| ObjectiveBone transport technology has been widely used in clinic since it was proposed by Llizarov,and it is one of the main methods for repairing bone defects in limbs.In the process of bone repair,the tensile stress stimulates the proliferation and differentiation of cells,thereby realizing the synchronous regeneration of bones and soft tissues.There are fibroblasts and mesenchymal stem cells in bone tissues and surrounding connective tissues,all of which have similar surface antigen markers and have the potential to differentiate into osteoblasts,adipocytes,and chondroblasts.Mechanical signal transduction is an important mechanism in regulating stem cell differentiation.Among the various regulatory pathways involved in mechanical transduction,the extracellular matrix-integrin-cytoskeleton axis is the main path for stem cells to respond to mechanical loading.Among them,integrin αVβ3 plays an important role by recruiting a variety of proteins to form focal adhesions and connecting the microfilament cytoskeleton to participate in mechanical signal transduction.This research explores how cyclic stretch stress affects the osteogenic differentiation of fibroblasts and mesenchymal stem cells through the integrinαVβ3-microfilament axis,and explores the specific mechanism by which stretch stress stimulates the osteogenic differentiation of fibroblasts and mesenchymal stem cells.Methods1.Human fibroblasts were loaded with tensile stress under different tensile deformations(5%,10%,15%)and at different tensile frequencies(0.1 Hz,0.5 Hz,1 Hz)for different lengths of time(2 h,4 h)every day.After loading for 7 days,the samples were extracted for detecting the expressions of osteogenic markers ALP and RUNX2 by using Western Blot and qRT-PCR.2.Osteoblast differentiation of fibroblasts,adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells was induced by chemical induction and tensile stress loading.The samples were extracted on 0 d,7 d and 14 d of chemical induction and on 0 d,4 d and 7 d of tensile stress loading.Then,the expressions of osteogenic markers ALP and RUNX2 were detected by Western Blot and qRT-PCR.3.Cyclic tensile stress(10%deformation,0.5 Hz,2 h/d,7 d)was applied to human fibroblasts,adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells.The protein and mRNA were extracted on 0 d,4 d and 7 d.The expression levels of integrin αVβ3,its downstream proteins and osteogenic markers were detected by Western Blot and qRT-PCR.4.Lentivirus transfection and peptide inhibitor c(RGDyk)were used to inhibit the function of integrin αVβ3.Then,fibroblasts,bone marrow mesenchymal stem cells and adipose-derived mesenchymal stem cells were subject to tensile stress loading,followed by the extraction of protein and mRNA on 0 d and 7 d.The expression levels of focal adhesion-associated proteins,microfilament,intra-nuclear YAP and osteogenic markers were detected by Western Blot and qRT-PCR.Western Blot was also applied to detect the phosphorylation level of FAK.The distribution of vinculin protein,microfilament and YAP protein were observed by immunofluorescence staining.In addition,the osteogenic effect was observed by ALP staining.5.Inhibitor Y-15,cytochalasin D and verteporfin were used to inhibit FAK protein phosphorylation,microfilament cytoskeleton polymerization and intra-nuclear YAP protein function,respectively.Fibroblasts,bone marrow mesenchymal stem cells and adipose-derived mesenchymal stem cells were subject to tensile stress loading,and then protein and mRNA were extracted on 0 d and 7 d.Western Blot was used to detect the expressions of osteogenic markers,and the expression of intra-nuclear YAP protein after the inhibition of microfilament formation by cytochalasin D.Results1.Through comparison of the expressions of osteogenic markers ALP and RUNX2 after fibroblasts were induced by tensile stress based on grouping according to different parameters,it was verified that cyclic tensile stress with 10%deformation and 0.5 Hz frequency could promote the differentiation of human fibroblasts into osteoblasts more significantly.2.Osteogenic induction medium and tensile stress loading were used to induce the expression levels of osteogenic markers in human fibroblasts,human adipose-derived stem cells and human bone marrow mesenchymal stem cells,respectively.Corresponding results of comparison suggested that the osteogenic differentiation potential of mesenchymal stem cells was superior to that of fibroblasts,while the osteogenic effect of stem cells induced by conventional chemical induction was better than that of tensile stress loading.3.During the process of tensile stress loading,there were increase in the expression levels of integrin αVβ3,focal adhesion associated-proteins talin,FAK,vinculin,β-actin and nuclear YAP protein increased,accompanied by upregulated phosphorylation level of FAK.Meanwhile,in fibroblasts,human adipose-derived stem cells and human bone marrow mesenchymal stem cells with inhibited function of integrin αVβ3,the levels of osteogenic markers and FAK phosphorylation were decreased after tensile stress loading,while decreased trends were observed in the expressions of downstream focal adhesion-associated protein,β-actin and nuclear Yap protein.Furthermore,based on the results of immunofluorescence,in the absence of tensile stress loading,the microfilaments of the cells showed straight and tight arrangement,were in the shape of slender filaments,with visible sporadic punctate focal adhesion complex around the cell membrane,and intranuclear Yap protein presented a fuzzy nuclear outline.While after tensile stress loading,actin formed thick stress filaments,showing irregular arrangement direction,the microfilament structure converged to the direction of cell membrane,and there were significantly increased focal adhesion complex and highly increased intranuclear Yap protein expression with clear outline.In addition,when tensile stress acted on the cells with inhibited function of integrin αVβ3,the microfilaments were in a thick filamentous structure,yet with tight and straight arrangement on the whole,accompanied by significantly reduced focal adhesion complex and the appearance of intranuclear Yap protein in the nuclear shape with fuzzy edges.4.Following the regulation of FAK phosphorylation level,microfilament formation and intranuclear Yap protein by Y-15,Cyto D and VP,respectively,the levels of osteogenic markers of cells induced by tensile stress were decreased compared with normal tensile stress loading group,with a decreased intranuclear Yap expression in Cyto D group.Conclusion1.Cyclic stretch stress(10%tensile deformation,0.5 Hz,2 h/d,7d)induced osteoblast differentiation of human fibroblasts,adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells and also promoted cell proliferation of human fibroblasts through integrin αVβ3.And cyclic stretch stress up-regulated YAP expression in downstream adhesion plaque associated protein microfilaments through integrin αVβ3.2.Phosphorylation of FAK and microfilaments and nuclear YAP protein respectively assisted osteoblast differentiation of human fibroblasts by cyclic stretch stress. |
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