Neural speech restoration at the cocktail party: Auditory cortex recovers masked speech of both attended and ignored speakers.

Humans are remarkably skilled at listening to one speaker out of an acoustic mixture of several speech sources. Two speakers are easily segregated, even without binaural cues, but the neural mechanisms underlying this ability are not well understood. One possibility is that early cortical processing performs a spectrotemporal decomposition of the acoustic mixture, allowing the attended speech to be reconstructed via optimally weighted recombinations that discount spectrotemporal regions where sources heavily overlap. Using human magnetoencephalography (MEG) responses to a 2-talker mixture, we show evidence for an alternative possibility, in which early, active segregation occurs even for strongly spectrotemporally overlapping regions. Early (approximately 70-millisecond) responses to nonoverlapping spectrotemporal features are seen for both talkers. When competing talkers' spectrotemporal features mask each other, the individual representations persist, but they occur with an approximately 20-millisecond delay. This suggests that the auditory cortex recovers acoustic features that are masked in the mixture, even if they occurred in the ignored speech. The existence of such noise-robust cortical representations, of features present in attended as well as ignored speech, suggests an active cortical stream segregation process, which could explain a range of behavioral effects of ignored background speech.

Engineering anisotropic 3D tubular tissues with flexible thermoresponsive nanofabricated substrates.

Tissue engineering aims to capture the structural and functional aspects of diverse tissue types in vitro. However, most approaches are limited in their ability to produce complex 3D geometries that are essential for tissue function. Tissues, such as the vasculature or chambers of the heart, often possess curved surfaces and hollow lumens that are difficult to recapitulate given their anisotropic architecture. Cell-sheet engineering techniques using thermoresponsive substrates provide a means to stack individual layers of cells with spatial control to create dense, scaffold-free tissues. In this study, we developed a novel method to fabricate complex 3D structures by layering multiple sheets of aligned cells onto flexible scaffolds and casting them into hollow tubular geometries using custom molds and gelatin hydrogels. To enable the fabrication of 3D tissues, we adapted our previously developed thermoresponsive nanopatterned cell-sheet technology by applying it to flexible substrates that could be folded as a form of tissue origami. We demonstrated the versatile nature of this platform by casting aligned sheets of smooth and cardiac muscle cells circumferentially around the surfaces of gelatin hydrogel tubes with hollow lumens. Additionally, we patterned skeletal muscle in the same fashion to recapitulate the 3D curvature that is observed in the muscles of the trunk. The circumferential cell patterning in each case was maintained after one week in culture and even encouraged organized skeletal myotube formation. Additionally, with the application of electrical field stimulation, skeletal myotubes began to assemble functional sarcomeres that could contract. Cardiac tubes could spontaneously contract and be paced for up to one month. Our flexible cell-sheet engineering approach provides an adaptable method to recapitulate more complex 3D geometries with tissue specific customization through the addition of different cell types, mold shapes, and hydrogels. By enabling the fabrication of scaled biomimetic models of human tissues, this approach could potentially be used to investigate tissue structure-function relationships, development, and maturation in the dish.

Regulation of skeletal myotube formation and alignment by nanotopographically controlled cell-secreted extracellular matrix.

Skeletal muscle has a well-organized tissue structure comprised of aligned myofibers and an encasing extracellular matrix (ECM) sheath or lamina, within which reside satellite cells. We hypothesize that the organization of skeletal muscle tissues in culture can affect both the structure of the deposited ECM and the differentiation potential of developing myotubes. Furthermore, we posit that cellular and ECM cues can be a strong determinant of myoblast fusion and morphology in 3D tissue culture environments. To test these, we utilized a thermoresponsive nanofabricated substratum to engineer anisotropic sheets of myoblasts which could then be transferred and stacked into multilayered tissues. Within such engineered tissues, we found that myoblasts rapidly sense topography and deposit structurally organized ECM proteins. Furthermore, the initial tissue structure was found to exert significant control over myoblast fusion and eventual myotube organization. These results highlight the importance of ECM structure on myoblast fusion and organization, and provide insights into substrate-mediated control of myotube formation in the development of novel, more effective, engineered skeletal muscle tissues. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1543-1551, 2018.

Novel Adult-Onset Systolic Cardiomyopathy Due to MYH7 E848G Mutation in Patient-Derived Induced Pluripotent Stem Cells.

A novel myosin heavy chain 7 mutation (E848G) identified in a familial cardiomyopathy was studied in patient-specific induced pluripotent stem cell-derived cardiomyocytes. The cardiomyopathic human induced pluripotent stem cell-derived cardiomyocytes exhibited reduced contractile function as single cells and engineered heart tissues, and genome-edited isogenic cells confirmed the pathogenic nature of the E848G mutation. Reduced contractility may result from impaired interaction between myosin heavy chain 7 and cardiac myosin binding protein C.

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells.

Understanding the phenotypic development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a prerequisite to advancing regenerative cardiac therapy, disease modeling, and drug screening applications. Lack of consistent hiPSC-CM in vitro data can be largely attributed to the inability of conventional culture methods to mimic the structural, biochemical, and mechanical aspects of the myocardial niche accurately. Here, we present a nanogrid culture array comprised of nanogrooved topographies, with groove widths ranging from 350 to 2000 nm, to study the effect of different nanoscale structures on the structural development of hiPSC-CMs in vitro. Nanotopographies were designed to have a biomimetic interface, based on observations of the oriented myocardial extracellular matrix (ECM) fibers found in vivo. Nanotopographic substrates were integrated with a self-assembling chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif. Using this platform, cell adhesion to peptide-coated substrates was found to be comparable to that of conventional fibronectin-coated surfaces. Cardiomyocyte organization and structural development were found to be dependent on the nanotopographical feature size in a biphasic manner, with improved development achieved on grooves in the 700-1000 nm range. These findings highlight the capability of surface-functionalized, bioinspired substrates to influence cardiomyocyte development, and the capacity for such platforms to serve as a versatile assay for investigating the role of topographical guidance cues on cell behavior. Such substrates could potentially create more physiologically relevant in vitro cardiac tissues for future drug screening and disease modeling studies.

Capillary force lithography for cardiac tissue engineering.

Cardiovascular disease remains the leading cause of death worldwide(1). Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart's extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale(2). Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering(3-5). A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling(2). Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart(6-9). Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)(8) and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function(10-14). Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively(15,16). Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (λ = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 °C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS(2).

Fabrication of poly(ethylene glycol): gelatin methacrylate composite nanostructures with tunable stiffness and degradation for vascular tissue engineering.

Although synthetic polymers are desirable in tissue engineering applications for the reproducibility and tunability of their properties, synthetic small diameter vascular grafts lack the capability to endothelialize in vivo. Thus, synthetically fabricated biodegradable tissue scaffolds that reproduce important aspects of the extracellular environment are required to meet the urgent need for improved vascular grafting materials. In this study, we have successfully fabricated well-defined nanopatterned cell culture substrates made of a biodegradable composite hydrogel consisting of poly(ethylene glycol) dimethacrylate (PEGDMA) and gelatin methacrylate (GelMA) by using UV-assisted capillary force lithography. The elasticity and degradation rate of the composite PEG-GelMA nanostructures were tuned by varying the ratios of PEGDMA and GelMA. Human umbilical vein endothelial cells (HUVECs) cultured on nanopatterned PEG-GelMA substrates exhibited enhanced cell attachment compared with those cultured on unpatterned PEG-GelMA substrates. Additionally, HUVECs cultured on nanopatterned PEG-GelM substrates displayed well-aligned, elongated morphology similar to that of native vascular endothelial cells and demonstrated rapid and directionally persistent migration. The ability to alter both substrate stiffness and degradation rate and culture endothelial cells with increased elongation and alignment is a promising next step in recapitulating the properties of native human vascular tissue for tissue engineering applications.

Thermoresponsive nanofabricated substratum for the engineering of three-dimensional tissues with layer-by-layer architectural control.

Current tissue engineering methods lack the ability to properly recreate scaffold-free, cell-dense tissues with physiological structures. Recent studies have shown that the use of nanoscale cues allows for precise control over large-area 2D tissue structures without restricting cell growth or cell density. In this study, we developed a simple and versatile platform combining a thermoresponsive nanofabricated substratum (TNFS) incorporating nanotopographical cues and the gel casting method for the fabrication of scaffold-free 3D tissues. Our TNFS allows for the structural control of aligned cell monolayers which can be spontaneously detached via a change in culture temperature. Utilizing our gel casting method, viable, aligned cell sheets can be transferred without loss of anisotropy or stacked with control over individual layer orientations. Transferred cell sheets and individual cell layers within multilayered tissues robustly retain structural anisotropy, allowing for the fabrication of scaffold-free, 3D tissues with hierarchical control of overall tissue structure.

Initiated chemical vapor deposition of thermoresponsive poly(N-vinylcaprolactam) thin films for cell sheet engineering.

Poly(N-vinylcaprolactam) (PNVCL) is a thermoresponsive polymer known to be nontoxic, water soluble and biocompatible. Here, PNVCL homopolymer was successfully synthesized for the first time by use of a one-step vapor-phase process, termed initiated chemical vapor deposition (iCVD). Fourier transform infrared spectroscopy results showed that radical polymerization took place from N-vinylcaprolactam monomers without damaging the functional caprolactam ring. A sharp lower critical solution temperature transition was observed at 31°C from the iCVD poly(N-vinylcaprolactam) (PNVCL) film. The thermoresponsive PNVCL surface exhibited a hydrophilic/hydrophobic alteration with external temperature change, which enabled the thermally modulated attachment and detachment of cells. The conformal coverage of PNVCL film on various substrates with complex topography, including fabrics and nanopatterns, was successfully demonstrated, which can further be utilized to fabricate cell sheets with aligned cell morphology. The advantage of this system is that cells cultured on such thermoresponsive surfaces could be recovered as an intact cell sheet by simply lowering the temperature, eliminating the need for conventional enzymatic treatments.

Nanotopography-guided tissue engineering and regenerative medicine.

Human tissues are intricate ensembles of multiple cell types embedded in complex and well-defined structures of the extracellular matrix (ECM). The organization of ECM is frequently hierarchical from nano to macro, with many proteins forming large scale structures with feature sizes up to several hundred microns. Inspired from these natural designs of ECM, nanotopography-guided approaches have been increasingly investigated for the last several decades. Results demonstrate that the nanotopography itself can activate tissue-specific function in vitro as well as promote tissue regeneration in vivo upon transplantation. In this review, we provide an extensive analysis of recent efforts to mimic functional nanostructures in vitro for improved tissue engineering and regeneration of injured and damaged tissues. We first characterize the role of various nanostructures in human tissues with respect to each tissue-specific function. Then, we describe various fabrication methods in terms of patterning principles and material characteristics. Finally, we summarize the applications of nanotopography to various tissues, which are classified into four types depending on their functions: protective, mechano-sensitive, electro-active, and shear stress-sensitive tissues. Some limitations and future challenges are briefly discussed at the end.

Patterning methods for polymers in cell and tissue engineering.

Polymers provide a versatile platform for mimicking various aspects of physiological extracellular matrix properties such as chemical composition, rigidity, and topography for use in cell and tissue engineering applications. In this review, we provide a brief overview of patterning methods of various polymers with a particular focus on biocompatibility and processability. The materials highlighted here are widely used polymers including thermally curable polydimethyl siloxane, ultraviolet-curable polyurethane acrylate and polyethylene glycol, thermo-sensitive poly(N-isopropylacrylamide) and thermoplastic and conductive polymers. We also discuss how micro- and nanofabricated polymeric substrates of tunable elastic modulus can be used to engineer cell and tissue structure and function. Such synergistic effect of topography and rigidity of polymers may be able to contribute to constructing more physiologically relevant microenvironment.

Exogenous mineralization of cell-seeded and unseeded collagen-chitosan hydrogels using modified culture medium.

Induced biomineralization of materials has been employed as a strategy to increase integration with host tissue, and more recently as a method to control cell function in tissue engineering. However, mineralization is typically performed in the absence of cells, since hypertonic solutions that lack the nutrients and culture components required for the maintenance of cell viability are often used. In the present study, we exposed fibroblast-seeded three-dimensional collagen-chitosan hydrogels to a defined culture medium modified to have specific concentrations of ions involved in biomineralization. The modified medium caused a significant increase in calcium deposition in collagen-chitosan gels, relative to constructs incubated in a standard medium, though serum supplementation attenuated mineral deposition. Collagen-chitosan constructs became opaque over 3 days of mineralization in modified Dulbecco's modified Eagle medium (DMEM), in contrast to translucent control gels incubated in standard DMEM. Histological staining confirmed increased levels of mineral in the treated constructs. Rheological characterization showed that both the storage and loss moduli increased significantly in mineralized materials. Mineralization of fibroblast-seeded constructs resulted in decreased cell viability and proliferation rate over 3 days of incubation in modified medium, but the cell population remained over 75% viable and regained its proliferative potential after rescue in standard culture medium. The ability to mineralize protein matrices in the presence of cells could be useful in creating mechanically stable tissue constructs, as well as to study the effects of the tissue microenvironment on cell function.