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      Comparison of In-Vitro and Ex-Vivo Wound Healing Assays for the Investigation of Diabetic Wound Healing and Demonstration of a Beneficial Effect of a Triterpene Extract

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          Abstract

          Diabetes mellitus is a frequent cause for chronic, difficult-to-treat wounds. New therapies for diabetic wounds are urgently needed and in-vitro or ex-vivo test systems are essential for the initial identification of new active molecules. The aim of this study is to compare in-vitro and ex-vivo test systems for their usability for early drug screening and to investigate the efficacy of a birch bark triterpene extract (TE) that has been proven ex-vivo and clinically to accelerate non-diabetic wound healing (WH), in a diabetic context. We investigated in-vitro models for diabetic WH, i.e. scratch assays with human keratinocytes from diabetic donors or cultured under hyperglycaemic conditions and a newly developed porcine ex-vivo hyperglycaemic WH model for their potential to mimic delayed diabetic WH and for the influence of TE in these test systems. We show that keratinocytes from diabetic donors often fail to exhibit significantly delayed WH. For cells under hyperglycaemic conditions significant decrease is observed but is influenced by choice of medium and presence of supplements. Also, donor age plays a role. Interestingly, hyperglycaemic effects are mainly hyperosmolaric effects in scratch assays. Ex-vivo models under hyperglycaemic conditions show a clear and substantial decrease of WH, and here both glucose and hyperosmolarity effects are involved. Finally, we provide evidence that TE is also beneficial for ex-vivo hyperglycaemic WH, resulting in significantly increased length of regenerated epidermis to 188±16% and 183±11% (SEM; p<0.05) compared to controls when using two different TE formulations. In conclusion, our results suggest that microenvironmental influences are important in WH test systems and that therefore the more complex hyperglycaemic ex-vivo model is more suitable for early drug screening. Limitations of the in-vitro and ex-vivo models are discussed. Furthermore our data recommend TE as a promising candidate for in-vivo testings in diabetic wounds.

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          Most cited references33

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          Keratinocyte-fibroblast interactions in wound healing.

          Cutaneous tissue repair aims at restoring the barrier function of the skin. To achieve this, defects need to be replaced by granulation tissue to form new connective tissue, and epithelial wound closure is required to restore the physical barrier. Different wound-healing phases are recognized, starting with an inflammation-dominated early phase giving way to granulation tissue build-up and scar remodeling after epithelial wound closure has been achieved. In the granulation tissue, mesenchymal cells are maximally activated, cells proliferate, and synthesize huge amounts of extracellular matrix. Epithelial cells also proliferate and migrate over the provisional matrix of the underlying granulation tissue, eventually closing the defect. This review focuses on the role of keratinocyte-fibroblast interactions in the wound-healing process. There is ample evidence that keratinocytes stimulate fibroblasts to synthesize growth factors, which in turn will stimulate keratinocyte proliferation in a double paracrine manner. Moreover, fibroblasts can acquire a myofibroblast phenotype under the control of keratinocytes. This depends on a finely tuned balance between a proinflammatory or a transforming growth factor (TGF)-beta-dominated environment. As the phenotype of fibroblasts from different tissues or body sites becomes better defined, we may understand their individual contribution in wound healing in more detail and possibly explain different clinical outcomes.
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            Clinical review: The role of advanced glycation end products in progression and complications of diabetes.

            Diabetic complications appear to be multifactorial in origin, but in particular, the biochemical process of advanced glycation, which is accelerated in diabetes as a result of chronic hyperglycemia and increased oxidative stress, has been postulated to play a central role in these disorders. Advanced glycation involves the generation of a heterogenous group of chemical moieties known as advanced glycated end products (AGEs), this reaction occurring as a result of a nonenzymatic reaction with glucose interacting with proteins, lipids, and nucleic acids, and involves key intermediates such as methylglyoxal. In this review we report on how these AGEs may exert deleterious effects in diabetes, as well as address current strategies to interrupt the formation or action of AGEs. First, AGEs act directly to induce cross-linking of long-lived proteins such as collagen to promote vascular stiffness, and, thus, alter vascular structure and function. Second, AGEs can interact with certain receptors, such as the receptor for AGE, to induce intracellular signaling that leads to enhanced oxidative stress and elaboration of key proinflammatory and prosclerotic cytokines. Over the last decade, a large number of preclinical studies have been performed, targeting the formation and degradation of AGEs, as well as the interaction of these AGEs with receptors such as the receptor for AGE. It is hoped that over the next few years, some of these promising therapies will be fully evaluated in the clinical context with the ultimate aim to reduce the major economical and medical burden of diabetes, its vascular complications.
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              TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays.

              Cell migration plays a major role in development, physiology, and disease, and is frequently evaluated in vitro by the monolayer wound healing assay. The assay analysis, however, is a time-consuming task that is often performed manually. In order to accelerate this analysis, we have developed TScratch, a new, freely available image analysis technique and associated software tool that uses the fast discrete curvelet transform to automate the measurement of the area occupied by cells in the images. This tool helps to significantly reduce the time needed for analysis and enables objective and reproducible quantification of assays. The software also offers a graphical user interface which allows easy inspection of analysis results and, if desired, manual modification of analysis parameters. The automated analysis was validated by comparing its results with manual-analysis results for a range of different cell lines. The comparisons demonstrate a close agreement for the vast majority of images that were examined and indicate that the present computational tool can reproduce statistically significant results in experiments with well-known cell migration inhibitors and enhancers.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 January 2017
                2017
                : 12
                : 1
                : e0169028
                Affiliations
                [1 ]University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, Hamburg, Germany
                [2 ]University Medical Center Hamburg-Eppendorf, Institute of Medical Biometry and Epidemiology, Hamburg, Germany
                [3 ]Fraunhofer Institute for Molecular Biology and Applied Ecology IME, ScreeningPort, Hamburg, Germany
                [4 ]Klinikum Stuttgart, Department for Endocrinology, Diabetology and Geriatrics, Stuttgart, Germany
                [5 ]University Medical Center Hamburg-Eppendorf, Department of Vascular Medicine, Hamburg, Germany
                [6 ]University Medical Center Hamburg-Eppendorf, Department and Clinic of Pediatric Surgery, Hamburg, Germany
                [7 ]Eberhard Karls University Tübingen, Department of Pharmaceutical Technology, Tübingen, Germany
                [8 ]Albert-Ludwigs-University Freiburg, Department of Pharmaceutical Biology and Biotechnology, Freiburg, Germany
                Children's Hospital Boston, UNITED STATES
                Author notes

                Competing Interests: This study was supported by Birken AG. However, the company was only involved in study design (for some of the experiments) but not in data collection, analysis, decision to publish or preparation of the manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. Therefore they declare that no competing interests exist.

                • Conceptualization: CU RD I. Merfort JMB.

                • Formal analysis: CU TV SS JMB.

                • Funding acquisition: JMB.

                • Investigation: CU TV PH SVyS BE PW.

                • Methodology: CU PH JMB SVyS BE PW.

                • Project administration: JMB.

                • Resources: BE AL-A KR IR KK RD I. Moll RL.

                • Supervision: JMB.

                • Validation: CU TV PH SVyS PW I. Merfort JMB.

                • Visualization: CU TV BE I. Merfort PW JMB.

                • Writing – original draft: CU BE I. Merfort RD JMB.

                • Writing – review & editing: CU TV SS BE RD I. Merfort JMB RL PH SVyS PW I. Moll KK IR KR AL-A.

                Article
                PONE-D-16-26989
                10.1371/journal.pone.0169028
                5207624
                28046026
                68258cb9-9c0a-4365-b636-7771477d9f18
                © 2017 Ueck et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 July 2016
                : 9 December 2016
                Page count
                Figures: 3, Tables: 2, Pages: 16
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100002765, Bundesministerium für Wirtschaft und Technologie;
                Award ID: KF239060SK1
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002765, Bundesministerium für Wirtschaft und Technologie;
                Award ID: KF239060SK1
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002765, Bundesministerium für Wirtschaft und Technologie;
                Award ID: KF239060SK1
                Award Recipient :
                J.M.B., I. Merfort, and R.D. were supported by a grant of the German Ministry of Economics and Technology (KF239060SK1). Birken AG was also supported by this grant. In addition, J.M.B., I. Merfort, and R.D. got additional funding by Birken AG for this study. The funders were involved in study design, but not in data collection, analysis, decision to publish or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Epithelial Cells
                Keratinocytes
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Epithelium
                Epithelial Cells
                Keratinocytes
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