POs-Ca Drives Osteogenic Differentiation of Human Dental Pulp Stem Cells Via AMPK-Dependent Autophagy Activation and Reciprocal Calcium-Autophagy Crosstalk.

Human dental pulp stem cells (hDPSCs) hold significant promise for bone regeneration, yet efficient osteogenic induction remains challenging. Phosphorylated oligosaccharides of calcium (POs-Ca), a novel calcium salt derived from potato starch, has recently attracted attention for its remineralization capabilities and potential to promote stem cell differentiation. Here, we investigated the impact of POs-Ca on the osteogenic differentiation of hDPSCs and its underlying mechanism. Isolated hDPSCs were characterized via flow cytometry based on mesenchymal surface markers. Biocompatibility and osteogenic differentiation were assessed via Cell Counting Kit-8 (CCK-8) assay, alkaline phosphatase (ALP) activity, Alizarin Red S staining, and protein levels of osteogenic (Collagen I, DSPP, DMP1, and RUNX2). Intracellular Ca2+ flux was monitored using Fluo-4 AM, while AMP-activated protein kinase (AMPK) signaling and autophagic flux were analyzed by western blot (p-AMPK, p-ULK1, and light chain 3 (LC3)-II), TEM, and LC3-GFP imaging. Mechanistic studies employed verapamil (Ca2+ channel blocker), Compound C (CC;AMPK inhibitor), and chloroquine (CQ;autophagy inhibitor). POs-Ca (5 mg/mL) exhibited excellent biocompatibility and significantly promoted osteogenic differentiation, as evidenced by a 3.22-fold increase in ALP activity and markedly enhanced mineralized nodule formation as shown by Alizarin Red S staining. Mechanistic studies revealed that POs-Ca triggers rapid intracellular Ca2+ influx, activating the AMPK pathway and inducing autophagic flux. Pharmacological inhibition established the essential causality of this cascade: verapamil abolished osteogenic enhancement, while CC and CQ suppressed ALP activity, mineralization, and osteogenic marker expression. Notably, CQ reciprocally attenuated POs-Ca-induced Ca2+ influx, revealing novel bidirectional Ca2+-autophagy crosstalk. In conclusion, POs-Ca might promote hDPSCs osteogenesis via a calcium influx-driven AMPK-autophagy axis, providing a foundation for novel biomaterials that exploit physiological calcium signaling. These findings offer immediate translational potential for developing minimally invasive, cost-effective strategies in dental pulp regeneration and bone defect repair.