Publications by authors named "Adam W Feinberg"

55 Publications

Gain-of-function mutation in ubiquitin-ligase KLHL24 causes desmin degradation and dilatation in hiPSC-derived engineered heart tissues.

J Clin Invest 2021 Jul 22. Epub 2021 Jul 22.

Department of Cardiology, University Medical Center Groningen, Groningen, Netherlands.

The start codon c.1A>G mutation in KLHL24, encoding ubiquitin-ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation, ultimately causing epidermolysis bullosa simplex (EBS). The majority of these EBS-patients are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homologue of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from two patients and three (non)familial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients' explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, that can be prevented by restoring desmin protein levels.
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http://dx.doi.org/10.1172/JCI140615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409593PMC
July 2021

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype.

Sci Transl Med 2021 07;13(603)

Regenerative Biomaterials and Therapeutics Group, Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.
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http://dx.doi.org/10.1126/scitranslmed.abd1817DOI Listing
July 2021

Engineering aligned human cardiac muscle using developmentally inspired fibronectin micropatterns.

Sci Rep 2021 06 1;11(1):11502. Epub 2021 Jun 1.

Department of Materials Science and Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania, 15213, USA.

Cardiac two-dimensional tissues were engineered using biomimetic micropatterns based on the fibronectin-rich extracellular matrix (ECM) of the embryonic heart. The goal of this developmentally-inspired, in vitro approach was to identify cell-cell and cell-ECM interactions in the microenvironment of the early 4-chambered vertebrate heart that drive cardiomyocyte organization and alignment. To test this, biomimetic micropatterns based on confocal imaging of fibronectin in embryonic chick myocardium were created and compared to control micropatterns designed with 2 or 20 µm wide fibronectin lines. Results show that embryonic chick cardiomyocytes have a unique density-dependent alignment on the biomimetic micropattern that is mediated in part by N-cadherin, suggesting that both cell-cell and cell-ECM interactions play an important role in the formation of aligned myocardium. Human induced pluripotent stem cell-derived cardiomyocytes also showed density-dependent alignment on the biomimetic micropattern but were overall less well organized. Interestingly, the addition of human adult cardiac fibroblasts and conditioning with T3 hormone were both shown to increase human cardiomyocyte alignment. In total, these results show that cardiomyocyte maturation state, cardiomyocyte-cardiomyocyte and cardiomyocyte-fibroblast interactions, and cardiomyocyte-ECM interactions can all play a role when engineering anisotropic cardiac tissues in vitro and provides insight as to how these factors may influence cardiogenesis in vivo.
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http://dx.doi.org/10.1038/s41598-021-87550-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169656PMC
June 2021

Recent Advances in Cellular and Molecular Bioengineering for Building and Translation of Biological Systems.

Cell Mol Bioeng 2021 May 25:1-16. Epub 2021 May 25.

Departments of Biomedical Engineering & Biological Sciences, Rensselaer Polytechnic Institute, Biotech 2147, 110 8th Street, Troy, NY 12180 USA.

In January of 2020, the Biomedical Engineering Society (BMES)- Cellular and Molecular Bioengineering (CMBE) conference was held in Puerto Rico and themed "Vision 2020: Emerging Technologies to Elucidate the Rule of Life." The annual BME-CMBE conference gathered worldwide leaders and discussed successes and challenges in engineering biological systems and their translation. The goal of this report is to present the research frontiers in this field and provide perspectives on successful engineering and translation towards the clinic. We hope that this report serves as a constructive guide in shaping the future of research and translation of engineered biological systems.
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http://dx.doi.org/10.1007/s12195-021-00676-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8147909PMC
May 2021

Intracellular action potential recordings from cardiomyocytes by ultrafast pulsed laser irradiation of fuzzy graphene microelectrodes.

Sci Adv 2021 Apr 7;7(15). Epub 2021 Apr 7.

Istituto Italiano di Tecnologia, Genova 16163, Italy.

Graphene with its unique electrical properties is a promising candidate for carbon-based biosensors such as microelectrodes and field effect transistors. Recently, graphene biosensors were successfully used for extracellular recording of action potentials in electrogenic cells; however, intracellular recordings remain beyond their current capabilities because of the lack of an efficient cell poration method. Here, we present a microelectrode platform consisting of out-of-plane grown three-dimensional fuzzy graphene (3DFG) that enables recording of intracellular cardiac action potentials with high signal-to-noise ratio. We exploit the generation of hot carriers by ultrafast pulsed laser for porating the cell membrane and creating an intimate contact between the 3DFG electrodes and the intracellular domain. This approach enables us to detect the effects of drugs on the action potential shape of human-derived cardiomyocytes. The 3DFG electrodes combined with laser poration may be used for all-carbon intracellular microelectrode arrays to allow monitoring of the cellular electrophysiological state.
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http://dx.doi.org/10.1126/sciadv.abd5175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026128PMC
April 2021

Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication.

APL Bioeng 2021 Mar 16;5(1):010904. Epub 2021 Feb 16.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.

In tissue engineering, an unresolved challenge is how to build complex 3D scaffolds in order to recreate the structure and function of human tissues and organs. Additive manufacturing techniques, such as 3D bioprinting, have the potential to build biological material with unprecedented spatial control; however, printing soft biological materials in air often results in poor fidelity. Freeform Reversible Embedding of Suspended Hydrogels (FRESH) is an embedded printing approach that solves this problem by extruding bioinks within a yield-stress support bath that holds the bioinks in place until cured. In this Perspective, we discuss the challenges of 3D printing soft and liquid-like bioinks and the emergence for FRESH and related embedded printing techniques as a solution. This includes the development of FRESH and embedded 3D printing within the bioprinting field and the rapid growth in adoption, as well as the advantages of FRESH printing for biofabrication and the new research results this has enabled. Specific focus is on the customizability of the FRESH printing technique where the chemical composition of the yield-stress support bath and aqueous phase crosslinker can all be tailored for printing a wide range of bioinks in complex 3D structures. Finally, we look ahead at the future of FRESH printing, discussing both the challenges and the opportunities that we see as the biofabrication field develops.
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http://dx.doi.org/10.1063/5.0032777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889293PMC
March 2021

3D Bioprinting using UNIversal Orthogonal Network (UNION) Bioinks.

Adv Funct Mater 2021 Feb 20;31(7). Epub 2020 Nov 20.

Department of Materials Science & Engineering, Stanford University, Stanford, CA 94305, USA.

Three-dimensional (3D) bioprinting is a promising technology to produce tissue-like structures, but a lack of diversity in bioinks is a major limitation. Ideally each cell type would be printed in its own customizable bioink. To fulfill this need for a universally applicable bioink strategy, we developed a versatile, bioorthogonal bioink crosslinking mechanism that is cell compatible and works with a range of polymers. We term this family of materials UNIversal, Orthogonal Network (UNION) bioinks. As demonstration of UNION bioink versatility, gelatin, hyaluronic acid (HA), recombinant elastin-like protein (ELP), and polyethylene glycol (PEG) were each used as backbone polymers to create inks with storage moduli spanning 200 to 10,000 Pa. Because UNION bioinks are crosslinked by a common chemistry, multiple materials can be printed together to form a unified, cohesive structure. This approach is compatible with any support bath that enables diffusion of UNION crosslinkers. Both matrix-adherent human corneal mesenchymal stromal cells and non-matrix-adherent human induced pluripotent stem cell-derived neural progenitor spheroids were printed with UNION bioinks. The cells retained high viability and expressed characteristic phenotypic markers after printing. Thus, UNION bioinks are a versatile strategy to expand the toolkit of customizable materials available for 3D bioprinting.
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http://dx.doi.org/10.1002/adfm.202007983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888563PMC
February 2021

Engineering Aligned Skeletal Muscle Tissue Using Decellularized Plant-Derived Scaffolds.

ACS Biomater Sci Eng 2020 05 6;6(5):3046-3054. Epub 2020 Apr 6.

To achieve organization and function, engineered tissues require a scaffold that supports cell adhesion, alignment, growth, and differentiation. For skeletal muscle tissue engineering, decellularization has been an approach for fabricating 3D scaffolds that retain biological architecture. While many decellularization approaches are focused on utilizing animal muscle as the starting material, decellularized plants are a potential source of highly structured cellulose-rich scaffolds. Here, we assessed the potential for a variety of decellularized plant scaffolds to promote mouse and human muscle cell alignment and differentiation. After decellularizing a range of fruits and vegetables, we identified the green-onion scaffold to have appropriate surface topography for generating highly confluent and aligned C2C12 and human skeletal muscle cells (HSMCs). The topography of the green-onion cellulose scaffold contained a repeating pattern of grooves that are approximately 20 μm wide by 10 μm deep. The outer white section of the green onion had a microstructure that guided C2C12 cell differentiation into aligned myotubes. Quantitative analysis of C2C12 and HSMC alignment revealed an almost complete anisotropic organization compared to 2D isotropic controls. Our results demonstrate that the decellularized green onion cellulose scaffolds, particularly from the outer white bulb segment, provide a simple and low-cost substrate to engineer aligned human skeletal muscle.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00058DOI Listing
May 2020

FRESH 3D Bioprinting a Full-Size Model of the Human Heart.

ACS Biomater Sci Eng 2020 11 23;6(11):6453-6459. Epub 2020 Oct 23.

Department of Biomedical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213, United States.

Recent advances in embedded three-dimensional (3D) bioprinting have expanded the design space for fabricating geometrically complex tissue scaffolds using hydrogels with mechanical properties comparable to native tissues and organs in the human body. The advantage of approaches such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) printing is the ability to embed soft biomaterials in a thermoreversible support bath at sizes ranging from a few millimeters to centimeters. In this study, we were able to expand this printable size range by FRESH bioprinting a full-size model of an adult human heart from patient-derived magnetic resonance imaging (MRI) data sets. We used alginate as the printing biomaterial to mimic the elastic modulus of cardiac tissue. In addition to achieving high print fidelity on a low-cost printer platform, FRESH-printed alginate proved to create mechanically tunable and suturable models. This demonstrates that large-scale 3D bioprinting of soft hydrogels is possible using FRESH and that cardiac tissue constructs can be produced with potential future applications in surgical training and planning.
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http://dx.doi.org/10.1021/acsbiomaterials.0c01133DOI Listing
November 2020

Hierarchical Machine Learning for High-Fidelity 3D Printed Biopolymers.

ACS Biomater Sci Eng 2020 12 20;6(12):7021-7031. Epub 2020 Nov 20.

Department of Biomedical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213, United States.

A hierarchical machine learning (HML) framework is presented that uses a small dataset to learn and predict the dominant build parameters necessary to print high-fidelity 3D features of alginate hydrogels. We examine the 3D printing of soft hydrogel forms printed with the freeform reversible embedding of suspended hydrogel method based on a CAD file that isolated the single-strand diameter and shape fidelity of printed alginate. Combinations of system variables ranging from print speed, flow rate, ink concentration to nozzle diameter were systematically varied to generate a small dataset of 48 prints. Prints were imaged and scored according to their dimensional similarity to the CAD file, and high print fidelity was defined as prints with less than 10% error from the CAD file. As a part of the HML framework, statistical inference was performed, using the least absolute shrinkage and selection operator to find the dominant variables that drive the error in the final prints. Model fit between the system parameters and print score was elucidated and improved by a parameterized middle layer of variable relationships which showed good performance between the predicted and observed data ( = 0.643). Optimization allowed for the prediction of build parameters that gave rise to high-fidelity prints of the measured features. A trade-off was identified when optimizing for the fidelity of different features printed within the same construct, showing the need for complex predictive design tools. A combination of known and discovered relationships was used to generate process maps for the 3D bioprinting designer that show error minimums based on the chosen input variables. Our approach offers a promising pathway toward scaling 3D bioprinting by optimizing print fidelity via learned build parameters that reduce the need for iterative testing.
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http://dx.doi.org/10.1021/acsbiomaterials.0c00755DOI Listing
December 2020

Fibronectin-based nanomechanical biosensors to map 3D surface strains in live cells and tissue.

Nat Commun 2020 11 18;11(1):5883. Epub 2020 Nov 18.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA.

Mechanical forces are integral to cellular migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution with minimal perturbation in living sytems. Here, we fabricate, calibrate, and test a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to the surface of cells and tissues to measure the magnitude, direction, and strain dynamics from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultra-thin FN lattice-mesh with spatial resolution tailored by adjusting the width and spacing of the lattice from 2-100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrates 2D and 3D surface strain tracking during mechanical deformation of known materials and is validated with finite element modeling. Analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles highlights the NMBS's ability to dynamically track microscopic tensile and compressive strains across diverse biological systems where forces guide structure and function.
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http://dx.doi.org/10.1038/s41467-020-19659-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675982PMC
November 2020

3D Printing Silicone Elastomer for Patient-Specific Wearable Pulse Oximeter.

Adv Healthc Mater 2020 08 16;9(15):e1901735. Epub 2020 Jun 16.

Department of Biomedical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA, 15213, USA.

Commercial pulse oximeters are used clinically to measure heart rate and blood oxygen saturation and traditionally made from rigid materials. However, these devices are unsuitable for continuous monitoring due to poor fit and mechanical mismatch. Soft materials that match the elastic properties of biological tissue provide improved comfort and signal-to-noise but typically require molding to manufacture, limiting the speed and ease of customizing for patient-specific anatomy. Here, freeform reversible embedding (FRE) 3D printing is used to create polydimethylsiloxane (PDMS) elastomer cuffs for use on the hand and foot. FRE enables printing liquid PDMS prepolymer in 3D geometries within a sacrificial hydrogel bath that provides support during cure. This serves as proof-of-concept for fabricating patient-specific pulse oximeters with pressure sensing, termed P -wearable. A sizing analysis establishes dimensional accuracy of FRE-printed PDMS compared to anatomical computer-aided design models. The P -wearable successfully outputs photoplethysmography (PPG) and pressure amplitude signals wirelessly to a tablet in real time and the PPG is used to calculate heart rate, blood oxygen content, and activity state. The results establish that FRE printing of PDMS can be used to fabricate patient-specific wearable devices and measure heart rate and blood oxygenation on par with commercial devices.
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http://dx.doi.org/10.1002/adhm.201901735DOI Listing
August 2020

Extracellular Matrix Structure and Composition in the Early Four-Chambered Embryonic Heart.

Cells 2020 01 24;9(2). Epub 2020 Jan 24.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

During embryonic development, the heart undergoes complex morphogenesis from a liner tube into the four chambers consisting of ventricles, atria and valves. At the same time, the cardiomyocytes compact into a dense, aligned, and highly vascularized myocardium. The extracellular matrix (ECM) is known to play an important role in this process but understanding of the expression and organization remains incomplete. Here, we performed 3D confocal imaging of ECM in the left ventricle and whole heart of embryonic chick from stages Hamburger-Hamilton 28-35 (days 5-9) as an accessible model of heart formation. First, we observed the formation of a fibronectin-rich, capillary-like networks in the myocardium between day 5 and day 9 of development. Then, we focused on day 5 prior to vascularization to determine the relative expression of fibronectin, laminin, and collagen type IV. Cardiomyocytes were found to uniaxially align prior to vascularization and, while the epicardium contained all ECM components, laminin was reduced, and collagen type IV was largely absent. Quantification of fibronectin revealed highly aligned fibers with a mean diameter of ~500 nm and interfiber spacing of ~3 µm. These structural parameters (volume, spacing, fiber diameter, length, and orientation) provide a quantitative framework to describe the organization of the embryonic ECM.
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http://dx.doi.org/10.3390/cells9020285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072588PMC
January 2020

Organ-on-e-chip: Three-dimensional self-rolled biosensor array for electrical interrogations of human electrogenic spheroids.

Sci Adv 2019 08 23;5(8):eaax0729. Epub 2019 Aug 23.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

Cell-cell communication plays a pivotal role in coordination and function of biological systems. Three-dimensional (3D) spheroids provide venues to explore cellular communication for tissue development and drug discovery, as their 3D architecture mimics native in vivo microenvironments. Cellular electrophysiology is a prevalent signaling paradigm for studying electroactive cells. Currently, electrophysiological studies do not provide direct, multisite, simultaneous investigation of tissues in 3D. In this study, 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field-effect transistors or passive microelectrodes were implemented to interface human cardiac spheroids in 3D. The arrays provided continuous and stable multiplexed recordings of field potentials with high sensitivity and spatiotemporal resolution, supported with simultaneous calcium imaging. Our approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment, such as arrhythmias.
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http://dx.doi.org/10.1126/sciadv.aax0729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707782PMC
August 2019

Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues.

Mater Today Chem 2019 Jun 14;12:61-70. Epub 2019 Jan 14.

Department of Biomedical Engineering, Case Western Reserve University.

Cell-laden microgels have been used as tissue building blocks to create three-dimensional (3D) tissues and organs. However, traditional assembly methods can not be used to fabricate functional tissue constructs with biomechanical and structural complexity. In this study, we present directed assembly of cell-laden dual-crosslinkable alginate microgels comprised of oxidized and methacrylated alginate (OMA). Cell-laden OMA microgels can be directly assembled into well-defined 3D shapes and structures under low-level ultraviolet light. Stem cell-laden OMA microgels can be successfully cryopreserved for long-term storage and on-demand applications, and the recovered encapsulated cells maintained equivalent viability and functionality to the freshly processed stem cells. Finally, we have successfully demonstrated that cell-laden microgels can be assembled into complicated 3D tissue structures via freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting. This highly innovative bottom-up strategy using FRESH 3D bioprinting of cell-laden OMA microgels, which are cryopreservable, provides a powerful and highly scalable tool for fabrication of customized and biomimetic 3D tissue constructs.
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http://dx.doi.org/10.1016/j.mtchem.2018.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377241PMC
June 2019

Large volume syringe pump extruder for desktop 3D printers.

HardwareX 2018 Apr 12;3:49-61. Epub 2018 Feb 12.

Department of Materials Science & Engineering, Carnegie Mellon University, United States.

Syringe pump extruders are required for a wide range of 3D printing applications, including bioprinting, embedded printing, and food printing. However, the mass of the syringe becomes a major challenge for most printing platforms, requiring compromises in speed, resolution and/or volume. To address these issues, we have designed a syringe pump large volume extruder (LVE) that is compatible with low-cost, open source 3D printers, and herein demonstrate its performance on a PrintrBot Simple Metal. Key aspects of the LVE include: (1) it is open source and compatible with open source hardware and software, making it inexpensive and widely accessible to the 3D printing community, (2) it utilizes a standard 60 mL syringe as its ink reservoir, effectively increasing print volume of the average bioprinter, (3) it is capable of retraction and high speed movements, and (4) it can print fluids using nozzle diameters as small as 100 µm, enabling the printing of complex shapes/objects when used in conjunction with the freeform reversible embedding of suspended hydrogels (FRESH) 3D printing method. Printing performance of the LVE is demonstrated by utilizing alginate as a model biomaterial ink to fabricate parametric CAD models and standard calibration objects.
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http://dx.doi.org/10.1016/j.ohx.2018.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258044PMC
April 2018

Natural Biomaterials for Corneal Tissue Engineering, Repair, and Regeneration.

Adv Healthc Mater 2018 08 29;7(16):e1701434. Epub 2018 May 29.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA.

Corneal blindness is a major cause of vision loss, estimated to affect over 10 million people worldwide. Once impaired through clouding or shape change, the best treatment option for restoring vision is corneal transplantation using full or partial thickness cadaveric grafts. However, donor corneas are globally limited and face rejection and graft failure, similar to other transplanted organs. Thus, there is a need for viable alternatives to donor corneas in order to increase supply, reduce rejection, and to minimize variability in tissue quality. To address this, researchers have developed new materials and strategies to tissue engineer full or partial thickness cornea grafts in order to repair, regenerate, or replace the diseased cornea. This progress report first reviews the anatomy and physiology of the cornea to frame the biological requirements and discuss the injuries and diseases that necessitate the need fortransplantation, as well as the requirements for a suitable donor tissue alternative. This is followed by recent progress using naturally derived biomaterials including silk, collagen, amniotic membranes, and decellularized corneas. Finally, remaining challenges in the field as they relate to the biomaterials discussed are identified, and the future research directions that should result in further advances in restoring corneal vision are highlighted.
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http://dx.doi.org/10.1002/adhm.201701434DOI Listing
August 2018

Graphene Microelectrode Arrays for Electrical and Optical Measurements of Human Stem Cell-Derived Cardiomyocytes.

Cell Mol Bioeng 2018 Oct 4;11(5):407-418. Epub 2018 May 4.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213 USA.

Introduction: Cell-cell communication plays a pivotal role in biological systems' coordination and function. Electrical properties have been linked to specification and differentiation of stem cells into targeted progeny, such as neurons and cardiomyocytes. Currently, there is a critical need in developing new ways to complement fluorescent indicators, such as Ca-sensitive dyes, for direct electrophysiological measurements of cells and tissue. Here, we report a unique transparent and biocompatible graphene-based electrical platform that enables electrical and optical investigation of human embryonic stem cell-derived cardiomyocytes' (hESC-CMs) intracellular processes and intercellular communication.

Methods: Graphene, a honeycomb sp hybridized two-dimensional carbon lattice, was synthesized using low pressure chemical vapor deposition system, and was tested for biocompatibility. Au and graphene microelectrode arrays (MEAs) were fabricated using well-established microfabrication methods. Au and graphene MEAs were interfaced with hESC-CMs to perform both optical and electrical recordings.

Results: Optical imaging and Raman spectroscopy confirmed the presence of monolayer graphene. Viability assay showed biocompatibility of graphene. Electrochemical characterization proved graphene's functional activity. Nitric acid treatment further enhanced the electrochemical properties of graphene. Graphene electrodes' transparency enabled both optical and electrical recordings from hESC-CMs. Graphene MEA detected changes in beating frequency and field potential duration upon β-adrenergic receptor agonist treatment.

Conclusion: The transparent graphene platform enables the investigation of both intracellular and intercellular communication processes and will create new avenues for bidirectional communication (sensing and stimulation) with electrically active tissues and will set the ground for investigations reported diseases such as Alzheimer, Parkinson's disease and arrhythmias.
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http://dx.doi.org/10.1007/s12195-018-0525-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816697PMC
October 2018

Expert-guided optimization for 3D printing of soft and liquid materials.

PLoS One 2018 5;13(4):e0194890. Epub 2018 Apr 5.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

Additive manufacturing (AM) has rapidly emerged as a disruptive technology to build mechanical parts, enabling increased design complexity, low-cost customization and an ever-increasing range of materials. Yet these capabilities have also created an immense challenge in optimizing the large number of process parameters in order achieve a high-performance part. This is especially true for AM of soft, deformable materials and for liquid-like resins that require experimental printing methods. Here, we developed an expert-guided optimization (EGO) strategy to provide structure in exploring and improving the 3D printing of liquid polydimethylsiloxane (PDMS) elastomer resin. EGO uses three steps, starting first with expert screening to select the parameter space, factors, and factor levels. Second is a hill-climbing algorithm to search the parameter space defined by the expert for the best set of parameters. Third is expert decision making to try new factors or a new parameter space to improve on the best current solution. We applied the algorithm to two calibration objects, a hollow cylinder and a five-sided hollow cube that were evaluated based on a multi-factor scoring system. The optimum print settings were then used to print complex PDMS and epoxy 3D objects, including a twisted vase, water drop, toe, and ear, at a level of detail and fidelity previously not obtained.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194890PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886457PMC
July 2018

Biohybrid actuators for robotics: A review of devices actuated by living cells.

Sci Robot 2017 11 29;2(12). Epub 2017 Nov 29.

The BioRobotics Institute, Scuola Superiore Sant'Anna, Pontedera, Pisa, Italy.

Actuation is essential for artificial machines to interact with their surrounding environment and to accomplish the functions for which they are designed. Over the past few decades, there has been considerable progress in developing new actuation technologies. However, controlled motion still represents a considerable bottleneck for many applications and hampers the development of advanced robots, especially at small length scales. Nature has solved this problem using molecular motors that, through living cells, are assembled into multiscale ensembles with integrated control systems. These systems can scale force production from piconewtons up to kilonewtons. By leveraging the performance of living cells and tissues and directly interfacing them with artificial components, it should be possible to exploit the intricacy and metabolic efficiency of biological actuation within artificial machines. We provide a survey of important advances in this biohybrid actuation paradigm.
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http://dx.doi.org/10.1126/scirobotics.aaq0495DOI Listing
November 2017

Measuring the Poisson's Ratio of Fibronectin Using Engineered Nanofibers.

Sci Rep 2017 10 17;7(1):13413. Epub 2017 Oct 17.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA.

The extracellular matrix (ECM) is a fibrillar protein-based network, the physical and chemical properties of which can influence a multitude of cellular processes. Despite having an important role in cell and tissue signaling, a complete chemo-mechanical characterization of ECM proteins such as fibronectin (FN) is lacking. In this study, we engineered monodisperse FN nanofibers using a surface-initiated assembly technique in order to provide new insight into the elastic behavior of this material over large deformations. FN nanofibers were patterned on surfaces in a pre-stressed state and when released from the surface underwent rapid contraction. We found that the FN nanofibers underwent 3.3-fold and 9-fold changes in length and width, respectively, and that the nanofiber volume was conserved. Volume was also conserved following uniaxial extension of the FN nanofibers of ~2-fold relative to the patterned state. This data suggests that the FN networks we engineered formed an incompressible material with a Poisson's ratio of ~0.5. While the Poisson's ratio of cells and other biological materials are widely estimated as 0.5, our experimental results demonstrate that for FN networks this is a reasonable approximation.
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http://dx.doi.org/10.1038/s41598-017-13866-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645409PMC
October 2017

Patterning on Topography for Generation of Cell Culture Substrates with Independent Nanoscale Control of Chemical and Topographical Extracellular Matrix Cues.

Curr Protoc Cell Biol 2017 Jun 19;75:10.23.1-10.23.25. Epub 2017 Jun 19.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania.

The cell microenvironment plays an important role in many biological processes, including development and disease progression. Key to this is the extracellular matrix (ECM), a complex biopolymer network serving as the primary insoluble signaling network for physical, chemical, and mechanical cues. In vitro, the ability to engineer the ECM at the micro- and nanoscales is a critical tool to systematically interrogate the influence of ECM properties on cellular responses. Specifically, both topographical and chemical surface patterning has been shown to direct cell alignment and tissue architecture on biomaterial surfaces, however, it has proven challenging to independently control these surface properties. This protocol describes a method termed Patterning on Topography (PoT) to engineer 2D nanopatterns of ECM proteins onto topographically complex substrates, which enables independent control of physical and chemical surface properties. Applications include interrogation of fundamental cell-surface interactions and engineering interfaces that can direct cell and/or tissue function. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpcb.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548430PMC
June 2017

Stretch-dependent changes in molecular conformation in fibronectin nanofibers.

Biomater Sci 2017 Jul;5(8):1629-1639

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA. and Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein that plays an important role in a wide range of biological processes including embryonic development, wound healing, and fibrosis. Recent evidence has demonstrated that FN is mechanosensitive, where the application of force induces conformational changes within the FN molecule to expose otherwise cryptic binding domains. However, it has proven technically challenging to dynamically monitor how the nanostructure of FN fibers changes as a result of force-induced extension, due in part to the inherent complexity of FN networks within tissue and cell-generated extracellular matrix (ECM). This has limited our understanding of FN matrix mechanobiology and the complex bi-directional signaling between cells and the ECM, and de novo FN fiber fabrication strategies have only partially addressed this. Towards addressing this need, we have developed a modified surface-initiated assembly (SIA) technique to engineer FN nanofibers that we can uniaxially stretch to >7-fold extensions and subsequently immobilize them in the stretched state for high resolution atomic force microscopy (AFM) imaging. Using this approach, we analyzed how the nanostructure of FN molecules within the nanofibers changed with stretch. In fully contracted FN nanofibers, we observed large, densely packed, isotropically-oriented nodules. With intermediate extension, uniaxially-aligned fibrillar regions developed and nodules became progressively smaller. At high extension, the nanostructure consisted of highly aligned fibrils with small nodules in a beads-on-a-string arrangement. In summary, we have established a methodology to uniaxially stretch FN fibers and monitor changes in nanostructure using AFM. Our results provide new insight into how FN fiber extension can affect the morphology of the constituent FN molecules.
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http://dx.doi.org/10.1039/c7bm00370fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549851PMC
July 2017

Scaffold-free tissue engineering of functional corneal stromal tissue.

J Tissue Eng Regen Med 2018 01 31;12(1):59-69. Epub 2017 May 31.

Department of Ophthalmology, University of Pittsburgh, PA, USA.

Blinding corneal scarring is predominately treated with allogeneic graft tissue; however, there is a worldwide shortage of donor tissue leaving millions in need of therapy. Human corneal stromal stem cells (CSSC) have been shown produce corneal tissue when cultured on nanofibre scaffolding, but this tissue cannot be readily separated from the scaffold. In this study, scaffold-free tissue engineering methods were used to generate biomimetic corneal stromal tissue constructs that can be transplanted in vivo without introducing the additional variables associated with exogenous scaffolding. CSSC were cultured on substrates with aligned microgrooves, which directed parallel cell alignment and matrix organization, similar to the organization of native corneal stromal lamella. CSSC produced sufficient matrix to allow manual separation of a tissue sheet from the grooved substrate. These constructs were cellular and collagenous tissue sheets, approximately 4 μm thick and contained extracellular matrix molecules typical of corneal tissue including collagen types I and V and keratocan. Similar to the native corneal stroma, the engineered corneal tissues contained long parallel collagen fibrils with uniform diameter. After being transplanted into mouse corneal stromal pockets, the engineered corneal stromal tissues became transparent, and the human CSSCs continued to express human corneal stromal matrix molecules. Both in vitro and in vivo, these scaffold-free engineered constructs emulated stromal lamellae of native corneal stromal tissues. Scaffold-free engineered corneal stromal constructs represent a novel, potentially autologous, cell-generated, biomaterial with the potential for treating corneal blindness. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432418PMC
January 2018

3D Printing PDMS Elastomer in a Hydrophilic Support Bath via Freeform Reversible Embedding.

ACS Biomater Sci Eng 2016 Oct 4;2(10):1781-1786. Epub 2016 May 4.

Department of Biomedical Engineering and Department of Materials Science and Engineering, Carnegie Mellon University , 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213 United States.

Polydimethylsiloxane (PDMS) elastomer is used in a wide range of biomaterial applications including microfluidics, cell culture substrates, flexible electronics, and medical devices. However, it has proved challenging to 3D print PDMS in complex structures due to its low elastic modulus and need for support during the printing process. Here we demonstrate the 3D printing of hydrophobic PDMS prepolymer resins within a hydrophilic Carbopol gel support via freeform reversible embedding (FRE). In the FRE printing process, the Carbopol support acts as a Bingham plastic that yields and fluidizes when the syringe tip of the 3D printer moves through it, but acts as a solid for the PDMS extruded within it. This, in combination with the immiscibility of hydrophobic PDMS in the hydrophilic Carbopol, confines the PDMS prepolymer within the support for curing times up to 72 h while maintaining dimensional stability. After printing and curing, the Carbopol support gel releases the embedded PDMS prints by using phosphate buffered saline solution to reduce the Carbopol yield stress. As proof-of-concept, we used Sylgard 184 PDMS to 3D print linear and helical filaments via continuous extrusion and cylindrical and helical tubes via layer-by-layer fabrication. Importantly, we show that the 3D printed tubes were manifold and perfusable. The results demonstrate that hydrophobic polymers with low viscosity and long cure times can be 3D printed using a hydrophilic support, expanding the range of biomaterials that can be used in additive manufacturing. Further, by implementing the technology using low cost open-source hardware and software tools, the FRE printing technique can be rapidly implemented for research applications.
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http://dx.doi.org/10.1021/acsbiomaterials.6b00170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059754PMC
October 2016

Engineered Basement Membranes for Regenerating the Corneal Endothelium.

Adv Healthc Mater 2016 11 10;5(22):2942-2950. Epub 2016 Oct 10.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213, USA.

Basement membranes are protein-rich extracellular matrices (ECM) that are essential for epithelial and endothelial tissue structure and function. Aging and disease cause changes in the physical properties and ECM composition of basement membranes, which has spurred research to develop methods to repair and/or regenerate these tissues. An area of critical clinical need is the cornea, where failure of the endothelium leads to stromal edema and vision loss. Here, an engineered basement membrane (EBM) is developed that consists of a dense layer of collagen IV and/or laminin ≈5-10 nm thick, created using surface-initiated assembly, conformally attached to a collagen I film. These EBMs are used to engineer a corneal endothelium (CE) that mimics the structure of Descemet's membrane with a thin stromal layer, toward use as a graft for lamellar keratoplasty. Results show that bovine and human CE cells form confluent monolayers on the EBM, express ZO-1 at the cell-cell borders, and achieve a density of ≈1600 cells mm for 28 and 14 d, respectively. These results demonstrate that the technique is capable of fabricating EBMs with structural and compositional properties that mimic native basement membranes and that EBM may be a suitable carrier for engineering transplant quality CE grafts.
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http://dx.doi.org/10.1002/adhm.201600488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354171PMC
November 2016

Understanding the Role of ECM Protein Composition and Geometric Micropatterning for Engineering Human Skeletal Muscle.

Ann Biomed Eng 2016 06 16;44(6):2076-89. Epub 2016 Mar 16.

Regenerative Biomaterials and Therapeutics Group, Department of Biomedical Engineering, Carnegie Mellon University, 700 Technology Dr., Pittsburgh, PA, 15219, USA.

Skeletal muscle lost through trauma or disease has proven difficult to regenerate due to the challenge of differentiating human myoblasts into aligned, contractile tissue. To address this, we investigated microenvironmental cues that drive myoblast differentiation into aligned myotubes for potential applications in skeletal muscle repair, organ-on-chip disease models and actuators for soft robotics. We used a 2D in vitro system to systematically evaluate the role of extracellular matrix (ECM) protein composition and geometric patterning for controlling the formation of highly aligned myotubes. Specifically, we analyzed myotubes differentiated from murine C2C12 cells and human skeletal muscle derived cells (SkMDCs) on micropatterned lines of laminin compared to fibronectin, collagen type I, and collagen type IV. Results showed that laminin supported significantly greater myotube formation from both cells types, resulting in greater than twofold increase in myotube area on these surfaces compared to the other ECM proteins. Species specific differences revealed that human SkMDCs uniaxially aligned over a wide range of micropatterned line dimensions, while C2C12s required specific line widths and spacings to do the same. Future work will incorporate these results to engineer aligned human skeletal muscle tissue in 2D for in vitro applications in disease modeling, drug discovery and toxicity screening.
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http://dx.doi.org/10.1007/s10439-016-1592-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880540PMC
June 2016

Spontaneous Helical Structure Formation in Laminin Nanofibers.

J Mater Chem B 2015 Oct 21;3(40):7993-8000. Epub 2015 Aug 21.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15219, USA ; Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, 15219, USA.

Laminin is a cross-shaped heterotrimer composed of three polypeptides chains that assembles into an insoluble extracellular matrix (ECM) network as part of the basement membrane, serving a vital role in many processes such as embryonic development, differentiation, and muscle and nerve regeneration. Here we engineered monodisperse laminin nanofibers using a surface-initiated assembly technique in order to investigate how changes in protein composition affect formation and structure of the network. Specifically, we compared laminin 111 with varying degrees of purity and with and without entactin to determine whether these changes alter biophysical properties. All the laminin types were reproducibly patterned as 200 μm long, 20 μm wide nanofibers that were successfuly released during surface-initiated assembly into solution. All nanofibers contracted upon release, and while initial lengths were identical, lengths of released fibers depended on the laminin type. Uniquely, the laminin 111 at high purity (>95%) and without entactin spontaneouly formed helical nanofibers at greater than 90%. Atomic force microscopy revealed that the nanofiber contraction was associated with a change in nanostructure from fibrillar to nodular, suggestive of refolding of laminin molecules into a globular-like conformation. Further, for the high purity laminin that formed helices, the density of the laminin at the edges of the nanofiber was higher than in the middle, providing a possible origin for the differential pre-stress driving the helix formation. Together, these results show that variation in the purity of laminin 111 and presence of entactin can have significant impact on the biophysical properties of the assembled protein networks. This highlights the fact that our understanding of protein assembly and function is still incomplete and that cell-free, in vitro assays can provide unique insights into the ECM.
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http://dx.doi.org/10.1039/C5TB01003ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675171PMC
October 2015

Biological Soft Robotics.

Authors:
Adam W Feinberg

Annu Rev Biomed Eng 2015 ;17:243-65

Department of Biomedical Engineering and Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213; email:

In nature, nanometer-scale molecular motors are used to generate force within cells for diverse processes from transcription and transport to muscle contraction. This adaptability and scalability across wide temporal, spatial, and force regimes have spurred the development of biological soft robotic systems that seek to mimic and extend these capabilities. This review describes how molecular motors are hierarchically organized into larger-scale structures in order to provide a basic understanding of how these systems work in nature and the complexity and functionality we hope to replicate in biological soft robotics. These span the subcellular scale to macroscale, and this article focuses on the integration of biological components with synthetic materials, coupled with bioinspired robotic design. Key examples include nanoscale molecular motor-powered actuators, microscale bacteria-controlled devices, and macroscale muscle-powered robots that grasp, walk, and swim. Finally, the current challenges and future opportunities in the field are addressed.
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http://dx.doi.org/10.1146/annurev-bioeng-071114-040632DOI Listing
September 2016

Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels.

Sci Adv 2015 Oct 23;1(9):e1500758. Epub 2015 Oct 23.

Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA. ; Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

We demonstrate the additive manufacturing of complex three-dimensional (3D) biological structures using soft protein and polysaccharide hydrogels that are challenging or impossible to create using traditional fabrication approaches. These structures are built by embedding the printed hydrogel within a secondary hydrogel that serves as a temporary, thermoreversible, and biocompatible support. This process, termed freeform reversible embedding of suspended hydrogels, enables 3D printing of hydrated materials with an elastic modulus <500 kPa including alginate, collagen, and fibrin. Computer-aided design models of 3D optical, computed tomography, and magnetic resonance imaging data were 3D printed at a resolution of ~200 μm and at low cost by leveraging open-source hardware and software tools. Proof-of-concept structures based on femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains were mechanically robust and recreated complex 3D internal and external anatomical architectures.
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http://dx.doi.org/10.1126/sciadv.1500758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646826PMC
October 2015
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