Orofacial clefts are the most common congenital craniofacial anomaly. Adverse healing following cleft palate repair can lead to oronasal fistula (ONF), a persistent connection between the oral and nasal cavities. Although human allograft tissues are currently the gold standard for ONF repair, these methods carry risks of infection and rejection, often requiring surgical revision. Immunoregenerative therapies present a novel alternative approach to harness the body's immune response and enhance the wound healing environment. An FDA‐approved immunomodulatory drug, FTY720, is repurposed to reduce lymphocyte egress and induce immune cell fate switching toward pro‐regenerative phenotypes. In this study, a bilayer biomaterial system is engineered using Tegaderm to secure and control the delivery of FTY720‐nanofiber scaffolds (FTY720‐NF). The release kinetics of the bilayer FTY720‐NF is optimized to maintain drug release for up to 7 days, ensuring safe transdermal absorption and tissue biodistribution. The comprehensive immunophenotyping results demonstrate a regenerative state transition in hybrid immune cells recruited to the wound site. Further, histological evaluations reveal a significant ONF closure in mice by day 7 following bilayer FTY720‐NF implantation. These findings demonstrate the utility of immunomodulatory strategies for oral wound healing, better positing the field to develop more efficacious treatment options in pediatric patients.

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 new insights into the use of potential FTY720P-treated macrophage therapies for oral wound healing and have clinical implications for cleft palate and oral surgery.

Mesenchymal stromal cells (MSCs) exert regenerative and immunomodulatory effects largely through secreted paracrine factors and extracellular vesicles (EVs), which transfer proteins, lipids, and nucleic acids to recipient cells. Lipid composition critically influences EV stability, uptake, and bioactivity. Sphingomyelinase (SMase), an enzyme that hydrolyzes sphingomyelin into ceramide, regulates EV biogenesis by inducing membrane curvature and initiating inward membrane budding. Here, MSCs were treated with SMase, and EVs were isolated and characterized by nanoparticle tracking analysis, miRNA sequencing, lipidomics, and proteomics. SMase treatment increased EV yield and altered lipid, protein, and miRNA cargo linked to TNF-α signaling, wound healing, and angiogenesis. Functionally, SMase-EVs suppressed TNF-α in macrophages, showed trending increased HUVEC tubular formation, and altered T cell populations following local delivery in a critical murine oral wound defect model. These findings highlight how enzymatic lipid remodeling modifies MSC-EVs, enhancing their therapeutic potential and informing strategies for optimized EV-based therapies and scalable production.

SUMMARY: Mesenchymal stromal cells (MSCs) exert regenerative and immunomodulatory effects largely through secreted paracrine factors and extracellular vesicles (EVs), which transfer proteins, lipids, and nucleic acids to recipient cells. Lipid composition critically influences EV stability, uptake, and bioactivity. Sphingomyelinase (SMase), an enzyme that hydrolyzes sphingomyelin into ceramide, regulates EV biogenesis by inducing membrane curvature and initiating inward membrane budding. Here, MSCs were treated with SMase, and EVs were isolated and characterized by nanoparticle tracking analysis, miRNA sequencing, lipidomics, and proteomics. SMase treatment increased EV yield and altered lipid, protein, and miRNA cargo linked to TNF-α signaling, wound healing, and angiogenesis. Functionally, SMase-EVs suppressed TNF-α in macrophages, showed trending increased HUVEC tubular formation, and altered T-cell populations following local delivery in a critical murine oral wound defect model. These findings highlight how enzymatic lipid remodeling modifies MSC-EVs, enhancing their therapeutic potential and informing strategies for optimized EV-based therapies and scalable production.

Mesenchymal stromal cells (MSCs) are widely studied for their immunomodulatory and tissue reparative capabilities, but clinical translation has been hampered by inconsistent efficacy and limited standardization in manufacturing. While cytokine-based priming methods, such as interferon-gamma (IFN-γ) stimulation, have shown promise in enhancing MSC potency, alternative approaches targeting distinct biological metabolism integral to secretome and membrane architecture have not been explored in MSCs. In this study, we investigate sphingomyelinase (SMase), an enzyme that generates ceramide from sphingomyelin, as a novel lipid-based priming strategy to modulate MSC function. Here, human MSCs were treated with SMase and high-content imaging and morphological profiling revealed that SMase-treated cells adopted a phenotype overlapping with IFN-γ–licensed MSCs, including increased cell compactness and solidity. Lipidomic analysis showed broad alterations in sphingolipid species, and dynamic flux estimation (DFE) modeling predicted distinct metabolic shifts in SMase-treated cells compared to untreated controls. These changes were sustained up to 35 hours post-stimulation, indicating stable metabolic reprogramming. SMase priming also altered the MSC secretome, enriching for factors implicated in immune regulation. Functionally, SMase-primed MSCs retained the ability to suppress T-cell activation and promote anti-inflammatory macrophage phenotypes. Collectively, these findings demonstrate that SMase stimulation induces a durable, immunomodulatory-like state in MSCs through coordinated changes in lipid metabolism and secretory activity. This lipid-centric priming approach represents a promising alternative to cytokine-based licensing strategies and may support therapeutic MSC products.