Surface Modification By Hydroxyapatite Loading With Osteogenic Active Peptides Of TixOs^?

The effective osteointegration is the assurance of oral implant technology.The surfacemodification of implant is an important factor affecting bone osteointegration.Studies have shown that titanium implants with superficial porous surface structure exhibits the ability to influence cellular behavior,including cvtoskeletal organization and cellular differentiation with matrix deposition.On the other hand,porous structures are able to reduce the elasticity mismatch between an implant and bone tissue.TixOs,(Leader-Novaxa,Milan,Italy)is a commercial,porous surface implant made up of laser metal direct forming technology.The TixOsR with 200-400 ?m micropores can closely combine with the bone tissue through mechanical intercalation.Moreover,its porous structure can provide sufficient 3D space for cell proliferation.Interestingly,the elastic modulus surface of the TixOsR is similar to that of the alveolar bone,which provides a suitable micro environment for osteoblasts to infiltrate into the surface of the implant.Consequently,as a successful implant,TixOsR have shown a high survival rate in the clinical study.However,the porous titanium implant also exhibits a weak bioactivity.In the present study we performed the process of HA adhesion onto the porous TixOsR with stoichiometry and crystallization by pulsed laser deposition(PLD)to improve the biological activity.P20,corresponding to residues 73-92 of BMP-2,could induce BMSCs osteoblast differentiation.RGD peptide promotes cell adhesion via cell transmembrane integrin receptors.(Glu)6 could distributed selectively to the bone,where it gradually releases the conjugated drug.The short peptide synthesis technology is used to link three short peptides together into a novel intelligent biosignal peptides,bio-adsorbed on the TixosR-HA surface.To sum up,we developed a new system TixosR-HA-(Glu)6-P20-RGD.We performed the experiment with MC3T3-E1,and proved it to show better bone-promoting effect than the TixosR.The specific research contents are as follows:1.The TixOsR surface is prepared with a stable,high crystallinity HA coating.We have attempted to addressed the process of HA adhesion onto the porous TixOsR with stoichiometry and crystallization,in this study,we have performed HA thin films coating onto the porous TixOsR using PLD.According to the results of morphology(SEM)and quasi-stoichiometry(EDS)of the HA on the implant,the optimal conditions for coating was 20K laser pulse and 300 mJ output energy.The bio stability and bioactivity of the obtained HA film coated on TixOsR was subsequently tested,and the results show that our PLD coating method may be promising in improving both of these qualities of TixOsR.2.To investigate the effect of(Glu)6-P20-RGD on bone formation of MC3T3-E1.Excluding the influence of porous materials on the behavior of cells,we used PLD technology to prepare films on 1cm2 smooth titanium(Ti)under optimal conditions,respectively adsorbing P20,(Glu)6-P20,(Glu)6.-RGD,(Glu)6-P20-RGD four peptides.The BCA method was used to test the polypeptide adsorption rate at different time intervals and the adsorption rate of polypeptides at different concentrations per square centimeter.The results showed that the peptides containing(Glu)6 had the ability of bone targeting.And the optimal adsorption condition of TiXosR-HA was 90?g/mL,60 mins.The surface morphology of MC3T3-E1 on the TiXosR-HA with four peptides was observed by 3D optical microscopy,The CCK-8 method was used to test the cell adhesion rate and the rate of increase,and it was concluded that RGD can increase the adhesion rate of the polypeptide to MC3T3-E1.By detecting the alkaline alkaline enzyme(ALP)activity and osteocalcin(OCN)level,P20 in the polypeptide can effectively promote osteogenic differentiation of MC3T3-E1.Studies have shown that(Glu)6-P20-RGD has the biological effects of sustained bone release,cell adhesion and proliferation,and osteogenic differentiation.3.To investigate the biological behavior and mechanism of TiXosR-HA-(Glu)6-P20-RGD in MC3T3-E1 cells.PLD was used to prepare the film on TiXosR under the optimal film formation conditions,and adsorbed P20,(Glu)6-P20,(Glu)6-RGD,(Glu)6-P20-RGD.Surface analysis by SEM and XPS confirmed that the polypeptide was successfully attached to Tixos-HA.Dynamic release experiments of peptides on Tixos-HA,Observation of cell adhesion morphology by hoechst fluorescence staining microscope after TiXoR,-HA-peptide was applied to MC3T3-E1,CCK-8 test cell adhesion and proliferation rate,ALP activity and OCN level detection,it is concluded that(Glu)6 has bone targeting and plays a role in sustained release of P20 on TiXosR-HA;RGD effectively promoted the adhesion and proliferation of MC3T3-E1,and improved the osteogenic efficiency of TiXosR-HA-(Glu)6-P20-RGD in mice;P20 can significantly improve the osteogenic differentiation of TiXosR-HA-(Glu)6-P20-RGD on MC3T3-E1.Real-time quantitative PCR(qRT-PCR)and Western Blotting(WB)experiments on osteogenic related genes and proteins is used to explore the osteogenic mechanism of TiXosR-HA-(Glu)6-P20-RGD.Studies have shown that TiXosR-HA-(Glu)6-P20-RGD promotes the osteogenic activity of MC3T3-E1 compared to TiXosR.It is possible to activate the Wnt/?-catenin signal transduction pathway of pre-osteoblastsOur study demonstrated the osteoinductive effect of TixosR-HA-(Glu)6-P20-RGD in mice,explored its mechanism of promoting bone formation.It provides preliminary support and theoretical basis for further clinical application.