The Effect of pH on the Extracellular Matrix and Biofilms.

Significance: Chronic wounds become caught in a state of inflammation causing an increase in levels of degrading proteases, which destroy components of the extracellular matrix (ECM) that are essential for the wound healing process. This review aims to highlight and provide readers with an overview of what is currently known about the role of pH and its effect on the ECM and biofilms within healing and nonhealing wounds. Recent Advances: The pH profiles of healthy skin, acute wounds, and chronic wounds differ significantly. Chronic wounds have an alkaline pH whereas healthy skin has a slightly acidic pH. Although there is evidence on the effect of pH on protease production and bacterial proliferation in wounds, there is little evidence to show its effect on ECM synthesis and degradation. Critical Issues: The implications for the complex nature of chronic wounds are that no single treatment is relevant for all wounds, but rather a combination of methodologies must be adopted. It is known that pH of a wound reduces throughout the stages of healing, suggesting that wound pH measurements could be beneficial to identify nonhealing wounds earlier and decide on the most appropriate course of treatment. Future Direction: Wound healing is a very complex process with multiple factors known to play a role. All aspects of the nonhealing wound (defective ECM, pH, microbial invasion, and excess proteases) need to be taken into account when investigating or clinically treating a chronic wound.

The role of polyphosphates in the sequestration of matrix metalloproteinases.

This study outlines the potential of a novel therapeutic dressing for the management of chronic wounds. The dressing incorporates polyphosphate, a non toxic compound with a number of beneficial characteristics in terms of wound healing, in a foam matrix. The aim of this study was to identify the potential of polyphosphate incorporated in the foam dressing to sequester the activity of matrix metalloproteinases (MMPs) and proteases derived from Pseudomonas aeruginosa. Methods used included gelatin zymography and milk-casein agar plate analysis. Results have shown that this dressing is effectively capable of reducing the levels of MMP-2 and MMP-9 in both their active and latent forms using an in vitro model. The dressing also demonstrated the compound's potential in the regulation of P. aeruginosa derived proteases.

The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing.

Cutaneous wound healing is orchestrated by a number of physiological pathways that ultimately lead to reformation of skin integrity and the production of functional scar tissue. The remodeling of a wound is significantly affected by matrix metalloproteinases (MMPs), which act to control the degradation of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and, as such, have a role to play in the healing of infected wounds. For example, the zinc-metalloproteinase, elastase, produced by Pseudomonas aeruginosa, induces degradation of fibroblast proteins and proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. Microbial extracellular enzymes have also been shown to degrade human wound fluid and inhibit fibroblast cell growth. It is now being acknowledged that host and bacterial MMPs may act synergistically to cause tissue breakdown within the wound bed. Several studies have suggested that bacterial-derived secreted proteases may act to up-regulate the levels of MMPs produced by the host cells. Together, these findings indicate that bacterial phenotype in terms of protease producing potential of bacteria should be taken into consideration during diagnostic and clinical intervention of infected wound management. Furthermore, both host MMPs and those derived from infecting bacteria need to be targeted in order to increase the healing capacity of the injured tissue. The aim of this review is to investigate the evidence suggestive of a relationship between unregulated levels of both host and bacterial proteases and delayed wound healing.

Chronic equine wounds: what is the role of infection and biofilms?.

Wound research is an evolving science in the equine species. In particular, interest is growing regarding the role that microorganisms play in delaying both acute and chronic wound healing. Equine wounds, particularly lower limb wounds, frequently display delayed healing and infection is commonly the underlying reason. This review will summarize the current research and knowledge surrounding equine wound healing and wound care. Particular focus is placed on the role that microbes play in chronic equine wounds and the significance of associated bacterial biofilms. .

Biofilm evidence and the microbial diversity of horse wounds.

Evidence of biofilms in human chronic wounds are thought to be responsible for preventing healing in a timely manner. However, biofilm evidence in horse wounds has not yet been documented. Consequently, this study aimed to determine whether biofilms could be detected in wounds, and to investigate the microbiology of chronic wounds in horses. Prior to analysis, wound surfaces were irrigated with 5 mL of sterile saline to remove debris. All wounds were swabbed twice (1 cm2 area) using sterile cotton-tipped swabs. In addition to this, 2 tissue biopsies were taken to investigate evidence of biofilm and the microbiology richness of the wounds. All swabs and 1 biopsy sample were transported to the laboratory in Robertson's cooked meat broth. Traditional culturable techniques and denaturing gradient gel electrophoresis with PCR were utilized to identify common bacteria isolated in all wounds. Following analysis of a number of the biopsy samples, biofilms could be clearly seen. The most common bacteria isolated from each wound analysed included Pseudomonas aeruginosa, Staphylococcus epidermidis, Serratia marcescens, Enterococcus faecalis, and Providencia rettgeri. Sequencing of the 16S ribosmonal DNAs, selected on the basis of DGGE profiling, enabled identification of bacterial species not identified using culturable technology. This study is the first to identify biofilms in the chronic wounds of horses. In addition, this study also demonstrated the importance of combining DGGE-PCR with culture techniques to provide better microbiology analysis of chronic wounds.

Silver resistance in MRSA isolated from wound and nasal sources in humans and animals.

Methicillin-resistant Staphylococcus aureus (MRSA) colonises skin, nasal passages and dermal wounds. Methods used to manage wounds infected and colonised with MRSA often include the use of topical antiseptics such as ionic silver and iodine. The objectives of this study were to determine the prevalence of silver-resistance (sil) genes in MRSA and methicillin-resistant coagulase-negative staphylococci (MR-CNS) isolated from wounds and nasal cavities of humans and animals, and also to determine the susceptibility of sil-positive and sil-negative MRSA isolates to a silver-containing Hydrofiber (SCH) wound dressing, on planktonic silE-positive and silE-negative MRSA. Polymerase chain reaction was used to determine the presence of three silver-resistance (sil) genes, silE, silP and silS in 33 MRSA and 8 methicillin-resistant staphylococci (MR-CNS). SilP and silS genes were absent in all isolates tested; however, two MRSA strains were found to contain the silE gene, together with one isolate of MR-CNS. Phenotypic resistance of the silE-positive strains and their susceptibility to the SCH dressing was evaluated using the zone of inhibition test on Mueller Hinton agar, and confocal laser microscopy using a live/dead fluorescent stain. Results confirmed that the SCH dressing was effective in killing all MRSA strains with and without the silE gene. First, this study showed that the prevalence of sil genes was low in the isolates investigated; and secondly, that the presence of a silver-resistance gene (silE) in MRSA and MR-CNS did not afford protection to the organism in the presence of a SCH wound dressing. The use of topical antiseptics in chronic wound care should be considered before the use of antibiotics that can result in their overuse and the risk of further resistance.

In vitro studies to show sequestration of matrix metalloproteinases by silver-containing wound care products.

Excess or "uncontrolled" proteinase activity in the wound bed has been implicated as one factor that may delay or compromise wound healing. One proteinase group--matrix metalloproteinases--includes collagenases, elastase, and gelatinases and can be endogenous (cell) or exogenous (bacterial) in origin. A study was conducted to assess the ability of five silver-containing wound care products to reduce a known matrix metalloproteinase supernatant concentration in vitro. Four silver-containing wound dressings (a carboxy-methyl cellulose, a nanocrystalline, a hydro-alginate, and a collagen/oxidized regenerated cellulose composite dressing), along with a 0.5% aqueous silver nitrate [w/v] solution and controls for matrix metalloproteinase-2 and matrix metalloproteinase-9 sourced from ex vivo dermal tissue and blood monocytes, respectively, were used. Extracts were separated and purified using gelatine-Sepharose column chromatography and dialysis and polyacrylamide gel electrophoretic zymography was used to analyze specific matrix metalloproteinase activity. All dressings and the solution were shown to sequester both matrix metalloproteinases. The silver-containing carboxy-methyl cellulose dressing showed significantly greater sequestration for matrix metalloproteinase-2 at 6 and 24 hours (P< 0.001) compared to the other treatments. For matrix metalloproteinase-9, both the carboxy-methyl cellulose dressing and the oxidized regenerated cellulose dressing achieved significant sequestration when compared to the other treatments at 24 hours (P <0.001), which was maintained to 48 hours (P < 0.001). Results from this study show that silver-containing dressings are effective in sequestering matrix metalloproteinase-2 and -9 and that this can be achieved without a sacrificial protein (eg, collagen). Although the varying ability of wound dressings to sequester matrix metalloproteinases has been shown in vitro, further in vivo evidence is required to confirm these findings.

Evaluating antibiotics for use in medicine using a poloxamer biofilm model.

BACKGROUND: Wound infections, due to biofilms, are a constant problem because of their recalcitrant nature towards antibiotics. Appropriate antibiotic selection for the treatment of these biofilm infections is important. The traditional in vitro disc diffusion method for antibiotic selection uses bacterial cultures grown on agar plates. However, the form of bacterial growth on agar is not representative of how bacteria grow in wounds and other tissue sites as here bacteria grow naturally in a biofilm. The aim of this research was to test a more appropriate method for testing antimicrobial efficacy on biofilms and compare with the standard methods used for antibiotic sensitivity testing. METHODS: Outer Membrane Protein analysis was performed on E.coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis and Acinetobacter juni when grown on Mueller Hinton agar ('quasi-biofilm state') and 30% Poloxamer hydrogel ('true- biofilm state). Susceptibility to antibiotics on 28 clinical isolates was determined using the modified Kirby Bauer disc diffusion method, on agar and 30% Poloxamer. RESULTS: Similar outer membrane proteins [OMPs] were identified in bacteria grown in a biofilm state and on a 30% poloxamer hydrogel, which were very different to the OMPs identified in bacteria grown on Mueller-Hinton agar and broth. There was a significant difference between the means of the clearance zones around the antibiotic discs on standard agar and poloxamer gels [P < 0.05]. The zones of clearance were generally smaller for poloxamer-grown bacteria than those grown on standard agar. Diffusion distances of various antibiotics through agar and 30% poloxamer showed no significant difference [P > 0.05]. CONCLUSION: The findings of this experiment suggest that poloxamer gel could be used as an appropriate medium on which to conduct biofilm antibiotic susceptibility tests as it enables bacteria to be grown in a state representative of the infected surface from which the culture was taken.

Silver deposition and tissue staining associated with wound dressings containing silver.

Argyria is the general term used to denote a clinical condition in which excessive administration and deposition of silver causes a permanent irreversible gray-blue discoloration of the skin or mucous membranes. The amount of discoloration usually depends on the route of silver delivery (ie, oral or topical administration) along with the body's ability to absorb and excrete the administered silver compound. Argyria is accepted as a rare dermatosis but once silver particles are deposited, they remain immobile and may accumulate during the aging process. Topical application of silver salts (eg, silver nitrate solution) may lead to transient skin staining. To investigate their potential to cause skin staining, two silver-containing dressings (Hydrofiber and nanocrystalline) were applied to human skin samples taken from electively amputated lower limbs. The potential for skin discoloration was assayed using atomic absorption spectroscopy. When the dressings were hydrated with water, a significantly higher amount of silver was released from the nanocrystalline dressing compared to the Hydrofiber dressing (P <0.005), which resulted in approximately 30 times more silver deposition. In contrast, when saline was used as the hydration medium, the release rates were low for both dressings and not significantly different (silver deposition was minimal). Controlling the amount of silver released from silver-containing dressings should help reduce excessive deposition of silver into wound tissue and minimize skin staining.

Models in vivo of wound healing in the horse and the role of growth factors.

Abstract Wound models attempt to simulate the natural healing processes in wounds. However, all models have significant limitations due to the complexity of the tissue repair process. Much can be learned from wound models in vitro by the use of cell culture techniques. The horse can provide a suitable naturally occurring model of chronic wound healing because it has many similarities to wound healing encountered in human medicine. The tissue architecture was investigated with regard to extracellular matrix and growth factor distribution during wound healing and growth factors were consistently present in the wound area. Biochemical investigations revealed increased levels of hydroxyproline, collagen, and TGFß1 in exuberant granulation tissue. Equine wound models were established in vitro using cell culture techniques and growth factors had significant effects on the growth of the cells and their ability to synthesize collagen. Two gelatinases (MMP-2 and MMP-9) were detected in the tissues and wound fluid samples investigated. Zusammenfassung Wundmodelle haben zum Ziel, die natürlichen Heilungsprozesse in Wunden zu simulieren. Allen Modellen sind durch den komplexen Gewebsheilungsprozess deutliche Grenzen gesetzt. Durch die Verwendung von Zellkulturtechniken kann von Wundmodellen in vitro viel abgeleitet werden. Das Pferd stellt wegen der vielen Ähnlichkeiten zur humanmedizinischen Wundheilung ein geeignetes Modell für chronische Wundheilung dar. Die Gewebsarchitektur wurde bezüglich der Verteilung von extrazellulärer Matrix und von Wachtumsfaktoren während der Wundheilung untersucht; Wachtsumsfaktoren waren ständig in der Wunde vorhanden. Biochemische Untersuchungen ergaben erhöhte Hydroxyprolin-, Kollagenund TGFß1-Spiegel. Wundmodelle beim Pferd in vitro und Zellkulturtechniken wurden entwickelt; Wachstumsfaktoren hatten deutliche Wirkung auf die Zellen und ihre Fahigkeit zur Kollagensynthese. Zwei Gelatinasen (MMP-2 und MMP-9) wurden im Gewebe identifiziert und Wundflüssigkeitsproben untersucht. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Wundheilungsmodelle in vivo beim Pferd und die Rolle von Wachstunisfaktoren). Veterinary Dermatology 1997; 8: 259-272] Resumen Los modelos de heridas intentan estimular el proceso natural de curación de heridas. Sin embargo, todos 10s modelos presentan limitaciones considerables debido a la complejidad del proceso de curación de heridas. Se puede aprender mucho de modelos de heridas in vitro mediante el uso de técnicas de cultivo celular. El caballo puede suponer un modelo natural adecuado de curación crónica de heridas ya que se asemeja a la curación de heridas en medicina humana. Se investigó la arquitectura tisular en referencia a la matriz extracelular y la distribución de factores de crecimento viendo que los factores de crecimiento se encontraban presentes de forma constante en el área de la herida. Las investigaciones bioquímicas revelaron incremento en los niveles de hidroxiprolina, colágeno y TGFbl en tejido de granulación exhuberante. Se establecieron modelos in vitro de heridas equinas utilizando técnicas de cultivo celular y los factores de crecimiento tuvieron efectos significativos en el crecimiento de las células y su capacidad de sintetizar colágeno. Se detectaron dos gelatinasas (MMP-2 y MMP-9) en 10s tejidos y muestras de fluidos de las heridas investigadas. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Modelos de curacion de heridas in vivo en el caballo y el papel de 10s factores de crecimiento). Veterinary Dermatology 1997; 8: 259-272] Résumé Les modèles de plaies tentent de simuler les processus naturels de cicatrisation. Cependant tous les modèles ont des limitations significatives dues à la complexité des processus de réparation des tissus. Beaucoup peut être étudié sur des modèles de plaies in vitro suite à l'utilisation des techniques de culture cellulaire. Le cheval peut constituer un modèle nature1 fiable de cicatrisation chronique de plaie de par les nombreuses similarités qu'il partage avec la cicatrisation des plaies en médecine humaine. L'architecture tissulaire a étéétudiée concernant la matrice extracellulaire et la distribution des facteurs de croissance pendant la cicatrisation, et les facteurs de croissance se sont montrés présents de façon consistante dans la zone de la plaie. Des investigations biochimiques ont révélé une élévation des taux d'hydroxyproline, collagène, et TGFß1 dans la granulation tissulaire exubérante. Des modèles équins de plaies ont étéétablis in vivo en utilisant des techniques de culture cellulaire et les facteurs de croissance ont montré des effets significatifs sur la croissance des cellules et leur capacité de synthétiser le collagène. Deux gélatinases (MMP-2 et MMP-9) ont été détectées dans les tissus et les échantillons de fluide des plaies ont été analysés. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Modeles in vivo de cicatrisation des plaies chez le cheval et role des facteurs de croissance). Veterinary Dermatology 1997; 8: 259-272].