Josue A. Goss

Nanofiber Assembly by Rotary Jet-Spinning

High-voltage electrical fields and low production rate limit electrospinning, the electrical charging of polymer liquids, as a means of nanofiber fabrication. Here, we show a facile method of fabrication of aligned three-dimensional nanofiber structures by utilizing high-speed, rotating polymer solution jets to extrude fibers. Termed rotary jet-spinning, fiber morphology, diameter, and web porosity can be controlled by varying nozzle geometry, rotation speed, and polymer solution properties. We demonstrate the utility of this technique for tissue engineering by building anisotropic arrays of biodegradable polymer fibers and seeding the constructs with neonatal rat ventricular cardiomyocytes. The myocytes used the aligned fibers to orient their contractile cytoskeleton and to self-organize into a beating, multicellular tissue that mimics the laminar, anisotropic architecture of the heart muscle. This technique may prove advantageous for building uniaxially aligned nanofiber structures for polymers which are not amenable to fabrication by electrospinning.

Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve

Endothelial-mesenchymal transformation (EMT) is a critical event for the embryonic morphogenesis of cardiac valves. Inducers of EMT during valvulogenesis include VEGF, TGF-β1, and wnt/β-catenin (where wnt refers to the wingless-type mammary tumor virus integration site family of proteins), that are regulated in a spatiotemporal manner. EMT has also been observed in diseased, strain-overloaded valve leaflets, suggesting a regulatory role for mechanical strain. Although the preponderance of studies have focused on the role of soluble mitogens, we asked if the valve tissue microenvironment contributed to EMT. To recapitulate these microenvironments in a controlled, in vitro environment, we engineered 2D valve endothelium from sheep valve endothelial cells, using microcontact printing to mimic the regions of isotropy and anisotropy of the leaflet, and applied cyclic mechanical strain in an attempt to induce EMT. We measured EMT in response to both low (10%) and high strain (20%), where low-strain EMT occurred via increased TGF-β1 signaling and high strain via increased wnt/β-catenin signaling, suggesting dual strain-dependent routes to distinguish EMT in healthy versus diseased valve tissue. The effect was also directionally dependent, where cyclic strain applied orthogonal to axis of the engineered valve endothelium alignment resulted in severe disruption of cell microarchitecture and greater EMT. Once transformed, these tissues exhibited increased contractility in the presence of endothelin-1 and larger basal mechanical tone in a unique assay developed to measure the contractile tone of the engineered valve tissues. This finding is important, because it implies that the functional properties of the valve are sensitive to EMT. Our results suggest that cyclic mechanical strain regulates EMT in a strain magnitude and directionally dependent manner.

Blast-induced phenotypic switching in cerebral vasospasm

Vasospasm of the cerebrovasculature is a common manifestation of blast-induced traumatic brain injury (bTBI) reported among combat casualties in the conflicts in Afghanistan and Iraq. Cerebral vasospasm occurs more frequently, and with earlier onset, in bTBI patients than in patients with other TBI injury modes, such as blunt force trauma. Though vasospasm is usually associated with the presence of subarachnoid hemorrhage (SAH), SAH is not required for vasospasm in bTBI, which suggests that the unique mechanics of blast injury could potentiate vasospasm onset, accounting for the increased incidence. Here, using theoretical and in vitro models, we show that a single rapid mechanical insult can induce vascular hypercontractility and remodeling, indicative of vasospasm initiation. We employed high-velocity stretching of engineered arterial lamellae to simulate the mechanical forces of a blast pulse on the vasculature. An hour after a simulated blast, injured tissues displayed altered intracellular calcium dynamics leading to hypersensitivity to contractile stimulus with endothelin-1. One day after simulated blast, tissues exhibited blast force dependent prolonged hypercontraction and vascular smooth muscle phenotype switching, indicative of remodeling. These results suggest that an acute, blast-like injury is sufficient to induce a hypercontraction-induced genetic switch that potentiates vascular remodeling, and cerebral vasospasm, in bTBI patients.

Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip

The lack of a robust pipeline of medical therapeutic agents for the treatment of heart disease may be partially attributed to the lack of in vitro models that recapitulate the essential structure–function relationships of healthy and diseased myocardium. We designed and built a system to mimic mechanical overload in vitro by applying cyclic stretch to engineered laminar ventricular tissue on a stretchable chip. To test our model, we quantified changes in gene expression, myocyte architecture, calcium handling, and contractile function and compared our results vs. several decades of animal studies and clinical observations. Cyclic stretch activated gene expression profiles characteristic of pathological remodeling, including decreased α- to β-myosin heavy chain ratios, and induced maladaptive changes to myocyte shape and sarcomere alignment. In stretched tissues, calcium transients resembled those reported in failing myocytes and peak systolic stress was significantly reduced. Our results suggest that failing myocardium, as defined genetically, structurally, and functionally, can be replicated in an in vitro microsystem by faithfully recapitulating the structural and mechanical microenvironment of the diseased heart.

Engineering hybrid polymer-protein super-aligned nanofibers via rotary jet spinning

Cellular microenvironments are important in coaxing cells to behave collectively as functional, structured tissues. Important cues in this microenvironment are the chemical, mechanical and spatial arrangement of the supporting matrix in the extracellular space. In engineered tissues, synthetic scaffolding provides many of these microenvironmental cues. Key requirements are that synthetic scaffolds should recapitulate the native three-dimensional (3D) hierarchical fibrillar structure, possess biomimetic surface properties and demonstrate mechanical integrity, and in some tissues, anisotropy. Electrospinning is a popular technique used to fabricate anisotropic nanofiber scaffolds. However, it suffers from relatively low production rates and poor control of fiber alignment without substantial modifications to the fiber collector mechanism. Additionally, many biomaterials are not amenable for fabrication via high-voltage electrospinning methods. Hence, we reasoned that we could utilize rotary jet spinning (RJS) to fabricate highly aligned hybrid protein-polymer with tunable chemical and physical properties. In this study, we engineered highly aligned nanofiber constructs with robust fiber alignment from blends of the proteins collagen and gelatin, and the polymer poly-ε-caprolactone via RJS and electrospinning. RJS-spun fibers retain greater protein content on the surface and are also fabricated at a higher production rate compared to those fabricated via electrospinning. We measured increased fiber diameter and viscosity, and decreasing fiber alignment as protein content increased in RJS hybrid fibers. RJS nanofiber constructs also demonstrate highly anisotropic mechanical properties mimicking several biological tissue types. We demonstrate the bio-functionality of RJS scaffold fibers by testing their ability to support cell growth and maturation with a variety of cell types. Our highly anisotropic RJS fibers are therefore able to support cellular alignment, maturation and self-organization. The hybrid nanofiber constructs fabricated by RJS therefore have the potential to be used as scaffold material for a wide variety of biological tissues and organs, as an alternative to electrospinning.

A Possible Role for Integrin Signaling in Diffuse Axonal Injury

Over the past decade, investigators have attempted to establish the pathophysiological mechanisms by which non-penetrating injuries damage the brain. Several studies have implicated either membrane poration or ion channel dysfunction pursuant to neuronal cell death as the primary mechanism of injury. We hypothesized that traumatic stimulation of integrins may be an important etiological contributor to mild Traumatic Brain Injury. In order to study the effects of forces at the cellular level, we utilized two hierarchical, in vitro systems to mimic traumatic injury to rat cortical neurons: a high velocity stretcher and a magnetic tweezer system. In one system, we controlled focal adhesion formation in neurons cultured on a stretchable substrate loaded with an abrupt, one dimensional strain. With the second system, we used magnetic tweezers to directly simulate the abrupt injury forces endured by a focal adhesion on the neurite. Both systems revealed variations in the rate and nature of neuronal injury as a function of focal adhesion density and direct integrin stimulation without membrane poration. Pharmacological inhibition of calpains did not mitigate the injury yet the inhibition of Rho-kinase immediately after injury reduced axonal injury. These data suggest that integrin-mediated activation of Rho may be a contributor to the diffuse axonal injury reported in mild Traumatic Brain Injury.

A simple model for nanofiber formation by rotary jet-spinning

Nanofibers are microstructured materials that span a broad range of applications from tissue engineering scaffolds to polymer transistors. An efficient method of nanofiber production is rotary jet-spinning (RJS), consisting of a perforated reservoir rotating at high speeds along its axis of symmetry, which propels a liquid, polymeric jet out of the reservoir orifice that stretches, dries, and eventually solidifies to form nanoscale fibers. We report a minimal scaling framework complemented by a semi-analytic and numerical approach to characterize the regimes of nanofiber production, leading to a theoretical model for the fiber radius consistent with experimental observations. In addition to providing a mechanism for the formation of nanofibers, our study yields a phase diagram for the design of continuous nanofibers as a function of process parameters with implications for the morphological quality of fibers.

Effect of Solvent Evaporation on Fiber Morphology in Rotary Jet Spinning

The bulk production of polymeric nanofibers is important for fabricating high-performance, nanoscale materials. Rotary jet spinning (RJS) enables the mass production of nanostructured fibers by centrifugal forces but may result in inconsistent surface morphologies. Because nanofiber performance is dependent upon its surface features, we asked which parameters must be optimized during production to control fiber morphology. We developed and tested a mathematical model that describes how the competition between fluid instability and solvent removal in RJS regulates the degree of beading in fibers. Our data suggest that solvent evaporation during the spinning process causes an increase in jet viscosity and that these changes inhibit both bead formation and jet thinning. The RJS was used to vary experimental parameters, showing that fiber beading can be reduced by increasing solvent volatility, solution viscosity, and spinning velocity. Collectively, our results demonstrate that nanofiber morphology and diameter can be precisely controlled during RJS manufacturing.

Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes

Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal–nuclear–chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations.

Laminar ventricular myocardium on a microelectrode array-based chip

Pharmaceutical screening based on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and multi electrode arrays (MEAs) have been proposed as a complementary method for electrophysiological safety and efficacy assessment in drug discovery and development. Contrary to animal models, these cells offer a human genetic background but, at present, fail to recapitulate the mechanical and structural properties of the native human myocardium. Here, we report that topographical cues on soft micromolded gelatin can coax hiPSC-CMs to form laminar cardiac tissues that resemble the native architecture of the heart. Importantly, using this method we were able to record tissue-level electrophysiological responses with a commercially available MEA setup. To validate this platform, we recorded cardiac field potentials at baseline and after pharmacological interventions with a β-adrenergic agonist (isoproterenol). Further, we tested the ability of our system to predict the response of laminar human cardiac tissues to a cardiotoxic pro-drug (terfenadine) and its non-cardiotoxic metabolite (fexofenadine). Finally, we integrated our platform with microfluidic components to build a heart-on-a-chip system that can be fluidically linked with other organs-on-chips in the future.