| OBJECTIVES:1.Construction of loading naringin osteochondral composite scaffolds and evaluated its relative performance;2.The biocompatibility of the composite scaffolds was evaluated by co-culture of loading naringin osteochondral composite scaffolds,cartilage layer and bone layer scaffold with BMSCs respectively;3.evaluated the effect of loading naringin osteochondral composite scaffolds for repairing knee osteochondral defects in rabbit.METHODS:1.Preparation of loading naringin microspheres,no loading naringin microspheres by water in oil in water phase method;Naringin as the sustained release drug for chondrocyte proliferation,TGF-?1 as positive control agent for chondrocyte proliferation,constructed the cartilage layer scaffold by mixing freeze dry method with type ? collagen respectively;Used attapulgite and type ? collagen by the method of solution casting and particle precipitation to construct the bone layer scaffold;By 3 layers sandwich method to construct no loading naringin osteochondral composite scaffolds ? loading naringin osteochondral composite scaffolds ? loading TGF-?1 osteochondral composite scaffolds.2.Morphology of microspheres and osteochondral composite scaffolds were observed by naked eye and SEM.Determinated the loading drug rate,encapsulation rate and in vitro drug release rate of loading naringin microspheres;Determinated the porosity rate?swelling rate of water absorption of cartilage layer and bone layer scaffold,and the drug release effect of loading naringin cartilage layer scaffold in vitro.3.BMSCs as seed cells,and evaluated the biocompatibility of loading naringin osteochondral composite scaffolds.SEM was used to observe the adhesion and growth of BMSCs on cartilage layer and bone layer scaffold respectively;CCK-8 method was used to detect the proliferation of BMSCs on cartilage layer and bone layer scaffold.4.48 clean grade male oryctolagus cuniculus were randomly divided into 4 groups A?B?C?D,and there were 12 rabbits in each group.After a 4.5 mm-diameter?4 mm-depth osteochondral defects were drilled in the intercondylar fossa of two femurs of rabbit,group A was defect(blank control)group,group B?C?D was implanted respectively no loading naringin osteochondral composite scaffolds(negative control group)?loading naringin osteochondral composite scaffolds(experimental group)and loading TGF-?1 osteochondral composite scaffolds(positive control group).5.Did 320 row dynamic volume CT testing at 3? 6 months after implantation,then the intercondylar fossa of two femurs was removed for the general?Micro CT?HE staining?toluidine blue staining and Immunohistochemical staining to observe the repairing effect respectively.RT-PCR and Western blot method respectively detected the expression of type II college mRNA and protein in newborn cartilage.RESULTS:1.Observation of loading naringin microspheres by naked eye was white powder,light;The microspheres were spherical and smooth,not bond to each other and the particle size was about 5-50 ?m by SEM.The loading drug rate of loading naringin microspheres was 5.41 % ±0.02 %,and the encapsulation efficiency was 27.03 % ± 0.07 %.The average drug release rate of loading naringin microspheres was 14.51 %;The average drug release rate of loading naringin osteochondral composite scaffolds was 4.39 %.2.The osteochondral composite scaffolds were white,cylindrical,with the diameter of 4.5 mm and the thickness of 4 mm,the thickness of the cartilage layer scaffold was 2 mm and the born layer scaffold was 2 mm.SEM revealed that the structure of loading naringin composite scaffolds was obvious,the cartilage layer scalffod was tightly connected with the bone layer scalffod;the cartilage layer scalffod was porous lamellar structure and the arrangement was irregular and connected with each other,and microspheres were dispersed in the scaffolds,its poraity rate was more than 95 % and swelling rate of water absorption was more than 150 %;bone layer scaffold structure was dense,aad had different sizes of aperture interlinke and the pore size ranging from 200-300 ?m.3.Cell proliferation rate of loading naringin cartilage layer scaffold was significantly increased compared with no loading naringin cartilage layer scaffold,and the difference was statistically significant(P<0.05).4.General observation after implantation revealed that defects range of group C?D was reduced significantly compared with group A?B at 3 months after implantation;At 6 months after implantation,defects of group C were covered by newborn cartilage,and that of group D was well integrated with the surrounding normal cartilage.Micro CT revealed that there was no significant difference(P>0.05)in the expression of bone mineral density(BMD)between group A?B?C?D at 6 months after implantation.The results of histological staining revealed that the defects were filled with a small amount of fibrous tissue in group A?B,and a small amount of newborn cartilage in group C?D at 3 months after implantation;The newborn cartilage in group C?D was similar to that of normal cartilage,the defects were filled with a large amount of fibrous tissue in group A?B at 6 months after implantation.5.RT-PCR results revealed that the expression of type II collagen mRNA in the group C?D were significantly increased compared with the group A?B,and the difference was statistically significant(P<0.05),and that there was no significant difference between the group C and D(P>0.05).Western blot results revealed that the expression of type II collagen in the group C?D were significantly increased compared with the group A?B,and the difference was statistically significant(P<0.05),and that there was no significant difference between the group C and D(P>0.05).CONCLUSION:1.Loading naringin osteochondral three layers composite scaffolds have high porosity?swelling rate of water absorption,and have good drug release rate in vitro;2.BMSCs can proliferate ?grow on composite scaffolds,and the composite scaffolds have good biocompatibility.3.Loading naringin composite scaffolds have good repair effect on rabbit knee articular osteochondral defects.The repairing efficiency is not different from that TGF-?1 osteochondral composite scaffold. |
PDF Full Text Request