New-bone formation by osteogenic protein-1 and autogenic bone marrow in a critical tibial defect model in sheep

AIM: Osteogenic Protein-1 (OP-1) is known to be a very potent osteoinductive growth factor. However, experimental studies using critical-size defect models in the weight-bearing lower extremity show non-uniform results. Therefore, we studied the osteoinductivity of OP-1 in a tibial worst-case defect model in sheep. Potential improvement of OP-1 induced new bone formation using a composite graft with autogenous bone marrow was to be investigated. METHOD: In 19 sheep a 5 cm segmental defect of the tibial diaphysis was treated by intramedullary nailing and filled with the following implants: 5 mg OP-1 + inactivated demineralized bone matrix (group 1; n = 6); 5 mg OP-1 + inactivated demineralized bone matrix + 5 ml autogenous bone marrow (group 2; n = 5); autogenous cancellous bone (group 3; n = 4), or inactivated demineralized bone matrix + 5 ml autogenous bone marrow (group 4; n = 4). RESULTS: In total, 3 out of 10 defect sites treated with OP-1 were completely bridged radiographically by 12 weeks. Initially, x-rays showed accelerated new bone formation by use of the composite grafts containing OP-1 and autogenous bone marrow. However, 12 weeks post surgery 3D-CT-volumetry could not detect significant differences of new bone formation within the defect sites treated by OP-1 with or without bone marrow, while new bone formation by autogenous cancellous bone was better than by OP-1. CONCLUSION: In our worst case defect model, the osteoinductive potential of OP-1 is initially accelerated but 12 weeks post surgery not increased when combined with autogenous bone marrow transplantation. So far, critical segmental bone defects of the weight-bearing lower extremity can not be bridged regularly in our model by use of OP-1. Therefore, for the treatment of such critical defects with rotational instability the examined application device of OP-1 can not yet be recommended.     

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.     

Dog bone less osteogenetic than rat bone Bone-matrix transplants in nude rats

Demineralized bone matrix and bone-matrix gelatin prepared from cortical rat bone, and from cortical and cancellous canine bone were implanted into muscle pouches of nude rats for 6 weeks. Evaluation was done by histology, histomorphometry, and determination of alkaline phosphatase. Rat matrix consistently induced new bone and high phosphatase levels. Canine matrix induced but small amounts of bone and lower phosphatase levels, with cortical matrix somewhat more inductive than cancellous matrix; demineralized cancellous bone matrix from the dog was the only material tested not showing any inductivity. Irrespective of bone type or species, gelatin had clearly higher induction capacity than demineralized bone matrix.     

Dog bone less osteogenetic than rat bone. Bone-matrix transplants in nude rats

Demineralized bone matrix and bone-matrix gelatin prepared from cortical rat bone, and from cortical and cancellous canine bone were implanted into muscle pouches of nude rats for 6 weeks. Evaluation was done by histology, histomorphometry, and determination of alkaline phosphatase. Rat matrix consistently induced new bone and high phosphatase levels. Canine matrix induced but small amounts of bone and lower phosphatase levels, with cortical matrix somewhat more inductive than cancellous matrix; demineralized cancellous bone matrix from the dog was the only material tested not showing any inductivity. Irrespective of bone type or species, gelatin had clearly higher induction capacity than demineralized bone matrix.     

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.     

Bone matrix and marrow versus cancellous bone in rabbit radial defects

Implants of demineralized bone matrix induce new bone formation. In order to estimate the possible clinical usefulness of this phenomenon, autologous cancellous bone grafts were compared with composite grafts of bone matrix and marrow. Cancellous bone from the tuber ischii of the rabbit was transplanted to a preformed radial defect in the same animal. On the opposite side, a similar defect was filled with a mixture of either allogenous or autogenous bone-matrix particles and autogenous bone marrow. After 25 days, calcium 45 was injected intravenously. Three days later the animals were killed. Standardized segments of the rabbit's forearms, containing the middle of the defect, were cut out, ashed, and analyzed for 45Ca activity. No side difference in 45Ca deposition was found. The callus ash weight of the allogenous matrix-transplanted side was approximately 60% of that of the cancellous bone side. This side difference of ash weights corresponds to the estimated initial mineral content of the cancellous graft. Nontransplanted defects had very low ash weight and 45Ca activity. Thus, in the rabbit, composite grafts of bone matrix and marrow produce a bone yield comparable to that of cancellous bone.     

A study of the mechanical strength of long bone defects treated with various bone autograft substitutes: an experimental investigation in the rabbit

This study was designed to determine which of several bone grafting materials would be the most efficacious substitute for autogenous bone graft in the treatment of segmental long bone defects. The experimental model was a 1-cm defect in the rabbit ulna. The control group had nothing implanted in the defect. The six grafts tested were: (a) autogenous iliac crest bone, (b) autogenous cortical bone (ulna), (c) hydroxylapatite, (d) hydroxylapatite-demineralized bone matrix (allograft) composite graft, (e) freeze-dried bone (allograft), and (f) demineralized bone matrix (allograft). At 6 weeks postoperatively, the ulnas were harvested, examined radiographically, and tested mechanically in torsion. The radiographic examination proved to be of little value because some materials were radiodense at the time of implantation. The rates (percentage) of union, torques at failure, and energy to failure values were statistically significantly higher than control for all groups except hydroxylapatite. We concluded that demineralized bone matrix and hydroxylapatite-demineralized bone matrix composite graft compare favorably with cortical replacement (autograft) in mechanical strength and rate of union and therefore may be satisfactory substitutes for bone grafting. Freeze-dried bone did not appear to be as satisfactory because of its low mean energy to failure, but statistical analysis failed to confirm this opinion. Hydroxylapatite graft, when used alone, does not appear to be a suitable material for grafting segmental bone defects.     

Bone matrix and marrow versus cancellous bone in rabbit radial defects

Summary Implants of demineralized bone matrix induce new bone formation. In order to estimate the possible clinical usefulness of this phenomenon, autologous cancellous bone grafts were compared with composite grafts of bone matrix and marrow. Cancellous bone from the tuber ischii of the rabbit was transplanted to a preformed radial defect in the same animal. On the opposite side, a similar defect was filled with a mixture of either allogenous or autogenous bone-matrix particles and autogenous bone marrow. After 25 days, calcium 45 was injected intravenously. Three days later the animals were killed. Standardized segments of the rabbit's forearms, containing the middle of the defect, were cut out, ashed, and analyzed for ⁴⁵Ca activity. No side difference in ⁴⁵Ca deposition was found. The callus ash weight of the allogenous matrix-transplanted side was approximately 60% of that of the cancellous bone side. This side difference of ash weights corresponds to the estimated initial mineral content of the cancellous graft. Nontransplanted defects had very low ash weight and ⁴⁵Ca activity. Thus, in the rabbit, composite grafts of bone matrix and marrow produce a bone yield comparable to that of cancellous bone.     

Qualitative and quantitative analysis of orthotopic bone regeneration by marrow

A rat model of a femoral segmental defect was used to specifically test the hypothesis that autogenous marrow has the osteogenic capability to heal a bone defect. The variables analyzed included the ratio of the marrow volume to the defect, implantation of live or dead marrow, and remodeling of established nonunions by implantation of live marrow. The uniqueness of this model allows biomechanical evaluation of the new bone formed by the implant. When live marrow was implanted, woven bone formed at 3 weeks, progressing to early lamellar bone at 6 weeks, with subsequent remodeling for as long as 12 weeks in a volumetric fashion (p < 0.05). Bone marrow, when placed in a fresh femoral defect and given in sufficient amounts, produced a rate of union comparable with that of autologous bone grafts. Mature lamellar bone formed by marrow was evaluated biomechanically: the results were statistically comparable with those of cancellous bone grafts at 12 weeks. Significant bone formation occurred when marrow was percutaneously injected in femoral nonunions, although union and remodeling did not take place in this rat model. Implantation of dead marrow resulted in rare cellular infiltration and minimal bone formation in a manner comparable with that of autogenous cancellous bone grafts. These results indicate that bone marrow can lead to structurally functional bone regeneration in an orthotopic location.     

Autogenous bone graft: donor sites and techniques

Autogenous cancellous bone graft provides an osteoconductive, osteoinductive, and osteogenic substrate for filling bone voids and augmenting fracture-healing.The iliac crest remains the most frequently used site for bone-graft harvest, but the proximal part of the tibia, distal end of the radius, distal aspect of the tibia, and greater trochanter are alternative donor sites that are particularly useful for bone-grafting in the ipsilateral extremity.The most common complication associated with the harvest of autogenous bone graft is pain at the donor site, with less frequent complications including nerve injury, hematoma, infection, and fracture at the donor site.Induced membranes is a method that uses a temporary polymethylmethacrylate cement spacer to create a bone-graft-friendly environment to facilitate graft incorporation, even in large segmental defects.     

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.     

Tissue-engineered bone formation in vivo using a novel sintered polymeric microsphere matrix

We have evaluated in vivo a novel, polymer-based, matrix for tissue engineering of bone. A segmental defect of 15 mm was created in the ulna of New Zealand white rabbits to determine the regenerative properties of a porous polylactide-co-glycolide matrix alone and in combination with autogenous marrow and/or the osteoinductive protein, BMP-7. In this study four implant groups were used: 1) matrix alone; 2) matrix with autogenous marrow; 3) matrix with 20 microg of BMP-7; and 4) matrix with 20 microg of BMP-7 and autogenous marrow. The results showed that the degree of bone formation was dependent on the properties of the graft material. The osteoconductive sintered matrix structure showed significant formation of bone at the implant-bone interface. The addition of autogenous marrow increased the penetration of new bone further into the central area of the matrix and also increased the degree of revascularisation. The osteoinductive growth factor BMP-7 induced penetration of new bone throughout the entire structure of the implant. The most effective treatment was with the combination of marrow cells and osteoinductive BMP-7.     

The biology of bone grafting

Many approaches are used to repair skeletal defects in reconstructive orthopaedic surgery, and bone grafting is involved in virtually every procedure. The type of bone graft used depends on the clinical scenario and the anticipated final outcome. Autogenous cancellous bone graft, with its osteogenic, osteoinductive, and osteoconductive properties, remains the standard for grafting. However, the high incidence of morbidity during autogenous graft harvest may make the acquisition of grafts from other sources desirable. The clinical applications for each type of bone graft are dictated by the structure and biochemical properties of the graft. An elegant cellular and molecular cascade follows bone transplantation. Bone graft incorporation within the host, whether autogenous or allogeneic, depends on many factors: type of graft (autogenous versus allogeneic, vascular versus nonvascular), site of transplant, quality of transplanted bone and host bone, host bed preparation, preservation techniques, systemic and local disease, and mechanical properties of the graft.     

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.     

Cranioplasty using allogeneic perforated demineralized bone matrix with autogenous bone paste

The efficacy of allogeneic perforated demineralized bone matrix with autogenous bone paste in the treatment of full-thickness cranial defects was evaluated in 10 consecutive patients between June 1998 and December 1998. The skull defects resulted from trauma in 9 patients and removal of a cranial tumor in 1 patient. The size of the skull defects ranged from 8 x 6 cm to 11 x 12.5 cm. Follow-up averaged 33 months for all patients. Postimplantation evaluations included serial photographs, repeated physical examination, and three-dimensional computed tomography for all patients. Visual inspection of the implanted biomaterial 6 months later was possible in 1 patient. The contour of the reconstructed skull was acceptable aesthetically without any secondary depression noted during the follow-up period. Three-dimensional computed tomographic scans taken 2 years after implantation indicated that the allogeneic perforated demineralized bone matrix provided a matrix for new bone formation with remarkable osteoinductive potential for new bone formation. The autogenous bone paste was able to caulk the demineralized bone matrix and fill the contour irregularities and gaps of the reconstructed cranium. The results from this clinical study indicated that allogeneic perforated demineralized bone matrix with autogenous bone paste is a promising alternative to an autogenous bone graft and or alloplastic material for cranioplasty.     

Bone graft materials. An overview of the basic science

Autograft, allograft, and synthetic bone graft substitute materials play an important role in reconstructive orthopaedic surgery, and understanding the biologic effects of these materials is necessary for optimum use. Although vascularized and cancellous autograft show optimum skeletal incorporation, host morbidity limits autograft availability. Experimental studies have confirmed an immune response to allograft bone, but the clinical significance of this response in humans still is unclear. Small amounts of cancellous allograft in humans usually are remodeled completely; large allografts become incorporated by limited, surface intramembranous bone formation suggesting that these graft are primarily osteoconductive. Several synthetic skeletal substitute materials also are osteoconductive, and may show remodeling characteristics similar to allograft. Demineralized bone matrix and some isolated or synthetic proteins can induce endochondral bone formation, and therefore are osteoinductive. The extent and distribution of remodeling of bone graft materials are influenced by many factors, including the quality of the host site and the local mechanical environment (strain). Graft materials are likely to become more specialized for use in specific clinical applications, and composite preparations may soon provide bone graft materials with efficacy that equals or exceeds that of autogenous grafts.     

Bridging large defects in bone by demineralized bone matrix in the form of a powder. A radiographic, histological, and radioisotope-uptake study in rats

Demineralized bone powder was used as an osteoinductive substance to bridge very large defects (more than 50 per cent of the total length of the bone) in one radius of each of thirty-three rats. An identical defect was produced in the contralateral radius of each animal for use as a control. The defect on the control side was left unbridged or was bridged by large chips of autologous bone or an autologous inlay graft. All rats showed formation of new bone throughout the length of the radial defect only on the side in which the demineralized bone powder had been implanted. The control side, in which an autologous graft in the form of chips or inlay had been implanted, showed resorption of the graft. The maximum rate of formation of bone occurred fifteen to twenty-one days after implantation of the demineralized bone powder. At thirty-five days, the experimental defect was fully bridged, forming solid bone, in 71 per cent of the rats, and the remaining 29 per cent showed bridging of 95.8 per cent of the length of the defect, with union on one side. Analysis of the sequential radiographs, technetium-99m scans, and histological findings showed that the formation of bone and bridging of the defect were superior on the side in which the demineralized bone powder had been implanted compared with the side in which pieces of autologous bone or an autologous inlay graft had been used.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evaluation of human demineralized bone matrices in a rat femoral defect model

The osteoconductive and osteoinductive potential of two human allogeneic demineralized bone matrix putties were compared in a critical-sized athymic rat femoral defect model. Defects were treated with (1) a demineralized bone matrix in a hyaluronic acid carrier, (2) a demineralized bone matrix in a glycerol carrier, (3) a hyaluronic acid carrier alone, or (4) with no implant. Radiographic examinations and histologic analyses were done at 4, 8, and 16 weeks postoperatively. Eight of the 48 defects treated with a demineralized bone matrix and none of the 36 surgical controls showed complete radiographic healing by 16 weeks and no statistically significant difference between the radiographic scores for the two demineralized bone matrix preparations was found. On histologic review, both preparations of demineralized bone matrix had passive remineralization. The largest foci of endochondral ossification were seen in limbs treated with a demineralized bone matrix in a hyaluronic acid carrier. The 8-mm rat femoral defect allows for stringent assessment of the osteoinductive potential of bone graft substitutes. Hyaluronic acid and glycerol are viable carriers for demineralized bone matrices. As both de-mineralized bone matrices tested provided an adequate osteoconductive matrix and showed some, although limited, osteoinductive capacity, these materials should be used in clinical practice only as bone graft extenders or enhancers.     

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.     

Osteogenesis in cranial defects and diffusion chambers: Comparison in rabbits of bone matrix, marrow, and collagen implants

In rabbits, we compared calcification and bone formation by bone marrow, acid-demineralized bone matrix and glutaraldehyde-cross-linked Type I collagen implanted in intramuscular diffusion chambers or in trephine skull defects. The rabbits were killed 4 weeks postimplantation and calcification and osteogenesis were evaluated radiographically and histologically, and by calcium and alkaline phosphatase assays. Bone marrow produced bone and fibrous tissue within the chambers and had high alkaline phosphatase levels. Bone matrix in chambers with intact filters failed to induce bone formation within and outside the chambers, while glutaraldehyde-cross-linked collagen produced only scant calcific deposits following implantation in either diffusion chambers or skull defects. Central areas of skull defects implanted with bone marrow were partially repaired with new bone and had high calcium and alkaline phosphatase levels, but not as high as defects implanted with demineralized bone matrix.