Occlusive dressings and wound healing

Over the last 15 years, there has been explosive growth in the use of occlusive dressings as an aid to wound healing. In 1980 synthetic wound dressings were rarely sold, whereas in 1987 it was estimated that $350 million was spent on these dressings.¹ The number and variety of dressings on the market have also increased dramatically over the past decade to the point where more than 70 different brands exist today (Table 1). A dressing is said to be “occlusive” if a moist wound surface is maintained when the dressing is in place. Occlusive dressings prevent wound desiccation by inhibiting the transmission of water vapor from the wound surface to the atmosphere. A dressing that transmits moisture vapor at a rate lower than the rate of production of moisture by the underlying tissue creates a moist wound environment.Besides increasing the rate of epithelialization of wounds, other benefits of occlusive dressings are improving the granulation tissue in chronic wounds, providing a means of painless, autolytic debridement of necrotic ulcers,² and protecting the wound bed from environmental toxins and microorganisms. These dressings reduce wound pain and help produce a better cosmetic appearance, that is, a less noticeable scar. Finally, occlusive dressings may be cost effective in the treatment of certain wounds by decreasing the nursing time required for wound care. In this review, we discuss the numerous types and uses of occlusive dressings. The proposed mechanisms responsible for the beneficial effects of wound occlusion and the microbiology of occluded wounds are also explored.     

Studies on cytokines related to wound healing in donor site wound fluid

This study was performed to evaluate cytokines in donor-site wound fluids and to determine their effect on wound healing. A film dressing was applied to the donor-site wound of 24 patients immediately after a split-thickness skin graft was taken. On the 5th day after treatment, 2–3 ml of the fluid retained under the film dressing was collected by means of puncture with a syringe. Growth factors and cytokines considered to accelerate wound healing were present in relatively large amounts in the exudate. Very low concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were detected by a commercially-available enzyme-linked immunosorbant assay (ELISA) kit. However, the presence of both growth factors in wound fluid could not be confirmed because of the possible cross-reactivity of the antibodies to other EGF and FGF family growth factors. In contrast, platelet derived growth factor (PDGF), interleukin-6 (IL-6), transforming growth factor- (TGF-) and TGF-β were present in relatively large amounts. The finding that certain cytokines coexist in a balanced state under the film dressing suggests that epithelization can proceed, since an adequate balance would insure proper regulation by the cytokine network. Our present study increases the likelihood that film or hydrocolloid dressings will be used more frequently in the future for treatment of burn wounds, ulcers or donor-site wounds since these dressings were shown to be more capable than ointments of retaining cytokines, particularly intrinsic growth factors secreted at the wound site.     

Stability of the cathelicidin peptide LL-37 in a non-healing wound environment

The endogenous cathelicidin peptide LL-37 is strongly expressed at the wound edge early in the process of acute wound healing, but only weakly expressed in chronic wounds. Excessive proteolysis may limit the therapeutic usefulness of exogenous LL-37, especially in ulcers colonized with Pseudomonas aeruginosa that produce elastase, which degrades LL-37. This study investigated the stability of synthetic LL-37 against two types of proteinases in the presence or absence of wound fluid samples (diluted to 10-20%) from nine non-healing venous leg ulcers. Incubation of LL-37 (10 μg/ml) at 37°C for 6 h resulted in complete degradation by the serine proteinase trypsin (≥ 10 ng/ml), while no degradation was observed with matrix metalloproteinase-9. LL-37 susceptibility to trypsin was diminished considerably in the presence of wound fluid, and there was no apparent cleavage of exogenous LL-37 incubated in wound fluid for up to 24 h at 37°C even when using fluids from ulcers with resident P. aeruginosa (n = 2). In conclusion, LL-37 was degraded by trypsin, but not by matrix metalloproteinase-9, and was fairly resistant to proteolytic cleavage ex vivo by incubation with wound fluid from non-healing venous leg ulcers. Thus, the proteolytic environment of chronic wounds does not seem to prevent the therapeutic use of topical LL-37.     

Wound fluid under occlusive dressings from diabetic patients show an increased angiogenic response and fibroblast migration

IntroductionMetabolic diseases like diabetes mellitus often show prolonged healing and chronic wounds. Occlusive wound dressings are known to support wound closure by creating a moist environment which supports collagen synthesis, epithelialization and angiogenesis. We aimed to assess the effect of occlusion on diabetic wound fluid on the cellular level regarding fibroblast activity and angiogenetic response.Material and methods22 split skin donor sites from 22 patients (11 patients with diabetes mellitus) were treated with occlusive dressings intraoperatively. On day 3, fluid and blood serum samples were harvested while changing the dressings. The influence of wound fluid on fibroblasts was assessed by measuring metabolic activity (Alamar Blue assay, Casey Counter), cell stress/death (LDH assay) and migration (in vitro wound healing assay) of fibroblasts. Angiogenesis of endothelial cells (HUVEC) was analyzed with the tube formation assay. Furthermore, a Magnetic Luminex Assay for multi-cytokines detection was performed focusing on inflammatory and pro-angiogenetic cytokines.ResultsThe influence of wound fluid under occlusive dressings from diabetic patients showed a significantly increased angiogenic response and fibroblast migration compared to the non-diabetic patient group. Additionally, cell stress was increased in the diabetic group. Cytokine analysis showed an increase in VEGF-A in the diabetic group.ConclusionOcclusive dressings may stimulate regenerative effects in diabetic wounds. Our in-vitro study shows the influence of wound fluid under occlusive dressings from diabetic patients on angiogenesis, migration and proliferation of fibroblasts, which are essential modulators of wound healing and scar modulation.     

Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid

Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non-infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non-infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections.     

Exogenous Smad3 accelerates wound healing in a rabbit dermal ulcer model

Exogenous administration of transforming growth factor-beta (TGF-beta) improves wound healing by affecting cellular and molecular events involved in tissue repair. But mice with a deficiency of a key TGF-beta signaling intermediate, Smad3, paradoxically showed accelerated cutanenous wound healing, suggesting that endogenous Smad3 had inhibitory effect on cutaneous wound healing. Here we investigated the effect of exogenous expression of Smad3 in dermal fibroblasts on cutaneous wound healing. Subcutaneous injection of adenovirus-containing Smad3 complementary DNA (AdCMV-Smad3) targeting mainly dermal fibroblasts accelerated tissue repair following full-thickness dermal round wounds in rabbit ear as judged by the size of granulation tissue area, number of capillaries, and re-epithelialization rate of the wounds. Expressions of alpha-smooth muscle actin (alpha-SMA), vascular endothelial growth factor (VEGF), and fibroblast growth factor receptor were upregulated in the wounded area injected with AdCMV-Smad3. Consistent with the in vivo findings, overexpression of Smad3 induced alpha-SMA, VEGF, and TGF-beta1 expression and augmented chemotactic response in cultured dermal fibroblasts. Therefore, exogenous administration of Smad3 targeting dermal fibroblasts accelerated tissue repair in a rabbit dermal ulcer model by affecting fibroblast responses associated with wound healing. The results suggest that Smad3, when overexpressed in dermal fibroblasts, can promote wound healing.     

Growth factors and chronic wounds: the need to understand the microenvironment.

The care of chronic wounds has become a major health issue in developed countries because of their increasingly elderly populations. There is hope that progress made in understanding and producing growth factors will lead to their successful use to induce faster and better healing of chronic wounds. This report will discuss growth factors in the context of their use in chronic wounds, and will focus on the importance of the wound microenvironment in determining the interactions between growth factors and wounds. We believe that a greater understanding of the chronic wound microenvironment will be of benefit in the optimal use of growth factors. In published studies, we have found that wound fluid taken from acute wounds stimulates fibroblast and endothelial cell proliferation, whereas fluid obtained from chronic non-healing wounds inhibits the growth of fibroblasts, endothelial cells, and keratinocytes. In this report, we describe the effect of these two types of wound fluid on the synthesis of extracellular matrix components. We hypothesize that the chronic wound microenvironment is generally non-conducive to cell growth, and that this may prevent a truly successful use of topical growth factors in chronic wounds. Novel approaches in the delivery of growth factors to wounds may be necessary to overcome these obstacles.     

Differences between fibroblasts cultured from oral mucosa and normal skin: implication to wound healing.

It is generally agreed that oral mucosa heals faster with less scar than skin does, and hypertrophic scar or keloid is very rare in the oral cavity. Fibroblasts are thought to play an important role in wound healing and scar formation, whose control is mediated by growth factors. We have studied whether there are any differences in the cellular behavior of fibroblasts between oral mucosa and skin, and in their response to growth factors. Oral mucosal fibroblasts proliferated slightly more than dermal fibroblasts on average. Dermal fibroblasts in collagen gel possessed greater contraction potency than oral mucosa fibroblasts, irrespective of the presence of growth factors; however, oral mucosa fibroblasts showed an earlier collagen gel contraction with or without TGF-beta1. There were no differences in basal collagen synthetic rate between dermal and oral mucosal fibroblasts, while the latter synthesized more collagen than dermal fibroblasts when they were stimulated with TGF-beta1. Our study showed that oral mucosal fibroblasts and dermal fibroblasts had selective differences in cellular behavior and in their responses to growth factors, which seems to contribute to the differences in wound healing.     

Dibutyryl cAMP influences endothelial progenitor cell recruitment during wound neovascularization

Delayed wound healing is one of the major complications of diabetes, and is caused by delayed cellular infiltration, reduced angiogenesis, and decreased formation and organization of collagen fibers. Recently, endothelial progenitor cells (EPC) isolated from peripheral blood were shown to accumulate at sites of neovascularization during wound healing. The present study tested the hypothesis that sodium N-6,2'-O-dibutyryl adenosine-3',5'-cyclic phosphate (DBcAMP), which has been shown to accelerate wound healing, promotes recruitment of EPC into wounds and contributes to the stimulation of neovascularization in genetically diabetic mice. Topical application of DBcAMP resulted in significant acceleration of wound healing and wound vascularization partly via enhanced recruitment of EPC. EPC in DBcAMP-treated wounds were mainly localized to cell clusters at the border of the granulation tissue, a site where blood supply is most insufficient. DBcAMP treatment increased the mRNA expression of angiogenic cytokines vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1alpha (SDF-1alpha) in vivo in wound tissue and in cultured fibroblasts and macrophages, in vitro. Culture supernatants of DBcAMP-treated cells enhanced EPC migration. Taken together, these results indicate that DBcAMP promotes neovascularization in wound healing, at least partly by increasing the accumulation of EPC at wound sites.     

Growth factors in wound healing

Normal wound healing requires the coordinated completion of a variety of cellular activities. The cellular activities involved in normal healing include phagocytosis, chemotaxis, mitogenesis, collagen synthesis, and the synthesis of other matrix components. After a wound is created, phagocytosis of bacteria and damaged tissue is a necessary prerequisite to most other healing processes. Neutrophils and macrophages are the primary phagocytic cells. Chemotaxis is the migration of cells against a gradient. Chemotactic agents for inflammatory cells, fibroblasts, and cells involved in angiogenesis contribute to the healing process. Mitogenesis-stimulating agents cause cells required for healing to divide. Mitogenesis generates more fibroblasts, epithelial cells, and angiogenic cells to carry out the healing process. New collagen and ground substance are required to give the healing wound strength and structure, and the synthesis of these proteins and glycoproteins, primarily by fibroblasts, is another critical component of the healing process. Wound contraction and scar remodeling are other major aspects of healing. These processes are most likely not purely cellular activities, though cells are required for wound contraction and scar remodeling to occur. All of these processes must be accomplished for a wound to heal.     

Accelerated epithelization under a highly vapor-permeable wound dressing is associated with increased precipitation of fibrin(ogen) and fibronectin

In a previous study we showed that the use of a newly developed, highly water vapor permeable, PEU wound dressing accelerates the epithelization of partial-thickness wounds more than an occlusive wound dressing (OpSite) in comparison with air exposure. The purpose of this study was to investigate the distribution of fibrin(ogen), fibronectin, and type IV collagen during the epithelization process under these three conditions. The breathable PEU film enabled coagulation of the wound exudate, preserving it into a semisolid gelatinous state. This coagulum layer contained an abundant amount of fibrin(ogen) and fibronectin. In wounds occluded with OpSite film, depositions of fibrin(ogen) and fibronectin were less extensive. Migrating keratinocytes contained intracellular depositions of fibrin(ogen), suggesting that these cells phagocytize components of the provisional fibrin matrix during wound healing. It was concluded that accelerated epithelization underneath the highly water vapor permeable polyetherurethane film dressing is associated with the presence of a gelatinous coagulum containing fibrin(ogen) and fibronectin. We speculate that the enhanced healing rate might be caused by an increased concentration of growth-promoting factors present in the residual exudate underneath the PEU dressing.     

The effect of thrombocytopenia on dermal wound healing

The immediate appearance of platelets in wounds and the ability of platelets to release growth factors suggest that platelets are an important trigger of the tissue repair process. To examine the effect of systemic thrombocytopenia on both the inflammatory and proliferative aspects of wound healing, adult mice were rendered thrombocytopenic by intraperitoneal administration of a rabbit antimouse platelet serum. Full-thickness excisional dermal wounds were prepared and analyzed for inflammatory cell content, growth factor production, reepithelialization, collagen synthesis, and angiogenesis at multiple time points after injury. Compared to control mice, thrombocytopenic mice exhibited significantly altered wound inflammation. Wounds of thrombocytopenic mice contained significantly more macrophages and T cells, yet exhibited neutrophil content similar to wounds from control mice. Surprisingly, thrombocytopenic mice exhibited no delay in the reparative aspects of wound healing. The rate of wound reepithelialization, collagen synthesis, and angiogenesis was nearly identical for thrombocytopenic and control mice. Analysis of vascular endothelial growth factor, fibroblast growth factor 2, transforming growth factor beta1, keratinocyte growth factor, and epidermal growth factor revealed no difference in the levels of these growth factors in the wounds of control and thrombocytopenic mice. Taken together, the results suggest that the presence of platelets may influence wound inflammation, but that platelets do not significantly affect the proliferative aspects of repair, including wound closure, angiogenesis, and collagen synthesis.     

Management of minor acute cutaneous wounds: importance of wound healing in a moist environment

Moist wound care has been established as standard therapy for chronic wounds with impaired healing. Healing in acute wounds, in particular in minor superficial acute wounds – which indeed are much more numerous than chronic wounds – is often taken for granted because it is assumed that in those wounds normal phases of wound healing should run per se without any problems. But minor wounds such as small cuts, scraps or abrasions also need proper care to prevent complications, in particular infections. Local wound care with minor wounds consists of thorough cleansing with potable tap water or normal saline followed by the application of an appropriate dressing corresponding to the principles of moist wound treatment. In the treatment of smaller superficial wounds, it appears advisable to limit the choice of dressing to just a few products that fulfil the principles of moist wound management and are easy to use. Hydroactive colloid gels combining the attributes of hydrocolloids and hydrogels thus being appropriate for dry and exuding wounds appear especially suitable for this purpose – although there is still a lack of data from systematic studies on the effectiveness of these preparations.     

Inducible lineage-specific deletion of TbetaRII in fibroblasts defines a pivotal regulatory role during adult skin wound healing

Previous attempts to delete type II TGFbeta receptor (TbetaRII) in fibroblasts have precluded examination of adult mice due to early mortality. We have selectively deleted TbetaRII postnatally in differentiated connective tissue fibroblasts using an inducible Cre-Lox strategy. Tamoxifen-dependent Cre recombinase linked to a fibroblast-specific regulatory sequence from the proalpha2(I)collagen gene permitted deletion of floxed TbetaRII alleles. After postnatal deletion of TbetaRII in fibroblasts, healing of excisional skin wounds in adults showed markedly attenuated dermal scar formation, defective wound contraction and enhanced epidermal proliferation. These findings support a pivotal role for transforming growth factor beta (TGFbeta) signalling in fibroblasts in regulating normal skin wound healing. Explanted dermal fibroblasts from TbetaRII-null-fib mice showed impaired migration and did not generate normal contractile biomechanical forces in fixed collagen gels nor develop alpha-smooth muscle antigen-rich stress fibers in response to TGFbeta1. Surprisingly, some TGFbeta-regulated proteins, including connective tissue growth factor (CTGF), were basally upregulated in TbetaRII-null fibroblasts and this was dependent on extracellular signal-regulated kinase 1/2 activity in these cells. This suggests that other intracellular pathways regulating CTGF expression may partially compensate for disruption of TGFbeta signalling in fibroblasts. Together, our data confirm that expression of TbetaRII in differentiated dermal fibroblasts is essential for normal wound healing and demonstrate a critical role in the development and function of myofibroblasts.     

S100A8/A9 deficiency in nonhealing venous leg ulcers uncovered by multiplexed antibody microarray profiling

Background Knowledge on the underlying mechanisms for nonhealing chronic wounds is fragmentary. Objectives To increase our understanding of the pathogenesis, the relationship between healing ability and a large panel of proteins was studied using a specially designed wound‐healing antibody‐based microarray. Methods Wound fluid from nondiabetic patients with nonhealing venous leg ulcers was compared with that from patients with healing open granulating acute wounds. The high‐throughput method enabled simultaneous measurement of the relative levels of 48 different proteins representing major categories of wound‐healing modulators. Results Unexpectedly, several of the examined proteins, including various proinflammatory cytokines, proteinases and antiproteinases, were not significantly (P > 0·001) changed in chronic wound fluid. For example, levels of matrix metalloproteinase‐9 and one of its substrates type IV collagen were similar in the two groups. The wound fluid samples displayed similar degrees of fragmentation of fibronectin by Western blot analysis and the total fibronectin levels were doubled (P < 0·001) in chronic compared with acute wounds. The increased fibronectin originated from α‐smooth muscle actin‐positive myofibroblasts and not from the circulation. S100A8/A9 was the sole protein that was reduced (P < 0·001) in wound fluid from venous ulcers [median 226 μg mL^?1 (interquartile range 213–278)] compared with healing wounds [455 μg mL^?1 (382–504)], probably reflecting a difference in inflammatory cell composition. Conclusion The molecular anomalies in chronic wounds are more subtle than the current paradigm and neither excessive proteinase activity nor deficiencies of examined extracellular matrix proteins, growth factors or angiogenic/angiostatic factors appear to contribute significantly to the nonhealing state of venous leg ulcers.     

Granulocyte-macrophage colony-stimulating factor is essential for normal wound healing

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent growth factor, which plays an important role during the process of wound healing. In clinical settings it has occasionally been employed in the treatment of cutaneous wounds of diverse etiologies. In a previous study, we have shown the positive influence of GM-CSF on full thickness excisional wounds in transgenic mice overexpressing GM-CSF in the basal layer of the epidermis. Direct GM-CSF action as well as indirect processes through the induction of secondary cytokines were proposed to contribute towards the beneficial effects. In this study, we analyzed the process of wound healing in transgenic mice overexpressing a GM-CSF antagonist in the epidermis. These mice not only exhibited a delayed scab rejection and reepithelialization but also neovascularization was reduced. The newly formed tissue was of poor quality as exhibited by the presence of extensive fibrosis. We suggest that the presence of GM-CSF in the repair process is of basic importance and its absence leads not only to delayed wound healing but it is also detrimental for the quality of the newly formed tissue.     

Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice

Degradation of the extracellular matrix, which is an indispensable step in tissue remodelling processes such as embryonic development and wound healing of the skin, has been attributed to collagenolytic activity of members of the matrix metalloproteinase family (MMPs). Here, we employed mmp13 knockout mice to elucidate the function of MMP13 in embryonic skin development, skin homeostasis, and cutaneous wound healing. Overall epidermal architecture and dermal composition of non-injured skin were indistinguishable from wild-type mice. Despite robust expression of MMP13 in the early phase of wound healing, wild-type and mmp13 knockout animals did not differ in their efficiency of re-epithelialization, inflammatory response, granulation tissue formation, angiogenesis, and restoration of basement membrane. Yet, among other MMPs also expressed during wound healing, MMP8 was found to be enhanced in wounds of MMP13-deficient mice. In summary, skin homeostasis and also tissue remodelling processes like embryonic skin development and cutaneous wound healing are independent of MMP13 either owing to MMP13 dispensability or owing to functional substitution by other collagenolytic proteinases such as MMP8.     

Fibroblast‐specific upregulation of Flightless I impairs wound healing

The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell‐specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue‐specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast‐specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue‐specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non‐inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non‐tissue‐specific upregulation of Flii suggesting that fibroblast‐derived Flii may have an important role in the wound healing response.