Preventing extrinsic mechanisms of bioprosthetic degeneration using polyphenols.

OBJECTIVES: The purpose of this study was to evaluate the impact of a polyphenols-based treatment on the extrinsic mechanisms responsible for early bioprosthetic heart valve (BHV) degeneration. Structural degeneration can be driven by both extrinsic and intrinsic mechanisms. While intrinsic mechanisms have been associated with inherent biocompatibility characteristics of the BHV, the extrinsic ones have been reported to involve external causes, such as chemical, mechanical and hydrodynamic, responsible to facilitate graft damage. METHODS: The chemical interaction and the stability degree between polyphenols and pericardial tissue were carefully evaluated. The detoxification of glutaraldehyde in commercial BHVs models and the protective effect from in vivo calcification were taken into relevant consideration. Finally, the hydrodynamic and biomechanical features of the polyphenols-treated pericardial tissue were deeply investigated by pulse duplicator and stress-strain analysis. RESULTS: The study demonstrated the durability of the polyphenols-based treatment on pericardial tissue and the stability of the bound polyphenols. The treatment improves glutaraldehyde stabilization's current degree, demonstrating a surprising in vivo anti-calcific effect. It is able to make the pericardial tissue more pliable while maintaining the correct hydrodynamic characteristics. CONCLUSIONS: The polyphenols treatment has proved to be a promising approach capable of acting simultaneously on several factors related to the premature degeneration of cardiac valve substitutes by extrinsic mechanisms.

Structural changes of rat mitral valve chordae tendineae during postnatal development.

BACKGROUND AND AIM OF THE STUDY: Mitral valve chordae tendineae are an essential component for correct functioning of the human heart. The microstructural make-up of the chordae is responsible for their tensile properties, and is seen gradually to change with age. However, little is known of the maturation of chordae tendineae and their microstructure. METHODS: To examine such maturation, structural changes in chordae tendineae were studied in rats at 1, 3, 7, 15 and 30 days of postnatal life, and in adult rats. Differences in the chordae microstructure of each age group were observed using light microscopy. The collagen fibril crimp period was determined using polarized light microscopy. RESULTS: At day 1 after birth the chordae had yet to develop, and the lateral sides of the mitral valve leaflets were completely attached to the papillary muscles. Chordae developed through the formation of gaps in the leaflet tissue. From day 7 on, numerous chordae were seen. As the chordae matured, crimped collagen fibrils were formed and began to align in a longitudinally packed core with increasing density. The collagen fibril crimp period increased significantly with the age of the animal. CONCLUSION: Rat chordae tendineae have yet to develop at postnatal day 1. Morphological development and microstructural maturation of the chordae are not completed until adulthood (>30 days). A further understanding of the development of mitral valve chordae tendineae will provide insight for the use of tissue-engineered chordae in surgical repair.

Transcatheter valves: a brave New World.

Over the past five years, transcatheter valves have stimulated the attention of physicians, engineers, and investors. Transcatheter valve design and implantation techniques depart from the time-proven features of surgical valves, and this has an important impact on the safety and efficacy of prosthetic valve therapy. Herein is reviewed the performance of transcatheter valve procedures in comparison to surgical valves, together with a summary of the specific design features of several emerging transcatheter valves. How the current and future generation transcatheter valves are likely to impact on patient treatment is also explored.

Ultrastructure of porcine mitral valve chordae tendineae.

BACKGROUND AND AIM OF THE STUDY: The chordae tendineae, which form an important component of the mitral valve apparatus, experience continuous cyclic loading and are thus well-adapted for effectively storing and dissipating energy. An understanding of their microstructure would be expected to shed light on the mechanism of their remarkable durability. METHODS: In these studies, porcine mitral valve chordae from freshly slaughtered pigs were used. Histological samples of Picrosirius Red-stained and Movat's pentachrome-stained chordae were examined with optical microscopy and laser scanning confocal microscopy. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to study the ultrastructure at high magnification. RESULTS: Both, optical microscopy and SEM revealed that the waviness of collagen fibers was uniform across the thickness of the chordae, with no straight fibers in the core. Wavy fibers and fiber bundles were found to be in skewed-register, rather than transverse. Collagen fiber bundles were found to undulate in a three-dimensional path, rather than the planar waveform, as reported previously. TEM showed that different types of chordae had different fibril configurations. Marginal chordae had smaller diameters but a higher fibril density than did basal and strut chordae. CONCLUSION: The configuration of collagen fibrils in the mitral valve chordae is more complex than initially thought, and different chordae have morphologies that are likely specific to their mechanical role in the mitral apparatus. These findings provide insight into possible improvements for chordal repair surgery, and form a structural basis for accurate computational modeling.

Heart valve tissue engineering.

Tissue-engineered heart valves have been proposed by physicians and scientists alike to be the ultimate solution for treating valvular heart disease. Rather than replacing a diseased or defective native valve with a mechanical or animal tissue-derived artificial valve, a tissue-engineered valve would be a living organ, able to respond to growth and physiological forces in the same way that the native aortic valve does. Two main approaches have been attempted over the past 10 to 15 years: regeneration and repopulation. Regeneration involves the implantation of a resorbable matrix that is expected to remodel in vivo and yield a functional valve composed of the cells and connective tissue proteins of the patient. Repopulation involves implanting a whole porcine aortic valve that has been previously cleaned of all pig cells, leaving an intact, mechanically sound connective tissue matrix. The cells of the patients are expected to repopulate and revitalize the acellular matrix, creating living tissue that already has the complex microstructure necessary for proper function and durability. Regrettably, neither of the 2 approaches has fared well in animal experiments, and the only clinical experience with tissue-engineered valves resulted in a number of early failures and patient death. This article reviews the technological details of the 2 main approaches, their rationale, their strengths and weaknesses, and the likely mechanisms for their failure. Alternative approaches to valvular tissue engineering, as well as the role of industry in shaping this field in the future, are also reviewed.

Skewness angle of interfibrillar proteoglycans increases with applied load on mitral valve chordae tendineae.

In highly aligned connective tissues, such as tendon, collagen fibrils are linked together by proteoglycans (PGs). Recent mechanical and theoretical studies on tendon micromechanics have implied that PGs mediate mechanical interactions between adjacent collagen fibrils. We used transmission electron microscopy to observe the collagen fibril-PG interactions in porcine mitral valve chordae under variable loading conditions and found that PGs attached to collagen fibrils perpendicularly in the load-free situation, and became skewed when the chordae were loaded. The average skewness angle of PGs increased with the applied load, and hence the strain in the chordae. The observation of PG skewing with the application of load demonstrates that, in mitral valve chordae, interfibrillar slippage occurs and that PGs play a role in fibril-to-fibril interaction and likely transfer force. The results of this study provide new insights into the mechanical role of PGs and support some recent theoretical models.

Apparently normal mitral valves in patients with heart failure demonstrate biochemical and structural derangements: an extracellular matrix and echocardiographic study.

OBJECTIVES: This study assessed apparently normal mitral valves from patients with congestive heart failure (CHF) using biochemical and echocardiographic measures of extracellular matrix (ECM) and anatomy. BACKGROUND: Mitral regurgitation (MR) is frequently found in patients with CHF. This MR is considered purely functional, yet animal studies suggest that altered left ventricular (LV) function leads to increased cellularity and fibrosis of the mitral valve. Therefore, we hypothesized that patients with CHF might have partly organic MR, via dysfunctional valvular remodeling. METHODS: Mitral valves from transplant recipient hearts of patients with CHF (23 dilated, 14 ischemic) were analyzed for deoxyribonucleic acid (DNA), collagen, glycosaminoglycan (GAG), and water concentrations and compared with autopsy controls. Cardiac dimensions and functional parameters (measured from recent echocardiograms) were compared with biochemical parameters using a repeated measures generalized linear model. RESULTS: The mitral valves in CHF had up to 78% more DNA (p <0.03), 59% more GAGs (p <0.02), and 15% more collagen (p <0.007), but 7% less water (p <0.05) than normal. The absence of anterior leaflet redundancy was associated with these deranged biochemical measures (p <0.03). Associations were found between leaflet thickness and DNA concentration (+, p=0.003), annular diameter and chordal collagen (+, p=0.03), and water concentration and both left atrial diameter (-, p=0.008) and LV collagen concentration (-, p=0.04). CONCLUSIONS: Mitral valves in CHF are biochemically different from normal, with ECM changes that are influenced by the altered cardiac dimensions. This remodeling suggests that MR in patients with CHF may not be purely functional, and that these valves are not "normal."

Novel geometries for tissue-engineered tendonous collagen constructs.

A promising approach to addressing the performance limitations of currently available mechanical and bioprosthetic heart valves lies in tissue engineering. Tissue-engineered valves should incorporate the complex microstructure of the native valves to mimic their unique mechanics. This would include a layered topology, mesh networks, and branched collagen fiber bundles. Our approach to heart valve tissue engineering is to develop the functional components of the aortic valve cusps separately in vitro and, once they are mature, integrate them into a composite valve structure. Here we report on our efforts to create more complex collagenous structures, suitable for heart valve tissue engineering. Collagen fiber bundles were fabricated using the principle of directed collagen gel contraction, using neonatal rat aortic smooth muscle cells and acid-soluble type I rat-tail tendon collagen. The collagen gels were cast into rectangular or branched wells with porous end holders that constrained the gels longitudinally but allowed contraction to occur transverse to the long axis. Pairs of such constructs were placed in direct contact with each other and cultured further to determine whether they integrated to form continuous tissue. After 6-8 weeks of culture, highly compacted and aligned collagen fiber bundles formed. Mechanical testing revealed that linear constructs (2 free ends) with an 8:1 aspect ratio were significantly stronger than similar constructs with an aspect ratio of 2:1 (mean +/- SD, 298 +/- 90 kPa vs. 152 +/- 49 kPa; p < .001). Branching reduced mechanical strength considerably. Constructs fabricated with 4 free ends were significantly weaker than constructs with 3 ends (31 +/- 32 kPa vs. 116 +/- 66 kPa; p < .003). Histologic images demonstrated the integration of the crossed collagen bundles, with a bonding strength of 2.1 +/- 1.1 g (0.02 N). We found that the geometry of the molds into which the collagen constructs are cast can greatly affect their mechanical strength: multibranched constructs were the weakest, and long, linear constructs were the strongest. We also found that integration of collagen constructs occurs in vitro and that the fabrication of a composite structure in vitro is probably feasible.

Glycosaminoglycan profiles of myxomatous mitral leaflets and chordae parallel the severity of mechanical alterations.

OBJECTIVES: This biochemical study compared the extracellular matrix of normal mitral valves and myxomatous mitral valves with either unileaflet prolapse (ULP) or bileaflet prolapse (BLP). BACKGROUND: Myxomatous mitral valves are weaker and more extensible than normal valves, and myxomatous chordae are more mechanically compromised than leaflets. Despite histological evidence that glycosaminoglycans (GAGs) accumulate in myxomatous valves, previous biochemical analyses have not adequately examined the different GAG classes. METHODS: Leaflets and chordae from myxomatous valves (n = 41 ULP, 31 BLP) and normal valves (n = 27) were dried, dissolved, and assayed for deoxyribonucleic acid, collagen, and total GAGs. Specific GAG classes were analyzed with selective enzyme digestions and fluorophore-assisted carbohydrate electrophoresis. RESULTS: Biochemical changes were more pronounced in chordae than in leaflets. Myxomatous leaflets and chordae had 3% to 9% more water content and 30% to 150% higher GAG concentrations than normal. Collagen concentration was slightly elevated in the myxomatous valves. Chordae from ULP had 62% more GAGs than those from BLP, primarily from elevated levels of hyaluronan and chondroitin-6-sulfate. CONCLUSIONS: The GAG classes elevated in the myxomatous chordae are associated with matrix microstructure and elastic fiber deficiencies and may influence the hydration-related "floppy" nature of these tissues. These abnormalities may be related to the reported mechanical weakness of myxomatous chordae. The biochemical differences between ULP and BLP confirm previous mechanical and echocardiographic distinctions.

Mitral valve stiffening in end-stage heart failure: evidence of an organic contribution to functional mitral regurgitation.

OBJECTIVE: Mitral regurgitation is a complication for many patients with congestive heart failure. Although this regurgitation is considered purely functional, we hypothesize that the alterations in cardiac geometry and function induce dysfunctional remodeling of the mitral valve, which can be demonstrated by alterations in the material behavior of the leaflets and chordae. METHODS: Mitral leaflets and chordae from 23 valves from transplant recipient hearts (11 with dilated and 12 with ischemic cardiomyopathy) and from 21 normal valves (from autopsy) were mechanically tested. RESULTS: Radially oriented anterior mitral leaflet strips from failing hearts were 61% stiffer and 23% less viscous on average than those from autopsy control hearts. The mean stiffness of circumferentially oriented anterior leaflet strips was 50% higher than that of control hearts. Leaflet extensibility was reduced 35% overall. Likewise, the failing heart chordae were an average of 16% stiffer (all P < or = .05). CONCLUSIONS: Mitral valves in congestive heart failure have significantly altered mechanics that suggest that the tissue is permanently distended and fibrotic and might be unable to stretch sufficiently to cover the valve orifice. These material changes in the valve tissues accompany the biochemical alterations in extracellular matrix composition that we have previously reported. Our finding of leaflet and chordal remodeling suggests that mitral regurgitation in patients experiencing heart failure might not be purely functional and that these mitral valves should not be considered normal. Moreover, there are implications for strategies of mitral valve surgery or percutaneous approaches in this patient population.

Mesostructures of the aortic valve.

BACKGROUND AND AIM OF THE STUDY: The aortic valve cusp is commonly described as a three-layered structure containing circumferentially aligned fiber bundles. Little is known, however, regarding fiber bundle sizes, branching patterns, or how they are connected. This is because previous morphological studies relied primarily on histological sectioning and staining techniques, which tend to affect all of the collagen, regardless of structure or orientation. METHODS: To address this problem, a novel system was developed for the visualization and analysis of the intermediate-scale 'mesostructures' of aortic valve cusps. Mesostructures are defined as the branching fiber bundle and membrane structures that make up the valve. This system uses elliptically polarized light to provide contrast between collagen mesostructures without the need for embedding, staining, or other contrast-enhancing techniques. Using this system, high-resolution images of 42 whole porcine aortic valve cusps were acquired in an unloaded (i.e. resting) condition and during application of controlled manipulation. Image-processing algorithms were developed to quantify fiber bundle morphological features and produce detailed maps of the fiber bundle patterns. RESULTS: Fiber bundle sizes and patterns were found to be significantly different for each of the three cusps. The non-coronary cusp had a significantly smaller bundle diameter (0.9 +/- 0.07 mm) than the left and right coronary cusps (1.1 +/- 0.08 mm). The left and non-coronary cusps appeared to be mirror images of each other, whereas the right coronary cusp was self-symmetric. When applying controlled loads to the cusp specimens, thin, overlapping, collagenous membranes were often found which connected the fiber bundles. Interesting pinnate fiber branching patterns were also found. CONCLUSION: These morphological results were strikingly different than the currently accepted three-layer description, and may provide valuable insight into aortic valve structure-function relationships.

Invariant formulation for dispersed transverse isotropy in aortic heart valves: an efficient means for modeling fiber splay.

Most soft tissues possess an oriented architecture of collagen fiber bundles, conferring both anisotropy and nonlinearity to their elastic behavior. Transverse isotropy has often been assumed for a subset of these tissues that have a single macroscopically-identifiable preferred fiber direction. Micro-structural studies, however, suggest that, in some tissues, collagen fibers are approximately normally distributed about a mean preferred fiber direction. Structural constitutive equations that account for this dispersion of fibers have been shown to capture the mechanical complexity of these tissues quite well. Such descriptions, however, are computationally cumbersome for two-dimensional (2D) fiber distributions, let alone for fully three-dimensional (3D) fiber populations. In this paper, we develop a new constitutive law for such tissues, based on a novel invariant theory for dispersed transverse isotropy. The invariant theory is derived from a novel closed-form 'splay invariant' that can easily handle 3D fiber populations, and that only requires a single parameter in the 2D case. The model fits biaxial data for aortic valve tissue as accurately as the standard structural model. Modification of the fiber stress-strain law requires no reformulation of the constitutive tangent matrix, making the model flexible for different types of soft tissues. Most importantly, the model is computationally expedient in a finite-element analysis, demonstrated by modeling a bioprosthetic heart valve.

Biomechanical and echocardiographic characterization of flail mitral leaflet due to myxomatous disease: further evidence for early surgical intervention.

BACKGROUND: Flail mitral leaflet (FML) is a common complication of mitral valve prolapse, often leading to severe mitral regurgitation (MR) and left ventricular dysfunction. In the absence of timely surgical correction, survival is significantly impaired. Early recognition of FML and identification of risk factors is important because early intervention increases the chances of survival. METHODS: We studied 123 patients undergoing mitral valve surgery for severe MR caused by myxomatous disease. Chart review, echocardiography, and tensile testing were performed. RESULTS: Thirty-eight patients had FML, and 85 patients had non-flail mitral leaflet (non-FML). Patients with FML were younger (53.7 +/- 1.8 vs 59.3 +/- 1.4 years, P =.02), had more severe MR (3.89 +/- 0.04 vs 3.76 +/- 0.04, P =.02), were less likely to be in New York Heart Association class III or IV heart failure (5% vs 20%, P =.037), and were less likely to have bileaflet mitral valve prolapse (5% vs 38%, P <.001) than non-FML patients. Valve tissue from patients with FML had less stiff chordae (23.5 +/- 3.6 vs 59.1 +/- 11.7 Mpa, P =.006) that tended to have a lower failure stress (3.8 +/- 0.9 vs 9.6 +/- 2.2 Mpa, P =.07) and had more extensible leaflets (56.4% +/- 7.9% vs 42.9% +/- 2.7% strain, P =.04) compared with that of non-FML patients. CONCLUSIONS: The development of FML may result from intrinsic tissue abnormalities and is associated with a distinct subset of the myxomatous population. Identification of such clinical characteristics in this population and knowledge of an implicit mechanical abnormality of valve tissue may further the argument for early surgical correction.

Clinical, echocardiographic, and biomechanical differences in mitral valve prolapse affecting one or both leaflets.

Mitral valve prolapse (MVP) is the most common cause of severe mitral regurgitation necessitating surgical correction. Unileaflet prolapse (ULP), usually involving the posterior leaflet, is more common than bileaflet prolapse (BLP), which is more difficult to repair. Little is known about clinical, echocardiographic, and biomechanical differences between ULP and BLP. In this study, biomechanical testing was performed on mitral valve leaflets and chordae obtained at operation for severe mitral regurgitation. Preoperative clinical characteristics and echocardiographic measurements were obtained on surgical patients (ULP = 88, BLP = 37). Men outnumbered women by a factor of 4:1 in ULP, and by 3:1 in BLP. Patients with BLP were younger (53.2 +/- 1.7 vs 59.5 +/- 1.1 years) than those with ULP, and this difference was greater in women (48.9 +/- 2.5 vs 62.9 +/- 2.2 years). BLP patients were less likely to be hypertensive, and more likely to undergo valve replacement rather than repair. Echocardiography showed that BLP leaflets were longer and thicker than ULP leaflets. The severity of mitral regurgitation was similar in both groups, although ULP patients had a much higher incidence of flail leaflets (45% vs 5% in BLP). Mechanical strength of chordae was greater in BLP than in ULP, although leaflet strength was similar. The increased chordal strength in BLP may be responsible for less flail. In patients with MVP and severe mitral regurgitation requiring surgery, ULP and BLP are distinct entities with substantial differences in the population affected, in echocardiographic manifestations including prevalence of flail, in chordal mechanics, and in the likelihood of surgical repair.

Fabrication of mitral valve chordae by directed collagen gel shrinkage.

The principles of tissue engineering are being used to explore numerous applications in reconstructive surgery. Mitral valve chordae are one such potential area, as mitral valve repair is increasing in popularity and synthetic materials have not been used widely. The use of cells, combined with reconstituted type I collagen, is an attractive option for fabricating materials for the replacement of thin tendonous structures such as mitral valve chordae. We have been using the principle of directed collagen gel shrinkage to fabricate tendinous structures with good mechanical properties. In this study, our objective was to maximize the strength of the collagen constructs by choosing cell type and optimizing cell-seeding density, culture time, and initial collagen concentration. A collagen-cell suspension was cast into silicone rubber wells with microporous anchors at the ends and cultured in an incubator. The anchors allowed shrinkage to occur only transverse to the long axis of the wells, thus creating highly aligned collagenous constructs. Collagen gel contraction increased with higher cell-seeding density. The optimal value was 10(6) cells/mL. The rate of gel contraction decreased with the initial collagen concentration. Fibril density increased with culture time, as the gel contracted. After the system was optimized, the mechanical strength of the constructs increased to 1.1 MPa, a value at least an order of magnitude greater than previously published results with similar systems. This study has demonstrated that collagen-cell constructs, with material properties similar to those of native mitral valve chordae, can be developed using the principle of directed collagen gel shrinkage. These structures may have application in other areas that require small-diameter tendons.

Characterization of statically loaded tissue-engineered mitral valve chordae tendineae.

Chordae tendineae are essential to the proper function of the mitral valve. Native chordae contain a dense collagenous core and an outer elastin sheath. We have been using the principle of directed collagen gel shrinkage to fabricate tissue-engineered mitral valve chordae. Because the microstructure of biologic tissues determines their mechanical behavior, the morphology of collagen and elastin in tissue-engineered chordae should mimic that of native chordae. The objective of this study, therefore, was to examine the morphology of our tissue-engineered constructs in comparison to native chordae. A collagen-cell suspension was cast into silicon rubber wells with microporous anchors at the ends and cultured in an incubator. The anchors allowed shrinkage to occur only transverse to the long axis of the wells, thus creating highly aligned collagen fibril constructs. The collagen constructs were cultured for 8 weeks and characterized mechanically, histologically, and biochemically at different culture time points. Histologic sections showed that in all mature constructs collagen fibers were oriented parallel to the long axis of the constructs. At the edge of the tissue collagen fibers were in general straight, whereas in the middle of the tissue they were wavy. Transmission electron microscopy showed a progressive increase in the density and longitudinal orientation of collagen fibrils with culture time. Light and scanning electron microscopy showed the presence of an elastin sheath around the collagen core. Immunostaining demonstrated that smooth muscle cells differentiate during tissue development and TUNEL assay showed that cells in the interior of the constructs undergo apoptosis. This study has demonstrated that collagen-cell constructs, with material properties and microstructure similar to native mitral valve chordae, can be developed using static culture.

Ultraviolet light-induced modification of crosslinked hyaluronan gels.

Hyaluronan (HA) gels (hylans) crosslinked with divinyl sulfone (DVS) are highly biocompatible and can be structurally modified to obtain desired mechanical properties that are attractive for their use as tissue-engineering scaffolds. However, unmodified hylan gels are not good substrates for cell attachment or infiltration, likely as a result of their smooth surface and the highly anionic nature of HA. This study investigated whether the cell-adhering characteristics of hylan gels could be enhanced by irradiation with ultraviolet (UV) light, with or without prior dehydration. The attachment and proliferation of neonatal rat smooth muscle cells atop these gels was compared with that on unmodified (control; C) or dehydrated (D) gels. UV-induced changes to gel structure and chemistry were characterized by confocal and electron microscopy, and fluorphore-assisted carbohydrate electrophoresis (FACE). Cell attachment was sparse on both unmodified (C) and dehydrated (D) gels. Significantly higher levels of cell attachment were observed on the surface of irradiated (UV) and dehydrated-irradiated (DUV) gels, likely because of texturing of the gel surface by UV light. In addition, dehydration of gels before UV irradiation created irregular pore-like structures through which cells appeared to migrate into the interior. FACE assays demonstrated that UV-irradiation alters the chemistry of HA, causing limited breakdown of HA chains and DVS crosslinks within gel and possibly creating new crosslinks that have not yet been identified. Because the hylan gels are altered structurally and chemically, binding of cells to the material is likely to be more permanent than possible by other approaches, such as coating of cell-adhesive matrix factors on the gel surface, described previously. The significance of this work is that we have developed a technique for the modification of DVS-crosslinked HA (hylans) to enhance their performance as a cellular scaffold for tissue-engineering applications.

Loss of chondroitin 6-sulfate and hyaluronan from failed porcine bioprosthetic valves.

Explanted porcine bioprosthetic valves have a thinned spongiosa, partially because of an overall loss of glycosaminoglycans (GAGs). We measured the concentrations of specific GAG classes in explanted bioprosthetic valves (n = 14, implanted 12.0 +/- 4.7 years) compared with glutaraldehyde-fixed porcine controls. After extraction with NaOH, GAGs were analyzed using either a hexuronic acid assay or fluorophore-assisted carbohydrate electrophoresis to quantify the individual GAG classes. The total GAG concentration in explants was 198 +/- 95 pmol/mg wet weight-93% less than freshly fixed controls. Explants also contained altered proportions of the different GAG classes relative to controls. The proportions of hyaluronan and chondroitin/dermatan-6-sulfate were reduced from 39 to 7% and 34 to 18% of total GAGs, respectively. The predominant explant GAG class was chondroitin/dermatan-4-sulfate (proportion elevated from 14 to 70%). This GAG is commonly found in the collagen-associated proteoglycan decorin, which is likely well crosslinked by glutaraldehyde. Chondroitin-6-sulfate is commonly found in the water- and hyaluronan-binding proteoglycan versican, which is likely poorly crosslinked. The loss of versican and its associated water-binding capacity is consistent with the thinned spongiosa. The resultant compromise of hydration, compressive resistance, and viscoelasticity may be responsible for the deterioration of the bioprosthesis in vivo.

A structural basis for the size-related mechanical properties of mitral valve chordae tendineae.

It has been reported previously that the mechanical properties of mitral valve chordae tendineae vary with chordal size and type. The popularity of mitral valve repair and chordal transposition warrant a better understanding of this phenomenon. The objectives of this study were to characterize the size- and type-related variations in chordal mechanics and explain them from the ultra-structural viewpoint. A total of 52 porcine mitral valve chordae from eight hearts were mechanically tested. We found that thicker chordae were more extensible than thinner chordae (4.2+/-1.5%, 8.1+/-2.5%, 15.7+/-3.9% and 18.4+/-2.8% strain corresponding to chordae with cross-sectional areas of 0.1-0.5, 0.5-1.0, 1.0-2.0, and 2.0-3.0mm(2), respectively), and had lower moduli (90.1+/-22.3, 83.7+/-18.5, 66.3+/-13.5 and 61.7+/-13.3 MPa corresponding to the same chordae groups). Polarized light microscopy was used to measure collagen fibril crimp. Thicker chordae had smaller crimp period than thinner chordae (11.3+/-1.4 microm vs. 14.8+/-3.0 microm), and were thus more highly crimped. Thicker chordae could therefore extend to greater strain before lock-up. Transmission electron microscopy (TEM) was used to measure choral fibril ultra-structure. Thinner chordae had lower average fibril diameter than thicker chordae but greater average fibril density. The cross-sectional area occupied by fibrils, however, was found to be constant at 49+/-2% regardless of chordal size or type. The difference in moduli between thick and thin chordae can therefore be explained by differences in fibril packaging and hence fibril-to-fibril interactions. According to a simple fibril interaction model, chordae with smaller diameter fibrils will have a greater number of fibril-to-fibril interactions, and hence a greater modulus.

A new method of estimating gauge length for porcine aortic valve test specimens.

Porcine aortic valve (PAV) cusps are folded and wrinkled in the in vitro state. In the tensile testing of PAV specimens, estimating gauge length (the length at which a specimen starts to offer measurable resistance to load) is often difficult and subjective. We have therefore developed a new method for estimating the gauge length of such tissues. The method is based on the observation that the specimen's gauge length can be associated with a stationary point on the slope of its load-length curve if loaded from a wrinkled state, or a state of slight compression. We represented the load-length response of test specimens in the low-load, high-compliance region by a cubic function and determined the stationary point on the slope of the function using elementary calculus. The cubic function representation is fine-tuned by reducing or expanding an originally selected "test region" until the correlation coefficient of the cubic fit is maximized. The new method was applied to data obtained from the tensile testing of strips of heart valve tissue and was found to be objective, repeatable and robust.