Mechanisms Of Rho GTPases/Wnt/?-catenin Mediated RBMSCs Morphology And Function On Micro/nanotextured Topography Of Titanium Surface

As our country enters an aging society and continues to improve the importance of oral health,there is an increasing demand for dental implants to repair missing teeth to improve the quality of life.Although a variety of commercial implants used in current clinic can achieve excellent osseointegration and meet the need of clinical implanting,part of patients need to restore missing teeth by early implanting or even immediate implanting.Therefore,shortening the healing period of implanting and obtaining more excellent osseointegration has become the core research on implanting materials,both in China and abroad.There are various ways to solve the problem above and micro/nanotextured topography of titanium surface is considered to be one of promising methods.Micro/nanotextured topography of titanium surface of this research is prepared by acid etching and anodic oxidation to simulate nature structure of bone tissue.Cytological experiments in vitro were confirmed to promote a series of biological functions of osteoblasts and r BMSCs.However,the lack of relevant molecular mechanisms might lead to bad design on topography of titanium implanting surface.Thus,it is important to explore the mechanisms of topography of titanium implanting surface and to provide the molecular basis for better design of implanting.First of all,we prepare micro/nanotextured topography of titanium surface of this research and analyze microstructures and properties of surface of the material.At the same time,r BMSCs are cultured identified.Then,r BMSCs are used to evaluate the effects of cell adhesion,proliferation,morphology,skeletal morphology and osteogenic differentiation on micro/nanotextured topography of titanium surface.Furthermore,m RNA expression of Rac1,Cdc42 and Rho A,and protein molecules of Wnt/?-catenin signaling pathway on micro/nanotextured topography were measured.Eventually,signal mechanism of Rho GTPases /Wnt/?-catenin or MAPK signaling pathway on micro/nanotextured topography might be discovered by immunofluorescence,western blot and real-time quantitative RCR to guide excellent design of titanium surface.Part? Preparation and analysis of micro/nanotextured topography and identification of r BMSCs[Objective] To prepare micro/nanotextured topography of titanium surface and identify r BMSCs as the basis of further experiments.[Methods] Micro/nanotextured topography of titanium surface and microstructures is prepare by acid etching and anodic oxidation to simulate nature structure of bone tissue and properties of surface of the material are analyzed by scanning electron microscopy(SEM),atomic force microscope(AFM)and Contact Angle Meter.r BMSCs are cultured and identified by measuring growth curve,clonal formation of cells and osteogenesis differentiation.[Results] There are a lot of uniform nanotubes on micro/nanotextured topography of titanium surface under the scanning electron microscopy(SEM).The average values of Contact Angle Meter on the samples of S,R,R5 and R20 decrease from 58.6� to 3.8�.rBMSCs cultured in vitro have the ability of proliferation,cell clonal formation and osteogenesis differentiation.[Conclusion] Prepared samples of micro/nanotextured topography of titanium surface and cultured r BMSCs is suitable for future experiments.Part ? Effects of r BMSCs morphology and function on micro/nanotextured topography of titanium surface[Objective]To evaluate the biological properties of micro/nanotextured topography of titanium surface by observing biological changes of morphology and function of r BMSCs.[Methods] Effects of r BMSCs morphology and function of osteogenic differentiation on micro/nanotextured topography of titanium surface are evaluated by cell adhesion counting,viability determination,cell morphology and cytoskeleton observation,alkaline phosphatase secretion,cell collagen secretion,extracellular matrix mineralization and osteogenic gene expression.[Results] Micro/nanotextured topography of titanium surface is significantly better than control group in cell adhesion counting,alkaline phosphatase secretion,cell collagen secretion,extracellular matrix mineralization and osteogenic gene expression,while there are no significant changes in cell viability between Micro/nanotextured topography and control group.r BMSCs on micro/nanotextured topography of titanium surface are more stretched,and lamellipodiums and filopodiums are significantly more.At the same time,the red fiber network structures on micro/nanotextured topography are more clearer under Confocal Laser Scanning Microscope(CLSM).[Conclusion] Micro/nanotextured topography of titanium surface not only significantly changes morphology and skeleton of r BMSCs,but also greatly promotes the osteogenic differentiation of r BMSCs.Part ? Mechanisms of Cdc42/Wnt/?-catenin Mediated r BMSCs Morphology and Function on Micro/nanotextured Topography of Titanium Surface[Objective]To discover signal mechanisms of Cdc42 /Wnt/?-catenin mediated r BMSCs morphology and function on micro/nanotextured topography and to provide basis and new ideas for guiding excellent design of titanium surface.[Methods] Effects of r BMSCs morphology and function of osteogenic differentiation on micro/nanotextured topography of titanium surface after transfection of Cdc42 si RNA are evaluated by cell adhesion counting,viability determination,cell morphology and cytoskeleton observation,alkaline phosphatase secretion,cell collagen secretion,extracellular matrix mineralization and osteogenic gene expression.Signal mechanisms of Cdc42 /Wnt/?-catenin mediated r BMSCs morphology and function on micro/nanotextured topography are detected by RT-q PCR and Western blot.[Results] Micro/nanotextured topography of titanium surface after transfection of Cdc42 si RNA is significantly decreased in alkaline phosphatase secretion,cell collagen secretion,extracellular matrix mineralization and osteogenic gene expression.r BMSCs are in a relatively atrophic state and lamellipodiums and filopodiums are significantly reduced.At the same time,the red fiber network structures on micro/nanotextured topography are stained under Confocal Laser Scanning Microscope(CLSM).Cdc42 si RNA transfection significantly reduces the expression of ?-catenin m RNA on micro/nanotextured topography(P <0.05).Cdc42 gene silencing significantly inhibites the expression of Nucleus ?-catenin and p-GSK3? on micro/nanotextured topography.The m RNA expressions of Wnt3 a,Wnt1,Integrin?1,Integrin?3 and Integrin?6 are significantly up-regulated on micro/nanotextured topography.[Conclusion] Cdc42/Wnt/?-catenin signaling pathway is involved in regulation of rBMSCs morphology and function on micro/nanotextured topography.Part ? Mechanisms of Rac1/MAPK Mediated r BMSCs Morphology and Function on Micro/nanotextured Topography of Titanium Surface[Objective]To discover another signal mechanisms of Rac1/MAPK mediated r BMSCs morphology and function on micro/nanotextured topography and to provide basis and new ideas for guiding excellent design of titanium surface.[Methods] Effects of r BMSCs morphology and function of osteogenic differentiation on micro/nanotextured topography of titanium surface after transfection of Rac1 si RNA are evaluated by cell adhesion counting,viability determination,cell morphology and cytoskeleton observation,alkaline phosphatase secretion,cell collagen secretion,extracellular matrix mineralization and osteogenic gene expression.Signal mechanisms of Rac1/MAPK mediated r BMSCs morphology and function on micro/nanotextured topography are detected by Western blot.[Results] Micro/nanotextured topography of titanium surface after transfection of Rac1 si RNA is significantly decreased in alkaline phosphatase secretion,cell collagen secretion,extracellular matrix mineralization and osteogenic gene expression.r BMSCs are in a relatively atrophic state and lamellipodiums are significantly reduced.At the same time,the red fiber network structures on micro/nanotextured topography are stained under Confocal Laser Scanning Microscope(CLSM).Rac1 si RNA transfection significantly inhibites the expression of p-p38 and p-ERK1/2 on micro/nanotextured topography.[Conclusion] Rac1/MAPK signaling pathway is also involved in regulation of r BMSCs morphology and function on micro/nanotextured topography.