Asian Cardiovasc Thorac Ann 2001;9:19-23
© 2001 Asia Publishing EXchange Pte Ltd
Calcification of Experimental Valve Bioprostheses
Chung Sung Woon, MD,
Kim Jong Won, MD,
Lee Hyung Ryul, MD,
Kim Yeong Dae, MD,
Chung Hwang Kiw, MD
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Department of Thoracic and Cardiovascular Surgery Pusan National University College of Medicine Pusan, Korea
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For reprint information contact: Chung Sung Woon, MD Tel: 82 51 240 7267 Fax: 82 51 243 9389 email: chungsungwoon{at}hanmail.net #1-10 Ami-dong, Seo-ku, Pusan 602-739, Korea.
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Abstract
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Bovine pericardial strips, porcine valve strips, and canine valve strips were treated with 0.625% glutaraldehyde and implanted subdermally in rats. Weight and calcium content were examined 9 weeks later. Bovine pericardial strips underwent calcification after implantation; electron microscopy showed concentric electron-dense calcified deposits in the collagen fibers. Implanted porcine valve strips also underwent calcification; electron microscopy showed concentric electron-dense calcified deposits in the interstitium. Calcification was also detected in canine valve strips after implantation, but the proportion of calcium was lower than in the other tissues; electron microscopy showed collagen bundles with speckled calcified granules. The process of calcification started on the surface of the collagen fibrils and in the interfibrillar space. It was concluded that preservation of collagen fibers would be helpful in preventing calcification. The use of canine cardiac valves might improve the durability of bioprostheses.
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Introduction
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Several conditions must be met in order to resect a diseased cardiac valve and implant an artificial valve in patients: the material must not alter the blood components so as to cause thrombosis and it must be durable and noiseless.1 While artificial valves made from glutaraldehyde-treated porcine valve and bovine pericardium are widely used today, they have some problems, primarily degradation from calcification and a lack of long-term durability. Many clinicians are reluctant to use tissue valves because of their lack of durability, in spite of their anticoagulation advantages over mechanical valves. Degradation was studied in porcine cardiac valves, bovine pericardium, and canine cardiac valves with the aim of improving the durability of artificial tissue valves. Because studying artificial valves in a clinical setting is difficult, tissue samples have been subdermally implanted into mice. This method has some drawbacks as there is no direct contact between the experimental tissue and circulating blood, but it has been deemed acceptable for studying xenograft calcification.2
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Materials and Methods
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Fresh bovine pericardium from 18-month-old cows was used. The fatty tissue was removed and the remaining tissue was delivered to the laboratory in normal saline solution at 4°C. Porcine and canine hearts were delivered in the same manner; mitral and aortic valves were obtained from these hearts. Penicillin (100,000 u•L-1), streptomycin (100 mg•L-1), and fungizone (250 µg•L-1) were used as antibiotics. Twenty samples measuring 1 cm2 of each tissue were treated with 0.625% glutaraldehyde for 2 hours and stored for 48 hours at room temperature. Two samples were implanted subdermally in each of the 30 male Sprague-Dawley rats weighing approximately 100 g (range, 93 to 110 g). Ketamine (10 mg•kg-1) was injected as a 10% solution into the peritoneal cavity of each rat as an anesthetic. After anesthetizing the rat, the two samples were inserted subdermally at least 2 cm apart. Before implantation, each sample was dried and weighed using micro scales. The samples were reweighed after 9 weeks. The weight of the rats was approximately 300 g each at that time.
The calcium contents of the degenerated tissues were measured by mixing them with a 1:2 solution of 11.33 M hydrochloric acid and 13.36 M nitric acid and heating in a Teflon flask. This solution was transferred to a 25-mL flask and distilled water was added to make up the volume. An atomic absorption spectrophotometer was used to measure calcium concentration at 422.7 nm. Additional samples of bovine pericardium, porcine valve, and canine valve, which were treated only with glutaraldehyde and not implanted in the rat, were used as controls. After hematoxylin and eosin staining, photomicroscopy and electron microscopy were used to compare the difference between pre- and post-implanted tissue.
SPSS version 6.13 software (SPSS, Inc., Chicago, IL, USA) was used to conduct paired t tests and analysis of variance on the resulting data. A p value of less than 0.05 was considered significant.
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Results
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The treated bovine pericardial strips weighed between 20 and 34 mg before implantation and between 24 and 41 mg after implantation (Table 1
). The 26.26% increase in weight after implantation was statistically significant. From the increase in the percentage of calcium in the bovine pericardial strips after implantation, it was estimated that calcification contributed 26.31% of the weight gain, which was statistically significant (Table 2).
Treated porcine valve strips weighed between 19 and 36 mg before implantation and increased to between 23 and 42 mg after implantation (Table 1
). This represents a 23.56% increase in weight after implantation, which was statistically significant. It was estimated that calcification contributed 28.94% to the weight gain, which was statistically significant (Table 2).
Treated canine valve strips weighed 6 to 11 mg before implantation and 8 to 14 mg after implantation. The canine valve strips increased 21.82% in weight after implantation, which was statistically significant (Table 1). It was estimated that calcification contributed 4.7% to the weight gain, which was not statistically significant (Table 2).
No evidence of calcification was found in the preimplanted strips of bovine pericardium (Figure 1). In an electron microscopic study, concentric calcified deposits were found in the midst of the collagen fibers in the implanted tissues (Figure 2). No evidence of calcification was found in the preimplantation strips of porcine valve (Figure 3). Electron deposits associated with collagen fiber were noted in the porcine valve after implantation (Figure 4). The canine valve was composed of loose myxoid stroma and scattered fibroblasts when examined by light microscopy in the preimplantation samples (Figure 5). Under electron microscopy, collagen bundles speckled with calcified granules were found after implantation (Figure 6).
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Figure 1. Before implantation, the collagenous tissue of bovine pericardium shows no evidence of calcification (hematoxylin and eosin stain, original magnification x100).
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Figure 2. A concentric calcified deposit is detected within the collagen fibers of the implanted tissue (transmission electron micrograph, original magnification x20,000).
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Figure 3. A section of porcine heart valve shows no evidence of calcification in preimplanted tissue (hematoxylin and eosin stain, original magnification x100).
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Figure 4. Fine electron-dense deposits associated with the collagen fibers are noted in the implanted porcine heart valve (transmission electron micrograph, original magnification x70,000).
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Figure 5. The valve is composed of loose myxoid stroma and scattered fibroblasts (hematoxylin and eosin stain, original magnification x100).
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Figure 6. Collagen bundles speckled with calcified granules (transmission electron micrograph, original magnification x25,000).
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Discussion
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In spite of the development of artificial cardiac valves, there is no ideal prosthesis. Among the available tissue valves, the Carpentier-Edwards valve and the Hancock valve are made from porcine valve, while the Inoescu-Shiley valve and the Edwards pericardial tissue heart valve are made from bovine pericardium. However, it has been observed that alterations of cell shape, early degeneration of tissue, tearing of collagen, and loss of elastic fibrils occurred when a xenograft was used in the human body.3,4 These problems are due to inadequate preservation of tissues during transportation from the organ harvesting center, delays before tissue treatment, and inappropriate processes for fixation. Calcified degeneration was affected by the xenograft itself and the recipient.5,6
In 1969, Carpentier and colleagues7 introduced the glutaraldehyde-fixed artificial prosthesis and subsequently, many artificial valves were used clinically, but tissue calcification remained a troublesome factor in the durability of the prosthesis. In 1978, Gallo and colleagues8 reported that glutaraldehyde-treated porcine pericardium could be implanted in dogs with good results. Subsequently, it was implanted into the human body with good results in regard to adhesion and immune response. Formalin, ethylene glycol, and metaperiodate were used as preservatives and fixatives but glutaraldehyde has been established as the best choice.3,9 Glutaraldehyde is weakly acidic and crosslinks with the amino groups of collagen in alkaline environments, which protects the tissue from degeneration, inhibits platelet aggregation, and has a strong antibiotic effect.10
In a study by Chanda,11 pathological changes were similar in bioprostheses made from bovine pericardium and porcine valves, but more severe alterations were found in bovine pericardium. This study found less calcification in canine valve than in bovine pericardium and porcine valve. Calcification occurs as a result of the breakdown of the collagen component by physical stimuli and subsequent exposure of the calcium-binding sites to circulating blood.12,13 Calcification affects the durability of artificial valves, and many studies on the prevention of calcification have been carried out, but no ideal method has been developed yet.14 Calcified deposits were found in experiments using both rats and rabbits.4,15 Calcification was also observed in experiments using large animals such as sheep and calves.11,16
The findings in this study of calcium deposits in bovine pericardium and porcine valves becoming evident after implantation, agree with the results obtained by Maxwell and colleagues17 in valves harvested after use in patients. Calcified deposits occurred in collagen fibers and the interfibrillar space, as noted by Fishbein and colleagues5 in earlier experiments. Calcification also occurred in the canine valve, but it has less dense myxoid stroma and, therefore, less calcification. Because of this, canine valves may be better for long-term durability as prostheses. Electron microscopy showed that the process of calcification primarily starts on the surface of collagen fibrils and in the interfibrillar space. It is evident that the breakdown of collagen fibers is an essential step in the mechanism of calcification. Therefore, preventing collagen fiber from breaking down will be helpful in the prevention of calcification.
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References
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Abstract
Introduction
