Effect of oral multispecies probiotic on wound healing, periodontitis and quality of life on patients with diabetes.

OBJECTIVE: Hard-to-heal (chronic) wounds are common in patients with diabetes and are associated with a decrease in quality of life (QoL). Pathogenic bacteria often colonise hard-to-heal wounds and hinder the healing process which poses a high risk for (systemic) infections. In this study, we aim to prove that probiotics are capable of displacing human pathogenic bacteria, ameliorating inflammation and positively influencing the microenvironment/microbiome of skin and mucosa. METHOD: In this pilot study, patients with diabetes and hard-to-heal wounds with a duration of 2-120 months received an oral multispecies probiotic daily for six months. Changes in oral, stool and wound microbiome were investigated, and the effects of the probiotic intervention on wound healing, periodontitis and wound-specific quality of life (Wound-QOL-17) were analysed throughout the course of this clinical study. RESULTS: In total, seven of the 20 patients included were unable to complete the study. After six months of oral probiotic intake supplementation in five out of the remaining 13 patients, the wounds had healed completely. Most patients reported an improvement in wound-specific QoL, with particular positive effects on pain and mobility. Microbiome analysis revealed a reduction in Staphylococcus aureus and Pseudomonas aeruginosa, and Staphylococcus epidermis in healed wounds. CONCLUSION: This findings of this study provide evidence for the beneficial effects of the oral application of a multispecies probiotic over six months in patients with diabetes and hard-to-heal wounds on wound closure, wound microbial pattern, QoL, and on dental health. A randomised, placebo-controlled, double-blinded clinical trial is required to verify the results.

Biodegradable Silk Fibroin Matrices for Wound Closure in a Human 3D Ex Vivo Approach.

In this study, the potential of silk fibroin biomaterials for enhancing wound healing is explored, focusing on their integration into a human 3D ex vivo wound model derived from abdominoplasties. For this purpose, cast silk fibroin membranes and electrospun nonwoven matrices from Bombyx mori silk cocoons were compared to untreated controls over 20 days. Keratinocyte behavior and wound healing were analyzed qualitatively and quantitatively by histomorphometric and immune histochemical methods (HE, Ki67, TUNEL). Findings reveal rapid keratinocyte proliferation on both silk fibroin membrane and nonwoven matrices, along with enhanced infiltration in the matrix, suggesting improved early wound closure. Silk fibroin membranes exhibited a significantly improved early regeneration, followed by nonwoven matrices (p < 0.05) compared to untreated wounds, resulting in the formation of multi-layered epidermal structures with complete regeneration. Overall, the materials demonstrated excellent biocompatibility, supporting cell activity with no signs of increased apoptosis or early degradation. These results underscore silk fibroin's potential in clinical wound care, particularly in tissue integration and re-epithelialization, offering valuable insights for advanced and-as a result of the electrospinning technique-individual wound care development. Furthermore, the use of an ex vivo wound model appears to be a viable option for pre-clinical testing.

Characterization of the Human Plasma Biofilm Model (hpBIOM) to Identify Potential Therapeutic Targets for Wound Management of Chronic Infections.

The treatment of chronic wounds still represents a major challenge in wound management. Recent estimates suggest that 60-80% of chronic wounds are colonized by pathogenic microorganisms, which are strongly considered to have a major inhibiting influence on the healing process. By means of an innovative biofilm model based on human plasma, the time-dependent behavior of various bacterial strains under wound-milieu-like conditions were investigated, and the growth habits of different cocci species were compared. Undescribed fusion events between colonies of MRSA as well as of Staphylococcus epidermidis were detected, which were associated with the remodeling and reorganization of the glycocalyx of the wound tissue. After reaching a maximum colony size, the spreading of individual bacteria was observed. Interestingly, the combination of different cocci species with Pseudomonas aeruginosa in the human plasma biofilm revealed partial synergistic effects in these multispecies organizations. RT-qPCR analyses gave a first impression of the relevant proteins involved in the formation and maturation of biofilms, especially the role of fibrinogen-binding proteins. Knowledge of the maturation and growth behavior of persistent biofilms investigated in a translational human biofilm model reflects a starting point for the development of novel tools for the treatment of chronic wounds.

Quantitative Insights and Visualization of Antimicrobial Tolerance in Mixed-Species Biofilms.

Biofilms are a major problem in hard-to-heal wounds. Moreover, they are composed of different species and are often tolerant to antimicrobial agents. At the same time, interspecific synergy and/or competition occurs when some bacterial species clash. For this reason, the tolerance of two dual-species wound biofilm models of Pseudomonas aeruginosa and Staphylococcus aureus or Enterococcus faecium against antimicrobials and antimicrobial dressings were analyzed quantitatively and by confocal laser scanning microscopy (CLSM). The results were compared to findings with planktonic bacteria. Octenidine-dihydrochloride/phenoxyethanol and polyhexamethylene biguanide (PHMB) irrigation solutions showed a significant, albeit delayed reduction in biofilm bacteria, while the PHMB dressing was not able to induce this effect. However, the cadexomer-iodine dressing caused a sustained reduction in and killed almost all bacteria down to 102 cfu/mL within 6 days compared to the control (1010 cfu/mL). By means of CLSM in untreated human biofilm models, it became evident that P. aeruginosa dominates over E. faecium and S. aureus. Additionally, P. aeruginosa appeared as a vast layer at the bottom of the samples, while S. aureus formed grape-like clusters. In the second model, the distribution was even clearer. Only a few E. faecium were visible, in contrast to the vast layer of P. aeruginosa. It seems that the different species avoid each other and seek their respective niches. These mixed-species biofilm models showed that efficacy and tolerance to antimicrobial substances are nearly species-independent. Their frequent application appears to be important. The bacterial wound biofilm remains a challenge in treatment and requires new, combined therapy options.

Human skin biofilm model: translational impact on swabbing and debridement.

OBJECTIVE: Wound biofilms are one of the greatest challenges in the therapy of hard-to-heal (chronic) wounds, as potent antimicrobial substances fail to eradicate bacteria within short incubation periods. Preclinical investigations using novel model systems that closely mimic the human wound environment and wound biofilm are required to identify new and effective therapeutic options. This study aims to identify bacterial colonisation patterns that are relevant for diagnosis and therapy. METHOD: In this study, a recently established human plasma biofilm model (hpBIOM) was incorporated into a wound within human dermal resectates after abdominoplasty. The interaction of the biofilm-forming bacteria meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa with the skin cells was investigated. Possible effects on wound healing processes in correlation with the persistence of the biofilm in the wound environment were analysed in patients with leg ulcers of different aetiologies and biofilm burden. RESULTS: Using haematoxylin and eosin staining, species-dependent infiltration modes of the bacteria into the wound tissue were determined for the pathogens MRSA and Pseudomonas aeruginosa. The spreading behaviour correlated with clinical observations of the spatial distributions of the bacteria. In particular, the clinically prominent Pseudomonas aeruginosa-specific distension of the wound margin was identified as epidermolysis due to persistent infiltration. CONCLUSION: The hpBIOM applied in this study represents a potential tool for preclinical analyses dealing with approval processes for new antimicrobial applications. In terms of clinical practice, a microbiological swabbing technique including the wound margin should be routinely applied to prevent wound exacerbation.

Composition of Challenge Substance in Standardized Antimicrobial Efficacy Testing of Wound Antimicrobials Is Essential to Correctly Simulate Efficacy in the Human Wound Micro-Environment.

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.

Pulsed low-intensity laser treatment stimulates wound healing without enhancing biofilm development in vitro.

OBJECTIVES: Treating infected or chronic wounds burdened with biofilms still is a major challenge in medical care. Healing-stimulating factors lose their efficacy due to bacterial degradation, and antimicrobial substances negatively affect dermal cells. Therefore, alternative treatment approaches like the pulsed low intensity laser therapy (LILT) require consideration. METHODS: The effect of pulsed LILT (904 nm, in three frequencies) on relevant human cells of the wound healing process (fibroblasts (BJ), keratinocytes (HaCaT), endothelial cells (HMEC), monocytes (THP-1)) were investigated in in-vitro and ex-vivo wound models with respect to viability, proliferation and migration. Antimicrobial efficacy of the most efficient frequency in cell biological analyses of LILT (3200 Hz) was determined in a human biofilm model (lhBIOM). Quantification of bacterial load was evaluated by suspension method and qualitative visualization was performed by scanning electron microscopy (SEM). RESULTS: Pulsed LILT at 904 nm at 3200 Hz ± 50% showed the most positive effects on metabolic activity and proliferation of human wound cells in vitro (after 72 h - BJ: BPT 0.97 ± 0.05 vs. 0.75 ± 0.04 (p = 0.0283); HaCaT: BPT 0.79 ± 0.04 vs. 0.59 ± 0.02 (p = 0.0106); HMEC: 0.74 ± 0.02 vs. 0.52 ± 0.04 (p = 0.009); THP-1: 0.58 ± 0.01 vs. 0.64 ± 0.01 (p > 0.05) and ex vivo. Interestingly, re-epithelialization was stimulated in a frequency-independent manner. The inhibition of metabolic activity after TNF-α application was abolished after laser treatment. No impact of LILT on monocytes was detected. Likewise, the tested LILT regimens showed no growth rate reducing effects on three bacterial strains (after 72 h - PA: -1.03%; SA: -0.02%; EF: -1,89%) and one fungal (-2.06%) biofilm producing species compared to the respective untreated control. Accordingly, no significant morphological changes of the biofilms were observed after LILT treatment in the SEM. CONCLUSIONS: Frequent application of LILT (904 nm, 3200 Hz) seems to be beneficial for the metabolism of human dermal cells during wound healing. Considering this, the lack of disturbance of the behavior of the immune cells and no growth-inducing effect on bacteria and fungi in the biofilm can be assigned as rather positive. Based on this combined mode of action, LILT may be an option for hard to heal wounds infected with persistent biofilms.

Antimicrobials cetylpyridinium-chloride and miramistin demonstrate non-inferiority and no "protein-error" compared to established wound care antiseptics in vitro.

Concern about microbial tolerance and resistance to established antimicrobials drives research into alternatives for local antiseptic wound treatment. Precise efficacy profiles are thereby important in the evaluation of potential alternative antimicrobials, and protein interference ("protein error") is a key factor. Here, the antimicrobial efficacy of cetylpyridinium chloride (CPC) and miramistin (MST) was compared to the established antimicrobials octenidine (OCT), povidon-iodine (PVP-I), polyhexamethylene-biguanide (PHMB) and chlorhexidine (CHX). Efficacy was evaluated after 0.5, 1, 3, 5 and 10 min against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecium and Candida albicans using an in vitro quantitative suspension method (based on DIN EN 13727). To investigate protein interference, 0.3% or 3% bovine albumin was used as the challenge. OCT and PVP-I demonstrated a significant efficacy within 0.5 min, regardless of the microbial organism and protein challenge (p < 0.01). CPC and MST showed no inferiority in efficacy, with only MST needing up to 3 min to achieve the same microbial reduction. PHMB and CHX also achieved significant reduction rates over the tested time-course, yet demonstrated a necessity for prolonged exposure (up to 10 min) for comparable reduction. A protein interference was predominantly observed for PHMB against S. aureus, but without statistically significant differences in antimicrobial efficacy between the 0.3% and 3% protein challenges. All other tested agents showed no relevant interference with the presence of protein. CPC and MST proved to be non-inferior to established wound antiseptics agents in vitro. In fact, CPC showed a more efficient reduction than PHMB and CHX despite there being an introduced protein challenge. Both agents demonstrated no significant "protein error" under challenging conditions (3% albumin), posing them as valid potential candidates for alternative antimicrobials in wound management.

Bacteria Are New Targets for Inhibitors of Human Farnesyltransferase.

Farnesyltransferase inhibitors (FTIs) are focus for the treatment of several diseases, particularly in the field of cancer therapy. Their potential, however, goes even further, as a number of studies have evaluated FTIs for the treatment of infectious diseases such as malaria, African sleeping sickness, leishmaniosis, and hepatitis D virus infection. Little is known about protein prenylation mechanisms in human pathogens. However, disruption of IspA, a gene encoding the geranyltranstransferase of Staphylococcus aureus (S. aureus) leads to reprogramming of cellular behavior as well as impaired growth and decreased resistance to cell wall-targeting antibiotics. We used an agar well diffusion assay and a time kill assay and determined the minimum inhibitory concentrations of the FTIs lonafarnib and tipifarnib. Additionally, we conducted cell viability assays. We aimed to characterize the effect of these FTIs on S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), Escherichia coli (E. coli), Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and Streptococcus pneumoniae (S. pneumoniae). Both the FTIs lonafarnib and tipifarnib were capable of inhibiting the growth of the Gram-positive bacteria S. aureus, MRSA, S. epidermidis, and S. pneumoniae, whereas no effect was observed on Gram-negative bacteria. The analysis of the impact of lonafarnib and tipifarnib on common human pathogens might lead to novel insights into their defense mechanisms and therefore provide new therapeutic targets for antibiotic-resistant bacterial infections.

In vitro Activity of Antimicrobial Wound Dressings on P. aeruginosa Wound Biofilm.

The treatment of acute and chronic infected wounds with residing biofilm still poses a major challenge in medical care. Interactions of antimicrobial dressings with bacterial load, biofilm matrix and the overall protein-rich wound microenvironment remain insufficiently studied. This analysis aimed to extend the investigation on the efficacy of a variety of antimicrobial dressings using an in vitro biofilm model (lhBIOM) mimicking the specific biofilm-environment in human wounds. Four wound dressings containing polyhexanide (PHMB), octendine di-hydrochloride (OCT), cadexomer-iodine (C-IOD) or ionic silver (AG) were compared regarding their antimicrobial efficacy. Quantitative analysis was performed using a quantitative suspension method, separately assessing remaining microbial counts within the solid biofilm as well as the dressing eluate (representing the absorbed wound exudate). Dressing performance was tested against P. aeruginosa biofilms over the course of 6 days. Scanning electron microscopy (SEM) was used to obtain qualitative visualization on changes in biofilm structure. C-IOD demonstrated superior bacterial reduction. In comparison it was the only dressing achieving a significant reduction of more than 7 log10 steps within 3 days. Neither the OCT- nor the AG-containing dressing exerted a distinct and sustained antimicrobial effect. PHMB achieved a non-significant microbicidal effect (1.71 ± 0.31 log10 steps) at day 1. Over the remaining course (6 days) it demonstrated a significant microbistatic effect compared to OCT, AG and the control. Quantitative results in the dressing eluate correlate with those of the solid biofilm model. Overall, AG- and OCT-containing dressings did not achieve the expected anti-biofilm efficacy, while C-IOD performed best. Chemical interaction with the biofilms extrapolymeric substance (EPS), visualized in the SEM, and dressing configuration (agent concentration and release pattern) are suspected to be responsible. The unexpected low and diverse results of the tested antimicrobial dressings indicate a necessity to rethink non-debridement anti-biofilm therapy. Focussing on the combination of biofilm-disruptive (for EPS structure) and antimicrobial (for residing microorganisms) features, as with C-IOD, using dehydration and iodine, appears reasonably complementary and an optimal solution, as suggested by the here presented in vitro data.

Antimicrobial Hypochlorous Wound Irrigation Solutions Demonstrate Lower Anti-biofilm Efficacy Against Bacterial Biofilm in a Complex in-vitro Human Plasma Biofilm Model (hpBIOM) Than Common Wound Antimicrobials.

Biofilms pose a relevant factor for wound healing impairment in chronic wounds. With 78% of all chronic wounds being affected by biofilms, research in this area is of high priority, especially since data for evidence-based selection of appropriate antimicrobials and antiseptics is scarce. Therefore, the objective of this study was to evaluate the anti-biofilm efficacy of commercially available hypochlorous wound irrigation solutions compared to established antimicrobials. Using an innovative complex in-vitro human plasma biofilm model (hpBIOM), quantitative reduction of Pseudomonas aeruginosa, Staphylococcus aureus, and Methicillin-resistant S. aureus (MRSA) biofilms by three hypochlorous irrigation solutions [two <0.08% and one 0.2% sodium hypochlorite (NaClO)] was compared to a 0.04% polyhexanide (PHMB) irrigation solution and 0.1% octenidine-dihydrochloride/phenoxyethanol (OCT/PE). Efficacy was compared to a non-challenged planktonic approach, as well as with increased substance volume over a prolonged exposure (up to 72 h). Qualitative visualization of biofilms was performed by scanning electron microscopy (SEM). Both reference agents (OCT/PE and PHMB) induced significant biofilm reductions within 72 h, whereby high volume OCT/PE even managed complete eradication of P. aeruginosa and MRSA biofilms after 72 h. The tested hypochlorous wound irrigation solutions achieved no relevant penetration and eradication of biofilms despite increased volume and exposure. Only 0.2% NaClO managed a low reduction under prolonged exposure. The results demonstrate that low-dosed hypochlorous wound irrigation solutions are significantly less effective than PHMB-based irrigation solution and OCT/PE, thus unsuitable for biofilm eradication on their own. The used complex hpBIOM thereby mimics the highly challenging clinical wound micro-environment, providing a more profound base for future clinical translation.

Comprehensive Analysis of Zinc Derivatives Pro-proliferative, Anti-Apoptotic and Antimicrobial Effect on Human Fibroblasts and Keratinocytes in a Simulated, Nutrient-deficient Environment In Vitro.

Zinc as therapeutic agent in skin and wound care has been known for centuries, but its role is controversial and comprehensive investigations in nutrient-deficient environments are lacking. We aimed to provide a broad analysis of different zinc derivatives on proliferation, apoptosis and antimicrobial properties in a simulated nutrient-deficient environment in vitro. Human fibroblasts (CRL2522) and keratinocytes (HaCaT) were treated with a broad concentration range (10 - 0.0001 µg/mL) of zinc-sulfate (ZnSO4), -gluconate (ZnGluc) and -histidine (ZnHis) for 1-6 days under nutrient-deficient media conditions. Cell proliferation was investigated by XTT assay. Targeted analyzes in proliferation (E2F1, PCNA) and apoptosis (TP53) associated genes were performed via qRT-PCR and apoptosis was determined via FACS (annexin V/7-AAD staining). Antimicrobial efficacy was investigated using a quantitative suspension method against S. aureus, P. aeruginosa, E. coli, and C. albicans. The results indicated that 0.1 to 0.001 µg/mL Zn increased cell proliferation in both cell lines. Fibroblasts were more susceptible with significant proliferation peaks on days 2 & 6, and days 1 & 4 for keratinocytes. No relevant changes in gene expression were detected for E2F1 and PCNA nor for TP53. Annexin-V/7-AAD-staining of fibroblasts revealed a small, yet insignificant reduction of apoptosis induction for ZnGluc and ZnSO4. ZnGluc and ZnSO4 (0.1%) achieved high microbial reductions (4-5 log10 reductions) against tested pathogens. ZnGluc and ZnSO4 showed relevant pro-proliferative and antimicrobial, as well as tendential anti-apoptotic features in a simulated nutrient-deficient microenvironment in vitro. This further validates a potential benefit of local zinc treatment in deficient wound microenvironments.

Side Effects of Frequently Used Antihypertensive Drugs on Wound Healing in vitro.

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 ß-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. ß-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.

Safety and efficacy profiles of different commercial sodium hypochlorite/hypochlorous acid solutions (NaClO/HClO): antimicrobial efficacy, cytotoxic impact and physicochemical parameters in vitro.

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.

Side effects of frequently used oral antidiabetics on wound healing in vitro.

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.

Effects of Vitamin B Complex and Vitamin C on Human Skin Cells: Is the Perceived Effect Measurable?

OBJECTIVE: Vitamins are essential for human health. In terms of local application for wound healing, vitamins' positive effect remains unclear. However, because of the regular appearance of nutritional deficiency in chronic wound patients, a favorable impact of locally applied vitamins can be hypothesized. METHODS: Vitamins B3, B5, B6, B7, B9, B10, B12, and C individually as well as different combinations of B vitamins were investigated regarding their ability to promote proliferation and migration of human skin fibroblasts and keratinocytes. Proliferation assays with and without bacterial challenge, immunocytochemical staining, and scratch assay were used to determine the most effective combination(s). MAIN RESULTS: Some vitamin combinations showed a positive impact on proliferation, especially for keratinocytes after 72 hours. In terms of wound closure, the combinations B9 and B12; B3, B5, B6, and B10; and B3, B5, and B7 improved closure rates by 25% to 30%. The improved closure rates are also reflected by immunocytochemically detected upregulation of the migration marker CXCR4 for several combinations. CONCLUSIONS: Certain combinations of B vitamins demonstrate a positive influence on human keratinocytes and fibroblasts. Vitamins especially promoted fibroblast migration, and a statistically significant induction of keratinocyte proliferation was observed. Therefore, local vitamin application could benefit the physiologic wound healing process.

Influence of human acute wound fluid on the antibacterial efficacy of different antiseptic polyurethane foam dressings: An in vitro analysis.

Treating infected acute and/or chronic wounds still represents a major challenge in medical care. Various interactions of antiseptic dressings with wound environments regarding antimicrobial efficacy remain unclear. Therefore, this work aimed to investigate the influence of human acute wound fluid (AWF) on the antimicrobial performance of different antiseptic foam dressings in vitro against typical bacterial wound pathogens. Eight antiseptic polyurethane foam dressings containing either a silver formulation or a polyhexamethylene-biguanide (PHMB) were assessed regarding their antimicrobial potency against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using a modified time-kill assay based on ISO EN 20743. The antiseptic efficacy was evaluated standardly as well as under the influence of human AWF after 2, 4, 6, and 24 hours. The specific chemical formulation and concentration of the antiseptic substance (ionic or nanocrystalline silver, silver sulfadiazine, PHMB 0.1%/0.5%) embedded within the dressings seemed to play a key role. For certain dressings (two nanocrystalline and one ionic silver dressing), the antimicrobial efficacy was significantly reduced under the influence of AWF compared to unchallenged test series. Unchallenged the efficacy of PHMB was comparable to silver against P. aeruginosa and even significantly superior against S. aureus and E. coli. Challenged with AWF the reduction rates for silver adjusted or even exceeded (P. aeruginosa) those of PHMB. Within a challenging wound environment, especially some silver formulations demonstrated a reduced bacterial reduction. Regarding the presented in vitro results, the biomolecular interactions of antiseptic wound dressings with wound fluid should be part of more extensive investigations, considering varying factors such as bacterial species and wound (micro)environment to develop targeted therapeutic regimes for the individual.

Comparing two polymeric biguanides: chemical distinction, antiseptic efficacy and cytotoxicity of polyaminopropyl biguanide and polyhexamethylene biguanide.

In this study, polyaminopropyl biguanide (PAPB) was compared to the molecularly closely related polyhexamethylene biguanide (PHMB) with respect to chemical relationship, antiseptic efficacy and cytotoxicity in vitro. Cytotoxicity for human keratinocytes (HaCaTs) and murine fibroblasts (L929) was determined according to ISO EN 10993-5 for both substances. Antimicrobial efficacy tests were performed via determination of the MBC, quantitative suspension method for substances and investigation of two PAPB- or PHMB-containing dressings against Staphyloccoccus aureus, Escherichia coli and Pseudomonas aeruginosa, according to international standards. Prior mass spectrometry was performed for chemical differentiation of the investigated substances. PHMB showed high toxicity even in low concentrations for both tested cell lines and a high antimicrobial efficacy against S. aureus and E. coli. In the case of PAPB, no or only low cytotoxicity was detected after 72 h, whilst comparable antibacterial features are lacking, as PAPB showed no relevant antimicrobial effects. Even though chemically closely related, PAPB proved to be ineffective in bacterial eradication, whilst PHMB showed a high efficacy. The discovery and establishment of safe and effective alternative antiseptics are important issues for the treatment of infected wounds. In particular, rising bacterial resistances to established agents, as well as ongoing discussions of potential toxic or carcinogenic effects emphasize this necessity. Nevertheless, the presented results highlight that even small changes in the chemical structure of related agents such as PHMB and PAPB can dramatically affect their efficacy and, therefore, need to be carefully distinguished and assessed side by side.

Comparison of hemostatic dressings for superficial wounds using a new spectrophotometric coagulation assay.

BACKGROUND: Due to demographical changes the number of elderly patients depending on oral anticoagulation is expected to rise. Prolonged bleeding times in case of traumatic injuries represent the drawback of these medications, not only in major trauma, but also in superficial wounds. Therefore, dressings capable of accelerating coagulation onset and shortening bleeding times are desirable for these patients. METHODS: The hemostatic potential and physical properties of different types of superficial wound dressings (standard wound pad, two alginates, chitosan, collagen (Lyostypt(®)), oxidized cellulose, and QuikClot(®)) were assessed in vitro. For this purpose the clotting times of blood under the influence of the named hemostatics from healthy volunteers were compared with Marcumar(®) or ASS(®) treated patients. For that, a newly developed coagulation assay based on spectrophotometric extinction measurements of thrombin activity was used. RESULTS: The fastest coagulation onset was observed for oxidized cellulose (Ø 2.47 min), Lantor alginate-L (Ø 2.50 min) and QuikClot(®) (Ø 3.01 min). Chitosan (Ø 5.32 min) and the collagen Lyostypt(®) (Ø 7.59 min) induced clotting comparatively late. Regarding physical parameters, QuikClot(®) showed the lowest absorption capacity and speed while chitosan and both alginates achieved the highest. While oxidized cellulose displayed the best clotting times, unfortunately it also revealed low absorption capacity. CONCLUSIONS: All tested specimens seem to induce clotting independently from the administered type of oral anticoagulant, providing the possibility to neglect the disadvantage in clotting times arising from anticoagulation on a local basis. QuikClot(®), oxidized cellulose and unexpectedly alginate-L were superior to chitosan and Lyostypt(®). Due to its additional well-known positive effect on wound healing alginate-L should be considered for further investigations.