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Circulation. 2006;114:261-264
doi: 10.1161/CIRCULATIONAHA.106.639005
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(Circulation. 2006;114:261-264.)
© 2006 American Heart Association, Inc.

Editorial

Bioprosthetic Valves and Laudable Inflammation?

Richard A. Hopkins, MD

From the Department of Cardiothoracic Surgery, Brown University and Rhode Island Hospital, Providence.

Correspondence to Richard Hopkins, MD, Karlson Professor and Chief Cardiothoracic Surgery, Brown University Medical School, Rhode Island Hospital, 2 Dudley St, MOC #500, Providence, RI 02905. E-mail rahopkins{at}lifespan.org


Key Words: Editorials • inflammation • prosthesis • tissue • transplantation • valves

In this issue of Circulation, Dr Manji and colleagues1 from the University of Alberta (Canada) report a series of animal experiments designed to test the mythic role of gluteraldehyde in preventing recognition of bioprosthetic heart valve antigenicity with subsequent rejection and failure. Gluteraldehyde–cross-linked xenograft tissues (initially porcine) have been used in the manufacturing of stented (and now stentless) heart valves since 1970. Original theories for the clinical efficacy of gluteraldehyde were based on its ability to irreversibly cross-link collagen and thus to increase mechanical strength and durability over fresh untanned cardiovascular tissues. Porcine valve leaflets, bovine pericardium, equine pericardium, and bovine jugular vein, among other structures, have been treated this way for clinical applications. Since its introduction, however, limitations and unexpected consequences of gluteraldehyde and similar chemical treatments have been recognized clinically. As pointed out by the authors, durability is quite variable, tending to be better in older patients whereas younger patients suffer early deterioration, calcification, and fibrocalcific failure.2,3 The good news has been that the failures tend to be progressive rather than catastrophic, leading to semielective reoperations. Additionally, the xenograft bioprostheses have been extremely helpful in avoiding warfarin in the very young and the very old, reducing the risk of thromboembolism. The traditional explanation for the fibrocalcific degeneration of the nonvital gluteraldehyde-treated bioprosthetic heart valves has been a combination of physical and chemical effects leading to calcification and a fixation of the structural proteins that prevent protein recycling and renewal. Mechanical theories of fatigue-induced wear resulting in calcification have been proposed, especially along the flexion lines of the cusps. Others have proposed that the gluteraldehyde has serendipitously "masked" the antigenicity of xenograft proteins, retained cells, and cellular debris, thus prolonging the period to calcification. This well-designed series of studies buttresses other studies demonstrating that gluteraldehyde fixation does not eliminate antigenicity of bioprosthetic heart valves and that one of the primary causes of fibrocalcific failure is immune rejection.4–6 Important implications are in 4 areas: (1) proper assessment of durability and clinical performance of manufactured valve xenograft bioprostheses; (2) recognition of inflammation-driven calcification potential of all types of biological valves, including allografts (homografts), autografts, and new treatments of xenograft tissues designed to make available donor structural proteins for turnover (eg, decellularized xenograft valves); (3) choice of appropriate preclinical surgical implant test species to qualify biological tissues for implantation in humans; and (4) thorough failure modes and effects analysis driving regulatory end points and clinical qualification of new valve options, including new treatments of cross-linked xenograft valves, decellularized allograft/xenograft valves, and "tissue-engineered" in vivo recellularized or bioreactor cell–seeded valves based on allograft or xenograft scaffolds.

Article p 318

The currently available xenograft bioprosthesis include multiple varieties of design, manufacturing, and source material. However, the vast majority are gluteraldehyde-treated bovine, equine, or porcine tissues. It is well known that these tissues calcify at some point in their natural history, often within 2 years in neonates and up to 12 to 14 years in older adults but essentially not at all in the very elderly.7 Manji and colleagues have a persuasive argument that this is a consequence of the strength of the immune response rather than the age-related calcium metabolism. They have made an excellent effort in trying to separate the 3 known, not mutually exclusive processes that operate in the clinical setting: (1) postsurgical wound healing, (2) nonspecific inflammation, also called foreign body or innate inflammatory reaction to even minimally provocative materials, and (3) immune-mediated rejection with consequential inflammation.8–10 The authors tested syngeneic transplants between rats and guinea pigs and then xenograft transplants of guinea pigs to rats. A xenogeneic transplant group also was treated with steroids that are both antiinflammatory and immunosuppressive. The steroid treatment decreased inflammation, humoral antibody rise, and T-cell/macrophage infiltration. These findings have previously been confirmed in the human whole-heart allograft transplant model in which donor valves appear pristine when there is a lack of rejection of the whole heart by transplant recipients.11,12 There was far greater inflammation, T-cell and macrophage infiltration, and antibody rise in serum antibody elevations in the xenogeneic valve transplants despite gluteraldehyde fixation. As would be expected by all surgeons, the syngeneic transplants also demonstrated some nonspecific wound-healing responses when gluteraldehyde fixation was used, suggesting that the implantation of toxic chemicals, suture damage of normal tissues, and prosthetic implants containing necrotic cells and cell debris are proinflammatory even in the absence of rejection.13

In the era in which decellularized heart valves are being proposed as a solution to the slow deterioration and fibrocalcification of cryopreserved allograft valve conduits, it is important to realize that these studies suggest that structural proteins alone can be proinflammatory. It has been well demonstrated that the presence of {alpha}1,3 Gal epitope, if retained in cellularized or decellularized constructs, provokes very strong and rapid rejection with fibrocalcific destruction (as is the case with porcine-to-human implants).14 These current studies, taken with other recently published reports, suggest that gluteraldehyde treatment alone does not shield porcine tissues from human or old-world monkey antibody and complement–independent or –dependent phagocytosis and cytolysis responses.15–17 Thus, any proposed decellularizing or tissue-engineering technique that is based on such {alpha}1,3 Gal–positive extracellular matrix must demonstrate total removal of such, whether or not gluteraldehyde is used.18–20

The authors make an interesting observation about the selection of various test species for evaluating these xenogeneic phenomena. Importantly, their calcium content studies were highly sensitive and quantifiable assays to nonspecific inflammation wound healing responses and immune-mediated inflammation. A mammalian species that has high calcification rates to smaller degrees of inflammation is thus ideal to test biological valves for all the inflammatory pathways as a summation effect predicting fibrocalcific failure; short of neonatal humans, the juvenile sheep model has withstood the test of time and qualifies as a preclinical "must do" even if old-world monkey studies are contemplated.21,22 The authors correctly point out that a model testing valve and conduit performance must be in the presence of flowing blood rather than subcutaneous tissues.23 Confirming some of our own observations, these authors have demonstrated that in the conduit wall macrophages and T cells tend to arise from the adventitia, whereas leaflets are more exposed to the blood flow within the lumen where circulating cells may play more of a role.24–27

The perfect or ideal valve implantation should theoretically be the autograft pulmonary valve to the aortic position in the so-called Ross operation (Table 1). There should be no immune issues, and the implant itself is out of the body for minutes rather than hours or days. In my operating room, the autograft is placed in oxygenated blood from the cardiopulmonary bypass machine (ie, autologous blood) while it awaits implantation into the aortic root (typical duration, ≤5 minutes). It gives one pause when the few available explants of chronically implanted clinical autograft valves have been examined. In our experience, years after the surgery and contrary to expectations, these valves are not normal histologically. There tends to be hypocellularity of the leaflet and fibrous sheathing of leaflets and portions of the wall, similar to the consequences of minimal inflammatory responses noted by Manji and colleagues and to a far lesser degree but not unlike the sheathing of cryopreserved allograft valves that are clearly immune provocative. This suggests that the minimal stress of autotransplantion is enough to trigger non–immune-related cell death. Most likely, this is due to the necrosis of cells dependent on the nourishment of the vasa vasorum (arterial wall and base of leaflet). These may be a contribution by stress triggering of valve interstitial cell apoptosis (should be noninflammatory). Ischemic cell necrosis could lead to wound-healing inflammatory responses generating fibrous scar formation (eg, pseudointima, smooth muscle and fibroblast proliferation).


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  		TABLE 1. Features of the Ideal Valve Replacement: Avoid Inflammation Provoked by Either Materials or Implantation Techniques

Numerous explants of cryopreserved or fresh allograft tissues have demonstrated a similar sheathing process late but with a more severe loss of native cellularity that begins very early28,29 (Figures 1 and 2Down). Because this happens fairly rapidly with marked loss of cellularity within 30 days and usually complete acellularity by 9 months, it often has been questioned as to why allografts (or homografts as they are historically known) do as well as they do, lasting 2 to 4 years in neonates and infants and up to 25 years in adults while retaining the design advantages of a normal semilunar heart valve (Tables 2 and 3Down). Current theory, which is consistent with the findings of Manji and colleagues, is that processing should strip the endothelium from the allograft while leaving the matrix cells intact. These valve interstitial or matrix cells are primarily myofibroblasts and are buried with the collagen matrix. If minimal cells are exposed to the bloodstream, then there can be a blunted but not absent humoral and cellular rejection response. It also has been demonstrated that the cell death in such a scenario is primarily by apoptosis rather than cell necrosis. The former is by definition a noninflammatory process.30,31 If the presence of exposed antigenic cellular material (collagen and elastin and nonantigenic within species) is minimized and cell necrosis is minimized, then the inflammatory response should be limited primarily to a nonspecific wound healing analogous to the fibrous encapsulation of metallic hip implants and capsule formation around silicone and Silastic breast implants (Table 4). If the "processing" is less effective, then graft durability is likely reduced. Thus, theories for the relative clinical success of processed allografts and now proposed decellularized allografts depend on the consequences of blunted, reduced, or eliminated inflammation. In any case, the final common pathway for all inflammation is partially cell in-migration, fibrous isolation of the foreign body, scar formation, and ultimately fibrocalcification.


Figure 1176961
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  		Figure 1. A, Fresh ovine semilunar valve leaflet. Normal cellularity. B, Explanted ovine cryopreserved semilunar valve after 30 days in functioning pulmonary valve position. Note hypocellularity and diminishing trilayer definition. Some of the apparent viable cells are actually macrophages. Magnification x100. Hematoxylin and eosin. F indicates fibrosa layer; s, spongiosa layer; and v, ventricularis. Reproduced from Cardiac Reconstructions With Allograft Tissues32 with kind permission from Springer Science and Business Media. Copyright 2005.


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  		Figure 2. Explanted cryopreserved semilunar valve leaflet that at time of explant (1 year) was functioning very well by echocardiography despite virtual total acellularity and loss of trilayer structure but with a viable, flexible fibrous sheath on both inflow and outflow surfaces. In this case, the semilunar valve has functioned as mandrel or mold for in vivo reconstruction of a functioning fibrous semilunar valve. Magnification x100.


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  		TABLE 2. Allograft, Autograft, and Putative Tissue-Engineered Semilunar Valves


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  		TABLE 3. Current Clinical Cryopreserved Allograft Disadvantages


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  		TABLE 4. Homografts: Theory About Why They Do So Well

In general, the simplification that wound-healing infiltrates composed primarily of macrophages are salubrious whereas those containing leukocytes (primarily T cells) are bad may hold a grain of truth in that the latter implies a less benign inflammatory process driven by immune or other pathways, presaging destruction and failure. Perhaps the key to success for manufactured, "minimally" processed, or tissue-engineered valves, whether xenogeneic or synthetic options such as polymer or hybrid-based extracellular matrix designs, is to avoid inflammation while not sacrificing material properties required for safety, durability, and design-driven performance. Just as pre- Pasteur and -Lister surgeons would differentiate "laudable pus" from wound discharges presaging poor outcomes, there now is a tendency to seek "laudable inflammation" and rename the inflammatory cell infiltration process with more designer terms such as constructive remodeling or adaptive remodeling. Just as Hippocrates (circa 460 to 370 BC) taught (predicted) that all pus is bad, we should be similarly wary of all inflammation. Unmodified, all inflammation can lead to the final common pathway of fibrocalcific destruction. In the development of new cardiovascular cell, gene, and tissue therapies and devices, inflammation of all types and relative putative consequences must be careful sought with highly sensitive and predictive qualifying tests, including surgical implant studies, with carefully chosen species that punish even low levels of inflammation with calcification and thus are highly sensitive (eg, sheep and rats) and, if successful, then progress to species in which the immune responses are most similar to those of humans despite a slower calcification response (eg, old-world monkeys).


*    Acknowledgments
 
Disclosures

None.


*    Footnotes
 
The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.


*    References
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 *References
 

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