Alveolar augmentation for implant dentistry: tissue engineering with rhBMP-2

Surgical placement of dental implants is primarily governed by the prosthetic design and secondarily by the morphology and quality of the alveolar bone. Often, implant placement may be difficult, if at all possible, due to alveolar ridge aberrations. In consequence, prostheticly dictated implant positioning often entails augmentation of the alveolar ridge. We herein review questions regarding current extensively evaluated and practiced bone augmentation technology in implant dentistry. We then focus the review to preclinical and clinical studies applying recombinant human bone morphogenetic protein-2 (rhBMP-2) technology to alveolar bone and peri-implant defects.

Current technology

Benefits from the osteogenic potential of autograft bone, the treatment of choice or « gold standard » for skeletal reconstruction are restricted due to limited donor tissue resources and morbidity (^{Weikel and Habal, 1977} ; ^{Marx and Morales, 1988}) besides the possibility that bone modeling may result in undesirable alterations in tissue volume and geometry. In perspective, allogeneic or xenogeneic bone derivatives such as decalcified or undecalcified, freeze-dried, allogeneic bone preparations or xenogeneic bone mineral preparations, commonly considered osteoconductive biomaterials, appear attractive to support bone reconstruction in the craniofacial skeleton. Briefly, osteoconduction delineates a property of a biomaterial that promotes ingrowth of bone into a defect from osteogenic tissue sources and is usually attributed to the geometry, porosity, and bioreactivity of the material.

Considerable work concerning the efficacy and safety of allogeneic bone preparations has been presented (^{Glowacki, 1992} ; ^{Malinin, 1992} ; ^{Mellonig, 1996}). It has been suggested that allogeneic bone, lyophilized and decalcified, may support regeneration of alveolar bone (^{Pinholt et al., 1990} ; ^{Simion et al., 1994}). However, a rapidly increasing body of evidence questions the clinical relevance, the osteoconductive and regenerative potential of such bone preparations. In brief, histologic studies evaluating lyophilized, decalcified, allogeneic bone in a variety of orthotopic models including long bones, calvaria, and the alveolar ridge in experimental animals and clinical cases provide little, if any, evidence of a short-or long-term benefit of these materials (^{Aspenberg et al., 1988} ; ^{Pinholt et al., 1994} ; ^{Becker et al., 1994} ; ^{Caplanis et al., 1997} and ^1998a, ^b ; ^{Kim et al., 1998a}, ^b). Xenogeneic bone mineral preparations appear to incorporate into bone however their slow resorption rates have impact on the quality of the newly formed bone and ultimately their clinical relevance (^{Skoglund et al., 1997} ; ^{Berglundh and Lindhe, 1997} ; ^{Young et al., 1999} ; ^{Merkx et al., 1999}). Moreover, public perception of allogeneic or xenogeneic cadaver materials reduces their acceptance for elective procedures. Thus, potential for immunologic reactions, fear of disease transmission, and uncertain outcomes, limit the acceptance and utility of allogeneic or xenogeneic bone derivatives.

Ceramic and polymeric bone substitutes, potentially osteoconductive, have also been suggested in the reconstruction of bone. A variety of resorbable or nonresorbable biomaterials including calcium-based ceramics, bioactive glass, and synthetic polymers are commercially available for alveolar reconstruction (^{Ouhayoun, 1997}). In selection of ceramic or polymeric bone substitutes, the clinician must carefully consider mechanical and biological qualities of these materials. Early resorption of an implanted material must not significantly interfere with bone formation. Late resorption must not significantly compromise bone maintenance. It appears critical that any implanted biomaterial, whether derived from allogeneic or xenogeneic bone, or being of ceramic or polymeric origin, does not compromise bone formation by obstructing the wound space, negating or delaying the native osteogenic potential of the site, nor should its long-term residence compromise mechanical properties of bone, including load-bearing and dental implant osseointegration.

Critical work by Karring and others have explored and taken advantage of the natural potential for regeneration of alveolar bone and periodontal attachment employing passive membrane devices that separate tissues during healing (^{Karring et al., 1993} ; ^{Buser et al., 1994}). This enabling technology has been termed guided tissue regeneration or guided bone regeneration (GTR or GBR). This work represents one of the basic tenets of tissue engineering : providing and maintaining a space to allow regeneration from specific tissue sources and preventing scar formation. Following much development of biomaterials and surgical techniques, GTR/GBR is today a widely accepted regenerative modality in periodontics and implant dentistry. However, clinical application generally restricted to defects offering space providing morphology and wound stability limits a broader utility (^{Buser et al., 1994}). Moreover, compromised wound closure or early mechanical wound failure, exposing the membrane, restricts regenerative outcomes even for well-defined defects (^{Lekholm et al., 1993} ; ^{Trombelli et al., 1997} ; Jovanovic et al., 1999).

Bone morphogenetic proteins

Historical perspectives

^{Levander (1938)} made one of the earliest suggestions of the existence of bone morphogenetic proteins (BMPs). Through implanting vital bone fragments subcutaneously or intramuscularly, he demonstrated new bone formation. He further showed that the bone formation was not dependent on the periosteum nor was it dependent on cells from the bone surface or within bone. Instead, he found that cells from the mesenchymal tissue surrounding the implanted bone fragments formed the new bone. Levander therefore proposed that the implanted bone material must contain soluble stimulating agents that support new bone formation. ^{Lacroix (1945)} later confirmed these findings of Levander by showing that alcoholic extract of the rabbit long bone cartilaginous epiphyses promoted bone formation. This phenomenon was attributed to a substance or substances within cartilage termed « osteogenin ».

Urist pursued this research and in 1965 reported that samples of untreated decalcified bone matrix implanted into muscle pouches in the rabbit and the rat resulted in new cartilage and bone formation. Urist hypothesized that a bone-inducing substance, which he subsequently termed « bone morphogenetic protein », was responsible for these observations. He later showed that protein extracts could be separated from decalcified bone and that these proteins were responsible for the new bone formation (^{Urist et al., 1982} ; ^{Mizutani and Urist, 1982}).

A deciding development in BMP research was the identification of a group of osteoinductive proteins from bovine bone (^{Wang et al., 1988}). ^{Wozney et al. (1988)} cloned the first recombinant BMPs (BMP-1 through BMP-4) as well as identified their biochemical and biological characteristics, and amino acid sequences. The isolation and characterization of additional BMPs (BMP-5 through BMP-8) followed. Presently more than 20 BMPs have been characterized.

rhBMP-2 osteoinduction

Evaluation of the osteoinductive potential of a material involves the formation of bone at an ectopic site ; a site remote from existing bone. Traditionally, the osteogenic potential of BMP extracts has been assayed using intramuscular implantation in mice or subcutaneous implantation in rats (^{Urist, 1965} ; ^{Sampath and Reddi, 1981}). The BMP is combined with a carrier to contain the protein at the implanted site and define the physical room to which new bone forms. The processes that lead to bone induction by rhBMP-2 are characterized by a complex series of events. These events commence with infiltration of the rhBMP-2 implant by undifferentiated mesenchymal cells followed by their differentiation into chondroblasts. The chondroblasts undergo hypertrophy and mineralization as the cartilaginous tissue is removed. Bone formation may be observed during the cartilage removal or earlier if high concentrations of rhBMP-2 are used. This series of events suggest bone induction through endochondral ossification.

^{Wozney (1992)} reported that implantation of BMP-2 may result in simultaneous formation of cartilage and bone, suggesting that BMP-2 may have an effect at multiple stages of bone formation. rhBMP-2 induced bone formation has also been observed as early as 5 days following implantation of high doses of rhBMP-2, suggesting direct formation of bone from the mesenchyme without an intermediate cartilaginous phase (^{Li et al., 1996}). Thus, it would appear that, at sufficiently high concentrations, rhBMP-2 induces bone formation through intramembranous ossification.

Recombinant technologies have allowed evaluation of the biological activities of a number of BMPs. Studies have shown BMP-2, -4, -5, -6 and -7 to be osteoinductive, with most of them showing similar abilities of bone induction in the rat ectopic model (^{Sampath et al., 1992} ; ^{Gitelman et al., 1994}). The exception appears to be BMP-5 that is required in considerably higher concentrations to produce comparable bone formation. Although most of the BMPs have been shown to induce cartilage and/or bone formation in adult animal models, it is not a universal phenomenon among the BMPs. For example, GDF 6 and GDF 7 (BMP-12 and BMP-13) have been shown to induce formation of a dense connective tissue resembling tendon and ligament (^{Wolfman et al., 1997}). Considering the number of BMPs and the homology of their primary amino acid sequence, it would be reasonable to assume that they act synergistically in development and in postfetal life. However, although several BMPs have been isolated from bone, it remains uncertain whether combinations of BMPs may exert a synergistic pharmacologic effect.

Cellular events associated with BMP osteoinduction have been examined in vitro. These studies indicate that treatment of mesenchymal cells, as well as cell lines derived from either adult or embryonic origin, with rhBMP-2 will result in differentiation of these cells and expression of chondroblastic or osteoblastic phenotypes (^{Sampath et al., 1992} ; ^{Thies et al., 1992}). While BMPs primarily act as differentiation factors, studies have shown BMPs to display mitogenic and chemotactic properties for certain cell types. Mitogenic properties of BMPs have been demonstrated using primary cultures of calvarial cells. Chemotaxis of osteoblastic cells by BMPs may assist in bringing cells into the area where BMP has been implanted.

Production of rhBMP-2

As discussed above, BMPs are naturally occurring substances derived from bone. They are unique in that they act as factors promoting differentiation of primitive, uncommitted stem cells, such as mesenchymal cells into specific cell types such as chondroblasts and osteoblasts that ultimately support formation of bone. One inherent difficulty encountered early on in BMP research was obtaining sufficient quantities of BMP. Isolating BMPs from cadaveric bone yields only small quantities relative to bone mass (0,1 µg BMP/kg bone), whereas recombinant BMPs may be readily produced in large quantities for evaluation including pharmacologic development.

Recombinant BMPs such as rhBMP-2 are manufactured through genetic engineering. This process entails cloning of the gene that encodes for the desired protein. Isolation of the gene is undertaken by identifying the messenger ribonucleic acid (mRNA) that has been replicated during protein synthesis. Once the mRNA has been isolated it may be transcribed back to DNA through use of an enzyme known as reverse transcriptase. It is the complementary DNA (cDNA) which is then used in a cell system for the production of the protein (^{fig. 1}).

Safety of rhBMP-2

In considering the application of any biologically active agent, there is always the concern that the introduction of the agent may induce or promote neoplastic growth. The effect of rhBMP-2 has been tested extensively on various cell lines including several osteosarcomas and failed to affect cell growth. Studies using primary human tumor cells exposed to rhBMP-2 have failed to show significant reaction, rather rhBMP-2 has been shown to have no effect or to inhibit tumor cell proliferation (^{Soda et al., 1998}). These findings support rhBMP-2's role as a differentiation factor rather than a growth factor as it does not appear to promote growth of tumor cells.

Current clinical application of rhBMP-2 involves its incorporation into an absorbable collagen sponge carrier (ACS) forming a rhBMP-2/ACS implant. A series of clinical studies have examined the safety and efficacy of rhBMP-2/ACS (^{Boyne et al., 1997} ; ^{Howell et al., 1997}). Neither study reported any adverse effects following surgical implantation of rhBMP-2/ACS.

Preclinical studies of rhBMP-2

Role of preclinical models

The continuous search for effective and safe therapies for reconstruction of bone requires preclinical evaluation to estimate their biologic potential, efficacy and safety prior to clinical application and introduction. Candidate therapies should first be evaluated in well-characterized rodent « screening models » for biologic potential and safety (^{Wang et al., 1990} ; ^{Zellin et al., 1995}). Therapies thus exhibiting biologic potential and a safe record may be evaluated for clinical potential and efficacy in discriminating preclinical models recognized as « critical-size defect models » in larger animals including canines or nonhuman primates. Critical-size defects are defects that must not spontaneously regenerate following reconstructive surgery without adjunctive measures (^{Schmitz and Hollinger, 1986}). Moreover, critical-size defects must allow clinically relevant regeneration induced or supported by implanted biologics and/or devices over that in a surgical control (^{Wikesjö et al., 1999a} ). In perspective, we have developed and characterized the « critical-size supraalveolar periodontal defect model » for periodontal reconstructive therapy (^{Wikesjö et al., 1994}). This model has proven to represent a « litmus test » for candidate therapies for periodontal regeneration (^{Wikesjö et al., 1999a}). Subsequently, we have modified the supraalveolar periodontal defect model to study regeneration of alveolar bone and dental implant osseointegration and thus characterized and introduced the « critical-size supraalveolar peri-implant defect model » (^{fig. 2}) (^{Wikesjö et al., 1999a}).

Once it has been established that a candidate therapy has a record of biologic potential and safety and that the effect has clinical relevance in a discriminating large animal model, successful therapies may become subject to « clinical modeling ». Clinical type defects that may not necessarily be discriminating critical-size defects but are recognized as difficult to successfully manage are produced in large animals to further evaluate efficacy and application of a candidate therapy. Examples of clinical modeling thus far used to evaluate rhBMP-2 in the craniofacial skeleton include mandibular segmental defects (^{Toriumi et al., 1991} ; ^{Boyne, 1996}), cleft palate defects (^{Mayer et al., 1996} ; ^{Boyne et al., 1998}), tooth eruption (^{Steinberg et al., 1999}), subantral augmentation for implant placement (^{Nevins et al., 1996} ; ^{Hanisch et al., 1997a}), alveolar ridge defects (^{Cochran et al., 1999} ; Barboza et al., 1999), and peri-implantitis defects (^{Hanisch et al., 1997b}). In the following, we present studies evaluating the effect of rhBMP-2 in discriminating critical-size defect models and following clinical modeling.

rhBMP-2 carrier systems

The use of a carrier appears essential for delivery, retention, and gradual release of BMPs at a defect site. Successful carrier systems must enable vascular and cellular invasion, allowing the BMP to act as a differentiation factor. Ideally, the carrier should be reproducible, nonimmunogenic, moldable, and space providing to define the contours of the resulting bone. Moreover, it should resorb completely following the initiation of bone induction ensuring bone formation. Various biomaterials have been tested as BMP carriers. These include bovine collagen (^{Hanisch et al., 1997a}, ^b ; ^{Cochran et al., 1999} ; ^{Wikesjö et al., 1999b}), decalcified bone matrix (^{Gerhart et al., 1993} ; ^{Giannobile et al., 1998}), hydroxyapatite (^{Ripamonti et al., 1992} ; ^{Herr et al., 1993}), calcium phosphate (^{Oda et al., 1997}), tricalciumphosphate (^{Urist et al., 1987}), a hydroxyapatite-collagen composite (^{Asahina et al., 1997}), various poly (α-hydroxy acids) (^{Miyamoto et al., 1993} ; ^{Miki et al., 1994} ; ^{Sigurdsson et al., 1995} ; ^{Mayer et al., 1996} ; ^{Kinoshita et al., 1997}), and titanium (^{Kawai et al., 1993} ; ^{Wang et al., 1993} and ¹⁹⁹⁴ ; ^{Jin et al., 1994} ; ^{Herr et al., 1996}).

We have evaluated candidate carriers for rhBMP-2 in the supraalveolar periodontal defect model (^{Sigurdsson et al., 1996}). The carrier systems included allogeneic, freeze-dried, decalcified bone matrix (DBM) with autologous blood, bovine de-organified bone matrix (Bio-Oss^®, Osteohealth^®) with autologous blood, absorbable type I bovine collagen sponge (ACS, Integra Life Sciences), DL-polylactid acid granules (PLA, Drilac, THM Biomedical) with autologous blood, fifty-fifty polylactic acid-polyglycolic acid-copolymer microparticles (BEP, Genetics Institute) with 6 % carboxymethyl cellulose in aqueous glycerol. In brief, contralateral jaw defects in 6 Beagle dogs were randomly assigned to receive either rhBMP-2/DBM, rhBMP-2/ACS, rhBMP-2/Bio-Oss^®, rhBMP-2/PLA, rhBMP-2/BEP, or DBM alone (control). Block sections of the implanted sites were subject to qualitative and quantitative histologic evaluation following an 8-week healing interval.

Treatment outcome following implantation of the rhBMP-2 constructs was carrier-dependent. The Bio-Oss^® carrier exhibited acceptable clinical handling, however Bio-Oss^® particles remained mostly unresorbed at the 8-week observation interval clearly obstructing bone formation (^{fig. 3a} and ^3b). Rather bone formation was observed much like an eggshell outside the boundaries of the Bio-Oss^® implant expanding the implanted site. This carrier property is unacceptable in that first the unresorbed Bio-Oss^® matrix compromises the quality of bone and second the volume of implanted site cannot be predicted.

The two polymers PLA and BEP exhibited poor clinical handling. While the BEP carrier supported an acceptable bone quality, bone quantity was variable, probably due to the poor space-providing capacity of this biomaterial. The PLA carrier exhibited poor bone quality and quantity (^{fig. 4a} and ^4b). This implant resulted in formation of sparsely trabeculated bone undergoing aggressive resorption. An intense accumulation of foamy macrophages dominated the implanted site most likely a response to fragmentation of the PLA material undergoing biodegradation. These characteristics make these polymers undesirable carriers for BMPs and probably for their overall use to support bone formation.

The DBM carrier combination exhibited desirable clinical handling. It was easily molded and maintained its shape. However, the histologic evaluation revealed extensive bone formation again expanding the boundaries of the original implant site (^{fig. 5a} and ^5b). The quality of the newly formed bone assumed characteristics of the immediate resident bone. Thus the DBM carrier exhibited advantages over the Bio-Oss^® and the PLGA and PLA carrier systems. Its clinical use, however, may be limited due to lack of an adequate and reproducible supply, and the possible transmission of infectious agents. Finally, the ACS biomaterial also did not fulfill the requirements of an ideal carrier in this model system. Despite desirable clinical handling, it lacked the ability to adequately provide space, resulting in limited bone formation.

In summary, none of the carriers evaluated can be considered optimal in all aspects. Since demands on a carrier system may differ between indications, the search for other carrier systems continues. For example, orthopedic indications may demand biomaterials that provide early mechanical stability in addition to support of bone formation. For other indications such as those in the craniofacial complex, a plastic material may be more suitable. Clearly, candidate biomaterials that are non-resorbable or that exhibit resorption profiles that may negatively influence bone formation and/or bone maintenance thereby compromising biomechanical properties of bone including weight bearing and dental implant osseointegration should not be considered suitable ideal carriers.

Alveolar augmentation

Prostheticly driven implant dentistry is often compromised by alveolar ridge deformities. Thus, alveolar ridge augmentation must be performed prior to, or concurrent with placement of the implants. In this section we present preclinical studies evaluating effects of surgical implantation of rhBMP-2 into clinically demanding alveolar ridge defects.

^{Sigurdsson et al. (1997)} evaluated the effect of rhBMP-2 on peri-implant bone formation in the critical-size, supraalveolar peri-implant defect model in 5 Beagle dogs. Ten-mm long endosseous dental implants were inserted 5 mm into the reduced edentulous mandibular ridge leaving 5 mm of the implants in a supraalveolar position. Then rhBMP-2/ACS (rhBMP-2 at 0,4 mg/ml) or buffer/ACS were implanted in the contralateral peri-implant defects. A 16-week healing interval followed. rhBMP-2 defects exhibited significantly increased bone formation along the exposed implant surface (4,2 ± 1,0 mm) compared to the control (0,5 ± 0,3 mm). The newly formed bone exhibited osseointegration to the exposed implant surfaces, however, bone-implant contact was, as may be expected, lower compared to that in resident alveolar bone following the relatively short healing interval.

^{Cochran et al. (1999)} examined bone formation in circumferential, intrabony, peri-implant defects following implantation of rhBMP-2 with or without provisions for GBR. Intrabony defects (4 mm) were created around endosseous dental implants in the edentulous mandible of 6 Fox hounds. rhBMP-2/ACS (rhBMP-2 at 0,2 mg/ml) or buffer/ACS were placed into the defects. Half of the sites were additionally prepared for GBR using an ePTFE membrane (WL Gore and Associates). Controls included carrier-treated defects with or without provisions for GBR. Animals were sacrificed at 4 and 12 weeks postsurgery. Implantation of rhBMP-2 resulted in increased defect resolution compared to control without rhBMP-2 (47 % vs. 34 %) already at 4 weeks postsurgery. The rhBMP-2 induced bone exhibited osseointegration to the previously exposed implant surfaces.

Jovanovic et al. (1999) examined bone formation in alveolar ridge defects following surgical implantation of rhBMP-2 and GBR. Surgically created, mandibular, alveolar ridge, full thickness, 15 × 10 mm saddle-type defects (2 defects/jaw quadrant) in 7 hound dogs were randomly assigned to receive rhBMP-2/ACS, rhBMP-2/ACS combined with GBR (rhBMP-2/GBR ; rhBMP-2 at 0,2 mg/ml), or control treatments. The GBR protocol included use of ePTFE membranes. The animals were euthanized at 12 weeks postsurgery for histologic evaluation. Postsurgery complications included wound failure in 7 of 16 defects (44 %) receiving rhBMP-2/GBR or GBR. Histologic analysis revealed bone fill averaging 101 % for defects receiving rhBMP-2/ACS or rhBMP-2/GBR, and 92 % for defects receiving GBR without wound failure. Bone fill for the surgical control averaged 60 %. Trabecular bone density ranged between 50 % to 57 % for defects receiving rhBMP-2/ACS or GBR, and in the surgical control. The results suggest that surgical implantation of rhBMP-2/ACS is an effective treatment of space-providing alveolar ridge defects without the potential for wound failure observed following GBR. Combining rhBMP-2 with GBR provides no additional value (^{fig. 6a to d}).

Barboza et al. (1999) created bilateral, chronic class III, alveolar ridge defects in 4 adult Mongrel dogs following extraction of the mandibular fourth premolar teeth. Contralateral alveolar ridge defects were subsequently surgically implanted with rhBMP-2/ACS or rhBMP-2/ACS combined with granular hydroxyapatite (HA ; OsteoGraf/LD ; rhBMP-2 at 0,2 mg/ml). The animals were euthanized at 12 weeks post-implantation and block biopsies processed for histologic evaluation. Limited alveolar ridge augmentation followed implantation of rhBMP-2/ACS (0,6 ± 0,7 mm). In contrast, sites receiving rhBMP-2/ACS/HA exhibited clinically relevant ridge augmentation (5,5 ± 1,6 mm). However, defects receiving rhBMP-2/ACS/HA exhibited sparse bone trabe-culae with the HA particles mostly encapsulated by fibrous connective tissue. There was no evidence of bone metabolic activity associated with the HA particles. The results suggest that rhBMP-2/ACS alone may have limited effect in class III alveolar ridge defects likely due to compression from or transmitted thorough the muco-gingival flaps. Inclusion of HA into the rhBMP-2 construct resulted in clinically relevant augmentation of this demanding alveolar ridge defect, however, the quality of the resulting bone appears inadequate for placement of dental implants (^{fig. 7}).

^{Sigurdsson et al. (1999)} evaluated alveolar ridge augmentation and subsequent osseointegration following implantation of rhBMP-2 and staged implant placement. Bilateral, critical-size, 5-6 mm, supraalveolar ridge defects in 5 Beagle dogs were implanted with a rhBMP-2/DBM/blood construct (rhBMP-2 at 0,2 mg/ml). Non-submerged, 10-mm, dental implants (Straumann/ITI) were placed at week 8 and 16 postsurgery into the new alveolar ridge (^{fig. 8a to d}). The animals were euthanized at week 24 postsurgery. Approximately 90 % of the bone-anchoring surface of the implants were invested in rhBMP-2 induced bone. Similar levels of bone-implant contact (55 %) was observed in induced and resident bone irrespective of healing interval (8 vs. 16 weeks). There was no significant difference in bone density between induced and resident bone. This study shows that rhBMP-2 in an allogeneic, freeze-dried DBM/autologous blood carrier has substantial utility to augment clinically demanding alveolar ridge defects to allow early placement of dental implants.

The observations reviewed demonstrate that rhBMP-2 may be used to stimulate bone formation in clinically relevant supraalveolar and intrabony peri-implant defects. The observations reviewed also point to the critical significance of space provision for rhBMP-2 induced bone formation. Nonspace-providing alveolar defects, i.e. the supraalveolar defect and the class III ridge defect, require rhBMP-2 constructs providing space for alveolar augmentation. In contrast, space-providing ridge defects such as the saddle-type defect and the intrabony peri-implant defect may be used to evaluate rhBMP-2/carrier combinations with lesser biomechanical properties. The addition of GBR barrier membranes to rhBMP-2 technology does not appear to provide additional value. Notably GBR membranes introduce an increased risk for wound failure and appear to decelerate the regenerative potential of the rhBMP-2 technology (^{Linde and Hedner, 1995} ; ^{Jovanovic et al., 2000} ; ^{Cochran et al., 1999}).

Peri-implantitis defects

^{Hanisch et al. (1997b)} first demonstrated bone fill and dental implant re-osseointegration in peri-implantitis defects using clinical modeling in nonhuman primates and surgical implantation of rhBMP-2/ACS. Ligature-enhanced peri-implantitis lesions were created around hydroxyapatite-coated dental implants in the posterior mandible and maxilla over 11 months in 4 adult Rhesus monkeys. The induced peri-implantitis lesions exhibited a microbiota similar to that of advanced human peri-implantitis and a complex, circumferential, vertical-horizontal defect morphology (^{Hanisch et al., 1997c}). The defects were surgically debrided and the implant surfaces properly cleaned prior to surgical implantation of rhBMP-2/ACS (rhBMP-2 at 0,4 mg/ml) and primary wound closure. Control defects in contralateral mandibular and maxillary jaw quadrants received buffer/ACS. A histologic analysis was performed following a 16-week healing interval. Vertical bone gain was 3-fold greater in rhBMP-2-treated defects compared to control. rhBMP-2 treated defects exhibited convincing evidence of re-osseointegration. The results from this demanding non-human primate model suggest that surgical implantation of rhBMP-2 may have significant clinical utility in the reconstruction of peri-implantitis defects and of alveolar defects of lesser complexity (^{fig. 9a to c}).

Subantral augmentation

Model selection appears critical in the study of bone-inducing agents for subantral augmentation. Differences between preclinical models and the human subantral space relative to morphologic characteristics, including the proximity of sinus walls and relationship between the maxillary sinus and the alveolar ridge, may complicate translation of preclinical observations to clinical application.

We utilized nonhuman primate clinical modeling to evaluate bone formation and dental implant osseointegration in the subantral space following implantation of rhBMP-2 (^{Hanisch, 1997a}). This preclinical model was chosen since the appositional bone formation rate in the Cynomolgus monkey closely parallels that in humans (^{Simmons, 1976} ; ^{Schenk, 1991}). For the bilateral sub-antral augmentation, rhBMP-2/ACS (rhBMP-2 at 0,4 mg/ml) or buffer/ACS was surgically implanted into the subantral space in 4 animals. After 12 weeks of healing, dental implants were placed into the augmented subantral space and anteriorly to the sinus in native bone (control). Histologic analysis following another 12 weeks of healing revealed significantly greater bone height in rhBMP-2 treated sites compared to control (6,0 ± 0,3 versus 2,6 ± 0,3 mm, respectively). Bone density and bone-implant contact were similar in rhBMP-2 augmented, control sites, and native bone surrounding the augmented sinus. This study provides evidence for considerable vertical bone gain in the subantral space following surgical implantation of rhBMP-2 allowing placement and osseointegration of dental implants (^{fig. 10a} and ^10b).

In another study, ^{Nevins et al. (1996)} demonstrated that rhBMP-2 stimulated bone growth in the maxillary sinus of 6 Alpine-Saanen goats. Bilateral maxillary sinus floor elevation procedures were performed and contralateral sites received rhBMP-2/ACS containing 1,7 mg rhBMP-2 or buffer/ACS (control). Animals were euthanized at 4, 8, and 12 weeks postsurgery. Computerized tomography over 3 months revealed increasing radiopacity in sites receiving rhBMP-2 with controls exhibiting unchanged or reduced radiopacity. Significant amounts of new bone compared to control were demonstrated histologically with normal progression of the bone forming process. Clinical observations could not reveal any serious adverse reactions towards the rhBMP-2/ACS implant including toxicity and significant immunologic reaction. This pilot study suggests that rhBMP-2 may be used safely to stimulate bone formation for maxillary sinus augmentation.

Clinical studies of rhBMP-2

At present there are 2 recombinant BMP molecules being tested in clinical trials : rhBMP-2 (Genetics Institute, Sofamor-Danek Group, Yamanouchi Pharmaceutical) and rhOP-1 (Creative BioMolecules, Stryker Biotech). Initial studies aim to establish the safety of rhBMP-2 containing implants, evaluate clinical handling of these implants, and determine the most appropriate assessment techniques so that these may be applied to larger clinical trials.

Augmentation of the maxillary sinus

A recent study evaluated the use of rhBMP-2/ACS (rhBMP-2 at 0,4 mg/ml) for bone augmentation of the maxillary sinus floor ^{(Boyne et al., 1997)}. The aims of this non-randomized, open-label study in 12 patients were to evaluate the local and systemic safety of rhBMP-2, test the handling of the rhBMP-2/ACS construct, and ascertain the most appropriate method for radiographic evaluation of induced bone within the maxillary sinus.

The report from this study indicates an ease of clinical handling of the rhBMP-2/ACS construct and absence of any adverse reactions. Patients were evaluated for antibodies against rhBMP-2, bovine and human collagen. No antibodies against rhBMP-2 or human collagen were detected. One patient exhibited low-titer anti-bovine collagen antibodies. At 16 weeks postsurgery, all 12 patients exhibited evidence of a radiodense bone fill within the sinus. Following the sinus augmentation procedure, the patients had dental implants placed into the rhBMP-2 augmented sites. This allowed for core biopsies to be obtained and histologic evaluation of the implanted areas. The biopsies were taken at different times after the 16-week healing period at the time endosseous implants were placed. Histological examination revealed newly formed trabecular bone at the implanted sites. Polarized light microscopy showed areas of active bone remodeling with areas of woven and lamellar bone. Specimens collected at later time points generally exhibited more lamellar bone compared to those obtained earlier, which tended to be characterized by numerous osteoblasts and marrow elements. None of the specimens contained residual ACS.

Alveolar ridge preservation

In a second clinical study, a rhBMP-2/ACS construct was utilized to augment or preserve the alveolar ridge following tooth extraction ^{(Howell et al., 1997)}. This study was primarily undertaken to evaluate the short-term safety and technical feasibility of the rhBMP-2/ACS construct for this indication as well as to evaluate the various tools to measure osseous changes. A total of 12 patients were included, 6 received rhBMP-2/ACS into extraction sites in an attempt at alveolar ridge preservation, and 6 patients received rhBMP-2/ACS as a ridge augmentation procedure. The concentration of rhBMP-2 used was 0,4 mg/ml, however due to the variation in volume of the rhBMP-2/ACS construct between sites and patients, the total rhBMP-2 dose per patient varied. A linear dose-response relationship was found between rhBMP-2 dose and bone height response in patients receiving rhBMP-2 for ridge preservation (^{fig. 11}). The study also bore out some of the inherent difficulties in assessing radiographic changes, concluding that computerized tomography was the most reliable means of reproducibly assessing quantitative bone height, width and density. The authors concluded that the clinical use of rhBMP-2/ACS was technically feasible and safe and that it had a positive effect on bone healing in human extraction sockets. These findings are significant when considering that this relatively straightforward procedure was able to promote reliable osseous healing of extraction sockets and ultimately aid in the subsequent placement of endosseous dental implants.

These initial studies provide a foundation for subsequent clinical trials. The results on the clinical use of rhBMP-2/ACS are promising and provide valuable information on the feasibility of its use and how best to critically evaluate the biological changes evident following application of rhBMP-2/ACS.

Conclusion

The preclinical studies reviewed herein show that rhBMP-2 induces normal physiologic bone in relevant defects in the craniofacial skeleton. The newly formed bone assumes characteristics of the adjacent resident bone, and allows placement and osseointegration of dental implants. Clinical studies optimizing dose, mechanism of delivery, and conditions for stimulation of bone growth will bring about a new era in implant dentistry. Thus, the ability to predictably promote osteogenesis through the use of rhBMP-2 technology is not far from becoming a clinical reality and will without doubt have a profound effect on the way in which dentistry is practiced.

The initial version of this text by the authors has been published and it has since undergone several revisions and updating for reviews in journals and book chapters.

Demande de tirés à part

Pr Ulf M.E. WIKESJÖ, 518 Morris Avenue, Bryn Mawr, PA 19010 -USA.

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