Experimental Study Of A Silicon-doped Modified Titanium Plate Surface On The Adhesion And Osteogenic Differentiation Of BMSCs

Objective:With the improvement of people’s living standard,dental implant has become the first choice for many patients with dental deficiency.Although titanium implants have good biocompatibility,they cannot promote the union with bone tissue.In this study,a surface modification strategy combining bioactive trace elements with surface micromorphology modification was used to enhance the biological properties of pure titanium.A new coating incorporatingsilicon micropore/microsphere topography was prepared on a titanium plate by micro arc oxidation（MAO）technology.The properties of the coating and its effects on the adhesion and osteogenic differentiation of rat bone marrow mesenchymal stem cells（BMSCs）were further analyzed.It provides a new idea for the design of implant surface treatment.Methods:1.The commercially obtained pure titanium material was cut into 10 mm x 10mm x 1 mm plates.The electrolyte was prepared with 0.03mmoL/L sodium dihydrogen phosphate pentahydrate（NaH₂PO₄·5H₂O）and 0.1mmoL/L sodium metasilicate nonahydrate（Na₂SiO₃·9H₂O）.A model WHD-20 MAO power supply was used（Harbin University of Technology Sino-Russian Science and Technology Co.,Ltd.,China）.The frequency and time were set to 50 Hz and 10 min respectively.The voltage was set to 320 V、350 V and 380 V for the MAO-320、MAO-350 and MAO-380 groups respectively.The Ti group was the control group.Scanning electron microscopy was used to observe the surface morphology of the material.X-ray diffraction was used to analyze the surface phase composition.an energy dispersive spectrometer was used to determine the element composition.ImageJ and Digimizer software were used to analyze the diameter and number of micropores and microspheres on the surfaces of the materials.The surface morphology of the samples was examined by scanning probe microscopy.The average roughness（Ra）of the coatings was calculated by means of SPM image analysis.Tibias and femurs from4-week-old SPF SD rats（provided by the Animal Laboratory Center,Medical College of Sun Yat-sen University）were used as sources of bone marrow mesenchymal stem cells（BMSCs）.The second-and third-generation cells were used in this study.2.To detect cell adhesion and extension,the number and extension of the cell adhesions were measured by fluorescence staining after 4 h of cell culture on the material surface.The nuclei were stained with 4’,6-diamidino-2-phenylindole（DAPI,1:1000 dilution）for 5 min,observed and photographed under a fluorescence microscope,and then counted with Image-Pro Plus software（IPP,USA）.The cytoskeleton were stained with FITC-phalloidin（Yeasen,China,1:200 dilution）observed and photographed under a fluorescence microscope and measured with ImageJ software.BMSCs from the four groups were inoculated onto the material surface.After dehydrated and sprayed gold on the surface of the materials,the cell morphology was observed by SEM.Live dead cell staining reagent was used to detect cell viability,cell counting kit-8 and EdU were used to detect the proliferation of cells on the metal surface.To measure the ALP activity,ALP reagent was added for staining.The total RNA was extracted with TRIzol reagent,the expression of the osteogenic-related genes ALP,OCN,OSX and BMP2 was detected by RT-qPCR.All the experimental results were expressed as the mean±standard deviation.Each experiment was repeated at least three times.ANOVA and the Student-Newman-Keuls method（SNK）were used to test the differences between the different groups.Differences were considered statistically significant when p<0.05and p<0.01.Results:1.The SEM results showed that a large variety of micropore/microsphere topographies were observed on the surfaces of the samples in the MAO-320,MAO-350 and MAO-380 groups.A uniform volcanic pore shape was observed for the micropores in the MAO-320 and MAO-350 groups,while the micropores in the MAO-380 group were irregular.Three-dimensional AFM images of the MAO coatings formed by using different applied voltages.The coating surface of the material was rough and showed a typical porous and spherical structure.The roughness in the Ti group was lower than that in the treated group,and the roughness increased with increasing applied voltage.The roughness in all treated samples was observed at the microscale.EDS was used to detect the material coating.Ti,O,Si and P were clearly detected.With increasing voltage,the weight%of Si increased gradually,while that of titanium decreased gradually.2.The MAO-350 group adhered to more cells in the initial 4 h,and the number of adherent cells was significantly higher than that in the MAO-380 and Ti groups.The average cell extension area in the MAO-350 group was the largest and was significantly greater than that of the MAO-380 and Ti groups.The results of live-dead staining and the statistics for the live-dead cells showed that there were no significant differences between the four groups,indicating that there was no significant difference between the surface toxicity of the treated groups and that of the Ti group.To detect the proliferation of cells on the surfaces of the samples,CCK-8 and EdU experiments were carried out.The EdU staining results showed that the proliferation of BMSCs on different samples was different during the initial 2 h.The proliferation effect in the MAO-350 group was the most obvious,and the statistical results showed significant differences.The results of the CCK-8 assay showed that the proliferation effect in the MAO-350 group was significantly improved at 1,3 and 7 days.The above results showed that the treated samples had no obvious toxicity towards cells,and the MAO-350 group was more conducive to cell proliferation.We detected the expression of ALP,OCN,BMP2 and OSX by RT-qPCR.According to the results,we can see that the expression of the four osteogenesis-related genes in the MAO-350group is significantly higher than that in the other three groups.Conclusion:In this study,a novel silicon-incorporating coating with a micropore/microsphere topography was developed on a titanium surface by adjusting the MAO voltage.The coating significantly promoted the adhesion,proliferation and osteogenic differentiation of BMSCs in vitro,and these cytological behaviors were attributed to the synergistic effects of the micropore/microspheretopography and silicon incorporation.These results indicate that silicon-incorporating implant coatings with micropore/microsphere topography have potential applications in dentistry and orthopedics.