The Effect Of Shear Stress On Mesenchymal Stem Cells And Possible Mechanisms

BackgroudTissue engineered heart valves (TEHV) exhibited many advantages compared to mechanical heart valves and conventional bioprosthetic heart valves. However, TEHV could not withstand the high blood pressure and stress after implanted in the arterial system and thus exhibited poor function and durability. The main reason of this limitation is that the seeding cells like mesenchymal stem cells (MSCs) could not survive under high shear stress and thus could not protect and remodel the tissue structures. So the key point for TEHV is to improve the cell survival and function under high shear stress.The stress of blood flow is an important initiator for heart valve development and plays an important role in forming and remodeling of valve structures [1-2]. The fluid stress of blood is gradually increased during the time of embryo to grown up. We hypothesized that by preconditioning MSCs with gradual increased shear stress, it could help TEHV to better withstand the high stress of blood flow.AimsThis study evaluated the effect of different levels as well as gradual increased shear stress on MSC. The mechanisms underlying was also explored. The hypothesis of shear stress preconditioning on MSCs was evaluated.Methods and ResultsPart Ⅰ:Rat MSCs were subjected to different levels of shear stress of0,1,3,8and15dyn/cm2for24h respectively. Cell morphology was observed by light microscopy. Cell growth and cell death or apoptosis were evaluated using MTT assay, flow cytometry analysis, Hoechst/PI staining and transmission electron microscopic observation. Results:Shear stress of1dyn/cm2has little effect on MSCs’growth, while the3dyn/cm2shear stress promoted cell proliferation. The shear stress of8and15dyn/cm2induced significant cell death. These results indicated MSCs could benefit from appropriate shear stress (3dyn/cm2) and could not withstand high shear stress (8and15dyn/cm2).Part Ⅱ:MSCs were subjected to gradual increased shear stress. MTT was used for evaluate of cell proliferation and viability. Flow cytometry was used to measure the proportion of cell death and apoptosis. The culture medium after shear stress exertion was collected for Enzyme-linked Immunosorbent Assay (ELISA) of bFGF, TGF-β1, PDGF and VEGF. Drugs loaded nanoparticles synthesized according to the Elisa results were added to the culture medium before shear stress exertion. Results:Gradual increased shear stress significantly improved the proliferation and viability of MSCs compared to high shear stress (8and15dyn/cm2), and also significantly decreased the proportion of cell death and increased the secretion of the growth factores (bFGF, TGF-β1, PDGF and VEGF). The bFGF and VEGF loaded nanoparticles improved cell survival after shear stress exertion. These results demonstrated that gradual increased shear stress could alleviate high shear stress induced cell death and growth factors’ reduction. The addition of growth factors could help cell survival during shear stress exertion.Part III:Phosphorylated ERK and Phosphorylated c-Jun (P-c-Jun) were immunobloted after MSCs were subjected to0,1,3,8,15and1-15dyn/cm2shear stress. Results:3dyn/cm2shear stress increased P-ERK while high shear stress (8and15dyn/cm2) could decreased P-ERK and increase the P-c-Jun.1-15dyn/cm2gradual increased shear stress could improve P-ERK and lower the level of P-c-Jun compared to the high shear stress groups. These results indicated that ERK and INK might be the possible signaling pathways involved in mechanisms by which shear stress affects the MSCs.Part IV:To design a pulsative bioreactor to mimic the chareacteristics of blood flow. The1-15dyn/cm2shear stress preconditioned MSCs were seeded on decellularized porcine aortic artery wall. The static cultured MSCs seeded artery walls were served as controls. Both the two groups were placed in an extracorporeal pulsative bioreactor under shear stress at15dyn/cm2for72h. The artery walls were collected for Hematoxylin and Eosin Staining and DNA quantification by spectrophotometry. The collagen I by MSCs was also measured by westernblot assay. Results:The extracorporeal pulsative bioreactor could yield pulsative blood flow similar to the blood flow of aorta. HE staining showed that preconditioned MSCs were more significantly preserved in the artery wall after72h stimulation at15dyn/cm2. Increased DNA content was observed in shear stress preconditioned MSCs seeded artery walls by spectrophotometry as well as collagen I by westernblot analysis. These results suggested that gradual increased shear stress preconditioned MSCs seeded grafts might better withstand high level fluid stress.ConclusionThis study systematically evaluated the different level of shear stress on MSCs. The part Ⅰ shows that appropriate shear stress (3dyn/cm2) could promote cell growth while low level of shear stress (1dyn/cm2) has no significant effect on MSCs and high shear stress (8and15dyn/cm2) could inhibit cell proliferation and even induce cell death. The results of part Ⅱ demonstrate that gradual increased shear stress could improve cell survival and increase growth factors secretion compared to high shear stress. The part Ⅲ indicates that ERK and INK activation are possible signaling pathways for MSCs survival and apoptosis. In part IV, gradual increased shear stress preconditioned MSCs could better survive and attach to decellularized artery walls and also secrete more collagen for tissue formation compared to MSCs without shear stress preconditioning.