脂肪干细胞复合PLGA体外诱导成软骨的实验研究

Chondrogenic differentiation of adipose derived stem cells in PLGA scaffolds

目的探讨脂肪干细胞接种于PLGA支架复合培养体外诱导成软骨的能力。方法收集脂肪干细胞,调整细胞悬液密度为4.0×1010/L,接种在PLGA支架材料上进行复合,在特定培养基条件下,对脂肪干细胞/PLGA进行成软骨诱导,于诱导3周后应用扫描电镜观察,藩红O/固绿染色和Ⅱ型胶原免疫组织化学检测成软骨表型;逆转录-聚合酶链反应(RT-PCR)检测前Ⅱ型胶原蛋白、aggrecan和Sox9的mRNA基因表达情况。结果脂肪干细胞接种于PLGA材料后,复合体表面逐渐光滑润泽,质地逐步增韧,体外诱导后细胞/PL-GA支架复合体基本保持原状;3周时取材行扫描电镜观察,可见细胞在材料表面附着生长,基质分泌明显,连串成片;石蜡切片藩红O/固绿染色可见诱导组细胞胞外基质分泌旺盛,和支架材料连接成片,呈强阳性红染;胞外基质Ⅱ型胶原免疫组织化学着色阳性;RT-PCR检测前Ⅱ型胶原蛋白、aggrecan和Sox9的mRNA基因均阳性表达。结论脂肪干细胞能够和PLGA复合培养,在特定诱导培养基条件下可以向软骨方向分化,形成类软骨样组织。

【Objective】 To explore chondrogenic differentiation capability of adipose derived stem cells cultured and induced in PLGA scaffolds.【Methods】 Adipose derived stem cells were cultured and collected and regulated into a concentration of 4.0×1010/L,still inoculated into PLGA,followed by the specific induction medium was set.The complex of cells and scoffolds was terminated in culture medium,embedded in paraffin and cut in section,still assessed using Safranin O/ Fast Green staining and collagen Ⅱ immunohistochemistry at 3 weeks after initial chondrogenic induction of the complex.The impanted cells and scaffolds were observed with scanning electron microscope.Still,the mRNA expressions of Col2al,Agc1 and Sox9 were detected by RT-PCR at 3 weeks after induction.【Results】 The tenacity of the complex of cells and scoffolds was increased and it＇s surface was lenitive,still it＇s shape was not changed in the induced groups.Scanning electron microscope showed many spherical cells distributed in pores and on the surface of the scaffolds and more extracellular matrices were found in the induced groups.The extracellular matrix was stained positively for Safranin O/Fast Green staining,and indicated collagen It was expressed positively,still a few cartilage lacunae to be found.RT-PCR indicated that the complex of cells and scoffolds could express Col2a1 mRNA,aggrecan mRNA and Sox9 mRNA at 3 weeks after induction.【Conclusion】 Adipose derived stem cells may be cultured with PLGA scaffolds,still form a material with part character of cartilaginous tissue and express cartilage phenotype under the specific inducing environment in vitro.