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Ann Thorac Surg 1997;64:50-58
© 1997 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Refinement of the Alpha Aminooleic Acid Bioprosthetic Valve Anticalcification Technique

Carlyle Fraser Heart Center, Crawford Long Hospital of Emory University, Emory University School of Medicine, and Biomedical Design, Inc, Atlanta, Georgia; University of Michigan Medical Center, Ann Arbor, Michigan; and Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Accepted for publication December 26, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
Background. Aminooleic acid treatment has been demonstrated to prevent porcine valve calcification and to protect valvular hemodynamic function. Initial enthusiasm was tempered by histologic studies of these AOA valves, which showed cuspal hematomas, structural loosening, and surface roughening. This prompted a systematic review of the AOA treatment process. Unsolubilized particles of alpha aminooleic acid present in the treatment solution were identified as the cause of mechanical abrasion of valve cusps during processing. These particles were eliminated with a revamped protocol, which included filtration of the AOA solution before valve preparation.

Methods. Porcine aortic valve cusps treated with this modified AOA protocol (AOA II) were studied in a rat subdermal implant model of mineralization. A juvenile sheep trial was then used to confirm the antimineralization effects of AOA II on glutaraldehyde-fixed porcine aortic roots in a circulatory model of accelerated calcification.

Results. Retrieved AOA II-treated cusps from the subdermal model were markedly less calcified than control cusps (AOA II, 1 ± 0, 17 ± 4, 23 ± 6, and 17 ± 10 versus control, 189 ± 14, 251 ± 16, 250 ± 14, and 265 ± 10 mg calcium/mg sample at 4, 8, 12, and 16 weeks, respectively; p < 0.0001). Morphologic examination of the AOA II cusps of the valves retrieved from the sheep demonstrated freedom from the structural loosening, surface roughening, and hematoma formation that had limited the utility of the original AOA preparation technique. Cusps from AOA II-treated porcine roots had significantly less calcium than control cusps (AOA II, 5.5 ± 3.0 mg/g; control, 91.2 ± 19.5 mg/g; p = 0.0004). The aortic walls had similar levels of calcification (AOA II, 156 ± 73 mg/g; control, 159 ± 10 mg/g; p = not significant).

Conclusions. These data suggest that the modified AOA technique warrants further evaluation as an antimineralization treatment for glutaraldehyde-fixed porcine bioprostheses.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
The inconvenience, expense, and potential hazard of anticoagulation associated with mechanical prosthetic valve use remain a strong impetus in the search for improved bioprosthetic valve longevity. The Achilles' heel of the glutaraldehyde-fixed bioprosthetic valve has been the variable onset of tissue calcification, which often causes gross structural and functional failure. The AOA* process was designed to address this important clinical problem, and has consistently demonstrated superior anticalcification properties in various animal models [1–7]. The utility of the original AOA technique was limited by apparent abrasion of the bioprosthetic tissue surface during valve processing [1]. This suggested a potential for premature mechanical failure, which led to a reevaluation of the AOA protocol.

This study outlines identification of the component of the original AOA method associated with the deleterious effects, and documents the rationale for modification of the technique. The efficacy of the new anticalcification process was then examined in two biological models of accelerated tissue mineralization: a rat subdermal implant model and a juvenile sheep circulatory implant model. This modified process will be designated AOA II in this communication.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
Isolation of the Cause of Surface Roughening
Multiple variables were systematically tested to identify the component of the AOA process that was associated with the induction of the deleterious tissue surface phenomena. The characteristics of the valve tissue incubation medium were varied by filtration, by altering pH, and by changing alpha aminooleic acid and NaCl concentrations. The effect of these variables on cuspal tissue was evaluated by scanning electron microscopy. Cusps were washed three times for 10 minutes in 0.1 mol/L sodium phosphate buffer, pH 7.2, rinsed three times with distilled water, immersed in a 1% osmium tetroxide solution for 1 hour, and rinsed again three times with distilled water. They then were dehydrated with increasing concentrations of ethanol, critical-point dried, and sectioned in half to expose the inner layers, and each half was coated at 10 mA four times for 15 seconds with palladium-gold. The sections were examined using a Hitachi 8800 scanning electron microscope (Hitachi Naka City, Japan).

	Confirmation of Persistent Anticalcification Efficacy

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
SUBDERMAL IMPLANT MODEL.
The rat subdermal implant model developed by Levy and associates [8] was used as the initial biological assay to demonstrate persistence of the anticalcification properties after alteration of the original AOA technique. Cusps were dissected from glutaraldehyde-fixed porcine aortic valves (clinical grade material provided by Medtronic Valve Division, Irvine, CA) and incubated in the orbital shaker at 37°C for 72 hours in the AOA II solution. Cusps not submitted to the AOA treatment were used as controls. All samples were then kept at room temperature until use in a 0.2% glutaraldehyde solution.

Immediately before implantation the cusps were rinsed for 2 minutes in normal saline solution three times. Four cusps per animal were implanted subdermally on the abdomen of 3-week-old male Sprague-Dawley rats (12 rats, 48 cusps). The rats were anesthetized with a subcutaneous 1:1 mixture of 0.1 mL/100 g body weight of ketamine hydrochloride (100 mg/mL) and xylazine (20 mg/mL).

After 4, 8, 12, and 16 weeks, the cusps were retrieved, and the surrounding tissue was removed. The samples were then washed five times for 5 minutes with distilled water, and submitted for quantitative calcium analysis as previously described [2, 8, 9].

CIRCULATORY MODEL.
The juvenile sheep model was chosen to evaluate anticalcification efficacy of the AOA II process in a biological system known for accelerated mineralization of glutaraldehyde-fixed bioprostheses [10]. Juvenile sheep (Ovis aries) underwent implantation of a left ventricle-to-descending thoracic aorta conduit, incorporating either a 19-mm control glutaraldehyde-fixed or a 19-mm AOA II-treated porcine aortic root at the aortic anastomosis. These animals were 16 to 22 weeks old at implantation with a body mass range of 17 to 33 kg. Age was verified by time of molar eruption and dental table morphology. There was no significant difference in mean age or mass between groups. Forty sheep underwent operation to yield 9 control and 7 AOA II-treated animals that survived the early postoperative period.

Three of the 9 control animals survived less than 3 weeks, leaving 6 controls for the final quantitative calcium analysis. One of the 7 AOA II-treated valves was misrouted in transfer to the analyzing facility, yielding 6 AOA II-treated valves for final quantitative calcium analysis.

After an overnight fast the animals were given atropine (0.2 mg/kg) and acetylpromazine (0.55 mg/kg) intramuscularly. The operative and intravenous access sites were then sheared. Parenteral administration of prophylactic antimicrobials (cephapirin and penicillin G benzathine suspension) was begun preoperatively and continued for 72 hours. The animals were anesthetized with ketamine (22 mg/kg) followed by halothane inhalation and neuromuscular blockade with pancuronium (0.1 mg/kg). A large-bore tube was passed orally for rumen decompression.

The animals were placed in the right lateral decubitus position, and a left fourth interspace thoracotomy was made. The left internal mammary artery was isolated and then cannulated with a 20-gauge catheter for arterial pressure monitoring. The left azygos vein was isolated for pulmonary artery catheter access for thermodilution measurement of cardiac output (Edwards Swan-Ganz; Baxter, Santa Ana, CA). The intrapericardial inferior vena cava was encircled with a cotton umbilical tape tourniquet. The ascending aorta just proximal to its first branch was encircled by a felt-pledgeted cotton umbilical tape. A felt-pledgeted 2-0 polypropylene pursestring suture was placed around the apex of the left ventricle followed by four interrupted, felt-pledgeted 3-0 polypropylene horizontal mattress sutures incorporating the sewing ring of the 16-mm right-angled apical connector (model 174A Hancock; Medtronic, Inc) (Fig 1A). After a small left apical ventriculotomy was made with a no. 11 blade, the Foley balloon catheter–16-mm trocar (model 1701A Hancock; Medtronic, Inc) assembly was inserted and the balloon was filled with 5 mL of saline solution. The animal was given intravenous lidocaine, atropine, bicarbonate, heparin, and a 500-mL bolus of crystalloid fluid. The inferior vena cava was occluded with a tourniquet while the trocar was used to core a circular portion of apical myocardium (Fig 1B). The trocar-Foley catheter assembly with the plug of excised myocardium was removed. The apical connector was inserted and then secured with interrupted horizontal mattress sutures. Additional pledgeted sutures were required to achieve hemostasis. After placement of a partial exclusion clamp, the control or AOA II-treated porcine aortic root was anastomosed to the descending thoracic aorta with a running 4-0 polypropylene suture (Fig 1C). The clamp was removed, hemostasis was achieved, and the apical connector-to-porcine aortic root anastomosis was made. The ascending aorta was ligated with a felt-pledgeted cotton umbilical tape (Fig 1D). Cardiac outputs and transvalvular gradients were measured and the chest was closed.

View larger version (61K): [in this window] [in a new window]   Fig 1. . (A) Placement of circumferential apical mattress sutures, which anchor the valved conduit. (B) A Foley catheter introduced through a left ventricular apical stab wound serves as a guide for the rotational cutting action of the myocardial coring trocar. (C) Partial exclusion technique for placement of valved conduit onto the descending aorta. (D) The ascending aorta is occluded after completion of apical-to-descending aortic valved conduit. (Reprinted with permission from Wise DL, Trantolo DJ, Altobelli DE, Yaszemski M, Gresser JD, Schwartz ER, eds. Encyclopedic handbook of biomaterials and bioengineering. New York: Marcel Dekker, 1995, by courtesy of Marcel Dekker Inc.)

The animals were segregated by sex, housed in a controlled indoor environment, exercised daily, and had access to food (Rumilab chow and sweet alfalfa) and water ad libitum. There was no attempt to limit calcium administration to either group. Postoperative chronic anticoagulation was not employed in any animal. The animals were closely monitored for evidence of endocarditis with routine complete blood counts and blood cultures. They were under close veterinary medical supervision during the preoperative, convalescent, and chronic phases of the study. The sheep were given levamisole nematocidal treatment preoperatively and vaccinated against tetanus, Clostridium perfringens C and D, Vibrio, Pasteurella, and ovine ecthyma.

After an approximate 20-week implantation period, the animals were anesthetized and instrumented through a median sternotomy for the hemodynamic studies. High-fidelity micromanometers (Millar Instruments, Houston, TX) were placed into the descending thoracic aorta via the left femoral artery and into the conduit just proximal to the aortic valve. Thermodilution cardiac outputs were obtained with balloon-tipped flow-directed pulmonary artery catheters. After completion of the hemodynamic studies, the animals were given heparin (300 units per kg) and euthanized with Beuthanasia-D (Schering Corporation, Kenilworth, NJ).

The sheep underwent a formal, complete postmortem examination by a board-certified veterinary pathologist (Dirck Dillehay, DVM, PhD, Emory University). The heart was excised in continuity with the porcine aortic root. The conduit and porcine root were carefully removed and examined for the presence of vegetations, hematoma, thrombotic material, signs of degradation, tears or perforations, fibrous tissue ingrowth, perivalvular leaks, and calcific deposits. The porcine root was fixed in 10% neutral-buffered formalin. A radial section of each valve cusp and its adjacent aortic wall were processed for histologic examination. Specimens were embedded in JB-4 glycol methacrylate medium (Polysciences, Inc, Warrington, PA). Sections 2 to 3 µm thick were cut with glass knives and stained with hematoxylin and eosin, von Kossa's stain (for calcium phosphates), and the Movat pentachrome stain (for collagen, elastin, and mucopolysaccharides). The remainder of the leaflet and corresponding aortic wall sections were rinsed in sterile saline solution, lyophilized, pulverized to a fine powder, dried to constant weight in a desiccator, and hydrolyzed under vacuum in 6 N HCl at 85°C for 24 hours. Calcium levels were then determined by atomic absorption. All animals (sheep and rat studies) received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

	Results

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
Isolation of the Cause of Surface Roughening
Of all parameters tested, only 0.45-µm filtration of the AOA suspension consistently eliminated the surface damage phenomena observed by scanning electron microscopy of unimplanted cusps (Fig 2). Deleterious effects of AOA (original, unfiltered) treatment on unimplanted cusps are seen as focal areas of surface roughening and nodules with lipid-like vesicles within the tissue (Fig 2B). The AOA II (modified, filtered) treated cusps were free of the rough surface and nodules on examination (Fig 2C).

View larger version (95K): [in this window] [in a new window]   Fig 2. . Scanning electron microscopy of radial cross-sections of (A) an unimplanted untreated control cusp, (B) an unimplanted cusp treated in the original unfiltered AOA solution, and (C) an unimplanted cusp treated in the modified filtered AOA II solution. Note the nodules and the rough surface produced by the unsolubilized particles of alpha aminooleic acid during treatment in the original AOA solution (B) (arrows), and the smooth surface and absence of nodules in both the control untreated cusp (A) (arrows) and the AOA II-treated cusp (C) (arrows). (Reprinted with permission from Wise DL, Trantolo DJ, Altobelli DE, Yaszemski M, Gresser JD, Schwartz ER, eds. Encyclopedic handbook of biomaterials and bioengineering. New York: Marcel Dekker, 1995, by courtesy of Marcel Dekker Inc.)

	Subdermal Implant Model

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

View larger version (16K): [in this window] [in a new window]   Fig 3. . Calcium levels in control and AOA II cusps at 4, 8, 12, and 16 weeks in a rat subdermal implant model (n = 6 for each condition; values are means ± standard error of the mean).

	Circulatory Model

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

Causes of interim mortality in 3 animals were pneumonia, gastrointestinal atony secondary to vagal nerve injury, and a hemothorax from a lacerated intercostal artery related to chest tube placement. There was no evidence that porcine aortic root valvular dysfunction contributed to these deaths. These animals are not included in the analysis of results.

A fourth animal with a control valve implant died suddenly of cardiopulmonary failure at 15 weeks after implantation before hemodynamic studies could be done. Postmortem examination of this animal demonstrated moderate pulmonary congestion and a tightly stenotic, heavily calcified valve with a right coronary cusp that was markedly thickened by thrombus and vegetations. There was no evidence of bacterial invasion on Gram stain of the leaflet section. This animal was included in the analysis of mineralization results.

The early death of this control animal appeared related to accelerated degeneration and stenosis; therefore, the control valves were explanted at a slightly earlier time (average duration of implantation, 133 ± 15 days and 146 ± 12 days for control and AOA II-treated, respectively; p = 0.11, not significant) to ensure that hemodynamic data on these animals would be obtained. Although the implantation duration between groups was not significantly different, bias may have been introduced by early explantation of the control valves. This bias, if present, would serve to minimize rather than maximize any intergroup difference in calcification as the AOA II material had the longer duration of exposure to the circulation.

One animal with an AOA II-treated porcine aortic root had both an elevated white blood cell count at explantation as well as a blood culture positive for gram-positive cocci. This valve also had evidence of focal destruction of the right coronary cusp by gram-positive cocci. This animal was included in the hemodynamic and calcium analyses.

Gross examination demonstrated extensive calcification of the control valve leaflets whereas all AOA II-treated valve leaflets were soft and pliable, with no gross sign of calcification (Fig 4). Both AOA II and the control walls were extensively calcified (see Fig 4).

View larger version (0K): [in this window] [in a new window]   Fig 4. . Gross photographs and radiographs of removed AOA II-treated and control nonstented porcine valves implanted as apicoaortic shunts. (A) Gross appearance and (B) radiograph of AOA II-treated valve; prominent calcification is present in aortic wall, but not in cusps. (C) Gross appearance and (D) radiograph of control valve. Radiographic calcification of aortic wall of control specimen (D) is equivalent to that of the AOA II-treated specimen shown in B, and is accompanied by cuspal calcification (white arrow). Gross calcification of aortic wall in both B and D predominates along the margins of the sinuses of Valsalva. In A and C, the open arrow delineates the proximal and the closed arrow the distal anastomosis; the noncoronary cusp is on the left and the right cusp is at the right.

View larger version (0K): [in this window] [in a new window]   Fig 5. . Microscopic histology of removed AOA II-treated and control nonstented porcine valves implanted as apicoaortic shunts. (A) Cusp and (B) aortic wall of AOA-treated valve. No calcification is present in the cusp, but calcification is diffuse in the aortic wall, involving its entire cross-section, most predominantly in the innermost and outermost 20% of the media. (C) Cusp and (D) aortic wall of control valve. In C, calcification of the cusp is designated by the arrow. Aortic wall mineralization in D is equivalent in extent and location to that of the AOA II-treated valve in B. (All stained with von Kossa's reagent [calcium phosphates black]; A and C originally approximately x100, B and D originally approximately x25.)

View larger version (20K): [in this window] [in a new window]   Fig 6. . Calcium content analysis of AOA II and control valvular tissue (values are expressed as mean ± standard error of the mean).

	Comment

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
Alpha aminooleic acid is derived from the naturally occurring oleic acid by addition of an amine group at the alpha position of the molecule (Fig 7). This amine allows covalent binding of the compound to the free aldehyde moieties of the glutaraldehyde present in the fixed tissue. Presence of an avid alpha aminooleic acid–glutaraldehyde interaction is supported by tissue binding studies that demonstrate that the level of alpha aminooleic acid is three times higher in glutaraldehyde-fixed than in fresh tissue [7]. Long-term in vitro extraction studies using various solvents have shown tenacious alpha aminooleic acid binding to glutaraldehyde-fixed tissue [4, 7]. There is an initial rapid alpha aminooleic acid release within 24 hours then a plateau is gradually approached over the next 30 days, which remains relatively flat for the remainder of the 8-month binding study. Approximately 40% of the alpha aminooleic acid remains bound to the cuspal tissue long term with bovine serum as the extraction medium [7]. Biocompatibility studies have demonstrated the nontoxic nature of alpha aminooleic acid [6].

View larger version (15K): [in this window] [in a new window]   Fig 7. . The alpha aminooleic acid molecule is an 18-carbon fatty acid derived from oleic acid by adding an amine in the alpha position for binding to glutaraldehyde.

Studies of the effect of two valve storage variables-time from glutaraldehyde fixation to AOA treatment and time from AOA treatment to implantation-support the idea that the free aldehyde groups in the glutaraldehyde-fixed tissue are prime instigators of calcification [5]. After 1 year of storage the presence of free aldehyde groups in the glutaraldehyde-fixed tissue is attenuated, perhaps by formation of more intricate molecules via condensation products or pyridinium complexes. This is associated with a diminished capacity for calcification when implanted in the subdermal model. This effect can be had immediately, without the need for a delay of 1 year, by treatment with alpha aminooleic acid. The Schiff reaction between the alpha aminooleic acid amino group and the tissue free aldehyde may block the initial calcium influx into the tissue, which is thought to be responsible for initiation of mineralization. Some of the difficulties with study of mechanisms of action of alpha aminooleic acid are outlined by Chen and associates [4, 7].

The successful initial AOA subdermal and sheep circulatory studies demonstrated superior anticalcification properties compared with previously reported antimineralization techniques [1, 2]. Histologic studies of unimplanted cusps revealed a problem. The initial AOA solution damaged the surface and induced the formation of lipid-like nodules within the cuspal tissue. These tissue surface effects now appear to have been due to the constant friction of unsolubilized alpha aminooleic acid particles on the cuspal surface during incubation in an orbital shaker. Alpha aminooleic acid particles penetrated below the surface of the tissue to form the lipid-containing nodules. These nodules likely resulted from the complex binding between glutaraldehyde, tissue proteins, and alpha aminooleic acid particles. The nodules presumably decreased in size after implantation and eventually disappeared, thus leaving lacunes that became available for deposition of formed elements of the blood through the damaged surface. This would explain the presence of the hematomas and erythrocytes seen in the morphologic examination in the sheep implant study of valves treated with the original process.

The morphologic studies that demonstrated loss of surface integrity and mechanical disruption prompted reevaluation of the AOA process. The mandate was to maintain or enhance the anticalcification effect and to eliminate elements of the process that jeopardized structural integrity.

An iterative process allowed isolation of the factor that appeared most likely to be responsible for the mechanical damage. Filtration of unsolubilized alpha aminooleic acid particles eliminated the tissue surface abrasive potential from the medium during treatment. Because this procedure decreased the alpha aminooleic acid availability in the medium, it became necessary to compensate by increasing the concentration. These modifications resulted in the altered protocol: AOA (proprietary process, Biomedical Design and Medtronic, Inc). Morphologic examination of valve cusps after the treatment process and before implantation was an effective screening step yielding important information about the surface damage phenomenon. Once we were confident that the structural problem was resolved by filtering the alpha aminooleic acid solution, the issue then became proof of sustained antimineralization effect. The rat subdermal implantation model mimics the bioprosthetic tissue mineralization seen clinically. It has the advantages of economy with quantitative results available within weeks. In this model the new AOA process exhibited the ongoing superior anticalcification effect that had characterized the original process.

The next logical step was large animal testing in a circulatory model. The value of this model is that the entire bioprosthetic device can be implanted and exposed to the effects of hydraulic forces and metabolism on the bioprosthetic tissue. It is a prerequisite to consideration of clinical implantation.

A previously described technique [11] was modified to allow implantation of a valve size that is clinically relevant in a circulatory model. The juvenile sheep model would not accommodate orthotopic implantation of a clinically relevant 19-mm bioprosthesis due to the size constraints of the left ventricular outflow tract of these young sheep; thus we used the heterotopic position of the valve for this study. The goal of confirmation of the antimineralization effect of the AOA II process in a large mammalian circulatory model was achieved.

The protective effect of the AOA II process on porcine aortic valve cusps was excellent and confirmed the ongoing anticalcification efficacy of this process. Of note was the lack of inhibition of calcification in the aortic wall. Calcium deposition in the AOA II aortic wall and in the control aortic wall was similar in extent and morphology. The reason for differing levels of protection against calcification seen in the AOA II aortic walls as compared with the cusps is uncertain. The time course of entry of the alpha aminooleic acid molecule into the delicate cuspal tissue as opposed to the thicker aortic wall was investigated. The AOA tissue diffusion study of Chen and associates [4, 7] comparing cuspal and aortic wall tissue demonstrated transport of alpha aminooleic acid to be tenfold faster in the cusps than in the aortic wall. The logical remedy would appear to be a longer AOA-tissue incubation period. This strategy has met with only limited success. Efforts to increase AOA aortic wall content were realized, but these higher levels of alpha aminooleic acid did not translate into substantial inhibition of calcification [3]. This suggested that perhaps the biochemical composition of the aortic wall as compared with the cuspal tissue was the issue rather than presence of a certain quantity of AOA.

The previously demonstrated deleterious mechanical effect associated with the original AOA process has been resolved. This report documents ongoing efficacy of the AOA antimineralization technique after modification of the original process. Significant protection of glutaraldehyde-fixed porcine aortic valve cuspal tissue by AOA treatment was demonstrated in a controlled, chronic animal circulatory model of accelerated bioprosthetic calcification.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
We acknowledge Mark Torrianni for his technical expertise; David Pearson, who successfully synthesized in large quantities the aminooleic acid compound; the technical expertise and histologic skills provided by Sara Murray, Elena Rabkin, and Helen Shing; Dr Pan-Chih, Sara Katzmark, and Dr William A. Cooper for technical and administrative support; Gail Nechtman and Kaye Callihan for secretarial support; and Mark Rigler and Jeannette Taylor (from Material Analytical Services, Norcross, GA) for expert assistance during the scanning electron microscopic studies.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 
Address reprint requests to Dr Guyton, Crawford Long Hospital of Emory University, Suite 7700, 550 Peachtree St, NE, Atlanta, GA 30365.

* AOA is a trademark of Biomedical Design Inc. This study was funded in part by Medtronic, Inc, and Biomedical Design, Inc.

Doctors Marie-Nadia and Jean-Marie D. Girardot are owners of Biomedical Design, Inc, and thus have a financial interest in the use of this technique.

	References

Top Footnotes Abstract Introduction Material and Methods Confirmation of Persistent... Results Subdermal Implant Model Circulatory Model Comment Acknowledgments References

 

1. Gott JP, Pan-Chih, Dorsey LM, et al. Calcification of porcine valves: a successful new method of antimineralization. Ann Thorac Surg 1992;53:207–16.[Medline]
2. Girardot MN, Girardot JM, Schoen FJ, et al. Development of the AOA process as antimineralization treatment for bioprosthetic heart valves. Trans Soc Biomater 1993;16:266.
3. Girardot MN, Torrianni M, Girardot JM. Effect of AOA on glutaraldehyde-fixed bioprosthetic heart valve cusps and walls: binding and calcification studies. Int J Artif Organs 1994;17:76–82.[Medline]
4. Chen W, Kim JD, Schoen FJ, Levy RJ. Effect of 2-amino oleic acid exposure conditions of inhibition of calcification of glutaraldehyde cross-linked porcine aortic valves. J Biomed Mat Res 1994;28:1485–95.[Medline]
5. Girardot MN, Girardot JM, Torrianni M. Alpha-aminooleic acid (AOA) anticalcification effect on glutaraldehyde fixed heart valves: shelf-life studies. In: Gabbay S, Frater RWM, eds. New horizons and the future of heart valve bioprostheses. First ed. Austin, TX: Silent Partners, 1994:41–52.
6. Girardot JM, Girardot MN, Gott JP, et al. Preclinical testing for antimineralization treatments of heart valve bioprostheses. In: Wise DL, Trantolo DJ, Altobelli DE, Yaszemski M, Gresser JD, Schwartz ER, eds. Encyclopedic handbook of biomaterials and bioengineering, part II: applications, vol 2. New York: Marcel Dekker, 1995:1289–330.
7. Chen W, Schoen FJ, Levy RJ. Mechanism of efficacy of 2-amino oleic acid for inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic heart valves. Circulation 1994;90:323–9.[Abstract/Free Full Text]
8. Levy RJ, Schoen FJ, Levy JT, et al. Biologic determinants of dystrophic calcification on osteocalcin deposition in glutaraldehyde-preserved porcine aortic valve leaflets implanted subcutaneously in rats. Am J Pathol 1983;13:143–55.
9. Paula WJ, Bernick S, Nimni ME. Calcification of implanted vascular tissues associated with elastin in an experimental animal model. J Biomed Mater Res 1992;26:1169–77.[Medline]
10. Jones M, Eidbo EE, Hilbert SL, et al. Anticalcification treatments of bioprosthetic heart valves: in vivo studies in sheep. J Cardiac Surg 1989;4:69–73.[Medline]
11. Brown JW, Salles CA, Kirsh MM. Extraanatomical bypass of the aortic root: an experimental technique. Ann Thorac Surg 1977;24:433–8.[Abstract]

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