Effect Of Strontium-incorporated Micro/Nano Rough Titanium Surfaces On Osseointegration Via Modulating Polarization Of Macrophages

Dental implant restoration is currently an important treatment for missing teeth and dentition defect.However,there is a 3-6 months waiting period for bone healing between implant placement and upper implant repair,which has brought many inconveniences to patients.In addition,there are still cases of poor osseointegration leading to implant failure.Therefore,it is an important goal for the researches of dental implant materials to improve osseointegration and shorten the period of bone healing.Previous studies have focused on the osteoblast spectrum series that are directly related to bone formation.The main strategy has been to optimize the characteristics of the implant to promote osteoblast osteogenesis or induce osteogenic differentiation of mesenchymal stem cells.However,the inconsistency between in vitro and in vivo studies suggests that the key factors affecting the process of implant osseointegration in vivo has not been fully elucidated.With the in-depth study of the post-implantation response,the role of immune response in osseointegration is receiving increasing attention.Among all immune cells,macrophages play a key role due to their high plasticity.They can exhibit different polarization states according to the microenvironment.Classically activated M1 macrophages and alternatively activated M2 macrophages are the both ends of polarization spectrum.The polarized state of macrophages directly participates in and affects the implant osseointegration.Therefore,it is necessary to further understand the effect of implant surface modification on macrophage polarization.Strontium is widely used in the modification of osteogenic biomaterials because of its ability to promote osteogenesis while inhibiting osteoclast.In this study,the surface of pure titanium strontium-doped micro/nano morphology was prepared by sandblasting,double-acid etching and hydrothermal synthesis.Macrophages were used as the research object,and the effect of strontium-incorporated micro/nano rough titanium surfaces on the function of macrophages was studied.Thorough research is conducted to provide certain theoretical and experimental basis for optimizing the design of implant material surface from the perspective of immune cells.Part I Preparation and Characterization of strontium-incorporated micro/nano rough titanium surfacesObjectives:To prepare and characterize the strontium-incorporated micro/nano rough titanium surfaces,and provide a research model for subsequent in vitro and in vivo biological evaluation.Methods:1.The surface of pure titanium was polished,sandbalsted with large particles,and double acid-etched to form a rough micron-sized titanium surface(SLA).Based on the SLA,a strontium hydroxide solution was used to prepare the strontium-incorporated micro/nano rough titanium surfaces(Sr-SLA).2.The surface morphology and chemical element compositions of samples were evaluated by scanning electron microscopy(SEM),energy dispersive spectrometer(EDS),X-ray diffractometer(XRD),and X-ray photoelectron spectroscopy(XPS).3.Surface roughness and hydrophilic properties of samples were evaluated by roughness test and contact angle detection.4.Strontium ion release was detected by inductively coupled plasma mass spectrometry(ICP-MS).Results:1.The SLA surface showed a micron-sized honeycomb porous structure.On the basis of this,the Sr-SLA surface added uniform and dense nanoparticles,showing a micro-nano surface.2.The strontium element on the surface of Sr-SLA is incorporated into the titanium surface through the formation of strontium titanate.3.The SLA surface and the Sr-SLA surface have similar roughness,and both are superhydrophilic surfaces.4.The surface of Sr-SLA can continuously release strontium ions into the solution.Conclusions:1.Strontium-incorporated micro/nano rough titanium surfaces could be fabricated through hydrothermal reaction.Part II Effect of strontium-incorporated micro/nano rough titanium surfaces on biological behavior and polarization of macrophagesObjectives:To evaluate the biological behavior and polarization status of macrophages including mouse monocyte-macrophage cell line RAW264.7 on SLA and Sr-SLA surfaces.Methods:1.The adhesion and cell morphology of RAW264.7 on different titanium surfaces were observed by immunofluorescence and SEM.Cell proliferation activity was evaluated by CCK8 proliferation experiment.2.Polarization status of RAW264.7 on different titanium surfaces were evaluated:flow cytometry(FCM)was used to detect the expression level of M1 marker(CD11c)and M2 marker(CD163);quantitative real-time real-time(q PCR)was used to detect the expression levels of inflammatory genes(IL-6?IL-1??TNF-??i NOS?IL10?CCL2?CCL5)and osteogenic genes(TGF-??BMP-2?VEGFa?PDGF);mouse cytokine array was used to detect the inflammatory factor secretion levels of RAW264.7;ELISA was used to detect secretion level of VEGFa and PDGF and western blot was used to detect the expression level of BMP-2.3.The expression of MAPK pathway protein in RAW264.7 was detected by western blot.Results:1.The results of cell proliferation experiments displayed similar proliferation rates between SLA surface and Sr-SLA surface;compared with SLA surfaces,RAW264.7showed more cytoplasmic extensions on Sr-SLA surfaces.2.Compared with the SLA surface,the Sr-SLA surface could inhibit M1 polarization and promote M2 polarization: FCM results showed that the Sr-SLA surface up-regulated the expression of M2 marker(CD163)and down-regulated the expression of M1 marker(CD11c);at the gene level,Sr-SLA significantly reduced the expression of proinflammatory-related genes(IL-6?IL-1??i NOS?TNF-??CCL2?CCL5),and up-regulated the expression of anti-inflammatory gene IL-10,also up-regulated the expression of osteogenic gene BMP-2;at the protein level,Sr-SLA surface significantly reduced the secretion level of inflammatory factors(TNF-?,CCL2,CCL5),and up-regulated the expression of BMP-2.3.Western blot displayed that Sr-SLA surface could enhance expression of ERK1/2protein in both RAW264.7.Conclusions:1.Compared with SLA surface,Sr-SLA surface inhibits M1 polarization of macrophages and improve M2 polarization.2.ERK pathway is involved in the macrophage polarization by Sr-SLA surface.Part III Effect of strontium-incorporated micro/nano rough titanium surfaces on osteogensis via affecting macrophages in vitro and in vivoObjectives:To evaluate the effects of macrophage secretion products induced by different surfaces on bone marrow mesenchymal stem cells(BMSC)in vtiro,and to estimate the effects of SLA and Sr-SLA implants on the polarization status of macrophages and osseointegration in vivo.Methods:1.A conditioned medium co-culture system model was established in vitro,and the groups was divided into five groups according to conditioned medium: DMEM group,Pure SLA group,Pure Sr-SLA group,SLA + RAW264.7 group and Sr-SLA +RAW264.7 group.Alkaline phosphatase(ALP)activity and osteogenic genes(ALP,TGF-?,BMP-2,RUNX2,Col-1)expressions were evaluated.2.In vivo,SLA and Sr-SLA implants were implanted near the metaphysis of the left and right tibia of rats,and samples were taken at 1,3,7,and 14 days after surgery.CCR7 positive cells(M1 macrophage surface markers)and CD163 positive cells(M2macrophage surface markers)were labeled by immunohistochemical staining,and the type and distribution of macrophages around the implant were observed and semi-quantitative analysed.3.Masson staining and morphological analysis were performed to evaluate bone regeneration around the implants.Results:1.In the conditioned medium co-culture system model,ALP activity was as follows: SLA + RAW264.7 group < Pure SLA group(p<0.05),Sr-SLA + RAW264.7group < Pure Sr-SLA group(p<0.05),Pure SLA group < Pure Sr-SLA group(p<0.05),SLA + RAW264.7group <Sr-SLA + RAW264.7 group(p<0.05).2.In the conditioned medium co-culture system model,osteogenic genes(ALP?TGF-??RUNX2?Col-1)expressions were lowest in SLA + RAW264.7 group.3.In vivo,both M1 and M2 macrophages could be observed around SLA implants and Sr-SLA implants.The number of M1 macrophages gradually decreased from day 1to day 7,while the number of M2 macrophages increased at day 7.Compared with SLA implants,the infiltration level of M1-type macrophages around Sr-SLA implants was significantly lower in the early stage of implantation(1 day after surgery).As the tissue healed(3 and 7 days after surgery),the infiltration level of M2 macrophages around Sr-SLA implants was significantly higher than SLA implants.4.Masson staining analysis showed that the bone area around the Sr-SLA implant was significantly higher than that of the SLA implant group at 14 days after implantation.Conclusions:1.Compared with SLA surface,Sr-SLA surface promots osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the function of macrophages.2.Compared with SLA implants,implantation of Sr-SLA implants in bone tissue can help reduce M1 macrophages infiltration in local tissues in the early stage and increase M2 macrophages at later stage3.Compared with SLA implants,Sr-SLA implants promoted the formation of new bone at the implantation site.