Topical Delivery of Immunosuppression to Prolong Xenogeneic and Allogeneic Split-Thickness Skin Graft Survival

Abstract

Cadaveric skin allograft is the current standard of treatment for temporary coverage of large burn wounds. Porcine xenografts are viable alternatives but undergo α-1,3-galactose (Gal)-mediated hyperacute rejection and are lost by post-operative day (POD) 3 because of naturally occurring antibodies to Gal in primate recipients. Using baboons, we previously demonstrated that xenografts from GalT-KO swine (lacking Gal) provided wound coverage comparable with allografts with systemic immunosuppression. In this study, we investigate topical immunosuppression as an alternative to prolong xenograft survival. Full-thickness wounds in baboons were created and covered with xenogeneic and allogeneic split-thickness skin grafts (STSGs). Animals were treated with slow-release (TyroSphere-encapsulated) topical formulations (cyclosporine-A [CSA] or Tacrolimus) applied 1) directly to the STSGs only, or 2) additionally to the wound bed before STSG and 1). Topical CSA did not improve either xenograft or allograft survival (median: treated grafts = 12.5 days, control = 14 days; P = 0.27) with similar results when topical Tacrolimus was used. Pretreatment of wound beds resulted in a significant reduction of xenograft survival compared with controls (10 vs 14 days; P = 0.0002), with comparable results observed in allografts. This observation was associated with marked reduction of inflammation on histology with Tacrolimus and not CSA. Prolongation of allograft and xenograft survival after application to full-thickness wound beds was not achieved with the current formulation of topical immunosuppressants. Modulation of inflammation within the wound bed was effective with Tacrolimus pretreatment before STSG application and may serve as a treatment strategy in related fields.

Figure 1.

Loading content and loading efficiency of FK506-loaded TyroSpheres with theoretical loading concentrations ranging from 10 to 60 wt%.

Figure 2.

Release profile of FK506 from dialysis cassettes in 1× PBS up to 7 days.

Figure 3.

Autografts were treated with empty TyroSpheres-gel to determine if skin graft healing would be affected by the vehicle. These photos are taken on POD 8. An untreated autograft was placed as a control (A). No detrimental healing effects were observed when the empty TyroSphere-gel was applied to an autograft every other day starting on POD 0 (B) or on POD 2 (C).

Figure 4.

No deleterious effect was observed clinically in autografts treated with 0.4% CSA-TyroSpheres-gel every other day. The grafts were observed on POD 0 (A), POD 2 (B), POD 4 (C), and POD 6 (D).

Figure 5.

A dose titration of autografts treated with 0.4% CSA-TyroSpheres-gel every other day demonstrated no adverse effect on graft healing when using 0.2 ml or 0.5 ml of the TyroSpheres-gel per graft. However, when 1.0 ml of the TyroSpheres-gel was used, the autografts became macerated and sloughed by POD 12. An untreated control was performed for comparison.

Figure 6.

A. Kaplan-Meier survival analysis for xenogeneic and allogeneic skin grafts treated with standard Bacitracin^TM or CSA/FK506-TyroSpheres-gel. Representative histology at 400× magnification showing hematoxylin and eosin–stained slide from a biopsy taken on POD 6 of an allograft treated with CSA-TyroSpheres-gel (B). Diffuse dermal rejection (*) with ischemic changes in the epidermis is seen in this graft. (C) Shows a 400× magnification hematoxylin and eosin–stained slide from a biopsy taken on POD 4 of an allograft treated with the standard wound care agent, Bacitracin^TM. This graft demonstrated early epidermal changes (†) consistent with ischemia and early rejection.

Figure 7.

A. Kaplan-Meier survival analysis for xenogeneic and allogeneic skin grafts treated with standard Bacitracin^TM or CSA/FK506-TyroSpheres-gel after initial treatment of the underlying wound bed. Representative histology at 400× magnification showing hematoxylin and eosin–stained slide from biopsies taken on POD 4 of xenografts treated with CSA- “underlay” TyroSphere-gel and topical CSA-TyroSpheres-gel (B) or Bacitracin^TM (C). The CSA-TyroSphere-gel–treated grafts demonstrated some ischemic changes in the epidermis (†), but there is evidence of epithelial reconstitution. Also, there is some mild dermal inflammation (*). The Bacitracin-treated control graft demonstrates diffuse dermal inflammation (*).