| The ideal bone implants would have the capacity to modulate immune response,inhibit bacterial growth and further enhance tissue repaire.Due to their good biocompatibility and mechanical properties,titanium and its alloys have been widely used in hard tissue implantation,such as bone and dental fields.However,they can hardly form bone-boding with tissue around because of their bioinert property,which may cause inflammatory response.Moreover,the lack of antibaterial ability can result in infection.Therefore,proper surface modification methods applied on titanium implants are necessary to improve their performance in inflammation regulation,anti-infection and further enhancing tissue repaire,increasing success rate of implants after implantation.In this study,hydrogel was employed as a drug delivery vehicle on titanium nanotubes for IL-4 cytokine or/and RGD peptides loading to regulate macrophage inflammatory response.This study further focused on how the macrophage phenotypic change and its subsequently influence on the osteo-immune response.Additionally,porous titanium with silver loaded microspheres to achieve silver long-time release,serving as antibacterial implants.Based on improving tissue repaire,this work maily concentrated on titanium surface modification from three sides:inflammatory regulation,immune osteogenesis and antibacterial,providing the basis of theoretical and technical for the clinical application of functional titanium implants.TNT arrays were fabricated on the Ti through anodization treatment.After that,double-layer sol–gel coatings were fabricated on TiO2 nanotubes.Based on a higher swelling capacity and degradability,the lower gel layer was loaded with IL-4 and the upper gel layer was used for controlled IL-4 delivery from the lower layer.An analysis of the release dynamics of IL-4 showed that the sample without the upper hydrogel layer showed significant release within the first 3 days?72.5%?.In contrast,the sample with the upper layer released only a small proportion of cytokine within the same period?12.5%?,which was followed by prolonged release over an extended period.Therefore,IL-4 programmed release has been achieved by the regulation of physical and chemical properties of hydrogel,and the spatial distribution of IL-4.In vitro study similar with physiological conditions was performed to evaluate whether IL-4 programmed release from the double-layer sol–gel coatings has the potential to regulate macrophage polarization.Culture medium was added with LPS and IFN-?for inflammatory micro-environment formation during three days.Macrophages were seeded on samples to evaluate the macrophage morphology and phenotypic changes.The results suggest that macrophages on all samples were activated,but only cells on samples with IL-4 loading had fused into multinucleated foreign body giant cells.Flow cytometry,quantified cytokine secretion and gene expression analysis revealed that after 3 days of culture in the presence of LPS+IFN-?,expression of the pro-inflammatory cytokines in M1 macrophages was higher than that of the anti-inflammatory cytokines,achieving macrophage polarization from M0 to M1.After 7 days,the anti-inflammatory cytokines significantly increased,especially on samples with IL-4.It suggests that macrophages polarization from M1 to M2.The results indicated that IL-4 controlled release fom double-layer hydrogel could modulate micro-environment from inflammation to anti-inflammation.To optimize double-layer sol–gel coatings and to relieaze double-factor-loading?IL-4 and RGD?,chemical modification of TiO2 with poly-dopamine was implemented to achieve IL-4 immobilization,and a hydrogel layer was fabricated on them for further RGD peptides incorporating.In vitro study suggests that both IL-4 and RGD peptides contributed to macrophage activation.When samples incorporated with RGD,macrophages exhibited needle-like filopodia.When samples immobilized with IL-4,macrophages showed the greatest degree of elongation with a tail morphology called lamellipodia.RGD and IL-4 work synergistically,which can stimulate the formation of lamellipodia and filopodia.It is generally accepted that macrophage morphologies are associated with their polarization states.T/DOP-IL4/CG-RGD exhibited higher expression of anti-inflammatory cytokines due to the immunoregulatory function of IL-4 and RGD.It suggests that RGD and IL-4 with specific spatial distributionand can simultaneously modulate M2 macrophage polarization.One transwell co-culture system was used to study how the double-layer gel with IL-4 and RGD spatial distribution regulate immune-osteogenesis.We investigate whether macrophages seeded on the designed biomaterials could create a favorable osteo-immune environment to regulate the osteogenic differentiation of MSCs.The results indicated samples with the immobilization of IL-4 and RGD can generate an immune environment that is favorable for osteogenesis;After that,MSCs were cultured on different samples to evaluate the effect of different sample surfaces on MSC osteogenic differentiation.The data revealed that RGD-peptide-modified samples can directly induce MSC adhesion and osteogenic differentiation;MSC osteogenesis ability can be accelerated by synergistic effect of IL-4 and RGD.Furthermore,significantly upregulated levels of osteogenic genes in the sample with IL-4 and RGD loading suggested that the early stage of immune-regulation and osteogenesis could be accelerated through the BMP2/SMAD/RUNX2 pathway.To improve antibacterial property of porous titanium,in this study,porous titanium scaffolds were prepared by powder metallurgy sintering technology,and then treated by the MAO to form the oxide layer and micro/nano structures,enhancing the surface activity.The gelatin mirospheres as silver carrier were incorporated into the porous titanium scaffold uniformly.With the swelling and degradation of the gelatin microspheres and ions diffusion,silver ions released from the microspheres at a controllable rate to achieve long-time antibacterial property.The results suggest that silver particles produced inside gelatin mirospheres within network structure besides on the surface of gelatin mirospheres.In vitro study also confirmed that porous titanium with silver loaded microspheres had an insignificant influence on cell viability,and great potential in antibacterial. |
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