To develop novel metformin carbon dots (MCDs) with superior performance to enhance the osteogenic differentiation potential of human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions.

Three types of MCDs-MCDsCA, MCDsAA, and MCDsCS were synthesized via a one-step hydrothermal method using metformin (Met) in combination with citric acid (CA), ascorbic acid (AA), and carboxymethyl chitosan (CS), respectively. Their physicochemical properties were characterized by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and UV-visible spectrophotometry. The biocompatibility and cellular uptake of the MCDs were assessed using CCK-8 assay and confocal laser scanning microscopy. Alkaline phosphatase (ALP) staining and ALP activity assay were conducted to identify the most effective MCDs type in promoting osteogenic differentiation of hPDLSCs under inflammatory conditions. Subsequently, ALP and Alizarin Red S (ARS) staining, qRT-PCR, and Western blotting were performed to assess the effects of the optimal concentration of MCDsCS and its raw material (Met+CS) on osteogenic differentiation under inflammatory conditions.

All 3 types of nanoscale MCDs exhibited favorable physicochemical characteristics and biocompatibility, and could be effectively internalized by hPDLSCs. Both MCDsCA and MCDsAA significantly promoted hPDLSCs proliferation at lower concentrations (0~0.50 mg/mL), while MCDsCS exhibited excellent cytocompatibility over a broader range (0~2.00 mg/mL). Under inflammatory conditions, MCDsCA and MCDsAA showed no significant effect on ALP expression in hPDLSCs, whereas MCDsCS significantly enhanced ALP expression in a dose-dependent manner, with the optimal effect observed at 0.10 mg/mL (P<0.05). Compared to Met+CS, 0.10 mg/mL MCDsCS significantly enhanced ALP expression and calcium nodule formation under inflammatory conditions. It also upregulated the expression of osteogenesis-related genes (ALP, RUNX2, COL-1, OCN) and proteins (ALP, RUNX2, OCN) (P<0.01), while reducing the levels of inflammatory cytokines IL-1β and IL-6 (P<0.01).

A novel MCDsCS with sound biocompatibility is successfully developed, and can effectively promote osteogenic differentiation of hPDLSCs and exerts anti-inflammatory effects under inflammatory conditions, indicating its potential as a nanotherapeutic agent for periodontitis-associated bone regeneration.