A Three Species Model to Simulate Application of Hyperbaric Oxygen Therapy to Chronic Wounds

Chronic wounds are a significant socioeconomic problem for governments worldwide. Approximately 15% of people who suffer from diabetes will experience a lower-limb ulcer at some stage of their lives, and 24% of these wounds will ultimately result in amputation of the lower limb. Hyperbaric Oxygen Therapy (HBOT) has been shown to aid the healing of chronic wounds; however, the causal reasons for the improved healing remain unclear and hence current HBOT protocols remain empirical. Here we develop a three-species mathematical model of wound healing that is used to simulate the application of hyperbaric oxygen therapy in the treatment of wounds. Based on our modelling, we predict that intermittent HBOT will assist chronic wound healing while normobaric oxygen is ineffective in treating such wounds. Furthermore, treatment should continue until healing is complete, and HBOT will not stimulate healing under all circumstances, leading us to conclude that finding the right protocol for an individual patient is crucial if HBOT is to be effective. We provide constraints that depend on the model parameters for the range of HBOT protocols that will stimulate healing. More specifically, we predict that patients with a poor arterial supply of oxygen, high consumption of oxygen by the wound tissue, chronically hypoxic wounds, and/or a dysfunctional endothelial cell response to oxygen are at risk of nonresponsiveness to HBOT. The work of this paper can, in some way, highlight which patients are most likely to respond well to HBOT (for example, those with a good arterial supply), and thus has the potential to assist in improving both the success rate and hence the cost-effectiveness of this therapy.     

Antimycotic ciclopirox olamine in the diabetic environment promotes angiogenesis and enhances wound healing

Diabetic wounds remain a major medical challenge with often disappointing outcomes despite the best available care. An impaired response to tissue hypoxia and insufficient angiogenesis are major factors responsible for poor healing in diabetic wounds. Here we show that the antimycotic drug ciclopirox olamine (CPX) can induce therapeutic angiogenesis in diabetic wounds. Treatment with CPX in vitro led to upregulation of multiple angiogenic genes and increased availability of HIF-1α. Using an excisional wound splinting model in diabetic mice, we showed that serial topical treatment with CPX enhanced wound healing compared to vehicle control treatment, with significantly accelerated wound closure, increased angiogenesis, and increased dermal cellularity. These findings offer a promising new topical pharmacologic therapy for the treatment of diabetic wounds.     

The role of oxidised regenerated cellulose/collagen in wound repair: effects in vitro on fibroblast biology and in vivo in a model of compromised healing

Irrespective of underlying chronic wound pathology, delayed wound healing is normally characterised by impaired new tissue formation at the site of injury. It is thought that this impairment reflects both a reduced capacity to synthesize new tissue and the antagonistic activities of high levels of proteinases within the chronic wound environment. Historically, wound dressings have largely been passive devices that offer the wound interim barrier function and establish a moist healing environment. A new generation of devices, designed to interact with the wound and promote new tissue formation, is currently being developed and tested. This study considers one such device, oxidised regenerated cellulose (ORC) /collagen, in terms of its ability to promote fibroblast migration and proliferation in vitro and to accelerate wound repair in the diabetic mouse, a model of delayed wound healing. ORC/collagen was found to promote both human dermal fibroblasts proliferation and cell migration. In vivo studies considered the closure and histological characteristics of diabetic wounds treated with ORC/collagen compared to those of wounds given standard treatment on both diabetic and non-diabetic mice. ORC/collagen was found to significantly accelerate diabetic wound closure and result in a measurable improvement in the histological appearance of wound tissues. As the diabetic mouse is a recognised model of impaired healing, which may share some characteristics of human chronic wounds, the results of this in vivo study, taken together with those relating the positive effects of ORC/collagen in vitro, may predict the beneficial use of this device in the clinical setting.     

Copper-induced vascular endothelial growth factor expression and wound healing

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. Whereas a direct role of copper to facilitate angiogenesis has been evident two decades ago, the specific targets of copper action remained unclear. This report presents first evidence showing that inducible VEGF expression is sensitive to copper and that the angiogenic potential of copper may be harnessed to accelerate dermal wound contraction and closure. At physiologically relevant concentrations, copper sulfate induced VEGF expression in primary as well as transformed human keratinocytes. Copper shared some of the pathways utilized by hypoxia to regulate VEGF expression. Topical copper sulfate accelerated closure of excisional murine dermal wound allowed to heal by secondary intention. Copper-sensitive pathways regulate key mediators of wound healing such as angiogenesis and extracellular matrix remodeling. Copper-based therapeutics represents a feasible approach to promote dermal wound healing.     

Application of decellularized human reticular allograft dermal matrix promotes rapid re-epithelialization in a diabetic murine excisional wound model

Background aimsThe treatment and care of human wounds represent an enormous burden on the medical system and patients alike. Chronic or delayed healing wounds are characterized by the inability to form proper granulation tissue, followed by deficiencies in keratinocyte migration and wound re-epithelialization, leading to increased likelihood of infection and poor wound outcomes. Human reticular acellular dermal matrix (HR-ADM) is one type of tissue graft developed to enhance closure of delayed healing wounds that has demonstrated clinical utility through accelerating closure of lower extremity diabetic ulcers, but the mechanisms underlying this clinical success are not well understood.MethodsThe authors utilized a diabetic murine splinted excisional wound model to investigate the effects of HR-ADM application on wound closure.ResultsThe authors demonstrate that application of HR-ADM served as a dermal scaffold and promoted rapid re-epithelialization and keratinocyte proliferation, resulting in accelerated wound closure while minimizing granulation tissue formation. HR-ADM-applied wounds also demonstrated evidence of cellular infiltration, neovascularization and collagen remodeling by the host organism.ConclusionsThese data suggest that HR-ADM supports epidermal closure in delayed healing wounds and remodeling of the matrix into host tissue, lending further support to the clinical success of HR-ADM described in clinical reports.     

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

It has been established that low concentrations of hydrogen peroxide (H₂O₂) are produced in wounds and is required for optimal healing. Yet at the same time, there is evidence that excessive oxidative damage is correlated with poor-healing wounds. In this paper, we seek to determine whether topical application of H₂O₂ can modulate wound healing and if its effects are related to oxidative damage. Using a C57BL/6 mice excision wound model, H₂O₂ was found to enhance angiogenesis and wound closure at 10 mM but retarded wound closure at 166 mM. The delay in closure was also associated with decreased connective tissue formation, increased MMP-8 and persistent neutrophil infiltration. Wounding was found to increase oxidative lipid damage, as measured by F₂-isoprostanes, and nitrative protein damage, as measured by 3-nitrotyrosine. However H₂O₂ treatment did not significantly increase oxidative and nitrative damage even at concentrations that delay wound healing. Hence the detrimental effects of H₂O₂ may not involve oxidative damage to the target molecules studied.     

Mesenchymal stem cells: Paracrine signaling and differentiation during cutaneous wound repair

Cutaneous wounds persist as a health care crisis in spite of increased understanding of the cellular and molecular responses to injury. Contributing significantly to this crisis is the lack of reliable therapies for treatment of wounds that are slow to heal including chronic wounds and deep dermal wounds that develop hypertrophic scars. This article will review the growing evidence demonstrating the promise of multipotent mesenchymal stem/stromal (MSCs) for the treatment of impaired wound healing. MSCs are often referred to as mesenchymal stem cells despite concerns that these cells are not truly stem cells given the lack of evidence demonstrating self-renewal in vivo. Regardless, abundant evidence demonstrates the therapeutic potential of MSCs for repair and regeneration of damaged tissue due to injury or disease. To date, MSC treatment of acute and chronic wounds results in accelerated wound closure with increased epithelialization, granulation tissue formation and angiogenesis. Although there is evidence for MSC differentiation in the wound, most of the therapeutic effects are likely due to MSCs releasing soluble factors that regulate local cellular responses to cutaneous injury. Important challenges need to be overcome before MSCs can be used effectively to treat wounds that are slow to heal.     

Non-adenine based purines accelerate wound healing

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5–2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8–3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.     

Hyperbaric Oxygen Therapy to Treat Diabetes Impaired Wound Healing in Rats

Wound healing in diabetes is frequently impaired and its treatment remains a challenge. Hyperbaric oxygen therapy (HBOT) receives a wide attendance and is often used as a last resort treatment option, however, its effectiveness for many conditions is unproven. We tested the effect of HBOT on healing of diabetic ulcers in an animal experimental setting. Experimental diabetes was induced by intraperitoneal injection of streptozotocin. Four weeks after diabetes induction, rats were ulcerated by clamping a pair of magnet disks on the dorsal skin for 16 h. After magnet removal, the animals received HBOT, daily on weekdays, for 4 weeks. To examine the effect of HBOT on diabetes impaired wound healing, the degree of wound tissue perfusion, inflammation, angiogenesis, and tissue breaking strength were evaluated. HBOT effects on the degree of inflammation and number of blood vessels could not be observed. HBOT improved the tissue breaking strength of the wound, however, this did not reach statistical significance. Twenty hours after ending the HBOT, a significantly improved oxygen saturation of the hemoglobin at the venous end of the capillaries and the quantity of hemoglobin in the micro-blood vessels was measured.     

Improved repair of dermal wounds in mice lacking microRNA‐155

Wound healing is a well-regulated but complex process that involves haemostasis, inflammation, proliferation and maturation. Recent reports suggest that microRNAs (miRs) play important roles in dermal wound healing. In fact, miR deregulation has been linked with impaired wound repair. miR-155 has been shown to be induced by inflammatory mediators and plays a central regulatory role in immune responses. We have investigated the potential role of miR-155 in wound healing. By creating punch wounds in the skin of mice, we found an increased expression of miR-155 in wound tissue when compared with healthy skin. Interestingly, analysis of wounds of mice lacking the expression of miR-155 (miR-155^−/−) revealed an increased wound closure when compared with wild-type animals. Also, the accelerated wound closing correlated with elevated numbers of macrophages in wounded tissue. Gene expression analysis of wounds tissue and macrophages isolated from miR-155^−/− mice that were treated with interleukin-4 demonstrated an increased expression of miR-155 targets (BCL6, RhoA and SHIP1) as well as, the finding in inflammatory zone-1 (FIZZ1) gene, when compared with WT mice. Moreover, the up-regulated levels of FIZZ1 in the wound tissue of miR-155^−/− mice correlated with an increased deposition of type-1 collagens, a phenomenon known to be beneficial in wound closure. Our data indicate that the absence of miR-155 has beneficial effects in the wound healing process.     

Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis

The biological roles of hyaluronan (HA) fragments in angiogenesis acceleration have been investigated recently. Studies have confirmed that oligosaccharides of HA (o-HA) are capable of stimulating neovascularization in vitro and promoting blood flow or angiogenesis in animal models. However, few laboratories have studied the function of o-HA as an exogenous treatment in injured tissue repair in vivo. It is thought that o-HA may lose its activities when used topically in vivo due to its small size, which may be absorbed quickly by the surrounding tissues. In this study, we prepared a special slow-releasing gel that contains a mixture of defined size of o-HA and studied the healing effects of o-HA by topical application to an acute wound model. We report that o-HA complex promotes the repair of tissue injury of a murine excisional dermal wound. The therapy by o-HA was compared with high molecular weight HA (HMW-HA) and the known angiogenesis stimulator, VEGF. At days 6 to 8 after treatment, significant differences were seen in wound closure rates between o-HA and control or HMW-HA groups, in which o-HA showed an increased wound recovery. Histological analysis revealed that increased neo-blood and lymph vessels were formed in wounded tissues treated by o-HA. In addition, treatments of wounds with o-HA resulted in more granulation production, collagen deposition, and fibroblast proliferation. Analysis of gene expression by real-time RT-PCR demonstrated a significant up-regulation of some cytokines or adhesion molecules in o-HA-treated wounds, which corresponds with the increased granulation tissue in these wounds. Our findings suggested that o-HA therapy may be useful in acute wound repair.     

A positive association of larval therapy and hyperbaric oxygen therapy in veterinary wound care

The treatment of cutaneous wounds is part of the veterinary routine from initial scientific reports due to being regularly present condition. Currently, several types of treatments are available to accelerate the healing process. This report presents the case of a dog with multiple lesions in the thoracic limbs resulting from a car accident, who underwent larval therapy and hyperbaric oxygen therapy (HBOT). The animal was a 2-year-old female mixed breed dog presenting severe skin degloving, fracture in the left thoracic limb (LTL), with abrasion lesions and dislocation in the right thoracic limb (RTL). The animal underwent multiple modality therapies, such as HBOT sessions associated with larval therapy; even after the LTL presented gangrene, this treatment resulted in optimal viability of the non-necrotic tissue adjacent to the gangrene. Due to chronic pain unresponsive to drug control and the presence of a fracture at a location where a possible exoprosthesis was supposed to be fixed, the LTL ended up being amputated. There are several reports of the use of HBOT or larval therapy in traumatized limbs; however, the combination of both therapies has not been previously described in the veterinary literature. Thus, we demonstrate through this report that it was possible to quickly recover the animal with good wound resolution through tissue oxygenation and a healthy granulation bed, both provided by the therapeutic combination.     

Efficacy of Butea monosperma on dermal wound healing in rats

Wound healing occurs as a fundamental response to tissue injury. Several natural products have been shown to accelerate the healing process. The present investigation was undertaken to determine the efficacy of topical administration of an alcoholic bark extract of Butea monosperma (B. monosperma) on cutaneous wound healing in rats. Full-thickness excision wounds were made on the back of rat and B. monosperma extract was administered topically. The granulation tissue formed on days 4, 8, 12 and 16 (post-wound) was used to estimate total collagen, hexosamine, protein, DNA and uronic acid. The extract increased cellular proliferation and collagen synthesis at the wound site, as evidenced by increase in DNA, total protein and total collagen content of granulation tissues. The extract treated wounds were found to heal much faster as indicated by improved rates of epithelialization and wound contraction, also confirmed by histopathological examinations. Also, the tensile strength of drug-treated wounds was increased significantly. In addition, we show that B. monosperma possesses antioxidant properties, by its ability to reduce lipid peroxidation. The results clearly substantiate the beneficial effects of the topical application of B. monosperma in the acceleration of wound healing.     

Human umbilical cord perivascular cells (HUCPVC)

Human bone marrow mesenchymal stem cells (hBM-MSC) have recently been employed in the clinical treatment of challenging skin defects. We have described an MSC population that can be easily harvested from human umbilical cord perivascular tissue, human umbilical cord perivascular cells (HUCPVC), which exhibit a higher proliferative rate and frequency than hBM-MSC. Our objective was to establish whether HUCPVC could promote healing of full thickness murine skin defects, and thus find utility as a cell source for dermal repair. To this end, bilateral full thickness defects were created on the dorsum of Balb/c nude mice. Fibrin was used as a delivery vehicle for 1 x 106 PKH-67 labeled HUCPVC with contralateral controls receiving fibrin only. Epifluorescent and brightfield microscopic evaluation of the wound site was carried out at 3 and 7 days while mechanical testing of wounds was carried out at 3, 7, and 10 days. Our results show that by 3 days, marked contraction of the wound was observed in the fibrin controls whilst the HUCPVC samples exhibited neither collapse nor contraction of the defect, and the dermal repair tissue was considerably thicker and more organized. By 7 days, complete re-epithelialization of the HUCPVC wounds was observed whilst in the controls re-epithelialization was limited to the wound margins. Wound strength was significantly increased in the HUCPVC treatment group by 3 and 7 days but no statistical difference was seen at 10 days. We conclude that HUCPVCs accelerate early wound healing in full thickness skin defects and thus represent a putative source of human MSCs for use in dermal tissue engineering.     

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.     

Human umbilical cord perivascular cells (HUCPVC): A mesenchymal cell source for dermal wound healing

Human bone marrow mesenchymal stem cells (hBM-MSC) have recently been employed in the clinical treatment of challenging skin defects. We have described an MSC population that can be easily harvested from human umbilical cord perivascular tissue, human umbilical cord perivascular cells (HUCPVC), which exhibit a higher proliferative rate and frequency than hBM-MSC. Our objective was to establish whether HUCPVC could promote healing of full thickness murine skin defects, and thus find utility as a cell source for dermal repair. To this end, bilateral full thickness defects were created on the dorsum of Balb/c nude mice. Fibrin was used as a delivery vehicle for 1 x 106 PKH-67 labeled HUCPVC with contralateral controls receiving fibrin only. Epifluorescent and brightfield microscopic evaluation of the wound site was carried out at 3 and 7 days while mechanical testing of wounds was carried out at 3, 7, and 10 days. Our results show that by 3 days, marked contraction of the wound was observed in the fibrin controls whilst the HUCPVC samples exhibited neither collapse nor contraction of the defect, and the dermal repair tissue was considerably thicker and more organized. By 7 days, complete re-epithelialization of the HUCPVC wounds was observed whilst in the controls re-epithelialization was limited to the wound margins. Wound strength was significantly increased in the HUCPVC treatment group by 3 and 7 days but no statistical difference was seen at 10 days. We conclude that HUCPVCs accelerate early wound healing in full thickness skin defects and thus represent a putative source of human MSCs for use in dermal tissue engineering.     

Amniotic mesenchymal stem cells enhance wound healing in diabetic NOD/SCID mice through high angiogenic and engraftment capabilities

Although human amniotic mesenchymal stem cells (AMMs) have been recognised as a promising stem cell resource, their therapeutic potential for wound healing has not been widely investigated. In this study, we evaluated the therapeutic potential of AMMs using a diabetic mouse wound model. Quantitative real-time PCR and ELISA results revealed that the angiogenic factors, IGF-1, EGF and IL-8 were markedly upregulated in AMMs when compared with adipose-derived mesenchymal stem cells (ADMs) and dermal fibroblasts. In vitro scratch wound assays also showed that AMM-derived conditioned media (CM) significantly accelerated wound closure. Diabetic mice were generated using streptozotocin and wounds were created by skin excision, followed by AMM transplantation. AMM transplantation significantly promoted wound healing and increased re-epithelialization and cellularity. Notably, transplanted AMMs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, indicating a direct contribution to cutaneous closure. Taken together, these data suggest that AMMs possess considerable therapeutic potential for chronic wounds through the secretion of angiogenic factors and enhanced engraftment/differentiation capabilities.