Zhang M, Powers R M, Wolfinbarger L
Center for Biotechnology, Old Dominion University, Norfolk, VA, USA.
J Periodontol. 1997 Nov;68(11):1076-84. doi: 10.1902/jop.1997.68.11.1076.
Demineralized bone matrix (DBM) is widely used in the repair of pathologies associated with skeletal defects and periodontal diseases. The present study was directed at establishing in vivo and in vitro models for a quantitative assessment of the osteoinductivity of DBM before clinical use. Athymic mice were used in an in vivo assay to overcome the species limitations (for human DBM) found in xenogeneic animal models. Calcium contents of explants, as an indicator of new bone formation, were assayed and expressed as a change in the weight percent calcium in the explant as compared to the weight percent of calcium in the implanted material. A total of 82 mice (2 implants per mouse) were used in this study. Significant amounts of new bone were induced in this animal model in response to implantation of DBM. Muscular implantation was found to be more osteoinductive (increases of 10.0 +/- 0.4 calcium weight percent of explant) than subcutaneous implantation (increases of 1.62 +/- 0.27 calcium weight percent of explant) and new bone formation in muscular implantation sites of athymic mice mimics endochondral bone formation. Between weeks 1 to 4, the weight of explanted materials did not significantly differ from the weight of the implanted material; however, by week 5 the explant weight began to increase. Calcium deposition over the 5 weeks of implantation increased in a nearly linear fashion. Consequently week 4 was chosen as the optimum time for explantation in the in vivo assay in that sufficient calcium levels had been achieved without a significant increase in explant dry weight. Aliquots of 10, 20, 30, and 40 mg per implantation site were used in dose response studies in the in vivo bioassay. Dose response curves with DBM exhibited maximal activity at the 20 mg DBM implant dose in the in vivo bioassay. An in vitro bioassay was also developed where human periosteal (HPO) cells were chosen because osteoprogenitor cells found in bone repair typically come from periosteal tissue. Alkaline phosphatase (ALP) activity in confluent cell cultures of HPO cells exposed to DBM, as an indicator of osteoblast induction, reached its highest level on day 5 of DBM treatment. Aliquots of 2, 5, 10, 20, 30, and 40 mg DBM per flask were chosen in dose response studies using the in vitro bioassay. These dose response studies with DBM revealed that quantities approximating 5 to 10 mg DBM in the in vitro model provided for maximal levels of ALP in cell extracts. A linear correlation (R2 = 0.7397) was demonstrated between the in vivo calcium remineralization assay and the in vitro ALP assay of osteoinductivity of DBM, suggesting that the in vitro assay can be used to quantitatively assess the osteoinductive potential of DBM where production and distribution of clinically usable DBM dictates rapid analysis.
脱矿骨基质（DBM）被广泛用于修复与骨骼缺损和牙周疾病相关的病理状况。本研究旨在建立体内和体外模型，以便在临床应用前对DBM的骨诱导活性进行定量评估。由于异种动物模型存在物种局限性（针对人DBM），因此在体内试验中使用了无胸腺小鼠。测定了外植体的钙含量，作为新骨形成的指标，并表示为外植体中钙的重量百分比相对于植入材料中钙的重量百分比的变化。本研究共使用了82只小鼠（每只小鼠植入2个植入物）。在该动物模型中，植入DBM后诱导出了大量新骨。发现肌肉植入比皮下植入更具骨诱导性（外植体钙重量百分比增加10.0±0.4），无胸腺小鼠肌肉植入部位的新骨形成类似于软骨内骨形成。在第1至4周期间，取出的材料重量与植入材料的重量无显著差异；然而，到第5周时，外植体重量开始增加。植入5周内钙沉积几乎呈线性增加。因此，第4周被选为体内试验中外植体取出的最佳时间，因为此时已达到足够的钙水平，而外植体干重没有显著增加。在体内生物测定的剂量反应研究中，每个植入部位使用10、20、30和40mg的等分试样。DBM的剂量反应曲线在体内生物测定中在20mg DBM植入剂量时表现出最大活性。还开发了一种体外生物测定方法，选择人骨膜（HPO）细胞，因为在骨修复中发现的骨祖细胞通常来自骨膜组织。作为成骨细胞诱导指标，暴露于DBM的HPO细胞汇合细胞培养物中的碱性磷酸酶（ALP）活性在DBM处理的第5天达到最高水平。在使用体外生物测定的剂量反应研究中，每个培养瓶选择2、5、10、20、30和40mg DBM的等分试样。这些DBM的剂量反应研究表明，在体外模型中，约5至10mg DBM的量可使细胞提取物中的ALP达到最高水平。DBM的体内钙再矿化测定与体外ALP骨诱导活性测定之间显示出线性相关性（R2 = 0.7397），这表明在临床可用DBM的生产和分布要求快速分析的情况下，体外测定可用于定量评估DBM的骨诱导潜力。
