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目的 观察脂肪干细胞(adipose tissue-derived stem cells)与软骨脱细胞基质构建兔耳软骨的效果.方法 从32例抽脂减肥患者的脂肪组织中提取脂肪干细胞体外传代培养.通过脱细胞方法用猪耳软骨制备天然生物支架材料,再用二代ADSC以5.0×107个/ml的细胞浓度接种于预成型的天然生物材料支架上,体外培养3d,形成细胞支架复合物.采用随机数字表法将32只新西兰大白兔分为对照组A、对照组B、对照组C和实验组,每组8只,均行耳软骨缺损建模.实验组、对照组A、对照组B分别将细胞支架复合物、脂肪干细胞、软骨脱细胞基质支架移植于耳软骨缺损处,对照组C则直接逐层缝合耳前组织和皮肤.术后16周后兔耳廓软骨取材,观察修复效果.新生软骨标本行甲苯胺蓝染色、HE染色、Ⅱ型胶原免疫组化及番红O染色观察,并行Ⅱ型胶原阳性染色结果定量分析以及耳软骨组织弹性检测.应用SPSS17.0软件进行统计学分析.结果 大体观察见对照组(A、B、C)软骨缺损处由肉芽样组织充填,而实验组软骨缺损修复良好.实验组与对照组软骨缺损修复区组织湿重比较,差异有统计学意义(P＜0.05).HE染色显示,3个对照组仅有纤维组织充填耳软骨缺损处,实验组耳软骨缺损处可见软骨陷窝形成.3个对照组修复区甲苯胺蓝染色、Ⅱ型胶原免疫组化及番红O染色均为阴性,而实验组为阳性.耳软骨弹性常数检测,实验组新生软骨与正常耳软骨比较,差异无统计学意义(P＞0.05).结论 脂肪干细胞与软骨脱细胞基质结合构建的兔耳新生软骨的力学和组织学已接近正常耳软骨.软骨脱细胞基质材料复合脂肪干细胞,对耳软骨缺损具有较好的修复重建能力,具有潜在的临床应用价值.

Objective To investigate the effects of the repair and restitution of ear-shaped cartilage by adipose tissue-derived stem cells(ADSC) and cartilage acellular extracellular matrix.Methods ADSC were extracted by digesting with collagenase type Ⅱ from the adipose tissue from 32 patients with adiposity whose fats were drawn,and were cultured and subcultured in vitro.The natural biological scaffolds were prepared by acellular method using porcine ear cartilage,and then the second generation ADSC(5.0× 107/ml) were inoculated on the preformed natural bio-scaffold scaffold by culturing in vitro for 3 days to form a cell scaffold complex.32 New Zealand white rabbits were randomly divided into the experimental groups,the control group A,the control group B and the control group C.All New Zealand white rabbits were modeled by ear cartilage defects.The cell scaffolds composite was implanted into the experimental group of the ear cartilage defects of rabbits,the ADSC were implanted into the control group A,the cartilage acellular extracellular matrix scaffold was implanted into the control group B and the control group C was modeled only by ear cartilage defects.After 16 weeks,the animals were sacrificed and the repair effect was observed by gross appearance and histological examinations including HE,Toluidine blue staining,Safranin O and type Ⅱ collagen staining.Its were quantitatively analyzed by positive staining results of type Ⅱ collagen.Ear cartilage tissue elasticity was detected.SPSS 17.0 software was used to analyze the data.Results The cartilage defects in the experimental group were repaired well by general shape observation and those in the control group was filled in with granulation tissue.There were significant differences between the experimental group and the control group in the wet weight(P＜0.05).HE staining showed that cartilage cavities formed in articular cartilage defects,and only the fibrous tissue was filled with the ear cartilage defect in the control groups.In the repair area,Toluidine blue staining,Safranin O and type Ⅱ collagen staining were positive in the experimental group,and negative in the control groups.There was no significant difference between the experimental group and the normal ear cartilage in the ear cartilage elastic constant detection(P＞0.05).Conclusions The mechanics and histology of rabbit ear neonatal cartilage constructed by ADSC combined with cartilage acellular matrix are close to normal ear cartilage.Cartilage acellular matrix material combined with adipose-derived stem cells has good repair and reconstruction ability for ear cartilage defects,which possesses potential clinical application value.