Study Of Microarc Oxidation And Electrochemical Treated Titanium To Enhance Bioactivity And Osseointegration In Canine

Objective:1.To fabricate unique hierarchical micro/nano-morphology surface on titanium via microarc oxidation(MAO group),and then the coating was electrochemically reduced in alkaline solution(MAO-AK group).2.To verify the beneficial effects of the innovative micro-arc oxidation process formed MAO and MAO-AK layers in vitro,the attachment,proliferation,and osteo-differentiation abilities of canine BMSCs on different structures were evaluated without any osteoinductive supplements.3.Canine femur implantation model was used to evaluate the in vivo osseointegration of MAO-AK and MAO treated Ti implants.The bone formation rate and the new bone formation of each group were observed.Material and Methods:1.The surface morphology of the three coatings(Ti,MAO and MAO-AK groups)was observed by a scanning electron microscopy.Chemical compositions of the coatings were analyzed by energy-dispersive spectrometry(EDS).The chemical states of Titanium(Ti)were determined by X-ray photoelectron spectroscopy(XPS).The components on the surface of these samples were detected using a thin-film x-ray diffractometer(XRD).The surface wettability of the coatings was assessed using a contact angle instrument.2.BMSCs were seeded on different substrates in vitro study.To assess cell viability,the Live-Dead cell staining was applied and cell proliferation was evaluated by MTT test.Serum protein adsorption test and immunofluorescent staining of integrinβ1 were applied to evaluate cell adhesion.The expression of osteogenic genes in BMSCs was tested by real-time PCR.Alkaline phosphatase(ALP)activity and alizarin red staining(ARS)were also detected to evaluate the osteogenic differentiation in BMSCs on different groups.3.Inserting titanium implants with different coatings in canine femur for 6 weeks.Radiographic observation,sequential fluorescent labeling and histological staining were used to the new bone formation speed and quality around implants.Results:1.Unique hierarchical micro/nano-morphology surface was fabricated on titanium by microarc oxidation(MAO group),and then the coating was electrochemically reduced in alkaline solution(MAO-AK group).From SEM results,MAO and MAO-AK coatings exhibited similar micro-structures but different nano-structures.From EDS and XRD results,it could be concluded that MAO and MAO-AK coatings show similar element components and even constituent compositions.However,the XPS results indicated the MAO surface was slightly reduced by the electrochemical treatment in alkaline solution,and trivalent titanium was found on the MAO-AK surface.Compared with Ti group,the contact angles of MAO group increased about 17°,while the MAO-AK samples exhibited the lowest contact angle compared with the other two groups.2.BMSCs were seeded on different substrates.After 24 h,cells were stained by “live-dead”kit and there were no significant differences in the percentages of viable cells after culturing on three substrates.Cell proliferation was measured by the MTT assay,there was a time dependent growth trend of BMSCs in all groups and the proliferation activities in both MAO group and MAO-AK group were significantly higher than that in the Ti group.3.Serum protein(FN)adsorption test inferred that the hierarchical hybrid micro/nano-structures on the MAO-AK plate surface facilitate the fibronectin immobilization.Quantitative assays of integrinβ1 expression were investigated via immunofluorescent staining,real-time PCR and western blotting tests.The expression levels of integrin β1 at two early time points in MAO group and MAO-AK group were significantly higher than that in Ti group,the highest expression of integrin β1 protein was observed in MAO-AK group.4.Cells on both MAO and MAO-AK group expressed more alkaline phosphatase activity according to the quantification results.Similar results were observed from Alizarin Red S staining and quantitative calculation via the calcium deposition assay.The expression of osteogenic-related genes including osterix,ALP,osteopontin(OPN)and osteocalcin(OCN)were quantified using real-time PCR after culturing,and the expression of these genes was up-regulated on the surface of MAO and MAO-AK group.5.From Radiographic observation,sequential fluorescent labeling and histological staining results,MAO-AK coating stimulates more new bone formation and accelerates the rate of mineralization.Conclusions:1.Titanium with unique hierarchical micro/nano-morphology surface was fabricated by novel duplex coating process,i.e.firstly a titanium oxide layer was coated by micro-arc oxidation,and then the coating was electrochemically reduced in alkaline solution.Hence,two types of titanium oxide coatings can be fabricated,namely the MAO group and the MAO-AK group.MAO-AK coatings are capable of facilitating aggregation of serum proteins such as fibronection.2.BMSCs on the MAO and MAO-AK groups exhibited enhanced adhesion,proliferation and osteogenic differentiation.Compared to the MAO coating,the MAO-AK coating enhanced the osteogensis activity of canine BMSCs significantly.3.The MAO-AK coatings have the significant potential to accelerate the process of bone formation and enhance the rapid osseointegration in canine femur implantation.