Effects of an angiotensin II type 1 receptor blocker on aortic valve sclerosis in a preclinical model.

BACKGROUND: Aortic valve sclerosis (AVS) is a chronic progressive disease involving lipid infiltration, inflammation, and tissue calcification. Despite its high prevalence, there are currently no clinically approved pharmaceuticals for the management of AVS. The objective of the current study was to elucidate the effects of an angiotensin II type 1 receptor blocker, alone or in combination with statin therapy, on the progression of AVS. METHODS: Male New Zealand white rabbits were fed an atherogenic diet for a period of 12 months to induce AVS. Once disease was established, rabbits were randomly assigned to receive no treatment, olmesartan medoxomil, atorvastatin calcium, or a combination of both drugs for a period of 6 months. Disease progression was monitored in vivo using clinically relevant magnetic resonance imaging, and aortic valve cusps were examined ex vivo using histologic and immunohistochemical methods. RESULTS: Cusp thickness significantly increased (0.58 ± 0.03 vs 0.39 ± 0.03 mm for cholesterol and control animals, respectively; P < 0.0001) and all classic hallmarks of disease progression-including lipid infiltration, inflammation, and tissue calcification-were observed after 12 months. Unfortunately, neither olmesartan medoxomil nor atorvastatin calcium were able to reverse or delay disease progression during the 6-month treatment period. However, several histologic changes were observed in the valvular microenvironment. CONCLUSIONS: The current study suggests that angiotensin receptor blockers, alone or in combination with statin therapy, may not be suitable for management of clinical AVS.

Exploring cell compatibility of a fibronectin-functionalized physically crosslinked poly(vinyl alcohol) hydrogel.

Physically crosslinked poly(vinyl alcohol) (PVA) hydrogels prepared using a low-temperature thermally cycled process have tunable mechanical properties that fall within the range of soft tissues, including cardiovascular tissue. An approach to render it hemocompatible is by endothelization, but its hydrophilic nature is not conducive to cell adhesion and spreading. We investigated the functionalization reaction of this class of PVA hydrogel with fibronectin (FN) for adhesion and spreading of primary porcine radial artery cells and vascular endothelial cells. These are cells relevant to small-diameter vascular graft development. FN functionalization was achieved using a multistep reaction, but the activation step involving carbonyl diimidazole normally required for chemically crosslinked PVA was found to be unnecessary. The reaction resulted in an increase in the elastic modulus of the PVA hydrogel but is still well within the range of cardiovascular tissue. Confocal microscopy confirmed the adhesion and spreading of both cell types on the PVA-FN surfaces, whereas cells failed to adhere to the PVA control. This is a first step toward an alternative for the realization of a synthetic replacement small-diameter vascular graft.

Statin treatment of hypercholesterolemic-induced aortic valve sclerosis.

BACKGROUND: Aortic valve sclerosis (AVS) is a common inflammatory heart valve disease prevalent in the population over the age of 65 years. Several published clinical and animal studies have examined the ability of statin treatment to modify disease progression. Clinical trials yielded conflicting results, and animal studies examined the effects of statins prior to the onset of disease. Our study assessed the effect of dietary modification and/or statin treatment on established aortic valve disease in a rabbit model of AVS to examine the tissue response to therapy. METHODS: Aortic valve sclerosis was induced in male New Zealand White rabbits by dietary cholesterol supplementation. Rabbits were followed over 2.5 years, with the introduction of statins and/or dietary changes for the second half of the study. At end point, valve function was examined by magnetic resonance imaging. Excised aortic valve cusp tissue was surveyed for thickness, lipid accumulation, protein deposition, calcification, and cellular infiltration. RESULTS: By 15 months, cholesterol-fed valves exhibited thickening due to significant lipid content, macrophage infiltration, and osteopontin expression. By 30 months, the untreated disease had progressed to include elevated collagen deposition, lymphocyte invasion, and calcification. With treatment, however, the valve cusps exhibited significant pathological changes including diminished immune cell infiltration and osteopontin expression. Unfortunately, lipid was retained and calcification persisted in all treated valves. CONCLUSIONS: In established AVS, the cellular response to statin therapy does not result in full regression of the sclerotic process.

Synthesis and in vitro biocompatibility assessment of a poly(amic acid) derived from ethylenediaminetetraacetic dianhydride.

Poly(amic acid) (PAA) derived from ethylenediaminetetracetic dianhydride shows great potential as a biomaterial suitable for biomedical applications. To evaluate this polymer class further, in vitro cell toxicity (WST-1/ECS, ELISA based) and cell compatibility (cell adhesion and cell proliferation) tests were conducted to establish structure-toxicity relationships. PAAs with a number-average molecular weight ranging between 100 to 200 kg/mol were synthesized at 37°C after 24 h. Porcine radial artery cells (RACs) and descending aorta endothelial cells (ECs) were seeded independently in a 96-well cell culture plate at a cell density of 5000 cells/cm(2) to observe toxic effects. Similarly, RACs and ECs were seeded independently onto PAA coated and uncoated cover slips at a cell density of 7000 cells/cm(2) to observe growth patterns. Our results showed no toxicity after 96 h of incubation and in addition, both RACs and ECs adhered and proliferated on the PAA films, preserving their phenotype during this time. The tested synthetic material seems promising as a future biomaterial and should elicit a desired cellular response upon implantation.

Angiotensin II type 1 receptor blocker inhibits arterial calcification in a pre-clinical model.

AIMS: Arterial calcification is a common complication of several disorders and is a strong predictor of mortality. The mechanism underlying arterial calcification is not fully understood and as such, no pharmaceutical therapies are currently available which impede its progression. The aim of this study was to investigate the effects of an angiotensin II (AngII) type 1 receptor blocker (ARB) on arterial calcification. METHODS AND RESULTS: Male New Zealand White rabbits were fed an atherogenic diet to induce atherosclerosis and arterial calcification over a period of 12 weeks, with an ARB administered in the final 4 weeks. Using clinically relevant micro-computed tomography, we found that animals fed the atherogenic diet displayed extensive arterial calcification when compared with control. In contrast, administration of the ARB completely inhibited calcification (2.80 ± 1.17 vs. 0.01 ± 0.01% calcified tissue in cholesterol and ARB-treated, respectively; n = 6 and 5; P < 0.05). Calcified regions were characterized by up-regulation of bone morphogenetic protein 2, osteocalcin, and the AngII type 1 receptor and concomitant down-regulation of α-smooth muscle actin, consistent with a phenotypic switch from vascular to osteoblast-like cells. CONCLUSION: These data provide the first evidence that angiotensin receptor blockade can inhibit arterial calcification by disrupting vascular osteogenesis and suggest that ARBs may be a novel treatment option for patients suffering from vascular calcification.

Synthesis, characterization and in vitro cell compatibility study of a poly(amic acid) graft/cross-linked poly(vinyl alcohol) hydrogel.

Although physically cross-linked poly(vinyl alcohol) (PVA) hydrogels have tunable mechanical properties to match that of soft tissues, such as vascular tissue, their hydrophilic nature is not conducive to cell adhesion and spreading. For applications such as small diameter vascular grafts for coronary bypass both mechanical matching and hemocompatibility are important. Poly(amic acid) (PAA), derived from ethylene diamine tetraacetic dianhydride, is a cell-compatible polymer. It was grafted/cross-linked onto physically cross-linked PVA to provide cell compatibility. Functionalization was achieved via a one-step esterification reaction using 1,3-dicyclohexylcarbodiimide as the coupling agent and 4-dimethylaminopyridine as the catalyst. The success of the grafting reaction was verified using Fourier transform infrared spectroscopy, solid-state nuclear magnetic resonance spectroscopy and X-ray photoelectron spectroscopy. The mechanical properties of the starting PVA hydrogel were largely preserved after the grafting reaction within the physiological strain range of vascular tissue. In vitro cell culture studies using primary porcine endothelial cells confirmed cell compatibility of the PAA graft PVA hydrogel, making it an attractive candidate for small diameter vascular graft development.

The effects of hammer pressure on cellular response in a porcine heart valve tissue.

Our objective was to design, develop, characterize and validate a prototype device for testing the response of aortic valve tissue to impact forces. With each cardiac cycle, the aortic valve, on closure, is subjected to a substantial impact force and the ability of valvular interstitial cells to withstand such forces without apoptosis has not been examined. Our aim was to correlate impact force with apoptosis, identifying the latter using a terminal transferase dUTP nick end-labelling (Tunel) assay. With our drop tower design, we created reproducible impact forces on heart valve tissue resulting in cellular trauma. The reliability of the impact tester design were verified and results showed that normal tissue can withstand impact forces more than 30× greater than the physiological forces to which the tissue is normally exposed. This provides a wide safety margin and indicates that bioengineered aortic valve tissue should have similar properties if it is to withstand physiologic forces long term.

Early identification of aortic valve sclerosis using iron oxide enhanced MRI.

PURPOSE: To test the ability of MION-47 enhanced MRI to identify tissue macrophage infiltration in a rabbit model of aortic valve sclerosis (AVS). MATERIALS AND METHODS: The aortic valves of control and cholesterol-fed New Zealand White rabbits were imaged in vivo pre- and 48 h post-intravenous administration of MION-47 using a 1.5 Tesla (T) MR clinical scanner and a CINE fSPGR sequence. MION-47 aortic valve cusps were imaged ex vivo on a 3.0T whole-body MR system with a custom gradient insert coil and a three-dimensional (3D) FIESTA sequence and compared with aortic valve cusps from control and cholesterol-fed contrast-free rabbits. Histopathological analysis was performed to determine the site of iron oxide uptake. RESULTS: MION-47 enhanced the visibility of both control and cholesterol-fed rabbit valves in in vivo images. Ex vivo image analysis confirmed the presence of significant signal voids in contrast-administered aortic valves. Signal voids were not observed in contrast-free valve cusps. In MION-47 administered rabbits, histopathological analysis revealed iron staining not only in fibrosal macrophages of cholesterol-fed valves but also in myofibroblasts from control and cholesterol-fed valves. CONCLUSION: Although iron oxide labeling of macrophage infiltration in AVS has the potential to detect the disease process early, a macrophage-specific iron compound rather than passive targeting may be required.

Vascular smooth muscle cells as a valvular interstitial cell surrogate in heart valve tissue engineering.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are a potential autologous cell source for aortic valve tissue engineering, but have a phenotype that differs from that of valvular interstitial cells in vivo. We hypothesized that combining basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF) with transforming growth factor beta-1 (TGF-beta1) would achieve a valvular interstitial cell-like phenotype of VSMCs. METHODS: VSMC phenotype was assessed by immunofluorescence, proliferation was measured by the tetrazolium reduction (MTT) assay, and extracellular matrix gene expression was determined by real-time polymerase chain reaction. RESULTS: Combinations of growth factors that included PDGF showed the greatest increases in proliferation. Immunofluorescence for alpha-smooth muscle actin demonstrated an inverse correlation between proliferation and a myofibroblast-like phenotype, while combinations of TGF-beta1+ EGF+bFGF (TEF) and TGF-beta1+EGF+PDGF (TEP) induced the greatest change of alpha-smooth muscle actin expression compared to untreated controls. Finally, TEP treatment showed an increase in versican, fibronectin, and type I collagen mRNA expression, while decreasing matrix metalloproteinase 1 expression. CONCLUSIONS: Combination of TGF-beta1 with EGF and PDGF induces VSMC proliferation and expression of extracellular matrix constituents found in the aortic valve. In vitro preconditioning of VSMCs provides a potentially viable surrogate cell source for developing a valve graft.

Rapid dynamic image registration of the beating heart for diagnosis and surgical navigation.

Dynamic cardiac magnetic resonance imaging (MR) and computed tomography (CT) provide cardiologists and cardiac surgeons with high-quality 4-D images for diagnosis and therapy, yet the effective use of these high-quality anatomical models remains a challenge. Ultrasound (US) is a flexible imaging tool, but the US images produced are often difficult to interpret unless they are placed within their proper 3-D anatomical context. The ability to correlate real-time 3-D US volumes (RT3D US) with dynamic MR/CT images would offer a significant contribution to improve the quality of cardiac procedures. In this paper, we present a rapid two-step method for registering RT3D US to high-quality dynamic 3-D MR/CT images of the beating heart. This technique overcomes some major limitations of image registration (such as the correct registration result not necessarily occurring at the maximum of the mutual information (MI) metric) using the MI metric. We demonstrate the effectiveness of our method in a dynamic heart phantom (DHP) study and a human subject study. The achieved mean target registration error of CT+US images in the phantom study is 2.59 mm. Validation using human MR/US volumes shows a target registration error of 1.76 mm. We anticipate that this technique will substantially improve the quality of cardiac diagnosis and therapies.

The in vivo diagnosis of early-stage aortic valve sclerosis using magnetic resonance imaging in a rabbit model.

PURPOSE: To use magnetic resonance imaging (MRI) to identify and monitor early aortic valve sclerosis (AVS) induced by cholesterol feeding in rabbits. AVS is a highly prevalent disease process, affecting more than 25% of the population over age 65. A major obstacle to early stage medical management of AVS has been the lack of an objective noninvasive technique to identify its presence and monitor its progress. MATERIALS AND METHODS: Retrospectively gated CINE fast spoiled gradient echo (fSPGR) images of aortic valve cusps were collected at 3-month intervals starting at 6 months using a 1.5 T MR scanner interfaced with a customized surface radiofrequency coil. At 16 months a subset of animals was sacrificed and excised cusps were examined with both high frequency ultrasound (US) and histopathological techniques to validate the MRI method. RESULTS: MR and US analysis identified significant thickening of diseased AV cusps when compared to control (P < 0.05). Histopathological analysis confirmed the presence of human-like AVS in diseased rabbit valves. CONCLUSION: Early AVS, exemplified by increased valve thickness, can be identified in vivo using high-resolution MRI.

Anisotropy of high-frequency integrated backscatter from aortic valve cusps.

The biaxial anisotropy of integrated backscatter from aortic valve cusps was characterized ex vivo as an initial assessment of the suitability of high-frequency ultrasound for nondestructive evaluation of fiber alignment in tissue-engineered heart valves. Apparent integrated backscatter (AIB) from eight fresh, intact porcine cusps was measured over an 80 degrees range of insonification angles using a 40-MHz ultrasound system. Angular dependence of backscatter was characterized by fitting a sinusoid to plots of AIB versus insonification angle for data acquired while rotating the transducer about the cusps in the circumferential and radial directions. Angular variations in backscatter were detected along both directions in individual specimens, although the mean amplitude of the fitted sinusoid was significantly greater for the circumferential data (12.1 +/- 2.6 dB) than the radial data (3.5 +/- 3.1 dB, p = 0.002). The higher angular variation of backscatter in the circumferential direction implies that collagen fibers in the fibrosa layer are the most prominent source of high-frequency scattering from porcine aortic valve cusps. The ability to characterize anisotropic backscattering from individual specimens demonstrates that high-frequency ultrasound can be used for nondestructive evaluation of fiber alignment in heart valve biomaterials.

Radial artery as an autologous cell source for valvular tissue engineering efforts.

To create a viable tissue-engineered aortic valve, it is important to identify suitable autologous cell sources that may be seeded onto a biocompatible scaffold. This study focused on the radial artery (RA) as one possible source, investigated optimal culture conditions, and determined the usefulness of small intestinal submucosa (SIS) as a scaffold for tissue-engineering. Porcine RA cells were cultured on either two-dimensional (2D) 100-mm dishes or three-dimensional (3D) 1-cm(2) SIS sheets, producing cell-scaffold composites (CSCs). Both 2D and 3D cultures were maintained in either Medium 199 (M199) or endothelial growth media (EGM) to determine optimal growth conditions. Cellular phenotype and matrix metalloproteinase (MMP) profiles were determined by immunoblotting of cell lysates and zymography of conditioned media, respectively. Cellular invasion was analyzed immunohistochemically on CSC tissue sections. We show that the RA contains phenotypes consistent with those found in the normal aortic valve. EGM, compared with M199, promotes the invasion and remodeling of SIS by RA cells, which is crucial in the process of replacing the foreign tissue scaffold prior to implantation. To our knowledge, this is the first study to show that the RA is a suitable source for the generation of a tissue-engineered valve.

Evaluation of an algorithm for semiautomated segmentation of thin tissue layers in high-frequency ultrasound images.

An algorithm consisting of speckle reduction by median filtering, contrast enhancement using top- and bottom-hat morphological filters, and segmentation with a discrete dynamic contour (DDC) model was implemented for nondestructive measurements of soft tissue layer thickness. Algorithm performance was evaluated by segmenting simulated images of three-layer phantoms and high-frequency (40 MHz) ultrasound images of porcine aortic valve cusps in vitro. The simulations demonstrated the necessity of the median and morphological filtering steps and enabled testing of user-specified parameters of the morphological filters and DDC model. In the experiments, six cusps were imaged in coronary perfusion solution (CPS) then in distilled water to test the algorithm's sensitivity to changes in the dimensions of thin tissue layers. Significant increases in the thickness of the fibrosa, spongiosa, and ventricularis layers, by 53.5% (p < 0.001), 88.5% (p < 0.001), and 35.1% (p = 0.033), respectively, were observed when the specimens were submerged in water. The intraobserver coefficient of variation of repeated thickness estimates ranged from 0.044 for the fibrosa in water to 0.164 for the spongiosa in CPS. Segmentation accuracy and variability depended on the thickness and contrast of the layers, but the modest variability provides confidence in the thickness measurements.

Dermal fibroblasts cultured on small intestinal submucosa: Conditions for the formation of a neotissue.

Small intestinal submucosa (SIS) is a naturally occurring, acellular biomaterial that has been used extensively as a soft tissue replacement, as a scaffold for tissue engineering, and as a substrate for the study of cells in 3D culture. The aim of this study is to define culture parameters that promote neotissue formation with the use of dermal fibroblasts and SIS. SIS sheets were seeded with dermal fibroblasts and cultured for 4 weeks. The resultant cell-scaffold composites (CSCs) were cultured with media alone, media supplemented with ascorbic acid, or fibronectin-pretreated SIS and ascorbic acid. CSCs were analyzed for cellular invasion into the scaffold, the rate of type I collagen production, MMP gelatinolytic activity, thickness, and ultrastructural morphology. CSCs treated with fibronectin and ascorbate showed an increase in Type I collagen production, no change in the MMP gelatinolytic activity, an increase in CSC thickness, and an organized neotissue on the surface of the SIS. Minimal cellular invasion was noted, suggesting that fibroblasts use the SIS as a template for neotissue growth rather than as a scaffold. These results indicate that fibronectin-treated SIS cultured with dermal fibroblasts in the presence of ascorbic acid will promote true neotissue formation for future cardiovascular tissue engineering efforts.

Development of aortic valve sclerosis in a rabbit model of atherosclerosis: an immunohistochemical and histological study.

BACKGROUND AND AIM OF THE STUDY: It has been suggested that aortic valve sclerosis (AVS) is an atherosclerotic disease process that can proceed to aortic stenosis. The absence of reports studying an animal model of the early stages of this disease has precluded the development of preventive therapeutic strategies. A cholesterol-fed (0.25% cholesterol in chow) rabbit model of atherosclerosis that is characterized by a moderate level of hypercholesterolemia was studied to determine its efficacy as a model of early AVS. Cellular, structural and morphological changes in the aortic valves of these rabbits were studied. METHODS: Twenty rabbits were assigned randomly to four experimental groups: Group 1 received normal chow for 40 weeks; group 2 received 0.25% cholesterol-supplemented chow for 20 weeks; group 3 received 0.25% cholesterol-supplemented chow for 40 weeks; and group 4 received 0.25% cholesterol-supplemented chow for 20 weeks followed by normal chow for an additional 20 weeks. The aortas and aortic valves were analyzed using immunohistochemical and histological methods to detect cellular and structural components of the developing lesions. RESULTS: All rabbits in groups 2, 3 and 4 developed atherosclerotic lesions in their aortas. Aortic valves from these animals demonstrated thickening, lipid deposition, a change in collagen content and organization, a reorganization of elastin, and the presence of both macrophage infiltrate and osteopontin. CONCLUSION: These findings were consistent with the suggestion of a link between atherosclerosis and AVS. Results were also similar to changes reported in human sclerotic aortic valves, suggesting the suitability of this rabbit model of atherosclerosis as a model for AVS.

Image-based cardiac gating for three-dimensional intravascular ultrasound imaging.

Three-dimensional (3-D) intravascular ultrasound (US), or IVUS, provides valuable insight into the tissue characteristics of the coronary wall and plaque composition. However, artefacts due to cardiac motion and vessel wall pulsation limit the accuracy and variability of coronary lumen and plaque volume measurement in 3-D IVUS images. ECG-gated image acquisition can reduce these artefacts but it requires recording the ECG signal and may increase image acquisition time. The goal of our study was to reconstruct a 3-D IVUS image with negligible cardiac motion and vessel pulsation artefacts, by developing an image-based gating method to track 2-D IVUS images over the cardiac cycle. Our approach involved selecting 2-D IVUS images belonging to the same cardiac phase from an asynchronously-acquired series, by tracking the changing lumen contour over the cardiac cycle. The algorithm was tested with IVUS images of a custom-built coronary vessel phantom and with patient images. The artefact reduction achieved using the image-gating approach was > 86% in the in vitro images and > 80% in the in vivo images in our study. Our study shows that image-based gating of IVUS images provides a useful method for accurate reconstruction of 3-D IVUS images with reduced cardiac motion artefact.

Dynamic 3D ultrasound and MR image registration of the beating heart.

Real-time three-dimensional ultrasound (RT3D US) is an ideal imaging modality for the diagnosis of cardiac disease. RT3D US is a flexible, inexpensive, non-invasive tool that provides important diagnostic information related to cardiac function. Unfortunately, RT3D US suffers from inherent shortcomings, such as low signal-to-noise ratio and limited field of view, producing images that are difficult to interpret. Multi-modal dynamic cardiac image registration is a well-recognized approach that compensates for these deficiencies while retaining the advantages of RT3D US imaging. The clinical application of multi-modal image registration methods is difficult, and there are a number of implementation issues to be resolved. In this work, we present a method for the rapid registration of RT3D US images of the beating heart to high-resolution magnetic resonance (MR) images. This method was validated using a volunteer image set. Validation results demonstrate that this approach can achieve rapid registration of images of the beating heart with fiducial landmark and registration errors of 1.25 +/- 0.63 and 1.76 mm respectively. This technique can potentially be used to improve the diagnosis of cardiac disease by augmenting RT3D US images with high-resolution MR images and to facilitate intra-operative image fusion for minimally invasive cardio-thoracic surgical navigation.

Design and manufacture of a polyvinyl alcohol (PVA) cryogel tri-leaflet heart valve prosthesis.

Although current artificial heart valves are life sustaining medical devices, improvements are still necessary to address deficiencies. Bioprosthetic valves have a compromised fatigue life, while mechanical valves have better durability but are prone to thromboembolic complications. A novel, one-piece, tricuspid valve, consisting of leaflets, stent and sewing ring, made entirely from the hydrogel, polyvinyl alcohol cryogel (PVA-C), has been developed and demonstrated. This valve has three thin leaflets attached to a cylindrical stent. In order to approximate the complex shape of the surface of the natural heart valve leaflets, two different geometries have been proposed: revolution about an axis of a hyperboloid shape and revolution about an axis of an arc subtending (joining) two straight lines. The parameters of both geometries were examined based on a compromise between avoiding sharp curvature of leaflets and minimization of the central opening of the valve when closed. The revolution of an arc subtending two straight lines was selected as the preferred geometry since it has the benefit of a smaller central opening when the value of the maximum curvature for the leaflets is the same for each valve geometry. A cavity mold has been designed and constructed to form the PVA-C heart valve. The three leaflets were formed and integrated into the stent and sewing ring in a single process. Prototype heart valves were manufactured in the mold from a solution of PVA and water, by controlled freezing and thawing cycles.

Three-dimensional visualization and thickness estimation of aortic valve cusps using high-frequency ultrasound.

High-frequency ultrasound techniques are introduced for three-dimensional imaging and thickness estimation of fresh heart valve cusps. Images of porcine aortic valve specimens were acquired within a 12 x 8 x 8 mm3 volume using a VisualSonics VS40 micro-imaging system operating at a 40 MHz centre frequency. Two image volumes were obtained from each of six left coronary cusps. One volume was acquired with the specimen submerged in distilled water and the second volume was acquired through either Hanks physiologic solution or coronary perfusion solution (CPS). The fibrosa, spongiosa and ventricularis were most readily distinguished when the specimen was imaged in distilled water. Colour thickness maps were computed from B-mode image data, and the mean and standard deviations of the thickness were determined for each cusp. In 11 of 12 trials, the image analysis algorithm yielded valid thickness estimates over greater than 98% of the region examined. Mean thickness estimates obtained with specimens submerged in Hanks solution or CPS ranged from 0.66 to 1.03 mm, and submersion in distilled water increased the mean thickness by 20-40%. This observation suggests that the cusps osmotically absorbed water. Information provided by high-frequency ultrasound is expected be valuable for characterizing the morphological properties of heart valves.