The Serial Researches Of Titanium Implant RNAi Functionalization And Topography Signaling Transduction

[Background] Titanium implant(Ti)is an ideal material for tooth missing restoration.Although numerous clinical cases indicate that the success rate of Ti is very high,there are still many failures due to poor osseointegration.Therefore,surface modifications of Ti to improve the osseointegration still needs to be developed in urgent.Biofunctionalization of Ti possesses the advantages of enhanced bioactivity and biocompatibility,which is extensively used nowadays.The RNAi technique is targeting,stable and consistent to silence gene expression,which represents a promising strategy for Ti functionalization.Optimizing Ti surface topography is also very common whereas the topography signaling transduction mechanism remains unclear.The previous work in our group has revealed several molecular signaling pathways during the osteogenic differentiation on micropits and nanotubes.However,more work is required to further elucidate this phenomenon.Therefore,my present study is divided into two parts.The first part is trying to explorethe feasibility of RNAi for Ti surface biofunctionalization,through different physical and chemical ways including simple adsorption,self-assembling and cathodic electrodeposition.The second part is to explain topography signaling transduction,mainly from subcellular organelles and cell stress responses.The study is to find out suitable surface modification techniques in order to improve osseointegration and to figure out the mechanisms of the topography mediated cell responses in order to guide surface design of biomaterials.[Aims] To explore the feasibility of RNAi biofunctionalization of Ti implant surface;To uncover the role of autophagy during si RNA delivery;To find out the influence of Ti surface topography on organelles;To figure out the influence of Ti topography on autophagy.[Methods] Part ?: RNAi biofunctionalization of Ti implant surface 1.Ti implant was either micro arc oxidized or anodized to formulate microscale pores and nanotubes.The delivery carrier was chitosan.Different mi RNAs(mi R-148 b,negative control)and si RNAs(si GFP,si Ckip-1)were used to formulate transfection complexes by direct mixing with chitosan.2.The CS/mi RNA complexes were immobilized onto MAO Ti surface by simple adsorption and freeze-dried.The layer-by-layer self-assembling method was performed via alternative depositing of positive-charged CS/si RNA and negative-charged hyaluronate onto smooth Ti surface.The cathodic electrodeposition procedure was performed in CS/siRNA solution and the pre-anodized Ti implant served as the cathode.3.The fabricated coatings were characterized by SEM,FM,CLSM and AFM.The loading efficiency and release profiles were determined by fluorescence intensity measurement.The CLSM was used to observe the cell uptake efficiency while the knockdown efficiency was measured by flow cytometry and real-time quantitative PCR.After osteogenic induction,the ALP activity,collagen secretion and mineralization were determined to evaluate the differentiation ability.4.The lipoplex and polyplex were fabricated by mixing si RNA with lipofectamine 2000and chitosan respectively.After transfection in H1299 cells,the MDC staining and western blot were performed to detect the autophagy levels.The uptake and knockdown efficiencies were determined by flow cytometry in the presence of different autophagy regulators.Meanwhile,the immunofluorescence was used to observe the distribution of autophagosomes.Part ?: Ti implant surface topography signaling transduction 1.The different topographies were fabricated on Ti implant surface via acid etching+anodizing,thermos alkali,hydrogen peroxide etching,respectively.2.Human osteoblast cell line MG63 was seeded directly onto Ti implant surface.The CLSM was used to observe the distribution of organelles after staining of organelle-specific probes.The related fluorescence intensity was quantified by flow cytometry.Real-time quantitative PCR and western blot were used to detect the expression of organelle-specific identities and PERK pathway.The ER lumen was observed by TEM.3.Murine osteoblast cell line MC3T3-E1 was seeded on Ti surface with different topographies.The MDC staining was used to check autophagy activity and the related protein expression was detected by western blot.TEM was used to observe the autophagosomes formation.The cytoskeleton was stained by rhodamine-conjugated phalloidine and the pseudopodia was observed by SEM.The intracellular Ca2+ concentration was indicated by fluorescence probe.The osteogenic differentiation was induced in the presence of autophagy inhibitors and the ALP activity as well as mineralization was determined to evaluate differentiation alterations.[Results] Part ?: RNAi biofunctionalization of Ti implant surface 1.The MAO treatment and anodization can generate microscale pores and nanotubes structures on Ti implant surface respectively.2.The CS/mi RNA complexes can be immobilized evenly on MAO Ti surface and penetrate into pores via simple adsorption procedure.They distribute homogeneously under SEM scanning.In the early stage,the release rate of mi RNA is fast,which slows downafterwards.The modified coating is able to transfect rat BMSCs and the osteogenic differentiation is significantly improved by using mi R-148 b without apparent cytotoxicity.3.The layer-by-layer self-assembling technique can formulate multilayered coating containing CS/si RNA on Ti implant surface.The AFM scanning indicates that the surface roughness is wavily changed and the chimerism structure between chitosan and hyaluronate can be clearly seen under SEM.The si RNA loading amount is increased with layers and the release rate is steadily slow.The GFP expressing in H1299 cells is constantly silenced by loading si GFP and the cytocomparibility is favorable.After loading si Ckip-1,the osteogenic differentiation of MG63 is significantly enhanced.4.The cathodic electrodeposition is able to rapidly establish CS/si RNA coating on Ti implant surface.With increasing of current density,the coating gradually covers the nanotubes.In a short term,the si RNA loading amount is in direct proportion to voltage and duration.The release profile varies in different p H conditions,which is fast in acidic condition and slow in neutral and alkali environment.The fabricated coating can continuously silence target gene with high transfection efficiency and neglected influence on adhesion or viability.The osteogenic differentiation is significantly improved by loading si Ckip-1.5.The m TOR-independent autophagy is induced after transfection with lipoplex and polyplex in H1299 cells.The cell uptake is not obviously influenced by different autophagy regulators.However,the knockdown efficiency is significantly enhanced by rapamycin and TG or inhibited by Li Br and 3-MA.The intracellular free si RNA proportion is also influenced by different autophagy regulators,which is in agree with the related knockdown efficiency.Part ?: Ti implant surface topography signaling transduction 1.The acid etching+anodizing,thermoalkali and hydrogen peroxide etching can generate micropits-nanotubes,nanofibers and nanopores topographies respectively.2.The intracellular organelle distribution and number of MG63 are not influenced significantly by the micro-nano topography.However,compared to smooth Ti surface,theorganelle-specific identities are temporally inhibited in the first day,which returns to normal level after 3 days.TEM depicts significant dilution of ER lumen and the PERK pathway is also activated.3.The nanotubes structure can induce temporal and reversible autophagy in MC3T3-E1 cells,which belongs to m TOR-independent category.The cytoskeleton spreads more obviously on nanotubes surface with abundant filopodia on the leading edge of cells and the intracellular Ca2+ concentration is also vastly increased.In addition,both nanofibers and nanopores can induce similar autophagy responses in MC3T3-E1 cells and both Hela and H1299 cells can be induced similar autophagy responses by nanotubes.After applying autophagy flux inhibitors,the ALP activity on Ti implant surface is significantly downregulated.The osteogenic differentiation promotion ability of nanotubes can be remained even after detaching with the cells.[Conclusions] 1.The RNAi biofunctionalization of Ti implant surface is practicable.With simple adsorption,layer-by-layer self-assembling and cathodic electrodeposition methods,the RNAi transfection complexes can be immobilized onto Ti surface to improve the osteogenic differentiation.2.The micro-nano topography on Ti surface has fewer influences on organelle number or distribution,whereas the organelle-specific identities are inhibited in the early stage,which triggers the ER stress response.3.Different Ti surface topographies can induce m TOR-independent autophagy response,which is temporal and reversible.The topography induced autophagy plays important role during the osteogenic differentiation.