Ann Thorac Surg 1997;64:1063-1066
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Prevention of Calcification in Glutaraldehyde-Treated Porcine Aortic and Pulmonary Valves
Jyotirmay Chanda, MD,
Ryosei Kuribayashi, MD,
Tadaaki Abe, MD
Department of Cardiovascular Surgery, Akita University School of Medicine, Akita, Japan
Accepted for publication April 12, 1997.
 |
Abstract
|
|---|
Background. The problem of calcification in porcine aortic (AVs) and pulmonary (PVs) valves and its relationship to glutaraldehyde (GA) is of current interest. We proposed an anticalcification treatment to develop noncalcifying porcine AVs and PVs.
Methods. Porcine AVs and PVs were cross-linked in GA. Partially degraded heparin was coupled to the GA-treated AVs and PVs through intermediate surface-bound substrate containing amino groups. Control AVs and PVs were cross-linked in 0.625% GA but had no heparin coupling. All specimens were implanted subdermally in 3-week-old rats for 5 months for calcification studies.
Results. Control AVs (Ca, 233.69 ± 42.61 mg/g) and PVs (Ca, 181.48 ± 4.06 mg/g) were severely calcified. Coupling of partially degraded heparin revealed complete prevention of calcification in GA-treated AVs (Ca, 0.73 ± 0.27) and PVs (Ca, 1.125 ± 0.22 mg/g) implanted subcutaneously in weanling rats for 5 months.
Conclusions. The proposed anticalcification treatment is effective in preventing calcification of GA-treated AVs and PVs implanted subcutaneously in weanling rats for 5 months.
|
Introduction
|
|---|
The pulmonary autograft is the best available aortic valve (AV) substitute in children although it is not possible to use it in every case [1, 2]. Early degeneration and progressive calcification have limited the durability of homograft valves in pediatric patients [1–5]. Glutaraldehyde (GA)-treated bioprosthetic valves exhibit rapid calcification and structural deterioration in the pediatric and young population [6–8].
The morphologic structure of the leaflets of porcine pulmonary valves (PVs) differs from those of the AV mainly with respect to thickness and collagen content. If the PV is used as a replacement device in the high-pressure aortic position or in the even higher pressure mitral position, the significantly lower collagen content compared with the aortic valve has the advantage of decreased stiffness, increased radial extensibility, and better preserved anisotropy after GA fixation [9]. Increased radial extensibility increases leaflet coaptation [10] and protects against both postimplantation reduction in extensibility [11, 12] and possible increases in aortic root diameter [9]. These features mean that the fixed porcine PV is mechanically more comparable with fresh AV.
Because calcification is the main cause of failure of all bioprostheses, proper anticalcification treatment should be developed before the introduction of any new bioprosthesis. In the hope of developing noncalcifying bioprostheses, we have evaluated our anticalcification treatment in porcine AVs and PVs.
|
Material and Methods
|
|---|
Groups 1 and 2
Porcine AVs and PVs were cross-linked in GA (Glutaraldehyde EM 25%, TAAB Laboratories Equipment Ltd., Reading, UK) in normal saline solution (pH 7.4) with gradually increasing concentrations of GA from 0.1% to 0.25% in normal saline solution at 37°C for a period of 1 month. Glutaraldehyde-treated AVs and PVs were coupled to 0.1% partially degraded heparin (depolymerized by deaminative cleavage with nitrous acid). For covalent binding of heparin to these AVs and PVs, an intermediate surface-bound substrate containing amino groups was used. This intermediate surface-bound substrate containing amino groups was prepared by coupling 0.1% chitosan (Sigma Chemical Co, St. Louis, MO) and 0.015% gentamicin sulfate (Schering-Plough, Osaka, Japan) to free aldehyde groups of GA already bound to the AVs and PVs. The partially degraded heparin was coupled with stable covalent bonds to aminated surfaces of GA-treated AVs and PVs by reduction with sodium borohydride at pH 8.4. Aortic valves and PVs without and with heparin bonding were grouped as follows: group 1 (GA-chitosan-gentamicin–treated AVs and PVs) and group 2 (GA-chitosan-gentamicin-heparin–treated AVs and PVs).
Group 3
Control AVs and PVs were treated with 0.625% GA in 0.067 mol/L phosphate-buffer solution (pH 7.4) at 2° to 4°C for 24 hours followed by preservation in 0.2% GA in the same buffer at 2° to 4°C for more than 4 weeks, and were washed in copious amounts of normal saline solution before implantation.
Thirty (15 for AVs and 15 for PVs) 3-week-old male Wistar rats (30 to 50 g) were used for this study. An entire AV leaflet (AVL) or PV leaflet (PVL) of 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: 2 pockets, upper pocket for AVLs or PVLs of group 1 and lower one for that of group 2; right side: one pocket for AVLs or PVLs of group 3) of each rat. 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 to 3 mm) was taken radially from midportion of each explanted cusp, and stained with hematoxylin and eosin and von Kossa stains for general morphology and calcification study, respectively. The remaining portion of the retrieved AVLs or PVLs was processed for quantitative calcium estimation by atomic absorption spectroscopy.
Calcium Estimation
The retrieved samples were dissected free of host tissue, rinsed with copious amounts of deionized water, and sent to SRL, Tokyo, Japan, for calcium estimation. 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 wave length 422.7 nm on aliquots of 60% (13.3 N) H2NO3 (Kanto Chemicals, Tokyo, Japan) hydrolysates of dried tissue, which were diluted with 1% lanthanum chloride solution (Wako Chemicals, Tokyo, Japan). A standard curve was obtained by using the calcium standard solution (Wako) in lanthanum solution. The amount of calcium was expressed as milligrams per gram of dry tissue.
|
Results
|
|---|
Although the calcium content of AVLs of group 1 (164.50 ± 15.58 mg/g) was less than that of control AVLs (group 3) (233.69 ± 42.61 mg/g; p < 0.001), morphologically, AVLs of both groups 1 and 3 were severely calcified when implanted subdermally in weanling rats (Fig 1
). As in AVLs, severe calcification was observed in practically all three layers of PVLs of groups 1 and 3 at 5 months of implantation (Fig 2). Unlike in AVLs, calcified deposits were less in PVL of group 1 when compared with those in PVLs of group 3. These pathologic features reflected the value of calcium level of PVL of both groups. Calcium content of PVLs of group 1 (63.86 ± 3.15 mg/g) was significantly less than that of PVLs of group 3 (181.48 ± 4.06 mg/g, p < 0.001). Both heparin-bonded AVLs and PVLs (group 2) did not calcify at all (Figs 3, 4). The level of calcium of AVLs and PVLs of group 2 achieved by 5 months was 0.73 ± 0.27 mg/g and 1.13 ± 0.22 mg/g, respectively.
View larger version (94K):
[in this window]
[in a new window]
|
Fig 1. . Light microscopic features of calcification of a leaflet of 0.625% glutaraldehyde-treated porcine aortic valve (group 3) implanted in a weanling rat for 5 months. Note the fairly diffuse involvement by irregular granular deposits of calcium practically in all three layers of the leaflet. (Von Kossa stain; x50 before 51% reduction.)
|
|
View larger version (82K):
[in this window]
[in a new window]
|
Fig 2. . Histologic features of calcification of 0.625% glutaraldehyde-treated porcine pulmonary valve (group 3) implanted in 3-week-old rat for 5 months. Note that like the aortic valve leaflet (Fig 1 ), all layers of the leaflet of PV are diffusely calcified. (Von Kossa stain; x50 before 51% reduction.)
|
|
View larger version (94K):
[in this window]
[in a new window]
|
Fig 3. . Light microscopic appearance of radial section of a cusp of heparin-bonded porcine aortic valve (group 2) implanted in 3-week-old rat for 5 months. No calcified deposit can be seen in the leaflet. (Von Kossa stain, x50 before 51% reduction.)
|
|
View larger version (100K):
[in this window]
[in a new window]
|
Fig 4. . Morphologic characteristics of a leaflet of heparin-coupled (group 2) porcine pulmonary valve show no calcification when implanted in 3-week-old rat for 5 months. (Von Kossa stain; x50 before 51% reduction.)
|
|
|
Comment
|
|---|
The present study reports that coupling of heparin prevents the calcification of GA-treated porcine AV and PV bioprostheses implanted subcutaneously in weanling rats for 5 months. Posttreatment with amino compounds (group 1) retards the calcification process to some extent, but it does not prevent the calcification of GA-treated bioprostheses implanted in weanling rats.
Direct mechanical comparisons between porcine PVs and AVs indicate that these valves have nearly identical mechanical properties [13, 14]. Although short-term results of aortic valve replacement with pulmonary homograft are satisfactory [15], a prediction of the future of the pulmonary allograft or treated xenograft must not be based on excellent behavior of the pulmonary autograft (Ross switch operation), for the autograft is fully viable and nonantigenic and excites no host reaction [9]. In vitro testing does not answer this question completely because the leaflet in vivo is progressively altered by the host response, potentially weakening the tissue by biologic process [9]. Porcine PVLs are thinner than AVLs and GA fixation did not affect this relationship [13]. The thinner PVLs are subject to higher stress if orthotopically implanted in the mitral or aortic position. Whether this will result in early leaflet failure is conjecture; the outcome depends on the presently unknown rate of tissue fatigue in vivo [9].
Heparin has an inhibitory effect on proliferation of vascular smooth muscle cells [16]. It has been shown that heparin binds to the surface of cells [17] and this may alter permeability to ions necessary for growth, change configuration of molecules to which it binds [18], or affect cell volume [19]. Furthermore, recent clinical and experimental studies suggest that heparin may be used as a preventive of atherosclerosis [20–22].
On the basis of previous experiments [23–25], to achieve better cross-linking we have promoted fixation of the tissue in GA with gradual increase in concentrations of GA from 0.1% to 0.25% for a period of 1 month at 37°C. Free aldehyde moieties of partially degraded heparin cannot bind with the same moieties of residual GA on the surface of the bioprosthesis. This is the reason an intermediate surface-bound substrate containing amino groups (chitosan and gentamicin) was used in the coupling reaction.
The exact role of heparin in the anticalcification process of bioprostheses is unknown. With the proposed anticalcification treatment, GA-treated porcine PVs with or without pulmonary artery may prove useful for right ventricular outflow tract reconstruction or as a replacement device in the low-pressure system (pulmonary circulation). If satisfactory results concerning hemodynamics and calcification prevention are achieved after orthotopic implantation in the mitral position in juvenile sheep, the GA-treated porcine AVs and PVs may become the desired heart valve substitutes.
|
Acknowledgments
|
|---|
This work was partly supported by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture (Monbusho) of Japan. We thank Professor Kentaro Yoshimura, Head and Chairman, Department of Parasitology, Akita University School of Medicine, Akita, Japan, for allowing us to use his biochemistry laboratory to carry out the present work.
|
Footnotes
|
|---|
Address reprint requests to Dr Chanda, Department of Cardiovascular Surgery, Akita University School of Medicine, Akita 010, Japan (e-mail: jchanda{at}med.akita-u.ac.jp).
|
References
|
|---|
- Monro JL, Salmon AP, Keeton BR. The outcome of antibiotic sterilised homografts used in Fontan procedure. Eur J Cardiothor Surg 1993;7:360–4.[Abstract]
- Cleveland DC, Williams WG, Razzouk AJ, et al. Failure of cryopreserved homograft valved conduits in the pulmonary circulation. Circulation 1992;86(Suppl 2):150–3.
- Gallo R, Kumar N, Prabhakar G, Al-Halees Z, Duran CMG. Accelerated degeneration of aortic homograft in an infant. J Thorac Cardiovasc Surg 1994;107:1161–2.[Free Full Text]
- Clarke DR, Campbell DN, Hayward AR, Bishop DA. Degeneration of aortic valve allografts in young recipients. J Thorac Cardiovasc Surg 1993;105:934–42.[Abstract]
- Yankah AC, Wottge HU, Müller-Hermelink HK, et al. Transplantation of aortic and pulmonary allografts, enhanced viability of endothelial cells by cryopreservation. Importance of histocompatibility. J Card Surg 1987;1(Suppl):209–20.
- Burdon TA, Miller DC, Oyer PE, et al. Durability of porcine valves at fifteen years in a representative North American patient population. J Thorac Cardiovasc Surg 1992;103:238–52.[Abstract]
- Jamieson WRE, Rosudo LJ, Munro AI, et al. Carpentier-Edwards standard porcine bioprosthesis: primary tissue failure (structural valve deterioration) by age groups. Ann Thorac Surg 1988;46:155–62.[Abstract]
- Fann JI, Miller DC, Moore KA, et al. Twenty-year clinical experience with porcine bioprostheses. Ann Thorac Surg 1996;62:1301–12.[Abstract/Free Full Text]
- Christie GW, Barratt-Boyes BG. Mechanical properties of pulmonary valve leaflets: how do they differ from aortic leaflets? Ann Thorac Surg 1995;60:S195–9.
- Christie GW. The anatomy of aortic heart valve leaflets: the influence of glutaraldehyde fixation on function. Eur J Cardiothorac Surg 1992;6(Suppl 1):S23–33.
- Christie GW, Barratt-Boyes BG. Identification of a failure mode of the antibiotic sterilized aortic allograft after 10 years: implications for their long-term survival. J Cardiac Surg 1991;6:462–7.[Medline]
- Barratt-Boyes BG, Christie GW, Raudkivi PJ. The stentless bioprosthesis: surgical challenges and implications for long-term durability. Eur J Cardiothorac Surg 1992;6(Suppl 1):S39–43.
- David H, Boughner D, Vesely I, Gerosa G. The pulmonary valve: is it mechanically suitable for use as an aortic valve replacement. ASAIO J 1994;40:206–12.[Medline]
- Leeson-Dietrich J, Boughner D, Vesely I. Porcine pulmonary and aortic valves: a comparison of their tensile viscoelastic properties at physiologic strain rates. J Heart Valve Dis 1995;4:88–94.[Medline]
- Gerosa G, Ross DN, Brucke PE, et al. Aortic valve replacement with pulmonary homografts: early experiences. J Thorac Cardiovasc Surg 1994;107:424–37.[Abstract/Free Full Text]
- Caplice NM, West MJ, Campbell GR, Campbell J. Inhibition of human vascular smooth muscle cell growth by heparin. Lancet 1994;344:97–8.[Medline]
- Hiebert LM, Jaques L. The observation of heparin on endothelium after injection. Thromb Res 1976;8:195–204.[Medline]
- Villanueva G, Danishefsky I. Evidence for a heparin-induced conformational change of antithrombin III. Biochem Biophys Res Commun 1977;74:803–9.[Medline]
- Norman M, Norrby K. Tumor cell volume changes in vivo induced by heparin. Pathol Eur 1971;6:268–77.[Medline]
- Ragazzi E, Chinellato A. Heparin: pharmacological potentials from atherosclerosis to asthma. Gen Pharmacol 1995;26:697–701.[Medline]
- Kanabrocki EL, Sothern RB, Bremner WF, 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;8:210–33.[Medline]
- Shulman AG. Heparin and atherosclerosis: an investigative report on the treatment of atherosclerosis. Biomed Pharmacother 1990;44:303–6.[Medline]
- Chanda J. Anticalcification treatment of pericardial prostheses. Biomaterials 1994;15:465–9.[Medline]
- Chanda J, Rao SB, Mohanty M, et al. Calcification prevention of tissue valve. Artif Organs 1994;18:752–7.[Medline]
- Chanda J. Prevention of calcification of heart valve bioprostheses: an experimental study in rat. Ann Thorac Surg 1995; 60:S339–42.
Top
Abstract
Introduction
