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

Abstract

Oral wound healing involves hemostasis, inflammation, proliferation, and tissue remodeling. The oral cavity is a complex wound healing environment due to 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, an FDA-approved sphingosine-1-phosphate (S1P) modulator, has been implicated in inducing the polarization of macrophages to the M2 phenotype. In this study, we investigated if macrophage pre-treatment with phosphorylated FTY720P (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, 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., IL-4, IL-13), induce macrophage M2 polarization (e.g., CCL6, CCL22), leukocyte migration (e.g., CXCL2, CCL2, CCL12, CCL22), angiogenesis (e.g.,VEGF), and epithelization (e.g., IL-1, IL17, IL22). In vitro, FTY720P-treated cells induced chemotaxis of macrophages and fibroblasts in transwell assays, 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 both 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 promoting tissue maturation. Our data provide new insights on the use of potential FTY720P-treated macrophage therapies for oral wound healing and have clinical implications for cleft palate and oral surgery.