Stem cell therapy is a new approach in veterinary medicine for the treatment of complicated disorders such as articular cartilage injuries in horses. Although the equine bone marrow-derived mesenchymal stem cells (eBMSCs) have a unique capacity in chondrogenic differentiation, it needs to be optimized. In the current study, we evaluated the enhancement of chondrogenic differentiation in eBMSCs using transforming growth factor beta-3 (TGF-β3) and bone morphogenic protein 6 (BMP-6) in vitro. The characterized eBMSCs at passage 3 (P3) were cultivated as micro-pellets for 21 days under different conditions: (1) basic culture medium (BCM), (2) chondrogenic differentiation medium (CDM), (3) CDM with BMP-6, (4) CDM with TGF-β3, and (5) CDM with BMP-6 and TGF-β3 to evaluate the chondrogenic differentiation by assessment of pellet size, histological characteristics, and gene expression profile. Pellet size in groups that contained growth factors had a significant difference in comparison with the control group or chondrogenic group without growth factors. The histological assessment showed that the cartilage progression score in groups containing TGF-β3, alone or combined with BMP-6, was significantly higher in comparison with that of the other groups (P < 0.05). Additionally, the aggrecan gene was expressed in all groups except the control group, while collagen type II was just expressed in groups containing TGF-β3, alone or combined with BMP-6. TGF-β3 may have more promising effects on the enhancement of chondrogenic differentiation of eBMSCs in comparison with BMP-6. However, the combination of TGF-β3 and BMP-6 promoted chondrogenic differentiation of eBMSCs in a synergistic manner.

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