Methods of Preparation

There is evidence that cryopreservation can result in significant cellular damage unless appropriate precautions are taken.³¹ During the cryopreservation process, the extracellular matrix freezes at a higher temperature than cellular cytoplasm. This leads to a vapor pressure gradient between the intracellular and extracellular components. When cooling occurs slowly, this gradient can result in cellular dehydration, whereas rapid cooling can lead to plasma membrane rupture. Work with cell suspensions, such as blood and semen, has revealed that certain substances, when added during the freezing process, can significantly improve cell viability.³² These substances, called cryoprotectants, include dimethylsulfoxide and glycerol. Their mechanism of action is to enter cellular cytoplasm and decrease the vapor pressure gradient that exists between the intracellular and extracellular components.²⁰

Over the last 20 years, cryopreservation techniques have been optimized and commercialized. Important variables inherent in modern cryopreservation processes include the type and amount of cryoprotectant used, freezing rate, storage temperature, duration of storage, and additives used.²⁰ The most common cryoprotectant in use today is dimethylsulfoxide at 10% to 20% dilution. The freezing rate varies among protocols, and there is some evidence that rapid freezing at 5°C/s may work best. Storage temperature may vary from −102°C to −196°C. The duration of cryopreservation may be important, and longer duration has been shown to have an adverse influence on vessel wall morphology but not on graft patency in one animal model.³³ Finally, there is evidence that the addition of certain additives such as chondroitin sulfate to the storage solution enhances vein viability and function.³⁴

Histology and Physiology

Cryopreserved arteries and veins are affected by both cryopreservation and immune rejection; a large body of research has been performed to define and dissect these processes from one another.

Cryopreservation has effects on the mechanical properties, histology, and physiology of the treated vessel. Elasticity and compliance of a vessel are important mechanical characteristics that affect its performance as a conduit. Changes in these properties lead to an increased difference in compliance between the conduit and host vessel, which can adversely affect graft patency. In vitro models comparing the mechanical properties of cryopreserved and freshly harvested arteries and veins reveal that cryopreservation does not significantly affect elasticity, contractility, compliance, and the mechanical buffering function of the treated vessel.^19,35,36

Cryopreservation of blood vessels leads to changes in the intima, media, and adventitia. Although appropriate cryopreservation does not affect the gross morphology of the endothelial layer, histologic changes such as focal microvillous projection, cytoplasmic vacuolization, nuclear prominence, and interruption of tight junctions have been visualized.^33,37 These changes increase with the duration of cryopreservation^33,37 and lead to partial endothelial cell loss.^37–39 Endothelial loss is significant when the cryopre­served graft is exposed to arterial flow. Although autoge­nous grafts re-establish an endothelial layer, only minimal re-endothelialization is observed in allografts.^20,29 Because of a compromise in intimal integrity, cryopreserved grafts accumulate low-density lipoprotein cholesterol at an accelerated rate as measured in an ex-vivo organ perfusion system.⁴⁰

Endothelial vasodilatory function, as measured by response to acetylcholine, thrombin, and calcium ionophore, appears to be retained, but is somewhat diminished, with cryopreservation.⁴¹ With regard to coagulation homeostasis, although cryopreservation of vein grafts is not associated with increased platelet deposition,⁴¹ it does cause decreased thrombomodulin activity.³⁸ Fibrinolytic activity appears to be similar in both fresh and cryopreserved canine jugular veins, but this activity may be adversely affected by the duration of cryopreservation.³⁷

The medial layer of cryopreserved vascular grafts appears to have grossly normal smooth muscle cells, although slight lysis and minimal mitochondrial edema were observed in a rabbit model. In that model, implantation of autologous veins into an arterial circuit led to the preservation of both smooth muscle cells and the elastic lamina. The smooth muscle cells displayed a synthetic, rather than a contractile, phenotype characterized by dilatation of the endoplasmic reticulum.³³ Despite these findings, collagen synthesis in cryopreserved veins, but not fresh veins, was diminished in a canine model.³⁹ Smooth muscle cells in cryopreserved canine saphenous autografts were noted to have a diminished relaxation response to nitric oxide,⁴¹ although contraction induced by norepinephrine, potassium chloride, and serotonin was unaltered.²⁰

Immunology

Allogeneic implantation of cryopreserved vessels leads to a different histologic and physiologic picture than that seen with cryopreserved autologous grafts. These observed changes are caused by immune mechanisms. Endothelial loss, encountered when an allograft is exposed to arterial flow, is not appreciably reversed, and exposed subendothelial elements are noted on electron microscopy.^20,29 Smooth muscle cell viability is lost,^23,42 severe medial fibrosis and disruption of elastic fibers occur,^23,29,42 and medial necrosis has been described.⁴³ Significant lymphocytic infiltration of the media and adventitia has been observed.²⁹ These alterations in vessel wall biology are not routinely observed with autologous conduits.⁴³

Although it is well known that transplanted allograft and xenograft organs elicit an immune response, it was initially believed that the host-mediated immune response of transplanted vessel allografts was minor^44,45 and could be successfully blunted by the cryopreservation process.²⁷ Recent literature, however, suggests that vascular allografts do trigger a significant immune response.^20,26 Endothelial cells present surface antigens that stimulate a cell-mediated immune response⁴⁶ against the donor graft. An immunoglobulin-G–mediated humoral immune response to donor-specific antigens has been described.^26,47 Transplanted canine venous allografts, but not autografts, demonstrated extensive medial fibrosis and lymphocytic infiltration consistent with immunologic rejection.⁴⁸ In a human model, analysis of 22 explanted cryopreserved saphenous vein (CSV) allografts revealed moderate to severe intimal, medial, and adventitial inflammatory infiltrates. Immunohistochemical analysis demonstrated an abundance of activated T lymphocytes containing cytotoxic granules.⁴⁹ In another experiment, cryopreservation did not alter antigenic expression and the immunologic response of a murine host to allograft transplantation in a number of studies.^50,51 Chronic immunologic rejection clearly plays a role in allograft biology and appears to be responsible for both diminished patency of cryopreserved vascular grafts and the predilection of these grafts to aneurysmal degeneration.⁴³

A number of investigators hypothesized that manipulating the host immune response to vascular allografts may attenuate immune rejection and improve graft patency. Matching of ABO blood groups was suggested by Ochsner et al,²⁵ who noted improved patency of allografts transplanted to ABO-matched patients. In animal models, immunosuppression with cyclosporine has been demonstrated to diminish immunologic rejection of aortic⁴³ and venous allografts.⁵² Azathioprine has likewise been shown to decrease the effects of rejection in venous allografts.²⁴

Based on these findings, attempts were made to improve the results of allograft use in humans by modulating the host immune response. Carpenter and Tomaszewski,⁵³ in a prospective, randomized trial of 40 CSV allografts implanted in patients treated with low-dose azathioprine, failed to show a significant improvement in graft patency at 1 year. Azathioprine immunosuppression, however, was associated with a decreased presence of T-lymphocyte cytotoxic granules in that study.⁴⁹ In another small human trial, a combination of low-dose cyclosporine, azathioprine, prednisone, warfarin, aspirin, and vasodilators was used in patients who underwent CSV bypass. Grafts treated with this immunosuppressive regimen demonstrated increased patency rates. This regimen, however, was associated with an increased incidence of complications and graft aneurysmal degeneration.⁵⁴ In one series of patients with prosthetic aortic infection, 10 of 30 patients who underwent aortic allograft replacement were concomitantly treated with cyclosporine. Although the measured humoral immune response was blunted in patients who received cyclosporine, no differences in graft patency or graft complication rates were appreciated.²⁶ In contrast, Randon et al⁵⁵ contended that a low-dose cyclosporine immunosuppressive regimen for lower extremity bypass using CSV was effective at reducing the risk of rejection while facilitating host cell repopulation of biograft endothelium.

Furthermore, an immunologic response evoked by a cryopreserved allograft can induce allosensitization, which may interfere with future organ transplantation. This mostly affects the use of cryopreserved femoral vein (CFV) allografts in hemodialysis access. A case-matched series of 20 patients who underwent creation of hemodialysis access with this graft demonstrated host allosensitization in all patients as measured by the panel-reactive antibody assay.⁵⁶ Allosensitization, however, did not occur when the CFV graft was processed to remove cellular elements.⁵⁷ Diminution of the immune response by removal of antigenic epitopes has led to multiple attempts to structurally modify biologic grafts.

Extremity Bypass

Acute or chronic ischemia of an extremity is caused by a number of conditions, including atherosclerosis, trauma, embolization, and in-situ thrombosis. Treatment of extremity ischemia involves revascularization by endovascular or surgical techniques. During infrainguinal surgical bypass, the choice of conduit is crucial to success of the operation. The autogenous great saphenous vein has proven to be the preferred conduit for infrainguinal revascularization.^70,71 When the autogenous great saphenous vein is not available, alternative autogenous conduits, such as an arm vein,⁷² the small saphenous vein,⁷³ and the composite autogenous vein,⁷⁴ have been used with good results. The ever-increasing age and complexity of patients with infrainguinal arterial occlusive disease has brought about increasingly frequent clinical scenarios in which the autogenous vein is not available and an alternative conduit must be found. Although prosthetic grafts have been used with moderate success above the knee, they have been disappointing when used for infrageniculate bypass.^75,76 Distal modification of prosthetic grafts with a vein cuff or distal AVF may improve patency rates,^77–79 but is more cumbersome.

Given the absence of reliable conduit options for infrageniculate bypass when a suitable autogenous vein is lacking, the feasibility of biologic grafts has been evaluated. In this setting, CSV allografts,⁸⁰ cryopreserved femoro-popliteal artery (CFA) allografts,⁴² HUV grafts,⁸¹ BCA xenografts,⁵⁸ and BMV xenografts⁸² have been used with varying degrees of success.

Arteriovenous Access

End-stage renal disease is a significant public health problem in the United States; its prevalence is increasing steadily, and it is forecast that by the year 2030, more than 2 million patients will be undergoing hemodialysis.^83,84 Long-term hemodialysis is best performed through a surgically created AVF that connects the arterial and venous circulations via a conduit. The ideal AV conduit carries high flow for efficient dialysis, is superficial enough for easy access, is sufficiently durable to withstand multiple cannulations, allows rapid sealing of cannulation sites, and is resistant to infection, stenosis, and thrombosis. A mature, native vein AVF comes closest to the ideal, and its use is strongly encouraged.¹¹ Unfortunately, many individuals lack suitable veins for native AVF construction because of small vein size, previous access procedures, or vein harvest for peripheral or coronary bypass. Furthermore, up to 60% of native AVFs fail to mature, and therefore, cannot be used successfully.^85,86 Although prosthetic AV grafts are widely used, they have lower patency rates, require more frequent revision, and are at higher risk than vein AVFs for infection.⁸⁷ The search for optimal hemodialysis access in patients who are not candidates for a native vein AVF has led to the use of biologic grafts. CFV allografts,⁸⁸ BCA xenografts,⁸⁹ bovine ureter xenografts,⁶⁹ and BMV xenografts⁶⁰ have been used in a variety of settings with variable results.

Replacement of Infected Prosthetic Grafts

Prosthetic graft infection, particularly when the aorta is involved, is one of the most dreaded complications in vascular surgery and is associated with high morbidity and mortality.⁹⁰ Treatment of an infected aortic prosthesis includes excision of the infected segment and extra-anatomic prosthetic bypass⁹⁰ or reconstruction with an antibiotic-soaked or antibiotic-bonded prosthetic graft,⁹¹ femoral vein,⁹² or aortic allograft.⁹³ Extra-anatomic bypass and aortic ligation are associated with long operative times, risk of remote infection, bypass thrombosis, and aortic stump rupture.^90,94 Antibiotic-soaked prosthetic grafts may work well for infections caused by relatively indolent Staphylococcus epidermidis, but are much less effective against more virulent organisms.⁹¹ Finally, use of the femoral vein for aortic reconstruction is tedious and associated with harvest-related complications.⁹²

Cryopreserved aortic allografts offer “off-the-shelf” availability, good handling properties, and the potential for expeditious in-situ repair. Cryopreserved aortic allografts were more resistant than prosthetic grafts to S. epidermidis infection in a canine model.⁹⁵ Resistance of vascular allografts to infection has led to the wide use of arterial allografts to treat aortoiliac infection,⁹⁶ CFV allografts to treat infection involving prosthetic AV grafts,⁹⁷ and CSV allografts to replace infected infrainguinal prosthetic bypass grafts.⁹⁸

Cryopreserved Allografts

A number of tissue banks and commercial companies prepare, store, and supply cryopreserved blood vessels. Despite similarities in conduit preparation, many have proprietary cryopreservation protocols.⁹⁹ The great saphenous vein, femoral vein, and arterial segments are harvested from multiorgan donors who are screened for an array of viral, bacterial, and fungal infections. Branches are suture ligated, and the allografts are sized with calibrated dilators. They are tested for presence of pathogens, rinsed in an antibiotic solution, placed in a proprietary cryoprotectant solution, and stored in the vapor phase of liquid nitrogen at −110°C to −196°C. Allografts are shipped and stored in a solution of dimethylsulfoxide at −96°C until needed. The vein, but not artery, is usually matched for ABO/Rh compatibility with the recipient to decrease the risk of rejection. At the start of the procedure, the allograft is rapidly thawed by submersion in a warm water bath at 37°C to 42°C for 20 minutes. After rinsing in a series of solutions provided by the manufacturer, it is ready for use.

CSV allografts are available in a number of lengths and diameters. Most commonly, the vein measures 3 to 5 mm in diameter. These grafts look, feel, and handle like the autogenous saphenous vein. During an infrainguinal bypass, the allograft is usually reversed and placed in a superficial tunnel for easy access.⁸⁰ Postoperative surveillance was not considered useful in one large series.⁸⁰

CFV allografts are usually less than 25 cm in length and have a diameter between 5 and 7 mm. They have most commonly been used in hemodialysis, particularly in the setting of prosthetic AV graft infection. When used for dialysis access, this allograft is appropriately reversed and tapered to a 5 mm diameter at the arterial anastomosis to decrease the incidence of ischemic steal syndrome.⁸⁸ It is allowed to mature for 3 to 4 weeks before it is accessed for hemodialysis. Revision of these grafts is very difficult because of their thin wall and surrounding fibrosis.⁹⁷

Arterial allografts have most frequently been used for aortic replacement in the setting of primary or prosthetic aortic infection. Given this clinical setting and the need to replace a large artery such as the aorta, these allografts have to withstand particularly hostile conditions. Technical modifications for the use of these allografts have been developed, including vigilance in following thawing instructions, use of appropriately long grafts, and construction of tension-free anastomoses, taking great care that suture ligation of branches is performed with polypropylene sutures that include the graft wall along with the branches, aggressive excision of infected tissue and wound drainage, circumferential anastomotic reinforcement with allograft strips, use of gentamicin-impregnated fibrin glue, and coverage of the graft with viable tissue, such as a pedicled omental or muscle flap.^93,100

Structurally Modified Allografts

The HUV graft (Fig. 94-1) is shipped and stored in 50% ethanol and provided on a glass mandril. It is irrigated up to 10 times with a low-molecular-weight dextran–containing solution and rinsed with a high-concentration heparin solution (10,000 U/L). This graft does not tolerate traction or the application of standard vascular clamps. To avoid injury, it needs to be passed through a metal or plastic conduit during tunneling and preferably controlled with a tourniquet. To decrease the risk of pseudoaneurysm formation during suturing, both the vein and the Dacron mesh need to be incorporated into the suture line. Finally, infusion of low-molecular-weight dextran has been recommended in the early postoperative period by the manufacturer to decrease the risk of early thrombosis.

Manufacturing of the umbilical vein graft by Synovis Life Technologies, Inc. (St. Paul, Minn) stopped in May 2005 in compliance with new U.S. Food and Drug Administration (FDA) guidelines governing combination tissue–medical devices.¹⁰¹ To date, a next-generation HUV graft that will fulfill FDA regulations is not yet available, although developers are actively seeking corporate industry support for such a venture (Herbert Dardik, personal communication, December 13, 2012).

The BCA graft (Fig. 94-2) is supplied in a specially designed tube containing a proprietary solution of 1% propylene oxide in 40% aqueous ethyl alcohol. It is naturally compliant, soft, and relatively easy to use. It is presently available in 6-, 7-, and 8-mm diameters and 15- to 50-cm lengths (Artegraft; Artegraft, Inc., New Brunswick, NJ). The BMV graft (Fig. 94-3) is shipped in a sterile saline solution and is available in 6-mm diameters and 10- to 40-cm lengths (ProCol Vascular Bioprosthesis, Hancock Jaffe Laboratories, Irvine, Calif). This graft is compliant and handles much like a saphenous vein.⁶⁰ Both xenografts require a series of rinsing steps in the operating room before use.