| Objective: Surface coating technology is one of the main methods to enhance the biological activity and improve the physical and chemical properties of dental implants and the important standard of measuring the quality of the coating is whether the coating has good bioactivity and lasting adhesion. There is research shows that hydroxyapatite (HA) has excellent biological compatibility and biological activity of inducing, but pure HA ceramic is brittleness, low compressive strength, poor wear resistance, the mechanical properties of difficult to achieve planting material loading requirements. The dental implants which combining HA with the substrate in a sintering way can be not only maintain the excellent mechanical properties of the plant, but also can effectively enhance the biological properties of the plant,effectively improve the integration rate between the coating and bone, promote cell proliferation and osteogenic differentiation, reduce implant direct stress impact. But more and more research found that after coating, implants were implanted in the surface coating prone to fracture. Resulting in degradation of HA and absorption,matrix separation, directly affect the implant stability and planting body inflammation around, resulting in the failure of the implant.This paper preliminary studies confirmed that with the nano hydroxyapatite high temperature sintering to ZrO2 ceramic surface formation of the coating that has strong shear bond strength, can significantly enhance the bonding effect, thus avoiding the coating disintegration and shedding. The purpose of this study is to test the biological properties of nano-HA after sintering at high temperature, and to explore the feasibility of the hydroxyapatite implant. Therefore, this experiment used dry pressing molding technology to powders nano hydroxyapatite compressed into ceramics Su embryo and application of high-temperature sintering technology to sinter it into ceramics, by detecting the nano hydroxyapatite ceramics on bone cell morphology,proliferation and osteogenic activity to explore the biological behavior, in order to provide experimental basis for further research and clinical application.Mathods :1.Preparation the test specimens of nano- hydroxyapatite ceramics: Put the 99%purity of nano-hydroxyapatite into homemade mold, measured the thickness of standing particles,inflated 150MPa with the universal testing machine,the speed was setted as lmm/min, steadily pressurized for 5 minutes, after pressing and forming,measured the thickness of nano- HA test specimens.2.Sintering: Take the nano- hydroxyapatite ceramics wafer which was made by press forming into Zirconia crystal furnace, preheated for 900℃,then , its were heated to 1550℃±10℃ with the heating rates of 5℃/min,kept warm for 4 hours,cold to room temperature along with the furnace. Take test piece out, measured the diameter of the sintered nano- HA ceramic wafer,and calculate the superficial area (S).3.Cell Culture and passage: Seleted the front of the rat osteoblast MC3T3-E3 as the function cell, we cultivated cells for four hours in inverted culture flask with 5% of carbon dioxide, 37℃, 95% of the air, complete humidity culture to grow well, trypsin digestion, go down to posterity by 1:2 scale. To the fourth generation, change to 1:3 ratio of passage, watch the cell growth, When the cells full of bottle bottom 80%,exhibited good growth, we digested the cell with panceratin, centrifuged, standby by cryopreserveded adding of DMSO.4. Cell culture experiment in vitro -Cytotoxicity-test in vitro: Take 20 test specimens that single average surface area after measurement and calculation is 1.0cm2. Specimens average into two culture dishes of the largest after treating with ultrasonic cleaning ,drying and autoclaved at 121℃ for 30 minutes. In accordance with the lcm2/3ml ratio of the material surface area and leaching medium were added to normal saline 30ml.Placed in 37℃ for extraction 72 hours , filtrated, packed.In 96 pore plate we are respectively arranged in the experimental group (nano ha dip extract), negative control group (containing 10% FBS alpha MEM) and positive control group (0.64%phenol containing complete medium) 3 group, (n = 5) complex hole, removed after continuous cultivation for 1d 3d, 5d, addition of CCK-8, detected the mean value of each groups, making cell proliferation curve, and calculate the groups of RGR toxicity rating.5. Cell culture experiment in vitro --Osteoblast morphology observation: Take four nano- HA ceramic specimens and ZrO2 respectively ,and we were observed osteoblast morphology in the 48 well plates, four holes per group ,0.64% phenol for positive control group, complete medium as blank control, continuous cultivation for 1d,3d,5d,7d,to determine fractionation with the observation of each material groups6.Alkaline phosphatase activity detection: Made titanium,ZrO2 and nano-HA ceramic specimens of 1.1cm diameter wafer respectively, took three groups into 48 orifice, each groups was set four holes, growed in five times ten to the four cells per hole. Culture 1d, 3d, 5d, 7d in the incubator, tested the alkaline phosphatase activity of three groups to determine the cell in vitro osteogenetic activity of each specimen group.Results:1.Cytotoxicity test showed that there was no significant difference (P > 0.05) between the nano- HA ceramic specimens and the negative control culture medium group, and the cell adherence was good. Compared with the positive control group (0.64%phenol), the difference was significant (P < 0.05).2 .Light microscope observation found that cell envelope of nano- HA ceramic specimens and ZrO2 group was complete, the cytoplasm was full, the granule was plump, the extension of the process was full, showed the good living condition. While,the positive control group which containing 0.64% phenol showed that the cell capsule rupture, cytoplasmic reduction, most of the cell suspension.3 .Three groups of different materials of ALP activity detection showed that in the certain time of incubation period (1-7 days), the ALP enzyme activity of three groups all showed increasing trend, ALP secretion reached the maximum peak on the 7th day and the ALP activity displayed in the same time ,nano-HA group ALP is higher than the other two groups,the osteogenic activity of cell is relatively good.Conclusion:1.With the 150MPa molding pressure, nano- HA has a certain strength ,hardness,and density nano- HA ceramics form a certain thickness at 1550℃ after sintering.2. High temperature sintering of nano hydroxyapatite ceramic cytology toxicity was rating for 0 grade, normal cell morphology and growth,, consistent with the requirements of the evaluation of the cytotoxicity of implanted materials in vivo.3.The alkaline phosphatase activity of the sintered compactness nano hydroxyapatite ceramic was better than that of the titanium group, and increased along with the growth of the time gradually. It showed that nano hydroxyapatite was more effective than the other two materials after high temperature sintering, which could promote the osteogenesis of osteoblasts. |
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