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Ann Thorac Surg 2001;71:S422-S427
© 2001 The Society of Thoracic Surgeons

Basic research

Stentless bioprosthetic heart valve research: sheep versus primate model

Ameli Trantina-Yates, MDa, Christoph Weissenstein, MDa, Paul Human, MSca, Peter Zilla, MD, PhDa

a Department of Cardiothoracic Surgery, Cape Heart Centre, University of Cape Town Medical School, Cape Town, South Africa

Address reprint requests to Dr Zilla, Cape Heart Centre, Faculty of Health Sciences, University of Cape Town, 7925 Observatory, Cape Town, South Africa
e-mail: ctszilla{at}samiot.uct.ac.za

Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Background. The mild inflammatory response against stented bioprosthetic heart valves in the sheep model is often opposed by a more distinct response in failing human implants. With the emergence of stentless root prostheses with their significantly larger proportion of tissue interacting with the immune system of the host, a more relevant animal model than the sheep may be needed.

Methods. Valved, porcine aortic roots of 5 cm length were fixed in 0.2% glutaraldehyde and implanted in the upper descending aorta of Merino sheep (n = 5; 43 ± 3 kg) and Chacma baboons (n = 5; 17 ± 3 kg). After 6 weeks of tissue calcification, pannus outgrowth and inflammation were assessed by atomic absorption spectrophotometry, histologic damage scoring (0 to 3), image analysis, and transmission electron microscopy.

Results. The main difference between the two animal models was in aortic wall calcification (64.8 ± 39.8 µg/mg in the sheep model versus 4.1 ± 5.9 µg/mg in the primate model; p > 0.005). In both models, leaflet calcification was negligible (2.6 ± 2.4 µg/mg in the sheep versus 2.5 ± 1.9 µg/mg in the primate), and the overall extent of inflammation was comparable (1.2 ± 0.8 versus 0.98 ± 0.7; p = 0.18 in the sheep and the primate, respectively). Qualitatively, the sheep demonstrated a macrophage-dominated reaction whereas the inflammatory demarcation often resembled a granulocyte-dominated xenograft response in the primate. Pannus outgrowth was comparable in length (8.4 ± 2.3 mm versus 9.1 ± 4.3 mm proximally and 7.1 ± 3.4 mm versus 7.4 ± 5.1 mm distally, in the sheep and baboon, respectively; p > 0.05).

Conclusions. Our results confirm the sheep as a significantly stronger calcification model for stentless aortic heart valves than the primate. Remaining antigenicity of porcine tissue as a result of incomplete cross-linking, however, elicits a distinctly stronger xenograft-type reaction in the primate model.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
The replacement of diseased heart valves with nonstented xenografts is a relatively recent development of the past decade. Apart from introducing a novel stress-dynamic concept promising extended longevity of the implant, a new dimension of interaction between graft and host tissue was broached. Other than the delicate leaflets of stented valves, which are only exposed to the blood, significantly more bioprosthetic material is interacting with both the blood and the surrounding host tissue. This abundance of bioprosthetic tissue is expected to naturally augment the overall calcification of such prostheses.

In the past the process of calcific degeneration was primarily seen as a result of passive chemical events initiated by the breakdown of the transmembrane calcium gradient of graft cells at the time of fixation and the presence of nucleation sites in the form of membrane phospholipids. Today, a much more complex picture emerges. While the almost identical calcification patterns of glutaraldehyde-fixed biologic root prostheses and homografts [1] have shifted the focus away from the long-standing villain role of the dialdehyde, the contribution of immune and inflammatory processes to tissue mineralization seems significantly higher than initially thought [2, 3].

Stentless heart valves with their distinct proportion of tissue exposed to the host may augment immune interactions between the host and the prosthesis, and among other consequences, one may see a hitherto unknown degree of tissue inflammation. For conclusively answering this question, however, too few clinical specimens have been retrieved as yet, and experimental work has primarily focused on calcification [4–7]. This shortcoming, together with the high proportion of patients already receiving stentless bioprosthetic heart valves, underlines the need for a relevant animal model.

The aim of the present study was to reassess the sheep model in its role as the gold standard of circulatory models. To address the rising awareness regarding the role of immune mechanisms, it seemed appropriate to compare it with a nonhuman primate model. A 6-week implantation period was chosen as a compromise when tissue calcification is already distinct [8] but inflammation not burned out yet [9].


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Tissue preparation and fixation
In accordance with common commercial fixation methods for stentless valves, porcine aortic roots were collected from the abattoir and kept at 4°C for 48 hours (phosphate-buffered saline, 0.1 mol/L; pH 7.4) before they were fixed in 0.2% glutaraldehyde (Saarchem electron microscope grade) for 1 week (15 g tissue/100 mL glutaraldehyde in phosphate-buffered saline; 4°C). After rinsing in high-volume phosphate-buffered saline (15 g tissue/100 mL) for 24 hours at 37°C, roots were stored in low-volume phosphate-buffered saline (800 µL/g tissue; 4°C) until implantation. The maximal length of storage was 7 days.

Implantation
All experimental procedures were approved by the Animal Research and Ethics Committee of the University of Cape Town and complied with the "Principles of Laboratory Care" and the "Guidelines for the Care and Use of Laboratory Animals" (National Institutes of Health publication 86-23).

Valves were inserted in the lower aortic arch of 5 juvenile Merino sheep (43 ± 3.25 kg) and 5 young Chacma baboons (16.6 ± 2.54 kg). Surgical access was by means of a left lateral thoracotomy through the fifth intercostal space under aseptic conditions. Implantations were performed under Gott-shunt protection. After 10 days the sheep were brought back to the farm where they were kept until tissue retrieval. Baboons were transferred to a large-cage area. Antibiotics (cephalexin, 30 mg/kg) were given intramuscularly for 3 days.

Tissue retrieval and sample processing
After 6 weeks of implantation, animals were euthanized, and the porcine roots together with parts of the adjacent host aorta were excised. The average pannus length was assessed from digital macro pictures using image analysis (Leica Q Win Pro, Leica Microsystems Imaging Solutions, Cambridge, UK). Leaflet shrinkage was determined by measuring the distance between the nodulus of Arantii and the nadir of the leaflet. Longitudinal sections of the entire root were stained with hematoxylin-eosin and Ham 56 or CD 66 (DAKO, Glostrup, Denmark; baboons) or hematoxylin-eosin and GSL-1 (Vector Laboratories, Burlingame, CA; sheep). The degree of inflammation was assessed using a scoring system from 0 to 3. This system ignored the cellular characteristics of inflammation, which were separately determined independent of the scoring. For transmission electron microscopy, samples were processed in standard fashion and analyzed in a JEM-100 S transmission electron microscope (Jeol, Tokyo, Japan).

Calcium analysis
Tissue calcium was measured quantitatively and assessed morphologically. For quantitative analysis, atomic absorption spectroscopy was used as previously described in detail [6]. Morphologic assessment was based on von Kossa stains.

Data analysis
Pannus length, leaflet shrinkage, and calcium data were expressed as mean ± standard deviation. Statistical analysis used the one-tailed Student’s t test with two-sample equal variance. The level of statistical significance was set at p less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
All sheep explants were hard and rigid, whereas those from baboons were almost as pliable as at the time of implantation. Leaflet shrinkage was distinct in both models (from 9.82 ± 0.004 mm at preimplantation to 4.04 ± 0.06 in the baboon [p < 0.0001] and 4.60 ± 0.05 in the sheep [p < 0.0001]). There was no significant difference between the animal models (p = 0.091).

Pannus outgrowth was comparable in both the baboon and the sheep (9.1 ± 4.3 mm versus 8.4 ± 2.3 mm proximally [p > 0.05] and 7.4 ± 5.1 mm versus 7.1 ± 3.4 mm distally [p > 0.05]).

Calcium analysis
A high level of calcification was found in the aortic wall of sheep (64.8 ± 39.8 µg/mg) as opposed to hardly any calcification in the baboon model (4.1 ± 5.9 µg/mg; p < 0.005; Fig 1). Calcification was practically absent in the leaflets of both the sheep (2.6 ± 2.4 µg/mg) and the baboon (2.5 ± 1.9 µg/mg) models.



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  		Fig 1. Calcium content in porcine aortic wall and leaflet tissue after 6 weeks of implantation in the lower aortic arch. Although aortic wall calcification showed a distinctly higher value in the sheep model, leaflet mineralization was negligible in both animal models.

 
Von Kossa stains showed two condensed mineralization zones close to the blood and adventitial surface in the aortic wall of sheep. The medial tissue layer between the two calcification zones contained fine granular calcium particles, which tapered down toward the center of the media. By contrast, no mineralization was seen in the aorta of the baboons. Leaflets were free of visible calcification in both animal models.

Histology
Histologically, a distinct degree of inflammatory demarcation and surface erosion was found in both animal models (Fig 2). Quantitatively, there was no difference in the inflammation, as reflected in the overall mean inflammatory score (1.2 ± 0.8 versus 0.98 ± 0.7 in the sheep and baboon, respectively; p = 0.18). Qualitatively, the sheep showed more the picture of a foreign body reaction to the implant, whereas in the primate, features of acute xenograft rejection were often found.



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  		Fig 2. Distinct inflammatory infiltration of the leaflets in both the baboon model and the sheep model. The leaflet infiltration in the baboon is dominated by granulocytes (A; CD 66, x200), which are sometimes aggressively penetrating into the adventitia (C; hematoxylin-eosin, x100). In the sheep model, macrophages penetrate deep into the fibrosa (B; GSL-1, x400), whereas the adventitial response resembles more a foreign body giant cell reaction (D; hematoxylin-eosin, x100).

 
On the adventitia side of the roots, the inflammation showed two distinct inflammation zones in the primate. The inner third was mostly dominated by polymorphonuclear granulocytes, often aggressively penetrating between the tissue planes of the prosthetic wall, whereas the outer two thirds of the inflammatory front consisted primarily of macrophages and lymphocytes (Fig 2C). The possibility of infection could be excluded on clinical grounds and on the basis of Gram stains of tissue sections. In the sheep model, adventitial inflammation was only vaguely double-layered (Fig 2D). A rim of foreign body giant cells and macrophages mildly eroded into the prosthesis whereas lymphatic follicles were occasionally found at the interface to the host tissue.



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  		Fig 3. (A) Typical polymorphonuclear granulocyte infiltration into the leaflet surface of the baboon (x6,000). (B) Macrophage phagocytosing collagen in the leaflet of the sheep model (x10,000). (C) Typical calcification in the membrane of aortic wall smooth muscle cell in the sheep (x6,000). (D) Nuclear calcification in the porcine aortic wall in the sheep model (x6,000).

 
On the blood surface, inflammation was most pronounced on the fibrosa of leaflets and underneath pannus tissue in both animal models. Particularly in the sheep, macrophages lined long stretches of the surface of the fibrosa, often superficially eroding into the tissue (Fig 2B). In the baboon, surface inflammation of the fibrosa did not differ much in its macrophage dominance but showed nests of aggressively infiltrating polymorphonuclear granulocytes (Fig 2A), particularly if sheltered from the bloodstream by small thrombi. In both models only scanty macrophages had penetrated the spongiosa. The ventricularis was least affected by inflammatory cells except for areas close to the leaflet base, which were covered by pannus tissue. This pannus-protected inflammation was again distinctly different in the sheep and the primate model: whereas foreign body giant cells and macrophages demarcated the pannus from the prosthetic tissue in the sheep model, macrophage-dominated areas alternated with zones of aggressively infiltrating polymorphonuclear granulocytes in the baboon. Overall, the inflammatory reaction within and underneath the pannus tissue showed a broad range from very mild to severe. In both models, the proximal and the distal pannus were only partly covered by an endothelium.

Transmission electron microscopy
Ultrastructurally, blood surfaces were distinctly eroded in both animal models. Plasma, platelets, and white blood cells were found penetrating deep between the rugged formations of degraded extracellular matrix and poorly preserved graft cells (Fig 3). Collagen often appeared fragmented and loose compared with the well-structured formation in the control leaflet. In the sheep model macrophages were sometimes found to phagocytose the collagen (Fig 3B) whereas clusters of polymorphonuclear granulocytes were occasionally found in the primate (Fig 3A). While leaflet tissue hardly showed ultrastructural signs of calcification, smooth muscle cells of the aortic wall of sheep implants showed both membrane and nuclear calcification (Fig 3C, D).


   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
For decades the sheep served as the gold standard of circulatory models in bioprosthetic heart valve research. Valves were stent-mounted, and the almost exclusive measurement of these studies was leaflet calcification. With the focus on mineralization, an entire era was preoccupied with antimineralization treatments. This type of univariant comparative study relied on the tandem of subcutaneous rat implants and circulatory sheep implants. With the advent of stentless xenograft heart valves, however, discrepancies began to arise between the two models. The dramatically reduced leaflet calcification found in stentless prostheses [5], for instance, as opposed to subcutaneous implants [4] or stented valves suggests that mechanical stress or the free communication of the cut spongiosa with the surrounding blood or tissue fluid may have been grossly underestimated in previous concepts. Our present study confirms this lack of calcification in leaflets of stentless valves—an observation that coincides with previous experience in allograft heart valves. In contrast, aortic wall calcification of porcine root prostheses was high in our sheep implants, again corresponding with the calcification pattern of homografts. Because homografts were never cross-linked with glutaraldehyde, a revised hypothesis for bioprosthetic heart valve degeneration seemed necessary, going beyond the vilification of glutaraldehyde. In the past 5 years, such a revised hypothesis reemerged on the forgotten ground of immune mechanisms. In the second half of the 1990s, an increasing number of studies provided evidence that contemporary heart valve prostheses are insufficiently cross-linked [2, 6–8] and therefore elicit an immune reaction [2, 6–8] that eventually facilitates degeneration [2, 6–8]. Together with the underappreciated fact that almost half of bioprostheses fail because of noncalcified degeneration, a new emphasis was placed on inflammatory and immune processes. In view of the manifold larger prosthetic tissue portions of stentless valves that interact with the host, it seemed justified to take inflammatory aspects into account when optimizing the animal model. Because an immune response was recently implied in both the inflammatory [10] and the calcific degeneration [2, 3] of bioprosthetic heart valves, the primate—which is immunologically closest to human—offered itself as a natural model.

When comparing the primate model with the sheep model, a foreign-body type inflammatory reaction associated with significant wall calcification was seen in the sheep model whereas a polymorphonuclear granulocyte-dominated inflammatory response with hardly any concomitant calcification dominated the baboon model. The combination of a mild xenograft-like rejection pattern with a lack of calcification in the primate model resembles the well-known phenomenon that unfixed bioprosthetic material generally gets resorbed rather than calcifies [11]. In view of the fact that contemporary bioprosthetic tissue is at least mildly cross-linked, such a response pattern indicates a very sensitive immune recognition of insufficiently cross-linked tissue in the primate model. Held against the distinct aortic wall calcification in the sheep model and the mild xenograft response in the baboon model, the clinical situation of stentless bioprosthetic heart valves seems to lie somewhere in between. On the one hand, stentless prostheses develop distinct aortic wall calcification at a relatively early stage, which would make the sheep model clearly the more relevant one. On the other hand, calcifying leaflets do not play the same role any longer that they used to play in stented valves. With the most dreaded failure mode of yesterday seemingly under control, inflammatory degeneration and immune responses may play a more significant role than previously perceived. In view of the increasing insight that bioprosthetic heart valves are under-cross-linked [2, 6–8], the baboon offers a sensitive detection model for discovering insufficiently capped antigens in alternative fixation. For calcific degeneration, however, our present study confirmed the sheep as a highly relevant model for stentless heart valve research.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
We thank Jenny Molde, Nazlia Samodien, and Phillip Christopher for their kind assistance with the microscopy slides and the image analysis, and the Animal Unit of the University of Cape Town for the care of the animals.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Cleveland D., Williams W., Razzouk A., et al. Failure of cryopreserved homograft valved conduits in the pulmonary circulation. Circulation 1992;86(Suppl 2):II150-II153.
  2. Human P, Zilla P. Inflammatory and immune processes: the neglected villain of bioprosthetic degeneration? J Long-Term Effects Med Implants 2001; in press.
  3. Vincentelli A., Latremouille C., Zegdi R., et al. Does glutaraldehyde induce calcification of bioprosthetic tissues?. Ann Thorac Surg 1998;66:S255-S258.
  4. Girardot M., Torrianni M., Dillehay D., Girardot J. Role of glutaraldehyde in calcification of porcine heart valves: comparing cusp and wall. J Biomed Mater Res 1995;29:793-801.[Medline]
  5. Herijgers P., Ozaki S., Verbeken E., et al. Calcification characteristics of porcine stentless valves in juvenile sheep. Eur J Cardiothorac Surg 1999;15:134-142.[Abstract/Free Full Text]
  6. Zilla P., Weissenstein C., Bracher M., et al. High glutaraldehyde concentrations reduce rather than increase the calcification of aortic wall tissue. J Heart Valve Dis 1997;6:502-509.[Medline]
  7. Weissenstein C., Human P., Bezuidenhout D., Zilla P. Glutaraldehyde detoxification in addition to enhanced amine cross-linking dramatically reduces bioprosthetic tissue calcification in the rat model. J Heart Valve Dis 2000;9:230-240.[Medline]
  8. Zilla P., Weissenstein C., Human P., Dower T., von Oppell U. High glutaraldehyde concentrations mitigate bioprosthetic root calcification in the sheep model. Ann Thorac Surg 2000;70:2091-2095.[Abstract/Free Full Text]
  9. Wilson G., Courtman D., Klement P., Lee J., Yeger H. Acellular matrix: a biomaterials approach for coronary artery bypass and heart valve replacement. Ann Thorac Surg 1995;60:S353-S358.
  10. Human P., Zilla P. Characterization of the immune response to valve bioprostheses and its role in primary tissue failure. Ann Thorac Surg 2001;71:S385-S388.[Abstract/Free Full Text]
  11. Vesely I., Noseworthy R., Pringle G. The hybrid xenograft/autograft bioprosthetic heart valve: in vivo evaluation of tissue extraction. Ann Thorac Surg 1995;60:S359-S364.



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