Bioactive carbon dots have shown significant progress in biomedical therapeutics due to their unique physicochemical characteristics and exceptional biocompatibility. This work focuses on the synthesis, characterization, and biomedical evaluation of carbon dots (CDs) extracted from waste and discarded marigold flowers for wound healing applications. The synthesized CDs exhibited a uniform size distribution of 2-5 nm and a zeta potential of -15.8 mV, indicating moderate stability against aggregation. Excitation-dependent photoluminescence was observed, with maximum intensity at 380 nm. In comparison, the UV-Vis adsorption spectrum showed a peak at 278 nm, corresponding to the π-π* transition of sp2 carbon domains. Additional FTIR and XPS analyses revealed the presence of functional groups, including hydroxyl, carbonyl, and aromatic domains, and confirmed nitrogen and oxygen doping, which enhanced hydrophilicity and reactivity, respectively. In vitro cytocompatibility assays using MTT and FDA showed maximum cell viability at 500 μg mL-1. Macroscopic examination of the scratch assay showed promising results, with cells and fibroblasts achieving a 98.97% wound healing rate at 48 h compared to control values. In vivo experiments on deep excisional full-thickness and burn injuries exhibited enhanced wound contraction, ECM deposition, angiogenesis, and faster healing with CD gel and spray compared to the control and standard treatments. Histology and immunohistochemistry supported enhanced collagen synthesis and remodeling in the treated tissue; however, the gel demonstrated marginally superior results compared to the spray, as it adhered more securely. These results show that the developed marigold-derived CDs are biocompatible, promote angiogenesis, and enhance healing of deep excisional and burn wounds.

The skin is our body's largest natural barrier, and daily, it undergoes numerous challenges, such as physical injury, chemical insults, and UV radiation. The significance of repairing breaches of this barrier as quickly as possible is especially highlighted in chronic nonhealing wounds, including those associated with diabetes, which affects one in ten people worldwide, with vascular disease and aging. Dying cells in the wound perpetuate inflammation, contribute to dysregulated tissue repair, and increase the risk of infection. Cutaneous wound healing typically consists of four phases, which are hemostasis, inflammation, growth, re-epithelialization, and remodeling. In this chapter, we will discuss how various cell types, immune and nonimmune cells that reside in the epidermis and dermis, die from apoptosis and how efficient dead cell clearance by professional and nonprofessional phagocytes contributes to effective skin wound healing. We will also consider a range of cell death modalities, such as ferroptosis, necroptosis, pyroptosis, and others, and present our current knowledge about their contribution to skin injury and repair.

Recently, seaweed extracts have been found to have potential in skin benefits. This study, therefore, aimed to explore phytochemical analysis, antimicrobial, antioxidant, and wound healing properties of brown seaweed Sargassum plagiophyllym ethanolic extract (SPEE) on human skin keratinocyte HaCaT cells and the possible mechanism involved. Our results indicated that SPEE contained flavonoid, phenolic, and carotenoid as the major active constituents. The HPLC chromatogram revealed C-phycocyanin and fucoidan presented in SPEE. SPEE demonstrated the antioxidant capability and significantly reduced wound space at 24 and 48 h in wound-healing assay. Treatment with SPEE (50 and 100 μg/mL) increased FAK and Src phosphorylation in western blotting. Moreover, SPEE also upregulated Akt and p38 MAPK phosphorylation but not ERK1/2. SPEE increased Rac1 protein expression. Interestingly, hyaluronan synthase (HAS1 and HAS2) as well as collagen type I and elastin were also significantly upregulated when compared with the control upon exposure to SPEE. In conclusion, our data suggested that SPEE promotes cutaneous wound healing by regulating FAK/Src-mediated Akt, p38 MAPK, and Rac1 signaling. These findings suggest the potential use of SPEE for skin wound treatment.

Wound healing is an essential repair process, and impaired wound healing is a common and sometimes debilitating medical problem. Despite advances in wound healing approaches, optimal management strategies are lacking, partly due to an incomplete understanding of the complex pathophysiology of this process. Here we show that Ang2, a previously known ligand for the Tie2 receptor, also binds to fibroblast growth factor receptor 2 (FGFR2) independently of Tie2 and attenuates FGF/FGFR2 signaling in endothelial cells. Functionally, Ang2 inhibits endothelial cell migration induced by FGF. In mouse, topical Ang2 delays the healing of skin wounds, associated with reduced wound angiogenesis and recruitment of mesenchymal-type cells. Additionally, topical AMG386, a blocker of Ang1/Ang2 binding to Tie2 and systemic REGN910, a blocker of Ang2 binding to Tie2, accelerate wound repair, associated with increased wound angiogenesis and recruitment of inflammatory cells. These results identify the tyrosine kinase FGFR2 as a previously unrecognized Ang2 receptor, explaining some of the context-dependent functions of Ang2 in endothelial cells. Since Ang2 is induced in cutaneous wounds and endogenous FGF/FGFR2 is essential for wound repair, Ang2 blockade holds promise as a new evidence-based therapeutic option to promote wound repair.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-025-09988-2.

Wound healing is a complex, multistep process involving sequential phases of inflammation, proliferation, and remodeling. Tenascin-C (TNC) is a matricellular protein actively involved in tissue regeneration. It is upregulated in response to tissue injury and plays an important role in the regulation of cell adhesion, migration, proliferation, and extracellular matrix protein synthesis. At the same time, during the early stages of wound healing, interleukin-1α (IL1α) exerts a significant effect as an alarmin that initiates inflammatory activation of fibroblasts. The objective of this study was to determine the optimal concentration of TNC stimulating both the migratory and synthetic activity of human dermal fibroblasts in vitro, including under conditions of preliminary inflammatory activation with IL1α. To this end, a comparative analysis of cell migration and proliferation was conducted, along with measurement of type I collagen synthesis using DF-1 human fibroblast cultures pre-incubated with IL1α (50 ng/mL) for 24 h, followed by the addition of recombinant TNC at concentrations of 0.05 μg/ml, 0.2 μg/ml, and 1 μg/mL. TNC exhibited a dose-dependent effect on fibroblasts: at a concentration of 0.2 μg/ml it stimulates cell migration and proliferation, accompanied by a statistically significant increase in type I collagen synthesis compared with the control. However, this level was lower than that observed at 1 μg/mL TNC, where a marked increase in collagen production was detected. Under conditions of IL1α pre-stimulation, the effects of TNC were amplified, particularly at concentrations of 0.2 μg/ml and 1 μg/ml, indicating the potential of TNC as a regulator of cellular activity within an inflammatory microenvironment. Higher concentrations did not further increase the effect. These findings may relevant in the context of to the development of biomaterials and therapeutic agents aimed at accelerating cutaneous wound healing by modulating cellular activity, which is especially relevant for the treatment of chronic or non-healing wounds.

Ozonized sunflower oil has been found to have a valuable antimicrobial activity against bacteria, viruses, and fungi. However, delayed cutaneous wound healing, severe adverse skin reactions such as pain, burns, irritation, and unsatisfactory storage stability have limited its clinical use as it is known that the performance of ozonized sunflower oil is reflected by core indicators such as iodine value (IV), peroxide value (PV), and acid value (AV). Aiming to improve the above characteristics, ozone oxidation conditions such as the oil water content of raw materials, stirring speed, and ozonized time were successfully optimized, and the optimized conditions were 5%, 300 rpm, and 6 h, respectively. Additionally, the UHPLC-Q-Exactive Orbitrap MS was used to identify the compounds of sunflower oil and ozonation sunflower oil, and 10 fatty acids were successfully identified. The results clearly indicated that the ozonized sunflower oil prepared under optimized ozonation conditions achieved a high degree of ozonation (II) and a satisfactory PV/AV ratio. These optimized conditions further proved to be valuable in improving not only the storage stability of ozonized sunflower oil for up to six months but also in ensuring its remarkable antimicrobial effects on Staphylococcus aureus and Candida albicans, with a PV of 165.75 mmol/kg achieving a 100% antibacterial efficacy.

1α,25-Dihydroxyvitamin D3 (VD3), the active form of vitamin D, plays a crucial role in wound healing. In this study, we aimed to investigate the effect of VD3 on the proliferation and differentiation of epidermal stem cells (EpSCs) and monitor its impact on re-epithelialization. We established a murine full-thickness skin defect model and applied four doses of VD3 (0, 5, 50, and 250 ng/mouse/day) to the wounds topically for three days. Immunostaining and flow cytometry confirmed the effect of VD3 on the proliferation and differentiation of EpSCs in wounds. This effect of VD3 (0, 1, 10, and 50 nM) on EpSCs and its possible mechanism were further confirmed in vitro by CCK8, westen blot, immunostaining, and flow cytometry. We found that on day five post-wounding, the means±SD length of the neo-epidermis was 195.88±11.57, 231.84±16.45, 385.80±17.50, and 268.00±8.22 μm in the control, 5, 50, and 250 ng groups, respectively, with a significant difference from the control (all P<0.05). Immunostaining and flow cytometry showed that VD3 improved the proliferation and differentiation of K15+ EpSC (vs control, all P<0.05), K14+ epidermal progenitor cells (vs control, all P<0.05), and K10+ epidermal terminal cells (vs control, all P<0.05) in vivo and in vitro. The PI3K signaling pathway appeared to underlie this response because significant inhibition of the response was found when inhibitors were used to inhibit PI3K. Our study demonstrated that VD3 is a potent promoter of cutaneous wound healing by stimulating EpSC proliferation and differentiation through PI3K activation.

Impaired wound healing affects the life quality of patients and causes a substantial financial burden. Hydrogen-rich medium is reported to have antioxidant and anti-inflammatory effects. However, the role of hydrogen-rich saline (HRS) in cutaneous wound healing remains largely unexplored, especially by metabolomics. Thus, untargeted metabolomics profiling was analysed to study the effects and mechanism of HRS combined with vacuum sealing drainage (VSD) in a rabbit full-thickness wound model. Our results indicated that the combination treatment of HRS and VSD could accelerate wound healing. Invitro experiments further confirmed its effects on HaCaT keratinocytes. We found that 45 metabolites were significantly changed between the VSD + HRS group and the VSD + saline-treated group. Pathway enrichment analysis indicated that biotin metabolism was the potential target pathway. The biochemical interpretation analysis demonstrated that combining HRS and VSD might enhance mitochondrial function, ATP synthesis, and GSH homeostasis by altering biotin metabolism. The detection of representative indicators of oxidative stress supported the critical metabolic pathway analysis as well. In summary, VSD combined with HRS might provide a new strategy to enhance wound healing.

Background: Healing is a survival process and essential for maintaining normal anatomical structure and function. Curcumin, the primary active compound in turmeric, and aloe vera, possess notable antioxidant and antimicrobial properties which aid in healing. Objective of this study was to investigate the effects of curcumin and aloe vera on cutaneous wound healing. Methods: This experimental study was conducted in Department of Pathology, Isra University, Hyderabad, from January to July 2023. Sixty-four healthy albino Wistar rats weighing 150–250 grams were procured from Sindh Agriculture University, Tando Jam. A full-thickness open excision-type wound was created and animals were divided into Group A (Control group, petroleum jelly applied to wound), Group B (Turmeric paste was applied on wound), Group C (aloe vera gel applied to wound), and Group D (Turmeric and aloe vera applied to wound). Four animals from each group were sacrificed on day 3, 5, 10, and 14 respectively. An excisional biopsy was taken and sections were examined for histological changes. Results: Markers of cutaneous wound healing such as congested blood vessels, inflammatory cells, number of fibroblasts, and amount of collagen deposition were best seen in Group D (p&lt;0.05). Group D also showed the earliest signs of wound healing (p&lt;0.05). Group A, B, and C showed some changes as congested blood vessels and inflammatory cells with signs of wound healing later. Conclusion: Combination therapy of curcumin and aloe vera exhibits better healing properties than individual therapy with either drug. Pak J Physiol 2024;20(4):13?6 DOI: https://doi.org/10.69656/pjp.v20i4.1652

The study evaluated the pancreatic cellular responses and histopathological changes associated with the cutaneous wound healing g of alloxan induced diabetic New Zealand White (NZW). Sixteen adult rabbits of either sex, weighing 1.8 – 3.2 kg were used for the study. They were divided into four group. s Group A is the control, it was -non diabetic and no wound. Group B was diabetic and no wound, whereas group C was non-diabetic and with wounded while group D was having wounded and diabetic. Diabetes induction was accomplished by administration of 100 mg/kg of alloxan monohydrate twice, 72 h apart in groups B and D only. A 3 cm 2 excisional skin wound was created at the dorsum of each of the rabbits in groups C and D. Skin tissue samples (wounded sites) and pancreas were harvested from two euthanized rabbits in each group on day 28 post induction of diabetes for histopathologic examinations. Diabetes was induced in groups B and D on day 3 post treatment. Histopathologic findings in the diabetic rabbits included hyperkeratosis, acanthosis, poor fibrogenesis of the injured skin site, as well as the necrosis and mononuclear cellular infiltration of the islet of Langerhans of the pancreas. Conclusion diabetes mellitus caused delayed skin wound healing up to day 28 post wound creation, evidenced by hyper cellularity and fibrous tissue proliferation pancreatic necrosis and cellular infiltration of the islets of Langerhans on day in the diabetic rabbit.

Cutaneous wound healing is a slow process that often terminates with permanent scarring while oral wounds, in contrast, regenerate after damage faster. Unique molecular networks in epidermal and oral epithelial keratinocytes contribute to the tissue-specific response to wounding, but key factors that establish those networks and how the keratinocytes interact with their cellular environment remain to be elucidated. The transcription factor PITX1 is highly expressed in the oral epithelium but is undetectable in cutaneous keratinocytes. To delineate if PITX1 contributes to oral keratinocyte identity, cell-cell interactions, and the improved wound healing capabilities, we ectopically expressed PITX1 in the epidermis of murine skin. Using comparative analysis of murine skin and oral (buccal) mucosa with single-cell RNA-Seq and spatial transcriptomics, we found that PITX1 expression enhances epidermal keratinocyte migration and proliferation and alters differentiation to a quasi-oral keratinocyte state. PITX1+ keratinocytes reprogrammed intercellular communication between skin-resident cells to mirror buccal tissue while stimulating the influx of neutrophils that establish a pro-inflammatory environment. Furthermore, PITX1+ skin healed significantly faster than control skin via increased keratinocyte activation and migration and a tunable inflammatory environment. These results illustrate that PITX1 programs oral keratinocyte identity and cellular interactions while revealing critical downstream networks that promote wound closure.

Abstract Background: Hypertrophic scars, resulting from alterations in the normal processes of cutaneous wound healing, are characterized by proliferation of dermal tissue with excessive deposition of fibroblast-derived extracellular matrix proteins, especially collagen, over long periods, and by persistent inflammation and fibrosis. The present study aim to compare the efficacy of silicone gel versus combination of a 577-nm diode laser and silicone gel in the treatment of post-surgery hypertrophic scar. Methods: This study is a randomized comparative study In the period from 14 January 2021 up to the end of January 2023. 30 patients with post-surgery hypertrophic scar divided into two groups: A: one half of scar was managed with 577 nm diode laser 3 sessions monthly combined with application of silicone gel at home. A + B: the entire scar was treated with silicone gel within six months who admitted to the Laser unit of the Dermatology and Andrology Department at Al-Azhar University Hospital, Assiut during the study period. Results: significant improvement in pigmentation, pliability, height and total Vancouver score. By comparing between silicone gel and combination of silicone gel and a 577 nm diode laser, the combination shows significant improving than silicone gel alone. The scar in the lip and breast area showed a significant higher improvement rate with yellow laser plus silicone gel compared to the other site. Conclusion: combination of silicone gel and yellow laser has significant effect in making surgical scars less distinct

528 Serotonin modulation improves cutaneous wound healing

BackgroundIncreasing evidence suggests that mesenchymal stem cells (MSCs) repair traumatized tissues primarily through paracrine secretion and differentiation into specific cell types. However, the role of epithelial differentiation of MSCs in cutaneous wound healing is unclear. This study aimed to investigate the epithelial differentiation potential of gingival tissue-derived MSCs (GMSCs) in epithelial cell growth medium and the mechanisms underlying their differentiation into an epithelial-like cell phenotype.MethodsWe used scanning electron microscopy to examine GMSCs for epithelial differentiation. Quantitative real-time polymerase chain reaction and Western blotting were respectively used to measure genes and proteins related to epithelial differentiation. Immunofluorescence was used to examine subcellular localization of KLF4, KRT19, and β-catenin proteins. Transcriptome sequencing was used to enrich the mechanisms underlying epithelial differentiation in GMSCs. An MSAB inhibitor was used to validate the Wnt signaling pathway further. The wound healing rate and re-epithelialization were assessed through macroscopical observation and hematoxylin and eosin staining.ResultsGMSCs cultured in epithelial cell growth medium from days 3 to 15 exhibited decreased expression of mesenchymal-epithelial transition and stemness-related proteins (N-cadherin, Vimentin, KLF4, and SOX2), increased expression of epithelial-related proteins (KRT12, KRT15, KRT19, and E-cadherin), and exhibited epithelial-like morphology. Mechanistically, high-throughput sequencing revealed that the Wnt and TGF-beta signaling pathways were inhibited during epithelial differentiation of GMSCs (Epi-GMSCs). MSAB-induced Wnt signaling pathway inhibition promoted epithelial-related gene and protein expression. Furthermore, we demonstrated the ability of Epi-GMSCs to facilitate wound healing by improving re-epithelialization in a full-thickness skin defect model.ConclusionsCollectively, this study uncovers that GMSCs have the ability to differentiate into epithelia and highlights a promising strategy for using Epi-GMSCs to improve cutaneous wound healing.

Cutaneous wound healing is a complex process involvingvariouscellular and molecular interactions, resulting in the formation ofa collagen-rich scar with imperfect function and morphology. Dermalfibroblasts are crucial to successful wound healing, migrating tothe wound site where they are activated to provide extracellular matrixremodeling and wound closure. Peripheral nerves have been shown toplay an important role in wound healing, with loss or damage to thesenerves often leading to impaired healing and the formation of chronicnonhealing wounds. Previous research has suggested that sensory nervessecrete trophic factors that can regulate wound healing, includingfibroblast activation; however, the direct cell–cell interactionbetween nerves and fibroblasts has not been extensively studied. Toaddress this knowledge gap, we developed an in vitro co-culture model using a device called the IFlowPlate. This modelsupports the long-term viability of multiple cell types while allowingfor direct contact between sensory nerve cells and dermal fibroblasts.Using the IFlowPlate, we demonstrate that co-culture of dorsal rootganglia with dermal fibroblasts increases fibroblast proliferation,collagen and α-smooth muscle actin expression, and secretionof pro-wound healing factors, suggesting that nerves can promote woundhealing by modulating fibroblast activation. The IFlowPlate offersa user-friendly and high-throughput platform to study the in vitrointeractions between nerves and a variety of cell types that can beapplied to wound healing and other important biological processes.

Fibroblasts are native residents in dermal layer of human skin which are important for dermal regeneration and essential during cutaneous wound healing by releasing inflammatory markers and actively migrate to close an open wound. Premature skin ageing due to methylglyoxal (MGO) has recently caught the attention considering its potential to accelerate the emergence of skin ageing signs, however previous studies were only focused in primary neonatal dermal fibroblast and NIH3t3 fibroblast cell line. Therefore, thorough investigation is required to study the impact of MGO on primary human dermal fibroblast isolated from adult subject (HDFa). In our experiments, short exposure of MGO was observed to induced significant reductions in cell viability at concentrations of 7.5, 10, 12.5, 15, and 17.5 mM (p < 0.005) after 3 hours of treatment. The cellular death of HDFa at 10, 12.5 and 15 mM of MGO were also marked by increased in intracellular ROS level, indicating the involvement of oxidative stress-induced death in these cells. We also observed enlarge scratch areas of cells exposed with 7.5 and 10 mM MGO compared to control after 26 hours, thereby suggesting a decline in cell migration and viability in this group. We propose the increased ROS as the consequence of AGE-RAGE activation which was marked by significant elevation of RAGE mRNA on cells exposed to 10 mM MGO. Our data also suggest the occurrence of DNA damage events via ROS-induced oxidation or mediated by decline in hTERT mRNA expression.

Introduction: To accelerate cutaneous wound healing and prevent scarring, regenerative approaches such as injecting a mechanically derived tissue stromal vascular fraction (tSVF) are currently under clinical and laboratory investigations. The aim of our study was to investigate a platform to assess the interaction between skin-derived extracellular matrix (ECM) hydrogels and tSVF and their effects on their microenvironment in the first ten days of culture. Material and Methods: A tSVF mixed with ECM hydrogel was cultured for ten days. After 0, 3, 5, and 10 days of culture viability, histology, immunohistochemistry, gene expression, and collagen alignment and organization were assessed. Results: The viability analysis showed that tSVF remained viable during 10 days of culture and seemed to remain within their constitutive ECM. The fiber analysis demonstrated that collagen alignment and organization were not altered. No outgrowth of capillaries was observed in (immuno)histochemical staining. The gene expression analysis revealed that paracrine factors TGFB1 and VEGFA did not change and yet were constitutively expressed. Pro-inflammatory factors IL1B and IL6 were downregulated. Matrix remodeling gene MMP1 was upregulated from day three on, while MMP14 was upregulated at day three and ten. Interestingly, MMP14 was downregulated at day five compared to day three while MMP2 was downregulated after day zero. Conclusions: Skin-derived ECM hydrogels appear to be a versatile platform for investigating the function of a mechanically isolated adipose tissue stromal vascular fraction. In vitro tSVF remained viable for 10 days and sustained the expression of pro-regenerative factors, but is in need of additional triggers to induce vascularization or show signs of remodeling of the surrounding ECM. In the future, ECM-encapsulated tSVF may show promise for clinical administration to improve wound healing.

Aloe vera is commonly used as traditional medicine for cutaneous wound healing. Nonetheless, the wound healing mechanisms of Aloe vera remain unclear. This review aims to provide insight into the molecular mechanisms of Aloe vera in promoting cutaneous wound healing, with particular emphasis on the mechanisms that stimulate cell proliferation and migration. Aloe vera has been shown to upregulate growth factors such as keratinocyte growth factor-1 (KGF-1), transforming growth factor-β (TGF-β), cyclin D1, insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF), and microfibril-associated glycoprotein 4 (MFAP4), as well as collagen, fibrillin, elastin, α-smooth muscle actin (α-SMA), integrins, and platelet endothelial cell adhesion molecule 1 (PECAM-1, also known as CD31), while downregulating the expression of matrix metalloproteinases (MMPs). In addition, Aloe vera was also found to upregulate PI3K/Akt and MAPK pathways, as well as the TGF-β signalling pathway via Smad proteins. Furthermore, molecular docking studies revealed that certain chemical constituents of Aloe vera bind to some of the forementioned growth factors or signalling molecules. With regards to current applications, although human clinical trials have reported positive results from using Aloe vera in healing open wounds and burns and alleviating some inflammatory skin diseases, the current commercial uses of Aloe vera remain largely focused on cosmetic products. Thus, greater advances are required to promote the use of Aloe vera products in clinical settings.

Chronic wounds, defined by their prolonged healing process, significantly impair patients' quality of life and impose a hefty financial burden on healthcare systems worldwide. Sex- and gender-specific mechanisms regulate inflammation and infection, angiogenesis, matrix synthesis and cell recruitment. All of these processes contribute to cutaneous wound healing but remain largely understudied. This review aims to spotlight the innovative realm of bioengineering and nanomedicine, which is at the helm of revolutionizing complex chronic wound care. It underscores the significance of integrating patient sex into the development and (pre)clinical testing of these avant-garde treatment modalities, in order to enhance healing prospects for all patients regardless of sex. Moreover, we explore the representation of male and female patients in clinical trials of bioengineered and nanomedicine products. Finally, we examine the primary reasons for the historical neglect in translating sex-specific wound healing research into clinical practice and propose strategic solutions. By tackling these issues, the article advocates advanced treatment frameworks that could significantly improve healing outcomes for individuals of all sexes, thereby optimizing both efficacy and inclusivity in chronic wound management.

Inducible T-cell costimulator (ICOS, CD278) is a costimulatory receptor primarily expressed by activated T cells. It binds to ICOS ligand (ICOSL, CD275), which is expressed by various immune and non-immune cell types, particularly in inflamed tissues. ICOSL can also bind to osteopontin (OPN), a protein that functions both as a component of the extracellular matrix and as a soluble pro-inflammatory cytokine. Previous studies, including ours, have shown that ICOS and ICOSL play a role in skin wound healing, as mice deficient in either ICOS or ICOSL exhibit delayed healing. The aim of this study was to investigate the involvement of the ICOS/ICOSL/OPN network in skin wound healing by analyzing mice that are single knockouts for ICOS, ICOSL, or OPN, or double knockouts for ICOS/OPN or ICOSL/OPN. Our results showed that wound healing is impaired in all single knockout strains, but not in the two double knockout strains. Cellular and molecular analyses of the wound healing sites revealed that the healing defect in the single knockout strains is associated with reduced neutrophil infiltration and decreased expression of α-SMA (a marker of myofibroblasts), IL-6, TNFα, and VEGF. In contrast, the normalization of wound closure observed in the double knockout strains was primarily linked to increased vessel formation. A local treatment with recombinant ICOS-Fc improved healing in all mouse strains expressing ICOSL, but not in those lacking ICOSL, and led to a local increase in vessel formation and macrophage recruitment, predominantly of the M2 type.