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.     

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.     

The effect of various sterilization procedures on the osteoinductive properties of demineralized bone matrix]

To minimize potential infection following the transplantation of allogeneic bone, extremely rigorous selection of donors and careful processing and storage of samples are required. Other major problems related to allogeneic transplants, such as reduced osteogenic properties and immunological reactions, led to the development of demineralized bone matrix (DBM). This osteoinductive bone extract is largely free of antigens and is easy to produce. However, to eliminate the potential risk of infection, DBM should be sterilized prior to implantation. The purpose of this study was to investigate the influence of different sterilization techniques on the osteoinductive properties of DBM. A series of 76 cortical defects (drill holes) 0.6 cm in diameter in the tibiae of 11 Merino sheep were filled with DBM in addition to autogeneic and allogeneic cancellous bone. Prior to implantation DBM was sterilized by autoclaving, gamma irradiation, or application of ethylene oxide or ethyl alcohol. A further 12 drill holes were left empty as controls. The formation of new bone was examined 3 and 6 weeks postoperatively, using histological, fluorescent-optical and microradiographical techniques. The amount of newly formed bone was also quantified. Apart from autoclaved DBM all matrix grafts showed excellent new bone formation following sterilization, by far exceeding the formation with allogeneic cancellous bone.     

Ethylene oxide does not extinguish the osteoinductive capacity of demineralized bone: A reappraisal in rats

We examined the influence of ethylene oxide (EO) and gamma irradiation on the osteoinductive capacity of demineralized bone. Demineralized bone powder prepared from Wistar rats was exposed to EO (55 °C or 40 °C) or gamma irradiation (25 KGy) or was preserved in ethanol. Sterilely-prepared bones served as controls. The powder was packed in a gelatin capsule and implanted for 6 weeks in muscles of 6-week-old female rats. Exposure of demineralized bone particles to EO 55 °C resulted in an almost complete loss of osteoinductivity. Irradiated bones lost about 40% of their osteoinductive capacity, while sterilization with EO at 40 °C resulted in only a slight alteration of the osteoinductivity, as assessed by the recovered weight ratio, calcium content, alkaline phosphatase activity measurements and histo-morphometry. Ethanol treatment had no influence on the new bone yield when compared to controls.

As EO exposure at 40 °C is a true sterilization procedure, it can be recommended in a clinical setting for its small effect on osteoinductive capacity as assessed experimentally in rats.     

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.     

Augmentation of osseous phenotypes in vivo with a synthetic peptide.

The synthetic peptide B2A2-K-NS augmented the in vitro expression of osseous phenotypes when cells were stimulated with BMP-2, an osteoinductive growth factor. B2A2-K-NS significantly enhanced the effects of BMP-2-induced alkaline phosphatase activity and mineralization. In the absence of BMP-2, B2A2-K-NS did not have an effect on these endpoints. Based on these observations, in vivo studies were conducted to evaluate if B2A2-K-NS could augment osseous phenotypes in an osteoinductive environment in which BMP-2 should be present. In one study, human demineralized bone matrix (DBM) was used to generate an osteoinductive environment and the effects of B2A2-K-NS on ectopic mineralization of subcutaneous implants evaluated. In the second study, a noncritical sized defect in rabbit ulnas with inherent reparative capacity was used as the osteoinductive environment and was treated with or without B2A2-K-NS. In the DBM studies, B2A2-K-NS augmented mineralization as determined using a combination of radiographic analysis and von Kossa staining at 4 weeks postimplant. In the rabbit ulna model, B2A2-K-NS significantly increased the radiographic bone density in the defects compared to carrier-only or no-treatment controls after 6 weeks. Histological staining confirmed that B2A2-K-NS generated a pronounced bone repair response. The results are consistent with the hypothesis that B2A2-K-NS augments osseous phenotypes in an osteoinductive environment, and suggests that B2A2-K-NS may have clinical utility.     

Effect of sterilization on osteoinduction Comparison of five methods in demineralized rat bone

The aim of this study was to find a safe, effective sterilization method that does not destroy the bone-inductive capacity of demineralized bone implants. Five sterilizing agents were tested in rats. Implants procured and processed under sterile conditions served as controls. New bone formation was evaluated by determining dry weight, calcium content, and Sr-85 incorporation of the induced ossicles.

Glutaraldehyde solution, formaldehyde gas, and ethylene oxide destroyed almost all the bone-inductive capacity. Irradiation by 2.5 Mrads Co-60 resulted in a loss of about half of the inductive capacity. Merthiolate (0.18 per cent) was the only sterilizing agent that did not reduce the bone-inductive capacity of the demineralized implants. Because merthiolate is not sporicidal, gamma irradiation appears to be the most appropriate sterilizing agent for demineralized bone in clinical use.     

Effect of sterilization on osteoinduction. Comparison of five methods in demineralized rat bone

The aim of this study was to find a safe, effective sterilization method that does not destroy the bone-inductive capacity of demineralized bone implants. Five sterilizing agents were tested in rats. Implants procured and processed under sterile conditions served as controls. New bone formation was evaluated by determining dry weight, calcium content, and Sr-85 incorporation of the induced ossicles. Glutaraldehyde solution, formaldehyde gas, and ethylene oxide destroyed almost all the bone-inductive capacity. Irradiation by 2.5 Mrads Co-60 resulted in a loss of about half of the inductive capacity. Merthiolate (0.18 per cent) was the only sterilizing agent that did not reduce the bone-inductive capacity of the demineralized implants. Because merthiolate is not sporicidal, gamma irradiation appears to be the most appropriate sterilizing agent for demineralized bone in clinical use.     

Dystrophic calcification and mineralization during bone induction: biochemical differences

The calcification of implants of glutaraldehyde-crosslinked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer-344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20-day-old embryo donors were introduced into the chambers prior to subcutaneous implantation. After 4 weeks of implantation in 26-month-old rats, the cylinders containing embryonic calvaria or muscle calls were found to be full of bone and/or cartilage.     

The effect of demineralized bone matrix gel on bone ingrowth and fixation of porous implants

The presence of demineralized bone matrix (DBM) gel did not enhance or accelerate attachment strength or bone ingrowth and resulted in a significant decrease in implant interface attachment strength at 3 weeks. Hydroxyapatite (HA) coating resulted in significant increases in interface shear strength and bone ingrowth compared with non-HA-coated porous implants at all time periods. The HA-coated implants achieved greater attachment strength and bone ingrowth at earlier time periods and maintained greater attachment strength at long-term periods. The results of this study indicate that in the presence of a good bone-implant interference fit, there is no beneficial effect in applying DBM gel to a porous-coated or HA-coated porous implant surface. The small amount that can be applied and the degree of osteoinductivity of DBM seem to preclude it from having a significant biologic effect.     

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.     

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.     

异体DBM复合rhBMP-2修复兔桡骨缺损的实验研究

目的探讨异体脱钙骨基质（demineralized bonematrix，DBM）复合重组人骨形态发生蛋白2（reconstruction humn bonemorphology protein-2，rhBMP-2）修复节段性骨缺损的能力。方法48只新西兰大白兔采用桡骨15mm节段性骨缺损模型，随机分为3组，A组植入异体DBM与rhBMP-2复合材料，B组植入异体DBM，C组为空白对照组。术后4周、8周、12周、16周．进行放射学和组织学检查。结果A组：术后4周宿主结缔组织长入植骨材料内的骨小梁间，并有岛状新生软骨、骨组织形成；术后8周，新生软骨及骨形成并融合成片；术后12周，新骨改建成熟，但仍能见到植骨材料；术后16周，管状骨结构形成，髓腔再通。B组：术后4周，植骨材料周围有软骨形成；术后8周，大量软骨形成；术后12周，大片状骨形成；术后16周，有髓腔形成。C组：各时问点仅见有纤维结缔组织，只在两端有新骨形成。X片示A组成骨量大，新骨改建、成熟迅速，术后16周全部达骨性愈合。B组成骨量少，仅2例达骨性愈合。C组未见骨性愈合。结论异体DBM复合rhBMP-2材料通过骨诱导和骨传导两种方式修复骨缺损，是一种较理想、具有高效成骨活性的植骨材料。     

Osteoinductive biomaterials for medical implantation

The search for the ideal implant material continues since presently available implants all have significant drawbacks. This paper reviews three studies that we recently completed with bone-inducing implants. Long-term clinical follow-up of 75 allogeneic demineralized bone implants showed an average degree of resorption of 49%. Implants used for dorsal nasal augmentation showed an average degree of resorption of 50.7%, increasing to 82.5% after a 24-month follow-up. The efficacy of demineralized bone implants is dependent on many factors including site of implantation, method of preparation, etc. Transforming growth factor-beta 1 (TGF-beta 1) is a regulator of bone formation. We combined recombinant TGF-beta 1 with demineralized bone powder in a rabbit facial augmentation model. At 6 weeks, there was evidence of increased bone formation in the implants containing TGF-beta 1. Even though TGF-beta 1 can increase bone formation in demineralized bone implants, the overall bone-inducing activity in these implants seems to be suboptimal. Osteoinductive factor extract (OFE) is a partially purified bone factor preparation that has been shown to form bone when implanted into rats. Using a collagen/ceramic carrier, we implanted OFE into a rabbit facial augmentation model. At 21 d, histomorphometry revealed numerous osteoblasts and bone formation in the OFE implants. The bone-inducing activity of many partially purified osteoinductive preparations such as OFE is probably due to Bone Morphogenetic Protein-2A (BMP-2A), which has been shown to induce bone formation in its recombinant form. Recombinant DNA methodology provides the technology necessary to produce these molecules in their homogeneous form, permitting evaluation of bone-inducing activity in a preparation free of contaminants. Finally, the ideal carrier must be devised to permit safe and effective delivery of recombinant bone-inducing factors.     

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.     

脱钙骨基质复合牛骨形态发生蛋白与单纯脱钙骨基质修复兔桡骨缺损比较

目的比较脱钙骨基质(DBM)复合牛骨形态发生蛋白(bBMP)和单纯DBM修复节段性骨缺损的能力.方法32只新西兰大白兔采用桡骨15 mm节段性骨缺损模型,随机分为2组:A组植入异体DBM与bBMP(10 mg)复合材料;B组植入异体DBM.术后4、8、12、16周,进行放射学检查、病理组织学检查和计算机图象分析新生骨面积.结果X线和组织学检查显示异体DBM与bBMP复合材料组的新骨生成、修复骨缺损能力优于异体DBM组,组织切片的计算机图象分析提示DBM bBMP组修复新骨面积大于DBM组,两者之间差异有显著性(4、8、12周P＜0.05,16周P＜0.01).结论异体DBM复合bBMP材料通过骨诱导和骨传导两种方式修复骨缺损,其修复骨缺损的能力要优于单纯DBM材料,是一种较为理想,具有高效成骨活性的植骨材料.     

Graft perforations favor osteoinduction. Studies of rabbit cortical grafts sterilized with ethylene oxide.

The healing of freeze-dried, ethylene oxide sterilized, segmental, allogenic cortical bone grafts was investigated in 15 rabbits using a 2-cm ulnar diaphyseal defect. Five different groups of bone grafts were evaluated: 1) unperforated undemineralized, 2) perforated undemineralized, 3) unperforated demineralized, 4) perforated demineralized, and 5) perforated demineralized grafts enclosed by silicone rubber (Silastic) sheets. There were 3 animals in each group. At 18 days, the study was terminated, and the implants were examined using radiographs and qualitative histologic preparations. We observed that healing of perforated demineralized bone was superior to unperforated demineralized bone, that undemineralized bone was partially sequestered in reactive lacunae, and that perforations in demineralized bone became centers of osteoinduction. Demineralized bone sterilized with ethylene oxide by this method vigorously formed new bone.     

Augmented demineralized bone matrix: a potential alternative for posterolateral lumbar spinal fusion

Variable osteoinductive potential has been reported between and within production lots of different demineralized bone matrix (DBM) products. This study compared fusion rates of different manufactured lots and augmented formulations of DBM with a dose-response curve of recombinant human bone morphogenetic protein 2 (rhBMP-2) on inactivated DBM carrier in a posterolateral fusion rat model. Lumbar fusions were performed in 145 rats. In the control rats, we implanted autograft, graft alternative, including inactivated DBM, or nothing (ie, no graft). In the study rats, we implanted 1 of 2 BioSETR (RTI Biologics, Alachua, Florida) DBM lots, growth factor-enriched DBM, and inactivated DBM plus rhBMP-2 in different concentrations. Manual palpation revealed fusion rates of 25% (autograft), 0% (inactivated DBM), 17% (DBM donor A), and 36% (DBM donor B). The fusion rate of the most enhanced donor B graft (83%) was higher (P<.05) than that of autograft or unenhanced DBM. Inactivated DBM plus rhBMP-2 fused between 45% and 100%. There was no significant difference between DBM plus rhBPM-2 and the highest enrichment group of donor B. Differences between 2 DBM lots in an athymic rat ectopic bone formation model also were found in the spine fusion model. Enhanced DBM formulations were comparable with inactivated DBM plus rhBMP-2 with respect to performance and could represent a bone graft alternative in spine fusion.     

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.