Effects of Drag-Reducing Polymers on Hemodynamics and Whole Blood-Endothelial Interactions in 3D-Printed Vascular Topologies.

Most in vitro models use culture medium to apply fluid shear stress to endothelial cells, which does not capture the interaction between blood and endothelial cells. Here, we describe a new system to characterize whole blood flow through a 3D-printed, endothelialized vascular topology that induces flow separation at a bifurcation. Drag-reducing polymers, which have been previously studied as a potential therapy to reduce the pressure drop across the vascular bed, are evaluated for their effect on mitigating the disturbed flow. Polymer concentrations of 1000 ppm prevented recirculation and disturbed flow at the wall. Proteomic analysis of plasma collected from whole blood recirculated through the vascularized channel with and without drag-reducing polymers provides insight into the effects of flow regimes on levels of proteins indicative of the endothelial-blood interaction. The results indicate that blood flow alters proteins associated with coagulation, inflammation, and other processes. Overall, these proof-of-concept experiments demonstrate the importance of using whole blood flow to study the endothelial response to perfusion.

Matching mechanical heterogeneity of the native spinal cord augments axon infiltration in 3D-printed scaffolds.

Scaffolds delivered to injured spinal cords to stimulate axon connectivity often match the anisotropy of native tissue using guidance cues along the rostral-caudal axis, but current approaches do not mimic the heterogeneity of host tissue mechanics. Although white and gray matter have different mechanical properties, it remains unclear whether tissue mechanics also vary along the length of the cord. Mechanical testing performed in this study indicates that bulk spinal cord mechanics do differ along anatomical level and that these differences are caused by variations in the ratio of white and gray matter. These results suggest that scaffolds recreating the heterogeneity of spinal cord tissue mechanics must account for the disparity between gray and white matter. Digital light processing (DLP) provides a means to mimic spinal cord topology, but has previously been limited to printing homogeneous mechanical properties. We describe a means to modify DLP to print scaffolds that mimic spinal cord mechanical heterogeneity caused by variation in the ratio of white and gray matter, which improves axon infiltration compared to controls exhibiting homogeneous mechanical properties. These results demonstrate that scaffolds matching the mechanical heterogeneity of white and gray matter improve the effectiveness of biomaterials transplanted within the injured spinal cord.

Transcriptomic analysis of a 3D blood-brain barrier model exposed to disturbed fluid flow.

Cerebral aneurysms are more likely to form at bifurcations in the vasculature, where disturbed fluid is prevalent due to flow separation at sufficiently high Reynolds numbers. While previous studies have demonstrated that altered shear stress exerted by disturbed flow disrupts endothelial tight junctions, less is known about how these flow regimes alter gene expression in endothelial cells lining the blood-brain barrier. Specifically, the effect of disturbed flow on expression of genes associated with cell-cell and cell-matrix interaction, which likely mediate aneurysm formation, remains unclear. RNA sequencing of immortalized cerebral endothelial cells isolated from the lumen of a 3D blood-brain barrier model reveals distinct transcriptional changes in vessels exposed to fully developed and disturbed flow profiles applied by both steady and physiological waveforms. Differential gene expression, validated by qRT-PCR and western blotting, reveals that lumican, a small leucine-rich proteoglycan, is the most significantly downregulated gene in endothelial cells exposed to steady, disturbed flow. Knocking down lumican expression reduces barrier function in the presence of steady, fully developed flow. Moreover, adding purified lumican into the hydrogel of the 3D blood-brain barrier model recovers barrier function in the region exposed to fully developed flow. Overall, these findings emphasize the importance of flow regimes exhibiting spatial and temporal heterogeneous shear stress profiles on cell-matrix interaction in endothelial cells lining the blood-brain barrier, while also identifying lumican as a contributor to the formation and maintenance of an intact barrier.

Oxygen gradients dictate angiogenesis but not barriergenesis in a 3D brain microvascular model.

A variety of biophysical properties are known to regulate angiogenic sprouting, and in vitro systems can parse the individual effects of these factors in a controlled setting. Here, a three-dimensional brain microvascular model interrogates how variables including extracellular matrix composition, fluid shear stress, and radius of curvature affect angiogenic sprouting of cerebral endothelial cells. Tracking endothelial migration over several days reveals that application of fluid shear stress and enlarged vessel radius of curvature both attenuate sprouting. Computational modeling informed by oxygen consumption assays suggests that sprouting correlates to reduced oxygen concentration: both fluid shear stress and vessel geometry alter the local oxygen levels dictated by both ambient conditions and cellular respiration. Moreover, increasing cell density and consequently lowering the local oxygen levels yields significantly more sprouting. Further analysis reveals that the magnitude of oxygen concentration is not as important as its spatial concentration gradient: decreasing ambient oxygen concentration causes significantly less sprouting than applying an external oxygen gradient to the vessels. In contrast, barriergenesis is dictated by shear stress independent of local oxygen concentrations, suggesting that different mechanisms mediate angiogenesis and barrier formation and that angiogenic sprouting can occur without compromising the barrier. Overall, these results improve our understanding of how specific biophysical variables regulate the function and activation of cerebral vasculature, and identify spatial oxygen gradients as the driving factor of angiogenesis in the brain.

Microindentation of Fluid-Filled Cellular Domes Reveals the Contribution of RhoA-ROCK Signaling to Multicellular Mechanics.

Cellular mechanics encompass both mechanical properties that resist forces applied by the external environment and internally generated forces applied at the location of cell-cell and cell-matrix junctions. Here, the authors demonstrate that microindentation of cellular domes formed by cell monolayers that locally lift off the substrate provides insight into both aspects of cellular mechanics in multicellular structures. Using a modified Hertz contact equation, the force-displacement curves generated by a micro-tensiometer are used to measure an effective dome stiffness. The results indicate the domes are consistent with the Laplace-Young relationship for elastic membranes, regardless of biochemical modulation of the RhoA-ROCK signaling axis. In contrast, activating RhoA, and inhibiting ROCK both alter the relaxation dynamics of the domes deformed by the micro-tensiometer, revealing an approach to interrogate the role of RhoA-ROCK signaling in multicellular mechanics. A finite element model incorporating a Mooney-Rivlin hyperelastic constitutive equation to describe monolayer mechanics predicts effective stiffness values that are consistent with the micro-tensiometer measurements, verifying previous measurements of the response of cell monolayers to tension. Overall, these studies establish microindentation of fluid-filled domes as an avenue to investigate the contribution of cell-generated forces to the mechanics of multicellular structures.

CD44 mediates shear stress mechanotransduction in an in vitro blood-brain barrier model through small GTPases RhoA and Rac1.

Fluid shear stress is an important mediator of vascular permeability, yet the molecular mechanisms underlying the effect of shear on the blood-brain barrier (BBB) have yet to be clarified in cerebral vasculature despite its importance for brain homeostasis. The goal of this study is to probe components of shear mechanotransduction within the BBB to gain a better understanding of pathologies associated with changes in cerebral perfusion including ischemic stroke. Interrogating the effects of shear stress in vivo is complicated by the complexity of factors in the brain parenchyma and the difficulty associated with modulating blood flow regimes. The in vitro model used in this study is compatible with real-time measurement of barrier function using a transendothelial electrical resistance as well as immunocytochemistry and dextran permeability assays. These experiments reveal that there is a threshold level of shear stress required for barrier formation and that the composition of the extracellular matrix, specifically the presence of high molecular weight hyaluronan, dictates the flow response. Gene editing to modulate the expression of CD44, a mechanosensitive receptor for hyaluronan, demonstrates that the receptor is required for the endothelial response to shear stress. Manipulation of small GTPase activity reveals CD44 activates Rac1 while inhibiting RhoA activation. Additionally, adducin-γ localizes to tight junctions in response to shear stress and RhoA inhibition and is required to maintain the barrier. This study identifies specific components of the mechanosensing complex associated with the BBB response to fluid shear stress and, therefore, illuminates potential targets for barrier manipulation in vivo.

Magnetic alignment of injectable hydrogel scaffolds for spinal cord injury repair.

Injectable hydrogels for cell delivery and tissue regeneration have several advantages over pre-fabricated scaffolds that require more invasive transplantation procedures, but lack the ability to implement tunable topologies. Here, we describe an approach to create patternable and injectable scaffolds using magnetically-responsive (MR) self-assembling peptide hydrogels, and validate their efficacy to promote and align axon infiltration at the site of a spinal cord injury. In vitro experiments reveal the parameters needed to align the fibers using the application of an external magnetic field. These results indicate that applying a 100-Gauss (G) field to the peptide hydrogels during polymerization causes fiber alignment as measured by electron microscopy, even in the presence of cells. In order to mimic infiltrating axons, neural progenitor cells (NPCs) are seeded on the surface of peptide hydrogels to interrogate the effects of both magnetic alignment and embedding human mesenchymal stem cells (hMSCs) in the scaffold. NPCs infiltrate peptide hydrogels seeded with hMSCs, and exhibit increased alignment and elongation in aligned gels. In order to evaluate these injectable and patternable scaffolds in vivo, hMSC-seeded peptide hydrogels are injected at the site of a contusion spinal cord injury with and without the presence of a magnetic field to align the resulting fibrous network. Measurements of axon growth and orientation as well as inflammation and glial scar formation indicate that these metrics are improved in magnetically aligned hMSC-seeded hydrogels. The results verify that MR hydrogels can dictate the orientation of infiltrating axons, providing a viable means to control the topology of injectable scaffolds.

SARS-CoV-2 Spike Protein Disrupts Blood-Brain Barrier Integrity via RhoA Activation.

The SARS-CoV-2 spike protein has been shown to disrupt blood-brain barrier (BBB) function, but its pathogenic mechanism of action is unknown. Whether angiotensin converting enzyme 2 (ACE2), the viral binding site for SARS-CoV-2, contributes to the spike protein-induced barrier disruption also remains unclear. Here, a 3D-BBB microfluidic model was used to interrogate mechanisms by which the spike protein may facilitate barrier dysfunction. The spike protein upregulated the expression of ACE2 in response to laminar shear stress. Moreover, interrogating the role of ACE2 showed that knock-down affected endothelial barrier properties. These results identify a possible role of ACE2 in barrier homeostasis. Analysis of RhoA, a key molecule in regulating endothelial cytoskeleton and tight junction complex dynamics, reveals that the spike protein triggers RhoA activation. Inhibition of RhoA with C3 transferase rescues its effect on tight junction disassembly. Overall, these results indicate a possible means by which the engagement of SARS-CoV-2 with ACE2 facilitates disruption of the BBB via RhoA activation. Understanding how SARS-CoV-2 dysregulates the BBB may lead to strategies to prevent the neurological deficits seen in COVID-19 patients.

The Use of Tissue Engineering to Fabricate Perfusable 3D Brain Microvessels in vitro.

Tissue engineering of the blood-brain barrier (BBB) in vitro has been rapidly expanding to address the challenges of mimicking the native structure and function of the BBB. Most of these models utilize 2D conventional microfluidic techniques. However, 3D microvascular models offer the potential to more closely recapitulate the cytoarchitecture and multicellular arrangement of in vivo microvasculature, and also can recreate branching and network topologies of the vascular bed. In this perspective, we discuss current 3D brain microvessel modeling techniques including templating, printing, and self-assembling capillary networks. Furthermore, we address the use of biological matrices and fluid dynamics. Finally, key challenges are identified along with future directions that will improve development of next generation of brain microvasculature models.

Implementation and characterization of a physiologically relevant flow waveform in a 3D microfluidic model of the blood-brain barrier.

Previous in vitro studies interrogating the endothelial response to physiologically relevant flow regimes require specialized pumps to deliver time-dependent waveforms that imitate in vivo blood flow. The aim of this study is to create a low-cost and broadly adaptable approach to mimic physiological flow, and then use this system to characterize the effect of flow separation on velocity and shear stress profiles in a three-dimensional (3D) topology. The flow apparatus incorporates a programmable linear actuator that superposes oscillations on a constant mean flow driven by a peristaltic pump to emulate flow in the carotid artery. The flow is perfused through a 3D in vitro model of the blood-brain barrier designed to induce separated flow. Experimental flow patterns measured by microparticle image velocimetry and modeled by computational fluid dynamics reveal periodic changes in the instantaneous shear stress along the channel wall. Moreover, the time-dependent flow causes periodic flow separation zones, resulting in variable reattachment points during the cycle. The effects of these complex flow regimes are assessed by evaluating the integrity of the in vitro blood-brain barrier model. Permeability assays and immunostaining for proteins associated with tight junctions reveal barrier breakdown in the region of disturbed flow. In conclusion, the flow system described here creates complex, physiologically relevant flow profiles that provide deeper insight into the fluid dynamics of separated flow and pave the way for future studies interrogating the cellular response to complex flow regimes.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system includes neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-COV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is expressed throughout various caliber vessels in the frontal cortex. Additionally, ACE2 was also detectable in primary human brain microvascular endothelial (hBMVEC) maintained under cell culture conditions. Analysis for cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48hr exposure window. However, when the viral spike proteins were introduced into model systems that recapitulate the essential features of the Blood-Brain Barrier (BBB), breach to the barrier was evident in various degrees depending on the spike protein subunit tested. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluid model of the human BBB, a platform that most closely resembles the human physiological conditions at this CNS interface. Subsequent analysis also showed the ability for SARS-CoV-2 spike proteins to trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

Disturbed flow disrupts the blood-brain barrier in a 3D bifurcation model.

The effect of disturbed flow profiles on the endothelium have been studied extensively in systemic vasculature, but less is known about the response of the blood-brain barrier (BBB) to these flow regimes. Here we investigate the effect of disturbed flow on the integrity of the BBB using a three-dimensional, perfusable bifurcation model consisting of a co-culture of endothelial cells with mural and glial cells. Experimental flow patterns predicted by computational fluid dynamics mimic in vivo flow regimes, specifically the presence of a recirculation zone immediately downstream of the bifurcation. Dextran permeability assays and immunostaining with markers for tight junctions show that barrier disruption is significantly greater in areas of disturbed flow compared to fully developed regions downstream of the bifurcation. Probing crosstalk between cell types suggests that disturbed flow causes barrier breakdown independent of endothelial-mural and endothelial-glial interaction. Overall, disturbed flow-induced disruption of the blood-brain barrier suggests that flow-mediated mechanisms may contribute to vascular pathologies in the central nervous system.

Fluid flow rate dictates the efficacy of low-intensity anti-vascular ultrasound therapy in a microfluidic model.

OBJECTIVE: Low-intensity anti-vascular ultrasound therapy is an effective means of disrupting the blood supply in the tumor microenvironment. Its diminished effect on the surrounding vasculature is thought to be due to higher blood flow rates outside the tumor that decreases the interaction time between the endothelial lining and the microbubbles, which transduce acoustic energy to thermal heat. However, investigating the effect of circulation rate on the response to low-intensity ultrasound is complicated by the heterogeneity of the in vivo vascular microenvironment. Here, a 3D microfluidic model is used to directly interrogate the dynamics of ultrasound stimulation. METHODS: A 3D in vitro vessel consisting of LifeACT transfected endothelial cells facilitate real-time analysis of actin dynamics during ultrasound treatment. Using an integrated testing platform, both the flow rate of microbubbles within the vessel and the magnitude of insonation can be varied. RESULTS: Morphological measurements and dextran transport assays indicate that lower flow rates exacerbate the effect of low-intensity ultrasound on vessel integrity. Additionally, immunostaining for VE-cadherin and transmission electron microscopy provide further insight into structural changes in cell-cell junctions following insonation. CONCLUSIONS: Overall, these results reveal that blood flow rate is an important parameter to consider during the refinement of anti-vascular low-intensity ultrasound therapies.