Effects Of Hydroxyapatite-Graphene Oxide Nanocoatings On Osteogenic Differentiation And Modulating Polarization Of Macrophages

Objective:Titanium implants have been widely used in medicine.However,its osteogenic induction ability is limited due to the bioinertness of titanium,It takes a long time for the implants to form bone union in vivo and increases the risk of treatment failure.Therefore,it is necessary to modify the titanium surface appropriately to promote bone bonding in titanium implants.At the same time,with the deepening of studies on the interaction between implants and relevant tissues in vivo,it is believed that the host’s inherent inflammatory response has a great influence on the success rate of implants.In particular,the M1/M2 polarization of macrophages and their inflammatory secretion function can directly affect the osteogenic differentiation which is a key regulatory factor for the prognosis of implants.Therefore,this study intends to prepare hydroxyapatite(HA)-graphene oxide(GO)nano coating material on pure titanium surface by electrophoresis method,taking bone marrow stem cells and macrophages as the research object,to further study the effect of HA-GO coating surface on bone marrow stem cells osteogenic differentiation and macrophage polarization regulation,so as to provide provides some theoretical and experimental basis for optimizing the surface design of plant materials.Methods:Part Ⅰ:(1)The surface of pure titanium is grinding,polished,and then washed with anhydrous ethanol,methylene chloride and acetone by ultrasonic swing in turn.(2)Prepare the electrolyte of the corresponding coating.(3)GO,HA,Ha-2wt%GO and Ha-5wt%GO groups were prepared on the surface of pure titanium sheet by DC power supply,and pure Ti was used as the control group.(4)Scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray photoelectron spectroscopy(XPS),X-ray diffractometer(XRD),infrared spectrometer(FTIR)and Raman spectroscopy were used to observe and characterize the surface morphology,elemental composition and surface chemical bonds of the coating materials.(5)The surface roughness and hydrophilicity of the sample were evaluated by atomic force microscopy(AFM)and contact Angle detection.(6)The surface corrosion resistance of each group of materials was tested by electrochemical workstation.Part Ⅱ:(1)The co-culture model of mouse bone marrow stem cells(BMSCs)was established in vitro.The proliferation rate of BMSCs on the surface of each group was detected by CCK-8,and the cell adhesion was observed by scanning electron microscope and fluorescence microscope.(2)Alkaline phosphatase(ALP),Alizarin Red activity and expression of osteogenic genes(ALP,TGF-β,BMP-2,RUNX2,Col-1)on BMSCs of each group were detected.(3)In vivo study,the implant with coating material was implanted in the epiphysis of the femoral shaft of the rat,and the position,direction and postoperative inflammation of the implant were observed by weekly X-ray.Samples were taken at 4 and 8w after surgery,and the bone union around the implant was observed and studied by X-ray,Micro-CT,tissue staining and immunohistochemistry.Part Ⅲ:(1)The co-culture model of mouse macrophage(RAW264.7)was established in vitro.The proliferation ability of RAW264.7 cells on the surface of each group was detected by CCK-8,and the cell adhesion was observed by scanning electron microscope and fluorescence microscope.Meanwhile,flow cytometry,immunofluorescence,enzyme-linked immunoadsorption(ELISA),reverse transcription polymerase linked reaction(RT-PCR)and other methods were used to detect the immunomodulatory effects of the coatings in vitro.(2)In vitro,conditional medium for co-culture RAW264.7 cells with samples from each group was established,and BMSCs were cultured to observe the effects of macrophage secretion products on the proliferation function and osteogenic differentiation of BMSCs.(3)A mouse back airbag model was established,and the coated titanium sheet was implanted into the airbag to activate the immune system of mouse.Through histological analysis of cytokines in the exudate of the airbag and the back skin,the immune regulatory ability of each group of coatings was verified and analyzed from the internal perspective of the body.Results:Part Ⅰ:(1)TEM observation showed that HA nanoparticles in HA-2wt%GO and HA-5wt%GO electrolyte were uniformly modified or grafted on the GO surface by van der Waals bonding,and only a small amount of HA nanoparticles accumulated at the GO sheet edge.(2)The surface of HA-GO coating showed a nanoscale honeycomb porous structure,and the surface of HA-2wt%GO showed no cracks,uniform and compact,presenting a surface with micro/nano morphology.(3)The successful preparation of HA-GO coating by electrochemical method(EPD)was confirmed by XPS,XRD and Raman.(4)FTIR,AFM and hydrophilic Angle test results confirmed that HA-2wt%GO has a high content of hydrophilic groups,nanometer roughness and superhydrophilic surface.(5)GO,HA,HA-2wt%GO and HA-5wt%GO groups can all increase the corrosion resistance of substrate Ti.Part Ⅱ:(1)The results of CCK-8 assay showed that the cell proliferation rate of HA-2wt%GO and HA-5wt%GO groups was significantly higher than that of Ti,HA and GO groups,which was also confirmed by fluorescence microscopy.(2)SEM observation showed that the distribution of BMSCs on the surfaces of HA-2wt%GO and HA-5wt%GO groups was wider,and the cell expansion area was larger than that on the surfaces of other materials,which indicated that the surfaces of HA-2wt%GO and HA-5wt%GO groups were more conducive to the adhesion and proliferation of these cells.The alkaline phosphatase and alizarin red activities of BMSCs co-cultured on the surface of HA-2wt%GO were significantly increased,and the expression of osteogenic-related genes was also significantly increased.(3)In vivo,Micro-CT results showed that the HA-2wt%GO group had the largest area of new bone around the implant.The HA-2wt%GO group had the thickest lamellar bone structure around the implants,and the calcium salt deposition and collagen fiber staining intensity were significantly higher than those of the control group.Part Ⅲ:(1)The results of CCK-8 assay showed that the proliferation rate of RAW264.7 macrophages on the surface of HA-2wt%GO and HA-5wt%GO groups was significantly higher than that of Ti,HA and GO groups.These results indicated that the HA-2wt%GO and HA-5wt%GO groups had relatively good surface biocompatibility and no cytotoxicity.(2)Through flow cytometry,immunofluorescence,enzyme-linked immunosorbent assay and other experiments,we found that HA-2wt%GO could induce the transformation of RAW264.7 cells to M2 phenotype,which was significantly better than that of HA,GO and Ti groups.In addition,the expression of anti-inflammatory related genes secreted by macrophages on the surface of HA-2wt%GO group was higher.It can create an immune microenvironment conducive to tissue repair.We cultured BMSCs in macrophage conditioned medium and found that BMSCs in HA-2wt%GO group showed the strongest ALP staining effect.At the same time,the immune microenvironment induced by macrophage conditioned medium was most favorable for the expression of osteogenesis-related genes.(3)The thickness of the skin fiber layer of mice in the GO group was the largest,and the inflammatory infiltration was relatively strong.However,the results of section staining of HA-2wt%GO group showed that the skin fiber layer was the thinnest,there was no obvious inflammatory stimulation,and the inflammatory cytokines in lavage fluid were lower,which further verified that the immune regulation function of HA-2wt%GO group was better.Conclusion:(1)Nano-hydroxyapatite-graphene oxide composite coating material was successfully prepared on pure titanium surface by electrochemical method.The coating properties can be precisely controlled by the voltage and electrolyte concentration,making the coating a nano-scale coating material with rich functional groups,strong hydrophilic ability and certain corrosion resistance.(2)Compared with Ti,HA,and GO groups,the surfaces of HA-2wt%GO and HA-5wt%GO groups were conducive to the osteogenic differentiation of BMSCs and promoted the osseointegration of the implants at the bone interface in vivo and shortened the healing process time after the implantation of surface implants.(3)Compared with Ti group,HA group and GO group,HA-2wt%GO group and HA-5wt%GO group were more conductive to the polarization of macrophages towards M2 direction,which could promote the osteogenic differentiation of BMSCs by regulating the function of macrophages.At the same time,the inflammatory infiltration of local tissue can be reduced in the back air-pouch of mice.At the same time,the cytokines secreted by macrophages in the air-pouch in the HA-2wt%GO group of mice were consistent with the results of in vitro experiments,and histological analysis showed that the coating could significantly inhibit the formation of fibrous tissue.