Silymarin Effects on Ovine Fetal Bone Marrow-Derived Mesenchymal Stem Cells Differentiation into Osteogenic Lineage

Cell therapy using mesenchymal stem cells (MSCs) can be a promising tool in regenerative medicine. One of the richest sources of mesenchymal stem cells is fetal bone marrow. Silymarin has strong antioxidant and anti-inflammatory activities with a positive effect on the proliferation of some cells as well as anti-osteoporosis properties. This study aimed to show the effect of silymarin on the differentiation of mesenchymal stem cells derived from the bone marrow of sheep embryos into the osteogenic line.

Mesenchymal stem cells were isolated from the bone marrow of sheep embryos. MTT test was performed to investigate the cytotoxicity of silymarin on cells at different concentrations for 24 and 72 hours. Then, cells in one of 8 groups 1: negative control; 2: treated with 10 μmol/liter silymarin in the usual environment, 3: treated with 20 μmol/liter silymarin in the usual environment, 4: treated with 100 μmol/liter estradiol in the usual environment, 5: positive control, 6: treatment treated with 10 μmol/liter silymarin in the differentiation medium, 7: treated with 20 μmol/liter silymarin in the differentiation medium, 8: treated with 100 μmol/liter in the differentiation medium, were cultured for 21 days. To determine the osteogenic differentiation of cells, the deposition of hydroxyapatite ions was examined using alizarin staining, and also, the amount of ALP enzyme secretion was also measured in the studied groups.

Comparing the average optical absorption of cells at different concentrations between 24 and 72 hours after treatment showed that the average optical absorption of cells at zero concentration of silymarin after 72 hours of treatment decreased in comparison with those treated for 24 hours (P<0.05), but no significant difference was observed in other concentrations (P>0.05). Examining the level of ALP enzyme secretion, 21 days after treatment with silymarin in the studied groups showed that the highest level of enzyme secretion was in group 8 (P≤0.05). The lowest amount of enzyme secretion was observed in group 1 (negative control) and then in group 2 and group 3 respectively (P<0.05). No significant difference was observed between groups 4, 5, and 6 (P>0.05). Based on the alizarin red staining results, calcium ions deposition was observed in all the groups related to the differentiation medium, which increased in groups 8, 7, 6, and 5, respectively. In the groups cultured in the usual environment, there was no calcification in group 1 and the amount of calcification increased in groups 2, 3, and 4, respectively. In total, the amount of calcification in the differentiation environment groups was higher in comparison with the usual environment.

During this study, Silymarin had no toxic effect on the mesenchymal stem cells derived from the bone marrow of sheep embryos in the studied concentrations after 24 and 72 hours of treatment. It increased the differentiation of the cells into the osteogenic lineage in a concentration-dependent manner. Therefore, it seems that with further studies and identification of the molecular pathways of silymarin's effect, it can be used in cell therapy in order to repair bone lesions.