可注射性藻酸钙凝胶载体组织工程性软骨的实验研究

Experimental study of in vitro culture of chondrocytes combined with injectable calcium alginate gel for neo-cartilage tissue engineering

目的 对经体外培养的软骨细胞与藻酸钙凝胶载体复合形成的组织工程性软骨进行组织形态学观察。方法 取 2周龄新西兰白兔的膝关节软骨细胞，经体外培养两次传代后与藻酸钠溶液复合，使细胞密度为 5× 10 6/ml、 NaCl 0.135 mol/L、 K 2HPO 4 0.1 mol/L、藻酸钠的质量分数为 1％，然后注入葡萄糖酸钙使终浓度为 40 mmol/L。在实验组 (A、 B组 )兔的背部皮下注入复合藻酸钙载体的软骨细胞悬液，对照组 (C、 D组 )动物或注入无载体的软骨细胞悬液或注入无细胞的藻酸钙载体，分别于注射后第 2周、第 4周取材，进行 HE和甲苯胺蓝染色。结果 实验组，于注射复合软骨细胞的藻酸钙凝胶载体第 2周时， HE染色结果显示皮下注射点出现大量软骨细胞增殖，第 4周时有软骨组织形成；甲苯胺蓝染色见有软骨细胞和软骨基质形成。在注入无载体的软骨细胞悬液组 (C组 )，部分动物的皮下注射点有少量软骨组织形成，而在注入无细胞的藻酸钙载体组 (D组 )则未见软骨形成。结论 应用可注射性藻酸钙凝胶载体复合软骨细胞可在同种异体动物体内形成新生软骨组织。

Objective To observe histomorphology of neo-cartilage obtained with tissue-engineering by combining calcium alginate gel and in vitro cultrue of chondrocytes. Methods Articular chondrocytes from the knee joints of 2-week-old New Zealand white rabbits were harvested, expanded in cell culture, and following the second generation of the culture, the chondrocyte suspension was mixed with calcium alginate gel resulting in a cell density of 5× 10 6/ml. Finally the mixture, which contained 1％ sodium alginate, 40 mmol/L calcium gluconate, 0.135 mol/L NaCl, 0.1 mol/L K 2HPO 4 and 5× 10 6 chondrocytes per milliliter, were injected into the dorsal subcutaneous tissue in rabbits of experimental groups (A and B). Animals of the control groups (C and D) were injected with calcium alginate without chondrocytes or chondrocytes without calcium alginate. Specimens were harvested at the 2nd and 4th week after injection, and stained with HE and toluidine blue. Results In the HE stained specimens in the experimental groups, proliferation of chondrocytes was demonstrated at the 2nd week and the formation of neo-cartilage at the 4th week after injection of calcium alginate chondrocyte-composite. Toluidine blue stained specimens showed positive staining of chondrocytes and cartilage matrix of neo-cartilage. In some animals injected with chondrocytes without calcium alginate, relative small amount of neo-cartilage was also formed and no neo-cartilage was observed in animals injected with calcium alginate without chondrocytes. Conclusion Injectable calcium alginate-chondrocyte-composite can induce tissue engineered neo-cartilage in allogenic animal.