The Study Of Molecular Mechanisms Involved In The Osteogenic Differentiation Of Rat Bone Mesenchymal Stem Cells On Nanoparticle Shaped Titanium Fabricated With Directed Cluster Beam Deposition

Recently,implant supported prostheses have become the best way for teeth restoration due to its advantages including beauty,comfort,masticatory efficiency and doing no harm to the adjacent teeth.The osseointegration between titanium implant and bone tissue around plays a key role in the success of implantation.Concerning the excellent physical and mechanical properties,chemical stability and biocompatibility of pure titanium,it has become the most common dental implant material clinically.It was reported that bone mesenchymal stem cells(BMSCs)were the earliest cells that migrated and adhered to the surface of implant among the cells in bone tissues.BMSCs could differentiate into osteoblasts,which promoted the formation of bones.However,the contact,adhesion,proliferation and differentiation of BMSCs are highly affected by the surface morphology,hydrophobicity,charge and hardness of pure titanium material.Recently,the surface morphology of nanoscale materials,which were similar with the morphology of extracellular matrix,have been determined to be beneficial for the adhesion and growth of BMSCs.Nano clusters beam technology,as a kind of nano material surface coating technology,emerged in recent years.It is featured with higher stability of coating particles,better controllability of particle size and coating thickness,stronger coating adhesion ability.Therefore,our study proposed to prepare a kind of nanoparticles morphology of pure titanium material using nano clusters beam technique,and further investigate of the molecular mechanism of nano-particle shaped pure titanium material on the adhesion,growth and osteogenic differentiation of BMSCs.Part ? The characterization of nanoparticle shaped titanium fabricated with directed cluster beam deposition and the identification of cultured BMSCsObjective1.Prepare a pure titanium material with uniform and stable nanoparticles morphology with directed cluster beam deposition,and characterize the surface of the material2,Isolation and identification of rat bone marrow mesenchymal stem cells(RBMSCs).Methods1.Surface treatment and characterization of pure titanium material1.1 Surface treatment of pure titanium material:prepare nanoparticles morphology pure titanium material via nano clusters beam technique,and measure the size and distribution of nano particles by transmission electron microscope.Determinate the surface morphology via atomic force acoustic microscope and prepare two control materials through polishing(Smooth)and sand blasting(Sblast)method.1.2 Characterization of pure titanium material:observe the surface morphology of the three pure titanium materials by field emission scanning electron microscope,and measure the roughness and contact angle to water of the three materials.2.The culture and identification of RBMSCs2.1 Separation,purification and culture of RMSCs via the method of differential attachment.2.2 Measure the expression of stem cell specific proteins by flow cytometry.2.3 Investigate the multi-directional differentiation ability of RBMSCs by differentiation medium.Results1.Using nano clusters beam technique could well control the size of nanoparticles at about 20 nm,and generate particles in the range of 40 to 160 nm in the surface of pure titanium material.Compared with the Smooth and Sblast pure titanium material,the roughness of nanoparticles was between them,and has the best hydrophilic.2.The RBMSCs isolated possess the character of mesenchymal stem cells,and have multiple differentiation potentials.Conclusion1.Using nano clusters beam technique could prepare pure titanium material with uniform and stable nanoparticle shaped surface.2.The method of differential attachment could successfully culture RBMSCs.Part ? Exploration of the mechanism of RBMSCs adhering to nanoparticle shaped titanium fabricated with directed cluster beam depositionObjectiveExplore the effects and mechanism of nano clusters beam technique treated pure titanium material on the adhesion and growth of RBMSCs,and find special proteins that play key roles in this process.Methods1.Measure the numbers of live cells that cultured on different materials by CCK-8.2.RBMSCs were incubated on different materials for 4 h,8h,12 h,48 h and 72 h,and cells' morphology were observed by SEM.3.Immunofluorescence was used to determine the rearrangement of the cytoskeleton on the surface of different materials.4.RT-PCR and Real time PCR were used to measure the expression of the family of integrin,and validate the specific upregulated integrin.5.The expressions of specific upregulated integrins were verified by Western blot and immunofluorescence.Results1.Nano clusters beam technique treated pure titanium material could imcrease the numbers of RBMSCs that adhered on the surface of the material.2,Nano clusters beam technique treated pure titanium material is beneficial for the RBMSCs morphology spreading and cytoskeleton rearrangement.3.RMSCs that incubated on the surface of nanoparticle shaped material mainly express integrin?1,?3,?5,?6,?8,?10,?v,?x,?1,?2,?3,?5 and ?6.4.Nano clusters beam technique treated pure titanium material could specific induce the expression of integrin ?5 and integrin ?3.And integrin ?1 has no change.5.Further verification by Western blot and immunofluorescence,Nano clusters beam technique treated pure titanium material could specific induce the expression of integrin ?5 and integrin ?1 in protein level.The upregulated integrin?5?1was mainly distributed in the area of focal adhesion and filopodia.Conclusion1.Nano clusters beam technique treated pure titanium material could promote RBMSCs' adhesion to the surface of the material,and this process was mediated by up regulating integrin ?5?1 and generating focal adhesion between cells and materials.Part ? Exploration of the mechanism of RBMSCs' osteogenic differentiation on nanoparticle shaped titanium fabricated with directed cluster beam depositionObjectiveExplore the effects and mechanism of nano clusters beam technique treated pure titanium material on the osteogenic differentiation of RBMSCs,and verify the function of ILK in this process.Methods1.RT-PCR was used to detect the expression of OCN and RUNX2,and the protein level of OPT,OCN and BSP were further measured by Western blot.2.RT-PCR was used to detect the expression Wnt signaling related factors including Wnt3a,?-catenin and GSK-3?.And the protein level of ?-catenin,GSK-3? and piGSK-3? were determined by Western blot.3.The expression of ILK was measured via RT-PCR,and the protein level of pi-ILK were detected by Western blot.4.The relationship between ITG? and ILK was validated by Co-IP.5.The expression of ?-Catenin,GSK-3?,pi-GSK-3?,OCN and OPT were detected by Western blot after activation and inhibition of the Wnt signaling pathway and the inhibition of ILK,to validate the function of Wnt signaling and ILK.Results1.Nano clusters beam technique treated pure titanium material could induce osteogenic differentiation of RBMSCs.2,Nano clusters beam technique treated pure titanium material could activate Wnt signaling in RBMSCs.3.Nano clusters beam technique treated pure titanium material could induce the expression of pi-ILK.4.ILK connected with integrin?1 through Vinculin and Paxillin,and ILK could connect with GSK-3? in RBMSCs.5.Activating Wnt signaling pathway could induce RBMSCs differentiation into osteoblasts.However,when RBMSCs were incubated with DKK-1 and Cpd22,Wnt signaling was suppressed and the expression of osteogenic related proteins were decreased.ConclusionNano clusters beam technique treated pure titanium material could induce RBMSCs differentiate into osteoblasts via ILK that could activate Wnt signaling pathway.Innovative outcome obtained in this study1.The introduction of advanced materials processing technology: nano clusters beam technique.2.Focus on Titanium-RBMSCs functional interface,a crossover study in the field of material science and biology.Our study focused on the intraction between titanium-RBMSCs,we took material,stem cell biology and molecular biology together,which could mutual infiltrate among the three disciplines.3.Start from the initial point of the interaction between material and cells,explore molecular mechanisms of the interaction between titanium and RBMSCs.We explored the relationship between cell adherion and osteogenic differentiation,which provide a theory support for implant prosthesis therapy.