Bioprosthetic heart valves have been widely used to replace diseased cardiac valves over the past 30 years because of their excellent hemodynamics, safety of insertion, low risk of infective endocarditis, and low rate of thromboembolism without long-term anticoagulation.1 These advantages, however, are frequently undermined by the relatively high rate of valve failure because of progressive calcification and degeneration of the valve cusps, particularly in younger patients (<65 years) and the pediatric population.2 One of the mechanistic factors responsible for bioprosthetic cuspal calcification is glutaraldehyde fixation.3,4 Manji et al.5 demonstrated a strong relationship between inflammatory infiltration (e.g., macrophages) and calcification in glutaraldehyde-fixed bioprosthetic valves. Because of the fact that studying artificial valves in a clinical setting is difficult, tissue samples have been subdermally implanted into rats. This method has some drawbacks as there is no direct contact between the experimental tissue and circulating blood; nevertheless, it has been deemed acceptable for studying xenograft calcification.6
Materials and Methods
Tissue Preparation and Rat Subdermal Implantation
Fresh cardiac valve tissue (aortic and pulmonary) and pericardium of Phoca groenlandica (Harp Seal) were harvested and transported in normal saline solution at 4°C to the laboratory. The hearts had been donated from the Department of Fisheries and Oceans, Iles de la Madeleine, Quebec, Canada, during the annual population-control program. The tissues were fixed with buffered glutaraldehyde solution 0.625% at pH 7.4 for at least 1 month. Fresh bovine pericardia were harvested from 18-month-old cows from a local slaughter house and were processed in the same manner as the Phoca groenlandica tissues. The pericardium of both Phoca groenlandica and bovine origin was cut into discs of diameter 8 mm. Leaflets from aortic and pulmonary valves of Phoca groenlandica were cut radially into three parts.
Four groups (eight rats/group) of juvenile Wistar rats, 12 days old and of female sex (Center for Experimental Surgery, Biomedical Research Foundation of the Academy of Athens), were selected along with their mother and had a free alimentation regime. Each rat received four fragments of tissue at the dorsum, through four separate incisions (two at each side) each of length 1 cm (Table 1). All tissues were rinsed three times in normal saline solution for 10 minutes each time before implantation.
The rats were anesthetized with continuous inhalation of 2% isoflurane, during the whole procedure. The operation was performed under sterile conditions using betadine (povidone iodine). After inserting the tissues at the pouches produced bilaterally, the incisions were closed with surgical clips. The rats were returned to their mother. After 21 days of free alimentation, the rats were euthanized by inhalation of CO2. All procedures were approved by the Animal Care Committee of the Academy of Athens and performed according to the Guide for the Care and Use of Laboratory Animals prepared by the Institutes of Laboratory Animal Resources, National Research Council and published by the National Academy Press, revised 1996 (NIH publication No. 85-23).
The explanted tissues were rinsed three times in distilled water for 10 minutes each time. They were then dried and cut into two parts. One part was used for Ca measurement and the other for histological analysis.
Calcium Content Analysis
Explanted cardiac valve (aortic and pulmonary leaflets) and pericardium of Phoca groenlandica and bovine pericardium (BP) were lyophilized at −40°C at low pressure (high vacuum: 10−3 Torr on pump head). This was to reduce moisture by 5%, which was further reduced to <1% by secondary drying. The calcium content was analyzed by flat atomic absorption (Perkin Elmer Aanalyst 300, Waltham, MA) after digestion with 70% nitric acid at 96°C for 10 minutes. The temperature of the air-acetylene mixture used was 2,300°C. Analysis was performed with a hollow cathode lamp (HCL), wave length at 422.7 nm. Concentration is expressed in milligrams per gram of dry tissue.
The representative explanted tissues of each group were fixed in 4% formaldehyde and embedded in paraffin. Sections (4 μm thick) were deparaffinized in xylene followed by a graded series of ethanol to water. Sections were stained with hematoxylin-eosin, Weigert Van Gieson, and for demonstrating calcification the von Kossa staining. The principle of the von Kossa method is that silver ions are displaced from solution by carbonate or phosphate ions, becuse of their respective positions in the electrochemical series. The argentaffin reaction is photochemical in nature, and the activation energy is supplied from ultraviolet light. As the demonstrable forms of tissue carbonate or phosphate ions are invariably associated with calcium ions, the method may be considered as demonstrating sites of tissue calcium deposition.7
All numeric data were expressed as mean ± standard deviation (SD). For statistical analysis, the commercially available software package analysis of variance (ANOVA) Origin 8.0 for Windows (OriginLab Corporation, Northampton, MA) was used. p values of 0.05 or less were defined as a statistically significant difference.
The results of calcium analysis of all groups are listed in Table 2. There was a significant difference between Ca content in preimplant tissues, when compared with the Ca content in postexplant tissues (p < 0.001). These results show that significant calcification occurs in the cardiac valves (aortic and pulmonary) and pericardium of Phoca groenlandica as well as in the BP during the rat subdermal implantation. Preimplant seal pericardium has significantly different means, when compared with the other tissues, and seal pericardium has also significantly different means, when compared with pulmonary valve in postexplant samples (Table 3).
This calcification was further corroborated by light microscopy studies. All preimplant samples had highly organized tissues. There was a distinct pattern with layers of elastin, collagen, and muscle tissue, neatly arranged as shown with hematoxylin-eosin (Figure 1, A–D) and Weigert Van Gieson staining (Figure 2, A–D) without obvious calcification as shown by von Kossa staining (Figure 3, A–D).
The postimplantation samples showed findings consistent with foreign-body response containing fibroblasts, mononuclear cells, neutrophils, macrophages (hematoxylin-eosin, Figure 1, E–H), with a disorganized pattern of collagen, elastin, and smooth muscle tissue (Weigert Van Gieson, Figure 2, E–H), and with obvious calcification (von Kossa, Figure 3, E–H).
Pinnipeds (seals) have been under protection for over 4 decades, which is why their population has increased significantly. This has led to an imbalance in the food chain with seals “competing” with the national fishing industries for fish. For this reason, many nations with a significant seal population have introduced population-control programs to keep their number steady. For this reason, Canada allows the harvest of 360,000 seals/yr; Namibia (South West Africa) 98,000; Russia 20,000; and Norway 18,000. In addition, approximately 80,000 seals are harvested annually during the hunting season in Nunavut (Canada), Greenland, and Scotland. Fur, meat, and subcutaneous fat for omega-3 production are the only use of these animals, and the totality of internal organs is considered to be of no value.
Seals are mammals and as such they have a lot in common with other mammals such as humans. Their cardiac anatomy is similar to the human heart. The possibility of using seal cardiac valves and pericardium to replace diseased human cardiac valves is currently being investigated in our institution.
Recently,8 our team examined the possibility of using Harp Seal (Phoca groenlandica) trachea to replace large tracheal defects, using a glutaradehyde-preserved trachea from Harp Seals to replace segments of pig tracheas. All the experimental animals survived the operation and lived up to 39 days (mean: 30.8 days). No immunosuppressant drugs were used. Macroscopic and histological analysis showed an intact bioprosthesis but near total occlusion of the native trachea by a ring of inflammatory infiltration at the site of distal anastomosis.
Since 1969, when Carpentier et al.9 presented a glutaraldehyde-fixed bioprosthetic valve, many other investigators published their experiences with artificial valves preserved in glutaraldehyde. In all of these the main problem remained that of the lack of durability due to the cusp calcification. Early degeneration of tissue, tearing of collagen, and loss of elastic fibers are observed after human implantation.10 Moreover, calcified degeneration is affected by the xenograft itself and the recipient.11
Many preservatives and fixatives have been tried such as formalin, ethylene glycol, alcohol, and metaperiodate, but glutaraldehyde is still conventionally used, because it is the only proven technology for long-term tissue stabilization with the beneficial properties of preserving and sterilizing tissue.12 Glutaraldehyde is weakly acidic and crosslinks with the amino groups of collagen in an alkaline environment, which protects the tissue from degeneration, inhibits platelet aggregation, and has a strong antibiotic effect.13 It is known that glutaraldehyde itself plays an important role in the calcification process because of resident aldehyde and cytotoxicity.14 Therefore, modification to glutaraldehyde fixation to eliminate its adverse effects (e.g., inflammatory reaction) may provide a promising alternative.
Wang et al.15 used 0.3% tannic acid (TA) as an additional treatment to 0.625% buffered glutaraldehyde (Glut) solution. BP strips were subdermally implanted in juvenile male Sprague-Dawley rats and explanted 21 days after implantation. The data from quantitative calcium analysis and von Kossa staining showed that Glut-fixed BP developed significantly more calcification than Glut/TA-fixed BP. TA exerts excellent anticalcification effects on Glut-fixed BP by inhibiting macrophage infiltration and expression of matrix metalloproteinase-9 and tenascin-C.
Connolly et al.16 investigated a novel polyepoxide crosslinker for conferring both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized by reacting epichlorohydrin and NH3. Rat subdermal implants (porcine aortic cusps/BP) pretreated with TGA demonstrated significantly less calcification than Glut-pretreated implants.
Lipids play a significant role in the process of calcification of bioprosthesis. The process of calcification is associated with the absorption of proteins and phospholipids,17,18 and ethanol preincubation of glut-treated porcine aortic valve bioprosthesis may mitigate calcification.19
Shen et al.20 assessed whether lipid extraction by ethanol, ether, or a surfactant could mitigate calcification of glut-treated bioprosthesis. For this reason, on BP samples pretreated with 0.6% glutaraldehyde, lipid extraction was carried out using ethanol, ether, or the Tween 80 surfactant, and combinations thereof. The treated tissues were implanted subcutaneously in 50 juvenile rats for 4 and 6 months. The most effective pretreatments were the combination of ethanol and surfactant or the combination of ethanol, ether, and surfactant when compared with surfactant alone. The authors concluded that ethanol or the combination of ethanol and ether added to the currently used glutaraldehyde-surfactant treatment further mitigates calcification.
Calcification affects the durability of artificial valves, and many studies on the prevention thereof have been carried out. Calcified deposits were found in experiments using both rats and rabbits.21 Calcification was also observed in experiments using large animals such as sheep and calves.22 Chung et al.23 found less calcification in a rat subdermal implantation model using canine valve strips when compared with BP and porcine valve strips. 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 breakdown will be helpful in the prevention of calcification.
From our own experience, after having dissected approximately 300 seals of various age (paper in preparation), we found no calcific deposits on the aortic leaflets, in the coronaries, or in the wall of the major arterial vessels. This may be due to a combination of reasons such as their dietary habits, physical activity, and/or the rich concentration of omega-3 lipids in their tissues. These observations, combined with the results of preliminary data derived from a rat subdermal implantation, which was performed in an outside institution with semiquantitative observation alone and failed to reveal calcific deposits in the seal tissues, misled us to conclude that bioprosthetic valves derived from Phoca groenlandica cardiac tissues might not exhibit any calcification in vivo.24
From this study, using the flat atomic absorption technique for accurate calcium measurement and histological analysis with three different stainings, we conclude that there is a significant calcification in the cardiac valve and pericardium of Phoca groenlandica, with a disorganized pattern of collagen, elastin, and muscle fibers along with a foreign body response, becoming evident after 21 days of rat subdermal implantation. These findings necessitate the use of an anticalcification treatment or a change of the preservation solution used if we can consider using these valves for long-term implantation in humans.
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