the cellular and molecular levels. In the following, we

Table 2. Studies of biological mediators on periodontal regeneration (reference number)

Preclinical studies Clinical studies Periodontal Implant Periodontal Implant

Platelet-derived growth factor + insulin growth factor 29, 30, 61, 62, 80 6,60 46 Platelet-derived growth factor + dexamethasone Platelet-derived growth factor

81 22, 30, 72, 99

Insulin growth factor + fibroblast growth factor +

Transforming growth factor-0 104

83 transforming growth factor-p

Bone morphogenetic proteina 77 50, 82, 100, 101 10

Enamel matrix 39 43,109 aBone-derived bone morphogenetic protein bRecombinant bone morphogenetic protein-7 = recombinant OP- 1

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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 combination of purified platelet-derived growth factor and recombinant insulin growth factor-I stimulated periodontal 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 defects. 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 observed with the newly formed bone lined by osteoblasts. 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, occurred within a two-week interval.

In a second report, Lynch et al. (61) described periodontal regeneration in 13 beagle dogs exhibiting natural periodontitis defects treated with a combination 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 periodontal ligament of appropriate dimensions was observed. Ankylosis was not observed in the growth factor-treated sites.

Following these first demonstrations of periodontal regeneration induced by growth factors in the dog, it was important to determine whether similar results could be obtained in nonhuman pri-

mates. Rutherford et al. (80) evaluated healing following surgical implantation of recombinant platelet-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 performed during the one-month healing interval. The histological analysis showed regeneration of approximately half of the attachment including bone formation in septa1 areas with horizontal bone loss. The results support a concept that exogenously applied 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 formation and integration.

In a subsequent study, Rutherford et al. (81) evaluated platelet-derived growth factor with dexamethasone in a collagen matrix carrier for periodontal regeneration. 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 cementum and periodontal ligament were observed with seven-fold increases in bone formation.

Giannobile et al. (29) compared regeneration following surgical implantation of recombinant platelet-derived growth factor-BB and insulin growth factor-I into canine natural periodontitis defects and into nonhuman primate ligature-induced periodontitis defects. Significant regeneration versus control occurred in both systems. Increased regeneration 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 nonhuman 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-

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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 combination, on periodontal regeneration in the nonhuman 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 similar to that in the control. Defects treated with platelet-derived growth factor-BB exhibited 70% new attachment at the three-month healing interval. 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 significantly stimulates regeneration similar to the combination of platelet-derived growth factor-BB and insulin growth factor-I.

Wang et al. (99) used autoradiography to evaluate the cellular response to topical platelet-derived growth factor. Bilateral periodontal fenestration defects were surgically created in the posterior mandible 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 provisions for guided tissue regeneration using expanded 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 proliferation was significantly enhanced in the presence of exogenous platelet-derived growth factor-BB. This enhancement also occurred in the presence or absence 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 implantation of platelet-derived growth factor-BB with guided tissue regeneration. Induced, chronic, through-and-through, horizontal, mandibular premolar furcation defects in six beagle dogs were sub- jected to reconstructive surgery including citric acid root conditioning, application of recombinant platelet-derived growth factor-BB (5 pg) and guided tissue regeneration using expanded polytetrafluoroethylene membranes. Control defects received the same protocol without growth factor. Two dogs each were

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/recombinant 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 significant 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, including immunological reactions. In conclusion, surgical implantation of recombinant platelet-derived growth factor-BB and recombinant insulin growth factor-I may support significant periodontal regeneration 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 implants 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-implant 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 control sites could be established at this observation interval, suggesting that the control sites had time to form bone in sufficient amounts. Thus, it appears that the combination of platelet-derived growth factor and insulin growth factor-I accelerated bone formation around endosseous dental implants.

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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 without growth factors. These results confirm the observations above regarding bone stimulation at endosseous dental implants.

A question arises as to why this approach to periodontal 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 approach 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 reason, this approach to periodontal regeneration is not actively pursued.

In other studies evaluating growth factors for periodontal regeneration, Selvig et al. (83) screened the effect of a insulin growth factor-II/basic fibroblast growth factor/transforming growth factor-pl combination 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 insulin 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

histological observations of healing at 3, 7, 10 and 14 days post-surgery. The results demonstrated no differences in fibroblast and collagen density in defects treated with the growth factor combination compared with control. Bone regeneration was significantly greater and occurred more frequently in the control compared with growth factor-treated defects, 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 carrier protected by an expanded polytetrafluoroethylene 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 approach 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, recombinant 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 reconstruction plates. Three groups of animals received treatment including recombinant bone morphogenetic 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

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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 minimal 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 fibrous scar tissue. This study demonstrates the potential 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 demineralized bone matrix or a bovine tendon-derived collagen matrix. Controls were either demineralized bone matrix or the collagen matrix alone. Both submerged and nonsubmerged wound closure techniques were used in the soft tissue management of the intrabony periodontal defects. Appropriate histological evaluation of block biopsies was performed following a six-month healing interval. Bone mor-

Fig. 1. Osseous repair of mandibular defects using recombinant bone morphogenetic protein-2 in the canine critical-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 absorbable collagen sponge (12 weeks post-surgery).

phogenetic protein-3 and demineralized bone matrix in a submerged environment resulted in significant periodontal regeneration. Bone morphogenetic protein-3 and demineralized bone matrix and demineralized bone matrix alone resulted in significantly more new tissue formation than bone morphogenetic protein-3 and collagen matrix or the collagen matrix alone in both submerged and nonsubmerged environments. No differences in tissue formation were found between bone morphogenetic 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 protein-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 protein fraction from bovine bone in combination with a collagen matrix to stimulate bone growth in monkey extraction sites in the presence or absence of endosseous 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 protein fractions containing bone morphogenetic protein might be useful in stimulating bone formation around endosseous implants.

Wang et al. (50, 100, 101) confirmed the observations by Rutherford et al. (82) and suggested that the rate of dental implant osseointegration may increase in the presence of bone morphogenetic protein. In this study, purified bovine bone morphogenetic 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 analyses of block biopsies harvested from three animals each at 1 , 2 , 4 , 8 and 12 weeks post-surgery revealed direct bone apposition after four weeks in sites treated with bone morphogenetic protein, while fibrous 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-

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results are emerging using bone morphogenetic proteins to stimulate regeneration, there is a need to identify the origin of the cells that respond to bone morphogenetic proteins more precisely, to understand the role of each of the different bone morphogenetic proteins and their combinations and to better understand delivery systems for the bone morphogenetic proteins.

Ishikawa et al. (49) used recombinant bone morphogenetic protein-2 to stimulate periodontal regeneration in surgically induced three-wall intrabony defects in dogs. Recombinant bone morphogenetic 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 compared to control.

Periodontal regeneration following surgical implantation of recombinant bone morphogenetic protein-2 has also been evaluated in the critical size, supra-alveolar periodontal defect model (86). This study used the same recombinant bone morphogenetic Fig. 2. Periodontal regeneration with recombinant bone

momhogenetic ~rotein-2 using canine SuDra-dveolar de- protein-2 delivery system as Ishikawa et al. (49). Con- - feet mozel (86): Buccal-lingual histological sections following implantation of buffer/carrier (left, A and C) or recombinant bone morphogenetic protein-2/carrier (right, B and D).

trols consisted of the carrier alone. Upon implantation of recombinant bone morphogenetic protein-2 (0.2 mg/ml), the gingival flaps were coronally advanced and sutured to submerge the 5-mm mandibu-

generation following surgical implantation of bone morphogenetic protein fractions obtained by ex- tracting bovine demineralized bone matrix. These preparations consisted primarily of bone morphogenetic 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 morphogenetic 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 ligament 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

lar premolar defects. The animals were killed at two months post-surgery, and block biopsies were pro- cessed for histometric analysis. The height of regenerated bone in recombinant bone morphogenetic protein-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 morphogenetic 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 protein-2. Ankylosis was found occasionally but was localized and appeared unrelated to the bone morphogenetic protein treatment.

Subsequently, Sigurdsson et al. (87) evaluated recombinant bone morphogenetic protein-2 with various carriers for periodontal regeneration. Canine demineralized bone matrix, bovine deorganified crys- talline bone matrix (Bio-Oss), an absorbable type I

Biological mediators .for periodontal regeneration

bovine collagen sponge, poly (D,L-lactide-co-glycolide) 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 morphogenetic protein-2. Cementum regeneration and ankylosis 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 significantly affected by the specific carrier system.

Ripamonti et al. (76) evaluated cementogenesis following application of recombinant bone morphogenetic 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 morphogenetic protein-7 in a bovine collagen matrix or the collagen 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 recombinant bone morphogenetic protein-2 induced periodontal regeneration to root surfaces with a history of dental plaque and calculus exposure. Ligature-induced, circumferential periodontal defects approxi- mating 50% of the root length in the mandibular premolar teeth in six beagle dogs received recombinant bone morphogenetic protein-2 (0.4 mg/ml) in gelatin and polylactic acid polyglycolide acid copolymer carrier or the carrier alone (control) in contralateral defects. The histometric evaluation revealed significantly more new bone and cementum with Sharpey’s fibers in defects treated with recombinant bone morphogenetic protein-2 compared with control following a three-month healing interval. Root resorption was a rare finding. Ankylosis was not observed in bone morphogenetic protein or control sites. The results suggest that periodontal regeneration induced by recombinant bone morphogenetic protein- 2 may also occur at root surfaces with a history of periodontal disease.

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 recombinant bone morphogenetic protein-2 in a collagen 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 morphogenetic 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 recombinant 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 between the sites. The results indicate that recombinant bone morphogenetic protein-2 accelerates bone formation and selectively enhances cementum formation 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 formation would not occur in absence of bone grafting. Bilateral maxillary sinus floor elevation procedures were performed in six adult goats. Each animal received 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 compared with control were demonstrated histologically, with normal progression of the bone formation process. 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 immune response.

The effect of recombinant bone morphogenetic protein-2 on peri-implant bone formation was reported 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 recombinant bone morphogenetic protein-2 and ab-

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Fig. 3. Histological analysis of peri-implantitis defect repair 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 morphogenetic protein-2 at 0.4 mg/ml) or buffer and absorbable 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 recombinant 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 regenerated bone averaged 4.2 mm for sites treated with recombinant bone morphogenetic protein-2 compared with 0.5 mm for the control. Average new bone area amounted to 6.1 mm2 and 0.2 mm2, respectively. Bone-to-implant contact within regenerated bone averaged 19% and 8% for recombinant bone morphogenetic protein-2 and control sites, respectively.

Hanisch et al. (41) were the first to demonstrate dental implant re-osseointegration using a nonhuman 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

defects received recombinant bone morphogenetic protein-2 and absorbable collagen sponge (recombinant 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 compared 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 protein-2 sites compared with 4% for controls. Within the area of new bone formation, bone-to-implant contact averaged 40% in bone stimulated with recombinant bone morphogenetic protein-2 compared with 9% for the control. Thus, recombinant bone morphogenetic protein-2 appears capable of stimulating bone formation to support dental implant 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 sinus 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 subantral space and anteriorly to the sinus (control placed in native bone). Histological analysis, following another three months of healing, revealed significantly 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 augmented, control sites and native bone surrounding the augmented sinus.

Cochran et al. (23) examined bone regeneration around endosseous dental implants following implantation of recombinant bone morphogenetic protein-2 with and without provisions for guided bone regeneration in a dog model. Standardized, intrabony 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

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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 significantly 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 regeneration compared with control. Bone fill was most enhanced in sites treated with recombinant bone morphogenetic protein-2 and guided bone regeneration followed by sites treated with recombinant bone morphogenetic protein-2 alone. When control 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 receiving 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 morphogenetic protein-2 induced bone within peri-implant defects is dependent on the healing interval, the carrier system employed and whether guided bone regeneration was used.

Recently, Cochran et al. (24) reported the histometric analyses from the peri-implant defect study (23). Induced bone area, bone-to-implant contact in the defect area, and defect bone fill were determined. Surgical implantation of recombinant bone morphogenetic protein-2 resulted in significantly increased induced bone area, bone-to-implant contact, 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-

trafluoroethylene membranes. In these sites increased bone formation was observed in the presence of recombinant bone morphogenetic protein- 2 compared with the absence of recombinant bone morphogenetic protein-2. Furthermore, bone-to-implant contact was significantly enhanced along the titanium implant surfaces. Guided bone regeneration and non-guided bone regeneration sites exhibited similar amounts of bone formation at three months, while at one month the guided bone regeneration 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 endosseous 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 collagen sponge in 12 patients. Computerized tomography was used to assess the effect of the augmentation procedure over a four-month healing interval. Eleven patients were available for the evaluation. All 11 patients implanted with recombinant bone morphogenetic 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 reactions 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 augmentation.

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 recombinant 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

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blood samples to determine serum chemistries, hematology and potential antibody formation. Re- combinant bone morphogenetic protein-2 and absorbable collagen sponge was found to be well toler- ated locally and systemically without serious adverse effects. Significant bone formation could not be established following the ridge augmentation procedure. 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 clinical trials demonstrate that recombinant bone morphogenetic 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 minipumps. 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 osmotic minipumps and controlled-release pellets to present prostaglandin E, subperiosteally next to the mandibular cortex. Dose-response curves were produced 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 increases 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 observed changes were a result of activated osseous re-

modeling. At the highest doses of prostaglandin El , 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 alveolar 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 histologically indistinguishable from native periodontal ligament. New attachment occurred in all 18 exper- imentally treated sites and in only one of seven control 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 ligament 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 demonstrate that the systemic administration of these molecules (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 arachidonic acid. These compounds are ubiquitous and found in a variety of tissues. The effect of the prostaglandins varies considerably from stimulating inflammation and bone resorption to the enhancement 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 periodontal literature focused on a model to evaluate bone resorption and the prostaglandins became known for their effects for stimulating bone resorption, as this was the effect this model system best evaluated. Reports on the systemic effects of prostaglandins 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

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as well as bone resorption might occur with these molecules. It appears that the addition of prostaglandin 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 formation in the oral cavity. It appears at this point, however, 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 application 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 demonstrated to increase the level of connective tissue attachment in a canine model (12). In this study using two beagle dogs with natural periodontal disease 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 concentrations of fibronectin were applied to surgically induced defects; no advantage was found to increasing the fibronectin concentration over plasma levels. Contrasting results were observed following root surface demineralization and fibronectin application in supra-alveolar periodontal defects in a 14-dog study by Wikesjo et al. (103). Animals receiving root surface application of fibronectin exhibited significantly less connective tissue attachment compared to control. These results were confirmed in a human histological case study by Alger et al. (1). In still another human study, the clinical effect of placement of fibronectin in periodontal defects following treatment of the root surfaces with citric acid demineralization was investigated (13). Groups treated with flap surgery alone, and flap surgery plus fibronectin and citric acid treatment, showed attachment level gains after one year. While there was a difference between the two treatments, the differ-

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 removed 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 performed following a two-month healing interval. Re- generation including acellular cementum with collagenous fibers connecting to new alveolar bone occurred when either homogenized enamel matrix or an extract containing amelogenins was used. Prep- arations without amelogenin resulted in minimal cementum 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 significant periodontal regeneration. The results of these experiments suggest that a cell-matrix interaction is permissive for tissue formation around teeth. In this study, amelogenin containing protein preparations as a matrix isolated from developing tooth enamel was combined with undifferentiated mesenchymal 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 applications (109). A total of 107 patients requiring periodontal surgery received the enamel matrix protein at two separate intrabony defects adjacent to single- rooted teeth. Within two to six weeks following treatment of the first defect, the second defect received surgery including application of enamel matrix proteins. 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 proteins. 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 significant difference between protein-treated and nontreated teeth. The enamel matrix derivative treatment 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-

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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 defect 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 radiographic 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 enamel matrix derivative, whereas the controls mini- mally differed from baseline values. No adverse effects of the treatment were noted. The results suggest that topical application of enamel matrix derivative during periodontal surgery may result in a significant gain in clinical attachment and radiographic bone fill.

Summary

A review of the literature on the use of growth-regulatory molecules in the oral cavity permits a model in which to consider approaches to oral tissue engin- eering. These concepts apply to periodontal regeneration 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 differentiation factors. Additionally, both hard and soft tissue stimulatory molecules appear to be permissive. Clas- sic receptor-mediate'd peptides or extracellular matrix 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-

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 polypeptides that stimulate soft tissue growth (such as platelet-derived growth factor) and both hard and soft tissue growth (such as transforming growth factor+) appear to have not led to successful enough outcomes to facilitate further work towards regulatory 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 derivative, a set of matrix proteins. Enamel matrix derivative appears to stimulate first acellular cementum formation, which may allow for functional periodontal 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 formation. 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 formation cascades.

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