A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Summary

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Abstract

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.

This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.

Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

Introduction

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants are now a clinical reality with numerous hand and face transplants performed worldwide¹². Despite the fact that early and intermediate results are favorable and highly encouraging², the requirement of chronic multidrug immunosuppression continues to limit its widespread clinical application. The advancements in murine models of VCA including super-microsurgical anastomoses and nonsuture cuff techniques^{13, 3} have paved the way to a better understanding of alloimmune responses in VCA. Myriads of immunomodulatory protocols have been proposed for clinical ap....

Protocol

In this video publication, all animal procedures were conducted in accordance with an animal protocol approved by the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC).

1. Preoperative Planning

1. We utilize MHC-defined MGH inbred miniature swine (15-20 kg) in our translational studies for limb allotransplantation wherein one male donor provides limbs for two female recipients (Figure 1). Prior to surgery, donor and recipient animals undergo thorough clinical evaluation by a veterinarian.
2. In order to minimize anesthesia and ischemia time, donor and recipient procedures are condu....

Results

Twenty-four SLA-defined swine heterotopic hind limb transplants were performed using our modified technique with a mean ischemia time of 78 min (Range: 62-94 min). Graft inset and dorsolateral skin paddle positioning were achieved without difficulty in all animals. Near infrared laser angiography showed excellent graft perfusion in all recipients. The initial twelve venous anastomoses were performed using conventional suture techniques while the last twelve venous anastomoses were performed using a vascular coupling devi.......

Discussion

Historically, the heterotopic hind limb transplant protocol included exteriorization of a skin paddle to the ventral abdominal wall and the vessels were anastomosed in an end-to-side manner (Hettiarachty 2004). However, in our modified method, an inverted flap insetting and end-to-end anastomoses bring the skin paddle more laterally and hence facilitates immune monitoring in a standing position of the animal. The identification of the sural artery and the zones of maximum perfusion using near infrared laser angiography f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
HTK	Custodial	N/A
EQUIPMENT
Electric Pen Drive	Synthes, Westchester PA	05.001.011	Reciprocating saw
Vascular Coupling device	Synovis, Newtown PA	21003B