Assessing the impact of extracellular matrix fiber orientation on breast cancer cellular metabolism.

The extracellular matrix (ECM) is a dynamic and complex microenvironment that modulates cell behavior and cell fate. Changes in ECM composition and architecture have been correlated with development, differentiation, and disease progression in various pathologies, including breast cancer [1]. Studies have shown that aligned fibers drive a pro-metastatic microenvironment, promoting the transformation of mammary epithelial cells into invasive ductal carcinoma via the epithelial-to-mesenchymal transition (EMT) [2]. The impact of ECM orientation on breast cancer metabolism, however, is largely unknown. Here, we employ two non-invasive imaging techniques, fluorescence-lifetime imaging microscopy (FLIM) and intensity-based multiphoton microscopy, to assess the metabolic states of cancer cells cultured on ECM-mimicking nanofibers in a random and aligned orientation. By tracking the changes in the intrinsic fluorescence of nicotinamide adenine dinucleotide and flavin adenine dinucleotide, as well as expression levels of metastatic markers, we reveal how ECM fiber orientation alters cancer metabolism and EMT progression. Our study indicates that aligned cellular microenvironments play a key role in promoting metastatic phenotypes of breast cancer as evidenced by a more glycolytic metabolic signature on nanofiber scaffolds of aligned orientation compared to scaffolds of random orientation. This finding is particularly relevant for subsets of breast cancer marked by high levels of collagen remodeling (e.g. pregnancy associated breast cancer), and may serve as a platform for predicting clinical outcomes within these subsets [3-6].

Mechanical properties of clot made from human and bovine whole blood differ significantly.

Thromboembolism - that is, clot formation and the subsequent fragmentation of clot - is a leading cause of death worldwide. Clots' mechanical properties are critical determinants of both the embolization process and the pathophysiological consequences thereof. Thus, understanding and quantifying the mechanical properties of clots is important to our ability to treat and prevent thromboembolic disease. However, assessing these properties from in vivo clots is experimentally challenging. Therefore, we and others have turned to studying in vitro clot mimics instead. Unfortunately, there are significant discrepancies in the reported properties of these clot mimics, which have been hypothesized to arise from differences in experimental techniques and blood sources. The goal of our current work is therefore to compare the mechanical behavior of clots made from the two most common sources, human and bovine blood, using the same experimental techniques. To this end, we tested clots under pure shear with and without initial cracks, under cyclic loading, and under stress relaxation. Based on these data, we computed and compared stiffness, strength, work-to-rupture, fracture toughness, relaxation time constants, and prestrain. While clots from both sources behaved qualitatively similarly, they differed quantitatively in almost every metric. We also correlated each mechanical metric to measures of blood composition. Thereby, we traced this inter-species variability in clot mechanics back to significant differences in hematocrit, but not platelet count. Thus, our work suggests that the results of past studies that have used bovine blood to make in vitro mimics - without adjusting blood composition - should be interpreted carefully. Future studies about the mechanical properties of blood clots should focus on human blood alone.

Sex- and age-dependent skin mechanics-A detailed look in mice.

Skin aging is of immense societal and, thus, scientific interest. Because mechanics play a critical role in skin's function, a plethora of studies have investigated age-induced changes in skin mechanics. Nonetheless, much remains to be learned about the mechanics of aging skin. This is especially true when considering sex as a biological variable. In our work, we set out to answer some of these questions using mice as a model system. Specifically, we combined mechanical testing, histology, collagen assays, and two-photon microscopy to identify age- and sex-dependent changes in skin mechanics and to relate them to structural, microstructural, and compositional factors. Our work revealed that skin stiffness, thickness, and collagen content all decreased with age and were sex dependent. Interestingly, sex differences in stiffness were age induced. We hope our findings not only further our fundamental understanding of skin aging but also highlight both age and sex as important variables when conducting studies on skin mechanics. STATEMENT OF SIGNIFICANCE: Our work addresses the question, "How do sex and age affect the mechanics of skin?" Answering this question is of both scientific and societal importance. We do so in mice as a model system. Thereby, we hope to add clarity to a body of literature that appears divided on the effect of both factors. Our findings have important implications for those studying age and sex differences, especially in mice as a model system.

Untangling the mechanisms of pulmonary arterial hypertension-induced right ventricular stiffening in a large animal model.

Pulmonary hypertension (PHT) is a devastating disease with low survival rates. In PHT, chronic pressure overload leads to right ventricle (RV) stiffening; thus, impeding diastolic filling. Multiple mechanisms may contribute to RV stiffening, including wall thickening, microstructural disorganization, and myocardial stiffening. The relative importance of each mechanism is unclear. Our objective is to use a large animal model to untangle these mechanisms. Thus, we induced pulmonary arterial hypertension (PAH) in sheep via pulmonary artery banding. After eight weeks, the hearts underwent anatomic and diffusion tensor MRI to characterize wall thickening and microstructural disorganization. Additionally, myocardial samples underwent histological and gene expression analyses to quantify compositional changes and mechanical testing to quantify myocardial stiffening. Finally, we used finite element modeling to disentangle the relative importance of each stiffening mechanism. We found that the RVs of PAH animals thickened most at the base and the free wall and that PAH induced excessive collagen synthesis, increased cardiomyocyte cross-sectional area, and led to microstructural disorganization, consistent with increased expression of fibrotic genes. We also found that the myocardium itself stiffened significantly. Importantly, myocardial stiffening correlated significantly with collagen synthesis. Finally, our computational models predicted that myocardial stiffness contributes to RV stiffening significantly more than other mechanisms. Thus, myocardial stiffening may be the most important predictor for PAH progression. Given the correlation between myocardial stiffness and collagen synthesis, collagen-sensitive imaging modalities may be useful for estimating myocardial stiffness and predicting PAH outcomes. STATEMENT OF SIGNIFICANCE: Ventricular stiffening is a significant contributor to pulmonary hypertension-induced right heart failure. However, the mechanisms that lead to ventricular stiffening are not fully understood. The novelty of our work lies in answering this question through the use of a large animal model in combination with spatially- and directionally sensitive experimental techniques. We find that myocardial stiffness is the primary mechanism that leads to ventricular stiffening. Clinically, this knowledge may be used to improve diagnostic, prognostic, and therapeutic strategies for patients with pulmonary hypertension.

Elasticity of whole blood clots measured via Volume Controlled Cavity Expansion.

Measuring and understanding the mechanical properties of blood clots can provide insights into disease progression and the effectiveness of potential treatments. However, several limitations hinder the use of standard mechanical testing methods to measure the response of soft biological tissues, like blood clots. These tissues can be difficult to mount, and are inhomogeneous, irregular in shape, scarce, and valuable. To remedy this, we employ in this work Volume Controlled Cavity Expansion (VCCE), a technique that was recently developed, to measure local mechanical properties of soft materials in their natural environment. Through highly controlled volume expansion of a water bubble at the tip of an injection needle, paired with simultaneous measurement of the resisting pressure, we obtain a local signature of whole blood clot mechanical response. Comparing this data with predictive theoretical models, we find that a 1-term Ogden model is sufficient to capture the nonlinear elastic response observed in our experiments and produces shear modulus values that are comparable to values reported in the literature. Moreover, we find that bovine whole blood stored at 4 °C for greater than 2 days exhibits a statistically significant shift in the shear modulus from 2.53 ± 0.44 kPa on day 2 (N = 13) to 1.23 ± 0.18 kPa on day 3 (N = 14). In contrast to previously reported results, our samples did not exhibit viscoelastic rate sensitivity within strain rates ranging from 0.22 - 21.1 s-1. By surveying existing data on whole blood clots for comparison, we show that this technique provides highly repeatable and reliable results, hence we propose the more widespread adoption of VCCE as a path forward to building a better understanding of the mechanics of soft biological materials.

Untangling the mechanisms of pulmonary hypertension-induced right ventricular stiffening in a large animal model.

Background: Pulmonary arterial hypertension (PHT) is a devastating disease with low survival rates. In PHT, chronic pressure overload leads to right ventricle (RV) remodeling and stiffening; thus, impeding diastolic filling and ventricular function. Multiple mechanisms contribute to RV stiffening, including wall thickening, microstructural disorganization, and myocardial stiffening. The relative importance of each mechanism is unclear. Our objective is to use a large animal model as well as imaging, experimental, and computational approaches to untangle these mechanisms. Methods: We induced PHT in eight sheep via pulmonary artery banding. After eight weeks, the hearts underwent anatomic and diffusion tensor MRI to characterize wall thickening and microstructural disorganization. Additionally, myocardial samples underwent histological and gene expression analyses to quantify compositional changes and mechanical testing to quantify myocardial stiffening. All findings were compared to 12 control animals. Finally, we used computational modeling to disentangle the relative importance of each stiffening mechanism. Results: First, we found that the RVs of PHT animals thickened most at the base and the free wall. Additionally, we found that PHT induced excessive collagen synthesis and microstructural disorganization, consistent with increased expression of fibrotic genes. We also found that the myocardium itself stiffened significantly. Importantly, myocardial stiffening correlated significantly with excess collagen synthesis. Finally, our model of normalized RV pressure-volume relationships predicted that myocardial stiffness contributes to RV stiffening significantly more than other mechanisms. Conclusions: In summary, we found that PHT induces wall thickening, microstructural disorganization, and myocardial stiffening. These remodeling mechanisms were both spatially and directionally dependent. Using modeling, we show that myocardial stiffness is the primary contributor to RV stiffening. Thus, myocardial stiffening may be an important predictor for PHT progression. Given the significant correlation between myocardial stiffness and collagen synthesis, collagen-sensitive imaging modalities may be useful for non-invasively estimating myocardial stiffness and predicting PHT outcomes.

Sex- and Age-dependent Skin Mechanics - A Detailed Look in Mice.

Skin aging is of immense societal and, thus, scientific interest. Because mechanics play a critical role in skin's function, a plethora of studies have investigated age-induced changes in skin mechanics. Nonetheless, much remains to be learned about the mechanics of aging skin. This is especially true when considering sex as a biological variable. In our work, we set out to answer some of these questions using mice as a model system. Specifically, we combined mechanical testing, histology, collagen assays, and two-photon microscopy to identify age- and sex-dependent changes in skin mechanics and to relate them to structural, microstructural, and compositional factors. Our work revealed that skin stiffness, thickness, and collagen content all decreased with age and were sex dependent. Interestingly, sex differences in stiffness were age induced. We hope our findings not only further our fundamental understanding of skin aging but also highlight both age and sex as important variables when conducting studies on skin mechanics.

An introduction to the Ogden model in biomechanics: benefits, implementation tools and limitations.

Constitutive models are important to biomechanics for two key reasons. First, constitutive modelling is an essential component of characterizing tissues' mechanical properties for informing theoretical and computational models of biomechanical systems. Second, constitutive models can be used as a theoretical framework for extracting and comparing key quantities of interest from material characterization experiments. Over the past five decades, the Ogden model has emerged as a popular constitutive model in soft tissue biomechanics with relevance to both informing theoretical and computational models and to comparing material characterization experiments. The goal of this short review is threefold. First, we will discuss the broad relevance of the Ogden model to soft tissue biomechanics and the general characteristics of soft tissues that are suitable for approximating with the Ogden model. Second, we will highlight exemplary uses of the Ogden model in brain tissue, blood clot and other tissues. Finally, we offer a tutorial on fitting the one-term Ogden model to pure shear experimental data via both an analytical approximation of homogeneous deformation and a finite-element model of the tissue domain. Overall, we anticipate that this short review will serve as a practical introduction to the use of the Ogden model in biomechanics. This article is part of the theme issue 'The Ogden model of rubber mechanics: Fifty years of impact on nonlinear elasticity'.

Biaxial mechanics of thermally denaturing skin - Part 1: Experiments.

The mechanics of collagenous soft tissues, such as skin, are sensitive to heat. Thus, quantifying and modeling thermo-mechanical coupling of skin is critical to our understanding of skin's physiology, pathophysiology, and its treatment. However, key gaps persist in our knowledge about skin's coupled thermo-mechanics. Among them, we haven't quantified the role of skin's microstructural organization in its response to superphysiological loading. To fill this gap, we conducted a comprehensive set of experiments in which we combined biaxial mechanical testing with histology and two-photon imaging under liquid heat treatment at temperatures ranging from 37∘C to 95∘C lasting between 2 seconds and 5 minutes. Among other observations, we found that unconstrained skin, when exposed to high temperatures, shrinks anisotropically with the principal direction of shrinkage being aligned with collagen's principal orientation. Additionally, we found that when skin is isometrically constrained, it produces significant forces during denaturation that are also anisotropic. Finally, we found that denaturation significantly alters the mechanical behavior of skin. For short exposure times, this alteration is reflected in a reduction of stiffness at high strains. At long exposure times, the tissue softened to a point where it became untestable. We supplemented our findings with confirmation of collagen denaturation in skin via loss of birefringence and second harmonic generation. Finally, we captured all time-, temperature-, and direction-dependent experimental findings in a hypothetical model. Thus, this work fills a fundamental gap in our current understanding of skin thermo-mechanics and will support future developments in thermal injury prevention, thermal injury management, and thermal therapeutics of skin. STATEMENT OF SIGNIFICANCE: Our work experimentally explores how skin reacts to being heated. That is, it measures how much skin shrinks, what forces it produces, and how its mechanical properties change; all as a function of temperature, but also of direction and time. Additionally, our work connects these measurements to changes in skin's microscopic make-up. This knowledge is important to our understanding of skin's function and dysfunction, especially during burn injuries or heat-dependent treatments.

Preparation and Mounting of Whole Blood Clot Samples for Mechanical Testing.

Studying and quantifying the mechanics of blood clots is essential to better diagnosis and prognosis of, as well as therapy for, thromboembolic pathologies such as strokes, heart attacks, and pulmonary embolisms. Unfortunately, mechanically testing blood clots is complicated by their softness and fragility, thus making the use of classic mounting techniques, such as clamping, challenging. This is particularly true for mechanical testing under large deformation. Here, we describe protocols for creating in vitro blood clots and securely mounting these samples on mechanical test equipment. To this end, we line 3D-printed molds with a hook-and-loop fabric that, after coagulation, provides a secure interface between the sample and device mount. In summary, our molding and mounting protocols are ideal for performing large-deformation mechanical testing, with samples that can withstand substantial deformation without delaminating from the apparatus. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Cube-shaped blood clot preparation Basic Protocol 2: Sheet-shaped blood clot preparation.

Hyper-viscoelastic damage modeling of whole blood clot under large deformation.

Blood clots play a diametric role in our bodies as they are both vital as a wound sealant, as well as the source for many devastating diseases. In blood clots' physiological and pathological roles, their mechanics play a critical part. These mechanics are non-trivial owing to blood clots' complex nonlinear, viscoelastic behavior. Casting this behavior into mathematical form is a fundamental step toward a better basic scientific understanding of blood clots, as well as toward diagnostic and prognostic computational models. Here, we identify a hyper-viscoelastic damage model that we fit to original data on the nonlinear, viscoelastic behavior of blood clots. Our model combines the classic Ogden hyperelastic constitutive law, a finite viscoelastic model for large deformations, and a non-local, gradient-enhanced damage formulation. By fitting our model to cyclic tensile test data and extension-to-failure data, we inform the model's nine unknown material parameters. We demonstrate the predictability of our model by validating it against unseen cyclic tensile test and stress-relaxation data. Our original data, model formulation, and the identified constitutive parameters of this model are openly available for others to use, which will aid in developing accurate, quantitative simulations of blood clot mechanics.

A whole blood thrombus mimic: Constitutive behavior under simple shear.

Deep vein thrombosis and pulmonary embolism affect 300,000-600,000 patients each year in the US. To better understand the highly mechanical pathophysiology of pulmonary embolism, we set out to develop an in-vitro thrombus mimic and to test this mimic under large deformation simple shear. In addition to reporting on the mechanics of our mimics under simple shear, we explore the sensitivity of their mechanics to coagulation conditions and blood storage time, and compare three hyperelastic material models for their ability to fit our data. We found that thrombus mimics made from whole blood demonstrate strain-stiffening, a negative Poynting effect, and hysteresis when tested quasi-statically to 50% strain under simple shear. Additionally, we found that the stiffness of these mimics does not significantly vary with coagulation conditions or blood storage times. Of the three hyperelastic constitutive models that we tested, the Ogden model provided the best fits to both shear stress and normal stress. In conclusion, we developed a robust protocol to generate regularly-shaped, homogeneous thrombus mimics that lend themselves to simple shear testing under large deformation. Future studies will extend our model to include the effect of maturation and explore its fracture properties toward a better understanding of embolization.

Right ventricular myocardial mechanics: Multi-modal deformation, microstructure, modeling, and comparison to the left ventricle.

The right ventricular myocardium, much like the rest of the right side of the heart, has been consistently understudied. Presently, little is known about its mechanics, its microstructure, and its constitutive behavior. In this work, we set out to provide the first data on the mechanics of the mature right ventricular myocardium in both simple shear and uniaxial loading and to compare these data to the mechanics of the left ventricular myocardium. To this end, we tested ovine tissue samples of the right and left ventricle under a comprehensive mechanical testing protocol that consisted of six simple shear modes and three tension/compression modes. After mechanical testing, we conducted a histology-based microstructural analysis on each right ventricular sample that yielded high resolution fiber distribution maps across the entire samples. Equipped with this detailed mechanical and histological data, we employed an inverse finite element framework to determine the optimal form and parameters for microstructure-based constitutive models. The results of our study show that right ventricular myocardium is less stiff then the left ventricular myocardium in the fiber direction, but similarly exhibits non-linear, anisotropic, and tension/compression asymmetric behavior with direction-dependent Poynting effect. In addition, we found that right ventricular myocardial fibers change angles transmurally and are dispersed within the sheet plane and normal to it. Through our inverse finite element analysis, we found that the Holzapfel model successfully fits these data, even when selectively informed by rudimentary microstructural information. And, we found that the inclusion of higher-fidelity microstructural data improved the Holzapfel model's predictive ability. Looking forward, this investigation is a critical step towards understanding the fundamental mechanical behavior of right ventricular myocardium and lays the groundwork for future whole-organ mechanical simulations.

The tricuspid valve also maladapts as shown in sheep with biventricular heart failure.

Over 1.6 million Americans suffer from significant tricuspid valve leakage. In most cases this leakage is designated as secondary. Thus, valve dysfunction is assumed to be due to valve-extrinsic factors. We challenge this paradigm and hypothesize that the tricuspid valve maladapts in those patients rendering the valve at least partially culpable for its dysfunction. As a first step in testing this hypothesis, we set out to demonstrate that the tricuspid valve maladapts in disease. To this end, we induced biventricular heart failure in sheep that developed tricuspid valve leakage. In the anterior leaflets of those animals, we investigated maladaptation on multiple scales. We demonstrated alterations on the protein and cell-level, leading to tissue growth, thickening, and stiffening. These data provide a new perspective on a poorly understood, yet highly prevalent disease. Our findings may motivate novel therapy options for many currently untreated patients with leaky tricuspid valves.

Nonlinear, dissipative phenomena in whole blood clot mechanics.

When a thrombus breaks off and embolizes it can occlude vital vessels such as those of the heart, lung, or brain. These thromboembolic conditions are responsible for 1 in 4 deaths worldwide. Thrombus resistance to embolization is driven by its intrinsic fracture toughness as well as other, non-surface-creating dissipative mechanisms. In our current work, we identify and quantify these latter mechanisms toward future studies that aim to delineate fracture from other forms of dissipation. To this end, we use an in vitro thrombus mimic system to produce whole blood clots and explore their dissipative mechanics under simple uniaxial extension, cyclic loading, and stress-relaxation. We found that whole blood clots exhibit Mullins-like effect, hysteresis, permanent set, strain-rate dependence, and nonlinear stress-relaxation. Interestingly, we found that performing these tests under dry or submerged conditions did not change our results. However, performing these tests under room temperature or body temperature conditions yielded differences. Importantly, because we use venous blood our work is most closely related to venous in vivo blood clots. Overall, we have demonstrated that whole blood clots show several dissipative phenomena - similarly to hydrogels - that will be critical to our understanding of thrombus embolization.

The regional-dependent biaxial behavior of young and aged mouse skin: A detailed histomechanical characterization, residual strain analysis, and constitutive model.

Skin fulfills several vital functions, many of which are dependent on its mechanical properties. Therefore, as mice have become an invaluable model for skin research, determining murine skin's mechanical properties is important. Specifically, skin's mechanical properties are important for functional tests as well as for prognostic and diagnostic purposes. Additionally, computational simulations of skin behavior are becoming commonplace, rendering accurate models of murine skin's constitutive behavior necessary. To date, our knowledge of mouse skin mechanics shows significant gaps. For example, there are no comprehensive reports correlating skin's mechanical properties with region, age, and direction. Moreover, mouse skin's residual strain behavior has not been reported on. In our current work, we set out to fill these gaps. Based on histology, 2-photon microscopy, and planar biaxial testing, while accurately tracking various reference configurations, we report on differences in gross structure, microstructural organization, and constitutive response of skin, and cast those properties into a versatile Fung-type hyperelastic constitutive law for three reference configurations. Our data is the most comprehensive report contrasting the mechanical properties of young (12 weeks) and aged (52 weeks) mouse skin and will, thus, be valuable to basic science as control data, and provide accurate constitutive laws for mouse skin modeling. STATEMENT OF SIGNIFICANCE: Our findings are significant as they fill several gaps in our understanding of mouse skin mechanics. This is particularly important as mouse skin is becoming a frequent and critical model of human skin for cosmetic and medical science. Specifically, we quantified how mechanical properties of mice skin vary with age, with location, and with direction. Additionally, we cast our findings into constitutive models that can be used by others for predictive computer simulations of skin behavior.

A detailed mechanical and microstructural analysis of ovine tricuspid valve leaflets.

The tricuspid valve ensures unidirectional blood flow from the right atrium to the right ventricle. The three tricuspid leaflets operate within a dynamic stress environment of shear, bending, tensile, and compressive forces, which is cyclically repeated nearly three billion times in a lifetime. Ostensibly, the microstructural and mechanical properties of the tricuspid leaflets have mechanobiologically evolved to optimally support their function under those forces. Yet, how the tricuspid leaflet microstructure determines its mechanical properties and whether this relationship differs between the three leaflets is unknown. Here we perform a microstructural and mechanical analysis in matched ovine tricuspid leaflet samples. We found that the microstructure and mechanical properties vary among the three tricuspid leaflets in sheep. Specifically, we found that tricuspid leaflet composition, collagen orientation, and valve cell nuclear morphology are spatially heterogeneous and vary across leaflet type. Furthermore, under biaxial tension, the leaflets' mechanical behaviors exhibited unequal degrees of mechanical anisotropy. Most importantly, we found that the septal leaflet was stiffer in the radial direction and not the circumferential direction as with the other two leaflets. The differences we observed in leaflet microstructure coincide with the varying biaxial mechanics among leaflets. Our results demonstrate the structure-function relationship for each leaflet in the tricuspid valve. We anticipate our results to be vital toward developing more accurate, leaflet-specific tricuspid valve computational models. Furthermore, our results may be clinically important, informing differential surgical treatments of the tricuspid valve leaflets. Finally, the identified structure-function relationships may provide insight into the homeostatic and remodeling potential of valvular cells in altered mechanical environments, such as in diseased or repaired tricuspid valves. STATEMENT OF SIGNIFICANCE: Our work is significant as we investigated the structure-function relationship of ovine tricuspid valve leaflets. This is important as tricuspid valves fail frequently and our current approach to repairing them is suboptimal. Specifically, we related the distribution of structural and cellular elements, such as collagen, glycosaminoglycans, and cell nuclei, to each leaflet's mechanical properties. We found that leaflets have different structures and that their mechanics differ. This may, in the future, inform leaflet-specific treatment strategies and help optimize surgical outcomes.