The Effects Of Sr-incorporated Micro/nano Rough Titanium Surface On BMSCs Migration And Osteogenic Differentiation For Rapid Osteointegration

Background and PurposeDental implant has become a widely popular method for replacing missing teeth.However,there is a 3-6 months waiting period which allows for the osseointegration process to be carried out.Besides,reduced bone mass and deterioration of bone microarchitecture may lead to reduction of bone healing around implants.These problems bring inconvenience to the patients and affect their acceptance of dental implant surgery.Hence,current research efforts are focused on implant surface modifications that shorten the healing period and enhance implant fixation strength.It is generally believed that the osteogenic cells that colonize the implant surfaces to induce new bone formation mainly originate from the bone marrow-derived mesenchymal stem cells(BMSCs).During the regeneration process,BMSCs are mobilized from adjacent tissue or remote bone marrow,migrate to the lesion site and participate in tissue regeneration by differentiation into osteogenic cells.The viability and functional activity of BMSCs are influenced by the surface characteristics of implants.Strontium(Sr)is known to direct the BMSCs commitment to the bone lineage and encourage bone formation.With respect to possible clinical applications,various Sr releasing systems have been locally applied to bone biomaterials,with the goal of enhancing osseointegration,however,the underlying mechanisms remain elusive.Till now,it still remains unknown whether Sr-incorporated titanium implant surface has recruitment effects on BMSCs.In the present study,Sr-incorporated micro/nano-rough titanium surface(MNT-Sr)was fabricated via sandblasted,double acid-etched method combined with a hydrothermal process.The physicochemical properties and biocompatibility of the surfaces were evaluated.And the recruitment and osteogenesis effects of MNT-Sr on BMSCs were explored for the first time,in order to provide theoretical basis for the development and application of this new implant surface.Part I Preparation and Biocompatibility Evaluation of Strontium-incorporated micro/nano-rough titanium surfaceObjective:Sr-incorporated micro/nano-rough titanium surface(MNT-Sr)were prepared,and its surface characterization and biocompatibility were evaluated.Methods:Sr-incorporated micro/nano-rough titanium surface(MNT-Sr)was fabricated via sandblasted,double acid-etched method(SLA)combined with a hydrothermal process.The micro-rough titanium(MT)was prepared as a control.The surface morphology and chemical compositions were evaluated by scanning electron microscopy(SEM),X-ray diffraction(XRD)and X-ray photoelectron spectroscopy(XPS),respectively.The roughness and hydrophilicity of the samples were evaluated by roughness test and contact angle experiments respectively.The bonding strength of MNT-Sr coating was evaluated by epoxy resin tensile test and in vivo screw-in and-out test.The corrosion resistance of MNT-Sr coating was evaluated by AC electrical impedance test.The in vitro release of Sr2+ ions were detected by inductively coupled plasma mass spectrometry(ICP-MS).Rat BMSCs were isolated and cultured on different Ti disks.The structure of actin filaments and the expression of integrin(31 in BMSCs were measured after seeding.The proliferation of BMSCs was evaluated using the AlamarBlue assay.Results:According to SEM images,MNT-Sr coating displayed micrometer-sized pits,which covered with dense nano-scale particles(040-50 nm).To investigate crystalline phase of MNT-Sr,XRD and XPS analyses of those Ti samples were recorded.Both MT and MNT-Sr displayed characteristic peaks of Ti and TiO2,and the peaks of SrTiO3 crystalline were observed in Sr-SLA surface.The Sr-containing oxide layer reached within a depth of approximately 500 nm of the entire sandblasted and acid-etched surface.Both MT and MNT-Sr surfaces were moderately rough(Ra:1.123±0.091 vs.1.218±0.103,p>0.05)and hydrophilic(CA:8.44±2.23°vs.5.11±1.98°,p>0.05).After 2-week exposure in air,the contact angles of MT and MNT-Sr surface increased,but the surfaces remained hydrophilic(65.17±14.46° vs.62.02±17.51°,p>0.05).The epoxy resin tensile test showed that the bondinging strength of MNT-Sr coating was 49±0.37 MPa,which was high enough to resistance the shear force generated during the implantation process.The corrosion resistance of MNT-Sr surface was significantly improved as compared to MT(p<0.01).The total Sr2+amount released from MNT-Sr surface was 1.34±0.09 mg/1 over the full 14-day range.The result of cell proliferation assay indicated that both MT and MNT-Sr exhibited good biocompatibility.Generally compared to those on MT,BMSCs formed better spread on MNT-Sr surface.The mRNA and protein expression of integrin ?1 were notably enhanced in BMSCs grown on MNT-Sr surface compared with those on MT.Conclusions:MNT-Sr surface exhibited excellent physicochemical properties and biocompatibility.Part ? The Effects of Sr-incorporated micro/nano rough titanium surface on BMSCs migration and osteogenic differentiation in vitroObjective:To evaluate the effects of MNT-Sr and its released Sr2+ ions on the migration and osteogenic differentiation of BMSCs in vitro.The effects of Sr2+ ions on expression level of SDF-1/CXCR4 axis were also explored.Methods:BMSCs were seeded on MT and MNT-Sr surfaces and cultured with their extracts respectively.The scratch wound healing assay was performed to study the effect of MT and MNT-Sr surfaces on BMSCs migration.Transwell assay was performed to further investigate the effect of Sr2+ions on BMSCs migration.The expression levels of HIF-l??SDF-1? and CXCR4 were tested by immunofluorescence,Western Blot,enzyme-linked immunosorbent assay(ELISA)and reverse transcription-polymerase chain reaction(RT-PCR).To determine the early osteogenic differentiation of rBMSCs,cells were cultured on different Ti disks and cultured in different Ti disk extracts without the supplementation of osteogenic induction media The relative mRNA expressions of osteogenic genes,including ALP,RUNX2,DLX5 and SP7 were detected by RT-PCR.ALP staining and ALP activity were assessed to evaluated the expression level of ALP.Results:The wound closure rate of BMSCs grown on MNT-Sr surface was 25.9±2.6%,which was significantly higher than that of MT group(20.1± 1.8%)after 18 h(p<0.05).Consistent with the scratch experiment,more BMSCs migrated into the lower chamber where MNT-Sr extracts was loaded(1.4-fold of MT group,p<0.01).The enhanced cell migration triggered by MNT-Sr extracts was largely attenuated by CXCR4 antagonist AMD3100.The expression levels of HIF-1?,SDF-1? and CXCR4 genes were up-regulated in cells cultured in MNT-Sr extracts.MNT-Sr extracts also promoted the expression of total CXCR4 and membrane CXCR4 in BMSCs,as well as the the secretion of SDF-la.According to the results of osteogenic differentiation experiments,MNT-Sr and the released Sr2+ ions promoted the expression of osteogenic-related genes(ALP,RUNX2,DLX5 and SP7)and the ALP activity in BMSCs.Conclusions:MNT-Sr possessed powerful BMSCs-recruiting capacity via up-regulated the SDF-1/CXCR4 axis by Sr2+ ions.MNT-Sr and its released Sr2+ ions promoted BMSCs osteogenic differentiation in vitro.Part III The effects of Sr-incorporated micro/nano rough titanium surface on BMSCs homing and implant osseointegration in vivo.Objective:To evaluated the effects of MNT-Sr implants on BMSCs homing and the expression levels of SDF-1/CXCR4 axis in tissues around the implants.The early osseointegration of MT and MNT-Sr implants was also compared in vivo.Methods:To visualize the migration of BMSCs in bone with MT and MNT-Sr implantation,wild-type rats were injected intravenously with GFP rBMSCs before implantation surgery.1,3,7 and 14 days after implantation,the percentage of GFP+cells in peripheral blood mononuclear cells was determined by FACS analysis.Immunohistochemistry was performed to detect the expression of HIF-la,SDF-la and CXCR4,as well as GFP cells.To confirm the injected GFP+rBMSCs had osteogenic differentiated,immunohistochemistry was performed to detect the GFP+/OCN+cells.Bone-implant contact(BIC)and bone area(BA)were calculated in undecalcified sections to evaluated the osseointegration.Removal torque test was performed to evaluate implant stability in the bone.Results:A small amount of GFP cells were detected in PBMNCs of recipient wild-type rats using flow cytometric analysis.The GFP signal decreased from day 1 to day 7,but was not detected at day 14,indicating that the injected GFP BMSCs had infused into the rat blood circulation and the amount of injected GFP BMSCs in x peripheral blood decreased over time.The expression levels of HIF-la(day 3 and 7)and SDF-la(day 3,7 and 14)in bone tissue surrounding MNT-Sr implants were increased,resulting in more recruited GFP+BMSCs and CXCR4+cells at 7 and 14 days after implantation,as compared with MT group.Some of the injected GFP+BMSCs were observed to differentiate into osteoblasts in vivo.Furthermore,the results of histological analysis and biomechanical testing indicated that BIC(61.8±5.2%vs.50.1±7.9%,p<0.05),BA(23.6±6.5%vs.17.8±3.5%,p<0.05)and removal torque values(10.5±3.2 vs.7.7±2.3 N/cm,p<0.05)of MNT-Sr implants were significantly higher than those of MT implants at 2 weeks after surgery,suggesting that MNT-Sr implants promoted early osseointegrationConclusions:MNT-Sr implant effectively improved the recruitment of exogenous BMSCs to the implantation site and promoted osseointegration through the delivery of Sr2+ ions.The activation of SDF-1/CXCR4 axis was involved in the Sr2+-induced BMSCs migration.