Epithelial mesenchymal transition traits in honey‐driven keratinocyte wound healing: Comparison among different honeys

Honey has been used since ancient times for wound repair, but the subjacent mechanisms are almost unknown. We have tried to elucidate the modulatory role of honey in an in vitro model of HaCaT keratinocyte re‐epithelialization by using acacia, buckwheat, and manuka honeys. Scratch wound and migration assays showed similar increases of re‐epithelialization rates and chemoattractant effects in the presence of different types of honey (0.1%, v/v). However, the use of kinase and calcium inhibitors suggested the occurrence of different mechanisms. All honeys activated cyclin‐dependent kinase 2, focal adhesion kinase, and rasGAP SH3 binding protein 1. However, vasodilator‐stimulated phosphoprotein, integrin‐β3, cdc25C, and p42/44 mitogen activated protein kinase showed variable activation pattern. Re‐epithelialization recapitulates traits of epithelial‐mesenchymal transition (EMT) and the induction of this process was evaluated by a polymerase chain reaction array, revealing marked differences among honeys. Manuka induced few significant changes in the expression of EMT‐regulatory genes, while the other two honeys acted on a wider number of genes and partially showed a common profile of up‐ and down‐regulation. In conclusion, our findings have shown that honey‐driven wound repair goes through the activation of keratinocyte re‐epithelialization, but the ability of inducing EMT varies sensibly among honeys, according to their botanical origin.     

Honey: An immunomodulator in wound healing

Honey is a popular natural product that is used in the treatment of burns and a broad spectrum of injuries, in particular chronic wounds. The antibacterial potential of honey has been considered the exclusive criterion for its wound healing properties. The antibacterial activity of honey has recently been fully characterized in medical‐grade honeys. Recently, the multifunctional immunomodulatory properties of honey have attracted much attention. The aim of this review is to provide closer insight into the potential immunomodulatory effects of honey in wound healing. Honey and its components are able to either stimulate or inhibit the release of certain cytokines (tumor necrosis factor‐α, interleukin‐1β, interleukin‐6) from human monocytes and macrophages, depending on wound condition. Similarly, honey seems to either reduce or activate the production of reactive oxygen species from neutrophils, also depending on the wound microenvironment. The honey‐induced activation of both types of immune cells could promote debridement of a wound and speed up the repair process. Similarly, human keratinocytes, fibroblasts, and endothelial cell responses (e.g., cell migration and proliferation, collagen matrix production, chemotaxis) are positively affected in the presence of honey; thus, honey may accelerate reepithelization and wound closure. The immunomodulatory activity of honey is highly complex because of the involvement of multiple quantitatively variable compounds among honeys of different origins. The identification of these individual compounds and their contributions to wound healing is crucial for a better understanding of the mechanisms behind honey‐mediated healing of chronic wounds.     

The effects of caffeine on wound healing

The purine alkaloid caffeine is a major component of many beverages such as coffee and tea. Caffeine and its metabolites theobromine and xanthine have been shown to have antioxidant properties. Caffeine can also act as adenosine‐receptor antagonist. Although it has been shown that adenosine and antioxidants promote wound healing, the effect of caffeine on wound healing is currently unknown. To investigate the effects of caffeine on processes involved in epithelialisation, we used primary human keratinocytes, HaCaT cell line and ex vivo model of human skin. First, we tested the effects of caffeine on cell proliferation, differentiation, adhesion and migration, processes essential for normal wound epithelialisation and closure. We used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) proliferation assay to test the effects of seven different caffeine doses ranging from 0·1 to 5 mM. We found that caffeine restricted cell proliferation of keratinocytes in a dose‐dependent manner. Furthermore, scratch wound assays performed on keratinocyte monolayers indicated dose‐dependent delays in cell migration. Interestingly, adhesion and differentiation remained unaffected in monolayer cultures treated with various doses of caffeine. Using a human ex vivo wound healing model, we tested topical application of caffeine and found that it impedes epithelialisation, confirming in vitro data. We conclude that caffeine, which is known to have antioxidant properties, impedes keratinocyte proliferation and migration, suggesting that it may have an inhibitory effect on wound healing and epithelialisation. Therefore, our findings are more in support of a role for caffeine as adenosine‐receptor antagonist that would negate the effect of adenosine in promoting wound healing.     

Hypoxia-enhanced wound-healing function of adipose-derived stem cells: Increase in stem cell proliferation and up-regulation of VEGF and bFGF

Adipose‐derived stem cells (ADSCs) have been shown to induce wound‐healing effects. Because inflammation near the wound area induces oxygen deficiency, it is interesting to elucidate the effect of hypoxia on the function of ADSCs. In this work, we asked: (1) does hypoxia alter the wound‐healing function of ADSCs? and (2) what are the major factors responsible for the alteration in the wound‐healing function? Effect of hypoxia on the proliferation of ADSCs was first examined that hypoxia (2% O₂) enhanced the proliferation of ADSCs in either the presence of serum or in the absence of serum. The conditioned medium of ADSCs harvested under hypoxia (hypoCM) significantly promoted collagen synthesis and the migration of human dermal fibroblasts, compared with that in normoxia (norCM). In the animal studies, hypoCM significantly reduced the wound area compared with norCM. Furthermore, mRNA and protein measurements showed that hypoxia up‐regulated growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Inhibition of VEGF and bFGF using neutralizing antibodies reversed the migration of the wounded human dermal fibroblasts and the healing of wounds in animal experiment. Collectively, these results suggest that hypoxia increases the proliferation of ADSCs and enhances the wound‐healing function of ADSCs, at least partly, by up‐regulating the secretion of VEGF and bFGF.     

Immunomodulatory potential of Nisin A with application in wound healing

Antimicrobial peptides can have a dual role with both antimicrobial activity against a broad range of bacteria and immunomodulatory effect, making them attractive as therapeutic treatment of difficult wounds. Nisin A is widely known for its antimicrobial activity, and a preliminary study demonstrated that it increased wound closure, but the mechanism behind its effect is unknown. The aim of this study is to elucidate the wound healing potential of Nisin A and the mechanism behind. First, an epithelial and endothelial cell line, human keratinocyte (HaCaT) and human umbilical vein endothelial cell, were used to demonstrate migration and proliferation effects in vitro. From HaCaT cells and peripheral blood mononuclear cell, changes in cytokine levels were shown by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. Second, the ex vivo porcine wound healing model was used to investigate the re‐epithelization potential of Nisin A. Finally, the model Galleria mellonella was used to confirm antimicrobial activity and to investigate potential immunomodulatory effects in vivo. Here, we demonstrated that Nisin A affected migration significantly of both human umbilical vein endothelial cell and HaCaT cells (p < 0.05) but not proliferation, potentially by decreasing the levels of proinflammatory cytokines tumor necrosis factor‐α, interleukin‐6, and interleukin‐8 (p < 0.001). Furthermore, Nisin A treatment diminished lipopolysaccharide‐induced tumor necrosis factor‐α levels from peripheral blood mononuclear cells and monocyte chemoattractant protein‐1 from HaCaT cells (p < 0.001). Furthermore, Nisin A did not affect proliferation ex vivo either but increased re‐epithelization of the porcine skin. Nisin A improved survival of G. mellonella significantly from Staphylococcus epidermidis (p < 0.001) but not from Escherichia coli, indicating that Nisin A did not help the larvae to survive the infection in a different than direct antimicrobial way. All together this makes Nisin A a potential treatment to use in wound healing, as it increases the mobility of skin cells, dampens the effect of lipopolysaccharide and proinflammatory cytokines, and decreases bacterial growth.     

Acute and chronic wound fluids influence keratinocyte function differently

Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT‐PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti‐proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP‐9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP‐9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing.     

Cell surface engineering using glycosylphosphatidylinositol anchored tissue inhibitor of matrix metalloproteinase‐1 stimulates cutaneous wound healing

The balance between matrix metalloproteinases and their endogenous tissue inhibitors (TIMPs) is an important component in effective wound healing. The biologic action of these proteins is linked in part to the stoichiometry of TIMP/matrix metalloproteinases/surface protein interactions. We recently described the effect of a glycosylphosphatidylinositol (GPI) anchored version of TIMP‐1 on dermal fibroblast biology. Here, cell proliferation assays, in vitro wound healing, electrical wound, and impedance measurements were used to characterize effects of TIMP‐1‐GPI treatment on primary human epidermal keratinocytes. TIMP‐1‐GPI stimulated keratinocyte proliferation, as well as mobilization and migration. In parallel, it suppressed the migration and matrix secretion of dermal myofibroblasts, and reduced their secretion of active TGF‐β1. Topical application of TIMP‐1‐GPI in an in vivo excisional wound model increased the rate of wound healing. The agent positively influenced different aspects of wound healing depending on the cell type studied. TIMP‐1‐GPI counters potential negative effects of overactive myofibroblasts and enhances the mobilization and proliferation of keratinocytes essential for effective wound healing. The application of TIMP‐1‐GPI represents a novel and practical clinical solution for facilitating healing of difficult wounds.     

Extracellular matrix-derived tripeptide proline-glycine-proline inhibits keratinocyte proliferation and migration

Keratinocytes are the predominant cell type in epidermis, and are primarily responsible for the epithelialization phase of wound healing. Previous studies by our group showed a positive correlation between IL‐8 concentration and delayed healing of porcine cutaneous partial‐thickness wounds. Interleukin‐8 and collagen‐breakdown product N‐acetyl‐Pro‐Gly‐Pro (PGP) are known as chemoattractant molecules for neutrophils during inflammation. The activity of both molecules is dependent on chemokine receptors CXCR1 and CXCR2. In addition to neutrophils, keratinocytes also express CXCR1 and CXCR2. Here we investigated the effects of IL‐8 and PGP on keratinocyte proliferation and migration. Our results showed that IL‐8 up to 100 ng/mL does not have any significant impact on keratinocyte proliferation or migration. ECM‐derived tripeptide PGP chemotactically attracts neutrophils but not keratinocytes. PGP strongly inhibits keratinocyte proliferation and migration in a cell‐type specific manner. Thus, collagen breakdown product PGP plays a key role in modulating both the inflammatory and epithelialization phases of wound healing.     

Vessel transformation in chronic wounds under topical negative pressure therapy: an immunohistochemical analysis

The underlying physiological mechanism of topical negative pressure (TNP) therapy is not yet completely understood. This prospective clinical study aims to clarify a potential influence of TNP therapy on vessel proliferation and hypoxia in chronic wounds. TNP was applied on chronic wounds of 16 patients (?125 mmHg) to prepare them for a plastic‐surgical reconstruction using free or pedicled flaps. Tissue biopsies were taken from the wound edge and wound bed at different time points. All samples were stained with haematoxylin and eosin, hypoxia‐induced factor‐1α and endothelial cell markers (CD31 and CD34) for the immunohistological analysis of inflammation, hypoxia and vessel proliferation. Between day 5 and day 8 of treatment, a considerable increase in blood vessel density could be observed, reaching a maximum of approximately 200% in contrast to the vessel density prior to treatment. In addition, the number of hypoxic and inflammatory cells was found to be increased at particular time points. This study demonstrates a stimulating effect on vessel proliferation under TNP treatment. TNP appears to support (neo‐) angiogenesis and transformation of chronic non‐healing wounds in a physiological wound healing process when combined with surgical debridement. This effect underlines the positive influence of TNP in the treatment of chronic wounds as shown by various clinical reports.     

Leucine‐rich glioma inactivated 3 promotes HaCaT keratinocyte migration

Our finding that human skin expresses leucine‐rich glioma inactivated 3 (LGI3) raises the question of the function of this cytokine in keratinocytes. We have shown that LGI3 stimulates human HaCaT keratinocyte migration without affecting viability or proliferation. Western blot analysis showed that LGI3 induced focal adhesion kinase activation, Akt phosphorylation, and glycogen synthase kinase 3β (GSK3β) phosphorylation in these cells. Using the scratch wound assay and a modified Boyden chamber, we found that LY294002, a selective phosphatidylinositol 3‐kinase inhibitor, and LiCl, a selective GSK3β inhibitor, abolished LGI3‐induced cell migration. We tested β‐catenin levels after LGI3 treatment because the Akt‐GSK3β pathway regulates β‐catenin accumulation, and β‐catenin promotes cell migration. LGI3 treatment increased β‐catenin protein and nuclear localization, whereas LY294002 prevented LGI3‐induced focal adhesion kinase and Akt activation as well as β‐catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following β‐catenin accumulation through the Akt pathway.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

A pre‐clinical functional assessment of an acellular scaffold intended for the treatment of hard‐to‐heal wounds

The majority of the population experience successful wound‐healing outcomes; however, 1–3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard‐to‐heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24‐hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound‐healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound‐healing purposes.     

Streptolysin O enhances keratinocyte migration and proliferation and promotes skin organ culture wound healing in vitro

ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is currently being investigated as a treatment for collagen-related disorders such as scleroderma and fibrosis. Furthermore, ML-05 may be effective in promoting wound healing and alleviating the formation of hypertrophic scars and keloids. To investigate the effects of ML-05 on wound-healing processes, in vitro wound-healing scratch assays (using human primary epidermal keratinocytes and dermal fibroblasts) and a human skin organ culture wound model were utilized. ML-05 markedly enhanced keratinocyte migration and proliferation in wound scratch assays. ML-05 did not affect either proliferation or migration of dermal fibroblasts, indicating that ML-05's effects on cell migration/proliferation may be keratinocyte-specific. ML-05 was tested in a dose-dependent manner in a skin organ culture wound model using two different application methods: Through the culture media (dermal exposure) or direct topical treatment of the wound surface. ML-05 was found to accelerate wound healing as measured by reepithelialization, particularly after topical application. Therefore, ML-05 may have potential as a wound-healing agent that promotes reepithelialization through stimulation of keratinocyte migration and proliferation.     

创面愈合评价指标进展

目的：建立客观,准确地评价创面愈合的标准。方法：对近年来国内外有关文献进行检索,总结。结果：筛选了创面愈合率,创面愈合时间,组织病理学分析,巨噬细胞定量分析,羟脯氨酸含量测定,细胞增殖情况,细胞DNA含量和细胞周期分析,转化生长因子－α水平,白细胞介素－1,白细胞介素－6和肿瘤坏死因子水平,角质细胞胶原酶－1含量测定,成纤维细胞生长因子受体－1水平,单核细胞化学诱导蛋白－1水平和角质细胞纤溶酶源活化抑制剂－2水平等十三种创面愈合评价指标。结论：创面愈合率,创面愈合时间及组织病理学分析仍是最直接而有效的创面愈合评价指标。     

Juglone ameliorates skin wound healing by promoting skin cell migration through Rac1/Cdc42/PAK pathway

Skin cell regeneration and wound healing are key processes in the recovery from skin injuries. Rapid cell migration and regeneration of skin cells lead to faster and better healing of wounded skin. In the present study, we aimed to investigate the wound healing potential of juglone, a naturally occurring Pin1 inhibitor found in walnuts. Cultured skin cells (NHDF and HaCaT) and hairless mice were treated with juglone after wound creation to examine its effects on cell migration and wound healing rate. The expressions of cell migration related proteins (Rac1, Cdc42, and α‐PAK), collagen deposition, and angiogenesis were analyzed. Juglone treatment resulted in faster rate of growth and migration and recovered cell morphology, particularly at a concentration of 5 µM, in skin cells compared to the untreated group. In vivo experiments showed that mice treated with juglone showed faster wound healing rate with better skin morphology and collagen deposition than the vehicle group. Furthermore, juglone increased the activation and/or expression of Cdc42, Rac1, and α‐pak in HaCaT cells, and resulted in enhanced angiogenesis in endothelial cells (HUVECs). Juglone also activated MAPKs signaling by activation of ERK, JNK, and p38 proteins. Taken together, these data suggest that juglone may be a potential candidate for wound healing and skin regeneration which ameliorates wound healing mainly by promoting skin cell migration through Rac1/Cdc42/PAK pathway.     

A simple microfluidic strategy for cell migration assay in an in vitro wound‐healing model

In vitro scratch wound assays are commonly used strategies to measure cell repair rate, facilitating the study of cell migration, tissue reorganization, and cell division. This work presented a simple and novel microfluidic device that allowed a quantitative investigation of the cell migration and cell proliferation behaviors in an in vitro wound‐healing model, especially focused on the scratch assay. The microfluidic device is composed of four units, which include cell growth regions and cell‐free regions created by micropillars. Using this device, we evaluated the proliferation and migration process of human gastric epithelial cells in the presence of different concentrations of the epidermal growth factor, and investigated the migration behavior of mesenchymal stem cells toward tumor cells as well. This approach has the unique capability to create localized cell‐free regions in parallel, and facilitate quantitative research on cell migration in the wound‐healing process, providing a powerful platform for elucidating the mechanism of cell migration in regeneration medicine.     

Bacterial wall products induce downregulation of vascular endothelial growth factor receptors on endothelial cells via a CD14-dependent mechanism: implications for surgical wound healing.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is a potent mitogenic cytokine which has been identified as the principal polypeptide growth factor influencing endothelial cell (EC) migration and proliferation. Ordered progression of these two processes is an absolute prerequisite for initiating and maintaining the proliferative phase of wound healing. The response of ECs to circulating VEGF is determined by, and directly proportional to, the functional expression of VEGF receptors (KDR/Flt-1) on the EC surface membrane. Systemic sepsis and wound contamination due to bacterial infection are associated with significant retardation of the proliferative phase of wound repair. The effects of the Gram-negative bacterial wall components lipopolysaccharide (LPS) and bacterial lipoprotein (BLP) on VEGF receptor function and expression are unknown and may represent an important biological mechanism predisposing to delayed wound healing in the presence of localized or systemic sepsis. MATERIALS AND METHODS: We designed a series of in vitro experiments investigating this phenomenon and its potential implications for infective wound repair. VEGF receptor density on ECs in the presence of LPS and BLP was assessed using flow cytometry. These parameters were assessed in hypoxic conditions as well as in normoxia. The contribution of CD14 was evaluated using recombinant human (rh) CD14. EC proliferation in response to VEGF was quantified in the presence and absence of LPS and BLP. RESULTS: Flow cytometric analysis revealed that LPS and BLP have profoundly repressive effects on VEGF receptor density in normoxic and, more pertinently, hypoxic conditions. The observed downregulation of constitutive and inducible VEGF receptor expression on ECs was not due to any directly cytotoxic effect of LPS and BLP on ECs, as measured by cell viability and apoptosis assays. We identified a pivotal role for soluble/serum CD14, a highly specific bacterial wall product receptor, in mediating these effects. The decreased VEGF receptor density on ECs accruing from the presence of bacterial wall products resulted in EC hyporesponsiveness to rhVEGF and significant abolition of VEGF-directed EC proliferation. CONCLUSION: These findings suggest that the well-recognized relationship between bacterial sepsis and attenuated wound healing may be due, in part, to the directly suppressive effects of bacterial wall components on EC VEGF receptor expression and, consequently, EC proliferation.     

Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells

Mesenchymal stem cells (MSCs), which can be isolated from bone marrow and other somatic tissues, are residing in an environment with relative low oxygen tension. The purpose of this study is to investigate the effects of hypoxia on MSCs, and we hypothesize that oxygen concentration regulates the intricate balance between cellular proliferation and commitment towards differentiation. In this study, human bone marrow‐derived MSCs were cultured under hypoxia with 1% O₂. The proliferation ability of MSCs was increased after a 7‐day hypoxic culture period. Migration assay showed that hypoxia enhanced the migration capabilities of MSCs. Moreover, expression of stemness genes Oct4, Nanog, Sall4 and Klf4 was increased under hypoxia. Furthermore, the differentiation ability of MSCs under hypoxia favored osteogenesis while adipogenesis was inhibited during a 4‐week induction period. Cytokine antibody array analysis showed that a number of growth factors were up‐regulated after a 7‐day hypoxic incubation and the differential expression of growth factors may account for the increased proliferation and osteogenic potentials of MSCs under hypoxic condition. Taken together, hypoxia provides a favorable culture condition to promote proliferation as well as osteogenesis of MSCs through differential growth factor production. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:260–266, 2012