Astaxanthin enhances migration, Type I and Type III collagen production, and osteogenic differentiation in periodontal ligament fibroblasts under hyperglycemic conditions.

BACKGROUND: Periodontal wound healing is impaired under hyperglycemic conditions. Astaxanthin (AST), a potent antioxidant carotenoid, has shown promise in tissue regeneration; however, its effects on human periodontal ligament fibroblasts (HPLFs) under high glucose conditions remain unclear. Therefore, this study aimed to examine the effects of AST on cytotoxicity, proliferation, migration, collagen production, osteogenic differentiation, and oxidative stress in HPLFs under hyperglycemic conditions. METHODS: Cytotoxicity and the optimal dose of AST were evaluated using a lactate dehydrogenase (LDH) assay and a Cell Counting Kit-8 (CCK-8) under normal glucose condition (5.5 mM). Subsequently, the effects of AST were investigated under hyperglycemic conditions (25 and 50 mM). Cell migration was analyzed using scratch and transwell assays. Collagen synthesis was assessed using picrosirius red staining and enzyme-linked immunosorbent assay (ELISA). Osteogenic potential was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining, and ALP activity. Collagen (COL3A1 and COL1A1) and osteogenic differentiation (runt-related transcription factor 2; RUNX2, osterix; OSX, ALP, and osteocalcin; OCN) genes were measured using real-time polymerase chain reaction (RT-qPCR). RUNX2 and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expressions were determined by western blotting. Reactive oxygen species (ROS) production was measured by the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. RESULTS: AST concentrations above 125 µM were cytotoxic to HPLFs, whereas 15 µM AST significantly enhanced cell proliferation from days 3 to 7. Under normal glucose, 15 µM AST effectively promoted collagen production, cell migration, and osteogenic differentiation. Under hyperglycemic conditions, AST partially mitigated the suppression of these cellular activities. AST improved cell migration and promoted collagen production at both mRNA and protein levels on day 14. AST also induced osteogenic differentiation, with upregulation of bone-related genes on day 14 and mineralization on day 21. Furthermore, AST reduced intracellular ROS levels and increased Nrf2 expression under both normal and hypoglycemic conditions. CONCLUSION: AST can increase cell migration, collagen production and stimulate bone differentiation under high glucose conditions, probably through its antioxidant properties. These findings support AST as a potential adjunctive agent to promote periodontal wound healing and bone formation in periodontal or other bone-related diseases.