Effects of uniaxial and biaxial mechanical loading on tenogenic differentiation of tendon-derived stem cells in mice
Qi Fangjie1, 2, Deng Zhantao2, Lyu Fengjuan1, Ma Yuanchen2, Zheng qiujian1, 2
1 School of Medicine, South China University of Technology, Guangzhou 510006, China;
2 Department of Orthopedics, Guangdong Provincial People's Hospital(Guangdong Academy of Medical Sciences), South China University of Technology School of Medicine, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China;
Abstract：Objective To investigate the effects of uniaxial mechanical loading and biaxial mechanical loading on the differentiation of mouse tendon-derived stem cells (TDSCs) and provide a theoretical basis for rehabilitation treatment after tendon injury. Methods Ten C57BL/6 mice aged 6-8 weeks were selected and exposed their hind legs to the soles of the feet under sterile conditions. Patellar tendon and Achilles tendon tissues were dissected and collected under microscope. TDSCs were isolated and cultured in vitro. The morphological characteristics of third-generation (P3) cells were observed. (1) Flow cytometry was used to detect mesenchymal stem cell markers (CD44, CD90, and Sca-1), endothelial cell markers (CD34 and Flk-1), and hematopoietic cell markers (CD45) in cells from the third generation to determine whether the cells were consistent with the characteristics of TDSCs. (2) TDSCs from the third generation were cultured into osteoblasts, chondrocytes, and adipocytes. Alizarin red, oil red, and alxin blue dyes were used to stain the cultured cells to determine whether the cells had the potential for multidirectional differentiation. (3) TDSCs from the third generation were inoculated on the silicone substrate and divided into three groups: uniaxial mechanical loading group, biaxial mechanical loading group, and control group. Cells in the biaxial group used Flexcell FX-4000TM flexible substrate tensile loading system, cells in the uniaxial group used self-made tensile force bioreactor, and cells in the control group had no tension. The TDSCs of the two mechanical stimulation groups were applied with 0.25 Hz and 6% biaxial cyclic tension or single-cycle cyclic tension for 8 h/d for 6 days. At the end of mechanical load stimulation on day 6, the three groups of cells were collected for real-time fluorescence quantitative polymerase chain reaction (qPCR) to detect the expression of tenogenic, osteogenic, adipogenic, and chondrogenic differentiation markers. Results Under the microscope, third-generation TDSCs were fusiform and had the same morphology. (1) Flow cytometry analysis showed the positive expression of mesenchymal stem cell markers CD44, CD90 and Sca-1, the negative expression of endothelial cell markers CD34 and Flk-1, and the negative expression of hematopoietic cell marker CD45. These findings confirmed the identity of TDSCs and conformed to the identification characteristics of TDSCs. (2) The three-line differentiation results showed that the extracted cells successfully differentiated into osteoblasts, adipocytes, and chondrocytes. The extracted cells had the potential to differentiate into osteoblasts, chondrocytes, and adipocytes. (3) The relative expression levels of tenogenic, osteogenic, adipogenic, and chondrogenic differentiation markers showed statistically significant differences among control, uniaxial, and biaxial groups (all P values＜0.05). The relative expression of tenogenic, osteogenic, and adipogenic differentiation marker PPARγ increased in the uniaxial group compared with that in the control group. The relative expression of chondrogenic differentiation markers decreased. All the differences were statistically significant (all P values＜0.05). However, no significant difference was found in the expression of adipogenic differentiation marker CEB/P (all P values＞0.05). Compared with control group, the expression of tenogenic differentiation markers Scx and Mohawk decreased in the biaxial group. The expression of osteogenic differentiation marker Runx2 increased, whereas the expression of alkaline phosphatase (ALP) decreased in the biaxial group. The expression of chondrogenic differentiation marker Sox9 increased, and the expression of Col2a1 decreased in the biaxial group. The expression of adipogenic differentiation markers all increased in the biaxial group. All the differences were statistically significant (all P values＜0.05). The relative expression of tenogenic differentiation marker Scx, Mohawk, and Col1a1 decreased, the relative expression of osteogenic differentiation marker ALP decreased, the relative expression of adipogenic and chondrogenic differentiation markers increased in the biaxial group compared with those in the uniaxial group. The differences were statistically significant (all P values＜0.05). Conclusions Uniaxial mechanical stimulation induced TDSCs to differentiate into tendon and osteoblasts, while biaxial mechanical stimulation induced TDSCs to differentiate into bone, fat, and chondrocytes. Uniaxial mechanical stimulation can promote the differentiation of TDSCs into tenocytes in vitro, which is beneficial to the regeneration of tendon tissues and tendon repair after injury. This work provides a theoretical basis for rehabilitation treatment of tendon injury in clinical applications.
祁方杰, 邓展涛, 吕凤娟, 马元琛, 郑秋坚. 单轴、双轴循环拉力对小鼠肌腱源性干细胞肌腱样分化的影响[J]. 中华解剖与临床杂志, 2020, 25(6): 683-690.
Qi Fangjie, Deng Zhantao, Lyu Fengjuan, Ma Yuanchen, Zheng qiujian. Effects of uniaxial and biaxial mechanical loading on tenogenic differentiation of tendon-derived stem cells in mice. Chinese Journal of Anatomy and Clinics, 2020, 25(6): 683-690.
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2020, Vol. 25 
