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.

Tyrosine kinase Eph receptor A6 sensitizes glioma-initiating cells towards bone morphogenetic protein-induced apoptosis.

Bone morphogenetic protein (BMP) signaling plays important roles in glioblastoma multiforme (GBM), a lethal form of brain tumor. BMP reduces GBM tumorigenicity through its differentiation- and apoptosis-inducing effects on glioma-initiating cells (GIC). However, some GIC do not respond to the tumor suppressive effects of BMP. Using a phosphoreceptor tyrosine kinase array, we found that EPHA6 (erythropoietin-producing hepatocellular carcinoma receptor A6) phosphorylation was regulated by BMP-2 signaling in some GIC. Analysis of The Cancer Genome Atlas showed that EPHA6 expression was lower in patients with GBM than in the normal brain, and that high EPHA6 expression was correlated with better prognosis. EPHA6 receptor increased the susceptibility of both sensitive and resistant GIC to BMP-2-induced apoptosis. The cooperative effect on apoptosis induction depended on the kinase activity of BMP type I receptor but was independent of EPHA6 kinase function. Overexpression of the EPHA6 receptor in GIC resulted in the formation of a protein complex of EPHA6 receptor and the BMP type I receptor ALK-2, which was associated with BMP-induced apoptosis in GIC. Intracranial injection of GIC into nude mice showed that gain-of-function of EPHA6 together with BMP-2 pretreatment slowed GBM tumor progression in the mouse brain and promoted mouse survival. In summary, EPHA6 together with BMP-2 signaling led to apoptotic cell death in GIC, and thus is a putative tumor suppressor in GBM.

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.

Adaptive growth factor delivery from a polyelectrolyte coating promotes synergistic bone tissue repair and reconstruction.

Traumatic wounds and congenital defects that require large-scale bone tissue repair have few successful clinical therapies, particularly for craniomaxillofacial defects. Although bioactive materials have demonstrated alternative approaches to tissue repair, an optimized materials system for reproducible, safe, and targeted repair remains elusive. We hypothesized that controlled, rapid bone formation in large, critical-size defects could be induced by simultaneously delivering multiple biological growth factors to the site of the wound. Here, we report an approach for bone repair using a polyelectrolye multilayer coating carrying as little as 200 ng of bone morphogenetic protein-2 and platelet-derived growth factor-BB that were eluted over readily adapted time scales to induce rapid bone repair. Based on electrostatic interactions between the polymer multilayers and growth factors alone, we sustained mitogenic and osteogenic signals with these growth factors in an easily tunable and controlled manner to direct endogenous cell function. To prove the role of this adaptive release system, we applied the polyelectrolyte coating on a well-studied biodegradable poly(lactic-co-glycolic acid) support membrane. The released growth factors directed cellular processes to induce bone repair in a critical-size rat calvaria model. The released growth factors promoted local bone formation that bridged a critical-size defect in the calvaria as early as 2 wk after implantation. Mature, mechanically competent bone regenerated the native calvaria form. Such an approach could be clinically useful and has significant benefits as a synthetic, off-the-shelf, cell-free option for bone tissue repair and restoration.

Surface-mediated bone tissue morphogenesis from tunable nanolayered implant coatings.

The functional success of a biomedical implant critically depends on its stable bonding with the host tissue. Aseptic implant loosening accounts for more than half of all joint replacement failures. Various materials, including metals and plastic, confer mechanical integrity to the device, but often these materials are not suitable for direct integration with the host tissue, which leads to implant loosening and patient morbidity. We describe a self-assembled, osteogenic, polymer-based conformal coating that promotes stable mechanical fixation of an implant in a surrogate rodent model. A single modular, polymer-based multilayered coating was deposited using a water-based layer-by-layer approach, by which each element was introduced on the surface in nanoscale layers. Osteoconductive hydroxyapatite (HAP) and osteoinductive bone morphogenetic protein-2 (BMP-2) contained within the nanostructured coating acted synergistically to induce osteoblastic differentiation of endogenous progenitor cells within the bone marrow, without indications of a foreign body response. The tuned release of BMP-2, controlled by a hydrolytically degradable poly(ß-amino ester), was essential for tissue regeneration, and in the presence of HAP, the modular coating encouraged the direct deposition of highly cohesive trabecular bone on the implant surface. In vivo, the bone-implant interfacial tensile strength was significantly higher than standard bioactive bone cement, did not fracture at the interface, and had long-term stability. Collectively, these results suggest that the multilayered coating system promotes biological fixation of orthopedic and dental implants to improve surgical outcomes by preventing loosening and premature failure.

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.

Divergent activities of osteogenic BMP2, and tenogenic BMP12 and BMP13 independent of receptor binding affinities.

Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.

Regulation of gene expression in human tendinopathy.

BACKGROUND: Chronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy. METHODS: We have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy. RESULTS: Diseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response. CONCLUSIONS: Identification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics.

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.

Mandibular reconstruction after gunshot trauma in a dog by use of recombinant human bone morphogenetic protein-2.

CASE DESCRIPTION: A 6-year-old German Shorthaired Pointer was evaluated for possible reconstruction of a mandibular defect resulting from gunshot trauma. CLINICAL FINDINGS: A 5-cm defect of the right mandibular body was evident. A segment of the mandibular body was removed 9 weeks earlier because of severe contamination and comminution associated with gunshot trauma. Subsequent right-sided mandibular drift resulted in malocclusion in which the left mandibular canine tooth caused trauma to mucosa of the hard palate medial to the left maxillary canine tooth. The right maxillary canine tooth caused trauma to gingiva lingual to the right mandibular canine tooth. TREATMENT AND OUTCOME: The right mandible was stabilized with a 2.0-mm maxillofacial miniplate positioned along the lateral alveolar margin and a 2.4-mm locking mandibular reconstruction plate placed along the ventrolateral mandible. An absorbable compression-resistant matrix containing collagen, hydroxyapatite, and tricalcium phosphate was soaked in recombinant human bone morphogenetic protein-2 (rhBMP-2; 7.2 mL of a 0.5 mg/mL solution for a dose of 3.6 mg) and placed in the defect. By 4 weeks after surgery, an exuberant callus was evident at the site of the defect. By 7 months after surgery, the callus had remodeled, resulting in normal appearance, normal occlusion, and excellent function of the jaw. CLINICAL RELEVANCE: Mandibular defects resulting from gunshot trauma can be treated by removal of contaminated tissue and comminuted bone fragments, followed by staged reconstruction. The combination of rhBMP-2 and compression-resistant matrix was effective in a staged mandibular reconstruction in a dog with a severe traumatic mandibular defect.

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.

Clinical use of recombinant human bone morphogenic protein-2 in a whooping crane (Grus americana).

OBJECTIVE: To report use of recombinant human bone morphogenetic protein-2 (rhBMP-2) as adjunctive therapy for treatment of a comminuted, open, proximal humeral fracture in an avian species. STUDY DESIGN: Clinical report. ANIMALS: A 3.5-month-old male whooping crane (Grus americana). METHODS: An open, severely comminuted humeral facture was stabilized with an intramuscular (IM) pin/type IA external skeletal fixator with tie-in configuration. rhBMP-2 was applied in a calcium phosphate matrix (CPM) paste directly to the fracture site as a bone graft substitute. Radiographic evidence of bone healing was monitored for 14 weeks. RESULTS: Substantial bony callus was evident at 4 weeks and at 8 weeks there was bridging callus with obvious bony remodeling. The fixation was destabilized at 9 weeks by IM pin removal, bone healing progressed and the fixator was removed at 11 weeks. By 14 weeks both cortices had been re-established with continued callus remodeling evident. CONCLUSION: rhBMP-2, applied in a CPM paste, was used as a bone graft substitute in the treatment of a comminuted, open humeral fracture in a whooping crane. CLINICAL RELEVANCE: Use of rhBMP-2/CPM should be considered in treatment of avian fractures.

Clinical application of recombinant human bone morphogenetic protein-2 in 4 dogs.

OBJECTIVE: To describe outcome in dogs with insufficient bone healing treated with recombinant human bone morphogenetic protein-2 (rhBMP-2). STUDY DESIGN: Retrospective study. ANIMALS: Four dogs clinically affected with delayed union or nonunion bone healing. METHODS: Medical records were reviewed for signalment, clinical problem, treatment, and outcome. RESULTS: Four dogs that had delayed- or nonunion of bone fracture, osteotomy, or arthrodesis were treated with either minimally invasive, fluoroscopically guided, percutaneous administration or direct surgical application of rhBMP-2. Doses used ranged from 0.2 to 1.6 mg of rhBMP-2. In 3 dogs, a calcium phosphate matrix (CPM) carrier was used whereas in 1 dog commercially prepared rhBMP-2 impregnated in an absorbable collagen sponge (INFUSE Bone Graft) was used. This latter dog had osteomyelitis associated with implant infection before rhBMP-2 administration. Rapid radiographic union was noted in all dogs with excellent long-term outcome. Adverse effects were minimal and included transient worsening of lameness after percutaneous administration of rhBMP-2 in 2 dogs. CONCLUSIONS: rhBMP-2 stimulated rapid bone formation at delayed- or nonunion sites resulting in radiographic bone union with minimal adverse effects and excellent long-term outcome in 4 dogs. CLINICAL RELEVANCE: Direct intraoperative administration or fluoroscopically guided, minimally invasive delivery of rhBMP-2 may be an effective treatment modality for bone delayed- or nonunions and could potentially be used to stimulate new bone production in a variety of orthopedic surgical conditions in dogs.

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.

Protein-based tissue engineering in bone and cartilage repair.

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.

Recombinant human bone morphogenetic protein-2 delivered in an injectable calcium phosphate paste accelerates osteotomy-site healing in a nonhuman primate model.

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 alpha-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/alpha-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/alpha-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/alpha-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/alpha-BSM-treated osteotomy sites but incomplete bridging of the paired-control osteotomy sites at ten weeks. CONCLUSIONS: A single percutaneous injection of rhBMP-2/alpha-BSM accelerates the healing of fibular osteotomy sites in nonhuman primates by approximately 40% compared with the healing of untreated osteotomy sites.

Percutaneous injection of recombinant human bone morphogenetic protein-2 in a calcium phosphate paste accelerates healing of a canine tibial osteotomy.

BACKGROUND: In this study, we evaluated the capacity of a single percutaneous injection of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in a rapidly resorbable calcium phosphate paste (alpha-BSM) to accelerate bone-healing in a canine tibial osteotomy model. We hypothesized that the osteotomy sites would heal faster after percutaneous delivery of rhBMP-2/alpha-BSM than they would after injection of alpha-BSM alone or after no treatment. METHODS: Bilateral tibial osteotomy was performed and the sites were stabilized with external fixators in sixteen dogs. Four hours after the surgery, one limb of each dog was treated with a single percutaneous injection of rhBMP-2/alpha-BSM paste or an equal volume of alpha-BSM alone. There were eight limbs in each group, and the osteotomy site in the contralateral limb served as an untreated control. The results were evaluated with serial radiography and force-plate analysis at four and eight weeks after surgery and with mechanical testing and histologic examination at eight weeks after the surgery. RESULTS: At four and eight weeks after the osteotomy and treatment, the scores for radiographic union were significantly greater for the rhBMP-2/alpha-BSM-treated limbs than they were for the alpha-BSM-treated or untreated, control limbs (p < 0.05). The callus area in the rhBMP-2/alpha-BSM-treated limbs was significantly greater than that in the alpha-BSM-treated and untreated, control limbs at four and eight weeks postinjection (p < 0.05). The time-integrated vertical force for the rhBMP-2-treated limbs was significantly greater than that for their contralateral controls at four weeks and significantly greater than that for the treated and control limbs of the alpha-BSM-treated dogs at four and eight weeks after the surgery (p

Bone consolidation is enhanced by rhBMP-2 in a rabbit model of distraction osteogenesis.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a differentiation factor which has been shown to induce bone formation and heal bony defects in a variety of animal models. A possible application of rhBMP-2 is to accelerate bone regeneration during distraction osteogenesis. which clinically is a long procedure, often involving significant complications. In this study we tested the ability of rhBMP-2 to accelerate the consolidation phase of distraction osteogenesis in a rabbit model of leg lengthening. Tibiae were lengthened 2 cm over a period of ten days. rhBMP-2 was administered at the end of the lengthening phase. Two modes of rhBMP-2 application were tested: surgical implantation of rhBMP-2/ACS (absorbable collagen sponge) into the regenerate (50 microl of 1.5 mg/ml rhBMP-2, total dose = 75 microg rhBMP-2), and percutaneous injection of rhBMP-2/buffer (0.1 ml of 0.75 mg/ml rhBMP-2. total dose = 75 microg rhBMP-2) into three sites within the regenerate. Also, there were three groups of control animals: (1) no surgical intervention, (2) surgical implantation of buffer/ACS and (3) percutaneous injection of buffer. Rabbits were sacrificed at 5, 14 and 28 days after the interventions. Radiographic evaluation indicated a significant increase in bony union of the distraction regenerate in the rhBMP-2 treated groups compared with the untreated groups at 5 and 14 days. At 28 days, formation of a cortex and reestablishment of the medullary canal was evident only in the rhBMP-2 treated groups. The bone mineral content (BMC) of the regenerate was significantly higher in the rhBMP-2 treated groups at 5 and 14 days. However, at 28 days, BMC of the regenerate was similar in all groups. The average volumetric density of the regenerate was significantly higher in the rhBMP-2 injection group at day 14. In Summary, both injection of rhBMP-2/buffer and implantation of rhBMP-2/ACS enhanced the consolidation stage of distraction osteogenesis in this rabbit model.