Comparison of rabbit bone marrow mesenchymal stem cells and chondrocytes co-cultured in vitro for the establishment of four kinds of cartilage without scaffold

Objective A large number of cells in four groups were cultured without scaffold in centrifuge tube, observing size and biological characteristics of aggregation co-culture system of cartilage tissue, and evaluate co-culture technology without support by centrifugal tube, which provides methodological guidance and experimental basis for the clinical repair of articular cartilage defect. Methods New Zealand white rabbits underwent puncture of bone marrow. Rabbit articular cartilage chondrocytes were separated, obtain relatively pure by type Ⅱ trypsin and collagenase, and then cultured. Isolation and culture of bone marrow mesenchymal stem cells by density gradient centrifugation to obtain the 3 passage of purified Bone marrow mesenchymal stem cells (BMSCs). Using alcian blue staining, safranin O-light green staining and type Ⅱ collagen immunohistochemical staining of Articular Chontrocytes (ACs) were identified. CD34, CD44, CD45 and CD105 molecular surface markers by immunofluorescence identification of BMSCs. BMSCs and ACs were mixed into co-culture (CO) groups according to the ratio of 1∶3 and 2∶2, the total number of cells was 4×107, 1 500 r/min centrifugation for 5 minutes, and the cell aggregation was co cultured in 15 ml centrifuge tube. The equivalent chondrocytes were taken as AC group, and the same amount of BMSCs was induced into the BMSCs induction group (transforming growth factor-beta 1, TGF-β1), and cultured in the centrifuge tube as comparative research. Four groups of high number of cells were cultured for 1 weeks, 2 weeks, 3 weeks and 4 weeks after paraffin tissue sections were dewaxed by alcian blue staining, saffron "O" -light green staining and immunohistochemical staining for type Ⅱ collagen. HE staining was used to observe the morphology and structure of cartilage tissue in each group under the condition of in vitro culture. Using alcian blue colorimetric method for quantitative detection of liquid supernatant of glycosaminoglycan content, enzyme-linked immunosorbent adsorption method was used to detect the liquid supernatant of type Ⅱ collagen content. Results ACs alcian blue, safranin O-light green and type Ⅱ collagen immunohistochemical staining were positive, the adherent subculture could obtain satisfactory purity of BMSCs and ACs. BMSCs immunofluorescence staining of CD44 (+ ), CD105 (+ ), CD45 (-), CD34 (-). AC group, 2∶2 and 1∶3 co-culture group, alcian blue staining, safranin O-light green staining and type Ⅱ collagen immunohistochemical staining were positive. The staining of BMSC induced group was weakly positive. HE showed that AC group and 1∶3 group were gradually fused into initial cartilage tissue. Gradually the integration of group cells induced by 2∶2 And BM, but no cartilage formed after four weeks. After 4 weeks, group AC [(179.43±59.19) μg/ml] and 2∶2 [(158.06±23.77) μg/ml], 1∶3 group [(160.06±30.58) μg/ml] GAG content had no significant difference (F=0.210, P=0.812). After 4 weeks, group AC [(185.13±35.96) ng/ml] and 2∶2 [(169.72±32.56) ng/ml], 1∶3 [(148.91±33.16) ng/ml] type Ⅱ collagen content had no significant difference (F=0.680, P=0.515); AC group [(179.43±59.19) μg/ml] and BM induced group [(70.74±40.17) μg/ml] GAG content was statistically significant (t=3.980, P=0.001); AC group [(185.13±35.96) ng/ml] and BM induced group [(116.83±40.17) ng/ml] type Ⅱ collagen content was statistically significant (t=3.434, P=0.003). Conclusion BMSCs and ACs were cultured in centrifuge tube by aggregation can induce differentiation of BMSCs to ACs, and get the biological characteristics of the AC. The AC group and 1∶3 co-culture group formed the rudiment of cartilage tissue after four weeks with HE staining, but BM induced group and 2∶2 group did not. Key words: Articular chondrocytes; Mesenchymal stem cells; Co-culture; No-scaffold; Tissue engineering cartilage