Plastic Surgery Research Council

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Ex-vivo Subnormothermic Oxygenated Machine Perfusion of Rodent Hindlimb: Feasibility Study to Elongate Preservation Time of Vascularized Composite Allograft
Laura C. Burlage, MD1,2, Alexandre G. Lellouch, MD3,4, Shannon N. Tessier, PhD1, Casie A. Pendexter, MSc1, Stephanie E. J. Cronin, MSc1, Ilse M. Schol, BSc3, Mark A. Randolph, M.A.Sc3, Robert J. Porte, MD, PhD2, Laurent A. Lantieri, MD, PhD4, Korkut Uygun, PhD1, Curtis L. Cetrulo, MD, FACS3.
1Massachusetts General Hospital/ Harvard Medical School, Department of Surgery, Boston, MA, USA, 2University Medical Center Groningen, Department of Transplant Surgery, Groningen, Netherlands, 3Massachusetts General Hospital/ Harvard Medical School, Department of Plastic Surgery, Boston, MA, USA, 4European George Pompidou Hospital, University of Paris, Department of Plastic Surgery, Paris, France.

PURPOSE: Over 2 million people in the U.S. are living with limb loss, and roughly 185,000 new amputations occur each year. To date, vascularized composite allograft (VCA) remains the only viable treatment option to restore motor and aesthetic function in these patients. A pressing challenge in the field of VCA is the methods of tissue preservation until transplantation. Protocols for tissue preservation successfully used in solid organ transplantation (static cold storage on ice [0-4C]) greatly jeopardize the quality of VCAs prior to transplantation resulting in hand allograft rejection. Ex-vivo subnormothermic oxygenated machine perfusion (SNMP) is a novel method of organ preservation which is known to improve the quality of cadaveric organs prior to transplantation. The aim of this study was to investigate the utility of SNMP on preservation time and resuscitation of ischemic hind limbs in a rat model.
METHODS: Nine rat hind limbs were procured and flushed with a mixed solution of saline and heparin (10U/mL) through both the cannulated femoral artery and vein. Warm ischemia time was approximately 10-15 minutes and cold ischemia was mitigated. During 3 hours of SNMP, the limbs were perfused by a pressure-controlled system through the femoral artery and the venous outflow was prepared for sample collection. The perfusion solution consisted of William's E medium, enriched with Pen-Strep, L-glutamine, heparin, insulin, hydrocortisone and dexamethasone. We also added bovine serum albumin (BSA) and polyethylene glycol (PEG) 2%. Hemodynamics of the limb were monitored by evaluation of arterial flow and vascular resistance. Perfusion samples were collected every 30-60 minutes for biochemical analysis. Lactate levels and oxygen consumption were monitored as markers of viability of the muscle tissue.
RESULTS: Arterial outflow and vascular resistance remained stable throughout the perfusion, between 0.5 and 2.0 mL/min and 20-40 mmHg/mL/min, respectively. After an initial rise in lactate levels, median lactate levels decreased significantly between the first and second hour of SNMP, 2.52 (IQR 2.11-3.96) and 1.40 (IQR 1.13-2.32) (p=0.02) respectively. After 1 hours of SNMP, median calculated oxygen uptake rate was 20.65 mL O2/min/gram limb (IQR 6.50-28.47) and remained stable thereafter. We also found that placement of the arterial cannula distally from the epigastric branch as well as addition of both BSA and PEG reduced weight gain due to edema.
CONCLUSIONS: This study shows that 3 hours ex vivo SNMP of rat hind limb is feasible. Furthermore, SNMP has the potential to both actively preserve and improve overall quality of hind limbs in a rat model. In the future, SNMP may enhance clinical outcome of VCA transplantation.


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