Piezo1 negatively regulates proliferation, but enhances mineralization in human cementoblasts

Objectives: Cementoblasts play essential roles in secreting collagenous and non-collagenous matrix proteins and in mineralization to produce cementum. Cementum is a mineralized tissue deposited in layers on the surface of the tooth root, where it is subjected throughout life to mechanical stresses including chewing and occlusal forces. To date, the detailed mechanosensitivity mechanisms regulating cementoblasts remain unclear. Methods: We investigated cellular functions driven by mechanosensitive processes in human cementoblasts (HCEM) by analyzing protein expression using immunofluorescence staining and mechanical stimulation-induced Ca²⁺signaling by measuring intracellular free Ca²⁺concentration ([Ca²⁺]_i) using the Ca²⁺indicator fura-2. We also assessed cell proliferation modulation using colony-forming unit fibroblast (CFU–F) analysis, and mineralization efficacy using Alizarin Red and von Kossa staining. Results: HCEM were immunopositive for cementoblast marker proteins, cementum attachment protein, and cementum protein 1, and the mechanosensitive cation channels Piezo1 and Piezo2. Both direct mechanical stimulation and pharmacological stimulation with the Piezo1 activator Yoda1 elicited transient increases in [Ca²⁺]_i, which were significantly suppressed by Dooku1, a pharmacological Yoda1 inhibitor. In the CFU-F assay, colony formation was significantly enhanced by treatment with pharmacological Piezo1 inhibitors, and by GsMTx4, Dooku1, and Piezo1 gene silencing, but was suppressed by Yoda1. HCEM mineralization efficacy was significantly promoted by Yoda1 but significantly suppressed by GsMTx4, Dooku1, and Piezo1 gene silencing. Conclusions: Piezo1 suppresses colony formation, but activates HCEM-mediated mineralization, suggesting that Piezo1-induced Ca²⁺signaling plays an important role in cementum mineralization and deposition by cementoblasts.