Nanotopography Of Zirconia Abutment Surfaces Regulate Gingival Fibroblasts Behavior Through Inducing Macrophage Polarization

Background Oral implant restoration has been considered as the preferred treatment for the patient with dentition defects or edentulous dentition since it can well restore the shape,beauty and function of teeth.At present,the 10-year success rate of dental implants is more than 90%.However,there are still some cases of implants which have poor osseointegration after being implanted into the alveolar bone.Peri-implantitis occurs,which leads to poor tissue healing around the implant and failure of the implant.In the past ten years,researches on improving the osteogenesis performance of implants by surface modification mainly focused on the biological response of the implant-osseointegration interface.In recent years,most scholars have paid more and more attention to the regulation of bone immunomodulation in implant osseointegration.When the implant material initially contacts the soft tissue,the immune response caused by the foreign body is a necessary step in the soft tissue defect repair process,and its occurrence,development and outcome are critical to the soft tissue defect repair.Mononuclear-macrophages are the first immune cells that recruit into the defect area after implantation of oral repair materials.Its function is the key to biological materials participating in immune response and regulating tissue repair.In particular,the M1/M2 polarization and inflammatory function of macrophages can affect the outcome of tissue healing,and act as key regulators that determine the prognosis of implants.Therefore,it is necessary to conduct further research about the effects of implant surface on the polarization and inflammatory function of macrophages.In this study,we constructed different nanotopography on the surface of zirconia.Mononuclear-macrophages and gingival fibroblasts were used as the research objects.The effect of zirconia abutment surfaces on the function of macrophages was studied,and the co-culture method was used to clarify the effect of the different nanotopography zirconia abutment surfaces on biological behavior of gingival fibroblasts under the regulation of macrophages.In this study,macrophages were introduced to simulate the immune microenvironment around the implant abutment in the oral cavity.In the in vitro study of soft tissue integration,the consideration of host inflammatory response was added to more accurately predict the cellular response after implant implantation.From the perspective of immune regulation,this study will provide an overview of manipulating the surface optimization design of implant abutment materials.Objective The purpose of this study was to investigate the regulatory effects of different nanotopography of zirconia abutment surfaces on the M1/M2 polarization of macrophages,and the effects of nanotopography of zirconia abutment surfaces alone or together with macrophage secretion on the HGFs’ behavior.Materials and methods The research was divided into the following three parts: 1.Preparation and characterization of nanotopography of zirconia surface.(1)Three kinds of different zirconia abutment surfaces were prepared into discs with a diameter of 15 mm and a thickness of 1 mm.All zirconia materials discs were divided into three groups for experiments: self-glazed zirconia(SGZ),coated self-glazed zirconia(CSGZ)and polished self-glazed zirconia(PSGZ).Scanning Electron Microscope(SEM),Atomic Force Microscope(AFM),Water Contact Angle and Energy Dispersive Spectrometer(EDS)were used to detect the physical and chemical properties of zirconia surface.2.The regulatory effect of different nanotopography of zirconia abutment surfaces on the M1/M2 polarization of macrophages.(1)Macrophage adhesion and proliferation: After the nuclears were stained with DAPI,10 fields of view were taken with a laser confocal microscope to calculate the cell adhesion number.CCK-8 experiment was used to study cell proliferation.(2)RT-PCR was used to detect the expression of macrophage polarization genes TNF-α,i NOS,CD86,IL-10,TGF-β and CD206.(3)Flow cytometry was used to detect the surface markers expression of CD86(M1 type)and CD206(M2 type).(4)ELISA method was used to detect the secretion of inflammatory cytokines TNF-α and IL-10 in the supernatant of macrophage culture.3.The surface nanotopography of different zirconia abutments affected the biological behavior of gingival fibroblasts by inducing the polarization of macrophage(1)Collection of conditioned medium: the supernatant of macrophage culture was collected and configured into conditioned medium(CM).DMEM medium was used as a negative control group.According to the culture medium and zirconia surface,the following experiments will be divided into 6 groups: SGZ,SGZ+CM,CSGZ,CSGZ+CM,PSGZ,PSGZ+CM.(2)CCK-8 method was used to detect the proliferation of HGFs.(3)Confocal laser microscope was used to observe the adhesion morphology and skeleton of HGFs.(4)RT-PCR was used to analyses the relative gene expression levels of collagen-I(COL-I),vinculin(VCL),and fibronectin(FN)of HGFs.(5)Immunofluorescence staining was used to detect the expression of extracellular matrix proteins COL-I,VCL and FN of HGFs.(6)Western blotting was performed to detect the expression of adhesion-related proteins COL-I,VCL and FN of HGFs.Results 1.SEM observed that SGZ surface showed irregular nano-grained structure,and the crystal grains on CSGZ surface were smaller,while PSGZ surface was relatively flat,with fine grooves.AFM showed that the surface roughness was SGZ surface>CSGZ surface>PSGZ surface.The water contact angle was SGZ surface<CSZG surface<PSGZ surface.EDS showed that surfaces of the three groups of zirconia had similar zirconium and oxygen content,with no obvious differences.2.The adhesion and proliferation of macrophages on the surface of CSGZ and PSGZ were higher than those on the surface of SGZ.Macrophages on SGZ,CSGZ and PSGZ surfaces all expressed the M1 and M2 polarization-related genes and surface markers.CSGZ and PSGZ surfaces can promote the secretion of growth factors and inhibit the secretion of inflammatory factors.3.Compared with the conventional culture medium,three zirconia surfaces under the conditioned medium were all conducive to HGFs proliferation.Under the effect of zirconia surface alone,the proliferation and adhesion of HGFs on PSGZ surface were better than that on SGZ and CSGZ(P <0.05).Under the combined modulation of the secretion of macrophages and the zirconia surface,gingival fibroblasts did not undergo obvious apoptosis.The secretion of macrophages could reduce the apoptosis rate of gingival fibroblasts.Compared with the CSGZ+CM and SGZ+CM groups,HGFs in the PSGZ+CM group highly expressed adhesion-related genes and adhesion-related proteins COL-I,VCL and FN.Conclusion(1)Nanocrystalline particles can be formed on the SGZ surface.Nanocrystalline particles on the CSGZ surface were more uniform and smaller.The PSGZ surface was smooth and flat,with small scratches visible.After coating and polishing,the surface roughness and wettability of CSGZ and PSGZ surfaces were reduced,but the surface chemical element composition has not changed significantly.(2)The three kinds of zirconia surface nanotopography can regulate the polarization of macrophages and the secretion of inflammatory cytokines,and all induced macrophages polarized into M1 and M2 types,of which M2 macrophages were the main ones.(3)The biological behavior of HGFs was regulated by the dual factors of zirconia abutment surface morphology and macrophage secretion mediated by zirconia surface topography,and there may be a synergistic effect between them.Three kinds of zirconia abutment surface morphology were shown to promote the biological behavior of HGFs.Among them,the PSGZ surface had the better effect of promoting the biological behavior of HGFs.