体外培养脂肪组织来源干细胞与胶原/壳聚糖复合支架的亲和性

Collagen-chitosan polymeric scaffolds for in vitro culture of human adipose tissue-derived stem cells

目的:制备适合脂肪组织来源的干细胞生长的支架,观察复合支架各组成的体积比率与细胞培养的亲和性。方法:实验于2006-09/2007-01在大连理工大学干细胞与组织工程研发中心完成。①实验方法:将3.55g/LⅠ型胶原和20g/L壳聚糖分别以9∶1,7∶3,5∶5,3∶7,1∶9的体积比混合冻干,碳化二亚胺/N-羟基琥珀酰亚胺交联后再次冻干并进行分析。②实验评估:扫描电镜观察不同材料交联前后的微观结构;IPP软件分析计算支架的平均孔径;测量支架的吸水性和孔隙率;通过胶原酶检测支架的体外生物可降解性;扫描电镜和苏木精-伊红染色观察脂肪组织来源的干细胞在复合支架上的生长情况。结果:①交联前后的微观结构:冻干后的各种支架材料呈白色,表面粗糙,材料内部呈海绵状多孔隙结构,其中以胶原/壳聚糖体积比为9∶1的复合支架最为疏松,1∶9的支架最致密。扫描电镜下支架的胶原含量越高,支架内的胶原丝越多,支架的孔与孔之间相互连通构成了通孔。交联前后支架的形态结构无明显改变。②支架的平均孔径:交联后体积比为9∶1,7∶3和5∶5的复合支架孔径50～200μm,可用于细胞的三维培养。③支架的吸水性和孔隙率:体积比为5∶5的复合支架的吸水性和含水量最高,而7∶3次之;多孔支架在水中未发生明显的溶胀现象;支架的孔隙率均在90%以上。④支架的体外生物可降解性:未交联的支架随着胶原含量的减少,支架的降解速率增加。而交联后随着胶原含量的减少,降解速率减慢,交联后的复合支架降解速度较未交联慢。⑤脂肪组织来源的干细胞在复合支架上的生长情况:脂肪组织来源的干细胞在支架上培养5d后扫描电镜观察细胞在7∶3的支架上爬行生长并融合成片,苏木精-伊红染色观察支架孔内及表面出现大量的细胞团,并融合成片状,而5∶5支架上黏附生长的细胞较少。结论:结合支架的孔径、吸水性、孔隙率、体外生物可降解性和细胞与支架的生物相容性,可知体积比为7∶3的复合支架对脂肪组织来源的干细胞的亲和性较好,适于脂肪组织来源的干细胞的三维培养。

AIM： To prepare polymeric scaffolds suitable for the growth of adipose tissue-derived stem cells （ADSCs）, and observe the affinity of composite scaffolds with volume ratio to cellular culture. METHODS： The study was completed in Research and Development Center for Stem Cell and Tissue Engineering, Dalian University of Technology between September 2006 and January 2007. ①20 g/L chitosan was fully mixed with 3.55 g/L tail type Ⅰ collagen with different volume ratios： 9：1, 7：3, 5：5, 3：7 and 1：9 （collagen：chitosan）, then freeze-dried and cross-linked with 1-ethyl-3-（3-dimethylaminopropyl） carbodiimide/N-hydroxysuccinimide, freeze-dried again and finally examined.②The microstructure of cross-linked scaffolds was observed via scanning electron microscopy. Average pore size was calculated by Image Pro Plus software. Bibulous ability, water content and interval porosity of scaffolds were measured before and after cross-linking. The collagenase detection was performed to evaluate the in vitro degradability of the scaffolds. The biocompatibility of ADSCs with composite scaffolds was observed by scanning electron microscopy and hematoxylin-eosin stain. RESULTS： ①After lyophilization, all scaffolds showed white color with coarse surface, and the inside of scaffolds was porous structure similar to sponginess. The most porous scaffolds were those with volume ratio 9：1, while the most compact were 1：9 group. Under scanning electron microscope, collagen fibers increased with the increase of collagen contents, holes were cross-connected with each other and formed the structure of through-hole. The morphology and structure of cross-linked scaffolds had no obvious difference with uncross-linked ones. ②The average pore sizes of cross-linked scaffolds with volume ratio 9：1, 7：3 and 5：5 were between 50 μm and 200 μm, which was suitable to the growth of cells. ③Scaffolds with the highest bibulous ability and water content were those with volume ratio 5：5, the second were 7：3; there was no obvious swelling of scaffolds in water and the interval porosity all exceeded 90%. ④In vitro degradation rate of uncross-linked scaffolds increased with the decrease of collagen content; while the degradation rate of cress-linked scaffolds decreased; uncross-linked composite scaffolds had a faster degradation rate than cross-linked ones. ⑤After ADSCs were cultured in scaffolds for five days, the result of scanning electron microscopy showed that, ADSCs expended and confluenced in scaffolds with volume ratio 7;3, mass of cell clumps in hole and surface of scaffolds could be seen after hematoxylin-eosin stain. There were fewer cells in scaffolds with volume ratio 5：5. CONCLUSION： Comprehensively considering pore size, bibulous ability, interval porosity, in vitro biodegradation and biocompatibility of all kinds of scaffolds, the affinity of composite scaffolds with volume ratio 7：3 to ADSCs is better than that of other scaffolds, and the composite scaffolds are suitable for the growth of ADSCs in three dimensions.