【摘要】 目的探讨兔脂肪来源干细胞(adipose-derived stem cells,ADSCs)作为种子细胞复合新型聚羟基乙酸(polylactic-co-glycolic acid,PLGA)/壳聚糖(chitosan,CS)支架材料的生物相容性,为进一步构建组织工程化脂肪组织提供实验依据。方法取雄性新西兰大白兔腹股沟脂肪组织,Ⅰ型胶原酶消化分离出ADSCs进行培养,贴壁细胞至3~5代,评价其多向分化能力。将干细胞收集重悬后,以l×107/mL的密度接种于多孔PLGA/CS支架,形成细胞-支架材料复合物。培养7天后,扫描电子显微镜观察细胞在支架材料上的黏附生长和基质分泌情况,评价细胞与支架材料的生物相容性;Dil荧光标记检测细胞在支架材料上的分布;Hochest 33258检测细胞在支架上的生长情况。接种1和7天后,分别对细胞-材料复合物行Annexin V/PI双染色法检测材料对细胞的毒性作用。用成脂诱导条件培养基诱导分化ADSCs及ADSCs支架材料复合物,7天后,尼罗红荧光染色液检测ADSCs在不同环境下的成脂分化能力。结果原代培养的ADSCs呈成纤维细胞样外观,在相应诱导条件下能够分化为脂肪细胞和骨细胞。细胞接种于PLGA/CS支架材料上第8天分裂增殖达到高峰,扫描电子显微镜下提示ADSCs在支架表面贴附生长良好,并向孔隙内壁充分延伸,细胞周围形成丰富的基质成分。活死双染结合共聚焦显微镜显示材料对细胞活性无影响,尼罗红染色可见成脂诱导后的ADSCs细胞质内有红色脂滴颗粒形成。结论多孔PLGA/CS支架与兔ADSCs具有良好的生物相容性,可作为构建组织工程脂肪组织的支架材料。更多还原

【Abstract】 Objective To investigate the biocompatibility of rabbit adipose-derived stem cells(ADSCs)and polylactic-co-glycolic acid(PLGA)/chitosan(CS)scaffold and to provide experiment basis for building tissue-engineered adipose tissue. Methods ADSCs were isolated from subcutaneous adipose tissue in groin area of New Zealand white rabbit with the method of enzymatic digestion.The cells were cultured and passaged to 3-5 generation and its differentiation ability was evaluated.After 3-5 generation ADSCs were harvested,re-suspended with a density of 1×107/mL and replanted into PLGA/CS scaffold,scanning electron microscope was used to reveal the microstructure of PLGA/CS and the growth of ADSCs on PLGA/CS scaffold 1 and 7 days after seeding for the evaluation of biocompatibility between ADSCs and PLGA/CS scaffold.The distribution of ADSCs on PLGA/CS scaffold appended with adipogenic inducing medium or basic culture medium was evaluated with Dil fluorescence and confocal microscopy.The proliferation of ADSCs on PLGA/CS scaffold appended with adipogenic inducing medium or basic culture medium was evaluated with hochest33258.At 1 and7 days post-seeding,the toxicity of PLGA/CS scaffold to ADSCs was assessed by Annexin V/PI influorescence fluid.ADSCs and ADSCs-PLGA/CS complex were cultivated in adipogenic inducing medium for 7 days and the differentiated cells were identified by Nile red stanning. Results Primarily cultured rabbit ADSCs presented fibroblast-like appearance and ADSCs cultured in adipogenic and ostogenic medium could differentiate into adipose cell and bone cells.The proliferation of cells on PLGA/CS scaffold with adipogenic inducing medium or basic culture medium went plateau phase on the 8 day.Scanning electron microscopy(SEM)showed that ADSCs could grow well on the PLGA/CS scaffold and abundant extracelluar matrix(ECM)both on the surface and interior pore of scaffold.Annexin V/PI influorescence staining and confocal microscopy demonstrated that the PLGA/CS scaffold could not affect the activity of ADSCs post-seeding.An amount of lipid droplet within cytoplasm of cells on the PLGA/CS scaffold was revealed by Nile red staining. Conclusions The porous PLGA/CS scaffold and rabbit ADSCs has excellent biocompatibility,which could be used as a tissue engineered scaffold for building adipose tissue.更多还原