Use of growth factors in bone regeneration

Introduction

Various advances have been made in periodontal regeneration treatments in the past few decades. Despite an armamentarium that includes bone grafts, root conditioning, and guided tissue regeneration, however, our ability to predictably regenerate periodontal tissues still needs improving. This has prompted researchers to focus a great deal of study in recent years on the basic nature of how periodontal tissues heal and regenerate on a cellular level, with an eye toward developing treatments that may help the process along.

Perhaps the most promising research currently underway involves the use of inductive proteins, known collectively as growth factors. These molecules - a class of polypeptide hormone - are key regulators of the cellular wound repair processes for nearly all of the body's tissues, including the periodontium ^{(Kiritsy and Lynch, 1993} ; ^{Lynch and Giannobile, 1994} ; ^{Ripamonti and Reddi, 1997)}. They stimulate a wide variety of cellular events, such as prolifération, chemotaxis, différentiation into mature cells that comprise various periodontal tissues, and the production of extracellular matrix proteins (^{Terranova and Wikesjo, 1987}).

When injury occurs, several growth factors are released from in and around the wound site. They work together to regulate cell activity and to repair and regenerate the various types of tissue. For instance, a prerequisite for periodontal regeneration is the adequate prolifération and migration of periodontal ligament cells, synthesis of extracellular matrix, and differentiation of cementoblasts and osteoblasts (^{Terranova and Wikesjo, 1987}). How well the growth factors that stimulate and orchestrate these important processes are expressed in the presence of progressing periodontal disease and associated bone and soft tissue injuries is not known, but this may profoundly influence the repair or regenerative process. Therefore, the goal of administering growth factors to treat chronic periodontitis or to spur osteoregeneration would be to enhance the normal wound healing process that may otherwise be insufficient to completely regenerate the tissues (^{American Academy of Periodontology, 1996}). This is the subject of current experimental research and human trials have also recently begun.

This article provides an overview of the various types of growth factors that have been identified and reviews the latest in vitro and in vivo research on their potential application to bone regeneration treatment.

Growth factors involved in bone regeneration

During the past decade, several growth factors have been identified and characterized. Those currently believed to contribute to bone regeneration include platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-ß), insulin-like growth factors (IGFs), fibroblast growth factor (FGF), and the BMPs (^{Howell et al., 1996}). These periodontal growth factors have some common features, including that they bind to specific receptors on the target cell surface, they are primarily locally acting, and they are multifunctional so they can stimulate and/or regulate a variety of cell activities (^{Graves et al., 1994}).

One important characteristic researchers have identified is that growth factors are cell specific. In other words, particular growth factors work only to stimulate specific cell types. Clearly, a combination of growth factors, including those that stimulate wound closure as well as the formation of periodontal tissues, is necessary. Several of the studies discussed here focus on combination treatments.

Insulin-like growth factors (IGF)

There are two types of IGFs, which function similarly but are independently regulated. They are produced to some degree by the liver and circulate in the vascular system. Large arnounts of inactive IGF-I and IGF-II are also found in bone (^{McCauley and Somerman, 1998}). The two have similar effects when tested in vitro, but most studies suggest IGF-Il is less potent than IGF-I in promoting bone formation ^{(American Academy of Periodontology, 1996} ; ^{McCarthy et al., 1989)}.

IGF-I is produced by osteoblasts and stimulates periodontal ligament and bone formation by increasing cellular proliferation, chemotaxis, differentiation, and bone matrix production ^{(American Academy of Periodontology, 1996} ; ^{Canalis et al., 1988} ; ^{Howell et al., 1995} ; ^{Park et al., 1995)}.

In vivo studies in dogs and nonhuman primates have found that IGF-I has little periodontal regenerative effect on its own ^{(Giannobile et al., 1994}, ^{(Giannobile et al., 1996} ; ^{Lynch et al., 1989)}. so most recent in vitro and in vivo studies have concentrated on its usefulness when combined with other growth factors. In vitro studies indicate that combining IGF-I with platelet-derived growth factor (PDGF) or transforming growth factor-ß1 (TGF-ß1) increases bone matrix apposition significantly more than any of these factors do individually ^{(Pfeilschifter et al., 1990)}. Other in vitro studies indicate that IGF-I increases osteoblast mitogenesis most significantly when combined with PDGF, TGF-ß1, and epidermal growth factor ^{(Giannobile et al., 1994} ; ^{Piche et Graves, 1989)}.

The most promising research has involved a combination of IGF-I and PDGF, which appear to act synergistically as discussed in the following section. Some data suggest that PDGF may promote IGF-I receptor expression ^{(American Academy of Periodontology, 1996)} and the bone cells stimulated, by PDGF downregulate IGF-I gene expression ^{(Becker et al., 1992)} and resulting protein levels ^{(Canalis et al., 1993)}. Thus, combining PDGF with exogenous IGF-I may maximize its metabolic response.

Platelet-derived growth factor (PDGF) (fig. 1, 2, 3 and 4)

PDGF, considered one of the principal wound healing hormones, has been the most studied growth factor. PDGF is a potent mitogen and chemotactic factor for cells of mesenchymal origin, including periodontal ligament cells and osteoblasts. There may be several cellular sources of PDGF at a wound site, including platelets, activated macrophages, bone matrix, and so forth. PDGF can exist as either a heterodimer (PDGF-AB) or a homodimer (PDGF-AA or -BB) and has been shown to stimulate the prolifération of osteoblasts and also periodontal ligament cells that have both fibroblastic and osteoblastic characteristics. When applied to root surfaces of teeth with surgically created defects in six dogs, PDGF enhanced fibroblast proliferation in the early healing stages ^{(Lynch et al., 1987)}.

Thus far, PDGF has shown great promise as an adjunct to regenerative therapy in animal and preliminary human studies. Most of the studies have examined the effect of PDGF combined with insulin-like growth factor-I (IGF-I), which appear to work synergistically. Several in vitro and in vivo studies suggest these two types of growth factors work better together than individually in promoting greater proliferation of various bone cells and fibroblasts, collagen deposition, soft tissue wound healing, and bone matrix formation ^{(Giannobile et al., 1994}, ^{(Giannobile et al., 1996} ; ^{Greehnalgh et al., 1993} ; ^{Lynch et al., 1987}, ^{Lynch et al., 1994} ; ^{Martin et al., 1998)}. When used alone, PDGF-BB has been shown in vivo to stimulate increased new attachment compared to controls but not significantly higher bone fill ^{(Giannobile et al., 1996)}.

In the first recently reported human trial, 38 patients with moderate-to-severe periodontitis were treated with either : a) 150 µg/mL each of PDGF-BB and IGF-1 in a methylcellulose vehicle ; b) vehicle alone ; or c) surgery alone. Those treated with the PDGF/IGF combination experienced significantly more bone fill within 9 month - 43 % on average versus an 18.5 % average fill for the control groups ^{(Howell et al., 1997)}. This growth factor « cocktail » treatment, using dosages of up to 150 µg/mL of each, was also deemed safe, causing only mild discomfort (according to VHO classification) in 15 patients ^{(Folkman et Klagsbrun, 1987)}.

These results regarding enhanced bone regeneration appear consistent with findings in several preclinical dog and nonhuman primate studies using the PDGF/IGF combination ^{(Giannobile et al., 1994}, ^{(Giannobile et al., 1996} ; ^{Lynch et al., 1989}, ^{Lynch et al., 1991)}. At least two studies also showed that this combination of growth factors enhances bone growth around implants placed into fresh extraction sockets ^{(Becker et al., 1992} ; ^{Lynch et al., 1991)}. Interestingly, the results of the in vivo studies have also been consistent with those of the in vitro studies on this combination of growth factors ^{(McCauley et Somerman, 1998)}.

The exact mechanism(s) by which this growth factor combination improves periodontal regeneration still needs to be proved in vivo as do precise amounts of the combination that would be optimally useful. In addition, some studies indicate that combining PDGF with guided tissue regeneration may also aid periodontal regeneration ^{(Becker et al., 1992} ; ^{Wang et al., 1995)}.

Transforming growth factor-beta (TGF-ß)

Latent TGF-ß is mainly stored in bone matrix, and the mechanism that activates it is unclear, although some speculate that a low pH level during osteoclastic bone resorption may be responsible ^{(Howell et al., 1996)} as well as gingival and periodontal inflammation and bacterial invasion ^{(Skaleric et al., 1997)}.

Almost every cell type can be stimulated by at least one of the five types of gene-encoded TGF-ß molecules. TGF-ß is a weak mitogen for osteoblasts ^{(Sporn et Roberts, 1992)} and periodontal ligament fibroblasts ^{(Dennison et al., 1994} ; ^{Howell et al., 1996} ; ^{Mailhot et al., 1995} ; ^{Oates et al., 1993)}. It also inhibits reepithelialization ^{(Lynch et al., 1989)},, and induces chemotaxis and bone matrix deposition and stimulates collagen type 1 synthesis ^{(Bonewald et Muncy, 1990} ; ^{Pfeilschifter et al., 1990)}. The effects of TGF-ß appear to depend largely on the bone cell source, the dosage applied, and the local environment ^{(Sporn et Roberts, 1992)}.

Recent in vitro studies suggest that the combination of TGF-ß and PDGF-BB may selectively stimulate periodontal ligament cells more than gingival fibroblasts, thus enhancing periodontal regeneration ^{(Dennison et al., 1994} ; ^{Oates et al., 1993)}. Further in vivo studies are needed to confirm this. Taken together, however, various research findings suggest that TGF-ß may hold some promise as an adjunct to periodontal regenerative treatment some day ^{(American Academy of Periodontology, 1996)}.

Fibroblast growth factor (FGF)

FGFs are classified as either acidic or basic, and both are mitogenic and chemotactic for fibroblasts, chondrocytes, osteoblasts, periodontal ligament, and endothelial cells in vitro ^{(Lynch, 1994} ; ^{Takayama et al., 1997)}. They are stored in the bone matrix ^{(Maihlot et al., 1995)}. Basic FGF is considered more potent than acidic FGF and may act, in part, by stimulating other growth factors ^{(McCauley et Somerman, 1998)}.

Although FGF increases the number of osteoblasts capable of regenerating bone, it decreases the amount of matrix each cell produces. The net effect, however, appears to be enhanced bone formation ^{(McCauley et Somerman, 1998)}.

An in vitro study showed that both acidic and basic FGF can act synergistically with TGF-ß1 and IGF-I to strengthen their inductive effects and promote gradients of cyological and functional changes in odontoblast-like cells ^{(Martin et al., 1998)}. FGFs are unique in that they are potent angiogenic factors, stimulating the formation of blood vessels, that are critical to wound healing and granulation tissue formation ^{(Denisson et al., 1994)}. Again, in vivo studies are needed to determine a treatment benefit.

Bone morphogenic proteins (BMPs)

BMPs are unique growth factors in that they have osteoinductive properties. BMPs, which are a structurally related member of the transforming growth factor (TGF) gene superfamily, are believed to play a significant role in recruiting osteoprogenitor cells to sites of bone formation ^{(O'Neal et al., 1994)}. They are able to stimulate the differentiation and prolifération of mesenchymal stem cells into chondroprogenitor and osteoprogenitor cells ^{(Reddi et Cunningham, 1993)}. BMPs have been the focus of much research in the past 20 years, and at least 15 BMPs have been identified thus far, including BMP-2, BMP-3 (osteogenin), BMP-4 and BMP-7 (osteogenic protein-1).

BMPs are abundant in bone and are produced by several cells including osteoblasts. BMPs are also found in commercial bone allograft materials used in dentistry ^{(Piche et Graves, 1989)} ; however, the levels vary considerably ^{(Schwartz et al., 1996)}, and this may in part explain why in vivo studies using demineralized freeze-dried bone allograft have provided inconsistent bone regeneration results ^{(Becker et al., 1995} ; ^{Mellonig, 1996} ; ^{Ripamonti et al., 1994)}. It appears that the more BMP the graft material contains, the better the bone regenerative results.

Various in vivo studies in animals have shown that BNPs induce significantly more cementum, periodontal ligament, and bone regeneration in surgically created defects than do untreated controls ^{(Giannobile et al., 1998} ; ^{Ripamonti et al., 1994} ; ^{Ripamonti et Reddi, 1997} ; ^{Sigurdsson et al., 1995)}. Similar to the PDGF/IGF combination, in vitro studies of BMP effectiveness in promoting bone regeneration parallel the in vivo findings ^{(Ripamonti et al., 1994)}. Currently, these two types of growth factor treatments, which have been best studied, show the greatest potential for use in bone regenerative treatment.

The use of recombinant BMP-2 (rhBMP-2), osteogenin (BMP-3) and osteogenic protein-1 (BMP-7), three of several different BMPs, have also, been show to, be promising in studies with dental implants. Hanisch et al. found that compared to controls, rh-BMP2 significantly promoted bone formation (2.6 mm versus 0.8 mm) and reosseointegration (29 % versus 3.5 %) in advanced peri-implantitis defects in nonhuman primates at 4 months postsurgery ^{(Hanisch et al., 1997)}. ^Cochran also noted significantly more bone fill and greater density around endosseous implants treated with rhBMP-2 thafi controls in a canine study. Interestingly, this study also reported that greater density was achieved when using a nonresorbable membrane barrier as opposed to no barrier and a collagen carrier as opposed to a polyactide/glycolide carrier.

In association with a bone-derived matrix, osteogenin (BMP-3) rapidly initiates bone formation in vitro ^{(Vukicevic et al., 1988)}. Bowers et al. (1991) found that combining osteogenin with DFDBA significantly enhanced regeneration of a new attachment apparatus (1.92 mm) compared with DFDBA alone (1.31 mm) when submerged, although the improvement was not statistically significant in a nonsubmerged environment. ^Wang found similar results noting that BNPs induced earlier bone apposition around implants in dogs. Rutherford noted that osteogenic protein-1 (BMP-7) induced peri-implant bone healing and promoted rapid osseointegration in implants placed in monkeys ^{(Rutherford et al., 1992)}. By accelerating osseointegration, BMPs might allow earlier loading of implants with permanent restorations.

Futures perspectives

The future clinical application of growth factors in regenerative therapy is promising. Indeed, some researchers have noted that they may eventually replace autogenous or allogenic grafts for promoting osteoinduction in defect sites.

As discussed, studies thus far indicate that growth factors can significantly influence cell behavior. Additional research at both the molecular and clinical levels is needed, however, to improve the predictability of their use in bone regenerative therapy. At this point, growth factor therapy is still in its experimental stage of development.

Much more is to be discovered regarding the biology of healing wound sites, the appropriate cells that should be optimally targeted with growth factor applications, ideal combinations, doses, and application sequences of factors for regenerative treatment, and optimal delivery systems. Ongoing trials will continue to assess the usefulness of growth factors for bone regeneration.

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