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Current standards insufficiently acknowledge the influence of the wound micro-environment on the efficacy of antimicrobial agents. To address this, octenidine/phenoxyethanol, polyhexanide, povidone-iodine, and sodium-hypochloride/hypochlorous acid solutions were submitted to standard-based (DIN-EN-13727) or modified peptide-based challenges and compared to a simulated clinical reference using human acute or chronic wound exudate (AWF/CWF). Antimicrobial efficacy against S. aureus and P. aeruginosa was compared using a quantitative suspension method. Agreement between methods were investigated using Bland-Altman (B&A) analysis. Different substances and challenges demonstrated diverging results, depending on class and concentration of agent and challenge. Highly concentrated antiseptics maintained a high efficacy under complex challenges, while especially chlorine-based irrigation solutions showed a remarkably reduced antimicrobial effect. Composition of challenge substance proved more relevant than pure concentration. Therefore, the current standard challenge conditions did not adequately reflect the wound micro-environment with over- or under-estimating antimicrobial efficacy, whilst the modified peptide-challenge showed a higher level of agreement with simulated realistic conditions (AWF/CWF). The results emphasize that a "one-fits-all" approach is not feasible to generalize antimicrobial efficacy, as certain aspects of the complex micro-environment pose a differing influence on varying agents. Based on these results, revision and target focused adaptation of the current standards should be considered.

Marine long-chain omega-3 polyunsaturated fatty acids (ω3 FA) are involved in numerous cell responses and therefore vital for the mammal organism. Because of the attribution of immunomodulatory effects, a favourable impact on the inflammatory response in chronic wounds and cells involved in wound healing can be suspected. In the experimental setup, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were investigated regarding their impact on metabolic activity, cell proliferation and migration of human keratinocytes (HaCaT) and newborn foreskin fibroblasts (CRL-2522). For simulation of the microenvironment of a chronic wound, human chronic wound fluid (CWF) was used in the experimental setup addressing the in vitro influence of DHA, EPA and CWF on regenerative processes. The results showed a significant increase in the metabolic activity of keratinocytes and fibroblasts after 72 h treatment with DHA and EPA. In contrast, treatment with ω3 FA had no significant positive effect on skin cell proliferation. Both ω3 FA had no influence on in vitro wound closure. CWF demonstrated significantly adverse effects, which ω3 FA were unable to mitigate. It can be concluded that CWF exhibited the expected adverse effect on both skin cell types, especially inhibiting in vitro wound closure. ω3 FAs showed a slightly positive, yet rarely significant effect on human skin cells. Overall, the addition of DHA or EPA showed no relevant benefit for skin cells challenged with human CWF, merely in combination with DHA an initial significant increase in cell metabolism (fibroblasts) and cell proliferation (keratinocytes) could be observed., (© 2021 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Biofilms are one of the greatest challenges in today's treatment of chronic wounds. While antimicrobials kill platonic bacteria within seconds, they are rarely able to harm biofilms. In order to identify effective substances for antibacterial therapy, cost-efficient, standardized and reproducible models that aim to mimic the clinical situation are required. In this study, two 3D biofilm models based on human plasma with immune cells (lhBIOM) or based on sheep blood (sbBIOM) containing S. aureus or P. aeruginosa, are compared with the human biofilm model hpBIOM regarding their microscopic structure (scanning electron microscopy; SEM) and their bacterial resistance to octenidine hydrochloride (OCT) and a sodium hypochlorite (NaOCl) wound-irrigation solution. The three analyzed biofilm models show little to no reaction to treatment with the hypochlorous solution while planktonic S. aureus and P. aeruginosa cells are reduced within minutes. After 48 h, octenidine hydrochloride manages to erode the biofilm matrix and significantly reduce the bacterial load. The determined effects are qualitatively reflected by SEM. Our results show that both ethically acceptable human and sheep blood based biofilm models can be used as a standard for in vitro testing of new antimicrobial substances. Due to their composition, both fulfill the criteria of a reality-reflecting model and therefore should be used in the approval for new antimicrobial agents., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Objectives: A major problem for wound healing is contamination with bacteria, often resulting in biofilm formation and wound infection, which, in turn, needs immediate intervention such as surgical debridement and through irrigation. A topical treatment with cold atmospheric pressure plasma (CAP) for wound disinfection may present an alternative and less painful approach., Methods: This study investigated the antibacterial effects of a cold atmospheric pressure argon plasma jet (kINPen® MED) as a CAP source, using the three-dimensional Staphylococcus aureus immunocompetent biofilm system hpBIOM in addition to a standard planktonic test. Furthermore, skin cell compatibility was evaluated using a keratinocyte (HaCat) model., Results: CAP treatment (0-240 s) followed by incubation (15, 120 min) within the CAP-treated media showed slight bactericidal efficacy under planktonic conditions but no effect on biofilms. However, indirect CAP treatment of keratinocytes performed under the same conditions resulted in a significant decrease in metabolic activity. Short CAP treatment and exposure time (30 s; 15 min) induced a slight increase in the metabolic activity; however, longer treatments and/or exposure times led to pronounced reductions up to 100%. These effects could partially be reversed by addition of catalase, indicating a dominant role of CAP-generated hydrogen peroxide., Conclusions: These results indicate that plasma treatment does not lead to the desired disinfection or significant reduction in the bacterial burden of Staphylococcus aureus in a wet milieu or in biofilms. Thus, treatment with CAP could not be recommended as a single anti-bacterial therapy for wounds but could be used to support standard treatments., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lifestyle diseases such as diabetes and arteriosclerosis are rising in the increasingly aging society, and the number of patients with daily intake of glucose-lowering medication has also increased. Interestingly, knowledge about oral antidiabetics with regard to wound healing is scarce. Therefore, the aim of this study was to identify possible (side) effects of the most frequently prescribed oral antidiabetics on skin cells and wound healing. Four oral antidiabetics of different substance classes (i.e., metformin, glibenclamide, sitagliptin, repaglinide) were investigated with regard to the promotion of cell metabolism and migration of human skin fibroblasts and keratinocytes by XTT and scratch assays. In addition, histological and immunohistochemical analyses were performed in a 3D wound model to address the impact of the antidiabetics on regeneration processes, such as cell migration, fibroblast activity, epidermal thickness, and cell apoptosis. In comparison to systemic application, metformin displayed the most adverse effects in vitro in nearly all analyses, interestingly at serum equivalent concentrations. In contrast, sitagliptin and glibenclamide had a slight but insignificant effect on fibroblasts compared with keratinocytes. Repaglinide tended to have a negative influence on keratinocyte metabolism. Interestingly, antidiabetics generally induced a significantly enhanced rate of apoptosis in fibroblasts, with the exception of repaglinide.Antidiabetics influenced key players in wound healing, namely, keratinocytes and fibroblasts. Particularly, metformin impaired human skin cells. These findings should be kept in mind in further studies because of their putative relevance in patients suffering from chronic wounds that do not respond to various wound therapies.

Background: Sodium hypochlorite (NaClO, SHC)/hypochlorous acid (HClO, HCA) wound irrigation solutions have experienced a renaissance in the prevention and treatment of low-level wound infections. They are attributed with lower cytotoxicity and have therefore gained increasing attention in daily clinical practice., Objectives: To determine the cytotoxicity and antimicrobial efficacy of six NaClO/HClO wound irrigation solutions., Methods: For cytotoxicity evaluation (based on DIN EN 10993-5), human keratinocytes (HaCaT) and human skin fibroblasts (BJ) were used. Staphylococcus aureus and Pseudomonas aeruginosa were used for antimicrobial efficacy evaluation (based on DIN EN 13727). Solutions were evaluated after 1, 5 and 15 min of exposure. Additionally, physicochemical properties (pH and oxidation-reduction potential values) were investigated., Results: Efficacy and cytotoxicity varied significantly between solutions. Generally, increasing antimicrobial activity was associated with decreasing cell viability. Furthermore, a concentration- and time-dependent impact on pathogens and cells was observed: cytotoxic and antimicrobial activity increased with rising NaClO/HClO solution concentrations and extended exposure times. Based on these in vitro evaluations, the following ranking (lowest to highest microbicidal effect and cytotoxic impact) was found: Microdacyn60® (SHC/HCA-M) < Granudacyn® (SHC/HCA-G) < Veriforte™ (SHC/HCA-V) < KerraSol™ (SHC-K) < Lavanox® (SHC-L) ≪ ActiMaris®forte (SHC/SM-A)., Conclusions: The presented results indicate that microbicidal effects are almost always associated with certain negative side effects on cell proliferation. Efficacy and biocompatibility of NaClO/HClO solutions depend on their specific formulation and physicochemical properties. The investigations also underline the necessity for exact product- and application-specific efficacy profiles.

Background: The number of patients who has a daily intake of antihypertensive drugs is rising, due to an also rising prevalence of lifestyle diseases. Interestingly, knowledge about effects of these drugs in terms of wound healing is low., Objective: Based on a few differing studies, the idea arose that antihypertensives may have side effects on wound healing., Methods: Five antihypertensive drugs from different substance classes (metoprolol, amlodipine, ramipril, hydrochlorothiazide, candesartan) were investigated, in terms of possible impacts on cell metabolism and migration of human skin fibroblasts and keratinocytes. Additionally, histological and immunohistochemical analyses were performed in a 3-dimensional (3D) wound model addressing the influence on regeneration processes, such as cell migration, metabolic activity, apoptosis and epidermal thickness., Results: Hydrochlorothiazide and ramipril exerted inhibiting effects in nearly all analyses, interestingly, in serum equivalent doses. In contrast, candesartan and amlodipine induced slight positive effects in 2D as well as in 3D models. The previously described positive effects of β-blockers could only partially be confirmed by metoprolol. Antihypertensive drugs affected fibroblasts more than keratinocytes - whether positively or negatively., Conclusion: Antihypertensive drugs have an influence on keratinocytes and fibroblasts; they are not neutral. Candesartan has the most positive effects on skin cells. For angiotensin-converting enzyme inhibitors and thiazide diuretics, wound healing in a 3D model is delayed. β-Receptor blockers seem to improve wound healing to a small extent just like calcium channel blockers. These results should be evaluated in a clinical trial to verify their clinical relevance., (© 2019 S. Karger AG, Basel.)

This paper presents a method of separating cells that are connected to each other forming clusters. The difference to many other publications covering similar topics is that the cell types we are dealing with form clusters of highly varying morphology. An advantage of our method is that it can be universally used for different cell types. The segmentation method is based on a growth simulation starting from the nuclei areas. To start the evaluation, the cells need to be made visible with a histological stain, in our case with the May-Grünwald solution. After the staining process has been completed, the nuclei areas can be distinguished from the other cell areas by a histogram backprojection algorithm. The presented method can, in addition to histological stained cells, also be applied to fluorescent-stained cells.