Topical p38 MAPK inhibition reduces bacterial growth in an in vivo burn wound model

BACKGROUND: Although the inflammatory response is a prerequisite for wound healing, excessive activation of the innate immune system can induce epithelial cell damage and apoptosis, which may further compromise dermal integrity. In a noninfectious burn wound model, we previously demonstrated that topical inhibition of p38 MAPK, an important inflammatory signaling pathway, attenuated epithelial cell damage and apoptosis. We now question whether attenuating local inflammation would weaken bacterial wound resistance and compromise host defense. METHODS: Rats received 30% total body surface area burn, and the wound was treated with topical application of a p38 MAPK inhibitor or vehicle. At 24 hours after injury, burn wounds were inoculated with Pseudomonas aeruginosa. At 48 hours postinjury, animals were sacrificed, and the burn wound was analyzed. RESULTS: Inoculating burn wounds induced significant bacterial growth. Dermal inflammatory changes were markedly accentuated in the inoculated animals. Topical p38 MAPK inhibition reduced the proinflammatory cytokine expression in the burn wounds and neutrophil sequestration with or without bacterial inoculation. Interestingly, the bacterial wound growth was significantly attenuated in animals treated with topical p38 MAPK inhibitor. CONCLUSIONS: Topical p38 MAPK inhibition attenuated wound inflammation without interfering with bacterial host defense. Attenuation of excessive burn wound inflammatory signaling may prevent secondary damage of the dermal barrier and reduce the growth of opportunistic pathogens.     

Invariant NKT cells promote skin wound healing by preventing a prolonged neutrophilic inflammatory response

The wound‐healing process consists of the inflammation, proliferation, and remodeling phases. In chronic wounds, the inflammation phase is prolonged with persistent neutrophil infiltration. The inflammatory response is critically regulated by cytokines and chemokines that are secreted from various immune cells. Recently, we showed that skin wound healing was delayed and the healing process was impaired under conditions lacking invariant natural killer T (iNKT) cells, an innate immune lymphocyte with potent immuno‐regulatory activity. In the present study, we investigated the effect of iNKT cell deficiency on the neutrophilic inflammatory response during the wound healing process. Neutrophil infiltration was prolonged in wound tissue in mice genetically lacking iNKT cells (Jα18KO mice) than in wild‐type (WT) control mice on days 1 and 3 after wounding. MIP‐2, KC, and IL‐17A were produced at a significantly higher level in Jα18KO mice than in WT mice. In addition, neutrophil apoptosis was significantly reduced in the wound tissue in Jα18KO mice than in WT mice. Treatment with anti‐IL‐17A mAb, anti‐Gr‐1 mAb, or neutrophil elastase inhibitor reversed the impaired wound healing in Jα18KO mice. These results suggest that iNKT cells may promote the wound healing process through preventing the prolonged inflammatory response mediated by neutrophils.     

Differential cutaneous wound healing in thermally injured MRL/MPJ mice

Adult wound repair occurs with an initial inflammatory response, reepithelialization, and the formation of a permanent scar. MRL/MpJ mice following ear‐hole punch biopsies display accelerated healing and tissue regeneration. In this study, we characterized the healing responses in both MRL/MpJ and BALB/c mice following a 15% total body surface area full‐thickness cutaneous burn injury. Macroscopic and histological observations show that delayed wound closure in MRL/MpJ mice is accompanied by an increase in edema, reduced neutrophil infiltration, and more prominent eschar. In vivo bromodeoxyuridine labeling showed no defect in keratinocyte proliferation and migration (reepithelialization). In comparison with BALB/c mice, MRL/MpJ wounds had greater collagen deposition, less granulation tissue formation, and contained fewer α‐smooth muscle actin‐positive myofibroblasts. An observed reduction in dermal neutrophil infiltration and myofibroblast development correlated with enhanced angiogenesis. Overall, BALB/c wounds contracted sooner and to a larger degree, resulting in a significant decrease in scar formation. Interestingly, MRL/MpJ mice showed overt abnormalities in hair follicle proliferation, morphogenesis, and subsequent hair regrowth postburn injury. No substantial evidence of tissue regeneration was observed in either BALB/c or MRL/MpJ wounds. Our results convincingly demonstrate that MRL/MpJ skin burn wounds heal with scar formation with delays in two critical wound healing events: wound closure, and myofibroblast development.     

Imaging mass spectrometry for accessing molecular changes during burn wound healing

The spatiotemporal analysis of the proteomic profile during human wound healing is a critical investigative step that can establish the complex interplay of molecular events that comprise the local response to burn injury. Partial‐thickness wound samples with adjacent “normal” skin were collected from twenty‐one patients with burn wounds and examined across a time spectrum ranging from the acute injury period at 3, 6, 11 days to the later hypertrophic scar period at 7 and 15 months. The techniques used for histology‐directed tissue analyses highlighted inflammatory protein markers at the early time points after injury with diminished expression as burn wounds progressed into the proliferative phase. The datasets show the usefulness of MALDI MS and imaging mass spectrometry as discovery approaches to identify and map the cutaneous molecular sequence that is activated in response to the unique systemic inflammatory response following burn trauma. This information has the potential to define the unique factors that predispose human burn victims to disfiguring hypertrophic scar formation.     

Extracorporeal shock wave therapy for chronic wounds: A systematic review and meta‐analysis of randomized controlled trials

A growing number of clinical studies demonstrate that extracorporeal shock wave therapy (ESWT) is a feasible noninvasive method for improving chronic wound healing. This systematic review and meta‐analysis aimed to assess the effectiveness of ESWT compared with that of the standard care treatment for the healing of chronic wounds, irrespective of etiology, in clinical practice. Randomized controlled trials that investigated the effect of ESWT on chronic wounds with different etiologies from 2000 to 2017 were included in this review. The methodological quality of each selected article was rated using the Jadad scale. A fixed or random effects model was used to calculate the pooled effect sizes according to the heterogeneity of the studies. The cumulative effect of ESWT on each outcome was illustrated using forest plots. Seven randomized controlled trials involving 301 subjects were included in this review. Meta‐analyses revealed that the use of ESWT as an adjunct to wound treatment could significantly accelerate the impaired healing process of chronic wounds. Compared with the control treatment, ESWT markedly increased the wound healing rate by 1.86‐fold (OR = 2.86, 95% CI: 1.63–5.03, p = 0.0003) and the percentage of the wound healing area by 30.46% (SMD = 30.46; 95% CI: 23.80–37.12; p < 0.00001). In addition, the wound healing time was reduced by 19 days (SMD = ?19.11, 95% CI: ?23.74–(–14.47), p < 0.00001) in chronic wound patients. No serious complications or adverse effects were observed secondary to the application of ESWT. The above data suggested that ESWT as an adjunct to wound treatment, could more significantly improve the healing process of chronic wounds than the standard care treatment alone. More high‐quality, well‐controlled randomized trials are needed to evaluate the efficacy of ESWT in clinical practice.     

Exacerbated and prolonged inflammation impairs wound healing and increases scarring

Altered inflammation in the early stage has long been assumed to affect subsequent steps of the repair process that could influence proper wound healing and remodeling. However, the lack of explicit experimental data makes the connection between dysregulated wound inflammation and poor wound healing elusive. To bridge this gap, we used the established rabbit ear hypertrophic scar model for studying the causal effect of dysregulated inflammation. We induced an exacerbated and prolonged inflammatory state in these wounds with the combination of trauma‐related stimulators of pathogen‐associated molecular patterns from heat‐killed Pseudomonas aeruginosa and damage‐associated molecular patterns from a dermal homogenate. In stimulated wounds, a heightened and lengthened inflammation was observed based on quantitative measurements of IL‐6 expression, tissue polymorphonuclear leukocytes infiltration, and tissue myeloperoxidase activity. Along with the high level of inflammation, wound healing parameters (epithelial gap and others) at postoperative day 7 and 16 were significantly altered in stimulated wounds compared to unstimulated controls. By postoperative day 35, scar elevation of stimulated wounds was higher than that of control wounds (scar elevation index: 1.90 vs. 1.39, p < 0.01). Moreover, treatment of these inflamed wounds with Indomethacin (at concentrations of 0.01, 0.1, and 0.4%) reduced scar elevation but with adverse effects of delayed wound closure and increased cartilage hypertrophy. In summary, successful establishment of this inflamed wound model provides a platform to understand these detrimental aspects of unchecked inflammation and to further test agents that can modulate local inflammation to improve wound outcomes.     

The anti‐inflammatory agent Propolis improves wound healing in a rodent model of experimental diabetes

Foot ulcers and poor wound healing are problematic for patients with diabetes. The beehive protectant Propolis can improve wound healing but whether it can improve healing in diabetic wounds has not been investigated. In this study, the effect of a single application of Propolis on epithelial closure, wound morphology, cellular infiltrate, and blood vessel density were investigated. Diabetes was induced in rats using streptozocin. After 6 weeks, diabetic and control animals were wounded and the wounds were treated with Propolis or saline as control. At days 6 and 12 animals were sacrificed and wounds were excised. Compared with controls, diabetes decreased epithelial closure and reepithelialization but had no effect on wound contraction. These delays were prevented by Propolis. At day 12, the impaired macrophage infiltration (C:1.49±0.09 vs. D:0.25±0.14), persistent neutrophil infiltration (C:0.22±0.19 vs. D:1.33±0.81), and increased myeloperoxidase activity (fourfold) in diabetic wounds were prevented by Propolis. Diabetes had no effect on wound volume, vessel number, or branch points. These novel data indicate that Propolis can accelerate wound healing in diabetes. As neutrophil infiltration is normalized, its mechanism of action may be through anti‐inflammatory pathways. This result and the established safety profile of Propolis provide a rationale for studying topical application of this agent in a clinical setting.     

Hyaluronan in human acute and chronic dermal wounds

There is growing interest in the relationship of hyaluronan and inflammation in a number of physiologic processes including wound healing. The objective of this study was to make a quantitative comparison of inflammation and hyaluronan expression in human normal healing open wounds and in pressure ulcers. Using an open dermal wound model, myeloperoxidase activity was found to peak at day 3. Hyaluronan levels showed a bimodal distribution with transient peaks occurring on days 1 and 7. Mean levels of myeloperoxidase activity in pressure ulcers were significantly higher than at any time in the acute wounds, whereas hyaluronan levels were significantly lower than at any time in the acute wounds. Levels of hyaluronidase activity increased slightly in the postwound period. Hyaluronidase activity in pressure ulcers was significantly elevated compared with the acute wounds. These results suggest a role for increased enzymatic degradation of hyaluronan as a function of inflammation during wound repair. This is the first reported quantitative examination of hyaluronan expression in human acute dermal wounds and in chronic pressure ulcers.     

Rapamycin reduces burn wound progression by enhancing autophagy in deep second‐degree burn in rats

Burn wound progression is caused by many mechanisms including local tissue hypoperfusion, prolonged inflammation, free radical damage, apoptosis, and necrosis in burn wounds. Autophagy, a homeostatic process by which cells break down their own components, was found to protect against ischemic injury, inflammatory diseases, and apoptosis in some cases. We tested whether rapamycin, an autophagy inducer, could ameliorate burn wound progression and promote wound healing through autophagy enhancement. Using a previously described deep second‐degree burn model, we first tested the effects of rapamycin on autophagic response in burn wound tissue. Autophagy levels in wound tissue of treated rats were increased as compared with controls. Furthermore, we found that laser Doppler flowmetry values and Na/K‐ATPase activities were markedly higher in the treated wounds. The content of interleukin‐8, methane dicarboxylic aldehyde, and myeloperoxidase activity in the wounds of treated rats were much lower than in controls. The apoptotic rates in treated wounds were much lower than controls as determined by terminal deoxynucleotidyl transferase mediated nick end labeling assay. Finally, histomorphological analysis showed that burn wound progression in the treatment group was ameliorated. The time to wound reepithelialization was shorter in the treated wounds than controls 22.5 ± 1.4 days vs. 24.8 ± 1.3 days (mean ± standard deviation, p < 0.01).     

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing

Transforming growth factor‐β (TGF‐β) is a multifunctional growth factor involved in all aspects of wound healing. TGF‐β accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol‐anchored protein, as a novel TGF‐β coreceptor and inhibitor of TGF‐β signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild‐type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin‐1α and monocyte chemoattractant protein‐1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.     

Enhanced wound‐healing performance of a phyto‐polysaccharide‐enriched dressing – a preclinical small and large animal study

Alginate is a natural rich anionic polysaccharide (APS), commonly available as calcium alginate (CAPS). It can maintain a physiologically moist microenvironment, which minimises bacterial infection and facilitates wound healing at a wound site. Patients with burn injuries suffer from pain and an inflammatory response. In this study, we evaluated the CAPS dressing and traditional dressing containing carboxymethyl cellulose (CMC) for wound healing and scar tissue formation in a burn model of rat and swine. In our pilot study of a burn rat model to evaluate inflammatory response and wound healing, we found that the monocyte chemoattractant protein (MCP)‐1 and transforming growth factor (TGF)‐β were up‐regulated in the CAPS treatment group. Next, the burn swine models tested positive for MCP‐1 in a Gram‐positive bacterial infection, and there was overproduction of TGF‐β during the burn wound healing process. Rats were monitored daily for 1 week for cytokine assay and sacrificed on day 28 post‐burn injury. The swine were monitored over 6 weeks. We further examined the pain and related factors and inflammatory cytokine expression in a rodent burns model monitored everyday for 7 days post‐burn. Our results revealed that the efficacy of the dressing containing CAPS for wound repair post‐burn was better than the CMC dressing with respect to natural wound healing and scar formation. The polysaccharide‐enriched dressing exerted an antimicrobial effect on burn wounds, regulated the inflammatory response and stimulated anti‐inflammatory cytokine release. However, one pain assessment method showed no significant difference in the reduction in levels of adenosine triphosphate in serum of rats after wound dressing in either the CAPS or CMC group. In conclusion, a polysaccharide‐enriched dressing outperformed a traditional dressing in reducing wound size, minimising hypertrophic scar formation, regulating cytokines and maximising antimicrobial effects.     

Topical insulin application improves healing by regulating the wound inflammatory response

Inflammation, the initiating stage of wound healing, is characterized by increased endothelial permeability, infiltration of inflammatory cells, and secretion of numerous growth factors and chemokines. By controlling wound contamination and infection, as well as inducing the repairing process, inflammatory response plays an irreplaceable role during wound healing. We utilized a variety of approaches to observe the effect of insulin on wound inflammatory response, specifically the effect of insulin on the function of wound macrophages. We also investigated whether insulin‐regulated inflammatory response contributed to insulin‐induced healing. Mice excisional wounds treated with insulin showed advanced infiltration and resolution of macrophages, which correlated with the expression of monocyte chemotactic protein‐1, a potent chemotactic factor for macrophages. Blockage of monocyte chemotactic protein‐1 resulted in reduced macrophages infiltration and impaired wound healing despite the presence of insulin. In vitro studies showed insulin‐facilitated monocytes/macrophages chemotaxis, pinocytosis/phagocytosis, and secretion of inflammatory mediators as well. Our study strongly suggests that insulin is a potent healing accelerant. Regulating wound inflammatory response, especially the quantity and function of macrophages, is one of the mechanisms explaining insulin‐induced accelerated wound healing.     

Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing

Inherent pathologies associated with diabetic wound microenvironment including increased proteolysis, inflammatory dysregulation, and impaired neovascularization prevent timely resolution of chronic diabetic ulcers. It is hypothesized that augmentation of local wound microenvironment with a stable provisional matrix formed by proteolysis-resistant angiogenic peptide nanofibers (NFs) will create permissive environment for attenuated inflammation, enhanced neovascularization, and improved diabetic wound healing. Using murine excisional wound healing models, full-thickness dorsal skin wounds were treated with either NFs or control solutions (phosphate buffered saline; hyaluronic acid) and analyzed for morphology, inflammatory response, neovascularization, and biomechanical properties. NF treatment of diabetic wounds stimulated formation of a robust pro-angiogenic in situ tissue-engineered provisional matrix leading to a significant decrease in wound inflammatory cell infiltration and proinflammatory interleukin-6 levels, a significant increase in endothelial and endothelial progenitor cell infiltration, vascular endothelial growth factor levels, and neovascularization (day 7), as well as improved wound morphology, accelerated wound closure, and significantly stronger repair tissue (day 28). These results suggest that appropriate design of provisional matrix may compensate for some of the complex disruptions in diabetic wound microenvironment and provide missing cues to cells and direct in situ responses toward improved healing, which is promising for future development of new therapies for diabetic ulcers.     

成纤维细胞生长因子在烧伤创面内变化的临床观察

目的 探讨临床烧伤创面愈合过程中成纤维细胞生长因子（ＦＧＦ）的变化及其作用。方法采用免疫组织化学方法,对烧伤创面肉芽中心部、肉芽与愈合皮肤的交界部、愈合皮肤和自身正常皮肤,以及Ⅲ度烧伤创面不同部位和Ⅱ度烧伤创面愈合过程中ＦＧＦ表达变化进行了观察。结果 Ⅲ度烧伤创面不同部位ＦＧＦ表达强度不同,以创面的肉芽中心部ＦＧＦ含量最多,肉芽与愈合皮肤的交界部ＦＧＦ含量次之,愈合皮肤ＦＧＦ含量较少;每个创面ＦＧ     

Blocking Interleukin-1β Induces a Healing-Associated Wound Macrophage Phenotype and Improves Healing in Type 2 Diabetes

Diabetes is associated with persistent inflammation and defective tissue repair responses. The hypothesis of this study was that interleukin (IL)-1β is part of a proinflammatory positive feedback loop that sustains a persistent proinflammatory wound macrophage phenotype that contributes to impaired healing in diabetes. Macrophages isolated from wounds in diabetic humans and mice exhibited a proinflammatory phenotype, including expression and secretion of IL-1β. The diabetic wound environment appears to be sufficient to induce these inflammatory phenomena because in vitro studies demonstrated that conditioned medium of both mouse and human wounds upregulates expression of proinflammatory genes and downregulates expression of prohealing factors in cultured macrophages. Furthermore, inhibiting the IL-1β pathway using a neutralizing antibody and macrophages from IL-1 receptor knockout mice blocked the conditioned medium–induced upregulation of proinflammatory genes and downregulation of prohealing factors. Importantly, inhibiting the IL-1β pathway in wounds of diabetic mice using a neutralizing antibody induced a switch from proinflammatory to healing-associated macrophage phenotypes, increased levels of wound growth factors, and improved healing of these wounds. Our findings indicate that targeting the IL-1β pathway represents a new therapeutic approach for improving the healing of diabetic wounds.Chronic wounds associated with diabetes, venous insufficiency, or pressure represent a major health problem, with millions of patients afflicted and the associated treatment costing billions of dollars per year (1). Despite the socioeconomic impact of chronic wounds, the underlying causes of impaired healing are not well-understood and effective treatments remain elusive. A common characteristic of these poorly healing wounds is a persistent inflammatory response, with prolonged accumulation of macrophages and elevated levels of proinflammatory cytokines (2–5). Translational research of the dysregulation of inflammation associated with impaired healing in diabetes should provide insight into the development of new therapeutic approaches.During normal wound healing in mice, inflammatory cells such as macrophages promote healing indirectly by killing pathogens and clearing the wound of damaged tissue, but also promote healing directly by producing growth factors that induce angiogenesis, collagen deposition, and wound closure (6–9). In contrast, during impaired healing of diabetic mice, wounds exhibit prolonged accumulation of macrophage associated with elevated levels of proinflammatory cytokines and proteases and reduced levels of various growth factors, all of which mimic chronic wounds in humans (10–12). We recently demonstrated that in wounds of diabetic mice, macrophages exhibit a sustained proinflammatory phenotype with an impaired upregulation of healing-associated factors that is observed in nondiabetic mice as healing progresses (13). However, the underlying causes of the dysregulation of macrophage in diabetic wounds remain to be elucidated.Multiple factors can influence macrophage phenotype and the actual phenotypes expressed in chronic wounds are likely determined by the balance of the proinflammatory and anti-inflammatory stimuli present in the wound environment. The proinflammatory environment observed in diabetic wounds has the potential to sustain a proinflammatory macrophage phenotype, which, in turn, would contribute to sustaining the proinflammatory environment. In fact, hyperglycemia is known to induce expression of interleukin (IL)-1β in a number of different cell types, including macrophages (14–16), and IL-1β, in turn, is known to induce a proinflammatory macrophage phenotype in part by inducing itself (17). Thus, the IL-1β pathway may be part of a positive feedback loop that sustains inflammation in chronic wounds and contributes to impaired healing. However, little is known about the actual role of IL-1β in diabetic wounds.The central hypothesis of this study is that sustained activity of the IL-1β pathway in diabetic wounds contributes to impaired healing of these wounds. The results of this study demonstrate that sustained IL-1β expression in wounds of diabetic humans and mice is associated with a proinflammatory macrophage phenotype, and that inhibiting the IL-1β pathway in wounds of diabetic mice induces the switch from proinflammatory to healing-associated macrophage phenotypes and improves healing of these wounds.     

Monitoring the healing of combat wounds using Raman spectroscopic mapping

Soldiers wounded in modern warfare present with extensive and complicated acute wounds, confounded by an overwhelming inflammatory response. The pathophysiology of acute wounds is unknown and timing of wound closure remains subjective. Collagen gene expression profiles are presented for 24 patients. Impaired healing wounds showed a twofold decrease in the up‐regulation of COL1A1 and COL3A1 genes in the beginning of the wound healing process, compared with normal healing wounds. By the final debridement, however, collagen gene expression profiles for normal and impaired healing wounds were similar for COL1A1 and COL3A1. In addition, Raman spectroscopic maps were collected of biopsy tissue sections, from the first and last debridements of 10 wounds collected from nine patients. Tissue components obtained for the debridement biopsies were compared to elucidate whether or not a wound healed normally. Raman spectroscopy showed a loss of collagen in five patients, indicated by a negative percent difference in the 1,665/1,445 cm^?1 band area ratios. Four healed patients showed an increased or unchanged collagen content. Here, we demonstrate the potential of Raman spectroscopic analysis of wound biopsies for classification of wounds as normal or impaired healing. Raman spectroscopy has the potential to noninvasively monitor collagen deposition in the wound bed, during surgical wound debridements, to help determine the optimal time for wound closure.     

Mature B cells accelerate wound healing after acute and chronic diabetic skin lesions

Chronic wounds affect 12–15% of patients with diabetes and are associated with a drastic decrease in their quality of life. Here, we demonstrate that purified mature naive B220⁺/CD19⁺/IgM⁺/IgD⁺ B cells improve healing of acute and diabetic murine wounds after a single topical application. B cell treatment significantly accelerated acute wound closure by 2–3 days in wild‐type mice and 5–6 days in obese diabetic mice. The treatment led to full closure in 43% of chronic diabetic wounds, as compared to only 5% in saline‐treated controls. Applying equivalent numbers of T cells or disrupted B cells failed to reproduce these effects, indicating that live B cells mediated pro‐healing responses. Topically applied B cell treatment was associated with significantly reduced scar size, increased collagen deposition and maturation, enhanced angiogenesis, and increased nerve growth into and under the healing wound. β‐III tubulin+ nerve endings in scars of wounds treated acutely with B cells showed increased relative expression of growth‐associated protein 43. The improved healing associated with B cell treatment was supported by significantly increased fibroblast proliferation and decreased apoptosis in the wound bed and edges, altered kinetics of neutrophil infiltration, as well as an increase in TGF‐β and a significant reduction in MMP2 expression in wound granulation tissue. Our findings indicate that the timeline and efficacy of wound healing can be experimentally manipulated through the direct application of mature, naive B cells, which effectively modify the balance of mature immune cell populations within the wound microenvironment and accelerate the healing process.