Glutaraldehyde-treated Porcine Aortic Valve Research Articles

Stress strain characteristics of glutaraldehyde treated porcine aortic valve tissue following ethanol treatment

Background Glutaraldehyde treatment of heterologous tissue valves is necessary to improve their durability. Anti-calcification treatment is required following glutaraldehyde fixation for tissue valves, to prevent bioprosthetic valve dysfunction due to degenerative changes. One of the simpler anti-calcification strategies utilises ethyl alcohol, and this has been shown to be highly effective. However, whether this treatment has an effect on the physical characteristics has not been studied. Present study was planned to evaluate the physical characteristics of the porcine aortic valve cusps using a Shimadzu AGS 500 G stress/strain tester.

Passive Biaxial Mechanical Properties of the Rat Bladder Wall After Spinal Cord Injury

Changes in the mechanical properties of the bladder wall after spinal cord injury can alter bladder compliance and wall tension, leading to changes in afferent nerve activity and to abnormal reflex mechanisms. To our knowledge we report the first application of biaxial mechanical testing to the normal bladder wall and demonstrate how these properties change after spinal cord injury. Whole bladders were harvested from mature female Sprague-Dawley rats weighing 250 to 300 gm. Test group animals underwent complete spinal cord transection at the T9 to T10 level and normal animals comprised the control group. The bladders were cut open longitudinally, the trigone and apex were removed and the remaining tissue was trimmed to a square of 9 to 13 mm. per side. Mechanical properties of the tissue sample were tested using planar biaxial testing, in which a stress was applied in the circumferential and longitudinal (base-apex) directions, and resulting axial strains were measured. In normal and spinal cord injured rats bladder wall tissue demonstrated isotropic mechanical behavior when equal stress levels were applied in anatomical directions. However, under nonequi-biaxial loading bladder specimens were not truly isotropic but displayed anisotropic-like behavior. Spinal cord injured tissues were consistently more compliant than normal controls. Biaxial mechanical testing can detect differences in normal control and hypertrophied rat bladders 10 and 14 days after spinal cord injury. These changes represent an important component of the bladder response to spinal cord injury.

Mineralization of glutaraldehyde-fixed porcine aortic valve cusps in the subcutaneous rat model: analysis of variations in implant site and cuspal quadrants.

When evaluating the efficacy of new antimineralization treatments for bioprosthetic heart valves, subcutaneous implantation in a rat model often is used as an initial test. Although this model is widely used, there still are many aspects of its implementation that have not been investigated. To further investigate several parameters that may affect mineralization in the subcutaneous rat model, portions of glutaraldehyde-treated porcine aortic valve cusps were implanted both ventrally and dorsally into 21-day-old male Sprague-Dawley rats. Cusp quadrants were explanted at 1,2, and 3 weeks postimplantation and the calcium levels determined by atomic absorption spectroscopy. The objective of this study was to determine whether or not different implant locations and/or regions of the cusps affect the degree to which tissue mineralizes in the subcutaneous rat model. A total of 270 tissue specimens were examined. While the specific portion of the cusp implanted did not significantly affect the degree of mineralization, dorsal implantation resulted in significantly more mineralization than abdominal implantation (p = 0.007). As expected, longer implantation time was associated with greater calcification (p = 0.0001). The results of this study indicate that inconsistent placement of tissues in the rat subcutaneous implant model can result in significant differences in the degree of mineralization.

Role of Glutaraldehyde in Calcification of Porcine Aortic Valve Fibroblasts

Glutaraldehyde-treated porcine aortic valve xenografts frequently fail due to calcification. Calcification in the prostheses begins intracellularly. In a previous study, various types of cell injury to canine valvular fibroblasts, including glutaraldehyde treatment, led to calcification. An influx of extracellular Ca2+ into the phosphate-rich cytosol was theorized to be the mechanism of calcification. To test the Ca2+ influx theory, cytosolic Ca2+ and Pi concentrations were assessed in glutaraldehyde-treated porcine aortic valve fibroblasts, and their relationship to a subsequent calcification was studied. Glutaraldehyde caused an immediate and sustained massive cytosolic Ca2+ increase that was dose dependent and a several-fold increase in Pi. Calcification of cells followed within a week. The earliest calcification was observed in blebs formed on glutaraldehyde-treated cells. Live control cells or cells fixed with glutaraldehyde in Ca2+-free solution did not calcify under the same conditions. Concomitant increases in Ca2+ and Pi in glutaraldehyde-treated cells appear to underlie the mechanism of calcification, and the presence of extracellular Ca2+ during glutaraldehyde fixation promotes calcification.

Valved conduit in the descending thoracic aorta in juvenile sheep: a useful, cost-effective model for accelerated calcification study in systemic circulation

To evaluate the efficacy of any new anticalcificant in bioprostheses, a cost-effective and easy circulatory model is proposed. Calcification of 0.625% glutaraldehyde-treated porcine aortic valved conduits implanted in the descending thoracic aorta in 11 juvenile sheep for 5 months was compared with that of leaflets of glutaraldehyde-treated porcine aortic valve implanted subcutaneously in 3-week-old male Wistar rats for the same period. Cusps of valved conduits (Ca, 205.41 ± 16.24 mg g −1) in sheep and aortic valve leaflets in rats (Ca, 235.21 ± 45.25 mg g −1) ( P = 0.0299) were severely calcified. Morphological characteristics of calcification of all explants were virtually identical. This model provides a model for testing calcification that lies between the subcutaneous weanling rat model and orthotopic whole valve replacement on the left side of the heart. It is less costly and easier to perform than the latter, but does provide exposure to the bloodstream under pressure, which the rat model does not.

Simultaneous assessment of bioprosthetic heart valve biomechanical properties and collagen crimp length.

A new morphologic method is described for the simultaneous quantitation of porcine aortic valve collagen crimp length and the assessment of biomechanical properties. This method utilizes the simultaneous real-time video recording of collagen crimp morphology and acquisition of crimp length data through the combination of polarized light microscopy and morphometry. We felt that the development of this method was warranted, due to the fundamental role played by collagen in porcine aortic valve performance. The development of this method involved the design and fabrication of a uniaxial microtensile stage, suitable for mounting on a standard microscope stage. The validation of our test method was accomplished by a comparison of untreated and glutaraldehydetreated porcine aortic valve leaflet tissue, because the biomechanical and morphologic characteristics of the native and fixed aortic valve have been extensively studied. The method described in this communication enables the collection of morphologic and biomechanical data from a single tissue specimen, eliminating the need for independent studies of multiple specimens. Furthermore, this method obviates the need for making assumptions, which may be difficult to verify, concerning the homogeneity of different test specimens with respect to their morphology and corresponding mechanical response to different experimental conditions.

Porcine aortic valve bioprostheses: A morphologic comparison of the effects of fixation pressure

It has been suggested that the mechanical durability of glutaraldehyde-treated porcine aortic valve (PAV) bioprostheses (BPs) may be increased if collagen crimp approximating that observed in native valvular tissue is maintained during preimplantation processing. Among the factors affecting PAV fixation, the magnitude of the pressure applied to the aortic valve during fixation influences the extent of collagen crimp. Polarized light microscopy is useful for both the qualitative assessment of collagen morphology and the quantitative measurement of collagen crimp in BPs. The purpose of this study was to determine the feasibility of using this optical method to detect quantitative differences in PAV collagen crimp following zero-, low-, and high-pressure fixation. The results of this study demonstrate the following: 1) coupling polarized light microscopy and morphometric measurements provides a quantitative method for the evaluation of the relationship between fixation pressure and collagen crimp in PAVs; 2) a significant association between increasing fixation pressure and absence of collagen crimp was observed in cords and the mid-cusp region; 3) collagen crimp length in the cords is affected by fixation pressure; 4) the alteration of collagen crimp by low- and high-pressure fixation does not uniformly affect all regions of the cusp; and, 5) collagen crimp was consistently present in all cusp regions in zero-pressure fixed PAVs.

Analysis of the bending behaviour of porcine xenograft leaflets and of natural aortic valve material: Bending stiffness, neutral axis and shear measurements

Flexibility of the materials used in the construction of bioprosthetic heart valves is essential for proper valve operation. We therefore examined the bending behaviour of glutaraldehyde treated porcine aortic valve cusps in comparison with fresh aortic valve tissue. We repeatedly bent a total of 35 strips of fresh and treated tissue to curvatures ranging from 0.2 to 2.2 mm −1. We compared the stiffness of the two materials between circumferential and radial bending, natural and reverse curvatures and constant or variable tensile stress (0.8–40 kPa). Our results showed a weak positive relationship between bending stiffness and applied tensile stress and a strong positive dependance of stiffness on tissue thickness ( t). For the fresh tissue, the bending stiffness increased in proportion to t 1.14 while for the glutaraldehyde treated tissue it increased with t 2.18. Fourteen strips of fresh and treated tissue were also histologically processed, sectioned and examined with polarized light microscopy. Collagen fiber wavelengths and shear deformations were measured utilizing the tissue banding patterns produced by polarized light microscopy. The neutral axis of bending was found to lie very close to the outer surface of the tissue, suggesting that aortic leaflets have a very low compressive elastic modulus. The shear strains measured in fresh tissue were 10 ± 2.7% vs 3 ± 4.4% for the treated, indicating a stiffening of the tissue following glutaraldehyde fixation. We conclude that both natural and bioprosthetic valve cusps have a complex flexural behaviour that cannot be modeled using simple bending principles, although the bioprosthetic material more closely approximates the simple beam than does the fresh. The non-linear elastic modulus, high compressibility and shearing between fiber layers are likely responsible for the observed behaviour of the fresh tissue, while the cross-linking and dehydrating effects of glutaraldehyde are believed to be responsible for the alteration in bending properties observed in the treated tissue. Our study suggests that bioprosthetic valve material does not adequately mimic the mechanics of the natural valve tissue, and that the current glutaraldehyde fixation process eliminates many of the beneficial, stress-reducing properties of the aortic leaflet.

Differences between heart valve allografts and xenografts in the incidence and initiation of dystrophic calcification

Following surgical removal because of primary tissue failure, 30 antibiotic-sterilized human aortic valve allografts and 27 glutaraldehyde-treated porcine aortic valve xenografts were examined for macroscopic and microscopic evidence of dystrophic calcification. These grafts had been mounted on stents and used for from 34 to 166 months to replace diseased mitral valves. After explantation the grafts were carefully examined then prepared for light microscopy, for transmission electron microscopy and for energy dispersive X-ray microanalysis. Gross calcification occurred significantly (p = 0.002) more frequently in xenografts (89%), and was more extensive than in allografts (53%). Calcification usually appeared as nodular excrescences on the cusps, although occasionally it formed plates within them. This reduced tissue pliability and was usually associated with either valvular stenosis or regurgitation. The calcified deposits contained calcium and phosphate in ratios approaching those of hydroxyapatite. In xenograft valves the smallest discrete deposits of calcification were spherical and usually associated with membranous debris of porcine donor fibroblasts, but allografts did not contain donor cell remnants and early calcification was linearly arranged along collagen fibres.

Tissue Buckling as a Mechanism of Bioprosthetic Valve Failure

Current reports indicate that collagen fiber disruption resulting from cyclic leaflet bending is a factor determining long-term durability of bioprosthetic heart valves. Examination of the opening characteristics of porcine xenografts has shown two areas of high bending curvature that correlate well with sites of leaflet tearing. These are at the free edge and near the attachment of the leaflets to the aortic root. To determine the potential effects of sharp bends in leaflet material, we examined 15 strips each of fresh and glutaraldehyde-treated porcine aortic valve tissue. Leaflet strips were bent to curvatures of 0.18 mm −1 to 6.67 mm −1, histologically processed, sectioned, and examined under a light microscope. We observed severe compressive buckling in the samples taken from bioprosthetic valves but little in the fresh-tissue samples. At physiological curvatures (less than 0.28 mm −1), no buckling occurred in the fresh tissue; at high bending curvatures (2.0 mm −1), the depth of buckling observed in the treated tissue was 100% greater than that in the fresh. We believe that porcine xenograft failure is related to compressive buckling of the aldehyde-treated tissue and is mediated by the systematic breaking of collagen fibers at the site of buckling. We suggest that alternative valve designs and preservation techniques be employed to prevent such abnormal leaflet deformations.

Thrombogenetic studies of bioprosthetic heart valve surfaces I. In vitro platelet adhesion in a static system

Contact angle measurements, histological examinations and platelet adhesion tests on native and glutaraldehyde-treated porcine aortic valve leaflets, are reported. Contact angles measured with water or saline on native and treated leaflets do not differ appreciably. Values of less than 10° for both the dry and wet leaflets were observed. Histological examination of the native and fixed tissue surfaces was carried out with both optical and scanning electron microscopy. Native leaflet surfaces appeared to be smoother with longer and straighter fibres beneath the surface than treated leaflets. Platelet adhesion tests were performed with fresh, non-anticoagulated human whole blood as well as with platelet suspension. Static platelet adhesion tests were carried out by depositing droplets of blood on the test surface. The blood droplets were washed off after a contact time of 2 minutes for whole blood and up to 32 minutes for platelet suspension. The adhering platelets were microscopically observed and counted. The platelet density on the native leaflets was found to be significantly higher than on the treated tissues. However, the platelet density did not vary with glutaraldehyde concentration in the range from 0.2 to 1.0% ( v v ).

Biomaterials-Associated Pathology of Cardiac Valve Prostheses: Clinical Explant Analysis and Studies of Tissue Valve Calcification

ABSTRACTStudies of the pathology of explanted clinical and experimental valve specimens were done to determine the role of biomaterials in the failure of heart valve prostheses. From 7/80 through 12/84, 129 patients had valve prostheses removed at our hospital. Thrombosis/thromboembolism was the most frequent cause of the 39 mechanical valve failures (23%). Structural failure (poor durability) caused 12%. In contrast, with tissue-derived bioprostheses (90 patients), primary tissue degenerative failure was frequent (70%), and was usually associated with calcification (CALC). Infection caused 19% of all valve failures with a frequency not differing among valve types. Other causes of failure were not biomaterials-related. Thus, although frank materials degradation necessitates only a minority of mechanical valve removals, the suboptimal biologic properties of contemporary heart valve biomaterials contribute to some frequent complications (such as thrombosis/thromboembolism). Furthermore, degeneration due to mineralization is the overwhelming mode of failure of bioprosthetic valves.To understand and potentially eliminate bioprosthetic valve CALC, the kinetics, morphology and host and implant determinants of this process were studied using subcutaneous implants in rats. In this model, in which the pathologic features were similar to those of actual experimental and clinical valve replacements, CALC of glutaraldehyde-treated porcine aortic valve and bovine pericardium began within 48 hours following implantation; mineral levels were comparable to those in failed clinical valves after 84–126 days. Calcific deposits were initially associated with connective tissue cells, but later involved collagen. Systemically-administered ethane-hydroxy-diphosphonate (EHDP), a drug used to treat some forms of metabolic bone disease, inhibited CALC by 97%, but caused significant bone growth retardation. However, EHDP given by site-specific controlled-release largely mitigated CALC at only 1% of the total effective systemic dose, but without adverse effects.

Elastic and viscoelastic material behaviour of fresh and glutaraldehyde-treated porcine aortic valve tissue

In order to obtain better insight into the changes in material properties of aortic valve tissue due to the treatment with glutaraldehyde, comparative tensile and relaxation experiments have been performed with strips taken from porcine aortic valve tissue in a fresh condition and after the treatment. To eliminate biological deviations between different strips, the comparative experiments were done on the same strip. The usual treatment for making a bioprosthesis, where an entire valve root is preserved under hydrostatic pressure of 13 kPa, was simulated by treating the strips under a uniaxial preload of 0.3 N. It was expected that the magnitude of the hydrostatic pressure over the valve during the treatment affects the material properties of the treated tissue. So, the treatments under hydrostatic pressures of 0 and 0.5 kPa were also simulated, by treating the tissue strips with uniaxial preloads of 0 and 0.01 N respectively. As a check leaflet strips of a Hancock bioprosthesis were also measured. To describe the elastic behaviour, two Young's moduli are defined: one in the low strain region ( E 0) and one in the high strain region ( E 13). For describing the viscoelastic behaviour, use was made of the model of Fung (1972, Biomechanics, its Foundations and Objectives; Fung, Perrone and Anliker, Prentice Hall.) to fit the results. Here, the viscoelastic parameters to determine, were K, θ 1 and θ 2. Due to the treatment the stress-strain curve shifted to the stress-axis for the strips treated under 0.3 N and in the opposite direction for the strips treated under 0 and 0.01 N. For all strips after treatment the total amount of relaxation decreased, being the most pronounced (60%) for the strips treated under 0.3 N. For the tissue treated with preload of 0.3 N the changes in the elastic and viscoelastic parameters E 0, E 13 and K were the most pronounced: E 0 changed from 0.18 × 10 6 to 1.7 × 10 6 N m −2, E 13 from 6.6 × 10 6 to 23.0 × 10 6 N m −2, K from 0.085 to 0.024. On the contrary, the variation in the time constants θ 1 and θ 2 (from 0.0054 to 0.0034 s and from 71 to 37 s respectively) was hardly significant. The elastic behaviour of the Hancock leaflet strips confirms that of the tissue strips treated under 0.3 N, while the viscoelastic behaviour differs somewhat.

Calcification of cardiac valve bioprostheses: Biochemical, histologic, and ultrastructural observations in a subcutaneous implantation model system

To study the process of calcification in bioprosthesis, 108 glutaraldehyde-treated porcine aortic valve leaflets were implanted subcutaneously in rabbits and removed 1 day to 6 months later; morphologic findings were correlated with biochemically determined levels of calcium (Ca++) and gamma-carboxyglutamic acid (Gla), a vitamin K-dependent Ca++-binding amino acid known to be present in a variety of tissues with pathological calcification. Gla and Ca++ levels began to increase about 2 months after implantation and increased progressively with time. Ca/Gla molar ratios were comparable to those in leaflets of bioprostheses explanted from patients, 22 to 64 months after implantation. Morphologically evident calcification began at the same time that Gla and Ca++ increases were detected biochemically and also increased in severity with time. Electron microscopy showed that calcification primarily involved the surface of collagen fibrils and the interfibrillar spaces. The biochemical and morphological findings in this experimental system are similar to those described in calcified porcine bioprosthetic valve leaflets removed from patients, but occurred much more rapidly. As with pathological calcification of other tissues, progressive calcification was accompanied by increased Gla levels, suggesting that Gla plays a role in the calcification of the leaflets. This model allows rapid comparative evaluation of large numbers of valve leaflets maintained under similar host conditions.

Comparative tensile and relaxation experiments on fresh and glutaraldehyde-treated porcine aortic valve tissue

Intracellular concentration gradients of substrate were obtained when rat-liver slices were incubated in Krebs-Ringer bicarbonate buffer with α-amino[1-14C]isobutyric acid (AIB) under conditions of aerobiosis but not anaerobically. Under aerobic conditions insulin did not affect the uptake of AIB. No concentration gradient was obtained when these slices were incubated in medium containing [1-14C]cycloleucine. No gradients were observed with various radioactive sugars including α- and β-methyl glycosidases, 3-O-methyl glucose, β-methyl thiogalactoside and potassium galactonate. Various attemps to influence the influx of α-methylglycoside by addition of possible competitive inhibitors, by the use of phlorizin, dinitrophenol and Na+-free media were without effect. It is concluded that concentration gradients involving an active transport process have been demonstrated by AIB but there was no evidence to confirm the presence of any active transport mechanism for sugars.

Spontaneous Heterograft Aortic Valve Failure

Glutaraldehyde-treated porcine aortic valve prostheses have been in clinical use for ten years. The long-term durability of these valves remains unknown although they have functioned well in most large clinical series for more than five years. At the present time, several manufacturers produce bioprostheses mounted on flexible stents. This report concerns the failure of an Edwards porcine xenograft in the aortic position 15 months following implantation. At reoperation, the right and left coronary leaflets of the explanted valve were torn from the aortic wall. The loss of wall integrity suggests that during the process of mounting the xenograft on the flexible stent, the aortic walls of the bioprosthesis may have been thinned beyond a critical point of maintenance of wall strength. The pathological and clinical findings of similar cases are reviewed.

98 Comparative Tensile- and Relaxation Experiments on Fresh and Glutaraldehyde-Treated Porcine Aortic Valve Tissue

98 Comparative Tensile- and Relaxation Experiments on Fresh and Glutaraldehyde-Treated Porcine Aortic Valve Tissue

An ‘in vitro’ study of mechanical fatigue in glutaraldehyde-treated porcine aortic valve tissue

Leaflet tissue specimens prepared from porcine aortic valves treated with glutaraldehyde at low and high pressures have been subjected to 0.45 × 10 9 accelerated fatigue cycles in Ringer's solution. The waveform or crimped property of the collagen fibres in the leaflet tissue is an essential requirement for its ability to resist localized deformation during repeated flexure. High pressure glutaraldehyde fixation of the whole valve eliminated the crimp structure and resulted in the formation of localized kink sites in the tissue specimen during repeated flexure. Eleven separate sites of serious tissue disruption were observed in the three fatigue specimens obtained from the high pressure-treated valve. In contrast to this only one site of serious disruption was observed in the three fatigue specimens obtained from the low pressure-treated valve. Here fixation preserved the fully crimped morphology of the collagen. It is expected that the long-term mechanical durability of glutaraldehyde treated aortic valves can be substantially increased if careful consideration is given to the pressure at which initial fixation is carried out.