Study of the Effect of Laser Etching and Micro-arc Oxidation on the Bioactivity of Pure Titanium Surface
|School||Southern Medical University,|
|Keywords||Implant Surface treatment Lasert Micro-arc oxidation PureTitanium Osseointegration Surface property Bioactivity Osteoconductive|
Background and Objection:"Osseointegration" theory was introduced by professor Branemark in the1960s, in1965, titanium implant was the first time to insert in human mandible and succeed to finish restoring the missing tooth. So far, the development of titanium implants surface treatment was becoming mature more increasingly, and titanium materials are widely used in the field of oral Medicine. With the development of oral implant technology is mature, gradually improve the life quality of the people, who with edentulous defect and wish implant as preferred repair program, the implants in oral medicine is increasingly widespread.A passivation film of TiO2was formed when pure titanium exposed in air under high temperature, the oxide film was a very stable, and present excellence biocompatibility, However, the oxide film can not promote the growth of bone tissue, can not be shorten osseointegration time. Although many surface treatment was claimed to be perfect, in Oral Implantology repair treatment, the applications of short implant expand the adaptation scope of the implant restoration, how to make the short implants become more reliable and more stable, these must be based on good surface treatment. So, faster osseointegration and more stable osseointegration were the pursuing goals of implant surface treatment. finest implant surface characteristics, promote new bone and integrate with impant. In order to shorten implant osseointegration time, enhanced osseointegration intensity, titanium implant surfaces usually need to do all kinds of surface treatment. By various surface treatment to change the surface properties of titanium, the implant surface obtain good bioactivity for improving osseointegration.After surface treatment, the surface of the implant has a better bioactivity. The bioactivity was mean, in vivo, biomaterials and bone integrated by produce chemical bonding, is an important indicator for evaluating biomaterials. There are two ways of new bone formation between implant and bone tissue:first, Distance osteogenesis, when implant newly inserted, there is a distance from tissue wounds to implant surface, new bone grow from bone tissue wounds to implant surface, and fulfilled the gap at last. This process, the new bone did not grow on the implant surface directly, in this osseointegration process the new bone formation is unidirectional. Second, Contact osteogenesis, when implant newly inserted, there is a distance from tissue wounds to implant surface, the gap was filled with blood and some bone fragment, the bone fragment as well as the osteoblast contact to the implant surface and anchored separatism, proliferation rapidly, new bone grow from implant surface to bone tissue wounds, while new bone grow from bone tissue wounds to implant surface, and fulfilled the gap at last. This process, the new bone grow on the implant surface directly, in this osseointegration process the new bone formation is bidirectional.In recent years, laser treatment has also been used for surface treatment of oral implants, laser treatment is a new surface treatment method, different intensity and different types of laser has different effects on titanium surface. Micropore and groove structure could be formed by laser etching, the depth and width can be adjusted by changing the laser energy, the precise processing can provide surface3D ideal morphology meet the need, but also can reduce the pollution of implant surface. In addition, these micropores and groove structure can significantly improve the the removal torque of titanium implant, make the implant become more stable and durable. Micro-arc oxidation is a kind of anodic oxidation technology, the early nineteen thirties, first reported by Gunterschulze and Betz, and recently, it has been used for surface treatment of oral implant. The surface with nanometer and micron microporous produced by micro-arc oxidation, that has a certain bioactivity, beneficial to osteoblast contacting, spreading and proliferation, which stimulate and speed up the formation of new bone.Titanium implant modified by both Micro-arc oxidation and laser etching has good biocompatibility, combined with laser pretreatment and other surface treatment methods have proven to be feasible, but the modification of laser etching and micro-arc oxidation almost no report. Therefore, this study will explore the effect of laser etching and micro-arc oxidation on the bioactivity of pure titanium surface.Objective:1. Pure titanium made into small titanium plates, compare the physicochemical properties of titanium plate surface modified by several surface treatment methods. Evaluate the feasibility of laser treatment and micro-arc oxidation as an implant surface treatment methods.2. Compare the effect of pure titanium surface modified by several treatment methods on adhesion, proliferation and differentiation of human osteosarcoma cell MG-63, provide a theoretical basis of laser etching and microarc oxidation technology as an implant surface treatment methods.3. Design and manufacture titanium cap, observe and compare the new bone augmentation in two kinds of titanium cap modified and non-modified by micro-arc oxidation in vivo, evaluate the osteoconductive ability of micro-arc oxidation film.4. Design and manufacture titanium screw implant, observe and compare bone response to both turn surface implant and titanium surface modified by laser etching and micro-arc oxidation, evaluate the osseointegration index of the implant modified by laser etching and micro-arc oxidation in vivo.Methods and materials:1. Titanium disc with a1.2mm thickness and15.0mm diameter were manufactured from rods of commercially pure titanium. And gradually polished by200and800silicon carbide sandpaper, then all polished discs were ultrasonically rinsed in a sequence of acetone, ethyl alcohol and distilled water for10minutes per rinsing step After dry in room temperature, the discs were modified by laser etching, micr-oarc oxidation, laser etching combined with micr-oarc oxidation, at last, The surface compositions were detected using an energy-dispersive X-ray and electronic probe; the topographies were characterised by scanning electron microscopy. The surface roughness was shown by3-dimensional interactive display.2. The titanium discs were randomly divided into four groups as follows:turn surface group (A group), laser group (B group), micr-oarc oxidation group (C group), laser etching combined with micr-oarc oxidation group (D group). Human osteosarcoma cell MG-63with a concentration of1×104/cm2were cultured on the disks of each group. At the time point of1h,3h and6h, SEM observe the morphology of cells. and At the time point of1h,3h and6h, the disks with cell were stain by immunity fluorescence, and laser confocal microscopy (CLSM) were used to and F-actin architectonic change.After human osteosarcoma cells MG63were cultured on disks for1d,3d and5d, the OD values was measured by MTS to evaluate the proliferation. Then compare all the different groups.A statistical analysis of OD values was performed with a One way-ANOVA (SPSS13.0version). The data were presented as the mean±standard deviation (x±s). The differences were considered statistically significant at P≤0.05.3. Titanium caps with a7.0mm height and5.0mm diameter were manufactured from rods of commercially pure titanium. The machined caps were ultrasonically rinsed in a sequence of acetone, ethyl alcohol and distilled water for15minutes per rinsing step. Twenty titanium caps were randomly divided into two groups as follows:10machined surface caps were used as the control group (CG). The other10machined surface implants were used as the test group (TG) and were further modified by micro-arc oxidation. All of the implants were ultrasonically rinsed with distilled water for15minutes, dried at room temperature for several hours and sterilised by an autoclave before being used. Two kinds of titanium caps were insert in calvarium of New Zealand white rabbits, Four weeks later, remove the caps from the calvarium, and measured the vertexes of the new bone for statistical analysis. The bone volume from the bottom line on the calvarium to the zenith of new bone was calculated by 3DX multi-imagine micro CT (the volume of new bone=bottom area×average height). The bone sample of the skull was fixed, dehydrated and demineralised before paraffin embedding; the samples were cut into slices and hematoxylin-eosin stained for histomorphology observation.A statistical analysis of the augmented bone was performed with a Student’s t-test (SPSS13.0version). The data were presented as the mean±standard deviation (x±s). The differences were considered statistically significant at P<0.05.4. Thread implants with3.4mm in diameter and8.0mm in length were manufactured from rods of commercially pure titanium, then all machined implant were ultrasonically rinsed in a sequence with acetone, ethyl alcohol and distilled water, each step lasted15minutes. In the present study,20titanium implants divided into two groups:10machined surface implants were used as control group. The other10machined surface implants used as test group were further pretreat by laser etching, successively, the implants were processed in an electrolytic solution containing3.5%glycerophosphate disodium salt pentahydrate (C3H7Na2O6P·5H2O) and1.2%calcium acetate monohydrate ((CH3COO)2Ca·H2O) by micr-oarc oxidation for15seconds. Then all implants were ultrasonically rinsed with distilled water for15minutes, and dry at room temperature for several hours and sterilized before use. The implants were inserted in the tibia of New Zealand white rabbits.13and14days before sacrificeing, all rabbits were hypodermic injected with fluorescently-labeled acheomycin for fluorescent labeling, and3and4days before sacrificeing calcein was also injected in the same way. Four weeks after the operation, all rabbits sacrifice to sampling, and the bone blocks with implants were embedded by plastic embedding techniques without decalcifying, then cut to slices and stained by methylene blue and acid fuchsine for histomorphology observation and histomorphometry of the bone-implant interface, eventually, the mineralization appositional rate and Osseointegration Index were measured and analyzed.Statistical analyses of the OI and the mineralization ratio were performed by using the Student’s test (SPSS13.0version). Data were presented as the mean±standard deviation(x±s).The differences were considered statistically significant at P<0.05. Results:1. The surface properties of the titanium surface Group A:Machined surface of the CG exhibited a smooth surface with a surface roughness (Ra) of0.179μm.Group B:Proportional spacing large craters formed by laser etching with diameter of100microns, depth of80to100microns and interval of100microns. Group C:Single MAO treatment surface without any craters provided surface roughness (Ra) was1.55μm.Group D:Both Large craters and porous structure were formed on the surface by laser etching and MAO treatment. The surfaces were a MAO film with a surface roughness of1.55μm.Through the analysis of X-ray diffraction, found that the main chemical composition is titanium in group A and B, while Ti and TiO2(anatase) were found in groupC and D. Through the analysis of chemical elements by electron probe, pure titanium was found in group A, oxygen and carbon were found in group B, C, and D. The component of carbon was found most in group D. That mean, both laser etching and MAO treatment would enhance the component of carbon.2. Cell morphology, proliferation activity and differentiation level(1) the SEM show that:within one hour, all the cells present quite small and the shape look like a rotundity, as the time increased the actins bundles appear to increase in the cell surface, at24h, cells spread more flatly and a few pseudopods contact the titanium surface in the group A. In other three groups, the cells stretched out pseudopods to anchor the edge of the porous surface, even some of them extends deep into the micropores.(2) The CLSM show:one hour after inoculation, cell actin turn to be a ring and wrap around the nucleolus in four groups of titanium disk. At3h, cell actin become more obvious in all group, mean while, microfilament bundles were found to grow as surrounding radiate in B, C, D group. At6h and24h, the cell spread to be a polygon, which was present especially obvious in the D group.(3) Compare OD values by MTS of the four groups:As the culture time increased, the activity of proliferation increased. At1d, there is no significant difference among A, C, D groups. At3d, group C and D is higher than group A and D, there are significant difference between group C and D, the proliferation rate of group C is higher than group D. At5d, group D was the highest one.3. After the caps were removed, new bone formation in the caps was observed; more new bone was observed in the TG caps compared with the CG caps. The amount of new bone increased along the inner wall of TG caps, whereas there was little in the CG caps. In the TG caps, the highest vertex of new bone reached3.0mm, whereas the new bone height of the CG caps was no more than0.7mm. A micro CT reconstruction of the rabbit calvarium is shown in Figure10. A histological section of the TG showed new bone growth along the inner surface of the TG cap and trabecular bone formation directly contacting the metal surface. New bone that grew along the inner surface of the caps appeared to be higher in the TG caps compared with that of the CG caps. In the TG, the new bone volume achieved was18.63±3.80mm3.There was significant difference between two groups.4. Four weeks later, new bone formation was displayed by the fluorescently-labeled acheomycin (yellow bands) and calcein (green bands), the spans of the two bands were measured by OsteoMeasure System, the mineralization rate of CG is0.56±0.09μm/d, while the. The fluorescein labeling showed that yellow bands of the TG had reached the implant surface, while the yellow bands of the CG still had a certain extent span to the implant surface, and the bands’ width of the TG were wider than that of the CG, which indicated that the new bone formation of the TG were more rapidly than that of the CG.The formation of trabeculas of new bone in four weeks, recognized by osteon formation, were also observed in all groups. New bone formation in direct contact with the implant interface appeared to be higher in the TG than that of the CG. The osseointegration index of each group in four weeks were measured, and the OI of the TG was higher than that of the CG (P=0.00).Conclusion:1. The titanium surface properties present different character when pure titanium modified by laser etching and micro-arc oxidation respectively. When two methods was combinated, both macro pore formed by laser and micro porous formed by MAO could be found on the the titanium surface. Tthis new surface has a certain roughness, rich in calcium, phosphorus ions.2. SEM and CLSM showed that an ordering, adhesion and spreading process of osteoblasts on titanium plate along with the time development, of law. adhesion process of cell and cytoskeletal distribution are interrelated with morphology and surface roughness of titanium surface. MTS showed the proliferation of osteoblasts on titanium substrate, first days to complete the initial adhesion, in this stage, cell proliferation of C, group D is not obvious. In the late stage of Proliferation, D group was the strongest, which indicates the surface present excellence biocompatibility.3. Bone formation is greater on an MAO surface compared with a machined surface. This study demonstrated that an MAO surface possesses excellent osteoconductive potential. Without any bone substitutes, caps’modified surfaces are beneficial for bone augmentation. Furthermore, the findings demonstrate that the surface of the titanium device for vertical bone augmentation should be modified. However, the cap inwall might present difficulties for some surface modifications; therefore, further investigations are required to design and optimise the titanium device.4. A new surface on pure titanium implant with craters, porous structure TiO2and some chemistry was formed by the surface modification of laser etching and micro-arc oxidation, it was demonstrated that the surface can enhance bone response of rabbits to implant surface. It indicated that surface chemistry and topography separately or together play an important role in the bone response to the implants. So, further investigations are required to analysis laser etching and MAO. Laser etching and micro-arc oxidation is a potential method for titanium implant surface treatment, and it is reliable and promising.