Investigation Into The Effects Of Mechanical Loading On The Differentiation Of Tendon Stem/Progenitor Cells And Regulatory Mechanism

Objective: Tendon stem cells(TSCs)maintain tendon homeostasis or lead to tendon injuries by sensing continuous mechanical loadings from sports and show different responses.However,it is still not clear about the effect of molecular mechanism from TSCs' differentiation yet.This project was aimed to investigate the molecular mechanism of TSCs' differentiation induced by different mechanical loading(movement).Findings will assist further revealing the physiological and pathological process of tendon regeneration,providing evidence for the development of exercise intensity post tendon injuries thus achieving best rehabilitation.In this present study,the biological characteristics of TSCs in vitro were studied.On this basis,Mechano Culture FX,periodic mechanical stimulation culture system were used,the effects of different mechanical loading to TSCs were studied by RNA-seq techniques and bioinformatics methods,the effect of mechanical stimulation on TSCs and the relevant signal pathways involved were studied,the key signal pathways and candidate genes were filtered.Method: Chose 6-week-old rats:(1)Enzymatical digestion were used to isolate TSCs from the Achilles tendon and patellar tendon tissues of rat.The expression of specific markers and genes of TSCs were identified by RT-PCR,immunofluorescence and flow cytometry.The proliferation potential of TSCs in vitro were studied by the growth curve and colony-forming cell assay,while the multi-differentiational potential was verified by induced differentiation experiment.(2)To investigate the effects of mechanical stimulation(movement)with different loading intensity ranged from 0%,2%,4%,6%,8% to 10% with frequency of 0.5 Hz and duration of 8h were applied to TSCs by Mechano Culture FX.CCK-8,RT-PCR and immunofluorescence was used to detect the proliferation,differentiation and cytoskeletal rearrangement ofTSCs;(3)RNA-sequencing analysis was performed in 0%(static culture group),4%(moderate-load)and 8%(high-load)mechanical loading group,the response of TSCs to mechanical loading was studied at the level of gene expression.At the same time,bioinformatics was used to screen the key signal pathways and candidate genes enriched by differentially expressed genes.Results:(1)Cells isolated in vitro were subcultured to 26 passages;Cells expressed specific markers of stem cell,such as Oct 4,Nanog,NS and SSEA4,specific markers of MSCs,CD166 and CD44,tenogenic markers Collagen I,Collagen III,TNC,Scx and Tnmd.The Clone formation assay and growth curve showed that colony-forming efficiency of cells at P3,P7,P15 and P23 were(32.18�1.21)%,(34.17�1.17)%,(28.84�1.24)% and(8.41�1.21)%,respectively.The PDT of cells at P3,P8,P15 and P22 were(14.18�1.11)h,(13.17�1.35)h,(18.87�2.61)h and(32.18�3.78),respectively.With the increase of passages,cells went into the stage of aging accompany with the decrease of self-renewal ability and proliferation potential.Multi-potent differentiation of cells indicated that cells incubated in adipogenic induction medium could be stained positively by oil red O staining and the cells expressed LPL and PPAR;Cells cultured in chondrogenic differentiation medium could be stained with the alcian blue staining and expressed ACAN and Sox9;Cells incubated in osteogenic induction medium could stained by alizarin red staining and express Runx2 and OPN,positively.(2)The morphology of TSCs under mechanical load showed that TSCs changed their shape along the direction perpendicular to the stress.The results of proliferation and differentiation potential showed that,4% mechanical loading significantly increased the proliferation activity of TSCs(P<0.01)and the expression of m RNA of tenogenic genes Tnmd and Scx increased significantly,Runx2 and Fabp4 m RNA expressions were significantly increased(P<0.01).The proliferation activity of TSCs was significantly inhibited in 8% mechanical strain(P<0.01)with the unregulated mRNA expression in Fabp4,Runx2,Scx,TNC and Col I.The results of immunofluorescence showed that mechanical loading caused rearrangement of cytoskeletal protein F-actin fiber,the degree of which depends on the loading intensity;(3)In virtue of filtrate the differentially expressed genes in different load groups(|log2FC|?2,Q-Value?0.001).Compared with control group,905 differentially expressed genes in 8% group significantly enriched in osteoclast differentiation,ECM-receptor interaction,focal adhesion,PI3K-AKT signaling pathway,while a total of 155 differentially expressed genes in the 4% group were significantly enriched in the focal adhesion and the PI3K-AKT signaling pathway.These genes in two group were mainly enriched in cell composition and biological processes.The candidate genes were screened according to the most significantly enriched pathwaysConclusion:(1)Isolated cells by enzyme digestion from Achilles tendon and patellar tendon possessed self-renewal capacity,colony-formation ability and multi-differentiation potential,and could express stem cell specific markers and tendon-related markers,indicated that isolated cells were TSCs;(2)Mechanical loading contributed TSCs to undergo morphological changes and cytoskeletal rearrangement,which was intensity dependent.4% mechanical loading(Moderate-load)promoted TSCs proliferation and increased expression of tenogenic genes,while 8% mechanical loading(high-load)inhibited TSCs' proliferation and increased the expression of m RNA of Runx2 and Fabp4;(3)Different mechanical loading contributes different biological effects to TSCs at the level of gene expression.The differentially expressed genes in 4% mechanical loading group were enriched in focal adhesion,ECM-receptor interaction,PI3k-AKT and other pathways,while genes in 8% were mainly enriched in osteogenic differentiation and signal transduction pathways.