The Role And Activation Mechanism Of TAZ In Microgrooves/Nanoporous Hierarchical Topography Titanium Surfaces Regulating Stem Cell Differentiation

ObjectivesFor intraosseous implants,micro/nanoscale hierarchical topography with bone bionic characteristics has better biological activity,which can enhance osteogenic and depress adipogenic differentiation of cells,and facilitate the formation of new bone on implant surfaces.However,the mechanical response process and potential response mechanisms of cells to micro/nanoscale hierarchical topography are currently unclear.The purpose of this study was to investigate the role of the mechanical signal response molecule TAZ and its activation mechanism in this process.The discussion of this problem will help to understand the regulation of micro/nanoscale hierarchical topography on cell biological behavior,and provide clues and ideas for targeted surface modification of planting materials.Methods1.To explore the role of TAZ in microgrooves/nanoporous hierarchical topography titanium surfaces regulating stem cell differentiationin.we fabricated titanium surfaces with a microgrooves/nanoporous hierarchical topography using selective laser melting fully melting(SLM)technology and alkali-heat treatment(AHT)and chose smooth titanium(Ti)and sandblasted,large-grit,acid-etched(SLA)titanium as control surface topography.In vitro experiments were performed to evaluate the effect of titanium surface morphology on osteogenic and adipogenic differentiation of periodontal stem cells(PDLSCs).PDLSCs were cultured on the surfaces of Ti,SLA and SLM-AHT titanium and induced by osteogenic and adipogenic conditional media respectively.Alkaline phosphatase(ALP)activity of cells cultured on titanium surfaces was detected by ALP-Kit.The mRNA expression levels of osteogenic related genes RUNX2 and ALP and adipogenic related genes PPARγ and CEBP/α were detected by real-time quantitative PCR(qRT-PCR).The protein expression levels of osteogenic related protein RUNX2 and adipogenic related protein PPARγ2 were detected by immunofluorescence and Western blot.TAZ localization on different topography titanium surfaces was detected by immunofluorescence.The mRNA expression levels of TAZ target genes CYR61 and CTGF were detected by qRT-PCR.TAZ shRNA lentiviral vector was constructed and stably transfected into PDLSCs,and knockdown efficiency was confirmed via qRT-PCR and Western blot.qRT-PCR was performed to detect the mRNA expression changes of osteogenic and adipogenic related genes of TAZ-shRNA-PDLSCs(TAZ knockdown group)and TAZ-Scramble-PDLSCs(control group)cultured on different topography titanium surfaces,and to verify the role of TAZ in microgrooves/nanoporous hierarchical topography titanium surfaces regulating PDLSCs differentiationin.2.To explore the activation mechanism of TAZ in microgrooves/nanoporous hierarchical topography titanium surfaces regulating PDLSCs differentiationin.The cytoskelet on of PDLSCs on different topography titanium surfaces was observed by immunofluorescence.After cytochalasin D treatment,TAZ distribution and the mRNA expression changes of TAZ target genes CYR61 and CTGF were observed by immunofluorescence and qRT-PCR to demonstrate the role of cytoskeleton in the activation of TAZ by microgrooves/nanoporous hierarchical topography.The protein expression levels of MAPK pathway key proterins JNK,ERK and p38 on different topography titanium surfaces were detected by Western blot.After MAPK inhibitor treatment,TAZ distribution and the mRNA expression changes of TAZ target genes CYR61 and CTGF were detected by immunofluorescence and qRT-PCR to demonstrate the role of MAPK pathway in the activation of TAZ by microgrooves/nanoporous hierarchical topography.Results1.In vitro ALP activity assay,qRT-PCR,immunofluorescence and Western blot results show that compared with Ti and SLA titanium surfaces,PDLSCs had highest ALP activity on SLM-AHT microgrooves/nanoporous hierarchical topography titanium surfaces and the expression levels of osteogenic related genes RUNX2 and ALP and osteogenic related protein RUNX2 were increased.However,the expression levels of adipogenic related genes PPARγ and C/EBPα and adipogenic related protein PPARγ2 were decreased.Immunofluorescence and qRT-PCR results showed that SLM-AHT microgrooves/nanoporous hierarchical topography titanium surfaces could promote TAZ nuclear translocation and increase the expression level of TAZ target genes CYR61 and CTGF.qRT-PCR results showed that after knockdown of TAZ compared with TAZ-Scramble-PDLSCs,TAZ-shRNA-PDLSCs increased RUNX2 and ALP levels and decreased PPARγ and C/EBPα levels on three titanium surfaces.SLM-AHT titanium surfaces osteogenic and adipogenic related genes had the largest changes and there was no significant difference between the three titanium surfaces.2.Immunofluorescence results showed that SLM-AHT microgrooves/nanoporous hierarchical topography titanium surfaces could promote the aggregation of cytoskeleton F-actin.Immunofluorescence and qRT-PCR results showed that Cytochalasin D could inhibit TAZ nuclear translocation and TAZ activation induced by micro/nanoscale hierarchical topography.Western blot showed that SLM-AHT microgrooves/nanoporous hierarchical topography titanium surfaces could upregulate p-ERK and p-p38 levels,that is,micro/nanoscale hierarchical topography could active ERK and p38 MAPK signal pathway.Immunofluorescence and qRT-PCR results showed that ERK and p38 inhibitors could inhibit TAZ nuclear translocation and TAZ activation induced by micro/nanoscale hierarchical topography.ConclusionsCompared with Ti and SLA titanium surfaces,SLM-AHT microgrooves/nanoporous hierarchical topography titanium surface has better ability to induce osteogenetic differentiation of PDLSCs in vitro.This surface can promote cytoskeleton aggregation and activate MAPK signaling pathway,thereby promoting TAZ nuclear translocation and increasing its activity,thereby promoting osteogenic differentiation and inhibiting adipogenic differentiation of PDLSCs.