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.     

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.     

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.     

DBM中BMP-2检测初步判断骨库供骨的活性

[目的]定量检测DBM小样中BMP-2含量,以初步评估骨库供体骨的骨诱导活性。[方法]深低温冷冻后的供体骨经过软组织剔除、脱脂、脱钙、干燥、粉碎等过程制成DBM样品。通过Elisa方法对36个供体DBM中BMP-2含量进行定量分析,并结合体内骨诱导实验,得出BMP-2含量与骨诱导活性的关系。[结果]不同供体DBM中BMP-2含量相差较大,最低为5.85 ng/g,最高为71.83 ng/g;体内骨诱导实验结果显示,随着BMP-2含量的增多,成骨现象越来越明显(P0.05);BMP-2含量30 ng/g以下时,成骨面积5%;BMP-2含量60 ng/g以上时,成骨面积25%。[结论] DBM样品中BMP-2的含量与供体骨诱导活性呈正性相关,提示可采用BMP-2的Elisa检测初步评估供体骨的活性。     

Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix.

A sensitive, rapid, reliable and quantitative method to check the bone forming potential of demineralized bone matrix (DBM) has been developed. The osteoinductivity of the bone morphogenetic proteins (BMPs), present in DBM, can be measured in vitro using a pluripotent myoblast C2C12 cell line. Alkaline phosphatase activity induced by co-incubation of DBM with C2C12 cells was dose-responsive and corresponds to the amount of active BMPs in DBM. Bone forming potential was simultaneously tested in vivo by implanting DBM intra-muscularly in nude rats. ALP activity induced in C2C12 cells, correlated with bone formation in vivo (r=0.88), determined by alkaline phosphatase activity, mineralization density and histomorphology of the DBM explants. Results from DBM batches, originating from five established Bone Banks, showed good consistency between in vitro and in vivo assays. However, DBM activity varied widely from bank to bank as well as from batch to batch within the same bank.     

Postmortem degradation of demineralized bone matrix osteoinductive potential. Effect of time and storage temperature

The osteoinductive growth factors present in demineralized bone are degraded by tissue enzymes. Storage of rat limbs at low temperature (4 degrees C) before harvesting of bones preserves the osteoinductive potential of such factors. Storage at room temperature for more than 24 hours causes the recovered bone matrix to be biologically inactive, presumably as the result of biodegradation.     

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.     

Aging effect on inductive capacity of human demineralized bone matrix

Demineralized bone powder (DBP) prepared from human cortical bone was implanted into subcutaneous pouches of athymic Nu/Nu mice for 28 days. The osteoinductive capacity was evaluated by histomorphometry of the induced cartilage and bone, and by alkaline phosphatase activity in the implant. Very small amounts of new bone and cartilage were found at histological analysis, confirming that human DBP is much less osteoinductive than that from other species. Whereas the morphometric data of the implants from the young and aged donors were not significantly different, the alkaline phosphatase activity was significantly lower in the implants from the old donors than from the younger ones. This difference between the morphometric and biochemical results could reflect the fact that the enzymatic activity is already present in the osteoprogenitor cells. At 28 days, the osteoblastic activity in contact with DBP from the aged group is characterized by a decrease in the enzymatic amount which is not yet visible at the tissue level. This tendency to a decrease in the osteoinductive capacity of bone matrix is an additional aspect of the age-related alterations which occur in bone tissue and could be attributed to modifications of different proteins of the bone matrix, including bone morphogenetic protein.     

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.     

冻干骨的骨诱导作用

目的 了解冻干骨的骨诱导特性，为冻干骨的应用提供依据。方法 取4例股骨头松质骨，每例分成2组。冻干照射组按骨制品制备要求处理后，与新鲜骨组一样，常规固定、脱钙，石蜡包埋切片，用免疫组织化学方法检测转化生长因子βl(TCFβl)和碱性成纤维细胞生长因子(bFCF、)。另用冻干骨和羟基磷灰石提取液对入骨髓间充质干细胞进行培养，用原位杂交方法检测细胞的碱性磷酸酶(AKP)、Ⅰ型胶原mRNA表达情况。结果 冻干照射组TCFβl和bFCF、检测阳性。但其阳性率和阳性信号要低于新鲜骨组。细胞经冻干骨提取液培养后，AKP、Ⅰ型胶原mRNA表达要明显高于经经基磷灰石提取液和单纯培养液培养的细胞。结论 骨组织经冻干照射后保留了一定的有生物活性的骨诱导成分，具有一定的骨诱导能力。     

Bioassayed demineralized bone matrix and calcium sulfate: use in bone-grafting procedures.

BACKGROUND AND AIMS: A combination product of bioassayed, demineralized bone matrix (AlloGro, AlloSource, Denver CO) and calcium sulfate pellets (OsteoSet, Wright Medical Technology, Arlington TN) was utilized in a prospective clinical study in 50 patients in need of bone-grafting procedures. It was proposed that the osteoinductive activity of the demineralized bone matrix combined with the osteoconduction and rapid dissolution of the calcium sulfate pellets would complement each other in promoting bone formation. MATERIALS AND METHODS: The patients were evaluated clinically and radiographically at regular intervals post-operatively by an independent clinician. A total 10-point healing score was used to determine healing characteristics and progress. Fifty patients (24 males and 26 females) were treated for benign bone lesions (35), nonunion (11), osteomyelitis (3), and acute fracture (1). The average age was 33 years (range, 3-64 years). Lesions were located in the femur (16), tibia (15), humerus (7), and other sites (12). RESULTS: The average length of follow-up was 14 months (range, 6-32 months). Forty-nine of 50 patients healed their lesions (98%), requiring an average time to heal of 11.8 weeks (range, 3-48 weeks). There were no graft-related complications. CONCLUSIONS: The results of this preliminary clinical study suggest that a combination of bioassayed demineralized bone matrix and calcium sulfate is very effective in treating benign lesions of bone, as well as nonhealing fractures, which is comparable to grafting with autograft. Future studies have been undertaken utilizing this combination in all acute operative settings and fracture management situations.     

Calcificationin vitro of demineralized bone matrix

Collagen was prepared from compact sheep bone by decalcification with EDTA and from rat tail tendons by acetic acid extraction and reconstitution with NaCl. The deposition of apatite in sheep bone collagen in a metastable calcification solution was studied chemically and by electron microscopy. The bone collagen was shown to be a good nucleation catalyst for mineral deposition, while rat tail collagen was a poor catalyst. Mineral deposition in bone collagen occured in two separate kinetic phases, a rapid phase of nucleation and crystal growth, giving rise to small calcified islands, and a second slow phase, ascribed to growth in regions not involving the catalytic sites. This second phase of mineral deposition is considered to be the result of impaired ion diffusion through the closely-aligned collagen fibrils, thus leaving large areas of the collagen free of mineral even though the buffer remains highly supersaturated. Electron micrographs suggested that the catalytic sites might be in some relationship to the 640 Å periodicity of collagen, but a role for non-collagenous material bound to the collagen has not been excluded.The poor catalytic activity of reconstituted collagen was not due to the presence of loosely-bound inhibitors, although inhibitors could be strongly bound to this type of collagen and be absent from bone collagen. The differences in catalytic activity may have a bearing on physiological calcification. A more general hypothesis for nucleation of a mineral phase in biological systems is required.This work was supported in part by the European Atomic Energy Community (EURA-TOM), Brussels, Belgium.     

一种高效制备工艺对脱钙骨基质性能的影响

目的 目前脱钙骨基质生产工艺耗时长效率低,浪费资源和成本.本研究在保存骨诱导活性的前提下,显著缩短了脱钙骨基质的工艺时间.方法 采用动态脱钙和二次换酸工艺制备脱钙骨基质,对pH值、钙含量进行评价.用裸鼠体内植入实验评价这种脱钙骨基质骨诱导活性.结果 材料的pH值为6.2±0.3,呈弱酸性.钙含量为(0.6±0.2)％,符合＜6％的标准.裸鼠体内植入后创面一期愈合,无不良反应发生.其骨诱导活性阳性,4周时可见大量的新生骨组织、骨髓样组织和软骨化成骨现象.结论 该工艺可以提高脱钙骨基质生产效率,不影响材料活性.     

Use of demineralized bone matrix in the extremities

Autologous bone graft is considered as the gold standard for all indications for bone grafting procedures but the limited availability and complications in donor site resulted in seeking other options like allografts and bone graft substitutes. Demineralized bone matrix (DBM) is an allograft product with no quantity limitation. It is an osteoconductive material with osteoinductive capabilities, which vary among different products, depending on donor characteristics and differences in processing of the bone. The purpose of the present review is to provide a critical review of the existing literature concerning the use of DBM products in various procedures in the extremities. Clinical studies describing the use of DBM alone or in combination with other grafting material are available for only a few commercial products. The Level of Evidence of these studies and the resulting Grades of Recommendation are very low. In conclusion, further clinical studies of higher quality are required in order to improve the Recommendation Grades for or against the use of DBM products in bone grafting procedures.     

辐照中脱矿骨基质粒径对胶原结构影响的实验研究

目的探讨不同粒径大小对γ辐照中脱钙骨基质(demineralized bone matrix,DBM)中胶原结构的影响以及辐照保护剂的有效性。方法取同一供体的冻干皮质骨,依据Urist改良法制备不同粒径的(0.5~1.0 mm、1.2~2.8 mm、3.3~4.7 mm及5.7~7.0 mm)DBM样品,按照不同剂量分为:0 kGy、15 kGy、25 kGy及25 kGy(辐照保护剂),真空密封后储存于-80℃冰箱待用。通过扫描电镜观察胶原表面形态,大体观察胶原表面结构损伤的程度;将样品按照0.2 g/ml生理盐水比例在50℃条件下72 h,利用浸提液颜色深度观察胶原被辐照损伤的程度;使用2,4-二硝基苯肼(吸光光度计法)测定样品中羰基含量;十二烷基硫酸钠聚丙烯酰胺钠凝胶电泳法(Sodium dodecyl sulfatepolyacrylamide gel electrophoresis,SDS-PAGE)测定样品中胶原分子量的变化;利用差示热量扫描法(differential scanning calorimetry,DSC)检测样品热变性温度以观察胶原热稳定性。结果样品浸提液颜色与γ辐照剂量相关度较高,未辐照样品浸提液颜色清亮,而在同粒径下随辐照剂量加大浸提液黄色逐渐加深,5.7~7.0 mm粒径组颜色相对较浅;25 kGy组相比于25 kGy+保护剂组浸提液颜色加深。扫描电镜观察到γ辐照导致胶原结构紊乱,纤维断裂,随着辐照剂量增大损伤区域增多,当粒径增大时,损伤区域有减少的趋势;相比于25 kGy组,25 kGy+保护剂组胶原结构性破坏减少。差示热量扫描法得出样品热交换曲线,随着粒径增大,热变性温度有增高的趋势,粒径间对比有统计学差异(F=189.4,P<0.001);同粒径间差异不明显。SDS-PAGE发现同粒径下γ辐照剂量愈大,胶原分子量愈小;同辐照条件下随粒径较小,高分子量胶原含量减少明显;225 kGy+保护剂组相比于25 kGy组,高分子量增多。羰基含量结果显示在同一粒径下,γ辐照使羰基含量增多,0.5~1.0 mm组(F=13.631,P=0.002),1.2~2.8 mm组(F=6.390,P=0.016),3.3~4.7 mm组(F=5.630,P=0.023),5.7~7.0 mm组(F=4.150,P=0.048)的差异均有统计学意义,不同粒径间随着粒径增大羰基含量逐渐减小但差异统计学意义(F=0.560,P=0.650)。结论γ辐照与胶原的氧化损伤具有明显的剂量反应关系,随着γ辐照剂量的增加,胶原损伤程度逐渐增加;DBM的粒径大小影响着胶原对γ辐照的敏感度,随着粒径的减小,DBM颗粒更易被γ辐照损伤;辐照保护剂在辐照过程中对胶原有一定程度的保护作用。     

Use of demineralized bone matrix in children

Eighty five clinical observations of the authors have shown autotransplants demineralized in the solution of hydrochloric acid to possess pronounced osteoinductive properties. Their ability to induce osteogenesis was mostly pronounced in simultaneous plasty with a demineralized and frozen bone. Reconstructive processes are perfectly completed within 18-24 months after operation.     

Effect of a diphosphonate on the osteoinductive activity of rat bone matrix

Implants of demineralized allogeneic bone were investigated to determine the effects of ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the osteoinductive effect of bone matrix. Implants of matrix prepared from bones of rats treated by EHDP (20 mg/kg/day) produced 40% less ash than implants of normal bone matrix. These results indicate that EHDP not only interferes with mineralization but also has a direct effect on protein metabolism of bone cells.     

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 and bone-marrow 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.     

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.