Experimental Study On The Effect Of Two Nano Morphologies Of Titanium On Inflammatory Activation Of Immune Cells & Case Report

Background and aims:Nowadays,titanium is broadly used in medical regeneration due to its excellent corrosion resistancem,mechanical properties,and biocompatibility.In clinical treatment in the field of dentistry,titanium is widely used to fabricate oral implants.However,immediately after natural titanium is implanted in the body,proteins from the body fluid adhere to the surface,thus forming a new layer of interface between the implant and the body fluid,which eventually causing an inflammatory reaction around the implant.In addition to this,the implant placement process generates a large number of damage-associated molecular patterns(DAMPs)which,by attaching to the surface of the material,influence the aggregation and inflammatory activation of immune cells,thus causing an implant placement-related inflammatory response.To this day,the excessive inflammatory activation of immune cells remains one of the main problems that prevent implants from functioning and even lead to implant failure.Immune cells are the first group of cells to respond to tissue damage and implant placement.After implant placement in the organism,the immune system recognizes the implant as a foreign body due to the adherent proteins and DAMPs on its surface,initiating a moderate inflammatory response mediated by immune cells that directs peri-implant tissue cell lysis,proliferation and remodeling,ultimately restoring intra-tissue homeostasis.However,excessive inflammatory response can lead to thickening of the fibrous layer of the peri-implant tissue,even into the bone and the implant,causing a failure of osseointegration and ultimately implant failure.Therefore,an excellent implant restoration healing process requires a balanced relationship between tissue repair and inflammatory response to be coordinated.How to influence the adsorption,conformational changes and functions of the material surface proteins through material surface modification to regulate the inflammatory activation of immune cells,so as to mitigate the adverse implant response and improve the implant efficiency has become a critical issue to be solved in current clinical practice.Current research has shown that the construction of nanostructures on the surface of implants can modulate cellular function and improve the performance of implants.Among other things,the construction of different nanostructures on the implant surface can have a significant impact on the inflammatory activation of immune cells,with changes on the scale of a few tens of nanometres leading to very different changes in the fate of immune cells.Although various surface nanostructure modification strategies have been promoted to modulate inflammation,the effects of different nanostructures on immune cell behaviour and the mechanisms of action remain unclear.Therefore,this thesis systematically constructs titanium plates with two different characteristic nanostructures to investigate the effects and mechanisms by which the different properties of nanostructures modulate the inflammatory activation behaviour of immune cells,laying the groundwork for the optimisation of surface modification strategies for implants.Methods:To address these issues,titanium nanostructures with two different nano-features were synthesised using the following methods: 1)titanium wafers with different pore size nanotubes were prepared by anodic oxidation and characterised by FE-SEM,AFM,XRD and XPS;2)titanium nanostructures with lower surface accessible area were obtained by magnetron sputtering of chromium on single crystal silicon wafers.The titanium surface with lower SAA as obtained by magnetron sputtering chromium on the surface to slow down the reaction rate of chloroauric acid with the silicon wafer.Titanium surfaces with different accessible areas were then obtained by magnetron sputtering on the surface and characterised by FE-SEM,AFM,XRD and XPS materials.In order to investigate the effect of titanium nanostructures with different nanometric characteristics on the inflammatory activation of immune cells,an animal model of mouse hind leg embedding was constructed: the material was embedded into the hind legs of mice,and the material was taken after 3 days,and the proportion of cell species around the material and the number of inflammatory activation were analysed by flowcytometry.Inflammatory infiltration around the material was analysed by immunohistochemical section results.In vitro,cells were cultured by DC2.4 and RAW264.7 cell lines and flowcytometry analysed for differences in cell activation on titanium surfaces with different nanometric characteristics.Inflammatory activation of cells on the surface of the material was analysed by immunofluorescence staining results.To investigate the specific mechanisms by which titanium nanostructures with different nanometric characteristics affect the inflammatory activation of immune cells,this paper first analyses the adsorption of various proteins on different material surfaces by ELISA and material surface proteomics.In order to examine the adsorption of proteins on different surfaces,the adsorption of proteins on different surfaces was measured using confocal microscopy,total internal reflection fluorescence microscopy(TIRF)and BCA assay kits.Finally,the adsorption and functional conformation of the proteins on the different material surfaces were modelled by molecular dynamics(MD)and the mechanisms of the adsorption and functional differences were explained.Results:1.Preparation and characterisation of titanium surfaces with different nano-featuresThe results of the material characterisation demonstrated that nanotubes with different pore sizes could be prepared by adjusting the voltage,with pore sizes of 30 nm at 5 V and 80 nm at 20 V.The results of the water contact angle experiments showed that the different surfaces exhibited good hydrophilicity after UV illumination.The surface roughness of the nanotube size materials was almost the same.The surface magnetron sputtering of Cr allows the preparation of titanium surfaces with different surface accessible areas,and the SEM and AFM results show a smaller surface accessible area on the surface of the Cr-plated material.For the lower surface accessible area group,the surfaces were labelled as Nano Coral(NC).The higher surface accessible area group is named Nano Ferns(NF).XRD and XPS showed no significant differences in the molecular composition and crystal structure of the different surface accessible areas of the titanium nanostructures,and AFM showed almost identical surface roughness for the different surface accessible areas.2.Effect of nanotubes sizes on titanium surfaces on the inflammatory activation of macrophagesResults from in vivo animal models showed significant differences in the proportion of macrophage infiltration and the degree of inflammatory activation around the different surfaces 3 days after implantation of the material.The proportion of macrophage inflammatory activation was lower on the 30 nm nanotube surface compared to the 80 nm nanotube and plane titanium surface.The location of fibronectin adsorption and exposure of active sites to different surfaces were determined by ELISA,confocal photographs of surface protein adsorption,and scanning electron microscopy photographs.Molecular dynamics(MD)simulations were used to model the movement of the RGD-containing fragments of fibronectin in different states,revealing the mechanism of different active site exposure of fibronectin in different nanotubes.Fibronectin exhibited a "size-limited" conformational change in 30 nm nanotubes,and 30 nm nanotubes matching the size of fibronectin resulted in better exposure of the RGD site,thereby activating the integrin-mediated adherens spot kinase(FAK)-phosphatidylinositol kinase γ(PI3Kγ)signalling pathway to inhibit nuclear-κB(NF)signalling.factor-κB(NF-κB)signalling and ultimately inhibit the inflammatory activation of macrophages.3.Effect of surface accessible area of the titanium nanostructures on the inflammatory activation of dendritic cellsResults from the in vivo animal model showed significant differences in the proportion of dendritic cell infiltration around the different surfaces 3 days after material implantation.Flowcytometry results showed a lower proportion of inflammatory activation of surface DCs in the NC group compared to the smooth plane and NF groups.ELISA results showed a significant difference in the adsorption of HMGB1 on different SAA surfaces only compared to other DAMPs.Total internal reflection microscopy(TIRF)and MD simulations showed that the exposure of cysteine(CYS)residues in HMGB1 was significantly lower on the surface of the NC group with lower SAA.Low exposure of CYS inhibited the activation of TLR4 on the surface of DCs,thereby down-regulating their myeloid differentiation factor(Myd88)-TNF receptor-associated factor 6(TRAF6)expression to suppress NF-κB signalling.Conclusion:By regulating the voltage by means of anodic oxidation,titanium nanotubes can be prepared with physical properties that differ almost exclusively in pore size,thus avoiding as far as possible the effects of hydrophobicity and roughness on the inflammatory activation of immune cells.By plating Cr on the surface of single crystalline silicon,it is possible to produce a titanium surface that differs in physical properties almost exclusively in terms of surface accessible area,thus avoiding as far as possible the influence of physical properties such as hydrophobicity and roughness on the inflammatory activation of immune cells.These materials are able to exclude the influence of other physical properties on the cells and focus more on the effect of a single physical variable on the cells,and can be used as a vehicle to study the effect of nanostructures on the surface of materials on the inflammatory activation of immune cells and the mechanisms.This thesis explores the mechanisms of how two nanostructures with different characteristics affect the inflammatory activation of immune cells at the protein and molecular level.The results show that,in addition to the amount of protein adsorbed,the exposure of active sites is also important.On the one hand,size limiting effects alter the distribution and exposure of protein functional peptides when the nanotube size is matched to the protein.The size fit between fibronectin and nanotubes allows for maximum exposure of the RGD structural domain,which in turn activates the integrin-FAK signalling pathway.Inhibition of NF-κB signalling into the nucleus through the downstream PI3Kγ-Akt pathway resulted in the expression of an inhibitory effect on macrophage inflammatory activation on the titanium surface of the 30 nm nanotubes.On the other hand,this thesis found that nanoscale SAA differences on titanium surfaces can influence the inflammatory activation of dendritic cells.Lower SAA induced a significant reduction in the exposure of CYS residues in HMGB1 and subsequently,this property could inhibit DC aggregation and inflammatory activation on low SAA titanium surfaces through downregulation of the TLR4-Myd88 pathway.The studies in this paper link the mechanical properties of implants to chemical/bio-signalling,explain the mechanisms of bio-signalling in nanostructures at the protein and molecular level,and propose two promising surface modification strategies to modulate the immune response of implants.The above work will provide new ideas for the fabrication of more biocompatible implants at the sub-micron and nano-scale.