Macrophage‐Pulp Fibroblast Interactions Modulate Initial Dental Pulp Regeneration In Vitro

AIM: Injured fibroblasts have been shown to play a significant role in controlling pulp inflammation and regeneration. Recent works have demonstrated that, depending on the stimulation type, fibroblasts induce macrophage differentiation into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. This work was set to study macrophage effects on the initial steps of pulp regeneration, namely on pulp stem cell (DPSC) proliferation/migration as well as on neo-angiogenesis. METHODOLOGY: Human pulp cells were isolated from third molars. DPSCs and pulp fibroblasts were obtained using cell sorting and characterisation. To mimic a deep carious lesion, fibroblasts were physically injured and incubated with Lipoteichoic Acid (LTA). Physically injured fibroblasts without adding LTA were used to simulate an inflammatory state without pathogen exposure. Undifferentiated macrophages (M0) were incubated with stimulated fibroblast supernatants to induce M1/M2 differentiation. After 24 h, the macrophage secretome was used to investigate the effects on DPSC viability with the MTT assay. Its effect on DPSC migration towards macrophages was performed using Boyden chambers. VEGF secretion by macrophages was quantified by ELISA. The macrophage secretome effect on endothelial cell viability was investigated using MTT assay, and that on neo-angiogenesis using endothelial cell organisation on Matrigel. RESULTS: The role played by macrophages in the initial steps of pulp inflammation and regeneration appears to be under the control of fibroblasts which determines macrophage differentiation into M1 or M2. Indeed, incubation of M0 with injured fibroblast supernatants induced a significant increase of DPSCs and endothelial cell viability, VEGF secretion, and endothelial cell organisation into tube-like structures. These effects are comparable to those of M2. However, incubating them with injured and LTA-stimulated fibroblasts induced similar effects to those of M1 macrophages including stem cell migration, VEGF secretion level and endothelial cell viability. CONCLUSION: This in vitro study shows that, depending on the type of stimulation, pulp fibroblasts induce macrophage differentiation into M1 or M2 which, in turn, modulate DPSC proliferation/migration and neo-angiogenesis. This highlights that interactions between different cell types play a significant role in the initial steps of pulp tissue regeneration.