The Impact of Antiseptic-Loaded Bacterial Nanocellulose on Different Biofilms-An Effective Treatment for Chronic Wounds?

Introduction: Pathogenic biofilms are an important factor for impaired wound healing, subsequently leading to chronic wounds. Nonsurgical treatment of chronic wound infections is limited to the use of conventional systemic antibiotics and antiseptics. Wound dressings based on bacterial nanocellulose (BNC) are considered a promising approach as an effective carrier for antiseptics. The aim of the present study was to investigate the antimicrobial activity of antiseptic-loaded BNC against in vitro biofilms. Materials and Methods: BNC was loaded with the commercially available antiseptics Prontosan® and Octenisept®. The silver-based dressing Aquacel®Ag Extra was used as a positive control. The biofilm efficacy of the loaded BNC sheets was tested against an in vitro 24-hour biofilm of Staphylococcus aureus and Candida albicans and a 48-hour biofilm of Pseudomonas aeruginosa. In vivo tests using a porcine excisional wound model was used to analyze the effect of a prolonged treatment with the antiseptics on the healing process. Results: We observed complete eradication of S. aureus biofilm in BNC loaded with Octenisept® and C. albicans biofilm for BNC loaded with Octenisept® or Prontosan®. Treatment with unloaded BNC also resulted in a statistically significant reduction in bacterial cell density of S. aureus compared to untreated biofilm. No difference on the wound healing outcome was observed for the wounds treated for seven days using BNC alone in comparison to BNC combined with Prontosan® or with Octenisept®. Conclusions: Based on these results, antiseptic-loaded BNC represents a promising and effective approach for the treatment of biofilms. Additionally, the prolonged exposure to the antiseptics does not affect the healing outcome. Prevention and treatment of chronic wound infections may be feasible with this novel approach and may even be superior to existing modalities.

Orchestrating the Dermal/Epidermal Tissue Ratio during Wound Healing by Controlling the Moisture Content.

A balanced and moist wound environment and surface increases the effect of various growth factors, cytokines, and chemokines, stimulating cell growth and wound healing. Considering this fact, we tested in vitro and in vivo water evaporation rates from the cellulose dressing epicitehydro when combined with different secondary dressings as well as the resulting wound healing efficacy in a porcine donor site model. The aim of this study was to evaluate how the different rates of water evaporation affected wound healing efficacy. To this end, epicitehydro primary dressing, in combination with different secondary dressing materials (cotton gauze, JELONET◊, AQUACEL® Extra ™, and OPSITE◊ Flexifix), was placed on 3 × 3 cm-sized dermatome wounds with a depth of 1.2 mm on the flanks of domestic pigs. The healing process was analyzed histologically and quantified by morphometry. High water evaporation rates by using the correct secondary dressing, such as cotton gauze, favored a better re-epithelialization in comparison with the low water evaporation resulting from an occlusive secondary dressing, which favored the formation of a new and intact dermal tissue that nearly fully replaced all the dermis that was removed during wounding. This newly available evidence may be of great benefit to clinical wound management.

Comparison of wound healing and patient comfort in partial-thickness burn wounds treated with SUPRATHEL and epictehydro wound dressings.

Among the available dressings for partial-thickness burn wound treatment, SUPRATHEL has shown good usability and effectiveness for wound healing and patient comfort and has been used in many burn centres in the last decade. Recently, bacterial nanocellulose (BNC) has become popular for the treatment of wounds, and many studies have demonstrated its efficacy. epicitehydro , consisting of BNC and 95% water, is a promising product and has recently been introduced in numerous burn centres. To date, no studies including direct comparisons to existing products like SUPRATHEL have been conducted. Therefore, we aimed to compare epicitehydro to SUPRATHEL in the treatment of partial-thickness burns. Twenty patients with partial-thickness burns affecting more than 0.5% of their total body surface area (TBSA) were enrolled in this prospective, unicentric, open, comparative, intra-individual clinical study. After debridement, the wounds were divided into two areas: one was treated with SUPRATHEL and the other with epicitehydro . Wound healing, infection, bleeding, exudation, dressing changes, and pain were documented. The quality of the scar tissue was assessed subjectively using the Patient and Observer Scar Scale. Wound healing in patients with a mean TBSA of 9.2% took 15 to 16 days for both treatments without dressing changes. All wounds showed minimal exudation, and patients reported decreased pain with the only significant difference between the two dressings on day 1. No infection or bleeding occurred in any of the wounds. Regarding scar evaluation, SUPRATHEL and epicitehydro did not differ significantly. Both wound dressings were easy to use, were highly flexible, created a safe healing environment, had similar effects on pain reduction, and showed good cosmetic and functional results without necessary dressing changes. Therefore, epicitehydro can be used as an alternative to SUPRATHEL for the treatment of partial-thickness burn wounds.

A 3D In Vitro Model for Burn Wounds: Monitoring of Regeneration on the Epidermal Level.

Burns affect millions every year and a model to mimic the pathophysiology of such injuries in detail is required to better understand regeneration. The current gold standard for studying burn wounds are animal models, which are under criticism due to ethical considerations and a limited predictiveness. Here, we present a three-dimensional burn model, based on an open-source model, to monitor wound healing on the epidermal level. Skin equivalents were burned, using a preheated metal cylinder. The healing process was monitored regarding histomorphology, metabolic changes, inflammatory response and reepithelialization for 14 days. During this time, the wound size decreased from 25% to 5% of the model area and the inflammatory response (IL-1ß, IL-6 and IL-8) showed a comparable course to wounding and healing in vivo. Additionally, the topical application of 5% dexpanthenol enhanced tissue morphology and the number of proliferative keratinocytes in the newly formed epidermis, but did not influence the overall reepithelialization rate. In summary, the model showed a comparable healing process to in vivo, and thus, offers the opportunity to better understand the physiology of thermal burn wound healing on the keratinocyte level.

Current Approaches of Preservation of Cells During (freeze-) Drying.

The widespread application of therapeutic cells requires a successful stabilization of cells for the duration of transport and storage. Cryopreservation is currently considered the gold standard for the storage of active cells; however, (freeze-) drying cells could enable higher shelf life stability at ambient temperatures and facilitate easier transport and storage. During (freeze-) drying, freezing, (primary and secondary) drying and also the reconstitution step pose the risk of potential cell damage. To prevent these damaging processes, a wide range of protecting excipients has emerged, which can be classified, according to their chemical affiliation, into sugars, macromolecules, polyols, antioxidants and chelating agents. As many excipients cannot easily permeate the cell membrane, researchers have established various techniques to introduce especially trehalose intracellularly, prior to drying. This review aims to summarize the main damaging mechanisms during (freeze-) drying and to introduce the most common excipients with further details on their stabilizing properties and process approaches for the intracellular loading of excipients. Additionally, we would like to briefly explain recently discovered advantages of drying microorganisms, sperm, platelets, red blood cells, and eukaryotic cells, paying particular attention to the drying technique and residual moisture content.

DMSO as new, counterintuitive excipient for freeze-drying human keratinocytes.

DMSO is widely used as powerful cryoprotectant for the storage and transport of frozen cells. Beyond this established application of DMSO, we could now show that it has also promising lyoprotectant effects in the field of lyophilisation of therapeutic cells. Freeze-drying of HaCaT keratinocytes in 10% HES, 5% HE and in presence of DMSO led to an increase in cell membrane integrity from 25.3 ± 2.7 % without DMSO to 41.4 ± 4.3 % with 2% DMSO, as determined by trypan blue exclusion. Interruption of the lyophilisation cycle at different sampling points showed a rapid decrease of cell membrane integrity below a critical residual moisture content. DMSO was able to stabilise cell membranes below this moisture level up to a final residual moisture content of less than 1%. Furthermore, DMSO increased the total protein content of cells after freeze-drying and subsequent SDS PAGE analysis indicated that certain abundant proteins were better preserved with the use of DMSO. Owed to its low vapour pressure, a significant part of DMSO is not removed during freeze-drying and remains as plasticiser in the lyophilised cake. However, a Tg above 60°C for 2% DMSO indicates that samples can still be stored at temperatures of 2-8°C. Also, no macroscopic or microscopic collapse can be observed by SEM or BET measurements and DMSO addition leads even to more elegant cakes with reduced cake cracking. With a better preservation of cell membranes and cellular structures, DMSO can contribute to the still unsolved problem of freeze-drying cells of higher complexity.

A novel human ex-vivo burn model and the local cooling effect of a bacterial nanocellulose-based wound dressing.

BACKGROUND: Burn wound progression is a significant problem as burns initially thought to be superficial can actually become full thickness over time. Cooling is an efficient method to reduce burn wound conversion. However, if the cooling agent is below room temperature, depending on the wound size the patient is at risk of hypothermia. Additionally, tissue perfusion is reduced leading to an aggravation of burn wound progression. We investigated if wound dressings based on non-pre-cooled bacterial nanocellulose (BNC) with a high water content cool a burn just by evaporation and reduce the intradermal damages in the skin. MATERIAL AND METHODS: In a human ex-vivo model, skin explants underwent contact burns using a 100 °C hot steel block. The burned areas were divided into two groups of which one was cooled with a BNC-based wound dressing. Intradermal temperature probes measured temperature in cooled and uncooled burn sites over 24 h. For histological assessments of the burned areas biopsies were taken at different time points. High mobility group box-1 (HMBG1) staining served as marker for cell vitality and necrosis in the different skin layers. RESULTS: Intradermal temperature measurement showed that application of the BNC-based wound dressing reduced temperature significantly in burned skin. This cooling effect resulted in a maximum temperature difference of 6.4 ± 1.9 °C and a significant mean reduction of the area under the curve in the first hour after burn of 62% (p < 0.0001). The histological results showed less necrosis and less dermal-epidermal separation in the cooled areas. The HMGB1 staining revealed more vital cells in the cooled group than in the uncooled group. CONCLUSION: Based on our results, BNC-based wound dressings cool a burn. Intradermal temperature as well as thermal damage of the tissue was reduced. The tested BNC-based wound dressing can be used without pre-cooling to cool a burn as well as to reduce the burn BNC-based wound progression through its evaporation cooling effect.

Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine.

BACKGROUND: Bacterial nanocellulose (BNC) is considered a promising carrier for various substances and novel approaches using BNC in combination with antiseptics are well documented. However, the difference in the molecular weight of these molecules influences their uptake by and release from BNC. Analysing the diffusion of standard molecules with different weight, e.g. dextrans, offers the possibility to investigate the mobility of various molecules. We aimed to test the use of BNC regarding uptake and release of different standard molecules as well as two commercially available antiseptics for possible applications in future wound dressings. MATERIAL AND METHODS: Diffusion profiles, uptake and release of three FITC-dextran molecules differing in weight as well as octenidine (Octenisept®) and povidone-iodine (Betaisodona®)-based antiseptics were tested with BNC-based wound dressings. Furthermore, the antiseptic efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested. RESULTS: Uptake and release capacity for FITC-dextran molecules showed a molecular weight-dependent mobility from BNC into an agarose gel. The loading capacity of BNC was also inversely proportional to the molecular weight of the antiseptics. The release test for octenidine showed a sustained and prolonged delivery into a solid matrix, whereas povidone-iodine was released faster. Both antiseptic solutions combined with BNC showed a good dose-dependent efficacy against S. aureus. CONCLUSION: Results obtained from the mobility of FITC-dextran molecules in the BNC matrix could open possible applications for the combination of BNC with other molecules for medical applications. Combination of both tested antiseptics with BNC showed to be an efficient approach to control bacterial infections.

Uptake of PHMB in a bacterial nanocellulose-based wound dressing: A feasible clinical procedure.

BACKGROUND: With the increase of antimicrobial resistance in recent decades, other methods of preventing and fighting infections must be considered. Burn patients, whose wound areas are often extensive, are especially prone to wound infections. The loading of bacterial nanocellulose (BNC) with antiseptics has already been successfully performed but unfortunately, the described procedure is time-consuming and thus not applicable in a clinical emergency setting. Therefore, a clinically feasible approach was established. MATERIAL AND METHODS: Sheets of BNC-based wound dressings were placed into antiseptic solutions containing PHMB (Prontosan® and LAVANID® 2) and were left to soak for up to two hours. At different time points, samples were analysed for their concentration of PHMB and antiseptic efficacy. RESULTS: Within 30min, clinically relevant concentrations of PHMB were achieved in the BNC-based wound dressing. The 30-min PHMB uptake for Prontosan® and LAVANID® 2 resulted in concentrations of 0.05% and 0.019%, respectively. Samples from the PHMB loaded dressing showed a dose dependent antiseptic efficacy for Staphylococcus aureus. CONCLUSION: This experiment showed that the loading of BNC-based wound dressings with PHMB-containing antiseptics was achieved by a simple and quick procedure. According to studies a PHMB concentration of 0.001% can already inhibits all bacterial growth, indicating that the concentrations of PHMB in the BNC-based wound dressings after 30min are higher than the minimal inhibitory concentration and the antiseptic efficacy after 120min loading analysed by an standardized bacterial disk diffusion assay was shown to be comparable to the clinically used Suprasorb® X+PHMB wound dressing.

Functionalization of bone implants with nanodiamond particles and angiopoietin-1 to improve vascularization and bone regeneration.

One of the major challenges in bone tissue engineering is adequate vascularization within bone substituents for nutrients and oxygen supply. In this study, the production and results of a new, highly functional bone construct consisting of a commercial three-dimensional ß-tricalcium phosphate scaffold (ß-TCP, ChronOS®) and hydrophilic, functionalized nanodiamond (ND) particles are reported. A 30-fold increase in the active surface area of the ChronOS + ND scaffold was achieved after modification with ND. In addition, immobilization of angiopoietin-1 (Ang-1) via physisorption within the ß-TCP + ND scaffold retained the bioactivity of the growth factor. Homogeneous distribution of the ND and Ang-1 within the core of the three-dimensional scaffold was confirmed using ND covalently labelled with Oregon Green. The biological responses of the ß-TCP + ND scaffold with and without Ang-1 were studied in a sheep calvaria critical size defect model showing that the ß-TCP + ND scaffold improved the blood vessel ingrowth and the ß-TCP + ND + ND + Ang-1 scaffold further promoted vascularization and new bone formation. The results demonstrate that the modification of scaffolds with tailored diamond nanoparticles is a valuable method for improving the characteristics of bone implants and enables new approaches in bone tissue engineering.

Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation.

Constitutive and induced protein SUMOylation is involved in the regulation of a variety of cellular processes, such as regulation of gene expression and protein transport, and proceeds mainly in the nucleus of the cell. So far, several hundred SUMOylation targets have been identified, but presumably they represent only a part of the total of proteins which are regulated by SUMOylation. Here, we used the Ubc9 fusion-dependent SUMOylation system (UFDS) to screen for constitutive and induced SUMOylation of 46 randomly chosen proteins with proven or potential nuclear localization. Fourteen new UFDS-substrate proteins were identified of which eight could be demonstrated to be SUMOylated in a UFDS-independent manner in vivo. Of these, three were constitutively SUMOylated (FOS, CRSP9 and CDC37) while the remaining five substrates (CSNK2B, TAF10, HSF2BP, PSMC3 and DRG1) showed a stimulation-dependent SUMOylation induced by the MAP3 kinase MEKK1. Hence, UFDS is appropriate for the identification and characterization of constitutive and, more importantly, induced protein SUMOylation in vivo.

Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation.

Although small ubiquitin-like modifier (SUMO) is conjugated to proteins involved in diverse cellular processes, the functional analysis of SUMOylated proteins is often hampered by low levels of specific SUMOylated proteins in the cell. Here we describe a SUMO-conjugating enzyme (Ubc9) fusion-directed SUMOylation (UFDS) system, which allows efficient and selective in vivo SUMOylation of proteins. Although SUMOylation of overexpressed p53 and STAT1 was difficult to detect in HEK293 cells, up to 40% of p53 and STAT1 were conjugated with endogenous SUMO when fused to Ubc9. We verified the specificity of UFDS using SUMOylation-site mutants and showed that the method is not dependent on SUMO ligases. Using UFDS we demonstrated that SUMOylation of STAT1 inhibits its phosphorylation at Tyr701 and discovered p53 multi-SUMOylation in vivo. We propose that UFDS will be useful for the analysis of function of SUMOylation in protein interactions, subcellular localization as well as enzymatic activity.