| BackgrondThe pure titanium is widely used in the field of dental implantology due to its good physical and mechanical properties and highly biocompatibility.Implant denture as human the third vice teeth is widely accepted in recent years for its beauty, comfort and can be a very good recovery on chewing function. Dental implant will face more challenges under the development of social aging. A large number of scholars became to strive for a deep rearch on the surface modification of dental implant since the Swedish scientists Branemark put forward the concept of osseointegration in1969.Micro-arc oxidation (MAO)is the dischage of anodic oxidation, a new anodic oxidation technique which can produce ceremic coating on the metal surface also is called micro-plasma oxidation(MPO). MAO technique became a hot spot on the surface modification of pure titanium in1990’s. Rencent years, the great biocompatibility and bone conduction of MAO technique has been well approved in the surface treatment of dental implant, which is favourable for the osteoblast adhere to the surface, and thus the MAO surfaces have a better ossteointegration ability than machined ones.Currently, According to reports in the literature, total osseointegrated implants area covered by bone remains (45±16)%at early stage, at most50%-75%. However, the BIC (bone-titanium contact percentage) of dental implant after MAO treatment are only (49±12)%,that are far below the idea100%. Thus, the bioactivity of implant surface using MAO technique still has a large room to be further improved for successful rate.In1972, light-induced titanium dioxide(TiO2) in the photovoltaic cell can cause photocatalysis which was reported by Fujishima. The light-induced superhydrophilicity of TiO2oxidation film was discovered in1997by Wang. The light-generation of a highly hydrophilic TiO2oxidation film is due to the altered surface structure of the hydrophilic phase caused by the light treatment. Two mechanisms have been proposed currently, one is UV treatment can alter the structure of surface TiO2by creating surface oxygen vacancies at bridging sites, resulting in the conversion of Ti4+sites to Ti3+sites, which are beneficial for adsorption of dissociative water riched in amount of active hydroxyl radicals. And the other mechanism is the UV light-mediated photofunctionalization of TiO2can decontaminate the hydrocarbon of surface, which enhanced wetting. The hydrophilicity of material surface is representative of the sign of surface energy, most of scholars believe that increasing the material surface hydrophilic would increase the surface energy, and a certain degree of protein adsorption ability to be increased, which is good for the further interaction between cells and matarials. The phenomenon of photofunctionalization of TiO2is commonly regarded as:TiO2is a semiconductor materials and its forbidden band is3.2ev, according to λR(nm)=1240/E8(ev), when the light energy greater than3.2ev is needed to induce TiO2photocatalytic activity,which corresponds to approximately the wavelenth range of Ultraviolet(UV), which matches to360nm wavelength of UVA, meanwhile UVC light around250nm wavelengthis also of interest.The superhydrophilicity was generated after UVC treatment on acid-etched surface have been reported, which enhanced attachment, proliferation and differentiation of osteoblast, the BIC was improved range from51.7%to98.2%after implantation at4weeks in the animal experiment, while there is of little investigation of UVA as a surface treatment. However, there is no research on UVA and UVC as a treatment on micro-arc oxidation titanium surfaces respectively. according to λR(nm)=1240/E8(ev),the shorter wavelength, the higher energy, and thus we hypothesized that UVC light-treated micro-arc oxidation titanium surface can generate stronger photocatalysis and bioactivity than UVA. To explore a new implant surface treatment method, we investigated the effects of different wavelength UV light-treated MAO titanium surfaces on the change of physiochemical properties and early cellular behaviors.ObjectiveTo investigate the UVA and UVC light-mediated photofunctionalization of micro-arc oxidation titanium surface on the effect of early biological behaviors of osteoblast (MG-63)Methods1.Pure grade-4titanium disks (15mm diametre) were cut from commercially pure titanium rods,ground with250#,400#,600#and1000#abrasive papers to remove rough scratches, then ultrasonically cheaned with acetone, alcohol and distilled water. According to the MAO processing reported in previous work, titanium discs were treated in an aqueous electrolyte containing calcium acetate and glycerophosphate disodium salt at a low temperature. Processing parameters such as: the applied voltage of350V, frequency of800HZ,duty cycle of20%for30s, then ultrasonically cleaned with distilled water and sterilized with Y ray fou use. Some MAO samples were irradiated by15-W UVC bactericidal lamp while others were irradiated by15-W UVA mercury lap for24h under sterile environment, a distance of10cm between samples and light sources. Without light-irradiated samples were controll groups MAO, while UVA irradiated samples were test groups MAO+UVA, UVC irradiated samples were test groups MAO+UVC. The surface hydrophilicity of samples were measured by an automatic OCA contact angle measuring device at the contact angle of lμl H2O, the surface morphology and surface elemental composition was evaluated using scanning electron microscopy (SEM) with energy spectrum analysis system, the surface crystalline phase was measured using X-ray diffraction (XRD),the surface roughness was evaluated using surface morphology instrument and the surface elements chemical state was measured by using X-ray photoelectron spectroscopy (XPS).2.The photocatalysis of micro-arc oxidation titanium surface light-induced by UVA and UVC ultraviolet was detected by degradation of methylene blue. The induced ability of generating apatite on the samples surface was evaluated by immersing samples in the stimulated body fluid after UVA and UVC ultraviolet light-treated.3. The amount of protein on the surface of samples were quantified using protein adsorption test after UVA and UVC ultraviolet light-treated; the attachment and stretch of MG-63cells on the samples were observed using fluorescent staining and scanning electron microscopy after UVA and UVC ultraviolet light-treated, the activity of cellular proliferation and Alkaine phosphatase (ALP) were detected with MTS,ALP kit,respectively.4. The number of samples were3for the contact angles test and cell attachment studies, each sample divided into3regions for contact angle measurement, while4fields for cell attachment test. The test of degradation of methylene blue, protein adsorption test and other cell studies which were evaluated in6samples, data is discribed as x±s, one-way ANOVA was used for statistical analysis with software SPSS13.0, P<0.05was considered statistically significant.Results1.There are no significant differences among the surface morphology, elemental composition, phase component and surface roughness of three groups of samples, all the pores on the surface like honeycomb and be well separated and distributed, different diameters vary from1-10μm. Exclude the unstability of C element, the main elements on the surface are Ti、O、Ca、P, while Ti、O elements count for the most, the total amount of Ca、P elements are approximate8%-15%, the atomic ratio of Ca/P is1.4-1.6. All the MAO film are composed of anatase, rutile and a large mount of titanium substrate. XPS spetra revealed that the groups of C-C and C-H markedly decreased after UVA and UVC treatment respectively, while the group of Ti-OH increased. The contact angle test showed that the surface hydrophilicity of MAO coating were more conspicuously improved after UVA and UVC treatment than the unlight-treated MAO surface, and the results are statistically significant (F=1431.521,P=0.000)2. All the MAO samples have the ability of degradation of methylene blue after UVA and UVC treatment respectively, and the value of OD (optical density) decreased over time, however, the group of MAO+UVC has a stronger ability than the group of MAO+UVA in degrating methylene blue. In the test of induced apatite formation, there are only a small amount of apatite formation on the surface of MAO group, while the amount of apatite on the surface of MAO+UVA and MAO+UVC groups have an increased trend over3weeks, and MAO+UVC group has the most amount.3. The protein adsorption rates of each group surface as follows:the group of MAO+UVC mounted to maximum value(45.50±1.63)%while the group of MAO and MAO+UVA were less than10%after2h incubation; both the group of MAO and MAO+UVA reached the peak value after6h incubation, but the protein adsorption rate was still less than20%, while the group of MAO+UVC maintained at a platform level after2h;the protein adsorption rate of MAO+UVC group was higher than the group of MAO and MAO+UVA in each period time, which are statistically significant(P<0.05),MAO group and MAO+UVA group have no statistically significant (P>0.05). The cellular morphology on each group surface observed using SEM as follows:three groups of cells had begun to adhere to all the surfaces after1h incubation, there were a small amount of dumpy pseudopodiums of MAO+UVC group cells stretched out around. All the groups of surface cells had many pseudopodiums extended around after4h incubation, some of which stretched deeply into surface pores, and formed mechanical lock with surface.While the cells on the surface of MAO+UVC group had a large number of sturdy pseudopodiums, and the cells just looked like polygons. The cells of group MAO and MAO+UVA were a thin layer on the material surface, while the cells of group MAO+UVC intergrated with MAO coating as a whole, without apparent boundary. The cellular attachment rate was increased over time within4h among three groups, MAO+UVC group was greater than the group of MAO and MAO+UVA in each period time, which are statistically significant (P<0.05), while MAO group and MAO+UVA group have no statistically significant (P>0.05). The effects of UV light-treated MAO surfaces on MG63cells proliferation:the OD values increased over time within5d among three groups, MAO+UVC group was higher than the MAO and MAO+UVA groups in each period time, which are statistically significant (P<0.05), while MAO group and MAO+UVA group have no statistically significant (P>0.05). The effects of UV light-treated MAO surfaces on MG63cells ALP activity as follows:the OD values increased over time within7d among three groups, but decreased on day14, MAO+UVC group was greater than the MAO and MAO+UVA groups at7and14days, which are statistically significant (P<0.05),while have no statistically significant on day3. MAO group and MAO+UVA group have no statistically significant in each period time (P>0.05)Conclusion1. Both UVA and UVC wavelength ultraviolet can enhance surface hydrophilicity and surface energy, and reduce the surface hydrocarbons on MAO film,while the amount of Ti-OH increased, especially on MAO coating after UVC treatment. However, the surface topography, roughness, elemental composition and TiO2crystal form did not changed after two wavelength ultraviolet treatment respectively.2. Both UVA and UVC wavelength ultraviolet light-treated MAO surface can cause degradation of methylene blue, and MAO+UVC group has a more stronger ability of degradation. The bioactivity was enhanced both in the group of MAO+UVC and MAO+UVA, while MAO surface after UVC treatment is more advantageous for apatite formation.3. Treatment of MAO surface with UVC ultraviolet conspicuously improved affinity for protein adsorption and MG63cellular attachment, stretch, proliferation and ALP activity than UVA ultraviolet. |
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