Recreating the heart's helical structure-function relationship with focused rotary jet spinning.

Helical alignments within the heart's musculature have been speculated to be important in achieving physiological pumping efficiencies. Testing this possibility is difficult, however, because it is challenging to reproduce the fine spatial features and complex structures of the heart's musculature using current techniques. Here we report focused rotary jet spinning (FRJS), an additive manufacturing approach that enables rapid fabrication of micro/nanofiber scaffolds with programmable alignments in three-dimensional geometries. Seeding these scaffolds with cardiomyocytes enabled the biofabrication of tissue-engineered ventricles, with helically aligned models displaying more uniform deformations, greater apical shortening, and increased ejection fractions compared with circumferential alignments. The ability of FRJS to control fiber arrangements in three dimensions offers a streamlined approach to fabricating tissues and organs, with this work demonstrating how helical architectures contribute to cardiac performance.

A bioinspired and hierarchically structured shape-memory material.

Shape-memory polymeric materials lack long-range molecular order that enables more controlled and efficient actuation mechanisms. Here, we develop a hierarchical structured keratin-based system that has long-range molecular order and shape-memory properties in response to hydration. We explore the metastable reconfiguration of the keratin secondary structure, the transition from α-helix to ß-sheet, as an actuation mechanism to design a high-strength shape-memory material that is biocompatible and processable through fibre spinning and three-dimensional (3D) printing. We extract keratin protofibrils from animal hair and subject them to shear stress to induce their self-organization into a nematic phase, which recapitulates the native hierarchical organization of the protein. This self-assembly process can be tuned to create materials with desired anisotropic structuring and responsiveness. Our combination of bottom-up assembly and top-down manufacturing allows for the scalable fabrication of strong and hierarchically structured shape-memory fibres and 3D-printed scaffolds with potential applications in bioengineering and smart textiles.

Mapping 2D- and 3D-distributions of metal/metal oxide nanoparticles within cleared human ex vivo skin tissues.

An increasing number of commercial skincare products are being manufactured with engineered nanomaterials (ENMs), prompting a need to fully understand how ENMs interact with the dermal barrier as a major biodistribution entry route. Although animal studies show that certain nanomaterials can cross the skin barrier, physiological differences between human and animal skin, such as the lack of sweat glands, limit the translational validity of these results. Current optical microscopy methods have limited capabilities to visualize ENMs within human skin tissues due to the high amount of background light scattering caused by the dense, ubiquitous extracellular matrix (ECM) of the skin. Here, we hypothesized that organic solvent-based tissue clearing ("immunolabeling-enabled three-dimensional imaging of solvent-cleared organs", or "iDISCO") would reduce background light scattering from the extracellular matrix of the skin to sufficiently improve imaging contrast for both 2D mapping of unlabeled metal oxide ENMs and 3D mapping of fluorescent nanoparticles. We successfully mapped the 2D distribution of label-free TiO2 and ZnO nanoparticles in cleared skin sections using correlated signals from darkfield, brightfield, and confocal microscopy, as well as micro-spectroscopy. Specifically, hyperspectral microscopy and Raman spectroscopy confirmed the identity of label-free ENMs which we mapped within human skin sections. We also measured the 3D distribution of fluorescently labeled Ag nanoparticles in cleared skin biopsies with wounded epidermal layers using light sheet fluorescence microscopy. Overall, this study explores a novel strategy for quantitatively mapping ENM distributions in cleared ex vivo human skin tissue models using multiple imaging modalities. By improving the imaging contrast, we present label-free 2D ENM tracking and 3D ENM mapping as promising capabilities for nanotoxicology investigations.

Biomimetic and estrogenic fibers promote tissue repair in mice and human skin via estrogen receptor ß.

The dynamic changes in estrogen levels throughout aging and during the menstrual cycle influence wound healing. Elevated estrogen levels during the pre-ovulation phase accelerate tissue repair, whereas reduced estrogen levels in post-menopausal women lead to slow healing. Although previous reports have shown that estrogen may potentiate healing by triggering the estrogen receptor (ER)-ß signaling pathway, its binding to ER-α has been associated with severe collateral effects and has therefore limited its use as a therapeutic agent. To this end, soy phytoestrogens, which preferentially bind to the ER-ß, are currently being explored as a safer therapeutic alternative to estrogen. However, the development and evaluation of phytoestrogen-based materials as local ER-ß modulators remains largely unexplored. Here, we engineered biomimetic and estrogenic nanofiber wound dressings built from soy protein isolate (SPI) and hyaluronic acid (HA) using immersion rotary jet spinning. These engineered scaffolds were shown to successfully recapitulate the native dermal architecture, while delivering an ER-ß-triggering phytoestrogen (genistein). When tested in ovariectomized mouse and ex vivo human skin tissues, HA/SPI scaffolds outperformed controls (no treatment or HA only scaffolds) towards promoting cutaneous tissue repair. These improved healing outcomes were prevented when the ER-ß pathway was genetically or chemically inhibited. Our findings suggest that estrogenic fibrous scaffolds facilitate skin repair by ER-ß activation.

Porous Biomimetic Hyaluronic Acid and Extracellular Matrix Protein Nanofiber Scaffolds for Accelerated Cutaneous Tissue Repair.

Recent reports suggest the utility of extracellular matrix (ECM) molecules as raw components in scaffolding of engineered materials. However, rapid and tunable manufacturing of ECM molecules into fibrous structures remains poorly developed. Here we report on an immersion rotary jet-spinning (iRJS) method to show high-throughput manufacturing (up to ∼1 g/min) of hyaluronic acid (HA) and other ECM fiber scaffolds using different spinning conditions and postprocessing modifications. This system allowed control over a variety of scaffold material properties, which enabled the fabrication of highly porous (70-95%) and water-absorbent (swelling ratio ∼2000-6000%) HA scaffolds with soft-tissue mimetic mechanical properties (∼0.5-1.5 kPa). Tuning these scaffolds' properties enabled the identification of porosity (∼95%) as a key facilitator for rapid and in-depth cellular ingress in vitro. We then demonstrated that porous HA scaffolds accelerated granulation tissue formation, neovascularization, and reepithelialization in vivo, altogether potentiating faster wound closure and tissue repair. Collectively, this scalable and versatile manufacturing approach enabled the fabrication of tunable ECM-mimetic nanofiber scaffolds that may provide an ideal first building block for the design of all-in-one healing materials.

Muscle tissue engineering in fibrous gelatin: implications for meat analogs.

Bioprocessing applications that derive meat products from animal cell cultures require food-safe culture substrates that support volumetric expansion and maturation of adherent muscle cells. Here we demonstrate scalable production of microfibrous gelatin that supports cultured adherent muscle cells derived from cow and rabbit. As gelatin is a natural component of meat, resulting from collagen denaturation during processing and cooking, our extruded gelatin microfibers recapitulated structural and biochemical features of natural muscle tissues. Using immersion rotary jet spinning, a dry-jet wet-spinning process, we produced gelatin fibers at high rates (~ 100 g/h, dry weight) and, depending on process conditions, we tuned fiber diameters between ~ 1.3 ± 0.1 µm (mean ± SEM) and 8.7 ± 1.4 µm (mean ± SEM), which are comparable to natural collagen fibers. To inhibit fiber degradation during cell culture, we crosslinked them either chemically or by co-spinning gelatin with a microbial crosslinking enzyme. To produce meat analogs, we cultured bovine aortic smooth muscle cells and rabbit skeletal muscle myoblasts in gelatin fiber scaffolds, then used immunohistochemical staining to verify that both cell types attached to gelatin fibers and proliferated in scaffold volumes. Short-length gelatin fibers promoted cell aggregation, whereas long fibers promoted aligned muscle tissue formation. Histology, scanning electron microscopy, and mechanical testing demonstrated that cultured muscle lacked the mature contractile architecture observed in natural muscle but recapitulated some of the structural and mechanical features measured in meat products.

Production-scale fibronectin nanofibers promote wound closure and tissue repair in a dermal mouse model.

Wounds in the fetus can heal without scarring. Consequently, biomaterials that attempt to recapitulate the biophysical and biochemical properties of fetal skin have emerged as promising pro-regenerative strategies. The extracellular matrix (ECM) protein fibronectin (Fn) in particular is believed to play a crucial role in directing this regenerative phenotype. Accordingly, Fn has been implicated in numerous wound healing studies, yet remains untested in its fibrillar conformation as found in fetal skin. Here, we show that high extensional (∼1.2 ×105 s-1) and shear (∼3 ×105 s-1) strain rates in rotary jet spinning (RJS) can drive high throughput Fn fibrillogenesis (∼10 mL/min), thus producing nanofiber scaffolds that are used to effectively enhance wound healing. When tested on a full-thickness wound mouse model, Fn nanofiber dressings not only accelerated wound closure, but also significantly improved tissue restoration, recovering dermal and epidermal structures as well as skin appendages and adipose tissue. Together, these results suggest that bioprotein nanofiber fabrication via RJS could set a new paradigm for enhancing wound healing and may thus find use in a variety of regenerative medicine applications.

Soy Protein/Cellulose Nanofiber Scaffolds Mimicking Skin Extracellular Matrix for Enhanced Wound Healing.

Historically, soy protein and extracts have been used extensively in foods due to their high protein and mineral content. More recently, soy protein has received attention for a variety of its potential health benefits, including enhanced skin regeneration. It has been reported that soy protein possesses bioactive molecules similar to extracellular matrix (ECM) proteins and estrogen. In wound healing, oral and topical soy has been heralded as a safe and cost-effective alternative to animal protein and endogenous estrogen. However, engineering soy protein-based fibrous dressings, while recapitulating ECM microenvironment and maintaining a moist environment, remains a challenge. Here, the development of an entirely plant-based nanofibrous dressing comprised of cellulose acetate (CA) and soy protein hydrolysate (SPH) using rotary jet spinning is described. The spun nanofibers successfully mimic physicochemical properties of the native skin ECM and exhibit a high water retaining capability. In vitro, CA/SPH nanofibers promote fibroblast proliferation, migration, infiltration, and integrin ß1 expression. In vivo, CA/SPH scaffolds accelerate re-epithelialization and epidermal thinning as well as reduce scar formation and collagen anisotropy in a similar fashion to other fibrous scaffolds, but without the use of animal proteins or synthetic polymers. These results affirm the potential of CA/SPH nanofibers as a novel wound dressing.

A tissue-engineered scale model of the heart ventricle.

Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 µl (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle), and ejection fractions and contractile work 50-250 times smaller and 104-108 times smaller than the corresponding values for rodent and human ventricles, respectively. We also measured tissue coverage and alignment, calcium-transient propagation and pressure-volume loops in the presence or absence of test compounds. Moreover, we describe an instrumented bioreactor with ventricular-assist capabilities, and provide a proof-of-concept disease model of structural arrhythmia. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings.

Cardiac microphysiological devices with flexible thin-film sensors for higher-throughput drug screening.

Microphysiological systems and organs-on-chips promise to accelerate biomedical and pharmaceutical research by providing accurate in vitro replicas of human tissue. Aside from addressing the physiological accuracy of the model tissues, there is a pressing need for improving the throughput of these platforms. To do so, scalable data acquisition strategies must be introduced. To this end, we here present an instrumented 24-well plate platform for higher-throughput studies of engineered human stem cell-derived cardiac muscle tissues that recapitulate the laminar structure of the native ventricle. In each well of the platform, an embedded flexible strain gauge provides continuous and non-invasive readout of the contractile stress and beat rate of an engineered cardiac tissue. The sensors are based on micro-cracked titanium-gold thin films, which ensure that the sensors are highly compliant and robust. We demonstrate the value of the platform for toxicology and drug-testing purposes by performing 12 complete dose-response studies of cardiac and cardiotoxic drugs. Additionally, we showcase the ability to couple the cardiac tissues with endothelial barriers. In these studies, which mimic the passage of drugs through the blood vessels to the musculature of the heart, we regulate the temporal onset of cardiac drug responses by modulating endothelial barrier permeability in vitro.