组织工程软骨体外构建的相关实验研究

In Vitro Construction of Tissue Engineered Cartilage Using Human Fetus Chondrocytes

目的探索组织工程软骨体外构建技术体系可行性。方法种子细胞选用胎儿软骨细胞(口服药物流产胎儿,胎龄3-6个月)。酶消化法获得第1代细胞,以50×106/ml浓度均匀接种于经聚乳酸(PLA)包埋聚乙醇酸(PGA)高分子聚合物支架,形成细胞-支架复合体,在体外静态培养。分别于2周、4周、8周进行大体观察、扫描电镜及组织学检测。结果体外构建的组织工程软骨,随培养时间延长,色泽由2周时的乳白色逐渐呈现半透明,8周时接近正常软骨外观。扫描电镜显示软骨细胞与材料具有良好相容性,培养7天PGA纤维之间有基质沉积。HE染色示2周有大量软骨陷窝形成和均匀嗜碱性基质分泌,Safranin’O染色示基质有酸性蛋白多糖分布,Massons’s trichome染色示基质有胶原成分,但含量较少,经免疫组织化学检测为特异Ⅱ型胶原。培养4周胶原成分开始明显增多,软骨陷窝形态接近成熟,8周细胞外基质蛋白多糖和Ⅱ型胶原含量丰富且分布均匀。结论以成熟软骨细胞为种子细胞,运用组织工程技术在体外能构建出具有正常软骨组织结构特征的人组织工程软骨。

Objective To evaluated the feasibility of growing tissue engineered cartilage in vitro using human fetus chondrocytes as seeding cells. Methods Full-thickness femoral and tibial epiphyseal articular cartilages were harvested from aborted human fetuses ,collected with informed parental consent within 18h postmortem . Chondrocytes were isolated by collagenase digestion and cultured in Ham' F-12 medium supplemented with 10% fetal bovine serum. Cell of Passage 1 were seeded onto PGA scaffold coated with PLA. The chondrocyte-polymer constructs were cultured statically in vitro for up to 8 weeks. At 2,4,8 weeks, the constructs were harvested respectively for gross inspection ,Scanning electron microscope (SEM) observation as well as histology and immunohistochemistry staining. Results After 8 weeks of cultivation, the constructs were nearly translucent as native cartilage and had intact shape compared with initial scaffold. The goodcompatibility with chondrocytes to PGA was observed through SEM. Histologically, constructs consisted of round cells embedded in homogeneous cartilaginous matrix. Massons's trichome and Safranin'O staining revealed collagen and proteoglycan deposition. Collagen type II expression in matrix was detected by Immunohistochemistry staining. Conclusion Human fetus chondrocytes possess the ability to form new cartilage when seeded onto a three-dimensional polymer scaffold in vitro. The results showed the feasibility of our method to construct human tissue engineered cartilage in vitro.