[Show abstract][Hide abstract] ABSTRACT: To determine if sequential treatment with Bevacizumab (Avastin), a monoclonal, VEGF antibody that blocks angiogenesis; Saratin, a 12 kD polypeptide with anti-inflammatory and anti-thrombotic properties; and Ilomastat, a matrix metalloproteinase (MMP) inhibitor, prolongs bleb life following glaucoma filtration surgery (GFS) in a rabbit model. Thirty-two New Zealand White rabbits (eight rabbits per group) underwent GFS in the left eye. Group 1 received a perioperative injection of both Saratin and Bevacizumab, and later, subconjuctival injections of Ilomastat on days 8 and 15. Group 2 received only Saratin perioperatively, and also received Ilomastat injections on days 8 and 15. Group 3, the negative control, received a single perioperative injection of Balanced Saline Solution (BSS) along with post-operative BSS injections on days 8 and 15. Group 4, the positive control, received topical treatment with Mitomycin-C (MMC) at the time of surgery with no further treatment. Blebs were evaluated by an observer masked to treatment every third day. Histology was obtained on two eyes in each group on post-op day twelve as well as all eyes following bleb failure. Eyes in group 1 had a mean bleb survival time of 29 ± 2.7 days, whereas those in group 2 that received the experimental treatment without Bevacizumab had a mean survival time of 25.5 ± 2.7 days. An ANOVA test showed that the Saratin/Ilomastat/Bevacizumab group demonstrated a significant prolongation of bleb survival compared to the BSS control-mean survival time of 19.7 ±2.7 days-(p = 0.0252) and was not significantly different from the MMC positive control group (p = 0.4238)-mean survival time of 32.5 ± 3.3. From tissue histology at day 12, the four different groups showed marked differences in the cellularity and capsule fibrosis. The MMC eyes showed minimal cellularity, were avascular and had minimal fibrous tissue. BSS group showed high cellularity, moderate to high fibrosis, and thicker and more defined capsules than either of the treatment groups and the positive control. Both the Saratin/Ilomastat/Bevacizumab and Saratin/Ilomastat only eyes showed moderate cellularity with minimal fibrosis, with less cellularity and fibrosis present in the triple treatment group. Sequential therapy with multiple agents, including Bevacizumab, prolonged bleb function following GFS in the rabbit model and were significantly better than the negative BSS control. The experimental group did not show the same surface tissue histological thinning and side effects associated with MMC treatment.
PLoS ONE 09/2015; 10(9):e0138054. DOI:10.1371/journal.pone.0138054 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to assess the potential differences in antibacterial activity and fluid flow rates Of 2 commonly used silver dressings: an alginate-only dressing and an alginate dressing with a nonadherent contact layer. Materials and Methods. The dressings' antibacterial activities were tested against 2 major bacterial pathogens for skin wounds Staphylococcus aureus and Pseudomonas aeruginosa using 2 in vitro models of skin wounds. The fluid flow rate through the 2 dressings was also measured and compared. Results. While materially similar, the choice to include a nonadherent layer leads to reduced antibacterial performance and a reduced rate of fluid flow into and through the dressing. Conclusion. The use of the silver antimicrobial alginate dressing with a nonadherent layer provides a welcome feature in that it does not adhere to surfaces; however, the results demonstrate lower rate Of fluid removal when compared to a silver antimicrobial alginate without the nonadherent layer.
[Show abstract][Hide abstract] ABSTRACT: Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, impact the lives of millions of people worldwide. These types of wounds represent a significant physical, social and financial burden to both patients and health care systems. Wound care has made great progress in recent years as a result of the critical research performed in academic, clinical and industrial settings. However, there has been relatively little translation of basic research discoveries into novel and effective treatments. One underlying reason for this paucity may be inconsistency in the methods of wound analysis and sample collection, resulting in the inability of researchers to accurately characterise the healing process and compare results from different studies. This review examines the various types of analytical methods being used in wound research today with emphasis on sampling techniques, processing and storage, and the findings call forth the wound care research community to standardise its approach to wound analysis in order to yield more robust and comparable data sets.
International Wound Journal 01/2015; DOI:10.1111/iwj.12399 · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulation of wound healing following GFS is a complex process and involves multiple growth factors, cytokines, and proteases, and the TGF-β and CTGF systems appear to play major roles in promoting scarring and contraction that result in bleb failure. An ideal outcome of GFS is a diffuse, but healthy, bleb that filters aqueous humor without leaking. Achieving this goal is challenging, as the currently available interventions utilize broadly acting anti-metabolites that can cause early complications, such as toxicity, and delayed complications, such as bleb leaks and scleral melts. The ideal modulator of GFS would, therefore, not only be cell-specific, but also, preferably, gene-specific to selectively alter the healing of the sub-Tenon's fibroblasts without off-target or delayed effects on the surrounding tissues and the fibroblasts themselves. Understanding the molecular regulation of healing following GFS in conjunctival/Tenon's capsule tissues facilitates the design of therapies that utilize drugs which selectively interfere with genes and proteins that promote scar formation and contraction without undesirable side effects.
[Show abstract][Hide abstract] ABSTRACT: Bacterial infection of acute and chronic wounds impedes wound healing significantly. Part of this impediment is the ability of bacterial pathogens to grow in wound dressings. In this study, we examined the effectiveness of a polyurethane foam wound dressings coated with poly diallyl-dimethylammonium chloride (pDADMAC-PU) to inhibit the growth and biofilm development by three main wound pathogens: Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, within the wound dressing. pDADMAC-PU inhibited the growth of all three pathogens. Time-kill curves were conducted both with and without serum to determine the killing kinetic of pDADMAC-PU. pDADMAC-PU killed S. aureus, A. baumannii, and P. aeruginosa. The effect of pDADMAC-PU on biofilm development was analyzed quantitatively and qualitatively. Quantitative analysis, (CFU) assay, revealed that pDADMAC-PU dressing produced more than 8 log reduction in biofilm formation by each pathogen. Visualization of the biofilms by either confocal laser scanning microscopy or scanning electron microscopy confirmed these findings. In addition, it was found that the pDADMAC-PU treated foam totally inhibited migration of bacteria through the foam for all three bacterial strains. These results suggest that pDADMAC-PU is an effective wound dressing that inhibits the growth of wound pathogens both within the wound and in the wound dressing.
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[Show abstract][Hide abstract] ABSTRACT: This study sought to determine whether silver-containing dressings and medical-grade honey gel interfere with one another in measurable ways.
Dressings applied together in clinical use were tested using in vitro and ex vivo methods to determine whether the combined modalities maintain their individual properties. In order to determine if the presence of silver dressings interfere with honey's osmotic strength, which is a key physical property of medical honey, changes in honey's 2 primary sugars were measured, as well as changes in its overall osmotic strength. Finally, the antibacterial barrier activity of the dressings were tested individually and in honey/silver pairs in 2 in vitro models with 2 clinical strains of bacteria.
The data demonstrate that honey with silver dressings resulted in an increased osmolarity, since both the concentration of the 2 primary sugars in honey as well as its overall osmolarity increased. The data also demonstrate that the in vitro antibacterial barrier activity seen with silver-containing dressings does not decrease with the addition of medical honey and in some instances increased.
Altogether, these data suggest that these 2 classes of dressings do not interfere with each other. Clinical evidence is still required to fully validate these findings.
[Show abstract][Hide abstract] ABSTRACT: Objective. This study sought to determine whether silver-containing dressings and medical-grade honey gel interfere with one another in measurable ways. Materials and Methods. Dressings applied together in clinical use were tested using in vitro and ex vivo methods to determine whether the combined modalities maintain their individual properties. In order to determine if the presence of silver dressings interfere with honey's osmotic strength, which is a key physical property of medical honey, changes in honey's 2 primary sugars were measured, as well as changes in its overall osmotic strength. Finally, the antibacterial barrier activity of the dressings were tested individually and in honey/silver pairs in 2 in vitro models with 2 clinical strains of bacteria. Results. The data demonstrate that honey with silver dressings resulted in an increased osmolarity, since both the concentration of the 2 primary sugars in honey as well as its overall osmolarity increased. The data also demonstrate that the in vitro antibacterial barrier activity seen with silver-containing dressings does not decrease with the addition of medical honey and in some instances increased. Conclusion. Altogether, these data suggest that these 2 classes of dressings do not interfere with each other. Clinical evidence is still required to fully validate these findings.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvβ6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-β1 activation in vitro.
CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver disease.
[Show abstract][Hide abstract] ABSTRACT: The effects of a triple combination of siRNAs targeting key scarring genes was assessed using an ex vivo organ culture model of excimer ablated rabbit corneas. The central 6 mm diameter region of fresh rabbit globes was ablated to a depth of 155 microns with an excimer laser. Corneas were excised, cultured at the air-liquid interface in defined culture medium supplemented with transforming growth factor beta 1 (TGFB1), and treated with either 1% prednisolone acetate or with 22.5 μM cationic nanoparticles complexed with a triple combination of siRNAs (NP-siRNA) targeting TGFB1, TGFB Receptor (TGFBR2) and connective tissue growth factor (CTGF). Scar formation was measured using image analysis of digital images and levels of smooth muscle actin (SMA) were assessed in ablated region of corneas using qRT-PCR and immunostaining. Ex vivo cultured corneas developed intense haze-like scar in the wounded areas and levels of mRNAs for pro-fibrotic genes were significantly elevated 3 to 8 fold in wounded tissue compared to unablated corneas. Treatment with NP-siRNA or steroid significantly reduced quantitative haze levels by 55% and 68%, respectively, and reduced SMA mRNA and immunohistostaining. This ex vivo corneal culture system reproduced key molecular patterns of corneal scarring and haze formation generated in rabbits. Treatment with NP-siRNAs targeting key scarring genes or an anti-inflammatory steroid reduced corneal haze and SMA mRNA and protein.
Experimental Eye Research 06/2014; 125. DOI:10.1016/j.exer.2014.06.014 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
This study aimed to elucidate the role of connective tissue growth factor (CTGF) in healthy eyes and wounded corneas of mice and rabbits. Conditional knockout mice were used to determine the role of CTGF in corneal healing.
CTGF expression was determined using transgenic mice carrying CTGF promoter driven-eGFP, quantitative RT-PCR, and immunofluorescent staining. Mice that carried two floxed CTGF alleles and a Cre/ERT2 transgene under the control of human ubiquitin C (ubc) promoter were used to conditionally delete CTGF gene in a tamoxifen-inducible manner. Phototherapeutic keratectomy (PTK) was used to generate an acute corneal wound and corneal re-epithelialization was assessed by fluorescein staining.
Connective tissue growth factor expression was found in multiple ocular tissues with relatively high levels in the corneal endothelium, lens subcapsular epithelium, and in the vasculature of the iris and retina. Wounded corneas responded with an immediate upregulation of CTGF in the epithelium at the wound margin and a sustained CTGF induction during re-epithelialization. At the onset of haze formation, CTGF protein becomes more focused in the basal epithelium. Deletion of the CTGF gene caused a 40% reduction (P < 0.01) in the cornea re-epithelialization rate in knockout mice compared with wild-type mice.
Connective tissue growth factor is expressed in the naïve cornea, lens, iris, and retina, and is expressed immediately after epithelial injury. Loss of CTGF impairs efficient re-epithelialization of corneal wounds.
[Show abstract][Hide abstract] ABSTRACT: Negative pressure wound therapy with instillation (NPWTi) is increasingly used as an adjunct therapy for a wide variety of infected wounds. However, the effect of NPWTi on mature biofilm in wounds has not been determined. This study assessed the effects of NPWTi using saline or various antimicrobial solutions on mature Pseudomonas aeruginosa biofilm using an ex vivo porcine skin explant biofilm model. Treatment consisted of six cycles with 10-minute exposure to instillation solution followed by 4 hours of negative pressure at -125 mm Hg over a 24-hour period. NPWTi using saline reduced bacterial levels by 1-log (logarithmic) of 7-log total colony-forming units (CFUs). In contrast, instillation of 1% povidone iodine (2-log), L-solution (3-log), 0·05% chlorhexidine gluconate (3-log), 0·1% polyhexamethylene biguanide (4-log), 0·2% polydiallyldimethylammonium chloride (4-log) and 10% povidone iodine (5-log), all significantly reduced (P < 0·001) total CFUs. Scanning electron micrographs showed disrupted exopolymeric matrix of biofilms and damaged bacterial cells that correlated with CFU levels. Compared with previous studies assessing microbicidal effects of topical antimicrobial dressings on biofilms cultured on porcine skin explants, these ex vivo model data suggest that NPWTi with delivery of active antimicrobial agents enhances the reduction of CFUs by increasing destruction and removal of biofilm bacteria. These results must be confirmed in human studies.
International Wound Journal 12/2013; 10(s1):48-55. DOI:10.1111/iwj.12180 · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Transforming growth factor β1 (TGFβ1), TGFβ receptor (TGFβR2), and connective tissue growth factor (CTGF) are key regulators of fibrosis in the cornea and in other tissues, including liver, skin, and kidney. We developed an antifibrotic treatment targeting these three critical scarring genes by using a combination of small interfering RNAs (siRNAs) and assessed its effect on downstream scarring genes, collagen I, and α smooth muscle actin (SMA).
Up to six individual siRNAs for each of the three target gene mRNAs were transfected into cultures of rabbit corneal fibroblasts at concentrations from 15 to 90 nM. The knockdown of target gene proteins was measured by ELISA, and the two most effective siRNAs were tested in dual combinations. Knockdown percentages of both individual and dual siRNA combinations were analyzed for synergy by using combination index to predict "effective" and "ineffective" triple siRNA combinations. Effects of both triple siRNA combinations on target and downstream mRNAs were measured by using quantitative RT-PCR, and levels of SMA protein were assessed by immunohistochemistry.
Single and dual siRNA combinations produced a wide range of protein knockdown of target genes (5%-80%). The effective triple siRNA combination significantly reduced mRNA levels of target genes (>80%) and downstream scarring genes (>85%), and of SMA protein (>95%), and significantly reduced cell migration without reducing cell viability.
Simultaneous targeting of TGFβ1, TGFβR2, and CTGF genes by effective triple siRNA combination produced high knockdown of target and downstream scarring genes without cell toxicity, which may have clinical applications in reducing corneal fibrosis and scarring in other tissues.
[Show abstract][Hide abstract] ABSTRACT: A bipedicle ischaemic rat skin flap model was used to study the effects of daily topical applications of platelet-derived growth factor (PDGF) on the healing of ischaemic wounds. Levels of tumour necrosis factor-alpha (TNFA), interleukin 1-beta (IL1B) and both the latent and active forms of matrix metalloproteinase 2 (MMP2) and 9 (MMP9) were measured. Full-thickness wounds were made on a total of 72 adult male Sprague-Dawley rats. Each group of 18 rats with normal and ischaemic wounds received either vehicle or 0·01% recombinant PDGF-BB. Additional applications were made on the wounds on a daily basis. Wound areas were measured at 0, 1, 3, 5, 7 9 and 13 days after wounding. Ischaemia caused a delay in wound healing as well as an increase in TNFA, IL1B and both the pro and active forms of MMP2 and MMP9. PDGF accelerated the rate of wound healing in both normal and ischaemic wounds and negated the effect of ischaemia. PDGF reduced the TNFA concentration in both normal and ischaemic wounds, and the rate of wound healing closely resembled the pattern of TNFA protein expression. PDGF also reduced both the magnitude and duration of the increases in IL1B and both the pro and active forms of MMP2 and MMP9 induced by ischaemia.
International Wound Journal 10/2013; 12(5). DOI:10.1111/iwj.12165 · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: The transforming growth factor beta1 (TGFB1) pathway has been linked to fibrosis in sev- eral tissues including skin, liver, kidney and the cor- nea. In this study, a RNA interference-based approach using siRNAs targeting three critical scarring genes, TGFB1, TGFB receptor 2 (TGFBR2) and connective tissue growth factor (CTGF), was tested for effects on reducing alpha smooth muscle actin (SMA) and cor- neal scarring (haze) in excimer laser ablated rabbit corneas. Methods: Levels of TGFB1 and CTGF mRNAs were measured using qRT-PCR in the epithelial and endothelial cell layers of normal and excimer ablated rabbit corneas at 30 minutes, 1 day and 2 days after ablation. Two different scarring models were utilized to assess the effects of the triple siRNA combination on corneal scarring. In the first model, rabbit corneas were unevenly ablated creating a mesh pattern then treated immediately with the triple siRNA combina- tion. After 1 day the ablated areas of corneas were collected and levels of mRNAs for TGFB1, TGFBR2 and CTGF were measured. After 14 days, levels of mRNA for SMA were measured and SMA protein im- munolocalized in frozen sections. In the second model, rabbit corneas were uniformly ablated to a depth of 155 microns followed by three daily doses of the triple combination of siRNA. After 14 days, corneas were photographed and images were analyzed using Image J software to assess corneal scarring. Corneas were also analyzed for levels of SMA mRNA. Results: In both unwounded and wounded corneas, levels of TGFB1 and CTGF mRNA were always significantly higher in endothelial cells than in epithelial cells (10 to 30 fold). Thirty minutes after injury, levels of both TGFB1 and CTGF mRNAs increased approximately 20-fold in both epithelial and endothelial cells, and further in- creased approximately 60-fold in 2 days. In the first therapeutic experiment with a single siRNA dose, two of three rabbits showed substantial reductions of all three target genes after 1 day with a maximum knock down of 80% of TGFb1, 50% reduction of TGFBR2 and 40% reduction of CTGF mRNA levels and reduc- ed SMA mRNA at day 14. In the second therapeutic experiment with multiple doses of siRNA treatment, both rabbits showed a ~22% reduction in scar forma- tion at day 14 as calculated by image analysis. There was also a corresponding 70% and 60% reduction of SMA RNA expression. Conclusion: These results de- monstrate that both TGFB1 and CTGF dramatically increase in rabbit corneal epithelial and endothelial cells after injury. Treatment of excimer ablated rab- bit corneas with a triple combination of siRNAs effec- tively reduced levels of the target genes and SMA, lead- ing to reduced corneal scarring at 14 days, suggesting tha t this triple siRNA combination may be an effective new approach to reducing scarring in cornea and other tissues.
Advances in Bioscience and Biotechnology 10/2013; 4(10):47-55. DOI:10.4236/abb.2013.410A4005
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes.
Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3' untranslated region (UTR) of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative real-time PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration.
At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at -14.33. Pre-miR-133b targeted the 3' UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor β1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFβ1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF.
Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation.
[Show abstract][Hide abstract] ABSTRACT: An ex vivo porcine skin explant biofilm model that preserves key properties of biofilm attached to skin at different levels of maturity (0-3 days) was used to assess the efficacy of commercially available antimicrobial dressings and topical treatments. Assays were also performed on the subpopulation of antibiotic tolerant biofilm generated by 24 hours of pre-treatment with gentamicin (120× minimal inhibitory concentration) prior to agent exposure. Five types of antimicrobial agents (iodine, silver, polyhexamethylene biguanide, honey and ethanol) and four types of moisture dressings (cotton gauze, sodium carboxymethylcellulose fibre, calcium alginate fibre and cadexomer beads) were assessed. Time-release silver gel and cadexomer iodine dressings were the most effective in reducing mature biofilm [between 5 and 7 logarithmic (log) of 7-log total], whereas all other dressing formulations reduced biofilm between 0·3 and 2 log in 24 or 72 hours with a single exposure. Similar results were found after 24-hour exposure to silver release dressings using an in vivo pig burn wound model, demonstrating correlation between the ex vivo and in vivo models. Results of this study indicate that commonly used microbicidal wound dressings vary widely in their ability to kill mature biofilm and the efficacy is influenced by time of exposure, number of applications, moisture level and agent formulation (sustained release).
International Wound Journal 09/2013; 12(4). DOI:10.1111/iwj.12142 · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Corneal opacification (i.e., haze) following a non-denaturing acute injury to the cornea is a process which takes about 5 days to manifest itself, indicating that it is the consequence of cellular and molecular biological processes. In order to obtain a better understanding of the haze development process, and to test candidate anti-haze therapies, we use a corneal scarring model whereby we create an excimer laser wound in the center of rabbit corneas. The primary data generated by this model are (1) changes in corneal thickness with time; (2) wound closure rates, or re-epithelialization; (3) changes in the location and density of corneal sub-epithelial haze; and (4) molecular and histological changes leading up to, during, and following the formation of haze.While the use of excimer lasers to generate consistent wounds in rabbit corneas is not a novel protocol for the study of corneal haze, the photographic technique presented here for the more objective recording and quantification of corneal haze is. At present, a qualitative, semiquantitative, grading system is employed whereby the amount of iris detail discernible through the scar is assigned a value between 0 and 4. Such a system makes direct comparisons amongst reported anti-haze trials nearly impossible. Furthermore, the additional "geographic" detail provided by the image provides a new layer of information about the formation of haze and the ability to troubleshoot dosing regimens. Altogether, with the information present herein, we believe that the study of corneal haze formation and the ability to compare and contrast candidate therapies are both greatly improved.
[Show abstract][Hide abstract] ABSTRACT: 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.