Search

Background Synovial membrane-derived factors are implicated in arthritis-related bone changes. The route that synovial factors use to access subchondral bone and the mechanisms responsible for these bone changes remain unclear. A safety study involving intra-articular injection of bone morphogenetic protein-2 (BMP-2)/calcium phosphate matrix (CPM) or CPM addresses these issues. Methods Knee joints in 21 monkeys were injected with CPM or 1.5 or 4.5 mg/mL BMP-2/CPM and were evaluated at 1 and 8 weeks. Contralateral joints were injected with saline solution. Knee joints in 4 animals each were injected with 1.5 or 4.5 mg/mL BMP-2/CPM. Contralateral joints were injected with corresponding treatments at 8 weeks. Both joints were evaluated at 16 weeks. Harvested joints were evaluated grossly and with histomorphometry. Knee joints in 3 animals were injected with 125I-labeled BMP-2/CPM and evaluated with scintigraphy and autoradiography at 2 weeks to determine BMP-2 distribution. Results All treatments induced transient synovitis and increased capsular vascularization, observed to anastomose with metaphyseal venous sinusoids, but did not damage articular cartilage. Both treatments induced unanticipated activation of vascular-associated trabecular bone remodeling compartments (BRCs) restricted to injected knees. Bone volume increased in BMP-2/CPM-injected knees at 8 and 16 weeks. Scintigraphy demonstrated metaphyseal 125I-labeled BMP-2 localization restricted to injected knees, confirming local rather than systemic BMP-2 release. Autoradiography demonstrated that BMP-2 diffusion through articular cartilage into the metaphysis was blocked by the tidemark. The lack of marrow activation or de novo bone formation, previously reported following metaphyseal BMP-2/CPM administration, confirmed BMP-2 and synovial-derived factors were not free in the marrow. The 125I-labeled BMP-2/CPM, observed within venous sinusoids of injected knees, confirmed the potential for capsular and metaphyseal venous portal communication. Conclusions This study identifies a synovitis-induced venous portal circulation between the joint capsule and the metaphysis as an alternative to systemic circulation and local diffusion for synovial membrane-derived factors to reach subchondral bone. This study also identifies vascular-associated BRCs as a mechanism for arthritis-associated subchondral bone changes and provides additional support for their role in physiological trabecular bone remodeling and/or modeling. Clinical relevance Inhibition of synovitis and accompanying abnormal vascularization may limit bone changes associated with arthritis.

Background Supraphysiologic bone morphogenetic protein (BMP)-2 concentrations are required to induce spinal fusion. In this study, a BMP-2/BMP-6/activin A chimera (BV-265), optimized for BMP receptor binding, delivered in a recombinant human collagen:CDHA [calcium-deficient hydroxyapatite] porous composite matrix (CM) or bovine collagen:CDHA granule porous composite matrix (PCM), engineered for optimal BV-265 retention and guided tissue repair, was compared with BMP-2 delivered in a bovine absorbable collagen sponge (ACS) wrapped around a MASTERGRAFT Matrix (MM) ceramic-collagen rod (ACS:MM) in a nonhuman primate noninstrumented posterolateral fusion (PLF) model. Methods In vivo retention of 125I-labeled-BV-265/CM or PCM was compared with 125I-labeled-BMP-2/ACS or BMP-2/buffer in a rat muscle pouch model using scintigraphy. Noninstrumented PLF was performed by implanting CM, BV-265/CM, BV-265/PCM, or BMP-2/ACS:MM across L3-L4 and L5-L6 or L3-L4-L5 decorticated transverse processes in 26 monkeys. Computed tomography (CT) images were acquired at 0, 4, 8, 12, and 24 weeks after surgery, where applicable. Manual palpation, μCT (microcomputed tomography) or nCT (nanocomputed tomography), and histological analysis were performed following euthanasia. Results Retention of 125I-labeled-BV-265/CM was greater than BV-265/PCM, followed by BMP-2/ACS and BMP-2/buffer. The CM, 0.43 mg/cm3 BMP-2/ACS:MM, and 0.05 mg/cm3 BV-265/CM failed to generate PLFs. The 0.15-mg/cm3 BV-265/CM or 0.075-mg/cm3 BV-265/PCM combinations were partially effective. The 0.25-mg/cm3 BV-265/CM and 0.15 and 0.3-mg/cm3 BV-265/PCM combinations generated successful 2-level PLFs at 12 and 24 weeks. Conclusions BV-265/CM or PCM can induce fusion in a challenging nonhuman primate noninstrumented PLF model at substantially lower concentrations than BMP-2/ACS:MM. Clinical relevance BV-265/CM and PCM represent potential alternatives to induce PLF in humans at substantially lower concentrations than BMP-2/ACS:MM.

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.

Background: Treatment of segmental bone loss remains a challenge in skeletal repairs. This study was performed to evaluate the efficacy of the use of recombinant bone morphogenetic protein-2 (rhBMP-2) delivered in an injectable calcium phosphate cement (alpha bone substitute material [α-BSM]) to bridge critical-sized defects in the rabbit radius. Methods: Unilateral 20-mm mid-diaphyseal defects were created in the radii of thirty-six skeletally mature New Zealand White rabbits. The defects in twelve rabbits each were filled with 0.166 mg/mL rhBMP-2/α-BSM cement, 0.033 mg/mL rhBMP-2/α-BSM cement, or buffer/α-BSM cement. Six rabbits from each group were killed at four weeks, and six were killed at eight weeks. Serial radiographs were made to monitor defect-bridging and residual α-BSM carrier. A semiquantitative histological scoring system was used to evaluate defect-bridging. Histomorphometry was used to quantify residual α-BSM; trabecular bone area; trabecular bone volume fraction; and cortical length, width, and area. Results: At four weeks, there had been more rapid resorption of α-BSM and filling of the defects with trabecular bone in the group treated with 0.166 mg/mL rhBMP-2/α-BSM than in the other two groups. Histomorphometry confirmed an increased trabecular area and volume fraction in this group compared with the other two groups. In both rhBMP-2/α-BSM-treated groups, the majority of the trabecular bone was formed by a direct process adjacent to the resorbing α-BSM. At eight weeks, complete cortical bridging and regeneration of the marrow space were present in all of the defects treated with 0.166 mg/mL rhBMP-2/α-BSM. That group also had reduced residual α-BSM and trabecular area and volume, compared with the other two groups, at eight weeks as a result of a rapid remodeling process. Conclusions: Treatment of a critical-sized defect in a rabbit radius with 0.166 mg/mL rhBMP-2/α-BSM injectable cement can result in bridging with cortical bone and a regenerated bone-marrow space by eight weeks. Site-specific remodeling appears to be responsible for corticalization and marrow regeneration. Clinical Relevance: RhBMP-2 delivered in a calcium phosphate cement may be useful to achieve bridging of critical-sized defects in patients. Its injectable properties may allow minimally invasive use. Delayed percutaneous administration would also be possible when augmentation is desired following an initial surgical procedure or when soft-tissue injuries preclude adequate initial treatment.

Objectives The potential of recombinant human bone morphogenetic protein-2 (rhBMP-2) to enhance bone healing following endodontic surgery was tested. The pattern and timing of de novo bone formation and cementum regeneration, and the potential for root resorption and ankylosis to accompany bone formation were evaluated. Study design Pulpal infections were induced in maxillary and mandibular incisor teeth in young adult Cynomolgus monkeys. The teeth received conventional endodontic treatment immediately followed by surgical root resection. In a randomized split-mouth design, contralateral apical bone defects received rhBMP-2 in absorbable collagen sponge (ACS) carrier or served as sham-surgery controls to provide histological and radiographic evaluations following 1 (mandibular incisors) and 4.5 (maxillary incisors) month(s) postsurgery. Results At 1 month postsurgery trabecular bone filled the apical bone defects. The newly formed bone appeared considerably more mature and had assumed characteristics of the contiguous resident bone at 4.5 months postsurgery. The resected root tips were almost completely covered by new cementum with a maturing functionally oriented periodontal ligament. Localized inflammatory infiltrates were associated with the filled root canals and extruded root-filling material. Root resorption and ankylosis were not observed. There were no apparent differences in healing patterns between sites implanted with rhBMP-2/ACS and those serving as sham-surgery controls. Conclusions Under conditions where the influence of infectious elements and irritation caused by root filling material are minimized, bone formation and cementum regeneration appears rapid following endodontic surgery. rhBMP-2/ACS did not offer an obvious benefit above and beyond that of the native osteogenic potential in this animal model.

Two major isoforms of the Runx2 gene are expressed by alternative promoter usage: Runx2 type I (Runx2-I) is derived from the proximal promoter (P2), and Runx2 type II (Runx2-II) is produced by the distal promoter (P1). Our previous results indicate that Dlx5 mediates BMP-2-induced Runx2 expression and osteoblast differentiation (Lee, M.-H., Kim, Y-J., Kim, H-J., Park, H-D., Kang, A-R., Kyung, H.-M., Sung, J-H., Wozney, J. M., Kim, H-J., and Ryoo, H-M. (2003) J. Biol. Chem. 278, 34387-34394). However, little is known of the molecular mechanisms by which Dlx5 up-regulates Runx2 expression in BMP-2 signaling. Here, Runx2-II expression was found to be specifically stimulated by BMP-2 treatment or by Dlx5 overexpression. In addition, BMP-2, Dlx5, and Runx2-II were found to be expressed in osteogenic fronts and parietal bones of the developing cranial vault and Runx2-I and Msx2 in the sutural mesenchyme. Furthermore, Runx2 P1 promoter activity was strongly stimulated by Dlx5 overexpression, whereas Runx2 P2 promoter activity was not. Runx2 P1 promoter deletion analysis indicated that the Dlx5-specific response is due to sequences between -756 and -342 bp of the P1 promoter, where three Dlx5-response elements are located. Dlx5 responsiveness to these elements was confirmed by gel mobility shift assay and site-directed mutagenesis. Moreover, Msx2 specifically suppressed the Runx2 P1 promoter, and the responsible region overlaps with that recognized by Dlx5. In summary, Dlx5 specifically transactivates the Runx2 P1 promoter, and its action on the P1 promoter is antagonized by Msx2.

Carriers for bone morphogenetic proteins (BMPs) are used to increase retention of these factors at orthopedic treatment sites for a sufficient period of time to allow regenerative tissue forming cells to migrate to the area of injury and to proliferate and differentiate. Carriers can also serve as a matrix for cell infiltration while maintaining the volume in which repair tissue can form. Carriers have to be biocompatible and are often required to be bioresorbable. Carriers also have to be easily, and cost-effectively, manufactured for large-scale production, conveniently sterilized and have appropriate storage requirements and stability. All of these processes have to be approvable by regulatory agencies. The four major categories of BMP carrier materials include natural polymers, inorganic materials, synthetic polymers, composites of these materials. Autograft or allograft carriers have also used. Carrier configurations range from simple depot delivery systems to more complex systems mimicking the extracellular matrix structure and function. Bone regenerative carriers include depot delivery systems for fracture repair, three-dimensional polymer or ceramic composites for segmental repairs and spine fusion and metal or metal/ceramic composites for augmenting implant integration. Tendon/ligament regenerative carriers range from depot delivery systems to three-dimensional carriers that are either randomly oriented or linearly oriented to improve regenerative tissue alignment. Cartilage regenerative systems generally require three-dimensional matrices and often incorporate cells in addition to factors to augment the repair. Alternative BMP delivery systems include viral vectors, genetically altered cells, conjugated factors and small molecules.

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.

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial.

Background: In recent clinical trials demonstrating the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2) for the acceleration of bone-healing, investigators used carriers requiring open surgery for administration. In this study, we used a nonhuman primate fibular osteotomy model to evaluate injectable rhBMP-2/carrier formulations that can be administered in closed fractures. Methods: The fibular osteotomy model was first characterized by evaluating surgically harvested fibular segments containing untreated osteotomy sites (controls) from seventy adult male Cynomolgus monkeys at eight weeks (twenty-four monkeys), ten weeks (thirty-four), twelve weeks (six), and fourteen weeks (six). Fibular segments, from twenty-four animals, in which an osteotomy had not been performed served as normal controls (intact). The contralateral limb of twenty-four of the animals was then used to evaluate the effect of rhBMP-2 administered, three hours after the osteotomy, in eight carrier formulations (buffer, calcium phosphate paste, and hyaluronan gel, hyaluronan paste, and gelatin foam formulated with and without tricalcium phosphate granules). Each carrier was used in three monkeys. At ten weeks, the fibulae with the treated osteotomy sites were harvested and were compared with the contralateral, untreated osteotomized fibulae (paired control). The most promising carrier, calcium phosphate paste (alpha bone substitute material, or α-BSM), was then evaluated in eleven additional animals. The outcomes included the findings on radiographs made weekly until the time of fibular harvest, the callus area, the biomechanical properties, and the histologic findings. Results: Radiographic and histologic studies confirmed complete bridging of the control osteotomy sites in most animals by fourteen weeks. The mean torsional stiffness and maximum torque of the control osteotomy sites were 42.7% and 53.7%, 55.2% and 60.4%, 66.7% and 66.4% of the mean torsional stiffness and maximum torque of the intact fibulae at eight, ten, and twelve weeks, respectively, but they were not substantially different from the mean torsional stiffness and maximum torque of the intact fibulae at fourteen weeks (82.3% and 79.8%). In the carrier screening study, outcome measures of healing were more consistently enhanced in the rhBMP-2/α-BSM-treated osteotomy sites. In the confirmatory study, the mean callus area, torsional stiffness, and maximum torque were 86%, 72%, and 68% greater in the rhBMP-2/α-BSM-treated osteotomy sites than in the paired-control osteotomy sites at ten weeks (p < 0.001). The torsional stiffness and maximum torque in the rhBMP-2/α-BSM-treated osteotomy sites were equal to those in the intact fibulae, whereas those parameters in the paired-control osteotomy sites were only 55% and 58%, respectively, of the torsional stiffness and maximum torque of the intact fibulae. Histologic analysis confirmed complete osseous bridging of the rhBMP-2/α-BSM-treated osteotomy sites but incomplete bridging of the paired-control osteotomy sites at ten weeks. Conclusions: A single percutaneous injection of rhBMP-2/α-BSM accelerates the healing of fibular osteotomy sites in nonhuman primates by approximately 40% compared with the healing of untreated osteotomy sites. Clinical Relevance: These results provide a rationale for evaluating rhBMP-2/α-BSM as a means with which to accelerate healing in humans with closed fractures.

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.

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2), surgically implanted with a matrix material, has been shown to induce bone formation and enhance fracture repair. The purpose of this investigation was to test the hypothesis that a single, local, percutaneous injection of rhBMP-2 would accelerate fracture-healing in a standard rat femoral fracture model. Methods: Fractures were created, following intramedullary pinning, in the femora of 144 male Sprague-Dawley rats. The animals were divided into three groups of forty-eight each. Six hours after the fracture, one group received an injection of 80 μg of rhBMP-2 in 25 μL of buffer vehicle, one received an injection of 25 μL of buffer vehicle alone, and one did not receive an injection. Twelve animals from each of these three groups were killed at one, two, three, and four weeks after treatment, and the femora were harvested for torsional biomechanical testing. An additional cohort of seventy-two animals, in which a fracture was also created, was divided into the same three treatment groups; six animals from each of these groups was killed at one, two, three, and four weeks; and the femora were processed for qualitative histological analysis. Results: Torsional biomechanical testing indicated that the stiffness of the rhBMP-2-treated fractures was twice that of both control groups at the two, three, and four-week time-points. The strength of the rhBMP-2-treated fractures was 34% greater than that of the buffer-treated controls (p = 0.03) at three weeks and, at four weeks, was 60% and 77% greater than that of the buffer-treated controls and that of the untreated controls, respectively (p < 0.005). At four weeks, the stiffness and strength of the rhBMP-2-treated fractures were equal to those of the intact contralateral femora, whereas the buffer-treated and untreated fractures were significantly weaker than the intact femora. At two and three weeks, large areas of bone formation, typically spanning the fracture, were observed histologically in the rhBMP-2-treated sites. In contrast, the control fractures exhibited primarily soft cartilaginous callus at these time-points. By four weeks, remodeling of the hard callus and recorticalization were observed in the rhBMP-2-treated fracture sites, whereas cartilage and/or soft tissue was still present in the control fracture sites. Conclusions: These data demonstrate that a single, local, percutaneous injection of rhBMP-2 accelerates fracture repair in this rat femoral fracture model. This effect appears to result from a combination of the induction of bone formation at the fracture site and acceleration of the rate at which the fracture callus matures. Clinical Relevance: The ability of an injection of rhBMP-2 to accelerate fracture repair provides a rationale for its use in fractures that do not require operative treatment or that are to undergo operative treatment without direct exposure of the fracture site. In addition, the finding that an injectable compound may produce a bone-graft-like effect suggests the need for additional studies to explore its application to other types of skeletal surgery.

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) delivered with an absorbable collagen sponge (ACS) has been used to both accelerate and ensure healing of open tibial fractures in human patients 1,2. Recombinant human osteogenic protein-1 (rhOP-1, rhBMP-7) delivered with type-1 bovine bone-derived collagen has also been used to treat tibial nonunions in patients 3. A major limitation of both of these biomaterials is the requirement for open surgical placement, which prevents treatment of patients with a closed fracture managed by closed reduction. This represents a substantial patient population, since closed fractures constitute the vast majority of the 6.3 million fractures seen annually in the United States 4. Development of an injectable carrier for osteogenic factors would allow treatment of closed fractures and would also overcome several other limitations of current implantable carriers. Placement of implantable products during the repair of open long-bone fractures is generally limited to areas of exposed bone caused by the injury, which often are not ideal environments for healing. For example, the anteromedial surface of the bone is typically exposed in open tibial fractures, and there is minimal soft tissue for interaction with osteogenic factors placed in this location. Elevation of normal muscle attachments to create a space for more optimal placement of implantable osteogenic factors is often not done because of the risk of further compromising the already damaged blood supply to the fractured bone. In contrast, injectable formulations could be delivered to these more advantageous sites with no additional soft-tissue exposure. Implantable products are also difficult to place into the sites of metaphyseal fractures, as access to these sites is sometimes limited. The increased use of minimally invasive surgical techniques further limits the utility of implantable osteogenic factor formulations. As a result, considerable clinical benefit would be derived from the …

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.

Study design A literature review was conducted. Objectives To review the discovery of the bone morphogenetic proteins and describe the bone morphogenetic protein products that will or may be available for clinical use. Summary of background data Bone morphogenetic proteins comprise the osteoinductive component of several tissue engineering products in late-stage development as replacements for autogenous bone graft, and for bone augmentation and repair. Methods The literature on bone morphogenetic proteins was reviewed. Results Bone morphogenetic proteins were discovered originally on the basis of their presence in osteoinductive extracts of bone matrix. Molecular cloning of bone morphogenetic proteins demonstrated that they are a family of related differentiation factors, each capable of inducing the formation of new bone tissue when implanted. Two of the molecules in clinical use, recombinant human bone morphogenetic protein-2 and recombinant human bone morphogenetic protein-7 (OP-1) are produced in a biotechnology process using recombinant deoxyribonucleic acid technology that offers unlimited supply and substantial control over purity and reproducible activity. A third material, bovine bone morphogenetic protein extract, is extracted from bone, and contains a mixture of bone morphogenetic protein molecules. Each of these molecules, although osteoinductive in vivo, has different physiologic roles and biologic activities in vivo and in vitro. Successful development of a product for use in spinal fusion involves selecting the osteoinductive molecule, the amount of the bone morphogenetic protein required, and the method of delivery, as well as conducting subsequent preclinical and clinical studies to evaluate its efficacy and safety. Conclusions On the basis of the data provided in this issue of Spine, some of these bone morphogenetic protein-based products provide for revolutionary therapies in orthopedic practice.

STUDY DESIGN A review was conducted. OBJECTIVES To review the rationale for the use of carrier systems to deliver bone morphogenetic proteins to sites of orthopedic repair, and to discuss commonly used carriers. SUMMARY OF BACKGROUND DATA Carriers for bone morphogenetic protein in spine fusion are used to increase the retention of these osteogenic factors at the treatment site, and to serve as an osteoconductive matrix for bone forming cells while maintaining a space or volume in which bone formation can occur. METHODS The literature is reviewed and discussed. RESULTS Although bone morphogenetic proteins can induce bone formation when delivered in formulation buffer in small animal models, carriers often are used in larger animal models and human clinical trials to maintain the concentration of osteogenic factors at the treatment site for a sufficient period to allow bone-forming cells to migrate to the area of injury and to proliferate and differentiate. For spine fusion, carriers also are required to serve as an osteoconductive matrix for bone-forming cells while maintaining a space or volume in which bone formation can occur. Four major categories of carrier materials are used for osteogenic factor delivery: inorganic materials, synthetic polymers, natural polymers, and composites of the first three materials. In addition, allograft bone has been used to deliver osteogenic factors to the site of orthopedic repairs. The efficacy of osteogenic carrier combinations often is site specific and species specific. The requirement for supraphysiologic concentrations of osteogenic factors may be related to the ability of the delivery system to increase the retention time at the treatment site and overcome tight regulation of these factors by their inhibitors. Dose escalation in large animal models also may be related to a decrease in the number of responding cells and a slower rate of bone formation. New delivery systems being evaluated include depot delivery systems, viral vector systems, conjugated osteogenic factor delivery systems, and oral small molecule targets. CONCLUSIONS Delivery systems play an important role in the use of osteogenic factors to augment spine fusions and other orthopedic repairs.

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.

Recombinant human bone morphogenetic protein-2 is an osteoinductive protein that plays a pivotal role in bone growth and regeneration. Several hundred studies were conducted in the past 7 years in numerous animal models to establish unequivocally the efficacy, safety, mechanism of action, pharmacokinetics, and surgical handling properties of recombinant human bone morphogenetic protein-2, building a solid foundation for clinical development programs. Pilot clinical trials have shown the feasibility and safety of recombinant human bone morphogenetic protein-2 treatment, and defined the effective dose for its use in open long bone fractures and for augmentation or preservation of the alveolar bone in the dental ridge. Prospective observational clinical studies helped define clinical efficacy end points, identify significant variables, and estimate appropriate population sample size for pivotal clinical trials. Pivotal clinical trials of recombinant human bone morphogenetic protein-2 are underway in patients with open tibial shaft fractures and in patients with a deficiency of the alveolar ridge.

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.

Background: Approximately 5% to 20% of fractures have delayed or impaired healing. Therefore, it is desirable to develop new therapies to enhance fracture-healing that can be used in conjunction with traditional treatment methods. The purpose of this study was to evaluate the ability of a single application of recombinant human bone morphogenetic protein-2 to accelerate fracture-healing in a rabbit ulnar osteotomy that heals spontaneously. Methods: Bilateral mid-ulnar osteotomies (approximately 0.5 to 1.0 mm wide) were created in seventy-two skeletally mature male rabbits. The limbs were assigned to one of three groups: those treated with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2, those treated with an absorbable collagen sponge containing buffer, and those left untreated. In the first two groups, an 8 20-mm strip of absorbable collagen sponge containing either 40 g of recombinant human bone morphogenetic protein-2 or buffer only was wrapped around the osteotomy site. The rabbits were killed at two, three, four, or six weeks after surgery. In addition, twenty-four age-matched rabbits were used to provide data on the properties of intact limbs. The retention of recombinant human bone morphogenetic protein-2 at the osteotomy site was determined with scintigraphic imaging of 125I-labeled recombinant human bone morphogenetic protein-2. After the rabbits were killed, the limbs were scanned with peripheral quantitative computed tomography to assess the area and mineral content of the mineralized callus. The limbs were then tested to failure in torsion, and undecalcified specimens were evaluated histologically. Results: Gamma scintigraphy of 125I-recombinant human bone morphogenetic protein-2 showed that 73% ± 6% (mean and standard deviation) of the administered dose was initially retained at the fracture site. Approximately 37% ± 10% of the initial dose remained at the site one week after surgery, and 8% ± 7% remained after two weeks. The mineralized callus area was similar in all groups at two weeks, but it was 20% to 60% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 than in either the ulnae treated with buffer or the untreated ulnae at three, four, and six weeks (p < 0.05). Biomechanical properties were similar in all groups at two weeks, but they were at least 80% greater in the ulnae treated with recombinant human bone morphogenetic protein-2 at three and four weeks than in either the ulnae treated with buffer (p < 0.005) or the untreated ulnae (p < 0.01). By four weeks, the biomechanical properties of the ulnae treated with recombinant human bone morphogenetic protein-2 were equivalent to those of the intact ulnae, whereas the biomechanical properties of both the ulnae treated with buffer and the untreated ulnae had reached only approximately 45% of those of the intact ulnae. At six weeks, the biomechanical properties were similar in all groups and were equivalent to those of the intact ulnae. The callus geometry and biomechanical properties of the ulnae treated with buffer were equivalent to those of the untreated ulnae at all time-points. Conclusions and Clinical Relevance: These findings indicate that treatment with an absorbable collagen sponge containing recombinant human bone morphogenetic protein-2 enhances healing of a long-bone osteotomy that heals spontaneously. Specifically, osteotomies treated with recombinant human bone morphogenetic protein-2 healed 33% faster than osteotomies left untreated. The results of this study provide a rationale for testing the ability of recombinant human bone morphogenetic protein-2 to accelerate healing in patients with fractures requiring open surgical management.

When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

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.

This study examines the hypothesis that recombinant human bone morphogenetic protein-2 can enhance bone ingrowth into a tendon graft placed into a bone tunnel. We transplanted the long digital extensor tendon into a drill hole in the proximal tibia in 65 adult mongrel dogs. We applied two different doses of the bone morphogenetic protein to the tendon-bone interface in one limb using an absorbable type I collagen sponge carrier and only the collagen sponge to the contralateral (control) limb. The healed tendon-bone attachment was evaluated at serial times between 3 days and 8 weeks using radiography, histologic examination, and biomechanical testing. At all time points, histologic and radiographic examination demonstrated more extensive bone formation around the tendon with closer apposition of new bone to the tendon in the protein-treated limb than in the paired control limb. Biomechanical testing demonstrated higher tendon pull-out strength in the protein-treated side at all time points, with a statistically significant difference between the low-dose-treated side and the control side at 2 weeks. The histologic and biomechanical data suggested superior healing at the lower protein dose. This study demonstrated that bone morphogenetic protein can accelerate the healing process when a tendon graft is transplanted into a bone tunnel.

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.

Bone morphogenetic proteins (BMPs) play an important role in nephrogenesis. The biologic effect and mechanism of action of these proteins in the adult kidney has not yet been studied. We investigated the effect of BMP2, a member of these growth and differentiation factors, on mitogenic signal transduction pathways induced by platelet-derived growth factor (PDGF) in glomerular mesangial cells. PDGF is a growth and survival factor for these cells in vitro and in vivo. Incubation of mesangial cells with increasing concentrations of BMP2 inhibited PDGF-induced DNA synthesis in a dose-dependent manner with maximum inhibition at 250 ng/ml. Immune complex tyrosine kinase assay of PDGF receptor beta immunoprecipitates from lysates of mesangial cells treated with PDGF showed no inhibitory effect of BMP2 on PDGF receptor tyrosine phosphorylation. This indicates that the inhibition of DNA synthesis is likely due to postreceptor events. However, BMP2 significantly inhibited PDGF-stimulated mitogen-activated protein kinase (MAPK) activity that phosphorylates the Elk-1 transcription factor, a component of the ternary complex factor. Using a fusion protein-based reporter assay, we also show that BMP2 blocks PDGF-induced Elk-1-mediated transcription. Furthermore, we demonstrate that BMP2 inhibits PDGF-induced transcription of c-fos gene, a natural target of Elk-1 that normally forms a ternary complex that activates the serum response element of the c-fos gene. These data provide the first evidence that in mesangial cells, BMP2 signaling cross-talks with MAPK-based transcriptional events to inhibit PDGF-induced DNA synthesis. One target for this inhibition is the early response gene c-fos.

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.

Bone morphogenetic protein-2 (BMP-2), a member of transforming growth factor-beta superfamily, inhibits the terminal differentiation of C2C12 myoblasts and changes their differentiation pathway into cells expressing osteoblast phenotypes such as alkaline phosphatase (ALP) activity and osteocalcin production (Katagiri et al., 1994, J. Cell Biol. 127, 1755-1766). Two type I receptors for BMP-2 (BMPR-IA and BMPR-IB) have been cloned, but the role of the respective receptors in signal transduction is not clear. In the present study, we examined the signal transduction of BMP-2 in C2C12 cells using constitutively activated mutant BMPR-IA and BMPR-IB. C2C12 cells expressed BMPR-IA and BMPR-II mRNAs, but not BMPR-IB mRNA at detectable levels in Northern blotting. When mutated BMPR-IA and BMPR-IB were transiently transfected into C2C12 cells, both BMPR-IA and BMPR-IB similarly induced ALP activity in the absence of BMP-2. We also established subclonal cell lines of C2C12 cells by stably transfecting mutated BMPR-IB. When the mutated BMPR-IB-transfected cells were cultured in medium with low serum (differentiation medium) without BMP-2, the cells differentiated into ALP-positive mononuclear cells and not into myosin heavy chain-positive myotubes. These mutated BMPR-IB-transfected cells expressed ALP activity and osteocalcin mRNA in a time-dependent manner, but neither muscle creatine kinase nor myogenin mRNAs. These results indicate that the mutated BMP-2 type I receptors can constitutively transduce BMP-2 signals in the absence of the ligand in C2C12 cells.

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.

Bone morphogenetic proteins (BMPs) induce cartilage and bone differentiation in vivo and promote osteoblast differentiation from calvarial and marrow stromal cell preparations. Functional differences between BMP-2, -4, and -6 are not well understood. Recent investigations find that these three closely related osteoinductive proteins may exert different effects in primary rat calvarial cell cultures, suggesting the possibility of unique functions in vivo. In this study, we use a fetal rat secondary calvarial cell culture system to examine the differential effects of BMP-2, -4, and -6 on early osteoblast differentiation. These cells do not spontaneously differentiate into osteoblasts, as do cells in primary calvarial cultures, but rather require exposure to a differentiation initiator such as glucocorticoid or BMP. We determined that BMP-6 is a 2- to 2.5-fold more potent inducer of osteoblast differentiation than BMP-2 or -4. BMP-6 induced the formation of more and larger bone nodules as well as increased osteocalcin secretion. The effects of all three of these BMPs were potentiated up to 10-fold by cotreatment or pretreatment with the glucocorticoid triamcinolone (Trm). The Trm effects were synergistic with those of BMP-2 or -4, suggesting that this glucocorticoid may increase the cell responsiveness to these BMPs. Finally, BMP-6 did not require either cotreatment or pretreatment with Trm to achieve greater amounts of osteoblast differentiation than seen with BMP-2 or BMP-4 treatment, suggesting that BMP-6 may act at an earlier stage of cell differentiation.

Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation. To that end, transgenic mice were produced using a 2.9-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene. The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells. From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned. One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules. Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules. 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization. The 2T3 cells also exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy. As with freshly isolated fetal rat calvarial cells, 1,25-dihydroxyvitamin D3 inhibited alkaline phosphatase activity and alkaline phosphatase mRNA expression, but stimulated osteocalcin mRNA expression, but stimulated osteocalcin mRNA expression. rhBMP-2 also accelerated the expression of alkaline phosphatase activity and mRNA, osteocalcin mRNA, and BMP-2 mRNA in 2T3 cells along with the formation of larger and more mineralized bone nodules. The 2T3 cell exhibits autoregulation at the mRNA and transcriptional levels. The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during osteoblast differentiation. 2T3 cells are reclonable and maintain their differentiation capabilities.

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-beta gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.

Background: Osteoporosis results in a decrease in bone density, bone quality, and strength throughout the skeleton. Despite systemic therapies, the morbidity and mortality that are associated with hip fractures remain a major consequence of osteoporosis. Methods: We used fourteen chronic ovariectomized female cynomolgus monkeys in this study. Six animals received an intraosseous injection of 0.5 mL of 1.5 mg/mL recombinant human bone morphogenetic protein-2/calcium phosphate matrix (rhBMP-2/CPM) into the femoral neck of one femur, and six animals received an intraosseous injection of 0.5 mL of CPM alone into the femoral neck of one femur. The contralateral femur of each of the animals was left untreated. The proximal aspect of each femur was evaluated monthly with use of radiography and at six months with use of peripheral quantitative computed tomography, microcomputed tomography, histological analysis, and mechanical testing. Two additional animals received an intraosseous injection of 0.5 mL of 1.5 mg/mL rhBMP-2/CPM into the femoral neck of one femur. The contralateral femur of each animal was left untreated. Bone formation in the intact specimens from these animals was histologically analyzed at one month in one animal and at three months in the other. Results: Radiographic evaluation over the six-month study period demonstrated an increase in cortical thickness and density in the rhBMP-2/CPM-treated femora as compared to the findings in the untreated contralateral femora or the femora that had been treated with CPM alone. At six months, the rhBMP-2/CPM-treated femora had decreased cortical density and increased cross-sectional area, cortical thickness, trabecular density, and trabecular volume fraction as compared with the contralateral untreated femora and the femora that had received CPM treatment alone, but the differences between the femora that had been treated with CPM alone and the contralateral untreated femora did not reach significance. Increases in bone structure resulted in a 13.7% ± 7.6% (p = 0.032) increase in the maximum bending force at the femoral neck as compared with that at the femoral neck of the contralateral untreated femora. The maximum bending force at the femoral neck was similar between the femora that had been treated with CPM alone and the contralateral untreated femora. De novo and appositional bone formation was present at one month after treatment in the rhBMP-2/CPM-treated femora. Conclusions: This study demonstrates an increase in bone structure and mechanical properties at six months following a single injection of rhBMP-2/CPM into the femoral neck of chronic ovariectomized nonhuman primates. Clinical Relevance: These results provide a rationale for evaluating intraosseous injection of rhBMP-2/CPM with regard to its value in reducing the incidence of osteoporotic hip fractures.

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)