FTY720P-treated macrophages in PEG-4MAL hydrogels promote oral wound healing

Oral wound healing involves hemostasis, inflammation, proliferation and tissue remodeling. The oral cavity is a complex wound healing environment because of the presence of saliva, a high bacterial burden and ongoing physical trauma from eating. The inflammatory component of wound healing balances the polarization of macrophages in healing tissues between M1 inflammatory macrophages and M2 anti-inflammatory macrophages. M2 macrophages secrete anti-inflammatory and pro-regenerative cytokines and chemokines, which aid wound healing. Fingolimod, or FTY720, a Food and Drug Administration–approved sphingosine-1-phosphate modulator, has been implicated in inducing the polarization of macrophages to the M2 phenotype. In this study, we investigated whether macrophage pre-treatment with phosphorylated FTY720 (FTY720P), the bioactive form of the drug, in a PEG-4MAL hydrogel promotes improved oral wound healing in a critically sized oral mucosal defect model. Using cytokine dot blots and Luminex cytokine assays (MilliporeSigma, Burlington, MA, USA), FTY720P-treated murine RAW 264.7 and human THP1-differentiated macrophages in PEG-4MAL hydrogels secreted chemokines and cytokines known to regulate inflammation (e.g., interleukin [IL] 4, IL-13) and induce M2 macrophage polarization (e.g., CCL6, CCL22), leukocyte migration (e.g., CXCL2, CCL2, CCL12, CCL22), angiogenesis (e.g., vascular endothelial growth factor) and epithelialization (e.g., IL-1, IL-17, IL-22). In vitro, FTY720P-treated cells induced chemotaxis of macrophages and fibroblasts in Transwell assays (Corning, Corning, NY, USA) and oral epithelial scratch wound closure. In a murine oronasal fistula (ONF) model of oral wound healing, the local application of FTY720P-treated macrophages in PEG-4MAL hydrogels significantly increased wound closure (75% closure) relative to non-treated cells (40% closure) and blank hydrogel controls (25% closure) (P < 0.0001). Flow cytometry of mouse palatal tissue showed that application of FTY720P-treated macrophage hydrogels to ONFs significantly increased day 7 percentage of M1 and M2 macrophages, mesenchymal stromal cells and CD19+ B cells. Significantly fewer neutrophils, monocytes, CD4+/CD8+ T cells and endothelial cells were observed in the FTY720P-treated macrophage defects, suggesting that FTY720P-treated macrophages in hydrogels promote oral wound healing via suppression of granulation and resolution of inflammation and promotion of tissue maturation. Our data provide ne