The Best Ratio Of Co-culture Of Chondrocytes And Bone Arrow Mesenchymal Stem Cells In Tissue-Engineered Cartilage

ObjectiveThis research was to establish a co-culture system of rabbit chondrcytes and bone marrow mesenchymal stem cells, to explore the best ratio of co-culture to enhance the expression of ECM in vitro, and to confirm the best ratio of co-culture onto PLGA to construct tissue engineered cartilage in vitro and in vivo.Methods1. The mononuclear cells were isolated by density gradient centrifugation through Percoll and then cultured in DMEM/F12 containing 10% fetal bovine serum. Light microscope was used to observe the cell morphology and flow cytometry was used to examine the expression of cell surface antigen. The well 2nd generation cells were divided into three groups: the first group was induced to an adipogenic lineage; the second group was induced to an osteogenic lineage; the third group was induced to a chondrogenic lineage. Intracellular lipid vesicles from the adipogenic monolayer cultures were stained with oil red O. Cytoplasmic alkaline phosphatase from differentiated osteoblasts was stained with ALP solution. Safranine O and toluidine blue were used to detect chondrogenesis of BMSCs.2. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte: BMSCs ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 3 weeks. BMSCs were cultured in chondrogenic induction medium. Equal numbers of cells (10~5) were plated for each condition in tissue-culture flasks. Specimens were harvested after 1, 2 and 3 weeks of in vitro culture. Quantitative real-time RT-PCR and Western blot were used to evaluate the results.3. Rabbit articular chondrocytes and BMSCs were cultured and then expanded separately in vitro. After the second passage of cells was digested and collected, cell density was adjusted to 4.0×l0~7/ml. The two distinct cells were mixed as the previous experiment. Equal numbers of cells (10~7) were seeded onto the 4×4×2mm PLGA scaffolds. After being cultured for 8 weeks in vitro, half of the scaffolds were transplanted subcutaneously in the dorsum of nude mice for another 8 weeks. All samples were observed macroscopically, histologically and immunohistochemically. The quantitation of GAG and DNA of the tissue-engineered cartilages was done.Result1. BMSCs were purified and all in the whirlpool shape. Flow cytometry showed that more than 98% cells expressed CD29 and CD44, while only 5% of the cells expressed CD34. The BMSCs exposed to osteogenic medium were strongly positive for ALP staining. Adipogenesis was confirmed by the presence of neutral lipid vacuoles that were strongly positive with oil red O staining. GAG and type II collagen were confirmed by toluidine blue and safranine O staining.2. In the co-cultures, type II collagen and aggrecan expression increased on weeks 2 and 3. At the mRNA level, the expression of type II collagen and aggrecan on week 3 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSCs group, and the best ratio of co-culture groups seems to be 2:1. Also on week 3, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than that in all other groups. On week 3, the expression of type I collagen in the 4:1, 1:1, 1:2, 1:4, and induced BMSCs groups was lower in the chondrocytes group, whereas there was no statistically significant difference between the 2:1 group and the chondrocyte group.3. The tissue engineered cartilage of 2:1 co-culture group was morphologically the thickest, and the most strongly positive for oil red O, ALP, safranine O and toluidine blue staining with all the scaffolds cultured for 8 weeks in vitro, and half of them cultured for another 8 weeks in vivo. The cells in tissue engineered cartilage of 2:1 co-culture group were obviously arranged in clusters, which were surrounded by ECM. So the cartilage-lacunae-like constructions could be observed. The GAG/DNA index in 2:1 group was much higher than that of all other groups (P<0.05). The quality of chondrocytes group and 4:1 group was less satisfied than that of 2:1 group. The quality of chondrocytes group and 4:1 group was less satisfied than that of 2:1 group. The quality of 1:1 group, 1:2 group and 1:4 group was reduced as the number of initial chondrocytes decreased with the tissue-engineered cartilages of the above three groups small and tough, and the GAG/DNA index of them much lower than that of 2:1 group (P<0.05). The tissue-engineered cartilage of the induced BMSCs group was similar to that of 1:1 group in vitro, however it was similar to that of 1:4 group in vivo. And at the end of the fifth week, the scaffold was dissolved, the cells flowed away and the tissue-engineered cartilage not formed in BMSCs group.ConclusionThe method of isolation and culture of rabbit BMSCs is simple and feasible. BMSCs could be induced into adipocytes, osteoblasts and chondrocytes by induced culture medium in vitro. The co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1. Furthermore, the best ratio of co-culture of chondrocytes and BMSCs on PLGA is also 2:1 after all scaffolds are cultured for 8 weeks in vitro, and half of them cultured for another 8 weeks in vivo.