A radiographical and biomechanical study of demineralized bone matrix implanted into a bone defect of rat femurs with and without bone marrow

Repair of large bone defects represents a challenge to orthopedic surgery since autogenous graft is not available in large amounts. Demineralized bone matrix (DBM) which contains bone morphogenic protein, a potent osteoinductive glycoprotein, and collagen, an osteoconductive matrix, may be an effective substitute for these graft materials. Bone marrow which contains osteoprogenitor cells could potentiate the osteoinductive and osteoconductive properties of demineralized bone matrix. This study tested the ability of demineralized bone matrix with and without bone marrow to bridge large segmental defects, and evaluated the results both radiographically and biomechanically as compared to autogenous (isogeneic) cancellous bone graft. Demineralized bone-matrix segments implanted into a plated femoral segmental defect in rats resulted in firm union in most animals. Bone marrow significantly enhanced bone formation of demineralized bone-matrix implants at an early stage but with time, differences between bone marrow-augmented and bone marrow-deprived demineralized bone implants were no longer demonstrable radiographically and biomechanically. Newly formed bone had about 50% of the strength of the contralateral control bones. Femurs implanted with cancellous bone isografts had similar evidence of absolute union rate, radiographic and mechanical properties as DBM-implanted femurs.     

Induction of bone by a demineralized bone matrix gel: A study in a rat femoral defect model

Demineralized bone matrix contains osteoinductive factors and stimulates filling of gaps and defects with bone; however, it is difficult to handle by itself and various preparations have been tested. Demineralized bone matrix with a gel consistency now is available for clinical use. We studied, in a femoral segment defect in the rat, the effects of rat demineralized bone matrix gel with and without a ceramic substratum. This preparation is analogous to the human demineralized bone matrix in the same carrier, used clinically for humans. One hundred adult male Fischer rats were divided into 10 experimental groups. Independent variables included the presence or absence of hydroxyapatite ceramic cylinders, the presence of demineralized bone matrix in carrier or carrier alone (glycerol), and the duration of observation (1, 2 and 4 months). Defects filled with the gel alone had significantly higher radiographic scores for host-graft union at 4 months compared with ceramic with the gel, ceramic alone, or carrier alone. Demineralized bone matrix gel significantly increased the total histologic score for host-graft union, whether ceramic was present or not, and a three-way interaction occurred among ceramic, the gel, and time. Demineralized bone matrix gel was an effective inducer of bone formation in this model. An additional substratum was not required; in fact, significantly more bone was formed in the absence of the ceramic cylinder. Neither the gel nor the ceramic were impediments to revascularization of the defect. Host-graft union was enhanced by demineralized bone matrix gel but not by the ceramic cylinder.     

An unexpected outcome during testing of commercially available demineralized bone graft materials: how safe are the nonallograft components?

STUDY DESIGN: Radiographic and histologic analyses of commercially available bone graft materials were performed. OBJECTIVE: To compare the osteoinductive efficacy of commercially available demineralized bone matrix material. SUMMARY OF BACKGROUND DATA: The relative in vivo bone formation and toxicology of the nonallograft components the make up various commercially available demineralized bone matrix products are not known. METHODS: An in vivo bone formation model was used in 30 athymic rats. Six different bone grafting materials were tested in subcutaneous and intermuscular locations. After 4 weeks, radiographic and histologic testing of bone formation was performed. RESULTS: Eight of nine rats implanted with Grafton demineralized bone matrix products died 1 to 4 days after implantation of the bone graft material. None of the remaining 10 animals implanted with the four other grafting materials died. The experiment was modified and completed with a lower dose of bone graft material. Pathologic analysis indicated that the cause of death was hemorrhagic necrosis of the kidneys, most likely caused by a toxic effect on the glomeruli and tubules. A possible causative factor may have been the glycerol in the graft material. CONCLUSIONS: Although the volume of Grafton product per kilogram of body weight used in this study was approximately eight times the maximum volume used in humans, the authors believe that this data must be reported because this product is used substantially in clinical settings. In addition, the osteoinductive performance and relative safety of the nonallograft components in all commercially available demineralized bone grafts are not known.     

Osteoinductivity of commercially available demineralized bone matrix. Preparations in a spine fusion model

BACKGROUND: Although autogenous bone is the most widely used graft material for spinal fusion, demineralized bone matrix preparations are available as alternatives or supplements to autograft. They are prepared by acid extraction of most of the mineralized component, with retention of the collagen and noncollagenous proteins, including growth factors. Differences in allograft processing methods among suppliers might yield products with different osteoinductive activities. The purpose of this study was to compare the efficacy of three different commercially available demineralized bone matrix products for inducing spinal fusion in an athymic rat model. METHODS: Sixty male athymic rats underwent spinal fusion and were divided into three groups of eighteen animals each. Group I received Grafton Putty; Group II, DBX Putty; and Group III, AlloMatrix Injectable Putty. A control group of six animals (Group IV) underwent decortication alone. Six animals from each of the three experimental groups were killed at each of three intervals (two, four, and eight weeks), and the six animals from the control group were killed at eight weeks. At each of the time-points, radiographic and histologic analysis and manual testing of the explanted spines were performed. RESULTS: The spines in Group I demonstrated higher rates of radiographically evident fusion at eight weeks than did the spines in Group III or Group IV (p < 0.05). Manual testing of the spines at four weeks revealed variable fusion rates (five of six in Group I, two of six in Group II, and none of six in Group III). At eight weeks, all six spines in Group I, three of the six in Group II, and no spine in Group III or IV had fused. Histologic analysis of the spines in Groups I, II, and III demonstrated varying amounts of residual demineralized bone matrix and new bone formation. Group-I spines demonstrated the most new bone formation. CONCLUSIONS: This study demonstrated differences in the osteoinductive potentials of commercially available demineralized bone matrices in this animal model.     

Current use of biologic graft extenders for spinal fusion

Use of biologic graft extenders in spinal fusions is increasing. Multiple allograft alternatives exist to the "gold-standard" autologous bone grafting. The ideal graft extender is osteoconductive, osteoinductive and has osteogenic potential. While the ideal graft extender has yet to be found, available bone graft extenders have varying degrees of predominantly osteoconductive and osteoinductive properties. This review will provide an update on available graft extenders including bone morphogenetic proteins, mesenchymal stem cells, and demineralized bone matrix. The goal is to provide a review of the current use in spinal fusions and future directions in biologics for spinal fusion.     

Effects of lathyritic drugs and lathyritic demineralized bone matrix on induced and sustained osteogenesis

Demineralized bone matrix was implanted in normal and lathyritic rats. At 2 weeks, the bone that formed in the lathyritic animals had an elevated alkaline phosphatase activity and a reduced calcium content compared with the controls. Four weeks after implantation, these biochemical parameters were reversed, with a decrease in alkaline phosphatase activity and an increase in calcium content to control levels. the histology of the recovered implants revealed new bone formation. Lathyritic demineralized bone matrix was prepared from bones of rats fed β-aminopropionitrile for 2 weeks (2-week BAPN-DBM) or 4 weeks (4-week BAPN-DBN), and was implanted in normal rats. Two weeks after implantation, both preparations of lathyritic demineralized bone matrix demonstrated early bone formation, although alkaline phosphatase activity and calcium content were reduced. By 4 weeks after implantation, no biochemical or histological evidence of bone formation remained at the site of the 4-week BAPN-DBM implants; continued but reduced bone formation was observed at the site of the 2-week BAPN-DBM implants. Reconstitution of inactivated normal demineralized bone matrix with the guanidine-soluble extracts restored the osteoinductive capacity. However, reconsistution of inactivated lathyritic demineralized bone matrix (4-week BAPN-DBM) failed to restore the osteoinductive capacity. These results indicate that the degree of crosslinking of the collagen matrix that acts as a carrier for osteoinductive proteins plays a key role in inducing and sustaining osteogenesis.     

Osteobiologics

"Osteobiologics" is the term that has been introduced to refer to the class of engineered materials that have been created and which promote healing of fractures and bone defects. The list of osteobiologics is rapidly expanding as new products incorporating osteoconductive materials are mixed with a variety of osteoinductive proteins, demineralized bone, and preparations of osteogenic cells. The growth in osteobiologics has been stimulated by the early success of osteoconductive materials as graft substitutes in the repair of fractures and by the increasing demand for grafts in all areas of orthopaedics. Although allografts have historically been employed with success, the number of donors has grown much slower than demand leading to the development of artificial materials. Manufactured bone graft substitutes, or osteobiologics, attempt to mimic the components of an autogeneous bone graft by reproducing the bone matrix, which is osteoconductive and osteoinductive. Other products aim to introduce osteogenic cells by concentrating bone marrow while others introduce differing growth factors from platelets in peripheral blood. Very few of these products have been supported by appropriate clinical studies and as such their value is unknown. Orthopaedic surgeons employing these products must understand the basic science principles behind their development in order to understand the indications and limitations of their application. Properly designed clinical studies should be performed to determine the usefulness and cost-effectiveness of both current and future products.     

Selection of bone grafts for revision total hip arthroplasty

The selection of bone grafts to reconstruct deficient bone for revision hip replacement requires an understanding of specific bone graft functions and the critical steps of the biologic incorporation of the graft into the host. Bone grafts provide functions of osteogenesis, either graft derived or by osteoinduction, osteoconduction, or both, and mechanical support. Autologous cancellous bone provides excellent osteogenesis and osteoconduction without structural support. Nonvascularized cortical autografts provide mechanical support and are somewhat osteogenic. Allogeneic cancellous bone is osteoconductive and minimally osteoinductive, whereas cortical allografts provide structural support, if not freeze-dried, and are somewhat osteoconductive. Allogeneic demineralization bone matrix is highly osteoinductive. The selection of the appropriate bone graft depends on the classification of the bone deficiency. Cavitary (contained) defects can be reconstructed with cancellous morselized autograft, frozen or freeze-dried allograft, or allogeneic demineralized bone matrix. Segmental defects require bulk corticocancellous and/or cortical autografts or allografts. The ultimate incorporation of the bone graft depends on the interaction of the graft and the host's mechanical and biologic environment, and host-bone graft contact and stability. Optimum bone graft selection will enhance the clinical outcomes in revision total hip arthroplasty.     

Efficacy of contained metaphyseal and periarticular defects treated with two different demineralized bone matrix allografts

The efficacies of two different allografts, Grafton (demineralized bone matrix [DBM] in a glycerol carrier) and Orthoblast (DBM in a reverse thermal poloxamer carrier) were examined from cases involving periarticular fractures. Demographic, perioperative, and outcome data for patients with periarticular fractures who underwent a prospectively designed protocol for bone grafting were compiled, with 15 cases using Orthoblast and 13 using Grafton. A successful graft was defined as healing on the first graft attempt without complications. Healing was determined by radiographic studies and clinical evaluation. The successful graft rates of Orthoblast and Grafton were 15/15 and 9/13, respectively.     

Effect of gas-plasma sterilization on the osteoinductive capacity of demineralized bone matrix.

The current study evaluated the effect of low-temperature hydrogen peroxide gas plasma sterilization on the osteoinductive capability of human demineralized bone matrix using a rat model. Twelve athymic rats received three separate implants consisting of steam-sterilized demineralized bone matrix (negative control), sterile-harvest demineralized bone matrix (positive control), and gas-plasma-sterilized demineralized bone matrix. A demineralized bone matrix pellet from each sterilization group was placed individually into one of three separate soft tissue pockets created in the epaxial musculature of each rat. All 12 rats were euthanized 9 weeks after implantation. Each implantation site was removed along with 0.5-cm normal tissue around the implant. Histologic examination was done on each implant site to determine the presence or absence of new bone, cartilage, or bone marrow elements. All 12 sterile harvest demineralized bone matrix sites histologically contained new bone elements, whereas none of the negative control or gas plasma sterilized demineralized bone matrix sites contained any of these same elements. The results of this study indicate that demineralized bone matrix sterilized with low-temperature, gas-plasma sterilization loses its osteoinductive capacity in a manner similar to that of steam-sterilized demineralized bone matrix, making low-temperature, gas- plasma sterilization unsuitable as a method of secondary sterilization of demineralized bone matrix.     

Demineralized bone matrix and hydroxyapatite/tri-calcium phosphate mixture for bone healing in rats

Purpose: Hydroxyapatite/tri-calcium phosphate (HA/TCP) mixture is an osteoconductive material used as a bone graft substitute, and demineralised bone matrix (DBM) is an osteoinductive material. A combination of DBM and HA/TCP mixture would probably create a composite with both osteoconductive and osteoinductive properties. The purpose of this study was to determine the effect of the combination of DBM and HA/TCP mixture on healing of rat radius segmental defects. Methods: Twenty-four adult male Wistar rats were used. Bilateral radial defects were created in each animal. Radial defects were implanted with DBM, HA/TCP mixture and a combination of both substances. Control defects were left unfilled. Ten weeks after implantation, the animals were sacrificed, and the radii were evaluated by radiograhic and histopathological studies. Results: The use of DBM alone demonstrated improved healing on radiographic and histological studies compared to other groups and the control group. There were no differences between the other two groups and the control group. Conclusion: The DBM group showed the best healing response. Combined use of DBM and HA/TCP mixture did not improve bone healing, and the osteoinductive properties of DBM were inhibited by HA/TCP mixture.     

Incorporation of impacted morselized bone allografts in rabbits

Morselized bone allografts have been used for the treatment of bone stock loss in orthopedic revision surgery with encouraging results. However, several parameters can influence the graft incorporation including the processing treatments. This experimental work used a cavitary bone defect in 90 rabbits to evaluate the sequence of incorporation of three different kinds of morselized bone allografts: uncryopreserved cancellous bone, freeze-dried cancellous bone, and totally demineralized cortical bone each of which were prepared in accordance with our rigid protocol. Revascularization and remodeling of the transplanted bone grafts were evident upon histological evaluation. Bone apposition and bone resorption resulted in a mixture of graft and new bone. Mineralized cancellous grafts showed great osteoconductive capacity, whereas demineralized cortical grafts showed an intense osteoinductive capacity and a weak osteoconductive capacity. In a general evaluation, cryopreserved cancellous bone grafts showed superior biological efficacy for reconstruction of experimental bone defects, closely followed by freeze-dried cancellous bone grafts, and, finally, by demineralized cortical bone grafts. These results validate our protocol for the processing and preservation of these three kinds of bone grafts.     

新型硫酸钙和脱钙骨基质混合物的临床应用

目的 评价新型硫酸钙和脱钙骨基质混合物作为骨移植替代物的临床应用效果. 方法 2005年2月至2008年2月采用新型人工骨移植治疗51例患者,按照植入物不同分为两组:人工骨与自体骨混合组21例,即植人硫酸钙和脱钙骨基质混合物加自体骨;单纯人工骨组30例,只植入硫酸钙和脱钙骨基质混合物.术后定期复查,观察人工骨吸收和新骨生长情况. 结果 51例切口一期愈合,无局部红肿、渗出.3例患者失访,48例随访6～36个月,平均16个月.单纯人工骨组术后4周可见人工骨部分吸收,颗粒形态模糊,术后8～12周(平均9.6周)完伞吸收,可见新生骨质,术后8～16周(平均11周)骨性愈合.人工骨与自体骨混和组术后8～12周(平均11.5周)人工骨颗粒完全吸收,骨折不愈合患者术后14～24周(平均19周)获骨性愈合,其余患者术后9～20周(平均13周)获骨性愈合. 结论 新型人工骨能够发挥增加移植物容量、促进骨生成的作用,无局部不良反应,是一种安全有效的骨移植替代物.     

Healing of a critical-sized defect in the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone morphogenetic protein

BACKGROUND: The purpose of this study was to evaluate the osseous healing of a critical-sized femoral defect in a rat model with use of recombinant human bone morphogenetic protein-2 (rhBMP-2), a matrix fabricated of D,D-L,L-polylactic and hyaluronan acid (OPLA-HY), and a vascularized periosteal flap. METHODS: The carrier matrix OPLA-HY with or without rhBMP-2 was implanted in a 1-cm-long femoral defect and secured with a plate and screws. In some groups, a vascularized periosteal flap was harvested from the medial surface of the tibia. In group 1, the femoral defects in the animals were filled with the OPLA-HY matrix alone; in group 2, the OPLA-HY matrix was covered by the vascularized periosteal flap; in group 3, 20 mug of rhBMP-2 was added to the OPLA-HY matrix; and in group 4, the femoral defect containing the OPLA-HY matrix and 20 mug of rhBMP-2 was wrapped circumferentially by the vascularized periosteal flap. The presence and density of new bone formation in the femoral defect were evaluated radiographically, histologically, and with histomorphometry at four and eight weeks postoperatively. RESULTS: Groups 1 and 2, which were not treated with rhBMP-2, showed no radiographic or histologic evidence of mature bone formation at four or eight weeks. Both groups 3 and 4, which were treated with rhBMP-2, demonstrated excellent bone formation. However, with the periosteal flap, group 4 demonstrated more bone formation on histomorphometric analysis at eight weeks (43.1%) than did group 3 (28.3%) (p < 0.01). Additionally, heterotopic bone formed outside the boundaries of the defect in eight of the fifteen animals in group 3, which had no periosteal flap. CONCLUSIONS: Bone-tissue engineering with use of the OPLA-HY matrix and rhBMP-2 produced good bone formation in the rat femoral defect model. However, the addition of a vascularized periosteal flap significantly increased bone formation within the boundaries of the defect and prevented heterotopic ossification.     

Current approaches to experimental bone grafting

A number of osteogenic, osteoinductive, and osteoconductive substances currently are being investigated for use in bone repair. It is conceivable that a selected combination of osteogenic cells, osteoinductive factors, and osteoconductive matrices can be combined and fabricated into an implantable material custom-suited to particular clinical demands. Consequently, it is crucial that potential graft substances be experimentally characterized in terms of their precise contribution to the bone-forming mechanisms. In this article, the authors review current areas of research in the realm of experimental grafting, including the current understanding of materials that manifest osteogenic, osteoinductive, or osteoconductive properties.     

Implants of heterologous demineralized bone matrix for induction of posterior spinal fusion in rats.

The authors tested the osteoinductive capacity of powdered heterologous (bovine) demineralized bone matrix in rats. The first part of the study concerned a monolateral posterior spinal implant after decortication of three vertebrae, using as a control area the animal's contralateral side, in which neither bone graft nor any other material were placed. In another group of rats, a comparative evaluation was made of powdered heterologous demineralized bone matrix and fresh autologous bone. In the same animal, autologous bone was implanted to realize a thoracic posterior fusion and demineralized bone matrix, to induce a posterior fusion in the lumbar area. All data obtained suggested a good osteoinductive activity of heterologous powdered demineralized bone matrix. The two posterior spinal fusions done in the same animal with heterologous demineralized bone matrix or autologous bone, respectively, had similar callus development and required the same time for formation.     

Antibacterial and osteoinductive properties of demineralized bone matrix treated with silver

The problems incurred by storage of demineralized bone allograft material and its potential use in contaminated operative sites make an antibacterial property desirable. Silver was considered for this role because of its wide spectrum of antibacterial susceptibility, low incidence of resistance, and its ability to persistently inhibit bacteria after binding to collagen matrices. Demineralized bone matrix prepared from rat diaphyseal bone segments was treated by exposure fo AgCl, AgNO3 and NaNO3 solutions prior to lyophilization. The resulting material was tested for bacterial inhibition after incubation in saline solutions for various times and showed inhibition persisting for at least four weeks (Ag-treated material only). Silver treating the matrix was found to partially inhibit the osteoinductive capacity at 10(-3) and 10(-2) M but not at 10(-5) M as measured by intramuscular implantation in the rat for six weeks. Control and NaNo3-treated specimens showed normal bone growth as measured by ashing and by 99mTc binding, and confirmed by radiologic densities. Histologic sections showed dense microdeposits on dense material predominately near the decalcified bone surfaces, but also within the matrix. The results suggest that pretreatment with silver at concentrations in the 10(-4) range would render the implant material antibacterial, protect its sterility, and leave the osteoinductive capacity intact.     

The Clinical Use of Allografts,Demineralized Bone Matrices,Synthetic Bone Graft Substitutes and Osteoinductive Growth Factors: A Survey Study

The emergence of new bone grafting options and alternatives has led to significant uncertainty when determining the most appropriate product for surgical procedures requiring bone graft in orthopedics. Allografts, demineralized bone matrices, synthetic bone graft substitutes, and osteoinductive growth factors are all viable options, yet there is a lack of data reporting clinical usage of these products. This correspondence reports on the use of bone grafting products at the Hospital for Special Surgery for a 27-month period and makes recommendations based on surgical usage, safety, and cost. Approximately half (48.6%) of all bone graft substitutes were implanted during spinal surgery. Arthroplasty, trauma, and foot/hand cases all used considerable amounts of bone grafting products as well (20.1%, 19.0%, 12.1%, respectively). Considerable differences were noticed in usage of bone grafting products among each orthopedic discipline. Of all bone graft substitutes used in arthroplasty, 14.4% were demineralized bone matrices, whereas 56.8% were allografts. Demineralized bone matrix grafts were used in 82% of trauma surgery and 89% of foot/hand cases. An increase in synthetic bone graft alternatives was noticed near the end of our investigation period.     

Anterior cervical interbody fusion with rhBMP-2 and tantalum in a goat model.

BACKGROUND CONTEXT: Tricortical autogenous iliac crest has long served as the gold standard for arthrodesis after cervical discectomy. The added morbidity resulting from bone graft harvest may be eliminated by the use of a biocompatible synthetic bone graft substitute with osteoconductive abilities, and when used with an osteoinductive agent, such as recombinant bone morphogenic protein (rhBMP)-2, it may facilitate arthrodesis similar to autograft. PURPOSE: To determine by radiographic and histologic analysis whether tantalum with and without rhBMP-2 can facilitate bony ingrowth and arthrodesis in an animal model. STUDY DESIGN/SETTING: Single-level anterior cervical discectomy and fusion was performed using a tantalum bone graft substitute with and without rhBMP-2 in a previously established goat model for anterior cervical fusion. METHODS: Eight goats underwent single-level anterior cervical discectomy and stabilization with a porous tantalum implant. There were four goats in each experimental group. Group A underwent anterior cervical stabilization with tantalum alone, whereas in Group B rhBMP-2 was added to the tantalum implant. The goats were sacrificed at 12 weeks, and their cervical spines were removed for histologic and radiological analysis. RESULTS: Only one of four goats in Group A had any bony ingrowth into the tantalum. Three of four goats in Group B demonstrated bony ingrowth. The average extent of bony ingrowth at the perimeter of the tantalum in Group A was 2.5% compared with 12.5% in Group B. Similarly, the volume of bony ingrowth within the tantalum was 2.5% in Group A and 10% in Group B. The difference was not statistically significant. CONCLUSIONS: The data in this pilot study suggest that tantalum may function as a synthetic osteoconductive bone graft substitute. The addition of rhBMP-2 may facilitate osteoinduction within a synthetic osteoconductive implant. The sample size in this study was too small for statistical significance. The present animal model as used in this study was inadequate for cervical arthrodesis where rigid implant fixation is desired.     

Effect of hydrogen peroxide on osteoinduction by demineralized bone

The osteoinductive capacity of demineralized bone matrix (DBM) has led to wide use of this material for surgical reconstruction. Preparation of DBM often includes sterilization with ethylene oxide, disinfection with various chemical agents, or irradiation. Exposure to hydrogen peroxide (H2O2) is used for both sterilization and bleaching of bone, the latter primarily for cosmetic reasons. We investigated the effect of H2O2, on the osteoinductive capacity of DBM. Cortical bone implants prepared from rat femurs were placed into 3% H2O2 solution. Control specimens were not exposed to H2O2. Bones were then lipid-extracted, demineralized, sterilized with ethylene oxide, and freeze-dried in an identical manner. Allografts were implanted into rat hosts for 1 to 3 weeks. Osteoinduction proceeded rapidly in implants not exposed to H2O2, with chondrocytes and new bone appearing in the implant. After 3 weeks, perforations in the implant were largely replaced with new bone. In contrast, osteoinduction did not occur in implants treated with H2O2. Perforations in H2O2-treated implants were filled with vascularized fibrous tissue, but no cartilage or bone. These findings reveal that H2O2 used for disinfection or bleaching of DBM can abolish its osteoinductive capacity in rats.