Tunable biomimetic materials elaborated by ice templating and self-assembly of collagen for tubular tissue engineering.

Synthetic tubular grafts currently used in clinical context fail frequently, and the expectations that biomimetic materials could tackle these limitations are high. However, developing tubular materials presenting structural, compositional and functional properties close to those of native tissues remains an unmet challenge. Here we describe a combination of ice templating and topotactic fibrillogenesis of type I collagen, the main component of tissues' extracellular matrix, yielding highly concentrated yet porous tubular collagen materials with controlled hierarchical architecture at multiple length scales, the hallmark of native tissues' organization. By modulating the thermal conductivity of the cylindrical molds, we tune the macroscopic porosity defined by ice. Coupling the aforementioned porosity patterns with two different fibrillogenesis routes results in a new family of tubular materials whose textural features and the supramolecular arrangement of type I collagen are achieved. The resulting materials present hierarchical elastic properties and are successfully colonized by human endothelial cells and alveolar epithelial cells on the luminal side, and by human mesenchymal stem cells on the external side. The proposed straightforward protocol is likely to be adapted for larger graft sizes that address ever-growing clinical needs, such as peripheral arterial disease or tracheal and bronchial reconstructions.

PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell Complex.

The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. PPP2R1A is found to associate at the lamellipodial edge with an alternative form of WAVE complex, the WAVE Shell Complex, that contains NHSL1 instead of the Arp2/3 activating subunit WAVE, as in the canonical WAVE Regulatory Complex. PPP2R1A is required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement is abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impair WAVE Shell Complex binding and migration regulation, suggesting that the coupling of PPP2R1A to the WAVE Shell Complex is essential to its function.

Physiological ramifications of constrained collective cell migration.

Constraining collective cell migration in vitro using different types of engineered substrates such as microstructured surfaces or adhesive patterns of different shapes and sizes often leads to the emergence of specific patterns of motion. Recently, analogies between the behavior of cellular assemblies and that of active fluids have enabled significant advances in our understanding of collective cell migration; however, the physiological relevance and potential functional consequences of the resulting migration patterns remain elusive. Here we describe the different patterns of collective cell migration that have been reported in vitro in response to geometrical constraints, explore the in vivo pertinence of the in vitro systems used to impose the geometrical constraints, and discuss the potential physiological ramifications of the collective migration patterns that emerge as a result of physical constraints. We conclude by highlighting key upcoming challenges in the exciting field of constrained collective cell migration.

Contractility-induced self-organization of smooth muscle cells: from multilayer cell sheets to dynamic three-dimensional clusters.

Smooth muscle cells (SMCs) are mural cells that play a vital contractile function in many tissues. Abnormalities in SMC organization are associated with many diseases including atherosclerosis, asthma, and uterine fibroids. Various studies have reported that SMCs cultured on flat surfaces can spontaneously form three-dimensional clusters whose organization resembles that encountered in some of these pathological settings. Remarkably, how these structures form remains unknown. Here we combine in vitro experiments and physical modeling to show that three-dimensional clusters initiate when cellular contractile forces induce a hole in a flat SMC sheet, a process that can be modeled as the brittle fracture of a viscoelastic material. The subsequent evolution of the nascent cluster can be modeled as an active dewetting process with cluster shape evolution driven by a balance between cluster surface tension, arising from both cell contractility and adhesion, and cluster viscous dissipation. The description of the physical mechanisms governing the spontaneous emergence of these intriguing three-dimensional clusters may offer insight into SMC-related disorders.

Impedance-based sensors discriminate among different types of blood thrombi with very high specificity and sensitivity.

BACKGROUND: Intracranial occlusion recanalization fails in 20% of endovascular thrombectomy procedures, and thrombus composition is likely to be an important factor. In this study, we demonstrate that the combination of electrical impedance spectroscopy (EIS) and machine learning constitutes a novel and highly accurate method for the identification of different human thrombus types. METHODS: 134 samples, subdivided into four categories, were analyzed by EIS: 29 'White', 26 'Mixed', 12 'Red' thrombi, and 67 liquid 'Blood' samples. Thrombi were generated in vitro using citrated human blood from five healthy volunteers. Histological analysis was performed to validate the thrombus categorization based on red blood cell content. A machine learning prediction model was trained on impedance data to differentiate blood samples from any type of thrombus and in between the four sample categories. RESULTS: Histological analysis confirmed the similarity between the composition of in vitro generated thrombi and retrieved human thrombi. The prediction model yielded a sensitivity/specificity of 90%/99% for distinguishing blood samples from thrombi and a global accuracy of 88% for differentiating among the four sample categories. CONCLUSIONS: Combining EIS measurements with machine learning provides a highly effective approach for discriminating among different thrombus types and liquid blood. These findings raise the possibility of developing a probe-like device (eg, a neurovascular guidewire) integrating an impedance-based sensor. This sensor, placed in the distal part of the smart device, would allow the characterization of the probed thrombus on contact. The information could help physicians identify optimal thrombectomy strategies to improve outcomes for stroke patients.

Mathematical modeling of intracellular calcium in presence of receptor: a homeostatic model for endothelial cell.

Calcium is a ubiquitous molecule and second messenger that regulates many cellular functions ranging from exocytosis to cell proliferation at different time scales. In the vasculature, a constant adenosine triphosphate (ATP) concentration is maintained because of ATP released by red blood cells (RBCs). These ATP molecules continuously react with purinergic receptors on the surface of endothelial cells (ECs). Consequently, a cascade of chemical reactions are triggered that result in a transient cytoplasmic calcium (Ca[Formula: see text]), followed by return to its basal concentration. The mathematical models proposed in the literature are able to reproduce the transient peak. However, the trailing concentration is always higher than the basal cytoplasmic Ca[Formula: see text] concentrations, and the Ca[Formula: see text] concentration in endoplasmic reticulum (ER) remains lower than its initial concentration. This means that the intracellular homeostasis is not recovered. We propose, herein, a minimal model of calcium kinetics. We find that the desensitization of EC surface receptors due to phosphorylation and recycling plays a vital role in maintaining calcium homeostasis in the presence of a constant stimulus (ATP). The model is able to capture several experimental observations such as refilling of Ca[Formula: see text] in the ER, variation of cytoplasmic Ca[Formula: see text] transient peak in ECs, the resting cytoplasmic Ca[Formula: see text] concentration, the effect of removing ATP from the plasma on Ca[Formula: see text] homeostasis, and the saturation of cytoplasmic Ca[Formula: see text] transient peak with increase in ATP concentration. Direct confrontation with several experimental results is conducted. This work paves the way for systematic studies on coupling between blood flow and chemical signaling, and should contribute to a better understanding of the relation between (patho)physiological conditions and Ca[Formula: see text] kinetics.

Mechanical regulation of the early stages of angiogenesis.

Favouring or thwarting the development of a vascular network is essential in fields as diverse as oncology, cardiovascular disease or tissue engineering. As a result, understanding and controlling angiogenesis has become a major scientific challenge. Mechanical factors play a fundamental role in angiogenesis and can potentially be exploited for optimizing the architecture of the resulting vascular network. Largely focusing on in vitro systems but also supported by some in vivo evidence, the aim of this Highlight Review is dual. First, we describe the current knowledge with particular focus on the effects of fluid and solid mechanical stimuli on the early stages of the angiogenic process, most notably the destabilization of existing vessels and the initiation and elongation of new vessels. Second, we explore inherent difficulties in the field and propose future perspectives on the use of in vitro and physics-based modelling to overcome these difficulties.

Modeling ATP-mediated endothelial cell elongation on line patterns.

Endothelial cell (EC) migration is crucial for a wide range of processes including vascular wound healing, tumor angiogenesis, and the development of viable endovascular implants. We have previously demonstrated that ECs cultured on 15-µm wide adhesive line patterns exhibit three distinct migration phenotypes: (a) "running" cells that are polarized and migrate continuously and persistently on the adhesive lines with possible spontaneous directional changes, (b) "undecided" cells that are highly elongated and exhibit periodic changes in the direction of their polarization while maintaining minimal net migration, and (c) "tumbling-like" cells that migrate persistently for a certain amount of time but then stop and round up for a few hours before spreading again and resuming migration. Importantly, the three migration patterns are associated with distinct profiles of cell length. Because of the impact of adenosine triphosphate (ATP) on cytoskeletal organization and cell polarization, we hypothesize that the observed differences in EC length among the three different migration phenotypes are driven by differences in intracellular ATP levels. In the present work, we develop a mathematical model that incorporates the interactions between cell length, cytoskeletal (F-actin) organization, and intracellular ATP concentration. An optimization procedure is used to obtain the model parameter values that best fit the experimental data on EC lengths. The results indicate that a minimalist model based on differences in intracellular ATP levels is capable of capturing the different cell length profiles observed experimentally.

Topography-induced large-scale antiparallel collective migration in vascular endothelium.

Collective migration of vascular endothelial cells is central for embryonic development, angiogenesis, and wound closure. Although physical confinement of cell assemblies has been shown to elicit specific patterns of collective movement in various cell types, endothelial migration in vivo often occurs without confinement. Here we show that unconfined endothelial cell monolayers on microgroove substrates that mimic the anisotropic organization of the extracellular matrix exhibit a specific type of collective movement that takes the form of a periodic pattern of antiparallel cell streams. We further establish that the development of these streams requires intact cell-cell junctions and that stream sizes are particularly sensitive to groove depth. Finally, we show that modeling the endothelial cell sheet as an active fluid with the microgrooves acting as constraints on cell orientation predicts the occurrence of the periodic antiparallel cell streams as well as their lengths and widths. We posit that in unconfined cell assemblies, physical factors that constrain or bias cellular orientation such as anisotropic extracellular matrix cues or directed flow-derived shear forces dictate the pattern of collective cell movement.

Distinct timing of neutrophil spreading and stiffening during phagocytosis.

Phagocytic cells form the first line of defense in an organism, engulfing microbial pathogens. Phagocytosis involves cell mechanical changes that are not yet well understood. Understanding these mechanical modifications promises to shed light on the immune processes that trigger pathological complications. Previous studies showed that phagocytes undergo a sequence of spreading events around their target followed by an increase in cell tension. Seemingly in contradiction, other studies observed an increase in cell tension concomitant with membrane expansion. Even though phagocytes are viscoelastic, few studies have quantified viscous changes during phagocytosis. It is also unclear whether cell lines behave mechanically similarly to primary neutrophils. We addressed the question of simultaneous versus sequential spreading and mechanical changes during phagocytosis by using immunoglobulin-G-coated 8- and 20-µm-diameter beads as targets. We used a micropipette-based single-cell rheometer to monitor viscoelastic properties during phagocytosis by both neutrophil-like PLB cells and primary human neutrophils. We show that the faster expansion of PLB cells on larger beads is a geometrical effect reflecting a constant advancing speed of the phagocytic cup. Cells become stiffer on 20- than on 8-µm beads, and the relative timing of spreading and stiffening of PLB cells depends on target size: on larger beads, stiffening starts before maximal spreading area is reached but ends after reaching maximal area. On smaller beads, the stiffness begins to increase after cells have engulfed the bead. Similar to PLB cells, primary cells become stiffer on larger beads but start spreading and stiffen faster, and the stiffening begins before the end of spreading on both bead sizes. Our results show that mechanical changes in phagocytes are not a direct consequence of cell spreading and that models of phagocytosis should be amended to account for causes of cell stiffening other than membrane expansion.

Luminal flow actuation generates coupled shear and strain in a microvessel-on-chip.

In the microvasculature, blood flow-derived forces are key regulators of vascular structure and function. Consequently, the development of hydrogel-based microvessel-on-chip systems that strive to mimic thein vivocellular organization and mechanical environment has received great attention in recent years. However, despite intensive efforts, current microvessel-on-chip systems suffer from several limitations, most notably failure to produce physiologically relevant wall strain levels. In this study, a novel microvessel-on-chip based on the templating technique and using luminal flow actuation to generate physiologically relevant levels of wall shear stress and circumferential stretch is presented. Normal forces induced by the luminal pressure compress the surrounding soft collagen hydrogel, dilate the channel, and create large circumferential strain. The fluid pressure gradient in the system drives flow forward and generates realistic pulsatile wall shear stresses. Rigorous characterization of the system reveals the crucial role played by the poroelastic behavior of the hydrogel in determining the magnitudes of the wall shear stress and strain. The experimental measurements are combined with an analytical model of flow in both the lumen and the porous hydrogel to provide an exceptionally versatile user manual for an application-based choice of parameters in microvessels-on-chip. This unique strategy of flow actuation adds a dimension to the capabilities of microvessel-on-chip systems and provides a more general framework for improving hydrogel-basedin vitroengineered platforms.

A compact integrated microfluidic oxygenator with high gas exchange efficiency and compatibility for long-lasting endothelialization.

We have developed and tested a novel microfluidic device for blood oxygenation, which exhibits a large surface area of gas exchange and can support long-term sustainable endothelialization of blood microcapillaries, enhancing its hemocompatibility for clinical applications. The architecture of the parallel stacking of the trilayers is based on a central injection for blood and a lateral injection/output for gas which allows significant reduction in shear stress, promoting sustainable endothelialization since cells can be maintained viable for up to 2 weeks after initial seeding in the blood microchannel network. The circular design of curved blood capillaries allows covering a maximal surface area at 4 inch wafer scale, producing high oxygen uptake and carbon dioxide release in each single unit. Since the conventional bonding process based on oxygen plasma cannot be used for surface areas larger than several cm2, a new "wet bonding" process based on soft microprinting has been developed and patented. Using this new protocol, each 4 inch trilayer unit can be sealed without a collapsed membrane even at reduced 15 µm thickness and can support a high blood flow rate. The height of the blood channels has been optimized to reduce pressure drop and enhance gas exchange at a high volumetric blood flow rate up to 15 ml min-1. The simplicity of connecting different units in the stacked architecture is demonstrated for 3- or 5-unit stacked devices that exhibit remarkable performance with low primary volume, high oxygen uptake and carbon dioxide release and high flow rate of up to 80 ml min-1.

Integration of substrate- and flow-derived stresses in endothelial cell mechanobiology.

Endothelial cells (ECs) lining all blood vessels are subjected to large mechanical stresses that regulate their structure and function in health and disease. Here, we review EC responses to substrate-derived biophysical cues, namely topography, curvature, and stiffness, as well as to flow-derived stresses, notably shear stress, pressure, and tensile stresses. Because these mechanical cues in vivo are coupled and are exerted simultaneously on ECs, we also review the effects of multiple cues and describe burgeoning in vitro approaches for elucidating how ECs integrate and interpret various mechanical stimuli. We conclude by highlighting key open questions and upcoming challenges in the field of EC mechanobiology.

eG Coated Stents Exhibit Enhanced Endothelial Wound Healing Characteristics.

PURPOSE: Despite their widespread use, a significant fraction of coronary stents suffer from in-stent restenosis and stent thrombosis. Stent deployment induces extensive injury to the vascular endothelium. Rapid endothelial wound closure is essential for the success of a stenting procedure. A recent study has demonstrated that the BuMA Supreme® sirolimus-eluting stent exhibits particularly attractive strut coverage characteristics. A unique feature of this stent is the presence of a thin brush layer of poly-butyl methacrylate (PBMA), covalently bonded to the stent's cobalt-chromium frame via electro-grafting (eG™). The present study aimed to determine whether the PBMA coating has an effect on endothelial cell wound healing and stent strut coverage. METHODS: We used an in vitro coronary artery model whose wall consisted of an annular collagen hydrogel and whose luminal surface was lined with a monolayer of endothelial cells. Mechanical wounding of the endothelial lining was preformed prior to deployment of a bare cobalt-chromium stent either with or without the PBMA layer. The migration of fluorescently labeled endothelial cells was monitored automatically over a period of 48 h to determine endothelial wound healing rates. RESULTS: Quantitative assessment of endothelial wound healing rates within the simulated arterial model is achievable using automated image analysis. Wound healing is significantly faster (44% faster at 48 h) for stents with the PBMA eG Coating™ compared to bare metal stents. CONCLUSION: The PBMA eG Coating™ has the effect of promoting endothelial wound healing. Future studies will focus on elucidating the mechanistic basis of this observation.

3D Printing for Cardiovascular Applications: From End-to-End Processes to Emerging Developments.

3D printing as a means of fabrication has seen increasing applications in medicine in the last decade, becoming invaluable for cardiovascular applications. This rapidly developing technology has had a significant impact on cardiovascular research, its clinical translation and education. It has expanded our understanding of the cardiovascular system resulting in better devices, tools and consequently improved patient outcomes. This review discusses the latest developments and future directions of generating medical replicas ('phantoms') for use in the cardiovascular field, detailing the end-to-end process from medical imaging to capture structures of interest, to production and use of 3D printed models. We provide comparisons of available imaging modalities and overview of segmentation and post-processing techniques to process images for printing, detailed exploration of latest 3D printing methods and materials, and a comprehensive, up-to-date review of milestone applications and their impact within the cardiovascular domain across research, clinical use and education. We then provide an in-depth exploration of future technologies and innovations around these methods, capturing opportunities and emerging directions across increasingly realistic representations, bioprinting and tissue engineering, and complementary virtual and mixed reality solutions. The next generation of 3D printing techniques allow patient-specific models that are increasingly realistic, replicating properties, anatomy and function.

Is there a universal mechanism of cell alignment in response to substrate topography?

Cell alignment and elongation in the direction of anisotropic and aligned topographies are key manifestations of cellular contact guidance and are observed in many cell types. Whether this observation occurs through a universal mechanism remains to be established. In this Views article, we begin by presenting the most widely accepted model of topography-driven cell alignment which posits that anisotropic topographies impose lateral constraints on the growth of focal adhesions and actin stress fibers, thereby driving anisotropic force generation and cellular elongation and alignment. We then discuss particular scenarios where alternative or complementary mechanisms of cell alignment appear to be at play. These include the cases of specific cell types such as amoeboid-like cells and neurons as well as certain topography sizes. Finally, we review the role of the actin cytoskeleton in modulating topography-driven cell alignment and underscore the need for elucidating the role that other cytoskeletal elements play. We close by identifying key open questions the responses to which will significantly enhance our understanding of the role of cellular contact guidance in health and disease.

Pericyte mechanics and mechanobiology.

Pericytes are mural cells of the microvasculature, recognized by their thin processes and protruding cell body. Pericytes wrap around endothelial cells and play a central role in regulating various endothelial functions, including angiogenesis and inflammation. They also serve as a vascular support and regulate blood flow by contraction. Prior reviews have examined pericyte biological functions and biochemical signaling pathways. In this Review, we focus on the role of mechanics and mechanobiology in regulating pericyte function. After an overview of the morphology and structure of pericytes, we describe their interactions with both the basement membrane and endothelial cells. We then turn our attention to biophysical considerations, and describe contractile forces generated by pericytes, mechanical forces exerted on pericytes, and pericyte responses to these forces. Finally, we discuss 2D and 3D engineered in vitro models for studying pericyte mechano-responsiveness and underscore the need for more evolved models that provide improved understanding of pericyte function and dysfunction.

Mueller polarimetric imaging for fast macroscopic mapping of microscopic collagen matrix remodeling by smooth muscle cells.

Smooth muscle cells (SMCs) are critical players in cardiovascular disease development and undergo complex phenotype switching during disease progression. However, SMC phenotype is difficult to assess and track in co-culture studies. To determine the contractility of SMCs embedded within collagen hydrogels, we performed polarized light imaging and subsequent analysis based on Mueller matrices. Measurements were made both in the absence and presence of endothelial cells (ECs) in order to establish the impact of EC-SMC communication on SMC contractility. The results demonstrated that Mueller polarimetric imaging is indeed an appropriate tool for assessing SMC activity which significantly modifies the hydrogel retardance in the presence of ECs. These findings are consistent with the idea that EC-SMC communication promotes a more contractile SMC phenotype. More broadly, our findings suggest that Mueller polarimetry can be a useful tool for studies of spatial heterogeneities in hydrogel remodeling by SMCs.