| BackgroundThe incidence rate of fracture and bone defect is high.In order to promote bone healing,implants are often applied in the damaged sites.Pure titanium materials are widely used clinically due to their excellent biocompatibility,corrosion resistance,and osteogenisis promoting ability.However,pure titanium implants often cause peri-implant inflammation,poor implant-bone integration,and a series of clinical problems after implantation,ultimately leading to implantation failure.Previous studies found that the morphology of implants plays an important role in the osteogenic process and the regulation of immune cells.Therefore,the modification of titanium implants is of great significance in promoting patient recovery.As the research went on,it has been found that when the diameter of titanium nanotube is between 30-120 nm,it can effectively promote osteogenesis and regulate monocytes/macrophages.Based on previous experiments,we constructed three different Ti O2 with different diameters,among which the control group P（polished）was only polished to the mirror surface;The diameter of NT5（nanotube 5 V）is 30 nm,which is anodized with 5 V voltage after polishing to mirror-like surface;The diameter of NT20（nanotube 20 V）is 80 nm,which is anodized with 20 V voltage after polishing to mirror surface.Peripheral blood monocytes can be divided into classical monocytes（also named inflammatory monocytes,i Mos）and non-classical monocytes（also named patrolling monocytes,p Mos）.i Mos and p Mos account for about 90%and 10%of peripheral blood monocytes,respectively.When tissue damage occurs,i Mos is chemotactic to the damaged site through CCR2 chemotactic receptor and participate in inflammatory response,while p Mos patrols the peripheral blood and tissue and can colonize the tissue under specific conditions,playing a regulatory role in the development and prognosis of inflammation.Being the largest proportion in peripheral blood monocytes and due to its involvement in inflammation through chemotaxis to the damage site,i Mos plays an important role in the occurrence and development of inflammation.However,the impact of different Ti O2nanomorphologies on i Mos is still unclear and needs to be elucidated.In recent years,many researches showed that cells transform external mechanical force signals into biological signals through the cytoskeleton,ultimately affecting cell functions.Previous studies have found that different Ti O2 nanostructures can affect cells,but whether Ti O2 nanostructures can further affect the phenotype and function of i Mos derived macrophages through cytoskeleton remains to be explored.Objective1.To investigate the effects of different Ti O2 nanostructures on the biological functions of human inflammatory monocytes（i Mos）derived macrophages.2.To figure out the effects of cytoskeleton on the biological function of human inflammatory monocytes（i Mos）derived macrophages on different Ti O2nanostructures.Methods1.NT5（with a diameter of 30 nm surface nanotubes）and NT20（with a diameter of diameter 80 nm surface nanotubes）were prepared through gradient grinding and anodizing.The control group was named after P（only polished to the mirror-like surface）,and then the three kinds of samples were conducted characterization in detail.2.The inflammatory monocytes（i Mos）were purified through magnetic beads and their purity and vitality were identified by flow cytometry.3.The i Mos were planted on sample P,NT5 and NT20 respectively,and cultured for 1,2,6 and 12 days.The corresponding samples were collected.The cell morphologies of i Mos-derived macrophages on different nanostructured Ti O2 were observed by field emission scanning electron microscopy and laser confocal microscopy;The expression of surface molecules was detected by flow cytometry to determine the differentiation of i Mos-derived macrophages on different nano-morphologies;The cytokine secretion of i Mos-derived macrophages on different nano morphologies was detected by ELISA,so as to analyze the influence of different nano-morphologies on the biological functions of i Mos-derived macrophages.4.Cytochalasin D was added to inhibit cytoskeleton.In presence of cytochalasin D,the effects of cytoskeleton on the change of biological functions of i Mos-derived macrophage on different nano-morphologies were investigated by immunofluorescence,ELISA and flow cytometry.Results1.The characterization analysis of different materials showed that the control group P presented a smooth surface and no nanotubes existed on its surface.The highly uniform nanotubes with a diameter of 30 nm were distributed on the sample NT5,and the highly uniform nanotubes with a diameter of 80 nm were distributed on the sample NT20.After anodic oxidation and UVA/C irradiation,the hydrophilicity of the sample NT5 and NT20was significantly improved compared with the control group.2.The purity of inflammatory monocytes obtained by magnetic beads sorting was more than 95%,and it presented a good vitality,which could be used for subsequent experiments.3.NT20 mainly promotes the secretion of pro-inflammatory cytokines such as,IL-6and IL-1βand induces the differentiation of i Mos-derived macrophages into M1 subtype macrophage.NT5 mainly promotes the secretion of anti-inflammatory cytokines like,IL-10 and TGF-βand also induces i Mos-derived macrophages into M2c macrophage.4.After cytoskeleton was inhibited,the inducing capacity of sample NT5 and sample NT20 on i Mos-derived macrophage were blunted and the secretion of cytokines was also significantly inhibited.ConclusionNT20 mainly promotes the differentiation of i Mos-derived macrophage into M1macrophages and the secretion of pro-inflammatory cytokines.Sample NT5 mainly promotes the differentiation of i Mos-derived macrophage into M2c macrophages and the secretion of anti-inflammatory cytokines.Notably,The cytokine secretion profile of i Mos-derived macrophage and cell polarization induced by different nanostructures require intact cytoskeleton reorganization. |
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