The Investigation Of Different Electrical Parameters On The Impact Of Micro-arc Of Titanium Surface

BackgroundTitanium and titanium alloy is the most widely used material in the medical field of Oral Implantology with stable chemical properties, excellent biocompatibility and mechanical performance, but also has problems of low strength and bone healing for a long time, which has a large impart on the clinical success rate of implanting. In recent years, with the rapid development of Oral Implantology, scholars all means to surface modification of pure titanium, in order to obtain a good implant-bone interface to enhance bone bonding strength, has also become research focus today.Micro-arc oxidation has been applied to the implant surface treatment, are a kind of the anodic oxidation, which breaking the limit of the anodizing voltage, directly generate ceramic layer in situ of non-ferrous metal surface. After treatment, the Titanium substrate not only improves the resistance, but also generates uniform porous in the surface, contains elements of calcium and phosphorus, which conducive to the induction of osteogenesis and significantly improves the biocompatibility of titanium implants and bone conduction.But micro-arc oxidation reaction system is complex, not yet unified electrical parameters in the processing of titanium implants. Biological materials for bone tissue engineering requires not only the surface composition, structure,morphology, and hydrophobic characteristics can impact on the material-bone,also could attract the cell adhesions on the material, may further the proliferation, migration, differentiation. Cell adhesions to the surface of material which play an important role for bone tissue regeneration and repair of the implants. However, because of the short timescale, the surface properties of implant materials on micro-arc oxidation is not deep enough.At present, the known electrical parameters, which contains of voltage, current density, duty cycle, frequency, reaction time, electrolyte concentration, etc. are on the impact of micro-arc oxidation. In this experiment,we investigated the impact on the micro-arc of titanium surface with different electrical parameters, which included voltage, duty cycle, electrolyte concentration,further discussed osteoblast adhesion, prolifertical, differentiation on the titanium surface which are treatment by some electrical parameters, in order to seek the law of micro-arc oxidation, and provide theoretical support foe optimization the surface treatment technology.Purpose1. Preliminary study:ompared the impact of different duty cycle, voltage, electrolyte concentration on the micro-arc oxidation of titanium surface characteristics, in order to generate a better electrical parameters which could get a performance coating on the surface of the micro-arc oxidized titanium.2. According to the preliminary findings, selected the appropriate duty cycle, voltage, and the formation of micro-arc oxidation of titanium in different electrolyte concentration, and further study of its film properties, as compared to human osteosarcoma cell line MG-63adhesion, proliferation,the different effects of differentiation, provide a theoretical basis for the optimization of micro-arc oxidation implant processing technology.MethodsMaterial preparation:Machining the commercially pure titanium into diameter of15mm, thickness1.2mm titanium wafer specimen, gradually polished using200and800silicon carbide sandpaper, respectively, in acetone, ethanol, deionized water ultrasonic swing wash for10min.1. Different duty cycle, voltage, electrolyte concentration on the impact of micro-arc oxidation of titanium surface, (1) Group1) The duty cycle of groups:the duty cycle of5%,25%,45%,65%under the conditions of microarc oxidation treatment of titanium plates, the remaining processing factors constant. The obtained titanium film in turn corresponds to a, b, c, d four groups;2) The voltage:voltage250V,300V,350V,400V conditions on the titanium plate micro-arc oxidation treatment, the remaining processing factors constant. Income corresponding to the titanium plate as a, b, c, d four groups;3) Electrolyte concentrations:in the electrolytes calcium and phosphorus concentrations were0.12,0.16,0.20the conditions on the titanium micro-arc oxidation treatment, the remaining processing factors constant. Income corresponding to the titanium plate for a, b, c.(2)Methods:Pure titanium disks are treated by MAO in accordance with the duty cycle, voltage, electrolyte concentration.The surface of the samples was observed and analyzed by scanning electron microscope (SEM), energy dispersive spectrum(EDS) and X-ray diffraction(XRD).2. To study different electrolyte concentrations of micro-arc oxidation of titanium properties, and its effect on human osteosarcoma cells MG-63shape, adhesion, proliferation and differentiation.(1) GroupPure titanium disks are treated by MAO in accordance with the different electrolyte concentration., Make the smooth titanium as A, define the other groups by the calcium and phosphorus molar ratio,group B is0.12,groupC is0.16, group D is0.20.(2)Methods:l)The surface characteristics were determined by light interferometer and OCA optical contact angle meter.2)After human osteoblastic sarcoma cellsMG63were cultured on disks after1h,3h and6h,SEM and laser confocal microscopy (CLSM) were used to observe the morphology of cells and F-actin architectonic change. The cell adhesion amount were counted to compare the adhesion rate.3) After human osteoblastic sarcoma cells MG63were cultured on disks after1d,4d and7d,MTS measured the proliferation by OD values. ALP activities were measured by P-Nitrophenyl phosphate Tablets after cells were cultured on disks after7d and14d.4)Each time-period above each group has six samples. Staistical analysis software is SPSS13.0.One way-ANOVA and Factorial analysis of variance were usedRESULT1. Different duty cycle, voltage, electrolyte concentration, processing micro-arc oxidation of titanium surface rough and porous as fire mountain pass like; containing Ca, P,O,Ti four elements; the crystalline structure of rutile TiO2and anatase TiO2.(1)Duty cycle1) a group: The surface pore size of about1～3μm size; calcium and phosphorus element content ratio of0.91; crystalline phase anatase TiO2, no rutile TiO2;2) b group:Surface pore size is about1～5μm size; calcium and phosphorus element content ratio1.18; crystalline phase of anatase TiO2, we can see a small amount of rutile TiO2;3) c group:The surface pore size is about1～8μm size, the microporous density decreases, the visible part of the region of cracks; calcium and phosphorus element content ratio of1.25;4) d group:Surface pore size of about1～10μm size, reduce the of microporous number in unit area, showing obvious surface cracks; calcium and phosphorus element content ratio was1.46; crystalline phase is mainly.2) voltage:1) a group:Surface pore size of about1～3μm size; calcium and phosphorus element content ratio1.14; crystalline phase containsof anatase TiO2, no rutile TiO2;2) b group:surface pore size of about1～5μm size; calcium and phosphorus element content ratio1.18; crystalline phase anatase TiO2, we can see a small amount of rutile peaks;3) c group:surface pore size of about1～5μm size, porous density decreases, the surface region of crack generation; calcium and phosphorus element content ratio1.23; crystalline phase of anatase TiO2, rutile T1O2increased significantly;4) d group:surface pore size of about1～1Oμm size, microporous reduction in the number of unit area, showing obvious surface cracks; calcium and phosphorus element content ratio of1.47; Increase in rutile TiO2.(3) Electrolyte concentration:1) a group::surface pore size is about1～5μm size; calcium and phosphorus element content ratio was1.17; crystalline phase of anatase TiO2, we can see a small amount of rutile TiO2;2) b group:surface pore size of about1～8μm size; calcium and phosphorus element content ratio of1.53; crystalline phase of anatase TiO2, visible part of the rutile TiO2;3) c group: surface pore size of about1～10μm size of the porous density decreases, the surface cracks; calcium and phosphorus element content ratio was1.77; crystalline phase contains of anatase TiO2and rutile TiO2.2. Different electrolyte concentrations of micro-arc oxidation of titanium properties, and its effect on human osteosarcoma cells MG-63shape, adhesion, proliferation and differentiation.(1) The surface of the four groups was determined by light interferometer, which shows different characteristics. The surface of group A shows more uniform, the height in B、C、D group shows different in varying degrees. The roughness test results shows Ra values:A<B<C<D; the water contact angle shows: A、C、D>B, the difference between C、D groups was not significant.(2)Cell morphology and adhesion rate is calculated as follows:1) SEM:with time increased the actins bundles appear to increase in the cell surface, at24h, cells spread to covered a few holes of the titanium surface; the cells tile in the group A; in the other three groups, the cells stretched out peseudopods to attached the edge of the holes in the surface, even extends into the holes. 2) CLSM:At1h, the cells’F-actin became in a ring wrapped the nucleus in all groups; the actin bundles wheel-like.The cell shows the long spindle in group A, but shows typical polygon in the other groups.3) Cell adhesion rate:The surface of cell adhesion rate all raise with time increased. At1h, groupA is lower than the other three groups, which means the rougher surface are more prone to cell adhesion in the early.At3h, the C, D group are higher than A group. At6h, among the four groups, there are on significant difference by analysis of variance.(3) Compare four groups of MTS OD values as cell proliferation level.1) with time increased, proliferation activity increased. At1d, there are on significant difference among A,C,D groups by analysis of variance., group B was the highest. At4d, group A is lower than C,D, there are on significant difference between C,D. At7d, group D was the highest.2) Compare four groups of ALP activity, at7d,there are statistically significant among the four groups, and group D was the highest, with time increased, differentiation activity increased. The results shows that, at14d,the C、D group which has higher roughness is still has higher differentiation than A、B group.Conclusion1. With the duty cycle, voltage and electrolyte concentration increased, the pore size increased in the titanium surface, but the number of the pore decreased. film surface Ca/P ratio increases; film rutile TiO2peaks gradually increase.(1) When the duty cycle is about25%, titanium surface did not crack, and at the same time containing rutile TiO2anatase TiO2can be used as a more appropriate electrical parameters.(2) When voltage reached about300V, the titanium plate oxide Ca/P ratios close to the high-voltage group c, and the surface and no cracks, containing both rutile TiO2and anatase TiO2, can be used as a more appropriate electrical parameters.(3) When the calcium and phosphorus electrolytes concentration ratio of0.16, calcium and phosphorus ratio greater surface and no obvious cracks, increase in rutile TiO2can be used as experimental reference. 2. Different electrolyte concentrations of micro-arc oxidation of titanium properties, and its effect on human osteosarcoma cells MG-63shape, adhesion, proliferation and differentiation.(1) After treatment by micro-arc oxidation, the titanium surface become more roughness and wettability. The titanium surface’s roughness and wettability is not the same after deal with different concentrations.(2) Cells in the roughness titanium surface stretching more microfilaments, abundant cytoplasm and more pseudopodia.Cell morphology has a certain relationshio with the properties of material surface. The surface with a certain degree of roughness and wettability are more suitable foe cell adhesion. At1h and3h,the titanium surface which roughness is more suitable for cell adhesion.(3)At first, the MTS of group B was the highest; at7d, group D was the highest. The ALP activity of group D which has high roughness,are higher than the other three groups. In this experiment, group D is rich in calcium and phosphors, which also could affect cell differentiation.In conclusion, when duty cycle is25%, voltage is300V, the calcium and phosphorus ratios in the electrolyte,concentrations higher than0.16, you can get better pure titanium surface morphology, which containing both rutile TiO2and anatase TiO2, an increase of titanium surface roughness and wettability, but also to promote the adhesion, proliferation and differentiation of human osteosarcoma cell MG-63on its surface.