CAJ | 학술논문

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种方法对孔隙率为92％、孔径为50 ～ 100 μm的PLGA支架进行细胞接种.48 h后,Hoechst33258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,各组比较差异均无统计学意义(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 ～ 100 μm的小孑L径PLGA支架,注射法是一种快捷、高效的细胞接种方法.
목적 탐토연골세포접충소공경취유산-간기을산공취물(PLGA)지가적최가접충방법. 방법 실험분3조(n=9):주사조、부압조、진탕조,취섬유단백원용액혼현제2대연골세포,채용상술3충방법대공극솔위92％、공경위50 ～ 100 μm적PLGA지가진행세포접충.48 h후,Hoechst33258법검측지가내DNA함량；접충병관찰지가내함이류청산형광소적무세포섬유단백원응효적분포；경조직절편、4’,6-이미기-2-분기신타(DAPI)염색관찰지가내세포분포.7d후,소묘전경관찰지가표면급내부적세포형태.각조부분지가(n=3)식입라서피하,이무세포PLGA지가위공백대조조,술후8주취출지가행갑분알람、Ⅱ형효원면역조직화학염색,병계산루적흡광도(IOD)치. 결과 주사조、부압조、진탕조평균DNA함량분별위(755.79±80.50)、(657.32±89.68)、(650.18±106.33)ng/mg,각조비교차이균무통계학의의(F=1.214,P=0.361).무세포섬유단백응효재각조중도균균분포,DAPI염색현시주사조세포분포교기타량조균균.소묘전경현시부압조화진탕조외주세포교주사조다,이지가공극내부주사조가견세포점부.주사조갑분알람、Ⅱ형효원면역조직화학염색적IOD치균우우기타3조,차이유통계학의의(P＜0.05). 결론 대우50 ～ 100 μm적소혈L경PLGA지가,주사법시일충쾌첩、고효적세포접충방법.
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 PLGA 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 fluorometric detection.FITC (fluorescein isothiocyanate) stained fibrin was also used to check distribution of the fibrin gel (without chondrocytes) in PLGA scaffolds.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 immunohistochemistry staining.Finally,the Image-Pro Plus 6.0 software was used to analyze the integrated optical density (IOD) 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 Ⅱ immunohistochemistry staining displayed that the injection group was better than the other 2groups (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.