Application Of A Strontium-loaded, Phase-transited Lysozyme Coating To A Titanium Surface To Enhance Osteogenesis And Osteoimmunomodulation

ObjectivesTitanium(Ti)and its alloys have been widely acknowledged as intraosseous implant materials in repair procedures of dentition defect and dentition loss.However,due to the lack of bioactivity and osteoinductivity,a minimum of 3 months is required to achieve sufficient osseointegrated fixation after implantation.Therefore,studies modifying the implant surface to accelerate osseointegration have been performed.Many efforts have demonstrated that the decoration of the implant with strontium(Sr)is one of the effective means.However,the traditional physical and chemical modification strategy is relatively complicated and costly.Thereupon,it is necessary to develop a simple,efficient and green method.Phase-transited lysozyme(PTL)is a novel amyloid-like interfacial modification material that can endow virtually arbitrary substrates functionalized coatings.In this study,the one-step PTL method was used to prepare the Sr-loaded coating,and its effects on osteogenesis and osteoimmunomodulation were explored.Methods1.Four groups of titanium specimens(PTL,PTL@10Sr,PTL@20Sr and PTL@50Sr groups)were prepared through PTL treatment,while the polished titanium sample was used as control group.The surface morphology of the specimens was characterized by field emission scanning electron microscopy(FE-SEM).The chemical composition of the specimens was analyzed by x-ray photoelectron spectroscopy(XPS).The release of Sr ions from the Sr-modified PTL coating was evaluated by inductively coupled plasma optical emission spectrometer(ICP-OES).2.Bone marrow mesenchymal stem cells(BMSCs)were cultured on the prepared specimens followed by the evaluation of cell behaviors.The number and morphology of BMSCs adhered on the surface of each group were observed by MTS assay and laser confocal microscope(CLSM),cell proliferation was analyzed by MTS assay,and cell migration experiments were performed via Transwell chambers.Osteogenic differentiation of BMSCs after osteoinduction were detected by alkaline phosphatase kit(ALP-kit),reverse transcription PCR(RT-PCR),immunofluorescence and western blot in each group.3.Macrophages RAW264.7 were cultured on the surface of each group,and the mRNA level of related secretion were detected to evaluate the response of macrophages to different surfaces.Co-culture model of macrophage and BMSCs was established,and the effects of macrophage conditioned medium on BMSCs migration and osteogenic differentiation were evaluated by Transwell chamber system,ALP-kit and RT-PCR assay.4.The femoral implant model was established for in vivo experiments,and the inflammation and bone formation around implants in different groups were detected through histological analysis to further verify the effects of prepared samples on osteogenesis.Results1.Sr-loaded PTL coatings were successfully prepared on titanium surfaces.Evenly distributed necklace-like fiber network structures on the modified surfaces were observed by FE-SEM.XPS results showed that PTL coatings and Sr-loaded PTL coatings were successfully deposited on the surface of titanium.And Sr-modified PTL coatings could achieve continuous and stable ions release.2.The proliferation experiments of BMSCs demonstrated that cells have good activity in each group.The results of MTS and CLSM revealed that PTL coating and Sr could promote the early adhesion and spread of BMSCs.Sr-loaded PTL groups,particullarly the PTL@20Sr group,exhibited stronger cell recruitment ability and proved more favorable for the osteogenic differentiation of BMSCs.3.The results of RT-PCR experiments indicated that PTL@20Sr group could suppress the expression of associated inflammatory factors,such as Tnfα and Il6,and promote the expression of osteogenic related genes,such as Bmp2 and Tgfβ1.The macrophage conditioned medium in the @20Sr group is more conducive to the migration and osteogenic differentiation of BMSCs compared with others.4.In vivo experiments showed that there was less inflammatory cell infiltration around the implants in PTL and PTL@20Sr groups after implantation.And more new bone formation could be observed in the PTL@20Sr group.ConclusionsIn this study,Sr was successfully incorporated into a PTL layer on a Ti surface.In vivo and in vitro experiments revealed that Sr-loaded PTL coating could promote BMSCs cell initial adhesion,spread,migration and osteogenic differentiation.The Srloaded PTL coating could also act on macrophages and affect cell microenvironment,thereafter invoked more osteogenic differentiation of BMSCs.