不同参数滚压模式对兔BMSCs向软骨细胞分化促进作用的研究

EFFECT OF ROLLING COMPRESSION LOADING BIOREACTOR ON CHONDROGENESIS OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS WITH DIFFERENT LOADING PARAMETERS

目的观察在不同滚压参数下,滚压载荷生物反应器对BMSCs-琼脂糖复合体中兔BMSCs向软骨细胞诱导分化的作用。方法分离2.5月龄新西兰兔BMSCs,培养至第3代后与低熔点琼脂糖复合成凝胶块,并在自制模具中形成直径4 mm、高4 mm的圆柱形BMSCs-琼脂糖复合体。将样本分别在0.4 Hz、3 h/d基本参数下比较压缩量10%、20%、30%(分别为A1、A2、A3组),以及0.4 Hz、20%压缩量基本参数下比较20 min、3 h、12 h滚压时间(分别为B1、B2、B3组)对BMSCs向软骨细胞分化的作用,以静态培养作为对照组。在培养24 h及7、14、21 d时取各组标本进行死活细胞检测、实时定量PCR检测、糖胺聚糖(glycosaminoglycan,GAG)含量检测及组织学染色观察。结果死活细胞检测:14、21 d时,A1、A2组活细胞比率均显著高于A3组,B1、B2组活细胞比率均显著高于B3组,差异均有统计学意义(P<0.05)。实时定量PCR检测:各实验组7、14、21 dⅡ型胶原及蛋白聚糖(Aggrecan)mRNA表达均高于对照组,差异有统计学意义(P<0.05);14、21 d时,A1、A2组Ⅱ型胶原、Aggrecan mRNA表达均高于A3组,B1、B2组均高于B3组,差异有统计学意义(P<0.05)。以上指标A1、A2组间及B1、B2组间比较,差异均无统计学意义(P>0.05)。GAG含量检测:14、21 d时,A1、A2组GAG含量均显著高于对照组和A3组,B1、B2组GAG含量也显著高于对照组和B3组,差异均有统计学意义(P<0.05);21 d时A1、A2组间差异均有统计学意义(t=2.830,P=0.027)。组织学观察:21 d时,A2组和B2组出现明显蓝染,可见软骨陷窝样结构;而A1、A3组和B1、B3组蓝染效果较差,分布稀少。结论滚压载荷生物反应器在0.4 Hz、20%压缩量、3 h滚压时间参数下,能更有效地促进BMSCs-琼脂糖复合体中兔BMSCs向软骨细胞诱导分化。

Objective To explore the effect of rolling compression loading bioreactor on chondrogenesis of rabbit bone marrow mesenchymal stem cells （BMSCs） with different loading parameters. Methods BMSCs were isolated from New Zealand rabbits, aged 2.5 months. BMSCs at passage 3 were used to prepare BMSCs-agarose gels （4 mm in diameter and height, respectively）. Samples were divided into 8 groups： 10% （group A1）, 20% （group A2）, and 30% （group A3） compression groups （0.4 Hz, 3 h/d） and 20 minutes （group B1）, 3 hours （group B2）, and 12 hours （group B3） rolling time groups and static culture （control groups）. The living cell rate, the collagen type II and Aggrecan gene expressions, and glycosaminoglycan （GAG） content were determined, and histological staining was done at 24 hours, 7 days, 14 days, and 21 days after culture. Results At 14 and 21 days, the living cell rates of groups A1 and A2 were significantly higher than that of group A3 （P 〈 0.05）, groups B1 and B2 were significantly higher than group B3 （P 〈 0.05）. Collagen type II and Aggrecan gene expressions of the experimental groups at each time point were significantly higher than those of the control groups （P 〈 0.05）; at 14 and 21 days, collagen type II and Aggrecan gene expressions of groups A1 and A2 were significantly higher than those of group A3, and groups B1 and B2 were also significantly higher than group B3 （P 〈 0.05）. At 14 and 21 days, the GAG contents of groups A1 and A2 were significantly higher than those of group A3 （P 〈 0.05）; groups B1 and B2 were also significantly higher than group B3 （P 〈 0.05）. At 21 days, toluidine blue staining showed that obvious blue-staining and even cartilage lacunae were seen in groups A2 and B2, but light and quite rare blue-staining in groups A1, A3, B1, and B3. Conclusion The rolling compression loading bioreactor has great promotion effect on chondrogenesis of rabbit BMSCs with rolling parameters of 0.4 Hz, 3 hours, and 20%