| ObjectiveTo explore the method and the result of repairing osteochondral defect of rabbits with tissue-engineered osteochondral graft composited by chondrocyte and nano-hydroxyapatite/chitosan(nano-HA/CS) scaffold and evaluate a new method to construct the tissue engineered osteochondral composite.Methods1.BMSCs were isolated from New Zealand rabbits through density-gradient centrifugation and cultured.2.Identify the BMSCs by morphology observations and observed the exterior antigen,cell cycle and cell activities by flow cytometry method.3.BMSCs were induced and differentiate into chondrocyte by using specific induction culture medium.They were identified by morphology,stained with Toluidine Blue.4.Nano-HA/CS was fabricated through a simple and effective technique combining with in situ hybridization and freeze-drying method.They were observed by scanning electron microscope.5.Chondrocyte and nano-HA/CS were co-cultured with the boot-shaped structure after compositing.They were observed by scanning electron microscope.6.The tissue-engineered osteochondral composite graft was implanted into the osteochondral defect in the rabbit's knee.The curative effect was evaluated by general observation,histologic analysis at 4th and 12th week.Result1.The BMSCs from rabbit could be obtained through density-gradient centrifugation. They could expand in vitro.2.BMSCs expressed CD29 and CD44 at 91.05%and 91.02%respectively while expressed CD34 and CD45 at 0.21%and 0.10%respectively.The cell activities was 95.27%and cells of G0-G1 phase were 94.68%.The shape of BMSCs were spindle. 3.The cell morphology changed from spindle to ellipsoid and triangle after cells been induced by specific induction cultural medium.Positive and strong reaction in Toluidine Blue staining.4.The nano-HA/CS with high porosity was observed by scanning electron microscope. The interstice ratio was 90%.The pore size ranged from 50～200μm,in the average of 150μm.5.Observed by the scanning electron microscope,the scaffold had good adhesion with chondrocyte.Cells could be found in the bottom of the graft which was isolated by the boot-shaped structure after 14 days while only few in the top part.6.The coupling between the bone and the cartilage in the experimental group is superior to the matched group obviously by morphology and histological grade after 4th and 12th week.Conclusion1.The BMSCs of rabbit can be obtained through density-gradient centrifugation.It can be cultured stably for long term.They are ideal for tissue engineering.2.BMSCs can be induced into chondrocyte by specific induction culture medium,3.Nano-HA/CS has good adhesion with BMSCs.4.Tissue-engineered osteochondral graft composited by boot-shaped structure can repair osteochondral defect in rabbits. |
