3种可注射性支架体内构建组织工程软骨的比较研究

Study on Construction of Tissue Engineered Articular Cartilage by Three Kinds of Rejectable Scaffold in Vivo

目的比较以纤维蛋白封闭剂(FS)、P luron ic-F 127(PF)、透明质酸(HA)3种可注射性支架体内构建组织工程软骨的效果。方法常规分离、消化、体外单层培养新生兔软骨细胞,分别将软骨细胞与3种支架材料混合接种于裸鼠皮下,并于第10周取材通过大体观察,湿重测定,GAG含量测定和组织化学检查来判断体内形成软骨的能力。结果三组均可形成软骨样组织块,其中FS组体积最大,质地较硬,表面光滑,软骨细胞位于成熟的陷窝中,阿尔新兰及Ⅱ型胶原免疫组化染色强阳性;PF组体积中等,质地柔韧,陷窝清楚,可以检测到阳性的阿尔新兰及Ⅱ型胶原表达。HA组只形成很小的组织块,硬度较差,含有的细胞数和分泌的基质最少,三组的GAG含量分别为(8.8±0.4)m g/g,(6.8±0.3)m g/g,(4.2±0.3)m g/g,各组间差异具有显著性(P<0.05,n=3)。结论3种可注射性支架中,FS成软骨作用最强,PF较好、HA组最差,FS和PF可以用于组织工程软骨的构建。

Objective To investigate the effect of Fibrin Sealant （FS）, Pluronic-F127 and HA as a carrier to create new cartilage in vivo. Methods Chondrocytes isolated by collagenase digestion from the newborn rabbit cartilage were cultured and expanded in monolayer system. The chondrocytes were collected and mixed with the three carriers respectively. Then the carrier-chondrocytes mixture was injected by a needle into the dorsum of nude mice. Specimens were harvested after 10 weeks, the ability of chondrogenesis in vivo was investigated by Alcian Blue staining, type Ⅱ collagen immunohistochemistry reaction. Results After injection of the mixtures, the specimens like cartilage were harvested after 10 weeks in all experimental groups. The constructs of FS group were largest and became firmer and glossier with a mature lacuna structure distributed in most of the areas. The results of histochemistry examination were strong positive with Alcian blue staining and collagen Ⅱ antigens expression. The specimens of PF group were smaller than these of FS group and were relative firm in texture. The formation had distinct lacuna structure and glycosaminoglycan （GAG）. Type Ⅱ Collagen was also detected. The specimens of HA group were the smallest ones and relative flexible in texture. The cell number and extracellular matrix were less. The content of GAG in the three groups were （8. 8±0.4） mg/g,（6. 8±0. 3） mg/g,（4.2±0. 3） mg/g respectively. There was significant difference among the three groups（P〈0.05,n=3）. Conclusion Among the three injectable carriers the FS is the best, the PF is the secondary one, the HA is the lattermost. The FS and PF can be used as a carrier of tissue engineered injectable cartilage.