Circadian rhythm disruption impairs time-dependent dentine regeneration and reprograms the late-stage dental proteome in a mouse pulp injury model.

Circadian rhythms coordinate metabolic and regenerative programs, yet their contribution to pulp healing and tertiary dentinogenesis remains unclear. Here, we investigated whether chronic circadian rhythm disruption (CRD) alters time-dependent dentine repair and proteomic remodeling after dental pulp injury (DPI) in male BALB/c mice. Animals were maintained either under a stable 12:12 h light-dark cycle or under a chronic jet-lag model with a daily 2 h phase advance. DPI was induced in the maxillary first molars, followed by immediate direct pulp capping with mineral trioxide aggregate and sealing with glass ionomer cement. Histological and histomorphometric analyses were performed at days 7, 28, and 56 post-injury, while open-field and light/dark transition tests were conducted on day 56. Untargeted LC-MS/MS proteomics was additionally performed on day-56 dental tissues to define injury-, circadian-, and dual-regulated molecular signatures. CRD produced a behavioral phenotype characterized by reduced locomotor activity and increased anxiety-like behavior. Histology revealed a canonical reparative pattern after DPI, with peripheral tertiary dentine formation surrounding a central connective tissue core; however, CRD consistently attenuated newly formed dentine area at all time points, indicating compromised odontoblast-like differentiation and mineral deposition. Proteomic profiling identified extensive remodeling of the dental proteome, with injury- and CRD-dependent signatures converging on metabolic regulation, cytoskeletal dynamics, immune pathways, and extracellular matrix organization. Integrated analyses delineated 204 injury-specific proteins, 128 CRD-specific proteins, and 86 co-regulated proteins, resolving the response into injury-dominant, circadian-dominant, dual-regulated, and condition-independent modules. Collectively, these results demonstrate that chronic circadian misalignment impairs dentine regenerative capacity and reprograms the dental proteome, suggesting that circadian status may be an underappreciated determinant of outcomes in vital pulp therapy and regenerative endodontic procedures.