Preparation And Biological Properties Analysis Of Trace Silicon,Strontium,Fluorine Doped Hydroxyapatite Coating

Part I Preparation and characterization of trace silicon,strontium and fluorine doped hydroxyapatite coatingObjective: to prepare and characterization of silicon,strontium and fluorine doped hydroxyapatite coating(Si+Sr-HA,Si-HA,Si+Sr+F-HA and HA)by hydrothermal method.Methods: The content of Si,Sr and F in the natural bone tissue was the same order of magnitude as the doping amount,Sr:87ppm;F:190 ppm;Si:56 ppm.HA,Si-HA,Si+Sr-HA,Si+Sr+F-HA materialswere prepared by hydrothermal method;Scanning electron microscope(SEM)was used to observe the surface morphology of the four groups of materials;the surface phase composition of the four groups were detected By X ray crystal diffraction(XRD)and infrared absorption spectroscopy.Results: SEM observation results are shown: four groups of samples with rank ordered morphology,Doping ions Si,Sr and F have little effect on the morphology;four groups of materials surface phase detection results are shown: Si,Sr and F doped,there was no observed phase except for the HA phase.Conclusion:HA,Si-HA,Si+Sr-HA,Si+Sr+F-HAwere preparedthrough the hydrothermal methodmaterial;No change of HA phase andcharacterization doped Si,Sr and F;The surface morphology of the four groups was consistent.Part II Studies on cytotoxicity and antibacterialactivity of trace silicon,strontium and fluorinedoped hydroxyapatite coatingObjective: to detect the biological safety and antibacterial activity of the four groups of HA,Si-HA,Si+Sr-HA and Si+Sr+F-HA.Methods: L929 cell was selected as the experimental cells.The cytotoxicity of different concentrations of the four groups of materials was studied by MTT method;phase contrast microscopewas used to observe The morphology of L929 cells cultured in four groups of extraction with different time;The antibacterial activity of four groups of materials to E.coli and Staphylococcus aureus were detected by the antibacterial test.Results:The cytotoxicity of the four groups of materials at different concentrations were 0 or 1,and there was no cell toxicity;With the extension of culture time,the growth of L929 cells was increased in the negative group and the four groups of materials;cultured for 1,4,7 days,the concentration of theextraction of 50mg/ml,100mg/ml Si-HA,Si+Sr-HA,Si+Sr+F-HA three group compared with the HA group to promote the proliferation of L929(P<0.05);cultured for7 days,the concentration of 200mg/ml,L929 cell growth and proliferation of HA group compared with Si+Sr+F-HA,Si+Sr-HA,there is a significant difference(P<0.05).Microscope was used to observe the morphology of L929 cells showed that four groups of materials of different leaching liquid concentration in culture of L929 cells as the culture time prolonged increase in cell number,density increased,the cell morphology and negative control group are more or less similar.Obviously inhibited the growth of Staphylococcus aureus bacteria,Escherichia coli compared with the traditional HA,the Escherichia coli inhibitory rates wererespectively7.4%,21.1%,32.2%;rate of Staphylococcus aureus were respectively4.5%,7.5%,38.3%.Conclusion: compared with the traditional HA,materials of Si-HA,Si+Sr-HA,Si+Sr+F-HA hade the same biological safety,in accordance with the biological material medical standard;Si,Sr,F doped hydroxyapatite improved the antibacterial properties of Escherichia coli and Staphylococcus aureus.Part III The influence of trace silicon,strontium and fluorinedoped hydroxyapatite coating on the adhesion ofosteoblastsObjective: To study the effects of four groups of HA,Si-HA,Si+Sr-HA and Si+Sr+F-HA on the osteoblast cell adhesion activity.Methods: MG63 cells were selected as the experimental cells.CCK8 method was used to detect the number of MG63 cells on the surface of the four groups of materials with culturedfor 1,6,12,24 hours;At 1,6 and 12 h,DAPI staining was used to observe the adhesion of MG63 cells on the surface of the four groups of materials;At 12 h,the morphology of MG63 cells on the surface of the four groups of materials were observed by SEM;At 1,6 and 12 h,Dynamic observation of morphology on the surface of MG63 four groups of materials of the cytoskeleton by FITC-Phalloidin and DAPI double staining method;Wound healing assay was performed to evaluate the migration of MG63 cells cultured on different surfaces.Results: At 6 h,Si-HA,Si+Sr-HA,Si+Sr+F-HA group,the number of cell adhesion was significantly higher than that of HA group(P < 0.05);At 12 h,the number of surface cell adhesion Si+Sr+F-HA>Si+Sr-HA> Si-HA > HA(P < 0.05);There was no significant difference between Si+Sr+F-HA and Si+Sr-HA(P > 0.05),but there were significant differences among the other groups(P < 0.05);The results of DAPI staining were similar to the results of quantitative analysis of CCK-8 cells;SEM morphology observation showed that: compared to HA and Si-HA,MG63 cell of Si+Sr-HA,and Si+Sr+F-HA were spreaded better and more flat.Cell spreading area and cell skeleton protein expression sequence of Si+Sr+F-HA>Si+Sr-HA>Si-HA group >HA;in Si+Sr-HA and Si+Sr+F-HA,the number of migratory cells was significantly higher than that of HA and Si-HA.Conclusion: Si,Sr,F doped hydroxyapatite promote adhesion,spreading and migration;The adhesion activity of the material was Si-HA >HA Si+Sr+F-HA>Si+Sr-HA>.Part IV The influence of trace silicon,strontium andfluoride doped hydroxyapatite coating on theproliferation and apoptosis of osteoblastsObjective: To study the effect of four groups of HA,Si-HA,Si+Sr-HA,Si+Sr+F-HAon osteoblast proliferation and apoptosis.Methods:At1,4,7and 14 d,the number of cell proliferation in four groups of materials was detected by CKK8 method;Flow cytometry was used to detect the cell cycle and apoptosis of MG63 cells on the surface of the four groups.Results:At 4 d,the number of MG63 cells on the surface of Si-HA,Si+Sr-HA and Si+Sr+F-HAwere significantly higher than that of HA group(P < 0.05).At 7 d,Si+Sr+F-HA>Si+Sr-HA>Si-HA>HA,The differences between the groups were statistically significant(P < 0.05);At 4 d,G0/G1:Si+Sr+F-HA<Si+Sr-HA<Si-HA< HA;S?G2/M:HA<Si-HA<Si+Sr-HA<Si+Sr+F-HA;At 7 d,G0/G1:Si+Sr+F-HA<Si+Sr-HA< Si-HA<HA,There was no significant difference between Si+Sr+F-HA and Si+Sr-HA;S : HA < Si-HA < Si+Sr-HA <Si+Sr+F-HA,There were statistically significant differences between the groups(P < 0.05);G2/M:Si+Sr+F-HA>Si+Sr-HA> Si-HA>HA,the difference between Si+Sr+F-HA and Si+Sr-HA was not significant;At 4and 7 d,Apoptosis rate: HA <Si-HA<Si+Sr-HA<Si+Sr+F-HA,there was no significant difference between Si+Sr-HA and Si+Sr+F-HA(P > 0.05).there were significant differences among the other groups(P < 0.05);At 14 d,The apoptosis rate of MG63 cells was significant difference between HA and Si+Sr-HA,Si+Sr+F-HA.Conclusion: Si,Sr and F can promote the proliferation of MG63 cells on the surface of materials,the proliferation activity of materials :Si+Sr+F-HA>Si+Sr-HA>.Si-HA >HA;By reducing the G0 / G1 phase and increase the cell volume ratio of S,G2/M,and MG63 in osteoblast apoptosis accelerated to promote MG63 cell proliferation.Part V The influence of trace silicon,strontium andfluoride doped hydroxyapatite coating onthe differentiationof osteoblastsObjective: To study the effect of four groups of HA,Si-HA,Si+Sr-HA,Si+Sr+F-HA on differentiation.Methods:MG63 cells were cultured on the surface of the four groups of materialsfor 1,4,7,14 days,the activity of ALP was detected;gene expression of COll-I,ALP,Run X2,OPN,BSP,OC were detected by real-time PCR.Results:At 4 and7 d,the activity of ALP: HA< Si-HA <Si+Sr-HA<Si+Sr+F-HA;At 14 d,The expression of ALP activity in HA group was significantly different from Si-HA,Si+Sr-HA,Si+Sr+F-HA three groups(P < 0.05);There was no significant difference between the three groups of Si-HA,Si+Sr-HA and Si+Sr+F-HA;At 4 d,gene expression of Coll-?:Si+Sr+F-HA>Si+Sr-HA> Si-HA> HA;Runx2 gene expression was no significant difference between HA and Si-HA,Si+Sr-HAand Si+Sr+F-HA,there were significant differences among the other groups(P < 0.05);At 7 d,gene expression of Coll-?and Runx2:Si+Sr+F-HA>Si+Sr-HA> Si-HA> HA,The difference of expression between the four groups was statistically significant(P < 0.05);At 14 d,There was significant difference in the expression of Coll-I and Runx2 between HA and the Si+Sr+F-HA;At 4 d,there was a significant difference in the expression of OC gene between HA and Si+Sr+F-HA(P < 0.05);At 7 and 14 d,gene expression of OC,OPN and BSP:Si+Sr+F-HA>Si+Sr-HA> Si-HA> HA,There were significant differences in gene expression between the four groups(P < 0.05).Conclusion: Si,Sr,Fcan not only promote MG63 bone cells early osteogenic differentiation markers.ALP,Coll-I,Runx2 expression level,but also improve the late differentiation and mineralization of OC,OPN and BSP protein gene expression,and the promoting enhancement effect and doping species was closed.Part VI The signal transduction passway mediatingosteoblastic activityon trace silicondoped hydroxyapatite coatingObjective:To study the possible signal transduction pathways of silicon doped hydroxyapatite to promote bone cell activity.Methods:MG63 cells were cultured on the surface of the materialrespectively for 1,4,12,24 hours and 1,4,7,14 days,the gene expression of the integrin,focal adhesion kinase(FAK),ERK1 / 2 andc-jun/c-fos of the downstream transcription factorwered detected byreal time quantitative PCR,otherwaylow density lipoprotein related protein(Lrp5)and Dkklof Wnt signaling pathway Molecular involved.Results:at 4,12,24 h,the expression of integrin ?5and FAK in Si-HA was significantly higher than that in HA(P<0.05);At 12,24 h,the expression ofintegrin?1in Si-HA was significantly higher than that of HA(P<0.05);At 4,7,14 d,the expression of ERK1 gene was significantly higher in Si-HA than that in HA(P<0.05);At1,4,7,14 d,the geneexpression of C-fos and C-jun in Si-HA group was significantly higher than that of HA group(P<0.05);At l 4,7,14,Lrp5 gene expression in Si-HA group was significantly higher than that in HA group(P<0.05),and the expression of DKK1 gene was significantly lower than that of HA group(P<0.05).Conclusion:Si-HA material promotes MG63 cell adhesion by up regulating the alpha 5,beta 1 and activation of FAK;FAK activates ERK1/2,regulates the expression of c-jun and c-fos,and enhances the proliferation and differentiation of MG63 cells;And,the activity of MG63 cells was promotedthrough inhibiting the expression of DKK1 gene in the classical Wnt signaling pathway and up-regulating the expression of Lrp5 gene.