Maresin-1/Resolvin-E1 Improve Regenerative Potential of Human Periodontal Ligament Stem Cells

Objectives: To evaluate the impact of Maresin-1(MaR1) and Resolvin-E1(RvE1) on regenerative functions of human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions
Methods: hPDLSCs were treated with MaR1(10nM) and/or RvE1(10nM) with or without IL-1β(10ng/mL) and/or TNF-α(10ng/mL) for 24h. Pluripotency markers (CD45,CD11b,CD73,CD90,CD105,CDHLA-ABC,Oct-4, and Sox-2) were evaluated by Flow cytometry (FC). Cell proliferation, apoptosis (AnnexinV staining), and in vitro wound healing were measured. Markers of periodontal ligament regeneration (Tenomodulin,α-SMA, and Periostin) were analyzed by qPCR, immunofluorescensce, and FC. After 7-days osteogenic differentiation treatment, alizarin red staining was performed, alkaline phosphatase measured by ELISA, cementum/osteogenesis markers (Runx2,Osteocalcin,CEMP1, and CAP) analysed by qPCR and Western blotting, and ChemR23 expression evaluated by FC. Quantitative phosphoproteomics, followed by principal component analysis and hierarchical clustering were performed and pathways identified by using KEGG, Reactome, PANTHER, and Wikipathway databases
Results: Pro-inflammatory stimuli downregulated pluripotency of hPDLSCs, impaired in vitro wound healing, induced cell death, and downregulated the expression of periodontal ligament, cementum, and osteogenic markers. MaR1/RvE1 restored hPDLSC pluripotency (e.g. Sox2 MFI: TNF-α=8211±225 vs TNF-α+RvE1=10444±890, p=0.012], apoptosis (e.g.%Annexin⁺ cells: TNF-α=15.1±0.7 vs MaR1+TNF-α=8.76±0.6, p<0.01), and increased cell migration and proliferation (e.g.IL-1β=1.8±0.14x10⁵ vs IL-1β+MaR1=2.9±0.1x10⁵ cells, p<0.01), ligament markers (e.g.%α-SMA⁺cells: TNF-α=72.1±1.5 vs TNF-α+MaR1=86.2±8,p<0.001), formation of calcified deposits (TNF-α=2±0.3 vs MaR1+TNF-α=12.6±0.7 nM,p<0.001), and cementum-osteogenic markers (e.g. Runx2 Fold-Change:TNF-α=1.1±0.3 vs MaR1+TNF-α=7.9±0.6,p<0.001). MaR1 reversed the effect of TNF-α stimulus through phosphorylation of intracellular cascades including anti-apoptosis, mRNA processing, cell cycle, PI3K-Akt, mTOR, Rho-GTPases, and Notch1 pathways
Conclusions: Mar1/RvE1 improved regenerative functions of hPDLSCs in an infammatory environment, increasing their pluripotency, proliferation, migration, and differentiation, suggesting a direct impact on regeneration of periodontal tissues