Patricia M. Mertz

THE PIG AS A MODEL FOR HUMAN WOUND HEALING

The medical literature describes numerous in vitro and in vivo wound‐healing models. The selection of an animal model depends on a number of factors including availability, cost, ease of handling, investigator familiarity, and anatomical/functional similarity to humans. Small mammals are frequently used for wound healing studies, however, these mammals differ from humans in a number of anatomical and physiological ways. Anatomically and physiologically, pig skin is more similar to human skin. The many similarities between man and pig would lead one to believe that the pig should make an excellent animal model for human wound healing. The purpose of this paper is to review the existing literature for evidence of this supposition and determine how well the various models correlate to human wound healing. Studies of wound dressings, topical antimicrobials, and growth factors are examined. Over 180 articles were utilized for this comparative review. Our conclusion is that the porcine model is an excellent tool for the evaluation of therapeutic agents destined for use in human wounds.

Microscopic and physiologic evidence for biofilm‐associated wound colonization in vivo

A biofilm is a collection of microbial cells that are attached to a surface and embedded in a self‐produced extrapolymeric substance. The understanding of the biofilm phenotype is important in the understanding of bacteria in vitro but it has been difficult to translate biofilm science to the clinical setting. More recently, preliminary criteria for defining biofilm associated diseases have been proposed and the purpose of this study was to create a biofilm‐associated wound model based on these criteria. Using a porcine model, partial thickness wounds were inoculated with a wound isolate Staphylococcus aureus strain. Wounds were then treated with either one of two topical antimicrobial agents (mupriocin cream or triple antibiotic ointment) within 15 minutes to represent planktonic bacteria or 48 hours after initial inoculation to represent biofilm‐associated wound infection. Using light microscopy, scanning electron microscopy and epifluorescence microscopy, we were able to observe biofilm‐like structures in wounds after 48 hours of inoculation and occlusion. The in vivo antimicrobial assay was used to demonstrate that both mupirocin cream and the triple antibiotic ointment were effective in reducing planktonic S. aureus but had reduced efficacy against biofilm‐embedded S. aureus. Our results demonstrated that S. aureus form firmly attached microcolonies and colonies of bacteria encased in an extracellular matrix on the surface of the wounds. These biofilm‐like communities also demonstrated increased antimicrobial resistance when compared with their planktonic phenotype in vivo. The structural and physiological results support the hypothesis that bacterial biofilms play a role in wound colonization and infection.

Wound healing. The effects of topical antimicrobial agents.

The effect of four commonly used topical antimicrobial agents on the rate of reepithelialization of clean wounds was evaluated in white domestic pigs. Neosporin Ointment was found to significantly increase the rate of reepithelialization by 25%, while Furacin significantly retarded the healing rate by 24%. Pharmadine, a preparation containing povidone-iodine, did not affect the rate of healing. Both Silvadene and its vehicle significantly increased the rate of reepithelialization by 28% and 21%, respectively. The effects of these agents cannot be explained on the basis of their antimicrobial activity.

Foot lesions associated with Pseudomonas cepacia

Summary Pseudomonas cepacia was isolated in small numbers from swamp waters and in large numbers from the toewebs of troops after training in swamp conditions. Colonisation with P. cepacia was associated with macerated, hyperkeratotic lesions of the feet. The isolates showed resistance to a wide range of antibiotics, but strains from these and other sources were sensitive in vitro to trimethoprim and sulphamethoxazole. The ability of P. cepacia to colonise sodden but otherwise intact skin is significant in view of the organisms potential as a cause of wound and other infections.

Second-degree burn healing: The effect of occlusive dressings and a cream

Because occlusive dressings and some creams have been found to speed epithelialization of blade-induced wounds, we studied the effect of two occlusive dressings and a polyglycerylmethacrylate cream containing low concentration of fibronectin on epithelialization in second-degree burn wounds. Cylindrical brass rods were heated in a boiling water bath, removed, wiped dry, and placed (6 sec) on the skin of domestic pigs. The burned epidermis was removed and each burn wound was assigned to one of the following treatment groups: (1) air-exposed, (2) DuoDERM (hydrocolloid dressing; Squibb Co., New Jersey), (3) Opsite (polyurethane dressing; Smith & Nephew, New Jersey), or (4) experimental cream. Several burn wounds were excised from each treatment group on Days 6 to 14 after wounding. The excised burn wounds were incubated in 0.5 M NaBr for 24 hr which allowed separation of the epidermis from the dermis. The epidermis was examined macroscopically for defects in the area of the burn. Specimens were considered healed when a defect was not present. Neither of the occlusive dressings changed the rate of epithelialization as compared to air exposure. Wounds which were treated with the experimental cream epithelialized faster than the air-exposed wounds (P less than 0.025).

The effect of electrical stimulation on the number of mast cells in healing wounds

Many cutaneous disorders are associated with activation or increased numbers of mast cells. Electrical stimulation has been shown to be effective in treating many of these disorders. This study is designed to examine the effect of electrical stimulation on mast cells in acute wounds. Four pathogen-free pigs received 20 wounds, each of which was subjected to biopsy at various times after wounding. Half of the wounds were treated with electrical stimulation and the other half were treated with a sham electrode. The biopsy specimens were fixed in Carnoys medium and stained with alcian blue and Nuclear Fast Red. Mast cells from both sets of wounds were counted and analyzed. Highly significant reductions in the number of mast cells were seen with electrical stimulation on days 1 and 2 compared with nonstimulated control wounds. Electron microscopy was performed to compare the stimulated and control mast cells for characteristic features in morphology, location, and evidence of degranulation. Electrical stimulation did not appear to induce degranulation. The ability of electrical stimulation to decrease the number of mast cells may be related to a reduction of either proliferation or migration of these cells and may prove to be a valuable therapeutic technique.

Collagen fiber orientation as quantified by small angle light scattering in wounds treated with transforming growth factor-beta2 and its neutalizing antibody.

The purpose of this study was determine quantitative differences in collagen fiber orientation in a wound healing model in the presence of transforming growth factor‐β2 and anti‐transforming growth factor‐β2,3 antibody. Full‐thickness wounds were made in the paravertebral area of two young pigs. Wounds were treated once, topically, with either transforming growth factor‐β2 or anti‐transforming growth factor‐β2 antibody, or with methylcellulose gel. Control wounds were left untreated. Tissue biopsies were obtained from each wound on days 7, 14 and 46 post wounding. Tissue sections were stained with hematoxylin and eosin, and collagen fiber preferred orientation was quantified using small angle light scattering. Our results indicated that wounds treated with transforming growth factor‐β2 and anti‐transforming growth factor‐β2,3 antibody had a significantly higher degree of orientation of collagen fibers than normal unwounded skin on days 7, 14 and 46 (p < 0.001). Transforming growth factor‐β2– treated wounds had a higher degree of orientation of collagen fibers than control wounds on days 7 and 14 (p < 0.001), and control wounds displayed a higher degree of orientation than wounds treated with anti‐transforming growth factor‐β2,3 and normal unwounded skin at all time points (p < 0.001). These results suggest that differences in the dermal collagen degree of orientation correlate with scarring, and show that small angle light scattering can be used quantitatively to assess differences in the collagen fiber architecture of dermal wounds.

The Effects of Topical Transforming Growth Factor-β2 and Anti-Transforming Growth Factor-β2,3 on Scarring in Pigs

Background: Transforming growth factor-β2 (TGF-β2) has been implicated in the inflammatory response and subsequent scarring during wound healing. Objective: The experiment was designed to study the effects of a topical application of TGF-β2 and mouse monoclonal anti-TGF-β2,3 neutralizing antibody (and TGF-β2,3) on the development of fibrosis during healing. Methods: Sixteen full-thickness excision wounds were made in the paravertebral and thoracic area of four domestic pigs. On day 0, three wounds each were treated with: a) 5 μg of TGF-β2, b) 5 μg of 2% methylcellulose (mc), or c) 1.2 mg of anti-TGF-β2,3. As a vehicle for treatment of each wound methylcellulose 2% was used. Four wounds served as the untreated air-exposed control. Wounds were biopsied and the tissue sectioned and stained with hematoxylin and eosin on days 7, 14, and 45. Three blinded observers evaluated the wound specimens. Results: Using computer-aided point count stereology on days 7, 14, and 45, we found a statistically significant increase (p < .05) in the number of nucleated cells in the TGF-β2-treated wounds as compared to the other control wounds. Wounds treated with anti-TGF-β2,3 had significantly (p < .05) fewer nucleated cells on days 7,14, and 45. Microscopically, the TGF-β2-treated wounds had a larger scar area as compared to anti-TGF-β2,3 and controls. Conclusion: Treating wounds with an antibody directed against TGF-β2 might be a useful clinical approach to reduce fibrosis.

Effects of an arginine-glycine-aspartic acid peptide-containing artificial matrix on epithelial migration in vitro and experimental second-degree burn wound healing in vivo.

Cells central to dermal tissue repair such as dermal fibroblasts and keratinocytes interact with arginine-glycine-aspartic acid (RGD)-containing proteins of the extracellular matrix such as fibronectin. It has been shown that synthetic peptides containing this RGD sequence can also support cell attachment and migration in vitro. We therefore set out to test whether the use of these peptides, when formulated as a synthetic RGD-peptide matrix consisting of peptide complexed with hyaluronic acid, would have an effect on the rate of epithelial migration and healing of experimental wounds. Evaluation consisted of measuring he extent of epithelial outgrowth from human dermal explants and the epithelization of experimental second-degree burn wounds in pigs. We show here that the RGD-peptide matrix supports epithelial sheet migration from explants in a dose-dependent manner. In second-degree burn wounds in pigs, wounds treated with daily applications of the RGD-peptide matrix under occlusion resurfaced at a significantly faster rate (day 7 = 57% completely epithelized) than wounds treated with hyaluronic acid under occlusion (day 7 = 13% completely epithelized, p < 0.01), occlusion alone (day 7 = 13% completely epithelized, p < 0.01), or air exposed (day 7 = 0% completely epithelized, p < 0.001). Histologic examination showed that wounds treated with the RGD-peptide matrix also had thicker epithelial covering and greater granulation tissue deposition than occluded, air-exposed, and hyaluronate-treated wounds. These data therefore show that the use of RGD-peptide matrix induces faster explant epithelial migration and results in faster healing of experimental second-degree burns.

Use of tissue-engineered skin to study in vitro biofilm development.

BACKGROUND Biofilms are aggregations of microorganisms that have been identified as potential pathogens in the chronicity of nonhealing wounds. OBJECTIVE To develop an in vitro wound model to study biofilms using Graftskin, a tissue‐engineered skin equivalent. MATERIALS AND METHODS Graftskin constructs were divided into sections, and wounds were created on each section. Bacterial suspensions with a concentration of 106 CFU/mL were prepared from cultures of pathogenic isolates of Pseudomonas aeruginosa and Staphylococcus aureus. A 25‐μL aliquot of each suspension was deposited in the center of wounds created on the Graftskin. Sections were incubated at various time points, and a biopsy was then taken from the wounded and inoculated area. Sections were visualized with light (hematoxylin and eosin) and epifluorescent microscopy (calcofluor white and ethidium bromide). RESULTS Biofilm was observed on the wound model. Biofilm formation was dependent on time of Graftskin exposure to the bacteria. Biofilm was visualized in the S. aureus group at an earlier time point than in the P. aeruginosa group. CONCLUSIONS We demonstrated biofilm formation in vitro using a wound model. This model may provide a basis on which future studies may explore therapeutic modalities to prevent and eradicate pathogenic bacterial biofilm.