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.

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.

Biaxial mechanics of thermally denaturing skin - Part 2: Modeling.

Understanding the response of skin to superphysiological temperatures is critical to the diagnosis and prognosis of thermal injuries, and to the development of temperature-based medical therapeutics. Unfortunately, this understanding has been hindered by our incomplete knowledge about the nonlinear coupling between skin temperature and its mechanics. In Part I of this study we experimentally demonstrated a complex interdependence of time, temperature, direction, and load in skin's response to superphysiological temperatures. In Part II of our study, we test two different models of skin's thermo-mechanics to explain our observations. In both models we assume that skin's response to superphysiological temperatures is governed by the denaturation of its highly collageneous microstructure. Thus, we capture skin's native mechanics via a microstructurally-motivated strain energy function which includes probability distributions for collagen fiber orientation and waviness. In the first model, we capture skin's response to superphysiological temperatures as a transition between two states that link the kinetics of collagen fiber denaturation to fiber coiling and to the transformation of each fiber's constitutive behavior from purely elastic to viscoelastic. In the second model, we capture skin's response to superphysiological temperatures instead via three states in which a sequence of two reactions link the kinetics of collagen fiber denaturation to fiber coiling, followed by a state of fiber damage. Given the success of both models in qualitatively and quantitatively capturing our observations, we expect that our work will provide guidance for future experiments that could probe each model's assumptions toward a better understanding of skin's coupled thermo-mechanics and that our work will be used to guide the engineering design of heat treatment therapies. STATEMENT OF SIGNIFICANCE: Quantifying and modeling skin thermo-mechanics is critical to our understanding of skin physiology, pathophysiology, as well as heat-based treatments. This work addresses a lack of theoretical and computational models of the coupled thermo-mechanics of skin. Our model accounts for skin microstructure through modeling the probability of fiber orientation and fiber stress-free states. Denaturing induces changes in the stress-free configuration of collagen, as well as changes in fiber stiffness and viscoelastic properties. We propose two competing models that fit all of our experimental observations. These models will enable future developments of thermal-therapeutics, prevention and management of skin thermal injuries, and set a foundation for improved mechanistic models of skin thermo-mechanics.

Texas TriValve 1.0 : a reverse­engineered, open model of the human tricuspid valve.

Nearly 1.6 million Americans suffer from a leaking tricuspid heart valve. To make matters worse, current valve repair options are far from optimal leading to recurrence of leakage in up to 30% of patients. We submit that a critical step toward improving outcomes is to better understand the "forgotten" valve. High-fidelity computer models may help in this endeavour. However, the existing models are limited by averaged or idealized geometries, material properties, and boundary conditions. In our current work, we overcome the limitations of existing models by (reverse) engineering the tricuspid valve from a beating human heart in an organ preservation system. The resulting finite-element model faithfully captures the kinematics and kinetics of the native tricuspid valve as validated against echocardiographic data and others' previous work. To showcase the value of our model, we also use it to simulate disease-induced and repair-induced changes to valve geometry and mechanics. Specifically, we simulate and compare the effectiveness of tricuspid valve repair via surgical annuloplasty and via transcatheter edge-to-edge repair. Importantly, our model is openly available for others to use. Thus, our model will allow us and others to perform virtual experiments on the healthy, diseased, and repaired tricuspid valve to better understand the valve itself and to optimize tricuspid valve repair for better patient outcomes.

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.

The effects of a simple optical clearing protocol on the mechanics of collagenous soft tissue.

Optical clearing of biological tissues improves imaging depth for light transmission imaging modalities such as two-photon microscopy. In studies that investigate the interplay between microstructure and tissue-level mechanics, mechanical testing of cleared tissue may be useful. However, the effects of optical clearing on soft tissue mechanics have not been investigated. Thus, we set out to quantify the effects of a simple and effective optical clearing protocol on the mechanics of soft collagenous tissues using ovine mitral valve anterior leaflets as a model system. First, we demonstrate the effectiveness of an isotonic glycerol-DMSO optical clearing protocol in two-photon microscopy. Second, we evaluate the mechanical effects of optical clearing on leaflets under equibiaxial tension in a dependent study design. Lastly, we quantify the shrinkage strain while traction-free and the contractile forces while constrained during clearing. We found the optical clearing protocol to improve two-photon imaging depth from ~100 µm to ~500-800 µm, enabling full-thickness visualization of second-harmonic generation, autofluorescent, and fluorophore-tagged structures. Under equibiaxial tension, cleared tissues exhibited reduced circumferential (p < 0.001) and radial (p = 0.009) transition stretches (i.e. stretch where collagen is recruited), and reduced radial stiffness (p = 0.031). Finally, during clearing we observed ~10-15% circumferential and radial compressive strains, and when constrained, ~2mN of circumferential and radial traction forces. In summary, we suggest the use of this optical clearing agent with mechanical testing be done with care, as it appears to alter the tissue's stress-free configuration and stiffness, likely due to tissue dehydration.

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.

Tricuspid Valve Annuloplasty Alters Leaflet Mechanics.

Tricuspid valve regurgitation is associated with significant morbidity and mortality. Its most common treatment option, tricuspid valve annuloplasty, is not optimally effective in the long-term. Toward identifying the causes for annuloplasty's ineffectiveness, we have previously investigated the technique's impact on the tricuspid annulus and the right ventricular epicardium. In our current work, we are extending our analysis to the anterior tricuspid valve leaflet. To this end, we adopted our previous strategy of performing DeVega suture annuloplasty as an experimental methodology that allows us to externally control the degree of cinching during annuloplasty. Thus, in ten sheep we successively cinched the annulus and quantified changes to leaflet motion, dynamics, and strain in the beating heart by combining sonomicrometry with our well-established mechanical framework. We found that successive cinching of the valve enforced earlier coaptation and thus reduced leaflet range of motion. Additionally, leaflet angular velocity during opening and closing decreased. Finally, we found that leaflet strains were also reduced. Specifically, radial and areal strains decreased as a function of annular cinching. Our findings are critical as they suggest that suture annuloplasty alters the mechanics of the tricuspid valve leaflets which may disrupt their resident cells' mechanobiological equilibrium. Long-term, such disruption may stimulate tissue maladaptation which could contribute to annuloplasty's sub-optimal effectiveness. Additionally, our data suggest that the extent to which annuloplasty alters leaflet mechanics can be controlled via degree of cinching. Hence, our data may provide direct surgical guidelines.

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.

The Effect of Downsizing on the Normal Tricuspid Annulus.

Tricuspid annuloplasty is a surgical procedure that cinches the valve's annulus in order to reduce regurgitant blood flow. One of its critical parameters is the degree of downsizing. To provide insight into the effect of downsizing, we studied the annulus of healthy sheep during suture annuloplasty. To this end, we implanted fiduciary markers along the annulus of sheep and subsequently performed a DeVega suture annuloplasty. We performed five downsizing steps in each animal while recording hemodynamic and sonomicrometry data in beating hearts. Subsequently, we used splines to approximate the annulus at baseline and at each downsizing step. Based on these approximations we computed clinical metrics of annular shape and dynamics, and the continuous field metrics height, strain, and curvature. With these data, we demonstrated that annular area reduction during downsizing was primarily driven by compression of the anterior annulus. Similarly, reduction in annular dynamics was driven by reduced contractility in the anterior annulus. Finally, changes in global height and eccentricity of the annulus could be explained by focal changes in the continuous height profile and changes in annular curvature. Our findings are important as they provide insight into a regularly performed surgical procedure and may inform the design of transcatheter devices that mimic suture annuloplasty.

Biomechanics of the Tricuspid Annulus: A Review of the Annulus' In Vivo Dynamics With Emphasis on Ovine Data.

The tricuspid annulus forms the boundary between the tricuspid valve leaflets and their surrounding perivalvular tissue of the right atrioventricular junction. Its shape changes throughout the cardiac cycle in response to the forces from the contracting right heart myocardium and the blood-valve interaction. Alterations to annular shape and dynamics in disease lead to valvular dysfunctions such as tricuspid regurgitation from which millions of patients suffer. Successful treatment of such dysfunction requires an in-depth understanding of the normal shape and dynamics of the tricuspid annulus and of the changes following disease and subsequent repair. In this manuscript we review what we know about the shape and dynamics of the normal tricuspid annulus and about the effects of both disease and repair based on non-invasive imaging studies and invasive fiduciary marker-based studies. We further show, by means of ovine data, that detailed engineering analyses of the tricuspid annulus provide regionally-resolved insight into the kinematics of the annulus which would remain hidden if limiting analyses to simple geometric metrics.

A fiduciary marker-based framework to assess heterogeneity and anisotropy of right ventricular epicardial strains in the beating ovine heart.

Quantifying ventricular deformation in health and disease is critical to our understanding of normal heart function, heart disease mechanisms, and the effect of medical treatments. Imaging modalities have been developed that can measure ventricular deformation non-invasively. However, because of the small thickness, complex shape, and anatomic position of the right ventricle, using these technologies to determine its deformation remains challenging. Here we develop a first fiduciary marker-based method to assess heterogeneity and anisotropy of right ventricular epicardial strain across the entire free wall. To this end, we combine a high-density array of sonomicrometry crystals implanted across the entire right ventricular epicardial surface with a subdivision surface algorithm and a large deformation kinematics framework. We demonstrate our approach on four beating ovine hearts and present a preliminary regional analysis of circumferential, longitudinal, and areal strain. Moreover, we illustrate maps of the same strains across the entire right ventricular epicardial surface to highlight their spatial heterogeneity and anisotropy. We observe in these animals that RV epicardial strains vary throughout the cardiac cycle, are heterogeneous across the RV free wall, and are anisotropic with larger compressive strains, i.e., contraction, in the longitudinal direction than in the circumferential direction. Average peak compressive strains vary by region between -3.34% and -8.29% in circumferential direction, and -4.02% and -10.57% in longitudinal direction. In summary, we introduce an experimental framework that will allow us to study disease- and device-induced deformations, and long-term consequences of these deformations, including heterogeneous and anisotropic effects.

The Effect of Acute Pulmonary Hypertension on Tricuspid Annular Height, Strain, and Curvature in Sheep.

The tricuspid annulus shows significant alterations in patients with functional tricuspid regurgitation-tricuspid valve dysfunction that is secondary to other diseases such as pulmonary hypertension. Early changes in annular shape and dynamics may provide an understanding of disease mechanisms and could predict disease progression. To gain a mechanistic insight into these early changes we perform a spatially-resolved analysis of the effect of acute pulmonary hypertension on the tricuspid annulus in sheep. To this end, we suture sonomicrometry crystals to the annulus of nine sheep and record their locations in the beating heart, before and after inducing acute pulmonary hypertension. Using least-squares cubic splines, we derive mathematical representations of the annulus to describe pulmonary hypertension-induced annular shape changes via strain, relative curvature, and relative annular height between the control group and the acute pulmonary hypertension group. Moreover, we determine hypertension-induced alterations to annular dynamics as within-group strains, relative curvature, and relative height throughout the cardiac cycle for each group. We confirm that the annulus in acute pulmonary hypertension dilates significantly, becomes more circular, and flattens. Our regional analysis reveals that annular dilation, circularization, and flattening are driven by highly localized changes in annular strains, curvature, and height. Additionally, we find that acute pulmonary hypertension alters annular dynamics, albeit minimally. This regionally-resolved analysis of acute hypertension-induced changes of annular shape and dynamics provides insight into early disease mechanisms, and may inspire future generations of annuloplasty devices and techniques that address spatial annular heterogeneities.

Implications of chronic daily anti-oxidant administration on the inflammatory response to intracortical microelectrodes.

OBJECTIVE: Oxidative stress events have been implicated to occur and facilitate multiple failure modes of intracortical microelectrodes. The goal of the present study was to evaluate the ability of a sustained concentration of an anti-oxidant and to reduce oxidative stress-mediated neurodegeneration for the application of intracortical microelectrodes. APPROACH: Non-functional microelectrodes were implanted into the cortex of male Sprague Dawley rats for up to sixteen weeks. Half of the animals received a daily intraperitoneal injection of the natural anti-oxidant resveratrol, at 30 mg kg(-1). The study was designed to investigate the biodistribution of the resveratrol, and the effects on neuroinflammation/neuroprotection following device implantation. MAIN RESULTS: Daily maintenance of a sustained range of resveratrol throughout the implantation period resulted in fewer degenerating neurons in comparison to control animals at both two and sixteen weeks post implantation. Initial and chronic improvements in neuronal viability in resveratrol-dosed animals were correlated with significant reductions in local superoxide anion accumulation around the implanted device at two weeks after implantation. Controls, receiving only saline injections, were also found to have reduced amounts of accumulated superoxide anion locally and less neurodegeneration than controls at sixteen weeks post-implantation. Despite observed benefits, thread-like adhesions were found between the liver and diaphragm in resveratrol-dosed animals. SIGNIFICANCE: Overall, our chronic daily anti-oxidant dosing scheme resulted in improvements in neuronal viability surrounding implanted microelectrodes, which could result in improved device performance. However, due to the discovery of thread-like adhesions, further work is still required to optimize a chronic anti-oxidant dosing regime for the application of intracortical microelectrodes.

A comparison of neuroinflammation to implanted microelectrodes in rat and mouse models.

Rat models have emerged as a common tool to study neuroinflammation to intracortical microelectrodes. While a number of studies have attempted to understand the factors resulting in neuroinflammation using rat models, a complete understanding of key mechanistic pathways remains elusive. Transgenic mouse models, however, could facilitate a deeper understanding of mechanistic pathways due to an ease of genetic alteration. Therefore, the goal of the present study is to compare neuroinflammation following microelectrode implantation between the rat and the mouse model. Our study suggests that subtle differences in the classic neuroinflammatory markers exist between the animal models at both two and sixteen weeks post implantation. Most notably, neuronal densities surrounding microelectrodes were significantly lower in the rat model at two weeks, while similar densities were observed between the animal models at sixteen weeks. Physiological differences between the species and slight alterations in surgical methods are likely key contributors to the observed differences. Moving forward, we propose that differences in the time course of neuroinflammation between the animal models should be considered when trying to understand and prevent intracortical microelectrode failure.