Preparation Of Hydroxyapatite/Chitosan-TGF-β₁ Composite Coatings Implants Implantation In Diabetic Rabbit In Vivo Osteogenesis Of Experimental Research

Objective:Using physical-chemical-biological joint method of Hydroxyapatite/Chitosan-transforming growth factor-β₁（HA/CS-TGF-β1）release microspheres composite coating was prepared on the surface of the implant.The implant was implanted into the animal model of diabetic rabbits and the osteogenesis effect was observed around the implant.To provide experimental basis for increasing the bone grafting rate of diabetes.Methods:Part I:Select 50 adult male New Zealand white rabbits,after adaptive feeding one week were randomly divided into three groups.Three groups were:one-time injection of drug group A（n=20）,two injections of drug group B（n=20）and blank control group C（n=10）.By ear vein injection of 5%alloxan induced diabetic animal models.Group A was given an injection dose of 100 mg/kg,group B was 50 mg/kg for the first injection,100 mg/kg for the second time,and group B for the same volume of saline.After 1 month,the blood glucose stabilized within the range of 19.0 mmol/l-24 mmol/l,and the model was considered successful.Postoperative monitoring of the two groups of animals in general,blood glucose changes,molding,mortality,blood glucose recovery rate and other information,compare the advantages and disadvantages of the two groups of models Using SPSS17.0 statistical software,the experimental data using chi-square test,repeated measurement of variance analysis of statistical data,P<0.05 for the difference was statistically significant.PartⅡ:After the implant was cleaned,the surface of the implant was subjected to micro-arc oxidation treatment using a sulfuric acid electrolyte system,dried,soaked dopamine,dried,soaked in HA/CS hydrogel,dried,The microspheres EDC and NHS containing TGF-β₁ protein were cross-linked to the implant surface.Using SEM,XRD,EDS were used to detect the implant surface coating morphology,crystal structure and chemical composition of elements,Test composite coating release microspheres release amount。The bioactivity and cytotoxicity of the implants were determined by MTT assay.PartⅢ:Twenty-seven diabetic rabbits were randomly divided into three groups:control group A（smooth implant group）,group B（micro-arc oxidation group）and compound group C（the HA/CS-TGF-β₁ composite coating was prepared by micro-arc oxidation）.The implant was implanted into the metaphyseal side of the bilateral tibia of the animal.At 4w,8w and 12w after operation,3 rats were sacrificed in each group.The bone specimens with implants were fixed in 4%neutral formalin,and then use methylamine blue-basic fuchsin,Measurement and analysis of bone implant contact rate（BIC）,the formation of new bone area ratio（BA）.Animals were injected with fluorescent dyes in sequence according to the sequence of fluorescent markers,after drawn using confocal mining,and measure the amount of mineralized bone deposition（MAP）,to observe the situation around the implant osseointegration.Results:The part I:Blood glucose values between the experimental groups was not statistically significant（P>0.05）.Blood glucose fluctuation in group A was less than that in group B,Blood glucose fluctuation in the second month after operation was earlier than that in group B,and the amplitude was less than that in group B,A group of more than group B,into the earlier stable blood sugar,There was no significant difference in the failure rate,mortality and modulus between the two groups（P>0.05）in the first month after modeling,but at the second month,there was statistical difference between the two groups（P<0.05）,The rate of formation,mortality and recovery were significantly lower in group A than in group B.PartⅡ:the micro-arc oxidation of the implant,the surface of the uniform formation of a layer of porous oxide layer,After the composite coating is visible between the modified release microspheres have a reunion phenomenon,cross each other into a network,For the success of bone cell crawling to provide a good space,through the Beckman laser particle size analyzer analysis and calculation,we can see that the microsphere particle size of about 8.78μm.For the success of bone cell crawling to provide a good space,through the Beckman laser particle size analyzer analysis and calculation,we can see that the microsphere particle size of about 8.78μm.Using EDX analysis,it was found that the material deposited a rich calcium phosphate salt,increased the biological activity of the implant surface.In vitro cell experiments of implants showed that the material showed better osteoblast activity and no significant cytotoxicity was observed.Part III:Test results showed a fluorescent label（P<0.05）,and the time of bone mineralization in group C was significantly higher than that in control group（P<0.05）.Methylamine blue-alkaline fuchsin staining results showed that:4w after each group have varying degrees of new bone formation,new bone arrangement irregular.Methylamine blue-alkaline fuchsin staining results showed that:4w after each group have varying degrees of new bone formation,new bone arrangement irregular.Methylamine blue-alkaline fuchsin staining results showed that:4w after each group have varying degrees of new bone formation,new bone arrangement irregular.The bone contact rate（BIC）of the implant on the group C was significantly different from that of the A and B groups（P<0.05）Far away from the implant there osteoblastsThe composite coating has protein release,which can promote the proliferation and differentiation of osteoblasts around the implant.In each group can be seen that after the 8 week different maturity Harvard bone plate,on the 12 week the bone tissue remodeling basically completed,showing a mature Haversian system..The difference of BIC between the A and B groups was not significant（P>0.05）.The difference was not significant in the bone contact rate between the two groups.（P<0.05）.There was no significant difference in BA between group A and group B（P>0.05）.The difference of BA between group A and group B was not significant（P>0.05）.In this study,single injection of the drug into the mold,significantly reduced blood sugar recovery rate of animal and improve the animal’s final molding rate,for the establishment of a long observation period,an animal model of diabetes blood sugar stable.The implants of HA/CS-TGF-β₁sustained-release micro-arc oxidation composite coating were implanted into the diabetic rabbits by in vivo experiments.The osteogenesis of the surroundings was observed,Gelatin microspheres can prolong the release of TGF-β₁ protein,can accelerate the formation of new bone around the implant,promote implant-bone combination.To promote diabetes planting osseointegration rate and provide experimental basis.