Advanced Methodology and Preliminary Measurements of Molecular and Mechanical Properties of Heart Valves under Dynamic Strain.

Mammalian heart valves are soft tissue assemblies with multi-scale material properties. This is because they are constructs comprising both muscle and non-contractile extracellular matrix proteins (such as collagens and proteoglycans) and transition regions where one form of tissue structure becomes another, significantly different form. The leaflets of the mitral and tricuspid valves are connected to chordae tendinae which, in turn, bind through papillary muscles to the cardiac wall of the ventricle. The transition regions between these tissue subsets are complex and diffuse. Their material composition and mechanical properties have not been previously described with both micro and nanoscopic data recorded simultaneously, as reported here. Annotating the mechanical characteristics of these tissue transitions will be of great value in developing novel implants, improving the state of the surgical simulators and advancing robot-assisted surgery. We present here developments in multi-scale methodology that produce data that can relate mechanical properties to molecular structure using scanning X-ray diffraction. We correlate these data to corresponding tissue level (macro and microscopic) stress and strain, with particular emphasis on the transition regions and present analyses to indicate points of possible failure in these tissues.

Effect of glutaraldehyde based cross-linking on the viscoelasticity of mitral valve basal chordae tendineae.

BACKGROUND: Mitral valve failure can require repair or replacement. Replacement bioprosthetic valves are treated with glutaraldehyde prior to implantation. The aim of this study was to determine the changes in mechanical properties following glutaraldehyde fixation of mitral valve chordae. METHODS: To investigate the impact of glutaraldehyde on mitral valve chordae, 24 basal chordae were dissected from four porcine hearts. Anterior and posterior basal (including strut) chordae were used. All 24 chordae were subjected to a sinusoidally varying load (mean level 2N, dynamic amplitude 2N) over a frequency range of 0.5-10 Hz before and after glutaraldehyde treatment. RESULTS: The storage and loss modulus of all chordal types decreased following glutaraldehyde fixation. The storage modulus ranged from: 108 to 119 MPa before fixation and 67.3-87.4 MPa following fixation for basal chordae; 52.3-58.4 MPa before fixation and 47.9-53.5 MPa following fixation for strut chordae. Similarly, the loss modulus ranged from: 5.47 to 6.25 MPa before fixation and 3.63-4.94 MPa following fixation for basal chordae; 2.60-2.97 MPa before fixation and 2.31-2.93 MPa following fixation for strut chordae. CONCLUSION: The viscoelastic properties of mitral valve chordae are affected by glutaraldehyde fixation; in particular, the reduction in storage moduli decreased with an increase in chordal diameter.

Targeting the finite-deformation response of wavy biological tissues with bio-inspired material architectures.

The Particle Swarm Optimization algorithm driven by a homogenized-based model is employed to target the response of three types of heart-valve chordae tendineae with different stiffening characteristics due to different degrees of waviness of collagen fibril/fiber bundles. First, geometric and material parameters are identified through an extensive parametric study that produce excellent agreement of the simulated response based on simplified unit cell architectures with the actual response of the complex biological tissue. These include amplitude and wavelength of the crimped chordae microstructure, elastic moduli of the constituent phases, and degree of microstructural refinement of the stiff phase at fixed volume fraction whose role in the stiffening response is elucidated. The study also reveals potential non-uniqueness of bio-inspired wavy microstructures in attaining the targeted response of certain chordae tendineae crimp configurations. The homogenization-based Particle Swarm Optimization algorithm, whose predictions are validated through the parametric study, is then shown to be an excellent tool in identifying optimal unit cell architectures in the design space that exhibits very steep gradients. Finally, defect criticality of optimal unit cell architectures is investigated in order to assess their feasibility in replacing actual biological tendons with stiffening characteristics.

Pathogenesis of mitral valve disease in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis VII (MPS VII) is due to the deficient activity of ß-glucuronidase (GUSB) and results in the accumulation of glycosaminoglycans (GAGs) in lysosomes and multisystemic disease with cardiovascular manifestations. The goal here was to determine the pathogenesis of mitral valve (MV) disease in MPS VII dogs. Untreated MPS VII dogs had a marked reduction in the histochemical signal for structurally-intact collagen in the MV at 6 months of age, when mitral regurgitation had developed. Electron microscopy demonstrated that collagen fibrils were of normal diameter, but failed to align into large parallel arrays. mRNA analysis demonstrated a modest reduction in the expression of genes that encode collagen or collagen-associated proteins such as the proteoglycan decorin which helps collagen fibrils assemble, and a marked increase for genes that encode proteases such as cathepsins. Indeed, enzyme activity for cathepsin B (CtsB) was 19-fold normal. MPS VII dogs that received neonatal intravenous injection of a gamma retroviral vector had an improved signal for structurally-intact collagen, and reduced CtsB activity relative to that seen in untreated MPS VII dogs. We conclude that MR in untreated MPS VII dogs was likely due to abnormalities in MV collagen structure. This could be due to upregulation of enzymes that degrade collagen or collagen-associated proteins, to the accumulation of GAGs that compete with proteoglycans such as decorin for binding to collagen, or to other causes. Further delineation of the etiology of abnormal collagen structure may lead to treatments that improve biomechanical properties of the MV and other tissues.

Determination of the mechanical properties of normal and calcified human mitral chordae tendineae.

The aim of the present research is to determine the influence of the calcification of human mitral valves on the mechanical properties of their marginal chordae tendineae. The study was performed on marginal chords obtained from thirteen human mitral valves, explanted at surgery, including six non-calcified, four moderately calcified and three strongly calcified valves. The mechanical response of the chords from the non-calcified and moderately calcified valves was determined by means of quasi-static tensile tests (the poor condition of the strongly calcified valves prevented them from being mechanically characterised). The material parameters that were obtained and analysed (the Young's modulus, the secant modulus, the proportional limit stress, the ultimate strength, the strain at fracture and the density of energy stored up to maximum load) revealed noticeable differences in mechanical behaviour between the two groups of mitral chordae tendineae. Large scatter was obtained in all cases, nevertheless, considering the mean values, it was observed that the normal chords are between three and seven times stiffer or more resistant than the moderately calcified ones. On the contrary, the results obtained for the strain at fracture showed a rather different picture as, in this case, no significant differences were observed between the two families of chords. A scanning electron microscopy study was conducted in order to find out the relevant features of the calcium deposits present in the calcified chordae tendineae. In addition, the general aspects appreciated in the stress vs. strain curves were correlated with the collagen morphological evidences determined microscopically. Finally, the calcium content present in the three groups of chords was quantitatively determined through atomic absorption spectroscopy; then, the relation between the mechanical properties of normal and moderately calcified chords as a function of its calcium content was obtained. This analysis confirmed the existence of a strong correlation between calcium content and stiffness or resistance whereas the influence on the ductility seems to be negligible.

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.

Correlation between clinical and histologic patterns of degenerative mitral valve insufficiency: a histomorphometric study of 130 excised segments.

The objectives of this study were to examine quantitatively the histological changes in incompetent degenerative mitral valves obtained at surgery for mitral valve repair, and to determine whether Barlow's disease (BD) and fibroelastic deficiency (FED) can be distinguished by histology. The billowing mitral leaflet syndrome (or Barlow's disease) and FED can be distinguished on the basis of clinical patterns and gross features, but their histologic patterns have not been described. One hundred thirty patients were studied. Thirty-nine (24 males) had BD; 44 (38 males) FED; 15 (7 males) Marfan's syndrome (MS); and 32 patients (25 males) a non-determined degenerative disease. Histological changes of the resected segment of the valve were quantitatively evaluated using scores of severity. A discriminant analysis was performed. The groups defined by the computer were checked for concordance with groups defined by the surgeon. Collagen alterations were found the most severe in MS patients. BD and MS had the most myxoid infiltration. MS and FED patients had the most elastic fiber alterations. No BD in males and only one in females were misclassified by the discriminant procedure into the FED group. Overall, the percentages of correct matchings were 54% in males and 62% in females. When the age of patients and the size of ring were added to histology to determine whether this additional information provided more discrimination, the percentages of correct matchings reached 90% in males and 100% in females. BD and FED are two fairly distinct entities, which can be distinguished by quantitative histology, whereas only modest differences were found in qualitative histology.

Heat-induced changes in the mechanics of a collagenous tissue: isothermal free shrinkage.

We present data from isothermal free-shrinkage tests (i.e., performed in the absence of mechanical loads) wherein bovine chordae tendineae were subjected to temperatures from 65 to 85 degrees C for 120 to 1200 s. These data reveal four new insights into heat-induced denaturation of a collagenous tissue. First, a characteristic time for the free shrinkage appears to exhibit an Arrhenius-type relationship with temperature. Second, scaling the actual heating time via the characteristic time results in a single correlation between free shrinkage and the duration of heating; this correlation suggests a time-temperature equivalence. Third, it is the cumulative, not current, heating time that governs the free shrinkage. And fourth, heat-induced free shrinkage is partially recovered when the tissue is returned to 37 degrees C, this recovery also being time-dependent. Although these findings will help guide future experimentation and constitutive modeling, as well as the design of new heat-based clinical therapies, there is a pressing need to collect additional isothermal data, particularly in the presence of well-defined mechanical loads.

Abnormalities in elastic fibers and other connective-tissue components of floppy mitral valve.

Histologic, immunohistochemical, and ultrastructural studies were performed on 12 floppy mitral valves, 4 mitral valves showing focal myxomatous changes without prolapse, and 3 normal mitral valves. All floppy mitral valves were thickened by deposits of proteoglycans and also showed diverse structural abnormalities in collagen and elastic fibers. From these observations we conclude that (1) the structure of all major components of connective tissue in floppy mitral valves is abnormal; (2) alterations in collagen and accumulations of proteoglycans are nonspecific changes that may be caused by the abnormal mechanical forces to which floppy mitral valves are subjected because of their excessively large surface area; (3) the presence of excessive amounts of proteoglycans may interfere with the normal assembly of collagen and elastic fibers; (4) abnormalities of elastic fibers resemble those in other conditions characterized by structural dilatation or tissue expansion; and (5) alterations in elastin could result from defective formation, increased degradation, or both.

Structure and protein composition of sites of papillary muscle attachment to chordae tendineae in avian hearts.

Most cardiac myocytes transmit force across fasciae adherentes, specialized sites of cell-cell adhesion. However, some cardiac myocytes in papillary muscle terminate on collagenous connective tissue in the chordae tendineae. These papillary myotendinous junctions (MTJs) are specialized for force transmission from myocytes to extracellular matrix. In the present study, we compared structural molecules at papillary MTJs to those at fasciae adherentes and skeletal MTJs. By using indirect immunofluorescence, we found that papillary MTJs more closely resemble skeletal MTJs in their molecular composition in that they are enriched in talin, vinculin, integrin, and fibronectin. Zeugmatin and alpha-actinin, both components of fasciae adherentes, are absent from papillary MTJs. Although papillary MTJs and skeletal MTJs display strong similarities in structural protein composition, ultrastructural organization of the two junctions is different. Papillary MTJs display little folding of the junctional membrane and, according to morphological criteria, more closely resemble sites of thin filament-membrane association in smooth muscle than skeletal MTJs. Thus, papillary MTJs display a combination of structural characteristics described previously in skeletal and smooth muscles but exhibit few structural features observed previously in cardiac fasciae adherentes.

Atrial natriuretic factor in Purkinje fibers of rabbit heart.

The Purkinje fibers of the rabbit false tendons (chordae tendineae spuriae) are endocrine cells containing immunoreactive atrial natriuretic factor (ANF) and ANF messenger RNA (mRNA). These cells, as visualized by immunocryoultramicrotomy, contain immunoreactive ANF in their secretory granules and their Golgi complex and exhibit ANF mRNA, as visualized by in situ hybridization with an ANF complementary RNA probe. The content of immunoreactive ANF and ANF mRNA of the Purkinje fibers is midway between that of atrial and ventricular working cardiocytes. High-pressure liquid chromatography analysis of immunoreactive ANF using antibodies against the C-terminal and N-terminal moieties of the molecule indicates that part of immunoreactive ANF contained in Purkinje fibers is the propeptide [Asn1,Tyr126]ANF whereas part was nonspecifically cleaved into C-terminal and N-terminal ANF. The chordae tendineae spuriae exhibit binding sites for ANF (Kd:approximately 1.0 nM; Bmax:approximately 2.3 fmol/mg). ANF profoundly decreases basal and stimulated (epinephrine, dopamine, isoproterenol, and forskolin) adenylate cyclase activity and cyclic adenosine monophosphate (AMP) levels. ANF has little effect on norepinephrine-stimulated adenylate cyclase activity or on norepinephrine-stimulated cyclic AMP levels. ANF produces only a slight increase in guanylate cyclase activity and cyclic guanosine monophosphate levels at high (10(7)-10(6) M) concentrations. These results suggest an autocrine function for ANF in the modulation of the impulse in the peripheral conduction cells (Purkinje fibers) of the rabbit through changes in second messenger levels.

Floppy mitral valve chordae tendineae: histopathologic alterations.

Pathologic studies of floppy or myxomatous mitral valves have focused primarily on changes in the valve cusps, with little attention given to the chordae tendineae. In a systematic study of the histopathology of floppy mitral valve chordae tendineae, 128 nonruptured chordae from 8 severely regurgitant floppy mitral valves were compared to 152 chordae from 10 normal control mitral valves and to 152 chordae from 8 control mitral valves with severe regurgitation due to ischemic heart disease. Collagen alterations were observed in 2% of normal mitral valve chordae and 3% of control regurgitant mitral valve chordae compared to 38% of floppy mitral valve chordae. Moderate or severe acid mucopolysaccharide accumulation was observed in 2% of normal mitral valve chordae and 3% of control regurgitant mitral valve chordae compared to 39% of floppy mitral valve chordae. Nonuniform histopathologic alterations, rare in normal and control regurgitant mitral valve chordae tendineae, were frequent in floppy mitral valve chordae tendineae (p less than 0.001). Histopathologic alterations provide the basis for abnormal physical properties previously demonstrated in floppy mitral valve chordae tendineae and may predispose to chordal elongation and rupture.

Altered collagen composition in a prolapsing mitral valve with ruptured chordae tendineae.

A patient presented with mitral valve prolapse, ruptured chordae tendineae, severe mitral regurgitation and congestive heart failure. Pathologic studies demonstrated myxomatous changes of both mitral leaflets. Collagen analysis of the valve and of the chordae tendineae revealed the presence of type I collagen in normal quantities and a striking absence of type III and AB collagens. The altered collagen in this patient's mitral valve probably contributed to the development of his mitral valve disorder.