幼年和成年猪松质骨脱细胞基质有机成分和成骨诱导能力的比较

A comparison in organic composition and osteogenic induction capacity of cancellous bone decellular matrix between the young and adult porcine

目的:比较幼年和成年猪松质骨脱细胞基质(DCBM)的成骨特性。方法:制备幼年和成年猪DCBM并验证脱细胞效果。胶原和糖胺聚糖(GAGs)定量检测、Masson三色和阿利新蓝染色评估脱细胞前后胶原和GAGs含量及分布。免疫组化检测松质骨基质中骨形态发生蛋白-2(BMP-2)、基质金属蛋白酶-13(MMP-13)和骨钙蛋白(OCN)的分布情况。q PCR检测幼年和成年DCBM诱导复植间充质干细胞(MSC)的I型胶原(ColI)、碱性磷酸酶(ALP)、BMP-2和OCN等成骨分化指标的基因表达。纳米液相色谱-串联质谱(LC-MS/MS)分析DCBM的蛋白组成。结果:定量分析显示,幼年松质骨较成年松质骨在脱细胞前后胶原和GAGs含量更高。幼年松质骨脱细胞前后BMP-2、MMP-13、OCN等的表达均高于成年松质骨。成骨诱导实验显示复植于幼年DCBM的MSC,ALP、Col-I等的m RNA的表达量高于复植于成年DCBM。蛋白质谱GO分析和丰度分析显示幼年DCBM较成年DCBM肽链种类丰富,包含成纤维细胞生长因子、转化生长因子、胰岛素样生长因子等促骨生长细胞因子。结论:同等条件下制备的幼年DCBM与成年猪DCBM比较含有更丰富的促骨生长细胞因子,具有更强的成骨诱导性能。

Objective:To confirm that young cancellous bone has better osteogenic properties than adult cancellous bone decellularized matrix at the cell-molecular level by comparing the organic composition and inducing capacity of MSC osteogenic differentiation in juvenile and adult decellularized cancellous bone matrix.Methods:Decellularized cancellous bone matrix(DCBM)was prepared for the young and adult cancellous bone and the decellularization efficacy was verified.Collagen and GAGs were quantified,and their content and distribution were assessed by Masson trichrome and Alcian blue staining before and after decellularization.Immunohistochemistry was performed to detect the distribution of matrix growth factors such as BMP-2,MMP-13 and OCN in the cancellous bone matrix.qPCR was performed to detect the gene expression of osteogenic differentiation indicators such as Col-I,ALP,BMP-2 and OCN in young and adult DCBM-induced replanted MSCs.LC-MS/MS was performed to analyze the protein composition of DCBM.Results:Quantitative analysis showed that young cancellous bone was higher in collagen and GAGs than adult cancellous bone before and after decellularization.The expression of cytokines such as BMP-2,MMP-13 and OCN were higher in young cancellous bone than in adult cancellous bone before and after decellularization.Osteogenesis induction assay showed that the expression of mRNA of ALP and Col-I was higher in MSC replanted in young DCBM than in adult DCBM.GO analysis and abundance analysis showed that young DCBM was richer in peptide chains than adult DCBM,including fibroblast growth factor,transforming growth factor,insulin-like growth factor and other bone growth promoting cytokines.Conclusion:Young DCBM,compared with adult porcine DCBM under the same conditions,contained more abundant osteogenic cytokines and had stronger osteogenic induction.