Biomechanical and biochemical changes in murine skin during development and aging.

Aging leads to biochemical and biomechanical changes in skin, with biological and functional consequences. Despite extensive literature on skin aging, there is a lack of studies which investigate the maturation of the tissue and connect the microscopic changes in the skin to its macroscopic biomechanical behavior as it evolves over time. The present work addresses this knowledge gap using multiscale characterization of skin in a murine model considering newborn, adult and aged mice. Monotonic uniaxial loading, tension relaxation with change of bath, and loading to failure tests were performed on murine skin samples from different age groups, complemented by inflation experiments and atomic force microscopy indentation measurements. In parallel, skin samples were characterized using histological and biochemical techniques to assess tissue morphology, collagen organization, as well as collagen content and cross-linking. We show that 1-week-old skin differs across nearly all measured parameters from adult skin, showing reduced strain stiffening and tensile strength, a thinner dermis, lower collagen content and altered crosslinking patterns. Surprisingly, adult and aged skin were similar across most biomechanical parameters in the physiologic loading range, while aged skin had lower tensile strength and lower stiffening behavior at large force values. This correlates with altered collagen content and cross-links. Based on a computational model, differences in mechanocoupled stimuli in the skin of the different age groups were calculated, pointing to a potential biological significance of the age-induced biomechanical changes in regulating the local biophysical environment of dermal cells. STATEMENT OF SIGNIFICANCE: Skin microstructure and the emerging mechanical properties change with age, leading to biological, functional and health-related consequences. Despite extensive literature on skin aging, only very limited quantitative data are available on microstructural changes and the corresponding macroscopic biomechanical behavior as they evolve over time. This work provides a wide-range multiscale mechanical characterization of skin of newborn, adult and aged mice, and quantifies microstructural correlations in tissue morphology, collagen content, organization and cross-linking. Remarkably, aged skin retained normal hydration and normal biomechanical function in the physiological loading range but showed significantly reduced properties at super-physiological loading. Our data show that age-related microstructural differences have a profound effect not only on tissue-level properties but also on the cell-level biophysical environment.

A novel bistable device to study mechanosensitive cell responses to instantaneous stretch.

The behavior of cells and tissues in vivo is determined by the integration of multiple biochemical and mechanical signals. Of the mechanical signals, stretch has been studied for decades and shown to contribute to pathophysiological processes. Several different stretch devices have been developed for in vitro investigations of cell stretch. In this work, we describe a new 3D-printed uniaxial stretching device for studying cell response to rapid deformation. The device is a bistable compliant mechanism holding two equilibrium states-an unstretched and stretched configuration-without the need of an external actuator. Furthermore, it allows multiple simultaneous measurements of different levels of stretch on a single substrate and is compatible with standard immunofluorescence imaging of fixed cells as well as live-cell imaging. To demonstrate the effectiveness of the device to stretch cells, a test case using aligned myotubes is presented. Leveraging material area changes associated with deformation of the substrate, changes in nuclei density provided evidence of affine deformation between cells and substrate. Furthermore, intranuclear deformations were also assessed and shown to deform non-affinely. As a proof-of-principle of the use of the device for mechanobiological studies, we uniaxially stretched aligned healthy and dystrophic myotubes that displayed different passive mechanical responses, consistent with previous literature in the field. We also identified a new feature in the mechanoresponse of dystrophic myotubes, which is of potential interest for identifying the diseased cells based on a quick mechanical readout. While some applications of the device for elucidating passive mechanical responses are demonstrated, the simplicity of the device allows it to be potentially used for other modes of deformation with little modifications.

A free-form patterning method enabling endothelialization under dynamic flow.

Endothelialization strategies aim at protecting the surface of cardiovascular devices upon their interaction with blood by the generation and maintenance of a mature monolayer of endothelial cells. Rational engineering of the surface micro-topography at the luminal interface provides a powerful access point to support the survival of a living endothelium under the challenging hemodynamic conditions created by the implant deployment and function. Surface structuring protocols must however be adapted to the complex, non-planar architecture of the target device precluding the use of standard lithographic approaches. Here, a novel patterning method, harnessing the condensation and evaporation of water droplets on a curing liquid elastomer, is developed to introduce arrays of microscale wells on the surface of a biocompatible silicon layer. The resulting topographies support the in vitro generation of mature human endothelia and their maintenance under dynamic changes of flow direction or magnitude, greatly outperforming identical, but flat substrates. The structuring approach is additionally demonstrated on non-planar interfaces yielding comparable topographies. The intrinsically free-form patterning is therefore compatible with a complete and stable endothelialization of complex luminal interfaces in cardiovascular implants.

A Novel Hybrid Membrane VAD as First Step Toward Hemocompatible Blood Propulsion.

Heart failure is a raising cause of mortality. Heart transplantation and ventricular assist device (VAD) support represent the only available lifelines for end stage disease. In the context of donor organ shortage, the future role of VAD as destination therapy is emerging. Yet, major drawbacks are connected to the long-term implantation of current devices. Poor VAD hemocompatibility exposes the patient to life-threatening events, including haemorrhagic syndromes and thrombosis. Here, we introduce a new concept of artificial support, the Hybrid Membrane VAD, as a first-of-its-kind pump prototype enabling physiological blood propulsion through the cyclic actuation of a hyperelastic membrane, enabling the protection from the thrombogenic interaction between blood and the implant materials. The centre of the luminal membrane surface displays a rationally-developed surface topography interfering with flow to support a living endothelium. The precast cell layer survives to a range of dynamically changing pump actuating conditions i.e., actuation frequency from 1 to 4 Hz, stroke volume from 12 to 30 mL, and support duration up to 313 min, which are tested both in vitro and in vivo, ensuring the full retention of tissue integrity and connectivity under challenging conditions. In summary, the presented results constitute a proof of principle for the Hybrid Membrane VAD concept and represent the basis for its future development towards clinical validation.

On the compressibility and poroelasticity of human and murine skin.

A total of 37 human and 33 murine skin samples were subjected to uniaxial monotonic, cyclic, and relaxation experiments. Detailed analysis of the three-dimensional kinematic response showed that skin volume is significantly reduced as a consequence of a tensile elongation. This behavior is most pronounced in monotonic but persists in cyclic tests. The dehydration associated with volume loss depends on the osmolarity of the environment, so that tension relaxation changes as a consequence of modifying the ionic strength of the environmental bath. Similar to ex vivo observations, complementary in vivo stretching experiments on human volar forearms showed strong in-plane lateral contraction. A biphasic homogenized model is proposed which allows representing all relevant features of the observed mechanical response.

The mechanical fingerprint of murine excisional wounds.

A multiscale mechanics approach to the characterization of murine excisional wounds subjected to uniaxial tensile loading is presented. Local strain analysis at a physiological level of tension uncovers the presence of two distinct regions within the wound: i) a very compliant peripheral cushion and ii) a core area undergoing modest deformation. Microstructural visualizations of stretched wound specimens show negligible engagement of the collagen located in the center of a 7-day old wound; fibers remain coiled despite the applied tension, confirming the existence of a mechanically isolated wound core. The compliant cushion located at the wound periphery appears to protect the newly-formed tissue from excessive deformation during the phase of new tissue formation. The early remodeling phase (day 14) is characterized by a restored mechanical connection between far field and wound center. The latter remains less deformable, a characteristic possibly required for cell activities during tissue remodeling. The distribution of fibrillary collagens at these two time points corresponds well to the identified heterogeneity of mechanical properties of the wound region. This novel approach provides new insight into the mechanical properties of wounded skin and will be applicable to the analysis of compound-treated wounds or wounds in genetically modified tissue. STATEMENT OF SIGNIFICANCE: Biophysical characterization of healing wounds is crucial to assess the recovery of the skin barrier function and the associated mechanobiological processes. For the first time, we performed highly resolved local deformation analysis to identify mechanical characteristics of the wound and its periphery. Our results reveal the presence of a compliant cushion surrounding a stiffer wound core; we refer to this heterogeneous mechanical behavior as "mechanical fingerprint" of the wound. The mechanical response is shown to progress towards that of the intact skin as healing takes place. Histology and multiphoton microscopy suggest that wounded skin recovers its mechanical function via progressive reconnection of the newly-deposited collagen fibers with the surrounding intact matrix.

A novel ultra-light suction device for mechanical characterization of skin.

Suction experiments have been extensively applied for skin characterization. In these tests the deformation behavior of superficial tissue layers determines the elevation of the skin surface observed when a predefined negative (suction) pressure history is applied. The ability of such measurements to differentiate between skin conditions is limited by the variability of the elevation response observed in repeated experiments. The scatter was shown to be associated with the force exerted by the observer when holding the instrument against the skin. We have developed a novel suction device and a measurement procedure aiming at a tighter control of mechanical boundary conditions during the experiments. The new device weighs only 3.5 g and thus allows to minimize the force applied on the skin during the test. In this way, it is possible to reliably characterize the mechanical response of skin, also in case of low values of suction pressure and deformation. The influence of the contact force is analyzed through experiments on skin and synthetic materials, and rationalized based on corresponding finite element calculations. A comparative study, involving measurements on four body locations in two subjects by three observers, showed the good performance of the new procedure, specific advantages, and limitations with respect to the Cutometer®, i.e. the suction device most widely applied for skin characterization. As a byproduct of the present investigation, a correction procedure is proposed for the Cutometer measurements, which allows to partially compensate for the influence of the contact force. The characteristics of the new suction method are discussed in view of future applications for diagnostic purposes.

Buckled Thin-Film Transistors and Circuits on Soft Elastomers for Stretchable Electronics.

Although recent progress in the field of flexible electronics has allowed the realization of biocompatible and conformable electronics, systematic approaches which combine high bendability (<3 mm bending radius), high stretchability (>3-4%), and low complexity in the fabrication process are still missing. Here, we show a technique to induce randomly oriented and customized wrinkles on the surface of a biocompatible elastomeric substrate, where Thin-Film Transistors (TFTs) and circuits (inverter and logic NAND gates) based on amorphous-IGZO are fabricated. By tuning the wavelength and the amplitude of the wrinkles, the devices are fully operational while bent to 13 µm bending radii as well as while stretched up to 5%, keeping unchanged electrical properties. Moreover, a flexible rectifier is also realized, showing no degradation in the performances while flat or wrapped on an artificial human wrist. As proof of concept, transparent TFTs are also fabricated, presenting comparable electrical performances to the nontransparent ones. The extension of the buckling approach from our TFTs to circuits demonstrates the scalability of the process, prospecting applications in wireless stretchable electronics to be worn or implanted.

Postoperative analysis of the mechanical interaction between stent and host tissue in patients after transcatheter aortic valve implantation.

The analysis is based on a finite element procedure to extract the contact forces between an implanted Nitinol stent and the surrounding host tissue using postoperative CT images. The methodology was applied for patients (N=46) which have undergone a TAVI procedure with the Medtronic CoreValve Revalving System (MCRS) to obtain corresponding deformation and force maps. The postoperative CT data were recorded for each patient in both systolic and diastolic phase of the heart cycle. Scalar parameters were defined, which map deformed geometry and contact force field to mechanically relevant quantities: radial dilatation, radial shape distortion, non-convex points, mean force, a force deviation measure and a pressure equivalent. The latter demonstrates that in the area of the aortic root, the added circumferential loading is of the same order as the baseline average blood pressure, thus leading to a doubling of the local mechanical load. Generally the force distribution along the stent is non-homogeneous. A comparison of systolic and diastolic data revealed slightly higher contact forces during the diastole, indicating that the stent has to carry more load in this phase. The geometrical and mechanical parameters were compared for two types of clinical complication: para-valvular leakage (PVL) and permanent pacemaker requirement (PPM). It was found that an increase in mean force can be associated with both complications; significantly for PVL and as a trend for PPM.

A discrete network model to represent the deformation behavior of human amnion.

A discrete network model (DNM) to represent the mechanical behavior of the human amnion is proposed. The amnion is modeled as randomly distributed points interconnected with connector elements representing collagen crosslinks and fiber segments, respectively. This DNM is computationally efficient and allows simulations with large domains. A representative set of parameters has been selected to reproduce the uniaxial tension-stretch and kinematic responses of the amnion. Good agreement is found between the predicted and measured equibiaxial tension-stretch curves. Although the model represents the amnion phenomenologically, model parameters are physically motivated and their effect on the tension-stretch and in-plane kinematic responses is discussed. The model is used to investigate the local response in the near field of a circular hole, revealing that the kinematic response at the circular free boundaries leads to compaction and strong alignment of the network at the border of the defect.

Stent Maps--Comparative Visualization for the Prediction of Adverse Events of Transcatheter Aortic Valve Implantations.

Transcatheter aortic valve implantation (TAVI) is a minimally-invasive method for the treatment of aortic valve stenosis in patients with high surgical risk. Despite the success of TAVI, side effects such as paravalvular leakages can occur postoperatively. The goal of this project is to quantitatively analyze the co-occurrence of this complication and several potential risk factors such as stent shape after implantation, implantation height, amount and distribution of calcifications, and contact forces between stent and surrounding structure. In this paper, we present a two-dimensional visualization (stent maps), which allows (1) to comprehensively display all these aspects from CT data and mechanical simulation results and (2) to compare different datasets to identify patterns that are typical for adverse effects. The area of a stent map represents the surface area of the implanted stent - virtually straightened and uncoiled. Several properties of interest, like radial forces or stent compression, are displayed in this stent map in a heatmap-like fashion. Important anatomical landmarks and calcifications are plotted to show their spatial relation to the stent and possible correlations with the color-coded parameters. To provide comparability, the maps of different patient datasets are spatially adjusted according to a corresponding anatomical landmark. Also, stent maps summarizing the characteristics of different populations (e.g. with or without side effects) can be generated. Up to this point several interesting patterns have been observed with our technique, which remained hidden when examining the raw CT data or 3D visualizations of the same data. One example are obvious radial force maxima between the right and non-coronary valve leaflet occurring mainly in cases without leakages. These observations confirm the usefulness of our approach and give starting points for new hypotheses and further analyses. Because of its reduced dimensionality, the stent map data is an appropriate input for statistical group evaluation and machine learning methods.

Image-based mechanical analysis of stent deformation: concept and exemplary implementation for aortic valve stents.

An approach for extracting the radial force load on an implanted stent from medical images is proposed. To exemplify the approach, a system is presented which computes a radial force estimation from computer tomography images acquired from patients who underwent transcatheter aortic valve implantation (TAVI). The deformed shape of the implanted valve prosthesis' Nitinol frame is extracted from the images. A set of displacement vectors is computed that parameterizes the observed deformation. An iterative relaxation algorithm is employed to adapt the information extracted from the images to a finite-element model of the stent, and the radial components of the interaction forces between the stent and the tissue are extracted. For the evaluation of the method, tests were run using the clinical data from 21 patients. Stent modeling and extraction of the radial forces were successful in 18 cases. Synthetic test cases were generated, in addition, for assessing the sensitivity to the measurement errors. In a sensitivity analysis, the geometric error of the stent reconstruction was below 0.3 mm, which is below the image resolution. The distribution of the radial forces was qualitatively and quantitatively reasonable. An uncertainty remains in the quantitative evaluation of the radial forces due to the uncertainty in defining a radial direction on the deformed stent. With our approach, the mechanical situation of TAVI stents after the implantation can be studied in vivo, which may help to understand the mechanisms that lead to the complications and improve stent design.

Simulation of transcatheter aortic valve implantation under consideration of leaflet calcification.

Transcatheter aortic valve implantation (TAVI) is a minimally invasive off-pump procedure to replace diseased aortic heart valves. Known complications include paravalvular leaks, atrioventricular blocks, coronary obstruction and annular rupture. Careful procedure planning including appropriate stent selection and sizing are crucial. Few patient-specific geometric parameters, like annular diameters, annular perimeter and measurement of the distance to the coronary ostia, are currently used within this process. Biomechanical simulation allows the consideration of extracted anatomy and material parameters for the intervention, which may improve planning and execution phases. We present a simulation workflow using a fully segmented aortic root anatomy, which was extracted from pre-operative CT-scan data and apply individual material models and parameters to predict the procedure outcome. Our results indicate the high relevance of calcification location and size for intervention planning, which are not sufficiently considered at this time. Our analysis can further provide guidance for accurate, patient-specific device positioning and future adaptations to stent design.