Microstructural alterations owing to handling of bovine pericardium to manufacture bioprosthetic heart valves: A potential risk for cusp dehiscence.

INTRODUCTION: Cross-linking and anti-calcification of prosthetic heart valves have been continuously improved to prevent degeneration and calcification. However, non-calcific structural deteriorations such as cuspal dehiscences along the stent still require further analysis. MATERIAL AND METHOD: Based upon the previous analysis of an explanted valve after 7 years, a fresh commercial aortic valve was embedded in poly(methyl methacrylate) (PMMA) and cut into slices to ensure the detailed observation of the assembly and material structures. A pericardial patch embossed to provide the adequate shape of the cusps was investigated after paraffin embedding and appropriate staining. The microstructural damages that occurred during manufacturing process were identified and evaluated by light microscopy, polarized microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: The wavy collagen bundles, the key structure of the pericardium patch, were damaged to a great extent at suture sites along the stent and in the compressed areas around the stent post. The fixation of the embossed pericardium patch along the plots of the stent aggravated the microstructural modifications. The damages mainly appeared as the elimination of collagen bundle waviness and delamination between the bundles. CONCLUSION: Considering the modes of failure of the explant, the damages to the collagen bundles may identify the vulnerable sites that play an important role in the cusp dehiscence of heart valve implants. Such information is important to the manufacturers. Recommendations to prevent in vivo cusp dehiscence can therefore be formulated.

Collagen-based Tissue Engineering as Applied to Heart Valves.

Using the method of directed collagen gel shrinkage, we have been fabricating heart valves and mitral valve chordae [1,2,3]. The principle involves mixing solubilized collagen with the appropriate cells. When the collagen-cell mixture is neutralized, soluble collagen reassembles into fibrils and a gel is created. When the gel is mechanically constrained, the collagen fibrils align in the direction of constraint. The generation of tensile force during contraction is crucial for the formation of highly aligned, compacted collagenous constructs. So far, inappropriate mechanical properties have been one of the main limitations of most collagen-based tissue equivalents. In this study, we focused on providing both biomechanical and biochemical stimuli to increase cellular proliferation, matrix synthesis, and hence improve the mechanical properties of the collagen constructs. We explored a number of holder materials and configurations, with an objective to maximize the lateral compaction of our constructs. We designed a bioreactor that can provide controlled static tension to our collagen constructs. We also developed a nutrition-fortified medium that includes trace elements (Zn2+, Cu2+, Fe2+ and Mn2+), various amino acids, and vitamins (A, B complex, and C). Our ultimate goal was to combine biomechanical and biochemical stimuli, and enhance the mechanical strength of our collagen constructs.

Progress in developing a composite tissue-engineered aortic valve.

This paper reviews the rationale for developing a tissue-engineered aortic valve by building up the complex microstructure from its basic components, and presents recent progress towards that goal. Over the past 4 years, we have been working on engineering the functional components of the composite valve the collagen fiber bundles, the elastin sheets, and the hyaluronan matrix that keeps the tissue hydrated. Most recently, we have been working on optimizing the geometry and material properties of the collagen constructs, by varying their size and aspect ratio, and the types of loading protocols the constructs experience during the culture process.

Computational modeling of vascular clamping: a step toward simulating surgery.

The objective of this study was to simulate clamping of the aorta. It is computationally demanding and involves contact between clamp and aorta, large deformations, and fluid-structure interactions (FSI). Models of the aortic root and clamp were created and solve in ADINA, a Finite Element Analysis package. The tissue model was created using a non-linear material. Fluid-structure interactions (FSI) were modeled. The deformation profile of the simulated aorta matched well with that of the real tissue. Clamping of a fluid-filled pressurized aorta, an important first step towards simulating of surgical procedures, was successfully modeled. The simulation was validated by clamping experiments. The modeling techniques developed are also applicable to pre-operative planning of cardiovascular surgery.

The effect of glycosaminoglycans and hydration on the viscoelastic properties of aortic valve cusps.

This paper reviews the rationale for developing a tissue-engineered aortic valve by building up the complex microstructure from its basic components, and presents recent progress towards that goal. Over the past 4 years, we have been working on engineering the functional components of the composite valve the collagen fiber bundles, the elastin sheets, and the hyaluronan matrix that keeps the tissue hydrated. Most recently, we have been working on optimizing the geometry and material properties of the collagen constructs, by varying their size and aspect ratio, and the types of loading protocols the constructs experience during the culture process.

Evaluation of the matrix-synthesis potential of crosslinked hyaluronan gels for tissue engineering of aortic heart valves.

Our goal is to fabricate continuous sheets of elastin atop non-biodegradable hydrogels (hylans) containing crosslinked hyaluronan, a glycosaminoglycan. Such elastin-hyaluronan composites may be useful to tissue engineer replacements for the glycosaminoglycan- and elastin-rich layers of the native aortic valve cusp. Neonatal rat aortic smooth muscle cells were cultured atop hylan gels with micro-textured surfaces, and on plastic, and the components of the extracellular matrix (collagen, elastin) were periodically analyzed. The hylan substrates induced the cells to proliferate more rapidly and over longer time periods (approximately 4 weeks) relative to those cultured on plastic (2-3 weeks). Consequently, at all assay times, the amounts of elastin was derived from the hylan-based cell cultures was 25% or more than that derived from cells cultured on plastic. However, when elastin content was normalized to the cell DNA content, no significant differences were found in the two substrates beyond the first two weeks of culture. Conversely, at culture times greater than 2 weeks, cells cultured atop hylan gels produced amounts of collagen/nanogram of DNA that were approximately 56% less than that synthesized by cells cultured on plastic. Cells grown on hylan deposited an unusual matrix layer, rich in elastin, at the hylan-cell interface. This elastin was found to be organized into fenestrated sheets and loose elastin fibers, structures that were also isolated from the elastin matrix of the ventricularis layer of porcine aortic valve cusps. We have thus demonstrated that hylan gels are useful as substrates to induce elastin synthesis in culture to obtain structures that resemble the elastin matrix of the native aortic valve.

Isolation of intact aortic valve scaffolds for heart-valve bioprostheses: extracellular matrix structure, prevention from calcification, and cell repopulation features.

Extracellular matrix (ECM) scaffolds isolated from valvulated conduits can be useful in developing durable bioprostheses by tissue engineering provided that anatomical shape, architecture, and mechanical properties are preserved. As evidenced by SEM, intact scaffolds were derived from porcine aortic valves by the combined use of Triton X-100 and cholate (TRI-COL) or N-cetylpyridinium (CPC) and subsequent nucleic acid removal by nuclease. Both treatments were effective in removing most cells and all the cytomembranes, with preservation of (1) endothelium basal membranes, (2) ECM texture, including the D-periodical interaction of small proteoglycans with normally D-banded collagen fibrils, and (3) mechanical properties of the treated valves. Ultrastructural features agreed with DNA, hexosamine, and uronic acid biochemical estimations. Calcification potential, assessed by a 6-week rat subdermal model, was significantly reduced by TRI-COL/nuclease treatment. This was not true for CPC only, despite better proteoglycan preservation, suggesting that nucleic acids also are involved in calcification onset. Human fibroblasts, used to repopulate TRI-COL samples, formed mono- or multilayers on surfaces, and groups of cells also were scattered within the valve leaflet framework. A biocompatible scaffolds of this kind holds promise for production of durable valve bioprostheses that will be able to undergo probable turnover and/or remodeling by repopulating recipient cells.

The evolution of bioprosthetic heart valve design and its impact on durability.

Bioprosthetic heart valves have evolved over the years into remarkably useful and predictable devices. During this process, a number of specific designs have come and gone, and a few have remained. Many design changes were successful, and many were not. This article will describe the successes and failures of the various bioprosthetic valve designs and will detail the specific reasons why a particular design change succeeded or failed to improve bioprosthetic valve performance.

Effect of specimen size and aspect ratio on the tensile properties of porcine aortic valve tissues.

The measurement of mechanical properties of biological tissues is subject to artifacts such as natural variability and inconsistency in specimen preparation. As a result, data cannot be easily compared across laboratories. To test the effects of variable specimen dimensions, we systematically modified the size and aspect ratio (AR) of porcine aortic valve tissues and measured their stiffness and extensibility. We found that: (i) as the AR of circumferential specimens increased from 1:1 to 5:1, their stiffness increased by 36% (p < 0.001) and their extensibility decreased by 21% (p < 0.001); (ii) as the AR of radial specimens increased from 0.8:1 to 4:1, their stiffness increased by 36% (p < 0.001) and their extensibility decreased by 34% (p < 0.001); (iii) as the size of circumferential specimens was reduced from 128 to 32 mm2 at fixed AR (2:1), their stiffness decreased by 6 (p = 0.05), and their extensibility increased by 17% (p < 0.001); and (iv) as the size of radial specimens was reduced from 72 to 32 mm2 at fixed AR (2:1), their stiffness decreased by 7% (p = 0.03) and their extensibility increased by 16% (p = 0.005). Thus, as specimens of constant length became narrower, they became stiffer and less extensible, and as specimens of fixed aspect ratio became smaller, they became less stiff and more extensible. Statistical models of these trends were predictive and can thus be used to integrate materials test data across different laboratories.

Mechanical properties of myxomatous mitral valves.

OBJECTIVE: We sought to characterize the mechanical properties of normal and myxomatous mitral valve tissues. METHODS: We tested 113 mitral valve sections from patients undergoing mitral valve repair or replacement for myxomatous mitral valve prolapse and sections from 33 normal valves obtained at autopsy. RESULTS: Myxomatous mitral valve leaflets were more extensible than normal leaflets when tested parallel to the free edge (41.2% +/- 18.5% vs 17.3% +/- 6.7% circumferential strain [mean +/- SD]; P <.001), as well as perpendicular to the free edge (43.2% +/- 19.4% vs 17.3% +/- 6.7% radial strain; P <.001). Myxoid leaflets were less stiff circumferentially (4.0 +/- 1.6 vs 6.1 +/- 1.4 kN/m; P <.001) and radially (4.5 +/- 1.1 vs 6.1 +/- 1.4 kN/m; P <.001) than normal leaflets. Leaflet strength, however, was similar in both groups. CONCLUSIONS: Myxomatous mitral valve leaflets are physically and mechanically different from normal mitral valve leaflets. They are more extensible and less stiff. Compared with chordae examined previously, however, they are affected much less. Myxomatous mitral valve disease may therefore affect the collagen in the chordae more severely than that in the leaflets.

Tissue damage and calcification may be independent mechanisms of bioprosthetic heart valve failure.

BACKGROUND AND AIM OF THE STUDY: Porcine bioprosthetic valves have excellent hemodynamics and do not require anticoagulation, but have limited durability. Cusp tearing is a major cause of bioprosthetic valve failure. It has been suggested that the mechanism of bioprosthetic valve failure is stiffening by calcification, which leads to elevated stresses and secondary collagen fiber damage and leaflet tearing. This thesis was tested in explanted porcine bioprostheses. METHODS: A total of 60 explanted porcine bioprosthetic valves was tested mechanically, and 15 explanted valves were examined grossly and histologically. Circumferentially and radially oriented samples of cusp tissue were tested uniaxially in a materials testing machine and compared with five controls. RESULTS: Mean (+/-SD) duration of implantation was 10.9+/-5.6 years. Circumferential specimens from explants were less extensible than controls (11.0+/-5.5% versus 24.5+/-2.8% strain, p <0.001), and failed at lower tensions (973+/-733 versus 3075+/-911 N/m, p = 0.001) and at lower strains (21.2+/-8.1% versus 47.3+/-7.1% strain, p <0.001). Radial specimens from explants were less extensible (28.7+/-6.8% versus 39.2+/-5.9% strain, p = 0.002) and failed at lower strains (60.3+/-17.3% versus 112.2+/-24.9% strain, p <0.001) than the controls. The stiffness of the explants was unchanged from controls in both circumferential and radial samples. There were no differences between explants and controls in radial and circumferential stiffness, and in radial failure strength. Calcification was mild and diffuse in most of the tested samples. Tears were found in areas without calcific deposits, along with breaks in collagen fiber bundles. CONCLUSION: These results do not support the thesis that calcification stiffens glutaraldehyde-fixed porcine bioprostheses, except when the entire cusp is transformed into a solid mass of mineral. Rather, leaflet tears may develop as a result of accumulated mechanical damage that is independent of calcification.

Myxomatous mitral valve chordae. I: Mechanical properties.

BACKGROUND AND AIM OF THE STUDY: Chordal rupture is the most common reason for severe mitral regurgitation requiring surgery. The features that predispose myxomatous chordae to rupture, however, have not been studied. Thus, the physical and mechanical properties of normal and myxomatous mitral valve chordae were measured. METHODS: Chordae from 24 normal and 59 myxomatous mitral valves were cut into 10 mm-long segments and mechanically tested to measure extensibility, modulus, failure stress, failure strain, and failure load. After testing, the specimens were weighed and their cross-sectional area and volume measured. RESULTS: Chordae from myxoid mitral valves were larger (1.9 +/- 0.1 mm2 versus 0.8 +/- 0.1 mm2, p < or = 0.001) and heavier (16.6 +/- 1.0 mg versus 6.5 +/- 0.4 mg, p < or = 0.001) than normal chordae. Myxoid chordae had significantly lower moduli (40.4 +/- 10.2 MPa versus 132 +/- 15 MPa, p < or = 0.001) and failed at significantly lower tensile stress (6.0 +/- 0.6 MPa versus 25.7 +/- 1.8 MPa, p < or = 0.001) and absolute load (728 +/- 50 g versus 1,450 +/- 135 g, p < or = 0.001) than normal chordae. Normal and myxoid chordae had similar measurements of extensibility and failure strain. CONCLUSION: Myxomatous degeneration severely affects the mechanical properties of mitral valve chordae. Most notably, myxoid chordae failed at loads one-half of those of normal chordae. This may explain why chordal rupture is the main indication for repair of myxoid mitral valves. These findings also suggest that chordal preservation should be carried out with caution, as myxoid chordae are clearly abnormal with compromised mechanical strength.

Myxomatous mitral valve chordae. II: Selective elevation of glycosaminoglycan content.

BACKGROUND AND AIM OF THE STUDY: Chordal rupture in myxomatous mitral valves is the leading cause of leaflet prolapse and regurgitation. Increased glycosaminoglycan (GAG) content has been reported in these valves. Therefore, the biochemical differences between myxomatous and control mitral valve chordae were investigated. METHODS: The contents of hexuronic acid, DNA, water, and collagen in chordae from 45 myxomatous valves and 10 control valves were measured. Collagen and hexuronic acid quantities were normalized to wet and dry weights, and to DNA content. Different GAG classes were measured using fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: Myxomatous chordae contained significantly more GAGs than controls after quantities were normalized for wet weight, dry weight, and DNA content. The FACE assay showed that the myxomatous chordae contained significantly more chondroitin/dermatan 6-sulfate when normalized to both wet and dry weight, and slightly more hyaluronan. In contrast to leaflets, which contain predominantly hyaluronan, the predominant GAG class in chordae was chondroitin/dermatan sulfate. Keratan sulfate, a GAG class previously unreported in valve tissues, was also discovered in the chordae. Myxomatous chordae contained more water and less collagen than control chordae, but equal quantities of DNA when normalized for wet weight. CONCLUSION: Cells in the chordae of myxomatous valves may produce more GAGs than cells in the chordae of control valves. The resulting accumulation of GAGs and bound water likely gives myxomatous valves their characteristic thickening and floppy, gelatinous nature, and may account for their reported mechanical weaknesses.

Case report: outer sheath rupture may precede complete chordal rupture in fibrotic mitral valve disease.

Rupture mechanics of mitral valve chordae have been difficult to elucidate because most surgical repairs and pathological examinations are performed after the rupture. In an excised anterior leaflet from a fibrotic mitral valve, chordae were observed in an initial phase of rupture. Microscopic sections showed that thinned, nearly ruptured chordal segments were actually chordal cores, containing highly aligned collagen fibers. The outer sheath of elastic fibers, disorganized circumferentially oriented collagen fibers, and endothelial cells that normally surrounds the collagen core apparently had retracted to the extreme ends of the thinned segment, resulting in a bulbous shape, as noted in the chordal rupture literature. In conclusion, these new observations lead us to propose that the rupture of mitral valve chordae is not spontaneous, but may occur over time. The failure of the outer sheath may represent the first phase in a slow, two-part process leading to eventual chordal rupture.

The effect of elastin damage on the mechanics of the aortic valve.

Porcine bioprosthetic heart valves degenerate and fail mechanically through a mechanism that is currently not well understood. It has been suggested that damage to the elastin component of prosthetic valve cusps could be responsible for changes in the mechanical function of the valve that would predispose it to increased damage and ultimate failure. To determine whether damage to elastin can produce the structural and mechanical changes that could initiate the process of bioprosthetic valve degeneration, we developed an elastase treatment protocol that fragments elastin and negates its mechanical contribution to the valve tissue. Valve cusps were mechanically tested before and after digestion to measure the mechanical changes resulting from elastin damage. Elastin damage produced a decrease in radial and circumferential extensibility (from 43 to 18% strain radially and 12 to 7% strain circumferentially), with a slight increase in stiffness (1.3-2.6kN/m for radial and 10.6-11.9kN/m for circumferential directions). Digestions with trypsin, which does not cleave elastin, confirmed that the changes in mechanics of the circumferential samples were likely due to the nonspecific removal of proteoglycans by elastase, while the changes in the radial samples were indeed due to elastin damage. Removing the mechanical contribution of elastin alters the mechanical behavior of the aortic valve cusp, primarily in the radial direction. This finding implies that damage to elastin will distend the cusps, reduce their extensibility, and increase their stiffness. Damage to elastin may therefore contribute to the degeneration and failure of prosthetic valves.

The influence of design on bioprosthetic valve durability.

Bioprosthetic heart valves have evolved over the years into remarkably useful and predictable devices. A number of specific designs have come and gone, and a few have remained. Many design changes were successful and many were not. This article describes the successes and failures of the various bioprosthetic valve designs and details the specific reasons why a particular design change succeeded or failed to improve bioprosthetic valve performance.

Role of preconditioning and recovery time in repeated testing of aortic valve tissues: validation through quasilinear viscoelastic theory.

We investigated how preconditioning history and specimen recovery time affect the accuracy of measurements of mechanical properties obtained from sequential, repeated materials testing of porcine aortic valve (PAV) cusps. Strain history protocols were modeled by quasilinear viscoelastic theory and the results compared with the experimental data. Assuming that the model was predictive, the accuracy of predicting experimental data was related to the suitability of the materials testing protocol. We found that the preconditioned state of the PAV material was not unique but was a function of the deformation history that had occurred before the preconditioning cycles. Preconditioning without an adequate rest period between tests increased predictive errors, whereas allowing the material to rest without preconditioning reduced errors. Modeling more of the strain history reduced errors for specimens briefly rested between tests but had no impact on specimens with long rest periods. The smallest predictive errors were obtained for a loading protocol with a 24 h specimen recovery period followed by material preconditioning. We recommend the use of this protocol for estimating material properties of PAV tissues.

Biaxial strain properties of elastase-digested porcine aortic valves.

BACKGROUND AND AIMS OF THE STUDY: Previous studies have suggested that elastin in porcine aortic valve cusps is responsible for restoring collagen fibers to their original configuration between loading-unloading cycles. METHODS: Biaxial loading tests were performed on intact aortic valves before and after elastase treatment to further investigate the role of elastin. RESULTS: Degradation of elastin caused an increase in the radial dimensions of the cusps (mean increase in gauge length, 29%), which corresponded to a significant decrease in radial extensibility (mean decrease, 61%) and a threefold increase in radial stiffness. Changes in circumferential extensibility and stiffness were smaller and, for most cusps, were not statistically significant. Control experiments, in which the valves were treated with buffer only, resulted in the opposite changes in radial dimensions and extensibility (7% decrease in gauge length and doubling of extensibility). CONCLUSION: Changes in the mechanical properties of the aortic valve cusps following incubation in elastase were due to elastin damage, and not incidental to soaking in buffer. As many explanted bioprosthetic valves have mechanical characteristics similar to those of the elastase-treated valves, elastin damage may be a factor in the progressive degeneration and ultimate failure of bioprosthetic heart valves.

Aortic root dilation prior to valve opening explained by passive hemodynamics.

BACKGROUND AND AIM OF STUDY: During the June 1999 World Symposium on Heart Valve Disease, the mechanism by which the aortic valve opens was discussed. It was suggested, indirectly, that the recently discovered contractile elements within the aortic valve may be responsible. We propose that aortic root dilation does not require any active mechanism within the leaflets or aortic wall, and provide an explanation based entirely on the passive hemodynamics of the aortic valve and root. METHODS AND RESULTS: Previous studies using cine fluoroscopy and sonomicrometry have reported a 5-7% expansion of the aortic root during ventricular contraction, prior to aortic valve opening. Simplified force calculations indicate that the mechanical interactions between the aortic valve and root produce an inward pull on the commissures, constraining the aorta from fully dilating. During systole, as the pressure in the ventricle increases and the aortic valve becomes unloaded, the inward pull on the commissures is reduced and the aorta is able to dilate fully. Aortic dilation therefore occurs before the aortic valve opens. CONCLUSION: We conclude that this mechanism of aortic root dilation prior to valve opening is purely passive, and does not require any active process by the aortic valve or the aortic root.

Mechanics of cryopreserved aortic and pulmonary homografts.

BACKGROUND AND AIM OF THE STUDY: The surgical placement of pulmonary valve grafts into the aortic position (the Ross procedure) has been performed for three decades. Cryopreserved pulmonary valves have had mixed clinical results, however. The objectives of this study were to compare the mechanics of cryopreserved human aortic and pulmonary valve cusps and roots to determine if the pulmonary root can withstand the greater pressures of the aortic position. METHODS: Six aortic and six pulmonary valve roots were obtained from the Oxford Valve Bank. They were harvested during cardiac transplantation from hearts explanted for dilated cardiomyopathy (mean patient age 68 years). The whole roots were initially stored frozen at -186 degrees C, then shipped packed on dry ice. After complete thawing, the roots were pressurized whole; test strips were then cut from the valve cusps, roots and sinuses and tested for stress/strain, stress relaxation, and ultimate failure strength. RESULTS: The pulmonary roots were more distensible (30% versus 20% strain to lock-up) and less compliant when loaded to aortic pressures. The pulmonary valve cusp and root tissue also showed greater extensibility and greater stiffness (lower compliance) when subjected to the same loads. CONCLUSION: We conclude that mechanical differences between aortic and pulmonary valve tissues are minimal. The pulmonary root should withstand the forces imposed on it when placed in the aortic position. However, if implanted whole, the pulmonary root will distend about 30% more than the aortic root when subjected to aortic pressures. These geometric changes may affect valve function in the long term and should be appreciated when implanting a pulmonary valve graft.