Oxygen concentration modulates HDAC1-Mediated regulation of osteogenic signaling pathways in dental pulp cells.

Background: Dental pulp regeneration represents a critical frontier in translational dentistry, with dental pulp stem cells (DPSCs) demonstrating exceptional reparative potential through their multipotent differentiation capacity. While oxygen tension is known to influence cellular physiology, its regulatory mechanisms on DPSC osteo/odontogenic differentiation remain poorly understood. Methods: We established physiologically relevant oxygen gradients (3%, 5%, 21% O2) to mimic developmental and pathological pulp microenvironments. Cellular proliferation and osteogenic capacity were assessed through flow cytometry, CCK-8 assays, and Live/Dead staining. Differentiation markers (RUNX2, OCN, ALP, DSPP) were quantified via qRT-PCR, immunoblotting, and enzymatic activity assays. Pharmacological inhibition studies using Oltipraz (HIF-1α inhibitor) and Valproic acid (HDAC inhibitor) elucidated pathway interactions. Publicly available transcriptomic datasets were analyzed to identify hypoxia-regulated pathways, and protein interactions were predicted using bioinformatics tools. Results: Moderate hypoxia (5% O2) significantly enhanced DPSC proliferation (p < 0.05 vs. normoxia) and upregulated osteogenic markers at transcriptional (1.8-3.2 fold) and translational levels. Severe hypoxia (3% O2) suppressed both proliferation (p < 0.01) and differentiation markers (0.4-0.7 fold). HIF-1α inhibition reversed 5% O2-mediated osteogenic enhancement (p < 0.01), while HDAC1 blockade with Valproic acid rescued differentiation capacity under 3% O2 (1.5-2.1 fold induction). Mechanistically, HDAC1 appeared to influence HIF-1α protein levels in an oxygen-dependent manner, and its inhibition affected pathways consistent with alterations in chromatin remodeling, influencing VEGFA-mediated osteogenic signaling. Conclusion: Our findings establish an oxygen-sensitive HDAC/HIF-1α regulatory axis governing DPSC fate determination. The biphasic response to hypoxia gradients suggests microenvironmental optimization strategies could enhance pulp regenerative outcomes. These insights provide mechanistic foundations for developing HDAC-targeted approaches in endodontic regeneration.