Research On Establishment Of An Icariin/aspirin Composite Coating On The Titanium Surface To Regulate Macrophage Behavior So As To Promote Osteoblast Activity

Objectives:Titanium and titanium alloys have become the most widely used implant materials due to their excellent characteristics.However,the biological inertness of untreated pure titanium implants is not conducive to early cell adhesion and rapid osseointegration.At the same time,with the concept of"Osteoimmunology"being put forward,more and more scholars are turning their attention to the influence of the establishment and maintenance of the bone immune microenvironment around the implant on the osseointegration of the implant.Therefore,it is of great theoretical and practical significance to modify implant surface in order to regulate the immune microenvironment around the implant,promote cell activity so as to promote osseointegration and improve implantation success rate.In this study,anodic oxidation was used to form a nano-morphology on the surface of the implant.Icariin and aspirin were loaded onto the nano-structured surface in order to construct a composite coating of icariin and aspirin.Our study aims to promote bone formation around the implant through regulating the immune microenvironment.Methods:1.First,titanium dioxide nanotubes structures were prepared on pure titanium plate by anodic oxidation.Then,icariin was loaded by soaking method,the aspirin and polylactic-co-glycolic acid copolymer（PLGA）mixture were further coated on the nano-structured surface to establish an icariin/aspirin composite coating on the titanium surface.Scanning electron microscope（SEM）was used to observe the surface morphology,X-ray photoelectron spectroscopy（XPS）was employed to detect the surface element content.Contact angle measuring instrument was applied to detect the surface hydrophilicity,while high performance liquid chromatography was used to evaluate the drug release profile in the established composite coating of icariin and aspirin.2.RAW 264.7 cell,the mouse macrophages,were seeded on different samples,and CCK-8 was used to detect the cell proliferation of different samples;SEM was used to observe the early cell adhesion pattern.Real-time PCR was used to detect gene expression levels in macrophages cultured on various surfaces induced by LPS.M1pro-inflammatory genes TNF-α（tumor necrosis factor-α）,IL-1β（interleukin-1β）and M2 pro-regenerative genes TGF-β（transforming growth factor-β）and HO-1（heme oxygenase-1）were examined.Moreover,enzyme linked immunosorbent assay（ELISA）was used to detect TNF-α,IL-1,TGF-βand HO-1 protein expression levels secreted by macrophages on different surfaces.3.MC3T3-E1 cells were seeded on different samples,and the proliferation of cells on different samples was detected by CCK-8 test.Early cell adhesion patterns were observed by SEM and real-time PCR was used to detect the gene expression level of osteocalcin in MC3T3-E1 cells.4.Under co-culture conditions of osteoblasts and macrophages,cell proliferation was examined using CCK-8 assay.SEM were used to observe the early adhesion morphologies of osteoblast cells.Real-time PCR and Western blot tests were used to detect the expression levels of the osteogenesis-related genes and proteins including OCN,OPN,COL1A1 and ALP.Results:1.SEM observation and XPS detection confirmed that the icariin/aspirin composite coating was successfully prepared,the hydrophilicity of composite coating was stronger than that of pure titanium.The release period of icariin reaches 30 days,and the release concentration is maintained at within 0.1-1×10^-5mol/L.The release period of aspirin is 7 days,and the release concentration maintained between 0.2 m M and 4 m M.2.Titanium discs loaded with icariin/aspirin composite coatings have no cytotoxicity on macrophages.Real-time PCR and enzyme-linked immunosorbent assay（ELISA）experiments have shown that the composite coating could inhibit the expression of pro-inflammatory genes and proteins including TNF-αand IL-1β.Whereas,the established ICA/ASP composite coating could promote the expression of pro-regenerative gene and proteins including TGF-βand HO-1.3.Titanium discs loaded with icariin/aspirin composite coatings could significantly promote osteoblast proliferation.Real-time PCR test proved that the osteogenesis-related gene OCN were significantly up-regulated in osteoblast cells cultured on the composite coating surface,compared with pure Ti group.4.Under co-culture conditions of osteoblasts and macrophages,in the presence of macrophage conditioned medium,titanium coated with the composite coating of icariin/aspirin significantly promotes osteoblast proliferation.SEM observations revealed that the pre-osteoblasts on the composite-coated titanium plate extend towards all directions,which revealed the satisfied cell adhesion ability.Real-time PCR and Western blot experiments demonstrated that the composite coating can up-regulate the expression of osteogenesis-related genes（OCN,OPN,COL1A1,ALP）and osteogenesis-related proteins（OCN,OPN,COL1A1,ALP）,compared to pure titanium.Conclusions:This experiment provides a new idea for the surface modification method of titanium implants.The anti-inflammatory and immunoregulatory effects and osteogenic function of icariin and aspirin were employed on the novel composite coating.Through controlling the release profiles of the two drugs,aspirin could exert anti-inflammatory effect in the early stage,while icariin could continue to exert osteogenic function.Therefore,the established composite coating of ASP and ICA could cooperate to create an immune microenvironment,which vavors bone formation,so as to promote implant osseointegration.