Effects of Y-27632 on the osteogenic and adipogenic potential of human dental pulp stem cells in vitro.

BACKGROUND

Human dental pulp stem cells (hDPSCs) possess mesenchymal stem cell properties, originating from migrating neural crest cells. hDPSCs have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and ability to differentiate in several cell phenotypes. In this study, we cultured hDPSCs with Y-27632 to observe their biological behaviors changes.

METHODS

The hDPSCs were separately cultured with Y-27632 (0, 0.156, 0.312, 0.625, 1.25, 2.50, 5, 10, 20, 40 μm) for 24, 48, 72 h to select the suitable concentration and time using CCK-8. Then, the hDPSCs were cultured with 2.50 μm Y-27632 for 48 h to analyzed the biological behaviors changes by 5-Ethynyl-2'-deoxyuridine (EdU), plate cloning, transwell, scratch, and Annexin V FITC/PI assays, separately. Additionally, osteogenic calcium nodules and lipid droplets were analyzed using alizarin red staining and oil red O staining, respectively. qRT-PCR was used to analyze the expression of osteogenesis, adipogenesis, stemness maintenance, and inflammation related genes.

RESULTS

The hDPSCs proliferation was significantly enhanced after cultured with 2.50 μm Y-27632 for 48 h, but there was no significant difference in migration and apoptosis. Observation of alkaline phosphatase (ALP) activity, osteogenic and adipogenic differentiation abilities of hDPSCs, Y-27632 treatment clearly decreased the ALP activity and osteogenic differentiation ability, increased the adipogenic differentiation ability. Furthermore, Y-27632 decreased the CD73, CD90, CD105, CD166, TLR4, and NF-κB p65 genes expression, but increased the IL-8 gene expression.

CONCLUSIONS

The biological behaviors of hDPSCs could be changed when they cultured with Y-27632.