Abstract

BACKGROUND Dental pulp inflammation triggers immune responses involving macrophages and dental pulp stem cells (DPSCs), which interact to regulate angiogenesis essential for tissue repair. M1 pro-inflammatory macrophages predominate early in pulpitis, and clarifying their angiogenic role is vital in identifying inflammatory regenerative mechanisms. METHODOLOGY THP-1 cells and peripheral blood monocyte (PBM)-derived macrophages were polarized to M1 or M2 phenotypes, characterized by qRT-PCR, ELISA, and angiogenesis arrays. A vasculature-on-a-chip comprising DPSCs, human umbilical vein endothelial cells (HUVECs) and THP-1-derived macrophages was imaged, and the vascular segments/sprouts were quantified using ImageJ. Density effects used 5 × 104 versus 7.5 × 104 M1 macrophages/device, with propidium iodide staining for cytotoxicity. IL-8 effects on DPSC VEGF secretion were assessed by ELISA (with/without Reparaxin 1 μM), Matrigel tube formation assays, and exogenous IL-8 (0.5 ng/mL). Transwell co-cultures underwent RNA sequencing and bioinformatics analysis, which identified candidate hub genes and signalling pathways; the results were validated by Western blotting (p-ERK, HIF-1α; ERK inhibitor SCH772984, 25 nM). Statistical testing was performed using ANOVA with Tukey's post hoc test (p < 0.05). RESULTS M1 macrophages at low density (5 × 104 cells/device) significantly enhanced vascularization in the vasculature-on-a-chip, increasing vascular segments (p < 0.0001) and free sprouts (p < 0.05-0.01) compared to M0 or no-macrophage controls, with effects comparable to M2. High-density M1 seeding (7.5 × 104 cells/device) reduced sprouts (p < 0.0001 day 4, p < 0.01 day 5) due to increased cytotoxicity (p < 0.0001). Both THP-1- and PBM-derived M1-conditioned media (CM) showed significantly elevated IL-8 levels.