骨骼肌无细胞基质的制备及其生物相容性研究

目的细胞外基质是脂肪组织工程材料的研究热点之一。通过探讨骨骼肌无细胞基质的制作方法及生物相容性,为其在脂肪组织工程中的应用奠定基础。方法取健康成年小香猪新鲜骨骼肌组织,横切成厚2～3 mm的组织块,采用低渗-去垢剂法脱细胞处理。处理后采用HE染色、Masson三色染色、免疫组织化学染色及扫描电镜检测骨骼肌无细胞基质是否有细胞成分残留,并观察其基本结构;应用MTT法检测骨骼肌无细胞基质细胞毒性。取乳腺癌患者自愿捐赠脂肪组织,分离培养人脂肪干细胞(human adipose-derived stem cells,hADSCs),从形态学、流式细胞学和成脂、成骨分化能力方面进行鉴定。将骨骼肌无细胞基质与第3代hADSCs共培养,于培养后第1、3、5、7天通过细胞活性检测材料上细胞黏附、扩散和增殖情况,了解其与细胞之间的相互作用。结果 HE、Masson、免疫组织化学染色及扫描电镜观察显示骨骼肌无细胞基质肌纤维去除完全,无细胞核残留,基质结构保留完整;大量连接成网状的胶原纤维呈多孔隙样结构,规则排列。MTT检测示骨骼肌无细胞基质细胞毒性为1级,细胞相容性好。细胞活性检测示hADSCs在骨骼肌无细胞基质上能很好地伸展,且能与周围基质黏附,进入基质内部并相互交织。结论经脱细胞处理的骨骼肌无细胞基质具有良好生物相容性,可能作为脂肪组织工程的支架材料。

Preparation and biocompatibility of porcine skeletal muscle acellular matrix for adipose tissue engineering

Objective Extracellular matrix is one of the focus researches of the adipose tissue engineering.To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix.Methods The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sliced into 2-3 mm thick sheets,which were treated by hypotonic-detergent method to remove the cells from the tissue.The matrix was then examined by histology,immunohistochemistry,and scanning electron microscopy.The toxic e ects of the matrix were tested by MTT.Human adipose-derived stem cells(hADSCs) were isolated from adipose tissue donated by patients with breast cancer,and identi ed by morphology,ow cytometry,and di erentiation ability.Then,hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix,and cultured in the medium.The cellular behavior was assessed by calcein-AM(CA) and propidium iodide(PI) staining at 1st,3rd,5th,and 7th days after culturing.Results Histology,immunohistochemistry,and scanning electron microscopy showed that the muscle bers were removed completely with the basement membrane structure;a large number of collagenous matrix presented as regular network,porous-like structure.The cytotoxicity score of the matrix was grade 1,which meant that the matrix had good cytocompatibility.The CA and PI staining showed the seeded hADSCs had the potential of spread and proliferation on the matrix.Conclusion Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial sca old for adipose tissue engineering.