Ethene oxide and bone induction: Controversy remains

There is controversy as to whether ethene oxide (“ethylene oxide”, EO) sterilization destroys the bone-inducing capacity of demineralized bone matrix (DBM) or not. Correctly performed studies seem to support both opinions. Bone conductive properties of fresh frozen, defatted bone grafts are greatly impaired by EO sterilization, whereas purified inductive proteins resist EO. Studies showing destruction of osteoinductive capacity used nonpulverized DBM, whereas the others used powder. This could be the key to resolving the controversy, because if EO treatment reduces the cells' ability to penetrate a cortical graft and to reach inductive proteins inside it, it may appear noninductive after EO sterilization, even though BMP molecules may be intact. On the other hand, cells could easily penetrate the powder implants.

We compared the effect of EO sterilization on the inductive capacity of demineralized cortical bone with that of DBM powder, using allogeneic material in rats. Cortical pieces lost all inductive capacity by EO sterilization, whereas the powder yielded a calcium content which was at best one fourth of the un-sterilized. The concentrations of residual EO, ethene chlorohydrin and ethene glucol at implantation were far below approved levels. Another difference between studies is the humidity during EO treatment. In our hands, humidification reduced bone yield by half.

In conclusion, EO sterilization may impair the biological performance of bone inductive implants by reducing cell penetration into bulk material. However, DBM powder, when correctly sterilized, also yielded scanty amounts of bone.     

In vivo analysis of the half-life of the osteoinductive potential of demineralized bone matrix using diffusion chambers

Summary Subcutaneous implantation of demineralized bone matrix (DBM) from rat initiates a sequence of developmental events that results in endochondral bone formation. This investigation examined the modification of the osteoinductive potential of DBM during the intial stages of this developmental cascade. Diffusion chambers (DC), constructed with filters of known pore size, permitting or excluding cells from entering the chambers, and containing DBM were subcutaneously implanted into Long-Evans male rats for specific time periods (1–7 days). DC were recovered and the osteoinductive potential of the matrix from these chambers was then tested by subcutaneous implantation and assaying the resulting day 11 plaque tissue enzymatically for alkaline phosphatase activity, and histologically for evidence of chondrogenesis and osteogenesis. The possible modification of DBM by local systemic factors (enzymatic degra-dation) or contact by polymorphonuclear leukocytes (PMNs) was also investigated. We have concluded from this study that the osteoinductive potential of DBM has a half-life of 5–7 days following implantation and although the enzymes collagenase, elastase, and trypsin abolished this activity, pepsin significantly enhanced it. Culture of PMNs with matrix prior to its implantation appeared to have little effect. Furthermore, during the initial stages of matrix-induced endochondral bone formation, DBM serves as both the instructive inducer and permissive substratum required in this process.     

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.     

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.     

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.     

同种异体脂肪干细胞复合脱钙骨修复大鼠尺骨缺损

目的探讨同种异体脂肪干细胞修复管状骨缺损的可行性。方法获取SD大鼠的腹股沟处脂肪,分离培养脂肪干细胞（Adipose-Derived Stem Cells,ADSCs）;鼠第3代ADSCs与脱钙骨复合,24 h后进行成骨诱导培养。检测细胞在材料表面的生长及成骨分化能力。建立鼠两侧尺骨缺损模型,分别植入鼠ADSCs-脱钙骨复合物（实验侧）和单纯脱钙骨材料（对照侧）;8周、24周后取样,行DR和组织学检测,观察成骨情况。结果 ADSCs能在脱钙骨上很好地黏附和生长,并维持成骨分化能力。细胞-材料复合物植入24周后,DR显示实验侧有新生骨基质长成,对照侧未见骨组织生成。组织学检测显示,实验侧缺损区被典型的骨组织取代,可见新生骨小梁附着于脱钙骨表面;对照侧只有少量的骨组织和纤维组织充填。结论 ADSCs-脱钙骨材料复合物植入,能成功修复临界大小的管状骨缺损。     

The matrix of endochondral bone differs from the matrix of intramembranous bone

Summary Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induced cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM.     

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.     

Influence of irradiation on the osteoinductive potential of demineralized bone matrix

Summary Samples of demineralized bone matrix (DBM) were exposed to graduated doses of radiation (1–15 Megarad) (Mrad) utilizing a linear accelerator and then implanted into the thoracic region of Long-Evans rats. Subcutaneous implantation of DBM into allogenic rats induces endochondral bone. In response to matrix implantation, a cascade of events ensues; mesenchymal cell proliferation on day 3 postimplantation, chondrogenesis on day 7, calcification of the cartilagenous matrix and chondrolysis on day 9, and osteogenesis on day 11 resulting in formation of an ossicle containing active hemopoietic tissue. Bone formation was assessed by measuring alkaline phosphatase activity, the rate of mineralization was determined by measuring⁴⁵Ca incorporation to bone mineral, and⁴⁰Ca content measured the extent of mineralization; acid phosphatase activity was used as a parameter for bone resorption. The dose of radiation (2.5 Mrad) currently used by bone banks for sterilization of bone tissue did not destroy the bone induction properties of DBM. Furthermore, radiation of 3–5 Mrad even enhanced bone induction, insofar as it produced more bone at the same interval of time than was obtained from unirradiated control samples. None of the radiation doses used in these experiments abolished bone induction, although the response induced by matrix irradiated with doses higher than 5 Mrad was delayed.     

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.     

Rapid quantitative bioassay of osteoinduction.

We developed a reproducible, relatively rapid bioassay that quantitatively correlates with the osteoinductive capacity of demineralized bone matrix obtained from human long bones. We have found that Saos human osteosarcoma cells proliferate in response to incubation with demineralized bone matrix and that an index of this proliferative activity correlates with demineralized bone matrix-induced osteogenesis in vivo. The bioassay (Saos cell proliferation) had an interassay coefficient of variation of 23 +/- 2% and an intra-assay coefficient of 11 +/- 1%. Cell proliferation was normalized to a standard sample of demineralized bone matrix with a clinically high osteoinductive capacity, which was assigned a value of one. The Saos cell proliferation for each sample was related to the standard and assigned a value placing it into the low (0.00-0.39), intermediate (0.40-0.69), or high (0.70-1.49) osteoinductive index group. Osteoinduction of human demineralized bone matrix was quantitated by expressing new bone formation as a function of the total bone volume (new bone plus the demineralized bone powder). The demineralized bone matrix was placed in pouches formed in the rectus abdominis muscles of athymic rats, and endochondral bone formation was assessed at 35 days following implantation, when marrow spaces in the ossicles were formed by new bone bridging the spaces between demineralized bone matrix particles. The proliferative index correlated with the area of new bone formation in histological sections of the newly formed ossicles. When the proliferative index (the osteoinductive index) was divided into low, intermediate, and high groups, the correlation between it and new bone formation (osteoinduction) was 0.850 (p < 0.0005) in 25 samples of demineralized bone matrix. There was no overlap in the osteoinduction stimulated between the samples with low and high osteoinductive indices. We conclude that the proliferation assay is useful for the routine screening of bone allograft donors for osteoinductive potential. Furthermore, the two-dimensional area of new bone formation, as it relates to total new bone area, is a quantitative measure of osteoinduction.     

Decalcified and undecalcified cancellous bone block implants do not heal diaphyseal defects in dogs

Summary In a previous study it has been shown that granulae of decalcified bone matrix do not induce bony healing of 8-week-old mid-diaphyseal defects in dogs. The aim of this study was to test whether osteoinduction combined with the osteoconductive mechanisms provided by the natural structure of cancellous bone blocks would yield better results. A 30-mm-long diaphyseal defect of the left ulna was created in eight adult mongrel dogs and the bone was stabilized with a plate. A Silastic spacer was inserted in the defect for 8 weeks, followed by implantation of frozen undecalcified or decalcified allogeneic cancellous bone blocks for 16 weeks. Healing was analyzed using morphologic methods. At 16 weeks after implantation all grafts had been resorbed. In the decalcified group one defect healed, while none in the other group did so. The implant material was bioassayed in nude rats for osteoinductivity, which was found to be low in decalcified matrix and not detectable in undecalcified bone. Conclusion: Allogeneic cancellous bone blocks, demineralized or not, have no osteoinductive capacity and no osteoconductive function that promotes healing of mid-diaphyseal bone defects in dogs.     

Treatment of nonunion by percutaneous injection of bone marrow and demineralized bone matrix. An experimental study in dogs

Successful treatment of nonunited fractures remains a major clinical challenge. Because bone marrow and demineralized bone matrix (DBM) are capable of stimulating osteogenesis, experiments were designed to test the effectiveness of bone marrow or DBM or both when injected percutaneously into a canine nonunion model. Six-millimeter segmental defects were created in the midtibial diaphysis of 24 adult mongrel dogs and held distracted by external fixation. For comparative purposes, a 0.5-mm osteotomy was created in five dogs. Five weeks later, the 6-mm defects were injected with either saline, autogeneic marrow, DBM powder, a composite of bone marrow and DBM, or treated by open grafting techniques with autogenic cancellous bone. Healing of the defect was evaluated roentgenographically, biomechanically (three-point bending), histologically, and biochemically 13 weeks postsurgery. Marrow and DBM stimulated defect healing. However, the combination of bone marrow with DBM produced a synergistic response in the defect, which was greater than the sum of either marrow or DBM alone. Healing in the composite-grafted dogs was comparable to those treated by standard cancellous bone grafting. These data suggest that percutaneous injection of bone marrow and DBM may be a potential alternative that offers numerous advantages over standard open grafting techniques in the treatment of fractures with nonunited defects.     

Heterotopic new bone formation causes resorption of the inductive bone matrix

The bone matrix of growing rats was labeled by multiple injections of 3H-proline, and demineralized bone matrix (DBM) was prepared. The DBM was allotransplanted heterotopically into growing rats. New bone formation was induced in and around the implants. The new bone formation was accompanied by a decrease in the content of 3H; 20 and 30 days after implantation, 72% and 46%, respectively, of the activity remained in the implants. Daily injections of indomethacin (2 mg/kg) inhibited calcium uptake by about 20% at 20 and 30 days and inhibited the release of 3H from the DBM to a similar degree. Heterotopic bone induction by DBM is accompanied by matrix resorption, and inhibition of the new bone formation decreases the resorption of DBM.     

Induced membranes secrete growth factors including vascular and osteoinductive factors and could stimulate bone regeneration.

Based on a new concept, a procedure combining induced membranes and cancellous autografts allows the reconstruction of wide diaphyseal defects. In the first stage of this procedure, a cement spacer is inserted into the defect; the spacer is responsible for the formation of a pseudo-synovial membrane. In the second stage, the defect is reconstructed two months later by an autologous cancellous bone graft. The aim of this study was to evaluate the histological and biochemical characteristics of these membranes induced in rabbits. Histological studies carried out two, four, six, and eight weeks following implantation revealed a rich vascularization. Qualitative and quantitative immunochemistry showed production of growth factors (VEGF, TGFbeta1) and osteoinductive factors (BMP-2). Maximum BMP-2 production was obtained four weeks after the implantation, and, at this time, induced membranes favored human bone marrow stromal cell differentiation to the osteoblastic lineage. Should these results be confirmed in humans, bone reconstruction could be carried out earlier than previously thought and in better conditions than expected, the membrane playing the role of an in situ delivery system for growth and osteoinductive factors.     

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.     

Safety and efficacy of commercially available demineralised bone matrix preparations: a critical review of clinical studies

Demineralised bone matrix (DBM), a form of allograft, possesses the properties of osteoinductivity and osteoconductivity. A large body of data obtained from extensive preclinical studies have clearly supported the utility of DBM in human clinical settings. However, it is now recognized that various DBM configurations may differ considerably with regard to their bone inductive activity. Several factors could account for such variability, including the biologic properties of the graft, the host environment, and the methods of allograft preparation. The differing efficacy of DBM products may also depend on differences in particle size and shape, donor selection criteria, protocols for collection and storage, as well as DBM carrier materials. Several comparative studies have confirmed the differences in the osteoinductive potential of various DBM preparations. The purpose of the present review is to provide a critical overview of the current applications of DBM in a clinical setting.     

Effects of indomethacin on demineralized bone-induced heterotopic ossification in the rat

Indomethacin inhibits bone formation when treatment is initiated before the implantation of demineralized bone matrix (DBM). For the inhibition of bone induction to occur, indomethacin treatment had to be initiated 6 h or more before implantation of DBM. Initiating the drug treatment at or after the time of DBM implantation had no effects on the amounts of new bone formed. The inhibition by indomethacin is dose related over a range between 0.04 and 4 mg/kg body weight. Recovered day-1 DBM implants, transplanted into indomethacin pre- and posttreated syngeneic rats, formed bone at the same rate as controls did. However, recovered day-1 DBM implants lyophilized before transplantation showed decreased bone formation but significant dystrophic calcification as judged by a lower alkaline phosphatase activity and an elevated calcium content.     

Effect of radiation sterilization on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing.

The effect of various doses of ionizing radiation on the osteoinductive properties of decalcified bone matrices implanted heterotopically and on the rate of remodeling of nondecalcified bone grafts implanted orthotopically in allogeneic systems was studied. Decalcified bone matrices and nondecalcified bone grafts were preserved by lyophilization or by deep-freezing and were subsequently irradiated with appropriate doses at room temperature or at -72 degrees. Lyophilized matrices irradiated at room temperature with 35 and 50 kGy, respectively, were completely resorbed five weeks after heterotopic implantation into the muscles and did not induce osteogenesis, whereas the resorption of deep-frozen ones irradiated with the same doses at -72 degrees was slower and new bone formation was induced. The preservation of the osteoinductive capacity of irradiated, deep-frozen matrices may depend on two factors: reduction of radiation damage on the inducing agents and collagen irradiated in the presence of water, which may diminish the rate of matrix resorption. The rate of remodeling of undecalcified deep-frozen bone implants irradiated at -72 degrees and grafted orthotopically was higher than that of lyophilized ones irradiated at room temperature. It is possible that the temperature during irradiation plays a critical role in protection against radiation damage.     

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.