Calcification characteristics of porcine stentless valves in juvenile sheep1

Paul Herijgersa, Shigeyuki Ozakia, Eric Verbekenb, Alfons Van Lommelb, Rozalia Racza,Miroslaw Zietkiewicza, Bartlomiej Pereka, Willem Flamenga,*

aCenter for Experimental Surgery and Anaesthesiology, Division of Experimental Cardiac Surgery, Provisorium I,Minderbroedersstraat 17, B-3000 Leuven, Belgium

bDepartment of Pathology, K.U. Leuven, B-3000 Leuven, Belgium

Received 21 September 1998; received in revised form 7 December 1998; accepted 16 December 1998

Abstract

Objective: To compare calcification characteristics of two porcine stentless valves (Toronto SPV and Freestyle) with differentdesigns, fixation and antimineralization techniques using a juvenile sheep model of valve implantation inside the circulation.Methods: The stentless valves (n = 2 × 6) were implanted in juvenile sheep in the pulmonary artery as an interposition, while thecirculation was maintained with a right ventricular assist device. The model was validated by the implantation of, clinically well-known, porcine (Hancock II) and pericardial (Pericarbon) valves. Half of the valves were explanted after 3 months, the rest after 6months. Valves were examined macroscopically, by X-ray, light microscopy (HE, Masson, Von Giesson, Von Kossa, PTAH stains),and transmission electron microscopy. Quantitative determination of the calcium content of the cusps was performed with atomicabsorption spectrometry.Results: After 3 months, the Freestyle had an extensively calcified aortic wall, most prominent at the outflowside of the porcine valve. After 6 months, calcification increased transmurally, but the valve cusps were free of calcification, and theinflow side was only slightly calcified. The Toronto SPV valve also started to calcify at the inflow side of the valve after 3 months withincreased calcification after 6 months. The base of the Toronto SPV valve cusps showed slight calcification after 6 months ofimplantation.Conclusions: The pattern of calcification of the porcine aortic wall differs between the two studied stentless valves,with calcification located predominantly at the outflow side in the Freestyle valve, but also at the inflow side in the Toronto SPVvalve. The cusps of the Freestyle valve were less prone to calcification than those from the Toronto SPV valve. 1999 Elsevier ScienceB.V. All rights reserved.

Keywords:Experimental; Juvenile sheep; Stentless aortic valve; Bioprosthesis; Cardiac surgery; Calcification

1. Introduction

Stentless bioprosthetic valves potentially have impor-tant advantages due to the low gradients over the valveand the fact that formal anticoagulation is not necessary,enabling the patient to regain a fully active life within ashort time period and causing fast regression of the leftventricular hypertrophy [1]. The durability of free-hand-sewn aortic valve homografts used for aortic valve

replacement in humans is greater than for stented aortichomografts [2]. In analogy with this, it is expectedthat the durability of stentless heterografts will be super-ior to that of its stented counterparts. This has been shownexperimentally by Hazekamp et al. [3], with the stentedIntact bioprosthesis (Medtronic, Irvine, CA), and itsclinically unavailable stentless counterpart in growingpigs.

Thus far, only the first medium- and long-term studies arebeing published concerning the clinical results of stentlessaortic bioprosthetic xenograft implantations [4]. Experi-mentally, juvenile sheep are a known model for biopros-thetic valve implantation, since they resemble the humansituation concerning annular sizes, heart rate, cardiac outputand intracardiac pressures. Most important, however, is the

European Journal of Cardio-thoracic Surgery 15 (1999) 134–142

1010-7940/99/$ - see front matter 1999 Elsevier Science B.V. All rights reserved.PII : S1010-7940(98)00313-3

* Corresponding author. Tel: +32-16-337298; fax: +32-16-337855.1 Presented at the 12th Annual Meeting of the European Association for

Cardio-thoracic Surgery, Brussels, Belgium, September 20–23, 1998.

fact that bioprostheses implanted for a few months in juve-nile sheep show changes comparable with those that takeseveral years to develop in bioprostheses implanted inpatients [5]. Bioprosthetic valve implantation in aortic [6],left ventricle apico-aortic conduit [7], mitral [8], or tricuspid[5,8] position in juvenile sheep is most frequently used totest the durability and calcification characteristics of bio-prosthetic valves. These models, however, are complicated,because full extracorporeal circulation is necessary in mostmodels, or important blood loss occurs, with a mortality ratefar over 50% [7,8]. The extracorporeal circulation, the long-term housing of the sheep for such experiments, and thehigh mortality make these models very expensive. Prob-ably for this reason, no experimental data are available atpresent comparing degeneration in several stentless biopros-thetic valves directly. Therefore, we decided to build aneasier and cheaper model of in vivo bioprosthetic valvetesting.

Several types of stentless valves are clinically available.They differ considerably in terms of design, composition ofmaterials, tissue preservation and anticalcification treat-ment. We have chosen to compare the calcification charac-teristics in two clinically widely available stentless aorticporcine valves, the Toronto SPV valve (St. Jude Medical, St.Paul, MN), and the Medtronic Freestyle valve (Medtronic,Irvine, CA).

2. Materials and methods

All animals received humane care in compliance withthe European Convention on Animal Care. The sudywas approved by the ethics committee of the Katho-lieke Universiteit Leuven. Juvenile sheep (less than 6months old) breeded specifically for this purpose wereselected.

2.1. Valves studied

Two clinically available stentless valves were selectedfor this study, the Toronto SPV valve (St. Jude Medical, St.Paul, MN) and the Freestyle valve (Medtronic, Irvine, CA).Both are porcine aortic roots, fixed with glutaraldehyde, butthe Toronto SPV valve has its three sinuses scalloped,whereas, the Freestyle is an intact root with both coronarystumps ligated. Furthermore, the outside of the TorontoSPV is fully covered with Dacron, whereas, the Freestylevalve is only partially covered at the base of the root overthe muscle bar that is most prominent near the right cor-onary cusp. The Freestyle valve is fixed without transvalv-ular gradient, with the aortic root under 40 mmHg, theToronto SPV under low pressure (about 2 mmHg). Theconcentration of the glutaraldehyde used for fixation is dif-ferent. The Freestyle valve is treated after fixation withalpha aminooleic acid (AOA), an agent known to reducecalcification [7,9].

2.2. Implantation

The sheep were fasted for 48 h. The animals were pre-medicated with ketamine (10–20 mg/kg intramuscularly).Anesthesia was induced with increasing concentrations ofhalothane in oxygen. Albipen LA (15 mg/kg, Mycofarm,Brussels, Belgium) was administered intramuscularly forantibiotic profylaxis. Anesthesia was maintained withhalothane and N2O. Fentanyl (Janssen, Beerse, Belgium)was administered in boluses as necessary. After endotra-cheal intubation, mechanical ventilation was instituted. Allventilation parameters were adjusted to keep the arterialblood gasses and pH within the physiological range. Theleft chest was shaved, prepped and draped, and a left thor-acotomy was performed in the second interspace. Afteradministration of 100 mg of lidocaine (Xylocard, Astra,Sodertalje, Sweden) intravenously, the pericardium wasincised taking care not to damage the phrenic nerve, andthe heart suspended in a pericardial cradle. The main pul-monary artery was completely isolated. After administra-tion of 3 mg/kg heparin (Novo, Bagsvaerd, Denmark)intravenously, a pneumatic right ventricular assist system(Medos HIA-VAD 54 ml ventricle, Medos-HelmholtzInstitute, Aachen, Germany) was installed with the inflowcannula in the right atrium and the outflow cannula 1 cmbefore the pulmonary bifurcation. The pulmonary arterywas clamped immediately above the pulmonary valve. Asecond clamp was placed immediately proximal to the out-flow cannula. In the mean time, the chosen valve was rinsedas prescribed in the manual. The valves were implanted asan interposition with running 5/0 polypropylene sutures.After removal of the clamps, the native pulmonary valvewas destroyed by tearing two cusps with a clamp intro-duced through a purse-string suture placed at the sinuses,and afterwards the Medos system was stopped. Carefulhemostasis was performed. The chest was closed in layerswith a chest drain in the left pleural space. After waking up,the animal was extubated and brought to the recoveryroom. Feeding was allowed immediately. Intravenousfluid administration was stopped after 2 h. The chestdrain was removed after 6 h. The animals received analge-sics (piritramide, Dipidolor, Janssen, Beerse, Belgium) forthe first 2 days on regular schemes and diuretics, as neces-sary. Albipen LA and low molecular weight heparin (enox-aparine, 20 mg twice daily, Clexane, Rhoˆne–PoulencRorer, Brussels, Belgium) were administered for 6 days.Afterwards, the sheep returned to the controlled animalfacility where the general health of the sheep was checkeddaily.

2.3. Explantation and analysis

Half of the valves were explanted after 3 months, theother half after 6 months. Sheep were premedicated andanesthetized in the way described before. The left thoracot-omy was reopened and the heart dissected free. Heparin 3

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mg/kg was administered, and after exsanguination, thevalve was excised together with a proximal and distal partof the sheep pulmonary artery.

2.3.1. Macroscopical examinationValves were grossly inspected and color photographs

were taken. Special attention was paid to retraction ofthe cusps, or any deformed or indurated parts of thevalve. Afterwards the valve was longitudinally tran-sected through the commissures. Each of the three speci-mens thus includes a pre- and postvalvular part of thesheep pulmonary artery, together with a part of the por-cine aortic wall (wall of the stentless valve), and res-pectively, a right coronary cusp (RCC), a non-coronarycusp (NCC), or a left coronary cusp (LCC). Color pictureswere taken again.

2.3.2. X-ray assessmentX-ray examination (face, profile) was performed under

mammography conditions to demonstrate and localizemacroscopical calcification.

2.3.3. HistologyFor histology, a longitudinal section of the specimen

through the middle of the LCC was embedded in paraffin.Four-micrometer thick sections were routinely stained withhematoxylin and eosin (HE), Masson’s trichrome stain forcollagen, an elastic Von Giesson stain, a phosphotungstic-acid-hematoxylin (PTAH) for fibrin, and a Von Kossa cal-cium staining.

2.3.4. Transmission electron microscopyFor transmission electron microscopy, the RCC was

divided in a basal part, a middle part, and the free edgeof the valve. Also the aorta inflow and outflow weresampled. From each of the five regions, three to ten samples(,1 mm) were embedded in Epon. The samples were nottaken from parts with massive calcification, since thisyielded, on the photographs, only large black areas withoutadditional information. One micrometer-thick sectionswere stained with Toluidine blue and examined by lightmicroscopy. From each of the three cusp fragments anarea of the outflow side, the inflow side and the middle(deep) part was dot marked. By analogy, from the aorticinflow and outflow, also the intimal inner medial and outermedial wall and adventitia were thus sampled. Ultra-thinsections were cut, stained with uranylacetate and leadcitrate. Sections were treated with 2% potassium pyroanti-monate to demonstrate calcium. Grids were examined in aPhilips CM 10 electron microscope. Random photographswere taken.

2.3.5. Quantitative calcium determinationHalf of every segment was used for quantitative calcium

determination. The cusps were divided in three parts: com-missural area, basal part and free edge. After lyophilization,

the tissue was pulverized, and desiccated to constant weightin an oven. Hydrolysates were made in 6 N HCl. Calciumcontent was measured by flame atomic absorption spectro-metry, and expressed as mg/mg of dry cuspal weight.

2.3.6. Data management and statistical analysisQuantitative data were expressed as median (range),

since the data did not follow a normal distribution. Compar-isons were made with the Mann–WhitneyU-test for com-parison between the two groups, and the Kruskal–WallisANOVA when more than two groups were compared. Thelevel for statistical significance was put at 0.05. Data man-agement and statistical analysis were done with Statistica4.5 (Statsoft, Tulsa, OK).

3. Results

Two sheep died during or shortly after the implantationprocedure. One died during the operation due to a tear in thepulmonary artery extending distal to the outflow cannula,with rapid exsanguination of the sheep. Another sheep dieddue to respiratory insufficiency within 6 h after the opera-tion. One sheep died 20 days postoperatively due to endo-carditis of the tricuspid valve and the bioprosthetic valve inpulmonary position. One sheep died 28 days postopera-tively, with necropsy showing pneumonia without evidenceof endocarditis. These four sheep were replaced by newexperiments and excluded from the study. Global mortalitywas thus 25%.

3.1. Macroscopical examination

The explanted Freestyle valves showed massive calcifi-cation of the aortic wall, forming an entirely rigid tubealready after 3 months, that remained macroscopicallyunchanged after 6 months. All cusps after 3 months werenicely pliable, without macroscopical signs of calcification.Occasionally, slight fibrous sheathing was seen near thecommissures and at the base of the cusps after 3 months.After 6 months the fibrous sheathing increased, somewhat,at the inflow side of the cusps, however never completefibrous overgrowth encapsulating the cusps was seen. Allcusps were functioning.

The explanted Toronto SPV valves showed also exten-sive calcification of the porcine aortic wall. A comparablefibrous reaction as in the Freestyle valve was also seen in theToronto SPV. Contrary to the situation in the Freestylevalves, some punctiform scattered calcification was seennear the commissures, and slight induration at the base ofthe cusps. This increased after 6 months.

3.2. X-ray examination

Already after 3 months, massive calcification of the Free-style aortic wall was visible. This calcification was exten-

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sive, in large homogenous plaques in the part of the aorticwall above the cusps (outflow part). Only minor calcifica-tion could be seen in the inflow part of the aortic wall in onevalve, whereas, in the two other valves it was completelyabsent. After 6 months, calcification in the outflow part ofthe Freestyle aortic wall became more dense. In two valves,minor calcification was seen in the inflow part. In two Free-style valves, calcification at the inflow suture line was seenafter 6 months (Fig. 1). No calcification of the cusps wasvisible at any time.

In the Toronto SPV valve, calcification of the outflow partwas also seen in large plaques. Remarkable was the muchmore extensive calcification however in the inflow part ofthe Toronto SPV when compared with the Freestyle valve(Fig. 2). This consisted of several types of calcification. Aplaque of calcification was seen under the right coronarycusp, sometimes extending in the adjacent part of the inflowportion of the aortic wall under the left and non-coronarycusp. A second was extensive calcification of the suture lineproximally, but also fully around the valve, marking the line

Fig. 1. A representative example is shown of an X-ray taken of a Freestyle valve explanted after 6 months. Large, continuous calcification of the outflowpartof the valve is clearly visible. Some calcification is also seen at the inflow suture line.

Fig. 2. A representative example is shown of an X-ray taken of a Toronto SPV valve explanted after 3 months. Calcification of the outflow part is seen.Remarkably when compared with Fig. 1 is the extensive calcification of the inflow part, consisting of calcification of the muscle bar, the suture lines, andsome additional irregular calcification.

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of attachment of the Dacron fabric to the valve. A third kindwas scattered irregular calcification, that occurred to a vari-able extent in all locations in the inflow part of the valve.Some very slight calcification of the base of the cusp wasseen after 3 months, and became more evident after 6months. No calcification was seen at any time in the centeror at the free edge of the cusp.

3.3. Light microscopy

In the Freestyle valves after 3 months of implantation,severe calcification was visible in the outflow part of theaortic wall in all sections. This was most pronounced in theinner layers of the media. After 6 months, calcificationincreased and extended also more in the outer layers ofthe media (Fig. 3). Only in one valve some calcificationwas visible in the inflow part of the Freestyle valve. Itwas located then completely at the outside of the porcineaortic wall, very close to the cloth covering. No calcification

was ever visible in the cusp. The cloth covering gave rise toa foreign body reaction with fibrosis and accumulation ofgiant cells. This foreign body reaction was equally presentafter 3 and 6 months. Thin fibrous tissue covered the insideof the aortic wall both in the inflow and outflow part. It alsocovered the inflow side of the base of the cusp. The thinfibrous tissue covering the inflow part had a higher cellular-ity than the fibrous tissue of the outflow part. The layerbecame thinner after 6 months when compared with thesituation after 3 months.

Different observations were made in the Toronto SPVvalve. Calcification was visible in all valves both after3 and 6 months of implantation in the outflow part,although somewhat less extensive than seen in the Freestylevalve, but also clearly in the inflow part of the aortic wall(Fig. 4). Here it was often located in the inner part ofthe wall. Furthermore, more calcification was seen closeto the cloth covering which covers the entire outer surfaceof the valve. The foreign body reaction was comparablewith the one seen in the Freestyle valve. The over-growth with fibrous tissue was somewhat thicker on theToronto SPV than on the Freestyle valve, and coveredmore surface of the cusp, often more than half of the out-flow surface. Also in the Toronto SPV, this layer was thin-ner after 6 months when compared with the situation after 3months.

3.4. Electron microscopy

Electron microscopical pictures were taken in parts thatwere not massively calcified, since this yielded only mas-sive black areas without additional information. With elec-tron microscopy, calcification was clearly seen in theinflow part of the aortic wall of the Freestyle valve after3 months, especially in the muscle bar, within the sarco-meres, in and around the mitochondria and the nucleus, inareas without macroscopical calcification. In the outflowpart of the aortic wall, calcification was evident aroundcollagen and elastic fibers. In the cusps, calcification wasbarely visible (Fig. 5). Some slight calcification was seenintracellularly, and rare dots of calcification between thecollagen fibers. The collagen fibers were well preserved,with a wavy appearance of the collagen bundles. No impor-tant qualitative differences were seen between valvesimplanted for 3 or 6 months.

In the Toronto SPV valves, the findings were very similarqualitatively concerning calcification of the aortic wall.Somewhat more calcification was seen in the cusps of theToronto SPV when compared with the Freestyle valves incellular elements, such as mitochondria, and also betweenthe collagen bundles (Fig. 6).

3.5. Calcium content

No statistically significant differences existed in cal-cification between the subsegments of the cusps, so that

Fig. 3. A representative example is shown of the light microscopicalappearance (H and E staining, overview) of a Freestyle valve explantedafter 6 months. Severe, almost transmural calcification is seen in the out-flow part of the valve. No calcification is seen in the inflow part. Note alsothe well preserved wavy appearance of the fibrosa of the leaflet.

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these subsegments were pooled for the rest of the analysis.Median calcium content in the cusp was 1.19 (0.005–17.04)mg/mg dry cuspal weight after 3 months in Toronto SPVvalves versus 0.50 (0.005–7.36)mg/mg in the Freestylevalves (P = 0.046). After 6 months this was 2.13 (0.005–96.36) versus 1.00 (0.125–33.36)mg/mg dry cuspal weightin Toronto SPV versus Freestyle valves, respectively(P = 0.021).

4. Discussion

The first aim of this study was to build an easier and (thus)cheaper model to study bioprosthetic valves inside the cir-culation in juvenile sheep. Such a model should allow thescientific community to test, more thoroughly, new bio-prostheses before the first clinical experiments, and thusavoiding potential clinical catastrophes. The model pre-sented here is certainly cheaper and easier than the pre-viously described ones [3,5–7]. The need to establish fullextracorporeal circulation with an oxygenator in the circuitis obviated. The 25% mortality in this series was muchlower than that reported in other series, reaching values ofover 50% [7,8]. This is probably caused by the less invasiveprocedure, which can be performed through a small thora-

cotomy of about 10 cm, with minimal blood loss, with mini-mal hemodilution due to the low priming volume of theright ventricular mechanical assist system, and performedwhile the lungs are continuously ventilated and perfused. Inmore recent series (unpublished) mortality went furtherdown to about 15%.

With this model, valves are implanted in right-sided, pul-monary position with evidently lower closing pressures andflow velocities over the tested valve than in left-sided posi-tion. The frequency of valve opening is certainly equal andthe range of excursion of the cusps is expected to be nearlythe same. The changed hemodynamic load might alter therate and pattern of calcification and degeneration of thebioprostheses. Thiene et al. [8], however, were unable tofind a significantly different rate of calcification when bio-prostheses were implanted in tricuspid or mitral position.Alterations of bioprosthetic valves implanted in tricuspidposition in juvenile sheep are clinically and pathologicallyvery similar to those occurring in human beings [5]. Tovalidate the model of implantation in pulmonary positionfurther, two series of stented bioprosthetic valves with well-known clinical behavior were implanted (Hancock II, Med-tronic, Minneapolis, MN,n = 4; and Pericarbon, Sorin,Saluggia, Italy,n = 4) for 3 and 6 months. Progressivedegeneration of these valves took place with a pattern of

Fig. 4. A representative example is shown of the light microscopical appearance (H and E staining, overview) of a Toronto SPV valve explanted after 6months. Calcification is visible at the inflow and outflow side of the aortic wall. Note also the calcification associated with the cloth covering. Somecalcification is also visible at the base of the leaflet. Remark the well preserved wavy appearance of the fibrosa of the leaflet.

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calcification mimicking the clinical findings. Calcificationwas especially visible in the aortic wall component of theHancock II valve, and in the basal parts of the cusps in boththe Hancock II and Pericarbon, but in time progressingtowards the middle of the cusps. A perforation in a cuspof a Hancock II was found immediately adjacent to a macro-scopically indurated zone after 6 months of implantation.

The severe fibrous overgrowth, called fibrous sheathing[5,8,10,11], encountered in bioprosthetic valves in sheepimplanted in tricuspid position, causing retraction andsometimes even complete immobility of the cusps, wasalso seen in pulmonary position after implantation of Han-cock II or stented Pericarbon valves (unpublished). Thiswas, however, minimal in our series of right-sidedimplanted Freestyle and Toronto SPV valves. This sheath-ing never caused restriction in movements of the cusps. Thismight indicate a very low thrombogenicity of these valves,

since it is believed that sheathing originates from fibrindeposition and thrombus organization [8].

In the literature, no detailed experimental data were avail-able concerning calcification of the Toronto SPV valves.The initial report by David et al. [12] concerning the pre-decessor of the Toronto SPV valve (with a somewhat dif-ferent fixation and anti-mineralization technology, andwithout cloth covering) implanted in aortic position insheep for up to 6 months, stated that all leaflets were mobilewithout any sign of calcification macroscopically or by his-tology. No information is given concerning calcification ofthe aortic wall. In our model, calcification is seen in theaortic wall, but also in the base of the leaflets. No finalexplanation for the difference with the findings of Davidet al., can be given, but it might be the fact that he usedsomewhat older sheep that is responsible for this. Further-more, it might be that addition of the cloth covering in the

Fig. 5. A representative example is shown of the transmission electron microscopical appearance of the basal part of a cusp of a Freestyle valve explantedafter 3 months. Well arranged bundles of collagen fibers are seen. No signs of calcification.

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Toronto SPV, or the differences in fixation technology areresponsible for this. An argument that favors this explana-tion is that the calcification in our model is often pro-nounced close to the cloth covering.

More data are available concerning the fate of Freestylevalves after implantation in juvenile sheep. In an apico-aor-tic conduit in seven juvenile sheep [7], all valve leafletswere soft and pliable after about 4 months without histolo-gical signs of important calcification, while the aortic wallwas severely calcified. This is concordant with our findings.Unexplained, is the reason why the valve cusps in the con-trol group of that series [7] were severely calcified, in thelight of our results with the Toronto SPV valves or thefindings of David et al. [12]. In aortic position in two grow-ing pigs, comparable findings were reported for the Free-style valve [13], with calcification of the aortic wall(although less extensive than seen in our juvenile sheepmodel), and without calcification of the cusps.

Our study is the first reported study to compare the dur-ability of two clinically available stentless valves directly inthe same experimental model. The outflow part of the aorticwall is severely calcified in both valves. The inflow part isconsiderably more calcified in the Toronto SPV valve whencompared with the Freestyle valve. Calcification of thecusps is limited in both valve types, but slightly more in

the Toronto valve. AOA treatment, less cloth covering, and/or differences in fixation techniques could be responsible forthis.

The severely calcified aortic wall is not without impor-tance. A stentless valve becomes in fact a stented valveagain, with its implications concerning the dynamical beha-vior, and incomplete opening of the valve. This might haveimplications on transvalvular gradients and regression ofventricular hypertrophy. Furthermore, calcification of theaortic wall might change the stress load on the cusps, within turn its implications for calcification and degeneration[14]. Finally, would reoperation become necessary, exten-sive calcification of the aortic wall part, might technicallycomplicate the operation.

We conclude that (1) this model is promising for precli-nical evaluation of bioprosthetic heart valves, and (2) calci-fication is less in the valve cusps and the inflow part of theaortic wall in the Freestyle valve than in the Toronto SPVvalve, but extensive calcification in the outflow part of theaortic wall remains problematic in both valves.

References

[1] Westaby S, Huysmans HA, David TE. Stentless aortic bioprostheses:compelling data from the second international symposium. AnnThorac Surg 1998;65:235–240.

[2] Angell WW, Angell JD, Oury JH, Lamberti JJ, Grehl TM. Long-termfollow-up of viable frozen aortic homografts. A viable homograftvalve bank. J Thorac Cardiovasc Surg 1987;93:815–822.

[3] Hazekamp MG, Goffin YA, Huysmans HA. The value of the stent-less biovalve prosthesis; an experimental study. Eur J Cardio-thoracSurg 1993;7:514–519.

[4] David TE, Feindel CM, Scully HE, Bos J, Rakowski H. Aortic valvereplacement with stentless porcine aortic valves: a ten-year ex-perience. J Heart Valve Dis 1998;7:250–254.

[5] Barnhart GR, Jones M, Ishihara T, Rose DM, Chavez AM, FerransVJ. Degeneration and calcification of bioprosthetic cardiac valves.Bioprosthetic tricuspid valve implantation in sheep. Am J Pathol1982;106:136–139.

[6] Ali ML, Kumar SP, Bjornstad K, Duran CMG. The sheep as ananimal model for heart valve research. Cardiovasc Surg 1996; 4:543–549.

[7] Gott JP, Girardot MN, Girardot JMD, Hall JD, Whitlark JD, HorsleyWS, Dorsey LMA, Levy RJ, Chen W, Schoen FJ, Guyton RA.Refinement of the alpha aminooleic acid bioprosthetic valve antic-alcification technique. Ann Thorac Surg 1997;64:50–58.

[8] Thiene G, Laborde F, Valente M, Gallix P, Talenti E, Calabrese F,Piwnica A. Morphological survey of a new pericardial valve pros-thesis (Pericarbon): long-term animal experimental model. Eur JCardio-thorac Surg 1989;3:65–74.

[9] Chen W, Schoen FJ, Levy RJ. Mechanism of efficacy of 2-aminooleic acid for inhibition of calcification of glutaraldehyde-pre-treated porcine bioprosthetic heart valves. Circulation 1994;90:323–329.

[10] Ishihara T, Ferrans VJ, Jones M, Boyce SW, Roberts WC. Occur-rence and significance of endothelial cells in implanted porcine bio-prosthetic valves. Am J Cardiol 1981;48:443–454.

[11] Arbustinini E, Jones M, Moses RD, Eidbo EE, Carroll RJ, FerransVJ. Modification by the Hancock T6 process of calcification of bio-prosthetic cardiac valves implanted in sheep. Am J Cardiol 1984;53:1388–1396.

Fig. 6. A representative example is shown of the transmission electronmicroscopical appearance of the basal part of a cusp of a Toronto SPVvalve explanted after 3 months. Well arranged bundles of collagen fibersare seen. Scattered calcification is seen throughout the sample.

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[12] David TE, Ropchan GC, Butany JW. Aortic valve replacement withstentless porcine bioprostheses. J Card Surg 1988;3:501–505.

[13] Hazekamp, M.G. Stentless biovalve prostheses. Addendum to Chap-ter 5, pp. 89-90, Thesis, Rijksuniversiteit Leiden, ISBN 90-9009262-5.

[14] Deiwick M, Glasmacher B, Zarubin AM, Reul H, Geiger A, vonBally G, Stargardt A, Rau G, Scheld HH. Quality control of biopros-thetic heart valves by means of holographic interferometry. J HeartValve Dis 1996;5:441–447.

Appendix A Conference discussion

Dr J.M. Hasenkam(Aarhus, Denmark): I noticed you did the implan-tations in the pulmonary artery where the hemodynamic load is obviouslyvery low. Could you speculate about the impact of the higher hemody-namic load on the left side of the circulation?

Dr Herijgers: Right-sided implantation was a prerequisite to operatewithout any extracorporeal circulation. The oxygenator is a major problemin experiments with sheep. This causes a high mortality. So we wanted toavoid this. This is why we chose the pulmonary artery. We destroyed thenative pulmonary valve to have a full load-bearing from the right side onthese implanted valves.

There is a nice study published by Professor Thiene from Padua whocompared the calcification in Pericarbon valves in tricuspid and mitralpositions, stented valves, and he showed that there was no significantdifference in the rate of calcification between the two implantation sites.If you see the absolute values in his publication, calcification is about two-thirds in the right-sided position than in the left side. So, probably pressureload has some effect but the difference was not statistically significant. Themajor problem in the right side is sheathing; this is why most of theimplantations are done on the left side. We did not see this in our experi-ments, at least not with the Toronto or the Freestyle valve, but we see thisalso with stented valves in the right side, also in the pulmonary position.

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142 P. Herijgers et al. / European Journal of Cardio-thoracic Surgery 15 (1999) 134–142