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.

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.