Osseointegrated dental implants offer predictable success rates for a variety of restorative procedures. Although dental implant placement and restoration continues to increase, the length of time necessary for osseointegration of dental implants may be a significant contraindication to treatment. Patients are often unwilling to enter into a treatment plan that necessitates up to eight months of healing time between implant fixture placement and uncovering. Therefore, predictable methods of accelerating osseointegration would be beneficial. In addition, methods of improving the prognosis for osseointegration in areas of poor bone quality, which are often encountered in the maxilla, would also benefit patients for whom dental implant treatment might otherwise be contraindicated. In order to attempt modification of the process of osseointegration, a thorough knowledge of the physiology and metabolism of bone formation and wound healing is necessary. In this way, predictable osseointegration-enhancement techniques (OETs) can be developed that are based upon sound scientific knowledge. One potential OET involves controlled application of bone growth factors known to locally influence bone formation and regeneration to either the prepared fixture site and/or the fixture surface prior to placement. In this review, locally produced bone growth factors that have anabolic effects on bone mass and/or on the cells involved in increasing and/or maintaining bone mass are discussed. Key words: osseointegration, dental implants, bone growth factors, PDGF, ILF, FGF,TGF-B, BMP
Bone growth factors (BGFs) are produced either by bone cells or by hematological cells, and their effects may either be autocrine (where the target cell and the cell producing the BGF are the same) or paracrine (where the target cell is different from, but near, the cell producing the BGF).The majority of data regarding each BGF originates from in vitro studies investigating individual BGFs, while in vivo information is relatively scarce. The classic confrontation exists between the in vitro isolation of BGF effects and the ultimately more relevant in vivo effects, which are concomitantly more challenging to investigate in a manner which provides meaningful data. Review of in vitro data is further complicated by the variety of model systems used to study BGF effects. Cell culture, enriched cell culture, bone explant, and fetal/neonatal calvariae models all are commonly employed, which hampers direct comparison between studies.
Human PDGF, a dimer of molecular weight (Mw) 28,000 to 31,000, contains two chains,A and B, each a result of a separate gene. PDGF exists in one of three isoforms which are combinations of the two chains: either as a homodimer PDGF-AA or PDGF-BB, or as a heterodimer PDGF-AB. First associated with platelets and subsequently wound healing it is now known that osteoblasts are also capable of PDGF production, but only of the PDGF-A chain. Of the three forms, PDGF-BB is the most active in terms of bone cell effects, producing the greatest increase in cell replication in calvariae models. In osteoblast-enriched cultures derived from fetal calvariae, PDGF-BB is approximately eight times more mitogenic than PDGF AA and three times more mitogenic than PDGF AB. Although increased collagen synthesis has been reported in bone and bone cell cultures after exposure to PDGF, individual cell production of collagen does not appear to be affected. Instead, the observed increase in collagen synthesis is related to the increase in cell numbers secondary to enhanced cell replication.
In contrast to fibroblasts which possess two types of cell surface PDGF receptors with differing affinities for the isoforms, Centrella et al. demonstrated that osteoblast-enriched cultures contain receptors only for PDGF-BB, which may account for the increased stimulatory activity in bone caused by the PDGF-B chain. Further research of this particular receptor may highlight methods of increasing cell numbers and overall collagen synthesis at implant fixture sites.
IGFs, Mr 7500, are considered to play a significant role in the regulation of cell growth and function. The majority of IGF-I is produced by the liver in response to serum levels of growth hormone (GH), but IGFs also are synthesized by other skeletal and non-skeletal tissues. IGF-I production by osteoblast-enriched cultures has been demonstrated, and such production may have a localized influence on bone metabolism. In contrast to PDGF, which causes only increased cell replication, IGF-I enhances cell replication, type I collagen production, and type I collagen mRNA synthesis, as well as decreasing collagen degradation. IGF-I, therefore, has positive anabolic and negative catabolic effects. Enhanced cell replication occurs predominantly in the periosteum, while the increased type I collagen synthesis and matrix apposition appears to be concentrated in the endosteum.
IGF-II, formerly referred to as skeletal growth factor, has approximately 60% sequence homology to IGF-I. Similar effects to IGF-I are observed with regard to increased cell replication, increased collagen synthesis, and decreased collagen breakdown. However, conflicting data exists with regard to the relative potencies of the two BGFs. In bone culture, IGF-II is four to seven times less potent than IGF-I,29 while in osteoblast-enriched cultures it has a greater stimulatory effect upon type I collagen synthesis than IGF-I. Therefore, at this time, the use of IGF-II as a growth factor appears to have limited potential since its main stimulatory effects compared to IGF-I have not been demonstrated in bone organ culture. In addition, the osteoblast-enriched model would appear to have limited relevance for study of IGF-II.
Centrella et al. demonstrated that three ligand/receptor complexes exist for IGF-I (Mr 130,000, 240,000, and 260,000). One of the three, Mr 240,000, appears to be the primary receptor for IGF-II, suggesting that the more significant cellular effects observed in bone organ culture are secondary to IGF-I binding at the other two receptor sites. Further research into ligand/receptor complexes at Mr 130,000 and 260,000 may permit influence of the effects on cell replication, collagen synthesis, and collagen degradation.
Two distinct forms of FGF exist that share 55% DNA sequence homology, acidic FGF (aFGF), and basic FGF (bFGF). Both forms have similar overall effects with quantitative differences likely due to variation in receptor affinity or binding. FGFs have been isolated from a variety of tissue types including bone matrix extracts. Both aFGF and bFGF have mitogenic effects on fibroblast-enriched and osteoblast-enriched cell cultures, with bFGF exhibiting greater potency. Using a calvariae model, Canalis et al. demonstrated enhanced DNA synthesis after exposure to either form. Similar findings have been reported in cell cultures prepared from fetal rat and bovine bone. Overall collagen production is increased as a consequence of hyperplasia. In addition to fibroblasts and osteoblasts, bFGF is mitogenic for endothelial cells, with a resultant increase in vascularization and, consequently, an enhanced nutrient supply to the wound healing site. When bFGF is combined with high levels of Transforming Growth Factor-Beta (IGF-B), a dose-dependent synergistic increase in DNA synthesis in serum-free fetal rat bone cell cultures is observed. Lower concentrations of TGF-B lead only to an additive response. In addition, Globus et al. have reported a synergistic increase of DNA synthesis in bovine bone cultures when chronically exposed to TGF-B and bFGF in the presence of serum.
TGF-D, formerly called bone-derived growth factor-I and now known to be cartilage-inducing factors A and B, is present in large quantities both in bone cells and in bone matrix, with the latter being the major reservoir of this polypeptide. There are five known isoforms of TGF-B (TGF-Bs 1, 2, 3, 4, and 5), with TGF-Bs 1, 2, and 3 found in humans. TGF-B is produced by platelets and bone cells in a latent form containing two mature sub-unit polypeptides, two amino-terminal precursor fragments, and a third protein. The active form contains 112 amino acids, nine cysteine residues, and a molecular weight of approximately 25,000. Daopin et al. used X-ray crystallography to investigate the molecular structure of TGF-B 2 and demonstrated that eight of the nine cysteine residues are arranged in a compact pattern by means of an array of disulfide bridges. This pattern has been termed a "TGF-S knot." Nuclear magnetic resonance analysis of TGF-B 1 in solution shows many similarities between the two isoforms and suggests that TGF-B may exist in several conformations. The exact conversion mechanism from latent to active form is not established. However, recent data indicates that the plasmin protease system may cleave and remove amino-terminal precursor fragments. Autoregulation may occur through this mechanism since an increase in levels of plasminogen activator and a decrease in the level of plasminogen activator inhibitors has been observed in isolated cultures of bone cells exposed to TGF-B. It is also known that latent TGF-B may be activated under acidic conditions. Complete release of active growth factor occurs at a pH of 2, a situation not normally encountered in human bone. However, activation still may occur at pH values up to 6. Milder acidity is typically observed associated with bone-resorbing areas, particularly under the ruffled borders of osteoclasts. Osteoclasts have been reported to activate the TGF-B complex, and so the possibility exists that osteoclasts may cause the release and activation of latent TGF-B during bone resorption.
Along with the numerous isoforms of TGF-B, there are four known TGF-B receptor binding sites. Type I and type II receptor sites bind TGF-Bs 1, 2, and 3. In some cells, unequal binding may occur, although this inequality does not necessarily result in differences in phenotypic expression. Type III binding sites also complex with TGF-Bs 1, 2, and 3, though to a lesser degree than with types I and II. Cellular effects after binding to type III receptors are minimally altered compared to the transduction of molecular signals associated with type I and type II receptors, and it has been suggested that the type III receptor acts as a pericellular site to locate the TGF-Ds of related molecules to the surface of cells for storage or subsequent activation. Type IV sites are poorly characterized. Osteoblast-enriched cultures contain all four sites, but it appears that although TGF-Bs 1, 2, and 3 do bind at type IV sites, the more distant members of theTGF-B family preferentially bind to these receptors.
TGF-B is a multifunctional cytokine with known effects on many cell types and, hence, on many tissues including epithelial cells, mesenchymal-epithelial cell interactions, cardiac myocytes, neutrophils, and astrocytes. For an overview of these functions the readers are referred to Sporn and Roberts. TGF-B effects upon bone cells are numerous and predominantly anabolic in nature. These effects appear to be caused by interaction at the transcriptional level, although TGF-B has not been shown to directly stimulate type I collagen mRNA production.
Studies using osteoblast-enriched cultures suggest that TGF-B has strong mitogenic effects, especially at concentrations below 100 picoMolar (pM). At higher concentrations, the mitogenic effects diminish somewhat but other effects are enhanced. Type I collagen polypeptide synthesis and type I collagen mRNA production is increased, and alkaline phosphatase activity is decreased. In the fetal rat calvariae model, TGF-B causes DNA and collagen synthesis to increase in a similar manner to that observed with osteoblast-enriched systems. In bone explant culture medium, TGF-B levels increase in the presence of other agents, notably parathyroid hormone (PTH, interleukin-1, and 1,25-dihydroxyvitamin D3. These effects are likely due to release of TGF-B from extracellular matrix, since the same increase in TGF-B levels is not observed in osteoblast-enriched cultures exposed to PTH. This extracellular release of TGF-8 demonstrates one potential avenue for the systemic influence on bone metabolism. In light of present knowledge, one would expect that surgical wound healing would be improved by the application of TGF-B and, indeed, Amento et al. have demonstrated that a single systemic dose of TGF-B 1 does enhance wound healing. In addition, Beck et al. described how a single topical administration of TGF-B, less than 1ug led to complete closure of a skull defect that did not heal without TGF-B treatment.These preliminary in vivo data show promise for the use of TGF-B to accelerate bone wound healing following implant placement.
The cascade of endochondral bone formation has been stated by Ripamonti and Reddi to include "activation and migration of undifferentiated mesenchymal cells by chemotaxis; anchorage dependent cell attachment to the matrix vie fibronectin; mitosis and proliferation of mesenchymal cells; differentiation of cartilage; mineralization of cartilage; vascular invasion and chondrolysis; differentiation of osteoblasts and deposition of bone matrix; and mineralization of bone and differentiation of hemopoietic marrow in the newly developed ossicle." Demineralized bone matrix (DBM) has been shown to induce cartilage formation at ectopic sites. Under certain controlled conditions, DBM could consistently cause osteoinduction, apparently by a diffusible substance. This factor was termed bone morphogenetic protein (BMP) by Urist and Strates, and there have been many attempts to isolate and purify BMP. Treatment of DBM with bacterial collagenase or non-polar ethylene glycol permitted solubilization of BMP. Wang et al. obtained highly purified BMP using guanidine HCl extracts of demineralized bovine bone, and production of recombinant human BMP is now possible. Seven BMP polypeptides have been identified and they are members of a family of growth factors and differentiation polypeptides that includes activins A and B, inhibins, TGF-B, Drosophila decapentaplegic gene complex, Mullerian inhibiting substance, and growth and differentiation factor-1. The seven BMPs can be divided into four groups based upon amino acid sequence analysis:
The mechanism of action of BMP on the endochondral bone formation cascade is still unclear. However, certain properties and effects of BMP have been identified that may explain its osteoinductive ability. Perhaps the most striking effect of BMP is its ability to induce differentiation of mesenchymal osteoblastic and chondrocytic osteoprogenitor cells. The other growth factors previously discussed either enhance mitogenesis or affect the cellular function of differentiated cells-they do not influence the process of cellular differentiation.
Vascular invasion and angiogenesis are critical factors in fracture healing and bone regeneration. Research into the role of the extracellular matrix and basement membrane components suggests that BMP-3/osteogenin binds to type I and type IX collagen. Ripamonti and Reddi have proposed that "type IV collagen and other matrix components around the endothelial cells of the invading capillaries may bind growth factors, and present them locally in an immobilized form to responding mesenchymal cells and osteoprogenitor cells to initiate osteogenesis." Paralkar et al. continue the hypothesis by suggesting that type IV collagen functions as a carrier or delivery system by binding to both initiators and promoters of endochondral bone differentiation, and releasing them by unknown mechanisms at sites of bone formation or regeneration. In addition, Vukicevic et al. has shown that osteoblastic cell lines are capable of recognizing the basement membrane components laminin and type IV collagen and undergo subsequent morphological changes. Therefore, basement membrane components also may play a modulating role in fracture repair and healing.
The dental implant literature currently offers promising but limited information regarding the use of bone growth factors. Pilot data using the beagle dog model suggests that the clinical use of PDGF-B in combination with IGF-I enhances bone regeneration around specially designed press-fit titanium implants. At seven days after fixture placement, treated implants had a greater percentage of bone fill in pert-implant spaces and a greater percentage of bone to implant contact than placebo or nontreated implants. At 21 days, only the percentage of bone fill in pert-implant spaces was still significantly increased over untreated and placebo sites.
Becker et al. tested the effects of PDGF/IGF-1 in combination with expanded polytetrafluoroethylene (ePTFE) membranes on bone formation around immediate extraction implants. PDGF/IGF-1 plus ePTFE treatment led to an approximate twofold increase in the percentage of implant surface and in the total length of the implant surface in contact with bone, compared to ePTFE treatment alone, according to histometric measurements at 18 weeks. Sites treated with PDGF-IGF-1 plus ePTFE had the highest bone density.
Presently, these two studies indicate that bone-growth factors may offer a method of influencing osseointegration. This promising preliminary data warrants further research to establish whether the gains observed at 21 days and 18 weeks are clinically pertinent in terms of ultimately establishing the necessary bone integrity to withstand functional loading, and whether such integrity can be predictably achieved in shorter time spans than currently accepted. In addition, there is a paucity of information, and hence the need for research, regarding the nature of interactions between the various growth factors. In vivo systemic effects of the local application of BGFs as described by Lynch et al. and Becker et al. are unknown. Pre-clinical studies utilizing animal model systems would permit the investigation of BGF use, their long-term effects, and the degree of predictability of success. To this end, Ripamonti has presented a promising nonhuman primate model for the study of calvarial reconstruction that may permit the investigation of the effects of BGFs. Interestingly, with this model, BMP-3/osteogenin appears to significantly enhance the wound healing/reparative process of calvarial defects.
The mechanism of action of BGFs is vital to our understanding of enhancing osseointegration, either to quicken bone formation or to increase the prognosis for osseointegration in areas of poor bone quality, and exciting research potential also exists to study methods of BGF application. Should BGFs be applied to surgically prepared fixture sites prior to fixture placement, or should implant fixture surfaces be treated with BGFs? Should a combination of the two techniques be utilized? In addition, investigation into the form of BGF carrier is necessary, i.e., how is a particular BGF to be introduced to an area of healing bone in order to best enhance osseointegration? Are different carriers necessary for different BGFs? For example, demineralized freeze-dried bone (DFDB) and ground dentin have innate osteogenic potential and perhaps are candidates for further treatment with BGFs to promote osseointegration. In addition, resorbable slow-release membranes impregnated with BGFs also may be potential carriers. The scientific community needs to investigate all these issues, and those that will undoubtedly arise, in order to provide information for the development of practical animal models. The use of BGFs as an osseointegration enhancement technique offers the dental profession an exciting and apparently promising method of improving clinically compromised situations in order to offer dental implant treatment options to patients devoid of bone of sufficient quantity or quality, and if a reduction in the time necessary for osseointegration to take place can be achieved, more patients may be willing to undergo dental implant treatment.