- Email: [email protected]
Heparin in calcification prevention of porcine pericardial bioprostheses
Biomoterials 16 (1997)1109-1113
ELSEVIER
0 1997 Elsevier Science Limited Printed in Great Britain. All rights reserved 0142-9612/97/$17.00
PII:SO142-9612(97)00033-l
Heparin in calcification prevention of porcine pericardial bioprostheses Jyotirmay Chanda, Ryosei Kuribayashi and Tadaaki Abe Department
of Cardiovascular
Surgery, Akita University School of Medicine, Akita 010, Japan
Calcific degeneration is the main cause of failure of glutaraldehyde-treated xenograft heart valve substitutes implanted in humans. Coupling of heparin through an intermediate surface-bound substrate containing amino groups showed complete prevention of calcification of glutaraldehydetreated porcine pericardium implanted subdermally in weanling rats for 5 months (heparin bonded pericardium: calcium, 0.625 f 0.24 mg g-‘; glutaraldehyde-only-treated pericardium: calcium, 228.32 I!Z37.39 mg g-‘; P < 0.0001). Conceivably, inactivation of unpaired aldehyde moieties present in bioprostheses after exposure to glutaraldehyde by amino compounds followed by blocking the potential binding sites of the graft with a surface modifying agent like heparin would be the key steps in the prevention of calcification and degeneration of glutaraldehyde-treated biological tissue grafts. 0 1997 Elsevier Science Limited. All rights reserved Keywords:
Heparin,
porcine
pericardium,
bioprosthetic
valve,
calcification
prevention
Received 6 November 1996; accepted 22 January 1997
Valve replacement surgery has been well established over the past 30 years and has been shared between mechanical and biological valves’. Valve repair instead of valve replacement is increasin ly attempted, but the procedure is not always feasible z?‘3; the haemodynamic results can be unsatisfactory4, the operation is techn:ically more difficult and the durability of the procedure is variable5. The alternative to mechanical valve replacement is the use of biological prostheses: there is no need for lifelong anticoagulation even in the presence of chronic atria1 fibrillation, and risk of systemic thromboembolism is very low. Furthermore, the quality of life can be considered very close to normal, allowing heavy work, sports, normal pregnancy and delivery, and life in remote areas6-8. The primary concern with bioprostheses, well documented over the previous years, is scepticism regarding durability and the potential morbidity and mortality related to degenerative prosthesesg-15. Glutaraldehyde (GA)-treated bioprosthetic valves result in rapid calcification and structural deterioration in paediatric and young populations12-‘6. The ideal cardiac valve for child-bearing patients is not yet available. Mechanical valves require lifelong anticoagulation, which may cause fetal deaths and birth defects7. Biological valves do not necessarily require lifelong anticoagulation, but their degeneration is accelerated during pregnancy6. To date, allografts are the best available biological heart valve substitutes for children17318. Though the long-term durability has been very satisfactory in adults, problems such as early degeneration and progressive Correspondence
calcification have resulted in the limited durability of the allograft valves in paediatric patientsl’-“. In previous works21-23, it was reported that chitosan post-treatment is effective in prevention of calcification of GA-treated bioprostheses implanted in adult rats (120-150g). GA cross-linked chitosan post-treated porcine pericardium (PP) functioned well as a pericardial substitute in mongrel dogsz4. Chitosan post-treatment is effective in calcification prevention of GA-treated porcine aortic non-coronary cusps implanted in the right ventricular outflow tract in dogsz5. Recently, we found that post-treatment with amino compounds including chitosan (present communication) is ineffective in prevention of calcification of GA-treated porcine heart valves implanted either in systemic circulation in juvenile sheep or subdermally in weanling rats. This fact prompted us to modify the previously proposed treatment21-23 for a better outcome. Like bovine pericardium, GA-treated PP is used in clinical practice26-28. PP is thinner than bovine and thicker than human pericardium, and may be used for construction of heart valve bioprostheses”. In this study, the efficacy of our newly developed chemical treatment for prevention of calcification of GA-treated PP implanted subdermally in weanling rats was evaluated.
MATERIALS
AND METHODS
PPs were cross-linked in GA (glutaraldehyde, EM 25%, TAAB Laboratories Equipment Ltd., Reading, UK) in normal saline (pH 7.4) with gradually increasing concentrations of GA from 0.1 to 0.25% in normal
to Dr J. Chanda.
1109
Biomaterials
1997, Vol. 18 No. 16
Heparin
1110
saline at 37°C for a period of 1 month. Heparin (sodium salt, 164.5 IUmggI; Nacalai Tesque Inc., Kyoto, Japan) was partially degraded by nitrous acid generated in situ by addition of 10mg sodium nitrite (Wako Pure Chemical Industries Ltd., Tokyo, Japan) and hydrochloric acid (Nacalai; as required to make the pH 2.0) to 1 g heparin in normal saline at 4°C and stirred for a period of 3 h. On completion of degradation, the pH of the solution was adjusted to 7.4 with 1 N sodium hydroxide (Nacalai). GA-treated PP was coupled to 0.1% partially degraded heparin at room temperature for a period of 1 week. For covalent binding of heparin to PP, an intermediate surface-bound substrate containing amino groups was utilized. To prepare this intermediate surface-bound substrate containing amino groups, 0.1% chitosan (Sigma Chemical Co., St Louis, MO, USA) and 0.015% gentamicin sulphate (ScheringPlough, Osaka, Japan) in deionized water (pH 6.5) were coupled to free aldehyde groups of GA already bound to the PP at room temperature for 1 week. The partially degraded heparin was coupled with stable covalent bonds to aminated surfaces of GA-treated PP by reduction with 0.0125% sodium borohydride (pH saline for 24 h at room 8.4-8.8) in normal GA-chitosan-gentamicin-treated PPs temperature. without heparin and with heparin bonding were as PP of group 1 (GA-chitosanconsidered gentamicin-treated) and group 2 (GA-chitosangentamicin-heparin-treated), respectively. Since commercially available valves are 0.625% GA treated, for a control group (group 3), PPs were crosslinked with 0.625% GA in 0.067M phosphate buffer solution (pH 7.4) at 24 for 24 h followed by preservation in 0.2% GA in the same buffer at 24°C for more than 4 weeks, and were washed in copious amounts of normal saline before implantation. PP (1.5 cm x 1.5 cm) of either group 1, group 2 or group 3 was implanted into each of three surgically created subcutaneous pouches created in either side of the back (left side: two pockets-upper pocket for PP of group 1 and lower one for that of group 2; right side: one pocket for PP of group 3) of 15 3-week-old male Wistar rats (305Og). Wounds were closed with 4-0 polypropylene. Samples were retrieved 5 months (n = 15 for each group) after implantation. Since there was no specific site of calcification, a small section (2-3 mm) was taken from the mid-portion of each explanted PP, and stained with haematoxylin-eosin and von Kossa stains for and calcification study, general morphology respectively. The remaining portion of the retrieved PP was processed for quantitative calcium estimation by atomic absorption spectroscopy.
in calcification
prevention:
J. Chanda
et al.
Chemicals, Tokyo, Japan). A standard curve was obtained by using a standard calcium solution (Wako) in lanthanum solution. The amount of calcium was expressed as milligrams per gram of dry tissue.
RESULTS Severe calcification was observed in PPs of both groups 1 and 3 at 5 months of implantation. Calcific deposits were diffuse and destructive to the tissue of PPs of both groups 1 and 3 (Figure ~a and b). These pathological features reflected the value of the calcium level of PPs of both groups. The calcium content of PP of group 1 (208.16 f 80.17mg per dry weight) was virtually identical to that of PP of group 3 (228.32 f 37.39mgg-I) (P = 0.5258) after 5 months of implantation in weanling rats. Whereas no calcium was detected in PP of group 2 (Figure 2) at 5 months, the level of calcium of heparin bonded PP (group 2) achieved by 5 months was 0.652 f 0.240mg per g dry weight (P < 0.0001).
a
Calcium estimation The retrieved samples were dissected free of host tissue, rinsed with copious amounts of deionized water and sent to the Special Reference Laboratory (SRL, Tokyo, Japan) for calcium estimation. At the SRL, specimens were freeze dried to a constant weight and weighed. The amount of calcium was determined by atomic absorption spectroscopy (Hitachi Z 6100, Tokyo, Japan) at a wavelength of 422.7 nm on aliquots of 60% (13.3~) H2N03 (Kant0 Chemicals, Tokyo, ztp) I$rolysates of dried tissue, which were diluted chloride solution (Wako OLl lanthanum Biomaterials
1997, Vol. 18 No. 16
b Histological features of calcification of: a, 0.625% glutaraldehyde-treated (group 3) and b, glutaraldehydechitosan-gentamicin-treated (group 1) porcine pericardia implanted subcutaneously in 3-week-old rats for 5 months. Notice that porcine pericardia of both groups are severely calcified (black), and the calcification process damaged the tissue in general. von Kossa stain. Original magnification x100.
Figure 1
Heparin in calcificatiorl
prevention:
J. Chanda et a/.
Figure 2 Morphological characteristics of glutaraldehydechitosan-gentamicin-heparin-treated (group 2) porcine pericardium implanted subdermally in a 3-week-old rat for 5 months. Morphological features of porcine pericardium remain unchanged, as in the unimplanted graft, and no calcific deposit can be seen. von Kossa stain. Original magnification x100.
DISCUSSION Though the exact mechanism is unknown, the present study reports for the first time that heparin plays an important role in prevention of calcification of GAtreated bioprostheses implanted subcutaneously in weanling rats. At 37”C, GA cross-links with collagen fibres of grafts much more quickly. The stabilization process of collagen fibres with GA may be completed within 1 month if the concentrations of GA would be gradually increased from 0.1% to 0.25%. Since allograft valves fail due to calcification and degeneration in children17-20, non-calcifying bioprosthetic valves are essential, especially for paediatric patients. Dahm et ~1.~’ demonstrated that GA-treated porcine aortic valve did not provoke immunogenic responses in tivo. The extent of GA cross-linking is clearly important, although the specific mechanisms by which GA fixation :facilitates mineralization are not understood31. It may be hypothesized that, after implantation of GA-treated bioprostheses, free aldehyde moieties on the surface of the implant undergo oxidation, followed by acid formation, and this acid probably traps the host plasma calcium. The slow release of residual (unbound) GA from the prosthesis over a period of time after implantation reinforces the host plasma calcium-acid bound complex. This acid-plasma calcium complex promotes further mineralization when GA plays no further role as the primary factor for nucleation of calcificationz3. In cells modified by aldehyde crosslinking or mechanical injury, cell membranes are disrupted (leading to increased permeability), and mechanisms for calcium extrusion are no longer fully functional. Moreover, high energy phosphates (particularly ATP) required to fuel these mechanisms are accumulation unavailable. Thus, calcium occurs unimpeded with a dramatic increase in intracellular calcium31~ 32. Inactivation of residual GA and unbound aldehyde moieties on the surface of the prostheses either with amino compounds like chitosan (a biopolymer) or glycine + gentamicin c:ompletely prevented the calcification of GA-treated bioprostheses implanted subdermally in adult rats (120-15Og, 8 weeks 01d)23,33. Post-treatment
1111
with amino compounds prevents the slow release of residual GA, and inactivates the free aldehyde moieties on the surface of the GA-treated prostheses. Chitosan and gentamicin may serve these purposes. Due to the presence of a large number of amino termini, one chitosan molecule may covalently cross-link with a number of free aldehyde moieties on the surface of the GA-treated bioprostheses and simultaneously may inter-link between other chitosan molecules with the help of residual GA, slowly released from the treated tissuez3. The age of experimental animals plays an important role in the evaluation of the efficacy of any new anticalcification agent in bioprostheses34. Heparin coupling post-treatment with amino compounds (group 1) is ineffective in the prevention of calcification of GA-treated PP implanted subdermally in weanling rats. It is a well established fact that heparin has a potent antigrowth effect in smooth muscle cells35. It has also been shown that heparin binds to the surfaces of cells3”, and this may alter the permeability to ions necessary for growth, change confirmation of molecules to which it binds3’ or affect cell volume38. Furthermore, recent clinical and experimental studies suggest that he arin may be used as a preventive of atherosclerosis3 !L . We attempted to find out whether heparin has an anticalcification effect in GA-treated bioprostheses. For this purpose, heparin should be immobilized by stable bonds. We developed a method for covalent binding of heparin to GA-treated heterograft. An intermediate surface-bound substrate containing amino groups was utilized in the coupling reaction. To prepare this intermediate surface-bound substrate containing amino groups, chitosan and gentamicin were coupled to free aldehyde groups of glutaraldehyde already bound to the prosthesis. Heparin is partially depolymerized by deaminative cleavage with nitrous acid, which converts a 2-amino-2-deoxy-D-glucopyranosyl residue to a 2,5anhydro-D-mannose residue, with concomitant release of the aglycone43. The resulting heparin fragments are terminated by 2,5-anhydro-D-mannose units. These units have aldehyde functions that are not involved in intramolecular hemiacetal formation and, hence, are more reactive than terminal residues of unmodified heparin. Compounds containing aldehyde functions react with primary amines of chitosan and gentamicin to give labile Schiff bases that can be converted to stable secondary amines by reduction. The partially degraded heparin was coupled with stable covalent bonds to aminated surfaces by reduction with sodium borohydride. Free aldehyde moieties of partially degraded heparin bind covalently with the free amino groups of chitosan and gentamicin. The exact role of heparin in the anticalcification process of bioprostheses still remains elusive. Conceivably, coupling of heparin to chitosan-gentamicin-treated grafts fills the intertropocollagen spaces, blocks the potential binding sites and modifies charges, and thus makes the prostheses impermeable to host plasma calcium.
ACKNOWLEDGEMENTS We are grateful to Professor Kentaro Yoshimura, Head, Department of Parasitology of Akita University School Biomaterials
1997, Vol. 18 No. 16
Heparin incalcification prevention: J. Chanda et al.
1112 of Medicine, Akita, Japan for allowing us biochemistry laboratory for our ongoing wish to express our sincere appreciation to Abe of the Department of Parasitology University School of Medicine for his during the course of this work.
to use his work. We Dr Tatsuya of Akita suggestions
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Ross, D., Biological valves: stented and unstented. In Cardiology and Cardiac Surgery: Current Topics, ed. A. Grossi, F. Donatelli, A. Corno and R. Brodman. Futura, Mount Kisco, NY, 1992, pp. 203-206. Angell, W. W., Oury, J. H. and Shah, P., A comparison of replacement and reconstruction in patients with mitral regurgitation. J. Thorac. Cardiovasc. Surg., 1987, 93, 665-674. Duran, C., Kumar, N., Gometza, B. and Alhalees, A., Indications and limitations of aortic valve reconstruction. Ann. Thorac. Surg., 1991,52,447-454. Duran, C.G., Revuelta, J.M. and Gaite, L., Stability of mitral reconstructive surgery at lo-12 years for predominantly rheumatic valvular disease. Circulation, 1988, Suppl. I, 91-96. Cosgrove, D. M., Chaver, A.M., Lytle, B. W. et al., Results of mitral valve reconstruction. Circulation, 1986,74 (Suppl. I), 182-187. Bortolotti, V., Milano, A., Mazzucco, A. et al., Pregnancy in patients with a porcine valve bioprosthesis. Am. J. Cardiol., 1982, 50,1051-1057. Larrea, J. L., Nune, L., Reque, J. A. et al., Pregnancy and mechanical valve prostheses: a high risk situation for mother and the fetus. Ann. Thorac. Surg., 1983, 36, 459-465. Badduke, B. R., Jamieson, W. R. E., Miyagishima, R. T. et al., Pregnancy and child bearing in a population with biological valvular prostheses. J. Thorac. Cardiovasc. Surg.,1991,102,179-186. Gallo, I., Ruiz, B., Nistal, F. and Duran, C.M. G., Degeneration in porcine bioprosthetic cardiac valves: incidence of primary tissue failure among 938 bioprostheses at risk. Am. J. Cardiol., 1984, 53, 10611065. Zussa, C., Ottino, G., di Summa, M. et al., Porcine cardiac bioprostheses: evaluation of long-term results in 990 patients. Ann. Thorac. Surg., 1985, 39, 243-250. Jamieson, W.R.E., Munro, I., Miyagishima, R.T. et al., Carpentier-Edwards supraannular porcine The bioprosthesis. A new generation tissue valve with excellent intermediate clinical performance. J. Thorac. Cardiovasc. Surg., 1988, 96, 652-666. Burdon, T. A., Miller, D. C., Oyer, P. E. et al., Durability of porcine valves at fifteen years in a representative North American patient population. J. Thorac. Cardiovast. Surg., 1992, 103,238-252. Jamieson, W.R.E., Rosudo, L. J., Munro, A.I. et al., Carpentier-Edwards standard porcine bioprosthesis: primary tissue failure (structural valve deterioration) by age groups. Ann. Thorac. Surg., 1988,46, 155-162. Magilligan, D. J. Jr, Lewis, J. W., Stein, P. and Alan, M., The porcine bioprosthetic heart valve: experience at fifteen years. Ann. Thorac. Surg., 1989,46, 324-330. Jones, E. L., Weintraub, W. S., Craver, J. M. et al., Tenyear experience with the porcine bioprosthetic valves: interrelationship of valve survival and patient survival in 1959 valve replacements. Ann. Thorac. Surg., 1990, 49, 370-384. Geha, A. S., Laks, H., Stansel, H. C. et al., Late failure of porcine valve heterografts in children. J. Thorac. Cardiovasc. Surg., 1979, 76,351-364.
Biomaterials 1997, Vol. 18 No. 16
17.
18.
19.
20.
21 22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Monro, J.L., Salmon, A.P. and Keeton, B.R., The outcome of antibiotic sterilised homografts used in Fontan procedure. Eur. J. Cardiothorac. Surg., 1993, 7, 360-364. Cleveland, D. C., Williams, W. G., Razzouk, A. J. et al., Failure of cryopreserved homograft valved conduits in the pulmonary circulation. Circulation, 1992, 86 (Suppl. 2),150-153. Clarke, D.R., Campbell, D.N., Hayward, A.R. and Bishop, D.A., Degeneration of aortic valve allografts in young recipients. J. Thorac. Cardiovasc. Surg., 1993, 105,934-942. Yankah, A. C., Wottge, H. U., Miiller-Hermelink, H. K. et al., Transplantation of aortic and pulmonary allografts, enhanced viability of endothelial cells by cryopreservation. Importance of histocompatibility. J. Card. Surg., 1987, 1 (Suppl.), 209-220. Chanda, J., Anticalcification treatment of pericardial prostheses. Biomateriak, 1994, 15,465-469. Chanda, J., Rao, S. B., Mohanty, M. et al., Prevention of calcification of tissue valve. Artif Organs, 1994, 18, 752-757. Chanda, J., Prevention of calcification of heart valve bioprosthesis: an experimental study in rat. Ann. Thorac. Surg., 1995, 60, S339-S342. Chanda, J., Kuribayashi, R. and Abe, T., Use of the glutaraldehyde-treated chitosan post-treated porcine pericardium as a pericardial substitute. Biomaterials, 1996,17,1087-1091. Kuribayashi, R., Chanda, J. and Abe, T., Efficacy of the chitosan posttreatment in calcification prevention of the glutaraldehyde treated porcine aortic noncoronary cusp implanted in the right ventricular outflow tract in dogs. Artif Organs, 1996, 20,761-766. Yamaguchi, M., Ohashi, H., Imai, M., Oshima, Y. and Hosokawa, Y., Bilateral enlargement of the aortic valve ring for valve replacement in children. New operative technique. J. Thorac. Cardiovasc. Surg., 1991, 102, 202-206. Oku, H., Matsumoto, T., Ueda, M., Saga, T. and Shirotani, H., Semilunar valve replacement with a cylindrical valve. J. Card. Surg., 1993, 8, 666-670. Matsuda, K., Oda, T., Terai, H., Hanyu, M. and Ban, T., New surgical technique for repair of ventricular septal perforation. Ann. Thorac. Surg., 1995, 60,1430-1431. Fentie, I. H., Allen, D. J., Schenck, M. H. and Didio, L. J., Comparative electron microscopic study of bovine, porcine and human parietal pericardium, as materials for cardiac valve bioprostheses. J. Submicrosc. Cytol., 1986,18,53-65. Dahm, M., Husmann, M., Mayer, E., Pruffer, D., Groh, E. and Oelert, H., Relevance of immunogenic reactions for tissue failure of bioprosthetic heart valves. Ann. Thorac. Surg., 1995, 60, S348-S352. Webb, C. L., Bendict, J. J., Schoen, F. J., Linden, J. A. and Levy, R. J., Inhibition of bioprosthetic heart valve calcification with aminodiphosphonate covalently bound to residual aldehyde groups. Ann. Thorac. Surg., 1988, 46, 309-316. Schoen, F. J., Kujovich, J.L., Levy, R. J. and St. John Sutton, M., Bioprosthetic heart valve pathology: clinicopathological features of valve failure and pathobiology of calcification. Cardiovasc. Clin., 1988, 18(2), 289-317. Chanda, J., Posttreatment with amino compounds effective in prevention of calcification of glutaraldehyde treated pericardium. Artif. Organs, 1994, l&408410. Schoen, F. J., Levy, R. J., Nelson, A. C., Bernhard, W. F., Nashef, A. and Hawley, M., Onset and progression of experimental bioprosthetic heart valve calcification. Lab. Invest., 1985, 52,523-532. Hoover, R.L., Rosenberg, R., Haering, W. and Karnovsky, M. J., Inhibition of rat arterial smooth
Heparin in calcification prevention: J. Chanda et al.
36.
37.
38.
39.
40.
muscle cell proliferation by heparin: II. In vitro studies. Circ. Res., 1980,47, 578-583. Hiebert, L. M. and Jaques, L., The observation of heparin on endothelium after injection. Thromb. Res., 1976, 8, 195-204. Villanueva, G. imd Danishefsky, I., Evidence for a heparin-induced conformational change of antithrombin III. Biochem. Biophys. Res. Commun., 1977, 74, 803-809. Norman, M. and Norrby, K., Tumor cell volume changes in vivo induced by heparin. Pathol. Eur., 1971, 6,268-277. Ragazzi, E. and Chinellato, A., Heparin: pharmacological potentials from atherosclerosis to asthma. Gen. Pharmacol., 1995, 26, 697-701. Herrmann, H.C., Okada, S. S., Hozakowska, E. et al., Inhibition of smooth muscle cell proliferation and
1113
41.
42.
43.
experimental angioplasty restenosis by beta-cyclodextrin tetradecasulfate. Arterioscler. Thromb., 1993, 13, 924-931. Kanabrocki, E. L., Sothern, R. B., Bremner, W. F. et al., Heparin as a therapy for atherosclerosis: preliminary observations on the intrapulmonary administration of low-dose heparin in the morning versus evening gauged by its effect on blood variables. Chronobiol. Int., 1991, 8, 210-233. Shulman, A. G., Heparin and atherosclerosis: an investigative report on the treatment of atherosclerosis. Biomed. Pharmacother., 1990,44, 303-306. Hoffman, J., Lann, 0. and Scholander, E., A new method for covalent coupling of heparin and other substances containing glycosaminoglycans to primary amino groups. Carbohydr. Res., 1983, 117, 328-331.
Biomaterials 1997. Vol. 18 No. 16
ELSEVIER
0 1997 Elsevier Science Limited Printed in Great Britain. All rights reserved 0142-9612/97/$17.00
PII:SO142-9612(97)00033-l
Heparin in calcification prevention of porcine pericardial bioprostheses Jyotirmay Chanda, Ryosei Kuribayashi and Tadaaki Abe Department
of Cardiovascular
Surgery, Akita University School of Medicine, Akita 010, Japan
Calcific degeneration is the main cause of failure of glutaraldehyde-treated xenograft heart valve substitutes implanted in humans. Coupling of heparin through an intermediate surface-bound substrate containing amino groups showed complete prevention of calcification of glutaraldehydetreated porcine pericardium implanted subdermally in weanling rats for 5 months (heparin bonded pericardium: calcium, 0.625 f 0.24 mg g-‘; glutaraldehyde-only-treated pericardium: calcium, 228.32 I!Z37.39 mg g-‘; P < 0.0001). Conceivably, inactivation of unpaired aldehyde moieties present in bioprostheses after exposure to glutaraldehyde by amino compounds followed by blocking the potential binding sites of the graft with a surface modifying agent like heparin would be the key steps in the prevention of calcification and degeneration of glutaraldehyde-treated biological tissue grafts. 0 1997 Elsevier Science Limited. All rights reserved Keywords:
Heparin,
porcine
pericardium,
bioprosthetic
valve,
calcification
prevention
Received 6 November 1996; accepted 22 January 1997
Valve replacement surgery has been well established over the past 30 years and has been shared between mechanical and biological valves’. Valve repair instead of valve replacement is increasin ly attempted, but the procedure is not always feasible z?‘3; the haemodynamic results can be unsatisfactory4, the operation is techn:ically more difficult and the durability of the procedure is variable5. The alternative to mechanical valve replacement is the use of biological prostheses: there is no need for lifelong anticoagulation even in the presence of chronic atria1 fibrillation, and risk of systemic thromboembolism is very low. Furthermore, the quality of life can be considered very close to normal, allowing heavy work, sports, normal pregnancy and delivery, and life in remote areas6-8. The primary concern with bioprostheses, well documented over the previous years, is scepticism regarding durability and the potential morbidity and mortality related to degenerative prosthesesg-15. Glutaraldehyde (GA)-treated bioprosthetic valves result in rapid calcification and structural deterioration in paediatric and young populations12-‘6. The ideal cardiac valve for child-bearing patients is not yet available. Mechanical valves require lifelong anticoagulation, which may cause fetal deaths and birth defects7. Biological valves do not necessarily require lifelong anticoagulation, but their degeneration is accelerated during pregnancy6. To date, allografts are the best available biological heart valve substitutes for children17318. Though the long-term durability has been very satisfactory in adults, problems such as early degeneration and progressive Correspondence
calcification have resulted in the limited durability of the allograft valves in paediatric patientsl’-“. In previous works21-23, it was reported that chitosan post-treatment is effective in prevention of calcification of GA-treated bioprostheses implanted in adult rats (120-150g). GA cross-linked chitosan post-treated porcine pericardium (PP) functioned well as a pericardial substitute in mongrel dogsz4. Chitosan post-treatment is effective in calcification prevention of GA-treated porcine aortic non-coronary cusps implanted in the right ventricular outflow tract in dogsz5. Recently, we found that post-treatment with amino compounds including chitosan (present communication) is ineffective in prevention of calcification of GA-treated porcine heart valves implanted either in systemic circulation in juvenile sheep or subdermally in weanling rats. This fact prompted us to modify the previously proposed treatment21-23 for a better outcome. Like bovine pericardium, GA-treated PP is used in clinical practice26-28. PP is thinner than bovine and thicker than human pericardium, and may be used for construction of heart valve bioprostheses”. In this study, the efficacy of our newly developed chemical treatment for prevention of calcification of GA-treated PP implanted subdermally in weanling rats was evaluated.
MATERIALS
AND METHODS
PPs were cross-linked in GA (glutaraldehyde, EM 25%, TAAB Laboratories Equipment Ltd., Reading, UK) in normal saline (pH 7.4) with gradually increasing concentrations of GA from 0.1 to 0.25% in normal
to Dr J. Chanda.
1109
Biomaterials
1997, Vol. 18 No. 16
Heparin
1110
saline at 37°C for a period of 1 month. Heparin (sodium salt, 164.5 IUmggI; Nacalai Tesque Inc., Kyoto, Japan) was partially degraded by nitrous acid generated in situ by addition of 10mg sodium nitrite (Wako Pure Chemical Industries Ltd., Tokyo, Japan) and hydrochloric acid (Nacalai; as required to make the pH 2.0) to 1 g heparin in normal saline at 4°C and stirred for a period of 3 h. On completion of degradation, the pH of the solution was adjusted to 7.4 with 1 N sodium hydroxide (Nacalai). GA-treated PP was coupled to 0.1% partially degraded heparin at room temperature for a period of 1 week. For covalent binding of heparin to PP, an intermediate surface-bound substrate containing amino groups was utilized. To prepare this intermediate surface-bound substrate containing amino groups, 0.1% chitosan (Sigma Chemical Co., St Louis, MO, USA) and 0.015% gentamicin sulphate (ScheringPlough, Osaka, Japan) in deionized water (pH 6.5) were coupled to free aldehyde groups of GA already bound to the PP at room temperature for 1 week. The partially degraded heparin was coupled with stable covalent bonds to aminated surfaces of GA-treated PP by reduction with 0.0125% sodium borohydride (pH saline for 24 h at room 8.4-8.8) in normal GA-chitosan-gentamicin-treated PPs temperature. without heparin and with heparin bonding were as PP of group 1 (GA-chitosanconsidered gentamicin-treated) and group 2 (GA-chitosangentamicin-heparin-treated), respectively. Since commercially available valves are 0.625% GA treated, for a control group (group 3), PPs were crosslinked with 0.625% GA in 0.067M phosphate buffer solution (pH 7.4) at 24 for 24 h followed by preservation in 0.2% GA in the same buffer at 24°C for more than 4 weeks, and were washed in copious amounts of normal saline before implantation. PP (1.5 cm x 1.5 cm) of either group 1, group 2 or group 3 was implanted into each of three surgically created subcutaneous pouches created in either side of the back (left side: two pockets-upper pocket for PP of group 1 and lower one for that of group 2; right side: one pocket for PP of group 3) of 15 3-week-old male Wistar rats (305Og). Wounds were closed with 4-0 polypropylene. Samples were retrieved 5 months (n = 15 for each group) after implantation. Since there was no specific site of calcification, a small section (2-3 mm) was taken from the mid-portion of each explanted PP, and stained with haematoxylin-eosin and von Kossa stains for and calcification study, general morphology respectively. The remaining portion of the retrieved PP was processed for quantitative calcium estimation by atomic absorption spectroscopy.
in calcification
prevention:
J. Chanda
et al.
Chemicals, Tokyo, Japan). A standard curve was obtained by using a standard calcium solution (Wako) in lanthanum solution. The amount of calcium was expressed as milligrams per gram of dry tissue.
RESULTS Severe calcification was observed in PPs of both groups 1 and 3 at 5 months of implantation. Calcific deposits were diffuse and destructive to the tissue of PPs of both groups 1 and 3 (Figure ~a and b). These pathological features reflected the value of the calcium level of PPs of both groups. The calcium content of PP of group 1 (208.16 f 80.17mg per dry weight) was virtually identical to that of PP of group 3 (228.32 f 37.39mgg-I) (P = 0.5258) after 5 months of implantation in weanling rats. Whereas no calcium was detected in PP of group 2 (Figure 2) at 5 months, the level of calcium of heparin bonded PP (group 2) achieved by 5 months was 0.652 f 0.240mg per g dry weight (P < 0.0001).
a
Calcium estimation The retrieved samples were dissected free of host tissue, rinsed with copious amounts of deionized water and sent to the Special Reference Laboratory (SRL, Tokyo, Japan) for calcium estimation. At the SRL, specimens were freeze dried to a constant weight and weighed. The amount of calcium was determined by atomic absorption spectroscopy (Hitachi Z 6100, Tokyo, Japan) at a wavelength of 422.7 nm on aliquots of 60% (13.3~) H2N03 (Kant0 Chemicals, Tokyo, ztp) I$rolysates of dried tissue, which were diluted chloride solution (Wako OLl lanthanum Biomaterials
1997, Vol. 18 No. 16
b Histological features of calcification of: a, 0.625% glutaraldehyde-treated (group 3) and b, glutaraldehydechitosan-gentamicin-treated (group 1) porcine pericardia implanted subcutaneously in 3-week-old rats for 5 months. Notice that porcine pericardia of both groups are severely calcified (black), and the calcification process damaged the tissue in general. von Kossa stain. Original magnification x100.
Figure 1
Heparin in calcificatiorl
prevention:
J. Chanda et a/.
Figure 2 Morphological characteristics of glutaraldehydechitosan-gentamicin-heparin-treated (group 2) porcine pericardium implanted subdermally in a 3-week-old rat for 5 months. Morphological features of porcine pericardium remain unchanged, as in the unimplanted graft, and no calcific deposit can be seen. von Kossa stain. Original magnification x100.
DISCUSSION Though the exact mechanism is unknown, the present study reports for the first time that heparin plays an important role in prevention of calcification of GAtreated bioprostheses implanted subcutaneously in weanling rats. At 37”C, GA cross-links with collagen fibres of grafts much more quickly. The stabilization process of collagen fibres with GA may be completed within 1 month if the concentrations of GA would be gradually increased from 0.1% to 0.25%. Since allograft valves fail due to calcification and degeneration in children17-20, non-calcifying bioprosthetic valves are essential, especially for paediatric patients. Dahm et ~1.~’ demonstrated that GA-treated porcine aortic valve did not provoke immunogenic responses in tivo. The extent of GA cross-linking is clearly important, although the specific mechanisms by which GA fixation :facilitates mineralization are not understood31. It may be hypothesized that, after implantation of GA-treated bioprostheses, free aldehyde moieties on the surface of the implant undergo oxidation, followed by acid formation, and this acid probably traps the host plasma calcium. The slow release of residual (unbound) GA from the prosthesis over a period of time after implantation reinforces the host plasma calcium-acid bound complex. This acid-plasma calcium complex promotes further mineralization when GA plays no further role as the primary factor for nucleation of calcificationz3. In cells modified by aldehyde crosslinking or mechanical injury, cell membranes are disrupted (leading to increased permeability), and mechanisms for calcium extrusion are no longer fully functional. Moreover, high energy phosphates (particularly ATP) required to fuel these mechanisms are accumulation unavailable. Thus, calcium occurs unimpeded with a dramatic increase in intracellular calcium31~ 32. Inactivation of residual GA and unbound aldehyde moieties on the surface of the prostheses either with amino compounds like chitosan (a biopolymer) or glycine + gentamicin c:ompletely prevented the calcification of GA-treated bioprostheses implanted subdermally in adult rats (120-15Og, 8 weeks 01d)23,33. Post-treatment
1111
with amino compounds prevents the slow release of residual GA, and inactivates the free aldehyde moieties on the surface of the GA-treated prostheses. Chitosan and gentamicin may serve these purposes. Due to the presence of a large number of amino termini, one chitosan molecule may covalently cross-link with a number of free aldehyde moieties on the surface of the GA-treated bioprostheses and simultaneously may inter-link between other chitosan molecules with the help of residual GA, slowly released from the treated tissuez3. The age of experimental animals plays an important role in the evaluation of the efficacy of any new anticalcification agent in bioprostheses34. Heparin coupling post-treatment with amino compounds (group 1) is ineffective in the prevention of calcification of GA-treated PP implanted subdermally in weanling rats. It is a well established fact that heparin has a potent antigrowth effect in smooth muscle cells35. It has also been shown that heparin binds to the surfaces of cells3”, and this may alter the permeability to ions necessary for growth, change confirmation of molecules to which it binds3’ or affect cell volume38. Furthermore, recent clinical and experimental studies suggest that he arin may be used as a preventive of atherosclerosis3 !L . We attempted to find out whether heparin has an anticalcification effect in GA-treated bioprostheses. For this purpose, heparin should be immobilized by stable bonds. We developed a method for covalent binding of heparin to GA-treated heterograft. An intermediate surface-bound substrate containing amino groups was utilized in the coupling reaction. To prepare this intermediate surface-bound substrate containing amino groups, chitosan and gentamicin were coupled to free aldehyde groups of glutaraldehyde already bound to the prosthesis. Heparin is partially depolymerized by deaminative cleavage with nitrous acid, which converts a 2-amino-2-deoxy-D-glucopyranosyl residue to a 2,5anhydro-D-mannose residue, with concomitant release of the aglycone43. The resulting heparin fragments are terminated by 2,5-anhydro-D-mannose units. These units have aldehyde functions that are not involved in intramolecular hemiacetal formation and, hence, are more reactive than terminal residues of unmodified heparin. Compounds containing aldehyde functions react with primary amines of chitosan and gentamicin to give labile Schiff bases that can be converted to stable secondary amines by reduction. The partially degraded heparin was coupled with stable covalent bonds to aminated surfaces by reduction with sodium borohydride. Free aldehyde moieties of partially degraded heparin bind covalently with the free amino groups of chitosan and gentamicin. The exact role of heparin in the anticalcification process of bioprostheses still remains elusive. Conceivably, coupling of heparin to chitosan-gentamicin-treated grafts fills the intertropocollagen spaces, blocks the potential binding sites and modifies charges, and thus makes the prostheses impermeable to host plasma calcium.
ACKNOWLEDGEMENTS We are grateful to Professor Kentaro Yoshimura, Head, Department of Parasitology of Akita University School Biomaterials
1997, Vol. 18 No. 16
Heparin incalcification prevention: J. Chanda et al.
1112 of Medicine, Akita, Japan for allowing us biochemistry laboratory for our ongoing wish to express our sincere appreciation to Abe of the Department of Parasitology University School of Medicine for his during the course of this work.
to use his work. We Dr Tatsuya of Akita suggestions
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Ross, D., Biological valves: stented and unstented. In Cardiology and Cardiac Surgery: Current Topics, ed. A. Grossi, F. Donatelli, A. Corno and R. Brodman. Futura, Mount Kisco, NY, 1992, pp. 203-206. Angell, W. W., Oury, J. H. and Shah, P., A comparison of replacement and reconstruction in patients with mitral regurgitation. J. Thorac. Cardiovasc. Surg., 1987, 93, 665-674. Duran, C., Kumar, N., Gometza, B. and Alhalees, A., Indications and limitations of aortic valve reconstruction. Ann. Thorac. Surg., 1991,52,447-454. Duran, C.G., Revuelta, J.M. and Gaite, L., Stability of mitral reconstructive surgery at lo-12 years for predominantly rheumatic valvular disease. Circulation, 1988, Suppl. I, 91-96. Cosgrove, D. M., Chaver, A.M., Lytle, B. W. et al., Results of mitral valve reconstruction. Circulation, 1986,74 (Suppl. I), 182-187. Bortolotti, V., Milano, A., Mazzucco, A. et al., Pregnancy in patients with a porcine valve bioprosthesis. Am. J. Cardiol., 1982, 50,1051-1057. Larrea, J. L., Nune, L., Reque, J. A. et al., Pregnancy and mechanical valve prostheses: a high risk situation for mother and the fetus. Ann. Thorac. Surg., 1983, 36, 459-465. Badduke, B. R., Jamieson, W. R. E., Miyagishima, R. T. et al., Pregnancy and child bearing in a population with biological valvular prostheses. J. Thorac. Cardiovasc. Surg.,1991,102,179-186. Gallo, I., Ruiz, B., Nistal, F. and Duran, C.M. G., Degeneration in porcine bioprosthetic cardiac valves: incidence of primary tissue failure among 938 bioprostheses at risk. Am. J. Cardiol., 1984, 53, 10611065. Zussa, C., Ottino, G., di Summa, M. et al., Porcine cardiac bioprostheses: evaluation of long-term results in 990 patients. Ann. Thorac. Surg., 1985, 39, 243-250. Jamieson, W.R.E., Munro, I., Miyagishima, R.T. et al., Carpentier-Edwards supraannular porcine The bioprosthesis. A new generation tissue valve with excellent intermediate clinical performance. J. Thorac. Cardiovasc. Surg., 1988, 96, 652-666. Burdon, T. A., Miller, D. C., Oyer, P. E. et al., Durability of porcine valves at fifteen years in a representative North American patient population. J. Thorac. Cardiovast. Surg., 1992, 103,238-252. Jamieson, W.R.E., Rosudo, L. J., Munro, A.I. et al., Carpentier-Edwards standard porcine bioprosthesis: primary tissue failure (structural valve deterioration) by age groups. Ann. Thorac. Surg., 1988,46, 155-162. Magilligan, D. J. Jr, Lewis, J. W., Stein, P. and Alan, M., The porcine bioprosthetic heart valve: experience at fifteen years. Ann. Thorac. Surg., 1989,46, 324-330. Jones, E. L., Weintraub, W. S., Craver, J. M. et al., Tenyear experience with the porcine bioprosthetic valves: interrelationship of valve survival and patient survival in 1959 valve replacements. Ann. Thorac. Surg., 1990, 49, 370-384. Geha, A. S., Laks, H., Stansel, H. C. et al., Late failure of porcine valve heterografts in children. J. Thorac. Cardiovasc. Surg., 1979, 76,351-364.
Biomaterials 1997, Vol. 18 No. 16
17.
18.
19.
20.
21 22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Monro, J.L., Salmon, A.P. and Keeton, B.R., The outcome of antibiotic sterilised homografts used in Fontan procedure. Eur. J. Cardiothorac. Surg., 1993, 7, 360-364. Cleveland, D. C., Williams, W. G., Razzouk, A. J. et al., Failure of cryopreserved homograft valved conduits in the pulmonary circulation. Circulation, 1992, 86 (Suppl. 2),150-153. Clarke, D.R., Campbell, D.N., Hayward, A.R. and Bishop, D.A., Degeneration of aortic valve allografts in young recipients. J. Thorac. Cardiovasc. Surg., 1993, 105,934-942. Yankah, A. C., Wottge, H. U., Miiller-Hermelink, H. K. et al., Transplantation of aortic and pulmonary allografts, enhanced viability of endothelial cells by cryopreservation. Importance of histocompatibility. J. Card. Surg., 1987, 1 (Suppl.), 209-220. Chanda, J., Anticalcification treatment of pericardial prostheses. Biomateriak, 1994, 15,465-469. Chanda, J., Rao, S. B., Mohanty, M. et al., Prevention of calcification of tissue valve. Artif Organs, 1994, 18, 752-757. Chanda, J., Prevention of calcification of heart valve bioprosthesis: an experimental study in rat. Ann. Thorac. Surg., 1995, 60, S339-S342. Chanda, J., Kuribayashi, R. and Abe, T., Use of the glutaraldehyde-treated chitosan post-treated porcine pericardium as a pericardial substitute. Biomaterials, 1996,17,1087-1091. Kuribayashi, R., Chanda, J. and Abe, T., Efficacy of the chitosan posttreatment in calcification prevention of the glutaraldehyde treated porcine aortic noncoronary cusp implanted in the right ventricular outflow tract in dogs. Artif Organs, 1996, 20,761-766. Yamaguchi, M., Ohashi, H., Imai, M., Oshima, Y. and Hosokawa, Y., Bilateral enlargement of the aortic valve ring for valve replacement in children. New operative technique. J. Thorac. Cardiovasc. Surg., 1991, 102, 202-206. Oku, H., Matsumoto, T., Ueda, M., Saga, T. and Shirotani, H., Semilunar valve replacement with a cylindrical valve. J. Card. Surg., 1993, 8, 666-670. Matsuda, K., Oda, T., Terai, H., Hanyu, M. and Ban, T., New surgical technique for repair of ventricular septal perforation. Ann. Thorac. Surg., 1995, 60,1430-1431. Fentie, I. H., Allen, D. J., Schenck, M. H. and Didio, L. J., Comparative electron microscopic study of bovine, porcine and human parietal pericardium, as materials for cardiac valve bioprostheses. J. Submicrosc. Cytol., 1986,18,53-65. Dahm, M., Husmann, M., Mayer, E., Pruffer, D., Groh, E. and Oelert, H., Relevance of immunogenic reactions for tissue failure of bioprosthetic heart valves. Ann. Thorac. Surg., 1995, 60, S348-S352. Webb, C. L., Bendict, J. J., Schoen, F. J., Linden, J. A. and Levy, R. J., Inhibition of bioprosthetic heart valve calcification with aminodiphosphonate covalently bound to residual aldehyde groups. Ann. Thorac. Surg., 1988, 46, 309-316. Schoen, F. J., Kujovich, J.L., Levy, R. J. and St. John Sutton, M., Bioprosthetic heart valve pathology: clinicopathological features of valve failure and pathobiology of calcification. Cardiovasc. Clin., 1988, 18(2), 289-317. Chanda, J., Posttreatment with amino compounds effective in prevention of calcification of glutaraldehyde treated pericardium. Artif. Organs, 1994, l&408410. Schoen, F. J., Levy, R. J., Nelson, A. C., Bernhard, W. F., Nashef, A. and Hawley, M., Onset and progression of experimental bioprosthetic heart valve calcification. Lab. Invest., 1985, 52,523-532. Hoover, R.L., Rosenberg, R., Haering, W. and Karnovsky, M. J., Inhibition of rat arterial smooth
Heparin in calcification prevention: J. Chanda et al.
36.
37.
38.
39.
40.
muscle cell proliferation by heparin: II. In vitro studies. Circ. Res., 1980,47, 578-583. Hiebert, L. M. and Jaques, L., The observation of heparin on endothelium after injection. Thromb. Res., 1976, 8, 195-204. Villanueva, G. imd Danishefsky, I., Evidence for a heparin-induced conformational change of antithrombin III. Biochem. Biophys. Res. Commun., 1977, 74, 803-809. Norman, M. and Norrby, K., Tumor cell volume changes in vivo induced by heparin. Pathol. Eur., 1971, 6,268-277. Ragazzi, E. and Chinellato, A., Heparin: pharmacological potentials from atherosclerosis to asthma. Gen. Pharmacol., 1995, 26, 697-701. Herrmann, H.C., Okada, S. S., Hozakowska, E. et al., Inhibition of smooth muscle cell proliferation and
1113
41.
42.
43.
experimental angioplasty restenosis by beta-cyclodextrin tetradecasulfate. Arterioscler. Thromb., 1993, 13, 924-931. Kanabrocki, E. L., Sothern, R. B., Bremner, W. F. et al., Heparin as a therapy for atherosclerosis: preliminary observations on the intrapulmonary administration of low-dose heparin in the morning versus evening gauged by its effect on blood variables. Chronobiol. Int., 1991, 8, 210-233. Shulman, A. G., Heparin and atherosclerosis: an investigative report on the treatment of atherosclerosis. Biomed. Pharmacother., 1990,44, 303-306. Hoffman, J., Lann, 0. and Scholander, E., A new method for covalent coupling of heparin and other substances containing glycosaminoglycans to primary amino groups. Carbohydr. Res., 1983, 117, 328-331.
Biomaterials 1997. Vol. 18 No. 16