William H. Eaglstein

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.

Tissue-Engineered Skin

Tissue-engineered skin is a significant advance in the field of wound healing and was developed due to limitations associated with the use of autografts. These limitations include the creation of a donor site which is at risk of developing pain, scarring, infection and/or slow healing.A number of products are commercially available and many others are in development. Cultured epidermal autografts can provide permanent coverage of large area from a skin biopsy. However, 3 weeks are needed for graft cultivation. Cultured epidermal allografts are available immediately and no biopsy is necessary. They can be cryopreserved and banked, but are not currently commercially available.A nonliving allogeneic acellular dermal matrix with intact basement membrane complex (Alloderm®) is immunologically inert. It prepares the wound bed for grafting allowing improved cultured allograft ‘take’ and provides an intact basement membrane. A nonliving extracellular matrix of collagen and chondroitin-6-sulfate with silicone backing (Integra®) serves to generate neodermis.A collagen and glycosaminoglycan dermal matrix inoculated with autologous fibroblasts and keratinocytes has been investigated but is not commercially available. It requires 3 to 4 weeks for cultivation. Dermagraft® consists of living allogeneic dermal fibroblasts grown on degradable scaffold. It has good resistance to tearing. An extracellular matrix generated by allogeneic human dermal fibroblasts (TransCyte™) serves as a matrix for neodermis generation.Apligraf® is a living allogeneic bilayered construct containing keratinocytes, fibroblasts and bovine type I collagen. It can be used on an outpatient basis and avoids the need for a donor site wound. Another living skin equivalent, composite cultured skin (OrCel™), consists of allogeneic fibroblasts and keratinocytes seeded on opposite sides of bilayered matrix of bovine collagen. There are limited clinical data available for this product, but large clinical trials are ongoing. Limited data are also available for 2 types of dressing material derived from pigs: porcine small intestinal submucosa acellular collagen matrix (Oasis™) and an acellular xenogeneic collagen matrix (E-Z-Derm™). Both products have a long shelf life.Other novel skin substitutes are being investigated. The potential risks and benefits of using tissue-engineered skin need to be further evaluated in clinical trials but it is obvious that they offer a new option for the treatment of 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.

Inhibition of cell proliferation by chronic wound fluid

It has been proposed that occlusive wound dressings may enhance chronic wound repair by the stimulatory action of the fluid accumulating beneath the dressings. In this report, we investigated the in vitro proliferative effects of chronic wound fluid obtained from under a polyurethane membrane applied for 24 hours to venous ulcers in the ambulatory setting. By measuring cell counts and DNA synthesis, we found that chronic wound fluid inhibited the proliferation of human dermal fibroblasts (p = 0.008) and failed to stimulate the proliferation of microvascular endothelial cells (p = 0.03) and keratinocytes (p = 0.03). The inhibitory activity of chronic wound fluid on fibroblast proliferation was blocked after the fluid was heated to 100° C, but not 56° C, and was mainly restricted to a fraction of chronic wound fluid enriched in components less than 30 kd in molecular weight (p = 0.028). At concentrations ranging from 1% to 4% and in the presence of serum, chronic wound fluid decreased the viability of fibroblasts, as shown by a decreased ability of the cells to exclude trypan blue (p = 0.02), and the viability of endothelial cells, as shown by an increased release of tritiated adenine (p = 0.03). We conclude that the wound fluid obtained from beneath occlusive dressings applied to chronic wounds inhibits cell proliferation.

Human wound fluid from acute wounds stimulates fibroblast and endothelial cell growth

One proposed mechanism for the beneficial effect of occlusive dressings on healing is the maintenance of contact between the wound bed and accumulated wound fluid, which is thought to contain growth stimulatory substances. We have examined the effect of human wound fluid on the in vitro growth of human dermal fibroblasts and umbilical vein endothelial cells. Acute wound fluid was collected from six patients undergoing split-thickness skin grafting. The acute wound fluid was sterilely collected daily from underneath a vapor-permeable membrane applied to the donor site and changed every 24 hours for 3 days postoperatively. After seeding in optimal growth media (control) on day 0, cultures of human dermal fibroblasts and umbilical vein endothelial cells were supplemented with or without acute wound fluid on the next day (day 1) and on day 3. As determined by cell counts, 2% acute wound fluid stimulated the growth of human dermal fibroblasts (p less than 0.05) and umbilical vein endothelial cells (p less than 0.01) when these cells were cultured in 2% fetal bovine serum and endothelial growth medium, respectively. Wound fluid from postoperative days 1 or 3 caused the same level of stimulation. The addition of an anti-platelet-derived growth factor antibody to wound fluid resulted in a 45% mean reduction in its stimulatory effect on fibroblast growth (p less than 0.02), suggesting that platelet-derived growth factor contributes to the observed effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue engineering and the development of Apligraf, a human skin equivalent.

In recent years, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This review details the pioneering work of numerous investigators that led to the following precursors of tissue-engineered skin replacement: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts; autologous/allogeneic composites, acellular collagen matrices, and cellular matrices. It also discusses the rationale for the development of the newer products and describes the technical advances leading to the development of Apligraf, a tissue-engineered human skin product.

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.

A composite skin substitute (graftskin) for surgical wounds. A clinical experience.

background Bioengineered skin substitutes offer tissue replacement without requiring a donor site and might produce better healing. objective To evaluate the recipients response to grafting a bioengineered skin equivalent onto acute surgical wounds. methods Graftskin, which is made of: 1) a bovine collagen matrix containing human fibroblasts, and 2) an overlying sheet of stratified human epithelium, was grafted onto the excision sites of 15 patients. results Blood and cell studies for toxicity were negative. Graftskin proved easy to handle, and a typical clinical appearance of the skin substitute during “take” was detected. Compared with expectations improved healing occurred. Twelve of 15 patients had initial clinical takes. conclusion Graftskin was not clinically rejected and was not toxic. It often appeared to take and produced better than expected healing.

Experiences with biosynthetic dressings

Almost all documented clinical experience shows that occlusively dressed wounds heal more quickly and with less pain, tenderness, and swelling than undressed wounds. Clinical experience with occlusive skin dressings on acute and chronic wounds, on diseased skin, and on normal skin is reviewed. This report presents the effects of some of the newest oxygen-permeable and oxygen-nonpermeable occlusive dressings: three polyurethane adhesive films (Bioclusive, Op-Site, and Tegaderm), a polyethylene oxide hydrogel dressing (Vigilon), and a hydroactive polymer dressing (DuoDerm).

The clinical spectrum of lipodermatosclerosis

Lipodermatosclerosis refers to the skin induration and hyperpigmentation of the legs that often occurs in patients who have venous insufficiency. Lipodermatosclerosis has also been termed hypodermitis sclerodermiformis and appears to be similar if not-identical to the recently described sclerosing panniculitis of the leg. There has been much confusion about the nature, clinical course, and treatment of lipodermatosclerosis. We believe that lipodermatosclerosis has an acute, inflammatory phase and a chronic, fibrotic stage, although a spectrum exists. Direct immunofluorescence studies of early and late lesions are helpful in that they show dermal pericapillary fibrin deposits without other immunoreactants. Treatment of lipodermatosclerosis consists of compression therapy with either graded stockings or elastic bandages. We and others have found that the anabolic steroid stanozolol improves this condition rapidly and consistently.