Skin grafts from genetically modified α-1,3-galactosyltransferase knockout miniature swine: A functional equivalent to allografts

Abstract

Burn is associated with a considerable burden of morbidity worldwide. Early excision of burned tissue and skin grafting of the resultant wound has been established as a mainstay of modern burn therapy. However, in large burns, donor sites for autologous skin may be limited. Numerous alternatives, from cadaver skin to synthetic substitutes have been described, each with varying benefits and limitations. We previously proposed the use of genetically modified (alpha-1,3-galactosyl transferase knockout, GalT-KO) porcine skin as a viable skin alternative. In contrast to wild type porcine skin, which has been used as a biologic dressing following glutaraldehyde fixation, GalT-KO porcine skin is a viable graft, which is not susceptible to loss by hyperacute rejection, and undergoes graft take and healing, prior to eventual rejection, comparable to cadaver allogeneic skin. In the current study we aimed to perform a detailed functional analysis of GalT-KO skin grafts in comparison to allogeneic grafts for temporary closure of full thickness wounds using our baboon dorsum wound model. Grafts were assessed by measurement of fluid loss, wound infection rate, and take, and healed appearance, of secondary autologous grafts following xenograft rejection. Comparison was also made between fresh and cryopreserved grafts. No statistically significant difference was identified between GalT-KO and allogeneic skin grafts in any of the assessed parameters, and graft take and function was not adversely effected by the freeze-thaw process. These data demonstrate that GalT-KO porcine grafts are functionally comparable to allogeneic skin grafts for temporary closure of full thickness wounds, and support their consideration as an alternative to cadaver allogeneic skin in the emergency management of large burns.

Keywords: Allogeneic skin; GalT-KO; Split-thickness skin graft; Temporary burn wound closure; Xenogeneic skin.

Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

Figures

Fig. 1

Control of fluid loss from full thickness wounds by GalT-KO porcine split thickness grafts is equivalent to allogeneic skin grafts. (A) Wound exudates measured by volume (mL) eluted from absorbent dressings. GalT-KO skin and allogeneic skin grafts both significantly reduced fluid loss in comparison to untreated full-thickness wounds over first three days following grafting (*, Day 1 Full thickness wound vs. allo graft p=0.0132; vs. GalT-KO p=0.0137: **, Day 2 Full thickness wound vs. allo graft p=0.0046; vs. GalT-KO p=0.0016: ***, Day 3 Full thickness wound vs. allo graft p=0.0017; vs. GalT-KO p=0.0005). (B) Wound exudates assessed daily as delta wet-dry weight (grams) of absorbent dressing. Exudates fell to undetectable levels after day 3. No statistical significance was observed between allo and GalT-KO grafts at any point (Day 1: p=0.7865; Day 2: p=0.1144; Day 3: p=0.0746). (C) Percentage wound infection rates for allogeneic and GalT-KO xenogeneic skin grafts. Clinical signs of wound infection were observed in 5.8% (1/17) grafts in each group. Common skin commensal organisms were cultured, and neither animal developed signs of systemic illness.

Fig. 2

Cryopreservation does not significantly effect survival of GalT-KO split-thickness skin grafts. (A) Kaplan–Meier survival curve demonstrating equivalent survival of GalT-KO split-thickness skin grafts whether placed immediately following harvest, or following period of cryopreservation at −80°C. (B) Representative images demonstrating visible appearance of fresh (left) and thawed cryopreserved (right) GalT-KO grafts four days after grafting; obvious pink coloration and fixed staining is event in fresh grafts. (C) Representative hematoxylin and eosin stained images of pre-implantation specimens of fresh (left) and thawed cryopreserved (right) GalT-KO grafts and (D) corresponding images from biopsies obtained 4days after grafting.

Fig. 3

Definitive closer of wounds with autologous split-thickness skin grafts is not affected by rejection of preceding GalT-KO grafts, nor allografts. (A) Time course of split-thickness skin graft appearance. Primary (left panel) GalT-KO porcine (upper row) and allografts (lower row) vascularized (early, POD 6) then rejected (late, POD 13) as expected within 14 days. Secondary autologous grafts (right panel) showed evidence of take and vascularization early (POD 2) and all healed to provide definitive wound closure (late, POD 48). Representative images are shown. (B) Mean cosmetic outcome scores for three autologous skin grafts post-GalT-KO and three autologous grafts post-allografts. A panel of blinded observers scored images of autologous grafts from 1 to 10 based upon cosmetic appearance. Statistical significance was determined using the student’s t-test (p=0.40). Two additional grafts are excluded from cosmetic outcome analysis due to technical failure and delayed re-grafting.