071TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms.
Funding: DOD BC99184 and NIH CA85492.     

Potential molecular mechanisms of negative pressure in promoting wound healing

Negative pressure wound therapy (NPWT) has been widely used in various lesions. This study aimed to explore the biological effects of negative pressure on the polymorphonuclear neutrophils (PMNs), macrophages, and epidermal keratinocyte cells involved in wound healing. PMNs differentiated from HL‐60, macrophages were derived from THP‐1 monocytes, and keratinocytes were cultured in vitro, and they were treated with 0, ?0.03 mp, and ?0.05 mp, respectively. Cell ultrastructure; viability; apoptosis; and protein factors such as tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), interleukin‐17 (IL‐17), and cell division cycle 42 (Cdc42) were determined by transmission electron microscopy (TEM), CCK8, flow cytometry (FCM), ELISA, and simple Western assays, respectively. After negative pressure stimulation, the cell ultrastructure of PMNs and macrophages cells was presented with a marked increase of lysosomes and a relative decrease of mitochondria. In addition, the cell viability was enhanced in PMNs and macrophages in a pressure‐dependent manner and apoptosis ratios were significantly reduced in PMNs and macrophages. In addition, under ?0.05 negative pressure, IFN‐γ and IL‐17 were significantly increased in PMNs or macrophages. Moreover, increased EGF and EGFR and Cdc42 levels in keratinocytes induced by the ?0.05 mpa were detected, indicating that the migration chemotaxis of keratinocyte cells was enhanced. Negative pressure might promote cell proliferation, accelerate inflammatory responses, and promote epithelialisation during wound healing by increasing IFN‐γ, IL‐17, Cdc42, EGF, and EGFR in PMNs, macrophages, or keratinocytes under different negative pressures.     

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.     

Coacervate delivery of HB‐EGF accelerates healing of type 2 diabetic wounds

Chronic wounds such as diabetic ulcers pose a significant challenge as a number of underlying deficiencies prevent natural healing. In pursuit of a regenerative wound therapy, we developed a heparin‐based coacervate delivery system that provides controlled release of heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) within the wound bed. In this study, we used a polygenic type 2 diabetic mouse model to evaluate the capacity of HB‐EGF coacervate to overcome the deficiencies of diabetic wound healing. In full‐thickness excisional wounds on NONcNZO10 diabetic mice, HB‐EGF coacervate enhanced the proliferation and migration of epidermal keratinocytes, leading to accelerated epithelialization. Furthermore, increased collagen deposition within the wound bed led to faster wound contraction and greater wound vascularization. Additionally, in vitro assays demonstrated that HB‐EGF released from the coacervate successfully increased migration of diabetic human keratinocytes. The multifunctional role of HB‐EGF in the healing process and its enhanced efficacy when delivered by the coacervate make it a promising therapy for diabetic wounds.     

UV fluorescence excitation imaging of healing of wounds in skin: Evaluation of wound closure in organ culture model

Background and Objective

Molecules native to tissue that fluoresce upon light excitation can serve as reporters of cellular activity and protein structure. In skin, the fluorescence ascribed to tryptophan is a marker of cellular proliferation, whereas the fluorescence ascribed to cross‐links of collagen is a structural marker. In this work, we introduce and demonstrate a simple but robust optical method to image the functional process of epithelialization and the exposed dermal collagen in wound healing of human skin in an organ culture model.

Materials and Methods

Non‐closing non‐grafted, partial closing non‐grafted, and grafted wounds were created in ex vivo human skin and kept in culture. A wide‐field UV fluorescence excitation imaging system was used to visualize epithelialization of the exposed dermis and quantitate wound area, closure, and gap. Histology (H&E staining) was also used to evaluate epithelialization.

Results

The endogenous fluorescence excitation of cross‐links of collagen at 335 nm clearly shows the dermis missing epithelium, while the endogenous fluorescence excitation of tryptophan at 295 nm shows keratinocytes in higher proliferating state. The size of the non‐closing wound was 11.4 ± 1.8 mm and remained constant during the observation period, while the partial‐close wound reached 65.5 ± 4.9% closure by day 16. Evaluations of wound gaps using fluorescence excitation images and histology images are in agreement.

Conclusions

We have established a fluorescence imaging method for studying epithelialization processes, evaluating keratinocyte proliferation, and quantitating closure during wound healing of skin in an organ culture model: the dermal fluorescence of pepsin‐digestible collagen cross‐links can be used to quantitate wound size, closure extents, and gaps; and, the epidermal fluorescence ascribed to tryptophan can be used to monitor and quantitate functional states of epithelialization. UV fluorescence excitation imaging has the potential to become a valuable tool for research, diagnostic and educational purposes on evaluating the healing of wounds. Lasers Surg. Med. 48:678–685, 2016. © 2016 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.     

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.     

Degradation of human collagen isoforms by Clostridium collagenase and the effects of degradation products on cell migration

Clostridium collagenase has been widely used in biomedical research to dissociate tissues and isolate cells; and, since 1965, as a therapeutic drug for the removal of necrotic wound tissues. Previous studies found that purified collagenase‐treated extracellular matrix stimulated cellular response to injury and increased cell proliferation and migration. This article presents an in vitro study investigating the digestive ability of Clostridium collagenase on human collagen types I, III, IV, V and VI. Our results showed that Clostridium collagenase displays proteolytic power to digest all these types of human collagen, except type VI. The degradation products derived were tested in cell migration assays using human keratinocytes (gold surface migration assay) and fibroblasts (chemotaxis cell migration assay). Clostridium collagenase itself and the degradation products of type I and III collagens showed an increase in keratinocyte and fibroblast migration, type IV‐induced fibroblast migration only, and the remainder showed no effects compared with the control. The data indicate that Clostridium collagenase can effectively digest collagen isoforms that are present in necrotic wound tissues and suggest that collagenase treatment provides several mechanisms to enhance cell migration: collagenase itself and collagen degradation products.     

Wound healing in skin promoted by inoculation with Pseudomonas aeruginosa PAO1: The critical role of tumor necrosis factor-α secreted from infiltrating neutrophils

Wound healing is promoted by the presence of replicating microorganisms adhering to the wounded tissue, but the precise mechanism is not fully understood. In the present study, using a rat model with full‐thickness dermal wounds, we examined the effect of Pseudomonas aeruginosa inoculation on wound healing and the role of neutrophils infiltrating the wound site. Within 3 days, inoculation with this bacterium had accelerated re‐epithelialization, epidermal cell proliferation, and neo‐vascularization, as well as the local infiltration of neutrophils, which reached a peak at 24 hours. Tumor necrosis factor (TNF)‐α was detected in the wound tissues on the mRNA and protein levels within 24 hours. Flow cytometry and immunohistochemical analyses detected higher levels of TNF‐α in the infiltrating neutrophils in rats inoculated with P. aeruginosa than in uninoculated rats. Neutropenic rats treated with anti‐neutrophil mAb or cyclophosphamide exhibited significant attenuation in re‐epithelialization, epidermal cell proliferation, neo‐vascularization, and TNF‐α synthesis compared with control; administration of TNF‐α reversed these attenuations. These wound‐healing responses were decelerated in rats treated with anti‐TNF‐α mAb, as was the infiltration of neutrophils. These results indicate that inoculation with P. aeruginosa promotes wound healing by inducing the infiltration of neutrophils, which play a critical role as a major source of TNF‐α.     

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.     

基质金属蛋白酶—1在表皮修复中的生物学作用

目的 阐明有关基质金属蛋白酶－1（MMP－1）在皮肤损伤后,表皮修复过程中的作用。方法 回顾近年来有关MMP－1在皮肤损伤修复中的作用及研究进展,总结其在上皮化局部微小环境内的表达及细胞分子生物学效应。结果 皮肤损伤信号直接引发表皮细胞于特定的部位及时间表达MMP－1;MMP－1通过特异性分解创面基质胶原蛋白,促进表皮细胞的增殖及迁移,由此影响表皮损伤创面的愈合结果。结论 皮肤损伤后表皮细胞表达MMP－1与创面重新上皮化直接相关。     

Keratin dressings speed epithelialization of deep partial‐thickness wounds

Keratin gene expression is regarded as a hallmark of epidermal biology. It demarcates the three keratinocyte phenotypes: basal (expressing KRT5 and KRT14), differentiating (expressing KRT1 and KRT10), and activated (wound healing), which is characterized by expression of KRT6, KRT16, and KRT17. Activated keratinocytes are among the first signals of epidermal wound healing. In addition, they are found deregulated in nonhealing chronic wounds. To examine keratins as a potential modality for wound‐healing disorders, we evaluated two different keratin dressings, liquid or solid, and assessed their effects of epithelialization and closure using porcine partial‐thickness wound‐healing model in vivo. We found that both forms of keratin dressings accelerated closure and epithelialization, achieving statistically significant differences on day 5. Evidence suggesting early onset of epithelialization was corroborated further by gene expression analyses revealing induction of KRT6A, KRT16, and KRT17 by day 2 postwounding. The data suggest that keratin dressings may stimulate epithelialization by enhancing the activation of keratinocytes. We conclude that keratin‐containing dressings can accelerate wound healing and closure. Further studies are needed to determine the molecular mechanisms of this activation.     

Basic fibroblast growth factor stimulates human keratinocyte motility by Rac activation

Topical application of human recombinant basic fibroblast growth factor (bFGF) promotes wound healing. bFGF, however, has been reported to have little in vitro effects on keratinocyte compared with other cell types such as endothelial cells or fibroblasts. The aim of this study was to investigate the mechanism(s) of bFGF-stimulated keratinocyte migration. Normal human keratinocytes, seeded on coverslips that were noncoated or coated with type I collagen or fibronectin, were stimulated with bFGF to evaluate their ability to spread. Keratinocyte migration was measured using a Boyden chamber assay. The lysates of keratinocytes, which were plated on noncoated, type I collagen-coated or fibronectin-coated plastic dishes and stimulated with bFGF, were subjected to pulldown assays to detect guanine triphosphate-loaded Rac. Morphologically, keratinocytes formed lamellipodia only when they were stimulated with bFGF on the collagen-coated coverslips. Keratinocyte migration was significantly enhanced by bFGF. Guanine triphosphate-loaded Rac was detected only in the lysate of bFGF-stimulated keratinocytes on collagen-coated dishes. This in vitro study shows that bFGF exerts a stimulatory effect on keratinocyte migration in the presence of type I collagen as a scaffold, and, at least, Rac activation is involved.     

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.     

Collagenase-2 (MMP-8) and matrilysin-2 (MMP-26) expression in human wounds of different etiologies

Wound healing involves highly controlled events including reepithelialization, neoangiogenesis, and reformation of the stromal compartment. Matrix metalloproteinases (MMPs) are a family of neutral zinc-dependent endopeptidases known to be essential for the wound-healing process. MMP-8 (collagenase-2) is a neutrophil-derived highly effective type I collagenase, recently indicated to be important for acute wound healing. MMP-26 is a more recent and less well-studied member of the MMP family. Our aim was to study the expression of MMP-8 and MMP-26 in human cutaneous wound repair and chronic wounds using histological methods and cell culture. MMP-8 expression was associated with epithelial cells, neutrophils, and other inflammatory cells in chronic human wounds. MMP-26 was prominently expressed in the extracellular compartment of most chronic wounds in close vicinity to the basement membrane area. MMP-26 was also expressed in acute day 1 wounds with declining expression thereafter. In vitro wound experiments showed that both MMP-8 and MMP-26 were expressed by migrating human mucosal keratinocytes. Inhibiting MMP-26 resulted in aberrant keratinocyte migration and proliferation. We conclude that MMP-8 and MMP-26 are differentially expressed in acute and chronic wounds.     

Expression of epithelial matrix molecules collagen and laminin and corresponding integrins in chronic wounds

Re-epithelialization of cutaneous wounds is a coordinated process of proliferation and migration of keratinocytes at the wound edge. The study objective was to identify the differences in epidermal morphology, keratinocyte proliferation and matrix molecules (laminin 1, laminin 5, type IV collagen) and their specific integrin (α3, α6) expression in biopsies of meshed split thickness grafted and chronic wounds. The mean mitotic index of keratinocytes (ratio of cell cycle associated antigen Ki-67 expressing keratinocytes to basal keratinocytes) was highest in chronic wounds (38.7%) compared to acute wounds (22.25%, range 5.7% to 54%). The mean thickness of the hyper-proliferative epithelium at the wound edge of chronic wounds was 0.69 mm compared to 0.15 mm at the wound margin of split thickness grafted wounds. Both chronic wounds and skin grafted wounds exhibited strong laminin 5 immunoreactivity at the basal side of the epithelium, which extended under the most forward keratinocytes. Laminin 1 and type IV collagen immunoreactivity did not extend to the wound margin in either skin grafted or chronic wounds. In both transplanted skin and chronic wounds, the integrin sub-units α3 and α6 exhibited a strong pericellular immunoreactivity on the leading keratinocytes of the wound margin. Our data demonstrates that the proliferation of keratinocytes and the expression of associated integrins are not impaired in chronic wounds. Presented at the 33rd Congress of the Association of German Plastic Surgeons, Germany, 18–21 September, 2002.     

Comparison of human umbilical cord blood‐derived mesenchymal stem cells with healthy fibroblasts on wound‐healing activity of diabetic fibroblasts

Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood‐derived mesenchymal stromal cells (hUCB‐MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB‐MSCs have a superior wound‐healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB‐MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB‐MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB‐MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB‐MSCs. After a 3‐day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB‐MSC‐treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast‐treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB‐MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing.     

The effect of pH on cell viability,cell migration,cell proliferation,wound closure,and wound reepithelialization: In vitro and in vivo study

Wound microenvironment plays a major role in the process of wound healing. It contains various external and internal factors that participate in wound pathophysiology. The pH is an important factor that influences wound healing by changing throughout the healing process. Several previous studies have investigated the role of pH in relation to pathogens but studies concentrating on the effects of pH on wound healing itself are inconclusive. The purpose of this study was to comprehensively and in a controlled fashion investigate the effect of pH on wound healing by studying its effect on human primary keratinocyte and fibroblast function in vitro and on wound healing in vivo. In vitro, primary human keratinocytes and fibroblasts were cultured in different levels of pH (5.5–12.5) and the effect on cell viability, proliferation, and migration was studied. A rat full‐thickness wound model was used to investigate the effect of pH (5.5–9.5) on wound healing in vivo. The effect of pH on inflammation was monitored by measuring IL‐1 concentrations from wounds and cell cultures exposed to different pH environments. Our results showed that both skin cell types tolerated wide range of pH very well. They further demonstrated that both acidic and alkaline environments decelerated cell migration in comparison to neutral environments and interestingly alkaline conditions significantly enhanced cell proliferation. Results from the in vivo experiments indicated that a prolonged, strongly acidic wound environment prevents both wound closure and reepithelialization while a prolonged alkaline environment did not have any negative impact on wound closure or reepithelialization. Separately, both in vitro and in vivo studies showed that prolonged acidic conditions significantly increased the expression of IL‐1 in fibroblast cultures and in wound fluid, whereas prolonged alkaline conditions did not result in elevated amounts of IL‐1 .