Bioengineered models of cardiovascular diseases.

Age-associated cardiovascular diseases (CVDs), predominantly resulting from artery-related disorders such as atherosclerosis, stand as a leading cause of morbidity and mortality among the elderly population. Consequently, there is a growing interest in the development of clinically relevant bioengineered models of CVDs. Recent developments in bioengineering and material sciences have paved the way for the creation of intricate models that closely mimic the structure and surroundings of native cardiac tissues and blood vessels. These models can be utilized for basic research purposes and for identifying pharmaceutical interventions and facilitating drug discovery. The advancement of vessel-on-a-chip technologies and the development of bioengineered and humanized in vitro models of the cardiovascular system have the potential to revolutionize CVD disease modelling. These technologies offer pathophysiologically relevant models at a fraction of the cost and time required for traditional experimentation required in vivo. This progress signifies a significant advancement in the field, transitioning from conventional 2D cell culture models to advanced 3D organoid and vessel-on-a-chip models. These innovative models are specifically designed to explore the complexities of vascular aging and stiffening, crucial factors in the development of cardiovascular diseases. This review summarizes the recent progress of various bioengineered in vitro platforms developed for investigating the pathophysiology of human cardiovascular system with more focus on advanced 3D vascular platforms.

A microfluidic model to study the effects of arrhythmic flows on endothelial cells.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and an important contributor to morbidity and mortality. Endothelial dysfunction has been postulated to be an important contributing factor in cardiovascular events in patients with AF. However, how vascular endothelial cells respond to arrhythmic flow is not fully understood, mainly due to the limitation of current in vitro systems to mimic arrhythmic flow conditions. To address this limitation, we developed a microfluidic system to study the effect of arrhythmic flow on the mechanobiology of human aortic endothelial cells (HAECs). The system utilises a computer-controlled piezoelectric pump for generating arrhythmic flow with a unique ability to control the variability in both the frequency and amplitude of pulse waves. The flow rate is modulated to reflect physiological or pathophysiological shear stress levels on endothelial cells. This enabled us to systematically dissect the importance of variability in the frequency and amplitude of pulses and shear stress level on endothelial cell mechanobiology. Our results indicated that arrhythmic flow at physiological shear stress level promotes endothelial cell spreading and reduces the plasma membrane-to-cytoplasmic distribution of ß-catenin. In contrast, arrhythmic flow at low and atherogenic shear stress levels does not promote endothelial cell spreading or redistribution of ß-catenin. Interestingly, under both shear stress levels, arrhythmic flow induces inflammation by promoting monocyte adhesion via an increase in ICAM-1 expression. Collectively, our microfluidic system provides opportunities to study the effect of arrhythmic flows on vascular endothelial mechanobiology in a systematic and reproducible manner.

Endothelial Response to the Combined Biomechanics of Vessel Stiffness and Shear Stress Is Regulated via Piezo1.

How endothelial cells sense and respond to dynamic changes in their biophysical surroundings as we age is not fully understood. Vascular stiffness is clearly a contributing factor not only in several cardiovascular diseases but also in physiological processes such as aging and vascular dementia. To address this gap, we utilized a microfluidic model to explore how substrate stiffness in the presence of shear stress affects endothelial morphology, senescence, proliferation, and inflammation. We also studied the role of mechanosensitive ion channel Piezo1 in endothelial responses under the combined effect of shear stress and substrate stiffness. To do so, we cultured endothelial cells inside microfluidic channels covered with fibronectin-coated elastomer with elastic moduli of 40 and 200 kPa, respectively, mimicking the stiffness of the vessel walls in young and aged arteries. The endothelial cells were exposed to atheroprotective and atherogenic shear stress levels of 10 and 2 dyn/cm2, respectively. Our findings show that substrate stiffness affects senescence under atheroprotective flow conditions and cytoskeleton remodeling, senescence, and inflammation under atherogenic flow conditions. Additionally, we found that the expression of Piezo1 plays a crucial role in endothelial adaptation to flow and regulation of inflammation under both atheroprotective and atherogenic shear stress levels. However, Piezo1 contribution to endothelial senescence was limited to the soft substrate and atheroprotective shear stress level. Overall, our study characterizes the response of endothelial cells to the combined effect of shear stress and substrate stiffness and reveals a previously unidentified role of Piezo1 in endothelial response to vessel stiffening, which potentially can be therapeutically targeted to alleviate endothelial dysfunction in aging adults.

Bioengineered Vascular Model of Foam Cell Formation.

Foam cell formation is a complex blood vessel pathology, which is characterized by a series of events, including endothelium dysfunction, inflammation, and accumulation of immune cells underneath the blood vessel walls. Novel bioengineered models capable of recapitulating these events are required to better understand the complex pathological processes underlying the development of foam cell formation and, consequently, advanced bioengineered platforms for screening drugs. Here, we generated a microfluidic blood vessel model, incorporating a three-dimensional (3D) extracellular matrix coated with an endothelial layer. This system enables us to perform experiments under a dynamic microenvironment that recapitulates the complexities of the native vascular regions. Using this model, we studied the effectors that regulate monocyte adhesion and migration, as well as foam cell formation inside vessel walls. We found that monocyte adhesion and migration are regulated by both the endothelium and monocytes themselves. Monocytes migrated into the extracellular matrix only when endothelial cells were cultured in the vessel model. In addition, the exposure of an endothelial layer to tumor necrosis factor α (TNF-α) and low shear stress both increased monocyte migration into the subendothelial space toward the matrix. Furthermore, we demonstrated the process of foam cell formation, 3 days after transmigration of peripheral blood mononuclear cells (PBMCs) into the vessel wall. We showed that pre-exposure of PBMCs to high shear rates increases their adhesion and migration through the TNF-α-treated endothelium but does not affect their capacity to form foam cells. The versatility of our model allows for mechanistic studies on foam cell formation under customized pathological conditions.

Fabrication of Nanocomposites with High Elasticity and Strength for the Load-Bearing Layer of Small-Diameter Vascular Grafts.

Compliance mismatch of commercially available artificial grafts, where the artificial graft and the native vessel are subject to different radial expansions, is a major issue that results in graft occlusion after implantation. A human artery possesses a nonlinear mechanical response to pulsatile pressure due to its nonlinear viscoelastic nature, which is difficult to replicate in artificial graft fabrication. Here, we fabricated nanocomposites with nonlinear mechanical responses for potential application as the load-bearing layer of vascular grafts, based on a poly(dimethylsiloxane) (PDMS)-casted nanofibrous film. The nanofibers consisted of a core-sheath structure with a PDMS elastomer reinforced with poly(methyl methacrylate) (PMMA) nanofibers as the sheath and thermoplastic polyurethane (TPU) elastomer as the core. The surface morphology and chemical composition together with the crystalline structure of the nanocomposites were characterized, and dynamic mechanical analysis was performed to select the graft with the most suitable properties as the load-bearing layer of a small-diameter vascular graft. The presence of the stiff polymer PMMA and elastic polymer TPU in the PMMA/PDMS/TPU combination resulted in a delayed dissipation of energy after exposure to a force corresponding to 180 mm Hg. Casting the PDMS/PMMA/TPU nanofibrous mat into a nanocomposite film improved the ultimate tensile strength of PDMS without compromising its elasticity. The compliance values of the nanocomposites were also found to be a close match to those of the greater saphenous vein, demonstrating a great potential of the nanocomposites as the load-bearing layer in a biostable vascular graft.

Recent developments in modeling, imaging, and monitoring of cardiovascular diseases using machine learning.

Cardiovascular diseases are the leading cause of mortality, morbidity, and hospitalization around the world. Recent technological advances have facilitated analyzing, visualizing, and monitoring cardiovascular diseases using emerging computational fluid dynamics, blood flow imaging, and wearable sensing technologies. Yet, computational cost, limited spatiotemporal resolution, and obstacles for thorough data analysis have hindered the utility of such techniques to curb cardiovascular diseases. We herein discuss how leveraging machine learning techniques, and in particular deep learning methods, could overcome these limitations and offer promise for translation. We discuss the remarkable capacity of recently developed machine learning techniques to accelerate flow modeling, enhance the resolution while reduce the noise and scanning time of current blood flow imaging techniques, and accurate detection of cardiovascular diseases using a plethora of data collected by wearable sensors.

Label-free macrophage phenotype classification using machine learning methods.

Macrophages are heterogeneous innate immune cells that are functionally shaped by their surrounding microenvironment. Diverse macrophage populations have multifaceted differences related to their morphology, metabolism, expressed markers, and functions, where the identification of the different phenotypes is of an utmost importance in modelling immune response. While expressed markers are the most used signature to classify phenotypes, multiple reports indicate that macrophage morphology and autofluorescence are also valuable clues that can be used in the identification process. In this work, we investigated macrophage autofluorescence as a distinct feature for classifying six different macrophage phenotypes, namely: M0, M1, M2a, M2b, M2c, and M2d. The identification was based on extracted signals from multi-channel/multi-wavelength flow cytometer. To achieve the identification, we constructed a dataset containing 152,438 cell events each having a response vector of 45 optical signals fingerprint. Based on this dataset, we applied different supervised machine learning methods to detect phenotype specific fingerprint from the response vector, where the fully connected neural network architecture provided the highest classification accuracy of 75.8% for the six phenotypes compared simultaneously. Furthermore, by restricting the number of phenotypes in the experiment, the proposed framework produces higher classification accuracies, averaging 92.0%, 91.9%, 84.2%, and 80.4% for a pool of two, three, four, five phenotypes, respectively. These results indicate the potential of the intrinsic autofluorescence for classifying macrophage phenotypes, with the proposed method being quick, simple, and cost-effective way to accelerate the discovery of macrophage phenotypical diversity.

Studying the Synergistic Effect of Substrate Stiffness and Cyclic Stretch Level on Endothelial Cells Using an Elastomeric Cell Culture Chamber.

Endothelial cells lining blood vessels are continuously exposed to biophysical cues that regulate their function in health and disease. As we age, blood vessels lose their elasticity and become stiffer. Vessel stiffness alters the mechanical forces that endothelial cells experience. Despite ample evidence on the contribution of endothelial cells to vessel stiffness, less is known about how vessel stiffness affects endothelial cells. In this study, we developed a versatile model to study the cooperative effect of substrate stiffness and cyclic stretch on human aortic endothelial cells. We cultured endothelial cells on elastomeric wells covered with fibronectin-coated polyacrylamide gel. Varying the concentrations of acrylamide and bis-acrylamide enabled us to produce soft and stiff substrates with elastic modules of 40 and 200 kPa, respectively. Using a customized three-dimensional (3D) printed cam-driven system, the cells were exposed to 5 and 10% cyclic stretch levels. This enabled us to mimic the stiffness and stretch levels that endothelial cells experience in young and aged arteries. Using this model, we found that endothelial cells cultured on a soft substrate had minimal cytoskeletal alignment to the direction of the stretch compared to the ones cultured on the stiff substrate. We also observed an increase in the cellular area and aspect ratio in cells cultured on the stiff substrate, both of which are positively regulated by cyclic stretch. However, neither cyclic stretch nor substrate stiffness significantly affected the nuclear circularity. Additionally, we found that the accumulation of NF-κB in the nucleus, endothelial proliferation, tube formation, and expression of IL1ß depends on the stretch level and substrate stiffness. Our model can be further used to investigate the complex signaling pathways associated with vessel stiffening that govern the endothelial responses to mechanical forces.

Dynamic Vortex Generation, Pulsed Injection, and Rapid Mixing of Blood Samples in Microfluidics Using the Tube Oscillation Mechanism.

Here, we describe the generation of dynamic vortices in micro-scale cavities at low flow rates. The system utilizes a computer-controlled audio speaker to axially oscillate the inlet tube of the microfluidic system at desired frequencies and amplitudes. The oscillation of the tube induces transiently high flow rates in the system, which facilitates the generation of dynamic vortices inside the cavity. The size of the vortices can be modulated by varying the tube oscillation frequency or amplitude. The vortices can be generated in single or serial cavities and in a wide range of cavity sizes. We demonstrate the suitability of the tube oscillation mechanism for the pulsed injection of water-based solutions or whole blood into the cavity. The injection rate can be controlled by the oscillation characteristics of the tube, enabling the injection of liquids at ultralow flow rates. The dynamic vortices facilitate the rapid mixing of the injected liquid with the main flow. The controllability and versatility of this technology allow for the development of programmable inertial microfluidic systems for performing multistep biological assays.

Tube Oscillation Drives Transitory Vortices Across Microfluidic Barriers.

Here, the generation of dynamic vortices across microscale barriers using the tube oscillation mechanism is demonstrated. Using a combination of high-speed imaging and computational flow dynamics, the cyclic formation, expansion, and collapse of vortices are studied. The dynamics of vortices across circular , triangular, and blade-shape barriers are investigated at different tube oscillation frequencies. The formation of an array of synchronous vortices across parallel blade-shaped barriers is demonstrated. The transient flows caused by these dynamic vortex arrays are harnessed for the rapid and efficient mixing of blood samples . A circular barrier scribed with a narrow orifice on its shoulder is used to facilitate the injection of liquid into the microfluidic channel, and its rapid mixing with the main flow through the dynamic vortices generated across the barrier. This approach facilitates the generation of vortices with desirable configurations, sizes, and dynamics in a highly controllable, programmable, and predictable manner while operating at low static flow rates.

Investigating the effects of low intensity visible light on human keratinocytes using a customized LED exposure system.

Photobiomodulation (PBM) refers to the use of light to modulate cellular processes, and has demonstrated utility in improving wound healing outcomes, and reducing pain and inflammation. Despite the potential benefits of PBM, the precise molecular mechanisms through which it influences cell behavior are not yet well understood. Inconsistent reporting of key light parameters has created uncertainty around optimal exposure profiles. In addition, very low intensities of light, < 0.1 J/cm2, have not been thoroughly examined for their use in PBM. Here, we present a custom-made compact, and modular LED-based exposure system for studying the effects of very low-intensity visible light (cell proliferation, migration, ROS production, and mitochondrial membrane potential) of three different wavelengths in a parallel manner. The device allows for six repeats of three different exposure conditions plus a non-irradiated control on a single 24-well plate. The immortalised human keratinocyte cell line, HaCaT, was selected as a major cellular component of the skin epidermal barrier. Furthermore, an in vitro wound model was developed by allowing the HaCaT to form a confluent monolayer, then scratching the cells with a pipette tip to form a wound. Cells were exposed to yellow (585 nm, 0.09 mW, ~ 3.7 mJ/cm2), orange (610 nm, 0.8 mW, ~ 31 mJ/cm2), and red (660 nm, 0.8 mW, ~ 31 mJ/cm2) light for 10 min. 48 h post-irradiation, immunohistochemistry was performed to evaluate cell viability, proliferation, ROS production, and mitochondrial membrane potential. The results demonstrate increased proliferation and decreased scratch area for all exposure conditions, however only red light increased the mitochondrial activity. Oxidative stress levels did not increase for any of the exposures. The present exposure system provides opportunities to better understand the complex cellular mechanisms driven by the irradiation of skin cells with visible light.

Piezo1 Response to Shear Stress Is Controlled by the Components of the Extracellular Matrix.

Piezo1 is a recently discovered Ca2+ permeable ion channel that has emerged as an integral sensor of hemodynamic forces within the cardiovascular system, contributing to vascular development and blood pressure regulation. However, how the composition of the extracellular matrix (ECM) affects the mechanosensitivity of Piezo1 in response to hemodynamic forces remains poorly understood. Using a combination of microfluidics and calcium imaging techniques, we probe the shear stress sensitivity of single HEK293T cells engineered to stably express Piezo1 in the presence of different ECM proteins. Our experiments show that Piezo1 sensitivity to shear stress is not dependent on the presence of ECM proteins. However, different ECM proteins regulate the sensitivity of Piezo1 depending on the shear stress level. Under high shear stress, fibronectin sensitizes Piezo1 response to shear, while under low shear stress, Piezo1 mechanosensitivity is improved in the presence of collagen types I and IV and laminin. Moreover, we report that α5ß1 and αvß3 integrins are involved in Piezo1 sensitivity at high shear, while αvß3 and αvß5 integrins are involved in regulating the Piezo1 response at low shear stress. These results demonstrate that the ECM/integrin interactions influence Piezo1 mechanosensitivity and could represent a mechanism whereby extracellular forces are transmitted to Piezo1 channels, providing new insights into the mechanism by which Piezo1 senses shear stress.

Low Temperature Nano Mechano-electrocatalytic CH4 Conversion.

Transforming natural resources to energy sources, such as converting CH4 to H2 and carbon, at high efficiency and low cost is crucial for many industries and environmental sustainability. The high temperature requirement of CH4 conversion regarding many of the current methods remains a critical bottleneck for their practical uptake. Here we report an approach based on gallium (Ga) liquid metal droplets, Ni(OH)2 cocatalysts, and mechanical energy input that offers low-temperature and scalable CH4 conversion into H2 and carbon. Mainly driven by the triboelectric voltage, originating from the joint contributions of the cocatalysts during agitation, CH4 is converted at the Ga and Ni(OH)2 interface through nanotribo-electrochemical reaction pathways. The efficiency of the system is enhanced when the reaction is performed at an increased pressure. The dehydrogenation of other nongaseous hydrocarbons using this approach is also demonstrated. This technology presents a possible low energy route for CH4 conversion without involving high temperature and harsh operating conditions.

Generation of dynamic vortices in a microfluidic system incorporating stenosis barrier by tube oscillation.

Microfluidic systems incorporating sudden expansions are widely used for generation of vortex flow patterns. However, the formation of vortices requires high flow rates to induce inertial effects. Here, we introduce a new method for generating dynamic vortices in microfluidics at low static flow rates. Human blood is driven through a microfluidic channel incorporating a semi-circular stenosis barrier. The inlet tube of the channel is axially oscillated using a computer-controlled audio-speaker. The tube oscillation induces high transient flow rates in the channel, which generates dynamic vortices across the stenosis barrier. The size of the vortices can be modulated by varying the frequency and amplitude of tube oscillation. Various vortex flow patterns can be generated by varying the flow rate. The formation and size of the vortices can be predicted using the Reynolds number of the oscillating tube. We demonstrate the potential application of the system for investigating the adhesion and phagocytosis of circulating immune cells under pathologically high shear rates induced at the stenosis. This approach facilitates the development of versatile and controllable inertial microfluidic systems for performing various cellular assays while operating at low static flow rates and low sample volumes.

Investigating the mechanotransduction of transient shear stress mediated by Piezo1 ion channel using a 3D printed dynamic gravity pump.

Microfluidic systems are widely used for studying the mechanotransduction of flow-induced shear stress in mechanosensitive cells. However, these studies are generally performed under constant flow rates, mainly, due to the deficiency of existing pumps for generating transient flows. To address this limitation, we have developed a unique dynamic gravity pump to generate transient flows in microfluidics. The pump utilises a motorised 3D-printed cam-lever mechanism to change the inlet pressure of the system in repeated cycles. 3D printing technology facilitates the rapid and low-cost prototyping of the pump. Customised transient flow patterns can be generated by modulating the profile, size, and rotational speed of the cam, location of the hinge along the lever, and the height of the syringe. Using this unique dynamic gravity pump, we investigated the mechanotransduction of shear stress in HEK293 cells stably expressing Piezo1 mechanosensitive ion channel under transient flows. The controllable, simple, low-cost, compact, and modular design of the pump makes it suitable for studying the mechanobiology of shear sensitive cells under transient flows.

Highly accurate and label-free discrimination of single cancer cell using a plasmonic oxide-based nanoprobe.

The detection of cancer cells at the single-cell level enables many novel functionalities such as next-generation cancer prognosis and accurate cellular analysis. While surface-enhanced Raman spectroscopy (SERS) has been widely considered as an effective tool in a low-cost and label-free manner, however, it is challenging to discriminate single cancer cells with an accuracy above 90% mainly due to the poor biocompatibility of the noble-metal-based SERS agents. Here, we report a dual-functional nanoprobe based on dopant-driven plasmonic oxides, demonstrating a maximum accuracy above 90% in distinguishing single THP-1 cell from peripheral blood mononuclear cell (PBMC) and human embryonic kidney (HEK) 293 from human macrophage cell line U937 based on their SERS patterns. Furthermore, this nanoprobe can be triggered by the bio-redox response from individual cells towards stimuli, empowering another complementary colorimetric cell detection, approximately achieving the unity discrimination accuracy at a single-cell level. Our strategy could potentially enable the future accurate and low-cost detection of cancer cells from mixed cell samples.

Studying the Mechanobiology of Aortic Endothelial Cells Under Cyclic Stretch Using a Modular 3D Printed System.

Here, we describe a motorized cam-driven system for the cyclic stretch of aortic endothelial cells. Our modular design allows for generating customized spatiotemporal stretch profiles by varying the profile and size of 3D printed cam and follower elements. The system is controllable, compact, inexpensive, and amenable for parallelization and long-term experiments. Experiments using human aortic endothelial cells show significant changes in the cytoskeletal structure and morphology of cells following exposure to 5 and 10% cyclic stretch over 9 and 16 h. The system provides upportunities for exploring the complex molecular and cellular processes governing the response of mechanosensitive cells under cyclic stretch.

Microfluidic models of the human circulatory system: versatile platforms for exploring mechanobiology and disease modeling.

The human circulatory system is a marvelous fluidic system, which is very sensitive to biophysical and biochemical cues. The current animal and cell culture models do not recapitulate the functional properties of the human circulatory system, limiting our ability to fully understand the complex biological processes underlying the dysfunction of this multifaceted system. In this review, we discuss the unique ability of microfluidic systems to recapitulate the biophysical, biochemical, and functional properties of the human circulatory system. We also describe the remarkable capacity of microfluidic technologies for exploring the complex mechanobiology of the cardiovascular system, mechanistic studying of cardiovascular diseases, and screening cardiovascular drugs with the additional benefit of reducing the need for animal models. We also discuss opportunities for further advancement in this exciting field.

Generation of programmable dynamic flow patterns in microfluidics using audio signals.

Customised audio signals, such as musical notes, can be readily generated by audio software on smartphones and played over audio speakers. Audio speakers translate electrical signals into the mechanical motion of the speaker cone. Coupling the inlet tube to the speaker cone causes the harmonic oscillation of the tube, which in turn changes the velocity profile and flow rate. We employ this strategy for generating programmable dynamic flow patterns in microfluidics. We show the generation of customised rib and vortex patterns through the application of multi-tone audio signals in water-based and whole blood samples. We demonstrate the precise capability to control the number and extent of the ribs and vortices by simply setting the frequency ratio of two- and three-tone audio signals. We exemplify potential applications of tube oscillation for studying the functional responses of circulating immune cells under pathophysiological shear rates. The system is programmable, compact, low-cost, biocompatible, and durable. These features make it suitable for a variety of applications across chemistry, biology, and physics.

High-k 2D Sb2O3 Made Using a Substrate-Independent and Low-Temperature Liquid-Metal-Based Process.

High dielectric constant (high-k) ultrathin films are required as insulating gate materials. The well-known high-k dielectrics, including HfO2, ZrO2, and SrTiO3, feature three-dimensional lattice structures and are thus not easily obtained in the form of distinct ultrathin sheets. Therefore, their deposition as ultrathin layers still imposes challenges for electronic industries. Consequently, new high-k nanomaterials with k in the range of 40 to 100 and a band gap exceeding 4 eV are highly sought after. Antimony oxide nanosheets appear as a potential candidate that could fulfill these characteristics. Here, we report on the stoichiometric cubic polymorph of 2D antimony oxide (Sb2O3) as an ideal high-k dielectric sheet that can be synthesized via a low-temperature, substrate-independent, and silicon-industry-compatible liquid metal synthesis technique. A bismuth-antimony alloy was produced during the growth process. Preferential oxidation caused the surface of the melt to be dominated by α-Sb2O3. This ultrathin α-Sb2O3 was then deposited onto desired surfaces via a liquid metal print transfer. A tunable sheet thickness between ∼1.5 and ∼3 nm was achieved, while the lateral dimensions were within the millimeter range. The obtained α-Sb2O3 exhibited high crystallinity and a wide band gap of ∼4.4 eV. The relative permittivity assessment revealed a maximum k of 84, while a breakdown electric field of ∼10 MV/cm was observed. The isolated 2D α-Sb2O3 nanosheets were utilized in top-gated field-effect transistors that featured low leakage currents, highlighting that the obtained material is a promising gate oxide for conventional and van der Waals heterostructure-based electronics.