The Interplay of <scp>M1</scp> Macrophages and Dental Pulp Stem Cells Promotes Angiogenesis Through <scp>IL</scp> ‐8‐Dependent <scp>VEGF</scp> Regulation: An In Vitro Study

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 × 10 4 versus 7.5 × 10 4 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 &lt; 0.05). Results M1 macrophages at low density (5 × 10 4 cells/device) significantly enhanced vascularization in the vasculature‐on‐a‐chip, increasing vascular segments ( p &lt; 0.0001) and free sprouts ( p &lt; 0.05–0.01) compared to M0 or no‐macrophage controls, with effects comparable to M2. High‐density M1 seeding (7.5 × 10 4 cells/device) reduced sprouts ( p &lt; 0.0001 day 4, p &lt; 0.01 day 5) due to increased cytotoxicity ( p &lt; 0.0001). Both THP‐1‐ and PBM‐derived M1‐conditioned media (CM) showed significantly elevated IL‐8 levels. M1 CM (THP‐1/PBM) induced DPSC VEGF secretion, blocked by Reparaxin ( p &lt; 0.01–0.0001), confirming IL‐8 mediation via CXCR1/2. Exogenous IL‐8 (0.5 ng/mL) upregulated DPSC VEGF protein/mRNA ( p &lt; 0.05) and Matrigel tube formation (segments/junctions p &lt; 0.05). M1‐DPSC CM enhanced vascular meshes/segments on Matrigel ( p &lt; 0.05), reduced by Reparaxin. RNA‐seq of M1 co‐cultured DPSCs identified 17 angiogenic genes (logFC &gt; 1.2), with HIF‐1α as a hub gene and an enriched MAPK/ERK pathway. Western blot analysis confirmed MAPK/ERK‐HIF‐1α as a contributory pathway in IL‐8‐induced upregulation of VEGF in DPSCs. Conclusion M1 macrophages promote angiogenesis via IL‐8‐induced DPSC VEGF secretion through CXCR1/2‐MAPK/ERK‐HIF‐1α signalling density‐dependently, suggesting that therapeutic modulation rather than total suppression of M1 activity could improve outcomes in vital pulp therapy.