Lipid extraction attenuates the calcific degeneration of bovine pericardium used in cardiac valve bioprostheses

J. Exp. Path. (1990) 71, I87-196 Lipid extraction attenuates the calcific degeneration of bovine pericardium used in cardiac valve bioprostheses Marcos A. Rossi, Domingo M. Braile, Maria D.R. Teixeira, Doroteia R.S. Souza and Luiz C. Peres Department of Pathology, Faculty of Medicine of Ribeirao Preto, University of Sdo Paulo, Ribeirdo Preto, and Institute of Cardiovascular Diseases, IMC-Biomedica, Sao lose do Rio Preto, Brazil Received for publication 20 June I989 Accepted for publication 9 October I989 Summary. Bovine pericardial bioprostheses frequently fail due to dystrophic calcification. Since (a) recent studies indicate that membrane-associated complexed acidic phospholipids play an important role in the process of both physiologic and pathologic calcification, and (b) cytoplasmic organelles and plasma membrane of interstitial cells seem to serve as initial sites of calcific degeneration of bioprosthetic bovine pericardial tissue, this investigation was undertaken to evaluate whether, and if so, to what extent,the mineralization of valve tissue could be attenuated by previous lipid extraction. Pretreatment of glutaraldehyde-preserved bovine pericardium with acidified sulphuric ether (ph 3.0-4.o) attenuated calcification significantly: 28 days after subcutaneous implantation in young rats the degree of mineral deposition was approximately equal to typical 7 days implants in this model. The mechanism of this beneficial effect is suggested to be due to partial extraction of tissue phospholipids, as demonstrated by electron microscopy, thus reducing the number of available sites for deposition of hydroxyapatite crystals. In addition, and importantly, the present results indicate that any attempt to reduce cardiac valve bioprosthesis mineralization will have to take into account the role of lipids and, particularly, the membranous phospholipids in the calcification mechanism. Keywords: bovine pericardium, calcific degeneration, cardiac valve bioprosthesis, lipid extraction, calcification Glutaraldehyde-preserved bovine pericar- post-operatively and 40-50% of children dium has been widely used for the fabrication after 4 years (Carpentier et al. I984). of bioprostheses to replace diseased human The development of preventive theracardiac valves since I971 (Ionescu et al. peutic methods to control the calcific degen- 1982). The bovine pericardial bioprostheses, eration of bioprosthetic valves depends on however, frequently fail due to dystrophic the knowledge of the primary sites where calcification. Reoperation is required in I0- calcium deposition occurs, which is essential 20% of adult recipients within 7-10 years to the understanding of the pathogenic Correspondence: Professor Marcos A. Rossi, Department of Pathology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, 14049-Ribeirao Preto, SP, Brazil. I87

I88 mechanism of this complication. Study from our laboratory has demonstrated that cytoplasmic organelles (mitochondria in particular) and plasma membrane of interstitial cells seem to serve as initial sites of the process of calcification in glutaraldehyde-preserved pericardial valvular xenografts implanted subcutaneously in young rats (Rossi et al. I986). Similarly, investigations evaluating subcutaneous implants of porcine aortic valve cusps (Schoen et al. I985) or bovine pericardial tissue (Schoen et al. I986) showed that the connective tissue cells are the earliest calcified structures. Previously, an electron microscopic study carried out in 26 explanted porcine bioprostheses revealed that debris and membrane fragments represent one of the initial nuclei of calcification (Valente et al. I985). Since in dystrophic calcification calcium and phosphate are likely attracted by mineralization nucleators represented by membranous phospholipids, precipitating as calcium phosphate when their levels are elevated (Anderson I983; Boskey et al. I988), we infer that one of the most promising and important approaches to producing a tissue valve with a reduced tendency to calcify would be to prevent initiation by eliminating the membranous phospholipids of the valve tissue. The present investigation was undertaken to evaluate whether, and if so, to what extent, the process of calcific degeneration of glutaraldehyde-preserved bovine pericardium implanted subcutaneously in young rats could be attenuated by previous lipid extraction with acidified sulphuric ether, without compromising the physicochemical stability of the tissue. Materials and methods Bovine parietal pericardium was collected at time of slaughter from i 8-month-old healthy animals, and immediately placed in 2.5% phosphate-buffered glutaraldehyde (ph 7.4) for 2 h. After removal of the excess of fat component from the external surface, pieces of tissue were submitted to lipid extraction M.A. Rossi et al. with acidified (i M HCI) sulphuric ether at ph 3.0-4.0, four changes of i h duration each, under continuous agitation. Neither unsaturated lipids nor phospholipids are rendered insoluble in organic solvents by aldehyde fixation (Pease I964). Aldehydes are, however, efficient cross-linking agents for protein (Golomb et al. I987). Following extraction, the pericardial tissue was submitted to the standardized procedure for the preparation of cuspal tissue of IMC bioprostheses (Institute of Cardiovascular Diseases, IMC-Biom& dica, Sao Jose do Rio Preto, Sao Paulo, Brazil), mounted in plastic mandrels with controlled tension and placed in o 5% purified aqueous glutaraldehyde in 0.1 3 M phosphate buffer (ph 7.4) for 3 h, at room temperature. The buffered glutaraldehyde is then renewed and the specimens are maintained in it for I 5 days at 4-80C, when they are stored in 4% buffered formalin (ph 5.4). Control pieces of pericardium, not submitted to lipid extraction, were processed as above after the initial fixation in 2.5% glutaraldehyde. To assess the physicochemical stability of control and extracted (submitted to lipid extraction) pericardia, tests were performed. The width of the tissue was measured with a Microtest IV Automatic apparatus. The efficacy of glutaraldehyde fixation was evaluated through the determination of the shrinkage temperature (normal < 8 30C). Mechanical strength parameters of pericardial samples were determined using an Instrom tensiometer. After pulling a sample a tensile force-elongation trace is obtained. From this mechanical trace can be calculated the tensile breaking strength, that is, the force necessary to rupture the tissue (normal = I.80 ± 0.3 5 kgf/mm2), the elongation, the deformation undergone by the tissue (normal = 22.43 + 5.4 3%), and the tenacity index, the energy necessary to break the material, represented by the area under the force-elongation curve (normal= 20.42 ± 8.93). Student's t-test was performed to determine statistical significance.

Lipid extraction attenuates calcific degeneration of pericardium For ultrastructural evaluation, small blocks of control and extracted pericardia were placed in o.i M phosphate-buffered 2.5% glutaraldehyde, washed with several changes of buffer, and postfixed with i% osmium tetroxide. Osmium tetroxide is known to react with the double bounds and the polar groups of lipids (Weakley I972). The specimens were then dehydrated in a graded series of ethanols and propylene oxide, and embedded in Epon. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined in a Zeiss EM IO9 electron microscope. Young Wistar albino rats, weighing 6o- 8o g, were used for the study. The animals were housed in polypropylene cages, maintained under controlled conditions, and given free access to a standard laboratory chow and tap water. Each animal was anaesthetized with ether and, after shaving the dorsal region, a small incision was made. Small samples of control and extracted pericardia, approximately o 5 cm2, after careful rinsing in sterile physiologic saline, were implanted subcutaneously (3-4 specimens per rat). Seven animals were selected randomly and sacrificed in light ether anesthesia at days 2, 7, 14, and 28. Twenty-one to 28 specimens were retrieved each time interval from both control and extracted groups, fixed in io% neutral buffered formalin, and embedded in paraffin. The blocks were then sectioned at 6 gum, stained with haematoxylin and eosin and von Kossa's stain (for calcium phosphates), and examined blindly under the light microscope. The average degree of calcification was estimated with a semiquantitative grading system 0-5 (o, none; i, slight; 2, slight to moderate; 3, moderate; 4, moderate to severe; and 5, severe). It has been pointed out that the morphologic assessment of the degree of calcification corresponds to the biochemically determined levels of calcium (Fishbein et a]. I982; Carpentier et a]. I984; Webb et al I988). Moreover, one small sample of control and extracted pericardia at each time interval was fixed immediately in o0i M phosphate-buffered 2.5% glutaraldehyde, postfixed in osmium tetroxide, dehydrated, embedded in Epon, sectioned, doublestained, and examined in the electron microscope. Results The results of the physicochemical stability tests performed on control and extracted pericardial samples did not differ, thus demonstrating the good physicochemical stability of the pericardium after lipid extraction with acidified sulphuric ether (Table i). Unimplanted control and extracted pericardia had two layers: a thick fibrosa, and a thin outer epipericardial connective tissue (Fig. I). The ultrastructural study revealed that both layers were composed of well preserved collagen fibrils, with normal cross- Table i. Physicochemical stability tests performed on both control and extracted pericardial samples Control Extracted Width (mm) 0.286±0.028 0.276±0.020 Tensile breaking strength (kgf/mm2) 2.24 ± 0.13 2.05 ± 0.29 Shrinkage temperature ( C) 86.60±0.24 87.00±0.00 Elongation (%) 23.75 ± 3.22 23.25 ±4.56 Tenacity index 29.97 ± 3.4 I 26.14± 8.20 Values are expressed as mean ± s.e.m. Seven samples tested in each group. No differences were significant by Student's t-test I89

Igo v ^#,,,, * s _,ext *- M.A. Rossi et al. Fig. i. Typical cross-sectional histologic appearance of unimplanted control glutaraldehyde-preserved bovine pericardium showing homogeneous collagenous structure. The formerly serosal internal (int) and external (ext) surfaces are indicated. Haematoxylin and eosin stain, X 270. banding periodicity, and elastic fibres; focally, the interstitium showed electronlucent spaces replacing the ground substance probably lost in the process offixation; the inner surface was devoid of the normal lining of mesothelial cells. However, the ultrastructural appearances of the connective tissue cells scattered among collagen bundles and elastic fibres were not the same in control and extracted tissues. In extracted pericardium, the plasmalemma and membranous cytoplasmic organelles of connective tissue cells were scarcely seen and poorly delineated; besides, they showed randomly distributed chromatin aggregates within the nuclear matrix. These findings contrasted with the well delineated and clearly seen membranes, and the normal distribution of nuclear chromatin of connective tissue cells in control pericardium (Fig. 2). Figure 3 summarizes the kinetics of mineralization, as evaluated by light microscopy with von Kossa's stain, of both control and extracted pericardia implanted subcutaneously in young rats. Intrinsic calcification of control implants was noted at 2 days, progressively becoming more extensive, as previously described (Rossi et al. I986); implants previously treated with acidified sulphuric ether, in contrast, showed no deposits of calcium at this time of experimentation, the same degree of calcification being reached only after 4 days; with I4 and 28 days of implantation the degree of mineral deposition in extracted tissues was approximately equal to that of typical 7-day controls (Fig. 4). It should be pointed out that, although fairly diffuse, calcification was not uniform in a definite specimen of pericardium, and that the extent of mineralization varied from specimen to specimen, even when retrieved from the same rat. Ultrastructural evaluation of the morphology of mineralization of both control and

Lipid extraction attenuates calcific degeneration of pericardium I9I Fig. 2. Electronmicrographs. A, Unimplanted control pericardium. Connective tissue cell showing welldelineated and clearly seen membranes (arrow heads) and normal distributed nuclear chromatin. B, Unimplanted pericardium pretreated with acidified sulphuric ether (extracted). The plasmalemma and membranous cytoplasmic organelles of interstitial cell are scarcely seen and poorly delineated (arrow heads). The nuclear matrix contains randomly distributed chromatin aggregates. nu, Nucleus; cf, collagen fibrils; el, elastic fibres. Ultrathin sections stained with uranyl acetate and lead citrate. x 9500. extracted pericardial samples subcutaneously revealed that the interstitial cells serve as the primary sites where calcium deposition occurs, similarly to our previous studies. Then the process progresses into the intercellular space, adjacent to collagen fibrils and elastic fibres. However, although morphologically similar, the degree of calcification was attenuated in ether-treated pericardia as compared to controls.

192 c 0 CU 5-4 CD 2 Ca) CD 2 4 7 14 28 Tirne after implantation (days) Fig. 3. Kinetics of calcification, as evaluated by light microscopy with von Kossa's stain, of o, control and *, sulphuric ether (ph 3.0-4.0)- extracted pericardia implanted subcutaneously in young rats. Discussion The validity of the subcutaneous implantation model for investigating the intrinsic calcification of tissues used in the fabrication of bioprosthetic heart valves has been supported by biochemical and morphologic data (Fishbein et al. I982; Shoen & Levy I984; Arbustini et al. I984; Carpentier et al. I984; Levy et al. I985; Schoen et al. I985; Johnston et al. I988), which suggest a similar physiopathologic mechanism when compared with clinically implanted valves (Schoen et a]. I988). The present study clearly demonstrated that glutaraldehyde-preserved bovine parietal pericardium can be chemically modified by treating preimplanted tissue with acidified M.A. Rossi et al. sulphuric ether to attenuate the tissue calcification: 28 days after subcutaneous implantation in young rats the degree of mineralization was approximately equal to that of typical 7 day implants in this model. Besides, and importantly, no adverse effects on the physicochemical stability of extracted tissues in comparison to controls were observed. The factors affecting calcification of bioprosthetic valve tissue may be separated into those related to the host and those related to the implant. Schoen, Levy and colleagues (Schoen et al. I988) believe that 'The most important determinant of the propensity of bioprosthetic tissue to calcify is probably the tissue preparation method'. Glutaraldehyde incorporation is required to confer material stability; however, the resulting disruption of tissular calcium regulation, due to paralysis of the energy metabolism and loss of the structural integrity of cell membranes, and protein cross-linking, would lead to its calcification (Schoen et al I988). Other factors have also been implicated such as dynamic mechanical stress and strain (Thubrikar et al. I983) and host metabolic factors (Levy et al. I985). In general, increased local concentration of calcium and phosphorus ions, modified or removed mineralization inhibitors, and mineralization nucleators are circumstances, not mutually exclusive, implicated in the physiologic as well as pathologic process of calcification (Boskey 198I; Wuttier I982). Membrane-bounded extracellular bodies, called matrix vesicles, which contain calcium-acidic phospholipid-phosphate complexes in their membranes, and possess significant alkaline phosphatase activity, have been identified as the sites of initial cartilage (Anderson I983; Anderson & Fig. 4. Light micrographs. Evolution of calcification in bovine pericardium implanted subcutaneously in young rats. A, Control implant removed after 2 days showing slight diffuse calcification. B, Extracted implanted pericardium removed after 2 days. No mineralization is apparent. C, Control implant removed after 28 days showing severe diffuse mineralization, with confluent areas. D, Extracted pericardium removed after 28 days. Moderate degree of calcification can be seen. Note markedly reduced calcific degeneration in B and D in comparison to A and C, respectively. Stained with von Kossa's stain (calcium phosphate is black). x 300.

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I94 Matsuzawa 19 70) or dental tissues (Almuddaris & Dougherty (I979) physiologic deposition of calcium (hydroxyapatite). On the other hand, in dystrophic calcification, associated with no abnormalities of the blood levels of calcium phosphate, the mineralization nucleators, represented by membrane debris associated with complexed acidic phospholipid and possessing significant alkaline phosphatase activity, seem to be crucially involved in initiating calcium deposition (Anderson I983; Boskey et al. I988). Thus, it appears that the membrane-associated complexed acidic phospholipids play an important role in the process of both physiologic and pathologic calcification, although other factors may contribute as well. The ultrastructural findings of the present study revealed that the initial calcium deposition was cell-associated in both control and extracted pericardial samples implanted subcutaneously, which is in consonance with the observation that the ether treatment did not significantly delay the onset of mineralization. This agrees with previous results from our laboratory indicating that cytoplasmic organelles and plasma membrane of interstitial cells are the initial sites of mineralization in glutaraldehyde-preserved bovine pericardium used in bioprosthetic cardiac valves implanted subcutaneously in young rats (Rossi et al. I986). Similar conclusions have been put forward by a few authors evaluating the calcification of subcutaneous implants of porcine aortic valve cusps (Schoen et al. I985), or bovine pericardial tissue (Schone et al. I986), or studying under the electron microscope explanted insufficient porcine bioprosthetic cardiac valves (Valente et al. I985). In bioprosthetic valve mineralization, serum calcium is probably attracted by membranous phospholipids within the valve-tissue structure, while serum phosphate is concentrated by alkaline phosphatase, working as a phosphotransferase. It has been recently demonstrated that glutaraldehyde-treated bovine pericardium retains the major fraction of original alkaline M.A. Rossi et a]. phosphatase activity (Maranto & Schoen I988). When the levels of calcium and phosphate are sufficiently elevated, calcium and phosphate deposition occurs, at first as a more soluble amorphous and granular compound and later as the more insoluble hydroxyapatite crystals. The successful attenuation of calcification of bovine pericardium treated with acidified sulphuric ether is very likely due to the partial removal of phospholipids. The ultrastructural observations of extracted, unimplanted pericardial tissues in the present study support this hypothesis: the plasmalemma and the membranous cytoplasmic organelles of connective tissue cells were scarcely seen and poorly delineated. Since lipids are not stabilized by aldehyde fixation (Pease I964), they were probably extracted with sulphuric ether and, thus, the spaces they originally occupied are visualized as negative images of cytomembranes. The development of preventive therapies to control the calcific degeneration of bioprosthetic valves depends on the knowledge of the pathogenetic mechanism of this complication. Since host and implant determinants are involved, several strategies have been adopted according to a specific point of view. It has been demonstrated that calcification of subcutaneous implants can be reduced by preimplantation treatment of porcine or pericardial valve tissues with surfactants, particularly sodium dodecyl sulphate (Arbustini et al. I 964; Carpentier et al. I984). The mechanisms of action of surfactants have been considered to depend on reduced insudation of phospholipids due to a hydrophobic barrier to their penetration into valve tissue (Carpentier et al. I984), extraction of phospholipids from the valve tissue (Arbustini et al. I984) and alteration of the membrane surface charge, the ground substance, or the collagen fibrils (Lentz et al. I982). In conclusion, pretreatment of bovine pericardium used in the fabrication of bioprosthetic heart valves with acidified sulphuric ether attenuates the intrinsic calcifi-

Lipid extraction attenuates calcific degeneration of pericardium cation of subcutaneous implants in young by the Hancock T6 process of calcification of rats, without any adverse effect on the bioprosthetic cardiac valves implanted in physicochemical stability of the tissue. The sheep. Am. J. Cardiol. 53, 1388-1396. mechanism of this attenuation is probably BOSKEY A.L. (I98I) Current concepts of the biochemistry and physiology of calcification. due to partial extraction of tissue membrane Clin. Orthop. Ret. Res. 157, I65-I96. phospholipids, as demonstrated by electron BOSKEY A.L., BULLOUGH P.G., VIGORITA V. & Di microscopy, thus reducing the number of CARLO E. (I988) Calcium-acidic phospholipidphosphate complexes in human hydroxyapa- sites available for deposition of hydroxyapatite crystals. These data should be validated tite-containing pathologic deposits. Am. J. by studies of intracardiac valve implantation Pathol. I33, 22-29. in large animals to evaluate the effectiveness CARPENTIER A., NASHEF A.,CARPENTIER S., AHMED A. & GOUSSEF N. (I984) Techniques for prevention of calcification of valvular bioprostheses. of this or a similar treatment in attenuating the process of calcific degeneration and the Circulation 70 (Suppl.I.), i 65-i68. mechanical durability of the valves. In addition, and importantly, the present results L.C., NASHEF A., GOODMAN A.P. & CARPENTIER FISHBEIN M.C., LEVY R.J., FERRANS V.J., DEARDEN indicate that any attempt to reduce cardiac A. (1982) Calcification of cardiac valve bioprostheses: Biochemical, histologic, and valve bioprosthesis mineralization will have ultrastruc- to take into account the role of lipids and, particularly, the membranous phospholipids in the calcification mechanism. Acknowledgements The authors express their gratitude to M.M.O. Rossi, M.E. Riul, M.H.L.N. Gomes, M.A. Abreu and L.G.V. Baroza for technical assistance. Professor M.A. Rossi is Senior Investigator of the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Proc. 30II09-79). Supported by a grant from the Fundaio de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (Proc. 89/26I6-8). References ALMUDDARIS M.F. & DOUGHERTY W.J. (I979) The association ofamorphous mineral deposits with the plasma membrane of pre- and young odontoblasts and their relationship to the origin of dentinal matrix vesicles in rat incisor teeth. Am. J. Anat. 155, 223-244. ANDERSON H.C. (I983) Calcific diseases: A concept. Arch. Pathol. Lab. Med. 107, 341-348. ANDERSON H.C. & MATSUZAWA T. (I 9 70) Membranous particles in calcifying cartilage matrix. Trans. N. Y. Acad. Sci. 32, 6I9-630. ARBUSTINI E., JONES M., MOSEs R.D., EIDBO E.E., CARROL R.J. & FERRANS V.J. (I984) Modification I95 tural observations in a subcutaneous implantation model system. J. Thorac. Cardiovasc. Surg. 83, 602-609. GOLOMB G., SCHOEN F.J., SMITH M.S., LINDEN J. DIXON M. & LEVY R.J. (I987) The role of glutaraldehyde-induced cross-links in calcification ofbovine pericardium used in cardiac valve bioprostheses. Am. J. Pathol. 127, 122-I30. IONESCU M.I., TANDON A.P., SAUNDERS N.R., CHI- DAMBARAM M. & SMITH D.R. (I982) Clinical durability of the pericardial xenograft valve: i i years' experience. In Cardiac Bioprostheses. Eds L.H. Cohn & V. Galucci. New York: Yorke Medical Books. pp. 42-60. JOHNSTON T.P., SCHOEN F.J. & LEVY R.J. (I988) Prevention of calcification of bioprosthetic heart valve leaflets by Ca2+ disphosphonate pretreatment. J. Pharm. Sci. 77, 740-744. LENTZ D.J., POLLOCK E.M., OLSEN D.B., ANDREWS E.J., MURASHITA J. & HASTINGS W.L. (I982) Inhibition of mineralization of glutaraldehydefixed Hancock bioprosthetic heart valves. In Cardiac Bioprostheses. Ed L.H. Cohn & V. Gallucci. New York: Yorke Medical Books. pp. 306-3I9. LEvY R.J., SCHOEN F.J., HOWARD S.L., LEVY J.T., OSHRY L. & HAWLEY M. (I985) Calcification of cardiac valve bioprostheses: Host and implant factors. In Calcium in Biological Systems. Eds R.P. Rubin, G.B. Weiss & J.W. Putney, Jr. New York: Plenum Publishing Corporation. pp. 66i-668. MARANTO A.R. & SCHOEN F.J. (I988) Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic cardiac valves. Circ. Res. 63, 844-848. PEASE D.C. (I964) Histological Techniques for Elec-

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