Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds C.-J. Gustafson, G. Kratz* Department of Reconstructive Plastic Surgery, Karolinska Hospital, 171 76 Stockholm, Sweden Accepted 5 January 1999 Abstract The purpose of this study was to establish a method for transplantation of autologous keratinocytes on an allogenic cell-free dermis. From four healthy volunteers two full thickness skin grafts, 1 Â 1 cm, were taken. The epidermis was separated from the dermis enzymatically and the cells of the dermal part were removed by incubation in Triton X-100. Keratinocytes were seeded on a cell-free dermis and the combined graft transplanted back to one of the wounds of the donor of the keratinocytes. The other wound was covered with cell-free dermis without keratinocytes. After 2, 3, 4 and 6 weeks, respectively, the grafted wounds were removed from the subjects and investigated histologically and immunohistochemically regarding re-epithelialisation, ®broblast ingrowth and angiogenesis. The wounds covered with cell-free dermis and keratinocytes showed a complete epidermal coverage 2 weeks after transplantation, in contrast to the wounds covered with un-seeded dermis which only showed epidermal coverage at the wound edges. There was also a marked dierence concerning ®broblast ingrowth and angiogenesis. In this study we have shown that autologous keratinocytes can be seeded on a cell-free dermis and transplanted as a kerato-dermal graft which stimulate re-epithelialisation as well as ®broblast ingrowth and angiogenesis in the wound. # 1999 Elsevier Science Ltd and ISBI. All rights reserved. 1. Introduction The de®nitive treatment for patients with full-thick- ness burns is autografting with tissue, which includes both dermal and epidermal components. Split-thick- ness autografts is the ®rst choice for permanent wound closure in these patients. However, conventional split thickness skin grafts are limited by the availability of donor sites. To circumvent this limitation of autolo- gous skin, Rheinwald and Green developed an in vitro technique to culture human keratinocytes into epider- mal sheets suitable for autotransplantation [1,2]. This technique has during the last decade been improved and is today a well-accepted treatment of massive burns all over the world. Although this method has been a tremendous step forward in the treatment of large burns there are indications that cultured epider- mal sheets by themselves do not provide a satisfactory skin cover on full thickness burns due to subsequent wound contraction, fragility of the grafted area and poor cosmetic result [3]. The lack of dermis when these grafts are used on full thickness wounds seems to be a major problem why research attempts have been di- rected towards the development of cultured or arti®cial dermis substitutes [4]. Furthermore, the culture methods used today involve at least 3 weeks of in vitro culture, complicated procedures for graft production and transplantation of a high percentage of dieren- tiated keratinocytes. Cadaver skin (fresh, cryopreserved or glycerol-pre- served) is frequently used as a temporary cover of extensive burn until donor sites or cultured autografts are available [5,6]. The allograft will start a host-ver- sus-graft reaction and is ultimately rejected. However, it has been shown that grafting cultured epithelial autografts to the remaining tissue after rejection of the allograft will accelerates biologic events in skin regen- Burns 25 (1999) 331±335 0305-4179/99/$20.00+0.00 # 1999 Elsevier Science Ltd and ISBI. All rights reserved. PII: S0305-4179(99)00004-2 * Corresponding author. Tel.: +46-8-5177-4152; fax: +46-8-5177- 2505. E-mail address: gunnar.kratz@kirurgi.ki.se (G. Kratz)