Resolution agonists are endogenous mediators that drive inflammation to homeostasis. We earlier demonstrated in vivo activity of resolvins and lipoxins on regenerative periodontal wound healing. The goal of this study was to determine the impact of resolvin D1 (RvD1) on the function of human periodontal ligament (PDL) fibroblasts, which are critical for wound healing during regeneration of the soft and hard tissues around teeth. Primary cells were cultured from biopsies obtained from three individuals free of periodontal diseases. Peripheral blood mononuclear cells were isolated by density gradient centrifugation from whole blood of healthy volunteers. PGE2, leukotriene B4 (LTB4), and lipoxin A4 (LXA4) in culture supernatants were measured by ELISA. The direct impact of RvD1 on PDL fibroblast proliferation was measured and wound closure was analyzed in vitro using a fibroblast culture "scratch assay." PDL fibroblast function in response to RvD1 was further characterized by basic FGF production by ELISA. IL-1β and TNF-α enhanced the production of PGE2. Treatment of PDL cells and monocytes with 0.1-10 ng/ml RvD1 (0.27-27 M) reduced cytokine induced production of PGE2 and upregulated LXA4 production by both PDL cells and monocytes. RvD1 significantly enhanced PDL fibroblast proliferation and wound closure as well as basic FGF release. The results demonstrate that anti-inflammatory and proresolution actions of RvD1 with upregulation of arachidonic acid-derived endogenous resolution pathways (LXA4) and suggest resolution pathway synergy establishing a novel mechanism for the proresolution activity of the ω-3 docosahexaenoic acid-derived resolution agonist RvD1.

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 hours in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 hours), but wound closure was equal to TGF-β1 alone at 48 and 72 hours. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function.

Background The resolution of inflammation is an active process mediated by specialized lipid mediators called lipoxins and resolvins. Periodontal ligament fibroblasts (PDLFs) play a significant role in periodontal regeneration. The purpose of the current study was to determine the impact of resolvin D1 (RvD1) on human PDLF cell wound healing and proliferation, receptor expression (G‐protein‐coupled receptor 32 [GPR32] and formyl peptide receptor 2 [ALX/FPR2]), and cytokine expression and release. Methods PDLFs were stimulated with interleukin‐1β (IL‐1β) (500 pg/ml) with and without RvD1 (100 nM). RvD1 receptor expression was determined by quantitative real‐time polymerase chain reaction (qPCR), immunofluorescence microscopy, and fluorescence‐activated cell sorting. Wound closure was measured by a scratch assay, and proliferation was determined by bromodeoxyuridine incorporation. Interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), monocyte chemoattractant protein‐1, cyclooxygenase‐2, matrix metalloproteinases‐1, ‐2, and ‐3 (MMP‐1, ‐2, and ‐3), tissue inhibitors of metalloproteinases‐1 and ‐2 (TIMP‐1 and ‐2), prostaglandin E2, and osteoprotegerin (OPG) gene expression and production were measured using qPCR and Western blotting, multiplex immunoassay, and enzyme‐linked immunosorbent assay. Results PDLF expressed GPR32 and ALX/FPR2. RvD1 reversed IL‐1β‐induced inhibition of wound healing and proliferation of PDLF. IL‐1β also induced the production of proinflammatory cytokines and MMPs. This effect was reversed by RvD1. RvD1 reversed IL‐1β‒induced inhibition of TIMP‐1, TIMP‐2, and OPG. Conclusion The data suggested that RvD1 has a pro‐wound healing, proliferative, and anti‐inflammatory impact on the PDLF that favors periodontal regeneration.