生物活性材料成骨活性的体外测定★◇

目的：运用动物体内实验的方法测定生物活性材料成骨活性有较多缺点，为快速有效地评价成骨活性的生物材料，探索一种新的体外测定成骨活性的方法。 方法：实验于2004-01/2005-05在解放军总医院骨科研究所完成。取羊脱钙骨基质和复合材料(粗制羊骨蛋白+羊脱钙骨基 质+牛腱Ⅰ型胶原)两种活性材料分别做免疫组织化学染色检查是否含有骨形成蛋白（bone morphogenetic protein，BMP）。并取脱钙骨基质、复合材料、rhBMP-2、3种材料与小鼠C2C12细胞共培养72 h后，用1% Triton将细胞裂解，取细胞裂解液测定碱性磷酸酶和总蛋白。利用所测定的碱性磷酸酶和总蛋白吸光度比值代表单位数量的细胞中所含碱性磷酸酶的含量。同时设立阴性对照（单纯细胞不加任何材料）。 结果：脱钙骨基质和复合材料中均含有BMP-2。用3种材料刺激C2C12细胞后，测定C2C12细胞中碱性磷酸酶含量高于阴性对照(P < 0.05)。rhBMP-2刺激C2C12细胞产生的碱性磷酸酶含量较其他材料明显增高(P < 0.001)。 结论：体外测定生物材料成骨活性在实际应用中具有操作简便、效果可靠的特点。

In vitro osteoinductivity assay of bioactive materials

AIM: The assay in animal body to detect osteoinductivity of bioactive materials has many defects. This study was designed to search for a new assay for osteoinductivity of biological activity materials in vitro more quickly and efficiently. METHODS: From January 2004 to May 2005, the experiment was carried out at the Orthopedics Research Institute in the Chinese PLA General Hospital. Bone morphogenetic protein-2 (BMP-2) in the goal demineralized bone matrix (DBM) and the composite material (DBM + crude protein extracted from goal DBM + type I collagen of bovine tendon) was determined by immunohistochemistry test. These three kinds of materials (DBM, composite material and BMP-2) were co-incubated with mouse C2C12 cells for 72 hours respectively, then the C2C12 cells was lysed in 1% Triton solution, and the lysate were assayed for alkaline phosphatase (ALP) and total protein content. The resulting absorbance about ALP and total protein was recorded. The relative ratio between absorbance of ALP and total protein was taken as an indicator of representing the quantity of ALP in the unit quantitative cells. Meanwhile the negative controls were set up without materials. RESULTS: Both the DBM and the composite material contained BMP-2. After the three kinds of materials were co-incubated with C2C12 cells, the quantity of ALP in the C2C12 cells showed a higher level compared with the negative controls (P < 0.05). The cells treated with recombinant human BMP-2 contain more ALP than ones treated with DBM or composite material (P < 0.001). CONCLUSION: The in vitro assay for detecting osteoinductivity of bioactive materials is feasible and reliable.