以可注射性纤维蛋白封闭剂为支架体内构建组织工程软骨

目的: 研究以纤维蛋白封闭剂（FS）为载体复合人胚关节软骨细胞体内构建可注射性组织工程软骨的可行性。方法:常规分离消化,体外单层培养胎儿关节软骨细胞，观察软骨细胞的生物学特性。分别将1×107、2×107、3×107第4代软骨细胞与FS混合接种于裸鼠皮下, 并于第10周取材判断体内形成软骨的能力。结果: 3~4代软骨细胞保持了很高的增殖和分泌基质的能力。软骨细胞与FS的复合物体内接种后各组均可形成软骨样组织块，其湿重、GAG含量随着接种细胞数量的增多而增高，各组之间差异具有显著性（p<0.05）。3×107细胞组GAG含量与正常人胚关节软骨没有差异（p>0.05）。组织切片显示软骨细胞位于类似正常软骨组织的陷窝中，阿尔新蓝染色及II型胶原表达阳性，细胞内富含高尔基体、粗面内质网及大量分泌泡。结论: FS和人胚关节软骨细胞可以作为理想的支架材料和种子细胞应用于可注射软骨组织的构建。

Construction of cartilage using injectable fibrin sealant with human embryo cartilages cells in vivo.

Objective: To investigate the feasibility of Fibrin Sealant (FS) as a carrier of human embryo chondrocytes to create new cartilage in vivo. Methods: Chondrocytes isolated by collagenase digestion from the human embryo cartilage were cultured and expanded in monolayer system. The biomechanical properties of the chondrocytes were investigated. 1×107、2×107、3×107 fourth passage chondrocytes were mixed with FS respectively. Then the FS-chondrocyte mixture was injected into the dorsum of nude mice，the ability of chondrogenesis in vivo was investigated 10 weeks later. Results: The proliferation and secretion of substance of the third-fourth passage were powerful. There are specimens like cartilage were harvested in all experimental groups. The average wet weight and GAG content has increased with the increase of cell numbers planted. The difference between any two groups is significant (p<0.05, n=3). There is no significant difference in GAG content in 3×107 group and the normal embryo cartilage (p>0.05, n=3). When the specimens were sectioned and stained, it was demonstrated that they hooked like normal cartilage tissue, with chondrocytes lying in a lacunae in basophilic ground-glass substance around it. The Alcian blue staining and Type II Collagen expressions were strong positive. Most cells appeared to contain several golgi zones and large areas of rough endoplasmic reticulum. Conclusion: This study demonstrates that FS and human embryo cartilage cell can be used as a constructing technique of tissue engineered injectable cartilage.