Mechanism Of Extracellular Matrix Stiffness Regulating Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix stiffness had an important influence on cell differentiation by regulating cell adhesion,proliferation,and migration.Different matrix stiffness can induce Bone Marrow Mesenchymal Stem Cells(BM-MSCs)into neural cells,adipocytes,myocytes and osteoblasts.However,mechanism of extracellular physical signals translated into intracellular signals that regulate stem cell differentiation is unclear.In this study,we used the dynamic mechanical reaction instrument to detect the matrix gel stiffness,real-time quantitative PCR to detect changes in the level of the gene during the experiment,Western Blot to detect protein level changes and iTRAQ Technique screening for proteomics differential protein.The focus is to explore the early proteomics changes induced by hard matrix-derived stem cells in osteogenic differentiation,aiming to study the molecular mechanisms of matrix stiffness in regulating stem cell osteogenic differentiation from the protein level.The results are as follows(a)ECM samples were prepared by mixing 8%acrylamide with various concentrations of diacrylamide to prepare materials with variable stiffness(13–16 kPa,35–38 kPa,48–53kPa,and 62–68 kPa).Because acrylamide monomers can affect cell proliferation,we extracted solution from the incubated gels and treated hMSCs for 24 h,48 h,and 72 h;however,no significant differences were found when compared to normal controls.Scanning electron microscopy(SEM)of the prepared ECM revealed a smooth gel surface with nanoscale pores.The gel was subsequently coated with 2ug/cm~2fibronectin to overcome it,as cell adhesion molecules can bind to the RGD fragment of fibronectin.SEM analysis of cells plated on the fibronectin-coated substrate showed clear pseudopod extensions that facilitate substrate binding.Thus,these data demonstrate that cells can survive on the formulated gel matrix with no apparent toxicity.(b)Mouse BM-MSCs were isolated and extracted from bone marrow,and cultured on different stiffness gels.Cells became more spread and more adhesive on substrates of higher stiffness.Similarly,the proliferation of BMMSCs increased as stiffness increased.Sox2expression was lower during 4h to 1 week on the 13-16 kPa and 62-68 kPa,in contrast,it was higher during 4h to 1 week on the 48-53 kPa.Oct4 expression on 13-16 kPa was higher than48-53 kPa at 4h,and it has no significant differences at other time point among three different stiffness groups.On 62-68 kPa,BMMSCs were able to be induced toward osteogenic phenotype and generated a markedly high level of RUNX2,ALP,and Osteopontin.The cells exhibited a polygonal morphology and larger spreading area.These results suggest that matrix stiffness modulates commitment of BMMSCs.Our findings may eventually aid in the development of novel,effective biomaterials for the applications in tissue engineering.(c)Osteogenic differentiation was increased on 62-68 kPa ECM,as evidenced by alpha-1type I collagen,Osteocalcin and RUNX2 gene expression and calcium deposition and ALP staining.In the process of differentiation,iTRAQ technology detected proteins in cultured cells at 62-68 kPa is more involved in intracellular regulation,especially cell-cell adhesion,and is associated with integrin binding.Gene and protein expression of integrin?5,?1increased,together with the protein expression of the downstream signaling molecules FAK,p-ERK,p-Akt,GSK-3?,p-GSK-3?and?-catenin,indicating that these molecules can affect the osteogenic differentiation of hMSCs.An antibody blocking integrin?5 suppressed the stiffness-induced expression of all osteoblast markers examined.In particular,alpha-1 type I collagen,RUNX2 and BGLAP were significantly down-regulated,indicating that integrin?5regulates hMSC osteogenic differentiation.Downstream expression of FAK,ERK,p-ERK and?-catenin protein was unchanged whereas Akt,p-Akt,GSK-3?and p-GSK-3?were up-regulated.At the same time,expressions of GSK-3?and p-GSK-3?were up-regulated and?-catenin levels had no difference between with or without anti-integrin?5 antibody groups.Expressions of p-Akt and p-GSK-3?were reduced effectively in the presence of the Akt inhibitor Triciribine.However,Akt,GSK-3?and?-catenin were unchanged.Results above suggested that expression of GSK-3?was regulated by Akt on 62–68 kPa ECM.In summary,four different stiffness matrices were constructed to modulate the fate of bone marrow mesenchymal stem cells and lay the foundation for the subsequent study of matrix stiffness in the field of stem cell regulation.High-hardness matrix not only regulates the self-renewal capacity of bone marrow mesenchymal stem cells,but also induces osteogenic differentiation of cells.During differentiation,integrin?5/PI3K and Wnt/?-catenin signaling pathways play an important regulatory role in the differentiation process.It is hoped that the mechanism of bone marrow mesenchymal stem cell osteogenic differentiation induced by matrix stiffness can be analyzed through the protein network in combination with the newly discovered protein molecules.