Extracellular Matrix Stiffness Regulates Osteogenic Differentiation Of Mesenchymal Stem Cells Through Piezo1

Background:Bone damage caused by trauma,disease or congenital defects is a major medical problem.Autogenous iliac crest bone graft(ICBG)is the primary choice for the treatment of bone defects.However,due to chronic pain at the transplantation site and the low survival rate of transplanted cells,alternative solutions are still needed.Cell therapy is one of the most promising alternatives to ICBG.Mesenchymal stem cells(MSCs)are the main candidate cells for cell therapy.Compared with embryonic stem cells,the advantages of MSCs proliferation,multi-lineage differentiation,pro-angiogenesis,immunomodulatory and anti-inflammatory effects,and little ethical controversy have great potential for tissue repair and regeneration therapy using cells.Umbilical cord mesenchymal stem cells(UC-MSCs)are easy to obtain and rich in content,and are ideal for MSCs.Matrix stiffness is a reaction of cells to external mechanical forces and plays an important role in the function and behavior of cells.Different types of tissue have different stiffness,and the stiffness of the matrix determines the differentiation direction of MSCs to a certain extent.The mechanically sensitive cation channel Piezol is a type of ion channel that is activated by mechanical force to cause cation circulation.As a mechanical converter,it plays an important role in responding to matrix stiffness and regulating stem cell differentiation.Studies have found that matrix stiffness can affect the direction of differentiation by adjusting Piezol.Hippo-YAP/TAZ is also a mechanically sensitive pathway that is also involved in sensing matrix stiffness and regulating stem cell differentiationMethods:Human umbilical cord mesenchymal stem cells(hUC-MSCs)were obtained and cultured from wharton jelly by the method of tissue mass cell culture,while flow cytometry for identification of surface markers,osteogenic and adipogenic differentiation experiments to detect multidirectional differentiation potential.Acrylamide and bisacrylamide are polymerized and crosslinked according to a certain ratio to prepare polyacrylamide gels with different stiffness.The hUC-MSCs were cultured on different stiffness for 1 day,7 days,and 14 days respectively.The osteogenic markers RUNX2,ALP,and COLI,adipogenic markers PPARy,and cEBPa were detected by qPCR.Fat droplets were identified by oil red o staining and calcium deposition by alizarin red staining.The localization and expression of YAP and p-YAP were detected by immunofluorescence and Western Blot;the expression difference of Piezo l protein was detected;Calcium ion fluorescence probe detected Ca2+levels.The lentiviral infection was used to construct the Piezol knockout model,and the Piezol activator Yodal was used to verify the efficiency of knockdown and activation.Piezol was activated to detect both the expression of differentiation markers and the expression of YAP and p-YAP.Results:This study prepared polyacrylamide gels with different stiffness(1-4 kPa and 62-68 kPa);hUC-MSCs were obtained by multidirectional differentiation potential experiment and surface marker identification.At softness,the cell morphology appeared as oval-shaped,which was similar to fat cells.At stiffness,the cell morphology showed polygonal shape,which was similar to osteoblasts,and the cell morphology is more elongated.qPCR showed high expression of osteogenic markers RUNX2 and ALP,but low expression of COLI,low expression of adipogenic markers PPARy and cEBPa in the stiff matrix compared with the soft matrix,with statistically significant differences(P<0.05).The expression of PPARy and cEBPa and COLI in soft matrix was higher than that in stiff matrix,while RUNX2 and ALP were lower,P<0.05.Oil red O staining showed lipid formation in softness at 7 days,suggesting the fat cells,alizarin red staining showed no calcium salt deposits.Different stiffness were differentially expressed Piezol,stiffness Piezol protein expression is higher than softness group,P<0.05.Immunofluorescence showed that Piezol expression increased over time,and that at 6h,the stiff matrix group began to be higher than the soft matrix group.The concentration of Ca2+ in the stiff matrix group was higher than that in the soft matrix group.p-YAP protein was highly expressed in the soft matrix group,while YAP protein was highly expressed in the stiff matrix group.Confocal laser microscope showed YAP translocation to the nucleus in the stiff matrix group.Western blot showed that the protein expression of Piezol in the lentivirus knockout group was significantly decreased(P<0.05),while the protein expression of Piezol in the Yodal group was not significantly different.In Yodal group,the osteogenic marker ALP was increased,while the lipid marker PPARy was decreased,with statistically significant difference(P<0.05),and YAP expression was increased and p-YAP expression was decreased.The p-YAP expression in the shPiezol group was increased.Conclusion:hUC-MSCs tend to osteogenic differentiation on the matrix stiffness of 62-68 kPa,and adipogenic differentiation on the soft matrix of 1-4 kPa.Piezol plays an important role in the differentiation of hUC-MSCs regulated by matrix stiffness.YAP is the downstream signaling molecule of Piezo1,and Ca2+ may be involved.