A BMP/Activin A Chimera Induces Posterolateral Spine Fusion in Nonhuman Primates at Lower Concentrations Than BMP-2.

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.

Activation of Bone Remodeling Compartments in BMP-2-Injected Knees Supports a Local Vascular Mechanism for Arthritis-Related Bone Changes.

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.

A BMP/activin A chimera is superior to native BMPs and induces bone repair in nonhuman primates when delivered in a composite matrix.

Bone morphogenetic protein (BMP)/carriers approved for orthopedic procedures achieve efficacy superior or equivalent to autograft bone. However, required supraphysiological BMP concentrations have been associated with potential local and systemic adverse events. Suboptimal BMP/receptor binding and rapid BMP release from approved carriers may contribute to these outcomes. To address these issues and improve efficacy, we engineered chimeras with increased receptor binding by substituting BMP-6 and activin A receptor binding domains into BMP-2 and optimized a carrier for chimera retention and tissue ingrowth. BV-265, a BMP-2/BMP-6/activin A chimera, demonstrated increased binding affinity to BMP receptors, including activin-like kinase-2 (ALK2) critical for bone formation in people. BV-265 increased BMP intracellular signaling, osteogenic activity, and expression of bone-related genes in murine and human cells to a greater extent than BMP-2 and was not inhibited by BMP antagonist noggin or gremlin. BV-265 induced larger ectopic bone nodules in rats compared to BMP-2 and was superior to BMP-2, BMP-2/6, and other chimeras in nonhuman primate bone repair models. A composite matrix (CM) containing calcium-deficient hydroxyapatite granules suspended in a macroporous, fenestrated, polymer mesh-reinforced recombinant human type I collagen matrix demonstrated improved BV-265 retention, minimal inflammation, and enhanced handling. BV-265/CM was efficacious in nonhuman primate bone repair models at concentrations ranging from 1/10 to 1/30 of the BMP-2/absorbable collagen sponge (ACS) concentration approved for clinical use. Initial toxicology studies were negative. These results support evaluations of BV-265/CM as an alternative to BMP-2/ACS in clinical trials for orthopedic conditions requiring augmented healing.

Intraosseous injection of rhBMP-2/calcium phosphate matrix improves bone structure and strength in the proximal aspect of the femur in chronic ovariectomized nonhuman primates.

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.

rhBMP-2/calcium phosphate matrix induces bone formation while limiting transient bone resorption in a nonhuman primate core defect model.

BACKGROUND: Transient bone resorption limits the use of recombinant human bone morphogenetic protein-2 (rhBMP-2)/absorbable collagen sponge in metaphyseal bone. The purpose of the present study was to evaluate the efficacy of rhBMP-2/calcium phosphate matrix (CPM) to induce bone formation while limiting transient bone resorption in nonhuman primate core defects. METHODS: Metaphyseal core defects were created in eighteen cynomolgus monkeys. rhBMP-2 retention was evaluated in the distal part of the radius. Bone formation was evaluated at eight weeks following treatment with 1.5 or 4.5-mg/mL rhBMP-2/CPM, CPM alone, or no treatment in the distal part of the radius, the proximal part of the tibia, and the proximal part of the femur; at twenty-four weeks following treatment with 1.5-mg/mL rhBMP-2/CPM or CPM alone in the proximal part of the tibia; and at one, two, and four weeks following treatment with 1.5-mg/mL rhBMP-2/CPM or no treatment in the distal part of the radius. Bone resorption was evaluated at four weeks following treatment with 1.5, 2.0, 3.0, and 4.5-mg/mL rhBMP-2/CPM or CPM alone in the distal part of the femur. Evaluations were performed with use of scintigraphy, radiographs, histological analysis, and computed tomography. RESULTS: Seventy-eight percent, 64%, 50%, 35%, and 12% of the rhBMP-2 was retained in the distal part of the radius at one, seven, fourteen, twenty-one, and forty-nine days after surgery. rhBMP-2/CPM increased bone formation within core defects and surrounding trabeculae compared with CPM alone or no treatment at all anatomic locations at eight weeks, and bone formation was ongoing in the rhBMP-2/CPM-treated proximal tibial sites at twenty-four weeks. Bone formation began in the trabeculae surrounding the core defects at one week and was observed adjacent to the resorbing CPM within the core defects and in the surrounding trabecular bone at two and four weeks in the rhBMP-2/CPM-treated distal radial sites. Bone formation was confined to the region immediately surrounding the core defects in the untreated distal radial sites at all time points. Transient bone resorption was only observed in the distal femoral sites treated with 4.5 mg/mL of rhBMP-2/CPM at two weeks. CONCLUSIONS: Treatment of nonhuman primate metaphyseal core defects with 1.5 to 3.0-mg/mL rhBMP-2/CPM resulted in bone formation without transient bone resorption. CLINICAL RELEVANCE: rhBMP-2/CPM may be useful to accelerate healing of metaphyseal bone defects in humans.

Evaluation of implants coated with recombinant human bone morphogenetic protein-2 and vacuum-dried using the critical-size supraalveolar peri-implant defect model in dogs.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (P >0.05). 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% (P >0.05). 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 (P <0.05); the corresponding bone-implant contact averaged 70.7% ± 6.1% versus 47.2% ± 6.0% (P <0.05). CONCLUSIONS: 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.

Evaluation of implants coated with rhBMP-2 using two different coating strategies: a critical-size supraalveolar peri-implant defect study in dogs.

BACKGROUND: Implants coated with recombinant human bone morphogenetic protein-2 (rhBMP-2) induce relevant bone formation but also resident bone remodelling. OBJECTIVES: To compare the effect of implants fully or partially coated with rhBMP-2 on new bone formation and resident bone remodelling. MATERIALS AND METHODS: 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 an rhBMP-2 solution (soak-load/six animals) for a total of 30 mug rhBMP-2/implant. All implants were air-dried. The animals were euthanized at 8 weeks for histometric evaluation. RESULTS: Clinical healing was uneventful. Supraalveolar bone formation was not significantly affected by the rhBMP-2 application protocol. New bone height and area averaged (+/- SE) 3.4 +/- 0.2 versus 3.5 +/- 0.4 mm and 2.6 +/- 0.4 versus 2.5 +/- 0.7 mm(2) for coronal-load and soak-load implants, respectively (p>0.05). The corresponding bone density and bone-implant contact (BIC) recordings averaged 38.0 +/- 3.8%versus 34.4 +/- 5.6% and 25.0 +/- 3.8%versus 31.2 +/- 3.3% (p>0.05). In contrast, resident bone remodelling was significantly influenced by the rhBMP-2 application protocol. Bone density outside the implants threads averaged 74.7 +/- 3.8% and 50.8 +/- 4.1% for coronal-load and soak-load implants, respectively (p<0.05); bone density within the thread area averaged 51.8 +/- 1.2% and 37.8 +/- 2.9%, and BIC 70.1 +/- 6.7% and 43.3 +/- 3.9% (p<0.05). CONCLUSION: Local application of rhBMP-2 appears to be a viable technology to support local bone formation and osseointegration. Coronal-load implants obviate resident bone remodelling without compromising new bone formation.

rhBMP-2 induces transient bone resorption followed by bone formation in a nonhuman primate core-defect model.

BACKGROUND: Bone resorption preceding bone formation has been reported following the administration of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in an absorbable collagen sponge (ACS) in metaphyseal bone. This study characterizes treatment with rhBMP-2/ACS in metaphyseal bone with use of a nonhuman primate core-defect model. METHODS: Unilateral proximal femoral core defects were treated with 360 microg of rhBMP-2/ACS or ACS alone or were left untreated in seven, five, and five adult male cynomolgus monkeys, respectively. Distal femoral core defects in seven of the above animals were treated with 360 microg of rhBMP-2/ACS in one limb and ACS alone in the contralateral limb. Retention of rhBMP-2 in the proximal part of the femora was determined with use of tracer amounts of (125)I-rhBMP-2 imaged with a gamma camera. The distal part of the femora was evaluated with in vivo computed tomography. Computed tomography and histological evaluation were performed on harvested segments in all animals at twenty-four weeks. The histological response in the proximal and distal parts of the femora containing core defects treated with 360 microg of rhBMP-2/ACS in one limb and ACS alone in the contralateral limb was evaluated at one, two, and four weeks in three animals per time point. RESULTS: Approximately 39.9%, 24.2%, 3.4%, and 0.5% of the rhBMP-2 was retained in the proximal part of the femora at one, seven, fourteen, and twenty-one days, respectively. The mineral density and trabecular volume fraction of the core defects treated with rhBMP-2/ACS, those treated with ACS alone, and untreated core defects in the proximal part of the femora were 81%, 54%, and 20%, respectively, and 94%, 36%, and 31%, respectively, of the corresponding region in the contralateral limbs at twenty-four weeks. The mineral density and trabecular volume fraction of the region surrounding the core defects treated with rhBMP-2/ACS, those treated with ACS alone, and untreated core defects were 112%, 105%, and 104%, respectively, and 117%, 108%, and 107%, respectively, of the corresponding region in the contralateral limbs. Treatment with rhBMP-2/ACS increased the size of the proximal and distal core defects compared with treatment with ACS alone. Histological evaluation of the rhBMP-2/ACS-treated limbs demonstrated that bone resorption was initiated at one week in association with osteoclasts and receptor activator of nuclear factor-kappaB ligand-positive stained spindle-shaped cells and peaked at two weeks. Bone formation was observed at two weeks and was ongoing at twenty-four weeks. CONCLUSIONS: Treatment of metaphyseal core defects with rhBMP-2/ACS resulted in bone resorption followed by bone formation in nonhuman primates.

Bone formation at recombinant human bone morphogenetic protein-2-coated titanium implants in the posterior mandible (Type II bone) in dogs.

BACKGROUND: Conventional oral/maxillofacial implants reach osseointegration over several months during which the titanium fixtures interact with alveolar bone. The objective of this study was to determine if adsorbing recombinant human bone morphogenetic protein-2 (rhBMP-2) onto a titanium porous oxide (TPO) implant surface might enhance or accelerate local bone formation and support osseointegration in a large animal oral/maxillofacial orthotopic model. MATERIAL AND METHODS: Endosseous implants with a TPO surface were installed into the edentulated posterior mandible in eight adult Hound Labrador mongrel dogs. The implant surface had been adsorbed with rhBMP-2 at 0.2 or 4.0 mg/ml. TPO implants without rhBMP-2 served as control. Treatments were randomized between jaw quadrants. Mucosal flaps were advanced and sutured leaving the implants submerged. Clinical and radiographic evaluations were made immediately post-surgery, at day 10 (suture removal), and week 4 and 8 post-surgery. The animals received fluorescent bone markers at week 3, 4, and at week 8 post-surgery, when they were euthanized for histologic analysis. RESULTS: TPO implants coated with rhBMP-2 exhibited dose-dependent bone remodelling including immediate resorption and formation of implant adjacent bone, and early establishment of clinically relevant osseointegration. The resulting bone-implant contact, although clinically respectable, appeared significantly lower for rhBMP-2-coated implants compared with the control [rhBMP-2 (0.2 mg/ml) 43.3+/-10.8%versus 71.7+/-7.8%, p<0.02; rhBMP-2 (4.0 mg/ml) 35.4+/-10.6%versus 68.2+/-11.0%, p<0.03]. CONCLUSIONS: rhBMP-2 adsorbed onto TPO implant surfaces initiates dose-dependent peri-implant bone re-modelling resulting in the formation of normal, physiologic bone and clinically relevant osseointegration within 8 weeks.

Bone formation at recombinant human bone morphogenetic protein-2-coated titanium implants in the posterior maxilla (Type IV bone) in non-human primates.

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<0.05). There were no statistically significant differences between rhBMP-2-coated and control implants relative to any other parameter including peri-implant and intra-thread bone density. CONCLUSION: rhBMP-2-coated TPO implants enhanced/accelerated local bone formation in Type IV bone in a dose-dependent fashion in non-human primates resulting in significant osseointegration. rhBMP-2-induced de novo bone formation did not reach the level of osseointegration observed in native resident bone within the 16-week interval.

Alveolar ridge augmentation using implants coated with recombinant human bone morphogenetic protein-2: histologic observations.

BACKGROUND: Studies using ectopic rodent, orthotopic canine, and non-human primate models show that bone morphogenetic proteins (BMPs) coated onto titanium surfaces induce local bone formation. The objective of this study was to examine the ability of recombinant human BMP-2 (rhBMP-2) coated onto a titanium porous oxide implant surface to stimulate local bone formation including osseointegration and vertical augmentation of the alveolar ridge. MATERIAL AND METHODS: Bilateral, critical-size, 5 mm, supra-alveolar, peri-implant defects were created in 12 young adult Hound Labrador mongrel dogs. Six animals received implants coated with rhBMP-2 at 0.75 or 1.5 mg/ml, and six animals received implants coated with rhBMP-2 at 3.0 mg/ml or uncoated control. Treatments were randomized between jaw quadrants. The mucoperiosteal flaps were advanced, adapted and sutured to submerge the implants for primary intention healing. The animals received fluorescent bone markers at weeks 3, 4, 7 and 8 post-surgery when they were euthanized for histologic evaluation. RESULTS: Jaw quadrants receiving implants coated with rhBMP-2 exhibited gradually regressing swelling that became hard to palpate disguising the contours of the implants. The histologic evaluation showed robust bone formation reaching or exceeding the implant platform. The newly formed bone exhibited characteristics of the adjoining resident Type II bone including cortex formation for sites receiving implants coated with rhBMP-2 at 0.75 or 1.5 mg/ml. Sites receiving implants coated with rhBMP-2 at 3.0 mg/ml exhibited more immature trabecular bone formation, seroma formation and peri-implant bone remodelling resulting in undesirable implant displacement. Control implants exhibited minimal, if any, bone formation. Thus, implants coated with rhBMP-2 at 0.75, 1.5 and 3.0 mg/ml exhibited significant bone formation (height and area) compared with the sham-surgery control averaging (+/-SD) 4.4+/-0.4, 4.2+/-0.7 and 4.2+/-1.2 versus 0.8+/-0.3 mm; and 5.0+/-2.2, 5.6+/-2.2 and 7.4+/-3.5 versus 0.7+/-0.3 mm(2), respectively (p<0.01). All the treatment groups exhibited clinically relevant osseointegration. CONCLUSIONS: rhBMP-2 coated onto titanium porous oxide implant surfaces induced clinically relevant local bone formation including vertical augmentation of the alveolar ridge and osseointegration. Higher concentrations/doses were associated with untoward effects.

rhBMP-12 accelerates healing of rotator cuff repairs in a sheep model.

BACKGROUND: The success rate of rotator cuff repairs is variable. This study was performed to evaluate the ability of recombinant human bone morphogenetic protein-12 (rhBMP-12), administered in several carriers, to accelerate healing in a sheep model of rotator cuff repair. METHODS: Local retention of tracer amounts of radiolabeled rhBMP-12, added to non-radiolabeled rhBMP-12 delivered in buffer, hyaluronan paste or sponges, or Type-I or Type-I/III collagen sponges was first evaluated with use of gamma scintigraphy in a pilot study of a rat intramuscular implant model. The rhBMP-12/paste and sponge combinations were then evaluated in eight sheep each with unilateral complete detachment and subsequent double-row reattachment of the infraspinatus tendon to the proximal part of the humerus. Contralateral, normal shoulders from sixteen sheep and shoulders in which a repair had been done without administration of rhBMP-12 in fourteen sheep were also evaluated. The rhBMP-12/Type-I and Type-I/III collagen sponge combinations were each evaluated in eight additional sheep on the basis of superior efficacy. The Type-I/III collagen sponge alone was evaluated in ten sheep to examine the effect of a collagen carrier. Ultrasound imaging was performed at four and eight weeks. Radiographic evaluation, mechanical testing, and biochemical evaluation were performed at eight weeks. Histological evaluation was performed on specimens from the sites of selected repairs following mechanical testing. RESULTS: The sponge carriers had longer local retention of rhBMP-12 than did the buffer or paste carriers in the rat models. All of the sheep shoulder-repair groups demonstrated ultrasound evidence of a gap between the tendon and the humeral insertion. The gap length and the cross-sectional area of the repair tissue decreased with time. The mechanical properties of the repairs treated with rhBMP-12 and hyaluronan paste were similar to those of the untreated repairs. The maximum loads for the rhBMP-12/hyaluronan sponge and rhBMP-12/collagen sponge-treated repairs were 2.1 and 2.7 times greater, respectively, than the loads for the untreated repairs and were 33% and 42% of the value for the normal tendon at eight weeks. The maximum loads for the repairs treated with rhBMP-12 and a Type-I or Type-I/III collagen sponge were 2.1 times greater than those for the repairs treated with the Type-I/III collagen sponge alone. Changes in maximum stiffness followed a similar pattern. Histological evaluation demonstrated accelerated healing of the rhBMP-12-treated repairs compared with the untreated repairs. Bone formation was observed in all repairs, and biochemical measurements were not equivalent to those of normal tendon at eight weeks. CONCLUSIONS: Delivery of rhBMP-12 in a collagen or hyaluronan sponge resulted in accelerated healing of acute full-thickness rotator cuff repairs in a sheep model. CLINICAL RELEVANCE: Delivery of rhBMP-12 in several sponge carriers has the potential to accelerate healing of rotator cuff repairs. Accelerated repair may allow shorter rehabilitation and an earlier return to occupational and recreational activities.

A phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy.

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 1 mg/kg; Cohort 2 at 3 mg/kg; Cohort 3 at 10 mg/kg; Cohort 4 at 30 mg/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 30 mg/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.

Surface-etching enhances titanium implant osseointegration in newly formed (rhBMP-2-induced) and native bone.

PURPOSE: The influence of surface modifications on osseointegration in newly formed bone is not well established. The purpose of this study was to compare osseointegration at acid-etched versus turned implants in newly formed and native bone. METHODS: Supra-alveolar peri-implant defects were created in 8 hound/Labrador mongrel dogs. Titanium implants 10 mm long (2 turned and 1 dual acid-etched) were placed 5 mm into the surgically reduced alveolar crest, creating 5-mm supra-alveolar peri-implant defects. Recombinant human bone morphogenetic protein-2 (rhBMP-2; 0.4 mg) in a collagen carrier was used to induce new bone formation. A macroporous, expanded polytetrafluoroethylene device was used to delineate new bone formation. The animals were euthanized at 8 weeks for histometric analysis of the experimental sites. RESULTS: There were no significant differences in rhBMP-2-induced bone density (mean +/- SD) at acid-etched versus turned implants (20.6% +/- 5.3% vs 23.8% +/- 4.7%; P = .232). However, there was a significant difference in bone-implant contact in favor of the acid-etched implants (12.3% +/- 6.8% vs 7.9% +/- 3.1%; P = .05). Native bone density averaged 63.9% +/- 7.5% and 64.5% +/- 9.0% for acid-etched and turned implants, respectively (P = .641). Nevertheless, bone-implant contact was significantly enhanced at acid-etched versus turned implants (59.7% +/- 11.3% vs 40.7% +/-21.2%; P =.005). CONCLUSIONS: Surface dual acid-etching of titanium implants has a positive effect on osseointegration in newly formed and native bone. Significant differences in bone density do not appear to influence this effect.

Role of RGM coreceptors in bone morphogenetic protein signaling.

BACKGROUND: The repulsive guidance molecule (RGM) proteins, originally discovered for their roles in neuronal development, have been recently identified as co-receptors in the bone morphogenetic protein (BMP) signaling pathway. BMPs are members of the TGFbeta superfamily of signaling cytokines, and serve to regulate many aspects of cellular growth and differentiation. RESULTS: Here, we investigate whether RGMa, RGMb, and RGMc play required roles in BMP and TGFbeta signaling in the mouse myoblast C2C12 cell line. These cells are responsive to BMPs and are frequently used to study BMP/TGFbeta signaling pathways. Using siRNA reagents to specifically knock down each RGM protein, we show that the RGM co-receptors are required for significant BMP signaling as reported by two cell-based BMP activity assays: endogenous alkaline phosphatase activity and a luciferase-based BMP reporter assay. Similar cell-based assays using a TGFbeta-induced luciferase reporter show that the RGM co-receptors are not required for TGFbeta signaling. The binding interaction of each RGM co-receptor to each of BMP2 and BMP12 is observed and quantified, and equilibrium dissociation constants in the low nanomolar range are reported. CONCLUSION: Our results demonstrate that the RGMs play a significant role in BMP signaling and reveal that these molecules cannot functionally compensate for one another.

Bone formation at rhBMP-2-coated titanium implants in the rat ectopic model.

BACKGROUND: The objective of this study was to evaluate local bone formation at titanium porous oxide (TPO) implant surfaces adsorbed with recombinant human bone morphogenetic protein-2 (rhBMP-2). METHODS: In vitro studies were used to estimate the kinetics of I125-labeled rhBMP-2 released from TPO surfaces with narrow (N) or open (O) pores. Machined/turned titanium (MT) surfaces served as control. The rat ectopic model was used to assess local bone formation. Briefly, TPO-N, TPO-O, and MT disc implants adsorbed with 5, 10, or 20 microg rhBMP-2, respectively, were implanted subcutaneously into the ventral thoracic region in 5-week-old male Long Evans rats. The animals were euthanized at day 14 postsurgery when implants with surrounding tissues were removed, radiographed, and gross observations recorded. The specimens were processed for histologic evaluation using conventional cut-and-grind techniques. TPO implants without rhBMP-2 included in a preliminary evaluation revealed no evidence of bone formation, tissue encapsulation, or vascularity, thus such controls were not further used. RESULTS: TPO and MT implant surfaces adsorbed with 5 microg rhBMP-2 retained 2.3-5.4% rhBMP-2 following immersion and rinse in buffer, and 1.1-2.2% rhBMP-2 following repeated immersions and rinses over 27 days. TPO implants retained the most rhBMP-2 and MT implants retained the least. Explants revealed increased hard tissue formation, tissue encapsulation, and vascularity at TPO compared with MT implants. Radiographic observations were consistent with the explant observations. The histologic analysis showed greater amounts of bone formation, osteoblastic cells, osteoid, marrow, tissue encapsulation, vascularity, and bone voids for implants adsorbed with 10 and 20 microg rhBMP-2, and for TPO implants at the 5-microg rhBMP-2 dose. The histometric analysis revealed significantly greater bone formation at TPO-O than at MT implants at the 5-microg rhBMP-2 dose. All surfaces showed significant bone formation at the 10- and 20-microg dose. CONCLUSIONS: rhBMP-2 adsorbed onto TPO implant surfaces executes an osteoinductive effect including bone contacting the implant surface. This effect is surface- and dose-dependent; the TPO-O surface yielding the most bone at the low discriminating rhBMP-2 dose.

Bone reconstruction following implantation of rhBMP-2 and guided bone regeneration in canine alveolar ridge defects.

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.

rhBMP-2 delivered in a calcium phosphate cement accelerates bridging of critical-sized defects in rabbit radii.

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 [alpha-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/alpha-BSM cement, 0.033 mg/mL rhBMP-2/alpha-BSM cement, or buffer/alpha-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 alpha-BSM carrier. A semiquantitative histological scoring system was used to evaluate defect-bridging. Histomorphometry was used to quantify residual alpha-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 alpha-BSM and filling of the defects with trabecular bone in the group treated with 0.166 mg/mL rhBMP-2/alpha-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/alpha-BSM-treated groups, the majority of the trabecular bone was formed by a direct process adjacent to the resorbing alpha-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/alpha-BSM. That group also had reduced residual alpha-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/alpha-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.

rhBMP-2/calcium phosphate matrix accelerates osteotomy-site healing in a nonhuman primate model at multiple treatment times and concentrations.

BACKGROUND: While recombinant human bone morphogenetic protein-2 (rhBMP-2) administered in a calcium phosphate cement accelerates osteotomy-site healing in animal models when administered three hours after surgery, definitive fracture treatment is often delayed. The present study evaluated the ability of rhBMP-2, administered in a new particulating calcium phosphate matrix, to accelerate nonhuman primate fibular osteotomy-site healing following treatment at multiple treatment times and concentrations. METHODS: The ability of 1.5-mg/mL rhBMP-2/calcium phosphate matrix to accelerate osteotomy-site healing when administered three hours, one day, one week, or two weeks after surgery was first evaluated with use of bilateral proximal and distal fibular osteotomy sites in adult male monkeys. In a second study, the healing of osteotomy sites that had been treated with the administration of calcium phosphate matrix alone and with different concentrations of rhBMP-2/calcium phosphate matrix (0.5 mg/mL, 1.5 mg/mL, or 4.5 mg/mL) seven days after surgery was compared with that of contralateral, untreated osteotomy sites. In a third study, the histologic progression of osteotomy-site healing following treatment with 1.5-mg/mL rhBMP-2/calcium phosphate matrix or calcium phosphate matrix alone, administered three hours or one week after surgery to the osteotomy site, was assessed at multiple time points for as long as twenty-four months after surgery. RESULTS: Radiographs demonstrated increased callus area and more rapid healing in response to 1.5-mg/mL rhBMP-2/calcium phosphate matrix administered over the range of treatment times after surgery as compared with the findings of previous reports on untreated osteotomy sites. Bone formation appeared at the osteotomy sites sooner following treatment at one and two weeks as compared with the findings at the earlier time-points. Scintigraphic imaging at one day and one week after surgery showed prolonged retention of rhBMP-2 at the osteotomy site following an initial burst release. In the second study, radiographic, peripheral quantitative computed tomographic, biomechanical, and microscopic evaluation demonstrated that administration of 1.5 and 4.5-mg/mL rhBMP-2/calcium phosphate matrix one week after surgery accelerated osteotomy-site healing by 40% to 50% compared with the findings in untreated controls. The magnitude of acceleration was less in response to 0.5-mg/mL rhBMP-2/calcium phosphate matrix, and calcium phosphate matrix alone did not accelerate osteotomy-site healing. Histological evaluation indicated that an increased cellular infiltrate and increased direct bone formation contributed to the accelerated osteotomy-site healing following administration of rhBMP-2/calcium phosphate matrix at one week compared with three hours after surgery. CONCLUSIONS: A single percutaneous injection of rhBMP-2/calcium phosphate matrix accelerated healing in nonhuman primate fibular osteotomy sites over a wide range of treatment times. Efficacy was optimized in association with the administration of 1.5-mg/mL rhBMP-2/calcium phosphate matrix. Delaying treatment for one week further accelerated healing because of an increase in the number of responding cells and an increase in direct bone formation.

Observations on healing following endodontic surgery in nonhuman primates (Macaca fascicularis): effects of rhBMP-2.

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.