Comparison of High-Speed Polarization Imaging Methods for Biological Tissues.

We applied a polarization filter array and high-speed camera to the imaging of biological tissues during large, dynamic deformations at 7000 frames per second. The results are compared to previous measurements of similar specimens using a rotating polarizer imaging system. The polarization filter eliminates motion blur and temporal bias from the reconstructed collagen fiber alignment angle and retardation images. The polarization imaging configuration dose pose additional challenges due to the need for calibration of the polarization filter array for a given sample in the same lighting conditions as during the measurement.

Deep thrombosis characterization using photoacoustic imaging with intravascular light delivery.

Venous thromboembolism (VTE) is a condition in which blood clots form within the deep veins of the leg or pelvis to cause deep vein thrombosis. The optimal treatment of VTE is determined by thrombus properties such as the age, size, and chemical composition of the blood clots. The thrombus properties can be readily evaluated by using photoacoustic computed tomography (PACT), a hybrid imaging modality that combines the rich contrast of optical imaging and deep penetration of ultrasound imaging. With inherent sensitivity to endogenous chromophores such as hemoglobin, multispectral PACT can provide composition information and oxygenation level in the clots. However, conventional PACT of clots relies on external light illumination, which provides limited penetration depth due to strong optical scattering of intervening tissue. In our study, this depth limitation is overcome by using intravascular light delivery with a thin optical fiber. To demonstrate in vitro blood clot characterization, clots with different acuteness and oxygenation levels were placed underneath ten-centimeter-thick chicken breast tissue and imaged using multiple wavelengths. Acoustic frequency analysis was performed on the received PA channel signals, and oxygenation level was estimated using multispectral linear spectral unmixing. The results show that, with intravascular light delivery, clot oxygenation level can be accurately measured, and the clot age can thus be estimated. In addition, we found that retracted and unretracted clots had different acoustic frequency spectrum. While unretracted clots had stronger high frequency components, retracted clots had much higher low frequency components due to densely packed red blood cells. The PACT characterization of the clots was consistent with the histology results and mechanical tests.

A structural-based computational model of tendon-bone insertion tissues.

Tendon-to-bone insertion provides a gradual transition from soft tendon to hard bone tissue, functioning to alleviate stress concentrations at the junction of these tissues. Such macroscopic mechanical properties are achieved due to the internal structure in which collagen fibers and mineralization levels are key ingredients. We develop a structural-based model of tendon-to-bone insertion incorporating such details as fiber preferred orientation, fiber directional dispersion, mineralization level, and their inhomogeneous spatial distribution. A python script is developed to alter the tapered tendon-bone transition zone and to provide spatial grading of material properties, which may be rather complex as experiments suggest. A simple linear interpolation between tendon and bone material properties is first used to describe the graded property within the insertion region. Stress distributions are obtained and compared for spatially graded and various piece-wise materials properties. It is observed that spatial grading results in more smooth stress distributions and significantly reduces maximum stresses. The geometry of the tissue model is optimized by minimizing the peak stress to mimic in-vivo tissue remodeling. The in-silico elastic models constructed in this work are verified and modified by comparing to our in-situ biaxial mechanical testing results, thereby serving as translational tools for accurately predicting the material behavior of the tendon-to-bone insertions. This model will be useful for understanding how tendon-to-bone insertion develops during tissue remodeling, as well as for developing orthopedic implants.

Tissue Level Mechanical Properties and Extracellular Matrix Investigation of the Bovine Jugular Venous Valve Tissue.

Jugular venous valve incompetence has no long-term remedy and symptoms of transient global amnesia and/or intracranial hypertension continue to discomfort patients. During this study, we interrogate the synergy of the collagen and elastin microstructure that compose the bi-layer extracellular matrix (ECM) of the jugular venous valve. In this study, we investigate the jugular venous valve and relate it to tissue-level mechanical properties, fibril orientation and fibril composition to improve fundamental knowledge of the jugular venous valves toward the development of bioprosthetic venous valve replacements. Steps include: (1) multi loading biaxial mechanical tests; (2) isolation of the elastin microstructure; (3) imaging of the elastin microstructure; and (4) imaging of the collagen microstructure, including an experimental analysis of crimp. Results from this study show that, during a 3:1 loading ratio (circumferential direction: 900 mN and radial direction: 300 mN), elastin may have the ability to contribute to the circumferential mechanical properties at low strains, for example, shifting the inflection point toward lower strains in comparison to other loading ratios. After isolating the elastin microstructure, light microscopy revealed that the overall elastin orients in the radial direction while forming a crosslinked mesh. Collagen fibers were found undulated, aligning in parallel with neighboring fibers and orienting in the circumferential direction with an interquartile range of -10.38° to 7.58° from the circumferential axis (n = 20). Collagen crimp wavelength and amplitude was found to be 38.46 ± 8.06 µm and 4.51 ± 1.65 µm, respectively (n = 87). Analyzing collagen crimp shows that crimp permits about 12% true strain circumferentially, while straightening of the overall fibers accounts for more. To the best of the authors' knowledge, this is the first study of the jugular venous valve linking the composition and orientation of the ECM to its mechanical properties and this study will aid in forming a structure-based constitutive model.

Strain state dependent anisotropic viscoelasticity of tendon-to-bone insertion.

Tendon-to-bone insertion tissues may be considered as functionally-graded connective tissues, providing a gradual transition from soft tendon to hard bone tissue, and functioning to alleviate stress concentrations at the junction of these tissues. The tendon-to-bone insertion tissues demonstrate pronounced viscoelastic behavior, like many other biological tissues, and are designed by the nature to alleviate stress at physiological load rates and strains states. In this paper we present experimental data showing that under biaxial tension tendon-to-bone insertion demonstrates rate-dependent behavior and that stress-strain curves for the in-plane components of stress and strain become less steep when strain rate is increased, contrary to a typical viscoelastic behavior, where the opposite trend is observed. Such behavior may indicate the existence of a protective viscoelastic mechanism reducing stress and strain during a sudden increase in mechanical loading, known to exist in some biological tissues. The main purpose of the paper is to show that such viscoelastic stress reduction indeed possible and is thermodynamically consistent. We, therefore, propose an anisotropic viscoelasticity model for finite strain. We identify the range of parameters for this model which yield negative viscoelastic contribution into in-plane stress under biaxial state of strain and simultaneously satisfy requirements of thermodynamics. We also find optimal parameters maximizing the observed protective viscoelastic effect for this particular state of strain. This model will be useful for testing and describing viscoelastic materials and for developing interfaces for dissimilar materials, considering rate effect and multiaxial loadings.

High-speed polarization imaging of dynamic collagen fiber realignment in tendon-to-bone insertion region.

A high-speed polarization imaging instrument is demonstrated to be capable of measuring the collagen fiber alignment orientation and alignment strength during high-displacement rate dynamic loading at acquisition rates up to 10 kHz. The implementation of a high-speed rotating quarter wave plate and high-speed camera in the imaging system allows a minimum measurement acquisition time of 6 ms. Sliced tendon-to-bone insertion samples are loaded using a modified drop tower with an average maximum displacement rate of 1.25 m / s, and imaged using a high-speed polarization imaging instrument. The generated collagen fiber alignment angle and strength maps indicate the localized deformation and fiber realignment in tendon-to-bone samples during dynamic loading. The results demonstrate a viable experimental method to monitor collagen fiber realignment in biological tissue under high-displacement rate dynamic loading.

Biaxial mechanical behavior of bovine saphenous venous valve leaflets.

Chronic venous disease is caused by chronic venous insufficiency (CVI), which results in significant symptoms such as venous ulcers, ankle eczema, leg swelling, etc. Venous valve incompetence is a major cause of CVI. When the valves of veins in the leg become incompetent (i.e., do not close properly), blood is able to flow backwards (i.e., reflux), which results in blood pooling in the lower extremities, distal venous hypertension, and CVI. Current clinical therapies, such as surgical venous valve reconstruction and bioprosthetic venous valve replacement, are highly invasive and only moderately successful. This is due, in part, to the scanty information available about venous valve leaflet structure and mechanical properties. To date, only one previous study by our research group has reported on the mechanical properties of venous valve leaflet tissue, and specifically in the case of jugular vein valves. In this study, we conducted equibiaxial tensile tests on bovine saphenous vein valve leaflet tissues to better understand their nonlinear, anisotropic mechanical behavior. By stretching the valvular tissues to 60% strain in both the circumferential and radial directions, we generated stress-strain curves for proximal (i.e., those closest to the heart) and distal (i.e., those furthest from the heart) valve leaflets. Histology and collagen assays were also conducted to study corresponding leaflet microstructures and the biochemical properties of the tissues. Results showed: (1) saphenous venous valve tissues possessed overall anisotropic properties. The tissues were stiffer in the circumferential direction than in the radial direction (p<0.01), and (2) saphenous venous valve tissues from the proximal end showed nonlinear isotropic mechanical properties, while those from the distal end showed nonlinear anisotropic mechanical properties. (3) Distal saphenous venous valve tissues appeared to be stiffer than proximal ones in the circumferential direction, p=0.04 (i.e., inter-valvular variability), and (4) the collagen concentration showed a decreasing trend from the proximal to the distal end. This study focuses on highly relevant animal (bovine) tissues to develop test protocols, establish biomechanical structure-function correlations, and to provide data critical to the design of clinical prosthetic venous valves. To the best of the author's knowledge, this is the first study reporting the biaxial mechanical properties of saphenous venous valve leaflet tissues and thus contributes toward refining our collective understanding of valvular tissue biomechanics.

Composition and structure of porcine digital flexor tendon-bone insertion tissues.

Tendon-bone insertion is a functionally graded tissue, transitioning from 200 MPa tensile modulus at the tendon end to 20 GPa tensile modulus at the bone, across just a few hundred micrometers. In this study, we examine the porcine digital flexor tendon insertion tissue to provide a quantitative description of its collagen orientation and mineral concentration by using Fast Fourier Transform (FFT) based image analysis and mass spectrometry, respectively. Histological results revealed uniformity in global collagen orientation at all depths, indicative of mechanical anisotropy, although at mid-depth, the highest fiber density, least amount of dispersion, and least cellular circularity were evident. Collagen orientation distribution obtained through 2D FFT of histological imaging data from fluorescent microscopy agreed with past measurements based on polarized light microscopy. Results revealed global fiber orientation across the tendon-bone insertion to be preserved along direction of physiologic tension. Gradation in the fiber distribution orientation index across the insertion was reflective of a decrease in anisotropy from the tendon to the bone. We provided elemental maps across the fibrocartilage for its organic and inorganic constituents through time-of-flight secondary ion mass spectrometry (TOF-SIMS). The apatite intensity distribution from the tendon to bone was shown to follow a linear trend, supporting past results based on Raman microprobe analysis. The merit of this study lies in the image-based simplified approach to fiber distribution quantification and in the high spatial resolution of the compositional analysis. In conjunction with the mechanical properties of the insertion tissue, fiber, and mineral distribution results for the insertion from this may potentially be incorporated into the development of a structural constitutive approach toward computational modeling. Characterizing the properties of the native insertion tissue would provide the microstructural basis for developing biomimetic scaffolds to recreate the graded morphology of a fibrocartilaginous insertion. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3050-3058, 2017.

Constitutive modeling of jugular vein-derived venous valve leaflet tissues.

Venous valve tissues, though used in vein reconstruction surgeries and bioprosthetic valves with moderate success, have not been extensively studied with respect to their structure. Their inherent anisotropic, non-linear behavior combined with severe diseases which affect veins, such as chronic venous insufficiency, warrant understanding the structure and material behavior of these tissues. Hence, before any bioprosthetic grafts may be used in place of tissues, it is of the utmost importance to understand the mechanical and structural properties of these tissues as this may lead to higher success rates for valve replacement surgeries. The longevity of the bioprosthetics may also increase if the manufactured grafts behave the same as native valves. Building on the scant information about the uniaxial and biaxial mechanical properties of jugular venous valves and wall tissues from previous studies, the current focus of our investigation lies in understanding the material behavior by establishing a phenomenological strain energy-based constitutive relation for the tissues. We used bovine veins to study the behavior of valve leaflet tissue and adjoining wall tissue (from the proximal and distal ends of the veins) under different biaxial testing protocols. We looked at the behavior of numerical partial derivatives of the strain energy to select a suitable functional form for the strain energy for wall and valve tissues. Using this strain energy descriptor, we determined the Cauchy stress and compared it with experimental results under additional sets of displacement-controlled biaxial testing protocols to find material specific model parameters by the Powell's method algorithm. Results show that whereas wall tissue strain energy can be explained using a polynomial non-linear function, the valve tissue, due to higher non-linearities, requires an exponential function. This study may provide useful information for the primary stages of bioprosthetic designs and replacement surgeries and may support future studies investigating structural models. It may also support the study of valvular diseases by providing a way to understand material properties and behavior and to form a continuum model when required for numerical analyses and computational simulations.

Biaxial mechanical properties of bovine jugular venous valve leaflet tissues.

Venous valve incompetence has been implicated in diseases ranging from chronic venous insufficiency (CVI) to intracranial venous hypertension. However, while the mechanical properties of venous valve leaflet tissues are central to CVI biomechanics and mechanobiology, neither stress-strain curves nor tangent moduli have been reported. Here, equibiaxial tensile mechanical tests were conducted to assess the tangent modulus, strength and anisotropy of venous valve leaflet tissues from bovine jugular veins. Valvular tissues were stretched to 60% strain in both the circumferential and radial directions, and leaflet tissue stress-strain curves were generated for proximal and distal valves (i.e., valves closest and furthest from the right heart, respectively). Toward linking mechanical properties to leaflet microstructure and composition, Masson's trichrome and Verhoeff-Van Gieson staining and collagen assays were conducted. Results showed: (1) Proximal bovine jugular vein venous valves tended to be bicuspid (i.e., have two leaflets), while distal valves tended to be tricuspid; (2) leaflet tissues from proximal valves exhibited approximately threefold higher peak tangent moduli in the circumferential direction than in the orthogonal radial direction (i.e., proximal valve leaflet tissues were anisotropic; [Formula: see text]); (3) individual leaflets excised from the same valve apparatus appeared to exhibit different mechanical properties (i.e., intra-valve variability); and (4) leaflets from distal valves exhibited a trend of higher soluble collagen concentrations than proximal ones (i.e., inter-valve variability). To the best of the authors' knowledge, this is the first study reporting biaxial mechanical properties of venous valve leaflet tissues. These results provide a baseline for studying venous valve incompetence at the tissue level and a quantitative basis for prosthetic venous valve design.

Biaxial stress relaxation of semilunar heart valve leaflets during simulated collagen catabolism: Effects of collagenase concentration and equibiaxial strain state.

Heart valve leaflet collagen turnover and remodeling are innate to physiological homeostasis; valvular interstitial cells routinely catabolize damaged collagen and affect repair. Moreover, evidence indicates that leaflets can adapt to altered physiological (e.g. pregnancy) and pathological (e.g. hypertension) mechanical load states, tuning collagen structure and composition to changes in pressure and flow. However, while valvular interstitial cell-secreted matrix metalloproteinases are considered the primary effectors of collagen catabolism, the mechanisms by which damaged collagen fibers are selectively degraded remain unclear. Growing evidence suggests that the collagen fiber strain state plays a key role, with the strain-dependent configuration of the collagen molecules either masking or presenting proteolytic sites, thereby protecting or accelerating collagen proteolysis. In this study, the effects of equibiaxial strain state on collagen catabolism were investigated in porcine aortic valve and pulmonary valve tissues. Bacterial collagenase (0.2 and 0.5 mg/mL) was utilized to simulate endogenous matrix metalloproteinases, and biaxial stress relaxation and biochemical collagen concentration served as functional and compositional measures of collagen catabolism, respectively. At a collagenase concentration of 0.5 mg/mL, increasing the equibiaxial strain imposed during stress relaxation (0%, 37.5%, and 50%) yielded significantly lower median collagen concentrations in the aortic valve (p = 0.0231) and pulmonary valve (p = 0.0183), suggesting that relatively large strain magnitudes may enhance collagen catabolism. Collagen concentration decreases were paralleled by trends of accelerated normalized stress relaxation rate with equibiaxial strain in aortic valve tissues. Collectively, these in vitro results indicate that biaxial strain state is capable of affecting the susceptibility of valvular collagens to catabolism, providing a basis for further investigation of how such phenomena may manifest at different strain magnitudes or in vivo.

Prediction of matrix-to-cell stress transfer in heart valve tissues.

Non-linear and anisotropic heart valve leaflet tissue mechanics manifest principally from the stratification, orientation, and inhomogeneity of their collagenous microstructures. Disturbance of the native collagen fiber network has clear consequences for valve and leaflet tissue mechanics and presumably, by virtue of their intimate embedment, on the valvular interstitial cell stress-strain state and concomitant phenotype. In the current study, a set of virtual biaxial stretch experiments were conducted on porcine pulmonary valve leaflet tissue photomicrographs via an image-based finite element approach. Stress distribution evolution during diastolic valve closure was predicted at both the tissue and cellular levels. Orthotropic material properties consistent with distinct stages of diastolic loading were applied. Virtual experiments predicted tissue- and cellular-level stress fields, providing insight into how matrix-to-cell stress transfer may be influenced by the inhomogeneous collagen fiber architecture, tissue anisotropic material properties, and the cellular distribution within the leaflet tissue. To the best of the authors' knowledge, this is the first study reporting on the evolution of stress fields at both the tissue and cellular levels in valvular tissue and thus contributes toward refining our collective understanding of valvular tissue micromechanics while providing a computational tool enabling the further study of valvular cell-matrix interactions.

Virtualisation of stress distribution in heart valve tissue.

This study presents an image-based finite element analysis incorporating histological photomicrographs of heart valve tissues. We report stress fields inside heart valve tissues, where heterogeneously distributed collagen fibres are responsible for transmitting forces into cells. Linear isotropic and anisotropic tissue material property models are incorporated to quantify the overall stress distributions in heart valve tissues. By establishing an effective predictive method with new computational tools and by performing virtual experiments on the heart valve tissue photomicrographs, we clarify how stresses are transferred from matrix to cell. The results clearly reveal the roles of heterogeneously distributed collagen fibres in mitigating stress developments inside heart valve tissues. Moreover, most local peak stresses occur around cell nuclei, suggesting that higher stress may be mediated by cells for biomechanical regulations.

Real-time strain mapping via biaxial stretching in heart valve tissues.

Previous studies show that the collagen fiber architecture is key to the heart valves tissue mechanical property. We report a real-time strain mapping approach that provides displacement vectors and principal strain directions during the mechanical characterization of heart valve tissues. The strain maps reported in the current study allows an individual to quickly identify the approximate strain imposed on a location of the sample. The result shows that when samples are biaxially stretched under 18% strain, less anisotropy is observed in both aortic and pulmonary valve leaflet samples. Moreover, when samples are stretched from 28% to 35%, pulmonary valves leaflet samples exhibits a stronger anisotropic effect than aortic valve. Therefore, a higher degree of straightening is required for collagen fibers to be fully aligned. This work provides an easy approach to quantify mechanical properties with the corresponding strain maps of heart valve tissues and potentially facilitates the developments of tissue engineering heart valves.

In-situ deformation of the aortic valve interstitial cell nucleus under diastolic loading.

Within the aortic valve (AV) leaflet resides a population of interstitial cells (AVICs), which serve to maintain tissue structural integrity via protein synthesis and enzymatic degradation. AVICs are typically characterized as myofibroblasts, exhibit phenotypic plasticity, and may play an important role in valve pathophysiology. While it is known that AVICs can respond to mechanical stimuli in vitro, the level of in vivo AVIC deformation and its relation to local collagen fiber reorientation during the cardiac cycle remain unknown. In the present study, the deformation of AVICs was investigated using porcine AV glutaraldehyde fixed under 0-90 mm Hg transvalvular pressures. The resulting change in nuclear aspect ratio (NAR) was used as an index of overall cellular strain, and dependencies on spatial location and pressure loading levels quantified. Local collagen fiber alignment in the same valves was also quantified using small angle light scattering. A tissue-level finite element (FE) model of an AVIC embedded in the AV extracellular matrix was also used explore the relation between AV tissue- and cellular-level deformations. Results indicated large, consistent increases in AVIC NAR with transvalvular pressure (e.g., from mean of 1.8 at 0 mm Hg to a mean of 4.8 at 90 mm Hg), as well as pronounced layer specific dependencies. Associated changes in collagen fiber alignment indicated that little AVIC deformation occurs with the large amount of fiber straightening for pressures below approximately 1 mm Hg, followed by substantial increases in AVIC NAR from 4 mm Hg to 90 mm Hg. While the tissue-level FE model was able to capture the qualitative response, it also underpredicted the extent of AVIC deformation. This result suggested that additional micromechanical and fiber-compaction effects occur at high pressure levels. The results of this study form the basis of understanding transvalvular pressure-mediated mechanotransduction within the native AV and first time quantitative data correlating AVIC nuclei deformation with AV tissue microstructure and deformation.

The effects of cellular contraction on aortic valve leaflet flexural stiffness.

The aortic valve (AV) leaflet contains a heterogeneous interstitial cell population composed predominantly of myofibroblasts, which contain both fibroblast and smooth muscle cell characteristics. The focus of the present study was to examine aortic valve interstitial cell (AVIC) contractile behavior within the intact leaflet tissue. Circumferential strips of porcine AV leaflets were mechanically tested under flexure, with the AVIC maintained in the normal, contracted, and contraction-inhibited states. Leaflets were flexed both with (WC) and against (AC) the natural leaflet curvature, both before and after the addition of 90 mM KCl to elicit cellular contraction. In addition, a natural basal tonus was also demonstrated by treating the leaflets with 10 microM thapsigargin to completely inhibit AVIC contraction. Results revealed a 48% increase in leaflet stiffness with AVIC contraction (from 703 to 1040 kPa, respectively) when bent in the AC direction (p=0.004), while the WC direction resulted only in 5% increase (from 491 to 516.5 kPa, respectively--not significant) in leaflet stiffness in the active state. Also, the loss of basal tonus of the AVIC population with thapsigargin treatment resulted in 76% (AC, p=0.001) and 54% (WC, p=0.036) decreases in leaflet stiffness at 5 mM KCl levels, while preventing contraction with the addition of 90 mM KCl as expected. We speculate that the observed layer dependent effects of AVIC contraction are primarily due to varying ECM mechanical properties in the ventricularis and fibrosa layers. Moreover, while we have demonstrated that AVIC contractile ability is a significant contributor to AV leaflet bending stiffness, it most likely serves a role in maintaining AV leaflet tissue homeostasis that has yet to be elucidated.