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Development of In Vitro and Ex Vivo Biofilm Models for the Assessment of Antibacterial Fibrous Electrospun Wound DressingsClick to copy article linkArticle link copied!
- Kairi LorenzKairi LorenzInstitute of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaMore by Kairi Lorenz
- Liis PreemLiis PreemInstitute of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaMore by Liis Preem
- Kadi SagorKadi SagorInstitute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaMore by Kadi Sagor
- Marta PutrinšMarta PutrinšInstitute of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaInstitute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaMore by Marta Putrinš
- Tanel TensonTanel TensonInstitute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaMore by Tanel Tenson
- Karin Kogermann*Karin Kogermann*Email: [email protected]Institute of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, EstoniaMore by Karin Kogermann
Molecular Pharmaceutics
Cite this: Mol. Pharmaceutics 2023, 20, 2, 1230–1246
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Abstract
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Increasing evidence suggests that the chronicity of wounds is associated with the presence of bacterial biofilms. Therefore, novel wound care products are being developed, which can inhibit biofilm formation and/or treat already formed biofilms. A lack of standardized assays for the analysis of such novel antibacterial drug delivery systems enhances the need for appropriate tools and models for their characterization. Herein, we demonstrate that optimized and biorelevant in vitro and ex vivo wound infection and biofilm models offer a convenient approach for the testing of novel antibacterial wound dressings for their antibacterial and antibiofilm properties, allowing one to obtain qualitative and quantitative results. The in vitro model was developed using an electrospun (ES) thermally crosslinked gelatin–glucose (GEL-Glu) matrix and an ex vivo wound infection model using pig ear skin. Wound pathogens were used for colonization and biofilm development on the GEL-Glu matrix or pig skin with superficial burn wounds. The in vitro model allowed us to obtain more reproducible results compared with the ex vivo model, whereas the ex vivo model had the advantage that several pathogens preferred to form a biofilm on pig skin compared with the GEL-Glu matrix. The in vitro model functioned poorly for Staphylococcus epidermidis biofilm formation, but it worked well for Escherichia coli and Staphylococcus aureus, which were able to use the GEL-Glu matrix as a nutrient source and not only as a surface for biofilm growth. On the other hand, all tested pathogens were equally able to produce a biofilm on the surface of pig skin. The developed biofilm models enabled us to compare different ES dressings [pristine and chloramphenicol-loaded polycaprolactone (PCL) and PCL−poly(ethylene oxide) (PEO) (PCL/PEO) dressings] and understand their biofilm inhibition and treatment properties on various pathogens. Furthermore, we show that biofilms were formed on the wound surface as well as on a wound dressing, indicating that the demonstrated methods mimic well the in vivo situation. Colony forming unit (CFU) counting and live biofilm matrix as well as bacterial DNA staining together with microscopic imaging were performed for biofilm quantification and visualization, respectively. The results showed that both wound biofilm models (in vitro and ex vivo) enabled the evaluation of the desired antibiofilm properties, thus facilitating the design and development of more effective wound care products and screening of various formulations and active substances.
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Copyright © 2023 The Authors. Published by American Chemical Society
1. Introduction
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Biofilms are reported to occur in many areas of medicine, being present in 80% of all known human infections. In wound care, microbial biofilms are recognized as one of the main causes of ineffective treatment and wound chronicity. (1) It is proposed that at least 78% of chronic wounds contain biofilms. (2) Mature biofilms develop already during the first 10 h and persist indefinitely when the wound remains untreated. Despite the increasing understanding of the mechanisms of biofilm formation in skin wounds, current strategies for wound biofilm and infection treatment are still far from ideal.
Clinical decisions for treatment are usually made based on planktonic bacteria in the wound, and this approach enables the treatment of acute wound infections. (1,3,4) However, to successfully treat wound infections in nonhealing wounds, the biofilm needs to be removed from the wound via surgical debridement. (2) As the next step, local antimicrobial therapy is needed to kill the released bacteria and attack the bacteria still residing in the biofilm residuals. Because bacterial biofilms in chronic and nonhealing wounds continue to be a medical challenge, the search for local therapies against biofilm formation and the treatment of formed biofilms are becoming increasingly important. (2)
Electrospinning (ES) is a popular method for creating advanced fibrous wound care preparations (e.g., wound dressings) for local antimicrobial drug delivery and wound healing. ES allows the incorporation of one or more antibacterial or antimicrobial agents into the fibrous matrices, and the drug release can be modified and controlled. (5−7) Polymeric ES dressings have shown potential for the treatment of chronic wounds, and there are different ES wound matrices already available in the market, such as Restrata and Phoenix. (8−10) However, the effectiveness of antibacterial ES fiber dressings has mainly been demonstrated with planktonic bacteria and using standardized antimicrobial assays, such as the Kirby–Bauer disk diffusion or broth microdilution methodologies and time–kill assays (e.g., EUCAST disk diffusion, American Association of Textile Chemists and Colorists (AATCC) methods; ASTM E2315-16). The mentioned antimicrobial assays are usually used to evaluate and screen the effect of wound care matrices against infection development. The most popular in vitro test for assessing the antimicrobial properties of wound dressings is the Kirby–Bauer disk diffusion method and its modifications. The prerequisite for the suitability of the agar plate method is that the drug must be released from the carrier and diffused into the agar. It has been shown that the carrier can vary with the formulation used, and it also differs between selected antimicrobial agents. (11) We have shown previously that ES fibrous matrices can have very different morphological, physicochemical, and mechanical properties, which consequently can modify the drug release and its antibacterial efficacy. (12,13) The surface structure and morphology of fibers and fibrous matrices highly influence biofilm formation. (14,15) Therefore, the antibiofilm activity of wound dressings needs to be separately demonstrated.
Various in vitro biofilm assays have been developed and compared. (16,17) Even a standardized method for the evaluation of biofilm resistance properties of tube, yarn, and fiber specimens has been proposed (ASTM E3151-18). These tests provide valuable information about the growth of biofilms and their quantification, and they have been used as such and/or with some modifications for the testing of wound dressings including ES dressings. (18−20)
There are various in vitro, ex vivo, and in vivo biofilm and wound infection models available for evaluating the antibacterial efficacy and antibiofilm properties of wound care products. (21,22) Indeed, in vivo wound infection models on animals (e.g., mice, rat, rabbit, and pig) have been reported, (23−25) but their use may be very expensive, and they provide low throughput for the initial formulation screening phase due to the high biological variability between animals. Furthermore, in vivo animal data cannot always be directly translated to humans, and there are differences in how biofilms are formed and how they interact with their hosts, not to mention the ethical aspects in line with the 3R rule. Therefore, to develop an effective wound care therapy, including antibiofilm therapy, cost-effective standardized and reproducible models that aim to mimic the clinical situation are required. (2) The use of in vitro and ex vivo infection and biofilm models has found its place. In vitro biofilm models have been developed, which enable one to study biofilm treatment properties of wound dressings under more wound-like conditions. (26−28) Also, various ex vivo wound infection models have been proposed where tissues or organs are extracted from animals and/or humans and further cultured under in vitro conditions while preserving their three-dimensional structure. (29−32) Different ex vivo models such as the burn wound infection model (33) or the wound biofilm model (32,34) on pig skin have been recently developed and published. Nevertheless, none of them are validated for the testing of ES wound dressings. Currently, there is a lack of appropriate protocols and biorelevant models or easy-to-use screening methods suitable for the characterization of such novel antimicrobial drug delivery system (DDS)-based wound dressings. Moreover, to test the antimicrobial and antibiofilm efficacy of a wound dressing, biofilm models with relevant pathogens (depending on the actual clinical problem) are needed.
The aim of the present study was to develop in vitro and ex vivo biofilm models and protocols for assessing the antibacterial and antibiofilm properties of ES wound dressings. Models were designed suitably for these novel fibrous DDSs, taking into consideration the formulation aspects such as materials and their concentrations (e.g., polymers and antibacterial agents), which determine the physicochemical as well as biopharmaceutical properties of the ES matrix, including its drug release behavior and efficacy against specific bacteria or biofilms. The created models were designed to test the effectiveness of ES wound dressings using different relevant wound pathogens and compare them with each other to find suitable wound-dressing candidates for further in vivo testing in an animal model and/or in humans.
2. Experimental Section
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2.1. Materials
2.1.1. Drugs, Polymers, and Supplies
Polycaprolactone (PCL) (Mn 80,000), poly(ethylene oxide) (PEO) (Mw 900,000), chloramphenicol (CAM) (PubChem CID: 5959), gelatin type A from porcine skin (GEL), anhydrous d-(+)-glucose (Glu), methanol (MET) (gradient grade), glycerol, chloroform (CHF), and acetic acid (99.8–100.5%, puriss p.a.), were purchased from Sigma-Aldrich Inc. (Darmstadt, Germany). All materials were of reagent grade or better and were used as received without any further purification.
2.1.2. Bacterial Strains and Growth Media
Three types of pathogenic bacteria relevant for skin and wound infections were used in the biofilm study: Gram-negative bacterium Escherichia coli DSM 1103 (ATCC 25922) and Gram-positive bacteria Staphylococcus aureus DSM 2569 (ATCC 29213) and Staphylococcus epidermidis DSM 28319 (ATCC 35984). For sterility testing, E. coli laboratory strain MG1566 (ATCC 700926) for aerobic conditions and Fusobacterium nucleatum spp polymorphum for anaerobic conditions were used as positive controls. All bacterial strains were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and stored at −80 °C. All bacteria were grown in BD Difco Lennox lysogeny broth (LB) (Becton, Dickinson and Company, Le Point de Claix, France) culture medium. For the biofilm studies, Dulbecco’s modified Eagle medium DMEM/F-12 (Sigma-Aldrich, Gillingham, United Kingdom), with 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, without l-glutamine and phenol red, was used together with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, Sao Paulo, Brazil). Phosphate-buffered saline (1 × PBS), pH adjusted to 7.4, was used in the biofilm assay. For some additional testing also 10 mM or 50 mM HEPES buffer was used after dilution from 1 M HEPES buffer solution (Corning, Manassas, USA). For European Pharmacopoeia (Ph.Eur, 10.0) sterility testing (monograph 2.6.1.), two different dehydrated culture media, soybean casein digest medium (also known as tryptic soy broth (TSB), Sigma-Aldrich, Bangalore, India) and fluid thioglycolate medium LAB025 (Lab M Limited, Lancashire, United Kingdom), were used. To create anaerobic conditions, anaerobic gas generation bags from BD GasPak, EZ Anaerobe Container System (Benex Limited, Dublin, Ireland) were used.
2.1.3. Histology Solutions
Tissue-freezing medium (Leica, Richmond, IL) was used for embedding and freezing the skin samples. Histological stains used were hematoxylin solution according to Harris (Carl-Roth, Karlsruhe, Germany) and eosin solution prepared according to the manufacturer′s instruction using eosin Y (Acros Organics, Geel, Belgium) (hematoxylin and eosin (H&E) staining).
2.1.4. Fluorescent Stains
To visualize biofilm formation by confocal fluorescence microscopy (CFM), nucleic acid stain Syto 9 (Invitrogen, Eugene) and the EBBABiolight 630 (Ebba Biotech AB, Solna, Sweden) stain binding to cellulose and amyloid components were used.
2.2. Preparation Methods
2.2.1. Preparation of Electrospun (ES) Artificial Skin and Fibrous Wound Dressings
2.2.1.1. GEL-Glu Matrix Artificial Skin
For the in vitro model, a previously published thermally crosslinked ES GEL-Glu matrix (35,36) was used as artificial skin. For the preparation of the ES solution, in 10M acetic acid, GEL 25% (w/v) was dissolved, and Glu 10% (w/w solid state) was added to the mixture as a crosslinking agent. (35) The ES solution was obtained after 24 h of stirring at room temperature (24 ± 2 °C) (RT). ES was carried out using an ESR200RD robotized ES system (NanoNC, Seoul, Republic of Korea). ES parameters were the following: needle 23G, voltage 18–19 kV, flow rate 15 μL/min, distance between the needle and the collector 15 cm, and roller speed 20 mm/min. The GEL-Glu matrix was crosslinked for 3 h at 170 °C after ES to achieve better mechanical properties and stability in aqueous conditions as shown in previous studies. (35−37)
2.2.1.2. Wound Dressings
Model wound dressings were prepared by ES to test the suitability of the in vitro and ex vivo models for analyzing the antibiofilm activity of ES drug delivery systems. The ES solution compositions, conditions, and method parameters of all four different types of wound dressings (two pristine PCL and PCL/PEO dressings and two antibacterial drug CAM-loaded CAM-PCL and CAM-PCL/PEO dressings) have been previously published by Preem et al. In brief, ES solutions were prepared by dissolving the polymer PCL 12.5% (w/V) alone or PCL 10% (w/V) together with PEO 2% (w/V) in a chloroform/methanol (CHF/MET) (3:1 V/V) solvent mixture. The concentration of the model drug CAM within drug-loaded dressings was 4% (w/w) based on the dry weight of the polymer(s). (38) To minimize the possible contamination of the produced ES wound dressings, clean preparation techniques were used. The folio for fiber collection was autoclaved before use; also, the ES robot chamber was cleaned with 70% ethanol solution before ES. Fibrous dressings were collected onto an aluminum foil and stored in ziplock bags at RT and 0% relative humidity (RH).
2.2.2. Preparation of Bacteria
The bacteria used in this study were stored in glycerol stocks at −80 °C. Then, the required bacteria were thawed and plated on LB agar plates for overnight incubation at 37 °C. For the biofilm assay, one bacterial colony was inoculated into 3 mL of liquid LB broth and grown for 20 h at 37 °C, with continuous movement at 200 rpm. Before every assay, the exact bacterial concentration in the overnight culture was fixed and confirmed by counting the colonies of colony forming unit (CFU) plating of 10-fold dilutions. For bacterial dilution, sterile distilled water was used.
2.2.3. Pig Ear Skin Collection, Preparation, and Storage
An ex vivo model was developed using pig ear skin, received from the local slaughterhouse (Rotaks-R Oy, Tartu, Estonia). All pigs were washed in a hot-water bath, during which superficial burn wounds were developed, and most of the hair on the skin was already removed. The ear skin was collected manually by separating the skin from the cartilage using surgical scalpels and cut into 10 × 10 cm pieces. In case needed, ears were first washed with 1 × PBS, and the remaining hair on the skin was removed by shaving. Cut pieces of the skin were treated differently for the histological study to determine the best skin preparation option (minimizing the modification of the skin). Half of the samples were placed in a 20% (V/V) glycerol/PBS solution for 2–4 h for cryoprotection purposes (39) and then placed into ziplock bags (LDPE); the other half of the samples were placed directly into ziplock bags. Liquid nitrogen for snap freezing of samples was used followed by γ sterilization. Sterilized samples were stored at −80 °C, as advised by the Central Tissue Bank. For the biofilm assay, the pig skin was defrosted and cut into 1 × 1 cm pieces. Pig ear skin had a uniform thickness of about 4 ± 1 mm. Before the test, pig ear skin samples were held in sterile 1 × PBS on ice for up to 2 h.
2.3. Sterilization
γ-Irradiation at a dose of 50 kGy was used to sterilize ES wound dressings, (40) GEL-Glu matrices, and pig ear skin. Sterilization was performed by the Scandinavian Clinics Estonia OÜ. Frozen pig ear skin samples on top of dry ice pellets were irradiated immediately after sample collection. The pig ear skin was subjected to different sterilization treatments (e.g., UV light for 30 min on both sides and 70% ethanol treatment), but skin samples were sterile only after γ-irradiation. In addition to the chemical indicators provided by the company, we also added biological controls. Bacillus sp. spores were prepared by first culturing bacteria on solid media, then suspending the solid-medium-grown cells in 50% ethanol, and incubating for 1 h to kill all vegetative cells. (41) The remaining spores were washed and suspended in distilled water. The number of spores in suspension was determined by plating and counting of CFUs. No growth after sterilization confirmed the efficacy of the methods. All of the liquids (growth media, 1 × PBS, distilled water) used in the sterility and antibacterial/antibiofilm studies were sterilized by filtration (filter membrane pore size of 0.22 μm) or autoclaved according to the manufacturer′s instruction. Autoclaving with the standardized program for 15 min at 121 °C was also used to sterilize filter paper disks (used in a biofilm model setup) and all other equipment and supplies needed for the sterility and antibiofilm tests.
2.4. Characterization Methods
2.4.1. Sterility Testing
After γ-irradiation, all samples (ES pristine wound dressings, ES GEL-Glu matrix, and pig ear skin) were tested in triplicates for sterility under aerobic and anaerobic conditions according to the Ph.Eur. (10.0, monograph 2.6.1.) guidelines. Testing was carried out under aseptic conditions. TSB was used for aerobic bacteria and fungi, and the fluid thioglycolate medium used for anaerobic bacteria was hydrated with distilled water before use according to the manufacturer′s instructions. Thereafter, previously cut and sterilized 1 × 1 cm samples in triplicate were inoculated into the test tubes with sterile forceps under a laminar-flow hood. The test samples were incubated for 14 days at RT and protected from light for aerobic bacteria and fungi and at 30 °C under anaerobic conditions for anaerobic bacteria. For anaerobic conditions, test tubes were placed in a plastic bag together with anaerobic gas generation bags and sealed airtight. According to the Ph.Eur. guidelines, samples were considered sterile if no growth was detected after 14 days of incubation. Negative controls, pure growth medium without any inoculation, had to remain clear, and positive controls, Gram-negative E. coli MG1655 for aerobic conditions and F. nucleatum spp for anaerobic conditions, inoculated at the same time as the samples, had to show bacterial growth. Results are documented as images.
2.4.2. Biofilm Assay
2.4.2.1. Biofilm Inhibition Model
A biofilm assay was designed to study the potential inhibition of biofilm formation in sterile nontreated flat-bottom 24-well plates (VWR International, LLC, Shanghai, China), and a one-well-based biofilm model was set up as follows. At the bottom of the well, three sterile filter paper disks cut to fit the well properly (diameter 13 mm) were placed. Dulbecco’s modified Eagle medium (DMEM/F-12) without l-glutamine and phenol red supplemented with heat-inactivated FBS (10% (v/v)) was used as a medium to mimic the wound exudate. Then, 250 μL of the medium was added into every well, which was an optimal amount to immerse the filter papers and keep the setup moist. Then, either a 1 × 1 cm GEL-Glu matrix (cut under aseptic conditions under a laminar-flow hood) as artificial skin for the in vitro setup or 1 × 1 cm pig skin for the ex vivo setup was positioned on top of the filter papers. A schematic representation is shown in the Results (Figure 1).
Figure 1
Figure 1. Schematics of the experimental work conducted for the development and validation of the biofilm models. Key: CAM, chloramphenicol; ES, electrospinning; GEL, gelatin; Glu, glucose; PCL, polycaprolactone; PEO, polyethylenoxide.
2.4.2.2. Biofilm Formation on Top of the Substrate
Different pathogenic bacteria were tested to create different monobacterial biofilms in the in vitro and ex vivo models. Namely, E. coli DSM 1103 (ATCC 25922), which is a clinical isolate, S. aureus DSM 2569 (ATCC 29213) isolated from wounds, and S. epidermidis DSM 28319 (ATCC 35984) isolated from catheter sepsis were used. An overnight culture (20 h, 37 °C, 200 rpm) in LB broth was diluted using 1 × PBS to the optimal dilution (106 CFU/mL) immediately before the experiment, and the exact concentration was fixed in every assay by CFU plating. About 10 μL of bacterial dispersion was added on top of the pig skin for the ex vivo setup or on the GEL-Glu matrix for the in vitro setup. These in vitro and ex vivo models were incubated for 24 and 48 h at 37 °C. For incubation, well plates were closed with parafilm and placed in ziplock bags to avoid drying.
To assess whether the selected bacterial strains use gelatin (GEL) or glucose (Glu) in the GEL-Glu matrix for nutritional purposes, an additional test was designed and performed. Bacteria were inoculated in 10 mM HEPES buffer (1 mL) alone and/or together with the GEL-Glu matrix (size of 1 × 1 cm). The buffer chosen was the same as that used in the medium for the developed biofilm assay. The bacterial concentration at time point 0 was 104 CFU/mL. Bacterial growth at all tested time points (24, 48, 72, and 96 h) was measured by CFU plating. After 1 week, biofilm disruption manipulations (as described in the following paragraph Biofilm Disruption and Quantification: (30 s sonication + 30 s vortexing) × 6) were performed on the samples to check for any biofilm formation.
In addition, to study the effect of a thicker layer of the GEL-Glu matrix in the test for biofilm formation, one modification was made to the in vitro biofilm model. The in vitro model was created as described above, but a thicker artificial skin was created by positioning three GEL-Glu matrices on top of each other. An S. epidermidis culture dispersion was used to create a biofilm, which was incubated for 24, 48, and 72 h. After incubation, the steps described in the following paragraph (Biofilm Disruption and Quantification) were used to disrupt and quantify the biofilm.
2.4.2.3. Biofilm Disruption and Quantification
Subsequently, planktonic bacteria were removed by washing the samples (S = 1 cm2) twice with 1 mL of 1 × PBS solution and placed into a new buffer (1 mL). Biofilm disruption was carried out similarly to that in previously published studies. (27,42) Shortly, vortexing (Vortex-Genie 2, Scientific Industries) of the samples for 30 s and sonification (Bandelin Sonorex digital 10 P, operating at 20% of the maximum power) for 30 s were performed 6 times. When not handling the samples, they were placed on ice. After that, 100 μL aliquots were used to make 10 times dilutions, plated on LB agar plates, and incubated overnight at 37 °C. All experiments were carried out in triplicate, and the mean value was obtained. Also, technical replicates were used for CFU plating. Data are presented as CFU/cm2, which is equal to the CFU of bacteria from the biofilm grown on a 1 × 1 cm sample.
2.4.2.4. Biofilm Model Validation
CAM-loaded ES wound dressings (CAM-PCL and CAM-PCL/PEO) and pristine wound dressings (PCL and PCL/PEO) were cut into 1 × 1 cm samples under aseptic conditions under a laminar-flow hood. All drug-loaded dressings were weighed on an analytical balance (RADWAG Wagi Elektroniczne XA 210.4Y, Radom, Poland) before the experiment. The average CAM-loaded dressing weight was 2 ± 0.7 mg; hence, the drug content in dressings (solid state 4%) was 0.08 ± 0.03 mg. All CAM-loaded dressings were always weighed before the experiment to calculate the exact drug content in every sample.
In the assay, ES wound dressings were placed on top of the GEL-Glu matrix or pig skin immediately after the bacterial dispersion. To ensure close and comparable contact between the ES wound dressing and the pig skin or GEL matrix, well plate inserts (CellCrown 24, Scaffdex Oy, Tampere Finland) were placed on top of the ES dressing which allows proper hydration of the wound dressing. For the biofilm assay, drug-loaded samples were always studied in comparison with pristine wound dressings and with uncovered substrates (GEL-Glu matrix or pig skin). For this, in every assay, three samples were covered with drug-loaded ES wound dressings, three with pristine wound dressings, and three were left uncovered, and they were incubated as described above. For biofilm quantification, the wound dressings were removed from the substrate, and both the substrate and the dressing were separately placed into 1 mL of 1 × PBS and washed twice with 1 × PBS; biofilm quantification was carried out as described above.
2.4.2.5. Biofilm Treatment Model
The biofilm model was modified to determine whether the model can be used to study the effect against already formed biofilms. A monobacterial biofilm was created on top of the GEL-Glu matrix or pig skin and incubated for 24 h. After 24 h, ES wound dressings were applied on top of the pregrown biofilm. To ensure direct contact, a Cell Crown insert was placed on top of the wound dressing, and an additional 250 μL of cell culture medium was added to provide nutrients for bacteria and ensure the desired wettability of the wound dressing. Incubation was performed for an additional 24 or 48 h, followed by biofilm quantification as described above.
2.4.2.6. Contamination Detection Controls
All sterile liquids (growth media, 1 × PBS, distilled water) were plated on agar plates before the first use and after the last use, which allowed us to determine whether the solutions were sterile and remained sterile during the assay. Also, the sterility of filter paper disks, the GEL-Glu matrix, pig skin, pristine ES wound dressings, and inserts was tested. For this, the tested object was placed on top of three filter paper disks, and 250 μL of medium was added. No bacterial dispersion was added to the controls, but otherwise the controls were incubated similarly to the samples. All manipulations performed with the samples were similarly performed with the controls. The sample results were considered to be valid only when controls were free of contamination.
2.5. Microscopic Analyses
2.5.1. Histological Evaluation of Pig Skin
Cryosections were made of differently treated pig ear skin samples for histological evaluation and confirmation of epidermis removal during heat treatment (development of superficial skin burn wounds). Skin samples (1 × 1 cm) were embedded perpendicularly to the surface of the tissue-freezing medium (Leica, Newcastle, United Kingdom) such that the sections were cut longitudinally through the epidermis and dermis using a cryostat (Leica, CM1850, Nussloch, Germany). Serial cross-sections, 50 μm thick, were cut at −23 °C. Sections were washed with 1 × PBS to get rid of the excess freezing medium. After that, H&E staining was carried out, and the samples were fixed with a cover glass on top of microscope slides. Micrographs were recorded using a Zeiss Stemi 508 light microscope together with a Zeiss Axiocam 208 color microscope camera (Suzhou, China), using program Zen 3.4. (Zen Lite).
2.5.2. Biofilm Imaging by Scanning Electron Microscopy (SEM)
SEM was used to visualize the formed bacterial biofilms. Micrographs were recorded by SEM (Zeiss EVO 15 MA, Germany) under 9000× and 2000× magnifications. Microscopy sample preparation for biofilms grown on both substrates (GEL-Glu matrix and pig skin) after the biofilm assay (24 h) was carried out. After incubation, substrates were rinsed twice with 1 × PBS and then fixed with 4% formaldehyde solution in 1 × PBS for 30 min at RT, and then rinsed again twice with 1 × PBS and dehydrated in increasing concentrations of ethanol (30, 60, and 96%). In the ethanol solution with the highest concentration, the samples were kept for 5 min. Dried samples were mounted on aluminum stubs using carbon tape and sputter coated with platinum in an argon atmosphere.
2.5.3. Biofilm Imaging by Confocal Fluorescence Microscopy (CFM)
Confocal fluorescence microscopy (CFM, LSM710, Carl Zeiss, Germany) was performed to visualize biofilm formation on top of the GEL-Glu matrix and pig skin at different time points (0, 24, and 48 h). Samples were prepared similarly to uncovered and untreated samples in the biofilm assay. For imaging, nucleic acid stain Syto 9 (Invitrogen, Eugene), an excitation laser of 488 nm, and emission recorded in the range of 503–542 nm were used to visualize bacteria and the stain EBBABiolight 630 (Ebba Biotech AB, Solna, Sweden), an excitation laser of 514 nm, and emission recorded in the range of 545–657 nm were used to visualize the biofilm matrix formed by E. coli. The EBBABiolight 630 orange optotracer molecule binds to cellulose and amyloid components in the biofilm matrix. For visualizing the GEL-Glu matrix fibers, an excitation laser of 405 nm and emission in the range of 410–502 nm were used to record autofluorescence. Stains were diluted 100× using 50 mM HEPES buffer, which was obtained by diluting 1 M HEPES buffer solution (Corning, Manassas). Samples for microscopy were prepared using 2 μL of both diluted stains, which were placed on top of the sample. Zen software was used for the analyses (Zeiss, Oberkochen, Germany).
2.5.4. Atomic Force Microscopy (AFM) of ES Wound Dressings
The surface topography and morphology of ES wound dressings were investigated with AFM (Autoprobe CP, ThermoMicroscopes). The surface topography images of the ES samples were obtained over a 20 μm × 20 μm area. The AFM mapping was performed in the contact mode with a cantilever of 0.12 N/m spring constant (Silicon cantilever, CSCH21A, NT-MDT Ltd., Russia) at a scan rate of 0.6 Hz. The measurements were carried out at RT using a large-area scanner (100 mm lateral scan size).
2.6. Statistical Analysis
Data are presented as the arithmetic mean (n = 3) ± standard deviation (SD). Statistical analyses were performed using either the pairwise t-test or the independent t-test. The variance was calculated using the f-test. CFU data were log10-transformed before statistical analyses. The log reduction was determined by subtracting the log of the bacterial concentrations in treated samples from the values quantified in untreated biofilms. The data were analyzed using Microsoft Excel (version 16.58). Scientifically significant differences were found using p-values, and a p-value of 0.05 was considered significant. In the case of multiple comparisons, p-values were adjusted using the Holm–Bonferroni method.
3. Results and Discussion
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3.1. Preparation, Storage, and Sterility Testing of Biofilm Substrates
The aim of the present study was to develop and compare in vitro and ex vivo biofilm models for assessing the antibiofilm properties of electrospun (ES) antibacterial wound dressings (Figure 1).
The in vitro model was created using a thermally crosslinked ES porcine gelatin and glucose (GEL-Glu) matrix as artificial skin. The ES GEL-Glu matrix displays a skin ECM-like fibrous structured surface with an average fiber diameter of 535 nm ± 60 nm; the degree of swelling was 290 ± 30%, and the thickness of the matrix was approximately 0.08 mm.
The ex vivo model was developed using pig ear skin as it resembles human skin the most. (43,44) The skin was subjected to a superficial burn wound in a hot-water bath (temperature 60 °C), collected, frozen using liquid nitrogen, and sterilized using γ-irradiation. To confirm the presence of a superficial burn wound (epidermis removal), skin samples were subjected to histological analyses by microscopy (Figure 2A). It can be seen that the epidermis layer was removed or partially removed during the thermal treatment in a water bath.
Figure 2
Figure 2. (A) Micrographs of cross-sections of heat-treated pig ear skin (with superficial burn wounds) with and without γ-irradiation and glycerol treatment; H&E stained. Scale bar: 500 μm. (B) AFM micrographs of ES fibrous wound dressings. (C) European Pharmacopoeia 10.0 sterility test results after 14 days of incubation of γ-irradiated (50 kGy) samples (ES pristine fibrous wound dressings and infection model substrates) under aerobic conditions at room temperature (RT) in the dark. Positive bacterial control (E. coli). Key: GEL, gelatin; Glu, glucose; PCL, polycaprolactone; PEO, polyethylenoxide. Scale bar 1 cm.
Different pathogenic bacteria isolated from infected wounds were used to develop monobacterial biofilms: Gram-negative bacterium E. coli DSM 1103 (ATCC 25922) and Gram-positive bacteria S. aureus DSM 2569 (ATCC 29213) and S. epidermidis DSM 28319 (ATCC 35984). Biofilm formation on top of the substrates (GEL-Glu matrix vs pig ear skin) was studied at first, and then, for model validation, four different previously developed and characterized ES wound dressings were used. (38) Two of them contained the antibacterial drug chloramphenicol (CAM) and the other two were without a drug. The biofilm model was used, and data were obtained at two time points: 24 and 48 h.
To visualize these ES wound dressings and better understand their interactions with bacteria, AFM micrographs were recorded (Figure 2B). All four ES dressings had uniform smooth fibers; for PCL and PCL/PEO dressings, the average fiber diameter was less than the micrometer range (fiber diameter varied across the mat), whereas the PCL/PEO dressing consisted of microfibers (fiber diameter was homogeneous across the mat). It has been previously shown that the porosity of ES wound dressings is the same in all cases (between 87 and 89%) (38) PCL dressings have more hydrophobic properties as the degree of swelling is only 5%, whereas PCL/PEO dressings are more hydrophilic, which allows the dressing to swell up to 250%. (38) Good wetting properties are important for sufficient drug release from wound dressings.
The design and development of a successful biofilm model require sterility. In this study, a combination of three sterilization methods was used, namely, filtration, autoclaving, and γ-irradiation. Filtration was used to sterilize all of the liquids (growth media, 1 × PBS, distilled water) used in the assay. Autoclaving was used to sterilize the filter paper disks and the equipment (scissors, forceps, pipettes, and pipette tips) used. In our preliminary experiments, UV treatment for 30 min on both sides for the sterilization of filter paper disks and GEL-Glu matrices was unsuccessful, and samples showed contamination. All ES wound dressings and GEL-Glu matrices were therefore sterilized using γ-irradiation. γ-Irradiation may cause changes in the molecular structures of the polymers and therefore affect the properties of ES wound dressings. (45) Therefore, γ-irradiation with a dose of 50 kGy was chosen, as it was previously shown to be suitable without any harmful effects and no reduction in the CAM content (no degradation of CAM). (40) The effectiveness of γ-sterilization was confirmed using the Ph.Eur sterility test, which showed that no bacterial growth occurred after 14 days of incubation (Figure 2C).
Identifying a suitable sterilization method for pig skin samples was not an easy task. The sterilization itself should successfully remove the contamination but should not damage the structure of the skin used for the model development. Preliminary testing showed that just wiping the surface of the skin gently with 70% ethanol solution was not enough for disinfection (Supporting Information, Figure S1). This has also been shown in previous studies. (33,46−48) Although even rinsing the pig skin samples with ultrapure water has previously been reported to be effective, (49) in our work, contamination was not removed using ultrapure water or ethanol. Often, 70% ethanol treatment is combined with the soaking of samples in an antibiotic solution. (50) This approach was not used in our study to avoid misinterpretation of the results later. Bathing the skin samples in ethanol for a longer time period seemed to be too destructive to the tissue, and for the same reason, UV sterilization was avoided. The chosen sterilization method for pig skin in this study was γ-irradiation as it has been previously used and recommended for the sterilization of pig ear skin samples for the ex vivo model. (51,52) The standard γ-irradiation dose for sufficient sterilization of biological samples is 25 kGy. (52,53) Unfortunately, for our samples, it was not effective enough. It has been previously established by Johnston et al. that the sterility assurance level is extremely dependent on the initial bioburden, and the use of standard irradiation levels will require a method to reduce the bioburden before irradiation. (39) In our study, pigs were placed in a hot-water bath after slaughter, which can also reduce the bioburden in addition to the formation of superficial burn wounds and the removal of hair. After this, sterilization with a higher dose of irradiation (50 kGy) was used. The effect of γ-irradiation is based on the water content of the samples; its absence leads to the breaking of collagen polypeptide chains and its presence leads to hydroxyl radical formation, which causes the crosslinking of collagen fibers. (51,54) Higher doses of γ-irradiation may also reduce tissue integrity. To understand the effect of γ-irradiation on the skin and confirm its suitability for sterilization, the initial skin samples and γ-sterilized samples were subjected to histological analyses by microscopy (Figure 2A). Comparing the micrographs, it was seen that the skin integrity was not reduced by γ-irradiation. No major differences were observed between the γ-sterilized and nonsterilized skin samples. It is likely that due to the protective measures taken, the skin integrity was preserved: samples were placed in 20% glycerol solution before sterilization and deeply frozen after sterilization, according to Rooney et al. (53)
During the biofilm model development, the relevance of sterility and detection of contamination was confirmed. For this reason, a contamination detection protocol for the assay was created, as described in the Methods section. Furthermore, negative controls without added bacterial dispersion were prepared in every assay in the same well plate to test the sterility of the filter paper disks, pig skin, and GEL-Glu matrix. These were incubated with the media similarly to the samples used in the assay. The 1 × PBS buffer and growth media used were also tested for sterility by CFU plating.
3.2. Biofilm Formation on Top of Different Substrates─ES GEL-Glu Matrix (In Vitro Model) vs Pig Skin (Ex Vivo Model)
The initial step in the model development was to determine whether monobacterial biofilms form in both models, using different pathogenic bacteria and at different time points. CFU enumeration was chosen to obtain appropriate and comparable results for biofilm formation. SEM micrographs were obtained to confirm the findings, and CFM results helped visualize the live biofilm matrix on different substrates (artificial skin vs pig skin).
The results showed that after 24 h, all three selected bacteria (E. coli DSM 1103, S. aureus DSM 2569, and S. epidermidis DSM 28319) had already adhered on these substrates and formed a biofilm consisting of up to 109 CFU/cm2 bacteria, being also visible in SEM and CFM micrographs (Figure 3).
Figure 3
Figure 3. (A) SEM micrographs of biofilm formation after 24 h on top of the substrates─gelatin–glucose matrix (GEL-Glu matrix) and pig skin. Arrows point out the bacteria. Three different wound bacteria were used: E. coli DSM 1103, S. aureus DSM 2569, and S. epidermidis DSM 28319. The scale bar is 3 μm for the overview image of the pig skin surface, and it is 10 μm for all other SEM micrographs. (B) CFM micrographs of biofilm formation (24 and 48 h) on top of the GEL-Glu matrix in the in vitro model. Biofilm matrix visualized using EbbaBiolight 630 is in pink; ES gelatin–glucose matrix (GEL-Glu matrix) fibers visualized by autofluorescence are in blue; and bacteria stained using SYTO-9 are in green. Three different wound bacteria were used: E. coli DSM 1103, S. aureus DSM 2569, and S. epidermidis DSM 28319. Scale bar: 10 μm. The sample depth shown is 14.4 μm. (C) CFM micrographs of biofilm formation (24 h) in the ex vivo model. Pig skin had nonspecific red autofluorescence; bacteria were stained with SYTO-9 in green. Two different wound bacteria were used: E. coli DSM 1103 and S. aureus DSM 2569. Scale bar: 10 μm. (D) Biofilm formation after 24 and 48 h on top of the substrates─gelatin–glucose matrix (GEL-Glu matrix) and pig skin. Three different wound bacteria were used: E. coli DSM 1103, S. aureus DSM 2569, and S. epidermidis DSM 28319. Results are shown in the logarithmic scale as the number of colony-forming units (CFUs), with standard deviation bars (n = 3). Statistical significance is shown as follows: *p < 0.05; **p < 0.01; and ***p < 0.001. Experiments were performed using at least three technical replicates. Key: GEL, gelatin; Glu, glucose.
SEM micrographs showed that both substrates were colonized with different bacteria already after 24 h (Figure 3A). It was also seen that in the in vitro model, bacteria grew between and inside the fibers, and in the ex vivo model, bacterial colonies were found more inside the skin pores. CFM micrographs also confirmed that all tested bacteria preferred to grow inside the GEL-Glu matrix, and by making z-stacks of our samples, the depth of colonized bacteria could be determined (Figure 3B). Differences between bacteria showed that E. coli microcolonies were formed more on the surface of the fibrous matrices, whereas S. aureus and S. epidermidis formed colonies far deeper inside the fiber matrix. CFM microscopy together with Ebba Biolight staining also provided additional and relevant information about live biofilm formation, which allowed us to visualize the biofilm matrix formed by E. coli on top of the developed artificial skin (GEL-Glu matrix) (Figure 3B). It was seen that the biofilm matrix was formed on top of the colonizing bacteria. For S. aureus and S. epidermidis, the biofilm matrix was not stained with Ebba Biolight, but larger microcolonies of both bacteria were observed at both time points (Figure 3B). Furthermore, the microcolonies were more frequently observed in the substrate after 48 h in comparison with after 24 h of testing.
The visualization of bacteria and the biofilm matrix on top of the pig skin by CFM was challenging. Stained pig skin without bacteria itself had a nonspecific red autofluorescence (Figure 3C). The biofilm matrix-specific red fluorescence was not detected on any of the CFM micrographs. Overall, it was challenging to visualize even colonizing bacteria on top of the pig skin by CFM due to their growth occurring mainly inside the skin pores, which are not easily reachable for visualization using CFM. SEM micrographs of E. coli and S. aureus colonies were successfully obtained on pig skin only at the edges of skin pores. For the ex vivo model, the SEM images were much more informative and can be used for better visualization of bacteria on top of pig skin (Figure 3A).
The results in Figure 3D indicate that all selected bacterial strains can be successfully used in the developed biofilm assays. Differences between the in vitro and ex vivo models in the amount of bacterial biofilm (measured as the number of biofilm-forming bacteria) formed on top of the substrates were observed after 24 h. For E. coli and S. aureus, there were statistically significant differences in the biofilm formation on the GEL-Glu matrix (in vitro model) compared with pig skin (ex vivo model), with a higher formation observed in the latter model. At the 48 h time point, the differences were not statistically relevant anymore for E. coli and S. aureus (Figure 3D). Differences between the in vitro and ex vivo models at the 48 h time point were observed with S. epidermidis, wherein the biofilm formation on top of the GEL-Glu matrix was significantly lower than that on top of the pig skin (Figure 3D). Overall, pig skin was the most favored substrate for biofilm formation for all three bacteria at any time point. Nevertheless, differences in the number of biofilm-forming bacteria among the three selected bacteria were seen only in the ex vivo model (Figure 3D). For example, in the ex vivo model, S. epidermidis formed less biofilm on pig skin compared with E. coli and S. aureus at both time points.
It is known that for a closed model, no continuous flow of the medium is possible, which leads to changes in experimental conditions as the nutrient content is reduced and metabolic products build up. (55) The latter results in a higher biofilm growth rate at the beginning of the assay when there are enough nutrients. (56) Nevertheless, with S. epidermidis, lower numerical values of biofilm bacteria were found in the in vitro biofilm model using the GEL-Glu matrix at the 48 h time point. This suggests that other bacteria (E. coli and S. aureus) might use other substances of the matrix for nutritional purposes. To confirm this statement, an additional test was performed, where S. aureus and S. epidermidis were inoculated into 10 mM HEPES buffer alone or together with the GEL-Glu matrix, and the use of the GEL-Glu matrix as a nutritional substrate was assessed (Figure 4A).
Figure 4
Figure 4. (A) Use of the gelatin–glucose matrix (GEL-Glu matrix) as a nutritional substrate in HEPES buffer. Bacteria were inoculated into 10 mM HEPES buffer alone and/or together with the GEL-Glu matrix for up to 96 h. S. aureus DSM 2569 and S. epidermidis DSM 28319 were used. The number of colony-forming units (CFUs), with standard deviation bars (n = 3), are shown in the logarithmic scale. Experiments were performed using at least three technical replicates. The detection limit of the assay is 2 log10 CFU/cm2. (B) S. epidermidis DSM 28319 biofilm formation on top of a single-layered gelatin–glucose matrix (1 × GEL-Glu) and triple-layered gelatin–glucose matrices (3× GEL-Glu matrix) in HEPES-buffered DMEM/F-12 growth media at different time points (24, 48, and 72 h). The number of colony-forming units (CFUs), with standard deviation bars (n = 3), are shown in the logarithmic scale. Statistical significance is shown as **P < 0.01. Experiments were performed using three technical replicates. Key: GEL, gelatin; Glu, glucose.
S. aureus survived in the testing environment when the GEL-Glu matrix was present for at least 96 h, but no bacteria were detected in pure HEPES buffer after 48 h (Figure 4A). For S. epidermidis, no bacteria were observed already after 24 h in both environments. These results enable us to explain the lower concentrations of the S. epidermidis biofilm bacteria on the GEL-Glu matrix in the developed in vitro biofilm model, as S. epidermidis does not use the GEL-Glu matrix as a nutritional substrate, unlike S. aureus. This is most likely due to the different virulence of these bacteria. S. aureus as well as E. coli have several virulence factors and are both pathogens capable of initiating infections in a human body, whereas S. epidermidis usually does not have aggressive virulence factors. (57)
Indeed, one of the possible reasons for the higher bacterial numbers on top of pig skin might also be related to the thickness of the substrate (5 mm for pig skin vs 0.08 mm for the GEL-Glu matrix). Taking this into consideration, a modification in the in vitro model was made. Instead of one GEL-Glu matrix, three GEL-Glu matrices on top of each other were used (a total thickness of 0.27 mm), and biofilm formation was tested with S. epidermidis (Figure 4B). To show the effect more clearly, we prolonged the study up to 72 h. In this assay, the S. epidermidis biofilm formation on top of the single-layered GEL-Glu matrix significantly decreased with every time point. Differences between the single- and triple-layered GEL-Glu matrices were not significant after 24 h but were significant in the subsequent time points. The results showed that when using triple-layered GEL-Glu matrices, the biofilm formation does not reduce with time at the same rate as it provides a larger three-dimensional surface for bacteria to grow. However, the number of biofilm-forming bacteria is still significantly lower after 72 h when compared with the 24 h time point (Figure 4B).
3.3. Biofilm Model Validation Using Drug-Loaded ES Fibrous Wound Dressings
Both in vitro and ex vivo biofilm models were used to study the effect of previously developed and characterized antibacterial ES wound dressings by Preem et al. on biofilm formation. (12,38,40)
The results showed that CAM-loaded wound dressings drastically inhibited biofilm formation on top of the substrates compared with non-CAM-loaded control dressings (Figure 5). Results were statistically significant in both models, showing up to 6 log reductions in the number of biofilm-forming bacteria at the 24 and 48 h time points. Both models behaved similarly, meaning that no statistically significant differences between the in vitro and ex vivo models in the number of biofilm-forming bacteria were found at any time point. As ex vivo models are mainly developed and used as a bridge between in vitro and in vivo models, it is a great finding to have similar results obtained using either artificial skin (GEL-Glu matrix) or pig skin. The ex vivo model is known to provide more valuable features for mimicking real “skin wound” conditions as most of the wound bed components are naturally occurring components in the skin. Ex vivo models do not require any supplementary agents to be added into the growth medium compared with in vitro models. (33) In our study, the GEL-Glu matrix in the in vitro model also provided nutritional supplements to some bacterial strains, acting similarly to the pig skin.
Figure 5
Figure 5. Model validation using electrospun (ES) wound dressings. For the in vitro model, the GEL-Glu matrix was used as artificial skin, and for the ex vivo model, pig skin was used. Three different bacteria were used: (A) E. coli DSM 1103, (B) S. aureus DSM 2569, and (C) S. epidermidis DSM 28319. Results are shown in the logarithmic scale as the number of colony-forming units (CFUs) cm2, with standard deviation bars (n = 3). The detection limit of the assay is 2 log10 CFU/cm2. CAM-loaded ES wound dressings were compared with pristine control wound dressings. A comparison between the PCL vs PCL/PEO formulations is also presented. Statistical significance is shown as follows: *P < 0.05; **P < 0.01; and ***P < 0.001. Key: CAM, chloramphenicol; PCL, polycaprolactone; and PEO, polyethylenoxide.
As expected, no effect was revealed with non-CAM-loaded control dressings, which showed biofilm formation up to 8 log10 CFU/cm2, similar to the nontreated dressing (uncovered substrate) (Figures 3D and 5). As shown by the results of our study, biofilm inhibition was somewhat less efficient against S. aureus as compared with E. coli and S. epidermidis, which might be due to the higher effective concentration of CAM needed. Nevertheless, antibacterial ES wound dressings had a statistically significant inhibiting effect on biofilm formation in both models at all time points and with all selected bacterial strains.
The aim of these developed in vitro/ex vivo models was not only to derive an assay to study the antimicrobial/antibiofilm properties of wound dressings but also to compare different formulations for their efficacy under more biorelevant conditions and predict their in vivo behavior. In this study, we on purpose tested ES wound dressings made from the hydrophobic polymer PCL and another one, where, in addition to the PCL hydrophilic polymer, PEO was added. As previously shown, the drug release kinetics into 10 mL 1 × PBS differ between the two wound dressings, although no differences in the disk diffusion assay (release into the gel) were seen. (38) Interestingly, previously reported biofilm assays revealed that the CAM-PCL dressing was more effective compared with the CAM-PCL/PEO dressing on E. coli CFT073, most likely due to the different release kinetics. (38) In the current assay, both formulations with the same drug load exhibited a similar antibiofilm effect, as a 6-log difference was observed in bacterial numbers for E. coli and S. epidermidis (Figure 5A,C). The effect was visible at all time points and in both models. The log reduction was the same or higher in ex vivo models compared with in vitro models, which can be explained by the overall higher bacterial numbers in the ex vivo model.
However, using the same dressings with S. aureus, differences between formulations were seen after 48 h (Figure 5B). The CAM-PCL/PEO dressing was statistically more effective in inhibiting biofilm formation than the CAM-PCL dressing. The drug load in the used ES wound dressings is approximately 80 μg, but the amount of drug released at specific time points from the matrix differs. As shown before for the CAM-PCL/PEO dressing, 90% of the drug is released in the first 60 min, but for the CAM-PCL dressing, the amount of drug released is only 40% after 24 h and up to 50% after 48 h. (38) Therefore, the available drug concentration after 24 h of treatment is different, being higher when using the CAM-PCL/PEO dressing.
3.4. Effect of Pristine ES Wound Dressings on Biofilm Formation and Bacterial Adhesion
It was also of interest to determine whether control dressings with no drug (pristine polymer dressings) have any effect on biofilm formation as carrier polymers can potentially inhibit or induce biofilm formation. The search for antibacterial/antibiofilm polymers and smart textiles with the same properties is evolving rapidly; (58−61) therefore, novel assays are needed to test their efficacy against biofilms. We proposed that the models developed by us could act as suitable tools for these evaluations. In a current study, all control dressings (pristine wound dressings without any antibacterial agent) were tested against uncovered substrates─GEL-Glu matrix or pig skin (shown as 100%)─to determine whether the number of bacteria in the formed biofilm differed (Figure 6). In addition, it was also investigated whether the bacteria adhered and formed the biofilm preferentially on the substrate or on the ES fibrous pristine wound dressing. For this assessment, the control dressing and the substrate were mechanically separated from each other after incubation, and the amounts of biofilm formed on both surfaces were studied separately.
Figure 6
Figure 6. Bacterial biofilm adhesion on top of the substrate−gelatin−glucose matrix (GEL-Glu matrix) or pig skin or onto the covering wound dressing (ES pristine wound dressings). Substrates were covered with pristine wound dressings and incubated for 24 h, after which the substrate and wound dressing were separated, and the biofilm formed on top of each part was studied independently. Three different bacteria were used: (A) E. coli DSM 1103, (B) S. aureus DSM 2569, and (C) S. epidermidis DSM 28319. Two different formulations were tested─PCL wound dressing and PCL/PEO wound dressing (control dressings); 100% is shown to mark the arithmetic mean of biofilm formation on top of the different uncovered substrates. Key: GEL, gelatin; Glu, glucose; PCL, pristine wound dressings made from polycaprolactone; PCL/PEO, pristine wound dressings made from polycaprolactone and polyethylenoxide.
Results showed that the amount of biofilm on top of the substrate when covered with the pristine control wound dressing does not exceed the amount of biofilm formed on top of the uncovered substrate. However, when the GEL-Glu matrix substrate is covered with the PCL dressing, then the amount of biofilm formed in combination with both (substrate and ES dressing) exceeds the amount of biofilm formed on uncovered substrates. For S. epidermidis, the results were different as it was also seen that the GEL-Glu matrix covered with PCL/PEO and pig skin covered with PCL dressing had higher amounts of combined biofilms than the uncovered samples.
The covered samples were studied further to determine whether the adhesion of the biofilm on top of the pristine wound dressing can be studied in the developed assay. It is known that bacterial biofilms are formed on almost all materials except bioinert and antibiofilm materials. Nanomaterials are no exception. (62) Results show that both PCL and PCL/PEO wound dressings were less favored for biofilm formation than the substrate (Figure 6). Differences are seen between bacteria and between substrates. The standard deviation is also quite wide, showing that the ex vivo model would be more favorable for studying the biofilm adhesion on top of the tested materials. Nevertheless, variability in this evaluation remains and most likely arises from the fact that mechanical separation was technically not easily reproducible.
Differences between formulations were evaluated, as it has been shown previously that PEO has antifouling properties that theoretically should avoid bacterial attachment. (63,64) In our formulation, only 2% of the PEO was added into the PCL polymer, but already changes in the biofilm-forming bacterial attachment were seen, showing that a lower amount of biofilm was formed on PCL/PEO dressings (Figure 6).
3.5. Model Modification to Study the Effect of ES Fibrous Wound Dressings on Preformed Biofilms
We also studied whether it is possible to grow the biofilm before applying a drug-loaded wound dressing and test its ability to treat already formed biofilms. For this, a modification was performed to the in vitro and ex vivo biofilm assays with E. coli. After 24 h, the biofilm was quantified (108 CFU/cm2) and then treated for 24 and 48 h with both CAM-loaded and pristine wound dressings. As the bacteriocidal activity of CAM is known to be related to the specific bacterial strain, no major biofilm treatment effect was expected. (65) Because CAM mainly has bacteriostatic effects, the results in Figure 7 are presented in a linear scale as the change in the initial count of biofilm bacteria.
Figure 7
Figure 7. Biofilm treatment model. Effect of the model CAM-loaded wound dressings (CAM-PCL and CAM-PCL/PEO dressings) on already formed E. coli DSM 1103 biofilms (24 h). A comparison is made with pristine wound dressings and uncovered substrates. The in vitro setup was created on top of the ES GEL-Glu matrix and the ex vivo setup was created on top of pig skin. The number of biofilm-forming bacteria before treatment was 108 CFU/cm2. Results are shown in a linear scale as the number of colony-forming units (CFUs), with standard deviation bars (n = 3). Changes in the threshold value (108 CFU/cm2) are presented. Statistical significance is shown as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Key: CAM, chloramphenicol-loaded wound dressings; PCL, pristine wound dressings made from polycaprolactone; PCL/PEO, pristine wound dressings made from polycaprolactone and polyethylenoxide.
The results clearly show that when substrates were left uncovered, the amount of biofilm increased; the same was seen with pristine wound dressings (Figure 7). For CAM-loaded wound dressings, the decrease in the number of biofilm-forming bacteria was detected after 24 h of treatment in both models and after 48 h of treatment in the in vitro model. In the ex vivo model, CAM-loaded wound dressings did not have any biofilm-inhibiting effect after 48 h of treatment, which can be explained by the higher growth in general on top of pig skin than that on top of the GEL-Glu matrix. Furthermore, previous studies suggested that the presence of efflux pumps in E. coli, upregulated specifically in bacterial biofilms, may remove toxic compounds, including antibiotics, from the bacterial intracytoplasmic space, which could explain the biofilm-specific recalcitrance. (66)
Differences between formulations were also observed: the inhibition of biofilm bacteria was more significant for CAM-PCL/PEO than for CAM-PCL wound dressings (Figure 7). Even though the drug load is the same for both formulations, the faster CAM release from CAM-PCL/PEO wound dressing and therefore higher amount of drug at the site of action at the beginning of the treatment was perhaps beneficial in this case. This might be the reason why a prolonged and sustained release of a higher amount of CAM would be needed for more effective treatment against already formed biofilms.
3.6. Comparison of the Developed Wound Infection and Biofilm Models with Currently Available Models
An increasing number of biorelevant models have been developed and published over the years for studying and evaluating already existing or novelly created wound treatments and wound preparations. Some of them are also validated to study the efficacy of different compounds to treat biofilms. Two previously published in vitro models and five ex vivo wound and biofilm models for obtaining quantitative results were chosen to be compared with the models developed by us in Table 1.
Table 1. Comparison between the Models Developed by Us and Previously Published Wound Infection and/or Biofilm Modelsa
a
Key: “+”, the listed feature was present in the model. More “+” signs show a better performance of the model for that evaluated parameter. CFM, confocal fluorescence microscopy; CLSM, confocal laser scanning microscopy; ES, electrospinning; GEL, gelatin; Glu, glucose; SEM, scanning electron microscopy. A blue background shows the best of the comparison.
Most of the published models used a specific substrate (e.g., silicon disks, collagen surface, pig skin, or human skin) to grow biofilms on top and also used microscopy to visualize the biofilms formed on the substrate. For example, the use of immunohistological staining and light microscopy of skin cross-sections is quite common. Fluorescence microscopy and confocal laser scanning microscopy images were also obtained from skin cross-sections and collagen surfaces (mimicking the skin surface in vitro), respectively. However, three-dimensional images were shown only by Werthen et al. (30) In the in vitro wound infection model developed by us, the bacterial biofilm formation on top of the GEL-Glu matrix was easily visualized using CFM, providing informative and easily understandable micrographs, which allowed the visualization of the bacteria and the biofilm matrix separately in three dimensions. Also, we were able to obtain SEM images that were not obtained in any of these published in vitro models. In previously published ex vivo models, the use of SEM to evaluate biofilms was quite common, together with light microscopy and/or fluorescence microscopy.
None of the previously published wound/biofilm models on substrates were validated for testing antimicrobial ES wound dressings. Hence, in the present study, we aimed to evaluate whether it is possible to use the ES wound dressings for these applications. The published in vitro models were promising, allowing a manageable size of the dressing to be used and allowing the properties of the dressings to be investigated. However, compared with the models developed by us, the amount of medium used was 5–10 times higher and the use of 15 mL tubes for one sample is much more inconvenient and operator unfriendly than using one 24-well plate for 24 samples. Among previously published ex vivo wound infection models, most of them were designed to use sample sizes (0.2–0.3 cm in diameter) smaller than the 1 × 1 cm squares used in our model. Larger sample sizes in our model provide the following key advantages for testing (considering the advantages for the operator as well as the test sample): (i) easy to reproduce, (ii) sufficient drug load with more biorelevant drug concentrations inside the dressing (closely mimicking the final application conditions), (iii) more precise measurement of the sample weight, and (iv) easy to handle while performing the assay.
Most of the published ex vivo models required some specific tools to create the wound (e.g., biopsy needle, electric soldering iron, or dermal tool), which were not necessary for our model as superficial burn wounds were self-created during the washing of pigs in a hot-water bath. For the in vitro model presented in this study, ES must be performed to create GEL-Glu matrices. However, this is most likely not a problem for facilities producing ES wound dressings.
One of the most important and key novelties is that our models enabled us to test biofilm inhibition and were validated using ES wound dressings. However, none of the published models were designed to evaluate the biofilm inhibition effect. Most of the published models allowed one to study the treatment of biofilms, similar to our model, quantifying the number of biofilm-forming bacteria after disrupting the biofilm. Sample collection for biofilm disruption was not always technically easy and was often challenging to reproduce. This was also discussed previously, and the latter was due to the variability of cutting out the pieces of the wound from the substrate. In our ex vivo model, no additional cutting needed to be performed as pretreated skin and the cut 1 × 1 cm substrate were entirely used to quantify the amount of biofilm formed.
4. Summary and Conclusions
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In conclusion, we herein demonstrate that the developed biorelevant in vitro gelatin–glucose (GEL-Glu) and ex vivo (pig ear skin) biofilm models can be successfully used to test the antibiofilm properties of novel ES fibrous antibacterial drug-loaded as well as pristine wound dressings. Depending on the model setup, the inhibition of biofilm formation as well as biofilm treatment can be explored. Pig skin with a superficial burn wound was the preferred substrate for biofilm formation in comparison with the GEL-Glu matrix. All three wound pathogens can be used in these models (E. coli, S. aureus, and S. epidermidis). The selection of pathogenic wound bacteria for model development is important, as E. coli and S. aureus are able to use the in vitro model substrate GEL-Glu matrix for nutrition. The sterility of the models must be assured to conduct successful experiments with wound pathogens. Pig skin is a more universal substrate to use, but its collection, sterilization, and storage are more challenging compared with the in vitro model. The electrospun (ES) GEL-Glu matrix can be easily and reproducibly produced and used as a substrate for biofilm growth and visualization, but when tested with ES wound dressings, its interactions (e.g., physical attachment) may affect the model results. This needs to be considered during model selection and design. Nevertheless, both tested chloramphenicol-loaded ES wound dressings exhibited an antibiofilm effect in both tested models. Therefore, it can be concluded that the developed in vitro and ex vivo models may be used as new diagnostic tools as well as to develop treatments for wound infections.
Supporting Information
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.2c00902.
Additional information about experimental methods and results; sterility test results (Figure S1) (PDF)
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Author Information
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- Karin Kogermann - Institute of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia;
https://orcid.org/0000-0002-6813-4828;
This manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript
This work was supported by the Estonian Research Council grant (PRG1507; K.K.). In addition, this research was funded by the European Regional Development Fund through the Centre of Excellence for Molecular Cell Technology (M.P. and T.T.)
Acknowledgments
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Prof K. Kirsimäe, MSc M. Külaviir, and Dr. G.-M. Lanno. (University of Tartu) are thanked for SEM measurements and MSc Anna Halenius (University of Helsinki) for the AFM measurements. The authors thank Ebba Biotech for not only providing a sample of the EbbaBiolight 520 optotracer for bacterial ECM component visualization but also assisting with technical support when necessary.
References
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This article references 66 other publications.
- 1Omar, A.; Wright, J.; Schultz, G.; Burrell, R.; Nadworny, P. Microbial Biofilms and. Chronic Wounds. Microorganisms 2017, 5, 9, DOI: 10.3390/microorganisms5010009Google Scholar1Microbial biofilms and chronic woundsOmar, Amin; Wright, J. Barry; Schultz, Gregory; Burrell, Robert; Nadworny, PatriciaMicroorganisms (2017), 5 (1), 9/1-9/15CODEN: MICRKN; ISSN:2076-2607. (MDPI AG)Background is provided on biofilms, including their formation, tolerance mechanisms, structure, and morphol. within the context of chronic wounds. The features of biofilms in chronic wounds are discussed in detail, as is the impact of biofilm on wound chronicity. Difficulties assocd. with the use of std. susceptibility tests (min. inhibitory concns. or MICs) to det. appropriate treatment regimens for, or develop new treatments for use in, chronic wounds are discussed, with alternate test methods specific to biofilms being recommended. Animal models appropriate for evaluating biofilm treatments are also described. Current and potential future therapies for treatment of biofilm-contg. chronic wounds, including probiotic therapy, virulence attenuation, biofilm phenotype expression attenuation, immune response suppression, and aggressive debridement combined with antimicrobial dressings, are described.
- 2Stuermer, E. K.; Besser, M.; Brill, F.; Geffken, M.; Plattfaut, I.; Severing, A. L.; Wiencke, V.; Rembe, J. D.; Naumova, E. A.; Kampe, A.; Debus, S.; Smeets, R. Comparative Analysis of Biofilm Models to Determine the Efficacy of Antimicrobials. Int. J. Hyg. Environ. Health 2021, 234, 113744 DOI: 10.1016/j.ijheh.2021.113744Google Scholar2Comparative analysis of biofilm models to determine the efficacy of antimicrobialsStuermer, E. K.; Besser, M.; Brill, F.; Geffken, M.; Plattfaut, I.; Severing, A. L.; Wiencke, V.; Rembe, J. D.; Naumova, E. A.; Kampe, A.; Debus, S.; Smeets, R.International Journal of Hygiene and Environmental Health (2021), 234 (), 113744CODEN: IJEHFT; ISSN:1438-4639. (Elsevier GmbH)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 clin. situation are required. In this study, two 3D biofilm models based on human plasma with immune cells (lhBIOM) or based on sheep blood (sbBIOM) contg. S. aureus or P. aeruginosa, are compared with the human biofilm model hpBIOM regarding their microscopic structure (SEM; SEM) and their bacterial resistance to octenidine hydrochloride (OCT) and a sodium hypochlorite (NaOCl) wound-irrigation soln. The three analyzed biofilm models show little to no reaction to treatment with the hypochlorous soln. 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 detd. effects are qual. reflected by SEM. Our results show that both ethically acceptable human and sheep blood based biofilm models can be used as a std. for in vitro testing of new antimicrobial substances. Due to their compn., both fulfill the criteria of a reality-reflecting model and therefore should be used in the approval for new antimicrobial agents.
- 3Stoffel, J. J.; Kohler Riedi, P. L.; Hadj Romdhane, B. A Multimodel Regime for Evaluating Effectiveness of Antimicrobial Wound Care Products in Microbial Biofilms. Wound Repair Regen. 2020, 28, 438– 447, DOI: 10.1111/wrr.12806Google Scholar3A multimodel regime for evaluating effectiveness of antimicrobial wound care products in microbial biofilmsStoffel Joseph J; Kohler Riedi Petra L; Hadj Romdhane BrittanyWound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society (2020), 28 (4), 438-447 ISSN:.Microbial biofilms have become increasingly recognized as a cause of wound chronicity. There are several topical antimicrobial wound care products available for use; however, their effectiveness has routinely been demonstrated with planktonic microorganisms. There is no target reference value for antimicrobial effectiveness of wound care products in biofilm models. In addition, data on antimicrobial activity of products in biofilm models are scattered across many test methods in a variety of studies. The aim of this work is to directly compare commercial products containing the commonly used topical antimicrobial agents iodine, silver, polyhexamethylene biguanide, octenidine, hypochlorous acid, benzalkonium chloride, and a surfactant-based topical containing poloxamer 188. Five different in vitro biofilm models of varied complexity were used, incorporating several bacterial pathogens such as Staphylococcus, Enterococcus, Streptococcus, Pseudomonas, Acinetobacter, Klebsiella, and Enterobacter. The fungal pathogens Candida albicans and Candida auris were also evaluated. A multispecies bacterial biofilm model was also used to evaluate the products. Additionally, C. albicans was used in combination with S. aureus and P. aeruginosa in a multikingdom version of the polymicrobial biofilm model. Statistically significant differences in antimicrobial performance were observed between treatments in each model and changing microbial growth conditions or combinations of organisms resulted in significant performance differences for some treatments. The iodine and benzalkonium chloride-containing products were overall the most effective in vitro and were then selected for in vivo evaluation in an infected immunocompromised murine model. Unexpectedly, the iodine product was statistically (P > .05) no different than the untreated control, while the benzalkonium chloride containing product significantly (P < .05) reduced the biofilm compared to untreated control. This body of work demonstrates the importance of not only evaluating antimicrobial wound care products in biofilm models but also the importance of using several different models to gain a comprehensive understanding of products' effectiveness.
- 4Wu, Y. K.; Cheng, N. C.; Cheng, C. M. Biofilms in Chronic Wounds: Pathogenesis and Diagnosis. Trends Biotechnol. 2019, 37, 505– 517, DOI: 10.1016/j.tibtech.2018.10.011Google Scholar4Biofilms in Chronic Wounds: Pathogenesis and DiagnosisWu, Yuan-Kun; Cheng, Nai-Chen; Cheng, Chao-MinTrends in Biotechnology (2019), 37 (5), 505-517CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)Chronic non-healing wounds have become a major worldwide healthcare burden. The impact of biofilms on chronic wound infection is well established. Despite increasing understanding of the underlying mechanism of biofilm formation in chronic wounds, current strategies for biofilm diagnosis in chronic wounds are still far from ideal. In this review, we briefly summarize the mechanism of biofilm formation and focus on current diagnostic approaches of chronic wound biofilms based on morphol., microbiol., and mol. assays. Innovative biotechnol. approaches, such as wound blotting and transcriptomic anal., may further shed light on this unmet clin. need. The continuous development of these sophisticated diagnostic approaches can markedly contribute to the future implementation of point-of-care biofilm detection in chronic wound care.
- 5Jannesari, M.; Varshosaz, J.; Morshed, M.; Zamani, M. Composite Poly(Vinyl Alcohol)/Poly(Vinyl Acetate) Electrospun Nanofibrous Mats as a Novel Wound Dressing Matrix for Controlled Release of Drugs. Int. J. Nanomed. 2011, 6, 993– 1003, DOI: 10.2147/IJN.S17595Google Scholar5Composite poly(vinyl alcohol)/poly(vinyl acetate) electrospun nanofibrous mats as a novel wound dressing matrix for controlled release of drugsJannesari, Marziyeh; Varshosaz, Jaleh; Morshed, Mohammad; Zamani, MaedehInternational Journal of Nanomedicine (2011), 6 (), 993-1003CODEN: IJNNHQ; ISSN:1178-2013. (Dove Medical Press Ltd.)The aim of this study was to develop novel biomedicated nanofiber electrospun mats for controlled drug release, esp. drug release directly to an injury site to accelerate wound healing. Nanofibers of poly(vinyl alc.) (PVA), poly(vinyl acetate) (PVAc), and a 50:50 composite blend, loaded with ciprofloxacin HCl (CipHCl), were successfully prepd. by an electrospinning technique for the first time. The morphol. and av. diam. of the electrospun nanofibers were investigated by SEM. X-ray diffraction studies indicated an amorphous distribution of the drug inside the nanofiber blend. Introducing the drug into polymeric solns. significantly decreased soln. viscosities as well as nanofiber diam. In vitro drug release evaluations showed that both the kind of polymer and the amt. of drug loaded greatly affected the degree of swelling, wt. loss, and initial burst and rate of drug release. Blending PVA and PVAc exhibited a useful and convenient method for electrospinning in order to control the rate and period of drug release in wound healing applications. Also, the thickness of the blend nanofiber mats strongly influenced the initial release and rate of drug release.
- 6Thakur, R. A.; Florek, C. A.; Kohn, J.; Michniak, B. B. Electrospun Nanofibrous Polymeric Scaffold with Targeted Drug Release Profiles for Potential Application as Wound Dressing. Int. J. Pharm. 2008, 364, 87– 93, DOI: 10.1016/j.ijpharm.2008.07.033Google Scholar6Electrospun nanofibrous polymeric scaffold with targeted drug release profiles for potential application as wound dressingThakur, R. A.; Florek, C. A.; Kohn, J.; Michniak, B. B.International Journal of Pharmaceutics (2008), 364 (1), 87-93CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)The authors have successfully fabricated a dual drug release electrospun scaffold contg. an anesthetic, lidocaine, and an antibiotic, mupirocin. Two drugs with different lipophilicities were electrospun from a poly-L-lactic acid (PLLA) soln. with a dual spinneret electrospinning app. into a single scaffold. The release of the drugs from the scaffold showed different profiles for the 2 drugs. Lidocaine hydrochloride exhibited an initial burst release (80% release within an hour) followed by a plateau after the first few hours. Mupirocin exhibited only a 5% release in the first hour before experiencing a more sustained release to provide antibacterial action for over 72 h. For comparative purposes, both drugs were spun from a single spinneret and evaluated to det. their release profiles. The scaffold maintained its antibiotic activity throughout the processes of electrospinning and gas sterilization and supported cell viability. It was reported in the literature that interactions between polymer and drug are known to govern the pattern of drug release from electrospun scaffolds. Here, it was found that the presence of the 2 drugs in the same polymer matrix altered the release kinetics of at least one drug. Based on the release profiles obtained, the dual spinneret technique was the preferred method of scaffold fabrication over the single spinneret technique to obtain a prototype wound healing device.
- 7Shirazaki, P.; Varshosaz, J.; Kharazi, A. Electrospun Gelatin/Poly(Glycerol Sebacate) Membrane with Controlled Release of Antibiotics for Wound Dressing. Adv. Biomed. Res. 2017, 6, 105, DOI: 10.4103/abr.abr_197_16Google Scholar7Electrospun gelatin/poly(glycerol sebacate) membrane with controlled release of antibiotics for wound dressingShirazaki, Parisa; Varshosaz, Jaleh; Kharazi, Anoushe ZargarAdvanced Biomedical Research (2017), 6 (Aug.), 105/1-105/11CODEN: ABRDD4; ISSN:2277-9175. (Medknow Publications)Background: The most important risk that threatens the skin wounds is infections. Therefore, fabrication of a membrane as a wound dressing with the ability of antibiotic delivery in a proper delivery rate is esp. important. Materials and Methods: Poly(glycerol sebacate) (PGS) was prepd. from sebacic acid and glycerol with 1:1 ratio; then, it was added to gelatin in the 1:3 ratio and was dissolved in 80% (vol./vol.) acetic acid, and fi nally, ciprofl oxacin was added in 10% (w/v) of polymer soln. The gelatin/PGS membrane was fabricated using an electrospinning method. The membrane was cross-linked using ethyl-3-(3-dimethylaminopropyl) carbodiimide ethyl-3-(3- dimethylaminopropyl)carbodiim (EDC) and N-hydroxysuccinimide (NHS) in different time periods to achieve a proper drug release rate. Fourier-transform IR (FTIR) spectroscopy was being used to manifest the peaks of polymers and drug in the membrane. SEM (SEM) was used to evaluate the morphol., fi bers diam., pore size, and porosity before and after crosslinking process. UV-visible spectrophotometry was used to show the ciprofl oxacin release from the cross-linked membrane. Results: FTIR anal. showed the characteristic peaks of gelatin, PGS, and ciprofl oxacin without any added peaks after the crosslinking process. SEM images revealed that nanofi bers' size increased during the crosslinking process and porosity was higher than 80% before and after crosslinking process. UV-visible spectrophotometry showed the proper rate of ciprofl oxacin release occurred from cross-linked membrane that remaining in EDC/NHS ethanol soln. for 120 min. Conclusion: The obtained results suggest that this recently developed gelatin/PGS membrane with controlled release of ciprofl oxacin could be a promising biodegradable membrane for wound dressing.
- 8Diller, R. B.; Tabor, A. J. The Role of the Extracellular Matrix (ECM) in Wound Healing: A Review. Biomimetics 2022, 7, 87, DOI: 10.3390/biomimetics7030087Google Scholar8The Role of the Extracellular Matrix (ECM) in Wound Healing: A ReviewDiller, Robert B.; Tabor, Aaron J.Biomimetics (2022), 7 (3), 87CODEN: BIOMJE; ISSN:2313-7673. (MDPI AG)A review. The extracellular matrix (ECM) is a 3-dimensional structure and an essential component in all human tissues. It is comprised of varying proteins, including collagens, elastin, and smaller quantities of structural proteins. Studies have demonstrated the ECM aids in cellular adherence, tissue anchoring, cellular signaling, and recruitment of cells. During times of integumentary injury or damage, either acute or chronic, the ECM is damaged. Through a series of overlapping events called the wound healing phases-hemostasis, inflammation, proliferation, and remodeling-the ECM is synthesized and ideally returned to its native state. This article synthesizes current and historical literature to demonstrate the involvement of the ECM in the varying phases of the wound healing cascade.
- 9Omer, S.; Forgách, L.; Zelkó, R.; Sebe, I. Scale-up of Electrospinning: Market Overview of Products and Devices for Pharmaceutical and Biomedical Purposes. Pharmaceutics 2021, 13, 1– 21, DOI: 10.3390/pharmaceutics13020286Google ScholarThere is no corresponding record for this reference.
- 10MacEwan, M. R.; MacEwan, S.; Kovacs, T. R.; Batts, J. What Makes the Optimal Wound Healing Material? A Review of Current Science and Introduction of a Synthetic Nanofabricated Wound Care Scaffold. Cureus 2017, 9, e1736 DOI: 10.7759/cureus.1736Google ScholarThere is no corresponding record for this reference.
- 11Wiegand, C.; Elsner, P.; Hipler, U.-C.; Abel, M.; Ruth, P. In Vitro Assessment of the Antimicrobial Activity of Wound Dressings: Influence of the Test Method Selected and Impact of the pH. J. Mater. Sci. Mater. Med. 2015, 26, 5343, DOI: 10.1007/s10856-014-5343-9Google Scholar11In vitro assessment of the antimicrobial activity of wound dressings: influence of the test method selected and impact of the pHWiegand Cornelia; Abel Martin; Ruth Peter; Elsner Peter; Hipler Uta-ChristinaJournal of materials science. Materials in medicine (2015), 26 (1), 5343 ISSN:.Antibacterial activity of dressings containing antimicrobials is mostly evaluated using in vitro tests. However, the various methods available differ significantly in their properties and results obtained are influenced by the method selected, micro-organisms used, and extraction method, the degree of solubility or the diffusability of the test-compounds. Here, results on antimicrobial activity of silver-containing dressings obtained by agar diffusion test (ADT), challenge tests (JIS L 1902, AATCC 100), and extraction-based methods (microplate laser nephelometry (MLN), luminescent quantification of bacterial ATP (LQbATP)) using Staphylococcus aureus and Pseudomonas aeruginosa were evaluated. Furthermore, the effect of the pH on antibacterial efficacy of these dressings was investigated. All silver-containing dressings exerted antimicrobial activity in all in vitro tests and results correlated considerably well. Differences were observed testing the agent-free basic materials. They did not exhibit any antimicrobial effects in the ADT, MLN or LQbATP, since these methods depend on diffusion/extraction of an active agent. However, they showed a strong antimicrobial effect in the challenge tests as they possess a high absorptive capacity, and are able to bind and sequester micro-organisms present. Therefore, it seems recommendable to choose several tests to distinguish whether a material conveys an active effect or a passive mechanism. In addition, it could be shown that release of silver and its antimicrobial efficacy is partially pH-dependent, and that dressings themselves affect the pH. It can further be speculated that dressings' effects on pH and release of silver ions act synergistically for antimicrobial efficacy.
- 12Preem Bock; Hinnu; Putrinš; Sagor; Tenson; Meos; Østergaard; Kogermann. Monitoring of Antimicrobial Drug Chloramphenicol Release from Electrospun Nano- and Microfiber Mats Using UV Imaging and Bacterial Bioreporters. Pharmaceutics 2019, 11, 487, DOI: 10.3390/pharmaceutics11090487Google Scholar12Monitoring of antimicrobial drug chloramphenicol release from electrospun nano- and microfiber mats using UV imaging and bacterial bioreportersPreem, Liis; Bock, Frederik; Hinnu, Mariliis; Putrins, Marta; Sagor, Kadi; Tenson, Tanel; Meos, Andres; Oestergaard, Jesper; Kogermann, KarinPharmaceutics (2019), 11 (9), 487CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)New strategies are continuously sought for the treatment of skin and wound infections due to increased problems with non-healing wounds. Electrospun nanofiber mats with antibacterial agents as drug delivery systems provide opportunities for the eradication of bacterial infections as well as wound healing. Antibacterial activities of such mats are directly linked with their drug release behavior. Traditional pharmacopoeial drug release testing settings are not always suitable for analyzing the release behavior of fiber mats intended for the local drug delivery. We tested and compared different drug release model systems for the previously characterized electrospun chloramphenicol (CAM)-loaded nanofiber (polycaprolactone (PCL)) and microfiber (PCL in combination with polyethylene oxide) mats with different drug release profiles. Drug release into buffer soln. and hydrogel was investigated and drug concn. was detd. using either high-performance liq. chromatog., UV-visible spectrophotometry, or UV imaging. The CAM release and its antibacterial effects in disk diffusion assay were assessed by bacterial bioreporters. All tested model systems enabled to study the drug release from electrospun mats. It was found that the release into buffer soln. showed larger differences in the drug release rate between differently designed mats compared to the hydrogel release tests. The UV imaging method provided an insight into the interactions with an agarose hydrogel mimicking wound tissue, thus giving us information about early drug release from the mat. Bacterial bioreporters showed clear correlations between the drug release into gel and antibacterial activity of the electrospun CAM-loaded mats.
- 13Zupančič, Š.; Preem, L.; Kristl, J.; Putrinš, M.; Tenson, T.; Kocbek, P.; Kogermann, K. Impact of PCL Nanofiber Mat Structural Properties on Hydrophilic Drug Release and Antibacterial Activity on Periodontal Pathogens. Eur. J. Pharm. Sci. 2018, 122, 347– 358, DOI: 10.1016/j.ejps.2018.07.024Google Scholar13Impact of PCL nanofiber mat structural properties on hydrophilic drug release and antibacterial activity on periodontal pathogensZupancic, Spela; Preem, Liis; Kristl, Julijana; Putrins, Marta; Tenson, Tanel; Kocbek, Petra; Kogermann, KarinEuropean Journal of Pharmaceutical Sciences (2018), 122 (), 347-358CODEN: EPSCED; ISSN:0928-0987. (Elsevier B.V.)Electrospinning enables to design and manuf. novel drug delivery systems capable of advancing the local antibacterial therapy. In this study, two hydrophilic drugs - metronidazole and ciprofloxacin hydrochloride - were loaded both individually and in combination into hydrophobic poly(ε-caprolactone) (PCL) matrix using electrospinning. We aimed to develop prolonged release drug delivery systems suitable for the treatment of periodontal diseases and understand how different rarely studied structural features, such as nanofiber mat thickness, surface area, wettability, together with intrinsic properties, like solid state and localization of incorporated drugs in nanofibers, affect the drug release. Furthermore, the safety of nanofiber mats was assessed in vitro on fibroblasts, and their antibacterial activity was tested on selected strains of periodontopathogenic bacteria. The results showed that the structural properties of nanofiber mat are crucial in particular drug-polymer combinations, affecting the drug release and consequently the antibacterial activity. The hydrophobicity of a PCL nanofiber mat and its thickness are the key characteristics in prolonged hydrophilic drug release, but only when wetting is the rate-limiting step for the drug release. Combination of drugs showed beneficial effects by inhibiting the growth of all tested pathogenic bacterial strains important in periodontal diseases.
- 14Lanno, G. M.; Ramos, C.; Preem, L.; Putrins, M.; Laidmaë, I.; Tenson, T.; Kogermann, K. Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing Applications. ACS Omega 2020, 5, 30011– 30022, DOI: 10.1021/acsomega.0c04402Google Scholar14Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing ApplicationsLanno, Georg-Marten; Ramos, Celia; Preem, Liis; Putrins, Marta; Laidmae, Ivo; Tenson, Tanel; Kogermann, KarinACS Omega (2020), 5 (46), 30011-30022CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Electrospun fiber scaffolds have a huge potential for the successful treatment of infected wounds based on their unique properties. Although several studies report novel drug-loaded electrospun fiber-based biomaterials, many of these do not provide information on their interactions with eukaryotic and bacterial cells. The main aim of this study was to develop antibacterial drug-loaded porous biocompatible polycaprolactone (PCL) fiber scaffolds mimicking the native extracellular matrix for wound healing purposes. Mech. property evaluation and different biorelevant tests were conducted in order to understand the structure-activity relationships and reveal how the surface porosity of fibers and the fiber diam. affect the scaffold interactions with the living bacterial and eukaryotic fibroblast cells. Cell migration and proliferation assays and antibiofilm assays enabled us to enlighten the biocompatibility and safety of fiber scaffolds and their suitability to be used as scaffolds for the treatment of infected wounds. Here, we report that porous PCL microfiber scaffolds obtained using electrospinning at high relative humidity served as the best surfaces for fibroblast attachment and growth compared to the nonporous microfiber or nonporous nanofiber PCL scaffolds. Porous chloramphenicol-loaded microfiber scaffolds were more elastic compared to nonporous scaffolds and had the highest antibiofilm activity. The results indicate that in addn. to the fiber diam. and fiber scaffold porosity, the single-fiber surface porosity and its effect on drug release, mech. properties, cell viability, and antibiofilm activity need to be understood when developing antibacterial biocompatible scaffolds for wound healing applications. We show that pores on single fibers within an electrospun scaffold, in addn. to nano- and microscale diam. of the fibers, change the living cell-fiber interactions affecting the antibiofilm efficacy and biocompatibility of the scaffolds for the local treatment of wounds.
- 15Bhattacharjee, A.; Khan, M.; Kleiman, M.; Hochbaum, A. I. Effects of Growth Surface Topography on Bacterial Signaling in Coculture Biofilms. ACS Appl. Mater. Interfaces 2017, 9, 18531– 18539, DOI: 10.1021/acsami.7b04223Google Scholar15Effects of Growth Surface Topography on Bacterial Signaling in Coculture BiofilmsBhattacharjee, Arunima; Khan, Mughees; Kleiman, Maya; Hochbaum, Allon I.ACS Applied Materials & Interfaces (2017), 9 (22), 18531-18539CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Bacteria form interface-assocd. communities, called biofilms, often comprising multiple species. Biofilms can be detrimental or beneficial in medical, industrial, and technol. settings, and their stability and function are detd. by interspecies communication via specific chem. signaling or metabolite exchange. The deterministic control of biofilm development, behavior, and properties remains an unmet challenge limiting the ability to inhibit formation of detrimental biofilms in biomedical settings and promote the growth of beneficial biofilms in biotechnol. applications. Here, the authors describe the development of growth surfaces that promote the growth of commensal Escherichia coli instead of the opportunistic pathogen Pseudomonas aeruginosa. Periodically patterned growth surfaces induced robust morphol. changes in surface-assocd. E. coli biofilms and influenced the antibiotic susceptibilities of E. coli and P. aeruginosa biofilms. Changes in the biofilm architecture resulted in the accumulation of a metabolite, indole, which controlled competition dynamics between the two species. The results show that the surface on which a biofilm grows has important implications for species colonization, growth, and persistence when exposed to antibiotics.
- 16Peeters, E.; Nelis, H. J.; Coenye, T. Comparison of Multiple Methods for Quantification of Microbial Biofilms Grown in Microtiter Plates. J. Microbiol. Methods 2008, 72, 157– 165, DOI: 10.1016/j.mimet.2007.11.010Google Scholar16Comparison of multiple methods for quantification of microbial biofilms grown in mictotiter platesPeeters, Elke; Nelis, Hans J.; Coenye, TomJournal of Microbiological Methods (2008), 72 (2), 157-165CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier B.V.)In the present study six assays for the quantification of biofilms formed in 96-well microtiter plates were optimized and evaluated: the crystal violet (CV) assay, the Syto9 assay, the fluorescein diacetate (FDA) assay, the resazurin assay, the XTT assay and the di-Me methylene blue (DMMB) assay. Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, Propionibacterium acnes and Candida albicans were used as test organisms. In general, these assays showed a broad applicability and a high repeatability for most isolates. In addn., the estd. nos. of CFUs present in the biofilms show limited variations between the different assays. Nevertheless, our data show that some assays are less suitable for the quantification of biofilms of particular isolates (e.g. the CV assay for P. aeruginosa).
- 17Azeredo, J.; Azevedo, N. F.; Briandet, R.; Cerca, N.; Coenye, T.; Costa, A. R.; Desvaux, M.; di Bonaventura, G.; Hébraud, M.; Jaglic, Z.; Kačániová, M.; Knøchel, S.; Lourenço, A.; Mergulhão, F.; Meyer, R. L.; Nychas, G.; Simões, M.; Tresse, O.; Sternberg, C. Critical Review on Biofilm Methods. Crit. Rev. Microbiol. 2017, 43, 313– 351, DOI: 10.1080/1040841X.2016.1208146Google Scholar17Critical review on biofilm methodsAzeredo, Joana; Azevedo, Nuno F.; Briandet, Romain; Cerca, Nuno; Coenye, Tom; Costa, Ana Rita; Desvaux, Mickael; Di Bonaventura, Giovanni; Hebraud, Michel; Jaglic, Zoran; Kacaniova, Miroslava; Knoechel, Susanne; Lourenco, Analia; Mergulhao, Filipe; Meyer, Rikke Louise; Nychas, George; Simoes, Manuel; Tresse, Odile; Sternberg, ClausCritical Reviews in Microbiology (2017), 43 (3), 313-351CODEN: CRVMAC; ISSN:1040-841X. (Taylor & Francis Ltd.)Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a neg. impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiol., structure and compn. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, compn. and physiol. are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a crit. perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.
- 18Demir, D.; Özdemir, S.; Ceylan, S.; Yalcin, M. S.; Sakım, B.; Bölgen, N. Electrospun Composite Nanofibers Based on Poly (ε-Caprolactone) and Styrax Liquidus (Liquidambar Orientalis Miller) as a Wound Dressing: Preparation, Characterization, Biological and Cytocompatibility Results. J. Polym. Environ. 2022, 30, 2462– 2473, DOI: 10.1007/s10924-022-02376-7Google Scholar18Electrospun Composite Nanofibers Based on Poly (ε-Caprolactone) and Styrax Liquidus (Liquidambar orientalis Miller) as a Wound Dressing: Preparation, Characterization, Biological and Cytocompatibility ResultsDemir, Didem; Ozdemir, Sadin; Ceylan, Seda; Yalcin, M. Serkan; Sakim, Burcu; Bolgen, NimetJournal of Polymers and the Environment (2022), 30 (6), 2462-2473CODEN: JPENFW; ISSN:1572-8919. (Springer)In this study, styrax liquidus (sweet gum balsam) extd. from Liquidambar orientalis Mill. incorporated PCL fibrous scaffolds were prepd. using the electrospinning method. The effects of the styrax liquidus content on the prepd. scaffolds were investigated using different physico-chem. and morphol. analyses. Then, the styrax-loaded nanofibers were examd. for their antioxidant activity, anti-biofilm, metal chelating, antimicrobial and DNA cleavage properties. The results obtained from these studies showed that the nanofibers exhibited effective biol. activity depending on the wt. ratio of the styrax liquidus. In light of the data obtained from the characterization and biol. studies, a sample with high ratio of balsam was built for detg. the cytocompatibility anal. in vitro. The cytotoxicity studies of the selected membrane were conducted using mouse embryonic fibroblast cells. The fibrous scaffolds lead to increase the cell no. as a result of high viability. According to the results, we propose a novel biocompatible electrospun hybrid scaffold with antioxidant and antimicrobial properties that can be used as wound healing material for potential tissue engineering applications.
- 19Serbezeanu, D.; Vlad-Bubulac, T.; Rusu, D.; Pircalabioru, G. G.; Samoilă, I.; Dinescu, S.; Aflori, M. Functional Polyimide-Based Electrospun Fibers for Biomedical Application. Materials 2019, 12, 3201, DOI: 10.3390/ma12193201Google Scholar19Functional polyimide-based electrospun fibers for biomedical applicationSerbezeanu, Diana; Vlad-Bubulac, Tachita; Rusu, Daniela; Pircalabioru, Gratiela Gradisteanu; Samoila, Iuliana; Dinescu, Sorina; Aflori, MagdalenaMaterials (2019), 12 (19), 3201CODEN: MATEG9; ISSN:1996-1944. (MDPI AG)The current study focuses on the application of cytotoxicity tests upon one membrane matrix based on electrospun polyimide fibers, appealing for biomedical application, such as scaffolds for cell growth, patches or meshes for wound healing, etc. Assays were performed in order to det. the viability and proliferation of L929 murine fibroblasts after they were kept in direct contact with the studied electrospun polyimide fibers. Increased cell viability and proliferation were detected for cells seeded on electrospun polyimide fibers membrane, in comparison with the control system, either after two or six days of evaluation. The no. of live cells was higher on the studied material compared to the control, after two and six days of cell seeding. The tendency of the cells to proliferate on the electrospun polyimide fibers was revealed by confocal microscopy. The morphol. stability of electrospun polyimide membrane was evaluated by SEM observation, after immersion of the samples in phosphate buffer saline soln. (PBS, 7.4 at 37 °C) at various time intervals. Addnl., the easy prodn. of electrospun polyimide fibers can facilitate the development of these types of matrixes into specific biomedical applications in the future.
- 20Gupta, S.; Prasad, P.; Roy, A.; Alam, M. M.; Ahmed, I.; Bit, A. Metallic Ion-Based Graphene Oxide Functionalized Silk Fibroin-Based Dressing Promotes Wound Healing via Improved Bactericidal Outcomes and Faster Re-Epithelization. Biomed. Mater. 2022, 17, 035010 DOI: 10.1088/1748-605X/ac64ddGoogle ScholarThere is no corresponding record for this reference.
- 21Parsons, D.; Meredith, K.; Rowlands, V. J.; Short, D.; Metcalf, D. G.; Bowler, P. G. Enhanced Performance and Mode of Action of a Novel Antibiofilm Hydrofiber Wound Dressing. Biomed. Res. Int. 2016, 2016, 7676471 DOI: 10.1155/2016/7616471Google ScholarThere is no corresponding record for this reference.
- 22Hill, K. E.; Malic, S.; McKee, R.; Rennison, T.; Harding, K. G.; Williams, D. W.; Thomas, D. W. An in Vitro Model of Chronic Wound Biofilms to Test Wound Dressings and Assess Antimicrobial Susceptibilities. J. Antimicrob. Chemother. 2010, 65, 1195– 1206, DOI: 10.1093/jac/dkq105Google Scholar22An in vitro model of chronic wound biofilms to test wound dressings and assess antimicrobial susceptibilitiesHill, Katja E.; Malic, Sladjana; McKee, Ruth; Rennison, Tracy; Harding, Keith G.; Williams, David W.; Thomas, David W.Journal of Antimicrobial Chemotherapy (2010), 65 (6), 1195-1206CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)The targeted disruption of biofilms in chronic wounds is an important treatment strategy and the subject of intense research. In the present study, an in vitro model of chronic wound biofilms was developed to assess the efficacy of antimicrobial treatments for use in the wound environment. Using chronic wound isolates, assays of bacterial coaggregation established that aerobic and anaerobic wound bacteria were able to coaggregate and form biofilms. A const. depth film fermenter (CDFF) was used to develop wound biofilms in vitro, which were analyzed using light microscopy and SEM. The susceptibility of bacteria within these biofilms was examd. in response to the most frequently prescribed chronic wound' antibiotics and a series of iodine- and silver-contg. com. antimicrobial products and lactoferrin. Defined biofilms were rapidly established within 1-2 days. Antibiotic treatment demonstrated that mixed Pseudomonas and Staphylococcus biofilms were not affected by ciprofloxacin (5 mg/L) or flucloxacillin (15 mg/L), even at concns. equiv. to twice the obsd. peak serum levels. The results contrasted with the ability of povidone-iodine (1%) to disrupt the wound biofilm; an effect that was particularly pronounced in the dressing testing where iodine-based dressings completely disrupted established 7 day biofilms. In contrast, only two of six silver-contg. dressings exhibited any effect on 3 day biofilms, with no effect on 7 day biofilms. Thus, this wound model emphasizes the potential role of the biofilm phenotype in the obsd. resistance to antibiotic therapies that may occur in chronic wounds in vivo.
- 23Dai, T.; Kharkwal, G. B.; Tanaka, M.; Huang, Y. Y.; Bil de Arce, V. J.; Hamblin, M. R. Animal Models of External Traumatic Wound Infections. Virulence 2011, 2, 296– 315, DOI: 10.4161/viru.2.4.16840Google Scholar23Animal models of external traumatic wound infectionsDai Tianhong; Kharkwal Gitika B; Tanaka Masamitsu; Huang Ying-Ying; Bil de Arce Vida J; Hamblin Michael RVirulence (2011), 2 (4), 296-315 ISSN:.BACKGROUND: Despite advances in traumatic wound care and management, infections remain a leading cause of mortality,morbidity and economic disruption in millions of wound patients around the world. Animal models have become standard tools for studying a wide array of external traumatic wound infections and testing new antimicrobial strategies. RESULTS: Animal models of external traumatic wound infections reported by different investigators vary in animal species used, microorganism strains, the number of microorganisms applied, the size of the wounds and for burn infections, the length of time the heated object or liquid is in contact with the skin. METHODS: This review covers experimental infections in animal models of surgical wounds, skin abrasions, burns, lacerations,excisional wounds and open fractures. CONCLUSIONS: As antibiotic resistance continues to increase,more new antimicrobial approaches are urgently needed.These should be tested using standard protocols for infections in external traumatic wounds in animal models.
- 24Seth, A. K.; Geringer, M. R.; Gurjala, A. N.; Abercrombie, J. A.; Chen, P.; You, T.; Hong, S. J.; Galiano, R. D.; Mustoe, T. A.; Leung, K. P. Understanding the Host Inflammatory Response to Wound Infection: An in Vivo Study of Klebsiella Pneumoniae in a Rabbit Ear Wound Model. Wound Repair Regen. 2012, 20, 214– 225, DOI: 10.1111/j.1524-475X.2012.00764.xGoogle Scholar24Understanding the host inflammatory response to wound infection: an in vivo study of Klebsiella pneumoniae in a rabbit ear wound modelSeth Akhil K; Geringer Matthew R; Gurjala Anandev N; Abercrombie Johnathan A; Chen Ping; You Tao; Hong Seok J; Galiano Robert D; Mustoe Thomas A; Leung Kai PWound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society (2012), 20 (2), 214-25 ISSN:.Wound infection development is critically dependent on the complex interactions between bacteria and host. Klebsiella pneumoniae has become an increasingly common wound pathogen, but its natural history within wounds has never been studied. Using a validated, in vivo rabbit ear model, wounds were inoculated with K. pneumoniae at different concentrations (102-107 colony-forming units) with measurement of viable and nonviable bacterial counts, histological wound-healing parameters, and host inflammatory gene expression at multiple time points postinoculation (48, 96, and 240 hours). Bacteria and wound morphologies were evaluated with scanning electron microscopy. Comparable experiments were performed in ischemic ears to model immune response impairment. All wounds, despite different inoculants, equilibrated to similar bacterial concentrations by 96 hours. With a 106 colony-forming units inoculant, wounds at 240 hours showed decreased bacterial counts (p < 0.01), with a corresponding improvement in healing (p < 0.01) and a decrease in inflammatory response (p < 0.05). In contrast, ischemic wounds revealed impaired inflammatory gene expression (p < 0.05) resulting in higher steady-state bacterial concentrations (p < 0.01), impaired healing (p < 0.05), and biofilm formation on scanning electron microscopy. We conclude that a normal inflammatory response can effectively stabilize and overcome a K. pneumoniae wound infection. An impaired host cannot control this bacterial burden, preventing adequate healing while allowing bacteria to establish a chronic presence. Our novel study quantitatively validates the host immune response as integral to wound infection dynamics.
- 25Mendes, J. J.; Leandro, C. I.; Bonaparte, D. P.; Pinto, A. L. A Rat Model of Diabetic Wound Infection for the Evaluation of Topical Antimicrobial Therapies. Comp. Med. 2012, 62, 37– 48Google Scholar25A rat model of diabetic wound infection for the evaluation of topical antimicrobial therapiesMendes, Joao J.; Leandro, Clara I.; Bonaparte, Dolores P.; Pinto, Andreia L.Comparative Medicine (2012), 62 (1), 37-48CODEN: COMEFT; ISSN:1532-0820. (American Association for Laboratory Animal Science)Diabetes mellitus is an epidemic multisystemic chronic disease that frequently is complicated by complex wound infections. Innovative topical antimicrobial therapy agents are potentially useful for multimodal treatment of these infections. However, an appropriately standardized in vivo model is currently not available to facilitate the screening of these emerging products and their effect on wound healing. To develop such a model, we analyzed, tested, and modified published models of wound healing. We optimized various aspects of the model, including animal species, diabetes induction method, hair removal technique, splint and dressing methods, the control of unintentional bacterial infection, sampling methods for the evaluation of bacterial burden, and aspects of the microscopic and macroscopic assessment of wound healing, all while taking into consideration animal welfare and the "3Rs" principle. We thus developed a new wound infection model in rats that is optimized for testing topical antimicrobial therapy agents. This model accurately reproduces the pathophysiol. of infected diabetic wound healing and includes the current std. treatment (i.e., debridement). The numerous benefits of this model include the ready availability of necessary materials, simple techniques, high reproducibility, and practicality for expts. with large sample sizes. Furthermore, given its similarities to infected-wound healing and treatment in humans, our new model can serve as a valid alternative for applied research.
- 26Geremias, T. C.; Batistella, M. A.; Magini, R. R. S.; Selene, S. M. A.; Franco, C.; Barbosa, L. C. A.; Pereira, U. A.; Hinestroza, J. P.; Pimenta, A. L.; Ulson de Souza, A. A. Functionalization of Poly(Lactic-Co-Glycolic Acid) Nanofibrous Membranes with Antibiofilm Compounds. Can. J. Chem. Eng. 2022, 100, S5– S15, DOI: 10.1002/cjce.24115Google Scholar26Functionalization of poly(lactic-co-glycolic acid) nanofibrous membranes with antibiofilm compoundsGeremias, Thaise C.; Batistella, Marcos A.; Magini, Ricardo R. S.; Guelli U. de Souza, Selene M. A.; Franco, Cesar V.; Barbosa, Luiz C. A.; Pereira, Ulisses A.; Hinestroza, Juan P.; Pimenta, Andrea L.; Ulson de Souza, Antonio A.Canadian Journal of Chemical Engineering (2022), 100 (S1), S5-S15CODEN: CJCEA7; ISSN:0008-4034. (John Wiley & Sons, Inc.)The aim of this research was to investigate the activity of functionalized PLGA electrospun membranes in preventing Streptococcus mutans biofilm formation. PLGA nanofibres were functionalized with the additives Melaleuca alternifolia and Coffea canephora essential oils, furan-2(5H)-one, and a novel synthetic butyrolactam, in three concns. (0.002%, 0.004%, and 0.008% w/v). Samples were characterized by SEM, FTIR-ATR, and GC-MS and exposed to S. mutans cultures. Planktonic growth was detd. following a 24 and 48-h incubation period by spectrophotometry and the biofilm formation was evaluated by counting colony forming units. Cytotoxicity of the new biomaterials was assessed by MTS assay, through the quantification of viable placenta-derived stem cells grown over the functionalized nanofibrous membranes. Observation of the electrospun membranes on SEM images revealed the smooth and bead-free morphol. of the nanofibres. No solvent residues were obsd. by FTIR-ATR, but the GC-MS results showed that N,N-dimethylformamide and dimethylsulfoxide were present as residues in the membranes after functionalization. Functionalization reduced bacterial attachment to membrane surfaces, with best results being obtained for M. alternifolia essential oil, furanone, and butyrolactam. Cytotoxicity results showed that furan-2(5H)-one-functionalized membranes demonstrated no statistically significant difference in the cell viability compared to the control membranes.
- 27Brackman, G.; Cos, P.; Maes, L.; Nelis, H. J.; Coenye, T. Quorum Sensing Inhibitors Increase the Susceptibility of Bacterial Biofilms to Antibiotics in Vitro and in Vivo. Antimicrob. Agents Chemother. 2011, 55, 2655– 2661, DOI: 10.1128/AAC.00045-11Google Scholar27Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivoBrackman, Gilles; Cos, Paul; Maes, Louis; Nelis, Hans J.; Coenye, TomAntimicrobial Agents and Chemotherapy (2011), 55 (6), 2655-2661CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)Although the exact role of quorum sensing (QS) in various stages of biofilm formation, maturation, and dispersal and in biofilm resistance is not entirely clear, the use of QS inhibitors (QSI) was proposed as a potential antibiofilm strategy. We have investigated whether QSI enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents. The QSI used in our study target the acyl-homoserine lactone-based QS system present in Pseudomonas aeruginosa and Burkholderia cepacia complex organisms (baicalin hydrate, cinnamaldehyde) or the peptide-based system present in Staphylococcus aureus (hamamelitannin). The effect of tobramycin (P. aeruginosa, B. cepacia complex) and clindamycin or vancomycin (S. aureus), alone or in combination with QSI, was evaluated in various in vitro and in vivo biofilm model systems, including two invertebrate models and one mouse pulmonary infection model. In vitro the combined use of an antibiotic and a QSI generally resulted in increased killing compared to killing by an antibiotic alone, although redns. were strain and model dependent. A significantly higher fraction of infected Galleria mellonella larvae and Caenorhabditis elegans survived infection following combined treatment, compared to treatment with an antibiotic alone. Finally, the combined use of tobramycin and baicalin hydrate reduced the microbial load in the lungs of BALB/c mice infected with Burkholderia cenocepacia more than tobramycin treatment alone. Our data suggest that QSI may increase the success of antibiotic treatment by increasing the susceptibility of bacterial biofilms and/or by increasing host survival following infection.
- 28Werthén, M.; Henriksson, L.; Jensen, P. Ø.; Sternberg, C.; Givskov, M.; Bjarnsholt, T. An in Vitro Model of Bacterial Infections in Wounds and Other Soft Tissues. Apmis 2010, 118, 156– 164, DOI: 10.1111/j.1600-0463.2009.02580.xGoogle Scholar28An in vitro model of bacterial infections in wounds and other soft tissuesWerthen Maria; Henriksson Lina; Jensen Peter Ostrup; Sternberg Claus; Givskov Michael; Bjarnsholt ThomasAPMIS : acta pathologica, microbiologica, et immunologica Scandinavica (2010), 118 (2), 156-64 ISSN:.There is growing evidence that bacteria play a crucial role in the persistence of chronic wounds. These bacteria are most probably present in polymer-embedded aggregates that represent the biofilm mode of growth. Much work has been carried out to study the development of biofilms in vitro, in particular in attachment to solid surfaces. The observations from the chronic wounds indicate that the bacteria are not attached to a solid surface. Consequently, a new in vitro model is required to investigate biofilms in more wound-like settings. This study describes such a novel in vitro model, with bacteria growing as biofilm aggregates in a collagen gel matrix with serum protein mimicking the wound bed of chronic wounds. The model was verified to comprise important hallmarks of biofilms such as the bacterial embedment in a matrix and increased antibiotic tolerance. Furthermore, we have verified the relevance of the model by comparing the organization of the bacteria in the model with the organization of the bacteria in a real chronic wound. We believe that we have developed an important new model for investigating bacterial biofilms in chronic wounds. This model may be used to study biofilm development in chronic wounds and to develop novel diagnostic tools as well as treatment strategies.
- 29McMahon, R. E.; Salamone, A. B.; Poleon, S.; Bionda, N.; Salamone, J. C. Efficacy of Wound Cleansers on Wound-Specific Organisms Using in Vitro and Ex Vivo Biofilm Models. Wound Manag. Prev. 2020, 66, 31– 42, DOI: 10.25270/wmp.2020.11.3142Google Scholar29Efficacy of Wound Cleansers on Wound-Specific Organisms Using In Vitro and Ex Vivo Biofilm ModelsMcMahon Rebecca E; Salamone Ann Beal; Poleon Suprena; Salamone Joseph C; Bionda NinaWound management & prevention (2020), 66 (11), 31-42 ISSN:.Biofilms are believed to be a source of chronic inflammation in non-healing wounds. PURPOSE: In this study, the pre-clinical anti-biofilm efficacy of several wound cleansers was examined using the Calgary minimum biofilm eradication concentration (MBEC) and ex vivo porcine dermal explant (PDE) models on Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans biofilms. METHODS: A surfactant-based cleanser and antimicrobial-based cleansers containing ionic silver, hypochlorous acid (HOCl), sodium hypochlorite (NaOCl), and polyhexamethylene biguanide (PHMB) were tested on the MBEC model biofilms with a 10-minute application time. Select cleansers were then tested on the mature PDE biofilms with 10-minute applications followed by the application of cleanser-soaked gauze. The PDE model was further expanded to include single and daily applications of the cleansers to mimic daily and 72-hour dressing changes. RESULTS: In the MBEC model, PHMB- and HOCl-based cleansers reduced immature MRSA, C albicans, and P aeruginosa biofilm regrowth by > 3× when compared with silver, surfactant, and saline cleansers. The major differences could be elucidated in the PDE model in which, after daily application, 1 PHMB-based cleanser showed a statistically significant reduction (3-8 CFU/mL log reduction) in all mature biofilms tested, while a NaOCl-based cleanser showed significant reduction in 2 microorganisms (3-5 CFU/mL log reduction, P aeruginosa and MRSA).The other PHMB-based cleanser showed a statistically significant 3 log CFU/mL reduction in P aeruginosa. The remaining cleansers showed no statistically significant difference from the saline control. CONCLUSION: Results confirm that there are model-dependent differences in the outcomes of these studies, suggesting the importance of model selection for product screening. The results indicate that 1 PHMB-based cleanser was effective in reducing mature P aeruginosa, MRSA, and C albicans biofilms and that sustained antimicrobial presence was necessary to reduce or eliminate these mature biofilms.
- 30Yang, Q.; Phillips, P. L.; Sampson, E. M.; Progulske-Fox, A.; Jin, S.; Antonelli, P.; Schultz, G. S. Development of a Novel Ex Vivo Porcine Skin Explant Model for the Assessment of Mature Bacterial Biofilms. Wound Repair Regen. 2013, 21, 704– 714, DOI: 10.1111/wrr.12074Google Scholar30Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilmsYang Qingping; Phillips Priscilla L; Sampson Edith M; Progulske-Fox Ann; Jin Shouguang; Antonelli Patrick; Schultz Gregory SWound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society (2013), 21 (5), 704-14 ISSN:.Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms.
- 31Steinstraesser, L.; Sorkin, M.; Niederbichler, A. D.; Becerikli, M.; Stupka, J.; Daigeler, A.; Kesting, M. R.; Stricker, I.; Jacobsen, F.; Schulte, M. A Novel Human Skin Chamber Model to Study Wound Infection Ex Vivo. Arch. Dermatol. Res. 2010, 302, 357– 365, DOI: 10.1007/s00403-009-1009-8Google Scholar31A novel human skin chamber model to study wound infection ex vivoSteinstraesser Lars; Sorkin M; Niederbichler A D; Becerikli M; Stupka J; Daigeler A; Kesting M R; Stricker I; Jacobsen F; Schulte MArchives of dermatological research (2010), 302 (5), 357-65 ISSN:.Wound infections with multi-drug resistant bacteria increase morbidity and mortality and have considerable socioeconomic impact. They can lead to impaired wound healing, resulting in rising treatment costs. The aim of this study was to investigate an ex vivo human wound infection model. Human full-thickness skin from the operating room (OR) was placed into the Bo-Drum and cultivated for 7 days in an air-liquid interphase. On day 8, the skin was inoculated with either (1) Pseudomonas aeruginosa, (2) Staphylococcus aureus (10(5) CFU, n = 3) or (3) carrier control. 1, 3 and 7 days after inoculation colony forming units in the tissue/media were determined and cytokine expression was quantified. A reliable and reproducible wound infection could be established for 7 days. At this time point, 1.8 x 10(8) CFU/g tissue of P. aeruginosa and 2 x 10(7) CFU/g tissue of S. aureus were detected. Immunohistochemical analysis demonstrated bacterial infection and epidermolysis in infected skin. RT-PCR analysis exhibited a significant induction of proinflammatory cytokines after infection. The BO-drum is a robust, easy-to-use, sterilizable and reusable ex vivo full-skin culture system. For investigation of wound infection, treatment and healing, the BO-drum presents a convenient model and may help to standardize wound research.
- 32Wilkinson, H. N.; McBain, A. J.; Stephenson, C.; Hardman, M. J. Comparing the Effectiveness of Polymer Debriding Devices Using a Porcine Wound Biofilm Model. Adv. Wound Care 2016, 5, 475– 485, DOI: 10.1089/wound.2015.0683Google Scholar32Comparing the Effectiveness of Polymer Debriding Devices Using a Porcine Wound Biofilm ModelWilkinson Holly N; Hardman Matthew J; McBain Andrew J; Stephenson ChristianAdvances in wound care (2016), 5 (11), 475-485 ISSN:2162-1918.Objective: Debridement to remove necrotic and/or infected tissue and promote active healing remains a cornerstone of contemporary chronic wound management. While there has been a recent shift toward less invasive polymer-based debriding devices, their efficacy requires rigorous evaluation. Approach: This study was designed to directly compare monofilament debriding devices to traditional gauze using a wounded porcine skin biofilm model with standardized application parameters. Biofilm removal was determined using a surface viability assay, bacterial counts, histological assessment, and scanning electron microscopy (SEM). Results: Quantitative analysis revealed that monofilament debriding devices outperformed the standard gauze, resulting in up to 100-fold greater reduction in bacterial counts. Interestingly, histological and morphological analyses suggested that debridement not only removed bacteria, but also differentially disrupted the bacterially-derived extracellular polymeric substance. Finally, SEM of post-debridement monofilaments showed structural changes in attached bacteria, implying a negative impact on viability. Innovation: This is the first study to combine controlled and defined debridement application with a biologically relevant ex vivo biofilm model to directly compare monofilament debriding devices. Conclusion: These data support the use of monofilament debriding devices for the removal of established wound biofilms and suggest variable efficacy towards biofilms composed of different species of bacteria.
- 33Andersson, M.; Madsen, L. B.; Schmidtchen, A.; Puthia, M. Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations. Int. J. Mol. Sci. 2021, 22, 1– 16, DOI: 10.3390/ijms22095045Google ScholarThere is no corresponding record for this reference.
- 34Wilkinson, H. N.; Iveson, S.; Catherall, P.; Hardman, M. J. A Novel Silver Bioactive Glass Elicits Antimicrobial Efficacy against Pseudomonas aeruginosa and Staphylococcus aureus in an Ex Vivo Skin Wound Biofilm Model. Front. Microbiol. 2018, 9, 1– 16, DOI: 10.3389/fmicb.2018.01450Google ScholarThere is no corresponding record for this reference.
- 35Siimon, K.; Reemann, P.; Põder, A.; Pook, M.; Kangur, T.; Kingo, K.; Jaks, V.; Mäeorg, U.; Järvekülg, M. Effect of Glucose Content on Thermally Cross-Linked Fibrous Gelatin Scaffolds for Tissue Engineering. Mater. Sci. Eng., C 2014, 42, 538– 545, DOI: 10.1016/j.msec.2014.05.075Google Scholar35Effect of glucose content on thermally cross-linked fibrous gelatin scaffolds for tissue engineeringSiimon, Kaido; Reemann, Paula; Poder, Annika; Pook, Martin; Kangur, Triin; Kingo, Kulli; Jaks, Viljar; Maeorg, Uno; Jarvekulg, MartinMaterials Science & Engineering, C: Materials for Biological Applications (2014), 42 (), 538-545CODEN: MSCEEE; ISSN:0928-4931. (Elsevier B.V.)Thermally cross-linked glucose-contg. electrospun gelatin meshes were studied as possible cell substrate materials. FTIR anal. was used to study the effect of glucose on crosslinking reactions. It was found that the presence of glucose increases the extent of crosslinking of fibrous gelatin scaffolds, which in return dets. scaffold properties and their usability in tissue engineering applications. Easy to handle fabric-like scaffolds were obtained from blends contg. up to 15% glucose. Maximum extent of crosslinking was reached at nearly 20% glucose content. Crosslinking effectively resulted in decreased soly. and increased resistance to enzymic degrdn. Preliminary short-term cell culture expts. indicate that such thermally cross-linked gelatin-glucose scaffolds are suitable for tissue engineering applications.
- 36Siimon, K.; Siimon, H.; Järvekülg, M. Mechanical Characterization of Electrospun Gelatin Scaffolds Cross-Linked by Glucose. J. Mater. Sci. Mater. Med. 2015, 26, 1– 9, DOI: 10.1007/s10856-014-5375-1Google Scholar36Mechanical characterization of electrospun gelatin scaffolds cross-linked by glucoseSiimon, Kaido; Siimon, Hele; Jarvekulg, MartinJournal of Materials Science: Materials in Medicine (2015), 26 (1), 1-9CODEN: JSMMEL; ISSN:0957-4530. (Springer)Nanofibrous gelatin scaffolds were prepd. by electrospinning from aq. acetic acid and cross-linked thermally by glucose. The effect of the amt. of glucose used as crosslinking agent on the mech. properties of gelatin fibers was studied in this paper. The elastic modulus of gelatin fibers cross-linked by glucose was detd. by modeling the behavior of the meshes during tensile test. The model draws connections between the elastic moduli of a fibrous mesh and the fiber material and allows evaluation of elastic modulus of the fiber material. It was found that crosslinking by glucose increases the elastic modulus of gelatin fibers from 0.3 GPa at 0 % glucose content to 1.1 GPa at 15 % glucose content. This makes fibrous gelatin scaffolds cross-linked by glucose a promising material for biomedical applications.
- 37Palo, M.; Kogermann, K.; Laidmäe, I.; Meos, A.; Preis, M.; Heinämäki, J.; Sandler, N. Development of Oromucosal Dosage Forms by Combining Electrospinning and Inkjet Printing. Mol. Pharmaceutics 2017, 14, 808– 820, DOI: 10.1021/acs.molpharmaceut.6b01054Google Scholar37Development of Oromucosal Dosage Forms by Combining Electrospinning and Inkjet PrintingPalo, Mirja; Kogermann, Karin; Laidmae, Ivo; Meos, Andres; Preis, Maren; Heinamaki, Jyrki; Sandler, NiklasMolecular Pharmaceutics (2017), 14 (3), 808-820CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)Printing technol. has been shown to enable flexible fabrication of solid dosage forms for personalized drug therapy. Several methods can be applied for tailoring the properties of the printed pharmaceuticals. In this study, the use of electrospun fibrous substrates in the fabrication of inkjet-printed dosage forms was investigated. A single-drug formulation with lidocaine hydrochloride (LH) and a combination drug system contg. LH and piroxicam (PRX) for oromucosal administration were prepd. The LH was deposited on the electrospun and cross-linked gelatin substrates by inkjet printing, whereas PRX was incorporated within the substrate fibers during electrospinning. The solid state anal. of the electrospun substrates showed that PRX was in an amorphous state within the fibers. Furthermore, the results indicated the entrapment and solidification of the dissolved LH within the fibrous gelatin matrix. The printed drug amt. (2-3 mg) was in good correlation with the theor. dose calcd. based on the printing parameters. However, a noticeable degrdn. of the printed LH was detected after a few months. An immediate release (over 85% drug release after 8 min) of both drugs from the printed dosage forms was obsd. In conclusion, the prepd. electrospun gelatin scaffolds were shown to be suitable substrates for inkjet printing of oromucosal formulations. The combination of electrospinning and inkjet printing allowed the prepn. of a dual drug system.
- 38Preem, L.; Mahmoudzadeh, M.; Putrinš, M.; Meos, A.; Laidmäe, I.; Romann, T.; Aruväli, J.; Härmas, R.; Koivuniemi, A.; Bunker, A.; Tenson, T.; Kogermann, K. Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-Implications for Designing Electrospun Drug Delivery Systems Countering Wound Infection. Mol. Pharmaceutics 2017, 14, 4417– 4430, DOI: 10.1021/acs.molpharmaceut.7b00524Google Scholar38Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-Implications for Designing Electrospun Drug Delivery Systems Countering Wound InfectionPreem, Liis; Mahmoudzadeh, Mohammad; Putrins, Marta; Meos, Andres; Laidmae, Ivo; Romann, Tavo; Aruvali, Jaan; Harmas, Riinu; Koivuniemi, Artturi; Bunker, Alex; Tenson, Tanel; Kogermann, KarinMolecular Pharmaceutics (2017), 14 (12), 4417-4430CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)Antibacterial drug-loaded electrospun nano- and microfibrous dressings are of major interest as novel topical drug delivery systems in wound care. In this study, chloramphenicol (CAM)-loaded polycaprolactone (PCL) and PCL/poly(ethylene oxide) (PEO) fiber mats were electrospun and characterized in terms of morphol., drug distribution, physicochem. properties, drug release, swelling, cytotoxicity, and antibacterial activity. Computational modeling together with physicochem. anal. helped to elucidate possible interactions between the drug and carrier polymers. Strong interactions between PCL and CAM together with hydrophobicity of the system resulted in much slower drug release compared to the hydrophilic ternary system of PCL/PEO/CAM. Cytotoxicity studies confirmed safety of the fiber mats to murine NIH 3T3 cells. Disk diffusion assay demonstrated that both fast and slow release fiber mats reached effective concns. and had similar antibacterial activity. A biofilm formation assay revealed that both blank matrixes are good substrates for the bacterial attachment and formation of biofilm. Importantly, prolonged release of CAM from drug-loaded fibers helps to avoid biofilm formation onto the dressing and hence avoids the treatment failure.
- 39Johnston, C.; Callum, J.; Mohr, J.; Duong, A.; Garibaldi, A.; Simunovic, N.; Ayeni, O. R.; on behalf of the Bioburden Steering Committee; Appleby, A.; Brubaker, S.; Callum, J.; Dowling, G.; Eastlund, T.; Fearon, M.; Germain, M.; Johnston, C.; Lotherington, K.; McTaggart, K.; Mohr, J.; Preiksaitis, J.; Strong, M.; Winters, M.; Young, K.; Zhao, J.; Callum, J.; Cartotto, R.; Davis, I.; Eastlund, T.; Gratzer, P.; Johnston, C.; Merkley, L.; Mohr, J. Disinfection of Human Skin Allografts in Tissue Banking: A Systematic Review Report. Cell Tissue Banking 2016, 17, 585– 592, DOI: 10.1007/s10561-016-9569-2Google Scholar39Disinfection of human skin allografts in tissue banking: a systematic review reportJohnston, C.; Callum, J.; Mohr, J.; Duong, A.; Garibaldi, A.; Simunovic, N.; Ayeni, O. R.; on behalf of the Bioburden Steering Committee and Skin Working groupCell and Tissue Banking (2016), 17 (4), 585-592CODEN: CTBAFV; ISSN:1389-9333. (Springer)The use of skin allografts to temporarily replace lost or damaged skin is practiced worldwide. Naturally occurring contamination can be present on skin or can be introduced at recovery or during processing. This contamination can pose a threat to allograft recipients. Bacterial culture and disinfection of allografts are mandated, but the specific practices and methodologies are not dictated by stds. A systematic review of literature from three databases found 12 research articles that evaluated bioburden redn. processes of skin grafts. The use of broad spectrum antibiotics and antifungal agents was the most frequently identified disinfection method reported demonstrating redns. in contamination rates. It was detd. that the greatest redn. in the skin allograft contamination rates utilized 0.1 % peracetic acid or 25 kGy of gamma irradn. at lower temps.
- 40Preem, L.; Vaarmets, E.; Meos, A.; Jõgi, I.; Putrinš, M.; Tenson, T.; Kogermann, K. Effects and Efficacy of Different Sterilization and Disinfection Methods on Electrospun Drug Delivery Systems. Int. J. Pharm. 2019, 567, 118450 DOI: 10.1016/j.ijpharm.2019.118450Google Scholar40Effects and efficacy of different sterilization and disinfection methods on electrospun drug delivery systemsPreem, Liis; Vaarmets, Ebe; Meos, Andres; Jogi, Indrek; Putrins, Marta; Tenson, Tanel; Kogermann, KarinInternational Journal of Pharmaceutics (Amsterdam, Netherlands) (2019), 567 (), 118450CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)Microbiol. quality of a pharmaceutical product is an essential requirement ensuring patient safety, thus effective sterilization/disinfection methods need to be found. The aim of this study was to evaluate the efficacy of different sterilization/disinfection methods on drug-loaded electrospun matrixes and the impact of these treatments on the functionality related characteristics of these matrixes. The sterilization efficacy of gamma-irradn., UV-irradn., in situ generated chlorine gas and low-pressure argon plasma treatment were evaluated on two different chloramphenicol-loaded electrospun matrixes using pristine polycaprolactone (PCL) as a carrier polymer or PCL in combination with polyethylene oxide. Drug stability, solid state properties, morphol., mech. properties, swelling, biodegrdn. and drug release kinetics were studied before and after the treatments. It was shown that all tested methods help to reduce bioburden and only plasma treated matrixes were not sterile. At the same time drug degrdn. after the treatment can be considerable and depends not only on the susceptibility of the drug to degrdn., but also on matrix properties (e.g. the nature of carrier polymers). Even though no morphol. changes were obsd., gamma sterilization increased the hardness and elasticity of PCL matrixes as a result of increased crystallinity of the polymer. Plasma treatment was able to significantly enhance water absorption to otherwise hydrophobic PCL/CAM matrix and had tremendous impact on its drug release kinetics as the drug was instantly released from otherwise prolonged release formulation.
- 41Koransky, J. R.; Allen, S. D.; Dowell, V. R. Use of Ethanol for Selective Isolation of Sporeforming Microorganisms. Appl. Environ. Microbiol. 1978, 35, 762– 765, DOI: 10.1128/aem.35.4.762-765.1978Google Scholar41Use of ethanol for selective isolation of sporeforming microorganismsKoransky, Jack R.; Allen, Stephen D.; Dowell, V. R., Jr.Applied and Environmental Microbiology (1978), 35 (4), 762-5CODEN: AEMIDF; ISSN:0099-2240.When mixed cultures contg. spore-forming bacteria were treated with heat or with EtOH [64-17-5], the latter consistently resulted in better recovery of Clostridium and Bacillus species. Both techniques were effective in eliminating vegetative cells. An EtOH concn. >25% and exposure for 45 min or longer were necessary to kill all vegetative cells in mixed-culture samples. EtOH treatment (50% EtOH for 1 h) was effective for isolating spore-forming bacteria from intestinal specimens. Seven different species of Clostridium were the only bacteria isolated from an EtOH-treated specimen of intestinal contents from the large bowel of a patient. Treatment with EtOH for 1 h is apparently an effective technique for selective isolation of spore-forming bacteria from mixed cultures and certain types of clin. specimens.
- 42Lipp, C.; Kirker, K.; Agostinho, A.; James, G.; Stewart, P. Testing Wound Dressings Using an in Vitro Wound Model. J. Wound Care 2010, 19, 220– 226, DOI: 10.12968/jowc.2010.19.6.48468Google Scholar42Testing wound dressings using an in vitro wound modelLipp C; Kirker K; Agostinho A; James G; Stewart PJournal of wound care (2010), 19 (6), 220-6 ISSN:0969-0700.OBJECTIVE: To determine whether or not there are any significant differences in the effects of wound dressings on bacterial bioburden. METHOD: A selection of non-occlusive, non-adhesive dressings was tested for their effect on bacterial bioburden. The dressings selected included two dressings with antimicrobial properties (one containing silver and one containing PHMB), a cotton-based dressing enclosed in a perforated sleeve of poly(ethylene terephthalate), a carboxymethyl cellulose-based dressing, a fibre-free alginate dressing, and a 12-ply 100% cotton gauze. Using the colony-drip flow reactor (DFR) model, a meticillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa biofilm was grown underneath a dressing sample. Biofilm growth was examined via plate counts, fluorescent microscopy and scanning electron microscopy. RESULTS: The dressings containing antimicrobial agents had the greatest effect on bacterial load. In the MRSA experiments, both antimicrobial dressings produced lower bacteria counts than the other dressings (p<0.001), while in the P. aeruginosa experiments, only the silver-containing sample had fewer bacteria (p<0.0001). However, neither antimicrobial dressing was able to completely eradicate the bacteria when testing with either microorganism. CONCLUSION: The results presented herein illustrate that bacteria can grow unchallenged within the dressing environment and that an antimicrobial dressing can limit this bacterial growth. DECLARATION OF INTEREST: None.
- 43Barbero, A. M.; Frasch, H. F. Pig and Guinea Pig Skin as Surrogates for Human in Vitro Penetration Studies: A Quantitative Review. Toxicol. In Vitro 2009, 23, 1– 13, DOI: 10.1016/j.tiv.2008.10.008Google Scholar43Pig and guinea pig skin as surrogates for human in vitro penetration studies: A quantitative reviewBarbero, Ana M.; Frasch, H. FrederickToxicology in Vitro (2009), 23 (1), 1-13CODEN: TIVIEQ; ISSN:0887-2333. (Elsevier Ltd.)A review. Both human and animal skin in vitro models are used to predict percutaneous penetration in humans. The objective of this review is a quant. comparison of permeability and lag time measurements between human and animal skin, including an evaluation of the intra- and interspecies variability. The authors limit the focus to domestic pig and rodent guinea pig skin as surrogates for human skin, and consider only studies in which both animal and human penetration of a given chem. were measured jointly in the same lab. When the in vitro permeability of pig and human skin were compared, the Pearson product moment correlation coeff. (r) was 0.88 (P < 0.0001), with an intraspecies av. coeff. of variation of skin permeability of 21% for pig and 35% for human, and an interspecies av. coeff. of variation of 37% for the set of studied compds. (n = 41). The lag times of pig skin and human skin did not correlate (r = 0.35, P = 0.26). When the in vitro permeability of guinea pig and human skin were compared, r = 0.96 (P < 0.0001), with an av. intraspecies coeff. of variation of 19% for guinea pig and 24% for human, and an interspecies coeff. of variation of permeability of 41% for the set of studied compds. (n = 15). Lag times of guinea pig and human skin correlated (r = 0.90, P < 0.0001, n = 12). When permeability data was not reported a factor of difference (FOD) of animal to human skin was calcd. for pig skin (n = 50) and guinea pig skin (n = 25). For pig skin, 80% of measurements fell within the range 0.3 < FOD < 3. For guinea pig skin, 65% fell within that range. Both pig and guinea pig are good models for human skin permeability and have less variability than the human skin model. The skin model of choice will depend on the final purpose of the study and the compd. under investigation.
- 44Simon, A.; Amaro, M. I.; Healy, A. M.; Cabral, L. M.; de Sousa, V. P. Comparative Evaluation of Rivastigmine Permeation from a Transdermal System in the Franz Cell Using Synthetic Membranes and Pig Ear Skin with in Vivo-in Vitro Correlation. Int. J. Pharm. 2016, 512, 234– 241, DOI: 10.1016/j.ijpharm.2016.08.052Google Scholar44Comparative evaluation of rivastigmine permeation from a transdermal system in the Franz cell using synthetic membranes and pig ear skin with in vivo-in vitro correlationSimon, Alice; Amaro, Maria Ines; Healy, Anne Marie; Cabral, Lucio Mendes; Pereira de Sousa, ValeriaInternational Journal of Pharmaceutics (Amsterdam, Netherlands) (2016), 512 (1), 234-241CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)In the present study, in vitro permeation expts. in a Franz diffusion cell were performed using different synthetic polymeric membranes and pig ear skin to evaluate a rivastigmine (RV) transdermal drug delivery system. In vitro-in vivo correlations (IVIVC) were examd. to det. the best model membrane. In vitro permeation studies across different synthetic membranes and skin were performed for the Exelon Patch (which contains RV), and the results were compared. Deconvolution of bioavailability data using the Wagner-Nelson method enabled the fraction of RV absorbed to be detd. and a point-to-point IVIVC to be established. The synthetic membrane, Strat-M, showed a RV permeation profile similar to that obtained with pig ear skin (R2 = 0.920). Studies with Strat-M resulted in a good and linear IVIVC (R2 = 0.991) when compared with other synthetic membranes that showed R2 values less than 0.90. The R2 for pig ear skin was 0.982. Strat-M membrane was the only synthetic membrane that adequately simulated skin barrier performance and therefore it can be considered to be a suitable alternative to human or animal skin in evaluating transdermal drug transport, potentially reducing the no. of studies requiring human or animal samples.
- 45Rediguieri, C. F.; Sassonia, R. C.; Dua, K.; Kikuchi, I. S.; de Jesus Andreoli Pinto, T. Impact of Sterilization Methods on Electrospun Scaffolds for Tissue Engineering. Eur. Polym. J. 2016, 82, 181– 195, DOI: 10.1016/j.eurpolymj.2016.07.016Google Scholar45Impact of sterilization methods on electrospun scaffolds for tissue engineeringRediguieri, Carolina Fracalossi; Sassonia, Rogerio Corte; Dua, Kamal; Kikuchi, Irene Satiko; Pinto, Terezinha de Jesus AndreoliEuropean Polymer Journal (2016), 82 (), 181-195CODEN: EUPJAG; ISSN:0014-3057. (Elsevier Ltd.)A review. Tissue engineering is a growing area within the regenerative medicine. The electrospun scaffolds are the most promising devices for translating engineered tissues into patients. However, in order to be used in clin. practice, one of the important fundamental aspects of the scaffold is to be sterile keeping the fact of patient safety in mind. Due to the various properties of electrospun fibers, such as high porosity and surface area, the effects of sterilization could have different outcomes than those obsd. in ordinary medical devices. Therefore, the present article provides an insight into the various sterilization methods that have been applied to electrospun scaffolds and their effects on scaffolds morphol., hydrophilicity, other physico-chem. and mech. properties and the performance of seeded cells after sterilization. In conclusion, the information provided in the review will help all scientists involved in this interdisciplinary field to understand and apply the knowledge in selection of appropriate sterilization method for the electrospun scaffolds.
- 46Myhrman, E.; Håkansson, J.; Lindgren, K.; Björn, C.; Sjöstrand, V.; Mahlapuu, M. The Novel Antimicrobial Peptide PXL150 in the Local Treatment of Skin and Soft Tissue Infections. Appl. Microbiol. Biotechnol. 2013, 97, 3085– 3096, DOI: 10.1007/s00253-012-4439-8Google Scholar46The novel antimicrobial peptide PXL150 in the local treatment of skin and soft tissue infectionsMyhrman, Emma; Hakansson, Joakim; Lindgren, Kerstin; Bjoern, Camilla; Sjoestrand, Veronika; Mahlapuu, MargitApplied Microbiology and Biotechnology (2013), 97 (7), 3085-3096CODEN: AMBIDG; ISSN:0175-7598. (Springer)Dramatic increase in bacterial resistance towards conventional antibiotics emphasizes the importance to identify novel, more potent antimicrobial therapies. Antimicrobial peptides (AMPs) have emerged as a promising new group to be evaluated in therapeutic intervention of infectious diseases. Here we describe a novel AMP, PXL150, which demonstrates in vitro a broad spectrum microbicidal action against both Gram-pos. and Gram-neg. bacteria, including resistant strains. The potent microbicidal activity and broad antibacterial spectrum of PXL150 were not assocd. with any hemolytic activity. Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance towards PXL150 during continued selection pressure. PXL150 caused a rapid depolarization of cytoplasmic membrane of S. aureus, and dissipating membrane potential is likely one mechanism for PXL150 to kill its target bacteria. Studies in human cell lines indicated that PXL150 has anti-inflammatory properties, which might be of addnl. benefit. PXL150 demonstrated pronounced anti-infectious effect in an in vivo model of full thickness wounds infected with MRSA in rats and in an ex vivo model of pig skin infected with S. aureus. S.c. or topical application of the peptide in rats did not lead to any adverse reactions. In conclusion, PXL150 may constitute a new therapeutic alternative for local treatment of infections, and further studies are warranted to evaluate the applicability of this AMP in clin. settings.
- 47Nilsson, E.; Björn, C.; Sjöstrand, V.; Lindgren, K.; Münnich, M.; Mattsby-Baltzer, I.; Ivarsson, M. L.; Olmarker, K.; Mahlapuu, M. A Novel Polypeptide Derived from Human Lactoferrin in Sodium Hyaluronate Prevents Postsurgical Adhesion Formation in the Rat. Ann. Surg. 2009, 250, 1021– 1028, DOI: 10.1097/SLA.0b013e3181b246a7Google Scholar47A novel polypeptide derived from human lactoferrin in sodium hyaluronate prevents postsurgical adhesion formation in the ratNilsson Elin; Bjorn Camilla; Sjostrand Veronika; Lindgren Kerstin; Munnich Mattias; Mattsby-Baltzer Inger; Ivarsson Marie-Louise; Olmarker Kjell; Mahlapuu MargitAnnals of surgery (2009), 250 (6), 1021-8 ISSN:.OBJECTIVE: The objective of the study was to evaluate whether a peptide derived from human lactoferrin, PXL01 could act safely to reduce the formation of peritoneal adhesions in the rat model and to map the molecular mechanisms of its action. SUMMARY BACKGROUND DATA: Adhesion formation is a significant problem within every surgical discipline causing suffering for the patients and major cost for the society. For many decades, attempts have been made to reduce postsurgical adhesions by reducing surgical trauma. It is now believed that major improvements in adhesion prevention will only be reached by developing dedicated antiscarring products, which are administrated in connection to the surgical intervention. METHODS: Anti-inflammatory as well as fibrinolytic activities of PXL01 were studied in relevant human cell lines. Using the sidewall defect-cecum abrasion model in the rat, the adhesion prevention properties of PXL01 formulated in sodium hyaluronate were evaluated. Large bowel anastomosis healing model in the rat was applied to study if PXL01 would have any negative effects on intestine healing. RESULTS: PXL01 exhibits an inhibitory effect on the most important hallmarks of scar formation by reducing infections, prohibiting inflammation, and promoting fibrinolysis. PXL01 formulated in sodium hyaluronate markedly reduced formation of peritoneal adhesions in rat without any adverse effects on wound healing. CONCLUSIONS: A new class of synthetically derived water soluble low molecular weight peptide compound, PXL01 showed marked reduction of peritoneal adhesion formation in an animal model without any negative effects on healing. On the basis of these data, a comprehensive adhesion prevention regimen in clinical situation is expected.
- 48Björn, C.; Mahlapuu, M.; Mattsby-Baltzer, I.; Håkansson, J. Anti-Infective Efficacy of the Lactoferrin-Derived Antimicrobial Peptide HLR1r. Peptides 2016, 81, 21– 28, DOI: 10.1016/j.peptides.2016.04.005Google Scholar48Anti-infective efficacy of the lactoferrin-derived antimicrobial peptide HLR1rBjorn Camilla; Mahlapuu Margit; Mattsby-Baltzer Inger; Hakansson JoakimPeptides (2016), 81 (), 21-8 ISSN:.Antimicrobial peptides (AMPs) have emerged as a new class of drug candidates for the treatment of infectious diseases. Here we describe a novel AMP, HLR1r, which is structurally derived from the human milk protein lactoferrin and demonstrates a broad spectrum microbicidal action in vitro. The minimum concentration of HLR1r needed for killing ≥99% of microorganisms in vitro, was in the range of 3-50μg/ml for common Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and for the yeast Candida albicans, when assessed in diluted brain-heart infusion medium. We found that HLR1r also possesses anti-inflammatory properties as evidenced by inhibition of tumor necrosis factor alpha (TNF-α) secretion from human monocyte-derived macrophages and by repression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) secretion from human mesothelial cells, without any cytotoxic effect observed at the concentration range tested (up to 400μg/ml). HLR1r demonstrated pronounced anti-infectious effect in in vivo experimental models of cutaneous candidiasis in mice and of excision wounds infected with MRSA in rats as well as in an ex vivo model of pig skin infected with S. aureus. In conclusion, HLR1r may constitute a new therapeutic alternative for local treatment of skin infections.
- 49Yan, W.; Banerjee, P.; Liu, Y.; Mi, Z.; Bai, C.; Hu, H.; To, K. K. W.; Duong, H. T. T.; Leung, S. S. Y. Development of Thermosensitive Hydrogel Wound Dressing Containing Acinetobacter Baumannii Phage against Wound Infections. Int. J. Pharm. 2021, 602, 120508 DOI: 10.1016/j.ijpharm.2021.120508Google Scholar49Development of thermosensitive hydrogel wound dressing containing Acinetobacter baumannii phage against wound infectionsYan, Wei; Banerjee, Parikshit; Liu, Yannan; Mi, Zhiqiang; Bai, Changqing; Hu, Haiyan; To, Kenneth K. W.; Duong, Hien T. T.; Leung, Sharon S. Y.International Journal of Pharmaceutics (Amsterdam, Netherlands) (2021), 602 (), 120508CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)With the emergence of multidrug resistance (MDR) bacteria, wound infection continues to be a challenging problem and represents a considerable healthcare burden. This study aims to evaluate the applicability of a phage loaded thermosensitive hydrogel in managing wound infections caused by MDR Acinetobacter baumannii, using IME-AB2 phage and MDR-AB2 as the model phage and bacteria, resp. Excellent storage stability of the IME-AB2 phage in a ∼18 wt% Poloxamer 407 (P407) hydrogel soln. was first demonstrated with negligible titer loss (∼0.5 log) in 24 mo at 4°C. The incorporated phage was released in a sustained manner with a cumulative release of 60% in the first 24 h. The in vitro bacterial killing efficiency of phage gel and phage suspension at 37°C demonstrated >5 log10 CFU/mL redn. against A. baumannii. A comparable biofilm elimination capacity was also noted between the phage gel and phage suspension (59% and 45% resp.). These results suggested that the incorporation of phage into the hydrogel not only had insignificant impacts on the bacterial killing efficiency of phage, but also act as a phage depot to maintain higher phage titer at the infectious site for a prolong period for more effective treatment. We also found that the hydrogel formulation significantly suppressed microbial survival in an ex vivo wound infection model using pig skin (90% redn. in bacterial counts was achieved after 4 h treatment). In summary, our results demonstrated that the P407-based phage-loaded thermosensitive hydrogel is a simple and promising phage formulation for the management of wound infections.
- 50Alhusein, N.; Blagbrough, I. S.; Beeton, M. L.; Bolhuis, A.; de Bank, P. A. Electrospun Zein/PCL Fibrous Matrices Release Tetracycline in a Controlled Manner, Killing Staphylococcus aureus Both in Biofilms and Ex Vivo on Pig Skin, and Are Compatible with Human Skin Cells. Pharm. Res. 2016, 33, 237– 246, DOI: 10.1007/s11095-015-1782-3Google Scholar50Electrospun Zein/PCL Fibrous Matrices Release Tetracycline in a Controlled Manner, Killing Staphylococcus aureus Both in Biofilms and Ex Vivo on Pig Skin, and are Compatible with Human Skin CellsAlhusein, Nour; Blagbrough, Ian S.; Beeton, Michael L.; Bolhuis, Albert; De Bank, Paul A.Pharmaceutical Research (2016), 33 (1), 237-246CODEN: PHREEB; ISSN:0724-8741. (Springer)Purpose: To investigate the destruction of clin.-relevant bacteria within biofilms via the sustained release of the antibiotic tetracycline from zein-based electrospun polymeric fibrous matrixes and to demonstrate the compatibility of such wound dressing matrixes with human skin cells. Methods: Zein/PCL triple layered fibrous dressings with entrapped tetracycline were electrospun. The successful entrapment of tetracycline in these dressings was validated. The successful release of bioactive tetracycline, the destruction of preformed biofilms, and the viability of fibroblast (FEK4) cells were investigated. Results: The sustained release of tetracycline from these matrixes led to the efficient destruction of preformed biofilms from Staphylococcus aureus MRSA252 in vitro, and of MRSA252 and ATCC 25923 bacteria in an ex vivo pig skin model using 1 × 1 cm square matrixes contg. tetracycline (30 μg). Human FEK4 cells grew normally in the presence of these matrixes. Conclusions: The ability of the zein-based matrixes to destroy bacteria within increasingly complex in vitro biofilm models was clearly established. An ex vivo pig skin assay showed that these matrixes, with entrapped tetracycline, efficiently kill bacteria and this, combined with their compatibility with a human skin cell line suggest these matrixes are well suited for applications in wound healing and infection control.
- 51Mrázová, H.; Koller, J.; Kubišová, K.; Fujeríková, G.; Klincová, E.; Babál, P. Comparison of Structural Changes in Skin and Amnion Tissue Grafts for Transplantation Induced by Gamma and Electron Beam Irradiation for Sterilization. Cell Tissue Banking 2016, 17, 255– 260, DOI: 10.1007/s10561-015-9536-3Google Scholar51Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilizationMrazova, H.; Koller, J.; Kubisova, K.; Fujerikova, G.; Klincova, E.; Babal, P.Cell and Tissue Banking (2016), 17 (2), 255-260CODEN: CTBAFV; ISSN:1389-9333. (Springer)Sterilization is an important step in the prepn. of biol. material for transplantation. The aim of the study is to compare morphol. changes in three types of biol. tissues induced by different doses of gamma and electron beam radiation. Frozen biol. tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10% formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, esp. in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradn. by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.
- 52Kairiyama, E.; Horak, C.; Spinosa, M.; Pachado, J.; Schwint, O. Radiation Sterilization of Skin Allograft. Radiat. Phys. Chem. 2009, 78, 445– 448, DOI: 10.1016/j.radphyschem.2009.03.078Google Scholar52Radiation sterilization of skin allograftKairiyama, E.; Horak, C.; Spinosa, M.; Pachado, J.; Schwint, O.Radiation Physics and Chemistry (2009), 78 (7-8), 445-448CODEN: RPCHDM; ISSN:0969-806X. (Elsevier Ltd.)In the treatment of burns or accidental loss of skin, cadaveric skin allografts provide an alternative to temporarily cover a wounded area. The skin bank facility is indispensable for burn care. The first human skin bank was established in Argentina in 1989; later, 3 more banks were established. A careful donor selection is carried out according to the national regulation in order to prevent transmissible diseases. As cadaveric human skin is naturally highly contaminated, a final sterilization is necessary to reach a sterility assurance level (SAL) of 10-6. The sterilization dose for 106 batches of processed human skin was detd. on the basis of the Code of Practice for the Radiation Sterilization of Tissue Allografts: Requirements for Validation and Routine Control (2004) and ISO 11137-2 (2006). They ranged from 17.6 to 33.4 kGy for bioburdens of >10-162.700 CFU/100 cm2. The presence of Gram neg. bacteria was checked for each produced batch. From the anal. of the exptl. results, it was obsd. that the bioburden range was very wide and consequently the estd. sterilization doses too. If this is the case, the detn. of a tissue-specific dose per prodn. batch is necessary to achieve a specified requirement of SAL. Otherwise if the dose of 25 kGy is preselected, a standardized method for substantiation of this dose should be done to confirm the radiation sterilization process.
- 53Rooney, P.; Eagle, M.; Hogg, P.; Lomas, R.; Kearney, J. Sterilisation of Skin Allograft with Gamma Irradiation. Burns 2008, 34, 664– 673, DOI: 10.1016/j.burns.2007.08.021Google Scholar53Sterilisation of skin allograft with gamma irradiationRooney P; Eagle M; Hogg P; Lomas R; Kearney JBurns : journal of the International Society for Burn Injuries (2008), 34 (5), 664-73 ISSN:0305-4179.The primary surgical requirement of skin allografts within the UK is for cryopreserved viable allografts as these engraft to the wound bed and gain a vascular supply, thus providing true wound closure and a superior clinical performance. Consequently the only disinfection treatment the skin receives is exposure to an antibiotic cocktail. However, antibiotic treatment does not reliably decontaminate skin allografts and 22% of cryopreserved skin fails microbial acceptance criteria and cannot be used clinically. We describe here a study which was carried out to determine a means of saving and using the microbiologically failed skin. Four different treatment regimens were investigated; treatment with 20%, 50% and 85% glycerol followed by 25 kGy irradiation at -80 degrees C, and treatment with 85% glycerol at ambient (30-40 degrees C) temperature and irradiation. Following treatment, the grafts were evaluated for their histological structure, in vitro cytotoxicity and handling properties. The radioprotective effects of the different glycerol concentrations and temperatures on microorganisms were also determined. The data indicate that 25 kGy irradiation of deep-frozen skin in 20% glycerol sterilised the tissue without any histological, cytotoxicological or physical alterations compared to normal cryopreserved skin. In contrast, irradiation of all other glycerol concentrations elicited some cytotoxicity and/or histological effect. These non-viable grafts can be made available for surgical use when cryopreserved viable grafts are not available or required.
- 54Mrázová, H.; Koller, J.; Fujeríková, G.; Babál, P. Structural Changes of Skin and Amnion Grafts for Transplantation Purposes Following Different Doses of Irradiation. Cell Tissue Banking 2014, 15, 429– 433, DOI: 10.1007/s10561-013-9407-8Google Scholar54Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiationMrazova, H.; Koller, J.; Fujerikova, G.; Babal, P.Cell and Tissue Banking (2014), 15 (3), 429-433CODEN: CTBAFV; ISSN:1389-9333. (Springer)An important part of the prepn. of biol. material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biol. tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was obsd. by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, esp. in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradn. than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochem. techniques.
- 55Cattò, C.; Cappitelli, F. Testing Anti-Biofilm Polymeric Surfaces: Where to Start?. Int. J. Mol. Sci. 2019, 20, 3794, DOI: 10.3390/ijms20153794Google Scholar55Testing anti-biofilm polymeric surfaces: where to start?Catto, Cristina; Cappitelli, FrancescaInternational Journal of Molecular Sciences (2019), 20 (15), 3794CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A review. Present day awareness of biofilm colonization on polymeric surfaces has prompted the scientific community to develop an ever-increasing no. of new materials with anti-biofilm features. However, compared to the large amt. of work put into discovering potent biofilm inhibitors, only a small no. of papers deal with their validation, a crit. step in the translation of research into practical applications. This is due to the lack of standardized testing methods and/or of well-controlled in vivo studies that show biofilm prevention on polymeric surfaces; furthermore, there has been little correlation with the reduced incidence of material deterioration. Here an overview of the most common methods for studying biofilms and for testing the anti-biofilm properties of new surfaces is provided.
- 56Yu, O. Y.; Zhao, I. S.; Mei, M. L.; Lo, E. C. M.; Chu, C. H. Dental Biofilm and Laboratory Microbial Culture Models for Cariology Research. Dent. J. 2017, 5, 21, DOI: 10.3390/dj5020021Google ScholarThere is no corresponding record for this reference.
- 57Otto, M. Staphylococcus epidermidis─The “accidental” Pathogen. Nat. Rev. Microbiol. 2009, 7, 555– 567, DOI: 10.1038/nrmicro2182Google Scholar57Staphylococcus epidermidis - the 'accidental' pathogenOtto, MichaelNature Reviews Microbiology (2009), 7 (8), 555-567CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Although nosocomial infections by Staphylococcus epidermidis have gained much attention, this skin-colonizing bacterium has apparently evolved not to cause disease, but to maintain the commonly benign relationship with its host. Accordingly, S. epidermidis does not produce aggressive virulence determinants. Rather, factors that normally sustain the commensal lifestyle of S. epidermidis seem to give rise to addnl. benefits during infection. Furthermore, we are beginning to comprehend the roles of S. epidermidis in balancing the epithelial microflora and serving as a reservoir of resistance genes. In this Review, I discuss the mol. basis of the commensal and infectious lifestyles of S. epidermidis.
- 58Ishihama, H.; Ishii, K.; Nagai, S.; Kakinuma, H.; Sasaki, A.; Yoshioka, K.; Kuramoto, T.; Shiono, Y.; Funao, H.; Isogai, N.; Tsuji, T.; Okada, Y.; Koyasu, S.; Toyama, Y.; Nakamura, M.; Aizawa, M.; Matsumoto, M. An Antibacterial Coated Polymer Prevents Biofilm Formation and Implant-Associated Infection. Sci. Rep. 2021, 11, 3602 DOI: 10.1038/s41598-021-82992-wGoogle Scholar58An antibacterial coated polymer prevents biofilm formation and implant-associated infectionIshihama, Hiroko; Ishii, Ken; Nagai, Shigenori; Kakinuma, Hiroaki; Sasaki, Aya; Yoshioka, Kenji; Kuramoto, Tetsuya; Shiono, Yuta; Funao, Haruki; Isogai, Norihiro; Tsuji, Takashi; Okada, Yasunori; Koyasu, Shigeo; Toyama, Yoshiaki; Nakamura, Masaya; Aizawa, Mamoru; Matsumoto, MorioScientific Reports (2021), 11 (1), 3602CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)To prevent infections assocd. with medical implants, various antimicrobial silver-coated implant materials have been developed. However, these materials do not always provide consistent antibacterial effects in vivo despite having dramatic antibacterial effects in vitro, probably because the antibacterial effects involve silver-ion-mediated reactive oxygen species generation. Addnl., the silver application process often requires extremely high temps., which damage non-metal implant materials. We recently developed a bacteria-resistant coating consisting of hydroxyapatite film on which ionic silver is immobilized via inositol hexaphosphate chelation, using a series of immersion and drying steps performed at low heat. Here we applied this coating to a polymer, polyetheretherketone (PEEK), and analyzed the properties and antibacterial activity of the coated polymer in vitro and in vivo. The ionic silver coating demonstrated significant bactericidal activity and prevented bacterial biofilm formation in vitro. Bio-imaging of a soft tissue infection mouse model in which a silver-coated PEEK plate was implanted revealed a dramatic absence of bacterial signals 10 days after inoculation. These animals also showed a strong redn. in histol. features of infection, compared to the control animals. This innovative coating can be applied to complex structures for clin. use, and could prevent infections assocd. with a variety of plastic implants.
- 59Peng, L.; de Sousa, J.; Su, Z.; Novak, B. M.; Nevzorov, A. A.; Garland, E. R.; Melander, C. Inhibition of Acinetobacter Baumannii Biofilm Formation on a Methacrylate Polymer Containing a 2-Aminoimidazole Subunit. Chem. Commun. 2011, 47, 4896– 4898, DOI: 10.1039/c1cc10691kGoogle Scholar59Inhibition of Acinetobacter baumannii biofilm formation on a methacrylate polymer containing a 2-aminoimidazole subunitPeng, Ling-Ling; De Sousa, Joseph; Su, Zhao-Ming; Novak, Bruce M.; Nevzorov, Alexander A.; Garland, Eva R.; Melander, ChristianChemical Communications (Cambridge, United Kingdom) (2011), 47 (17), 4896-4898CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A polymeric composite contg. a 2-aminoimidazole deriv. was synthesized. It was found that this polymer was resistant to biofilm colonization by Acinetobacter baumannii, no leaching of the 2-aminoimidazole deriv. was obsd. after 2 wk of treatment with deionized water, and the resulting polymer was not hemolytic.
- 60Vishwakarma, A.; Dang, F.; Ferrell, A.; Barton, H. A.; Joy, A. Peptidomimetic Polyurethanes Inhibit Bacterial Biofilm Formation and Disrupt Surface Established Biofilms. J. Am. Chem. Soc. 2021, 143, 9440– 9449, DOI: 10.1021/jacs.1c02324Google Scholar60Peptidomimetic Polyurethanes Inhibit Bacterial Biofilm Formation and Disrupt Surface Established BiofilmsVishwakarma, Apoorva; Dang, Francis; Ferrell, Allison; Barton, Hazel A.; Joy, AbrahamJournal of the American Chemical Society (2021), 143 (25), 9440-9449CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Over 80% of all chronic bacterial infections in humans are assocd. with biofilms, which are surface-assocd. bacterial communities encased within a secreted exopolysaccharide matrix that can provide resistance to environmental and chem. insults. Biofilm formation triggers broad adaptive changes in the bacteria, allowing them to be almost 1000-fold more resistant to conventional antibiotic treatments and host immune responses. The failure of antibiotics to eliminate biofilms leads to persistent chronic infections and can promote the development of antibiotic-resistant strains. Therefore, there is an urgent need to develop agents that effectively prevent biofilm formation and eradicate established biofilms. Herein, we present water-sol. synthetic peptidomimetic polyurethanes that can disrupt surface established biofilms of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, all of which show tolerance to the conventional antibiotics polymyxin B and ciprofloxacin. Furthermore, while these polyurethanes show poor antimicrobial activity against planktonic bacteria, they prevent surface attachment and stimulate bacterial surface motility to inhibit biofilm formation of both Gram-pos. and Gram-neg. bacteria at subinhibitory concns., without being toxic to mammalian cells. Our results show that these polyurethanes show promise as a platform for the development of therapeutics that target biofilms and modulate surface interactions of bacteria for the treatment of chronic biofilm-assocd. infections and as antibiofilm agents.
- 61Zakrzewska, A.; Bayan, H.; Nakielski, M. A.; Petronella, P.; de Sio, F.; Pierini, L. F. Nanotechnology Transition Roadmap toward Multifunctional Stimuli-Responsive Face Masks. ACS Appl. Mater. Interfaces 2022, 14, 46123– 46144, DOI: 10.1021/acsami.2c10335Google Scholar61Nanotechnology Transition Roadmap toward Multifunctional Stimuli-Responsive Face MasksZakrzewska, Anna; Haghighat Bayan, Mohammad Ali; Nakielski, Pawel; Petronella, Francesca; De Sio, Luciano; Pierini, FilippoACS Applied Materials & Interfaces (2022), 14 (41), 46123-46144CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)A review. In recent times, the use of personal protective equipment, such as face masks or respirators, is becoming more and more critically important because of common pollution; furthermore, face masks have become a necessary element in the global fight against the COVID-19 pandemic. For this reason, the main mission of scientists has become the development of face masks with exceptional properties that will enhance their performance. The versatility of electrospun polymer nanofibers has detd. their suitability as a material for constructing "smart" filter media. This paper provides an overview of the research carried out on nanofibrous filters obtained by electrospinning. The progressive development of the next generation of face masks whose unique properties can be activated in response to a specific external stimulus is highlighted. Thanks to addnl. components incorporated into the fiber structure, filters can, for example, acquire antibacterial or antiviral properties, self-sterilize the structure, and store the energy generated by users. Despite the discovery of several fascinating possibilities, some of them remain unexplored. Stimuli-responsive filters have the potential to become products of large-scale availability and great importance to society as a whole.
- 62Lencova, S.; Svarcova, V.; Stiborova, H.; Demnerova, K.; Jencova, V.; Hozdova, K.; Zdenkova, K. Bacterial Biofilms on Polyamide Nanofibers: Factors Influencing Biofilm Formation and Evaluation. ACS Appl. Mater. Interfaces 2021, 13, 2277– 2288, DOI: 10.1021/acsami.0c19016Google Scholar62Bacterial Biofilms on Polyamide Nanofibers: Factors Influencing Biofilm Formation and EvaluationLencova Simona; Svarcova Viviana; Stiborova Hana; Demnerova Katerina; Zdenkova Kamila; Jencova Vera; Hozdova KristynaACS applied materials & interfaces (2021), 13 (2), 2277-2288 ISSN:.Electrospun polyamide (PA) nanofibers have great potential for medical applications (in dermatology as antimicrobial compound carriers or surgical sutures). However, little is known about microbial colonization on these materials. Suitable methods need to be chosen and optimized for the analysis of biofilms formed on nanofibers and the influence of their morphology on biofilm formation. We analyzed 11 PA nanomaterials, both nonfunctionalized and functionalized with AgNO3, and tested the formation of a biofilm by clinically relevant bacteria (Escherichia coli CCM 4517, Staphylococcus aureus CCM 3953, and Staphylococcus epidermidis CCM 4418). By four different methods, it was confirmed that all of these bacteria attached to the PAs and formed biofilms; however, it was found that the selected method can influence the outcomes. For studying biofilms formed by the selected bacteria, scanning electron microscopy, resazurin staining, and colony-forming unit enumeration provided appropriate and comparable results. The values obtained by crystal violet (CV) staining were misleading due to the binding of the CV dye to the PA structure. In addition, the effect of nanofiber morphology parameters (fiber diameter and air permeability) and AgNO3 functionalization significantly influenced biofilm maturation. Furthermore, the correlations between air permeability and surface density and fiber diameter were revealed. Based on the statistical analysis, fiber diameter was confirmed as a crucial factor influencing biofilm formation (p ≤ 0.01). The functionalization of PAs with AgNO3 (from 0.1 wt %) effectively suppressed biofilm formation. The PA functionalized with a concentration of 0.1 wt % AgNO3 influenced the biofilm equally as nonfunctionalized PA 8% 2 g/m(2). Therefore, biofilm formation could be affected by the above-mentioned morphology parameters, and ultimately, the risk of infections from contaminated medical devices could be reduced.
- 63Wang, Z.; Scheres, L.; Xia, H.; Zuilhof, H., 1; Wang, Z.; Scheres, L.; Xia, H.; Zuilhof, H. Developments and Challenges in Self-Healing Antifouling Materials. Adv. Funct. Mater. 2020, 30, 1908098 DOI: 10.1002/adfm.201908098Google Scholar63Developments and Challenges in Self-Healing Antifouling MaterialsWang, Zhanhua; Scheres, Luc; Xia, Hesheng; Zuilhof, HanAdvanced Functional Materials (2020), 30 (26), 1908098CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Self-healing antifouling materials have gained rapidly increasing interest over the past decade and have been studied and used in a rapidly increasing range of applications. Recent developments and challenges in self-healing antifouling materials are summarized in four sections: first, the different mechanisms for both antifouling and self-healing are briefly discussed. Second, three main categories of self-healing antifouling materials based on surface replenishing and dynamic covalent and noncovalent interactions are discussed, with a focus on the prepn., characterization, and central characteristics of different self-healing antifouling materials. Third, different types of potential applications of self-healing antifouling materials are summarized, such as injectable hydrogels and oil/water sepns. Finally, a summary of future development of the field is provided, and a no. of crit. limitations that are still outstanding are highlighted.
- 64Patterson, A. L.; Wenning, B.; Rizis, G.; Calabrese, D. R.; Finlay, J. A.; Franco, S. C.; Zuckermann, R. N.; Clare, A. S.; Kramer, E. J.; Ober, C. K.; Segalman, R. A. Role of Backbone Chemistry and Monomer Sequence in Amphiphilic Oligopeptide- and Oligopeptoid-Functionalized PDMS- and PEO-Based Block Copolymers for Marine Antifouling and Fouling Release Coatings. Macromolecules 2017, 50, 2656– 2667, DOI: 10.1021/acs.macromol.6b02505Google Scholar64Role of Backbone Chemistry and Monomer Sequence in Amphiphilic Oligopeptide- and Oligopeptoid-Functionalized PDMS- and PEO-Based Block Copolymers for Marine Antifouling and Fouling Release CoatingsPatterson, Anastasia L.; Wenning, Brandon; Rizis, Georgios; Calabrese, David R.; Finlay, John A.; Franco, Sofia C.; Zuckermann, Ronald N.; Clare, Anthony S.; Kramer, Edward J.; Ober, Christopher K.; Segalman, Rachel A.Macromolecules (Washington, DC, United States) (2017), 50 (7), 2656-2667CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)Poly(dimethylsiloxane) (PDMS)- and poly(ethylene oxide) (PEO)-based block copolymer coatings functionalized with amphiphilic, surface-active, and sequence-controlled oligomer side chains were studied to directly compare the effects of hydrophilicity, hydrogen bonding, and monomer sequence on antifouling performance. Utilizing a modular coating architecture, structurally similar copolymers were used to make direct and meaningful comparisons. Amphiphilic character was imparted with non-natural oligopeptide and oligopeptoid pendant chains made from oligo-PEO and surface-segregating fluoroalkyl monomer units. Surface anal. revealed rearrangement for all surfaces when moved from vacuum to wet environments. XPS spectra indicated that the polymer backbone and oligomer interactions play key roles in the surface presentation. Biofouling assays using the macroalga Ulva linza showed that the presence of peptoid side chains facilitated the removal of sporelings on the PDMS block copolymer, with removal matching that of a PDMS elastomer std. The lack of a hydrogen bond donor in the peptoid backbone likely contributed to the lower adhesion strength of sporelings to these surfaces. Both the initial attachment and adhesion strength of the diatom Navicula incerta were lower on the coatings based on PEO than on those based on PDMS. Surprisingly, on the PEO coating bearing the blocky peptoid sequence, initial attachment of N. incerta showed no measurable cell d.
- 65Putrinš, M.; Kogermann, K.; Lukk, E.; Lippus, M.; Varik, V.; Tenson, T. Phenotypic Heterogeneity Enables Uropathogenic Escherichia coli to Evade Killing by Antibiotics and Serum Complement. Infect. Immun. 2015, 83, 1056– 1067, DOI: 10.1128/IAI.02725-14Google Scholar65Phenotypic heterogeneity enables uropathogenic Escherichia coli to evade killing by antibiotics and serum complementPutrins, Marta; Kogermann, Karin; Lukk, Eliisa; Lippus, Markus; Varik, Vallo; Tenson, TanelInfection and Immunity (2015), 83 (3), 1056-1067, 12 pp.CODEN: INFIBR; ISSN:1098-5522. (American Society for Microbiology)Uropathogenic strains of Escherichia coli (UPEC) are the major cause of bacteremic urinary tract infections. Survival in the bloodstream is assocd. with different mechanisms that help to resist serum complement-mediated killing. While the phenotypic heterogeneity of bacteria has been shown to influence antibiotic tolerance, the possibility that it makes cells refractory to killing by the immune system has not been exptl. tested. In the present study, we sought to det. whether the heterogeneity of bacterial cultures is relevant to bacterial targeting by the serum complement system. We monitored cell divisions in the UPEC strain CFT073 with fluorescent reporter protein. Stationary-phase cells were incubated in active or heat-inactivated human serum in the presence or absence of different antibiotics (ampicillin, norfloxacin, and amikacin), and cell division and complement protein C3 binding were measured by flow cytometry and immunofluorescence microscopy. Heterogeneity in the doubling times of CFT073 cells in serum enabled three phenotypically different subpopulations to be distinguished, all of them being recognized by the C3 component of the complement system. The population of rapidly growing cells resists serum complement-mediated lysis. The dominant subpopulation of cells with intermediate growth rate is susceptible to serum. The third population, which does not resume growth upon diln. from stationary phase, is simultaneously protected from serum complement and antibiotics.
- 66Lebeaux, D.; Ghigo, J.-M.; Beloin, C. Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics. Microbiol. Mol. Biol. Rev. 2014, 78, 510– 543, DOI: 10.1128/MMBR.00013-14Google Scholar66Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibioticsLebeaux David; Ghigo Jean-Marc; Beloin ChristopheMicrobiology and molecular biology reviews : MMBR (2014), 78 (3), 510-43 ISSN:.Surface-associated microbial communities, called biofilms, are present in all environments. Although biofilms play an important positive role in a variety of ecosystems, they also have many negative effects, including biofilm-related infections in medical settings. The ability of pathogenic biofilms to survive in the presence of high concentrations of antibiotics is called "recalcitrance" and is a characteristic property of the biofilm lifestyle, leading to treatment failure and infection recurrence. This review presents our current understanding of the molecular mechanisms of biofilm recalcitrance toward antibiotics and describes how recent progress has improved our capacity to design original and efficient strategies to prevent or eradicate biofilm-related infections.
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Molecular Pharmaceutics
Cite this: Mol. Pharmaceutics 2023, 20, 2, 1230–1246
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Abstract
Figure 1
Figure 1. Schematics of the experimental work conducted for the development and validation of the biofilm models. Key: CAM, chloramphenicol; ES, electrospinning; GEL, gelatin; Glu, glucose; PCL, polycaprolactone; PEO, polyethylenoxide.
Figure 2
Figure 2. (A) Micrographs of cross-sections of heat-treated pig ear skin (with superficial burn wounds) with and without γ-irradiation and glycerol treatment; H&E stained. Scale bar: 500 μm. (B) AFM micrographs of ES fibrous wound dressings. (C) European Pharmacopoeia 10.0 sterility test results after 14 days of incubation of γ-irradiated (50 kGy) samples (ES pristine fibrous wound dressings and infection model substrates) under aerobic conditions at room temperature (RT) in the dark. Positive bacterial control (E. coli). Key: GEL, gelatin; Glu, glucose; PCL, polycaprolactone; PEO, polyethylenoxide. Scale bar 1 cm.
Figure 3
Figure 3. (A) SEM micrographs of biofilm formation after 24 h on top of the substrates─gelatin–glucose matrix (GEL-Glu matrix) and pig skin. Arrows point out the bacteria. Three different wound bacteria were used: E. coli DSM 1103, S. aureus DSM 2569, and S. epidermidis DSM 28319. The scale bar is 3 μm for the overview image of the pig skin surface, and it is 10 μm for all other SEM micrographs. (B) CFM micrographs of biofilm formation (24 and 48 h) on top of the GEL-Glu matrix in the in vitro model. Biofilm matrix visualized using EbbaBiolight 630 is in pink; ES gelatin–glucose matrix (GEL-Glu matrix) fibers visualized by autofluorescence are in blue; and bacteria stained using SYTO-9 are in green. Three different wound bacteria were used: E. coli DSM 1103, S. aureus DSM 2569, and S. epidermidis DSM 28319. Scale bar: 10 μm. The sample depth shown is 14.4 μm. (C) CFM micrographs of biofilm formation (24 h) in the ex vivo model. Pig skin had nonspecific red autofluorescence; bacteria were stained with SYTO-9 in green. Two different wound bacteria were used: E. coli DSM 1103 and S. aureus DSM 2569. Scale bar: 10 μm. (D) Biofilm formation after 24 and 48 h on top of the substrates─gelatin–glucose matrix (GEL-Glu matrix) and pig skin. Three different wound bacteria were used: E. coli DSM 1103, S. aureus DSM 2569, and S. epidermidis DSM 28319. Results are shown in the logarithmic scale as the number of colony-forming units (CFUs), with standard deviation bars (n = 3). Statistical significance is shown as follows: *p < 0.05; **p < 0.01; and ***p < 0.001. Experiments were performed using at least three technical replicates. Key: GEL, gelatin; Glu, glucose.
Figure 4
Figure 4. (A) Use of the gelatin–glucose matrix (GEL-Glu matrix) as a nutritional substrate in HEPES buffer. Bacteria were inoculated into 10 mM HEPES buffer alone and/or together with the GEL-Glu matrix for up to 96 h. S. aureus DSM 2569 and S. epidermidis DSM 28319 were used. The number of colony-forming units (CFUs), with standard deviation bars (n = 3), are shown in the logarithmic scale. Experiments were performed using at least three technical replicates. The detection limit of the assay is 2 log10 CFU/cm2. (B) S. epidermidis DSM 28319 biofilm formation on top of a single-layered gelatin–glucose matrix (1 × GEL-Glu) and triple-layered gelatin–glucose matrices (3× GEL-Glu matrix) in HEPES-buffered DMEM/F-12 growth media at different time points (24, 48, and 72 h). The number of colony-forming units (CFUs), with standard deviation bars (n = 3), are shown in the logarithmic scale. Statistical significance is shown as **P < 0.01. Experiments were performed using three technical replicates. Key: GEL, gelatin; Glu, glucose.
Figure 5
Figure 5. Model validation using electrospun (ES) wound dressings. For the in vitro model, the GEL-Glu matrix was used as artificial skin, and for the ex vivo model, pig skin was used. Three different bacteria were used: (A) E. coli DSM 1103, (B) S. aureus DSM 2569, and (C) S. epidermidis DSM 28319. Results are shown in the logarithmic scale as the number of colony-forming units (CFUs) cm2, with standard deviation bars (n = 3). The detection limit of the assay is 2 log10 CFU/cm2. CAM-loaded ES wound dressings were compared with pristine control wound dressings. A comparison between the PCL vs PCL/PEO formulations is also presented. Statistical significance is shown as follows: *P < 0.05; **P < 0.01; and ***P < 0.001. Key: CAM, chloramphenicol; PCL, polycaprolactone; and PEO, polyethylenoxide.
Figure 6
Figure 6. Bacterial biofilm adhesion on top of the substrate−gelatin−glucose matrix (GEL-Glu matrix) or pig skin or onto the covering wound dressing (ES pristine wound dressings). Substrates were covered with pristine wound dressings and incubated for 24 h, after which the substrate and wound dressing were separated, and the biofilm formed on top of each part was studied independently. Three different bacteria were used: (A) E. coli DSM 1103, (B) S. aureus DSM 2569, and (C) S. epidermidis DSM 28319. Two different formulations were tested─PCL wound dressing and PCL/PEO wound dressing (control dressings); 100% is shown to mark the arithmetic mean of biofilm formation on top of the different uncovered substrates. Key: GEL, gelatin; Glu, glucose; PCL, pristine wound dressings made from polycaprolactone; PCL/PEO, pristine wound dressings made from polycaprolactone and polyethylenoxide.
Figure 7
Figure 7. Biofilm treatment model. Effect of the model CAM-loaded wound dressings (CAM-PCL and CAM-PCL/PEO dressings) on already formed E. coli DSM 1103 biofilms (24 h). A comparison is made with pristine wound dressings and uncovered substrates. The in vitro setup was created on top of the ES GEL-Glu matrix and the ex vivo setup was created on top of pig skin. The number of biofilm-forming bacteria before treatment was 108 CFU/cm2. Results are shown in a linear scale as the number of colony-forming units (CFUs), with standard deviation bars (n = 3). Changes in the threshold value (108 CFU/cm2) are presented. Statistical significance is shown as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Key: CAM, chloramphenicol-loaded wound dressings; PCL, pristine wound dressings made from polycaprolactone; PCL/PEO, pristine wound dressings made from polycaprolactone and polyethylenoxide.
References
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- 1Omar, A.; Wright, J.; Schultz, G.; Burrell, R.; Nadworny, P. Microbial Biofilms and. Chronic Wounds. Microorganisms 2017, 5, 9, DOI: 10.3390/microorganisms50100091Microbial biofilms and chronic woundsOmar, Amin; Wright, J. Barry; Schultz, Gregory; Burrell, Robert; Nadworny, PatriciaMicroorganisms (2017), 5 (1), 9/1-9/15CODEN: MICRKN; ISSN:2076-2607. (MDPI AG)Background is provided on biofilms, including their formation, tolerance mechanisms, structure, and morphol. within the context of chronic wounds. The features of biofilms in chronic wounds are discussed in detail, as is the impact of biofilm on wound chronicity. Difficulties assocd. with the use of std. susceptibility tests (min. inhibitory concns. or MICs) to det. appropriate treatment regimens for, or develop new treatments for use in, chronic wounds are discussed, with alternate test methods specific to biofilms being recommended. Animal models appropriate for evaluating biofilm treatments are also described. Current and potential future therapies for treatment of biofilm-contg. chronic wounds, including probiotic therapy, virulence attenuation, biofilm phenotype expression attenuation, immune response suppression, and aggressive debridement combined with antimicrobial dressings, are described.
- 2Stuermer, E. K.; Besser, M.; Brill, F.; Geffken, M.; Plattfaut, I.; Severing, A. L.; Wiencke, V.; Rembe, J. D.; Naumova, E. A.; Kampe, A.; Debus, S.; Smeets, R. Comparative Analysis of Biofilm Models to Determine the Efficacy of Antimicrobials. Int. J. Hyg. Environ. Health 2021, 234, 113744 DOI: 10.1016/j.ijheh.2021.1137442Comparative analysis of biofilm models to determine the efficacy of antimicrobialsStuermer, E. K.; Besser, M.; Brill, F.; Geffken, M.; Plattfaut, I.; Severing, A. L.; Wiencke, V.; Rembe, J. D.; Naumova, E. A.; Kampe, A.; Debus, S.; Smeets, R.International Journal of Hygiene and Environmental Health (2021), 234 (), 113744CODEN: IJEHFT; ISSN:1438-4639. (Elsevier GmbH)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 clin. situation are required. In this study, two 3D biofilm models based on human plasma with immune cells (lhBIOM) or based on sheep blood (sbBIOM) contg. S. aureus or P. aeruginosa, are compared with the human biofilm model hpBIOM regarding their microscopic structure (SEM; SEM) and their bacterial resistance to octenidine hydrochloride (OCT) and a sodium hypochlorite (NaOCl) wound-irrigation soln. The three analyzed biofilm models show little to no reaction to treatment with the hypochlorous soln. 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 detd. effects are qual. reflected by SEM. Our results show that both ethically acceptable human and sheep blood based biofilm models can be used as a std. for in vitro testing of new antimicrobial substances. Due to their compn., both fulfill the criteria of a reality-reflecting model and therefore should be used in the approval for new antimicrobial agents.
- 3Stoffel, J. J.; Kohler Riedi, P. L.; Hadj Romdhane, B. A Multimodel Regime for Evaluating Effectiveness of Antimicrobial Wound Care Products in Microbial Biofilms. Wound Repair Regen. 2020, 28, 438– 447, DOI: 10.1111/wrr.128063A multimodel regime for evaluating effectiveness of antimicrobial wound care products in microbial biofilmsStoffel Joseph J; Kohler Riedi Petra L; Hadj Romdhane BrittanyWound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society (2020), 28 (4), 438-447 ISSN:.Microbial biofilms have become increasingly recognized as a cause of wound chronicity. There are several topical antimicrobial wound care products available for use; however, their effectiveness has routinely been demonstrated with planktonic microorganisms. There is no target reference value for antimicrobial effectiveness of wound care products in biofilm models. In addition, data on antimicrobial activity of products in biofilm models are scattered across many test methods in a variety of studies. The aim of this work is to directly compare commercial products containing the commonly used topical antimicrobial agents iodine, silver, polyhexamethylene biguanide, octenidine, hypochlorous acid, benzalkonium chloride, and a surfactant-based topical containing poloxamer 188. Five different in vitro biofilm models of varied complexity were used, incorporating several bacterial pathogens such as Staphylococcus, Enterococcus, Streptococcus, Pseudomonas, Acinetobacter, Klebsiella, and Enterobacter. The fungal pathogens Candida albicans and Candida auris were also evaluated. A multispecies bacterial biofilm model was also used to evaluate the products. Additionally, C. albicans was used in combination with S. aureus and P. aeruginosa in a multikingdom version of the polymicrobial biofilm model. Statistically significant differences in antimicrobial performance were observed between treatments in each model and changing microbial growth conditions or combinations of organisms resulted in significant performance differences for some treatments. The iodine and benzalkonium chloride-containing products were overall the most effective in vitro and were then selected for in vivo evaluation in an infected immunocompromised murine model. Unexpectedly, the iodine product was statistically (P > .05) no different than the untreated control, while the benzalkonium chloride containing product significantly (P < .05) reduced the biofilm compared to untreated control. This body of work demonstrates the importance of not only evaluating antimicrobial wound care products in biofilm models but also the importance of using several different models to gain a comprehensive understanding of products' effectiveness.
- 4Wu, Y. K.; Cheng, N. C.; Cheng, C. M. Biofilms in Chronic Wounds: Pathogenesis and Diagnosis. Trends Biotechnol. 2019, 37, 505– 517, DOI: 10.1016/j.tibtech.2018.10.0114Biofilms in Chronic Wounds: Pathogenesis and DiagnosisWu, Yuan-Kun; Cheng, Nai-Chen; Cheng, Chao-MinTrends in Biotechnology (2019), 37 (5), 505-517CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.)Chronic non-healing wounds have become a major worldwide healthcare burden. The impact of biofilms on chronic wound infection is well established. Despite increasing understanding of the underlying mechanism of biofilm formation in chronic wounds, current strategies for biofilm diagnosis in chronic wounds are still far from ideal. In this review, we briefly summarize the mechanism of biofilm formation and focus on current diagnostic approaches of chronic wound biofilms based on morphol., microbiol., and mol. assays. Innovative biotechnol. approaches, such as wound blotting and transcriptomic anal., may further shed light on this unmet clin. need. The continuous development of these sophisticated diagnostic approaches can markedly contribute to the future implementation of point-of-care biofilm detection in chronic wound care.
- 5Jannesari, M.; Varshosaz, J.; Morshed, M.; Zamani, M. Composite Poly(Vinyl Alcohol)/Poly(Vinyl Acetate) Electrospun Nanofibrous Mats as a Novel Wound Dressing Matrix for Controlled Release of Drugs. Int. J. Nanomed. 2011, 6, 993– 1003, DOI: 10.2147/IJN.S175955Composite poly(vinyl alcohol)/poly(vinyl acetate) electrospun nanofibrous mats as a novel wound dressing matrix for controlled release of drugsJannesari, Marziyeh; Varshosaz, Jaleh; Morshed, Mohammad; Zamani, MaedehInternational Journal of Nanomedicine (2011), 6 (), 993-1003CODEN: IJNNHQ; ISSN:1178-2013. (Dove Medical Press Ltd.)The aim of this study was to develop novel biomedicated nanofiber electrospun mats for controlled drug release, esp. drug release directly to an injury site to accelerate wound healing. Nanofibers of poly(vinyl alc.) (PVA), poly(vinyl acetate) (PVAc), and a 50:50 composite blend, loaded with ciprofloxacin HCl (CipHCl), were successfully prepd. by an electrospinning technique for the first time. The morphol. and av. diam. of the electrospun nanofibers were investigated by SEM. X-ray diffraction studies indicated an amorphous distribution of the drug inside the nanofiber blend. Introducing the drug into polymeric solns. significantly decreased soln. viscosities as well as nanofiber diam. In vitro drug release evaluations showed that both the kind of polymer and the amt. of drug loaded greatly affected the degree of swelling, wt. loss, and initial burst and rate of drug release. Blending PVA and PVAc exhibited a useful and convenient method for electrospinning in order to control the rate and period of drug release in wound healing applications. Also, the thickness of the blend nanofiber mats strongly influenced the initial release and rate of drug release.
- 6Thakur, R. A.; Florek, C. A.; Kohn, J.; Michniak, B. B. Electrospun Nanofibrous Polymeric Scaffold with Targeted Drug Release Profiles for Potential Application as Wound Dressing. Int. J. Pharm. 2008, 364, 87– 93, DOI: 10.1016/j.ijpharm.2008.07.0336Electrospun nanofibrous polymeric scaffold with targeted drug release profiles for potential application as wound dressingThakur, R. A.; Florek, C. A.; Kohn, J.; Michniak, B. B.International Journal of Pharmaceutics (2008), 364 (1), 87-93CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)The authors have successfully fabricated a dual drug release electrospun scaffold contg. an anesthetic, lidocaine, and an antibiotic, mupirocin. Two drugs with different lipophilicities were electrospun from a poly-L-lactic acid (PLLA) soln. with a dual spinneret electrospinning app. into a single scaffold. The release of the drugs from the scaffold showed different profiles for the 2 drugs. Lidocaine hydrochloride exhibited an initial burst release (80% release within an hour) followed by a plateau after the first few hours. Mupirocin exhibited only a 5% release in the first hour before experiencing a more sustained release to provide antibacterial action for over 72 h. For comparative purposes, both drugs were spun from a single spinneret and evaluated to det. their release profiles. The scaffold maintained its antibiotic activity throughout the processes of electrospinning and gas sterilization and supported cell viability. It was reported in the literature that interactions between polymer and drug are known to govern the pattern of drug release from electrospun scaffolds. Here, it was found that the presence of the 2 drugs in the same polymer matrix altered the release kinetics of at least one drug. Based on the release profiles obtained, the dual spinneret technique was the preferred method of scaffold fabrication over the single spinneret technique to obtain a prototype wound healing device.
- 7Shirazaki, P.; Varshosaz, J.; Kharazi, A. Electrospun Gelatin/Poly(Glycerol Sebacate) Membrane with Controlled Release of Antibiotics for Wound Dressing. Adv. Biomed. Res. 2017, 6, 105, DOI: 10.4103/abr.abr_197_167Electrospun gelatin/poly(glycerol sebacate) membrane with controlled release of antibiotics for wound dressingShirazaki, Parisa; Varshosaz, Jaleh; Kharazi, Anoushe ZargarAdvanced Biomedical Research (2017), 6 (Aug.), 105/1-105/11CODEN: ABRDD4; ISSN:2277-9175. (Medknow Publications)Background: The most important risk that threatens the skin wounds is infections. Therefore, fabrication of a membrane as a wound dressing with the ability of antibiotic delivery in a proper delivery rate is esp. important. Materials and Methods: Poly(glycerol sebacate) (PGS) was prepd. from sebacic acid and glycerol with 1:1 ratio; then, it was added to gelatin in the 1:3 ratio and was dissolved in 80% (vol./vol.) acetic acid, and fi nally, ciprofl oxacin was added in 10% (w/v) of polymer soln. The gelatin/PGS membrane was fabricated using an electrospinning method. The membrane was cross-linked using ethyl-3-(3-dimethylaminopropyl) carbodiimide ethyl-3-(3- dimethylaminopropyl)carbodiim (EDC) and N-hydroxysuccinimide (NHS) in different time periods to achieve a proper drug release rate. Fourier-transform IR (FTIR) spectroscopy was being used to manifest the peaks of polymers and drug in the membrane. SEM (SEM) was used to evaluate the morphol., fi bers diam., pore size, and porosity before and after crosslinking process. UV-visible spectrophotometry was used to show the ciprofl oxacin release from the cross-linked membrane. Results: FTIR anal. showed the characteristic peaks of gelatin, PGS, and ciprofl oxacin without any added peaks after the crosslinking process. SEM images revealed that nanofi bers' size increased during the crosslinking process and porosity was higher than 80% before and after crosslinking process. UV-visible spectrophotometry showed the proper rate of ciprofl oxacin release occurred from cross-linked membrane that remaining in EDC/NHS ethanol soln. for 120 min. Conclusion: The obtained results suggest that this recently developed gelatin/PGS membrane with controlled release of ciprofl oxacin could be a promising biodegradable membrane for wound dressing.
- 8Diller, R. B.; Tabor, A. J. The Role of the Extracellular Matrix (ECM) in Wound Healing: A Review. Biomimetics 2022, 7, 87, DOI: 10.3390/biomimetics70300878The Role of the Extracellular Matrix (ECM) in Wound Healing: A ReviewDiller, Robert B.; Tabor, Aaron J.Biomimetics (2022), 7 (3), 87CODEN: BIOMJE; ISSN:2313-7673. (MDPI AG)A review. The extracellular matrix (ECM) is a 3-dimensional structure and an essential component in all human tissues. It is comprised of varying proteins, including collagens, elastin, and smaller quantities of structural proteins. Studies have demonstrated the ECM aids in cellular adherence, tissue anchoring, cellular signaling, and recruitment of cells. During times of integumentary injury or damage, either acute or chronic, the ECM is damaged. Through a series of overlapping events called the wound healing phases-hemostasis, inflammation, proliferation, and remodeling-the ECM is synthesized and ideally returned to its native state. This article synthesizes current and historical literature to demonstrate the involvement of the ECM in the varying phases of the wound healing cascade.
- 9Omer, S.; Forgách, L.; Zelkó, R.; Sebe, I. Scale-up of Electrospinning: Market Overview of Products and Devices for Pharmaceutical and Biomedical Purposes. Pharmaceutics 2021, 13, 1– 21, DOI: 10.3390/pharmaceutics13020286There is no corresponding record for this reference.
- 10MacEwan, M. R.; MacEwan, S.; Kovacs, T. R.; Batts, J. What Makes the Optimal Wound Healing Material? A Review of Current Science and Introduction of a Synthetic Nanofabricated Wound Care Scaffold. Cureus 2017, 9, e1736 DOI: 10.7759/cureus.1736There is no corresponding record for this reference.
- 11Wiegand, C.; Elsner, P.; Hipler, U.-C.; Abel, M.; Ruth, P. In Vitro Assessment of the Antimicrobial Activity of Wound Dressings: Influence of the Test Method Selected and Impact of the pH. J. Mater. Sci. Mater. Med. 2015, 26, 5343, DOI: 10.1007/s10856-014-5343-911In vitro assessment of the antimicrobial activity of wound dressings: influence of the test method selected and impact of the pHWiegand Cornelia; Abel Martin; Ruth Peter; Elsner Peter; Hipler Uta-ChristinaJournal of materials science. Materials in medicine (2015), 26 (1), 5343 ISSN:.Antibacterial activity of dressings containing antimicrobials is mostly evaluated using in vitro tests. However, the various methods available differ significantly in their properties and results obtained are influenced by the method selected, micro-organisms used, and extraction method, the degree of solubility or the diffusability of the test-compounds. Here, results on antimicrobial activity of silver-containing dressings obtained by agar diffusion test (ADT), challenge tests (JIS L 1902, AATCC 100), and extraction-based methods (microplate laser nephelometry (MLN), luminescent quantification of bacterial ATP (LQbATP)) using Staphylococcus aureus and Pseudomonas aeruginosa were evaluated. Furthermore, the effect of the pH on antibacterial efficacy of these dressings was investigated. All silver-containing dressings exerted antimicrobial activity in all in vitro tests and results correlated considerably well. Differences were observed testing the agent-free basic materials. They did not exhibit any antimicrobial effects in the ADT, MLN or LQbATP, since these methods depend on diffusion/extraction of an active agent. However, they showed a strong antimicrobial effect in the challenge tests as they possess a high absorptive capacity, and are able to bind and sequester micro-organisms present. Therefore, it seems recommendable to choose several tests to distinguish whether a material conveys an active effect or a passive mechanism. In addition, it could be shown that release of silver and its antimicrobial efficacy is partially pH-dependent, and that dressings themselves affect the pH. It can further be speculated that dressings' effects on pH and release of silver ions act synergistically for antimicrobial efficacy.
- 12Preem Bock; Hinnu; Putrinš; Sagor; Tenson; Meos; Østergaard; Kogermann. Monitoring of Antimicrobial Drug Chloramphenicol Release from Electrospun Nano- and Microfiber Mats Using UV Imaging and Bacterial Bioreporters. Pharmaceutics 2019, 11, 487, DOI: 10.3390/pharmaceutics1109048712Monitoring of antimicrobial drug chloramphenicol release from electrospun nano- and microfiber mats using UV imaging and bacterial bioreportersPreem, Liis; Bock, Frederik; Hinnu, Mariliis; Putrins, Marta; Sagor, Kadi; Tenson, Tanel; Meos, Andres; Oestergaard, Jesper; Kogermann, KarinPharmaceutics (2019), 11 (9), 487CODEN: PHARK5; ISSN:1999-4923. (MDPI AG)New strategies are continuously sought for the treatment of skin and wound infections due to increased problems with non-healing wounds. Electrospun nanofiber mats with antibacterial agents as drug delivery systems provide opportunities for the eradication of bacterial infections as well as wound healing. Antibacterial activities of such mats are directly linked with their drug release behavior. Traditional pharmacopoeial drug release testing settings are not always suitable for analyzing the release behavior of fiber mats intended for the local drug delivery. We tested and compared different drug release model systems for the previously characterized electrospun chloramphenicol (CAM)-loaded nanofiber (polycaprolactone (PCL)) and microfiber (PCL in combination with polyethylene oxide) mats with different drug release profiles. Drug release into buffer soln. and hydrogel was investigated and drug concn. was detd. using either high-performance liq. chromatog., UV-visible spectrophotometry, or UV imaging. The CAM release and its antibacterial effects in disk diffusion assay were assessed by bacterial bioreporters. All tested model systems enabled to study the drug release from electrospun mats. It was found that the release into buffer soln. showed larger differences in the drug release rate between differently designed mats compared to the hydrogel release tests. The UV imaging method provided an insight into the interactions with an agarose hydrogel mimicking wound tissue, thus giving us information about early drug release from the mat. Bacterial bioreporters showed clear correlations between the drug release into gel and antibacterial activity of the electrospun CAM-loaded mats.
- 13Zupančič, Š.; Preem, L.; Kristl, J.; Putrinš, M.; Tenson, T.; Kocbek, P.; Kogermann, K. Impact of PCL Nanofiber Mat Structural Properties on Hydrophilic Drug Release and Antibacterial Activity on Periodontal Pathogens. Eur. J. Pharm. Sci. 2018, 122, 347– 358, DOI: 10.1016/j.ejps.2018.07.02413Impact of PCL nanofiber mat structural properties on hydrophilic drug release and antibacterial activity on periodontal pathogensZupancic, Spela; Preem, Liis; Kristl, Julijana; Putrins, Marta; Tenson, Tanel; Kocbek, Petra; Kogermann, KarinEuropean Journal of Pharmaceutical Sciences (2018), 122 (), 347-358CODEN: EPSCED; ISSN:0928-0987. (Elsevier B.V.)Electrospinning enables to design and manuf. novel drug delivery systems capable of advancing the local antibacterial therapy. In this study, two hydrophilic drugs - metronidazole and ciprofloxacin hydrochloride - were loaded both individually and in combination into hydrophobic poly(ε-caprolactone) (PCL) matrix using electrospinning. We aimed to develop prolonged release drug delivery systems suitable for the treatment of periodontal diseases and understand how different rarely studied structural features, such as nanofiber mat thickness, surface area, wettability, together with intrinsic properties, like solid state and localization of incorporated drugs in nanofibers, affect the drug release. Furthermore, the safety of nanofiber mats was assessed in vitro on fibroblasts, and their antibacterial activity was tested on selected strains of periodontopathogenic bacteria. The results showed that the structural properties of nanofiber mat are crucial in particular drug-polymer combinations, affecting the drug release and consequently the antibacterial activity. The hydrophobicity of a PCL nanofiber mat and its thickness are the key characteristics in prolonged hydrophilic drug release, but only when wetting is the rate-limiting step for the drug release. Combination of drugs showed beneficial effects by inhibiting the growth of all tested pathogenic bacterial strains important in periodontal diseases.
- 14Lanno, G. M.; Ramos, C.; Preem, L.; Putrins, M.; Laidmaë, I.; Tenson, T.; Kogermann, K. Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing Applications. ACS Omega 2020, 5, 30011– 30022, DOI: 10.1021/acsomega.0c0440214Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing ApplicationsLanno, Georg-Marten; Ramos, Celia; Preem, Liis; Putrins, Marta; Laidmae, Ivo; Tenson, Tanel; Kogermann, KarinACS Omega (2020), 5 (46), 30011-30022CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)Electrospun fiber scaffolds have a huge potential for the successful treatment of infected wounds based on their unique properties. Although several studies report novel drug-loaded electrospun fiber-based biomaterials, many of these do not provide information on their interactions with eukaryotic and bacterial cells. The main aim of this study was to develop antibacterial drug-loaded porous biocompatible polycaprolactone (PCL) fiber scaffolds mimicking the native extracellular matrix for wound healing purposes. Mech. property evaluation and different biorelevant tests were conducted in order to understand the structure-activity relationships and reveal how the surface porosity of fibers and the fiber diam. affect the scaffold interactions with the living bacterial and eukaryotic fibroblast cells. Cell migration and proliferation assays and antibiofilm assays enabled us to enlighten the biocompatibility and safety of fiber scaffolds and their suitability to be used as scaffolds for the treatment of infected wounds. Here, we report that porous PCL microfiber scaffolds obtained using electrospinning at high relative humidity served as the best surfaces for fibroblast attachment and growth compared to the nonporous microfiber or nonporous nanofiber PCL scaffolds. Porous chloramphenicol-loaded microfiber scaffolds were more elastic compared to nonporous scaffolds and had the highest antibiofilm activity. The results indicate that in addn. to the fiber diam. and fiber scaffold porosity, the single-fiber surface porosity and its effect on drug release, mech. properties, cell viability, and antibiofilm activity need to be understood when developing antibacterial biocompatible scaffolds for wound healing applications. We show that pores on single fibers within an electrospun scaffold, in addn. to nano- and microscale diam. of the fibers, change the living cell-fiber interactions affecting the antibiofilm efficacy and biocompatibility of the scaffolds for the local treatment of wounds.
- 15Bhattacharjee, A.; Khan, M.; Kleiman, M.; Hochbaum, A. I. Effects of Growth Surface Topography on Bacterial Signaling in Coculture Biofilms. ACS Appl. Mater. Interfaces 2017, 9, 18531– 18539, DOI: 10.1021/acsami.7b0422315Effects of Growth Surface Topography on Bacterial Signaling in Coculture BiofilmsBhattacharjee, Arunima; Khan, Mughees; Kleiman, Maya; Hochbaum, Allon I.ACS Applied Materials & Interfaces (2017), 9 (22), 18531-18539CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Bacteria form interface-assocd. communities, called biofilms, often comprising multiple species. Biofilms can be detrimental or beneficial in medical, industrial, and technol. settings, and their stability and function are detd. by interspecies communication via specific chem. signaling or metabolite exchange. The deterministic control of biofilm development, behavior, and properties remains an unmet challenge limiting the ability to inhibit formation of detrimental biofilms in biomedical settings and promote the growth of beneficial biofilms in biotechnol. applications. Here, the authors describe the development of growth surfaces that promote the growth of commensal Escherichia coli instead of the opportunistic pathogen Pseudomonas aeruginosa. Periodically patterned growth surfaces induced robust morphol. changes in surface-assocd. E. coli biofilms and influenced the antibiotic susceptibilities of E. coli and P. aeruginosa biofilms. Changes in the biofilm architecture resulted in the accumulation of a metabolite, indole, which controlled competition dynamics between the two species. The results show that the surface on which a biofilm grows has important implications for species colonization, growth, and persistence when exposed to antibiotics.
- 16Peeters, E.; Nelis, H. J.; Coenye, T. Comparison of Multiple Methods for Quantification of Microbial Biofilms Grown in Microtiter Plates. J. Microbiol. Methods 2008, 72, 157– 165, DOI: 10.1016/j.mimet.2007.11.01016Comparison of multiple methods for quantification of microbial biofilms grown in mictotiter platesPeeters, Elke; Nelis, Hans J.; Coenye, TomJournal of Microbiological Methods (2008), 72 (2), 157-165CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier B.V.)In the present study six assays for the quantification of biofilms formed in 96-well microtiter plates were optimized and evaluated: the crystal violet (CV) assay, the Syto9 assay, the fluorescein diacetate (FDA) assay, the resazurin assay, the XTT assay and the di-Me methylene blue (DMMB) assay. Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, Propionibacterium acnes and Candida albicans were used as test organisms. In general, these assays showed a broad applicability and a high repeatability for most isolates. In addn., the estd. nos. of CFUs present in the biofilms show limited variations between the different assays. Nevertheless, our data show that some assays are less suitable for the quantification of biofilms of particular isolates (e.g. the CV assay for P. aeruginosa).
- 17Azeredo, J.; Azevedo, N. F.; Briandet, R.; Cerca, N.; Coenye, T.; Costa, A. R.; Desvaux, M.; di Bonaventura, G.; Hébraud, M.; Jaglic, Z.; Kačániová, M.; Knøchel, S.; Lourenço, A.; Mergulhão, F.; Meyer, R. L.; Nychas, G.; Simões, M.; Tresse, O.; Sternberg, C. Critical Review on Biofilm Methods. Crit. Rev. Microbiol. 2017, 43, 313– 351, DOI: 10.1080/1040841X.2016.120814617Critical review on biofilm methodsAzeredo, Joana; Azevedo, Nuno F.; Briandet, Romain; Cerca, Nuno; Coenye, Tom; Costa, Ana Rita; Desvaux, Mickael; Di Bonaventura, Giovanni; Hebraud, Michel; Jaglic, Zoran; Kacaniova, Miroslava; Knoechel, Susanne; Lourenco, Analia; Mergulhao, Filipe; Meyer, Rikke Louise; Nychas, George; Simoes, Manuel; Tresse, Odile; Sternberg, ClausCritical Reviews in Microbiology (2017), 43 (3), 313-351CODEN: CRVMAC; ISSN:1040-841X. (Taylor & Francis Ltd.)Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a neg. impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiol., structure and compn. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, compn. and physiol. are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a crit. perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.
- 18Demir, D.; Özdemir, S.; Ceylan, S.; Yalcin, M. S.; Sakım, B.; Bölgen, N. Electrospun Composite Nanofibers Based on Poly (ε-Caprolactone) and Styrax Liquidus (Liquidambar Orientalis Miller) as a Wound Dressing: Preparation, Characterization, Biological and Cytocompatibility Results. J. Polym. Environ. 2022, 30, 2462– 2473, DOI: 10.1007/s10924-022-02376-718Electrospun Composite Nanofibers Based on Poly (ε-Caprolactone) and Styrax Liquidus (Liquidambar orientalis Miller) as a Wound Dressing: Preparation, Characterization, Biological and Cytocompatibility ResultsDemir, Didem; Ozdemir, Sadin; Ceylan, Seda; Yalcin, M. Serkan; Sakim, Burcu; Bolgen, NimetJournal of Polymers and the Environment (2022), 30 (6), 2462-2473CODEN: JPENFW; ISSN:1572-8919. (Springer)In this study, styrax liquidus (sweet gum balsam) extd. from Liquidambar orientalis Mill. incorporated PCL fibrous scaffolds were prepd. using the electrospinning method. The effects of the styrax liquidus content on the prepd. scaffolds were investigated using different physico-chem. and morphol. analyses. Then, the styrax-loaded nanofibers were examd. for their antioxidant activity, anti-biofilm, metal chelating, antimicrobial and DNA cleavage properties. The results obtained from these studies showed that the nanofibers exhibited effective biol. activity depending on the wt. ratio of the styrax liquidus. In light of the data obtained from the characterization and biol. studies, a sample with high ratio of balsam was built for detg. the cytocompatibility anal. in vitro. The cytotoxicity studies of the selected membrane were conducted using mouse embryonic fibroblast cells. The fibrous scaffolds lead to increase the cell no. as a result of high viability. According to the results, we propose a novel biocompatible electrospun hybrid scaffold with antioxidant and antimicrobial properties that can be used as wound healing material for potential tissue engineering applications.
- 19Serbezeanu, D.; Vlad-Bubulac, T.; Rusu, D.; Pircalabioru, G. G.; Samoilă, I.; Dinescu, S.; Aflori, M. Functional Polyimide-Based Electrospun Fibers for Biomedical Application. Materials 2019, 12, 3201, DOI: 10.3390/ma1219320119Functional polyimide-based electrospun fibers for biomedical applicationSerbezeanu, Diana; Vlad-Bubulac, Tachita; Rusu, Daniela; Pircalabioru, Gratiela Gradisteanu; Samoila, Iuliana; Dinescu, Sorina; Aflori, MagdalenaMaterials (2019), 12 (19), 3201CODEN: MATEG9; ISSN:1996-1944. (MDPI AG)The current study focuses on the application of cytotoxicity tests upon one membrane matrix based on electrospun polyimide fibers, appealing for biomedical application, such as scaffolds for cell growth, patches or meshes for wound healing, etc. Assays were performed in order to det. the viability and proliferation of L929 murine fibroblasts after they were kept in direct contact with the studied electrospun polyimide fibers. Increased cell viability and proliferation were detected for cells seeded on electrospun polyimide fibers membrane, in comparison with the control system, either after two or six days of evaluation. The no. of live cells was higher on the studied material compared to the control, after two and six days of cell seeding. The tendency of the cells to proliferate on the electrospun polyimide fibers was revealed by confocal microscopy. The morphol. stability of electrospun polyimide membrane was evaluated by SEM observation, after immersion of the samples in phosphate buffer saline soln. (PBS, 7.4 at 37 °C) at various time intervals. Addnl., the easy prodn. of electrospun polyimide fibers can facilitate the development of these types of matrixes into specific biomedical applications in the future.
- 20Gupta, S.; Prasad, P.; Roy, A.; Alam, M. M.; Ahmed, I.; Bit, A. Metallic Ion-Based Graphene Oxide Functionalized Silk Fibroin-Based Dressing Promotes Wound Healing via Improved Bactericidal Outcomes and Faster Re-Epithelization. Biomed. Mater. 2022, 17, 035010 DOI: 10.1088/1748-605X/ac64ddThere is no corresponding record for this reference.
- 21Parsons, D.; Meredith, K.; Rowlands, V. J.; Short, D.; Metcalf, D. G.; Bowler, P. G. Enhanced Performance and Mode of Action of a Novel Antibiofilm Hydrofiber Wound Dressing. Biomed. Res. Int. 2016, 2016, 7676471 DOI: 10.1155/2016/7616471There is no corresponding record for this reference.
- 22Hill, K. E.; Malic, S.; McKee, R.; Rennison, T.; Harding, K. G.; Williams, D. W.; Thomas, D. W. An in Vitro Model of Chronic Wound Biofilms to Test Wound Dressings and Assess Antimicrobial Susceptibilities. J. Antimicrob. Chemother. 2010, 65, 1195– 1206, DOI: 10.1093/jac/dkq10522An in vitro model of chronic wound biofilms to test wound dressings and assess antimicrobial susceptibilitiesHill, Katja E.; Malic, Sladjana; McKee, Ruth; Rennison, Tracy; Harding, Keith G.; Williams, David W.; Thomas, David W.Journal of Antimicrobial Chemotherapy (2010), 65 (6), 1195-1206CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)The targeted disruption of biofilms in chronic wounds is an important treatment strategy and the subject of intense research. In the present study, an in vitro model of chronic wound biofilms was developed to assess the efficacy of antimicrobial treatments for use in the wound environment. Using chronic wound isolates, assays of bacterial coaggregation established that aerobic and anaerobic wound bacteria were able to coaggregate and form biofilms. A const. depth film fermenter (CDFF) was used to develop wound biofilms in vitro, which were analyzed using light microscopy and SEM. The susceptibility of bacteria within these biofilms was examd. in response to the most frequently prescribed chronic wound' antibiotics and a series of iodine- and silver-contg. com. antimicrobial products and lactoferrin. Defined biofilms were rapidly established within 1-2 days. Antibiotic treatment demonstrated that mixed Pseudomonas and Staphylococcus biofilms were not affected by ciprofloxacin (5 mg/L) or flucloxacillin (15 mg/L), even at concns. equiv. to twice the obsd. peak serum levels. The results contrasted with the ability of povidone-iodine (1%) to disrupt the wound biofilm; an effect that was particularly pronounced in the dressing testing where iodine-based dressings completely disrupted established 7 day biofilms. In contrast, only two of six silver-contg. dressings exhibited any effect on 3 day biofilms, with no effect on 7 day biofilms. Thus, this wound model emphasizes the potential role of the biofilm phenotype in the obsd. resistance to antibiotic therapies that may occur in chronic wounds in vivo.
- 23Dai, T.; Kharkwal, G. B.; Tanaka, M.; Huang, Y. Y.; Bil de Arce, V. J.; Hamblin, M. R. Animal Models of External Traumatic Wound Infections. Virulence 2011, 2, 296– 315, DOI: 10.4161/viru.2.4.1684023Animal models of external traumatic wound infectionsDai Tianhong; Kharkwal Gitika B; Tanaka Masamitsu; Huang Ying-Ying; Bil de Arce Vida J; Hamblin Michael RVirulence (2011), 2 (4), 296-315 ISSN:.BACKGROUND: Despite advances in traumatic wound care and management, infections remain a leading cause of mortality,morbidity and economic disruption in millions of wound patients around the world. Animal models have become standard tools for studying a wide array of external traumatic wound infections and testing new antimicrobial strategies. RESULTS: Animal models of external traumatic wound infections reported by different investigators vary in animal species used, microorganism strains, the number of microorganisms applied, the size of the wounds and for burn infections, the length of time the heated object or liquid is in contact with the skin. METHODS: This review covers experimental infections in animal models of surgical wounds, skin abrasions, burns, lacerations,excisional wounds and open fractures. CONCLUSIONS: As antibiotic resistance continues to increase,more new antimicrobial approaches are urgently needed.These should be tested using standard protocols for infections in external traumatic wounds in animal models.
- 24Seth, A. K.; Geringer, M. R.; Gurjala, A. N.; Abercrombie, J. A.; Chen, P.; You, T.; Hong, S. J.; Galiano, R. D.; Mustoe, T. A.; Leung, K. P. Understanding the Host Inflammatory Response to Wound Infection: An in Vivo Study of Klebsiella Pneumoniae in a Rabbit Ear Wound Model. Wound Repair Regen. 2012, 20, 214– 225, DOI: 10.1111/j.1524-475X.2012.00764.x24Understanding the host inflammatory response to wound infection: an in vivo study of Klebsiella pneumoniae in a rabbit ear wound modelSeth Akhil K; Geringer Matthew R; Gurjala Anandev N; Abercrombie Johnathan A; Chen Ping; You Tao; Hong Seok J; Galiano Robert D; Mustoe Thomas A; Leung Kai PWound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society (2012), 20 (2), 214-25 ISSN:.Wound infection development is critically dependent on the complex interactions between bacteria and host. Klebsiella pneumoniae has become an increasingly common wound pathogen, but its natural history within wounds has never been studied. Using a validated, in vivo rabbit ear model, wounds were inoculated with K. pneumoniae at different concentrations (102-107 colony-forming units) with measurement of viable and nonviable bacterial counts, histological wound-healing parameters, and host inflammatory gene expression at multiple time points postinoculation (48, 96, and 240 hours). Bacteria and wound morphologies were evaluated with scanning electron microscopy. Comparable experiments were performed in ischemic ears to model immune response impairment. All wounds, despite different inoculants, equilibrated to similar bacterial concentrations by 96 hours. With a 106 colony-forming units inoculant, wounds at 240 hours showed decreased bacterial counts (p < 0.01), with a corresponding improvement in healing (p < 0.01) and a decrease in inflammatory response (p < 0.05). In contrast, ischemic wounds revealed impaired inflammatory gene expression (p < 0.05) resulting in higher steady-state bacterial concentrations (p < 0.01), impaired healing (p < 0.05), and biofilm formation on scanning electron microscopy. We conclude that a normal inflammatory response can effectively stabilize and overcome a K. pneumoniae wound infection. An impaired host cannot control this bacterial burden, preventing adequate healing while allowing bacteria to establish a chronic presence. Our novel study quantitatively validates the host immune response as integral to wound infection dynamics.
- 25Mendes, J. J.; Leandro, C. I.; Bonaparte, D. P.; Pinto, A. L. A Rat Model of Diabetic Wound Infection for the Evaluation of Topical Antimicrobial Therapies. Comp. Med. 2012, 62, 37– 4825A rat model of diabetic wound infection for the evaluation of topical antimicrobial therapiesMendes, Joao J.; Leandro, Clara I.; Bonaparte, Dolores P.; Pinto, Andreia L.Comparative Medicine (2012), 62 (1), 37-48CODEN: COMEFT; ISSN:1532-0820. (American Association for Laboratory Animal Science)Diabetes mellitus is an epidemic multisystemic chronic disease that frequently is complicated by complex wound infections. Innovative topical antimicrobial therapy agents are potentially useful for multimodal treatment of these infections. However, an appropriately standardized in vivo model is currently not available to facilitate the screening of these emerging products and their effect on wound healing. To develop such a model, we analyzed, tested, and modified published models of wound healing. We optimized various aspects of the model, including animal species, diabetes induction method, hair removal technique, splint and dressing methods, the control of unintentional bacterial infection, sampling methods for the evaluation of bacterial burden, and aspects of the microscopic and macroscopic assessment of wound healing, all while taking into consideration animal welfare and the "3Rs" principle. We thus developed a new wound infection model in rats that is optimized for testing topical antimicrobial therapy agents. This model accurately reproduces the pathophysiol. of infected diabetic wound healing and includes the current std. treatment (i.e., debridement). The numerous benefits of this model include the ready availability of necessary materials, simple techniques, high reproducibility, and practicality for expts. with large sample sizes. Furthermore, given its similarities to infected-wound healing and treatment in humans, our new model can serve as a valid alternative for applied research.
- 26Geremias, T. C.; Batistella, M. A.; Magini, R. R. S.; Selene, S. M. A.; Franco, C.; Barbosa, L. C. A.; Pereira, U. A.; Hinestroza, J. P.; Pimenta, A. L.; Ulson de Souza, A. A. Functionalization of Poly(Lactic-Co-Glycolic Acid) Nanofibrous Membranes with Antibiofilm Compounds. Can. J. Chem. Eng. 2022, 100, S5– S15, DOI: 10.1002/cjce.2411526Functionalization of poly(lactic-co-glycolic acid) nanofibrous membranes with antibiofilm compoundsGeremias, Thaise C.; Batistella, Marcos A.; Magini, Ricardo R. S.; Guelli U. de Souza, Selene M. A.; Franco, Cesar V.; Barbosa, Luiz C. A.; Pereira, Ulisses A.; Hinestroza, Juan P.; Pimenta, Andrea L.; Ulson de Souza, Antonio A.Canadian Journal of Chemical Engineering (2022), 100 (S1), S5-S15CODEN: CJCEA7; ISSN:0008-4034. (John Wiley & Sons, Inc.)The aim of this research was to investigate the activity of functionalized PLGA electrospun membranes in preventing Streptococcus mutans biofilm formation. PLGA nanofibres were functionalized with the additives Melaleuca alternifolia and Coffea canephora essential oils, furan-2(5H)-one, and a novel synthetic butyrolactam, in three concns. (0.002%, 0.004%, and 0.008% w/v). Samples were characterized by SEM, FTIR-ATR, and GC-MS and exposed to S. mutans cultures. Planktonic growth was detd. following a 24 and 48-h incubation period by spectrophotometry and the biofilm formation was evaluated by counting colony forming units. Cytotoxicity of the new biomaterials was assessed by MTS assay, through the quantification of viable placenta-derived stem cells grown over the functionalized nanofibrous membranes. Observation of the electrospun membranes on SEM images revealed the smooth and bead-free morphol. of the nanofibres. No solvent residues were obsd. by FTIR-ATR, but the GC-MS results showed that N,N-dimethylformamide and dimethylsulfoxide were present as residues in the membranes after functionalization. Functionalization reduced bacterial attachment to membrane surfaces, with best results being obtained for M. alternifolia essential oil, furanone, and butyrolactam. Cytotoxicity results showed that furan-2(5H)-one-functionalized membranes demonstrated no statistically significant difference in the cell viability compared to the control membranes.
- 27Brackman, G.; Cos, P.; Maes, L.; Nelis, H. J.; Coenye, T. Quorum Sensing Inhibitors Increase the Susceptibility of Bacterial Biofilms to Antibiotics in Vitro and in Vivo. Antimicrob. Agents Chemother. 2011, 55, 2655– 2661, DOI: 10.1128/AAC.00045-1127Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivoBrackman, Gilles; Cos, Paul; Maes, Louis; Nelis, Hans J.; Coenye, TomAntimicrobial Agents and Chemotherapy (2011), 55 (6), 2655-2661CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)Although the exact role of quorum sensing (QS) in various stages of biofilm formation, maturation, and dispersal and in biofilm resistance is not entirely clear, the use of QS inhibitors (QSI) was proposed as a potential antibiofilm strategy. We have investigated whether QSI enhance the susceptibility of bacterial biofilms to treatment with conventional antimicrobial agents. The QSI used in our study target the acyl-homoserine lactone-based QS system present in Pseudomonas aeruginosa and Burkholderia cepacia complex organisms (baicalin hydrate, cinnamaldehyde) or the peptide-based system present in Staphylococcus aureus (hamamelitannin). The effect of tobramycin (P. aeruginosa, B. cepacia complex) and clindamycin or vancomycin (S. aureus), alone or in combination with QSI, was evaluated in various in vitro and in vivo biofilm model systems, including two invertebrate models and one mouse pulmonary infection model. In vitro the combined use of an antibiotic and a QSI generally resulted in increased killing compared to killing by an antibiotic alone, although redns. were strain and model dependent. A significantly higher fraction of infected Galleria mellonella larvae and Caenorhabditis elegans survived infection following combined treatment, compared to treatment with an antibiotic alone. Finally, the combined use of tobramycin and baicalin hydrate reduced the microbial load in the lungs of BALB/c mice infected with Burkholderia cenocepacia more than tobramycin treatment alone. Our data suggest that QSI may increase the success of antibiotic treatment by increasing the susceptibility of bacterial biofilms and/or by increasing host survival following infection.
- 28Werthén, M.; Henriksson, L.; Jensen, P. Ø.; Sternberg, C.; Givskov, M.; Bjarnsholt, T. An in Vitro Model of Bacterial Infections in Wounds and Other Soft Tissues. Apmis 2010, 118, 156– 164, DOI: 10.1111/j.1600-0463.2009.02580.x28An in vitro model of bacterial infections in wounds and other soft tissuesWerthen Maria; Henriksson Lina; Jensen Peter Ostrup; Sternberg Claus; Givskov Michael; Bjarnsholt ThomasAPMIS : acta pathologica, microbiologica, et immunologica Scandinavica (2010), 118 (2), 156-64 ISSN:.There is growing evidence that bacteria play a crucial role in the persistence of chronic wounds. These bacteria are most probably present in polymer-embedded aggregates that represent the biofilm mode of growth. Much work has been carried out to study the development of biofilms in vitro, in particular in attachment to solid surfaces. The observations from the chronic wounds indicate that the bacteria are not attached to a solid surface. Consequently, a new in vitro model is required to investigate biofilms in more wound-like settings. This study describes such a novel in vitro model, with bacteria growing as biofilm aggregates in a collagen gel matrix with serum protein mimicking the wound bed of chronic wounds. The model was verified to comprise important hallmarks of biofilms such as the bacterial embedment in a matrix and increased antibiotic tolerance. Furthermore, we have verified the relevance of the model by comparing the organization of the bacteria in the model with the organization of the bacteria in a real chronic wound. We believe that we have developed an important new model for investigating bacterial biofilms in chronic wounds. This model may be used to study biofilm development in chronic wounds and to develop novel diagnostic tools as well as treatment strategies.
- 29McMahon, R. E.; Salamone, A. B.; Poleon, S.; Bionda, N.; Salamone, J. C. Efficacy of Wound Cleansers on Wound-Specific Organisms Using in Vitro and Ex Vivo Biofilm Models. Wound Manag. Prev. 2020, 66, 31– 42, DOI: 10.25270/wmp.2020.11.314229Efficacy of Wound Cleansers on Wound-Specific Organisms Using In Vitro and Ex Vivo Biofilm ModelsMcMahon Rebecca E; Salamone Ann Beal; Poleon Suprena; Salamone Joseph C; Bionda NinaWound management & prevention (2020), 66 (11), 31-42 ISSN:.Biofilms are believed to be a source of chronic inflammation in non-healing wounds. PURPOSE: In this study, the pre-clinical anti-biofilm efficacy of several wound cleansers was examined using the Calgary minimum biofilm eradication concentration (MBEC) and ex vivo porcine dermal explant (PDE) models on Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans biofilms. METHODS: A surfactant-based cleanser and antimicrobial-based cleansers containing ionic silver, hypochlorous acid (HOCl), sodium hypochlorite (NaOCl), and polyhexamethylene biguanide (PHMB) were tested on the MBEC model biofilms with a 10-minute application time. Select cleansers were then tested on the mature PDE biofilms with 10-minute applications followed by the application of cleanser-soaked gauze. The PDE model was further expanded to include single and daily applications of the cleansers to mimic daily and 72-hour dressing changes. RESULTS: In the MBEC model, PHMB- and HOCl-based cleansers reduced immature MRSA, C albicans, and P aeruginosa biofilm regrowth by > 3× when compared with silver, surfactant, and saline cleansers. The major differences could be elucidated in the PDE model in which, after daily application, 1 PHMB-based cleanser showed a statistically significant reduction (3-8 CFU/mL log reduction) in all mature biofilms tested, while a NaOCl-based cleanser showed significant reduction in 2 microorganisms (3-5 CFU/mL log reduction, P aeruginosa and MRSA).The other PHMB-based cleanser showed a statistically significant 3 log CFU/mL reduction in P aeruginosa. The remaining cleansers showed no statistically significant difference from the saline control. CONCLUSION: Results confirm that there are model-dependent differences in the outcomes of these studies, suggesting the importance of model selection for product screening. The results indicate that 1 PHMB-based cleanser was effective in reducing mature P aeruginosa, MRSA, and C albicans biofilms and that sustained antimicrobial presence was necessary to reduce or eliminate these mature biofilms.
- 30Yang, Q.; Phillips, P. L.; Sampson, E. M.; Progulske-Fox, A.; Jin, S.; Antonelli, P.; Schultz, G. S. Development of a Novel Ex Vivo Porcine Skin Explant Model for the Assessment of Mature Bacterial Biofilms. Wound Repair Regen. 2013, 21, 704– 714, DOI: 10.1111/wrr.1207430Development of a novel ex vivo porcine skin explant model for the assessment of mature bacterial biofilmsYang Qingping; Phillips Priscilla L; Sampson Edith M; Progulske-Fox Ann; Jin Shouguang; Antonelli Patrick; Schultz Gregory SWound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society (2013), 21 (5), 704-14 ISSN:.Bacterial biofilms have been proposed to be a major factor contributing to the failure of chronic wounds to heal because of their increased tolerance to antimicrobial agents and the prolonged inflammation they cause. Phenotypic characteristics of bacterial biofilms vary depending on the substratum to which they attach, the nutritional environment, and the microorganisms within the biofilm community. To develop an ex vivo biofilm model that more closely mimics biofilms in chronic skin wounds, we developed an optimal procedure to grow mature biofilms on a central partial-thickness wound in 12-mm porcine skin explants. Chlorine gas produced optimal sterilization of explants while preserving histological properties of the epidermis and dermis. Pseudomonas aeruginosa and Staphylococcus aureus developed mature biofilms after 3 days that had dramatically increased tolerance to gentamicin and oxacillin (∼100× and 8,000× minimal inhibitory concentration, respectively) and to sodium hypochlorite (0.6% active chlorine). Scanning electron microscopy and confocal microscopy verified extensive exopolymeric biofilm structures on the explants. Despite a significant delay, a ΔlasI quorum-sensing mutant of P. aeruginosa developed biofilm as antibiotic-tolerant as wild-type after 3 days. This ex vivo model simulates growth of biofilms on skin wounds and provides an accurate model to assess effects of antimicrobial agents on mature biofilms.
- 31Steinstraesser, L.; Sorkin, M.; Niederbichler, A. D.; Becerikli, M.; Stupka, J.; Daigeler, A.; Kesting, M. R.; Stricker, I.; Jacobsen, F.; Schulte, M. A Novel Human Skin Chamber Model to Study Wound Infection Ex Vivo. Arch. Dermatol. Res. 2010, 302, 357– 365, DOI: 10.1007/s00403-009-1009-831A novel human skin chamber model to study wound infection ex vivoSteinstraesser Lars; Sorkin M; Niederbichler A D; Becerikli M; Stupka J; Daigeler A; Kesting M R; Stricker I; Jacobsen F; Schulte MArchives of dermatological research (2010), 302 (5), 357-65 ISSN:.Wound infections with multi-drug resistant bacteria increase morbidity and mortality and have considerable socioeconomic impact. They can lead to impaired wound healing, resulting in rising treatment costs. The aim of this study was to investigate an ex vivo human wound infection model. Human full-thickness skin from the operating room (OR) was placed into the Bo-Drum and cultivated for 7 days in an air-liquid interphase. On day 8, the skin was inoculated with either (1) Pseudomonas aeruginosa, (2) Staphylococcus aureus (10(5) CFU, n = 3) or (3) carrier control. 1, 3 and 7 days after inoculation colony forming units in the tissue/media were determined and cytokine expression was quantified. A reliable and reproducible wound infection could be established for 7 days. At this time point, 1.8 x 10(8) CFU/g tissue of P. aeruginosa and 2 x 10(7) CFU/g tissue of S. aureus were detected. Immunohistochemical analysis demonstrated bacterial infection and epidermolysis in infected skin. RT-PCR analysis exhibited a significant induction of proinflammatory cytokines after infection. The BO-drum is a robust, easy-to-use, sterilizable and reusable ex vivo full-skin culture system. For investigation of wound infection, treatment and healing, the BO-drum presents a convenient model and may help to standardize wound research.
- 32Wilkinson, H. N.; McBain, A. J.; Stephenson, C.; Hardman, M. J. Comparing the Effectiveness of Polymer Debriding Devices Using a Porcine Wound Biofilm Model. Adv. Wound Care 2016, 5, 475– 485, DOI: 10.1089/wound.2015.068332Comparing the Effectiveness of Polymer Debriding Devices Using a Porcine Wound Biofilm ModelWilkinson Holly N; Hardman Matthew J; McBain Andrew J; Stephenson ChristianAdvances in wound care (2016), 5 (11), 475-485 ISSN:2162-1918.Objective: Debridement to remove necrotic and/or infected tissue and promote active healing remains a cornerstone of contemporary chronic wound management. While there has been a recent shift toward less invasive polymer-based debriding devices, their efficacy requires rigorous evaluation. Approach: This study was designed to directly compare monofilament debriding devices to traditional gauze using a wounded porcine skin biofilm model with standardized application parameters. Biofilm removal was determined using a surface viability assay, bacterial counts, histological assessment, and scanning electron microscopy (SEM). Results: Quantitative analysis revealed that monofilament debriding devices outperformed the standard gauze, resulting in up to 100-fold greater reduction in bacterial counts. Interestingly, histological and morphological analyses suggested that debridement not only removed bacteria, but also differentially disrupted the bacterially-derived extracellular polymeric substance. Finally, SEM of post-debridement monofilaments showed structural changes in attached bacteria, implying a negative impact on viability. Innovation: This is the first study to combine controlled and defined debridement application with a biologically relevant ex vivo biofilm model to directly compare monofilament debriding devices. Conclusion: These data support the use of monofilament debriding devices for the removal of established wound biofilms and suggest variable efficacy towards biofilms composed of different species of bacteria.
- 33Andersson, M.; Madsen, L. B.; Schmidtchen, A.; Puthia, M. Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations. Int. J. Mol. Sci. 2021, 22, 1– 16, DOI: 10.3390/ijms22095045There is no corresponding record for this reference.
- 34Wilkinson, H. N.; Iveson, S.; Catherall, P.; Hardman, M. J. A Novel Silver Bioactive Glass Elicits Antimicrobial Efficacy against Pseudomonas aeruginosa and Staphylococcus aureus in an Ex Vivo Skin Wound Biofilm Model. Front. Microbiol. 2018, 9, 1– 16, DOI: 10.3389/fmicb.2018.01450There is no corresponding record for this reference.
- 35Siimon, K.; Reemann, P.; Põder, A.; Pook, M.; Kangur, T.; Kingo, K.; Jaks, V.; Mäeorg, U.; Järvekülg, M. Effect of Glucose Content on Thermally Cross-Linked Fibrous Gelatin Scaffolds for Tissue Engineering. Mater. Sci. Eng., C 2014, 42, 538– 545, DOI: 10.1016/j.msec.2014.05.07535Effect of glucose content on thermally cross-linked fibrous gelatin scaffolds for tissue engineeringSiimon, Kaido; Reemann, Paula; Poder, Annika; Pook, Martin; Kangur, Triin; Kingo, Kulli; Jaks, Viljar; Maeorg, Uno; Jarvekulg, MartinMaterials Science & Engineering, C: Materials for Biological Applications (2014), 42 (), 538-545CODEN: MSCEEE; ISSN:0928-4931. (Elsevier B.V.)Thermally cross-linked glucose-contg. electrospun gelatin meshes were studied as possible cell substrate materials. FTIR anal. was used to study the effect of glucose on crosslinking reactions. It was found that the presence of glucose increases the extent of crosslinking of fibrous gelatin scaffolds, which in return dets. scaffold properties and their usability in tissue engineering applications. Easy to handle fabric-like scaffolds were obtained from blends contg. up to 15% glucose. Maximum extent of crosslinking was reached at nearly 20% glucose content. Crosslinking effectively resulted in decreased soly. and increased resistance to enzymic degrdn. Preliminary short-term cell culture expts. indicate that such thermally cross-linked gelatin-glucose scaffolds are suitable for tissue engineering applications.
- 36Siimon, K.; Siimon, H.; Järvekülg, M. Mechanical Characterization of Electrospun Gelatin Scaffolds Cross-Linked by Glucose. J. Mater. Sci. Mater. Med. 2015, 26, 1– 9, DOI: 10.1007/s10856-014-5375-136Mechanical characterization of electrospun gelatin scaffolds cross-linked by glucoseSiimon, Kaido; Siimon, Hele; Jarvekulg, MartinJournal of Materials Science: Materials in Medicine (2015), 26 (1), 1-9CODEN: JSMMEL; ISSN:0957-4530. (Springer)Nanofibrous gelatin scaffolds were prepd. by electrospinning from aq. acetic acid and cross-linked thermally by glucose. The effect of the amt. of glucose used as crosslinking agent on the mech. properties of gelatin fibers was studied in this paper. The elastic modulus of gelatin fibers cross-linked by glucose was detd. by modeling the behavior of the meshes during tensile test. The model draws connections between the elastic moduli of a fibrous mesh and the fiber material and allows evaluation of elastic modulus of the fiber material. It was found that crosslinking by glucose increases the elastic modulus of gelatin fibers from 0.3 GPa at 0 % glucose content to 1.1 GPa at 15 % glucose content. This makes fibrous gelatin scaffolds cross-linked by glucose a promising material for biomedical applications.
- 37Palo, M.; Kogermann, K.; Laidmäe, I.; Meos, A.; Preis, M.; Heinämäki, J.; Sandler, N. Development of Oromucosal Dosage Forms by Combining Electrospinning and Inkjet Printing. Mol. Pharmaceutics 2017, 14, 808– 820, DOI: 10.1021/acs.molpharmaceut.6b0105437Development of Oromucosal Dosage Forms by Combining Electrospinning and Inkjet PrintingPalo, Mirja; Kogermann, Karin; Laidmae, Ivo; Meos, Andres; Preis, Maren; Heinamaki, Jyrki; Sandler, NiklasMolecular Pharmaceutics (2017), 14 (3), 808-820CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)Printing technol. has been shown to enable flexible fabrication of solid dosage forms for personalized drug therapy. Several methods can be applied for tailoring the properties of the printed pharmaceuticals. In this study, the use of electrospun fibrous substrates in the fabrication of inkjet-printed dosage forms was investigated. A single-drug formulation with lidocaine hydrochloride (LH) and a combination drug system contg. LH and piroxicam (PRX) for oromucosal administration were prepd. The LH was deposited on the electrospun and cross-linked gelatin substrates by inkjet printing, whereas PRX was incorporated within the substrate fibers during electrospinning. The solid state anal. of the electrospun substrates showed that PRX was in an amorphous state within the fibers. Furthermore, the results indicated the entrapment and solidification of the dissolved LH within the fibrous gelatin matrix. The printed drug amt. (2-3 mg) was in good correlation with the theor. dose calcd. based on the printing parameters. However, a noticeable degrdn. of the printed LH was detected after a few months. An immediate release (over 85% drug release after 8 min) of both drugs from the printed dosage forms was obsd. In conclusion, the prepd. electrospun gelatin scaffolds were shown to be suitable substrates for inkjet printing of oromucosal formulations. The combination of electrospinning and inkjet printing allowed the prepn. of a dual drug system.
- 38Preem, L.; Mahmoudzadeh, M.; Putrinš, M.; Meos, A.; Laidmäe, I.; Romann, T.; Aruväli, J.; Härmas, R.; Koivuniemi, A.; Bunker, A.; Tenson, T.; Kogermann, K. Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-Implications for Designing Electrospun Drug Delivery Systems Countering Wound Infection. Mol. Pharmaceutics 2017, 14, 4417– 4430, DOI: 10.1021/acs.molpharmaceut.7b0052438Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-Implications for Designing Electrospun Drug Delivery Systems Countering Wound InfectionPreem, Liis; Mahmoudzadeh, Mohammad; Putrins, Marta; Meos, Andres; Laidmae, Ivo; Romann, Tavo; Aruvali, Jaan; Harmas, Riinu; Koivuniemi, Artturi; Bunker, Alex; Tenson, Tanel; Kogermann, KarinMolecular Pharmaceutics (2017), 14 (12), 4417-4430CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)Antibacterial drug-loaded electrospun nano- and microfibrous dressings are of major interest as novel topical drug delivery systems in wound care. In this study, chloramphenicol (CAM)-loaded polycaprolactone (PCL) and PCL/poly(ethylene oxide) (PEO) fiber mats were electrospun and characterized in terms of morphol., drug distribution, physicochem. properties, drug release, swelling, cytotoxicity, and antibacterial activity. Computational modeling together with physicochem. anal. helped to elucidate possible interactions between the drug and carrier polymers. Strong interactions between PCL and CAM together with hydrophobicity of the system resulted in much slower drug release compared to the hydrophilic ternary system of PCL/PEO/CAM. Cytotoxicity studies confirmed safety of the fiber mats to murine NIH 3T3 cells. Disk diffusion assay demonstrated that both fast and slow release fiber mats reached effective concns. and had similar antibacterial activity. A biofilm formation assay revealed that both blank matrixes are good substrates for the bacterial attachment and formation of biofilm. Importantly, prolonged release of CAM from drug-loaded fibers helps to avoid biofilm formation onto the dressing and hence avoids the treatment failure.
- 39Johnston, C.; Callum, J.; Mohr, J.; Duong, A.; Garibaldi, A.; Simunovic, N.; Ayeni, O. R.; on behalf of the Bioburden Steering Committee; Appleby, A.; Brubaker, S.; Callum, J.; Dowling, G.; Eastlund, T.; Fearon, M.; Germain, M.; Johnston, C.; Lotherington, K.; McTaggart, K.; Mohr, J.; Preiksaitis, J.; Strong, M.; Winters, M.; Young, K.; Zhao, J.; Callum, J.; Cartotto, R.; Davis, I.; Eastlund, T.; Gratzer, P.; Johnston, C.; Merkley, L.; Mohr, J. Disinfection of Human Skin Allografts in Tissue Banking: A Systematic Review Report. Cell Tissue Banking 2016, 17, 585– 592, DOI: 10.1007/s10561-016-9569-239Disinfection of human skin allografts in tissue banking: a systematic review reportJohnston, C.; Callum, J.; Mohr, J.; Duong, A.; Garibaldi, A.; Simunovic, N.; Ayeni, O. R.; on behalf of the Bioburden Steering Committee and Skin Working groupCell and Tissue Banking (2016), 17 (4), 585-592CODEN: CTBAFV; ISSN:1389-9333. (Springer)The use of skin allografts to temporarily replace lost or damaged skin is practiced worldwide. Naturally occurring contamination can be present on skin or can be introduced at recovery or during processing. This contamination can pose a threat to allograft recipients. Bacterial culture and disinfection of allografts are mandated, but the specific practices and methodologies are not dictated by stds. A systematic review of literature from three databases found 12 research articles that evaluated bioburden redn. processes of skin grafts. The use of broad spectrum antibiotics and antifungal agents was the most frequently identified disinfection method reported demonstrating redns. in contamination rates. It was detd. that the greatest redn. in the skin allograft contamination rates utilized 0.1 % peracetic acid or 25 kGy of gamma irradn. at lower temps.
- 40Preem, L.; Vaarmets, E.; Meos, A.; Jõgi, I.; Putrinš, M.; Tenson, T.; Kogermann, K. Effects and Efficacy of Different Sterilization and Disinfection Methods on Electrospun Drug Delivery Systems. Int. J. Pharm. 2019, 567, 118450 DOI: 10.1016/j.ijpharm.2019.11845040Effects and efficacy of different sterilization and disinfection methods on electrospun drug delivery systemsPreem, Liis; Vaarmets, Ebe; Meos, Andres; Jogi, Indrek; Putrins, Marta; Tenson, Tanel; Kogermann, KarinInternational Journal of Pharmaceutics (Amsterdam, Netherlands) (2019), 567 (), 118450CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)Microbiol. quality of a pharmaceutical product is an essential requirement ensuring patient safety, thus effective sterilization/disinfection methods need to be found. The aim of this study was to evaluate the efficacy of different sterilization/disinfection methods on drug-loaded electrospun matrixes and the impact of these treatments on the functionality related characteristics of these matrixes. The sterilization efficacy of gamma-irradn., UV-irradn., in situ generated chlorine gas and low-pressure argon plasma treatment were evaluated on two different chloramphenicol-loaded electrospun matrixes using pristine polycaprolactone (PCL) as a carrier polymer or PCL in combination with polyethylene oxide. Drug stability, solid state properties, morphol., mech. properties, swelling, biodegrdn. and drug release kinetics were studied before and after the treatments. It was shown that all tested methods help to reduce bioburden and only plasma treated matrixes were not sterile. At the same time drug degrdn. after the treatment can be considerable and depends not only on the susceptibility of the drug to degrdn., but also on matrix properties (e.g. the nature of carrier polymers). Even though no morphol. changes were obsd., gamma sterilization increased the hardness and elasticity of PCL matrixes as a result of increased crystallinity of the polymer. Plasma treatment was able to significantly enhance water absorption to otherwise hydrophobic PCL/CAM matrix and had tremendous impact on its drug release kinetics as the drug was instantly released from otherwise prolonged release formulation.
- 41Koransky, J. R.; Allen, S. D.; Dowell, V. R. Use of Ethanol for Selective Isolation of Sporeforming Microorganisms. Appl. Environ. Microbiol. 1978, 35, 762– 765, DOI: 10.1128/aem.35.4.762-765.197841Use of ethanol for selective isolation of sporeforming microorganismsKoransky, Jack R.; Allen, Stephen D.; Dowell, V. R., Jr.Applied and Environmental Microbiology (1978), 35 (4), 762-5CODEN: AEMIDF; ISSN:0099-2240.When mixed cultures contg. spore-forming bacteria were treated with heat or with EtOH [64-17-5], the latter consistently resulted in better recovery of Clostridium and Bacillus species. Both techniques were effective in eliminating vegetative cells. An EtOH concn. >25% and exposure for 45 min or longer were necessary to kill all vegetative cells in mixed-culture samples. EtOH treatment (50% EtOH for 1 h) was effective for isolating spore-forming bacteria from intestinal specimens. Seven different species of Clostridium were the only bacteria isolated from an EtOH-treated specimen of intestinal contents from the large bowel of a patient. Treatment with EtOH for 1 h is apparently an effective technique for selective isolation of spore-forming bacteria from mixed cultures and certain types of clin. specimens.
- 42Lipp, C.; Kirker, K.; Agostinho, A.; James, G.; Stewart, P. Testing Wound Dressings Using an in Vitro Wound Model. J. Wound Care 2010, 19, 220– 226, DOI: 10.12968/jowc.2010.19.6.4846842Testing wound dressings using an in vitro wound modelLipp C; Kirker K; Agostinho A; James G; Stewart PJournal of wound care (2010), 19 (6), 220-6 ISSN:0969-0700.OBJECTIVE: To determine whether or not there are any significant differences in the effects of wound dressings on bacterial bioburden. METHOD: A selection of non-occlusive, non-adhesive dressings was tested for their effect on bacterial bioburden. The dressings selected included two dressings with antimicrobial properties (one containing silver and one containing PHMB), a cotton-based dressing enclosed in a perforated sleeve of poly(ethylene terephthalate), a carboxymethyl cellulose-based dressing, a fibre-free alginate dressing, and a 12-ply 100% cotton gauze. Using the colony-drip flow reactor (DFR) model, a meticillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa biofilm was grown underneath a dressing sample. Biofilm growth was examined via plate counts, fluorescent microscopy and scanning electron microscopy. RESULTS: The dressings containing antimicrobial agents had the greatest effect on bacterial load. In the MRSA experiments, both antimicrobial dressings produced lower bacteria counts than the other dressings (p<0.001), while in the P. aeruginosa experiments, only the silver-containing sample had fewer bacteria (p<0.0001). However, neither antimicrobial dressing was able to completely eradicate the bacteria when testing with either microorganism. CONCLUSION: The results presented herein illustrate that bacteria can grow unchallenged within the dressing environment and that an antimicrobial dressing can limit this bacterial growth. DECLARATION OF INTEREST: None.
- 43Barbero, A. M.; Frasch, H. F. Pig and Guinea Pig Skin as Surrogates for Human in Vitro Penetration Studies: A Quantitative Review. Toxicol. In Vitro 2009, 23, 1– 13, DOI: 10.1016/j.tiv.2008.10.00843Pig and guinea pig skin as surrogates for human in vitro penetration studies: A quantitative reviewBarbero, Ana M.; Frasch, H. FrederickToxicology in Vitro (2009), 23 (1), 1-13CODEN: TIVIEQ; ISSN:0887-2333. (Elsevier Ltd.)A review. Both human and animal skin in vitro models are used to predict percutaneous penetration in humans. The objective of this review is a quant. comparison of permeability and lag time measurements between human and animal skin, including an evaluation of the intra- and interspecies variability. The authors limit the focus to domestic pig and rodent guinea pig skin as surrogates for human skin, and consider only studies in which both animal and human penetration of a given chem. were measured jointly in the same lab. When the in vitro permeability of pig and human skin were compared, the Pearson product moment correlation coeff. (r) was 0.88 (P < 0.0001), with an intraspecies av. coeff. of variation of skin permeability of 21% for pig and 35% for human, and an interspecies av. coeff. of variation of 37% for the set of studied compds. (n = 41). The lag times of pig skin and human skin did not correlate (r = 0.35, P = 0.26). When the in vitro permeability of guinea pig and human skin were compared, r = 0.96 (P < 0.0001), with an av. intraspecies coeff. of variation of 19% for guinea pig and 24% for human, and an interspecies coeff. of variation of permeability of 41% for the set of studied compds. (n = 15). Lag times of guinea pig and human skin correlated (r = 0.90, P < 0.0001, n = 12). When permeability data was not reported a factor of difference (FOD) of animal to human skin was calcd. for pig skin (n = 50) and guinea pig skin (n = 25). For pig skin, 80% of measurements fell within the range 0.3 < FOD < 3. For guinea pig skin, 65% fell within that range. Both pig and guinea pig are good models for human skin permeability and have less variability than the human skin model. The skin model of choice will depend on the final purpose of the study and the compd. under investigation.
- 44Simon, A.; Amaro, M. I.; Healy, A. M.; Cabral, L. M.; de Sousa, V. P. Comparative Evaluation of Rivastigmine Permeation from a Transdermal System in the Franz Cell Using Synthetic Membranes and Pig Ear Skin with in Vivo-in Vitro Correlation. Int. J. Pharm. 2016, 512, 234– 241, DOI: 10.1016/j.ijpharm.2016.08.05244Comparative evaluation of rivastigmine permeation from a transdermal system in the Franz cell using synthetic membranes and pig ear skin with in vivo-in vitro correlationSimon, Alice; Amaro, Maria Ines; Healy, Anne Marie; Cabral, Lucio Mendes; Pereira de Sousa, ValeriaInternational Journal of Pharmaceutics (Amsterdam, Netherlands) (2016), 512 (1), 234-241CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)In the present study, in vitro permeation expts. in a Franz diffusion cell were performed using different synthetic polymeric membranes and pig ear skin to evaluate a rivastigmine (RV) transdermal drug delivery system. In vitro-in vivo correlations (IVIVC) were examd. to det. the best model membrane. In vitro permeation studies across different synthetic membranes and skin were performed for the Exelon Patch (which contains RV), and the results were compared. Deconvolution of bioavailability data using the Wagner-Nelson method enabled the fraction of RV absorbed to be detd. and a point-to-point IVIVC to be established. The synthetic membrane, Strat-M, showed a RV permeation profile similar to that obtained with pig ear skin (R2 = 0.920). Studies with Strat-M resulted in a good and linear IVIVC (R2 = 0.991) when compared with other synthetic membranes that showed R2 values less than 0.90. The R2 for pig ear skin was 0.982. Strat-M membrane was the only synthetic membrane that adequately simulated skin barrier performance and therefore it can be considered to be a suitable alternative to human or animal skin in evaluating transdermal drug transport, potentially reducing the no. of studies requiring human or animal samples.
- 45Rediguieri, C. F.; Sassonia, R. C.; Dua, K.; Kikuchi, I. S.; de Jesus Andreoli Pinto, T. Impact of Sterilization Methods on Electrospun Scaffolds for Tissue Engineering. Eur. Polym. J. 2016, 82, 181– 195, DOI: 10.1016/j.eurpolymj.2016.07.01645Impact of sterilization methods on electrospun scaffolds for tissue engineeringRediguieri, Carolina Fracalossi; Sassonia, Rogerio Corte; Dua, Kamal; Kikuchi, Irene Satiko; Pinto, Terezinha de Jesus AndreoliEuropean Polymer Journal (2016), 82 (), 181-195CODEN: EUPJAG; ISSN:0014-3057. (Elsevier Ltd.)A review. Tissue engineering is a growing area within the regenerative medicine. The electrospun scaffolds are the most promising devices for translating engineered tissues into patients. However, in order to be used in clin. practice, one of the important fundamental aspects of the scaffold is to be sterile keeping the fact of patient safety in mind. Due to the various properties of electrospun fibers, such as high porosity and surface area, the effects of sterilization could have different outcomes than those obsd. in ordinary medical devices. Therefore, the present article provides an insight into the various sterilization methods that have been applied to electrospun scaffolds and their effects on scaffolds morphol., hydrophilicity, other physico-chem. and mech. properties and the performance of seeded cells after sterilization. In conclusion, the information provided in the review will help all scientists involved in this interdisciplinary field to understand and apply the knowledge in selection of appropriate sterilization method for the electrospun scaffolds.
- 46Myhrman, E.; Håkansson, J.; Lindgren, K.; Björn, C.; Sjöstrand, V.; Mahlapuu, M. The Novel Antimicrobial Peptide PXL150 in the Local Treatment of Skin and Soft Tissue Infections. Appl. Microbiol. Biotechnol. 2013, 97, 3085– 3096, DOI: 10.1007/s00253-012-4439-846The novel antimicrobial peptide PXL150 in the local treatment of skin and soft tissue infectionsMyhrman, Emma; Hakansson, Joakim; Lindgren, Kerstin; Bjoern, Camilla; Sjoestrand, Veronika; Mahlapuu, MargitApplied Microbiology and Biotechnology (2013), 97 (7), 3085-3096CODEN: AMBIDG; ISSN:0175-7598. (Springer)Dramatic increase in bacterial resistance towards conventional antibiotics emphasizes the importance to identify novel, more potent antimicrobial therapies. Antimicrobial peptides (AMPs) have emerged as a promising new group to be evaluated in therapeutic intervention of infectious diseases. Here we describe a novel AMP, PXL150, which demonstrates in vitro a broad spectrum microbicidal action against both Gram-pos. and Gram-neg. bacteria, including resistant strains. The potent microbicidal activity and broad antibacterial spectrum of PXL150 were not assocd. with any hemolytic activity. Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance towards PXL150 during continued selection pressure. PXL150 caused a rapid depolarization of cytoplasmic membrane of S. aureus, and dissipating membrane potential is likely one mechanism for PXL150 to kill its target bacteria. Studies in human cell lines indicated that PXL150 has anti-inflammatory properties, which might be of addnl. benefit. PXL150 demonstrated pronounced anti-infectious effect in an in vivo model of full thickness wounds infected with MRSA in rats and in an ex vivo model of pig skin infected with S. aureus. S.c. or topical application of the peptide in rats did not lead to any adverse reactions. In conclusion, PXL150 may constitute a new therapeutic alternative for local treatment of infections, and further studies are warranted to evaluate the applicability of this AMP in clin. settings.
- 47Nilsson, E.; Björn, C.; Sjöstrand, V.; Lindgren, K.; Münnich, M.; Mattsby-Baltzer, I.; Ivarsson, M. L.; Olmarker, K.; Mahlapuu, M. A Novel Polypeptide Derived from Human Lactoferrin in Sodium Hyaluronate Prevents Postsurgical Adhesion Formation in the Rat. Ann. Surg. 2009, 250, 1021– 1028, DOI: 10.1097/SLA.0b013e3181b246a747A novel polypeptide derived from human lactoferrin in sodium hyaluronate prevents postsurgical adhesion formation in the ratNilsson Elin; Bjorn Camilla; Sjostrand Veronika; Lindgren Kerstin; Munnich Mattias; Mattsby-Baltzer Inger; Ivarsson Marie-Louise; Olmarker Kjell; Mahlapuu MargitAnnals of surgery (2009), 250 (6), 1021-8 ISSN:.OBJECTIVE: The objective of the study was to evaluate whether a peptide derived from human lactoferrin, PXL01 could act safely to reduce the formation of peritoneal adhesions in the rat model and to map the molecular mechanisms of its action. SUMMARY BACKGROUND DATA: Adhesion formation is a significant problem within every surgical discipline causing suffering for the patients and major cost for the society. For many decades, attempts have been made to reduce postsurgical adhesions by reducing surgical trauma. It is now believed that major improvements in adhesion prevention will only be reached by developing dedicated antiscarring products, which are administrated in connection to the surgical intervention. METHODS: Anti-inflammatory as well as fibrinolytic activities of PXL01 were studied in relevant human cell lines. Using the sidewall defect-cecum abrasion model in the rat, the adhesion prevention properties of PXL01 formulated in sodium hyaluronate were evaluated. Large bowel anastomosis healing model in the rat was applied to study if PXL01 would have any negative effects on intestine healing. RESULTS: PXL01 exhibits an inhibitory effect on the most important hallmarks of scar formation by reducing infections, prohibiting inflammation, and promoting fibrinolysis. PXL01 formulated in sodium hyaluronate markedly reduced formation of peritoneal adhesions in rat without any adverse effects on wound healing. CONCLUSIONS: A new class of synthetically derived water soluble low molecular weight peptide compound, PXL01 showed marked reduction of peritoneal adhesion formation in an animal model without any negative effects on healing. On the basis of these data, a comprehensive adhesion prevention regimen in clinical situation is expected.
- 48Björn, C.; Mahlapuu, M.; Mattsby-Baltzer, I.; Håkansson, J. Anti-Infective Efficacy of the Lactoferrin-Derived Antimicrobial Peptide HLR1r. Peptides 2016, 81, 21– 28, DOI: 10.1016/j.peptides.2016.04.00548Anti-infective efficacy of the lactoferrin-derived antimicrobial peptide HLR1rBjorn Camilla; Mahlapuu Margit; Mattsby-Baltzer Inger; Hakansson JoakimPeptides (2016), 81 (), 21-8 ISSN:.Antimicrobial peptides (AMPs) have emerged as a new class of drug candidates for the treatment of infectious diseases. Here we describe a novel AMP, HLR1r, which is structurally derived from the human milk protein lactoferrin and demonstrates a broad spectrum microbicidal action in vitro. The minimum concentration of HLR1r needed for killing ≥99% of microorganisms in vitro, was in the range of 3-50μg/ml for common Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and for the yeast Candida albicans, when assessed in diluted brain-heart infusion medium. We found that HLR1r also possesses anti-inflammatory properties as evidenced by inhibition of tumor necrosis factor alpha (TNF-α) secretion from human monocyte-derived macrophages and by repression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) secretion from human mesothelial cells, without any cytotoxic effect observed at the concentration range tested (up to 400μg/ml). HLR1r demonstrated pronounced anti-infectious effect in in vivo experimental models of cutaneous candidiasis in mice and of excision wounds infected with MRSA in rats as well as in an ex vivo model of pig skin infected with S. aureus. In conclusion, HLR1r may constitute a new therapeutic alternative for local treatment of skin infections.
- 49Yan, W.; Banerjee, P.; Liu, Y.; Mi, Z.; Bai, C.; Hu, H.; To, K. K. W.; Duong, H. T. T.; Leung, S. S. Y. Development of Thermosensitive Hydrogel Wound Dressing Containing Acinetobacter Baumannii Phage against Wound Infections. Int. J. Pharm. 2021, 602, 120508 DOI: 10.1016/j.ijpharm.2021.12050849Development of thermosensitive hydrogel wound dressing containing Acinetobacter baumannii phage against wound infectionsYan, Wei; Banerjee, Parikshit; Liu, Yannan; Mi, Zhiqiang; Bai, Changqing; Hu, Haiyan; To, Kenneth K. W.; Duong, Hien T. T.; Leung, Sharon S. Y.International Journal of Pharmaceutics (Amsterdam, Netherlands) (2021), 602 (), 120508CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)With the emergence of multidrug resistance (MDR) bacteria, wound infection continues to be a challenging problem and represents a considerable healthcare burden. This study aims to evaluate the applicability of a phage loaded thermosensitive hydrogel in managing wound infections caused by MDR Acinetobacter baumannii, using IME-AB2 phage and MDR-AB2 as the model phage and bacteria, resp. Excellent storage stability of the IME-AB2 phage in a ∼18 wt% Poloxamer 407 (P407) hydrogel soln. was first demonstrated with negligible titer loss (∼0.5 log) in 24 mo at 4°C. The incorporated phage was released in a sustained manner with a cumulative release of 60% in the first 24 h. The in vitro bacterial killing efficiency of phage gel and phage suspension at 37°C demonstrated >5 log10 CFU/mL redn. against A. baumannii. A comparable biofilm elimination capacity was also noted between the phage gel and phage suspension (59% and 45% resp.). These results suggested that the incorporation of phage into the hydrogel not only had insignificant impacts on the bacterial killing efficiency of phage, but also act as a phage depot to maintain higher phage titer at the infectious site for a prolong period for more effective treatment. We also found that the hydrogel formulation significantly suppressed microbial survival in an ex vivo wound infection model using pig skin (90% redn. in bacterial counts was achieved after 4 h treatment). In summary, our results demonstrated that the P407-based phage-loaded thermosensitive hydrogel is a simple and promising phage formulation for the management of wound infections.
- 50Alhusein, N.; Blagbrough, I. S.; Beeton, M. L.; Bolhuis, A.; de Bank, P. A. Electrospun Zein/PCL Fibrous Matrices Release Tetracycline in a Controlled Manner, Killing Staphylococcus aureus Both in Biofilms and Ex Vivo on Pig Skin, and Are Compatible with Human Skin Cells. Pharm. Res. 2016, 33, 237– 246, DOI: 10.1007/s11095-015-1782-350Electrospun Zein/PCL Fibrous Matrices Release Tetracycline in a Controlled Manner, Killing Staphylococcus aureus Both in Biofilms and Ex Vivo on Pig Skin, and are Compatible with Human Skin CellsAlhusein, Nour; Blagbrough, Ian S.; Beeton, Michael L.; Bolhuis, Albert; De Bank, Paul A.Pharmaceutical Research (2016), 33 (1), 237-246CODEN: PHREEB; ISSN:0724-8741. (Springer)Purpose: To investigate the destruction of clin.-relevant bacteria within biofilms via the sustained release of the antibiotic tetracycline from zein-based electrospun polymeric fibrous matrixes and to demonstrate the compatibility of such wound dressing matrixes with human skin cells. Methods: Zein/PCL triple layered fibrous dressings with entrapped tetracycline were electrospun. The successful entrapment of tetracycline in these dressings was validated. The successful release of bioactive tetracycline, the destruction of preformed biofilms, and the viability of fibroblast (FEK4) cells were investigated. Results: The sustained release of tetracycline from these matrixes led to the efficient destruction of preformed biofilms from Staphylococcus aureus MRSA252 in vitro, and of MRSA252 and ATCC 25923 bacteria in an ex vivo pig skin model using 1 × 1 cm square matrixes contg. tetracycline (30 μg). Human FEK4 cells grew normally in the presence of these matrixes. Conclusions: The ability of the zein-based matrixes to destroy bacteria within increasingly complex in vitro biofilm models was clearly established. An ex vivo pig skin assay showed that these matrixes, with entrapped tetracycline, efficiently kill bacteria and this, combined with their compatibility with a human skin cell line suggest these matrixes are well suited for applications in wound healing and infection control.
- 51Mrázová, H.; Koller, J.; Kubišová, K.; Fujeríková, G.; Klincová, E.; Babál, P. Comparison of Structural Changes in Skin and Amnion Tissue Grafts for Transplantation Induced by Gamma and Electron Beam Irradiation for Sterilization. Cell Tissue Banking 2016, 17, 255– 260, DOI: 10.1007/s10561-015-9536-351Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilizationMrazova, H.; Koller, J.; Kubisova, K.; Fujerikova, G.; Klincova, E.; Babal, P.Cell and Tissue Banking (2016), 17 (2), 255-260CODEN: CTBAFV; ISSN:1389-9333. (Springer)Sterilization is an important step in the prepn. of biol. material for transplantation. The aim of the study is to compare morphol. changes in three types of biol. tissues induced by different doses of gamma and electron beam radiation. Frozen biol. tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10% formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, esp. in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradn. by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.
- 52Kairiyama, E.; Horak, C.; Spinosa, M.; Pachado, J.; Schwint, O. Radiation Sterilization of Skin Allograft. Radiat. Phys. Chem. 2009, 78, 445– 448, DOI: 10.1016/j.radphyschem.2009.03.07852Radiation sterilization of skin allograftKairiyama, E.; Horak, C.; Spinosa, M.; Pachado, J.; Schwint, O.Radiation Physics and Chemistry (2009), 78 (7-8), 445-448CODEN: RPCHDM; ISSN:0969-806X. (Elsevier Ltd.)In the treatment of burns or accidental loss of skin, cadaveric skin allografts provide an alternative to temporarily cover a wounded area. The skin bank facility is indispensable for burn care. The first human skin bank was established in Argentina in 1989; later, 3 more banks were established. A careful donor selection is carried out according to the national regulation in order to prevent transmissible diseases. As cadaveric human skin is naturally highly contaminated, a final sterilization is necessary to reach a sterility assurance level (SAL) of 10-6. The sterilization dose for 106 batches of processed human skin was detd. on the basis of the Code of Practice for the Radiation Sterilization of Tissue Allografts: Requirements for Validation and Routine Control (2004) and ISO 11137-2 (2006). They ranged from 17.6 to 33.4 kGy for bioburdens of >10-162.700 CFU/100 cm2. The presence of Gram neg. bacteria was checked for each produced batch. From the anal. of the exptl. results, it was obsd. that the bioburden range was very wide and consequently the estd. sterilization doses too. If this is the case, the detn. of a tissue-specific dose per prodn. batch is necessary to achieve a specified requirement of SAL. Otherwise if the dose of 25 kGy is preselected, a standardized method for substantiation of this dose should be done to confirm the radiation sterilization process.
- 53Rooney, P.; Eagle, M.; Hogg, P.; Lomas, R.; Kearney, J. Sterilisation of Skin Allograft with Gamma Irradiation. Burns 2008, 34, 664– 673, DOI: 10.1016/j.burns.2007.08.02153Sterilisation of skin allograft with gamma irradiationRooney P; Eagle M; Hogg P; Lomas R; Kearney JBurns : journal of the International Society for Burn Injuries (2008), 34 (5), 664-73 ISSN:0305-4179.The primary surgical requirement of skin allografts within the UK is for cryopreserved viable allografts as these engraft to the wound bed and gain a vascular supply, thus providing true wound closure and a superior clinical performance. Consequently the only disinfection treatment the skin receives is exposure to an antibiotic cocktail. However, antibiotic treatment does not reliably decontaminate skin allografts and 22% of cryopreserved skin fails microbial acceptance criteria and cannot be used clinically. We describe here a study which was carried out to determine a means of saving and using the microbiologically failed skin. Four different treatment regimens were investigated; treatment with 20%, 50% and 85% glycerol followed by 25 kGy irradiation at -80 degrees C, and treatment with 85% glycerol at ambient (30-40 degrees C) temperature and irradiation. Following treatment, the grafts were evaluated for their histological structure, in vitro cytotoxicity and handling properties. The radioprotective effects of the different glycerol concentrations and temperatures on microorganisms were also determined. The data indicate that 25 kGy irradiation of deep-frozen skin in 20% glycerol sterilised the tissue without any histological, cytotoxicological or physical alterations compared to normal cryopreserved skin. In contrast, irradiation of all other glycerol concentrations elicited some cytotoxicity and/or histological effect. These non-viable grafts can be made available for surgical use when cryopreserved viable grafts are not available or required.
- 54Mrázová, H.; Koller, J.; Fujeríková, G.; Babál, P. Structural Changes of Skin and Amnion Grafts for Transplantation Purposes Following Different Doses of Irradiation. Cell Tissue Banking 2014, 15, 429– 433, DOI: 10.1007/s10561-013-9407-854Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiationMrazova, H.; Koller, J.; Fujerikova, G.; Babal, P.Cell and Tissue Banking (2014), 15 (3), 429-433CODEN: CTBAFV; ISSN:1389-9333. (Springer)An important part of the prepn. of biol. material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biol. tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was obsd. by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, esp. in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradn. than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochem. techniques.
- 55Cattò, C.; Cappitelli, F. Testing Anti-Biofilm Polymeric Surfaces: Where to Start?. Int. J. Mol. Sci. 2019, 20, 3794, DOI: 10.3390/ijms2015379455Testing anti-biofilm polymeric surfaces: where to start?Catto, Cristina; Cappitelli, FrancescaInternational Journal of Molecular Sciences (2019), 20 (15), 3794CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)A review. Present day awareness of biofilm colonization on polymeric surfaces has prompted the scientific community to develop an ever-increasing no. of new materials with anti-biofilm features. However, compared to the large amt. of work put into discovering potent biofilm inhibitors, only a small no. of papers deal with their validation, a crit. step in the translation of research into practical applications. This is due to the lack of standardized testing methods and/or of well-controlled in vivo studies that show biofilm prevention on polymeric surfaces; furthermore, there has been little correlation with the reduced incidence of material deterioration. Here an overview of the most common methods for studying biofilms and for testing the anti-biofilm properties of new surfaces is provided.
- 56Yu, O. Y.; Zhao, I. S.; Mei, M. L.; Lo, E. C. M.; Chu, C. H. Dental Biofilm and Laboratory Microbial Culture Models for Cariology Research. Dent. J. 2017, 5, 21, DOI: 10.3390/dj5020021There is no corresponding record for this reference.
- 57Otto, M. Staphylococcus epidermidis─The “accidental” Pathogen. Nat. Rev. Microbiol. 2009, 7, 555– 567, DOI: 10.1038/nrmicro218257Staphylococcus epidermidis - the 'accidental' pathogenOtto, MichaelNature Reviews Microbiology (2009), 7 (8), 555-567CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)A review. Although nosocomial infections by Staphylococcus epidermidis have gained much attention, this skin-colonizing bacterium has apparently evolved not to cause disease, but to maintain the commonly benign relationship with its host. Accordingly, S. epidermidis does not produce aggressive virulence determinants. Rather, factors that normally sustain the commensal lifestyle of S. epidermidis seem to give rise to addnl. benefits during infection. Furthermore, we are beginning to comprehend the roles of S. epidermidis in balancing the epithelial microflora and serving as a reservoir of resistance genes. In this Review, I discuss the mol. basis of the commensal and infectious lifestyles of S. epidermidis.
- 58Ishihama, H.; Ishii, K.; Nagai, S.; Kakinuma, H.; Sasaki, A.; Yoshioka, K.; Kuramoto, T.; Shiono, Y.; Funao, H.; Isogai, N.; Tsuji, T.; Okada, Y.; Koyasu, S.; Toyama, Y.; Nakamura, M.; Aizawa, M.; Matsumoto, M. An Antibacterial Coated Polymer Prevents Biofilm Formation and Implant-Associated Infection. Sci. Rep. 2021, 11, 3602 DOI: 10.1038/s41598-021-82992-w58An antibacterial coated polymer prevents biofilm formation and implant-associated infectionIshihama, Hiroko; Ishii, Ken; Nagai, Shigenori; Kakinuma, Hiroaki; Sasaki, Aya; Yoshioka, Kenji; Kuramoto, Tetsuya; Shiono, Yuta; Funao, Haruki; Isogai, Norihiro; Tsuji, Takashi; Okada, Yasunori; Koyasu, Shigeo; Toyama, Yoshiaki; Nakamura, Masaya; Aizawa, Mamoru; Matsumoto, MorioScientific Reports (2021), 11 (1), 3602CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)To prevent infections assocd. with medical implants, various antimicrobial silver-coated implant materials have been developed. However, these materials do not always provide consistent antibacterial effects in vivo despite having dramatic antibacterial effects in vitro, probably because the antibacterial effects involve silver-ion-mediated reactive oxygen species generation. Addnl., the silver application process often requires extremely high temps., which damage non-metal implant materials. We recently developed a bacteria-resistant coating consisting of hydroxyapatite film on which ionic silver is immobilized via inositol hexaphosphate chelation, using a series of immersion and drying steps performed at low heat. Here we applied this coating to a polymer, polyetheretherketone (PEEK), and analyzed the properties and antibacterial activity of the coated polymer in vitro and in vivo. The ionic silver coating demonstrated significant bactericidal activity and prevented bacterial biofilm formation in vitro. Bio-imaging of a soft tissue infection mouse model in which a silver-coated PEEK plate was implanted revealed a dramatic absence of bacterial signals 10 days after inoculation. These animals also showed a strong redn. in histol. features of infection, compared to the control animals. This innovative coating can be applied to complex structures for clin. use, and could prevent infections assocd. with a variety of plastic implants.
- 59Peng, L.; de Sousa, J.; Su, Z.; Novak, B. M.; Nevzorov, A. A.; Garland, E. R.; Melander, C. Inhibition of Acinetobacter Baumannii Biofilm Formation on a Methacrylate Polymer Containing a 2-Aminoimidazole Subunit. Chem. Commun. 2011, 47, 4896– 4898, DOI: 10.1039/c1cc10691k59Inhibition of Acinetobacter baumannii biofilm formation on a methacrylate polymer containing a 2-aminoimidazole subunitPeng, Ling-Ling; De Sousa, Joseph; Su, Zhao-Ming; Novak, Bruce M.; Nevzorov, Alexander A.; Garland, Eva R.; Melander, ChristianChemical Communications (Cambridge, United Kingdom) (2011), 47 (17), 4896-4898CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)A polymeric composite contg. a 2-aminoimidazole deriv. was synthesized. It was found that this polymer was resistant to biofilm colonization by Acinetobacter baumannii, no leaching of the 2-aminoimidazole deriv. was obsd. after 2 wk of treatment with deionized water, and the resulting polymer was not hemolytic.
- 60Vishwakarma, A.; Dang, F.; Ferrell, A.; Barton, H. A.; Joy, A. Peptidomimetic Polyurethanes Inhibit Bacterial Biofilm Formation and Disrupt Surface Established Biofilms. J. Am. Chem. Soc. 2021, 143, 9440– 9449, DOI: 10.1021/jacs.1c0232460Peptidomimetic Polyurethanes Inhibit Bacterial Biofilm Formation and Disrupt Surface Established BiofilmsVishwakarma, Apoorva; Dang, Francis; Ferrell, Allison; Barton, Hazel A.; Joy, AbrahamJournal of the American Chemical Society (2021), 143 (25), 9440-9449CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Over 80% of all chronic bacterial infections in humans are assocd. with biofilms, which are surface-assocd. bacterial communities encased within a secreted exopolysaccharide matrix that can provide resistance to environmental and chem. insults. Biofilm formation triggers broad adaptive changes in the bacteria, allowing them to be almost 1000-fold more resistant to conventional antibiotic treatments and host immune responses. The failure of antibiotics to eliminate biofilms leads to persistent chronic infections and can promote the development of antibiotic-resistant strains. Therefore, there is an urgent need to develop agents that effectively prevent biofilm formation and eradicate established biofilms. Herein, we present water-sol. synthetic peptidomimetic polyurethanes that can disrupt surface established biofilms of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, all of which show tolerance to the conventional antibiotics polymyxin B and ciprofloxacin. Furthermore, while these polyurethanes show poor antimicrobial activity against planktonic bacteria, they prevent surface attachment and stimulate bacterial surface motility to inhibit biofilm formation of both Gram-pos. and Gram-neg. bacteria at subinhibitory concns., without being toxic to mammalian cells. Our results show that these polyurethanes show promise as a platform for the development of therapeutics that target biofilms and modulate surface interactions of bacteria for the treatment of chronic biofilm-assocd. infections and as antibiofilm agents.
- 61Zakrzewska, A.; Bayan, H.; Nakielski, M. A.; Petronella, P.; de Sio, F.; Pierini, L. F. Nanotechnology Transition Roadmap toward Multifunctional Stimuli-Responsive Face Masks. ACS Appl. Mater. Interfaces 2022, 14, 46123– 46144, DOI: 10.1021/acsami.2c1033561Nanotechnology Transition Roadmap toward Multifunctional Stimuli-Responsive Face MasksZakrzewska, Anna; Haghighat Bayan, Mohammad Ali; Nakielski, Pawel; Petronella, Francesca; De Sio, Luciano; Pierini, FilippoACS Applied Materials & Interfaces (2022), 14 (41), 46123-46144CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)A review. In recent times, the use of personal protective equipment, such as face masks or respirators, is becoming more and more critically important because of common pollution; furthermore, face masks have become a necessary element in the global fight against the COVID-19 pandemic. For this reason, the main mission of scientists has become the development of face masks with exceptional properties that will enhance their performance. The versatility of electrospun polymer nanofibers has detd. their suitability as a material for constructing "smart" filter media. This paper provides an overview of the research carried out on nanofibrous filters obtained by electrospinning. The progressive development of the next generation of face masks whose unique properties can be activated in response to a specific external stimulus is highlighted. Thanks to addnl. components incorporated into the fiber structure, filters can, for example, acquire antibacterial or antiviral properties, self-sterilize the structure, and store the energy generated by users. Despite the discovery of several fascinating possibilities, some of them remain unexplored. Stimuli-responsive filters have the potential to become products of large-scale availability and great importance to society as a whole.
- 62Lencova, S.; Svarcova, V.; Stiborova, H.; Demnerova, K.; Jencova, V.; Hozdova, K.; Zdenkova, K. Bacterial Biofilms on Polyamide Nanofibers: Factors Influencing Biofilm Formation and Evaluation. ACS Appl. Mater. Interfaces 2021, 13, 2277– 2288, DOI: 10.1021/acsami.0c1901662Bacterial Biofilms on Polyamide Nanofibers: Factors Influencing Biofilm Formation and EvaluationLencova Simona; Svarcova Viviana; Stiborova Hana; Demnerova Katerina; Zdenkova Kamila; Jencova Vera; Hozdova KristynaACS applied materials & interfaces (2021), 13 (2), 2277-2288 ISSN:.Electrospun polyamide (PA) nanofibers have great potential for medical applications (in dermatology as antimicrobial compound carriers or surgical sutures). However, little is known about microbial colonization on these materials. Suitable methods need to be chosen and optimized for the analysis of biofilms formed on nanofibers and the influence of their morphology on biofilm formation. We analyzed 11 PA nanomaterials, both nonfunctionalized and functionalized with AgNO3, and tested the formation of a biofilm by clinically relevant bacteria (Escherichia coli CCM 4517, Staphylococcus aureus CCM 3953, and Staphylococcus epidermidis CCM 4418). By four different methods, it was confirmed that all of these bacteria attached to the PAs and formed biofilms; however, it was found that the selected method can influence the outcomes. For studying biofilms formed by the selected bacteria, scanning electron microscopy, resazurin staining, and colony-forming unit enumeration provided appropriate and comparable results. The values obtained by crystal violet (CV) staining were misleading due to the binding of the CV dye to the PA structure. In addition, the effect of nanofiber morphology parameters (fiber diameter and air permeability) and AgNO3 functionalization significantly influenced biofilm maturation. Furthermore, the correlations between air permeability and surface density and fiber diameter were revealed. Based on the statistical analysis, fiber diameter was confirmed as a crucial factor influencing biofilm formation (p ≤ 0.01). The functionalization of PAs with AgNO3 (from 0.1 wt %) effectively suppressed biofilm formation. The PA functionalized with a concentration of 0.1 wt % AgNO3 influenced the biofilm equally as nonfunctionalized PA 8% 2 g/m(2). Therefore, biofilm formation could be affected by the above-mentioned morphology parameters, and ultimately, the risk of infections from contaminated medical devices could be reduced.
- 63Wang, Z.; Scheres, L.; Xia, H.; Zuilhof, H., 1; Wang, Z.; Scheres, L.; Xia, H.; Zuilhof, H. Developments and Challenges in Self-Healing Antifouling Materials. Adv. Funct. Mater. 2020, 30, 1908098 DOI: 10.1002/adfm.20190809863Developments and Challenges in Self-Healing Antifouling MaterialsWang, Zhanhua; Scheres, Luc; Xia, Hesheng; Zuilhof, HanAdvanced Functional Materials (2020), 30 (26), 1908098CODEN: AFMDC6; ISSN:1616-301X. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Self-healing antifouling materials have gained rapidly increasing interest over the past decade and have been studied and used in a rapidly increasing range of applications. Recent developments and challenges in self-healing antifouling materials are summarized in four sections: first, the different mechanisms for both antifouling and self-healing are briefly discussed. Second, three main categories of self-healing antifouling materials based on surface replenishing and dynamic covalent and noncovalent interactions are discussed, with a focus on the prepn., characterization, and central characteristics of different self-healing antifouling materials. Third, different types of potential applications of self-healing antifouling materials are summarized, such as injectable hydrogels and oil/water sepns. Finally, a summary of future development of the field is provided, and a no. of crit. limitations that are still outstanding are highlighted.
- 64Patterson, A. L.; Wenning, B.; Rizis, G.; Calabrese, D. R.; Finlay, J. A.; Franco, S. C.; Zuckermann, R. N.; Clare, A. S.; Kramer, E. J.; Ober, C. K.; Segalman, R. A. Role of Backbone Chemistry and Monomer Sequence in Amphiphilic Oligopeptide- and Oligopeptoid-Functionalized PDMS- and PEO-Based Block Copolymers for Marine Antifouling and Fouling Release Coatings. Macromolecules 2017, 50, 2656– 2667, DOI: 10.1021/acs.macromol.6b0250564Role of Backbone Chemistry and Monomer Sequence in Amphiphilic Oligopeptide- and Oligopeptoid-Functionalized PDMS- and PEO-Based Block Copolymers for Marine Antifouling and Fouling Release CoatingsPatterson, Anastasia L.; Wenning, Brandon; Rizis, Georgios; Calabrese, David R.; Finlay, John A.; Franco, Sofia C.; Zuckermann, Ronald N.; Clare, Anthony S.; Kramer, Edward J.; Ober, Christopher K.; Segalman, Rachel A.Macromolecules (Washington, DC, United States) (2017), 50 (7), 2656-2667CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)Poly(dimethylsiloxane) (PDMS)- and poly(ethylene oxide) (PEO)-based block copolymer coatings functionalized with amphiphilic, surface-active, and sequence-controlled oligomer side chains were studied to directly compare the effects of hydrophilicity, hydrogen bonding, and monomer sequence on antifouling performance. Utilizing a modular coating architecture, structurally similar copolymers were used to make direct and meaningful comparisons. Amphiphilic character was imparted with non-natural oligopeptide and oligopeptoid pendant chains made from oligo-PEO and surface-segregating fluoroalkyl monomer units. Surface anal. revealed rearrangement for all surfaces when moved from vacuum to wet environments. XPS spectra indicated that the polymer backbone and oligomer interactions play key roles in the surface presentation. Biofouling assays using the macroalga Ulva linza showed that the presence of peptoid side chains facilitated the removal of sporelings on the PDMS block copolymer, with removal matching that of a PDMS elastomer std. The lack of a hydrogen bond donor in the peptoid backbone likely contributed to the lower adhesion strength of sporelings to these surfaces. Both the initial attachment and adhesion strength of the diatom Navicula incerta were lower on the coatings based on PEO than on those based on PDMS. Surprisingly, on the PEO coating bearing the blocky peptoid sequence, initial attachment of N. incerta showed no measurable cell d.
- 65Putrinš, M.; Kogermann, K.; Lukk, E.; Lippus, M.; Varik, V.; Tenson, T. Phenotypic Heterogeneity Enables Uropathogenic Escherichia coli to Evade Killing by Antibiotics and Serum Complement. Infect. Immun. 2015, 83, 1056– 1067, DOI: 10.1128/IAI.02725-1465Phenotypic heterogeneity enables uropathogenic Escherichia coli to evade killing by antibiotics and serum complementPutrins, Marta; Kogermann, Karin; Lukk, Eliisa; Lippus, Markus; Varik, Vallo; Tenson, TanelInfection and Immunity (2015), 83 (3), 1056-1067, 12 pp.CODEN: INFIBR; ISSN:1098-5522. (American Society for Microbiology)Uropathogenic strains of Escherichia coli (UPEC) are the major cause of bacteremic urinary tract infections. Survival in the bloodstream is assocd. with different mechanisms that help to resist serum complement-mediated killing. While the phenotypic heterogeneity of bacteria has been shown to influence antibiotic tolerance, the possibility that it makes cells refractory to killing by the immune system has not been exptl. tested. In the present study, we sought to det. whether the heterogeneity of bacterial cultures is relevant to bacterial targeting by the serum complement system. We monitored cell divisions in the UPEC strain CFT073 with fluorescent reporter protein. Stationary-phase cells were incubated in active or heat-inactivated human serum in the presence or absence of different antibiotics (ampicillin, norfloxacin, and amikacin), and cell division and complement protein C3 binding were measured by flow cytometry and immunofluorescence microscopy. Heterogeneity in the doubling times of CFT073 cells in serum enabled three phenotypically different subpopulations to be distinguished, all of them being recognized by the C3 component of the complement system. The population of rapidly growing cells resists serum complement-mediated lysis. The dominant subpopulation of cells with intermediate growth rate is susceptible to serum. The third population, which does not resume growth upon diln. from stationary phase, is simultaneously protected from serum complement and antibiotics.
- 66Lebeaux, D.; Ghigo, J.-M.; Beloin, C. Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics. Microbiol. Mol. Biol. Rev. 2014, 78, 510– 543, DOI: 10.1128/MMBR.00013-1466Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibioticsLebeaux David; Ghigo Jean-Marc; Beloin ChristopheMicrobiology and molecular biology reviews : MMBR (2014), 78 (3), 510-43 ISSN:.Surface-associated microbial communities, called biofilms, are present in all environments. Although biofilms play an important positive role in a variety of ecosystems, they also have many negative effects, including biofilm-related infections in medical settings. The ability of pathogenic biofilms to survive in the presence of high concentrations of antibiotics is called "recalcitrance" and is a characteristic property of the biofilm lifestyle, leading to treatment failure and infection recurrence. This review presents our current understanding of the molecular mechanisms of biofilm recalcitrance toward antibiotics and describes how recent progress has improved our capacity to design original and efficient strategies to prevent or eradicate biofilm-related infections.
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Additional information about experimental methods and results; sterility test results (Figure S1) (PDF)
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