Emre Tomin

Use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model.

The ability of the osteoinductive protein and recombinant human bone morphogenetic protein-2, combined with polylactic glycolic acid porous microspheres and autologous blood clot to heal a large segmental defect was tested in a rabbit diaphyseal defect model. Two centimeter nonuniting defects were surgically created in the bilateral ulnae of 50 male New Zealand white rabbits. Each defect was then implanted with a pastelike polylactic glycolic acid/blood clot combination that was mixed with 5 different concentrations of recombinant human bone morphogenetic protein-2. The forearms were radiographically assessed on a biweekly schedule for 8 weeks. At 8 weeks, all animals were sacrificed and forearms radiographed. Radiographs were then scored by 3 independent observers for bone formation and union rates. United limbs were tested in torsion for mechanical strength using a Burstein torsion tester. All nonunited limbs were analyzed histologically as were 2 united limbs from each dosage group. Radiographic evaluation revealed that there was a dose dependent response in healing of the ulnar defect with a higher bone formation rate in the 2 higher dose limbs than in the lower dose limbs. Union was achieved in 100% of the highest dose limbs, whereas only 50% of the lowest dose limbs achieved bony union. No defects implanted with carrier alone achieved union. Biomechanical studies revealed significantly stiffer bone than age matched controls. Histologic analysis demonstrated normal bone formation with abundant normal appearing osteoid. These dose response data further support the role of recombinant human bone morphogenetic protein-2 as a potent morphogen in bone regeneration.

Biosynthetic bone grafting.

The regeneration of bone remains an elusive yet important goal in the field of orthopaedic surgery. Despite its limitations, autogenous cancellous bone grafting continues to the most effective means by which bone healing is enhanced clinically. Biosynthetic bone grafts currently are being developed as an alternative to autogenous bone grafting. These grafts generally contain one or more of three critical components: (1) osteoprogenitor cells; (2) an osteoconductive matrix; and (3) osteoinductive growth factors. The importance of each of these components based on preclinical data supports their use in biosynthetic bone grafts. The use of growth factors such as bone morphogenetic proteins, transforming growth factor, platelet derived growth factor, and fibroblastic growth factor is reviewed in preclinical long bone defect and spinal fusion models. The use of bone marrow in preclinical and clinical settings is presented with specific emphasis given to the use of bone marrow as a source of osteoprogenitor cells and how the use of these cells can be enhanced with the use of bone morphogenetic protein-2. These data support the concept that although products that contain only one of the three key components of a bone graft may regenerate bone successfully, composites of the three key components will be more successful clinically.

Morphology and matrix composition during early tendon to bone healing.

This study outlines the early morphologic phenomenon of tendon to bone healing in the rabbit model. Twelve skeletally mature, male New Zealand White rabbits received transplantation of the hallucis longus tendon into a 2-mm calcaneal bone tunnel. The morphologic characteristics of the healing tendon to bone interface were evaluated at 1, 2, 4, and 6 weeks after surgery by the use of conventional histology and immunohistochemical localization of collagen Types I, II, and III. Histologic analysis illustrated progressive maturation and reorganization of the tendon to bone interface with subsequent development of tissue collagen fiber continuity between the tendon and bone. Initially, diffuse immunolocalization of all three collagen types was observed within the scar tissue filling the space between the tendon and bone. During a 6-week period, reorganization of the scar tissue into an interface occurred, similar to an indirect insertion. Although a definitive fibrocartilage region did not form, Type II collagen was localized at the remodeling insertion site throughout the first 6 weeks of repair. In addition, Type III collagen fibers, resembling Sharpeys fibers, were noted to span this interface. The characterization of the insertion between tendon and bone is important to the understanding of healing in commonly used orthopaedic grafting procedures, such as anterior cruciate ligament reconstructions.

CLINICAL APPLICATIONS OF BONE GRAFT SUBSTITUTES

Autogenous bone grafting remains the gold standard for osseous reconstruction in clinical practice. It is associated with several limitations. The search for an alternative bone graft substitute with combined osteoinductive, osteoconductive, and osteogenic properties continues. This article highlights the properties of the various bone grafting materials currently available and discusses their efficacy in clinical practice.

Bone marrow and recombinant human bone morphogenetic protein-2 in osseous repair.

Bone marrow stem cells and recombinant human bone morphogenetic protein-2 each has the capacity to repair osseous defects. Recombinant human bone morphogenetic proteins require the presence of progenitor cells to function. It is hypothesized that a composite graft of recombinant human bone morphogenetic protein-2 and marrow would be synergistic and could result in superior grafting to autogenous bone graft. Syngeneic Lewis rats with a 5-mm critical sized femoral defect were grafted with recombinant human bone morphogenetic protein-2 and marrow, recombinant human bone morphogenetic protein-2, marrow, syngeneic cancellous bone graft, or carrier alone (control). Serial radiographs (3, 6, 9, 12 weeks) and torque testing (12 weeks) were performed. Bone formation and union were determined. The recombinant human bone morphogenetic protein-2 and marrow composite grafts achieved 100% union at 6 weeks. Recombinant human bone morphogenetic protein alone achieved 80% union by week 12. Both groups yielded a higher union rate and superior mechanical properties than did either syngeneic bone graft (38%) or marrow (47%) alone. The superior performance of recombinant human bone morphogenetic protein-2 combined with bone marrow in comparison with each component alone strongly supports a biologic synergism. This experimentation shows the clinical importance of establishing operative site proximity for the osteoinductive factors and responding progenitor cells.

Bisphosphonate therapy in fibrous dysplasia.

Fibrous dysplasia is proliferation of fibrous tissue within the bone marrow causing osteolytic lesions and pathologic fractures. Recently, second generation bisphosphonates have shown promise in the treatment of patients with fibrous dysplasia. In the current study, six patients with fibrous dysplasia were treated with either oral alone or oral and intravenous bisphosphonates. The participants were observed for changes in N-telopeptide, pain score, and radiographic changes. In the current study, the combination bisphosphonate therapy diminished pain, prevented fractures, lowered N-telopeptide values, and led to partial resolution of fibrous dysplasia lesions.

Use of bone morphogenetic protein 2 on ectopic porous coated implants in the rat.

The ability of recombinant human bone morphogenetic protein 2 to remain osteoinductive and stimulate appositional bone formation on a porous coated implant was tested in a rat quadriceps muscle pouch. Implants with or without hydroxyapatite were used to compare the effects on bone formation of two different doses (23 μg or 46 μg) of recombinant human bone morphogenetic protein 2 against controls as evidenced by contact radiography, histologic examination, and backscatter scanning electron microscopic analysis. Cylindrical plasma sprayed porous titanium implants were placed bilaterally within a muscle pouch surgically created in 48 Lewis rats. Implants treated with recombinant human bone morphogenetic protein 2 formed significantly more bone than did control implants independent of the dose or presence of hydroxyapatite. In all implants with bone formation, osteoinduction via endochondral ossification began within 7 days. By 21 days, cartilage largely was replaced by bone and marrow. The results of this ectopic, nonweightbearing in vivo assay suggest that recombinant human bone morphogenetic protein 2 remains biologically active after application to a titanium implant and may be used to enhance appositional bone formation by direct application to the implant surface.

Parathyroid Hormone (1-34) Augments Spinal Fusion, Fusion Mass Volume, and Fusion Mass Quality in a Rabbit Spinal Fusion Model

Study Design. The posterolateral rabbit spinal fusion model was used to assess the effect of intermittent parathyroid hormone on spinal fusion outcomes. Objective. To test the hypothesis that intermittent parathyroid hormone (PTH) improves spinal fusion outcomes in the rabbit posterolateral spinal fusion model. Summary of Background Data. Spinal fusion is the definitive management for spinal deformity or instability, yet despite current technology, 5% to 40% of lumbar fusions result in pseudarthrosis. Animal studies have demonstrated enhanced fracture healing with the use of PTH, but the effect of PTH on spinal fusion is poorly described. Methods. Forty-four male New Zealand white rabbits underwent bilateral posterolateral spine fusion (L5–L6 level). Twenty-two rabbits received daily subcutaneous injections of PTH (1–34) (10 &mgr;g/kg) and 22 received an injection of saline fluid. All were killed 6 weeks after surgery. L5–L6 vertebral segments were removed and analyzed with manual bending, faxitron radiography, microCT, and histomorphometry. Results. Manual bending identified fusion in 30% (control) versus 81% (PTH) animals (P < 0.001). A radiographic scoring system (“0” = no bone formation, “5” = full fusion) resulted in an average score of 3.36 (control) versus 4.51 (PTH) (P < 0.001). MicroCT analysis demonstrated a median mass of 3.5 cc (control) (range, 2.25–5.40 cc) versus 6.03 cc (PTH) (range, 4.34–10.58 cc) (P < 0.001). Histology showed a median percentage bone area of 14.3% (control) (n = 12) versus 29.9% (PTH) (n = 15) (P < 0.001). The median percentage cartilage was 2.7% (control) (n = 5) versus 26.6% (PTH) (n = 5) (P < 0.01). Osteoclast quantification revealed median values of 140.5 (control) (n = 6) and 345.0 (PTH) (n = 8) (P < 0.001) respectively, and the percentage of osteoblasts revealed a median value of 31.4% (control) (n = 6) versus 64.4% (PTH) (n = 8) (P < 0.001). Conclusion. Intermittent PTH administration increased posterolateral fusion success in rabbits. Fusion bone mass and histologic determinants were also improved with PTH treatment. PTH has promise for use as an adjunctive agent to improve spinal fusion in clinical medicine.

Osseous regeneration in preclinical models using bioabsorbable delivery technology for recombinant human bone morphogenetic protein 2 (rhBMP-2)

Abstract Novel delivery systems for the bone morphogenetic protein rhBMP-2, consisting of poly( d,l -lactide-co-glycolide) porous microspheres as bioabsorbable filling material with either autologous blood clot or carboxymethylcellulose as binding agent, were tested in several preclinical models including a rat calvarial defect model, a rabbit radius segmental defect model, and a rabbit ulna segmental defect model. In the rat calvarial defect model, these novel delivery systems were shown to be comparable to inactivated collagenous bone matrix as a matrix for rhBMP-2 delivery at the appropriate dose. The incidence of union in the rabbit long bone studies exhibited a rhBMP-2 dose-response, achieving an incidence of union of nearly 100% at the higher doses tested. Histological evaluation showed remodelling of newly generated bone, and biomechanical testing found that the healed limbs were as strong as untreated control limbs. These studies demonstrate that it is possible to obtain osseous regeneration of critical-size defects by combining rhBMP-2 with synthetic delivery systems.

Resorbable posterolateral graft containment in a rabbit spinal fusion model.

The effect of a resorbable graft containment device was evaluated in a rabbit posterolateral lumbar spinal fusion model. Twenty rabbits were divided into four groups: autogenous bone graft (ABG), ABG with the MacroPore containment device (MacroPore Biosurgery Inc, San Diego, Calif) (ABG+MP), demineralized bone matrix (DBM), and DBM with the containment device (DBM+MP). Fusion mass was assessed at 6 weeks with high resolution radiographs and volumetric computed tomography (CT). The graft containment device was associated with alteration of the fusion mass structure and significant enhancement of fusion mass volume (ABG versus ABG+MP, P=.027; DBM versus DBM+MP, P=.043). A bioabsorbable, protective graft containment device successfully enhanced posterolateral spinal fusion mass volume.