Abstract: BACKGROUND: The capability of repairing articular cartilage damage is very limited, and tissue engineering technology provides new therapeutic options for repairing damaged cartilage, in which the interaction and induction between chondrocytes, bone marrow mesenchymal stem cells, and synovial mesenchymal stem cells is the basis of autologous healing of cartilage damage.
OBJECTIVE: To construct the chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell co-culture system to simulate the in vitro microenvironment of chondrocytes, and to explore the optimal cell inoculation ratio, meanwhile to observe the effects of icariin-containing serum on the proliferation of chondrocytes and the chondrogenic differentiation of stem cells in the system.
METHODS: Rat knee chondrocytes, bone marrow mesenchymal stem cells and synovial mesenchymal stem cells were extracted, cultured and identified, and a chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell non-contact co-culture system was constructed according to different cell inoculation ratios. After 72 hours of co-culturing, the chondrocyte proliferative activity and phenotypic ability were observed, and the co-culture system with the best overall effect was selected. New Zealand white rabbits were gavaged with icariin solution (0.25 mg/mL) to prepare icariin-containing serum, and cultured in conventional complete medium (high sugar DMEM culture medium containing 10% fetal bovine serum and 1% double antibody by volume) as the control group, while the experimental group was intervened by adding 10% icariin-containing serum by volume on the basis of above. The proliferative activity of chondrocytes and the expression of collagen type II were tested for the two groups after 24 and 48 hours. The differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells into chondrocytes in the co-culture system was tested by immunofluorescence staining after 14 days. 
RESULTS AND CONCLUSION: (1) The three kinds of cells grew normally adherently to the wall in different ratios of co-culture, where chondrocytes showed the best proliferative activity and phenotypic ability in the co-culture system when chondrocytes: bone marrow mesenchymal stem cells: synovial mesenchymal stem cells = 2:1:1. (2) Compared with the control group, the proliferative activity and type II collagen expression of chondrocytes in the experimental group were significantly increased after 24 hours (P < 0.01), and the two groups still had difference after 48 hours (P < 0.05). The two groups showed obvious chondrogenic differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells after 14 days (P < 0.01), and some of the cells appeared round or oval, and the cytoplasmic type II collagen immunofluorescence staining was positive. The fluorescence intensity of the experimental group was significantly higher than that of the control group (P < 0.01). (3) The results showed that the chondrocyte-bone marrow mesenchymal stem cell-synovial mesenchymal stem cell co-culture system could be successfully established by the non-contact co-culture method, and the best chondrocyte proliferative activity and phenotypic ability could be obtained when the cell ratio was 2:1:1. Icariin-containing serum had the promoting effect on chondrocyte proliferation, and chondrogenic differentiation of bone marrow mesenchymal stem cells and synovial mesenchymal stem cells in the system. 

Key words: icariin-containing serum, chondrocyte, bone marrow mesenchymal stem cell, synovial mesenchymal stem cell, co-culture, cell proliferation, type II collagen, chondrogenic differentiation