| Research Background:In various oral restoration methods of dentition defects and edentulous,the traditional fixed and activity restoration methods can basically meet the requirements of patients,but the effect is not satisfied.The emergence of implant restoration means more choices of restoration methods can be selected.From the perspective of clinical effects,the effect of implant restoration is incomparable to other forms of restoration.At present,titanium implants used in clinical have good biocompatibility,but the biological activity still needs to be improved.The surface of titanium implants cannot be chemically combined with new bone tissue quickly,so it takes a long time to achieve osseointegration.The study of surface modification of implants by scholars from various countries has never stopped.Research on surface modification of implants at home and abroad is mainly focused on biological activity and antibacterial coating research,so as to make implants more convenient and widely used.Micro arc oxidation(MAO)is a method applied to the surface modification of metals,which can form a loose porous structure oxide coating on the surface of titanium implants.This technique also has the additional advantage that the composition of the electrolyte can be adjusted to change the chemical composition of the coating,so that the coating can be rich in calcium ions and phosphorus ions,which increases the biological activity of the micro arc oxidation coatings,and is more conducive to newborn bone cells attachment.Dandelion polysaccharide is one of the main active ingredients of Chinese herbal dandelion,accounting for 30%~50%of the dry weight of dandelion.Dandelion polysaccharide,which is abundant in nature with advanced extracting process,has biological activity and is cheap.However,the researches on dandelion polysaccharide used in implant coatings are hardly found.Polysaccharides has been found that it can react with metal cations to form corresponding compounds.Under the heated conditions,dandelion polysaccharides are introduced into the coatings to allow certain chemical reactions with titanium and calcium ions to produce dandelion polysaccharide metal compounds.The coatings were stably loading with dandelion polysaccharide and assessed its biological evaluation.In this study,we prepared the coatings by means of micro arc oxidatino on the surface of the pure titanium as they were rich in calcium and phosphorus and loading with dandelion polysaccharide.Studying the effects on the biological activity of bone marrow mesenchymal cells,can be provided a new idea to construct biologically active implant coatings..Research Purpose:1.Successfully to prepare the coatings rich in calcium and phosphorus on titanium surface by micro arc oxidation.2.Study the effects of bone marrow mesenchymal stem cell on the oxidation coatings after loading with dandelion polysaccharide.3.Detect the safety of the coatings after loading with dandelion polysaccharide in animals experiment.Experimental method:In this study,micro arc oxidation was used to prepare porous coatings on the surface of pure titanium.The dandelion polysaccharide was reacted with calcium ions into the layer to produce dandelion polysaccharide calcium compouds.All experimental data,including early cell adhesion tests,DAPI staining,cell proliferation experiment,and alkaline phosphatase assays for BMSCs in each experimental group and control group,was statistical analysed to assess the effects of the early cell adhesion,cell proliferation and alkaline phosphatase activity.The test level was a=0.05.Finally,in animal implantation experiments in vivo,12 male SD rats were randomly divided into 3 groups on average.According to the surgical procedure,the MAO loaded group and the Ti group titanium specimens were respectively implanted into the muscle bags,and the layers were sutured and observed closely.The three groups of animals were fed in the cage for 2 weeks.Two weeks later,collect the 2 ml of arterial blood and specimen sampling from each group.The experimental animals were sacrificed and tissue blocks containing the implant and its surrounding tissues were got.Blood routine tests and blood biochemistry were performed,gross specimens were observed,and tissues were observed by HE stained.Result:1.The surface morphology of the titanium specimen after micro arc oxidation was observed by SEM.The phase and elemental composition of the coatings on the surface of the titanium after loading with dandelion polysaccharide were determined by XRD and EDS.After the experimental treatment,the surface of the pure titanium sample forms a distinct“volcanic reactor" morphology.The phases determined by XRD are titanium,anatase,rutile and calcium titanate.The main elements of the layer are O and Ti.It also contains Ca,P,Si,and C.The calculated Ca/P ratio of the oxide layer is 1.53.2.Early adhesion of cells:At 2h and 4h,the number of cells adhered to MAO group and MAO loaded group was greater than the pure titanium group(Ti group).The difference was statistically significant(P<0.05),but there was no significant difference between micro arc oxidation treatment of titanium group(MAO group)and the coatings loaded with dandelion polysaccharide group(MAO loaded group)(P>0.05).3.DAPI staining:Inverted fluorescent light microscopy showed the three groups of cell morphology.We found that the three groups'nucleus were bright blue,round or oval,and the cell volume was basically the same.There were obvious filamentous feet.In the MAO group and MAO loaded group,the cell density is denser than the Ti group,and there are more filamentous pseudopods protruded.The morphological change of cells after dandelion polysaccharide loading was not obvious.4.Cell proliferation:At day 3,the number of surface cells in the MAO loaded group was significantly higher than that in the MAO group and the Ti group(P<0.05),and at the day 5 and 7,the number of cells on the surfaces of the MAO group and the MAO loaded group were significantly higher than those of Ti group(P<0.05),but there was no significant difference between MAO group and MAO loaded group(P>0.05).5.At day 7 and 14,the ALP activity on the surface of MAO loaded group was higher than that of MAO group(P<0.05).At day 14,the ALP activity of the two groups was not significantly different(P>0.05).6.Blood routine:The blood routine parameters of the three groups of animals after 2 weeks of the experiment are shown in Table 3-2.There was no significant difference among the three groups of blood routine indicators(P>0.05).7.Blood biochemical indicators:Table 3-3 for results of blood biochemical indicators of the three groups of animals after 2 weeks of the experiment.There was no significant difference between the three groups on ALT,BUN,Cr and UA(P>0.05).8.Gross tissue observation:The general observation showed that there was no significant difference between the groups after 2 weeks of implantation.There was a fibrous tissue surrounded by titanium in the sarcomere and there was no infection and no inflammatory reaction in the surrounding tissue.9.Observation of histological stain of HE:There was a annular fibrous wrapping the titanium in Ti group and MAO loaded group.Epithelial and keratinized epithelial debris were brought into the surgical area.Most cyst walls were smooth and localized with a small amount of lymphocyte infiltration.No vascular hemorrhage and other pathological changes appeared.Conclusion1.Micro arc oxidation was successfully used to prepare coatings rich in calcium and phosphorus elements and loading with dandelion polysaccharide.2.The coatings maded by micro arc oxidation on pure titanium could promote the early adhesion,proliferation and differentiation of BMSCs after loading with dandelion polysaccharide.3.Acorrding to the animal safety testing in vivo,it suggested that the coatings maded by micro arc oxidation on pure titanium had good biocompatibility after loading with dandelion polysaccharide. |
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