Abstract: Objective    To establish an in vitro mechanical culture model of rat condylar chondrocytes and investigate the effects of different cyclic tensile stress magnitude and loading duration on osteogenic differentiation. Methods    The study was conducted in the laboratory of Zhejiang Chinese Medical University from June 2021 to December 2022. The condylar chondrocytes were isolated after the Sprague-Dawley（SD）rats were sacrificed and dissected under sterile conditions，and condylar chondrocytes were cultured and identified by type Ⅱ collagen immunofluorescence staining. The second-generation condylar chondrocytes cultured in vitro were used for application of cyclic tensile stress. For the experimental group，the conditions were set as follows：the applied force was a 5%，10%，and 15% deformation rate gradient，the duration gradient was 6 h，12 h，and 24 h，and the applied force frequency was 1 Hz. The control group was the cells without any tensile stress applied. The mRNA expression of Runt-related transcription factor 2（Runx2），osteocalcin（OC），and Osterix（Osx）in rat condylar chondrocytes was detected by real-time fluorescence quantitative PCR. Results    Immunofluorescence staining of type Ⅱ collagen showed that the condylar chondrocytes of the second generation were stained uniformly. In addition，the cells were extended well and arranged in order. Two-factor analysis of variance showed that the relative mRNA expression levels of OC，Osx，and Runx2 in condylar chondrocytes were all statistically different under different tensile stress and duration（P < 0.05），and there was a significant interaction effect between the tensile stress and duration（P < 0.05）. When the tensile stress was 15%，the relative mRNA expression level of OC，Osx and Runx2 in condylar chondrocytes at different duration in the experimental groups was lower than that in the control group. The relative mRNA expression level of Runx2 in condylar chondrocytes at different duration in the experimental groups had no statistical difference compared with the control group when the tensile stress was 5%. The relative mRNA expression levels of OC，Osx，and Runx2 in condylar chondrocytes were higher than those in the control group after 24 h when the tensile stress was 10%. Moreover，among the three experimental groups，when the tensile stress was 10%，the relative mRNA expression level of OC and Osx in condylar chondrocytes of all the experimental groups was the highest after 24 h，and the differences were statistically significant（P < 0.05）. Conclusion    Different cyclic tensile stress and different duration have different osteogenic effect on rat condylar chondrocytes. The osteogenic effect of condylar chondrocytes may be better under the 10% tensile stress for 24 h.

Key words: cyclic tensile stress, application duration, condylar cartilage, osteogenesis

摘要： 目的    建立大鼠髁突软骨细胞体外力学培养模型，探讨不同大小循环张应力及加力时间对大鼠髁突软骨细胞成骨的影响。方法    研究于2021年6月至2022年12月在浙江中医药大学实验室进行。无菌条件下处死并解剖分离SD大鼠髁突，培养并使用Ⅱ型胶原免疫荧光染色鉴定髁突软骨细胞。取第2代髁突软骨细胞施加循环张应力，加力频率为1 Hz，加力大小包括5%、10%、15%形变率，加力时间为6、12、24 h，作为实验组；同时设置不施加张应力的细胞作为对照组。实时荧光定量PCR检测各组骨特异性转录因子2（Runt-related transcription factor 2，Runx2）、骨钙素（osteocalcin，OC）和成骨细胞特异性转录因子Osterix（Osx）的mRNA相对表达量。结果    Ⅱ型胶原免疫荧光染色显示，第2代髁突软骨细胞染色均匀，细胞伸展较好、排列有序。经两因素析因设计资料的方差分析显示，不同加力大小、加力时间作用下髁突软骨细胞中OC、Osx、Runx2 mRNA相对表达量比较，差异均有统计学意义（均P < 0.05）；且加力大小与加力时间这2个因素间存在相互效应（均P < 0.05）。其中，当加力大小为15%形变率时，髁突软骨细胞中OC、Osx、Runx2的mRNA相对表达量均较相同加力时间对照组低，差异均有统计学意义（均P < 0.05）；当加力大小为5%形变率时，髁突软骨细胞中Runx2的mRNA相对表达量与相同加力时间对照组比较，差异均无统计学意义（均P > 0.05）。当加力大小为10%形变率时，加力24 h后髁突软骨细胞中OC、Osx、Runx2的mRNA相对表达量均较对照组高；且在3个实验组中，加力24 h后10%形变率实验组髁突软骨细胞中OC、Osx的mRNA相对表达量最高，差异均有统计学意义（均P < 0.05）。结论   不同大小循环张应力及加力时间对大鼠髁突软骨细胞成骨具有差异性影响，在10%形变率、24 h加力时间条件下髁突软骨细胞成骨效果可能更佳。

关键词: 循环张应力, 加力时间, 髁突软骨, 成骨