| Titanium and titanium alloy are widely used in dental implants for their good physicochemical performance (such as good corrosion resistance, light weight, high ductility and melting point). However, high possibility of peri-implantitis and lack of implant-osseous tissue integration may result in dental implants failure Researches show that:Surface modification can improve the implants success rate. Recently, surface modification researches focus on these points:improving implant-osseous tissue integration by changing the material surface wettability, devising the surface roughness and morphology, coating the surface with material similar to bone tissue; Reducing the incidence of peri-implantitis by modifying the surface with anti-bacterial agent, manufacturing the functional coat as the anti-bacterial elements. However, surface modification method of improving osteocyte proliferation and meanwhile reducing peri-implantitis have not been reported.In this study, femtosecond laser structuring technique was applicated to alter the titanium alloy surface pattern, different types of microstructure formed by changing the laser parameter, and silver doped titanium technique was applicated with the femtosecond laser to alter the alloy surface pattern, to form a antibacterial layer on the implant surface, the antibacterial activity and biocompatibility of different silver doped quality was tested, screened the femtosecond laser energy and the silver doped quality, optimized the best testing parameters, to testify the practicability of the femtosecond laser and the silver doped technique. The purpose is to find a brand new technique to restrain the bacterial adhesion, promote the osteoblast adhesion and proliferation, promote the implant anti-infection of the implant, promote the osteointegration, then provide statistics for the implant success.1Femtosecond laser can induce the taitanium alloy surface periodically structuring.In this study, different laser parameters of femtosecond laser were used to form a three-dimensional topography of different sizes, and observed by scanning electron microscope(SEM), and the X-ray energy dispersive spectrum(EDS) was used to analyse the elemental composition, and accessed the wettability. The titanium alloy disk used in the study was8mm in diameter,1mm in thickness, grinded and polished by biaxial lens grinding machine, and the surface was treated by Ti:sappire laser system which the width of the pulse energy was100fs, the central length was800nm at a1-kHz repetition. The result shows that when the laser power was0.07J/cm periodical stripe-like structure of400nm width was formed on the surface, and when the laser power was1.4J/cm2, a composite structure of micron process and nano-stripe-the processes were even with a diameter of20um, and a secondary structure with a400nm periodical stripes. The sample surface was studied by X-ray energy dispersive spectrum(EDS) and found that the elemental composition did not vary a lot, and the contact-angle of the processed samples was detected by the penent drop method, the contact-angle of the polished samples was50.02°, and the contact-angel of the lased processed sample was7.30, reached suprahyrophilic.2Biological evaluation of the periodical structure on the titanium alloy induceed by the femtosecond laser.2.1Cell adhesion and proliferation testIn this study, osteoblast cell line MG63of human and fibroblast L929of mouse were used and cultured with samples together, scanned by SEM to observe the cells morphology adhered to the material surface and accessed the adherence characteristic and ability. Acridine orange staining was used to count the MG63cells and L929cells adhered to the sample surface. Methylthiazolyldiphenyl-tetrazolium bromide(MTT) was used to test the proliferation ability of the cells adhered to the sample surface. PI single staining flow cytometry was used to analyze the variation of cell cycle. The control group(C-Ti) was titanium disks polished only, low-energy group(LE) was disks processed with0.07J/cm2laser power, high-energy group(HE) was disks process with1.4J/cm2laser power.The results shows:The C-Ti samples surface were smooth without process or stripe, there were polygon-like L929cells which diameter were15-20um tiled to the surface, protruding plate-like pseudopods and lots of tiny microvilli on the margins. The LE samples surface was full of nano-stripe structues, the fusiform-like cells which diameter were20-30um, protruding a small amout of pseudopods and microvilli, the adhering cells protruded on the surface. On the micron-nano complex structure surface of the HE samples, cells were hanging on the side wall with the strong protruding pseudopods. The MG63cells adhered to the C-Ti were spherical, spharse and the single cell morphology was not clear;LE were coverd partially by the adhering cells, HE were covered mostly by cells joined to a membrane sticking to the protrudes, leaving top of protrude apexes exposing.Using MTT to analyze the proliferation ability of the MG63cells and L929cells on the surfaces of C-Ti, LE and HE. The MG63and L929cells were made into5×103cells/ml suspension, cultured for24hours, made sure that had attached to the wall, then used enzyme-labeled instrument to test the absorbance in24h and48h. The result shows that the L929cells absorbance in every group increased over time which means that there was a division growth. At24h, the absorbance of LE and HE were close but lower than C-Ti; At48h,the absorbance of HE was obviously higher than C-Ti and LE (P<0.05), which means that the cell proliferation ability was better in HE than C-Ti and LE. The MG63cells absorbance in every group increased over time which means that there was a division growth. At24h, the absorbance of LE and HE were higher than C-Ti but the difference was not significant(P>0.05); At48h,the absorbance of HE was obviously higher than C-Ti and LE (P<0.05), which means that the cell proliferation ability was better in HE than C-Ti and LE.PI single staining flow cytometry was used to analyze the variation of cell cycle on different surfaces, cultured the C-Ti, LE and HE samples with L929cells for48h, tested with cytometry, a hypodiploid peak appeared, cells of S phase are18.75%in C-Ti,20.44%in LE,25.99%in HE, this manifests that titanium alloy processed by femtosecond laser is able to suppress L929fibroblast apoptosis and promote proliferation. Cultured the C-Ti, LE and HE samples with MG63cells for48h tested with cytometry, hypodiploid peak appeared, cells of S phase are25.83%in C-Ti,19.96%in LE,27.63%in HE, which manifests that titanium alloy processed by femtosecond laser is able to suppress MG63fibroblast apoptosis and promote proliferation.2.2Formation and expression of fibroblast extracellular matrixDetected the expression of type I collagen mRNA with real time RT-PCR, the result shows that LE and HE had an influence on the expression of human osteoblast MG63Coll gene:after LE and HE had effected human osteoblast MG63for24h, the expression of Coll gene increased. After LE and HE had effected human fibroblast L929for24h, the expression of Coll gene did not have a significant change. This reveals that the periodical micron-nano complex structure formed from femtosecond laser in HE is beneficial to the adhesion and proliferation of osteoblast, and can promote the biocompatibility of the titanium alloy surface.3The biocompatibility assess of femtosecond laser structuring and silver doped pure titaniumIn this experiment, vacuum coating equipment was used to evaporate a layer of silver on the samples, the thickness of silver was Onm,25nm,100nm,200nm,300nm,400nm and500nm in different groups, than processed with femtosecond laser which power was1.4J/cm2, a silver doped micron-nano structure formed on the samples, the quality of silver increased while the thickness of silver increased Samples were devided into7groups based on the silver thickness:Ag-Ti-Onm, Ag-Ti-25nm, Ag-Ti-100nm, Ag-Ti-200nm, Ag-Ti-300nm, Ag-Ti-400nm and Ag-Ti-500nm. Assess the biocompatibility with the human osteoblast cell line MG63and mouse fibroblast L929.3.1Cellular poison experimentAnalyze the cellular poison with MTT in group Ag-Ti-Onm, Ag-Ti-25nm, Ag-Ti-100nm, Ag-Ti-200nm, Ag-Ti-300nm, Ag-Ti-400nm and Ag-Ti-500nm. The MG63and L929cells were made into5×103cells/ml suspension, then used enzyme-labeled instrument to test the absorbance in24h,48h and72h with490nm wavelength. Results showed that:For L929, at24h, the absorbance had no significant difference in different groups; At48h, absorbance of group Ag-Ti-25nm, Ag-Ti-100nm, Ag-Ti-200nm, Ag-Ti-300nm, Ag-Ti-400nm and Ag-Ti-500nm were lower than group Ag-Ti-Onm, which means a minor but not significant cellular poison showed up(P>0.05); At72h, the absorbance of Ag-Ti-300nm, Ag-Ti-400nm and Ag-Ti-500nm were lower than group Ag-Ti-Onm, which means that cellular poison, while absorbance of group Ag-Ti-500nm is the highest and the cellular poison is the strongest.For MG63, at24h, the absorbance increased while the silver thickness increased, while group Ag-Ti-400nm and Ag-Ti-500nm were close, significantly higher than other groups (P<0.05); At48h, absorbance of these groups was higher than group Ag-Ti-Onm, which means there was no cellular poison in these groups and the absorbances had no significant difference(P>0.05); At72h, the absorbances had no significant difference(P>0.05), Ag-Ti-400nm and Ag-Ti-500nm were lower than group Ag-Ti-Onm, which means that cellular poison, while absorbance of group Ag-Ti-500nm is the highest and the cellular poison is the strongest.3.2Experiment of cell adhesion and proliferationCultured MG63and L929with the samples in each group for48h then detect the adhered cell morphology with SEM. Under low power microscope, L929covered the micron protrudes on the material with a membrane-like shape, the cell body protruded, however, under high power microscope, cells link to each other with pseudopods but had few links to the material protrudes; Under low power microscope, MG63covered the micron protrudes on the material with a membrane-like shape, leaving the protrudes apexes out of the membrane. Under high power microscope, the fusiform-like cells protruding lots of strong pseudopods with which the cells adhered to the micron protrudes.Test the quality of the adhered cells with acridine orange staining method on the material surface, under low power microscope, L929spreaded on the surface evenly, while cells of group Ag-Ti-500nm were obviously less than others, and under high power microscope, spherical cells could be observed. MG63spreaded on the surface evenly, adhered cells quantity were close in these groups, and under high power microscope,fusiform cells could be observed.Using MTT to test the cell proliferation, the absorbance of L929at24h,48h and72showed that:the absorbance in24h,48h and72h with490nm wavelength. Results showed that:At24h, the proliferation of group Ag-Ti-200nm and Ag-Ti-300nm were higher than other groups, and the absorbance of group Ag-Ti-25nm and Ag-Ti-500nm were close and weaker than others. At48h, the absorbance of group Ag-Ti-Onm, Ag-Ti-25nm and Ag-Ti-100nm were higher than that of24h, which means that cells in these groups have the proliferation ability, while the absorbance of group Ag-Ti-200nm, Ag-Ti-300nm, Ag-Ti-400nm and Ag-Ti-500nm were lower than that of24h(P>0.05) which means the proliferation ability was inhibited. At72h, the absorbance of each group was higher than48h, which means the cells have the proliferation ability and the absorbance has no significant difference h(P>0.05).Tthe absorbance,of L929at24h,48h and72showed that:At24h, the absorbance of group Ag-Ti-400nm and Ag-Ti-500nm were close and significantly higher than other groups (P<0.05) which means strong proliferation ability. At48h, the absorbance of each group was higher than24h, which means the proliferation ability was strong. At72h, the absorbance of each group has no significant difference(P>0.05), while the absorbance of group Ag-Ti-25nm, Ag-Ti-400nm and Ag-Ti-500nm were lower than that of48h which means the proliferation ability was inhibited. however, the absorbance of group Ag-Ti-Onm, Ag-Ti-100nm, Ag-Ti-100nm and Ag-Ti-500nm were higer than48h which means the cells have the proliferation ability, the absorbance of Ag-Ti-100nm was the highest means that the cells in this group have the strongest proliferation ability.4Experiment of the antibacterial ability of femtosecond laser structuring and silver doped pure titaniumAfter applied femtosecond laser structuring and silver doped step, inoculated Porphyromonas gingivalis onto the titanium samples in these groups, detestes the bacterial morphology with SEM, tested the quantity and morphology of the bacteria colonies on the culture medium, to discuss the influence on the adherence and morphology of Porphyromonas gingivalis. The result shows:the bacteria colonies were more in group Ag-Ti-Onm spreading all over the culture medium and the bacteria morphololy was small; In Ag-Ti-25nm, group Ag-Ti-100nm, Ag-Ti-200nm and Ag-Ti-300nm, the quantity of bacteria colony decreased while the silver thickness increased, while single colony became bigger since the silver thickness increased; Bacterial colony quantity in group Ag-Ti-400nm was less than other groups, while there are barely no bacteria colony in group Ag-Ti-500nm.In conclusion, femtosecond laser can form periodical morphology in different sizes on the titanium surface. The biocompatibility of micron-nano complex structure is better; Applying silver doped technique with femtosecond laser can modify the material morphology while the chemical characteristic is also changed, the antibacterial ability increases while the silver thickness increases, proper silver thickness will promote cell adherence and proliferation, as a result, the material biocompatibility will be promoted. |
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