Biologic Grafts - Clinical Tree

In the field of vascular surgery, the use of surgical bypass is fundamental to the treatment of a wide variety of arterial and venous disorders. In turn, the technical conduct and success of surgical bypass are directly dependent on the conduit used. The ideal conduit should be readily available, easy to handle, resistant to thrombosis and infection, durable, inexpensive, and should have characteristics similar to the vessel that it is replacing.

Although the perfect conduit does not exist, autogenous blood vessels are closest to the ideal. Autogenous arterial conduits, such as the internal mammary, radial, and gastroepiploic arteries, have been used with great success in the coronary circulation. The internal iliac and radial arteries have been used in the visceral vascular bed, ^, and the superficial temporal artery has been used for extracranial–intracranial bypass. Unfortunately, short conduit length and invasive harvest have limited the use of autogenous arterial grafts to a relatively small number of clinical scenarios.

Autogenous vein has been the preferred conduit for infrainguinal bypass because long lengths of vein can be harvested, removal is inconsequential, and harvest complexity is minimal. Autogenous veins have been used for the bypass of upper extremity, carotid, coronary, and visceral arterial beds. They have also been preferentially used in the construction of arteriovenous fistulae (AVFs) for hemodialysis.

Although autogenous vascular grafts perform well, there are multiple clinical situations in which these conduits are inadequate, unavailable, or improperly matched to the recipient vascular bed. These unmet demands led to the development of artificial grafts. Although multiple materials have been tried, polyethylene terephthalate (Dacron, DuPont, Wilmington, DE) and polytetrafluoroethylene (PTFE) have emerged as the standard materials for prosthetic vascular grafts. These grafts have been used with excellent success for the bypass of large vessels, such as the aorta and iliac arteries, and medium-sized vessels, such as the subclavian artery. Prosthetic grafts have also been used extensively for dialysis access and with mixed results for infrainguinal revascularization. Dacron and PTFE grafts offer “off-the-shelf” availability and a variety of sizes to permit replacement of even the largest vessels. In general, however, they cannot be used in infected fields and, compared with autogenous conduits, are at increased risk for infection, structural deterioration, and occlusion. Although patency rates are acceptable for aortoiliac reconstruction because of high flow rates and low outflow resistance, bypass to smaller targets, such as the tibial arteries, is associated with low graft patency.

Limitations of autogenous and prosthetic grafts have fueled exploration for other potential conduits, and this investigative effort has led to the evaluation of biologic grafts for bypass. Biologic grafts, or biografts, are bypass conduits made of nonautogenous biologic vessels modified for use in clinical practice. Allografts or homografts refer to arteries or veins that are transplanted from one individual to another within the same species. Xenografts or heterografts are vessels transplanted from an individual of one species to an individual of another species.

Carrel was first to experiment with fresh allografts and xenografts in dogs during the first decade of the 20th century. The first recorded human use of allografts, obtained from casualties, occurred during World War I. In 1948, Gross et al. described the first clinical series of fresh arterial allografts, and less than a decade later, Linton published his series of fresh venous allografts. Various methods of allograft cryopreservation were developed in the 1950s, refined in the 1970s, ^, and standardized and commercialized in the late 1980s. In parallel, enzymatically treated and tanned bovine carotid artery (BCA) xenografts were evaluated and first described in a clinical setting in 1966. Application of similar techniques to human vessels led to development of the human umbilical vein (HUV) graft by Dardik and Dardik in 1976.

Theoretically, biografts promise to be the optimal vascular conduit. They can potentially offer “off-the-shelf” availability, a wide variety of sizes, excellent handling characteristics, and patency rates similar to those of autogenous vessels. These attractive features prompted scientific investigation and clinical use of these conduits that has spanned the course of almost a century. Although this collective experience with an assortment of biografts in a variety of clinical settings led to specific clinical indications for their use, biologic grafts have failed to become the “Holy Grail” of vascular surgery.

Graft Properties

Fresh Vascular Allografts

Fresh arterial and venous allografts have been studied in animal experiments. ^, ^, In one canine model, fresh venous allografts had a patency rate of 69% at 20 months. Pathologic analysis of explanted veins revealed intimal proliferation, medial inflammation, medial degeneration, and periadventitial fibrosis. In another canine venous allograft experiment, dogs that were immunosuppressed with azathioprine demonstrated slightly better graft patency than did those that were not. Conversely, in a murine model, fresh venous allografts implanted in rats had excellent patency rates and minimal intimal thickening on histologic analysis.

In humans, fresh venous allografts used for infrainguinal bypass had a failure rate of 55% in one series; failed grafts either occluded or became aneurysmal. Patency rates of allografts appeared to be higher in patients whose grafts were harvested from blood types ABO compatible donors. In another study, fresh arterial allografts placed in the aortic position were noted to be highly immunogenic, with evidence of both a humoral and cellular immune response. These animal and human data suggest that fresh vessel allografts initiate a host immune response. Furthermore, the patency of these grafts appears to vary among species.

Aside from their immunogenicity, the use of fresh vascular allografts in the clinical setting has been hampered by logistic factors. Scarce availability of fresh arteries and veins and a need to successfully store such vessels for future use have led to the development of a number of preservation and modification techniques. These techniques can be divided into those that involve preservation without a planned significant change in graft integrity and those in which the graft is intentionally chemically altered. Cryopreservation is the most common example of the preservation technique, whereas proteolytic enzymatic digestion and dialdehyde starch tanning are examples of the modification technique. In addition to creating a conduit that would be more readily available, it is hoped that these techniques will inhibit the host immune response and thereby increase graft patency.

Over the past century, multiple vessel preservation techniques have been tested. Grafts were stored in a number of solutions, including nutrient broths, glycerol, and plasminate. A variety of storage temperatures ranging from room temperature to −70°C were tried. ^, Finally, a number of adjunctive sterilization techniques, including ethyl dioxide and irradiation, were attempted. Early techniques focused on preservation without much regard to viability of the vascular tissue. Initial results with preserved vascular grafts were inconsistent, probably because significant cellular and structural damage occurred in many of these vessels and made them nonviable. ^,

Cryopreserved Allografts

Methods of Preparation

There is evidence that cryopreservation can result in significant cellular damage unless appropriate precautions are taken. 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. 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.

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. 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. Finally, there is evidence that the addition of certain additives such as chondroitin sulfate to the storage solution enhances vein viability and function.

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. ^, ^,

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. ^, These changes increase with the duration of cryopreservation ^, and lead to partial endothelial cell loss. 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. ^, 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.

Endothelial vasodilatory function, as measured by response to acetylcholine, thrombin, and calcium ionophore, appears to be retained, but is somewhat diminished, with cryopreservation. With regard to coagulation homeostasis, although cryopreservation of vein grafts is not associated with increased platelet deposition, it does cause decreased thrombomodulin activity. 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.

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. Despite these findings, collagen synthesis in cryopreserved veins was diminished in a canine model. Smooth muscle cells in cryopreserved canine saphenous autografts were noted to have a diminished relaxation response to nitric oxide, although contraction induced by norepinephrine, potassium chloride, and serotonin was unaltered.

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. ^, Smooth muscle cell viability is lost, ^, severe medial fibrosis and disruption of elastic fibers occur, ^, ^, and medial necrosis has been described. Significant lymphocytic infiltration of the media and adventitia has been observed. These alterations in vessel wall biology are not routinely observed with autologous conduits.

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 ^, and could be successfully blunted by the cryopreservation process. Recent literature, however, suggests that vascular allografts do trigger a significant immune response. ^, Endothelial cells present surface antigens that stimulate a cell-mediated immune response against the donor graft. An immunoglobulin-G-mediated humoral immune response to donor-specific antigens has been described. ^, Transplanted canine venous allografts, but not autografts, demonstrated extensive medial fibrosis and lymphocytic infiltration consistent with immunologic rejection. 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. 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. ^, 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. 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., who noted improved patency of allografts transplanted to ABO-matched patients. However, a recent study of seventy-two implanted allografts revealed that ABO-mismatch had no effect on death, thrombosis, rupture, stenosis, or aneurysmal degeneration. In animal models, immunosuppression with cyclosporine has been demonstrated to diminish immunologic rejection of aortic and venous allografts. Azathioprine has likewise been shown to decrease the effects of rejection in venous allografts.

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, 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. 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. 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. In contrast, Randon et al. 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. Allosensitization, however, did not occur when the CFV graft was processed to remove cellular elements. Diminution of the immune response by removal of antigenic epitopes has led to multiple attempts to structurally modify biologic grafts.

Structurally Modified Biologic Grafts

In parallel with the development of cryopreservation techniques, further research was conducted to modify blood vessels so that an acceptable vascular substitute could be developed. The goal was to transform a harvested blood vessel into a durable nonimmunogenic graft that could be easily produced and stored. During early experiments in the 1950s, animal arteries were modified by enzymatic digestion of the musculoelastic portion of the vessel wall with ficin, a proteolytic enzyme isolated from figs, to remove immunologically reactive proteins. The resultant collagenous vascular skeleton was strengthened by collagen cross-linking through subsequent tanning with dialdehyde starch. ^, This modified graft was then sterilized and stored in a 1% propylene oxide-50% ethanol solution.

In the earliest experiments, modified BCA grafts were implanted as xenografts first in dogs and then in patients with symptomatic lower extremity occlusive disease. Although no graft ruptures had occurred at 3 years of follow-up, early neointimal hyperplasia and diminished patency were observed. An unacceptable late rate of graft infection and aneurysmal degeneration led to a change to glutaraldehyde-based tanning protocols. ^,

Bovine mesenteric veins (BMVs) have also been modified by a patented process of glutaraldehyde cross-linking and sterilized by γ radiation. Both BCAs and BMVs have been used as xenografts in a number of clinical applications.

HUV is a modified biologic conduit that was first evaluated in baboons in the early 1970s and subsequently used in humans ^, in 1975. Umbilical vessels are uniform in caliber, valveless, and branchless. The umbilical vein was removed from the umbilical cord by a variety of techniques, including enzymatic digestion and mechanical stripping. Polyester fiber mesh was then sutured in place about the length and outside circumference of the graft for added support. The reduced immunogenicity of this graft was hypothesized to be secondary to pretreatment with glutaraldehyde, which was thought to bind to graft histocompatibility antigen sites and thereby shielded them from the host immune response.

Other Grafts

The search for an ideal blood vessel substitute led to the investigation of a number of nonconventional biologic grafts in animal models. Vascular prostheses fashioned from pericardium and small intestinal mucosa have been evaluated. Chemically modified human and bovine ureters have been used as vascular conduits with some success. Modified bovine ureters have been used clinically with acceptable patency rates in femoral to popliteal bypass in one small Australian series. In addition, a small randomized trial claimed clinical equivalence between bovine ureters and PTFE used for hemodialysis access in patients with no vein options.

Clinical Use In Vascular Surgery

Indication

Biologic grafts have been used in modern vascular surgery mostly in three distinct clinical settings: extremity bypass in the absence of suitable autogenous conduit, arteriovenous (AV) access for hemodialysis, and replacement of infected prosthetic grafts.

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