the effect of ex situ perfusion in a swine limb ...cmendias/publications/ozerjhands...we used an...

SCIENTIFIC ARTICLE

The Effect of Ex Situ Perfusion in a Swine LimbVascularized Composite Tissue Allograft on

Survival up to 24 HoursKagan Ozer, MD,* Alvaro Rojas-Pena, MD,† Christopher L. Mendias, PhD,* Benjamin S. Bryner, MD,†

Cory Toomasian, BS,‡ Robert H. Bartlett, MD†

Purpose To test the potential for the ex situ limb perfusion system to prolong limb allograftsurvival up to 24 hours.

Methods We used 20 swine for the study. In group 1 (control), 4 limbs were perfused withheparin solution and preserved at 4!C for 6 hours. In group 2, 4 limbs were perfused withautologous blood at 27!C to 32!C for 24 hours. In both groups, limbs were transplantedorthotopically to recipients and monitored for 12 hours. In addition to perfusion parameters,we recorded perfusate gases and electrolytes (pH, pCO2, pO2, O2 saturation, Na, K, Cl, Ca,HCO3, glucose, and lactate) and obtained functional electrostimulation hourly throughout theexperiment. Histology samples were obtained for TUNEL staining and single-muscle fibercontractility testing.

Results In both groups, hemodynamic variables of circulation remained stable throughout theexperiment. Neuromuscular electrical stimulation remained intact until the end of reperfusionin group 2 vs no response in group 1. In group 2, a gradual increase in lactate levels duringpump perfusion returned to normal after transplantation. Compared with the contralateral limbin group 2, single-muscle fiber contractility testing showed no significant difference at the endof the experiment.

Conclusions We demonstrated extended limb survival up to 24 hours using normothermicpulsatile perfusion and autologous blood.

Clinical relevance Successful prolongation of limb survival using ex situ perfusion methods pro-vides with more time for revascularization of an extremity. (J Hand Surg Am. 2016;41(1):3e12.Copyright ! 2016 by the American Society for Surgery of the Hand. All rights reserved.)Key words Ex situ, perfusion, reperfusion, swine, vascularized composite tissue allograft.

U PPER EXTREMITY LOSS PROXIMAL TO the wrist is amajor disability.1 In case of major upper ex-tremity amputations, replantation needs to be

performed within 4 to 6 hours to prevent permanent

muscle damage. Delay in transportation, lack of anavailable microsurgery team, or concomitant life-threatening injuries results in limb loss.2 Vascularizedcomposite tissue allograft (VCA) transplantation is alsosubject to time constraints.3,4 Once a donor is available,preoperative preparation, transfer of the extremity part,and revascularization need to be completedwithin a fewhours to succeed.

After traumatic or elective amputations (as in VCAtransplantation), the extremity is preserved in coldstorage solutions at 4!C. Although this reduces themetabolism and lactate production in the short term,beyond a certain critical point it leads to exten-sive damage to the cell membrane, resulting in cold

From the *Department of Orthopaedic Surgery and the †Department of Surgery, Universityof Michigan, Ann Arbor; and the ‡Wayne State University Medical School, Detroit, MI.

Received for publication October 7, 2015; accepted in revised form November 1, 2015.

No benefits in any form have been received or will be received related directly orindirectly to the subject of this article.

Corresponding author: Kagan Ozer, MD, Department of Orthopaedic Surgery, Universityof Michigan, 2098 South Main Street, Ann Arbor, MI 48103; e-mail: [email protected].

0363-5023/16/4101-0002$36.00/0http://dx.doi.org/10.1016/j.jhsa.2015.11.003

! 2016 ASSH r Published by Elsevier, Inc. All rights reserved. r 3

mailto:[email protected]

http://dx.doi.org/10.1016/j.jhsa.2015.11.003

ischemia-reperfusion injury and accelerated graftloss. Such damage is well documented in solid organsand hand allografts.5e12

To optimize the timing of surgery and minimizecold ischemia-reperfusion injury, normothermic exsitu perfusion systems have been used successfully insolid organ transplantations.13e20 Overall, experiencewith VCA is limited. Various authors have prolongedlimb allograft survival successfully up to 12 hoursin preclinical studies.21e23 These were statisticallysignificant compared with native ischemic toleranceof a limb (4 hours) but more time is needed to make aclinically meaningful difference. In this study, westudied the potential to achieve such a goal up to24 hours.

MATERIALS AND METHODSOur university committee on the use and care ofanimals approved this study, and its guidelines andpolicies for the humane use of animals in researchalong with those from the United States Departmentof Agriculture and the Association for Assessmentand Accreditation of Laboratory Animal Care werefollowed in this study.

Experimental groupsWe disarticulated 4 forelimbs in each of 2 groups atthe level of the elbow, perfused them with 10,000 Uheparin on 100 mL of saline solution, and weightedthem. In group 1 (control), limbs were stored at 4!Cfor 6 hours before transplantation. In group 2, limbswere immediately connected and perfused for 24hours according to the protocol described subse-quently. At the end of the cold storage in group 1 andperfusion in group 2, all limbs were transplanted toanother animal orthotopically and monitored for anadditional 12 hours.

Animal modelWe used an established swine limb replantation/transplantation model.23 After induction of anes-thesia, mechanical ventilation was set to keep PCO2

at 35 to 45 mm Hg and peak inspiratory pressures lessthan 20 cm H2O. The right carotid artery and rightinternal jugular vein were used to monitor meanarterial blood pressure, heart rate, central venouspressure, and fluid administration (80e120 mL/h)and to collect blood samples.

Surgical techniquePreparation of the forelimb on donor: All donor fore-limbs were disarticulated at the elbow to minimizeblood loss.23 After amputation, we cannulated the

brachial artery and marked the median and ulnarnerves for monitoring.

Preparation of the recipient and transplantation:Before the end of cold storage or perfusion, each re-cipient swine was anesthetized and the ipsilateralforelimb was amputated for orthotopic transplantationusing the same technique.

Limbs in both groups were weighted before trans-plantation. We then used 2 Steinmann pins to fix theolecranon to the humerus. All origins of the flexor andextensor tendons on the humerus were approximatedusing 2-0 suture. This took the tension off the vascularbundle. Under microscopic magnification, the brachialartery and vein and the cephalic and basilic veins wererepaired using end-to-end technique and 8-0 nylonsuture. Finally, skin was approximated. We used themedian nerve to monitor neuromuscular stimulationuntil the end of the experiment. Before opening thevascular clamps, each recipient was administered200 mg/kg heparin subcutaneously.

Ex situ perfusion protocolThe perfusion apparatus consisted of a perfusion pump(Waters Medical Systems, Minneapolis, MN) and anoxygenator (Baby CAPIOX RX, Terumo Cardiovas-cular Systems Co, Ann Arbor, MI). The oxygenatorhad 0.5-m2 surface area connected to a 70% O2e5%CO2 gas tank. The gas mixture contained 5% CO2 and95% O2. Pulsatile perfusion pressure was limited to60 to 80 mm Hg. During perfusion, the limb wasmaintained in an antiseptic diluted sodium hypochlo-rite solution dressing (Fig. 1).

Preparation of the perfusate: We first centrifugeddonor blood and removed white blood cell andplatelet fractions. Packed red blood cells and plasmawere mixed with dextran in a ratio of 1:2 to reach acolloid osmotic pressure of 20 to 30 mm Hg. Duringthe experiment, perfusate was replaced at a constantrate (80 mL/h) to remove toxic metabolites from thesystem in the absence of liver and kidney functions.We also measured perfusate glucose levels every 2hours and substituted the perfusate either with glu-cose (1 mL dextrose 50%) (if the level was < 4.5mmol/L) or with insulin (2 U) (if glucose was > 14mmol/L). The perfusate was maintained at 27! to 32!

C throughout the experiment.

MonitoringThe following variables were measured throughoutthe experiment:

" From the limb on pump perfusion: pressure, flow,resistance, temperature, O2 delivery and consump-tion, acid-base status, and blood gases.

4 EX SITU PERFUSION PROLONGS ALLOGRAFT SURVIVAL

J Hand Surg Am. r Vol. 41, January 2016

" From the perfusate and the recipient (every 2 h):hemoglobin lactate, Kþ, Naþ, Cle, HCO3

e, pH,pCO2, pO2, and glucose.

" Neuromuscular electrical stimulation every hourusing a nerve stimulator.

" Limb weight: After the amputation, before thetransplantation, and at the end of reperfusion (at 12hours).

HistologySingle-muscle fiber contractility testing: Muscle biopsies(10 $ 5 mm) were placed in a relaxing solutioncontaining Ca2þ chelating agents in a pH-buffered

solution.24 We then placed muscle bundles in skin-ning solution to further the permeability of the cellmembranes for 30 minutes and stored them at e80!Cuntil the day of testing. On the day of testing, wethawed the specimens and placed them in a chambercontaining relaxing solution. The bundles weresecured at one end to a servomotor (Aurora Scien-tific, Aurora, Ontario, Canada) and at the other endto a force transducer (Aurora Scientific) using 2 tiesof 10-0 monofilament nylon suture at each end. Fiberlength was adjusted to obtain a sarcomere length of2.7 mm using a laser diffraction system. We thencalculated the fiber cross-sectional area at 5 different

FIGURE 1: Diagram showing the entire setup for the experiment. Red and blue lines depict oxygenated and deoxygenated blood in thesystem.

FIGURE 2: Muscle fiber contractility testing diagram. A Overview of general orientation of fiber in the testing system and representativeimage demonstrating the midportion of porcine muscle fiber in the system. B Calculation of muscle fiber cross-sectional area. Five widthmeasurements from top and side views of midportion fiber are fitted to the formula of an ellipse to calculate fiber cross-sectional area.(Reprinted with permission from Ozer K, Rojas-Pena A, Mendias CL, Bryner B, Toomasian C, Bartlett RH. Ex situ limb perfusionsystem to extend vascularized composite tissue allograft survival in swine. Transplantation. 2015;99(10):2095-2101)23

EX SITU PERFUSION PROLONGS ALLOGRAFT SURVIVAL 5


positions. Maximum fiber isometric force was eli-cited by immersing the fiber in a solution containinga high concentration of Ca2þ and adenosinetriphosphate (ATP). The specific force of fiberswas calculated by dividing the fiber isometric forceby the fiber cross-sectional area. Fibers were cate-gorized as fast or slow by examining their forceresponse to rapid, constant-velocity shortening,and 10 fast fibers were tested from each biopsysample (Fig. 2).

Terminal deoxynucleotidyl transferase deoxyuridine-triphosphatase nick end labeling stainingFirst, we placed muscle biopsies in TissueTek OCT(Sakura Finetek Europe, Alphen aan den Rijn, TheNetherlands) and snap-froze them in liquid nitro-gen. We then put 10-mm sections on a cryostat andlabeled them with a Click-It terminal deoxy-nucleotidyl transferase deoxyuridine-triphosphatasenick end labeling (TUNEL) assay (Life Technolo-gies, Grand Island, NY), which marked apoptotic

FIGURE 3: Data on the right show perfusion parameters in group 2. Limbs in this group were connected to the ex situ perfusion systemfor 24 hours whereas limbs in group 1 were stored using conventional methods of cold preservation at 4!C.



nuclei with the green fluorophore AF488. Sectionswere also treated with the blue fluorescent nucleardye Hoechst 33342 to identify total nuclei andthe red fluorescent extracellular matrix markerWGA-lectin-AF555 to identify intramyocellularversus extramyocellular nuclei.25

Statistical analysisData are presented as means % SD. We tested dif-ferences between the control and perfusion groupsusing the Student t test. Using the SD generated by

the primary outcome variable (single-muscle fibercontractility testing), a power of 80% required aminimum of 4 animals to be tested in each groupwhen statistical significance was set to .05.

RESULTSLimbs on pump perfusion in group 2 showedincreased peripheral vascular resistance within thefirst hour but returned to lower values after the sec-ond hour and vascular resistance remained low untilthe end of the perfusion at 24 hours (Fig. 3, left

FIGURE 4: Graphs in the left column show metabolic parameters of limb perfusion when the limb was attached to the ex situ perfusionsystem. Data in the right column show the same parameters in the recipient animal after transplantation.



column). This occurred without a significant increasein flow or mean arterial pressures. Recipients in bothgroups remained hemodynamically stable throughoutthe experiment. Mean arterial pressure, hematocrit(not shown in Fig. 3), and heart rate did not show astatistically significant difference between groups (P> .05) (Fig. 3, right column).

In group 1, the brachial and radial arteries hadblood flow throughout the experiment. Capillary refillon the skin remained more than 4 seconds (comparedwith 2 seconds in group 2). Skin temperature did notreturn to normal despite warming. Lactate and po-tassium levels did not increase after revascularization.

In group 2, among metabolic parameters of perfu-sion, lactate increased steadily until the end of perfu-sion at 24 hours (Fig. 4, left column). Potassium, pH,and other parameters remained stable, likely owing toperiodic perfusion exchange. After transplantation,lactate in group 2 returned to normal levels. Lactate,potassium, pH, and other parameters did not show astatistically significant difference between groups afterreperfusion (P > .05) (Fig. 4, right column).

Single-muscle fiber contractility testing valuesshowed a statistically significant drop between 18 and

24 hours while limbs were on the pump in group 2(P < .05). This returned to normal at 30 and 36 hoursduring the reperfusion phase (6 and 12 h aftertransplantation) (P > .05). In contrast, the controlgroup showed a steady decline throughout the ex-periment (Fig. 5, right column). Muscle samples ob-tained for TUNEL staining showed a limited number ofapoptotic cell nuclei between group 2 and controlsamples, which suggested a normal cell cycle after 12hours of reperfusion (Fig. 6).

DISCUSSIONUsing an ex situ perfusion system, we demonstratedthe possibility of extending limb allograft survival upto 24 hours as evidenced by metabolic, functional,and histology parameters of limb perfusion. Sincehand transplantation became a reality, the standard ofcare in preserving the procured VCA has been to usestatic cold storage methods.26e28 Although thisapproach successfully reduces the initial ATP con-sumption and metabolism of the muscle cell, iteventually leads to anaerobic metabolism and com-plete depletion of ATP stores.29e31 Studies on cardiacallografts in particular demonstrated that the primary

FIGURE 4: (continued).



site of cold injury is the cell membrane of endothe-lium and myocytes.8,10,11 The magnitude of theseinjuries seems to correlate with the duration of coldischemia.32,33 Cardiac allografts subjected to 6 hoursof cold ischemia had 25% greater graft loss thanthose subjected to 2 hours.33 At the cellular level,cold ischemic injury is characterized by coagulativemyonecrosis (CMN), which coexists with or mimicsacute cellular rejection but also heals with a scar thatis indistinguishable from ischemic cell death or ne-crosis.34 In hand allografts, Landin et al12 reportedperioperative ischemic injury in a patient with bilat-eral hand transplantation. They observed the devel-opment of a slowly progressive wrist contracture over7 months on the left side, which was subjected to 3.5hours of cold ischemia (7 hours total ischemia) before

revascularization. The authors thought that this didnot result from a localized compartment syndrome orallograft rejection because they found extensiveCMN on histology. On both cardiac and hand allo-grafts, CMN develops in days to weeks. In the currentstudy design, we terminated our experiments 12hours after reperfusion. This was not enough to detectCMN. Instead, we used TUNEL staining to distin-guish apoptotic cell death from ischemic necrosis.With single-muscle fiber contractility testing, wefurther confirmed the presence of healthy myocyteunits after 24 hours on pump perfusion.

Normothermic perfusion methods were introducedto circumvent problems associated with cold storageand have been successfully applied in solid organtransplantations.14,16,18,19,35,36 In experimental studies,

FIGURE 5: After transplantation, biopsies were obtained at 1, 6, and 12 hours. Graphs in the left column show data regarding musclecontractility after 24 hours on pump perfusion and 12 hours after transplantation. Data on the right show muscle contractility in controlgroup, which was subjected to 6 hours of cold ischemia and transplantation afterward. Arrows on the x axis indicate the time oftransplantation in all graphs. Fo, maximum fiber isometric force; sFo, specific force of fibers.



FIGURE 6: Samples in the left column were obtained from normal muscle without ischemia. Samples on the right were taken fromanimals after 24 hours of pump perfusion followed by 12 hours of reperfusion. Apoptotic nuclei, which are the sign of pending tissuenecrosis, would appear in green fluorescent dye. A comparison of muscle samples (top row) shows no difference between the 2 sites.WGA, wheat germ agglutinin.



cardiac allografts kept in completely beating statuswith normothermic blood perfusion displayed excel-lent myocardial ultrastructure on electron microscopyimages.37 In clinical trials, cardiac allografts have beenreported to have fewer primary graft failures and acuterejection when preserved on normothermic ex vivoperfusion compared with cold preservation using his-tidineetryptophaneketoglutarate solution.36 Overallexperience with the use of normothermic ex situperfusion in VCAs is only preclinical at this point.Two authors have shown successful prolongationof allograft survival on swine models.21,23 Con-stantinescu et al21 used a modern perfusion technique,autologous blood at 32!C, and reported survival up to12 hours on swine forelimbs. Ozer et al23 showedsimilar results using pulsatile perfusion and dilutedblood as the oxygen carrier at 27!C to 32!C. Theyreported minor increases in limb weight with ongoingcontraction after 12 hours on the pump and 12 hours ofreperfusion. In the current study, we perfused limbs for24 hours and observed continuous neuromuscularelectrical stimulation and normal force generation onsingle-fiber contractility testing. Limb weightincreased 20% at the end of pump perfusion and thenreturned to 15% after 12 hours of reperfusion withexcellent contraction on all muscles tested. At thispoint, using the technique described in this article, 24hours seems to be reliable for ex situ limb perfusion.

Limitations of the current study include a limitedobservation period after transplantation. As a result,we do not know the effect of the reperfusion injurybeyond 12 hours in either group. No conclusions canbe drawn regarding the long-term function of thetransplanted extremity, because this would depend onthe effect of ex situ perfusion on nerve regeneration inthe presence of long-term immunosuppressive ther-apy. Despite the presence of statistically significantdifferences between the control and experimentalgroups in single-muscle fiber contractility testing, it isunknown whether this translates into a difference inthe function of the extremity. These limitations can beaddressed in another experiment with long-termfollow-up using chronic immunosuppression,possibly leading to clinical application.

ACKNOWLEDGMENTSThis study was funded in part by the AmericanFoundation for Surgery of the Hand.

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