Nanostructured Titanium Surface Improves Implant Osseointegration By Mediating Macrophage Polarization

Dental implant has become an important tactics to restore missing tooth anddentition defect. However, implant failure occurs due to the delayed bone formationaround implant and impaired bone-implant interface. Hence, the rapid bone formation andstable bone-implant integration is the main target of implantable materials designing.Former biological researches focused on the osteogenic differentiation of osteogenesisrelated cells on micro/nano-modified implant surface. With the development of theresearch on tissue response after implantation, the host innate inflammation, especially theM1/M2polarization and inflammatory secretion of macrophages, has been recognized toplay the key role of regulating the bone formation and the balance of osteogenesis/osteolysis.Therefore, it is necessary to deeply clarify how nano-textured surfacetopography influence the inflammatory functions of macrophages. In this research, weconstruct super-hydrophilic TiO2nanotubular surface topography(NT) with differenttube size by anodization and UVA/C irradiation compound methods. Human macrophageswere seeded on the Ti samples and the inflammatory functions of macrophages wereassessed. Additionally, the co-culture system was built to clarify the interaction betweenmacrophages and bMSCs. Finally, Ti implants with different surface nano-topographies were implanted in rats. The inflammation and bone formation around implants wereobserved and analyzed. Our study focuses on the M1/M2polarization of macrophages andmakes the in vitro research model of bone-implant integration closer to the environment invivo. Thereby, the performance of implants can be predicted more precisely, and thedesign of implantable materials will be optimized from the perspective of modulating hostinflammation.Part I Fabrication and characterization of super-hydrophilic TiO2nanotubular surface topography on titanium surface[Objective] To fabricate TiO2nanotubular surface topography on Ti samples for followingbiological experiments[Methods] After polishing, pure titanium samples were anodized with DC power supply at5V and20V for45min to fabricate nanotubular surface topography. The anodized sampleswere treated with UVA/C irradiation. The morphology of Ti samples was inspected byfield-emission scanning electron microscopy(FE-SEM). The surface profile and roughnessof Ti samples were measured by atom force microscopy(AFM). The protein adsorption ofsamples were also detected by eluent assay.[Results] Super-hydrophilic nanotubular surface topography was formed on Ti samplesafter anodization and UV irradiation. The average diameter of nanotubes was30nm(NT5)and80nm(NT20) respectively according to different anodization voltages.[Conclusions] Super-hydrophilic TiO2nanotubular topographies with different diameterscan be fabricated on Ti samples by anodization and UV irradiation. Part II The effects of TiO2nanotubular surface topography on themorphological and survival behaviors of human monocyte-macrophage[Objectives] To evaluate the influence of TiO2nanotubular surface topography on the attachment, morphology and apoptosis behaviors of monocytes-macrophages.[Methods] Human monocytes were isolated from peripheral blood by gradientcentrifugation and purified by rapid adhesion methods. Monocytes were seeded on Tisamples. Laser confocal scanning microscopy(LCSM) and FE-SEM were employed toobserve the adhesion and morphology of macrophages. The flow cytometry was used todetect the cell apoptosis.[Results] Monocytes-macrophages were obviously repulsed by nanotubular surfaces andNT5surface showed the lowest cell adhesion. NT5induced macrophages spreading tooval-like shape and NT20compel the macrophage stretching to spindle-like shape. Theapoptosis of macrophages were not affected by surface nano-topographies.[Conclusions] The attachment and morphology of macrophages were affected bynanotubular surface topographies with different tube size. Cell apoptosis is independent ofsurface topography of Ti samples. Part III The effects of TiO2nanotubular surface topography on theM1/M2polarization of human monocyte-macrophage[Objective] To detect the influence of TiO2nanotubular surface topography on the M1/M2polarization and inflammatory factors secretion of monocytes-macrophages, and to clarifywhether the detached macrophages retain their pro-inflammatory functions[Methods] Fresh isolated monocytes-macrophages were seeded on Ti samples. Flowcytometry was used to identify the M1/M2polarization of macrophages with membraneand intracellular markers. Real-time PCR was employed to detect theinflammation-related genes expression of macrophages adherent or detached respectively.[Results] NT5surface topography induced the M2polarization of macrophages andinhibited the secretion of inflammatory mediators. NT20surface topography induced the M1polarization and enhanced the secretion of pro-inflammatory factors. The polarizationof macrophages was induced directly by surface nano-topographies of Ti samples ratherthan response to cytokines produced by macrophages themselves. Adherent macrophagesshowed high level of IL-1β、 IL-6and IL8mRNA expression whereas thesepro-inflammatory genes expression of detached macrophages were hardly detected.[Conclusions] Ti samples directly control the polarization of macrophages and thesecretion of inflammatory factors. Macrophages detached from the surface of Ti sampleswill lose their functions of inflammatory secretion. Part IV TiO2nanotubular surface topography regulating bone-implantintegration by mediating macrophage polarization in vitro/vivo[Objective] To detect the interaction between macrophages and bMSCs on differentnano-structured surfaces. To evaluate and analyze the bone formation around Ti implantswith different surface topographies.[Methods] Built a conditioned medium co-culture system model. Seeded primary humanbMSCs on transwell, culture plate and Ti samples with different surface topographies. Themigration, proliferation and osteogenic differentiation of bMSCs were detected. Thefeedback regulation of bMSCs in conditioned mediums against macrophage osteoclastdifferentiation through OPG/RANKL and M-CSF secretion was also evaluated.[Results] Both nanotubular surfaces induced macrophage conditioned medium couldenhance the homing, proliferation and ostegenic differentiation of bMSCs. bMSCsinhibited osteoclast differentiation in NT5induced conditioned medium whereas enhancedosteoclast differentiation in NT20induced conditioned medium. NT5implant showedoptimized bone formation and the lowest inflammation infiltration around implant. Thebone formation around NT20implant was partly impaired by the enhanced inflammation elicited by NT20surface topography.[Conclusions] The interaction between macrophages and bMSCs through paracrinepathway plays the key role in the formation of bone-implant integration.