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Ann Thorac Surg 2005;79:905-910
© 2005 The Society of Thoracic Surgeons
a Chris Barnard Division of Cardiothoracic Surgery, Cape Heart Center, University of Cape Town, Cape Town, South Africa
Accepted for publication December 10, 2003.
* Address reprint requests to Dr Zilla, Cape Heart Center, Faculty of Health Sciences, University of Cape Town, Anzio Rd, Observatory 7925, Cape Town, South Africa
peter.zilla{at}uct.ac.za
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Abstract |
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-NH2 groups of tissue were seen to significantly inhibit bioprosthetic calcification. In the current study, the potential ability of 
-amino oleic acid (AOA) to act as a bridging molecule between -NH2- and COOH-dependent cross-links was hypothesized to similarly augment the anticalcification effect of the AOA molecule. METHODS: Porcine aortic wall tissue from Medtronic Freestyle valve bioprostheses incorporating the AOA anticalcification process additionally underwent carboxyl-group cross-linking with Jeffamine (poly[propylene glyco]-bis-[aminopropyl ether]) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Tissue was subdermally implanted into 5-week-old Long-Evans rats for 60 days. Standard 0.2% GA-fixed tissue served as a control. To further assess the impact of storage solution on AOA tissue, samples were either stored in GA (0.2%GA) or EDC (25 mmol/L carbodiimide) before implantation. Tissue calcification was assessed by atomic absorption spectroscopy and histochemical staining.
RESULTS: Aldehyde end-capping with AOA achieved only a modest reduction of calcification in GA-treated aortic wall tissue (–20.0%; p < 0.05). Replacing GA with EDC as a storage solution led to a further 32.4% (p < 0.01) mitigation of calcification in Freestyle tissue. Incorporating an intermediate EDC/Jeffamine cross-linking step achieved a distinct additional reduction of calcification by 40.4% (p < 0.05). Overall, aortic wall calcification was 59.7% (p < 0.0001) lower if commercial Freestyle tissue underwent an additional EDC/Jeffamine cross-linking step and subsequent storage in EDC. Relative to control GA-fixed tissue, this represented a 67.8% (p < 0.0001) reduction. Incorporation of AOA was essential for the beneficial effect of the additional EDC/Jeffamine cross-linking step.
CONCLUSIONS: Potentially utilizing both the amino- and the carboxyl moieties of AOA for tissue binding dramatically reduces aortic wall calcification of GA-fixed tissue.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Once it was apparent that a higher cross-link density represented a protective principle against bioprosthetic tissue calcification, a more profound emphasis on actual cross-link chemistry naturally ensued. Although initially confined to GA-based cross-linking, these studies soon revealed a more complex role for tissue fixation in the quest for long-lasting bioprosthetic heart valves. By introducing additional long-range GA cross-links by diamine bridges between terminal aldehyde groups [4], a stronger and partly different mitigation of bioprosthetic degeneration was found, hinting at a possible synergism between different cross-link types [5]. Subsequent experiments combining GA-based amino-group cross-links and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/Jeffamine (poly[propylene glyco]-bis-[aminopropyl ether])-based carboxyl-group cross-links conclusively proved such a synergism and thereby opened the field for further permutations [6]. By adding diamine-bridged GA bonds to amino-carboxyl double fixation, for instance, a distinct further reduction in bioprosthetic calcification could be achieved.
The present study went one step further by investigating whether the synergistic effect of different cross-link types could be additionally augmented by incorporating so called "anticalcification" molecules into the cross-links. For two reasons the anticalcification substance chosen for our experiments was -amino oleic acid (AOA). Firstly, it has been successfully used in clinically implanted heart valves for several years but, restricted to GA chemistry, did not develop the same anticalcification potential in aortic wall tissue as seen in leaflets. Secondly, it potentially offered itself as a highly hydrophobic molecule for "end-capping" of amino-groups in the tissue, thus avoiding terminal aldehyde residuals, a principle that proved highly successful before [16].
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Tissue Fixation
Glutaraldehyde (0.2% GA) fixed porcine aortic roots treated with the AOA anticalcification process were obtained as nonstented Freestyle aortic root prostheses from the Heart Valve Division of Medtronic (Santa Ana, CA) and served as the basis for all AOA-treated tissue. Non-AOA glutaraldehyde-fixed aortic roots served as controls. After rinsing, circular coupons of 12 mm diameter were punched from the supracommissural aortas of both Freestyle and control valves and transferred into either fresh GA (0.2%; weight:volume ratio = 1 g per 30 mL fixative solution) or, in the case of tissue destined to include EDC/Jeffamine cross-linking, placed in sterile-filtered Jeffamine/Aldrich/230D: 60 mmol/L in morpholino-ethane-sulfonic acid buffer/Aldrich: 0.27 mol/L, pH5) and agitated for 30 minutes at room temperature. In the latter, cross-linking was performed by placing the tissue into freshly prepared Jeffamine solution now containing EDC/Aldrich: 300 mmol/L) and NHS (N-hydroxysuccinimide/Aldrich: 0.1 mol/L) and agitating for 2.5 hours at room temperature. Subsequently, this tissue was rinsed five times with sterile, double-distilled, deionized water. Control tissue was stored in GA (0.2% GA in phosphate buffered saline [PBS]). Thus five groups were defined: 0.2% GA stored in GA (n = 6); Freestyle stored in GA (n = 6); Freestyle stored in EDC (n = 6); GA + EDC/Jeffamine stored in EDC (n = 6); and Freestyle + EDC/Jeffamine stored in EDC (n = 6).
Rat Implants
Anesthetic and surgical procedures were approved by the Research Ethics Committee of the University of Cape Town. Subdermal ventral abdominal implants were performed in 5-week old male Long-Evans rats (200 to 250 g) with each animal receiving maximally four aortic wall coupons that remained implanted for 60 days [7]. Treatment groups were not duplicated in the same animal and were rotated with respect to their relative implant positions. After retrieval of the implants, each tissue sample was divided into two equal halves before the calcium content was assessed both quantitatively (atomic absorption spectrophotometry) and qualitatively (light microscopy of von Kossa stains).
Calcium Analysis
To avoid the complication of an edge effect, a 1 mm wide circumferential ring was removed from all explanted samples destined for atomic absorption spectroscopy [7]. After liquid nitrogen milled tissue was dried at 104°C for 24 hours, it was weighed, ashed in a muffle furnace at 560°C for 12 hours, and dissolved in a 20% hydrochloric acid solution (10 mg dried tissue: 1 mL HCl). A lanthanum chloride solution (0.5%) was used for the final dilution. Absorption was measured at 422.7 nm on an atomic absorption spectrophotometer (Varian AA1275). Calcium levels were expressed as µg/mg of dry mass of tissue. Histologically, von Kossa stains with van Gieson counterstaining were made on paraffin wax sections of the tissue. Additionally, histochemical double-stains were prepared combining von Kossa calcium stains with either Victoria Blue stains for elastin or Azan stains for collagen.
Statistical Analysis
Atomic absorption spectroscopy calcium data were expressed as means ± standard error of the means. Inferential statistical analyses involved the Tukey-Kramer HSD (honestly significant difference) test (JMP version 4.02 software; SAS, Cary, NC). The Tukey-Kramer HSD test is sized for all differences among the means, thus allowing for protection across multiple inferences. A significance level of 0.05 (two-tailed) or less was accepted as being statistically significant.
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Results |
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Quantitative Calcium Analysis
If tissue was stored in 0.2% GA, the efficacy of the anticalcification agent AOA (as represented by Freestyle tissue) was only mildly discernable from GA-fixed control tissue (–20%, p < 0.05; Figs 3 and 4). However, simply substituting the GA storage solution with a 25 mmol/L EDC storage solution reduced calcium levels of the Freestyle tissue by 32.4% (p < 0.01). Thus, AOA treatment led to a 46.0% (p < 0.0001) reduction in calcification of 0.2%GA fixed aortic wall provided the tissue was stored in EDC. If Freestyle tissue additionally underwent a thorough cross-linking step with EDC/Jeffamine before storage in 25 mmol/L EDC, a further 40.4% (p < 0.05) mitigation in calcification could be achieved. In contrast, double fixation in GA and EDC/Jeffamine without accompanying AOA treatment had no effect on aortic wall calcification.
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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-amino-oleic acid dramatically mitigate leaflet calcification, they are ineffective in preventing the mineralization of the prosthetic aortic wall [8]. In the present study, we could eliminate this shortcoming by demonstrating that AOA, albeit able to significantly reduce calcification relative to standard GA fixed tissue, was also capable of more distinctly reducing aortic wall calcification if the treatment was followed by an EDC-dependent carboxyl-group activation step. By comparing various cross-link combinations with and without AOA, four main principles emerged: (1) neither AOA nor EDC—with or without Jeffamine—had any dramatic effect on aortic wall tissue on its own if used on top of conventional GA fixation and storage; (2) no remarkable effect of any combined treatment could be observed without the inclusion of AOA in the process; (3) if AOA represented an interim step between GA-based amino-group cross-linking and subsequent EDC-based carboxyl-group activation/cross-linking, a highly significant mitigation of aortic wall calcification was achieved; and (4) if AOA incorporation into GA-fixed tissue was followed by an EDC- based fixation and storage step, a dramatic and complete change in calcification pattern was seen.
It is this complete conversion of the well-known sandwich pattern of surface calcification into almost the reverse pattern that suggests that a fundamental change in calcification mechanisms is at the core of events. This possibility encourages one to discount certain conventional explanations for what is happening. Better tissue incorporation of AOA, for instance, falls to some degree into this category. In spite of the fact that it is likely to occur, it does not offer a satisfying explanation for the inversion of calcification patterns. It is well known that apart from a low efficiency of AOA incorporation into aortic wall tissue, there is a distinct leaching effect of the detergent over time [9]. Furthermore, it is widely believed that the chemical binding of AOA into the tissue occurs through the formation of a Schiff base between the -amino group of AOA and a terminal aldehyde residual of GA. If EDC exposure follows AOA treatment, not only tissue carboxyl groups are activated but also the carboxyl group of the AOA. Given the close proximity of both the NH2 and the COOH groups within the AOA molecule, dissolution of the unstable Schiff base is very likely. Among other options, a much more stable amide bond between the carboxyl group and a tissue amine group or inversely between an activated tissue carboxyl group and the alpha amino group of AOA is highly probable. In view of the high "excess" of carboxyl groups in the tissue, it is reasonable to assume that up to 60% of AOA molecules, which are thought to eventually leach out of the aortic wall tissue, may be retained through such amide bonds. Nevertheless, with reagent concentrations of the AOA process being optimized through commercial fine-tuning, the amount of originally introduced AOA could be defined as being titrated to a minimum. Therefore, although the tissue chemistry does suggest firmer incorporation of AOA in the course of EDC double-fixation, this would still only result in a relatively moderate increase in AOA in the tissue.
As much as an increase in tissue-bound AOA could explain a quantitative mitigation of calcification, it seems highly unlikely to cause the dramatic change in calcification pattern observed. This applies even more so in view of the likely shift of AOA molecules from terminal aldehyde residues toward amide bonds with the tissue, thereby "uncapping" the aldehydes. One of the assumed ways through which AOA was thought to act as an anticalcification molecule was its ability to end-cap these aldehyde residues. "Uncapping" would certainly rather contribute to an augmentation of the typical "sandwich pattern" of calcification than to its abolition. This is supported by recent studies in which the intrinsically pro-calcific effect of GA and its condensation products was confirmed. By optimizing the extraction procedure for excess GA [10] a distinct mitigation of aortic wall calcification could be achieved [11, 12]. Since the AOA process results in the undiminished presence of the "surface bands" of calcification [8] it can hardly have a strong end-capping effect on GA.
Alternatively, AOA was considered to act as a detergent, whereby a surfactant-extraction of phospholipids would reduce the initial nucleation sites in the cell membranes [13, 14]. Such an increased surfactant activity of AOA near the tissue surfaces is even more unlikely to explain the complete disappearance of calcification in the outer two thirds of AOA/EDC treated aortic wall tissue than its role as a GA end-capping molecule. Apart from the fact that the "freeing-up" of AOA from terminal aldehyde residues would not result in more "free" AOA but rather the opposite, Chen et al reported relatively little phospholipid extraction by AOA [15], suggesting that the usual detergent mechanism is not operative. However, even if the two-stage reaction of EDC/Jeffamine treatment could temporarily create a situation in which the activated carboxyl group of the AOA molecule reacts with the Jeffamine molecule and thus creates a hydrophilic tail on the AOA which would facilitate its penetration into the depth, it would not explain the distinct change of calcification pattern near the surfaces.
Therefore, it is likely that certain changes in the underlying cross-link chemistry have created a completely new situation for calcification. After almost two decades of focusing on "anticalcification" treatments, scientists have increasingly begun to elucidate the relationship between cross-link chemistry and bioprosthetic degeneration. Two recent observations may help to explain the effect of combined AOA/EDC treatment and both may relate to changes in the intermolecular tissue spaces: On the one hand it was shown that a high cross-link density inhibits calcification. This was facilitated by adding additional long-range bonds suggesting that some sort of spatial inhibition of crystal growth could play a role. On the other hand, this "space filling" effect may not require the physical presence of actual molecules dividing the intermolecular space but rather a functional equivalent. In a recent study EDC/Jeffamine was used to cross-link the carboxyl groups of bioprosthetic tissue after end-capping of the amines. It appeared that, rather than the actual length of "capping molecules" protruding into the intermolecular space, its hydrophobicity determined the degree of inhibition of calcification [16]. Moreover, the calcification pattern in aortic wall tissue blocked with the most hydrophobic molecule bore a striking resemblance to the "centralised" AOA/EDC pattern seen in our present study. Therefore it seems feasible that both actual space-filling and microstructure-enhanced hydrophobicity may play a role in the inhibition of surface calcification in AOA/EDC treated tissue.
Since both the zero-length cross-links between adjacent NH2 and COOH groups initiated by storage in EDC or the combined creation of zero-length cross-links as well as Jeffamine-based cross-links between carboxyl groups in the EDC/Jeffamine treatment did not seem to have any effect on calcification, it leaves the reaction products of AOA as the crucial elements responsible for the near abolition of calcification in the surface two-thirds of the tissue. Within the amide-bonds between AOA and the tissue, the abundance of COOH groups in the tissue - particularly after preceding GA fixation –makes those bonds between the NH2 of AOA and the COOH groups of the tissue more likely to occur. Furthermore, the EDC activation of the carboxyl group on the AOA molecule could also lead to polymerization of the molecule, potentially creating long hydrophobic "end-capping" chains.
Last not least –even if the likelihood is low because of the proximity of the NH2 and the COOH groups on the AOA molecule - one cannot exclude the possibility of creating long "bridging-cross-links" whereby the amino group of the AOA molecule binds to an activated tissue COOH group and the activated carboxyl group of the AOA molecule to either an amino group in the tissue or—through Jeffamine—to another carboxyl group in the tissue. All these possibilities make both explanations—the space filling and the increased presence of hydrophobic "capping" entities—a feasible option. Whether the preferential binding by the carboxyl group of the AOA molecule additionally alters the hydrophobicity of the molecule remains speculative. The fact that the only calcification sites found were deeply buried in the tissue indicates that the chemical change near the surface is even capable of overriding the presence of unblocked GA. The suggested preferential calcification of elastin indicates that the chemical reactions beneficial for other tissue components may not occur as readily in this unique structure, which is known to be resistant against even conventional fixation.
In summary, our study addressed a very practical and a theoretical issue. By breaking through the limitations of AOA in its use for stentless aortic bioprostheses, it may have contributed to a significant extension of the longevity of these modern replacement heart valves. By highlighting a dramatic effect of a change in tissue cross-link chemistry, it may help to further explain a variety of phenomena recently observed in connection with alternative tissue fixation.
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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