TAZ And YAP Induced Regulation Of Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells By Surface Topography Of Scaffold

Background:In recent years, studies demonstrated that the applications of stem cell in the field of medical biotechnology were prospective but still challenging. One of the most important problems that need to be solved for successfully tissue engineering is the biomaterials.. Tissue engineering includes three elements:seed cell,growth factor and Scaffold which plays a main role. In addition to providing structural support for the specific cell, the three-dimensional surface topography of the Scaffold is extremely important in terms of regulating cell adhesion, spreading and gene expression, inducing tissue regeneration, and controlling tissue structure. Meanwhile, signaling pathway also plays an important role in the process of cell growth. Hippo signaling demonstrated as an important signaling of cell proliferation, differentiation and apoptosis..Studies have shown that TAZ and YAP, essential molecules in the Hippo signaling,were not only to maintain pluripotency of human and mouse embryonic stem cells, but also important regulatory factors of stem cells proliferation. Although some studies have investigated cell behaviors on micro-topography, the underlying mechanism of cell sensing, intercellular signaling, or long-term response to surface topography is still uncertain.Objective:This project plans to study the effects and mechanisms of the topography of scaffold with Hippo signaling on the proliferation and differentiation of mesenchymal stem cells in vitro; The purposes of this project are to define the effects and mechanism of the micro-topography on the proliferation and osteogenetic differentiation of mesenchymal stem cells, the effects and mechanism of the micro-topography on the proliferation and osteogenetic differentiation of mesenchymal stem cells based on the Hippo signaling pathway. This project is expected to provide a deeper understanding of the mechanism of the micro-topography increasing the proliferation and osteogenetic differentiation of mesenchymal stem cells, and to support therapeutic and experimental design of the safer and more effective scaffold for bone tissue engineering.Methods:1.The construction of hydroxyapatite scaffoldCa3(PO4)2and CaCO3proportionally reacted at high temperature and injected of high pressure steam, then washed dried powder by NH4Cl aqueous solution,sintered it together at1250℃2. The grinding of the surface morphologyAfter construction hydroxyapatite scaffold, we polished the surface with the type of P180, P240, P600, P800, P1200and P2000, in corresponding particle diameter82μm,58.5μm,25.8μm,21.8μm,15.3μm and10.3μm.3. The meassurement of surface roughnessSurface roughness measurement observed the changes in the parameter of surface roughness of the microscopic unevenness about height characteristics—roughness amplitude Ra and surface roughness of the microscopic unevenness about distance characteristics—distance between surface features Sm. Scanning electron microscopy (SEM) shows different thickness and depth grooves.4. The proliferative effect on surface roughness of bone marrow mesenchymal stem cell.BMSC were seeded on scaffold with the density of5*10＾4/scaffold,Alamar Blue reagent detect cell proliferation at the time piont of12h,24h,36h,48h,60h,72h,84h, and96h after inoculation.5. The differentiation effect on surface roughness of bone marrow mesenchymal stem cell.BMSC were cultured on scaffold with the density of5*10＾4/scaffold in osteogenic medium. RT-PCR for the detection of several markers for osteoblastic differentiation was done at2and7days. ALP staining of differentiating BMSC on the scaffold were done at7days. Alizarin red staining of differentiating BMSC on the scaffold were done at28days.6. The machanism of the behavior effects on surface roughness of bone marrow mesenchymal stem cell.BMSC were cultured on scaffold with the density of5*10＾4/scaffold in osteogenic medium. RT-PCR for the detection of several markers for osteoblastic differentiation and the key factors of hippo signaling-TAZ and YAP were done at2days.Osteogenic differentiation of BMSC on scaffold were detected after TAZ and YAP were knockout including ALP staining, alizarin red staining and the mRNA expression levels of several markers for osteoblastic differentiationResults:1. Scanning electron microscopy (SEM) shows different thickness and depth grooves. Surface roughness measurement observed the identical changes with polished particle diameter in the parameter of surface roughness of the microscopic unevenness about height characteristics—roughness amplitude Ra and surface roughness of the microscopic unevenness about distance characteristics—distance between surface features Sm.2. Bone marrow mesenchymal stem cell displayed stronger proliferation in the roughness surface..3. Alkaline phosphatase staining and alizarin red staining showed the roughness surface increased osteogenesis differentiation of marrow mesenchymal stem cell.4. The micro-topography of hydroxyapatite scaffold boost the osteogenesis differentiation of mesenchymal stem cells, and increased the levels of YAPand TAZ, the key factors of Hippo signaling pathway. ALP staining, alizarin red staining and the mRNA expression levels of several markers for osteoblastic differentiation of BMSC seeded on roughness surface did not show variances after TAZ and YAP were knockout with lentivirus as compared to unpolished surface.Conclusion:In this study, we designed a hydroxyapatite scaffold, and polished the surface with different kinds of the sand paper. BMSC seeded on the rough surface showed stronger ability of proliferation and osteogenic differentiation that directly demonstrated the effects of surface morphology on behavior of mesenchymal stem cells. The micro-topography of hydroxyapatite scaffold boosted the osteogenesis differentiation of mesenchymal stem cells, and increased the levels of TAZ and YAP, the key factors of Hippo signaling pathway. Interestingly, the mRNA level of TAZ and YAP and osteogenesis gene expression level presented the same trend. After having knocked out TAZ and YAP, the ability of proliferation of BMSC had no significant difference between cultured on the polished rough surface and unpolished smooth surface of hydroxyapatite scaffold. The surface morphology did regulate BMSC to differentiate into osteoblast mediated by TAZ and YAP. This project provided a deeper understanding of the mechanism that the micro-topography increased the proliferation and osteogenetic differentiation of mesenchymal stem cells, and therapeutic and experimental support for the design of the safer and more effective scaffold for bone tissue engineering.