聚己内酯/壳聚糖与聚己内酯/聚乳酸支架与人脂肪来源干细胞的生物相容性研究

Biocompatibilty analysis of human adipose-derived stem cells and Polycaprolactone/chitosan or Polycaprolactone/ Polylactide scaffold

目的 对比人脂肪来源干细胞（Human adipose derived stem cells,hADSCs）与聚己内酯/聚乳酸（Polycaprolactone/Polylactide,PCL/PLA）和聚己内酯/壳聚糖（Polycaprolactone/chitosan,PCL/CS）复合支架的生物相容性,为进一步体内组织修复提供依据.方法 体外分离、培养和扩增hADSCs,分别制备PCL/PLA、PCL/CS复合支架,扫描电镜观察支架材料的表面情况.取材料浸提液培养hADSCs,CCK-8法检测细胞活力,评价支架的细胞毒性.hADSCs传代扩增后,接种到支架材料上,裸鼠皮下培养2周,HE染色观察细胞在支架上的生长情况.结果 hADSCs与成纤维细胞相似,呈“梭形”,并以集落形式生长.扫描电镜观察PCL/CS孔径在100μm左右,孔隙率为88.76％,而PCL/PLA支架孔径则在40μm左右,孔隙率为91.45％.hADSCs在PCL/CS浸提液中培养1、3、7天的相对增殖率分别为98.6％、101.6％、110.3％,而PCL/PLA组为98.1％、100.7％、108.4％,说明hADSCs在两种浸提液中均保持较高的增殖率,即两种合成支架均无细胞毒性.hADSCs复合两种支架体内培养后,HE染色后可见有大量细胞长入PCL/CS支架内部,同时免疫组化HLA-Ⅰ检测发现,支架材料内部分细胞阳性表达,说明PCL/CS支架内该部分的细胞来源于人,即hADSCs.而在PCL/PLA内部渗透进入的细胞不如PCL/CS支架组.结论 PCL/CS和PCL/PLA支架安全无毒,hADSCs在PCL/CS支架上显示更好的细胞相容性,该支架可以作为hADSCs的载体材料,用于组织工程膀胱修复的研究.

Objective To observe the growth of human adipose derived stem cells cultured on Polycaprolactone/ chitosan （PCL/CS） and Polycaprolactone/Polylactide（PCL/PLA） biomaterials in vitro and in vivo. Methods hADSCs were isolated from human subcutaneous adipose tissue after collagenase digesting, filtrating and centrifuging. The scaffoldwas prepared by freeze-drying technique and the method of vacuum thermal cross-linking. The cytotoxicity of scaffold was evaluated by cell growth. The third passage of hADSCs were seeded onto the PCL/CS and PCL/PLA scaffolds and were subcutaneously planted into nude mice for observing hADSCs growth in vivo. Results hADSCs growed faster in the leaching solution of the PCL/CS or PCL/PLA.The PCL/CS,indicating that PCL/PLA scaffolds were non-toxic for hADSCs growth.The histological analysis showed that hADSCs could grow in the space of the scaffolds no matter in vitro or in vivo culture.When cultured in vitro, some cells adhered at the edge of the scaffolds 1 week later and more cells grew into the inside of the scaffolds after 2 weeks. When cultured in vivo, a great deal of ceils grew into the scaffolds. Conclusion Both of PCL/CS and PCL/PLA scaffolds are non-toxic, but former has a better biocompatibility with hADSCs than later, suggesting that it can be used as a vehicle for hADSCs in tissue engineering repair of bladder defect reconstruction.