Interkingdom Endodontic Biofilm Supernatant Induces Biphasic Inflammatory and Metabolic Reprogramming in Dental Pulp Stem Cells

This study investigate how soluble by-products derived from a four-species endodontic biofilm model impacts upon the viability, transcriptomic profile, and inflammatory response of human dental pulp stem cells (DPSCs). The sterile-filtered supernatant was extracted from an established interkingdom endodontic biofilm model comprising Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, and Candida albicans. DPSCs were exposed to the microbial biofilm supernatant (BSN) for 4 and 24 hours. Cellular responses were evaluated via MTT, CCK-8, LDH assays, and Annexin V/PI staining. Whole transcriptomic sequencing was performed to assess gene expression dynamics, with GO and KEGG pathway enrichment analyses. IL6 and IL8 expression was validated by qPCR and ELISA.It was found that BSN significantly suppressed DPSC metabolic activity without inducing apoptosis or necrosis. RNA-seq revealed 723 significantly differentially expressed genes at 4 h and 1667 at 24 h. Early responses were dominated by upregulation of inflammatory mediators (e.g., CXCL8, IL6, TNFAIP2), with enrichment of TNF, NF-κB, and JAK-STAT signalling pathways. At 24 h, the expression profile shifted toward redox regulation and metabolic suppression, including downregulation of glycolytic and purine metabolism pathways. IL6 and IL8 expression was markedly increased at both transcript and protein levels. Indeed, soluble factors produced by a biofilm model representative of deep caries and carious pulp exposures are capable of reprogramming DPSC function in a time-dependent manner. The soluble factors initiate a biphasic response characterized by early immune activation and later metabolic adaptation. The findings underscore the potential role of biofilm-derived products associated with deep caries and carious pulp exposures in compromising DPSC function. In addition, they emphasise the need for the development of vital pulp therapy strategies that not only neutralise microorganisms but also their secreted products.