外周血来源的间充质干细胞在脱矿松质骨支架中的增殖和成软骨能力的实验研究

Proliferative and Chondrogenic Potential of Peripheral Blood Mesenchymal Stem Cells in Demineralized Cancellous Bone Scaffolds

目的:研究外周血来源的间充质干细胞(PBMSCs)在3D多孔支架中的生物学行为。方法:将兔PBMSCs接种到猪脱矿松质骨(DCB)支架中,并以骨髓间充质干细胞(BMMSCs)以及关节软骨细胞(ACCs)进行对照,通过扫描电镜观察3D环境中的细胞形态和分布;通过Live/Dead染色检测细胞活性,Hoechst33258法检测DNA含量,二甲基亚甲基蓝(DMMB)法检测糖胺聚糖(GAG)含量,酶联免疫吸附试验(ELISA)和免疫荧光法检测二型胶原(COL 2)表达、RT-PCR法定量分析软骨分化相关基因的表达。结果:扫描电镜(SEM)观察显示3种细胞在支架中均匀贴附,Live/Dead染色显示接种3天后3种细胞存活率相似(P>0.05),7天后3种细胞增殖能力以及DNA含量无明显差异(P>0.05)。PBMSCs和BMMSCs均较ACCs分泌更多GAG,但COL 2分泌量相似。此外,PBMSCs和BMMSCs的聚集蛋白聚糖(AGC),COL 2和碱性磷酸酶(ALP)等基因表达均明显上调(P<0.05);而ACCs组这些基因的表达显著下调(P<0.05)。MSCs组的COL 1表达趋于增加,而ACCs组中的表达趋于减少(P>0.05)。在成软骨诱导21天时,PBMSCs和BMMSCs的COL 2和ALP表达较ACCs升高(P<0.05),AGC和COL 1表达与ACCs相比无显著差异(P>0.05)。结论:外周血来源的间充质干细胞在异种脱矿松质骨支架中具有与骨髓间充质干细胞相似的良好的增殖和成软骨能力,但体外培养条件下依然存在肥大软骨细胞基因表达的现象,需要对培养条件进一步优化。

Objective To study the biological behavior of peripheral blood mesenchymal stem cells（PBMSCs） in 3 D composite scaffolds. Methods The proliferation and chondrogenesis of rabbit PBMSCsseeded on porcine cancellous bone（DCB） scaffolds were evaluated,and bone marrow mesenchymal stem cells（BMMSCs） and articular chondrocytes（ACCs） were used as controls. Cell morphology anddistribution in scaffolds were observed using scanning electron microscopy（SEM）. Live/Dead stainingwas employed to detect cell viability,Hoechst 33258 method to measure DNA content,dimethylmeth-ylene blue（DMMB） assay to detect glycosaminoglycan（GAG）,enzyme-linked immunosorbent assay（ELISA） and immunofluorescence to detect the content of type 2 collagen（COL 2）,and RT-PCR toanalyze chondrogenesis-related gene expression. Results SEM showed that three kinds of cells uniformlyadhered and evenly distributed in DCB scaffolds. Live/Dead staining observed the similar viability ofthe three kinds of cells three days after seeding（P〈0.05）. There was no significant difference in theproliferation ability and DNA content among three kinds of cells after seven days of in vitro culture.After 21 days of chondrogenic culture,both PBMSCs and BMMSCs secreted more GAGs than ACCs,while the secretion of COL 2 was similar to that of ACCs. Moreover,the gene expression of AGC,COL2 and alkaline phosphatase（ALP） were significantly up-regulated（P〈0.05） in PBMSCs and BMMSCsbut significantly down-regulated in ACCs（P〈0.05）. The expression of COL 1 in MSCs groups dis-played an increasing trend but a decreasing trend in ACCs group（P〈0.05）. The gene expression ofCOL 2 and ALP,but not of AGC and COL 1,in PBMSCs and BMMSCs was higher than those inACCs（P〈0.05）. Conclusions PBMSCs and BMMSCs have similarly excellent proliferation and chondro-genesis potential in 3 D porous DCB scaffolds. However,hypertrophic gene expression is still observedunder in vitro culturing condition,suggesting the need to further optimize the culture system.