软骨微粒复合仿生溶胶构建组织工程软骨

Study on the feasibility of tissue engineering cartilage constructed by cartilage particle and bionic sol matrix composite

目的 探讨软骨微粒复合Ⅱ型胶原蛋白/透明质酸/硫酸软骨素（COLⅡ/HA/CS）仿生基质溶胶体外构建组织工程软骨可行性.方法 从5个月龄贵州小香猪关节获取软骨微粒,用不同孔径的微粒筛过滤后分别培养,在1、3、7 d观察软骨细胞脱落、增殖;将COLⅡ/HA/CS按质量比8.0∶1.5∶0.5配置成终质量浓度为10 g/L的仿生溶胶,观察成胶效果;选用增殖能力最强的软骨微粒,按5×10^7/L与溶胶混合,培养1周后进行大体、活细胞免疫荧光、组织学观察.结果 不同粒径软骨微粒均可见软骨细胞脱落及增殖,粒径小于120μm的软骨微粒中细胞增殖能力最强,差异有统计学意义（P＜0.05）;仿生基质溶胶低温条件中和后具有流动性,交联并升温至37℃、6 min后成胶性能良好;软骨微粒与仿生溶胶共培养1周后,外观呈透明软骨样,表面光滑并具有一定弹性;免疫荧光见软骨细胞活性良好;组织学观察见软骨细胞从软骨微粒中脱落、增殖,微粒基质与凝胶融合.结论 粒径小于120μm的微粒中软骨细胞脱落增殖能力最强;软骨微粒与COLⅡ/HA/CS仿生基质凝胶生物相容性好,构建组织工程软骨具有可行性.

Objective To explore the feasibility of constructing tissue engineered cartilage by using the cartilage particle as seed ceils and collagen Ⅱ-hyaluronic acid-chondroitin sulfate （COL Ⅱ-HA-CS） bionic matrix sol.Methods Cartilage particles were acquired from 5 months old Guizhou minipig knee joint;particles with different diameters were acquired by sieve filtration.The proliferation and adhesion of chondrocytes were evaluated at day 1,3,and 7.The bionic sol matrix at final concentration of 10 g/L was prepared by dissolving COL Ⅱ-HA-CS （mass ratio：8.0∶1.5∶0.5） in 0.15 mol/L HCl;The quality of prepared gel was examined.The cartilage particles with the diameter of strongest proliferation rate was seeded into COL Ⅱ/HA/CS sol mix at 5 × 10^7/L the in vitro results were evaluated by Living cell immunofluorescence and histological observation after 1 week.Results Cartilage particles with different sizes all developed visible chondrocytes adherence and proliferation,and particle size less than 120 μm showed strongest chondrocytes proliferation ability,the difference is statistically significant （P 〈 0.05）.Neutralized COL Ⅱ/HA/CS bionic sol matrix showed good flowability at low temperature;and transferred to gel at 37 ℃ for 6 min.After 1 week the cultured cartilage particles/gel complex showed transparent chondroid appearance,smooth surface and certain level of flexibility;Immunofluorescence observation showed good chondrocytes activity;Histological observation demonstrated chondrocytes were evenly distributed with good viability.Conclusion It reached the best proliferation of chondrocytes when particle size was less than 120 μm;Cartilage particles and COL Ⅱ/HA/CS bionic matrix sol are feasible to culture and construct bionic cartilage in vitro.