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Co-Culture With Bone Marrow Mesenchymal Stem Cells And Cartilage Cells In Centrifuge Tubes For The Repair Of Caritilage In Rabbit Osteochondral Defect Model

Posted on:2017-05-24Degree:MasterType:Thesis
Country:ChinaCandidate:B HuFull Text:PDF
GTID:2284330488460809Subject:Academy of Pediatrics
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Part Ⅰ:In vitro foundation of co-culture system in centrifuge with Bone marrow mesenchymal stem cells and chondrocytesObjective Explore bone marrow mesenchymal stem cells( BMSCs) and Articular Chontrocytes(ACs)in vitro by centrifugal gathered to build co-culture system,and its ability of BMSCs differentiate into chondrocytes phenotypes in this system.Methods Rabbit BMSCs and ACs are harvested from 4-week-old New Zealand white rabbits in a sterile environment, respectively isolated 、 expanded and cultured.Accumulated and mixed the fourth passages(P4) for BMSCs and the first passages(P1)for ACs in a ratio of 1:3, the total number of cells for 4x107,in centrifuge tube at 500 g centrifugal, centrifuged for 5 minutes, It was defined as group CO. In addition, equal amounts of ACs were cultivated with the above method in the centrifugal tubes as group AC. The culture medium was changed every two days. Collect the culture medium,quantitatively detected the GAG and the collagen type Ⅱ(col-Ⅱ) with Alcian blue binding assay and enzyme-linked immunosorbent assay( ELISA).Results BMSCs were uniformly positive for CD44 and negative for CD31 and ACs could be identified with Alcian blue reaction and Saffron O dyeing. Via adherent culture method can obtain satisfactory purity BMSCs and ACs. The differences in secretion of GAG and col-Ⅱ between group CO and group AC were no statistical significance(P > 0.05).Conclusion Co-cultured BMSCs with ACs through centrifugal gathered could induce BMSCs differentiation into chondrocyte phenotypes.Part II: Transplantation of scaffold-free spheroids composed of bone marrow mesenchymal stem cells and chondrocytes for the treatment of osteochondral defects of the knee in rabbitsObjective Research the ability of repair osteochondral defects of the knee using scaffold-free spheroids of BMSCs and ACs cultured in centrifuge tube.Compared co-culture System with BMSCs and ACs and spheroids composed of them respectively in repairment for osteochondral defects in rabbits.Methods To build the osteochondral defects model of knee in rabbit : product 4 mm in diameter osteochondral defect between femoral condyle concave bilaterally in rabbit knee with hand drill. Isolated ACs and BMSCs were labeled with PKH-67 and PKH-26 respectively, prior to spheroid culture. Fabricated the cellular scaffold-free constructs centrifugal gathered in Centrifugal in ratio of 1:3,a total of 4 x 107,at 500 g for5min.Additionly, equal amounts of BMSCs and ACs were centrifugal to spheroids with the above method in the centrifugal tubes. Repair osteochondral defects of the knee in rabbits with these spheroids. Rabbits were classified into four recipient groups: a group implanted spheroids of BMSCs and ACs(group CO),a group implanted spheroids of ACs(group AC),a group implanted spheroids of BMSCs(group BMSC) and a non-transplanted control group(group Empty). After repaired articular cartilage defect in 4weeks, 8 weeks, and 12 weeks, the distal parts of the femur were harvested respectively,the caritilage defects were examined and scored macroscopically. Then the harvested specimens were cut into halves, one half were for frozen section to observe fluorescence,and the other half were for permanent paraffin section.These sections were stained with hematoxylin and eosin(H-E) and Safranin O and immuno-histochemical staining. Histological scoring were performed based on Wakitani’s histological score.Results All rabbit knee osteochondral defects gained repaired. The surface of knees in group CO and AC were better than that in group BMSC and the Empty. Cook ’s score results were statistically significant(p < 0.05) between them.The difference between Group CO and AC of Cook ’s score had no statistical significance(p > 0.05). Frozen section fluorescent displayed BMSCs and ACs jointly involved in repairing the osteochondral defects but exogenous BMSCs in group BMSC limited in their ability to repair the osteochondral defects. The difference of Wakitani’s histological grading between group CO and group Empty was statistically significant(p<0.05).And there was no statistically significant difference in Wakitani’s histological grading(p > 0.05)between group BMSC and Empty.Conclusion 1. Co-culture system in centrifuge with Bone marrow mesenchymalstem cells and chondrocytes can be used to repair the osteochondral defect in rabbit.2. BMSCs could be differentiation into chondrocyte phenotypes by ACs in co-culture system and participate in the repair of osteochondral defect, and the pre-differentiation of BMSCs were limited to repair the osteochondral defects.3.Co-culture with BMSCs and ACs formed scaffold-free spheroids was comparable to chondrocytes spheroids for repair osteochondral defect. It could be an alternation to save chondrocytes.
Keywords/Search Tags:Bone marrow mesenchymal stem cells, carticular chondrocyte cartilage, co-culture, Scaffold-free, Tissue engineering
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