Research On Molecular Mechanism Of Stem Cell Osteoblastic Differentiation Induced By Biomaterial Micro/Nanotopography

Biomaterial micro/nanotopography has important biological functions that promote osseointegration.The molecular mechanism of micro/nanotopography-induced bone marrow mesenchymal stem cells(BMSC)can provide a theoretical basis for the development of new biomaterials.Through literature research,it was found that physical pathway-related signaling pathways are involved in the regulation of BMSC osteoblastic differentiation during the differentiation of BMSCs induced by micro/nanotopography.Mechanical stress is an important member of physical factors.It regulates the differentiation,proliferation and migration of stem cells by activating mechanical signaling pathways.However,the mechanism of mechanical stress in the process of osteoblastic differentiation of BMSC induced by micro/nanotopography is still unclear.In this study,the role of mechanical signals in the osteoblastic differentiation of BMSC induced by micro/nanotopography was investigated based on titanium dioxide nanotubes with different pore sizes.We studied the mechanism of action of the mechanical pathway-related proteins F-actin and YAP in response to micro-nanotopography.At the same time,it was found that AMOT130,a potential participant in the mechanical signaling pathway,was up-regulated in response to micro/nanotopography.Using AMOT130 agonists,it was confirmed that AMOT130 participates in BMSC osteoblastic differentiation through mechanical signaling pathway and confirms its mechanism of action in this process.This study focused on the role of the AMOT130 / YAP pathway in osteoblastic differentiation of bone marrow mesenchymal stems cultured on titanium(Ti)carrying micro/nanotopography,and cell behaviors(i.e.,morphology,F-actin formation,osteoblastic differentiation,YAP localization)were studied.Ti O2 nanotubes with different pore sizes were prepared by anodization as a substrate material to study the molecular mechanism of osteoblastic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)on micro/ nanotopography surfaces.The methods,results and conclusions are as follows.(1)Through transcriptome sequencing(RNA-seq)analysis,investigate the role of BMP,Wnt,Notch and mechanical signaling pathways in morphologically induced BMSC osteoblastic differentiation.The results showed that the expression levels of Notch and Wnt non-canonical signaling pathways as well as mechanical signaling pathway members were up-regulated.It is concluded that Notch,Wnt non-classical and mechanical signaling pathways are involved in nanomorphology-induced BMSC osteoblastic differentiation.(2)Immunofluorescence,q RT-RCR were used to detect F-actin formation,osteoblast differentiation,and YAP nuclear localization.It was found that micro/nanotopography induced osteoblastic differentiation of BMSC,and F-actin formation,and YAP nuclear localization also increased.After inhibition of mechanical stress transduction,osteoblast gene expression,F-actin,and YAP nuclear localization were decreased.It is concluded that the mechanical transduction signaling pathway is deeply involved in the regulation of BMSC differentiation induced by micro/nanotopography.(3)BMSCs on flat titanium were treated with AMOT130 agonist.Immunofluorescence,q RT-RCR and western blot were used to detect F-actin formation,osteoblast differentiation,YAP nuclear localization and AMOT130 expression.It was found that with the up-regulation of AMOT130,the expression of osteoblastic genes on flat titanium sheets increased,F-actin formation,and the amount of YAP increased.It is concluded that,AMOT130/YAP is an important pathway mediating the translation of micro/nanotopography signals to BMSC osteoblastic commitment,likely via the cascade: The micro/nanotopography up-regulated AMOT130 gene expression.AMOT130 stabilizes F-actin and promotes the formation of the latter.F-actin causes YAP to enter the nucleus.YAP acts as a co-transcription factor to promote downstream osteoblastic-related gene expression.