Chondrogenic differentiation of peripheral blood-derived mesenchymal stem cells induced by transforming growth factor beta 3: a dose-effect relationship

doi:10.12307/2022.008

Abstract

Abstract: BACKGROUND: With the development of cartilage tissue engineering technology, there have been in vitro culture and animal experiments on repairing cartilage defects with peripheral blood-derived mesenchymal stem cells, and good progress has been made. Transforming growth factor beta 3 is often used in cell culture in vitro to induce chondrogenic differentiation of mesenchymal stem cells. However, in the experimental application, the dose-effect study of transforming growth factor beta 3 is rarely involved, and the cell culture experiments involving dose-effect often misuse one-way analysis of variance or t-test to analyze this kind of data.
OBJECTIVE: To analyze the dose-effect relationship of chondrogenic differentiation potency of peripheral blood-derived mesenchymal stem cells induced by transforming growth factor beta 3 by repeated measurement, in order to obtain a better dosage of transforming growth factor beta 3, and obtain a better effect of chondrogenic differentiation of peripheral blood-derived mesenchymal stem cells.
METHODS: After mobilization, extraction and culture of peripheral blood-derived mesenchymal stem cells of Diannan small eared pigs, transforming growth factor beta 3 at different concentrations (25-500 μg/L) was added for chondrogenic differentiation or not added (control group). Cell proliferation in each group was detected by CCK8 assay at 2, 4, 6, 8, 10, 12 and 14 days. At 3, 7, 14 and 21 days of culture, the supernatant of each group was collected, and the level of type II collagen in the supernatant was detected by ELISA. At 21 day of culture, toluidine blue staining was used to observe the expression of the aggregated proteoglycan. At 21 days, immunocytochemical staining was performed to observe the expression of type II collagen.
RESULTS AND CONCLUSION: In the range of 25-500 μg/L, the proliferation ability of peripheral blood-derived mesenchymal stem cells was the strongest at the concentration of transforming growth factor beta 3 at 40 μg/L. The concentration of transforming growth factor beta 3 was more likely to promote the chondrogenic differentiation of peripheral blood-derived mesenchymal stem cells in 40 and 80 μg/L.

Key words: stem cells, peripheral blood derived mesenchymal stem cells, cytokines, transforming growth factor beta 3, repeated measurements, cartilage tissue engineering, collagen type II, aggrecan, cartilage repair

CLC Number:

Yang Tengyun, Li Yanlin, Liu Dejian, Wang Guoliang, Zheng Zhujun. Chondrogenic differentiation of peripheral blood-derived mesenchymal stem cells induced by transforming growth factor beta 3: a dose-effect relationship[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(1): 45-51.

Figures/Tables 8

2.2.2 酶联免疫吸附法检测Ⅱ型胶原A1水平对照组未加入诱导液培养，其他实验组加入含不同质量浓度转化生长因子β3的诱导液之后，采用统计学两因素重复测量方差分析方法比较各组各时间段细胞培养上清中Ⅱ型胶原A1水平，结果如下：球形检验结果显示P > 0.05，证明此次研究数据对称，以“主体内效应检验结果”为此次研究最终结果。主体内效应检验结果显示：时间效应显著(F=3 425.986，P < 0.001)，表明各质量浓度转化生长因子β3组Ⅱ型胶原A1水平随培养天数的递增而出现明显变化；时间*浓度分组交互效应显著(F=42.395，P < 0.001)，表明不同质量浓度转化生长因子β3组在不同培养天数Ⅱ型胶原A1水平的变化趋势也有显著差异；主体间效应检验结果显示浓度分组效应显著(F=324.553，P < 0.001)，表明Ⅱ型胶原A1水平随转化生长因子β3质量浓度的变化而出现明显变化。各组PBMSCs分化曲线基本相似，第一时点细胞处于增殖期，未见明显分化，与对照组之间差异无显著性意义(P > 0.05)，从第二时间点开始，各组间差异逐渐显著，各组Ⅱ型胶原A1水平随培养时间的增长逐步递增。随着转化生长因子β3质量浓度增加，在7-14 d时Ⅱ型胶原A1水平增长较明显，在第14天后增长减缓，第21天时达到分化高峰，此时40，80 μg/L转化生长因子β3组Ⅱ型胶原A1水平差异无显著性意义(P > 0.05)，但明显优于其他质量浓度组。以上结果表明，高于2.5 μg/L时转化生长因子β3均能促进PBMSCs分化，并且在5-40 μg/L范围有一定时间浓度依赖，随着转化生长因子β3质量浓度增高和培养时间延长，分化产生Ⅱ型胶原A1更多；80 μg/L转化生长因子β3促进 PBMSCs分化能力与40 μg/L相当，高于80 μg/L时促进PBMSCs 分化能力又减弱。各组相比较之下转化生长因子β3质量浓度在40，80 μg/L时更容易促进PBMSCs成软骨分化，见表2。"

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