Nanotubular Titanium Surface Mediate Osteogenesis And Angiogenisis By Modulating The Osteoimmune Response Of Macrophages

Objective:Anodically oxidation was used to modify the commercially pure titanium(cp Ti)surfaces to fabricate titania nanotubes.The behavior of macrophages on the titanium surfaces was evaluated,and their conditioned medium(CM)was collected to stimulate MC3T3 and HUVECs in vitro symbolizing the surrounding microenvironmentMaterials and methods:1.Taking cp Ti as substrates,anodic oxidation was carried out for 30 minutes at 10V(NT 10)and 20V(NT20)respectively to fabricate different dimesional nanotubular surface topography2.The surface morphology,chemical composition and crystal structure of the surfaces were characterized by field-emission scanning electron microscopy(FE-SEM),atomic force microscopy(AFM),X-ray diffractometer(XRD)and X-ray photoelectron spectroscopy(XPS).The surface wettability was examined using a surface—contact angle machine and the protein asorption was measured3.The murine-derived macrophage cell line RAW 264.7 cells were used to evaluate cell bioactivity including cell adhesion,spreading and proliferation.The macrophage polarizaiton was examined by real-time PCR(IL-1p,TNF-a,INOS,IL-10 and ARG)and immunofluorescence.The cell morphology was detected by FE-SEM and cytoskeleton staining.The AMPK protein in macrophages was detected by Western Blot4.The osteogenic capacity of mouse preosteoblast MC3T3 in CM from macrophages on different surfaces was investegated by real-time PCR(OPN,OCN and COLX),ALP staining and ALP semi-quantification5.The angiogenic capacity of human umbilical vein endothelial cells(HUVECs)in CM from macrophages on different surfaces was detected by wound healing assay,tube formation and real-time PCR(vWF and SDF)Results:1.Different dimensional nanotubular surface topography with high hydrophilicity was formed on Ti samples after anodization.The dimension of nanotubes was 30-40nm(NT 10)and 70-80nm(NT20)respectively.The surface roughness decreased and the hydrophilicity increased on nanotubular topography.The protein adsorption capacity is NT20>NT10>cp Ti2.The adhesion and proliferation ability of macrophage on nanotubular surfaces were reduced compared to cp Ti.NT 20 induced macrophage activation similar to the anti-inflammatory M2-like state with spindle shape while NT 10 was associated with Ml phenotype in oval shape,and the expression of AMPK and p-AMPK protein increased with the increase of nanotube diameter3.The osteogenic capacity of MC3T3 to CM from NT 20 was enhanced4.CM from both nanotubular surfaces promote tube formation capacity of HUEVCs with increasing tube size(NT 10<NT 20)Conclusion:1.A uniform nanotubular surfaces with high hydrophilicity were fabricated using anodic oxidation at 10V and 20V for 30 minutes2.Nanotubular surfaces with large dimension(NT20,70nm)were benificial to the local microenvironment for tissue repairing and macrophages on NT20 promote osteogenesis and angiogenisis in vitro3.Anodic oxidation,a potentially excellent dental implant fabrication method,is low-cost,effective and relatively simple/facile to produce nanotubular surfaces which perform well in the biocompatibility and the capacity to control the local inflammatory microenvironment.