CAJ | 학술논문

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.
목적 설계일충가동시진행성골화성연골유도적쌍강교반식생물반응기,탐토기제공적역학자격능부촉진β-린산삼개(β-TCP)지가내산양골수기질간세포(BMSCs)적증식여분화. 방법 분리배양산양BMSCs,전지제3대,이5×107/mL접충우β-TCP지가,재쌍강교반식생물반응기중진행성연골화성골유도.실험분위동태배양조화정태배양조.우배양1、3、7、10、14 d채용Alamar blue법검측세포적증식정황(n=5),우배양7、14d응용소묘전경관찰지가내세포적형태,우배양7、14、21d응용실시정량-취합매련반응(PCR)검측Ⅱ형효원、단백취당、골교단백(OPN)、골개소(OC)기인적표체(n=12). 결과 배양1、3、7、10、14 d동태배양조적세포량균고우정태배양조,차이유통계학의의(P＜0.05).배양7、14d동태배양조지가내적세포분포화기질분비교정태배양조다.배양14、21d동태배양조지가내Ⅱ형효원、단백취당、OPN、OC기인표체균고우정태배양조[배양21d시분별위(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)],차이유통계학의의(P＜0.05). 결론 본연구설계적쌍강교반식생물반응기가대β-TCP지가내적BMSCs동시진행성골화성연골쌍향유도,종이촉진지가내BMSCs적증식여분화.
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 (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.