The Study On The Biological Activity And Antimicrobial Activity Of Dandelion Polysaccharide Loaded On TiO₂ Nanotubes

Research purpose:1.To study the effect of TiO₂ nanotube coating modified by dandelion polysaccharides on bone marrow mesenchymal stem cells.2.To study the effect of TiO₂ nanotube coating modified by dandelion polysaccharides on gingival porphyromonas.3.To evaluate the bioactivity and antibacterial activity of modified TiO₂nanotubes coated with dandelion polysaccharides.Research methods1.Preparation of titanium dioxide nanotubes:in the self-made electrolyte,nanotube arrays with uniform diameter were prepared on the surface of titanium sheets by anodic oxidation method.The surface of the successfully oxidized titanium sheets showed multicolor.2.Loading of dandelion polysaccharide:dandelion polysaccharide was dissolved with anhydrous ethanol/DMSO as an organic solvent,then the anodized titanium sheets were placed in polysaccharide solutions of different concentrations.The dandelion polysaccharide was loaded by simple physical adsorption.3.Experimental grouping:the anodic titanium oxide tablets were set as the control group（group A）,and the anodic titanium oxide tablets loaded with different concentrations of dandelion polysaccharide were set as the experimental group:polysaccharide concentrations were 50mg/mL（group B）,100mg/mL（group C）,and200mg/mL（group D）.4.Cell and bacterial experimental in vitro:bone marrow mesenchymal stem cells were cultured on the surface of titanium plates in the experimental group and the control group to observe cell adhesion,proliferation and differentiation on the surface of sheets.Porphyromonas gingivalis was cultured on the surface of titanium plates in the experimental group and the control group to detect bacterial activity on the surface of sheets.5.Detection methods:FESEM was used to observe the surface morphology,XPS was used to detect the surface element composition,DAPI staining was used to observe the cell adhesion and extension on the surface of the specimen,CCK-8 kit was used to detect the cell adhesion and proliferation ability on the surface of the specimen,ALP kit was used to detect the cell alkaline phosphatase activity on the surface of the specimen and trypan blue staining was used to observe the bacterial activity on the surface of sheets.Results:1.The surface morphology of the specimens was observed by FESEM:the nanotube arrays with uniform diameter can be seen on the surface of successfully anodized titanium sheet.The wall of the nanotube is smooth and its diameter is about80-100 nm.In the experimental group（B/C/D）,more white millet particles were deposited on the surface and inside the nanotube and the amount of particles was positively correlated with the concentration of dandelion polysaccharide,that is,the higher concentration of dandelion polysaccharide,the more polysaccharide loaded on the surface of the titanium sheet.Group D>group C>group B.2.Element composition on the surface of the specimens was detected by XPS:the surface of titanium plates in the experimental group and the control group were all composed of N,C and O elements.The characteristic peak value of C element in the experimental group was significantly increased,which was positively correlated with the concentration of dandelion polysaccharide.That is,the higher the concentration of dandelion polysaccharide,the higher the characteristic peak value of C element.Group D>group C>group B.3.DAPI staining results:the morphology of bone marrow mesenchymal stem cells on the surface of the four groups of specimens was observed under an inverted fluorescence microscope.It was found that the cells were evenly spread,the nuclei were bright blue,round or oval,and the cytoplasm was irregular with many filamentous pseudopodia extending out and connecting with each other.No significant changes in cell morphology were observed between the experimental group and the control group.4.Detection of early cell adhesion by cck-8 kits:with the extension of culture time,the number of cell adhesion on the surface of specimens in the experimental group and the control group increased gradually.At 1 hour,there was no significant difference in the number of cell adhesion on the surface between the experimental group and the control group.At 2 hour and 4 hour,the number of cell adhesion on the surface of specimens in the experimental group was significantly higher than that in the control group,and the difference was statistically significant（P<0.05）.There was no significant difference in the number of cell adhesion between the experimental groups.5.Detection of cell proliferation by cck-8 kits:with the extension of culture time,the number of cell proliferation on the surface of specimens in the experimental group and the control group increased gradually.When the cells were cultured for 3 days and 5 days,the number of cell proliferation on the surface of specimens in the experimental group was significantly higher than that in the control group,and the difference was statistically significant（P<0.05）.At 7 days of cell culture,there was no significant difference in the number of cell proliferation between the experimental group and the control group,or between the experimental groups.6.Detection of ALP activity:with the extension of culture time,the ALP activity of cells on the surface of the experimental group and the control group has an increasing tendency.At day 7,the cell ALP activity on the surface of specimens in the experimental group was higher than that in the control group,and the difference was statistically significant（P<0.05）.At day 14,there was no significant difference in cell ALP activity between the control group and the experimental group.And at day 7 and14,there was no significant difference in cell ALP activity between the experimental groups.7.Detection of bacterial activity:after trypan blue staining,the bacterial activity was observed under the microscope,and it was found that the living bacteria were transparent and the dead bacteria were dyed blue.The number of dead bacteria on the surface of specimens in the experimental group（C/D）was significantly higher than that in the control group（P<0.05）.There was no significant difference in the number of dead bacteria in group B and the control group.The number of dead bacteria between experimental groups was group D>group C>group B,and the bacterial activity between four groups was group D<group C<group B<group A.Conclusion:1.TiO₂ nanotubes loaded with dandelion polysaccharide active coating have good biocompatibility and bioactivity,which can promote the adhesion and proliferation of bone marrow mesenchymal stem cells,and may involve the cells’differentiation.2.TiO₂ nanotubes loaded with dandelion polysaccharide active coating have certain antibacterial properties,and have a strong killing and inhibiting effect on porphyromonas gingivalis.