双腔搅拌式生物反应器促进β-磷酸三钙支架内骨髓基质干细胞增殖与分化的研究

Dual-chamber stirred bioreactor improves proliferation and differentiation of bone marrow stromalcells in B-tricaicium phosphate scaffolds

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价促进B-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5X107／mL接种于B—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、14d采用Alamarblue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、2ld也用实时定量一聚合酶链反应（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、14d动忿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到胶原、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）rs（6．384-0．06）、（7．124-0．10）m（4．77±0．06）、（6．234-0．25）VS．（3．784-0．14）、（5。254±0．06）vs（3．504-0．12）I，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B—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。

Objective 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 （[3-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-cuhured to passage 3. The third passage BMSCs were seeded into [3-TCP scaffohts at the density of 5 x 107/mL. The complex of cell and seafibld was put into the dual-chamber stirred bioreactor where the BMSCs umterwent in- duction of osteogenesis and chondrogenesis simuhaneously. The experiment was conducted in a dynamic cuhure 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 B-TCP scaflbld; at 7, 14 and 21 days, real-tirue 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 Jgarding the gene expressions of Col- lI, 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. 06versus3.50±0. 12inOC （P 〈0.05） . Conclusion Thedtml-chamber stirred bioreactor we have designed can induce sinmltaneously osteogenesis and chundrogenesis of BMSCs and also promote the proliferation and differentiation of BMSCs in a B-TCP scaffold.