| Objective: 1 To investigate the feasibility of cocultures of autogenic bone marrow-derived mesenchymal stem cells(BMSCs) with homogeneity variant chondrocytes, in order to provide experiment basis for optimizing and extending seeding cells resources in cartilage tissue engineering. 2 To repaire full-thickness articular cartilage defects of rabbits by cocultures of autogenic BMSCs with homogeneity variant chondrocytes seeded into acellular derma matrix(ADM),grade the repaired tissues and evaluate repairing effect of defects in order to provide experimental and theoretical basis for clinical application.Methods: Thirty-six Newzealand rabbits (the age between 4 - 6 months) and twelve yong rabbits (the age between 2 - 4 weeks) were chosen in this experiment. Autogenic BMSCs were collected by density gradient centrifugalization and chondrocytes were collected by enzymatic digestion. BMSCs and chondrocytes at the concentration of 3×105/ml were collected from the second passage cells. Cocultures of BMSCs with chondrocytes at the ratio of 2 to 1 were served as experimental group A(the ultimate concentration is 3×105/ml), simple cultures of chondrocytes at the concentration of 3×105/ml as group B and simple cultures of chondrocytes at low concentration of 1×105/ml as group C(concentration equals to concentration of chondrocytes in group A) , draw cell proliferative curves and measured the contents of glycosaminoglycan(GAG)in each digestive medium. Cell carrier(ADM) were prepared by calf derma. After the cocultures were seeded into ADM and cultured for 3 days in vitro, cocultures/ADM were transplantated into full-thickness defects of the cartilages at intercondylar fossa. Thirty-six healthy Newzealand rabbits were divided into three groups randomly. There were 12 rabbits in each group. The cartilage defects in the intercondylar fossa were filled with cocultures/ADM in experimental group(cocultures/ADM group) , with only in negative control group(ADM control group),and with nothing in blank control group (blank control group). Four rabbits were killed at 4 weeks, 8 weeks and 12 weeks after transplantation in each group, repaired tissues in the zones of articular cartilage defects were observed with macroscopic views, histological scores and immunohistochemistrical stains. The data were input SPSS 13.0 software to proceed statistical analysis.Results: The average population doubling time was 3 days in group A, 7 days in group B, 8 days in group C respectively, and the differences were significant(P<0.05). The differences of the contents of glycosaminoglycan(GAG) were significant in each group (P<0.05). In the cocultures/ADM experimental group repaired tissues represented hyaline-like, integrated with peripheral cartilages and subchondral bones excellently, but repaired tissues in ADM group and blank group showed fibrous repair and no repair at 12 weeks after transplantation. Gross grading scale of repaired tissues showed that cocultures/ADM experimental group excelled control group and blank control group, differences were statistically significant(p<0.05), ADM control group excelled blank group(p<0.05); Histological grading scale of repaired tissues indicated that cocultures/ADM group excelled ADM group and blank group, differences were significant(p<0.05),but diffrences between ADM 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 hones satisfactorily in the cocultures/ADM experimental group.Conclusion: BMSCs in cultures 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 chondroctes. ADM can fit for adhesion and proliferation of cocultures. It is a kind of ideal scaffold in cartilage tissue engineering. Cocultures seeds into ADM can repair articular cartilage defects effectively.
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