Antibacterial Modification Of Micro-/Nano-structured Titanium And Their Biological Properties

Titanium and its alloys are widely used in orthopedic implants.Nevertheless,the studies of clinical cases showed that infection,lack of integration and prolonged chronic inflammation might lead to the implant failure.After adhering to implant surfaces,bacteria secrete exopolysaccharides to form biofilms which protect them from phagocytic uptake and antibiotics.Titanium was lack of bioactivity because of the inert titanium dioxide（Ti O₂）film on the surface.Meanwhile,poor bone-implant osseointegration was originated from granuloma and fibrous encapsulation induced by prolonged chronic inflammation.Furthermore,many repots reveal that the inflammatory response to implants is a key stage of promoting angiogenesis,wound healing and regulating osteogenesis.Altering the surface topography could inhibit bacterial attachment,promote cell adhesion,proliferation and differentiation,and regulate inflammatory response.Silver has antibacterial properties against a broad spectrum of bacteria and fungi with low incidence of resistance.In vivo infection model,silver nanopartices（Ag NPs）could promote the bone healing.In order to improve the integration between implant and bone tissue,and antibacterial activity,we modified titanium surface via combining topography modification with Ag NPs to obtain micro-/nano-structured titanium decorated with Ag NPs,and then explored its effect on macrophage polarization.Nano-structured（TNT）,micro-structured（AE）and micro-/nano-structured（MNT,AE-TNT and AE-MNT）titanium were fabricated via anodic oxidation,micro-arc oxidiation and acid etching.Compared with pretreated titanium（PT）,the roughness of TNT and MNT increased.Afer acid etching treatment,the roughness increased to microscale.The roughness of AE-TNT and AE-MNT were higher than that of AE.Hydrophilicity increased with roughness and MNT showed mid-hydrophobic because of mixed rutile and anatase Ti O₂ on the surface.Roughness played a dominant role in the amount of protein adsorbed on the surface rather than hydrophilic and crystalline form.AE provided more adhesion points and space protection for bacteria,which was not benefit to preventing infection.AE-TNT,AE-MNT,TNT and MNT suppressed the gene expressions of attachment（fnb A and Clf B）and biofilm（ica A and ica D）in Staphylococcus aureus to inhibit bacterial attactment and biofilm formation.Silver nanoparticles（Ag NPs）with a diameter of 10 nm were synthesized by a chemical reduction method and then were loaded onto MNT by vacuum absorption,generating Ag NP-decorated MNT（Ag MN）,to improve the antibacterial activity.Ag MN had no adverse affect fibroblast proliferation and morphologies,even cells covered the sample surface completely after 7 days,which indicated that no significant cytotoxicity associated with Ag MN.The expressions of fnb A and Clf B were downregulated by Ag MN.Most bacteria closed to the Ag MN surface were prevented from attaching to the surface,and some attached bacteria directly touched the Ag NPs immobilized on the surface,and then were dead as a result of the significant cytoderm permeability and damage.Ag MN also inhibited biofilm formation.Hence,Ag MN showed excellent antibacterial activity.In vivo infection model,cells and tissues adhered to the implant surfaces prior to bacteria.It was difficult to observe that bacteria attacted on Ag MN after 7-day implantation.Moreover,the secretions of proinflammatory cytokines TNF-αand IL-6 induced by Ag MN were less than those by MNT suggesting that Ag MN suppressed inflammation and improved wound healing.Dopamine was used to prepare Ag NPs,and then immobilized Ag NPs onto MNT via covalent and ionic bonds,yielding Ag PD-MNT to reduce the release of silver（<8ppb,21days）.In cytotoxicity test,there were no obvious differences in proliferation and morphology between MNT,MNT with polydopamine（PD-MNT）and Ag PD-MNT.Therefore,Ag PD-MNT exhibited high antibacterial efficacy against Escherichia coli without cytotoxicity.To investigate inflammatory respose to modified titanium surfaces,macrophages were applied in this work due to their high plasticity.The results of macrophage proliferation and morphology found that the numbers of cells increased within 5 days,apoptosis occurred after 7 days.The experimental results manifested that macrophages on various surfaces polarized to mixed macrophage phenotypes,the proportion of M1 macrophages was higher after 3 days.More macrophages were activated and polarized to M2 phenotype with upregulating expressions of Arg-1,PDGF-BB,TGF-β,BMP2 and BMP6.It suggested that macrophage phenotype switched from M1 to M2 during 3 to 7 day,which was beneficial for inflammatory inhibition and tissue reconstruction.Among material groups,Ag PD-MNT showed the most significant regulation in macrophage polarization.Ag PD-MNT induced adherent-macrophage apoptosis,regulated the gene expressions of NF-κB（IκB and IL-1ra）and TLR（Myd88,Ticam1 and Ticam2）signaling pathways cascades and activated autophagy to reduce inflammation.Then,we investigated the mineralization ability of samples in vitro,and osteogenic differentiation of rat bone mesenchymal stem cells（r BMSCs）on MNT,PD-MNT and Ag PD-MNT in complete and conditioned medium.Hydroxyapatite formed on the MNT surface after soaking in simulated body fluid（SBF）.PD-MNT and Ag PD-MNT enhanced their ability to promote mineralization in vitro because the abundant hydroxyl groups chelated calcium ions in SBF.MNT upregualed the osteogenic gene expressions of Runx2,ALP,COL-I,OPN and OCN,indicaitng that MNT induced osteogenic differentiation.PD-MNT and Ag PD-MNT were significantly enhenced in osteogenesis.The conditioned medium（CM）,which was prepared by collected medium from macrophage culturing on materials and r BMSCs complete medium,were used to culture r BMSCs on various surfaces.Cytokines TGF-βand BMP2 in CM could activate TGF-β/BMP/SMADS signaling pathways in r BMSCs to upregulated osteogenic gene expressions and then promote osteogenic differentiation cooperating with materials.Ag PD-MNT contributed to the best ability of osteogenic differentiation.After that,Transwell co-culture system was used to study the interaction of various surfaces with macrophages and r BMSCs for simulated microenvironment of coexistence between r BMSCs and immune cells in vivo.Cytokines secreted by r BMSCs could affect macrophage polarization.At 3d,TGF-βsecreted by r BMSCs and macrophages response to r BMSCs promoted macrophage polarized to pro-inflammatory M1 phenotype.At 7d,r BMSCs induced macrophages switch to M2 phenotype through secretion of M-CSF and activation of TGF-β/TGF-βR2 signaling pathway in macrophages.Besides,cytokines secreted by macrophages stimulated by materials and r BMSCs promoted the osteogenic differentiation of r BMSCs via paracrine in Transwell.Finally,an implantation experiment in rats was applied to investigate the regulation of MNT,PD-MNT and Ag PD-MNT on the early inflammatoty response and later osseointegration in vivo.With the increasing implantation time,the expression of M2marophage marker（Arg-1）and the population of M2 macrophage around implants were upregulated,demonstrating that macrophage phenotype had been switched from M1 to M2,which was favourable in suppression of inflammatory immune reactions and the tissue remodeling.Due to the difference in inflammatory response,the fibrous layers were observed on MNT and PD-MNT at 2 weeks,which caused osseointegration delay.After 8 weeks,the Ag PD-MNT showed best osseointegration among modified surfaces with highest bone fraction and bone-implant contact ratio.In conclusion,Ag NPs immobilized on micro-/nano-structured titanium with excellent antibacterial property,osteoimmunomodulatory ability,promoting osteogenic differentiation of bone marrow mesenchymal stem cells and osseointegration is therefore a promising candidate bone substitution material for orthopedic surgery.