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Ann Thorac Surg 2001;71:S401-S405
© 2001 The Society of Thoracic Surgeons

Basic research

Calcification characteristics of porcine stented valves in a juvenile sheep model

Willem J. Flameng, MD, PhDa, Shigeyuki Ozaki, MDa, Jessa Ypermana, Paul Herijgers, MD, PhDa, Bart Meuris, MDa, Alfons Van Lommel, PhDb, Erik Verbeken, MD, PhDb

a Department of Cardiac Surgery, Katholieke Universiteit Leuven, Leuven, Belgium
b Department of Pathology, Katholieke Universiteit Leuven, Leuven, Belgium

Address reprint requests to Dr Flameng, C. E. H. A., Provisorium I, Minderbroedersstraat 17, B-3000 Leuven, Belgium
e-mail: willem.flameng{at}med.kuleuven.ac.be

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


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. Different antimineralization treatments of stented porcine bioprostheses were evaluated: ethanol (Epic), {alpha}-amino-oleic acid (AOA) (Mosaic), and sodium dodecyl sulfate (SDS) (Hancock II). A nontreated, glutaraldehyde-fixed valve (Labcor) served as control.

Methods. For each treatment, six valves were implanted in juvenile sheep in the pulmonary position. Valves were explanted after 3 and 6 months and examined macroscopically, by roentgenogram and light and transmission electron microscopy. Calcium content (µg/mg) was determined by atomic absorption spectrometry.

Results. The Labcor valves revealed small calcium deposits in the cusps, although calcium content remained low (median value 0.4 ± 0.8 µg/mg). SDS did not prevent cusp calcification as assessed by histology and calcium content measurement, which was higher than in all other valves: 1.9 ± 4.6 µg/mg (p < 0.05). Cusp retraction and rupture were occasionally found in the Hancock. The Mosaic and Epic valves showed no cusp calcification and had low calcium contents (0.3 ± 2.4 µg/mg and 0.7 ± 0.6 µg/mg, respectively). Epic showed less pannus formation, but had hematoma or iron staining in the cusps.

Conclusions. SDS is inefficient as an antimineralization treatment, in contrast to ethanol or AOA. Cusp hematoma after ethanol treatment needs further investigation.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
Tissue calcification is a common cause of clinical failure of stented glutaraldehyde-fixed porcine aortic bioprosthetic valves [1]. Several antimineralization treatments of glutaraldehyde-fixed valves have been developed and processed in porcine bioprostheses. These strategies include treatment of the fixed tissue with compounds such as sodium dodecyl sulfate (SDS) [2], {alpha}-amino-oleic acid (AOA) [3, 4], and ethanol [5, 6]. These substances have different properties: ethanol and SDS extract phospholipids from biological tissue, whereas AOA neutralizes free aldehyde moieties that remain present after glutaraldehyde fixation. All these procedures showed some benefit in terms of reduction of tissue calcification in several experimental models [2, 4, 5].

To compare the efficacy of these antimineralization treatments, we implanted glutaraldehyde-fixed, stented porcine aortic bioprosthetic valves in our juvenile sheep model [7, 8]. These valves were either SDS-, AOA- or ethanol-treated and were compared with a nontreated, glutaraldehyde-fixed valve.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
All animals were cared for by a veterinarian in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). The study was approved by the Ethics Committee of the Katholieke Universiteit Leuven. Young sheep bred especially for this purpose were selected.

Valves studied
Four types of stented porcine aortic valves were selected for this study: the Epic (St. Jude Medical, St. Paul, MN), the Mosaic (Medtronic, Minneapolis, MN), the Hancock II (Medtronic, Minneapolis, MN), and the Labcor (Sulzer Carbomedics, Austin, TX). These valves are all fixed by glutaraldehyde. The Epic (n = 7) is treated with ethanol and has a silver-coated fabric. The Mosaic (n = 6) is treated with AOA. The Hancock II (n = 6) has SDS as anticalcification treatment. The Labcor (n = 6) has no antimineralization treatment.

Implantation
All valves were implanted by previously described methods [7, 8]. Through a left thoracotomy at the second intercostal space, the main pulmonary artery was isolated, and a pneumatic right ventricular assist device (Medos-HIA VAD 54-mL ventricle, Medos-Helmholtz Institute, Aachen, Germany) was installed. After proximal and distal clamping of the pulmonary artery, the artery was opened and the stented valves were implanted using a continuous 4-0 polypropylene suture. After removal of the clamps, the native pulmonary valve was destroyed by tearing two cusps with a clamp introduced through a purse-string suture placed at the sinuses. The animals received analgesic, antibiotic, and diuretic agents as necessary. Low molecular weight heparin (enoxaparin sodium, 20 mg twice daily, Clexane, Rhone-Poulenc Rorer, Brussels, Belgium) was administered for 6 days. Afterwards, the sheep returned to the controlled animal facility where the general health of the sheep was checked daily.

Explantation and analysis
Three of the six valves in each series were explanted after 3 months, the remaining three after 6 months. Sheep were premedicated and anesthetized as described previously. The left thoracotomy was reopened and after heparinization and exsanguination, the valve was excised.

Gross examination
After gross inspection, the valve was transected longitudinally through the commissures. Each of the three resulting specimens thus included one complete cusp with a small part of porcine aortic wall and a pre- and postvalvular part of the sheep pulmonary artery.

Roentgenogram assessment
Roentgenogram examination was performed on every cusp in two directions under mammography conditions. The degree of calcification was scored semiquantitatively with three categories: 0 = no calcification, 1 = slight calcification, and 2 = severe calcification.

Histology
For histology, paraffin sections (4 µm thick) were routinely stained with hematoxylin and eosin, Masson’s trichrome stain for collagen, an elastic Von Giesson stain, a phosphotungstic-acid-hematoxylin for fibrin, and a Von Kossa calcium staining.

Transmission electron microscopy
From each valve, several small samples were taken and embedded in dow exposy resin. One-micrometer thick sections were stained with toluidine blue and examined by light microscopy. Ultrathin sections were cut, stained with uranylacetate and lead citrate. Sections were treated with 2% potassium pyroantimonate to demonstrate calcium. Grids were examined in a Philips CM10 electron microscope. Random photographs were taken. Afterwards, every photograph was scored in a similar semiquantitative manner as for the roentgenograms: 0 = no calcification, 1 = slight calcification, and 2 = severe calcification.

Quantitative calcium determination
The cusps were divided into three parts: the commissural area, basal part, and free edge. The aortic wall portion was also divided in two parts. This resulted in nine cusp samples and six aortic wall samples in every valve. After lyophilization, the tissue was pulverized and desiccated. Pulverized tissue was diluted in 20% hydrochloric acid at a ratio of 10 mg dried tissue/1 mL HCl. Calcium content was measured by flame atomic absorption spectrometry, and expressed as microgram per milligram of dry weight.

Data management and statistical analysis
Normal probability plots and the Shapiro–Wilks test for normality showed that the calcium content data had a nonnormal distribution. Consequently, data were expressed as median ± interquartile range. The Kruskal–Wallis analysis of variance test was used for comparison between the four groups. When the Kruskal–Wallis test yielded a p value less than 0.05, pairwise comparison between groups was performed using the Mann–Whitney U test, with Bonferroni correction.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Gross examination
In the Epic group, 1 animal was lost at 2 months after implantation because of severe endocarditis. This animal had to be replaced by a new implantation. In all other valves, no infection occurred and valve thrombosis was never found. One of the Hancock valves showed a small cusp rupture.

Pannus formation was seen predominantly in the Labcor (5 of 6 animals) and the Hancock (4 of 6). It was less frequent in the Mosaic (3 of 6) and rare in the Epic valves (1 of 6). This neointima formation induced cusp retraction mainly in the Hancock (4 of 6). Cusp retraction was found in two of six valves in the Labcor and Mosaic. The results are summarized in Table 1.


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  		Table 1. Macro- and Microscopical Findings in Explanted Stented Bioprostheses

 
Roentgenogram examination
Calcification on roentgenogram was mainly seen in both the Hancock and Labcor valves. The other valves showed only minimal radiographic signs of calcification. The result of the semiquantitative roentgenogram analysis is given in Figure 2.



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  		Fig 2. Semiquantitative score for the roentgenogram (A) and the electron microscopy (B) analyses. ({square} = proportion of roentgenogram or electron microscopic photographs with no calcification (score 0), = proportion with slight calcification (score 1), {blacksquare} = proportion with severe calcification (score 2).)

 
Light microscopy
Microscopically, calcification of the cusp or the aortic wall portion was demonstrated in all Labcor and Hancock valves. Cuspal calcification in these valves was present in the form of small, diffuse calcific deposits. Large accumulations of calcium in the cusp were never seen. The wall portion showed calcium aggregation in the tunica media. In the Mosaic valves, calcification was found only occasionally. Only two of six Mosaic valves showed a small calcium deposit in the cusps. The Epic showed no calcium deposition at all.

Both Labcor and Hancock showed significant neointima formation, which sometimes completely covered the cusp and caused cusp retraction. Foreign body reaction with accumulation of giant cells near the wall portion was also present in these valve types. Neointima formation was also found in the Mosaic valves, but to a lesser extent (Fig 1). The layer of fibrous tissue reached the base of the leaflets, but almost never extended further toward the free edge of the cusp.



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  		Fig 1. (A) Perls staining for iron in a cusp of an explanted Epic valve: cuspal hematoma (group of red blood cells) with positive iron staining (black dots) in iron macrophages (x200 before 25% reduction). (B) Hematoxylin-eosin staining of an explanted Mosaic with neointima covering the base of the cusp (x12.5 before 25% reduction); (C) Electron microscopy of an explanted Labcor cusp: visible calcification (black dots) between an ultrastructurally preserved environment (x24,400 before 25% reduction).

 
Neointima formation was practically absent in the Epic valves. These valves, however, frequently showed iron deposits in the leaflets. A fresh hematoma was found in the cusp of one Epic valve. A sample of the histologic image is shown in Figure 1. The iron staining is most likely the result of resorbed hemoglobin, coming from intracuspal red blood cells from a small cusp hematoma.

Transmission electron microscopy
In all valves, the main ultrastructural tissue pattern was well preserved. In all valves, except for the Epic valve, small dots of calcium deposits were found in the cusps, both extracellularly spread between collagen fibers and intracellularly in cell remnants. These calcifications were variable in degree and incidence. The result of the semiquantitative analysis of cusp calcification is shown in Figure 2. Cusp calcification was highest in the Hancock and Labcor valves, and low in the Mosaic and Epic valves.

Calcium content
When the results of calcium content were compared between valves explanted at 3 months and those explanted at 6 months, no statistically significant difference was found. Therefore, the data of the 3- and 6-month explants were pooled in every group. The results are listed in Table 2. The calcium content in the cusps of all studied valves were low, except for the Hancock valve. In this valve, calcium content in the cusps was 1.9 ± 4.6 µg/mg (median value ± interquartile range), which is significantly higher (p < 0.05) than in all other valves. Calcium content in the aortic wall portion was also significantly higher (p < 0.05) in the Hancock valve, compared with all other valve types.


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  		Table 2. Calcium Content (µg/mg) in the Cusps and Aortic Wall Portion of Stented Bioprostheses

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
In stented porcine aortic bioprostheses, a small portion of the aortic wall, contiguous with the cusps, is trimmed and incorporated into the prosthesis. This aortic wall portion is known to calcify after implantation of the valve and many antimineralization treatments are obviously unable to prevent this calcification process [9]. However, in contrast to stentless valves, stented valves have only a small portion of aortic wall, located close to the stents, so that stiffening is not expected to change the hemodynamics of the valve. Furthermore, it is known in human explants that calcification of the aortic wall portion of stented porcine aortic bioprostheses is less than that of the corresponding cusps [10]. Therefore, anticalcification approaches need not be effective beyond the cusps of the stented valves. This fact is why we focused our analysis of calcification characteristics of the four stented valves on changes occurring predominantly in the cusps.

In a glutaraldehyde-fixed, nontreated valve (Labcor), we found relatively low calcium contents in the cusps. However, cuspal calcification was clearly seen in light microscopy, but only in small deposits. Also, transmission electron microscopy clearly revealed the presence of calcium aggregates in the cusp. This contradiction between histologic findings and calcium content in the Labcor can be related to sampling error because cusp calcification is never homogeneous.

Although roentgenogram assessment and light and electron microscopy revealed cusp calcification in the Labcor valve, the extent of calcification was still less than that found in the Hancock. This valve underwent a treatment with SDS to inhibit mineralization. Although this treatment, based on phospholipid extraction, has been shown to inhibit calcification in experimental animals, it was not efficient in our juvenile sheep model. Cusp calcification was clearly demonstrated by roentgenogram, light and electron microscopy, and atomic absorption spectrometry. In our case, also a small cusp rupture was found. In a recent clinical study on explants of the Hancock valve, it was shown that tears of the cusps (possibly caused by collagen disruption) and cusp calcification are the most common causes of valve failure in this stented valve [11].

{alpha}-Amino-oleic acid is known to inhibit calcification of glutaraldehyde-pretreated porcine aortic valve cusps, but not the aortic wall portion of bioprosthetic valves [4]. In our study, we found that AOA is much more efficient as an antimineralization agent than SDS. Cusp calcification in the Mosaic valves could be shown only by transmission electron microscopy and was only rarely found in the light microscopic sections of the cusps.

The Labcor, Hancock, and, to a certain extent, the Mosaic valves, showed neointima formation. This finding was always pronounced at the aortic wall portion and at the base of the leaflets, and extended sometimes to the free edges of the cusps. These valves also showed a clear foreign body reaction in the structures surrounding the stent and sewing ring. Both phenomena were rarely found in the ethanol-treated Epic valve. The exact reason for this lack of chronic inflammatory reaction and reduced neointima formation is not known. It cannot be excluded that the incorporation of silver in the polyester fabric plays a certain role.

Cusp calcification was clearly inhibited by the ethanol treatment and confirms results from previous experiments [5]. This inhibition of calcification of valvular tissue is related to selective lipid removal from the tissue [12]. In our study, light and electron microscopy as well as roentgenogram examination and calcium content determination all demonstrated almost optimal prevention of valve mineralization in the Epic. However, the Epic showed consistent iron deposits and small hematomas in the cusps. The iron deposits are most likely the remnants of a previous intracuspal bleeding. Cuspal hematomas were also described after AOA treatment [3, 4] and may result from small tears in the surface related to either unsoluble AOA crystals [4] or lipid extraction [12]. This feature needs further evaluation.

In conclusion, these results show that by using the juvenile sheep model, different antimineralization treatments can be compared and their efficiency determined.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

  1. Schoen F.J., Levy R.J. Tissue heart valves: current challenges and future research perspectives. J Biomed Mater Res 1999;47:439-465.[Medline]
  2. Hirsh D., Drader J., Thomas T.J., Schoen F.J., Levy J.T., Levy R.J. Inhibition of calcification of glutaraldehyde pretreated porcine aortic valve cusps with sodium dodecyl sulfate. J Biomed Mater Res 1993;27:1477-1484.[Medline]
  3. Gott J.P., Pan-Chih, Dorsey L.M., et al. Calcification of porcine valves: a successful new method of antimineralization. Ann Thorac Surg 1992;53:207-216.
  4. Gott J.P., Girardot M.N., Girardot J.M., et al. Refinement of alpha amino-oleic acid bioprosthetic valve anticalcification technique. Ann Thorac Surg 1997;64:50-58.[Abstract/Free Full Text]
  5. Vyavahare N., Hirsch D., Lerner E., et al. Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Circulation 1997;95:479-488.[Abstract/Free Full Text]
  6. Langanki D., Ogle M.F., Cameron J.D., et al. Evaluation of a novel bioprosthetic heart valve incorporating anticalcification and antimicrobial technology in a sheep model. J Heart Valve Dis 1998;7:633-638.[Medline]
  7. 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]
  8. Ozaki S., Herijgers P., Verbeken E., et al. The influence of stenting on the behaviour of amino-oleic acid treated, glutaraldehyde fixed porcine aortic valves in a sheep model. J Heart Valve Dis 2000;9:552-560.[Medline]
  9. Girardot M.N., Torrianni M., Girardot J.M. Effect of AOA on glutaraldehyde-fixed bioprosthetic heart valve cusps and walls: binding and calcification studies. Int J Artif Organs 1994;17:76-82.[Medline]
  10. Biedrzycki L.M., Lerner E., Levy R.J., Schoen F.J. Differential calcification of cusps and aortic wall of failed stented porcine bioprosthetic valves. J Biomed Mater Res 1997;34:411-415.[Medline]
  11. Butany J., Yu W., Silver M.D., David T.E. Morphologic findings in explanted Hancock II porcine bioprostheses. J Heart Valve Dis 1999;8:4-15.[Medline]
  12. Jorge-Herrero E., Fernandez P., de la Torre N., et al. Inhibition of the calcification of porcine valve tissue by selective lipid removal. Biomat 1994;15:815-820.



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