Effect Of Diameter-controlled Ti-TiO₂Nanotubes On The Biological Behavior Of Fibroblast And Osteoblast

Diameter-controlled TiO₂nanotubes formed on Ti surface by anodization atdifferent potentials could simulate nano-size environment cell natural in vivo，with potentials of loading medicine and biotic factor and so on. Early studies,both in vitro and in vivo, have shown that TiO₂nanotubes promoted implantosseointegration. As a special through oral mucosal structure, dental implant hastwo distinct interfaces----implant-soft tissue interface and implant-bone tissueinterface. However, few studies have yet examined the effect of TiO₂nanotubeson implant-soft tissue interface. Numerous studies have indicated that cells weresensitive to nano-size and that different type of cells responded differently toidentical size of nano-topography. However, the size of TiO₂nanotubes that ismore favorable for the tow most common cells around dental implant-fibroblastand osteoblast-respectively, and possible mechanisms of TiO₂nanotubes affecting on them are still not clear. Diameter-controlled TiO₂nanotube arraywere formed on the polished pure titanium using anodization, and their effect onthe biological behavior of osteoblast and fibroblast were studied to provide atheoretical basis for screening of a nanotube-like structure that was suitable to thedifferent parts of the dental implant.The polished pure titanium was anodized at different anodizing voltage1、5、10and20V; the surface topography of samples was observed using Fieldemission Scanning electron microscope （FE-SEM）; Contact angle measurementswere performed by an contact angle measuring system and surface energy werecalculated using the Acid-Base method; Fibroblast and osteoblast adhesion wasassessed by the method of staining and counting with fuorescence microscope;cell spreading morphology was observed using FE-SEM; cell proliferation wasassessed using3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay; Collagen production of fibroblasts was assayed by Alizarin Redstaining and Alkaline phosphatase （ALP） activity of osteoblasts was assessedusing commercial kits.The results showed that:1. After treatment of anodization, a nanonet-like topography was formedat1V and the average diameter of the pores was about15nm; a nanotubulartopography was formed at5、10and20V and the average diameter of the hollownanotubes was about30、70and100nm respectively.2. The contact angle results obtained with deionized water、 formamideand diiodomethane. The contact angles of liquid on TiO₂nanotube surface weresmaller than the polished surface, while the surface energy of samples showedno significant difference.3. It was found that all anodized surfaces presented a significantly inhibition fibroblast adhesion and proliferation compared with polishedsurfaces（p <0.05）, and significantly lower cell adhesion and proliferation wereobserved on the30nm TiO₂nanotube surface formed at5V compared with otheranodized surfaces（p <0.05）.4. After1h culture, the fibroblasts were cylindrical and spherical, not fullystretched on anodized surfaces and polished surfaces. Then up to2h, the cellpresented typical spindle-shaped on polished surface, well spreading withobvious lamellopodias on nanotubes formed at1V, spherical with shortlamellopodias and fillopodias on nanotubes formed at5V and longspindle-shaped spreading out a large amount of varying lengths fillopodias onnanotubes formed at20V, less spreading compared with polished surface.5. Anodized surfaces promoted the collagen production of fibroblastcompared with polished surfaces, and nanotubes of larger diameter presented astronger promotion.6. All anodized surfaces presented a significantly enhancement ofosteoblast adhesion and proliferation compared with polished surfaces（p <0.05）,a significantly better cell adhesion and proliferation were observed on the30nmTiO₂nanotube surface formed at5V compared with any other anodizedsurfaces（p <0.05）.7. After1h culture, the osteoblasts presented lamellopodias on polishedsurfaces and nanotube surfaces anodized at1V and5V, while a few fillopodiason nanotube surfaces anodized at10V and20V. Then up to2h, the cell spreadfurther and presented differently; the osteoblasts spread well with obviouslamellopodias on polished surfaces and nanotube surfaces anodized at1V and5V; the cell polarized with the ends of Long axis spreading out a large amount ofshort fillopodias on nanotubes formed at10V and20V. 8. All anodized surfaces promoted ALP activity of osteoblast comparedwith polished surfaces, and the larger diameter nanotubes presented a strongerpromotion.Conclusion:Uniform TiO₂nanotubes arrays were formed on Ti surface by anodizationand higher the anodizing potential, larger the TiO₂nanotubes diameter;compared with the polished surfaces, all anodized surfaces presented inhibitionfibroblast adhesion、spreading and proliferation, while the30nm TiO₂nanotubesurface formed at5V had the most inhibition; nanotubes surfaces presented asignificantly enhancement of osteoblast adhesion and proliferation comparedwith polished surfaces, and the adhesion and proliferation were best on the30nm TiO₂nanotube surface formed at5V; small nanotubes of15and30nmpromoted osteoblast spreading while larger nanotubes of70and100nminhabited the preading; the anodized nanotubes surfaces promoted the respectivefunction differentiation of fibroblasts and osteoblasts, and the nanotubessurfaces with a diameter of100nm presented the most promotion.