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.     

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.     

Decreased osteoinductive potential of bone matrix from ovariectomized rats

The effect of estrogen deficiency on matrix-induced bone formation was investigated. Female rats were ovariectomized and given demineralized bone matrix (DBM) intramuscularly 3 weeks before termination. the DBM was taken from previously ovariectomized and from sham-operated on rats. the animals were killed at various times after ovariectomy (6-27 weeks). Implants were processed undemineralized for histologic and biochemical studies.

Normal DBM implanted in ovariectomized or normal rats induced extensive bone formation 6 weeks postovariectomy. the amount of newly formed bone decreased with the age of host rats. Bone matrix taken from ovariectomized rats was incompletely resorbed in both ovariectomized and normal hosts, therefore reducing the extent of osteogenesis andbone-marow formation. Instead, chondrogenesis was intensive, but delayed. the calcium, magnesium, and zinc contents were decreased in implants taken from ovariectomized rats when compared with implants taken from normal animals.

Normal osteoinduction with DBM taken from normal rats and implanted in ovariectomized rats and the absence of osteogenesis with DBM taken from ovariectomized rats indicate that an estrogen-deficient environment is not crucial for altered matrix-induced endochondral bone formation in ovariectomized rats. An altered composition of matrix from ovariectomized rats and a subsequent abnormality in the cell-matrix interaction should be considered responsible.     

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.     

The Bone Inductive Capacity of Decalcified Bone Matrix Modified by Diphenylhydantoin

Decalcified bone matrix was prepared from cortical bones of rats premedicated with I) Diphenylhydantoin (DPH), II) DPH + Vitamin D₃, III) Vitamin D₃ or IV) no premedication for 10 days.

In the donor animals, DPH lowered the serum calcium level, caused a weight loss of 10 per cent, and stopped the growth of the long bones. Vitamin D₃ supplementation normalized the serum calcium concentration but had no effect on the other parameters. Vitamin D₃ alone caused hypertrophy of the growth cartilage, while the bone growth and structure was normal.

The bone inductive capacity of decalcified bone matrix was highest in the DPH group, and the DPH + D₃ group also showed significantly higher values than the D₃, and control groups.

The results of the present study show that the bone inductive capacity of the decalcified bone matrix is independent of Vitamin D₃ metabolism.     

Bone formation enhanced by induction: Bone growth in titanium implants in rats

Bone-inducing materials have been investigated for the purpose of augmenting bone formation in implants made of porous fiber titanium. The bone-inducing materials used were: (1) Bone from the iliac crest of inbred rats (isografts), (2) Antigen-extracted, autolyzed, demineralized bone from outbred rats (AAA bone a.m. Urist), and (3) AAA bone combined with bone marrow from inbred rats. Tubes of fiber titanium were packed with bone-inducing materials and implanted in the back musculature of inbred rats. Bone formation was assessed by labelling with ³H-proline (collagen synthesis) and ⁴⁷Ca (mineral deposit) and by content of calcium of the harvested implants.

Isografts and AAA bone with marrow yielded a substantial amount of new bone. Without the marrow, AAA bone yielded very small amounts of new bone.     

Indomethacin inhibits bone formation in inductive allografts but not in autografts: studies in rat

Background Non-steroidal anti-inflammatory drugs (NSAIDs) are known to have inhibitory effects on new bone formation. We wanted to analyze some effects of the NSAID indomethacin on different inductive stimuli for bone formation.

Methods We experimentally induced heterotopic new bone with demineralized allogeneic bone matrix (DABM) and with bone autografts in rats, in order to study the effects of indomethacin on new bone formation at 3 and 6 weeks.

Results Indomethacin inhibited net bone formation in DABMs by 30% at 6 weeks. At 6 weeks, the mineral accretion rate was unaffected, indicating that it is at the early phase of the inductive process that mineral accretion is sensitive to inhibition by indomethacin, but not at the later stages.

In traumatized skeletal bone, the ⁴⁵Ca-specific activity was higher than in non-traumatized bone, while indomethacin significantly reduced the ⁴⁵Ca uptake at 3 weeks, but not at 6 weeks.

In the autografts, a net mineral loss occurred, but neither mineral content nor ⁴⁵Ca incorporation was affected by Indomethacin treatment.

Interpretation Indomethacin inhibited the early phase of new bone formation in heterotopic DABMs and the early bone healing process in traumatized skeletal bone, but did not affect resorption or bone formation in heterotopic autografts. ▪     

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.     

Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation.

Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation.

The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.     

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.     

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。     

Bone induction by fetal and adult human bone matrix in athymic rats

In the rat the intramuscular implantation of demineralized rat bone matrix induces local bone formation. In adult primates, however, allogenous bone matrix induces little or no bone formation in extraskeletal sites. To assay inductive properties, human demineralized bone matrix from 6 adult donors and 4 fetuses was implanted intramuscularly in athymic rats for 6 weeks. Fetal and adult matrix implants yielded about the same amount of bone: about half of the bone yield from rat or rabbit matrix in the same model. We conclude that human bone matrix has inductive properties and that failures to induce bone formation in adult primates may be due to an inability by the recipients to respond to inductive stimuli of adult bone matrix.     

Bone induction by fetal and adult human bone matrix in athymic rats

In the rat the intramuscular implantation of demineralized rat bone matrix induces local bone formation. In adult primates, however, allogenous bone matrix induces little or no bone formation in extraskeletal sites. To assay inductive properties, human demineralized bone matrix from 6 adult donors and 4 fetuses was implanted intramuscularly in athymic rats for 6 weeks. Fetal and adult matrix implants yielded about the same amount of bone: about half of the bone yield from rat or rabbit matrix in the same model. We conclude that human bone matrix has inductive properties and that failures to induce bone formation in adult primates may be due to an inability by the recipients to respond to inductive stimuli of adult bone matrix.     

Osteogenic enhancement of diaphyseal reconstruction: Comparison of bone grafts in the rabbit

We investigated incorporation of autoclaved autografts in segmental defects of rabbit humeri for comparison with a previous study on similar grafts supplemented with demineralized allogeneic bone matrix (DABM). We also made similar reconstructions with frozen allografts—both DABM and nonsupplemented allografts.

Before the animals were killed at 8 months, they underwent scintigraphy, showing that all 28 humeral reconstructions were metabolically active. Faxitrone radiography showed nonunion in three of nine with autoclaved autografts and in two of eight with frozen allografts, whereas all 11 DABM-supplemented frozen allografts had incorporated. Taking into account only the 23 healed reconstructions, the mean torsional strength in relation to the contralateral nonoperated on humeri was 0.81 for all three groups. Histologically, new bone enveloping, partly replacing, the implants was more abundant in DABM-supplemented reconstructions.

Our study shows that osteogenic enhancement is more important than the type of nonviable bone chosen for diaphyseal repair. However, if healing is obtained, osteogenic enhancement per se does not increase the strength.     

Irradiation has no effect on the incorporation of impacted morselized bone: a bone chamber study in goats

Background Gamma irradiation has been widely used for sterilization of bone allografts. However, gamma irradiation alters proteins. This is favorable when it reduces immunogenicity, but is undesirable when osteoinductive proteins are damaged. Although the effect of gamma irradiation on BMPs has been studied, the effect of irradiation on the process of incorporation of morselized bone chips remains unclear. We studied the effects of sterilization by gamma irradiation on the incorporation of impacted morselized allografts.

Methods Bone chambers with impacted allografts, rinsed impacted allografts, allografts that were rinsed and subsequently irradiated, and an empty control were implanted in proximal medial tibiae of goats. Incorporation was evaluated using histology and histomorphometry.

Results Histology revealed evidence of bone graft incorporation, which proceeded in a similar way in unprocessed, rinsed, and both rinsed and irradiated bone grafts. After 12 weeks, no difference in bone and tissue ingrowth was found between the unprocessed, the rinsed, and the rinsed and subsequently irradiated allografts. The amount of unresorbed graft remnant was highest in the unprocessed bone grafts.

Interpretation We conclude that sterilization with gamma irradiation does not influence the incorporation of impacted rinsed bone allografts.     

Juxta-Articular Bone Cyst

Juxta-articular bone cyst was first described as a distinct clinical entity by Hicks in 1956 and called “intraosseous ganglion”. Hitherto, some 150 similar cases have been described.

The cyst, which is usually solitary, occurs in the epiphysis near a weight-bearing joint in middle-aged persons. It may cause persistent pain but is easily overlooked on ordinary X-rays. The histogenesis is uncertain.

Six patients treated by thorough curettage and autologous bone transplantation are presented. All achieved immediate relief from pain.

Some theoretical aspects concerning the histogenesis are discussed.     

The effect of aging on bone formation in rats: Biochemical and histological evidence for decreased bone formation capacity

Summary Ectopic bone formation by subcutaneously implanted demineralized bone matrix powder (DBM) was assessed biochemically and histologically in Fischer 344 rats of different ages. The total calcium accumulated in implants was greatly depressed in older rats, as was the rate of⁴⁵Ca deposition. High alkaline phosphatase activity appeared later in the 10- and 16-month-old rats compared with 1-month-old rats, and the magnitude of the alkaline phosphatase activity was decreased in 16-month-old rats. The accumulation of the bone-specific vitamin K-dependent bone protein (bone gla protein, BGP) was decreased in the implants in older rats. Histological examination of the implants confirms the decreased ability of aged animals to produce bone in response to DBM. Measurements of total calcium, alkaline phosphatase, and BGP at the site of demineralized bone matrix implants clearly demonstrates that bone formation decreases dramatically with increasing age. Significant differences in total calcium can be detected even between 1-month-old and 3-month-old rats. Serum BGP shows a marked decrease (47%) between 1-month- and 3-month-old rats, a decrease not paralleled by a similar decrease in BGP present in calvarial or tibial 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.     

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.     

Osteogenic Phenomena Across Endosteal Bone-Implant Spaces with Porous Surfaced Intramedullary Implants

Porous surfaced femoral components of hip prostheses stabilized by tissue ingrowth are often situated a certain distance away from the endosteal cortex in the diaphysis. The purpose of this study was to examine the significance of this space between an implant and the cortex on bone growth into the porous surface of the implant.

Intramedullary rods of different diameters with porous surface regions made of powder metal were inserted into the femurs of adult beagles. The rods had outside diameters of 2.5, 3.2, 4.5, and 5.5 millimeters; this variation produced endosteal bone-implant surface spaces ranging from 0 to 4 millimeters. The animals were sacrificed at 4, 8, 12, and 16 weeks.

Histological sections revealed that by 12 weeks the implants became generally surrounded by a thin shell of spongy bone which was joined to the endosteal cortex by bony trabeculae. This feature was most prominent for implants which were approximately 2 millimeters or less from the endosteum. Denser, more haversian-like bone developed up to and within those areas of implants which were in contact with the cortex.

The development of this intramedullary type of bone could significantly contribute to the fixation strength of clinical porous surfaced prostheses whose stems do not completely fill the medulla.