Cryopreserved Arterial Homografts:Preliminary Results in InfrageniculateArterial Reconstructions

Manuel Alonso, MD, Ramon J. Segura, MD, Carlos Prada, MD, Santiago Caeiro, MD,Jose Antonio Cachaldora, MD, Eduardo Diaz, MD, Salvador Lujan, MD, Lina Cal, MD, andJuan Vidal, MD, A Coruna, Spain

The aim of this report is to present our preliminary experience using cryopreserved arterialhomografts in below-knee revascularization. We carried out a retrospective study at the PublicHealth Hospital of the Servicio Galego da Saude (SERGAS) from October 1995 to March 1997in which cryopreserved arterial homografts were used for revascularization of 17 lower limbs in16 patients. The clinical indications were limb-threatening ischemia in 15 lower extremities (7with rest pain and 8 with ischemic ulcers or gangrene), and large aneurysms of femoropoplitealarteries in 2. In addition, 75% of the patients had undergone previous surgical procedures forrevascularization on the involved extremity. No patient had a suitable greater saphenous vein inthe ipsilateral extremity and all patients required a below-knee arterial reconstruction procedure.There was just one runoff vessel in 11 of 17 extremities (65%). A histological exam was per-formed in four patients who died (1 case) or had homograft-related complications (3 cases). Theresults of this study indicated that cryopreserved arterial homografts could be a promisingalternative when below-knee revascularization is required in patients lacking suitable greatersaphenous vein, especially in those with limited life expectancy, but despite early acceptableresults, many aspects must be clarified. Close follow-up is mandatory. (Ann Vasc Surg 1999;13:261-267).

INTRODUCTION

Currently, the autogenous saphenous vein is con-sidered the most suitable conduit for peripheral ar-terial reconstructions, especially when below-kneesurgical procedures are required, because in suchcircumstances, bypass with synthetic prostheses hashad disappointing results and other surgical tech-niques employing different graft materials have alsohad limited success. Frequently, the greater saphe-

nous vein is unavailable because of disease or pre-vious use, and determining the best vascular con-duit to use represents a challenging problem for thevascular surgeon.

After initial experimental studies in dogs carriedout at the beginning of this century by Carrel, inwhich the thoracic aorta was replaced with a sec-ond dog’s jugular vein preserved in cold 24 hr be-fore transplantation, other authors used arterial ho-mografts for lower-limb revascularization but witha high rate of complications and poor results.1 As aresult, the development of synthetic graft materialsbegan. The renewed interest in using cryopreservedarterial homografts in the infrageniculate arterialsurgery is mainly due to recent advances in cryobi-ology (strict control of the curve of cooling, the use

From the Service of Vascular Surgery (M.A., R.J.S., S.C.,J.A.C., E.D., S.L., L.C., J.V.), and Pathology (C.P.), HospitalJuan Canalejo, A Coruna, Spain.

Correspondence to: M.A. Perez, MD, Avenida de Galicia, No.12-2°D, 33860-Salas, Asturias, Spain.

261

of a penetrating cryoprotectant such as dimethyl-sulfoxide for partial preservation of cellular viabil-ity) and the acceptable preliminary results reportedby Barral’s group using them for limb salvage.2

We have been using cryopreserved arterial ho-mografts for ischemic lower limbs revascularizationand aortoiliac reconstruction in the setting of vas-cular infection.3 The purpose of this report is toshow our early experience with cryopreserved ar-terial homografts in below-knee arterial surgery.

MATERIALS AND METHODS

From October 1995 to March 1997, cryopreservedarterial homografts were used for bypass surgery in17 lower limbs of 16 patients. The age range wasbetween 44 and 87 years (mean 69 years). Riskfactors and clinical characteristics of the patientswere those usually found in cases with advancedatherosclerotic disease and are shown in Table I. In9 out of 16 patients (56%) three or more risk factorswere present and 75% of the patients (12/16) hadhad previous revascularization procedures on theipsilateral extremity.

The clinical indications were (1) threateningischemia in 15 lower extremities, 7 with rest painand 8 with ischemic ulcers or gangrene; and (2)large aneurysms of the femoropopliteal arterieswithout symptoms in two extremities of differentpatients (one of them underwent bilateral recon-struction because he suffered from critical leg isch-emia due to a distal embolism on the opposite side,and was already included above). No patient had asuitable greater saphenous vein in the limb thatneeded to be operated on, and all of them requireda below-knee revascularization procedure. Therewas just one runoff vessel in 11 of 17 extremities(65%).

A routine preoperative study was done in allcases including serological test for viruses (HIV, Band C hepatitis, CMV), toxoplasmosis, and lues.

ABO incompatibility was not considered a reasonprecluding surgical repair with a cryopreserved ar-terial homograft in these patients when it was notpossible to find a compatible one.

Arterial homografts were procured under strictaseptic technique from non-heart-beating cadaversor multiorgan donors while arterial perfusion withcold saline from the hypogastrics or the aorta wasperformed. Thoracic and abdominal aorta, iliac, andfemoropopliteal arterial segments were removed.Harvested arteries were kept in sterile bottles withEurocollins and broad-spectrum antibiotics solutionand transferred to tissue banks in isothermal boxesfilled with ice. Cryopreservation was started within24 hr after harvesting. Under a laminar flow, hutarteries were transferred into sterile bags containingcold Eurocollins and antibiotics solution. Dimethyl-sulfoxide was gradually added reaching a final con-centration of 10%. The bags were labeled and trans-ferred into a programmable freezer for cryopreser-vation according to an electronically monitoredprogram that allows a cooling rate of −1°C/min to−40°C and after at −5°C/min to −120°C. The cryo-preserved arterial homografts were stored in the va-por phase of liquid nitrogen at −140°C. Bags withthe cryopreserved homografts were sent to the hos-pital when required in a cryogenic dry shipper.They were thawed in the operating room by sub-merging the bags in saline at 37°C, and after di-methylsulfoxide was gradually removed, washingthe homografts with Eurocollins repeatedly.Samples for histological and bacteriological studieswere obtained. Finally, after all collaterals had beensutured, the arterial homograft was implanted.

A surgical bypass was performed to the cruralarteries in 15 extremities (88%) and to the distalpopliteal artery in 2 limbs (12%) (see Table II). Thecryopreserved arterial homografts were implantedpreferably in a superficial extraanatomic position(65%, 11/17 lower limbs) which facilitates the fol-low-up and treatment of possible complications. An

Table I. Risk factors and clinical characteristics

Risk factor n (%)

Tobacco use 12 (75)Hypertension 4 (25)Diabetes 7 (44)Hyperlipidemia 6 (38)Coronary artery disease 3 (19)Pulmonary obstructive disease 4 (25)Renal failure 1 (5)Hepatitis 1 (5)

Table II. Type of arterial bypass performed

Type of bypass n (%)

Common femoral-anterior tibial 6 (35)External iliac-anterior tibial 2 (12)Common femoral-posterior tibial 4 (24)Common femoral-peroneal 2 (12)External iliac-peroneal 1 (6)External iliac-distal popliteal 1 (6)Common femoral-distal popliteal 1 (6)

262 Alonso et al. Annals of Vascular Surgery

anastomotic suture between two cryopreserved ar-terial fragments was necessary in 9 of the 17 pro-cedures (53%) in order to achieve adequate length.A minor amputation (toes or forefoot) was also per-formed in 5 out of 15 lower limbs with critical isch-emia (33%). Immediately after surgery patientswere given treatment with low-molecular-weightheparin, and during the first 48 hr they were alsogiven dextran 40. Afterward, nine patients (56%)received oral anticoagulation because they hadbeen taking it previously, had very bad runoff, orhad undergone many failed revascularization at-tempts in the past; seven (44%) patients were givenantiplatelet therapy. No patients received immuno-suppressive treatment.

All patients had angiography before discharge(an intraoperative arteriogram or a postoperativeintravenous digital subtraction angiogram). For fol-low-up, patients were seen at 3, 6, and 12 monthsand every 6 months thereafter duplex scanning wasperformed; an intravenous digital angiography wasalso carried out in the sixth month. A histological

study with light microscopy was performed afterimplantation in four patients because they died orhad complications related to the homograft. Pri-mary and secondary cumulative patency rates werecalculated using the Kaplan-Meier method.

RESULTS

There was no surgical mortality and no graft suf-fered occlusion during the first 30 postoperativedays. A young diabetic patient needed a below-knee amputation on the seventh postoperativeday because of persistent infection and failure of afoot amputation, although the tibial bypass withcryopreserved arterial homograft remained open(Fig. 1).

During a mean follow-up of 12.4 months (be-tween 2.5 and 20 months), no patients died ofgraft-related causes. Two elderly patients with pat-ent grafts died in the third and ninth months fromcauses not related to the operation. Three patientssuffered cryopreserved arterial homograft thrombo-

Fig. 1. Patent anterior tibial bypass with cryopreservedarterial homograft in spite of poor runoff in a patient whorequired leg amputation.

Fig. 2. Angiography showing distal dilatation of an ante-rior tibial bypass with cryopreserved arterial allograft.

Vol. 13, No. 3, 1999 Cryopreserved arterial homografts 263

sis: one had a previous below-knee amputation 4months before as detailed above (a thrombectomywas done without success), another had occlusionin the 17th month and was not reoperated on be-cause his limb no longer had critical ischemia, andone patient suffered occlusion of a peroneal arterybypass in the tenth month and needed another op-eration in which a lower distal anastomosis withcryopreserved arterial homograft was performed. Inthe last two cases, angiography showed atheroscle-rotic disease progression in the distal arterial tree.

One case with an anterior tibial bypass developeda distal dilatation of the graft that required its re-section and prolongation with a new segment ofcryopreserved homograft in the 13th postoperativemonth (Fig. 2). Of the four patients who had ho-mograft-related complications three (75%) werenot ABO group compatible.

The 17-month cumulative primary patency was51% and secondary patency was 74% (Tables IIIand IV). Limb salvage was possible in 14 of the 15lower extremities with critical leg ischemia (93%).

Histological exam in some patients who died(one case) or had homograft thrombosis (two pa-tients) showed loss of endothelial integrity, intimalthickening, loss of smooth muscle medial cells, me-dial fibrosis with calcifications in some cases, andinflammatory infiltrate affecting mainly the adven-titia and the external part of the media. The dilatedhomograft had the same changes but with the fol-lowing more striking features: almost complete dis-appearance of endothelial and smooth muscle cells,big subendothelial plaques of fibrosis, very signifi-cant thinning of the media, and a decrease in elasticfibers (Fig. 3).

DISCUSSION

Arterial homografts were accepted in the earlyyears of vascular surgery as arterial substitutes in

humans;4 however, when they were implanted asan infrainguinal bypass an unacceptable short-termand mid-term thrombosis rate1 led to their aban-donment. When below-knee arterial surgery is re-quired and there is an unsuitable or unavailableautogenous vein, options are limited and successvariable.5-7 In the case of unavailable ipsilateralgreater saphenous vein, we are not in favor of usingvein from the contralateral limb, especially if thissuffers from ischemic disease, and because theprobability that this limb will require a distal bypasscan reach 60% within 3 years.8 Although it is pos-sible that some cases lacking a greater saphenousvein could be managed with an all-autogenous tis-sue procedure (arm veins, lesser saphenous vein,and remnants of greater saphenous vein) with ac-ceptable results,8,9 composite vein grafts are fre-quently necessary and in these cases, patency islower. Londrey et al. reported a 1-year cumulativesecondary patency rate of only 56%, with graft re-vision required in nearly 20% of the cases.7 More-over, for some of these procedures general anaes-thesia is mandatory and two surgical teams are rec-ommended. Other alternatives for obtainingautogenous material, such as harvesting of the su-perficial femoral vein, involve more complex tech-niques and higher operative mortality.10

With recent advances in cryobiology enablingpreservation of living cells and tissues for a longperiod of time, and given the high rate of short-term occlusions and poor results with cryopre-served homografts veins,11-13 a renewed interesthas developed in cryopreserved arterial homografts,resulting in the creation of arterial banks. Construc-tion of long bypasses with cryopreserved arterialhomografts is less traumatic and is a technicallysimple procedure for lower-limb revascularization.Recently, Barral’s group reported use of cryopre-served arterial homografts in redo operations for

Table IV. Secondary cumulative patency (Kaplan-Meier)

Time (months) Failed (n) Censored (n) Remain exposed (n) Patency (%) Standard error

4 1 4 12 92.3 7.317 1 7 4 74.0 17.5

Table III. Primary cumulative patency (Kaplan-Meier)

Time (months) Failed (n) Censored (n) Remain exposed (n) Patency (%) Standard error

4 1 4 12 92.3 7.310 1 3 8 82.0 11.613 1 2 5 68.4 15.817 1 1 3 51.3 18.9

264 Alonso et al. Annals of Vascular Surgery

limb salvage with a 2-year cumulative secondarypatency rate of 72%.2 In our experience, none ofthe cryopreserved arterial homografts sufferedthrombosis during the early postoperative period,and even in one case that needed a below-kneeamputation, the anterior tibial bypass remainedopen for 4 months, despite poor runoff (Fig. 1).These features suggest that cryopreserved arterialhomografts have low thrombogenicity in the short

term, especially in view of the fact that redo infrain-guinal bypass surgery is associated with worse re-sults and with a reported immediate occlusion rateas high as 14% using autogenous vein grafts and29% for synthetic prostheses.6 The 17-month cu-mulative primary and secondary patency rates of51% and 74%, respectively, in our group of pa-tients are acceptable clinical results in light of theabove factors. The cause of thrombosis in our series

Fig. 3. A Intraluminalthrombus (*) with nearabsence of endotheliallayer and thinning of themedia, (hematoxylin andeosin, ×40magnification).B Disappearance ofinternal elastic laminaeand elastic fibers,(Orcein, ×40magnification).C Decreased number ofsmooth muscle medialcells (*) andinflammatory infiltrate,with polymorphonuclearcells and lymphocytesmainly in the adventitiaand external part of themedia (hematoxylin andeosin, ×40magnification). D Loss ofsmooth muscle medialcells with areas of fibrosisand iron accumulation(*) (Pearls, ×100magnification).

Vol. 13, No. 3, 1999 Cryopreserved arterial homografts 265

remains unclear, because all patients had poor run-off. Although the histological exam showed patho-logical changes, it was not possible to differentiatebetween immune rejection and a nonspecific in-flammatory response or to specify the contributionof each to graft occlusion.

An important observation is that of the four pa-tients who had homograft-related complications,ABO blood group incompatibility was present inthree, and the other patient who suffered occlusionof a compatible graft had justified reasons for failureafter a below-knee amputation (Fig. 1). This canstress the importance of at least considering ABOgroup matching in arterial homografting in the fu-ture. However, some studies with cryopreservedveins did not find that ABO matching influencedtheir results.14 Although undue traction and care-less technique during harvesting could damage thearterial wall and cause aneurysmal dilatation after-wards, an immune mechanism may be involved. Inour case of graft dilatation, the histopathologicalexam can suggest such a mechanism.

The 5-year survival rate in patients undergoing aredo operation for limb-threatening ischemia hasbeen reported to be as low as 12%.15 Two of ourpatients with critical limb ischemia died after 3 and9 months with an open graft and their limb pre-served, (93% limb salvage rate), so at the momentcryopreserved arterial allografts could be indicatedmainly for elderly patients or those with limited lifeexpectancy who require below-knee arterial sur-gery and who do not have a suitable saphenousvein.

Long-term success of cryopreserved arterial ho-mografts depends on obtaining a less antigenic graftthat maintains viability of arterial cells after cryo-preservation.16 Cellular components, endothelium,and smooth muscle cells can express major histo-compatibility antigens. When these cells were re-moved using detergents, a reduced adventitial in-flammatory infiltration was observed, but with anunacceptable high rate of graft thrombosis.17,18

Although some authors found almost the samefeatures of rejection for fresh and cryopreserved al-lografts,18 many other reports suggest that cryo-preservation may decrease antigenicity of arterialhomografts by a yet poorly defined mechanism.19

Elimination of some arterial wall cells by cryopres-ervation might limit rejection until repopulationwith cells of host origin occurs,20,21 and preserva-tion of areas with viable endothelium after thawingcould diminish intimal hyperplasia and earlythrombosis.17,20 However, rejection continues to bean unsolved problem. Even though the use of im-munosuppressive drugs could maintain the struc-

tural integrity and functional characteristics of cryo-preserved homografts,22 long-term immunosup-pression, except when the patient has already beenreceiving it for other reasons, seems a very highprice to pay for lower-limb revascularization inmost patients with critical leg ischemia.23,24 Tem-porary treatment (until repopulation of the homo-graft with host cells) may be a more feasible clinicalapplication.25

In conclusion, cryopreserved arterial allograftscould be a promising alternative for below-knee ar-terial revascularization when the greater saphenousvein is unsuitable or unavailable, especially in pa-tients with limited life expectancy. Despite earlypromising results, many aspects of arterial homo-grafting must be clarified and further clinical expe-rience obtained. Close follow-up of these patients ismandatory.

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