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Antimineralization treatments for bioprosthetic heart valves
Antimineralization treatments for bioprosthetic heart valves Assessment of efficacy and safety Since calcification limits the durability of contemporary bioprosthetic heart valves, antimineralization treatments are being widely investigated. Potential antimineralization treatments must have sustained prevention of mineralization without adverse effects. The preclinical investigation of efficacy and safety of antimineralization treatments comprises four essential steps: (1) subcutaneous implantation in smaU animals, (2) in vitro biomechanical studies of hemodynamics and durability, (3) morphology of unimplanted valves, and (4) circulatory implants in large animals. (J 'fHORAC CARDIOVASC SURG 1992;104:1285-8)
Frederick J. Schoen, MD, Phl)," Boston, Mass., Robert J. Levy, MD,b Ann Arbor, Mich., Stephen L. Hilbert, Phl),? Rockville, Md., and Richard W. Bianco," Minneapolis, Minn.
Calcification is the most frequent factor contributing to the failure of contemporary glutaraldehyde pretreated porcine aortic valve bioprostheses'>' and is an important cause of pericardia I valve failure.t' To enhance the durability and extend the functional life of bioprosthetic valves, several types of tissue antimineralization treatments have been proposed. Some have been investigated in experimental and or clinical studies.v'? However, a general approach to the preclinical evaluation of potential antimineralization treatments is lacking. From the Department of Pathology, Brigham and Women's Hospital, and Harvard Medical School, Boston"; Departments of Pediatrics, Communicable Diseases, and Pharmaceutics, University of Michigan Medical School, Ann Arbor"; Office of Science and Technology, Center for Devices and Radiological Health, Food and Drug Administration, Rockville"; and Cardiac Surgical Research Laboratory, Department of Surgery, University of Minnesota, Minne-
apolis," Received for publication Aug. 16, 1991. Accepted for publication Jan. 22, 1992. Opinions or assertions contained herein are the private views of the authors, not to be construed as conveying either official endorsement or criticism by the U.S. Department of Health and Human Services or the Food and Drug Administration. Address for reprints: Frederick J. Schoen, MD, PhD, Department of Pathology, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
12/1/36846
Most anticalcification strategies encompass systemic or localized therapy with pharmacologic agents or tissue pretreatment to modify cuspal chemistry or physical characteristics, or both. However, the mechanisms of action of most of the current antimineralization treatments for bioprosthetic tissue calcification are unknown. Of the various antimineralization treatments investigated thus far, sodium dodecyl sulfate (SDS) pretreatment is the first under consideration for approval by the U.S. Food and Drug Administration, as the T6 pretreatment of the Medtronic Hancock II bioprosthetic heart valve. SDS has been shown to be efficacious for inhibiting calcification, although incompletely, in subdermal and circulatory animal model studies. Nevertheless, despite extensive clinical and experimental investigation, the mechanism of action of SDS pretreatments for preventing bioprosthetic calcification has not been established. Because calcification of porcine or pericardial bioprosthetic tissue is potentiated by glutaraldehyde cross-linking and is primarily intrinsic, involving the cuspal connective tissue cells and collagen,20-25 possible mechanisms for calcification inhibition include nonaldehyde preservation, extraction of calcifiable material, ionic and/or macromolecular binding to putative nucleation sites, neutralization of critical mineralization cofactors, interference with growth of initial calcific microcrystals, charge modification, alteration of interstitial tissue spaces, prevention
1285
The Journal of Thoracic and Cardiovascular Surgery
I 2 8 6 Schoen et al.
Table I. Criteria for efficacy and safety of antimineralization treatments Efficacy • Effective and sustained calcification inhibition • Adequate valve performance (i.e., unimpaired hemodynamics and durability)
Safety • Does not ca use adverse blood-surface interactions (e.g., hemolysis, platelet adhesion, coagulation protein activation, complement activation, inflammatory cell activation, binding of vital serum factors) • Does not enhance local or systemic inflammation (e.g., foreign body reaction, immunologic reactivity, hypersensitivity) • Does not cause local or systemic toxicity • Does not potentiate infection
of serum insudation by an impervious coating, and restoration of natural inhibitors. For any approach efficacy and safety must be demonstrated (Table I). Subcutaneous implantation ofbioprosthetic tissue in small animals (e.g., rats, mice, and rabbits) reproduces bioprosthetic tissue mineralization noted clinically and provides a well-characterized calcification model that is economical, quantitative, and rapid. This calcification model, suitable for biochemical, kinetic, and morphologic investigations,2o.23,25 can be used to assess antimineralization treatment mechanisms of action, dose-response relationships, and toxicity.8-1O, 26, 27 Nevertheless, effectiveness of antimineralization treatment could be overestimated in subcutaneous implants because specimens in this model are not subjected to mechanical and dynamic stresses, and neither circulatory washout of beneficial factors nor reaccumulation of previously extracted calcifiable material can be easily assessed with this model. Moreover, there is no blood-surface contact, thereby preventing assessment of interactions with bloodborne substances, particularly platelets, coagulation factors, lipoproteins, and lipids. Although investigation of mechanisms and dose-response pharmaceutics may be difficult to justify from a commercial point of view, such studies are strongly encouraged, to provide a scientific data base that would direct and enhance further valve development and to anticipate clinical complications. Antimineralization treatments demonstrated to be effective should be further evaluated with in vitro accelerated wear testing (e.g., 25 million cycles), followed by subcutaneous implantation, to ensure that the antimineralization treatment effect remains present after cycling. In vitro hydrodynamic testing and accelerated wear
studies must similarly ensure that an antimineralization treatment neither impairs cuspal durability nor alters tissue biomechanics by stiffening or enhancing prolapse. For example, porcine bioprosthetic valve pretreatment with polyoxyethylene ether (Triton X-IOO) and N-Iauryl sarcosine caused marked tissue weakening that resulted in cuspal perforations after 20 weeks of implantation in the mitral position in sheep." This problem likely could have been predicted by in vitro durability testing. Certainly the functional integrity of the cuspal matrix should not be degraded and the surface not roughened or made more permeable by an antimineralization treatment. Moreover, careful structural and ultrastructural analysis on unimplanted tissue 28,29 and then in vitro, accelerated wear testing should be done to ensure that no morphologically evident structural degradation has occurred. The most important areas of safety evaluation are blood-surface interactions, local or systemic toxicities, and inflammatory effects. Assessment requires in vitro studies, careful preclinical animal testing, and close patient follow-up. Contemporary porcine bioprostheses generally have acceptable blood-surface interaction, with minimal hemolysis, thrombosis, and serum protein activation. Antimineralization treatments must not potentiate hemolysis, excessiveplatelet adhesion, or activation of complement or other contact factors. Neither immunologic reactivity, local or systemic toxicity (particularly that related to defective skeletal mineralization), inflammation, hypersensitivity, or binding or activation of serum proteins should be enhanced by an antimineralization treatment. Local or systemic toxicity may be initially evaluated by a conventional in vitro toxicologic screen followed by detailed pathologic studies in animal models, with careful morphologic study of explanted specimens. Hypersensitivity is likely species dependent, and the human allergic response could be difficult to predict with certainty from animal studies. It must be also demonstrated that the antimineralization treatment itself neither predisposes the tissue to infection nor impairs the use of certain bactericidal or bacteriostatic substances used in the commercial valve preparation process. The most important phase of preclinical animal testing involves orthotopic valve replacement in large animals, usually juvenile sheep or calves. In such model systems features of valve performance and safety not otherwise obtainable can be assessed, including device configuration, surgical technique, in vivo hemodynamics, and explant valve disease, including overall durability and calcification, thrombi, thromboembolism and hemolysis, and cardiac and systemic disease. In our estimation the sheep is more satisfactory as a large animal model for
Volume 104 Number 5 November 1992
Efficacy and safety of antimineralization treatments
anticalcification agents than the calf because of more favorable size and growth considerations. Moreover, congenital atrial septal defects frequently complicate cardiac operations in calves (but not sheep); the resultant suture line repair, only millimeters from the experimental valve, could influence valve inflow healing. Although hemodynamic monitoring, angiography, and both transthoracic and transesophageal echocardiography are no more difficult in calves during the operation, these studies become difficult in calves after about 3 months because of their large size; moreover, calves are more difficult to maintain long-term because of rapid growth. Nevertheless, because of a narrow and fragile aortic root in sheep, calves are superior for orthotopic aortic valve replacement with either stented or unstented valves. In sheep a 25 or 27 mm mitral valve is used; a mitral valve of up to 31 mm can be used in calves. In conclusion, an antimineralization treatment considered for clinical use must be shown to yield sustained prevention of mineralization onset, without adverse effects. Preclinical efficacy and safety investigation of antimineralization treatments comprises four essential steps: (I) subcutaneous implantation in small animals, (2) in vitro biomechanical studies of hemodynamics and durability, (3) morphology of unimplanted valves, and (4) circulatory implants in large animals. Each step must involve an assessment of differences between valves with antimineralization treatment and control bioprostheses. Clearly, however, bioprosthetic heart valve durability and performance can be assessed only by long-term (> 10 years) clinical evaluation.
REFERENCES I. Schoen FJ, Hobson CEo Anatomic analysis of removed prosthetic heart valves: causes of failure of 33 mechanical valves and 58 bioprostheses, 1980-1983. Hum Pathol 1985;16:549-59. 2. Milano A, Bortolotti U, Talenti E, et al. Calcific degeneration as the main cause of porcine bioprosthetic valve failure. Am J CardioI1984;53:1066-70. 3. Ferrans VJ, Hilbert SL, Fujita S, et al. Morphologic abnormalities in explanted bioprosthetic heart valves. In: Virmani R, Atkinson JB, FenoglioJJ, eds. Cardiovascular pathology. Philadelphia: WB Saunders, 1991:373. 4. Reul GJ, CooleyDA, Duncan JM, et al. Valvefailure with the Ionescu-Shileybovine pericardial bioprosthesis: analysis of 2680 patients. J Vase Surg 1985;2:192-204. 5. Schoen FJ, Fernandez J, Gonzalez-Lavin L, Cernaianu A. Cause of failure and pathologic findings in surgicallyremoved Ionescu-Shileystandard bovine pericardial heart valve bioprostheses: emphasis on progressive structural deterioration. Circulation 1987;76:618-27.
I 287
6. Oury JH, Angell WW, KoziolJA. Comparison of Hancock I and Hancock II bioprostheses. J Cardiac Surg 1988; 3:375-81. 7. Jones M, Eidbo EE, Hilbert SL, et al. Anticalcification treatments of bioprosthetic heart valves: in-vivo studies in sheep. J Cardiac Surg 1989;4:69-73. 8. Levy RJ, Wolfrum J, Schoen FJ, et al. Inhibition of calcification of bioprosthetic heart valves by controlled-release diphosphonate. Science 1985;229:190-2. 9. Levy RJ, Hawley MA, Schoen FJ, et al. Inhibition by diphosphonate compounds of calcification of porcine bioprosthetic heart valves implanted subcutaneously in rats. Circulation 1985;71:344-56. 10. Webb CL, Schoen FJ, Flowers WE, et al. Inhibition of mineralization of glutaraldehyde-pretreated bovine pericardium by AlCI3: mechanisms and comparisons with FeCh, LaCh, and Ga(N03h in rat subdermal model studies. Am J PathoI1991;138:971-81. II. Williams MA. The intact bioprosthesis: early results. J Cardiac Surg 1988;3:347-51. 12. Arbustini E, Jones M, Moses RD, et al. Modification of the Hancock T6 process of calcification of bioprosthetic cardiac valves implanted in sheep. Am J Cardiol 1984;53: 1388-96. 13. Thiene G, Laborde F, Valente M, et al. Experimental evaluation of porcine-valved conduits processedwith a calciumretarding agent (T6). J THORAC CARDIOVASC SURG 1986;91:215-24. 14. Okashi T, Noishiki Y, Tomizawa Y, et al. A new bioprosthetic cardiac valve with reduced calcification. ASAIO Trans 1990;36:M411-4. 15. Petite H, Rault I, Hue A, et al. Use of the acyl azide method for cross-linkingcollagen-rich tissues such as pericardium. J Biomed Mater Res 1990;24:179-87. 16. Golomb G, Ezra V. Prevention ofbioprosthetic heart valve tissue calcification by charge modification: effects of protamine binding by formaldehyde. J Biomed Mater Res 1991;25:85-98. 17. Carpentier A, Nashef A, Carpentier S, et al. Prevention of tissue valve calcification by chemical techniques. In: Cohn LH, Gallucci V, eds. Cardiac bioprostheses. New York: Yorke, 1982:320-7. 18. Tsao JW, Schoen FJ, Shankar R, et al. Calcification of bovine pericardium used in bioprosthetic heart valves is retarded by the physiologic inhibitor, phosphocitrate, and a synthetic analog, administered locally. Biomaterials 1988; 9:393-7. 19. Song T, VeselyI, Boughner D. Effects of dynamic fixation on sheer behaviour of porcine xenograft valves. Biomaterials 1990;11:191-6. 20. Fishbein MC, Levy RJ, Ferrans VJ, et al. Calcification of cardiac valve bioprostheses: biochemical, histologic and ultrastructural observationsin a subcutaneous implantation model system. J THORAC CARDIOVASC SURG 1982;83: 602-9. 21. Schoen FJ, Levy RJ, Nelson AC, et al. Onset and progres-
1 2 8 8 Schoen et al.
22.
23.
24.
25.
sion of experimental bioprosthetic heart valve calcification. Lab Invest 1985;52:523-32. Levy RJ, Schoen FJ, Levy JT, et al. Biologicdeterminants of dystrophic calcification and osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats. Am J PathoI1983;113:14355. Ferrans FJ, Boyce SW, Billingham ME, et al. Calcific deposits in porcine bioprostheses: structure and pathogenesis. Am J CardioI1980;46:721-34. Valente M, Bortolotti U, Thiene G. Ultrastructural substrates of dystrophic calcification in porcine bioprosthetic valve failure. Am J PathoI1985;119:12-21. Schoen FJ, Tsao JW, Levy RJ. Calcification of bovine pericardium used in cardiac valve bioprostheses: implications for the mechanisms of bioprosthetic tissue mineralization. Am J Pathol 1986;123:134-45.
The Journal of Thoracic and Cardiovascular Surgery
26. Golomb G, Langer R, Schoen FJ, et al. Controlled release of diphosphonate to inhibit bioprosthetic heart valve calcification: dose-response and mechanistic studies. J Control Rei 1987;4:181-94. 27. Levy RJ, Schoen FJ, Lund SA, Smith MS. Prevention of leaflet calcification of bioprosthetic heart valves with diphosphonate injection therapy: experimental studies of optimal dosage and therapeutic durations. J THORAC CARDIOVASC SURG 1987;94:551-7. 28. Hilbert SL, Barrick MK, Ferrans VJ. Porcine aortic valve bioprostheses: a morphologic comparison of the effects of fixation pressure. J Biomed Mater Res 1990;24:773-87. 29. Flomenbaum MA, Schoen FJ. Effects of fixation backpressure and antimineralization treatment on the morphology of porcine aortic bioprosthetic valves. J THORAC CARDIOVASC SURG [In press).
Frederick J. Schoen, MD, Phl)," Boston, Mass., Robert J. Levy, MD,b Ann Arbor, Mich., Stephen L. Hilbert, Phl),? Rockville, Md., and Richard W. Bianco," Minneapolis, Minn.
Calcification is the most frequent factor contributing to the failure of contemporary glutaraldehyde pretreated porcine aortic valve bioprostheses'>' and is an important cause of pericardia I valve failure.t' To enhance the durability and extend the functional life of bioprosthetic valves, several types of tissue antimineralization treatments have been proposed. Some have been investigated in experimental and or clinical studies.v'? However, a general approach to the preclinical evaluation of potential antimineralization treatments is lacking. From the Department of Pathology, Brigham and Women's Hospital, and Harvard Medical School, Boston"; Departments of Pediatrics, Communicable Diseases, and Pharmaceutics, University of Michigan Medical School, Ann Arbor"; Office of Science and Technology, Center for Devices and Radiological Health, Food and Drug Administration, Rockville"; and Cardiac Surgical Research Laboratory, Department of Surgery, University of Minnesota, Minne-
apolis," Received for publication Aug. 16, 1991. Accepted for publication Jan. 22, 1992. Opinions or assertions contained herein are the private views of the authors, not to be construed as conveying either official endorsement or criticism by the U.S. Department of Health and Human Services or the Food and Drug Administration. Address for reprints: Frederick J. Schoen, MD, PhD, Department of Pathology, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
12/1/36846
Most anticalcification strategies encompass systemic or localized therapy with pharmacologic agents or tissue pretreatment to modify cuspal chemistry or physical characteristics, or both. However, the mechanisms of action of most of the current antimineralization treatments for bioprosthetic tissue calcification are unknown. Of the various antimineralization treatments investigated thus far, sodium dodecyl sulfate (SDS) pretreatment is the first under consideration for approval by the U.S. Food and Drug Administration, as the T6 pretreatment of the Medtronic Hancock II bioprosthetic heart valve. SDS has been shown to be efficacious for inhibiting calcification, although incompletely, in subdermal and circulatory animal model studies. Nevertheless, despite extensive clinical and experimental investigation, the mechanism of action of SDS pretreatments for preventing bioprosthetic calcification has not been established. Because calcification of porcine or pericardial bioprosthetic tissue is potentiated by glutaraldehyde cross-linking and is primarily intrinsic, involving the cuspal connective tissue cells and collagen,20-25 possible mechanisms for calcification inhibition include nonaldehyde preservation, extraction of calcifiable material, ionic and/or macromolecular binding to putative nucleation sites, neutralization of critical mineralization cofactors, interference with growth of initial calcific microcrystals, charge modification, alteration of interstitial tissue spaces, prevention
1285
The Journal of Thoracic and Cardiovascular Surgery
I 2 8 6 Schoen et al.
Table I. Criteria for efficacy and safety of antimineralization treatments Efficacy • Effective and sustained calcification inhibition • Adequate valve performance (i.e., unimpaired hemodynamics and durability)
Safety • Does not ca use adverse blood-surface interactions (e.g., hemolysis, platelet adhesion, coagulation protein activation, complement activation, inflammatory cell activation, binding of vital serum factors) • Does not enhance local or systemic inflammation (e.g., foreign body reaction, immunologic reactivity, hypersensitivity) • Does not cause local or systemic toxicity • Does not potentiate infection
of serum insudation by an impervious coating, and restoration of natural inhibitors. For any approach efficacy and safety must be demonstrated (Table I). Subcutaneous implantation ofbioprosthetic tissue in small animals (e.g., rats, mice, and rabbits) reproduces bioprosthetic tissue mineralization noted clinically and provides a well-characterized calcification model that is economical, quantitative, and rapid. This calcification model, suitable for biochemical, kinetic, and morphologic investigations,2o.23,25 can be used to assess antimineralization treatment mechanisms of action, dose-response relationships, and toxicity.8-1O, 26, 27 Nevertheless, effectiveness of antimineralization treatment could be overestimated in subcutaneous implants because specimens in this model are not subjected to mechanical and dynamic stresses, and neither circulatory washout of beneficial factors nor reaccumulation of previously extracted calcifiable material can be easily assessed with this model. Moreover, there is no blood-surface contact, thereby preventing assessment of interactions with bloodborne substances, particularly platelets, coagulation factors, lipoproteins, and lipids. Although investigation of mechanisms and dose-response pharmaceutics may be difficult to justify from a commercial point of view, such studies are strongly encouraged, to provide a scientific data base that would direct and enhance further valve development and to anticipate clinical complications. Antimineralization treatments demonstrated to be effective should be further evaluated with in vitro accelerated wear testing (e.g., 25 million cycles), followed by subcutaneous implantation, to ensure that the antimineralization treatment effect remains present after cycling. In vitro hydrodynamic testing and accelerated wear
studies must similarly ensure that an antimineralization treatment neither impairs cuspal durability nor alters tissue biomechanics by stiffening or enhancing prolapse. For example, porcine bioprosthetic valve pretreatment with polyoxyethylene ether (Triton X-IOO) and N-Iauryl sarcosine caused marked tissue weakening that resulted in cuspal perforations after 20 weeks of implantation in the mitral position in sheep." This problem likely could have been predicted by in vitro durability testing. Certainly the functional integrity of the cuspal matrix should not be degraded and the surface not roughened or made more permeable by an antimineralization treatment. Moreover, careful structural and ultrastructural analysis on unimplanted tissue 28,29 and then in vitro, accelerated wear testing should be done to ensure that no morphologically evident structural degradation has occurred. The most important areas of safety evaluation are blood-surface interactions, local or systemic toxicities, and inflammatory effects. Assessment requires in vitro studies, careful preclinical animal testing, and close patient follow-up. Contemporary porcine bioprostheses generally have acceptable blood-surface interaction, with minimal hemolysis, thrombosis, and serum protein activation. Antimineralization treatments must not potentiate hemolysis, excessiveplatelet adhesion, or activation of complement or other contact factors. Neither immunologic reactivity, local or systemic toxicity (particularly that related to defective skeletal mineralization), inflammation, hypersensitivity, or binding or activation of serum proteins should be enhanced by an antimineralization treatment. Local or systemic toxicity may be initially evaluated by a conventional in vitro toxicologic screen followed by detailed pathologic studies in animal models, with careful morphologic study of explanted specimens. Hypersensitivity is likely species dependent, and the human allergic response could be difficult to predict with certainty from animal studies. It must be also demonstrated that the antimineralization treatment itself neither predisposes the tissue to infection nor impairs the use of certain bactericidal or bacteriostatic substances used in the commercial valve preparation process. The most important phase of preclinical animal testing involves orthotopic valve replacement in large animals, usually juvenile sheep or calves. In such model systems features of valve performance and safety not otherwise obtainable can be assessed, including device configuration, surgical technique, in vivo hemodynamics, and explant valve disease, including overall durability and calcification, thrombi, thromboembolism and hemolysis, and cardiac and systemic disease. In our estimation the sheep is more satisfactory as a large animal model for
Volume 104 Number 5 November 1992
Efficacy and safety of antimineralization treatments
anticalcification agents than the calf because of more favorable size and growth considerations. Moreover, congenital atrial septal defects frequently complicate cardiac operations in calves (but not sheep); the resultant suture line repair, only millimeters from the experimental valve, could influence valve inflow healing. Although hemodynamic monitoring, angiography, and both transthoracic and transesophageal echocardiography are no more difficult in calves during the operation, these studies become difficult in calves after about 3 months because of their large size; moreover, calves are more difficult to maintain long-term because of rapid growth. Nevertheless, because of a narrow and fragile aortic root in sheep, calves are superior for orthotopic aortic valve replacement with either stented or unstented valves. In sheep a 25 or 27 mm mitral valve is used; a mitral valve of up to 31 mm can be used in calves. In conclusion, an antimineralization treatment considered for clinical use must be shown to yield sustained prevention of mineralization onset, without adverse effects. Preclinical efficacy and safety investigation of antimineralization treatments comprises four essential steps: (I) subcutaneous implantation in small animals, (2) in vitro biomechanical studies of hemodynamics and durability, (3) morphology of unimplanted valves, and (4) circulatory implants in large animals. Each step must involve an assessment of differences between valves with antimineralization treatment and control bioprostheses. Clearly, however, bioprosthetic heart valve durability and performance can be assessed only by long-term (> 10 years) clinical evaluation.
REFERENCES I. Schoen FJ, Hobson CEo Anatomic analysis of removed prosthetic heart valves: causes of failure of 33 mechanical valves and 58 bioprostheses, 1980-1983. Hum Pathol 1985;16:549-59. 2. Milano A, Bortolotti U, Talenti E, et al. Calcific degeneration as the main cause of porcine bioprosthetic valve failure. Am J CardioI1984;53:1066-70. 3. Ferrans VJ, Hilbert SL, Fujita S, et al. Morphologic abnormalities in explanted bioprosthetic heart valves. In: Virmani R, Atkinson JB, FenoglioJJ, eds. Cardiovascular pathology. Philadelphia: WB Saunders, 1991:373. 4. Reul GJ, CooleyDA, Duncan JM, et al. Valvefailure with the Ionescu-Shileybovine pericardial bioprosthesis: analysis of 2680 patients. J Vase Surg 1985;2:192-204. 5. Schoen FJ, Fernandez J, Gonzalez-Lavin L, Cernaianu A. Cause of failure and pathologic findings in surgicallyremoved Ionescu-Shileystandard bovine pericardial heart valve bioprostheses: emphasis on progressive structural deterioration. Circulation 1987;76:618-27.
I 287
6. Oury JH, Angell WW, KoziolJA. Comparison of Hancock I and Hancock II bioprostheses. J Cardiac Surg 1988; 3:375-81. 7. Jones M, Eidbo EE, Hilbert SL, et al. Anticalcification treatments of bioprosthetic heart valves: in-vivo studies in sheep. J Cardiac Surg 1989;4:69-73. 8. Levy RJ, Wolfrum J, Schoen FJ, et al. Inhibition of calcification of bioprosthetic heart valves by controlled-release diphosphonate. Science 1985;229:190-2. 9. Levy RJ, Hawley MA, Schoen FJ, et al. Inhibition by diphosphonate compounds of calcification of porcine bioprosthetic heart valves implanted subcutaneously in rats. Circulation 1985;71:344-56. 10. Webb CL, Schoen FJ, Flowers WE, et al. Inhibition of mineralization of glutaraldehyde-pretreated bovine pericardium by AlCI3: mechanisms and comparisons with FeCh, LaCh, and Ga(N03h in rat subdermal model studies. Am J PathoI1991;138:971-81. II. Williams MA. The intact bioprosthesis: early results. J Cardiac Surg 1988;3:347-51. 12. Arbustini E, Jones M, Moses RD, et al. Modification of the Hancock T6 process of calcification of bioprosthetic cardiac valves implanted in sheep. Am J Cardiol 1984;53: 1388-96. 13. Thiene G, Laborde F, Valente M, et al. Experimental evaluation of porcine-valved conduits processedwith a calciumretarding agent (T6). J THORAC CARDIOVASC SURG 1986;91:215-24. 14. Okashi T, Noishiki Y, Tomizawa Y, et al. A new bioprosthetic cardiac valve with reduced calcification. ASAIO Trans 1990;36:M411-4. 15. Petite H, Rault I, Hue A, et al. Use of the acyl azide method for cross-linkingcollagen-rich tissues such as pericardium. J Biomed Mater Res 1990;24:179-87. 16. Golomb G, Ezra V. Prevention ofbioprosthetic heart valve tissue calcification by charge modification: effects of protamine binding by formaldehyde. J Biomed Mater Res 1991;25:85-98. 17. Carpentier A, Nashef A, Carpentier S, et al. Prevention of tissue valve calcification by chemical techniques. In: Cohn LH, Gallucci V, eds. Cardiac bioprostheses. New York: Yorke, 1982:320-7. 18. Tsao JW, Schoen FJ, Shankar R, et al. Calcification of bovine pericardium used in bioprosthetic heart valves is retarded by the physiologic inhibitor, phosphocitrate, and a synthetic analog, administered locally. Biomaterials 1988; 9:393-7. 19. Song T, VeselyI, Boughner D. Effects of dynamic fixation on sheer behaviour of porcine xenograft valves. Biomaterials 1990;11:191-6. 20. Fishbein MC, Levy RJ, Ferrans VJ, et al. Calcification of cardiac valve bioprostheses: biochemical, histologic and ultrastructural observationsin a subcutaneous implantation model system. J THORAC CARDIOVASC SURG 1982;83: 602-9. 21. Schoen FJ, Levy RJ, Nelson AC, et al. Onset and progres-
1 2 8 8 Schoen et al.
22.
23.
24.
25.
sion of experimental bioprosthetic heart valve calcification. Lab Invest 1985;52:523-32. Levy RJ, Schoen FJ, Levy JT, et al. Biologicdeterminants of dystrophic calcification and osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats. Am J PathoI1983;113:14355. Ferrans FJ, Boyce SW, Billingham ME, et al. Calcific deposits in porcine bioprostheses: structure and pathogenesis. Am J CardioI1980;46:721-34. Valente M, Bortolotti U, Thiene G. Ultrastructural substrates of dystrophic calcification in porcine bioprosthetic valve failure. Am J PathoI1985;119:12-21. Schoen FJ, Tsao JW, Levy RJ. Calcification of bovine pericardium used in cardiac valve bioprostheses: implications for the mechanisms of bioprosthetic tissue mineralization. Am J Pathol 1986;123:134-45.
The Journal of Thoracic and Cardiovascular Surgery
26. Golomb G, Langer R, Schoen FJ, et al. Controlled release of diphosphonate to inhibit bioprosthetic heart valve calcification: dose-response and mechanistic studies. J Control Rei 1987;4:181-94. 27. Levy RJ, Schoen FJ, Lund SA, Smith MS. Prevention of leaflet calcification of bioprosthetic heart valves with diphosphonate injection therapy: experimental studies of optimal dosage and therapeutic durations. J THORAC CARDIOVASC SURG 1987;94:551-7. 28. Hilbert SL, Barrick MK, Ferrans VJ. Porcine aortic valve bioprostheses: a morphologic comparison of the effects of fixation pressure. J Biomed Mater Res 1990;24:773-87. 29. Flomenbaum MA, Schoen FJ. Effects of fixation backpressure and antimineralization treatment on the morphology of porcine aortic bioprosthetic valves. J THORAC CARDIOVASC SURG [In press).