Wound Administration of M2-Polarized Macrophages Does Not Improve Murine Cutaneous Healing Responses

Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds.     

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100?mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.     

Application of Electron Paramagnetic Resonance (EPR) Oximetry to Monitor Oxygen in Wounds in Diabetic Models

A lack of oxygen is classically described as a major cause of impaired wound healing in diabetic patients. Even if the role of oxygen in the wound healing process is well recognized, measurement of oxygen levels in a wound remains challenging. The purpose of the present study was to assess the value of electron paramagnetic resonance (EPR) oximetry to monitor pO₂ in wounds during the healing process in diabetic mouse models. Kinetics of wound closure were carried out in streptozotocin (STZ)-treated and db/db mice. The pO₂ was followed repeatedly during the healing process by 1 GHz EPR spectroscopy with lithium phthalocyanine (LiPc) crystals used as oxygen sensor in two different wound models: a full-thickness excisional skin wound and a pedicled skin flap. Wound closure kinetics were dramatically slower in 12-week-old db/db compared to control (db/+) mice, whereas kinetics were not statistically different in STZ-treated compared to control mice. At the center of excisional wounds, measurements were highly influenced by atmospheric oxygen early in the healing process. In pedicled flaps, hypoxia was observed early after wounding. While reoxygenation occurred over time in db/+ mice, hypoxia was prolonged in the diabetic db/db model. This observation was consistent with impaired healing and microangiopathies observed using intravital microscopy. In conclusion, EPR oximetry using LiPc crystals as the oxygen sensor is an appropriate technique to follow wound oxygenation in acute and chronic wounds, in normal and diabetic animals. Nevertheless, the technique is limited for measurements in pedicled skin flaps and cannot be applied to excisional wounds in which diffusion of atmospheric oxygen significantly affects the measurements.     

Validation of a model for the study of multiple wounds in the diabetic mouse (db/db)

The genetically diabetic db/db mouse exhibits symptoms that resemble human type 2 diabetes mellitus, demonstrates delayed wound healing, and has been used extensively as a model to study the role of therapeutic topical reagents in wound healing. The purpose of the authors' study was to validate an excisional wound model using a 6-mm biopsy punch to create four full-thickness dorsal wounds on a single db/db mouse. Factors considered in developing the db/db wound model include reproducibility of size and shape of wounds, the effect of semiocclusive dressings, comparison with littermate controls (db/-), clinical versus histologic evidence of wound closure, and cross-contamination of wounds with topically applied reagents. The size of wounds was larger, with less variation in the db/db mice (31.11 +/- 3.76 mm2) versus db/- mice (23.64 +/- 4.78 mm2). Wounds on db/db mice that were covered with a semiocclusive dressing healed significantly more slowly (mean, 27.75 days) than wounds not covered with the dressing (mean, 13 days; p < 0.001), suggesting the dressings may splint the wounds open. As expected, wounds healed more slowly on db/db mice than db/- mice (covered wounds, 27.75 days versus 11.86 days, p < 0.001; wounds not covered, 13 days versus 11.75 days, p = 0.39). Covered wounds, thought to be closed by clinical examination, were confirmed closed by histology only 62 percent of the time in the db/db and 100 percent of the time in the db/- mice. Topical application of blue histologic dye or soluble biotinylated laminin 5 to one of the four wounds did not spread locally and contaminate adjacent wounds. Multiple, uniform, 6-mm wounds in db/db mice heal in a relatively short time, decrease the number of animals needed for each study, and allow each animal to serve as its own control. The db/db diabetic mouse appears to be an excellent model of delayed wound healing, particularly for studying factors related to epithelial migration.     

Burn injury-induced alterations in wound inflammation and healing are associated with suppressed hypoxia inducible factor-1alpha expression

A major complication associated with burn injury is delayed wound healing. While healing of the burn injury site is essential, healing of distal injury sites caused by surgical interventions and other processes also is important. The impact of burn injury on healing of these distal wound sites is not understood clearly. To study this, mice were subjected to major burn injury or a sham procedure. Immediately following, excisional wounds were made on the dorsal surface caudal to the burn site and wound closure was monitored over a 7-d period by planimetry. In a second series of experiments, plasma and excisional wounds were collected for in vitro analysis of cyto- and chemokine levels, L-arginine metabolism, and hypoxia-inducible factor (HIF)-1alpha expression. At 1-7 d post-injury, a significant inflammatory response was evident in both groups, but the healing process was delayed in the burn-injured mice. At 3 d post-injury, wound levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and keratinocyte-derived chemokine were suppressed in the burn group. This difference in the wound inflammatory response was independent of changes in L-arginine metabolism (nitrate levels, inducible nitric oxide synthase expression, arginase activity), but correlated with a marked reduction in HIF-1alpha protein levels. In conclusion, these findings suggest that HIF-1alpha and the inflammatory response play a significant role in wound healing, and reduced levels of HIF-1alpha contribute to the impaired healing response post-burn.     

Adiponectin Regulates Cutaneous Wound Healing by Promoting Keratinocyte Proliferation and Migration via the ERK Signaling Pathway

Diabetic patients are at high risk of developing delayed cutaneous wound healing. Adiponectin plays a pivotal role in the pathogenesis of diabetes and is considered to be involved in various pathological conditions associated with diabetes; however, its role in wound repair is unknown. In this study, we elucidated the involvement of adiponectin in cutaneous wound healing in vitro and in vivo. Normal human keratinocytes expressed adiponectin receptors, and adiponectin enhanced proliferation and migration of keratinocytes in vitro. This proliferative and migratory effect of adiponectin was mediated via AdipoR1/AdipoR2 and the ERK signaling pathway. Consistent with in vitro results, wound closure was significantly delayed in adiponectin-deficient mice compared with wild-type mice, and more importantly, keratinocyte proliferation and migration during wound repair were also impaired in adiponectin-deficient mice. Furthermore, both systemic and topical administration of adiponectin ameliorated impaired wound healing in adiponectin-deficient and diabetic db/db mice, respectively. Collectively, these results indicate that adiponectin is a potent mediator in the regulation of cutaneous wound healing. We propose that upregulation of systemic and/or local adiponectin levels is a potential and very promising therapeutic approach for dealing with diabetic wounds.     

Comparison of botulinum toxin type A and aprotinin monotherapy with combination therapy in healing of burn wounds in an animal model

Burns are one of the most common injuries that are complicated by many challenges including infection, severe inflammatory response, excessive expression of proteases, and scar formation. The aim of this study was to investigate the effect of botulinum toxin type A (BO) and aprotinin (AP) separately or in combination (BO-AP) in healing process. Four burn wounds were created in each rat and randomly filled with silver sulfadiazine (SSD), BO, AP and BO-AP. The rats were euthanized after 7, 14, and 28 days, and their harvested wound samples were evaluated by gross pathology, histopathology, gene expression, biochemical testing, and scanning electron microscopy. Both BO and AP significantly reduced expression of interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) at the 7th post wounding day. Moreover, they inhibited scar formation by reducing the TGF-β1 level and increasing basic fibroblast growth factor (bFGF) at the 28th day. AP by decreasing protease production showed more effective role than BO in wound regeneration. AP increased tissue organization and maturation and improved cosmetic appearance of wounds, at 28 days. The best results gained when combination of BO and AP were used in healing of burn wounds. Treatment by BO-AP significantly subsided inflammation compared to the BO, AP, and SSD treated wounds. Treatment with BO-AP also reduced collagen density and led to minimal scar formation. Combination of botulinum toxin type A and aprotinin considerably increased structural and functional properties of the healing wounds by reducing scar formation and decreasing production of proteases.

     

MSC attenuate diabetes-induced functional impairment in adipocytes via secretion of insulin-like growth factor-1

The function of subcutaneous adipocytes in promoting wound healing is significantly suppressed in diabetic wounds. Recent studies have demonstrated the ability of mesenchymal stem cell (MSC) to ameliorate impaired diabetic wound healing. We hypothesized that MSC function may involve subcutaneous adipocytes. The abnormal function of subcutaneous adipocytes from STZ induced diabetic mice including glucose uptake and free fatty acid (FFA) secretion level were assessed. Then these cells were co-cultured with MSC via a transwell system to observe the changes of metabolic index and glucose transporter four (GLUT4) as well as phosphoinositide 3-kinase/protein kinase (PI3K/AKT) signaling pathway expression. The results of metabolic index suggest that MSC obviously attenuated the diabetes-induced functional impairment. Both mRNA and protein expression analyses showed that PI3K/AKT insulin signaling pathway and GLUT4 expression were up-regulated. These changes were substantially associated with a increased level of insulin-like growth factor-1 (IGF-1) secretion from MSC. These findings suggest that MSC could attenuate abnormal function of diabetic adipocytes by IGF-1secretion, which was more or less associated with the beneficial effects of MSC on improving diabetic wound healing.     

PDGF-BB Does Not Accelerate Healing in Diabetic Mice with Splinted Skin Wounds

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epeithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.     

Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice

Background

Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.

Methodology/Principal Findings

Macrophages isolated from wounds of diabetic mice showed significant impairment in efferocytosis. Impaired efferocytosis was associated with significantly higher burden of apoptotic cells in wound tissue as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines. Observations related to apoptotic cell load at the wound site in mice were validated in the wound tissue of diabetic and non-diabetic patients. Forced Fas ligand driven elevation of apoptotic cell burden at the wound site augmented pro-inflammatory and attenuated anti-inflammatory cytokine response. Furthermore, successful efferocytosis switched wound macrophages from pro-inflammatory to an anti-inflammatory mode.

Conclusions/Significance

Taken together, this study presents first evidence demonstrating that diabetic wounds suffer from dysfunctional macrophage efferocytosis resulting in increased apoptotic cell burden at the wound site. This burden, in turn, prolongs the inflammatory phase and complicates wound healing.     

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2^nd and 3^rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.     

Effects of CD100 promote wound healing in diabetic mice

Diabetes is a condition that causes delayed wound healing and results in chronic wounds. CD100 has been reported to promote and induce potent and obvious angiogenesis both in vivo and in vitro studies, the absence of which are a main cause of the diabetic chronic wound. In the present study, we investigated the effects of application of soluble CD100 on wound healing in diabetic mice. Four 5-mm full-thickness dermal wounds were made on each male db/db mouse. 12 mice from CD100 group were subcutaneously injected with 250 ng of CD100 (50 µl) per wound, in addition, 12 mice were injected with the same volume phosphate-balanced solution as the control. The animals were treated every other day until the wounds healed completely. Images were obtained to calculate the area ratio of the original area. HE and Masson’s trichrome staining were used for histological examination. Collagen remodeling, angiogenesis and wound bed inflammation were evaluated by immunohistochemical staining and western blot. We demonstrated that CD100 had distinct functions during the wound healing process. Histological and western blotting analysis showed a more organized epithelium and dermis, more collagen fibers, higher angiogenesis and lower inflammation in the CD100 group than in the PBS group. These findings suggest that CD100 may accelerate wound healing in diabetic mice by promoting angiogenesis in the wound and by reducing the inflammatory response.     

Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome

Converging clinical data suggest that peripheral inflammation is likely involved in the pathogenesis of the neuropsychiatric symptoms associated with metabolic syndrome (MetS). However, the question arises as to whether the increased prevalence of behavioral alterations in MetS is also associated with central inflammation, i.e. cytokine activation, in brain areas particularly involved in controlling behavior. To answer this question, we measured in a mouse model of MetS, namely the diabetic and obese db/db mice, and in their healthy db/+ littermates emotional behaviors and memory performances, as well as plasma levels and brain expression (hippocampus; hypothalamus) of inflammatory cytokines. Our results shows that db/db mice displayed increased anxiety-like behaviors in the open-field and the elevated plus-maze (i.e. reduced percent of time spent in anxiogenic areas of each device), but not depressive-like behaviors as assessed by immobility time in the forced swim and tail suspension tests. Moreover, db/db mice displayed impaired spatial recognition memory (hippocampus-dependent task), but unaltered object recognition memory (hippocampus-independent task). In agreement with the well-established role of the hippocampus in anxiety-like behavior and spatial memory, behavioral alterations of db/db mice were associated with increased inflammatory cytokines (interleukin-1β, tumor necrosis factor-α and interleukin-6) and reduced expression of brain-derived neurotrophic factor (BDNF) in the hippocampus but not the hypothalamus. These results strongly point to interactions between cytokines and central processes involving the hippocampus as important contributing factor to the behavioral alterations of db/db mice. These findings may prove valuable for introducing novel approaches to treat neuropsychiatric complications associated with MetS.     

The effects of topical treatment with acidified nitrite on wound healing in normal and diabetic mice

The effects of the application of a nitric oxide generating acidified nitrite cream comprising sodium nitrite and citric acid, on the healing of incisional wounds in mice, has been investigated. The effects of acidified nitrite on wound healing were critically dependent on the time of application after wounding. Application of acidified nitrite starting on the day of wounding and on consecutive days thereafter significantly inhibited both half time to closure and extent of wound closure. Conversely, application starting on days 1-4 after wounding and on consecutive days thereafter significantly augmented the rate and extent of wound healing. Optimal effects on improving wound healing were observed with cream concentrations of 3.0% (w/v) sodium nitrite and 4.5% (w/v) citric acid. Starting application on day 5 after wounding had no effect on the rate or extent of wound healing. In diabetic Lepr db/db mice, starting treatment at day 2 after wounding, acidified nitrite at 3.0% (w/v) sodium nitrite and 4.5% (w/v) citric acid significantly increased the rate and extent of wound healing. This suggests that acidified nitrite is effective in improving wound healing against a diabetic background. The present data shows that acidified nitrite cream, a clinically effective means of topically delivering nitric oxide, augments the wound healing process and may be of clinical benefit.     

A novel composition for in vitro and in vivo regeneration of skin and connective tissues

The particular combination of polydeoxyribonucleotides, l-carnitine, calcium ions, proteolytic enzyme and other ingredients acts in a synergetic way in the regeneration of skin and connective tissues. This new formulation of active principles was tested in vitro as a cell and tissue culture medium and in vivo for various preparations in support of tissue regeneration. In vitro, the new blend allowed the maintenance of skin biopsies for more than 1 year in eutrophic conditions. Immunocytochemical analyses of fibroblasts isolated from these biopsies confirmed a significant increase of the epidermal and connective wound-healing markers such as collagen type I, collagen type IV, cytokeratin 1 (CK1), CK5, CK10 and CK14 versus controls. To examine the effects of the new compound in vivo, we studied impaired wound healing in genetically diabetic db/db mice. At day 18, diabetic mice treated with the new composition showed 100% closure of wounds and faster healing than mice treated with the other solutions. This complex of vital continuity factors or life-keeping factors could be used as a tissue-preserving solution or a cosmetic/drug/medical device to accelerate wound healing in the treatment of patients with deficient wound repair to promote the regeneration of cutaneous and connective tissues (injuries-wound, dermatitis) and prevent the recurrent relapses.     

14S,21R-dihydroxydocosahexaenoic acid remedies impaired healing and mesenchymal stem cell functions in diabetic wounds

Treatment of diabetes-impaired wound healing remains a major unresolved medical challenge. Here, we identified suppressed formation of a novel reparative lipid mediator 14S,21R-dihydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14S,21R-diHDHA) in cutaneous wounds of diabetic db/db mice. These results indicate that diabetes impedes the biosynthetic pathways of 14S,21R-diHDHA in skin wounds. Administration of exogenous 14S,21R-diHDHA to wounds in diabetic animals rescued healing and angiogenesis. When db/db mesenchymal stem cells (MSCs) were administered together with 14S,21R-diHDHA to wounds in diabetic animals, they coacted to accelerate wound re-epithelialization, granulation tissue formation, and synergistically improved vascularization. In the pivotal cellular processes of angiogenesis, 14S,21R-diHDHA enhanced VEGF release, vasculature formation, and migration of db/db dermal microvascular endothelial cells (DMVECs), as well as remedied paracrine angiogenic functions of db/db MSCs, including VEGF secretion and the promotion of DMVEC migration and vasculature formation. Our results show that 14S,21R-diHDHA activates the p38 MAPK pathway in wounds, db/db MSCs, and DMVECs. Overall, the impeded formation of 14S,21R-diHDHA described in this study suggests that diabetes could affect the generation of pro-healing lipid mediators in wound healing. By restoring wound healing and MSC functions, 14S,21R-diHDHA is a new lead for the development of better therapeutics used in treating wounds of diabetics.     

Thymosin beta4 promotes matrix metalloproteinase expression during wound repair

Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.     

Xanthine Oxidoreductase Function Contributes to Normal Wound Healing

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H₂O₂, 0.15%) or allopurinol (30 μg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H₂O₂ and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H₂O₂ restored wound healing in tungsten-fed mice. In vitro, nitrite and H₂O₂ both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.