| Background:The regeneration of osteochondral defects is scientifically challenging,due to the insufficient natural healing of the defects.Tissue engineering and regenerative medicine provides an alternative method to restore the osteochondral defects.Many studies using biological or synthetic scaffold promotes cell attachment,proliferation,differentiation.The composite scaffold that a three-layer structure(layer of bone/bone and cartilage interface layer/layer of cartilage),which were Modelled on morphology and structure of joints,were prepared by combination of poly(hydroxybutyrate-co-hydroxyoctanoate),with excellent performance and independent intellectual property rights,and collagen-type I.Early experiments have confirmed that the poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold,the pore structure and biocompatibility was desirable,could support BMSCs attachment,proliferation,and facilitate their differentiation.But the poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold combined with bone marrow mesenchymal stem cells in repairing articular osteochondral defects in weight-bearing area effect needs to be further research.Objective:To investigate the effect of poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold combined with bone marrow mesenchymal stem cells on repair of weight-bearing area of articular osteochondral defects in rabbits.Methods:1.The poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold of a three-layer structure were made of poly(hydroxybutyrate-co-hydroxyoctanoate)and collagen-type I at different Particle size(150~220?m in the cartilage layer,60~150?m in the middle layer,220~450?m in the bone layer),fabricated using the solvent casting-particulate leaching method;We use the adherent culture method for isolation and culture of bone marrow mesenchymal stem cells,three of the differentiation for identification its biological activity;The third-passage BMSCs cell suspensions(3×10~5cells/mL)were seeded drop by drop into the poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold;BMSCs cultured on the scaffolds were analyzed for proliferation by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.2.Cone-shaped osteochondral defects(4.5mm in depth,4.5mm in length,3mm in width)were created in the femoral medial condyle of right knee of 45 New Zealand white rabbits and were placed a hinged external fixator,The animals were randomly divided into three groups: the scaffold-cell group,in which poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold combined with bone marrow mesenchymal stem cells were implanted into the defects;the scaffold group,in which poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold were implanted alone into the defects;and the control group,in which osteochondral defects were filled with no implantation.3.At 4,8,12 weeks after surgery,animals were sacrificed for gross observation and macroscopically analyzed using ICRS system,Micro-CT scanning and three-dimensionally reconstructed,HE staining,safranin O staining,and immunohistochemical staining of collagen II,histologically analyzed using Wakitani's scoring system,in order to comprehensive judgment defect repairing.Results:1.ICRS cartilage defect grade: At each time point,The macroscopical score of the scaffold-cell group was higher than those of the other two groups(P<0.05),and the scaffold group had a higher score than the control group(P<0.05).2.Micro-CT scanning and threedimensionally reconstruction suggested that bone formations were present at the peripheral areas and developed toward the central area.From 4 weeks to 12 weeks after surgery,the scope of subchondral bone defect decreases at each group.By 12 weeks after surgery,the subchondral bone formation in the scaffold group and the control group were incomplete at the central area,endochondral bone formation in the scaffold-cell group progressed,and almost the whole area corresponding to the subchondral bone was replaced by trabecular bone.3.Histological staining: Until 12 weeks after the operation,repair tissue almost entirely fibrous histiocytoma in the control group;For simple group,after 12 weeks,the reconstruction of the subchondral bone is not sufficient,the repairing cartilage was fibrocartilage,cartilage and subchondral bone layer boundaries is not obvious;at 12 weeks after surgery,Compared with the scaffold group and control group,in the scaffold-cell group,almost the whole area corresponding to the subchondral bone was replaced by bone structures,tidemark appears,the cell distribution and morphology of regenerative cartilage in the scaffold-cell group,whose contains more glycosaminoglycan,was quite close to the normal cartilage.4.The immunohistochemistry of collagen-II: Until 12 weeks after the operation,repair tissue of collagen-II in the control group did not express;from 4 weeks to 12 weeks,the expression of scaffold group and scaffold-cell group showed an increasing trend,at each time point,the expression of the scaffold-cell group were higher than that the scaffold group;at 12 weeks after surgery,the expression of scaffoldcell group were similar to the normal cartilage.5.Wakitani cartilag repair and score showed that over time,Wakitani scores in each group were decreasing,but compared with 4 weeks,the Wakitani score lower in the control group at 8 weeks was not statistically significant(P>0.05);at each time point,the Wakitani score of scaffold-cell group was lower than those of the other two groups(P<0.05),and the scaffold group had a lower score than the control group(P<0.05).Conclusion:The poly(hydroxybutyrate-co-hydroxyoctanoate)/collagen composite osteochondral scaffold exactly contributes to the weight-bearing area of articular osteochondral defects repair,combined with bone marrow mesenchymal stem cells further promote the repair of osteochondral defect. |
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