生物骨替代材料成骨活性的体外测定(英文)  

Determining in vitro osteogenic activites of bone substitute biomaterials

作　　者：左进步[1] 孙明学[2] 

机构地区：[1]武警北京总队医院骨科,北京市100027 [2]解放军总医院骨科研究所,北京市100853

出　　处：《中国组织工程研究与临床康复》2008年第32期6385-6388,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:传统骨替代材料成骨活性的检测方法为体内检测,在可靠性、时效性、直观性等方面存在着不足,特别是在检测大批量生物替代材料时其缺点尤为突出。目的:探索一种能够在体外检测骨移植替代材料成骨活性的方法。设计:对比观察的细胞学实验。时间及地点:实验于2006-08/2007-05在解放军总医院全军骨科研究所进行。材料:C2C12细胞由北京协和医科大学细胞中心提供,人脱钙骨基质、人骨提取的骨蛋白由解放军总医院骨科研究所提供,牛腱Ⅰ型胶原由北京益尔康生物工程开发中心提供,重组人骨形态发生蛋白2由杭州华东基因研究所提供。方法:将重组人骨形态发生蛋白2、人脱钙骨基质、复合材料(人骨提取的骨蛋白与人脱钙骨基质、牛腱Ⅰ型胶原以透析的方法复合)3种材料与小鼠肌肉C2C12细胞共培养72h,阴性对照为单纯细胞,不加任何材料。细胞裂解后用比色法分别检测裂解液中成骨细胞特异性标记物碱性磷酸酶及总蛋白的吸光度值,以两者的比值表示单位数量的C2C12细胞所含有的碱性磷酸酶含量,以此衡量待测生物材料成骨活性的高低。主要观察指标:碱性磷酸酶与总蛋白的吸光度值。结果:重组人骨形态发生蛋白2材料中碱性磷酸酶含量最高,人脱钙骨基质材料合复合材料次之,阴性对照最低。3种材料中碱性磷酸酶含量与阴性对照比较差异均有显著性意义(P<0.05)。结论:体外检测生物材料诱导C2C12细胞产生碱性磷酸酶含量,可作为评定材料成骨活性的指标。BACKGROUND： Conventionally, the osteoinduction of bone substitute materials is detected in vivo, which is unsatisfactory regarding the reliability, chronergy and precision, especially for a large amount of substitute materials. OBJECTIVE： To search an in vitro assay for determining in vitro osteogenic activities of bone graft substitutes. DESIGN： Controlled cytological trials. TIME AND SETTING： The experiments were carried out in the Institute of Orthopaedics, Chinese PLA General Hospital （Beijing, China） from August 2006 to May 2007. MATERIALS： C2C12 cells was offered by the Cell Center of Peking Union Medical College; Human decalcified bone matrix and bone protein were offered by the Institute of Orthopaedics in Chinese PLA General Hospital; Type Ⅰ collagen extracted from bovine tendon was purchased from Beijing Yierkang Bioengineering Development Center; Recombinant human bone morphogenetic protein 2 was purchased from Hangzhou Gene Technology of Huadong Medicine Group. METHODS： By means of dialysis, a composite material was prepared with the bone protein extracted from human, human decalcified bone matrix and type Ⅰ collagen of bovine tendon. The samples of decalcified bone matrix, composite material and recombinant human bone morphogenetic protein 2 were respectively co-incubated with C2C12 cells for 72 hours. Negative control group comprised pure cells without materials. Then C2C12 cells were lysed and the lysate were assayed for the absorbance of alkaline phosphatase （ALP） and total protein by chromatometry. ALP is the specific mark of osteoblastic phenotype. The relative ratio of absorbance value between ALP and total protein could represent ALP activity in the unit quantitative C2C12 cells. MAIN OUTCOME MEASURES： The ratio of absorbance value between ALP and total protein. RESULTS： The ALP activity was the highest in the recombinant human bone morphogenetic protein 2 group, then in the decalcified bone matrix group and composite material group, and the lowest in

关 键 词：脱钙骨基质 诱导成骨活性 测定 体外 评价 

分 类 号：R318[医药卫生—生物医学工程]