The Biological Study And Construction Of Micro-nano Titanium Surface

Pure titanium, because of its good biocompatibility and mechanical properties which ensure its combination with bone tissue, becomes the preferred implant materials and has been widely used clinically. for over 40 years. However, at present the main clinical problem is that the bone-implant healing time is unfavorably long, extending the patient's treatment period. Studies have shown that the surface characteristics of titanium implants influence the rate of bone formation around the implant, bone formation quantity and quality. Therefore, the implant surface modification draws the scholars'eyes, particularly in the geometric morphology of the surface, such as the impact of micro-and nano-scale morphology on the bone integration has become the main focus. Studies have shown that micro-morphology can increase the area of bongding between bone and implant,make the blood clot and extracellular matrix protein firm, afford stable micro-environment for osteoblast; Nano-structure of the surface can promote adhesion, proliferation and differentiation of osteoblast. The hybrid micro-nano topography may be the future tendency of implant surface. In this study, the hierarchical micro-nano surface topographies were produced on titanium by Electrolytic Etching (EE) methods, surface physical-chemical properties were analyzed, the bioactivity of materials was evaluated in vitro using osteoblast. This study provides insight to the mechanism that the hierarchical micro-nano topography effects on cell behaviors and bone formation.The aim of this study is to develop a new implant surface topography and surface treatment technology, to improve the surface properties of implant materials, shorten the implant-bone healing time and enhance the implant early-loading capacity and the clinical success rate in weak bones.1.The construction and study of the micro-and nanostructure titanium surfaceIn this study Electrolytic etching technique was preformed, with pure titanium as the anode and platinum foil as cathode. Through the pre-exploration and the two orthogonal design, field emission scanning electron microscope (FSEM) evaluation and analysis helped determining the optimum conditions for EE, and in this condition an uniform titanium surface topography was fabricated, with micro-nanometer three-dimensional structure. The EE surface was analyzed by Field emission scanning electron microscopy, X-ray photoelectron spectroscopy (XPS), laser scanning confocal microscope (CFM), compared with sand blasting and acid etching (SLA) and machined (M) surface. The results showed that:①surface topography:The EE surface consisted of bowl-like micropits(diameter of about 30-50μm, depth of about 10-20μm), some irregular dish-like pits (diameter of about 8-10μm) and scattered micropores (diameter of about 2-4μm) were observed in the bowl-like micropits. Nanopits, nanopores and nanostairs could be clearly seen in the micropits. SLA showed irregularly structure, cracks and a large number of microholes (1-2μm), and some fusion or break, sharp edges, also a few of residual particles. On the M surface, the shallow grooves ware parallel arranged.②Surface roughness, contact angle:the Sa of EE,SLA, M surface were 5.56±1.00μm,3.50±0.81μm,0.77±0.23μm respectively; water contact angles of EE, SLA, M were 56.60°±3.81°,120.27°±20.70°,60.77°±1.07°; sodium chloride contact angles were 73.77°±2.86°,130.22°±8.42°,71.18°±4.04°; plasma contact angles were 69.10°±1.73°,116.23°±9.64°,67.68°±6.04°. These data indicated that, EE and M surface were hydrophilic, while the SLA was hydrophobic. The chemical composition of the three surfaces were all titania, the surface of EE contained few fluoride ions, while SLA with a small amount of Si, and C.2. The biocompatibility of EE titanium surface2.1 Cytotoxicity of EE titanium surfaceCytotoxicity test involved MTT test and hemolysis test. In the MTT test, vat liquor with different concentrations from EE surfaces were used to culture MG-63 cells. After 1d,3d and 5d absorbance A was detected and proliferation rate was evaluated. In the hemolysis test, the absorbance A was measured by healthy human fresh blood, distilled water as positive control, saline as negative control, then hemolytic percentage was calculated. The results showed that:the EE titanium surface was non-toxic for MG-63 cells, which can be a reference value for clinical application. The hemolysis rate of EE specimen is 0.257%, less than 5%, which met the requirements of biological material in hemolysis experiments.2.2 The effects of EE titanium surface on cell proliferation, adhesion, cell morphology and alkaline phosphatase activity(ALP)The MG-63 cells were cultured respectively on EE, SLA, and M surface, using MTT method to evaluate the cell proliferation rate and count the number of living cells under a microscope by trypan blue staining, on 1 d,3 d,5 d and 7 d.1～24 h cells adhesion rate in different the surface was calculated; the cell morphology and structure was observed by FSEM after vaccination 1 d,3 d,5 d; and alkaline phosphatase detection kit was used to detect the change of activity of alkaline phosphatase on 1 d,3 d,5 d, and 7 d. The results showed that:With the extension of time, MG-63 cells cultured on EE, SLA, and M the surface were all proliferated, MG-63 cells proliferation in EE group was almost as same as normal cells, cell proliferation in SLA group and M group was lower than normal cells;MG-63 cell proliferation in EE group was significantly higher than SLA group and M group and cell proliferation capacity in M group was the lowest among the three. MG-63 cells were inoculated on the surface after 1～24 h, with time prolonged, adhesion rate gradually increased,1 h cell adhesion rate of EE group was significantly higher than that SLA group and M group (P<0.05) and M group showed the lowest rate of cell adhesion; cells adhesion rates of each group in 2 h,6 h,12 h and 24 h were significantly increased compared with in 1 h (P<0.05). And still the greatest rate was observed in EE group, while M group showed the smallest; of these, adhesion rate of EE group and SLA group had no significant difference in 12 h. FSEM showed that: the first 1d, MG-63 cell attached on the bowl-shaped concave of EE surface, showing polygonal, about 25μm cell body stretched out a number of pseudopodium and microvilli to attach to the side wall, and some cells across the top of the bowl concave attached to the edge of the ridge or the nano-structure of sidewall; MG-63 cells attached on SLA surface were mainly spindle-shaped and few spherical, pseudopodium and microvillus were less, shape and stretch of cells is not as good as EE; MG-63 cells on M surface were mainly spherical, few spindle-shaped, close to the surface, mainly in microvilli adhesion, pseudopod was rare; On 3d, EE surface were fully covered with the MG-63 cells, bowl-shaped concave and other forms were covered by cells. the cells closely attached to the EE titanium surface and became more flat. The pseudopodium became wider and longer. On the surface there were many processes and a large segment of about 1μm mitochondria -like and other microstructures;SLA surface micro-morphology was covered by MG-63 cells, which were spherical, spindle-shaped and irregular shaped. The cells connected to each other and had no obvious protrusions; MG-63 cells on M surface were spherical and not firmly attached to surface. They showed mainly microvilli but rare pseudopodia; on 5 d, cells closed to the EE surface was more flat and a few cells showed spherical. The entire bowl concave was covered by cells and some particulate matter was observed; MG-63 cells on SLA surface also presented sheet-like and flat shape and there were a small number of spindle-shaped and spherical cells.cells with microvilli protruding and connecting to each other, closely sticked onto the surface; the cells on M surface of were still spherical, but had already stretched, the cells extended microvilli, which already connected, closely attached to the surface and there were rare pseudopodia. ALP activity of of EE, SLA and the M group on 1 d,3 d,5 d and 7d were time-dependent increasing, EE group was significantly higher than the other groups (P <0.05), in which the highest was EE group, and M group was the lowest; ALP activity of SLA and the M group was significantly lower than normal group (P<0.05).2.3 The effect of EE surface on osteogenesis-related gene expressionFluorescent quantitative real time PCR technical was used to analyze the expression of osteogenesis-related genes:osteopontin (OPN),key osteoblast transcription factor (RUNX2) osteocalcin (OCN) and typeⅠcollagen (COLLⅠ) on EE, SLA, and M surface.The results indicated that:①In MG-63 cells on different titanium surfaces, OPN mRNA expressed diversely. OPN mRNA of EE group was higher than SLA group, much more higher than M group, and was time-dependent, which on 5 d expression amount in EE group came to the largest;②The mRNA expression of RUNX2 gene showed various differences between the titanium surfaces, most in EE group and least in M group. Moreover, The mRNA expression of RUNX2 gene on the EE surface increased largely on 1d,3d, and 5 d, while the increase rate on SLA and M surface were much less;③COLL ImRNA expressed in MG-63 cells also varied between different surfaces, EE group cells expressed higher COLL I mRNA than SLA group,much more higher than M group and the peak of expression appeared on 3 d EE group;④OCN mRNA expressed in MG-63 cells showed differences between different surfaces, OCN mRNA in EE Group were higher than SLA group, much more higher than M group, on 5 d EE group expressed the largest amount. These results indicated that electrolytic etching surface was best for osteoblast-like cell differentiation, while the machining surface was worst.3. The study of bone inserting experimentPathology and biomechanics research were involved. The titanium specimen, with a diameter of 4 mm and thickness 2mm, treated by EE, SLA and M methods, were implanted in rabbit tibia. The histological observation was taken at 4 weeks,8 weeks and 12 weeks and the maximum pullout force was tested at 4 weeks,7 weeks, 10 weeks and 13 weeks. The histological results showed that:At 4 weeks, lamellar bone had formed on the EE surface with most bone mass than others and at 8 weeks, bone tissue nearly matured, and a good osseointegration was found on the surface. The SLA surface, the formation of bone tissue was relatively small and late-matured; the bone matured time and quality of the M surface were far less than SLA surface and EE surface. This indicated EE surface acted as a more significant role in accelerating the healing of bone tissue than SLA and M surface. Biomechanics results showed that:At 4 weeks, the pull-out force varied among three groups of the surface. The pull-out force of EE and SLA surface increased more significantly than the M surface, the difference was significant (P<0.05). It suggested the surface with concave and micropore could promote bone formation at an early stage, which might be related to a large concave and nano-structure. At 7 weeks,10 weeks and 13 weeks pull-out force showed an uptrend in each group, but at 7 weeks EE group increased significantly and its pull-out force was significantly higher than SLA and the M group (P<0.05). At 10 weeks and 13 weeks the increase were less and had no significant difference, but were still significantly higher than the SLA and M group (P<0.05), implied that at 7 weeks, bone tissue on EE surface was already quite matured, with the capacity of promoting early osteogenic.at 7 weeks,10 weeks and 13 weeks the pull-out force of SLA group was higher than the M group. The pull-out force of EE surface at 7 weeks were even higher than the SLA group and M group at 13 weeks, showing that the bone maturity of EE surface was at least 6 weeks earlier than M surface and SLA surface. EE surface topography, with large bowl-micron concaves and holes and nano-structure, can significantly promote bone formation and shorten the bone healing time.In a word, the hierarchical micro-nano surface topographies were produced on titanium by Electrolytic Etching (EE) methods. This surface have favorable biocompatibility and non-toxic effects in vivo, which can promote osteoblast adhesion, proliferation and differentiation and the expression of osteogenesis-related gene such as osteopontin (OPN), transforming osteoblast transcription factor (RUNX2), osteocalcin (OCN) and typeⅠcollagen (COLLⅠ).It can accelerate bone growth and promote bone formation at early stage, and guarantee the immediate loading and early stage loading in clinical practice.