Uncultured bone marrow mononuclear cells delay the dedifferentiation of unexpanded chondrocytes in pellet culture

Uncultured bone marrow mononuclear cells (BMMC) were recently used to successfully repair damaged cartilage. However, the effect of BMMCs on the proliferation and differentiation of chondrocytes that are critical to cartilage repair is unclear. Here, we investigate the influence of BMMCs on chondrocyte dedifferentiation in pellet culture. We isolated and mixed BMMCs and chondrocytes in a 1:1 (BMMC/C) ratio and cultured in pellets (1.6 × 10 6 cells per pellet) for 2, 4, or 8 weeks. Chondrocyte differentiation was evaluated using macrography, histological examination, immunohistochemistry and gene expression analysis. While a transparent and smooth surface was observed in both BMMC/C and chondrocyte cultures over time, the former was smaller in size after 2 and 4 weeks of culture. Interestingly, after 8 weeks, BMMC/C cultures became significantly larger than chondrocyte cultures ( P = 0.003). The distribution of a cartilage-specific extracellular matrix (ECM), that includes components like glycosaminoglycan (GAG) and type II collagen, was gradually reduced in chondrocyte cultures. On the other hand, while we found no obvious differences in the ECM in BMMC/C cultures between 2 and 4 weeks in vitro, after 8 weeks the concentration of ECM components decreased significantly. Further, we detected an upregulation of cartilage-specific genes in BMMC/C cultures, when compared with chondrocytes. Altogether, we demonstrate that co-culture with BMMCs delays the dedifferentiation of chondrocytes in pellet cultures in vitro. This suggests that uncultured BMMC, which can be quickly and safely obtained, could serve as a potential alternative cell source for engineering of cartilage tissue.