CAJ | 학술논문

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、直观性等方而存在着不足,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牛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:将重组人骨形态发牛蛋白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72 h,阴性对照为单纯细胞,不加仟何材料.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:重组人骨形态发牛蛋白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:体外榆测生物材料诱导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.
배경:전통골체대재료성골활성적검측방법위체내검측,재가고성、시효성、직관성등방이존재착불족,특별시재검측대비량생물체대재료시기결점우위돌출.목적:탐색일충능구재체외검측골이식체대재료성골활성적방법.설계:대비관찰적세포학실험.시간급지점:실험우2006-08/2007-05재해방군총의원전군골과연구소진행.재료:C2C12세포유북경협화의과대학세포중심제공,인탈개골기질、인골제취적골단백유해방군총의원골과연구소제공,우건Ⅰ형효원유북경익이강우물공정개발중심제공,중조인골형태발생단백2유항주화동기인연구소제공.방법:장중조인골형태발우단백2、인탈개골기질、복합재료(인골제취적골단백여인탈개골기질、우건Ⅰ형효원이투석적방법복합)3충재료여소서기육C2C12세포공배양72 h,음성대조위단순세포,불가천하재료.세포렬해후용비색법분별검측렬해액중성골세포특이성표기물감성린산매급총단백적흡광도치,이량자적비치표시단위수량적C2C12세포소함유적감성린산매함량,이차형량대측생물재료성골활성적고저.주요관찰지표:감성린산매여총단백적흡광도치.결과:중조인골형태발우단백2재료중감성린산매함량최고,인탈개골기질재료합복합재료차지,음성대조최저.3충재료중감성린산매함량여음성대조비교차이균유현저성의의(P＜0.05).결론:체외유측생물재료유도C2C12세포산생감성린산매함량,가작위평정재료성골활성적지표.
BACKGROUND:Conventjonally,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:C2C 12 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 C2C12cells for 72 hours.Negative control group comprised pure cells without materials.Then C2C 1 2 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 the negative control group.There were significant differences in the ALP activity between the three trial groups and the negative control group(P＜0.05).CONCLUSION:The assay in vitro is effective to detect the ALP activity and it can be used to determine the osteoinduction of bone graft substitutes.