| ObjectSr/Si/F trace-doped hydroxyapatite?DHA?was prepared by hydrothermal method,and the mass fraction of the three elements was consistent with that of natural bone apatite.DHA coating and zirconia reinforced Sr/Si/F trace-doped hydroxyapatite?ZrO2-DHA?coatings were prepared on zirconia substrate by plasma spraying.The phase composition and surface/internal structure of coatings were characterized,and the mechanical properties and in vitro biological properties were investigated.By analyzing the experimental results,the mechanism of osteogenesis promotion of DHA coating was discussed,and the potential coating of component proportion and the relation between the proportion of zirconia in ZrO2-DHA coating and property change was explored.This study is intended to provide new evidence for surface modification applications of zirconia implants.MethodThe first part:the preparation and characteristics of DHA coating and ZrO2-DHA coatings.HA and DHA were synthesized by hydrothermal method in which Ca?OH?2 and Ca?H2PO4?2·H2O were used as raw materials,and SrO,SiO2 and CaF2 were used as trace element introduction agents.The mass fractions of three elements in DHA were guaranteed consistent with the natural bone apatite by hydrogen synthesis.The thermal spray powders were prepared by the spray granulation method,wherein DHA and zirconia are uniformly mixed and granulated according to a mass ratio of 7:3/5:5/3:7.The zirconia substrate is blasted with corundum powder to increase the surface roughness,and then the HA/DHA coatings and the three kinds of ZrO2-DHA coatings?named as 7DHA,5DHA and 3DHA respectively according to the proportion of 70%DHA,50%DHA and 30%DHA?with different proportion were prepared according to the preseted spraying parameters.The surface and internal microstructure of the coating were observed by scanning electron microscope,and the phase composition of the powder and the coating was examined by X-ray diffractometry and Fourier transform infrared spectroscopy.The second part:the mechanical and in vitro biological properties of DHA coating and ZrO2-DHA coatings.Microhardness and bond strength of DHA coating and ZrO2-DHA coatings were tested by Vickers hardness tester and universal testing machine.In vitro biological properties of DHA coating and ZrO2-DHA coatings consisted of?1?bacteriostasis test:the growth inhibition of Escherichia coli and Staphylococcus aureus on different coatings by plate culture method,?2?cell viability test:the toxicity grades and the effects of different coatings on the viability of MC3T3-E1 were tested by MTT,?3?cell adhesion test:the numbers of cells adhering on different coatings at different time after seeding were counted by DAPI fluorescence staining method,evaluating the effects of coatings on cell adhesion of mouse osteogenic precursor cells,?4?cell osteogenic differentiation test:the relative activity of alkaline phosphatase and the mineralization effect of different coatings at different time after seeding were investigated by PNPP method and alizarin red staining respectively,evaluating the effect of coatings on osteogenic differentiation of mouse osteogenic precursor cells;?5?Cell osteogenic differentiation mechanism test:the relatiave expression of osteogenic differentiation-related genes Alp and Col-I of coatings at different time after seeding were tested by real-time fluorescent quantitative PCR method,evaluating the effect of coatings on osteogenic differentiation of mouse osteogenic precursor cells in terms of mechanism.ResultsIn the first part,the surface morphology of the typical plasma sprayed coating was seen by SEM.The element mapping test showed that the DHA and zirconia are interlaced and wrapped by each other both in the plane or cross section of the coatings,forming three-dimensional network structure.XRD and FTIR tests showed that the XRD peaks of DHA are consistent with the characteristics of pure phase HA.As to the ZrO2-DHA coatings,the XRD diffraction peaks of DHA and zirconia are obvious,and the peaks of zirconia are enhanced with the increasing of the content of zirconia.In the second part,for the mechanical properties,the microhardness and bond strength of ZrO2-DHA coatings increased with the increment of zirconia content.The hardness of DHA,7DHA,5DHA and 3DHA were 250.8±18.9kgf/mm2?320.1±22.5kgf/mm2?418.4±17.3kgf/mm2?526.2±23.6kgf/mm2 respectively,and the bond strength of these coatings were 25.1±2.6Mpa,31.8±2.8Mpa,39.4±2.3Mpa and 46.2±4.3Mpa.The fracture section analysis of the coatings showed that there were failures at the substrate-coating interface,inside the coating,and at the coating-glue interface,belonging to a mixed mode failure of cohesive and adhesive failure.As to the in vitro biological properties,bacteriostasis test suggested that DHA coating and ZrO2-DHA coatings inhibited the growth of Escherichia coli and Staphylococcus aureus to varying degrees compared to ZrO2 substrate,among which the inhibition rate of DHA coating on the two bacteria were 29.76%and 30.23%higher than that of the HA coating,and the corresponding parameters of the 7DHA coating were 22.62%and25.91%higher than the HA coating.MTT test indicated that the toxicity level of DHA coating and ZrO2-DHA coatings was 0 grade,and compared to zirconia substrate,these coatings promoted osteoblast viability to varying degrees;Among them,the relative proliferation rate of DHA coating and 7DHA coating relative to ZrO2 substrate were 116.2%and 111.3%,respectively.In the cell adhesion test,cell adhesion of the mouse osteogenic precursor cells on DHA coating and ZrO2-DHA coatings were superior to zirconia substrate.The results of the ALP activity test and the alizarin red staining test were similar to the cell adhesion results,among which the performance of DHA coating and 7DHA coating were better than that of HA coating.The gene expression test explained and verified the results of the above tests:Alp and Col-I expression of DHA coating were 3.34 and 3.11 times that of ZrO2 substrate,and the relative gene expression of the coatings with 7DHA significantly higher than that of HA coating.ConclusionIn this study,the DHA coating and ZrO2-DHA coatings prepared on the zirconia substrate by plasma spraying method provide a new evidence for the surface modification of zirconia implants.Through the study of the mechanical properties and in vitro biological properties of the coating,the following conclusions were obtained:?1?The early bone-promoting property of DHA coating is superior to the HA coating and zirconia substrate.?2?The early bone-promoting properties of the ZrO2-DHA coatings are significantly better than those of ZrO2 substrate,which is determined by the DHA component of the coating.The bone-promoting properties of ZrO2-DHA coatings are positively correlated with DHA content.?3?The mechanical properties of ZrO2-DHA coatings are significantly better than those of the DHA coating.The composite of DHA and ZrO2 forms the three-dimensional structure.The mechanical propertoes of the ZrO2-DHA coatings are positively correlated with the zirconia content.?4?Compared with HA coating,7DHA coating has obvious advantages in bone-promoting in vitro and mechanical property,which is a promising zirconia implant coating material. |
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