Comparison of oxidative stress biomarker profiles between acute and chronic wound environments

Increasing evidence implicates excessive reactive oxygen species (ROS) generation and ROS-derived degradation products in the pathogenesis of many skin diseases. While numerous attempts have been made to identify prognostic biomarkers of wound healing in skin, these have met with limited success. This study examined the profiles of various oxidative stress biomarkers, namely total protein carbonyl content (from protein oxidation), malondialdehyde content (from lipid peroxidation), and the total antioxidant capacities, in acute wound fluid (n= 10) and chronic wound fluid (n= 12), using a rapid, noninvasive collection technique. Protein carbonyl content was quantified spectrophotometrically and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting, following 2,4-dinitrophenylhydrazine derivitization. Malondialdehyde levels were similarly quantified, following N-methyl-2-phenylindole derivitization. Total antioxidant capacity was determined via wound fluid inhibition of cytochrome C reduction by a superoxide radical flux. Acute wound fluid contained higher protein carbonyl content than chronic wound fluid, particularly evident following sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis under nonreducing and reducing conditions (p < 0.001 and p < 0.02, respectively), related to significantly higher protein levels (p = 0.0005) in acute wound fluid. Human serum albumin ( approximately 66 kDa) was identified as the most prominent protein oxidized in both acute and chronic wound fluid, which may contribute to the reduced albumin and total protein levels in chronic wound fluid. No significant difference (p > 0.1) in malondialdehyde levels or total antioxidant capacities were determined between acute and chronic wound fluids, although chronic wound fluid exhibited significantly higher total antioxidant capacities (p < 0.005), accounting for variations in wound fluid protein content. These findings suggest an adaptation in the antioxidant profiles of chronic wound fluid to counteract the loss of consumed antioxidants in the chronic wound environment. This study highlights the roles of ROS/antioxidants in skin wound healing, their possible involvement in chronic wounds and the potential value of ROS-induced biomarkers in wound healing prognosis.     

Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers

The purpose of this study was to determine the biochemical composition of fluid taken from chronic wounds, to compare these values with that of serum, and therefore to assess whether the wound fluid is representative of the extracellular environment of the wound. Paired wound fluid and blood samples were collected from eight patients with chronic leg ulcers in a nonhealing and healing phase. Wound fluid and serum samples were screened for a profile of general biochemical analyses, including electrolytes, lactate, glucose, and protein analyses. Electrolyte levels were essentially identical in wound fluid and serum samples. Lactate and lactate dehydrogenase levels were significantly greater and glucose and bicarbonate levels were significantly lower in wound fluid when compared with the paired serum samples. Albumin and total protein levels in wound fluid were on average half those of serum levels. In this small sample of eight patients, wound fluid collected from chronic leg ulcers is an exudate with the biochemical composition expected in extracellular fluid. In addition, bicarbonate and glucose levels increase and C-reactive protein levels decrease in wound fluid, but remain unchanged in serum, during healing. These results suggest changes in the state of hypoxia and the inflammatory process in the healing wound.     

Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors

To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 microg MMP Eq/ml) compared to acute wounds (median 0.76 microg MMP Eq/ml) (p < 0.001). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0. 001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.     

Surgical wound bed preparation of chronic and acute wounds

Our aging population has presented us with many new challenges. One such challenge is the need to manage an increase in wound-related problems effectively and efficiently. Over time, two parallel, yet divergent, management systems have developed. One strategy, used by medical specialists, uses a variety of dressings, topical enzymes, and local and systemic medications ultimately aimed at the promotion of healing by secondary intention or, in some cases, optimization of the wound for surgical reconstruction. In the second strategy, used by surgeons, early surgical intervention is used to prepare the wound for reconstruction in a timelier manner while promoting the healing process. This article reviews the development of these two distinct management systems and their areas of commonality and sets forth a new model to support the role of surgery in the treatment of problematic wounds.     

负压治疗技术在急、慢性骨感染中的应用

目的 探讨负压治疗技术在治疗急、慢性骨感染中的价值.方法 采用负压治疗技术.即在一段时间内将伤口置于密闭强力负压状态,治疗急、慢性骨感染患者30例(33个部位),辅助清创手术,应用敏感抗生素,应用植皮、肌皮瓣转移等方法闭合创面.结果 29个部位通过1次负压治疗就可达到创面闭合条件.所有患者均获随访,随访时间6～23个月,平均13.6个月,感染无复发.结论 负压治疗技术能有效控制急、慢性骨感染,缩短治疗时间,在骨感染治疗中有较好的应用前景.     

A novel option in negative pressure wound therapy (NPWT) for chronic and acute wound care

Introduction

Negative pressure wound therapy (NPWT) has become a widely accepted technique in treatment of all kinds of wounds. After a long period of clinical application of the V.A.C.™ system (KCI Inc., San Antonio, Texas, USA) a number of options for delivery of NPWT are now commercially available. An urgent need exists for evidence demonstrating clinical efficacy of these new devices to support clinicians regarding their choice of NPWT.

Methods

42 patients with an acute or chronic wound were randomly assigned to either treatment by V.A.C.™ (group A) or therapy with an alternative newly available polyurethane foam-based NPWT system (RENASYS GO™ – F/P, Smith &; Nephew GmbH) (group B). In both groups NPWT was applied after surgical debridement to prepare the wound bed for skin grafting. After skin grafting NPWT was applied additionally to secure skin grafts and improve grafts survival. Primary outcome measures were the time to complete healing (days) and duration of the NPWT application (days). Secondary outcome measures were the number of dressing changes and reported complications. In addition, we evaluated the cost-benefit in the clinical implementation.

Results

There were no significant differences comparing the investigated parameters between both groups. Especially average time to complete healing and average time NPWT was applied did not differ (p > 0.05). No complications occurred in either group. By an almost identical supply agreement of both providers for our hospital RENASYS™ system appeared to be more cost-effective.

Conclusion

After a long period of preserving a monopoly market position of the V.A.C.™ system, a new comparable option was successfully tested in this preliminary study. The polyurethane foam-based NPWT system (RENASYS GO™ – F/P, Smith &; Nephew GmbH) is an efficient and cost-effective alternative NPWT system, which we effectively implemented in therapeutic management of different kinds of wounds.     

Damage control in trauma: laparotomy wound management acute to chronic

Damage control surgery is fundamental to operative trauma care. Prophylactic application of open abdomen techniques has led to avoidance of a great deal of the organ dysfunction associated with abdominal compartment syndrome. Surgeons are learning about management of large open abdominal wounds. There seems to be a general consensus regarding acute management of these wounds. Institutions are using staged techniques of management. Getting open wounds closed as soon as possible leads to fewer complications. The acute use of vacuum wound may provide for early secondary closure. There is less study focused on optimal definitive reconstructive techniques. Further study in all of these areas will lead to improved outcomes.     

A statistical analysis of murine incisional and excisional acute wound models

Mice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation.     

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.     

Biofilms and chronic wound inflammation

In contrast to the commonly accepted hypothesis of host-centred pathology, it is possible that surface bacteria, not host dysfunction, cause the chronicity and perpetual inflammation associated with chronic non-healing wounds.     

The effect of a new wound dressing on wound healing: Biochemical and histopathological evaluation

Electrospinning process has gained importance in the production of wound dressings in recent years. The wound dressings prepared by electrospinning method provide many advantages over conventional wound dressings. The aim of this study was to assess the histological, biochemical, and immunohistochemical evaluation of collagen/doxycycline loaded nanofiber wound dressing in both acute and chronic wound healing. Full-thickness wound model was created on rats and rats were divided in two main groups: normoglycemic (acute) and hyperglycemic (chronic) groups. Each group was divided into three sub groups: not treated (control) group, treated with nanofiber wound dressing group and treated with commercial product group. Wound closure rates were measured. Oxidative events were investigated by biochemical analyses. In addition to histological studies, matrix metalloproteinase, tissue inhibitor of metalloproteinase, vascular endothelial growth factor, basic-fibroblast growth factor, and von Willebrand factor levels were investigated with immunohistochemical studies. According to the biochemical analyses, it was concluded that the nanofiber wound dressing helps to increase antioxidant capacity and decrease lipid peroxidation. Immunohistochemical studies showed that nanofiber wound dressing enhanced angiogenesis and shortened the inflammatory phase. It was concluded that an effective and safe prototype nanofiber wound dressing, which has similar wound healing effect to the commercial product, has been developed to be used in acute or chronic wound treatment.     

An in silico approach to the analysis of acute wound healing

The complex interactions that characterize acute wound healing have stymied the development of effective therapeutic modalities. The use of computational models holds the promise to improve our basic approach to understanding the process. By modifying an existing ordinary differential equation model of systemic inflammation to simulate local wound healing, we expect to improve the understanding of the underlying complexities of wound healing and thus allow for the development of novel, targeted therapeutic strategies. The modifications in this local acute wound healing model include: evolution from a systemic model to a local model, the incorporation of fibroblast activity, and the effects of tissue oxygenation. Using these modifications we are able to simulate impaired wound healing in hypoxic wounds with varying levels of contamination. Possible therapeutic targets, such as fibroblast death rate and rate of fibroblast recruitment, have been identified by computational analysis. This model is a step toward constructing an integrative systems biology model of human wound healing.     

慢性前列腺炎在急性附睾炎发病中的关联度分析

目的 探讨慢性前列腺炎(CP)在急性附睾炎(acute epididymitis.AE)发病中的作用及两者发病的关联度.方法 将1998年1月至2009年1月年以来222例慢性前列腺炎与181例急性附睾炎患者随机分为急性附睾炎组及慢性前列腺炎组,比较AE伴有CP发病率与CP伴发AE的发病率,分析两者发病率差异性及关联度.结果 急性附睾炎组同时伴发慢性前列腺炎发病率为96.68%(175/181例),未伴发CP者6例.慢性前列腺炎组急性附睾炎发病率为13.96%(31/222例),未伴发CP者191例.附睾炎组CP发病率高于慢性前列腺炎组AE发病率,采用t检验两者差异有统计学意义.结论 附睾炎组CP发病率明显高于慢性前列腺炎组AE发病率,CP的存在是急性附睾炎发病的重要基础性因素,但不能排除急性附睾炎引起前列腺炎的可能,两者有重要关联度.     

Temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor and gelatinase-B in chronic wound fluid switches from a chronic to acute wound profile with progression to healing

The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor-1 and gelatinase-B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor-1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor- bound urokinase plasminogen activator-plasminogen activator inhibitor-1 complex ( congruent with 80-116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor-1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator-plasminogen activator inhibitor-1 complex in blood- and plasma-derived serum and mastectomy wound fluid. Plasminogen activator inhibitor-1 was detected in blood-derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase-9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase-9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood-derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme- inhibitor complex, the level of expression of matrix metalloproteinase-9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase-9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase-9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase-9 appear to be useful biomarkers to determine clinical wound healing status.     

Acute wound healing: the biology of acute wound failure

Acute wound healing failure is an important source of morbidity and mortality for surgical patients. Many incisional hernias, gastrointestinal anastomotic leaks, and vascular pseudoaneurysms occur despite patient optimization and standardized surgical technique. Modern surgical experience suggests that biologic and mechanical pathways overlap during "normal" acute wound healing. The cellular and molecular processes activated to repair tissue from the moment of injury are under the control of biologic and mechanical signals. Successful acute wound healing occurs when a dynamic balance is met between the loads placed across a provisional matrix and the feedback and feed-forward responses of repair cells.     

The structure and composition of chronic wound eschar

Wounds with a covering of eschar require debridement before optimal wound healing can proceed. There are several different methods available but these have been derived empirically with no direct evidence of the structure or composition of the tissue they are designed to remove, or of the potential autolytic mechanisms which are the targets for some of these treatments. The aim of this study therefore was to determine the composition of chronic wound eschar and hence identify potential targets for the induction of autolytic debridement. Chronic wound eschar was removed by surgical debridement and analysed using immunohistochemistry, polyacrylamide gel electrophoresis (PAGE) and gelatin zymography. Immunohistochemistry using antibodies specific for extracellular matrix (ECM) proteins revealed a definite tissue structure, consisting of many fibrous regions and fine fibrillar elements separated by areas of tissue which were of a more irregular and amorphous nature. An antibody specific for all leucocytes revealed the presence of leucocytes in the region of tissue closest to the wound bed. The presence of this leucocyte population correlated to elevated levels of gelatinase activity as identified by gelatin zymography. PAGE analysis identified various protein species in the range 3.5-60 kDa molecular weight. These data indicate that wound debridement is likely to require multiple enzyme specificities to degrade the eschar and that these enzymes may be supplied by inflammatory leucocytes infiltrating the eschar from the wound bed. The various protein species demonstrated by PAGE may represent ECM proteins, those with lower molecular weight possibly representing the degradation products of autolytic debridement.     

急性化脓性阑尾炎切口感染的危险因素分析及预防对策

目的：分析急性化脓性阑尾炎患者术后切口感染的危险因素,探讨有效的预防对策。方法回顾性分析我院2011年1月～2014年2月间收治的219例急性化脓性阑尾炎患者的临床资料,观察术后切口感染情况,对术后切口感染的危险因素进行分析,总结预防经验。结果本组219例急性化脓性阑尾炎患者,术后切口感染16例,切口感染率为7.3%。经对症处理后,切口达到Ⅱ期愈合。切口感染率在不同年龄、合并基础疾病、术前应用抗生素以及手术时间方面的差异有统计学意义（P＜0.05）,而不同性别、切口长度及术后抗生素应用时间方面的差异无统计学意义（P＞0.05）。结论高龄、合并有基础疾病、术前未应用抗生素以及手术时间过长是急性化脓性阑尾炎术后切口感染的危险因素,术前合理应用抗生素,术中合理操作,缩短手术时间,术后加强观察是预防切口感染的关键。     

Biochemical assessment of acute and chronic treatment of Paget's bone disease with calcitonin and calcium with and without biphosphonate

Response to acute and chronic administration of calcitonin and calcium and of biphosphonates (EHDP) was evaluated in 14 patients with Paget's bone disease who were grouped on the basis of homogeneous disease activity, as appraised by bone involvement and alkaline phosphatase and hydroxyproline levels. At first, 100 MRC U of calcitonin followed 4 hours later by 500 mg of elemental calcium were given for 10 days; a significant (p less than 0.001; paired and unpaired Student t test) reduction in alkaline phosphatase (-25%) and hydroproline (-55%) was observed. Subsequently, 5 mg/kg/day of EHDP was given for 20 days. Both parameters increased to levels similar to basal values. These increases were significant (p less than 0.001 for the paired and unpaired Student test) compared with those obtained after calcitonin administration; alkaline phosphatase rose +27% and hydroxproline +135%. After this, patients were divided into 2 groups (A and B). Group A was treated with calcitonin and calcium, at the dosage indicated above, for 10 days a month during 6 months. Group B continued with the same protocol with the addition of EHDP for the 20 days during which calcitonin and calcium were not given. The results of 6 months of treatment showed that calcitonin was more active and suggested that EHDP diminishes hormonal effects. These results also demonstrate a short-term absence of EHDP activity.