脱钙骨基质中BMP-2含量与其体内/外成骨活性的相关性研究

Study on the correlation between the content of bone morphogenetic protein 2 in demineralized bone matrix and its osteogenic activity in vitro and in vivo

目的探讨脱钙骨基质(demineralized bone matrix,DBM)中BMP-2含量与其体内/外成骨活性的相关性,以期选择一种简易、便捷的评价DBM成骨活性的方法。方法取9具人尸体左侧股骨中段组织,采用动态脱钙工艺制备DBM(S1~S9),同时制备灭活DBM(对照)。分别采用蛋白酶抑制剂法、胶原酶法、盐酸胍/EDTA法、RIPA裂解液法提取S1~S9以及灭活DBM中BMP-2,测量比较不同DBM间BMP-2含量差异。取S1~S9以及灭活DBM,分别与小鼠胚胎成骨细胞MC3T3-E1共培养,采用MTT法及荧光染色检测细胞增殖,同时检测ALP活性。取30只BALB/c雄性裸小鼠分为10组,分别为S1~S9 DBM组(S1~S9组)以及灭活DBM组(对照组),每组3只。于各组小鼠双侧后肢大腿中部制备肌袋,植入对应DBM材料,术后4周取材行HE染色观察并半定量评价(新骨形成评分)。结果不同供体骨制备的DBM,其BMP-2含量存在差异;同一供体骨制备的DBM,经不同提取方法获得的BMP-2含量也不同,其中盐酸胍/EDTA法提取效率最高。体外成骨性能评价,共培养后各时间点S4、S6组细胞增殖、ALP活性均较其他组明显增高(P<0.05),且7 d时S4组细胞增殖最显著(P<0.05);荧光染色示各组成骨细胞状态均良好,但S1、S2、S3、S4、S6组成骨细胞多于其他组。体内异位成骨诱导实验示,术后4周S1~S6组植骨区域均可见软骨和新骨生成,并且随着BMP-2含量增加,材料诱导新骨生成越多,新骨形成评分较高(P<0.05);其中S4、S6组骨新生区域含有大量软骨细胞和成骨细胞。结论 BMP-2定量检测结合体外成骨细胞增殖分化实验可用于评估DBM成骨活性。

Objective To investigate the correlation between the content of bone morphogenetic protein 2(BMP-2) in demineralized bone matrix(DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM. Methods The left mid-femoral tissues of 9 donors were taken, and DBMs(S1-S9) were prepared by dynamic decalcification process, and inactivated DBM(control group)was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid(EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3 T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase(ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups(S1-S9 groups) and inactivated DBM group(control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated. Results The BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation(P<0.05). Moreover, the cell proliferation of S4 group was the most significant at7 days(P<0.05);fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material(P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area. Conclusion The osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.