Study On Full-Thickness Articular Cartilage Defects Repaired By Cocultures Of Bone Arrow-Derived Mesenchymal Stem Cells With Chondrocytes Seeded Into Allogenic Demineralized Bone Matrix

OBJECTIVE: 1. To investigate the feasibility of cocultures of autogenic bone marrow-derived mesenchymal stem cells (BMSCs) with chondrocytes, elucidate the probable interactive mechanism between BMSCs and chondrocytes in order to provide experimental basis for optimizing and extending seeding cells resources in cartilage tissue engineering. 2. To detect the degradable rate, porosity and adhesive rate of demineralized bone matrix(DBM) in vitro₃/To repaire full-thickness articular cartilage defects of rabbits by cocullures of autogenic BMSCs with chondrocytes seeded into allogenic DBM ,grade the repaired tissues and evaluate repairing effect of defects in order to provide experimental and theoretical basis for clinical application. METHODS: 1._Autogenic BMSCs and chondrocytes at the concentration of 3×10⁵/ml were collected from the second passage cells. Cocultures of BMSC_s with chondrocytes at the ratio of 2 to 1 were served as experimental group A, simple cultures of chondrocytes at the concentration of 3×10⁵ cells/ml as group B and simple cultures of chondrocytes at low concentration of 1×10⁵ cells/ml as group C(its concentration equals to concentration of chondrocytes in cocultures) , drew cell proliferative curves and measured the contents of glycosaminoglycan(GAG)in each digestive medium. 2.Allogenic DBM were prepared according to Urist's method. Ultrastructure of DBM were observation with scanning electron microscope. DBM were put into PBS solution and tested its degradable rate and porosity dy liquid replacement method. Adhesive rate of cocultures into DBM were detcted. 3.After the cocultures were seeded into DBM and cultured for 3 days in vitro, cocultures/DBM were transplantated into full-thickness defects of the cartilages atintercondylar fossa. Thirty-six healthy Qingzilan rabbits were divided into three groups randomly. There were 12 rabbits, in each group. The cartilage defects in the intercondylar fossa were filled with autogenic cocultures/DBM in experimental group(cocultures/ DBM group), with only DBM in negative^control group(DBM group), and with nothing in blank control group(blank group).Six rabbits were killed at 6 weeks and 12 weeks after transplantation in each group, repaired tissues in the zones of articular cartilage defects w<>re observed with macroscopic views, histological scores and immunohistochemistrical stains. The data were input SPSS 10.0 software to proceed statistical analysis.RESULTS: L The average population doubling time was 3 days in group A , 7 days in group B ,8 days in group C respectively.The contents of glycosaminoglycan (GAG) were 81.00 + 3. 52(Mg/ml) in group A, 61. 67 + 6. 71 (ug/ml) in group B, 20. 33±2. 50(Mg/ml) in group C , the differences were significant in each group(P<0. 05). 2/'DBM showed spongy-like structure with scanning electron microspe .diameter of pore arranged from 210Mra to 390Mm, porosity were 88.00+1.83. The degradation increase with the time prolonged .complete degradation required over 12 weeks in vitro. After cocultures were seeded into DBM, the shapes of BMSCSand chondrocytes maintained premixed state, adhesive rate of cocultures into DBM were the highest at the third day, adhesive rates of cocultures into DBM had no significant differenc.es between third day and fifth day(/->0. 05), but adhesive rate occurred degressive tendency while coculture continued. 3^ In the cocultures/DBM experimental group repaired tissues represented hyaline-like, integrated with peripheral cartilages and subchondral bones excellently, but repaired tissues in DBM group and blank group showed fibrous repair and no repair at 16 weeks after transplants! ion. Macroscopic scores of .repaired tissues showed that cocultures/DBM experimental group excelled DBM control group and blank control' group, differences were statistically significant(/X0. 01), DBM control group excelled blank group (p<0. 01);Histolog i cal scores of repaired tissues indicated that cocultures/ DBM group excelled DBM group and blank group, differences were statisticallysignificant (/XO. 01), but differences between DBM control group and blank control group were not significant (p>0. 05). Immunohistochemistrical stains of repaired tissues showed that chondrocytes in the zones of repaired tissues arranged columnnedly, riched in type-II collagen matrix and integrated with adjacent cartilages and subchondral bones satisfactorily in the cocultures/DBM experimental group.CONCLUSIONS: L_BMSCs in cocultures can promote proliferation of chondrocytes and production of chondral matrix. Cocultures as seeding cells can shorten culturing periods, reduce subculture times of chondrocytes and save a large number of chondrocytes. 2. Ultrastructure of DBM can fit for adhesion and proliferation of cocultures. DBM is compatible with cocultures. Biodegradation of DBM in vivo can synchronize cartilage formation in articular cartilage defect zone in phase. So DBM is a kind of ideal scaffold in cartilage tissue engineering. 3. Cocultures embedded.into DBM can repair articular cartilage defects effectively.