Introduction: development of skin substitutes

D.P. Orgill    Brigham and Women’s Hospital, USA

C. Blanco    Joseph M. Still Research Foundation, USA

1.1 Historical development

Wounds, defined as a disruption in the integument, have long plagued mankind because, if left untreated, they can result in sepsis and death. During the American Civil War, amputation allowed for prevention of death by halting localized infection from spreading. Aseptic techniques and antibiotics were major advances in the 20th century that contributed to increased survival following large wounds and thermal injury. Advances in burn care in the 1960s and 1970s, including early excision and grafting, fluid resuscitation, mechanical ventilation, topical antimicrobials and skin grafting techniques allowed burn victims with large total body surface area burns to survive. The development of the powered dermatome provided uniform thickness sheet skin grafts that were efficient for closing large wounds, but created partial thickness wounds that also needed to heal. As technology improved and larger burns were treated, the available donor sites were reduced. Innovations including widely meshed skin grafts and micro-grafting helped close the wound but had a poor aesthetic outcome. The inability to achieve autologous skin coverage of large burns effectively was a major incentive for the National Institutes of Health to invest in skin substitute development in the 1970s and 1980s. Investment in these projects produced a better understanding of the requirements of skin substitutes and also led to the commercialization of several technologies that are currently used today to treat burn patients and have been extended, in some cases, to their use in reconstructive surgery and the treatment of wounds (Table 1.1).1,2

Despite great advances in burn care, the resulting severe scarring and deformity continues to be one of the greatest challenges facing burn victims and their families. Research in recent years has focused on methods to improve regeneration while limiting scarring. The following monograph will review many of the current technologies available to clinicians, highlight some treatments that are in early development phases and point to areas of potential improvement for the future.

A thorough understanding of the biology of skin and its response to injury is essential for designing skin substitutes (Table 1.2). For centuries man has understood the important functions of skin in providing a barrier to bacteria and moisture loss as well as a strong and elastic integument that drapes over complex surfaces. Historically, physicians have turned first to biological membranes that have these basic properties including cadaver skin, pigskin and amniotic membrane. Xenografts, such as pigskin, showed good temporary coverage but the very high antigenicity resulted in predictable failure over the long term. Human cadaver skin works well as a temporary skin substitute but tends to reject between days 10 and 14 after application. In addition, the supply can be erratic and there is also a possibility of bacterial or other disease transmission. Clearly better techniques were needed to treat very large wounds more optimally.

1.2 Skin regeneration

An injured epidermis heals by spontaneous regeneration, leading to formation of a new, intact epidermis. In contrast, the injured adult dermis generally does not regenerate spontaneously and heals instead by wound contraction and scarring. A superficial injury in the dermis may show restoration as described in an experimental incisional scar model in humans showing that incisions made at a depth of 0.53 mm or less (approximately the top one-third of the dermis) showed no long-term visible scar.3 In contrast, deep partial thickness burns, full-thickness burns and full-thickness traumatic wounds heal exclusively with scarring and wound contraction.

In the late 1970s and 1980s three groups at the Massachusetts Institute of Technology (MIT) worked independently on three different methods to treat the skin substitute problem. The results of their research provide the foundation for most skin substitute research done today. Howard Green, working with James Rheinwald pioneered cell culture techniques including culturing of keratinocytes.4 Prior to their innovations, culturing keratinocytes was difficult. Their contributions included specific culture media and the addition of irradiated fibroblasts as a feeder layer for keratinocytes. From a small biopsy of normal skin, taken shortly after the burn injury, they were able to grow rapidly large quantities of keratinocyte sheets referred to as cultured epithelial autografts (CEAs) which could be grafted onto the burn wound within three weeks. This technique became famous when Gallico and O’Connor applied it to two severely burned children at The Shriners Burns Institute in Boston who were able to survive a massive burn injury.5 At the time, there was a debate about whether or not dermis was a necessary component for the long term success of the technique. Despite the remarkable achievements of Gallico and O’Connor, others found that using CEAs alone resulted in a very fragile skin. Cuono later showed that applying allograft sheets first, and then removing just the epidermis prior to the application of CEAs, resulted in more stable coverage.6 This technology formed the basis for the company Advanced Tissue Sciences that was later sold to Genzyme® Tissue Repair (Cambridge, Massachusetts). CEAs are still an important adjunct in treating very large burns. Because the number of large burns is decreasing in the USA, the market size for this technique has not grown significantly in the last several years.

Eugene Bell developed a fibroblast seeded collagen lattice and then covered this with keratinocytes.7 The collagen lattice contracted significantly in vitro after being seeded with keratinocytes. These lattices could then be covered with a keratinocyte layer to perform a ‘skin equivalent’. This technique never really caught on in the burn community and now is most often used with allogenic cells derived from neonatal foreskins. Subsequently, Organogenesis Corporation (Canton, Massachusetts) made these sheets for a successful clinical trail in diabetic foot infections.8 Today, the resulting product, ‘Apligraf’, is used mostly to treat chronic wounds. Early thoughts were that some of this material actually ‘took’ into the wounds. Most clinicians today believe Apligraf works as a very advanced dressing, providing both a barrier and a rich source of growth factors to the wound.

Ioannis V. Yannas and John F. Burke worked together to develop a dermal template composed of bovine collagen and chondroiten-6-sulfate derived from shark cartilage. They believed that dermis was the most difficult part of skin to regenerate and if they could solve the problems of scarring and contraction that the epidermal problem would then be less of an issue over the long term. Using a guinea pig wound contraction model, they defined optimal characteristics of the matrix including average pore size, cross-link density and percentage of glycosaminoglycan. This matrix was covered by a silicone elastomer which mimicked the natural permeability of skin. One of the reasons for the success of this technique was the way burns were treated at the Massachusetts General Hospital and Shriners Burns Institute in Boston. Burke was an advocate of early excision and grafting. As such, after excision of the burn, there was a clean sterile bed to apply the matrix. The matrix was left...