Two of the challenges of caring for wounds, whether they are secondary to ischemia or other causes, are infection prevention and stimulation of new epidermal tissue growth. Please welcome back Dr. Caroline Fife, the Director of the Hermann Center for Wound Healing, part of the Memorial Hermann Hospital System, who will discuss the exciting new area of the use of synthetic and semi-synthetic skin for wound management.

-- Richard W. Smalling, M.D., Ph.D., Cardiovascular Moderator

Despite the increasing sophistication of medical science to elucidate the biochemical basis of many diseases and, despite the numerous medical and surgical treatment options available for chronic conditions, certain basic wound healing problems remain essentially unchanged. The development of new medical and surgical treatments for peripheral arterial disease, for example, has not slowed the incidence of venous stasis ulceration, a common chronic wound problem affecting millions of individuals each year. Similarly, despite general medical progress, neuropathic plantar foot wounds will probably continue to be one of the most common types of indolent, problem wounds.

Plantar foot wounds and venous stasis ulceration alone result in the expenditure of millions of health care dollars annually. Moreover, though the most seriously "limb-threatening" wounds are those caused by or complicated by arterial insufficiency, many relatively well-vascularized wounds also fail to heal. While repeated trauma or swelling may contribute to the chronic nature of these lesions, it is accepted that there are probably defects in the process of healing occurring in certain disease processes.

Tissue Engineering -- What's the Point?

Very large wounds, whether from venous ulceration, burns or trauma, represent significant problems because of the relatively slow nature of epithelial migration. In many cases, the most expeditious way to effect wound closure is via autografting, the use of the patient's own skin for coverage, usually by split thickness skin grafting. This process, while highly effective in properly prepared wounds, usually requires hospitalization or immobilization, anesthesia and the surgical creation of another, often very painful wound (the donor site). Skin grafts are sometimes not incorporated into the wound bed, resulting in the need for long term wound care despite surgery. Some areas of the body are not amenable to grafting, such as the plantar foot. As a result of all these factors, a cost-effective alternative to autografting would be highly desirable.

The creation of human-like skin in the laboratory is a logical evolution of grafting technology. Xenografts (from a nonhuman donor, e.g., pigskin) can provide useful temporary coverage but are always rejected. Allografts (the use of donor skin from another person) have had the obvious disadvantage of immunological rejection unless there is a genetic match. The disadvantages of autografts (the patient's own skin) have been discussed.

Acellular preparations act as a "scaffold" to promote cell migration and act as a temporary covering. There are currently four types of acellular skin substitutes available: AlloDerm^® (LifeCell Corporation: dermal matrix derived from cadaveric skin), Biobrane^® (Dow-Hickam: nylon mesh coated with porcine collagen on a silicone film), Integra Artificial Skin Replacement^® (Integra LifeSciences Corporation: silicone over a dermal layer of bovine tendon collagen), Dermagraft-Temporary Covering^® (Advanced Tissue Sciences Inc: silicone bonded to synthetic matrix embedded with non-viable fibroblasts). Unfortunately, acellular skin substitutes can not provide the same biochemical stimulus or mechanical coverage as skin. Cellular preparations can actually synthesize growth factors and other materials that can aid in the healing process. Cells cultured from the patient's own skin, however, take weeks to grow and prepare and do not provide actual mechanical coverage. Epicel^® (Genzyme Tissue Repair) is an example of a single layer epidermal autograft grown from the patient's own skin.

The ideal coverage material would elicit no immunologic response, would not require the creation of another wound, would be easily transportable and readily available, would be simple to store and apply, would compare favorably with the cost of surgical wound closure, would structurally resemble skin and would be at least as effective as allografting. Apligraf^® (Novartis) is an entirely new category of product, the first bi-layer skin substitute developed in the laboratory to simulate the structure of human skin. It is the only FDA approved semi-synthetic human skin currently available.

While Apligraf^® does not satisfy all of the above criteria, it does represent the next step in tissue engineering technology. It is safe to assume that, as tissue engineering technology advances, similar, perhaps even more sophisticated products, will become available in the future.

Brief Overview of the Healing Process

Normal wounds heal in a predictable period of time. This healing process is the result of a complex collaboration between many cell types beginning with coagulation. Complete epithelialization can not occur in the absence of adequate granulation tissue. The "beefy red" appearance of healthy granulation tissue is actually the result of capillary budding. Keratinocytes then migrate across the wound surface and, once they entirely cover the wound, differentiation occurs and a stratified epidermis develops.(1) All the aspects of the healing process, including angiogenesis, collagen deposition and epithelialization, are controlled by the interaction of numerous growth factors (cytokines).(2) In fact, the mechanism by which skin grafts achieve wound closure involves more than just mechanical coverage. The grafted skin probably contributes cytokines that promote the synthesis of matrix and angiogenesis.

Healing at any stage can be inhibited by many factors, including ischemia, infection or heavy bacterial colonization, nutritional deficiency, bleeding, edema, a dry wound environment or toxic topical preparations.(3) It is imperative that those factors that can be controlled are addressed. Arterial supply must be adequate. Nutritional deficiencies must be corrected, blood glucose and edema must be controlled and infection must be treated. A comprehensive outline of wound care is beyond the scope of this Cyberounds^®. The following information regarding the clinical use of semi-synthetic human skin assumes that all appropriate adjunctive measures have been performed and a granulated but unepithelialized wound remains.

What Is Apligraf^®?

The unique feature of Apligraf^® is that, like human skin, it has two primary layers.(4) In fact, microscopic cross sections of human skin, when compared to Apligraf^®, look strikingly similar. The dermis is created using bovine collagen seeded with human fibroblasts. The epidermal layer has a stratum corneum formed of living human keratinocytes. These human keratinocytes and fibroblasts were isolated originally from neonatal foreskins and serially cultured. In addition to using pedigree donors, cells are subjected to numerous tests at several stages for the presence of pathogens and contaminants.

To manufacture a piece of Apligraf^®, the cryopreserved cell stocks of fibroblasts are thawed, placed over the bovine collagen matrix and incubated for six days, during which the fibroblasts multiply, contract the collagen filaments and produce human matrix proteins. Living keratinocytes are then seeded onto this "dermis" where they attach, proliferate and differentiate, forming an epidermal layer. In 20 days, the developing bilayer is exposed to air, which enhances the maturation of the keratinocytes and produces a stratum corneum.(4) The mature semi-synthetic human skin is placed in a nutrient medium, individually packaged, sealed in a polybag and shipped overnight to the clinic. Apligraf^® is supplied as a circular disc of translucent skin about 7.5 cm in diameter and only about 0.075 cm thick. Visually, it looks like a thin piece of pink skin.

Clinical Data

In a randomized venous stasis ulcer trial, semi-synthetic skin, Apligraf^®, with compression, was compared to compression and standardized wound care (n=120).(5) Apligraf^® was three times more effective than compression therapy alone in achieving closure of ulcers of greater than a year in duration (p=.048). More semi-synthetic human skin patients achieved full closure at six weeks than after six months of compression alone. Furthermore, there were significantly higher healing rates at all time points after week four. A particularly well-designed FDA trial in diabetic neuropathic plantar foot wounds was recently completed. This trial compared the healing rate of patients off-loaded by an excellent custom-made walking sandal to those utilizing the sandal and receiving Apligraf^®. Preliminary reports describe a statistically significant improvement in the rate of closure with this semi-synthetic human skin for these chronic neuropathic wounds compared to off-loading alone.

In some cases, partial or full "take" of the semi-synthetic human skin may occur, similar to that observed with autologous skin.(5) These living and metabolizing cells also release growth factors which may stimulate the underlying wound bed and encourage tissue regeneration.(6) It must be remembered that both of these wound models (stasis ulcers and well-vascularized neuropathic foot wounds) involved completely granulated wound beds.

The Logistics of Obtaining and Using Semi-synthetic Skin

A potentially significant disadvantage to the clinician is that the shelf life of Apligraf^® is only five days from the day of packaging. By the time the Apligraf^® arrives in the office, allowing a day for shipping, only 72 hours remain in which to apply it to a prepared wound before it must be disposed of. This restriction poses logistical challenges for the clinic and potential economic ones as well, since semi-synthetic human skin must be purchased directly by the facility. In a hospital-based facility, the Medicare allowable charges for wound debridement and placement of the semi-synthetic skin (using standard skin graft codes) can cover the approximately $1,200 per disc cost of purchase. However, issues relating to insurance pre-authorization, if required by the payor, must be taken care of well ahead of time. It is imperative to have a mechanism by which missed appointments are kept to a minimum since the cost of wasted tissue must be borne by the clinic.

The importance of adequate wound preparation can not be over emphasized. In our experience, the likelihood that this semi-synthetic human skin product will actually be integrated into the wound increases as the quantitative bacterial load decreases (see clinical hints below). It is well known that quantitative bacterial cultures with >10(5) organisms per gram of tissue appear to retard wound repair(7) and high bacterial loads are also known to affect the rate of split thickness skin graft take. Adequate management of other factors, such as pressure in the case of plantar wounds or edema in the case of venous stasis, is also necessary, both before and after the use of semi-synthetic human skin. Apligraf^® is not a substitute for good standard wound protocols. Infection has occurred in patients receiving Apligraf^® but this has not been directly attributed to the semi-synthetic skin. There has been no evidence of rejection or other type of immune reaction to date.

Practical Clinical Guidelines

The following suggestions are based on our clinical experience:

1. Apligraf^® is for clean, well-vascularized, well-granulated wounds. It is FDA approved, specifically, for venous stasis ulcers. FDA approval for well-vascularized, diabetic neuropathic foot wounds is expected soon, based on the favorable results of Phase III pharmaceutical trials. Although not specifically FDA approved for other types of wounds, any well-vascularized wound that has failed to epithelialize after appropriate management could be considered for semi-synthetic skin.
2. Wounds must be adequately debrided. Non-viable tissue prevents contact between the semi-synthetic human skin and the vascularized wound bed, its nutritional supply, and may promote infection. Sometimes, surgical debridement is necessary.
3. Do not use on infected wounds. Treat infection with adequate doses of antibiotics directed at specific pathogens.
4. Minimize bacterial colonization before applying. Small tissue biopsies for quantitative bacterial counts are a cost-effective way to evaluate the wound bed prior to using semi-synthetic human skin (or split thickness skin grafting). Use topical agents, such as Iodosorb^® (cadexamer iodine is non toxic to granulation tissue; do not use cyto-toxic povodine preparations or peroxide), Arglaes^® (Maersk Medical LTD), Acticotem^® (Westaim) or even 25% acetic acid, for a week before applying semi-synthetic human skin may significantly improve results by lowering bacterial colony counts.
5. Apply to the wound immediately after removing from packaging, with the same orientation (it is packaged "epidermis up"). Position over the wound bed and then trim with sterile scissors to match the contour of the wound. Remove air pockets by rolling a sterile cotton swab along the surface.
6. Immobilize the semi-synthetic human skin with a non-adherent primary dressing, then a bolster of some sort of absorbent dressing (gauze works well, or Exudry pads). Some overlay of the semi-synthetic skin margins with the intact skin at the wound periphery may also be helpful.
7. After application, wounds should not be traumatized (see the description of the normal appearance of semi-synthetic human skin after several days in photos). Resist the temptation to aggressively "clean" the wound bed as the semi-synthetic skin changes its appearance.
8. Edema must be controlled both before and after the application. Mild to moderate compression with bandages or stockings can be applied over the semi-synthetic skin but some decrease in activity for the week following application is highly advisable.
9. Fenestrating the Apligraf^® may significantly improve results. Fluid tends to collect under the semi-synthetic human skin, decreasing contact with the wound bed. Fenestrating with a scalpel or a surgical skin mesher prior to application allows fluid to be absorbed by the secondary dressings.
10. The semi-synthetic human skin should be left alone for at least four days after application, although the absorptive dressings over the graft can be changed.

The Question of Cost Effectiveness

The most interesting issue relating to Apligraf^® is the question of cost effectiveness. No independent clinical trials have been performed to assess this. Patient charges for application of this semi-synthetic skin certainly exceed the cost of the product alone and if the clinical situation necessitates application in the operating room rather than the clinic, charges for Apligraf^® can equal the cost of traditional split thickness skin grafting. However, the likelihood of a primary "take" with Apligraf^® is, in our experience, less than that of a split thickness skin graft, although most of the chronic wounds in which we have used semi-synthetic human skin have healed within six weeks of a single application. Therefore, it is likely that some type of continued wound care will be necessary for several days or weeks after semi-synthetic human skin is used. This must also be considered in determining the cost benefit of the procedure.

In wounds where conservative management will likely result in wound closure over time (such as compression bandaging in stasis ulcers or total contact casting in neuropathic foot wounds), it is necessary to show not only that semi-synthetic human skin substantially reduces time for healing but also the total cost of care. Modalities, such as compression bandaging and total contact casting, are relatively cheap, so in a wound that is progressing well with such treatments, the cost-effectiveness of semi-synthetic skin may be low. However, a recent publication demonstrated that the cost of 10 weeks of outpatient treatment with compression bandaging ranged from $1,444 to $2711,(8) so even with relatively inexpensive wound care options, costs can quickly escalate, particularly when lost productivity, infection risk and reduced quality of life are taken into account. For wounds in which the usual conservative modalities have failed to effect epithelialization after an appropriate period of time, semi-synthetic human skin would seem to represent substantial cost savings over autografting. In such cases, proper wound preparation is essential in order to increase the likelihood of benefit.

In wounds which are amenable to split thickness skin grafting, the cost-effectiveness of substituting semi-synthetic human skin would seem to be reduced if multiple discs are required for coverage of a large surface area or multiple applications are required over the same area. However, in cases where autografting is not possible or is unlikely to be successful (extensive burns, primary skin disorders, etc.), even the cost of multiple discs or multiple applications may be easily justifiable.

We suggest that the following questions are considered before semi-synthetic human skin is applied:

1. Is the wound well granulated (i.e., would it be considered favorably for traditional autografting)?
2. Has the wound failed to respond to a reasonable trial of appropriate conservative management (e.g., adequate compression or adequate off-loading, preferably over at least four weeks duration)?
3. Is there a clinical reason why an autograft is NOT a good option at this time (concurrent medical problems, skin disorder, etc.)?
4. Can the number of semi-synthetic human skin discs needed for wound coverage be justified in comparison to an autograft?

Independent analyses are needed to evaluate the complex issues of cost effectiveness. Until further data are available, if the above questions can be answered in the affirmative, then the cost of Apligraf is probably justifiable. Proper patient selection, proper application technique and proper wound care before and after the application are all necessary to optimize benefit from this new product. The need for such products to aid healing is likely to increase as the population ages and the prevalence of chronic wounds further increases. Despite its current limitations, bilayer human skin substitutes represent the next step in the evolution of tissue bioengineering.