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Endosseous implants coated with recombinant human bone morphogenetic protein-2 (rhBMP-2) in a laboratory bench setting and air-dried induce relevant bone formation but also resident bone remodeling. Thus, the objective of this study is to evaluate the effect of implants fully or partially coated with rhBMP-2 and vacuum-dried using an industrial process on local bone formation and resident bone remodeling.Twelve male adult Hound Labrador mongrel dogs were used. Critical-size, supraalveolar, peri-implant defects received titanium porous oxide surface implants coated in their most coronal aspect with rhBMP-2 (coronal-load, six animals), or by immersion of the entire implant in a rhBMP-2 solution (soak-load, six animals) for a total of 30 μg rhBMP-2 per implant. All implants were vacuum-dried. The animals were sacrificed at 8 weeks for histometric evaluation.Clinical healing was unremarkable. Bone formation was not significantly affected by the rhBMP-2 application protocol. New bone height and area averaged (± SE) 3.2 ± 0.5 versus 3.6 ± 0.3 mm, and 2.3 ± 0.5 versus 2.6 ± 0.8 mm(2) for coronal-load and soak-load implants, respectively (P0.05). The corresponding bone density and bone-implant contact registrations averaged 46.7% ± 5.8% versus 31.6% ± 4.4%, and 28% ± 5.6% versus 36.9% ± 3.4% (P0.05). In contrast, resident bone remodeling was significantly influenced by the rhBMP-2 application protocol. Peri-implant bone density averaged 72.2% ± 2.1% for coronal-load versus 60.6% ± 4.7% for soak-load implants (P0.05); the corresponding bone-implant contact averaged 70.7% ± 6.1% versus 47.2% ± 6.0% (P0.05).Local application of rhBMP-2 and vacuum-drying using industrial process seems to be a viable technology to manufacture implants that support local bone formation and osseointegration. Coronal-load implants obviate resident bone remodeling without compromising local bone formation.

In addition to structural proteins of bone, such as type I collagen, bone cells synthesize a number of growth regulatory peptides that are also stored in the bone matrix, presumably as a consequence of local production by osteoblasts. Among the bone growth regulatory peptides found in the bone matrix are the recently described bone morphogenetic proteins (BMPs). These factors were purified from bone matrix by their capacity to stimulate ectopic bone formation, but it is not known whether they are produced by normal bone cells and influence normal bone formation. To determine whether they are expressed by normal osteoblasts during differentiation, we used the technique of prolonged primary culture of fetal rat calvarial osteoblasts. These cultures have been shown to be an informative model for studying expression of bone-related genes by cultured osteoblasts, since specific genes are expressed as the cells undergo proliferation and differentiation. We found that the bone morphogenetic proteins 1, 2, 4, and 6 are expressed by cultures of fetal rat calvarial osteoblasts before they form mineralized bone nodules and as they express alkaline phosphatase, osteocalcin, and osteopontin. This model can be used for study of regulation of expression of bone morphogenetic proteins by osteoblasts.

BACKGROUND Studies using ectopic rodent and orthotopic canine models (Type II bone) have shown that titanium porous oxide (TPO) surface implants adsorbed with recombinant human bone morphogenetic protein-2 (rhBMP-2) induce local bone formation including osseointegration. The objective of this study was to evaluate local bone formation and osseointegration at such implants placed into Type IV bone. MATERIAL AND METHODS rhBMP-2-coated implants were installed into the edentulated posterior maxilla in eight young adult Cynomolgus monkeys: four animals each received three TPO implants adsorbed with rhBMP-2 (2.0 mg/ml) and four animals each received three TPO implants adsorbed with rhBMP-2 (0.2 mg/ml). Contra-lateral jaw quadrants received three TPO implants without rhBMP-2 (control). Treatments were alternated between left and right jaw quadrants. Mucosal flaps were advanced and sutured to submerge the implants. The animals received fluorescent bone markers at weeks 2, 3, 4, and at week 16 when they were euthanized for histologic analysis. RESULTS Clinical healing was uneventful. Extensive local bone formation was observed in animals receiving implants adsorbed with rhBMP-2 (2.0 mg/ml). The newly formed bone exhibited a specific pinpoint bone-implant contact pattern regardless of rhBMP-2 concentration resulting in significant osseointegration; rhBMP-2 (2.0 mg/ml): 43% and rhBMP-2 (0.2 mg/ml): 37%. Control implants exhibited a thin layer of bone covering a relatively larger portion of the implant threads. Thus, TPO control implants bone exhibited significantly greater bone-implant contact ( approximately 75%; p

Objective: Myostatin is an endogenous negative regulator of muscle growth and a novel target for muscle diseases. We conducted a safety trial of a neutralizing antibody to myostatin, MYO-029, in adult muscular dystrophies (Becker muscular dystrophy, facioscapulohumeral dystrophy, and limb-girdle muscular dystrophy). Methods: This double-blind, placebo-controlled, multinational, randomized study included 116 subjects divided into sequential dose-escalation cohorts, each receiving MYO-029 or placebo (Cohort 1 at 1mg/kg; Cohort 2 at 3mg/kg; Cohort 3 at 10mg/kg; Cohort 4 at 30mg/kg). Safety and adverse events were assessed by reported signs and symptoms, as well as by physical examinations, laboratory results, echocardiograms, electrocardiograms, and in subjects with facioscapulohumeral dystrophy, funduscopic and audiometry examinations. Biological activity of MYO-029 was assessed through manual muscle testing, quantitative muscle testing, timed function tests, subject-reported outcomes, magnetic resonance imaging studies, dual-energy radiographic absorptiometry studies, and muscle biopsy. Results: MYO-029 had good safety and tolerability with the exception of cutaneous hypersensitivity at the 10 and 30mg/kg doses. There were no improvements noted in exploratory end points of muscle strength or function, but the study was not powered to look for efficacy. Importantly, bioactivity of MYO-029 was supported by a trend in a limited number of subjects toward increased muscle size using dual-energy radiographic absorptiometry and muscle histology. Interpretation: This trial supports the hypothesis that systemic administration of myostatin inhibitors provides an adequate safety margin for clinical studies. Further evaluation of more potent myostatin inhibitors for stimulating muscle growth in muscular dystrophy should be considered. Ann Neurol 2008;63:561–571

Background: Alveolar ridge aberrations commonly require bone augmentation procedures for optimal placement of endosseous dental implants. The objective of this study was to evaluate local bone formation following implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier with or without provisions for guided bone regeneration (GBR) as potential treatment modalities for alveolar augmentation. Methods: Surgically induced, large, mandibular alveolar ridge saddle-type defects (2 defects/jaw quadrant) in seven young adult Hound dogs were assigned to receive rhBMP-2/ACS, rhBMP-2/ACS combined with GBR (rhBMP-2/GBR), GBR, and surgery controls. The animals were euthanized at 12 weeks post-surgery when block sections of the defect sites were collected for histologic analysis. Results: Clinical complications included swelling for sites receiving rhBMP-2 and wound failure with exposure of the barrier device for sites receiving GBR (4/6) or rhBMP-2/GBR (3/7). The radiographic evaluation showed substantial bone fill for sites receiving rhBMP-2/ACS, rhBMP-2/GBR, and GBR. In particular, sites receiving rhBMP-2/GBR presented with seroma-like radiolucencies. The surgery control exhibited moderate bone fill. To evaluate the biologic potential of the specific protocols, sites exhibiting wound failure were excluded from the histometric analysis. Sites receiving rhBMP-2/ACS or rhBMP-2/GBR exhibited bone fill averaging 101%. Bone fill averaged 92% and 60%, respectively, for sites receiving GBR and surgery controls. Bone density ranged from 50% to 57% for sites receiving rhBMP-2/ACS, GBR, or surgery controls. Bone density for sites receiving rhBMP-2/GBR averaged 34% largely due to seroma formation encompassing 13% to 97% of the sites. Conclusion: rhBMP-2/ACS appears to be an effective alternative to GBR in the reconstruction of advanced alveolar ridge defects. Combining rhBMP-2/ACS with GBR appears to be of limited value due to the potential for wound failure or persistent seromas.

The objective of this study was to evaluate alveolar ridge augmentation following surgical implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) using two novel space-providing carrier technologies in the baboon (Papio anubis) model.Standardized alveolar ridge defects ( approximately 15 x 8 x 5 mm) were surgically produced in maxillary and mandibular edentulous areas in four baboons. The defect sites were implanted with rhBMP-2 (0.4 mg/mL) in a tricalcium phosphate/hydroxyapatite/ absorbable collagen sponge composite (TCP/HA/ACS) or calcium phosphate cement (alpha-BSM). Control treatments were TCP/HA/ACS and ?-BSM without rhBMP-2 and sham surgery. Stainless steel pins were placed at the mid-apical and coronal level of the defect sites to provide landmarks for clinical measurements pre- and post-implantation. Impressions were obtained pre- and postimplantation to determine changes in alveolar ridge volume. Radiographic registrations were obtained pre- and post-implantation. Block sections of the defect sites were harvested at week 16 postimplantation and processed for histometric analysis including new bone area and bone density. Statistical comparisons between treatments were made using a mixed effect generalized linear model using least squares estimation.The carrier systems without rhBMP-2 provided a modest ridge augmentation. The addition of rhBMP-2 resulted in an almost 2-fold increase in alveolar ridge width, including a greater percentage of trabecular bone and a higher bone density compared to controls (Por =0.05) without significant differences between the two rhBMP-2 protocols.TCP/HA/ACS and alphaBSM appear to be suitable carrier technologies for rhBMP-2. Alveolar augmentation procedures using either technology combined with rhBMP-2, rather than stand-alone therapies, may provide clinically relevant augmentation of alveolar ridge defects for placement of endosseous dental implants.

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to induce clinically relevant bone formation for orthopedic, craniofacial, and oral indications. It appears critical, in particular for onlay indications, that the associated carrier technology exhibits structural integrity to offset compressive forces in support of rhBMP-2-induced bone formation. The objective of this study was to evaluate a calcium phosphate (CP) cement, Ceredex™, as a candidate carrier for rhBMP-2 in a defect model with limited osteogenic potential. Materials: Bilateral, critical size, 6-mm, supra-alveolar, periodontal defects were created in six, adult, male, Hound Labrador mongrels. Three animals received rhBMP-2/Ceredex™ (rhBMP-2 at 0.20 and 0.40 mg/ml) in contralateral defect sites (implant volume/defect ∼1 ml). One defect site in each of the three remaining animals received Ceredex™ without rhBMP-2 (control). The animals were euthanized at 12 weeks postsurgery for histologic and histometric analysis. Results: Mean induced bone height exceeded 80% of the defect height for supra-alveolar periodontal defects receiving rhBMP-2/Ceredex™ without major differences between rhBMP-2 concentrations compared with approximately 40% for the control. The newly formed bone, a mixture of lamellar and woven bone in fibrovascular tissue, circumscribed relatively large portions of the residual Ceredex™ biomaterial. Inflammatory lesions were associated with limited bone formation in some sites. From a periodontal perspective, sites receiving rhBMP-2/Ceredex™ exhibited increased cementum formation compared with control, but without a functionally oriented periodontal ligament, and increased ankylosis and root resorption. Control sites exhibited early wound failure and exposure, loss of the Ceredex™ biomaterial, and limited bone formation. Conclusions: The Ceredex™ CP cement appears a potentially promising carrier technology for rhBMP-2 onlay indications. However, a slow resorption rate may prevent its wider use. This study does not support use of the rhBMP-2/Ceredex™ combination for periodontal indications.

Objectives: Subcutaneous and intramuscular implants of bone morphogenetic protein-12 (BMP-12) have been shown to induce formation of tendon and ligament tissue. BMP-12 induced a new attachment with a distinct fibrocartilaginous zone at the tendon-bone interface in the rat tendon-bone attachment model. Surgical controls showed poor healing and failure to reform the appropriate tendon–bone attachment morphologically. Application of recombinant human BMP-12 (rhBMP-12) to periodontal defects suggests that rhBMP-12 has the potential to support regeneration of the periodontal ligament (PDL). The objective of this pilot study was to evaluate this effect of rhBMP-12 in a tooth replantation model. Methods: Six, young adult, male Hound Labrador mongrel dogs were used. Maxillary and/or mandibular incisor and premolar teeth were extracted and the PDL was either left “intact” or removed by root planing. rhBMP-12 (1.0 mg/ml) or a buffer control was topically applied to teeth with “intact” PDL in contralateral jaw quadrants in each of 3 animals. The teeth were immersed in 1.0 ml of the rhBMP-12 or the buffer solution for 10 min and then replanted. The remaining three animals received rhBMP-12 (1.0 mg/ml) and the buffer control in a similar fashion applied to teeth instrumented to remove the PDL and cementum, and surface demineralized with citric acid. The animals were euthanized at 8 weeks postsurgery and block sections were collected and processed for histopathologic analysis. Results: No dramatic differences were found between teeth receiving topical rhBMP-12 and the buffer control. Application of rhBMP-12 did not have an apparent effect on new cementum and PDL formation in the tooth replantation model. Moreover, application of rhBMP-12 did not increase nor did it decrease the apparent presence and extent of ankylosis along the root surface compared to the control. Conclusions: The observations from this study do not support the use of topical rhBMP-12 to support the reestablishment of the PDL including regeneration of cementum and functionally oriented fibers, and to prevent ankylosis and root resorption following replantation of teeth.

Background: Previous studies have shown a limited potential for bone augmentation following guided bone regeneration (GBR) in horizontal alveolar defects. Surgical implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge carrier (ACS) significantly enhances bone regeneration in such defects; however, sufficient quantities of bone for implant dentistry are not routinely obtained. The objective of this study was to evaluate the potential of rhBMP-2/ACS to enhance GBR using a space-providing, macro-porous expanded polytetrafluoroethylene (ePTFE) device. Methods: Bilateral, critical size, supra-alveolar, peri-implant defects were surgically created in four Hound Labrador mongrel dogs. Two turned and one surface-etched 10-mm titanium dental implant were placed 5 mm into the surgically reduced alveolar ridge creating 5-mm supra-alveolar defects. rhBMP-2/ACS (rhBMP-2 at 0.2 mg/ml) or buffer/ACS was randomly assigned to left and right jaw quadrants in subsequent animals. The space-providing, macro-porous ePTFE device was placed to cover rhBMP-2/ACS and control constructs and dental implants. Gingival flaps were advanced for primary wound closure. The animals were euthanized at 8 weeks postsurgery for histologic and histometric analysis. Results: Bone formation was significantly enhanced in defects receiving rhBMP-2/ACS compared to control. Vertical bone gain averaged (±SD) 4.7±0.3 and 4.8±0.1 mm, and new bone area 10.3±2.0 and 8.0±2.5 mm2 at turned and surface-etched dental implants, respectively. Corresponding values for the control were 1.8±2.0 and 1.3±1.3 mm, and 1.8±1.3 and 1.2±0.6 mm2. Bone–implant contact in rhBMP-2-induced bone averaged 6.4±1.4% and 9.6±7.5% for turned and surface-etched dental implants, respectively (P=0.399). Corresponding values for the control were 14.6±19.4% and 23.7±9.7% (P=0.473). Bone–implant contact in resident bone ranged between 43% and 58% without significant differences between dental implant surfaces. Conclusions: rhBMP-2/ACS significantly enhances GBR at turned and surface-etched dental implants. The dental implant surface technology does not appear to substantially influence bone formation.

Alveolar ridge aberrations commonly compromise optimal dental implant installation. To offset any variance between an aberrant alveolar ridge and prosthetic designs, bone augmentation procedures become necessary. The objective of this study was to evaluate bone formation and osseointegration at alveolar dehiscence defects following augmentation of the defect site with recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge carrier (ACS) at dental implant installation including transmucosal positioning of the dental implant.Four adult male Cynomolgus monkeys received dental implants in contralateral extraction socket sites with surgically created 6 x 4 mm buccal dehiscence defects following elevation of mucoperiosteal flaps. Contralateral sites received rhBMP-2/ACS (rhBMP-2 at 1.5 mg/ml; 0.1 mg/defect) or served as sham-surgery controls. The flaps were adapted and sutured around the healing abutments leaving the implants in a transmucosal position. The animals were sacrificed at 16 weeks postsurgery and block sections of the implant sites were harvested and prepared for histometric analysis.One dental implant from each treatment group failed to osseointegrate. Another 3 dental implants (sham-surgery controls) failed to osseointegrate with newly-formed bone in the defect area. Thus, 7 of 8 defect sites (4/4 animals) receiving rhBMP-2/ACS compared to 4 of 8 sites (2/4 animals) receiving sham-surgery exhibited evidence of osseointegration with newly formed bone in the defect area. Mean +/- SD defect height amounted to 5.3 +/- 0.2 and 5.4 +/- 0.1 mm for the rhBMP-2/ACS and sham-surgery sites, respectively. Vertical bone gain in rhBMP-2/ACS treated defects (3.9 +/- 0.3 mm) did not differ significantly from that in the sham-surgery control (3.7 +/- 0.4 mm; P0.05; paired t-test, N = 4). There were also no significant differences noted for coronal bone-implant contact (3.0 +/- 0.6 versus 3.6 +/- 0.5 mm), and bone-implant contact within the defect site (28.5% +/- 15.1% versus 27.4% +/- 31.7%) and within resident bone (46.9% +/- 26.8% versus 47.8% +/- 39.4%) for the rhBMP-2/ACS and control sites, respectively.The observations in this study point to a substantial native osteogenic potential of the alveolar process that has previously not been explored and show that surgical reentry observations of new bone formation may not necessarily indicate that osseointegration has occurred. Bone formation in control defects was substantially greater than predicted, limiting the value of adding an osteoinductive biologic construct.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) technologies have been shown to significantly support alveolar bone formation. Biomaterial limitations, however, have restricted the biologic potential for onlay indications. The objective of this study was to evaluate regeneration of alveolar bone and periodontal attachment, and biomaterials reaction following surgical implantation of a spaceproviding, bioabsorbable, macroporous, polyglycolic acid-trimethylene carbonate (PGA-TMC) membrane combined with a rhBMP-2 construct in a discriminating onlay defect model.Routine supraalveolar periodontal defects were created at the mandibular premolar teeth in 9 beagle dogs. Contralateral jaw quadrants in subsequent animals were randomly assigned to receive the domeshaped PGA-TMC (100 to 120 μm pores) membrane with rhBMP-2 (0.2 mg/mL) in a bioresorbable hyaluronan (Hy) carrier or the PGA-TMC membrane with Hy alone (control). The gingival flaps were advanced to submerge the membranes and teeth and sutured. Animals were euthanized at 8 and 24 weeks postsurgery for histologic observations.Jaw quadrants receiving the PGA-TMC membrane alone experienced exposures at various time points throughout the study. Jaw quadrants receiving the PGA-TMC/rhBMP-2 combination remained intact, although one site experienced a late minor exposure. Newly formed alveolar bone approached and became incorporated into the macroporous PGA-TMC membrane in sites receiving rhBMP-2. The PGA-TMC biomaterial was occasionally associated with a limited inflammatory reaction. Residual PGA-TMC could not be observed at 24 weeks postsurgery. Residual Hy could not be observed at any time interval. Regeneration of alveolar bone height (means ± SD) was significantly increased in sites receiving the PGA-TMC/rhBMP 2 combination compared to control (3.8 ± 1.3 versus 0.7 ± 0.5 mm at 8 weeks and 4.6 ± 0.8 versus 2.1 ± 0.4 mm at 24 weeks; P0.05). Limited cementum regeneration was observed for PGA-TMC/rhBMP-2 and PGA-TMC control sites. Ankylosis compromised regeneration in sites receiving PGA-TMC/rhBMP-2.The bioabsorbable, space-providing, macroporous PGA-TMC membrane appears to be compatible biomaterial for bone augmentation procedures. rhBMP-2 significantly enhances alveolar bone augmentation and soft tissue healing when combined with the PGA-TMC membrane. J Periodontol 2003; 74:635-647.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable sponge (ACS) carrier is currently being evaluated as candidate therapy for periodontal regeneration. The objective of this study was to characterize, in some detail, tissue reactions following surgical implantation of rhBMP-2/ACS into periodontal defects.Four young adult, male beagle dogs with surgically induced, bilateral, critical size, supra-alveolar, mandibular premolar defects sequentially received rhBMP-2/ACS (rhBMP-2 at 0.2 mg/ml) in right and left jaw quadrants. After 4 or 8 weeks of healing, experimental teeth with surrounding tissues were harvested and processed for light and transmission electron microscopy.Surgical implantation of rhBMP-2/ACS into large supra-alveolar periodontal defects resulted in a variable tissue response without marked difference between 4- and 8-week observations. New bone, exceeding the volume of the normal alveolar process, had formed within 4 weeks. The regenerated bone tissue consisted of finely trabeculated woven bone. Marrow spaces exhibited a continuous lining of osteoblasts, osteoclasts, and resting cells. The marrow spaces contained numerous large, thin-walled vessels but were almost devoid of collagen fibrils or fibroblasts. Large voids (seromas) encountered in the newly formed bone were free of structured elements except for occasional aggregates of effete erythrocytes. A variety of tissue reactions were observed along the root surface including areas of resorption, areas of hard tissue deposition, and areas without resorptive or appositional activity. Ankylosis was a frequent observation, although areas showing characteristics of a periodontal ligament with a fine layer of acellular fiber cementum and occasional inserting Sharpey's fibers were also observed. Osteoblasts facing the root surface often appeared to be in a highly active state judged by their cuboidal shape, well-developed endoplasmic reticulum and numerous mitochondria, and the presence of an adjacent layer of preosteoblasts. Conspicuous bundles of wide collagen fibrils near the dentin surface as well as within the marrow spaces were considered to represent remnants of the ACS. These fibrils were associated with areas of mineralization as verified by examination of undecalcified specimens.rhBMP-2/ACS elicits a rapid osteoinductive process throughout the implant as well as along and onto the instrumented adjacent root surface. Lamellated trabecular bone was the predominant regenerated tissue. A typical cementum-periodontal ligament-alveolar bone relationship was a rare observation. The great variability in histological tissue response along the instrumented root surface indicates that the stimulus to hard tissue formation resided primarily in the rhBMP-2/ACS implant rather than in the root surface.

The objective of this study was to evaluate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) dose on alveolar ridge augmentation and dental implant osseointegration. Bilateral, 5 mm supraalveolar, peri-implant defects were surgically created in 6 beagle dogs. rhBMP-2 (0.05, 0.1 or 0.2 mg/ml) in an absorbable collagen sponge (ACS) carrier was molded around the fixtures and wounds were closed. Treatment variations were alternated between animals (incomplete block design). Animals were sacrificed at week 8 postsurgery. Nine of twelve jaw quadrants healed uneventfully. Two jaw quadrants exhibited wound failure by week 4 and one by week 8 postsurgery. Radiographic bone regeneration was observed in defects without wound failure from week 4 postsurgery. Radiolucent voids of variable size and shape were observed and regressed over time. In weeks 6 through 8, there was an apparent increase in bone density and trabecular structure, while bone height and volume decreased. Histometric analysis revealed limited differences in bone regeneration between experimental conditions. Bone regeneration area averaged (+/- SD) 1.0 +/- 0.5, 3.5 +/- 1.4 and 2.3 +/- 0.4 mm2 for the 0.05, 0.1 and 0.2 mg/ml dose, respectively. There were no significant differences in osseointegration. Osseointegration in newly formed bone averaged 19 +/- 4%, 18 +/- 10% and 21 +/- 6% for the 0.05, 0.1 and 0.2 mg/ml rhBMP-2 sites, respectively. Collectively, the data suggest that there are no dramatic differences in bone induction and osseointegration within the selected dose and observation interval.

The objectives of this study were to evaluate the effect of chronic prednisolone treatment on osteotomy healing in rabbits and to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) would enhance healing in the presence of chronic glucocorticoid therapy. Forty-nine skeletally mature, male rabbits were injected with either prednisolone (n = 26; 0.35 mg/kg per day, three times a week) or saline (n = 23). After a 6-week pretreatment period, bilateral ulnar osteotomies were created surgically. One osteotomy was treated with rhBMP-2 (0.2 mg/ml of rhBMP-2, 40 μg of rhBMP-2 total) delivered on an absorbable collage sponge (ACS), whereas the contralateral osteotomy remained untreated. Prednisolone or saline treatment was continued until the rabbits were killed either 6 weeks or 8 weeks after creation of the osteotomy. Osteotomy healing was evaluated by radiography, peripheral quantitative computed tomography (pQCT), torsional biomechanics, and undecalcified histology. Because we observed similar responses to both prednisolone and rhBMP-2/ACS treatment in the 6-week and 8-week cohorts, the results from these time points were combined. Serum osteocalcin and vertebral trabecular bone density were lower in the prednisolone-treated rabbits. Prednisolone treatment dramatically inhibited osteotomy healing. In the untreated ulnas, callus area and torsional strength were 25% and 55% less, respectively, in the prednisolone-treated rabbits than in the saline group (p < 0.001 for both). rhBMP-2/ACS enhanced healing in both the prednisolone- and the saline-treated groups, although the effect was larger in the prednisolone-treated rabbits. In the prednisolone-treated rabbits, callus area and torsional strength were 40% and 165% greater (p < 0.001 for both), respectively, in osteotomies treated with rhBMP-2/ACS compared with the contralateral, untreated osteotomies. Histological evaluation confirmed that osteotomy healing was inhibited by prednisolone and accelerated by rhBMP-2/ACS. In summary, a single application of rhBMP-2/ACS counteracted the inhibition of osteotomy healing caused by prednisolone exposure. These results suggest that rhBMP-2/ACS may be a useful treatment for enhancing fracture healing in patients who are undergoing chronic glucocorticoid therapy.

Temperature-sensitive polymers were prepared for in vivo delivery of recombinant human Bone Morphogenetic Protein-2 (rhBMP-2). The polymers were designed to overcome a critical limitation of current rhBMP-2 delivery systems, namely to provide a mechanism for in situ retention of the protein. The polymers were based on N-isopropylacrylamide (NiPAM). Ethyl methacrylate (EMA) and N-acryloxysuccinimide (NASI) were incorporated into the NiPAM polymer to reduce the lower critical solution temperature and to conjugate to proteins, respectively. To test capacity of polymers to retain rhBMP-2, rhBMP-2 were labeled with 125 I, formulated with the polymers and were either implanted with a collagen sponge or injected directly into an intramuscular site in rats. The results indicated that implantation with a relatively low polymer concentration (3.9 mg/mL) did not result in significant rhBMP-2 retention, but increasing the polymer concentration (28.7 mg/mL) gave a better retention with NiPAM/NASI polymers. In the injectable mode, NiPAM/NASI and NiPAM/EMA gave ∼200-fold better retention after 9 days in vivo. We conclude that synthetic, temperature-sensitive polymers can be engineered to sequester and retain osteoinductive proteins at a site of administration. Devices with an enhanced osteopotency should result by delivering rhBMP-2 with the prepared biomaterials.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) was biotinated, and the bioactivity of biotinated protein was assessed in vitro (alkaline phosphate induction in limb bud cells) and in vivo (osteoinduction in the rat ectopic assay). Amino-biotinated rhBMP-2 exhibited an increase in bioactivity whereas carboxy-biotinated rhBMP-2 did not exhibit any changes in bioactivity in vitro. Avidin inhibited the bioactivity of amino-biotinated but not carboxyl-biotinated rhBMP-2. Both amino- and carboxy-modified rhBMP-2 induced bone at an equivalent level to that of unmodified rhBMP-2 in vivo. The presence of avidin did not affect the osteoinductive activity of both types of biotinated rhBMP-2. The overall results indicated that binding to a large protein, avidin, might affect rhBMP-2 activity in vitro depending on the binding site; however, in vivo activity was unaffected by the avidin binding. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 668–672, 1999.

Objective: To determine the degree of bone resorption and stability of 3-cm, full-thickness canine mandibular defects reconstructed with recombinant human bone morphogenetic protein 2 (rhBMP-2) and a bioerodible particle carrier followed for 30 months after reconstruction. Study Design: Nine dogs, divided into three groups, underwent reconstruction of surgically created 3-cm, full-thickness defects of the body of the mandible. Methods: Mandibular reconstruction was performed via a combined intraoral and extraoral approach. Using standard plating techniques, a unilateral full-thickness, 3-cm defect was created in the body of the mandible. After stabilizing the defects with titanium reconstruction plates, test implants composed of rhBMP-2 and poly(lactide-co-glycolide) bioerodible particles were placed in the mandibular defects of six animals. Reconstruction plates were removed from test animals at 10 weeks. Three short-term test animals were sacrificed 3 months after reconstruction. Three long-term test animals were sacrificed 30 months after reconstruction to determine the degree of resorption and long-term stability of the rhBMP-2-induced bone. Control implants (carrier without rhBMP-2) were used in three animals and were sacrificed at 3 months. At 9 months, long-term animals were advanced to a solid diet. Masticatory function and body weight were monitored periodically to assess diet tolerance. Roentgenographic photodensitometry was performed on serial dental roentgenograms of the reconstructed segments to determine bone density and the degree of bone resorption over 30 months. After sacrifice, reconstructed segments were harvested and embedded in plastic for histological analysis and histomorphometry to determine the percentage of the defect replaced by mineralized bone (area density) and degree of resorption from 3 to 30 months after reconstruction. The main outcome measures were bone density and bone height determined from serial roentgenograms and percentage of the reconstructed segment replaced by mineralized bone (area density) determined from histomorphometry. Results: Control animals without rhBMP-2 showed no evidence of bone formation across the defect. Histological examination revealed good bone formation in two of three of the short-term test animals with a mean area density of 41.0%. The long-term test animals treated with rhBMP-2 demonstrated good bone formation that was comparable to that of normal host bone by 3 months. The roentgenographic photodensity measurements stabilized at 5 months without evidence of persistent bone resorption. The height of the reconstructed segment (rhBMP-2-induced bone) initially decreased, then stabilized by 11 months after reconstruction with no indication of resorption or failure. Histological examination of the long-term test animals revealed good bone formation across the mandibular defects. However, there were localized areas of thinning of the cortical bone as compared with the short-term test animals sacrificed at 3 months. Histological examination verified the loss of height of the bone in the reconstructed segments. The area density (mean) of the long-term test animals was 56.5%. Despite the decrease in height of the induced bone, there was an increase in area density of the bone over time. Conclusions: This study demonstrated that rhBMP-2 in a bioerodible particle carrier induced new host bone formation across critical-size mandibular defects. The newly formed bone successfully integrated with existing host bone creating a stable union capable of withstanding the forces of masticatory function in a canine. There was some evidence of early bone resorption (thinning of the cortical bone and decrease in height) in the rhBMP-2-induced bone. The rhBMP-2-induced bone stabilized by 11 months after reconstruction and no further resorption was noted. The percentage of area of the defect replaced by rhBMP-2-induced bone (area density) increased over 30 months. Both roentgenographic photodensitometry and histomorphometry supported the ability of rhBMP-2 to induce structurally stable bone without evidence of bone failure or persistent bone resorption.

Several different biodegradable bone graft materials are in clinical or preclinical use for the repair of bone defects in orthopedics, maxillofacial surgery, and periodontics. This study tested the hypothesis that poly-D,L-lactide-co-glycolide copolymer (PLG) can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) and that the composite has osteoinductive ability. Porous PLG rods were shredded to a particle size ranging from 250 to 850 μm. Active and inactive demineralized freeze-dried bone allografts (DFDBA) with a comparable particle size were used as positive and negative controls, respectively. PLG particles were treated with vehicle or with 5 or 20 μg rhBMP-2. DFDBA and PLG particles were placed in gelatin capsules, mixed with vehicle or rhBMP-2, and implanted at intramuscular sites in male Nu/Nu (nude) mice. Each mouse underwent bilateral implantation with implants of the same formulation, resulting in five groups of four mice per group: active DFDBA, inactive DFDBA, PLG, PLG + 5 μg rhBMP-2, and PLG + 20 μg rhBMP-2. After 56 days, the implants were recovered and processed for histology. Bone induction was assessed by use of a semiquantitative scoring system based on the amount of new bone formed in representative histological sections. Histomorphometry was also used to measure the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induced the formation of ossicles containing new bone with bone marrowlike tissue, whereas inactive DFDBA or PLG particles alone did not induce new bone. The addition of rhBMP-2 to PLG particles resulted in new bone formation that had a greater bone induction score than active DFDBA. Moreover, the histomorphometric analysis showed that the addition of rhBMP-2 to PLG particles induced the formation of a greater area of new bone and bone marrowlike tissue than active DFDBA. The resorption of the PLG particles was markedly increased with the addition of rhBMP-2, suggesting that rhBMP-2 may attract and regulate resorptive cells at the implantation site. The results of the present study indicate that PLG copolymers are good carriers for BMP and promote the induction of new bone formation. Further, the PLG copolymers with rhBMP-2 had a greater effect in inducing new bone formation and resorbing the implanted material than active DFDBA alone. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 46, 51–59, 1999.

The objective of this study was to evaluate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) concentration on regeneration of alveolar bone and cementum, and on associated root resorption and ankylosis. Contralateral, critical size, supra-alveolar, periodontal defects were surgically produced and immediately implanted with rhBMP-2 in an absorbable collagen sponge (ACS) carrier in 8, young adult, male, beagle dogs. 6 animals received rhBMP-2/ACS (rhBMP-2 at 0.05, 0.10, or 0.20 mg/mL; total construct volume/defect approximately 4.0 mL) in contralateral defects following an incomplete block design. 2 animals received rhBMP-2/ACS (rhBMP-2 at 0 and 0.10 mg/mL) in contralateral defects (controls). The animals were euthanised at 8 weeks post-surgery and block sections of the defects were collected for histologic and histometric analysis. Supra-alveolar periodontal defects receiving rhBMP-2 at 0.05, 0.10, or 0.20 mg/ml exhibited extensive alveolar regeneration comprising 86%, 96%, and 88% of the defect height, respectively. Cementum regeneration encompassed 8%, 6%, and 8% of the defect height, respectively. Root resorption was observed for all rhBMP-2 concentrations. Ankylosis was observed in almost all teeth receiving rhBMP-2. Control defects without rhBMP-2 exhibited limited, if any, evidence of alveolar bone and cementum regeneration, root resorption, or ankylosis. Within the selected rhBMP-2 concentration and observation interval, there appear to be no meaningful differences in regeneration of alveolar bone and cementum. There also appear to be no significant differences in the incidence and extent of root resorption and ankylosis, though there may be a positive correlation with rhBMP-2 concentration.

To determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) can be adsorbed onto porous ceramic hydroxyapatite (HA) and promote the integration of HA to host bone, 54 subperiosteal pockets were created on the skulls of 19 adult Pasteurella-free white rabbits. Fourteen HA implants were saturated with saline and placed in subperiosteal pockets (control), 22 HA implants were saturated with saline and placed into subperiosteal pockets after burring 1-2 mm of calvarium to expose bleeding cancellous bone, and 18 HA implants were saturated with rhBMP-2 and placed into subperiosteal pockets. The animals were sacrificed at 1 month with examination to determine implant mobility. Histology was used to determine the amount of bone growth into the implant. Of the 14 control sites, 10 implants were found to be freely mobile, five demonstrated host bone resorption, and only one exhibited bone growth into the implant. Of the 22 burred sites, eight were freely mobile and 10 demonstrated bone growth into the implant (p = 0.04). Of the 18 rhBMP-2 sites, only two were freely mobile, none demonstrated host bone resorption, and 16 exhibited bone growth into the implant (p = 0.00002). This study supports the use of porous ceramic HA as a biocompatible, osteoconductive implant material for use in craniomaxillofacial augmentation and reconstruction. It also provides evidence that rhBMP-2 enhances osseointegration, thereby fixing the implant in position against the host-bone interface. In the clinical setting, osseous fixation of the implant should aid in preventing displacement, minimizing host bone resorption, and decreasing the incidence of extrusion.

Originally identified as the active components within osteoinductive extracts derived from bone, the BMPs now are known to include a large family of proteins within the TGF-beta superfamily of growth and differentiation factors. Members of the BMP family have been determined to be key signaling molecules in embryogenesis, in species ranging from Drosophila to humans. They are involved in delivering positional information, the development of hard tissues (both bones and teeth), as well as soft tissue types. When implanted into adult animals, several of the BMPs have been shown to initiate the complex cellular process resulting in the induction of bone through both the endochondral and intramembranous bone formation pathways. Preclinical studies have shown the ability of these factors to induce bone and heal large bony defects in a variety of models relevant to clinical problems in orthopedics, as well as the oral and maxillofacial/dental areas. For example, implantation of recombinant human BMP-2 (rhBMP-2) has been shown to augment the alveolar ridge in several animal models, and when placed in a periodontal environment to restore not only new bone, but also the attachment tissues. Results from ongoing clinical studies support the ability of rhBMP-2 implants to induce physiologic bone.

The study reported describes a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) and collagen (C) to regenerate bone. Unilateral critical-sized defects (CSDs) were prepared in radii of 32 skeletally mature New Zealand white rabbits. Rabbits were divided evenly among four treatments: autograft, absorbable C (Helistat), 35 microg of rhBMP-2 combined with absorbable C (rhBMP-2/C), and untreated CSDs. The two euthanasia periods were 4 and 8 weeks. Radiographs were taken the day of surgery, every 2 weeks, and at term and the percent of radiopacity was measured. Data analysis revealed a time-dependent increase in the percent radiopacity with rhBMP-2/C. Histological examination revealed the rhBMP-2/C treatment regenerated osseous contour by 8 weeks. According to quantitative histomorphometry, the CSD and C groups had significantly less new bone than either autograft or rhBMP-2/C (p < or = 0.05). The results suggest that rhBMP-2/C could be an effective therapy to restore segmental bone defects.

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor β (TGF-β) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-β binding to its type I receptors. Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-β binding or function. BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment. In addition, BMP-2 suppressed the effects of cortisol on TGF-β activity, on TGF-β binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting. J. Cell. Biochem. 67:528–540, 1997. © 1997 Wiley-Liss, Inc.

We examined the mRNA expression of scleraxis, a non-myogenic helix-loop-helix type transcription factor in C2C12 myogenic cells. Scleraxis mRNA has been shown to be expressed in sclerotome and perichondrium of the embryos. We found that C2C12 cells express 1.2 kb scleraxis mRNA constitutively. Since BMP was reported to induce ectopic bone formation when implanted in muscle, we examined the effects of BMP on scleraxis expression. Scleraxis mRNA expression in C2C12 cells was suppressed by the treatment with BMP2. This suppression was observed at 200 ng/ml but not at the lower concentrations. BMP2 treatment suppressed scleraxis mRNA level within 24 h and lasted at least up to 48 h. Electrophoresis mobility shift assay showed that the proteins in the crude nuclear extracts prepared from C2C12 cells bound to an Scx-E-box sequence, CATGTG, which is preferentially recognized by scleraxis. This binding was competed out by 100-fold molar excess of cold Scx-E-box sequence but not by the one with mutations in the E-box. This band was supershifted by the addition of antiserum raised against scleraxis. BMP2 treatment suppressed the Scx-E binding activity in C2C12 cells. This suppression of the Scx-E-box binding activity was in parallel to the BMP2 suppression of the transcriptional activity of the Scx-E-CAT reporter gene transfected into C2C12 cells. These data indicated that although the default pathway for C2C12 cells is to differentiate into muscle cells, these cells do express non-myogenic transcription factor, scleraxis, whose expression is suppressed by BMP2. J. Cell. Biochem. 67:66–74, 1997. © 1997 Wiley-Liss, Inc.

Axial skeletons such as vertebrae, ribs, and scapulae develop from the embryonic somitic mesoderm through interactions with neural tube/notochord and skin ectoderm. Bone morphogenetic proteins (BMPs) seem to play important roles in these tissue interactions; however, the relationship between BMP signaling and the early development of axial skeletons is poorly understood. In this report, we investigated possible roles of BMP signaling in axial skeletal formation. First, we describe the expression patterns of BMP4 and type I receptors for BMP during somitogenesis in chick embryos based on whole mount in situ hybridization. Next, the effects of BMP on axial skeletal morphogenesis were investigated by implantation of BMP proteins into the dorsal mesoderm at the time of somitogenesis. Transcripts for both BMP4 ligand and its receptors are expressed in the dorsal ectoderm and mesoderm. Implantation of BMP4 and BMP2 into the dorsal regions of embryos result in subsequent anomalies of vertebrae, ribs, and scapulae. The effects of BMP implantation on the skeleton are shown to be dependent upon the somitic stage. Vertebral anomalies are restricted to the dorsolateral elements of the vertebrae and specifically observed after BMP implantation into embryonic day 2 (E2) embryos, but not E3 embryos. These results indicate that implantation of BMP into the dorsal part of embryos where endogenous BMP ligand and BMP receptors are expressed perturbs BMP signaling and causes axial skeletal malformations. The findings presented here suggest that BMP signaling may be involved in the early developmental process of the axial skeleton.

Growth factors and cytokines are currently under investigation as potential therapeutics for the site-specific regeneration of alveolar bone. Many of these factors, including TGF-beta, PDGF, IGF-I, IGF-II, and FGF influence bone growth and resorption, and as such may be useful in the regeneration process. However, these molecules have effects on many other tissue and cell types. In contrast, the bone morphogenetic proteins (BMPs) represent a unique set of differentiation factors that induce new bone formation at the site of implantation instead of changing the growth rate of pre-existing bone. Recombinant human BMP-2 (rhBMP-2), for example, has been shown to induce ectopic bone formation in an in vivo setting. Cell culture studies indicate that rhBMP-2 can cause mesenchymal precursor cells to differentiate into cartilage- and bone-forming cells. Additional animal studies have shown that rhBMP-2 is capable of replacing large (2.5 cm) defects in canine mandibles, healing a variety of long bone defects in orthopedic animal models, and repairing bony defects in animal models of bone lost due to periodontal disease. These results suggest that rhBMP-2 has broad therapeutic potential for dental and cranio/maxillofacial reconstruction.

This study evaluated bone and cementum regeneration following periodontal reconstructive surgery using recombinant human bone morphogenetic protein-2 (rhBMP-2) in six beagle dogs. Surgically created mandibular supraalveolar premolar tooth defects in contralateral jaw quadrants were randomly assigned to receive rhBMP-2 or control vehicle. Clinical defect height was prepared to 5 mm. rhBMP-2 was applied with synthetic bioerodable particles and autologous blood using 20 micrograms rhBMP-2 per 100 microliters implant volume. Flaps were advanced to submerge the teeth and sutured. The dogs were sacrificed 8 weeks postsurgery. Histometric recordings included defect height, height and area of alveolar bone regeneration, height of cementum regeneration, root resorption, and ankylosis. Group means, standard deviations, and P values are shown (Student t test; n = 6). Histometric defect height for rhBMP-2 and control defects was 3.7 +/- 0.3 and 3.9 +/- 0.4 mm, respectively (P = 0.446). Height of alveolar bone regeneration amounted to 3.5 +/- 0.6 and 0.8 +/- 0.6 mm for rhBMP-2 and control defects, respectively (P = 0.000). Corresponding values for bone area were 8.4 +/- 4.5 and 0.4 +/- 0.5 mm2, respectively (P = 0.006). Cementum regeneration was observed in all experimental defects (17/17) and in 15 out of 17 controls, averaging 1.6 +/- 0.6 and 0.4 +/- 0.3 mm for rhBMP-2 and control defects, respectively (P = 0.005). Small amounts of root resorption were seen in rhBMP-2 defects, whereas controls exhibited substantial resorption (0.2 +/- 0.1 and 1.1 +/- 0.3 mm, respectively; P = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Background. Bone morphogenetic proteins (BMP) induce ectopic bone formation in vivo and may play a role in normal bone development. In addition, bone morphogenetic activity, as measured in a bone-forming assay in immunodeficient, athymic nu/nu mice, is present in a proportion of osteosarcomas; this activity, which may be mediated by BMP, is correlated with a poor prognosis. Methods. The development of a monoclonal antibody against recombinant human BMP-2, AbH3b2/17, has allowed immunohistochemical localization of BMP in tumor tissues. Cryostat sections of osteosarcomas (21 tumor samples), chondrosarcomas (5 samples), and Ewing's sarcomas of bone (5 samples) were examined with AbH3b2/17 using the avidin-biotin-immunoperoxidase method. Results. The authors found AbH3b2/17 immunoreactivity in 12 of the 21 osteosarcoma samples (57% sensitivity) obtained from 20 patients. For one patient, samples of the primary lesion and a subsequent metastasis were tested, and only the latter showed AbH3b2/17 immunore-activity. The chondrosarcomas and Ewing's sarcomas examined showed no immunoreactivity. In antigen-positive osteosarcomas, AbH3b2/17 immunostaining was localized predominantly in the cytoplasm of tumor cells. Moreover, the proportion of AbH3b2/17-reactive cells varied among osteosarcomas with disparate histologic features. Conclusions. The authors identified a rapid and widely applicable method for detecting BMP expression in intact tissues, which may complement and enhance the bone-forming assay in nu/nu mice as a prognostic procedure in osteosarcomas.

The BMPs (bone morphogenetic proteins) are a group of related proteins originally identified by their presence in bone-inductive extracts of demineralized bone. By molecular cloning, at least six related members of this family have been identified and are called BMP-2 through BMP-7. These molecules are part of the TGF-beta superfamily, based on primary amino acid sequence homology, including the absolute conservation of seven cysteine residues between the TGF-betas and the BMPs. The BMPs can be divided into subgroups with BMP-2 and BMP-4 being 92% identical, and BMP-5, BMP-6, and BMP-7 being an average of about 90% identical. To examine the individual activities of these molecules, we are producing each BMP in a mammalian expression system. In this system, each BMP is synthesized as a precursor peptide, which is glycosylated, processed to the mature peptide, and secreted as a homodimer. These reagents have been used to demonstrate that single molecules, such as BMP-2, are capable of inducing the formation of new cartilage and bone when implanted ectopically in a rodent assay system. Whether each of the BMPs possesses the same inductive activities in an animal is the subject of ongoing research. Based on the chondrogenic and osteogenic abilities of the BMPs in the adult animal, the expression of the mRNAs for the BMPs has been examined in the development of the embryonic skeleton by in situ hybridization. These studies demonstrate that the BMP mRNAs are spatially and temporally expressed appropriately for the proteins involved in the induction and development of cartilage and bone in the embryonic limb bud.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcutaneous implants of a recombinant human form of the bone-inducing protein rhBMP-2 (recombinant human bone morphogenetic protein-2) in rats have resulted in the local induction of endochondral bone formation. To test the osteoinductive activity of rhBMP-2 in an osseous location, we created five-millimeter segmental defects in the femora of forty-five adult male Sprague-Dawley rats. Two doses of lyophilized rhBMP-2 (1.4 or 11.0 micrograms) were implanted in each defect, together with guanidine-hydrochloride extracted demineralized rat-bone matrix as a carrier, and the results were compared with those in rats that had implantation of guanidine-hydrochloride extracted demineralized rat-bone matrix only. The formation and healing of bone were determined by radiographic, histological, and mechanical analysis. Both doses of rhBMP-2 induced formation of endochondral bone in the osseous defects in a dose-related manner. Implantation of 11.0 micrograms of rhBMP-2 yielded significant (p less than 0.05) bone formation, resulting in radiographic, histological, and mechanical evidence of union. Despite new-bone formation in the defects that had received 1.4 micrograms of rhBMP-2, no instances of union were observed.