CAJ | 학술논문

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5×107/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.
목적 탐토연골미립복합Ⅱ형효원단백/투명질산/류산연골소(COLⅡ/HA/CS)방생기질용효체외구건조직공정연골가행성.방법 종5개월령귀주소향저관절획취연골미립,용불동공경적미립사과려후분별배양,재1、3、7 d관찰연골세포탈락、증식;장COLⅡ/HA/CS안질량비8.0∶1.5∶0.5배치성종질량농도위10 g/L적방생용효,관찰성효효과;선용증식능력최강적연골미립,안5×107/L여용효혼합,배양1주후진행대체、활세포면역형광、조직학관찰.결과 불동립경연골미립균가견연골세포탈락급증식,립경소우120μm적연골미립중세포증식능력최강,차이유통계학의의(P＜0.05);방생기질용효저온조건중화후구유류동성,교련병승온지37℃、6 min후성효성능량호;연골미립여방생용효공배양1주후,외관정투명연골양,표면광활병구유일정탄성;면역형광견연골세포활성량호;조직학관찰견연골세포종연골미립중탈락、증식,미립기질여응효융합.결론 립경소우120μm적미립중연골세포탈락증식능력최강;연골미립여COLⅡ/HA/CS방생기질응효생물상용성호,구건조직공정연골구유가행성.
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 × 107/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.