Researches On The Mechanism Of Osteogenic Differentiation Of Human Mesenchymal Stem Cells Induced By Fluid Shear Stress

Human mesenchymal stem cells (hMSCs) are tissue stem cells that reside in bone marrow microenvironment and have been widely used in studies of tissue engineering because of their multiple differentiation potent and low immunogenicity, especially bone tissue engineering. Various factors can induce the osteogenic differentiation of hMSCs, such as hormones, cytokines and mechanical stimulation. Since bone is a porous tissue, mechanical loading can lead to the deformation of bone, induce strain gradients as well as local pressure gradients, and accordingly lead to the flow of marrow which causes fluid shear stress (FSS) loading on cells residing in bone marrow including MSCs. FSS is very important to the growth and homeostasis of bone and can promote the proliferation and osteogenic differentiation of preosteoblasts. Therefore, biomaterials and bioreactors are used to imitate the micro-structure of bone and produce FSS exerting on hMSCs to promote the formation of engineering bone tissue. Although it has been proved that FSS can induce hMSCs to differentiate into osteoblasts, the mechano-transduction of FSS in hMSCs remains largely unknown.In this study we investigated the effects of different FSS on the osteogenic differentiation of hMSCs and the mechanism of FSS promoting the ostoegenic differentiation using perfusion culture system and PLGA-3D scaffolds. Firstly, we examined the effects of intermittent FSS at4.2dynes/cm2, continuous FSS at4.2 dynes/cm2and low FSS at0.34dynes/cm2on the osteogenic differentiation of hMSCs. The phosphorylaton levell of ERK1/2was also measured to investigate the effects of different FSS application manners on the activation of signaling molecules using Western-blot. RT-PCR was used to measure the mRNA level of ALP, Runx2, COLIa and OCN. The ALP activity was examined with ALP measurement kit and Modified Gomori Staining. The results showed that intermittent FSS was favored to the osteogenesis of hMSCs compared with continuous FSS and low FSS. The expression level of osteogenic genes and the ALP activity in hMSCs cultured under intermittent FSS application were significantly higher than those in hMSCs cultured under continuous FSS and low FSS application. Moreover, intermittent FSS could more markedly up-regulated the activation of ERK1/2in hMSCs than continuous FSS and low FSS.Secondly, it was investigated whether β1integrins acted as mechano-receptors in FSS-induced osteogenic differentiation of hMSCs to sense the stimulation of FSS and activate ERK1/2by activating FAK and the effect of ERK1/2on the activation of Runx2was also measured. Western-blot was used to evaluate the phosphorylation level or protein level of ERK1/2, FAK and β1integrins. And immunoprecipitation was performed to detect the phospho-Runx2level. To access the roles of ERK1/2and β1integrins in the FSS-induced osteogenic differentiation of hMSCs, PD98059and RGDS peptide were used to individually block ERK1/2pathway and β1integrin pathway. The results showed that FSS enhanced significantly the phosphorylation of ERK1/2, Runx2and FAK. And FSS-induced activation of ERK1/2and FAK was inhibited by the blockade of the connection between β1integrins and ECM with RGDS peptide. Additionally, our study also found that FSS could up-regulate the expression level of β1integrins, and this up-regulation could be abolished by PD98059. Therefore, it could be inferred that β1integrins should detect the stimulation of FSS and thus activate ERK1/2through activating of FAK, and the FSS-activated ERK1/2feed back to up-regulated the expression of β1integrins.Finally, it was estimated how FSS-activated ERK1/2modulated the expression of β1integrins and Runx2as it was proved that FSS-induced up-regulation of β1integrins and Runx2was dependent on the activation of ERK1/2in the previous two sections of this study. It is s widely accepted that BMPs/Smad pathway modulates the expression of Runx2and NFkB also acts as important transcription factor of BMP2, BMP4and β1integrins. We suspect that FSS-activated ERK1/2should mediate the expression of BMP2, BMP4and β1integrins by modulating the activation of NFkB, and high expression of BMP2and BMP4would consequently lead to the activation of BMPs/Smad1/5/8pathway to initiate the expression of Runx2. To verify above suspicion, a series of works were done in this study:determining whether FSS could increase the expression of BMP2and BMP4and activate Smad1,5and8; examining the effect of FSS on the nuclear translocation of p65NF-kB and the activation of p65 and IKBa; using PD98059to inhibit ERK1/2activation to determine whether ERKl/2had effect on FSS-induced activation of NF-kB and Smadl/5/8; determining whether BMPs/Smad pathway was essential to FSS-induced upregulation of Runx2by the inhibition of human recombinant noggin to BMPs; testing the requirement of NF-kB for FSS-induced up-regulation of Runx2and integrin β1by blockade of NF-kB nuclear translocation with BAY11-7082. The results of our study showed that BMPs/Smad pathway was involved in the mechanotransduction of FSS in hMSCs and was critical to FSS-induced upregulation of Runx2. The activation of NF-kB p65increased significantly with FSS application and was mediated by ERKl/2. Furthermore, blockade of the activation of NF-kB inhibited FSS-induced up-regulation of β1integrins, Runx2and BMPs. Such, our data supports an opinion that ERKl/2mediates the expression of β1integrins by modulating the activation of NFkB and regulates the expression of Runx2via crosstalking with BMPs/Smad pathway by mediating the activation of NFkB.In summary, our study has not only confirmed that intermittent FSS could more effectively induce the osteogenic differentiation of hMSCs than continuous FSS but also demonstrated that two novel important signaling pathways involved in the mechanotransduction of FSS in hMSCs, combined with the classical signaling pathway, formed a moecular signaling network to enhance osteogenesis of hMSCs: First, β1integrins act as an important mechanoreceptor to sense the stimulation of extracelular FSS and in turn activate ERK1/2by activating FAK. The activated ERK1/2leads to the phosphorylation of Runx2, and the phosphorylated Runx2initiates the transcription of osteogenic genes to promote hMSCs to differentiate into osteoblasts. Second, the FSS-activated ERK1/2increases the expression of BMPs via activating NFkB, the increased BMPs results in the activation of BMPs/Smad pathway and finally leads to the expression of Runx2. Third, the FSS-activated ERK1/2influences the expression of integrin β1by mediating the activation of NFkB. Our study has provided better understanding how FSS promotes the osteogenic differentiation of hMSCs through a molecular signaling network and important information to elucidate the mechanism of FSS mechanotransduction in hMSCs. The understanding on the mechanism of FSS inducing the osteogenic differentiation of hMSCs will not only be helpful to develop the bone tissue engineering but also provide new targets for drug discovery for treatment of osteoporosis and related bone-wasting diseases.