ABSTRACT
Objective:To investigate the feasibility of construction of tissue engineered cartilage by co-culture of bone marrow mesenchymal stem cells (BMSCs) and costal chondrocytes (CCs),and to provide theoretical basis and experimental basis for clinical repair of articular cartilage defects by Wuzhishan miniature pig knee cartilage defects with co-cultured cells.Methods:Density gradient centrifugation method was used to isolate BMSCs from Wuzhishan miniature pig.The double enzyme digestion method was used to isolate CCs.The passage 3 generation of BMSCs and passage 2 generation of CCs were randomly divided into 3 groups:a co-culture group of BMSCs∶CCs for 1∶2 (Group A),a simple CCs (Group B),and a simple BMSCs (Group C).The cell growth curve was drawn,and the content of glycosaminoglycan (GAG) of external separation in chondrocytes was determined.The 12 Wuzhishan miniature pigs were randomly divided into a co-culture cells/collagen membrane experimental group,a collagen membrane control group and the blank group.In the co-culture cells/collagen membrane experimental group,the co-cultured cells/collagen membrane were implanted into the cartilage defects of the mandibular condyle;in the collagen membrane control group,only collagen membrane was implanted;while in the blank group,nothing was implanted.Six animals were sacrificed at 8 and 16 weeks after surgery respectively (2 animals in each group).General observation,cartilage histological score and histopathological examination were carried out.Results:The BMSCs and co-culture cells grew well.The biological activity of CCs was good.After 16 weeks of operation,the repair tissues in the co-cultured cells/collagen membrane experimental group showed hyaline cartilage features:smooth,flat,and integrated well with the surrounding cartilage and subchondral bone.The collagen membrane in the collagen membrane control group was fibrously repaired.Repair tissue gross score in the co-culture cells/collagen membrane experimental group was significantly better than that in the collagen membrane control group and the blank group (both P0.05).Conclusion:BMSCs,CCs and co-cultured cells can function as the seed cells for cartilage tissue engineering,and the co-culture cells (BMSCs∶CCs=1∶2) possess more advantages;the short-term effect of co-culture cells with collagen membrane on repairing cartilage defects is satisfied.
ABSTRACT
Objective To explore inhibition of nicotine on apoptosis of chondrocytes induced by monosodium iodoacetate ( MIA) .Methods Rat primary chondrocytes were isolated by enzyme digestion, and the cells were treated with 10 -8 , 10 -7 , 10 -6 , 10 -5 mol/L nicotine for 48 h.The cases were randomly divided into five groups, except for normal group, the other four groups were treated with 4μmol/L MIA 24 h, and three groups were treated 10 -8 , 10 -7 , 10 -6 mol/L nicotine.The viability of chondrocytes was detected by MTT assay.The apoptosis of chondrocytes was examed by Annexin V-FITC/PI flow dual-staining method.The activity of cysteinyl aspartate specific proteinase 3 ( Caspase 3 ) was measured by spectrophotography method.The activation of phosphatidylinositol 3 kinase ( PI3K)/protein kinase B ( AKT) and the expression of down-stream molecule Bax, Bcl-2 was assayed by western blot.Results 10 -7 , 10 -6 mol/L nicotine increased chondrocytes' viability (P0.05).10 -8, 10 -7, 10 -6 mol/L nicotine could increase MIA-induced chondrocytes' viability (P<0.05), suppress MIA-induced chondrocytes' apoptosis and the activity of MIA-induced Caspase 3 (P <0.05).Moreover, 10 -7, 10 -6 mol/L nicotine could increase the expression of PI3K and phosphorylation of AKT ( P<0.05) , down-regulate the expression of Bax and up-regulate the expression of Bcl-2 in MIA-induced rat chondrocytes ( P<0.05 ) .Conclusion These results suggested nicotine could exert anti-apoptosis in MIA-induced rat chondrocytes, which might be related to PI3K/AKT signal pathway.