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

Effects of uniaxial and biaxial mechanical loading on tenogenic differentiation of tendon-derived stem cells in mice

目的探讨单轴、双轴循环拉力对小鼠肌腱源性干细胞(TDSCs)分化的影响,为临床肌腱损伤后的康复治疗提供理论基础。方法取6~8周龄C57BL/6小鼠10只,无菌条件下暴露双侧后腿至脚掌,显微镜下解剖收集小鼠髌腱和跟腱组织块,体外分离培养细胞,观察第3代细胞的形态特点。(1)取传代至第3代的细胞,采用流式细胞术检测间充质干细胞标志物(CD44、CD90、Sca-1)、内皮细胞标志物(CD34、Flk-1)、造血细胞标志物(CD45),鉴定细胞是否符合TDSCs特点。(2)取传代至第3代的TDSCs进行成骨细胞、软骨细胞和脂肪细胞分化培养,分别采用茜素红、油红和阿尔新蓝染料对培养的三系细胞进行染色,鉴定细胞是否具有多向分化潜能。(3)取传代至第3代的TDSCs接种到硅胶底培养皿上,分为双轴循环拉力组、单轴循环拉力组、对照组3组。双轴循环拉力组细胞使用Flexcell? FX-4000TM柔性基底拉伸加载系统,单轴循环拉力组细胞使用自制拉伸力生物反应器,对照组细胞无拉力。双轴循环拉力组、单轴循环拉力组施加机械负荷组的参数均设置为0.25 Hz、6%的循环拉力,在培养期间进行机械负荷加载,每天加载8 h,共加载6 d。第6天机械负荷刺激结束后,收集3组细胞进行实时荧光定量PCR (qPCR),检测肌腱、成骨、脂肪和软骨相关转录因子的表达。结果显微镜下观察第3代TDSCs形态一致,呈梭形纤维状。(1)流式细胞技术检测结果显示,间充质干细胞标志物CD44、CD90和Sca-1表达阳性、内皮细胞标志物CD34和Flk-1表达阴性、造血细胞标志物CD45表达阴性,符合TDSCs标记鉴定特点。(2)三系分化细胞检测结果显示,提取的细胞成功分化为成骨细胞、脂肪细胞和软骨细胞,验证了提取的细胞具有向成骨细胞、软骨细胞和脂肪细胞分化的潜能。(3)对照组、单轴循环拉力组、双轴循环拉力组3组间比较,肌腱、成骨、软骨、脂肪相关转录因子的相对表达量差异均有统计学意义(P值均<0.05)。组间两两比较:单轴循环拉力组与对照组比较,肌腱、成骨相关转录因子以及脂肪相关转录因子PPARγ的相对表达均增高,软骨相关转录因子的相对表达均降低,差异均有统计学意义(P值均<0.05),而脂肪相关转录因子CEB/P的相对表达差异无统计学意义(P>0.05);双轴循环拉力组与对照组比较,肌腱相关转录因子Scx、Mohawk的相对表达降低,成骨相关转录因子Runx2的相对表达增高、碱性磷酸酶(ALP)的相对表达降低,软骨相关转录因子Sox9相对表达增高、Col2a1的相对表达降低,脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均<0.05);单轴循环拉力组与双轴循环拉力组比较,双轴循环拉力组肌腱相关转录因子Scx、Mohawk、Col1a1的相对表达均降低,成骨相关转录因子ALP的相对表达降低,软骨、脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均<0.05)。结论单轴循环拉力诱导TDSCs向肌腱细胞、成骨细胞分化,而双轴循环拉力诱导TDSCs向成骨细胞、脂肪细胞、软骨细胞分化。单轴循环拉力的作用下可以促进体外TDSCs向肌腱细胞分化,有利于肌腱组织的再生和损伤后的修复,为临床肌腱损伤后的康复治疗提供了理论依据。

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 PPARγincreased 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.