摘要目的 设计一种可同时进行成骨和成软骨诱导的双腔搅拌式生物反应器,探讨其提供的力学刺激能否促进β-磷酸三钙(β-TCP)支架内山羊骨髓基质干细胞(BMSCs)的增殖与分化. 方法 分离培养山羊BMSCs,传至第3代,以5×107/mL接种于β-TCP支架,在双腔搅拌式生物反应器中进行成软骨和成骨诱导.实验分为动态培养组和静态培养组.于培养1、3、7、10、14 d采用Alamar blue法检测细胞的增殖情况(n=5),于培养7、14d应用扫描电镜观察支架内细胞的形态,于培养7、14、21d应用实时定量-聚合酶链反应(PCR)检测Ⅱ型胶原、蛋白聚糖、骨桥蛋白(OPN)、骨钙素(OC)基因的表达(n=12). 结果 培养1、3、7、10、14 d动态培养组的细胞量均高于静态培养组,差异有统计学意义(P＜0.05).培养7、14d动态培养组支架内的细胞分布和基质分泌较静态培养组多.培养14、21d动态培养组支架内Ⅱ型胶原、蛋白聚糖、OPN、OC基因表达均高于静态培养组[培养21d时分别为(8.62±0.08)vs.(6.38±0.06)、(7.12±0.10) vs.(4.77±0.06)、(6.23±0.25)vs.(3.78 ±0.14)、(5.25 ±0.06) vs.(3.50±0.12)],差异有统计学意义(P＜0.05). 结论 本研究设计的双腔搅拌式生物反应器可对β-TCP支架内的BMSCs同时进行成骨和成软骨双向诱导,从而促进支架内BMSCs的增殖与分化.

abstractsObjective To explore whether the mechanical stimulation from a dual-chamber stirred bioreactor can promote the proliferation and differentiation of goat bone marrow stromal cells (BMSCs) in 3-tricalcium phosphate (β-TCP) scaffolds.Methods A dual-chamber stirred bioreactor which can induce osteogenesis and chondrogenesis at the same time was designed.Goat BMSCs were isolated and sub-cultured to passage 3.The third passage BMSCs were seeded into β-TCP scaffolds at the density of 5 × 107/mL.The complex of cell and scaffold was put into the dual-chamber stirred bioreactor where the BMSCs underwent induction of osteogenesis and chondrogenesis simultaneously.The experiment was conducted in a dynamic culture group (group A) and a static culture group (group B).At 1,3,7,10 and 14 days,Alamarblue assay was performed to detect cell proliferation (n =5); at 7 and 14 days scanning electron microscopy (SEM) was done to observe the morphology of BMSCs in the β-TCP scaffold; at 7,14 and 21 days,real-time quantitative PCR was performed to detect the gene expressions of Col-Ⅱ,aggrecan,osteopontin (OPN) and osteocalcin (OC) (n =12).Results At 1,3,7,10 and 14 days,the amount of cells in group A was significantly higher than in group B (P ＜ 0.05).At 7 and 14 days there were more cell distribution and matrix secretion in group A than in group B.At 14 and 21 days,group A were all significantly higher than group B regarding the gene expressions of Col-Ⅱ,aggrecan,OPN and OC (P ＜ 0.05).At 21 days,the gene expressions in groups A and B were 8.62 ± 0.08 versus 6.38 ±0.06 in Col-Ⅱ,7.12 ±0.10 versus 4.77 ±0.06 in aggrecan,6.23 ±0.25 versus 3.78 ± 0.14inOPN,and5.25±0.06 versus 3.50 ± 0.12inOC (P ＜0.05).Conclusion Thedual-chamber stirred bioreactor we have designed can induce simultaneously osteogenesis and chondrogenesis of BMSCs and also promote the proliferation and differentiation of BMSCs in a β-TCP scaffold.