软骨细胞接种小孔径聚乳酸-羟基乙酸共聚物支架的方法选择

An optimal method to seed chondrocytes into PLGA scaffolds with tiny pores

目的探讨软骨细胞接种小孔径聚乳酸－羟基乙酸共聚物（PLGA）支架的最佳接种方法。方法实验分3组（n=9）：注射组、负压组、振荡组，取纤维蛋白原溶液混悬第2代软骨细胞，采用上述3种方法对孔隙牢为92％、孔径为50～100μm的PLGA支架进行细胞接种。48h后，Hoechst 33258法检测支架内DNA含量；接种并观察支架内含异硫氰酸荧光素的无细胞纤维蛋白原凝胶的分布；硬组织切片、4’，6-二脒基-2-苯基吲哚（DAPI）染色观察支架内细胞分布。7d后，扫描电镜观察支架表面及内部的细胞形态。各组部分支架（n=3）植入裸鼠皮下，以无细胞PLGA支架为空白对照组，术后8周取出支架行甲苯胺蓝、Ⅱ型胶原免疫组织化学染色，并计算累积吸光度（IOD）值。结果注射组、负压组、振荡组平均DNA含量分别为（755．79±80．50）、（657．32±89．68）、（650．18±106．33）ng／mg，各组比较差异均尤统计学意义（F=1．214，P=0．361）。无细胞纤维蛋白凝胶在各组中都均匀分布，DAPI染色显示注射组细胞分布较其他两组均匀。扫描电镜显示负压组和振荡组外周细胞较注射组多，而支架孔隙内仅注射组可见细胞黏附。注射组甲苯胺蓝、Ⅱ型胶原免疫组织化学染色的IOD值均优于其他3组，差异有统计学意义（P〈0．05）。结论对于50～100μm的小孔径PLGA支架，注射法是一种快捷、高效的细胞接种方法。

Objective To explore an optimal method for seeding chondrocytes into a poly （lactic-co-glycolic acid） （PLGA） scaffold of 50-100 μm pore size. Methods The experiment was conducted in three groups （ n = 9）： injection, low-pressure and orbital shaker ones. Fibrin gel containing chondrocytes was seeded into PI,GA scaffolds with a porosity of 92% and pore size of 50-100 μm. Forty-eight hours later, the DNA content in each construct was analyzed by Hoechst33285 flurometric detection. FITC （fluorescein isothiocyanate） stained fibrin was also used to check distribution of the fibrin gel （without chondmeytes） in PLGA scaffohts. The distribution of chondrocytes in the scaffolds was assessed by hard tissue slice and DAPI （4＇, 6-diamidino-2-phenylindole） nucleus staining. Seven days later, SEM detection was used to observe the presence of chondrocytes in the outer periphery and the interior of the scaffolds. Some constructs were implanted subcutaneously into nude mice （ n = 3）. PLGA scaffolds with no chondrocytes were set as blank control group. After 8 weeks, the slices were examined by toluidine blue and collagen type II immunohisto- chemistU staining. Finally, the hnage-Pro Plus 6.0 software was used to analyze the integrated optical density （1OD） of the images. Results The DNA content was 755.79± 80.50 ng/mg in the injection group,657.32 ±89.68 ng/mg in the low-pressure group and 650. 18 ± 106. 33 ng/mg in the orbital shaker group, with no significant difference between the 3 groups （ F = 1. 214, P = 0. 361 ） . The fibrin gel was uniformly distributed in the PLGA scaffolds in all the 3 groups. DAPI staining indicated that the distribution of cells in the injection group was more uniform than in the other 2 groups. SEM detection demonstrated that more cells distributed in the outer periphery in the low-pressure and orbital shaker groups than in the injection group, and only the inside pores in the injection group had visible cell adhesion. The IOD values of toluidine blue and collagen type 11 immunohistoehemistry staining displayed that the injection group was better than the other 2 groups （ P 〈 0. 05） . Conclusion For PLGA scaffolds with a pore size of 50-100 μm, injection is a quick and efficient method for cell seeding.