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Ann Thorac Surg 2001;71:S417-S421
© 2001 The Society of Thoracic Surgeons

Basic research

Tissue characterization and calcification potential of commercial bioprosthetic heart valves

Crystal M. Cunanan, MSa, Christine M. Cabiling, BSa, Tan T. Dinh, BSa, ShihHwa Shen, BSa, Phihoa Tran-Hata, BSa, James H. Rutledge, III, PhDa, Michael C. Fishbein, MDb

a Heart Valve Therapy Research, Edwards Lifesciences, Irvine, California, USA
b UCLA Center for Health Sciences, Department of Pathology and Laboratory Medicine, Los Angeles, California, USA

Address reprint requests to Ms Cunanan, Edwards Lifesciences, Heart Valve Therapy Research, One Edwards Way, Irvine, CA 92614
e-mail: crystal_cunanan{at}edwards.com

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


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. Tissue properties may contribute to intrinsic calcification of bioprosthetic heart valves. Phospholipids have been proposed as potential nucleation sites for calcification. Other tissue properties might also be important in calcification.

Methods. Commercial and control bioprosthetic valve tissues were characterized by shrinkage temperature, moisture content, free amine content, phospholipid content, and calcification level after 90-day rat subcutaneous implantation as described.

Results. Shrinkage temperature, moisture content, and free amine content were typical for glutaraldehyde–cross-linked tissues. Phospholipid and calcium levels varied considerably among valve types. There was a significant correlation between phospholipid levels and calcification (r = 0.63, p = 0.04). Sulzer Carbomedics Mitroflow and Toronto SPV valve tissues had significantly more calcification than other commercial bioprostheses in this study (p < 0.01). Carpentier-Edwards Duraflex, CE SAV, and CE PERIMOUNT valve tissues had significantly less calcification than Medtronic Mosaic in this animal model (p < 0.02).

Conclusions. Processes that reduce phospholipid levels are associated with reduced calcification in the rat subcutaneous model. Significant differences in calcification level were found among commercially available valves. The clinical significance of these results is unknown.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
Calcification is an important factor in clinical dysfunction of bioprosthetic heart valves [1]. Morphologic examination of explanted bioprostheses indicates that calcification is frequently associated with collagen fibrils, organelles such as mitochondria and nuclei of devitalized cells (including connective tissue cells of the leaflets, and cells of the muscle shelf, and aortic wall of porcine aortic valves), thrombi, and vegetations [1–3].

The onset of calcification has been postulated to originate from an electrostatic attraction between the acid phospholipids of the connective tissue and calcium [4]. In this study we defined and measured some basic properties of commercial bioprosthetic tissues and examined their potential role in calcification.


   Material and methods
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Forty-eight commercial bioprosthetic valves of eight different types were obtained from suppliers. Six Carpentier-Edwards Duraflex, three SAV, and six PERIMOUNT valves were obtained from Edwards Lifesciences. Seven Mosaic, nine Freestyle, and eight Hancock II valves were obtained from Medtronic. Eight Toronto SPV valves were obtained from St. Jude Medical. One Mitroflow Model 12 valve was obtained from Sulzer Carbomedics. Control pericardial tissues were prepared by fixation and storage in 0.625% wt/vol glutaraldehyde solution only. Fresh porcine leaflet and pericardial tissues were tested as received from approved slaughterhouses and represented commercial-quality tissue. Tissue samples were excised using aseptic technique and tested as outlined below.

Tissue characterization techniques
Shrinkage temperature
Porcine leaflet and pericardial tissue strips measuring 5 mm x 15 mm were excised and loaded into the fixture. The fixture was lowered into a bath, and the solution was heated at 0.8°C/min while a strain gauge measured the strain in the tissue. The shrinkage temperature was taken as the temperature at which the tissue length shrank by 1%.

Moisture content
Porcine leaflet and pericardial tissues were weighed, then lyophilized to dryness and reweighed to calculate moisture content.

Free amine content
Porcine leaflet and pericardial tissues were analyzed using a modification of the ninhydrin method. Briefly, tissues were incubated with a colorimetric reagent that reacts with free {alpha}-amines to form a purple complex. The complex was detected using standard spectroscopy methods and quantitated using a standard curve of N-{alpha}-acetyl lysine. Values are reported as nanomoles free amine per milligram dry tissue weight.

Phospholipid content
After the above characterization tests were conducted and implant samples were prepared, the remaining tissues were pooled in groups of three to five valves per group, and the phospholipids were extracted in chloroform-methanol 2:1 (vol/vol), separated using thin layer chromatography techniques [5], detected using primulin dye, and quantitated after scanning the plate using purified phospholipid standards (Avanti Polar Lipids, Alabaster, AL). Image analysis techniques (ImageQuant, Molecular Dynamics, Sunnyvale, CA) were used to quantitate the levels of sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidic acid. The average value from duplicate runs was used for each sample. These results were then summed to provide a total quantity of phospholipids for each valve type. Phospholipid levels are reported as microgram phospholipid per milligram dry tissue weight.

Only the phospholipid assay was performed on the Mitroflow tissue because of limited sample.

Calcification assay
Rat subcutaneous implantation
All implantation studies were conducted in compliance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals." Eight-millimeter disks prepared aseptically were rinsed according to manufacturer’s recommendations and implanted into the subcutaneous pocket of male Sprague Dawley rats, 21 to 28 days old, for 90 days. A total of 262 implants was performed.

Calcium content
Samples were retrieved and analyzed for calcium content using standard atomic absorption spectroscopy methods. Briefly, disks were removed from host tissue, filmed with x-rays, then hydrolyzed in 70% nitric acid. Samples were analyzed using a Varian 200 AAS Spectrometer (Varian Instruments, Walnut Creek, CA) and quantitated using calcium standards. Results are reported as microgram calcium per milligram dry tissue weight. The nonparametric Mann-Whitney U—Wilcoxon rank sum test was used to determine any significant differences among valve types. The relationship between phospholipid levels and calcium content was also examined using nonparametric methods (Spearman’s correlation).

Histology
Representative samples were taken from each valve type while still contained in host tissue and processed using paraffin-embedding techniques. Slides were stained with hematoxylin and eosin, trichrome, and von Kossa stains. In each specimen, both the implant tissue and the surrounding host tissue were evaluated and scored for signs of inflammation (acute, chronic, and granulomatous), granulation tissue, scar tissue, hemorrhage, and calcium. The implanted tissues were also examined for signs of collagen degeneration. In all cases, a 0 to +3 system was used. Nonimplanted tissues were also evaluated. Mitroflow tissue was not examined using this technique.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Tissue characterization results
Table 1 contains the overall average and standard deviation for each of the valve types for shrinkage temperature, moisture content, and free amine content. These results indicate all valves tested were well cross-linked, yielding values typical of glutaraldehyde-treated tissues. Only moisture content differed significantly among the valves, with the moisture content of the porcine leaflet tissues being approximately 92% and the bovine pericardium, approximately 79%. This difference reflects differences in the tissues rather than any effects of processing.


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  		Table 1. Shrinkage Temperature, Moisture Content, and Free Amine Content for Valve Tissues

 
Table 2 lists the phospholipids measured in each valve type. Because phospholipids are a minor component in tissue (< 3% in porcine leaflet and < 1% in bovine pericardium), the tissues remaining after conducting the other tests had to be pooled into only two samples per valve type. The average of these two pooled samples is reported in Table 2, and no statistical comparisons are possible.


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  		Table 2. Phospholipid Content of Bioprosthetic Tissues

 
Examination of the data in Table 2 reveals marked differences between porcine and pericardial tissues as well as among different processes. Fresh porcine leaflet tissue contains three times more phospholipids compared with fresh bovine pericardial tissues. The distribution of phospholipids also differed between porcine leaflet tissues and bovine pericardium. Finally, fresh pericardium contains detectable levels of phosphatidic acid whereas porcine leaflet tissues contain only trace amounts of phosphatidic acid.

All commercial processes reduced the phospholipid levels in the tissues compared with fresh tissue, with the lowest value of total phospholipids contained in the CE PERIMOUNT tissue (0.25 µg/mg) and the highest level of phospholipids found in the Toronto SPV leaflet tissue (7.53 µg/mg). Among the porcine leaflet tissues, the CE SAV, Mosaic, and CE Duraflex valves had the lowest phospholipid levels (0.87, 1.78, and 1.88 µg/mg, respectively). The Freestyle and Hancock II leaflets had somewhat higher levels of phospholipids (2.44 and 2.75 µg/mg, respectively), and the Toronto SPV leaflets had the highest levels of phospholipids. Among the two pericardial valves tested, the Mitroflow tissue had higher levels of total phospholipids compared with the CE PERIMOUNT (1.78 and 0.25 µg/mg, respectively).

Calcification assay results
Folding
On explantation, some samples were found folded whereas others were not. In this study, tissues that were found folded were associated with higher calcification levels (data not shown). Therefore, the results from the 68 folded samples were excluded from the statistical analysis.

Calcium content
Table 3 contains the calcium data from the 194 nonfolded implants for each valve type tested. The tissues with the lowest calcium levels were CE SAV, CE Duraflex, and CE PERIMOUNT (0.8, 2.1, and 3.3 µg calcium/mg dry tissue weight, respectively). Tissues with an intermediate level of calcium were the Hancock II, Freestyle, and Mosaic leaflets (8.2, 9.5, and 25.4 µg calcium/mg dry tissue weight, respectively). Tissues with the highest levels of calcium were the Mitroflow, Toronto SPV, and the glutaraldehyde-only control (215, 244, and 259 µg calcium/mg dry tissue weight, respectively).


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  		Table 3. Calcium Content of Bioprosthetic Heart Valve Tissues

 
Note that not only is the average calcium content of interest, but the variability in the calcium content for each valve type may also provide useful information. Table 3 also contains the range measured for each valve type, calculated as the maximum value minus the minimum value. Note the greatest ranges were seen in the Toronto SPV, Mosaic, Freestyle, and Hancock II tissues (all > 100 µg calcium/mg dry wt). Mitroflow, CE PERIMOUNT, CE Duraflex, and CE SAV all had smaller ranges, less than 50 µg calcium/mg dry wt.

Table 4 contains the p values for the comparisons among each of the valve types. In the rat subcutaneous model, CE Duraflex, CE PERIMOUNT, and CE SAV all calcified significantly less than Mosaic, Mitroflow, and Toronto SPV and are not significantly different from the Hancock II. In this study, the Freestyle tended to calcify more than the CE Duraflex and CE PERIMOUNT, although this did not reach statistical significance (p = 0.06 and 0.09, respectively). Freestyle and Mosaic were not statistically significantly different. Mitroflow and Toronto SPV calcified significantly more than all other commercial valve tissues in this animal model.


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  		Table 4. Statistical Analysis of Calcium Data for Each Valve Type

 
There was a statistically significant correlation between phospholipid levels and calcification (r = 0.63, p = 0.04), with lower phospholipid levels being associated with lower calcium levels.

Histology
No signs of acute inflammation were found in either the implanted or host tissue. Signs of chronic inflammation were found in both implanted and host tissues, although it was frequently more severe in the implanted tissue. Mild to moderate collagen degeneration was noted intermittently in all groups tested. All nonimplanted specimens appeared similar, suggesting collagen degeneration was occurring as a result of implantation.


   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
In this study we characterized four properties of commercial bioprosthetic heart valve tissues and examined their relationship to calcification. We found a correlation between the phospholipid content of tissues and the severity of calcification. The CE Duraflex, CE SAV, and CE PERIMOUNT tissues had the lowest phospholipid contents and calcium levels. All three of these valves are treated with the XenoLogiX Treatment containing ethanol and Tween-80. Vyavahare and associates [6] have also reported that ethanol can extract phospholipids and lead to reduced calcification in animal models. We found that the XenoLogiX Treatment removed more than 90% of the phospholipids in both porcine leaflet and pericardial tissues.

Interestingly, although in this study the Mosaic tissue has a lower phospholipid content than the Freestyle tissue, it had a significantly higher calcium content, thus suggesting that other factors in the design or processing of bioprosthetic valves of this type may also be important in calcification. Ozaki and colleagues [7] have reported a higher calcification rate in Mosaic valves when compared with Freestyle valves in the sheep model. Other process variables such as incubation volume [8] and incubation time [9] have also been reported to play a role in amino oleic acid incorporation.

The Mitroflow and Toronto SPV tissues calcified significantly more than the other commercial valves in this study and were not significantly different from the pericardial glutaraldehyde-only control. These tissues retained 25% and 33% of the lipids, respectively, compared with fresh tissues. Although the phospholipid levels in the Mitroflow and Mosaic tissues are the same, the Mitroflow tissue calcified significantly more than the Mosaic tissue in this study. The reasons for this are unknown.

Chemical treatments of bioprosthetic tissues exhibit reduced calcification in animal studies. Surfactants, such as polysorbate (Tween) and sodium dodecyl sulfate, have reportedly decreased calcification in bioprosthetic leaflets implanted subcutaneously [10, 11]. This inhibition may occur by removing nucleation sites (phospholipids) from the substrate [11]. The phospholipid thin layer chromatography data reported here would support that hypothesis, although it has also been reported that phosphatidylinositol acts as a membrane binding site for alkaline phosphatase, an enzyme required for proper mineralization of cartilage and bone [12]. Thus, the efficient extraction of phospholipids such as phosphatidylinositol might also remove binding sites for alkaline phosphatase and other factors important in calcification.

Although phospholipids appear to be a significant contributor to the calcification of bioprosthetic tissue in this experiment, there may be other mechanisms that also contributed to the observed calcification. Residual aldehyde toxicity and mechanical and cellular factors [2, 13, 14] have all been implicated as important factors in calcification.

The relationship between bioprosthetic heart valve calcification mitigation in animal model studies and clinical outcomes has also not been established. Different lipoprotein profiles, potential differences in calcium metabolism, hematologic differences, and coexisting cardiovascular or valvular disease may play a role in the onset of calcification.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 
Drs Rutledge and Fishbein are paid consultants of Edwards Lifesciences LLC. Crystal M. Cunanan, Christine M. Cabiling, Tan T. Dinh, ShihHwa Shen, and Phihoa Tran-Hata are all employees of Edwards Lifesciences LLC.

Edwards Lifesciences, Edwards, Carpentier-Edwards, PERIMOUNT, SAV, Duraflex, XenoLogiX Treatment, and the stylized E logo are trademarks of Edwards Lifesciences Corporation. Hancock, Mosaic, and Freestyle are trademarks of Medtronic, Inc. Toronto SPV is a trademark of St. Jude Medical, Inc. Mitroflow is a trademark of Mitroflow International. Mitroflow Model 12 is distributed by Sulzer Carbomedics.


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

  1. Schoen F.J., Levy R.J. Pathology of substitute heart valves: new concepts and developments. J Card Surg 1994;9(Suppl):222-227.[Medline]
  2. Schoen F.J., Levy R.J. Tissue heart valves: current challenges and future research perspectives. J Biomed Mater Res 1999;47:439-465.[Medline]
  3. Ferrans V.J., Tomita Y., Hilbert S.L., et al. Pathology of bioprosthetic cardiac valves. Hum Pathol 1987;18:586-595.[Medline]
  4. Herrero E.J., Gutierrez M.P., Cunanan C.M., et al. Inhibition of bovine pericardium calcification: a comparative study of Al3+ and lipid removing treatments. J Mater Sci Mater Med 1991;2:86-88.
  5. Dugan E.A. Analysis of phospholipids by one-dimensional thin-layer chromatography. LC/GC North Am 1985;3:126-128.
  6. Vyavahare N., Hirsch D., Lerner E., et al. Prevention of bioprosthetic heart valve calcification by ethanol preincubation: efficacy and mechanisms. Circulation 1997;95:479-488.[Abstract/Free Full Text]
  7. Ozaki S., Herijgers P., Verbeken E., et al. The influence of stenting on the behavior of amino-oleic acid-treated, glutaraldehyde-fixed porcine aortic valves in a sheep model. J Heart Valve Dis 2000;9:552-560.[Medline]
  8. Girardot M.N., Torrianni M., Girardot J.M.D. 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]
  9. Chen W., Kim J.D., Schoen F.J., Levy R.J. Effect of 2-amino oleic acid exposure conditions on the inhibition of calcification of glutaraldehyde cross-linked porcine aortic valves. J Biomed Mater Res 1994;28:1485-1495.[Medline]
  10. Carpentier A., Nashef A., Carpentier S., et al. Techniques for prevention of calcification of valvular bioprostheses. Circulation 1984;70(Suppl 1):165-168.[Abstract/Free Full Text]
  11. Lentz D.J., Pollock E.M., Olsen D.B., et al. Inhibition of mineralization of glutaraldehyde-fixed Hancock bioprosthetic heart valves. In: Cohn L.H., Gallucci V., eds. Cardiac bioprostheses. New York, NY: Yorke, 1982:306-319.
  12. Harrison G., Shapiro I.M., Golub E.E. The phosphatidylinositol-glycolipid anchor on alkaline phosphatase facilitates mineralization initiation in vitro. J Bone Miner Res 1995;10:568-573.[Medline]
  13. Grimm M., Eybl E., Grabenwoger M., et al. Glutaraldehyde affects biocompatibility of bioprosthetic heart valves. Surgery 1992;111:74-78.[Medline]
  14. Grabenwoger M., Grimm M., Eybl E., et al. New aspects of the degeneration of bioprosthetic heart valves after long-term implantation. J Thorac Cardiovasc Surg 1992;104:14-21.[Abstract]



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