Vitamin D Effects on Osteoblastic Differentiation of Mesenchymal Stem Cells from Dental Tissues

Abstract

1α,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃), the active metabolite of vitamin D (Vit D), increases intestinal absorption of calcium and phosphate, maintaining a correct balance of bone remodeling. Vit D has an anabolic effect on the skeletal system and is key in promoting osteoblastic differentiation of human Mesenchymal Stem Cells (hMSCs) from bone marrow. MSCs can be also isolated from the immature form of the tooth, the dental bud: Dental Bud Stem Cells (DBSCs) are adult stem cells that can effectively undergo osteoblastic differentiation. In this work we investigated the effect of Vit D on DBSCs differentiation into osteoblasts. Our data demonstrate that DBSCs, cultured in an opportune osteogenic medium, differentiate into osteoblast-like cells; Vit D treatment stimulates their osteoblastic features, increasing the expression of typical markers of osteoblastogenesis like RUNX2 and Collagen I (Coll I) and, in a more important way, determining a higher production of mineralized matrix nodules.

Figure 1

ALP positivity and mineralized nodules formation. (a) ALP histochemical assay (purple staining) performed on DBSCs seeded and differentiated for 7 days with vehicle (−) and 1,25(OH)₂D₃ (+). The graph shows quantification of positive staining as percentage compared to untreated cells (−) (^∗ P < 0.05) and is representative for 3 independent donors. Data are presented as mean ± SEM. Student's t-test was used for single comparisons. (b) Alizarin Red (red staining) shows mineralized nodules formation by DBSCs incubated for 21 days in osteogenic medium with vehicle (−) and 1,25(OH)₂D₃ (+). The graph shows quantification of ARS with the Optical Density (OD) at 405 nm (b); ^∗indicates statistical significance (P < 0.001). Data are representative for 3 independent donors and are presented as mean ± SEM. Student's t-test was used for single comparisons.

Figure 2

Protein expression. Immunoblots showing the trend expression of RUNX2 (a), Coll I (b), Pro-Coll I (c), BSP (d), and OPN (e) during the osteogenic differentiation process (0–21 days) of DBSCs cultured in osteogenic medium with vehicle (−) and 1,25(OH)₂D₃ (+). Each graph represents means ± SEM of 3 independent experiments. ^∗ P < 0.01 compared to undifferentiated samples (und) and ^# P < 0.05 compared to untreated samples (−). Student's t-test was used for single comparison.