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1.
J Biomed Mater Res A ; 2024 May 16.
Article in English | MEDLINE | ID: mdl-38752415

ABSTRACT

Tissue engineering can provide in vitro models for drug testing, disease modeling, and perhaps someday, tissue/organ replacements. For building 3D heart tissue, the alignment of cardiac cells or cardiomyocytes (CMs) is important in generating a synchronously contracting tissue. To that end, researchers have generated several fabrication methods for building heart tissue, but direct comparisons of pros and cons using the same cell source is lacking. Here, we derived cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) and compare the assembly of these cells using three fabrication methods: cardiospheres, muscle rings, and muscle strips. All three protocols successfully generated compacted tissue comprised of hiPSC-derived CMs stable for at least 2 weeks. The percentage of aligned cells was greatest in the muscle strip (55%) and the muscle ring (50%) compared with the relatively unaligned cardiospheres (35%). The iPSC-derived CMs within the muscle strip also exhibited the greatest elongation, with elongation factor at 2.0 compared with 1.5 for the muscle ring and 1.2 for the cardiospheres. This is the first direct comparison of various fabrication techniques using the same cell source.

2.
Biomaterials ; 302: 122317, 2023 11.
Article in English | MEDLINE | ID: mdl-37717406

ABSTRACT

Damage that affects large volumes of skeletal muscle tissue can severely impact health, mobility, and quality-of-life. Efforts to restore muscle function by implanting tissue engineered muscle grafts at the site of damage have demonstrated limited restoration of force production. Various forms of mechanical and biochemical stimulation have been shown to have a potentially beneficial impact on graft maturation, vascularization, and innervation. However, these approaches yield unpredictable and incomplete recovery of functional mobility. Here we show that targeted actuation of implanted grafts, via non-invasive transcutaneous light stimulation of optogenetic engineered muscle, restores motor function to levels similar to healthy mice 2 weeks post-injury. Furthermore, we conduct phosphoproteomic analysis of actuated engineered muscle in vivo and in vitro to show that repeated muscle contraction alters signaling pathways that play key roles in skeletal muscle contractility, adaptation to injury, neurite growth, neuromuscular synapse formation, angiogenesis, and cytoskeletal remodeling. Our study uncovers changes in phosphorylation of several proteins previously unreported in the context of muscle contraction, revealing promising mechanisms for leveraging actuated muscle grafts to restore mobility after volumetric muscle loss.


Subject(s)
Muscular Diseases , Tissue Engineering , Mice , Animals , Muscle, Skeletal , Muscle Contraction/physiology , Prostheses and Implants
3.
Proc Natl Acad Sci U S A ; 119(35): e2200741119, 2022 08 30.
Article in English | MEDLINE | ID: mdl-36001689

ABSTRACT

The next robotics frontier will be led by biohybrids. Capable biohybrid robots require microfluidics to sustain, improve, and scale the architectural complexity of their core ingredient: biological tissues. Advances in microfluidics have already revolutionized disease modeling and drug development, and are positioned to impact regenerative medicine but have yet to apply to biohybrids. Fusing microfluidics with living materials will improve tissue perfusion and maturation, and enable precise patterning of sensing, processing, and control elements. This perspective suggests future developments in advanced biohybrids.


Subject(s)
Biomimetic Materials , Cells , Microfluidics , Robotics
4.
APL Bioeng ; 6(1): 010903, 2022 Mar.
Article in English | MEDLINE | ID: mdl-35274072

ABSTRACT

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell-cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the "black box" of living cells.

5.
Sci Adv ; 6(39)2020 09.
Article in English | MEDLINE | ID: mdl-32978160

ABSTRACT

Neurochemical dysregulation underlies many pathologies and can be monitored by measuring the composition of brain interstitial fluid (ISF). Existing in vivo tools for sampling ISF do not enable measuring large rare molecules, such as proteins and neuropeptides, and thus cannot generate a complete picture of the neurochemical connectome. Our micro-invasive platform, composed of a nanofluidic pump coupled to a membrane-free probe, enables sampling multiple neural biomarkers in parallel. This platform outperforms the state of the art in low-flow pumps by offering low volume control (single stroke volumes, <3 nl) and bidirectional fluid flow (<100 nl/min) with negligible dead volume (<30 nl) and has been validated in vitro, ex vivo, and in vivo in rodents. ISF samples (<1.5 µL) can be processed via liquid chromatography-tandem mass spectrometry. These label-free liquid biopsies of the brain could yield a deeper understanding of the onset, mechanism, and progression of diverse neural pathologies.


Subject(s)
Brain , Extracellular Fluid , Biomarkers/analysis , Extracellular Fluid/chemistry , Hydrogels , Specimen Handling
6.
Sci Adv ; 6(3): eaay0065, 2020 01.
Article in English | MEDLINE | ID: mdl-32010768

ABSTRACT

Triggerable materials capable of being degraded by selective stimuli stand to transform our capacity to precisely control biomedical device activity and performance while reducing the need for invasive interventions. Here, we describe the development of a modular and tunable light-triggerable hydrogel system capable of interfacing with implantable devices. We apply these materials to two applications in the gastrointestinal (GI) tract: a bariatric balloon and an esophageal stent. We demonstrate biocompatibility and on-demand triggering of the material in vitro, ex vivo, and in vivo. Moreover, we characterize performance of the system in a porcine large animal model with an accompanying ingestible LED. Light-triggerable hydrogels have the potential to be applied broadly throughout the GI tract and other anatomic areas. By demonstrating the first use of light-degradable hydrogels in vivo, we provide biomedical engineers and clinicians with a previously unavailable, safe, dynamically deliverable, and precise tool to design dynamically actuated implantable devices.


Subject(s)
Gastrointestinal Tract/physiology , Hydrogels/radiation effects , Light , Animals , Biocompatible Materials/pharmacology , Caco-2 Cells , Esophagus/physiology , HT29 Cells , Humans , Hydrogels/chemical synthesis , Stents , Swine
7.
Adv Mater ; 32(13): e1901969, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31271257

ABSTRACT

Precision medicine requires materials and devices that can sense and adapt to dynamic physiological and pathological conditions. This motivates the design and manufacture of biohybrid materials that mimic the responsive behaviors demonstrated by natural biological systems. Two parallel approaches to biohybrid design are presented-biomimetics and biointegration. Biohybrid hydrogels that mimic the form and function of natural materials, or that integrate living cells or bioactive moieties, can respond to a range of environmental stimuli in parallel, including heat, light, pH, hydration, enzymes, and electric, mechanical, and magnetic forces. A range of examples that illustrate the tremendous potential of this nascent discipline are presented, and ongoing technical challenges related to manufacturing, storage, transport, and external noninvasive control of these materials that will need to be overcome in the coming years are outlined. The ethical, educational, and regulatory challenges that will govern translation of biohybrid design into medical applications are also discussed. Personalized medical therapies that target the precise needs of patients are a critically needed and expanding market. Biohybrid design offers the unique ability to manufacture materials and devices that match the dynamic and patient-specific in vivo environment, promising to generate more effective and safe therapies that enable personalized care.


Subject(s)
Biomimetic Materials/therapeutic use , Biomimetics/methods , Precision Medicine/methods , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/therapeutic use , Biomimetic Materials/chemistry , Humans , Hydrogels/chemistry , Hydrogels/therapeutic use , Prostheses and Implants
9.
Tissue Eng Part A ; 25(13-14): 1023-1036, 2019 07.
Article in English | MEDLINE | ID: mdl-30412045

ABSTRACT

IMPACT STATEMENT: The ability to freeze, revive, and prolong the lifetime of tissue-engineered skeletal muscle without incurring any loss of function represents a significant advancement in the field of tissue engineering. Cryopreservation enables the efficient fabrication, storage, and shipment of these tissues. This in turn facilitates multidisciplinary collaboration between research groups, enabling advances in skeletal muscle regenerative medicine, organ-on-a-chip models of disease, drug testing, and soft robotics. Furthermore, the observation that freezing undifferentiated skeletal muscle enhances functional performance may motivate future studies developing stronger and more clinically relevant engineered muscle.


Subject(s)
Cryopreservation , Muscle, Skeletal/physiology , Tissue Engineering/methods , Animals , Biomechanical Phenomena , Cell Differentiation/drug effects , Cell Line , Cell Survival/drug effects , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Freezing , Leucine/analogs & derivatives , Leucine/pharmacology , Mice , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/drug effects , Muscle, Skeletal/ultrastructure , Proteolysis/drug effects , Time Factors
10.
Adv Healthc Mater ; 6(20)2017 Oct.
Article in English | MEDLINE | ID: mdl-28881469

ABSTRACT

The discipline of biological design has a relatively short history, but has undergone very rapid expansion and development over that time. This Progress Report outlines the evolution of this field from biomimicry to biofabrication to biohybrid systems' design, showcasing how each subfield incorporates bioinspired dynamic adaptation into engineered systems. Ethical implications of biological design are discussed, with an emphasis on establishing responsible practices for engineering non-natural or hypernatural functional behaviors in biohybrid systems. This report concludes with recommendations for implementing biological design into educational curricula, ensuring effective and responsible practices for the next generation of engineers and scientists.


Subject(s)
Biocompatible Materials/chemistry , Biomimetics , Tissue Engineering , Animals , Drug Carriers/chemistry , Hydrogels/chemistry , Microfluidics , Printing, Three-Dimensional
11.
Adv Healthc Mater ; 6(12)2017 Jun.
Article in English | MEDLINE | ID: mdl-28489332

ABSTRACT

A deeper understanding of biological materials and the design principles that govern them, combined with the enabling technology of 3D printing, has given rise to the idea of "building with biology." Using these materials and tools, bio-hybrid robots or bio-bots, which adaptively sense and respond to their environment, can be manufactured. Skeletal muscle bioactuators are developed to power these bio-bots, and an approach is presented to make them dynamically responsive to changing environmental loads and robustly resilient to induced damage. Specifically, since the predominant cause of skeletal muscle loss of function is mechanical damage, the underlying mechanisms of damage are investigated in vitro, and an in vivo inspired healing strategy is developed to counteract this damage. The protocol that is developed yields complete recovery of healthy tissue functionality within two days of damage, setting the stage for a more robust, resilient, and adaptive bioactuator technology than previously demonstrated. Understanding and exploiting the adaptive response behaviors inherent within biological systems in this manner is a crucial step forward in designing bio-hybrid machines that are broadly applicable to grand engineering challenges.


Subject(s)
Muscle, Skeletal/physiology , Optogenetics/methods , Tissue Engineering/methods , Wound Healing , Animals , Cell Line , Mice , Stress, Mechanical
12.
Nat Protoc ; 12(3): 519-533, 2017 03.
Article in English | MEDLINE | ID: mdl-28182019

ABSTRACT

Biological machines consisting of cells and biomaterials have the potential to dynamically sense, process, respond, and adapt to environmental signals in real time. As a first step toward the realization of such machines, which will require biological actuators that can generate force and perform mechanical work, we have developed a method of manufacturing modular skeletal muscle actuators that can generate up to 1.7 mN (3.2 kPa) of passive tension force and 300 µN (0.56 kPa) of active tension force in response to external stimulation. Such millimeter-scale biological actuators can be coupled to a wide variety of 3D-printed skeletons to power complex output behaviors such as controllable locomotion. This article provides a comprehensive protocol for forward engineering of biological actuators and 3D-printed skeletons for any design application. 3D printing of the injection molds and skeletons requires 3 h, seeding the muscle actuators takes 2 h, and differentiating the muscle takes 7 d.


Subject(s)
Biomimetics/instrumentation , Muscle, Skeletal/physiology , Animals , Biomechanical Phenomena , Cell Line , Electric Stimulation , Equipment Design , Humans , Mice , Printing, Three-Dimensional , Tissue Engineering
13.
Microsyst Nanoeng ; 3: 17015, 2017.
Article in English | MEDLINE | ID: mdl-31057862

ABSTRACT

A complex and functional living cellular system requires the interaction of one or more cell types to perform specific tasks, such as sensing, processing, or force production. Modular and flexible platforms for fabrication of such multi-cellular modules and their characterization have been lacking. Here, we present a modular cellular system, made up of multi-layered tissue rings containing integrated skeletal muscle and motor neurons (MNs) embedded in an extracellular matrix. The MNs were differentiated from mouse embryonic stem cells through the formation of embryoid bodies (EBs), which are spherical aggregations of cells grown in a suspension culture. The EBs were integrated into a tissue ring with skeletal muscle, which was differentiated in parallel, to create a co-culture amenable to both cell types. The multi-layered rings were then sequentially placed on a stationary three-dimensional-printed hydrogel structure resembling an anatomical muscle-tendon-bone organization. We demonstrate that the site-specific innervation of a group of muscle fibers in the multi-layered tissue rings allows for muscle contraction via chemical stimulation of MNs with glutamate, a major excitatory neurotransmitter in the mammalian nervous system, with the frequency of contraction increasing with glutamate concentration. The addition of tubocurarine chloride (a nicotinic receptor antagonist) halted the contractions, indicating that muscle contraction was MN induced. With a bio-fabricated system permitting controllable mechanical and geometric attributes in a range of length scales, our novel engineered cellular system can be utilized for easier integration of other modular "building blocks" in living cellular and biological machines.

14.
Sci Robot ; 2(12)2017 11 29.
Article in English | MEDLINE | ID: mdl-33157905

ABSTRACT

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.

15.
J Biol Eng ; 10: 10, 2016.
Article in English | MEDLINE | ID: mdl-27688801

ABSTRACT

BACKGROUND: The rapidly evolving discipline of biological and biomedical engineering requires adaptive instructional approaches that teach students to target and solve multi-pronged and ill-structured problems at the cutting edge of scientific research. Here we present a modular approach to designing a lab-based course in the emerging field of biofabrication and biological design, leading to a final capstone design project that requires students to formulate and test a hypothesis using the scientific method. RESULTS: Students were assessed on a range of metrics designed to evaluate the format of the course, the efficacy of the format for teaching new topics and concepts, and the depth of the contribution this course made to students training for biological engineering careers. The evaluation showed that the problem-based format of the course was well suited to teaching students how to use the scientific method to investigate and uncover the fundamental biological design rules that govern the field of biofabrication. CONCLUSIONS: We show that this approach is an efficient and effective method of translating emergent scientific principles from the lab bench to the classroom and training the next generation of biological and biomedical engineers for careers as researchers and industry practicians.

16.
Biomed Microdevices ; 18(3): 49, 2016 06.
Article in English | MEDLINE | ID: mdl-27215416

ABSTRACT

Multifunctional particles with distinct physiochemical phases are required by a variety of applications in biomedical engineering, such as diagnostic imaging and targeted drug delivery. This motivates the development of a repeatable, efficient, and customizable approach to manufacturing particles with spatially segregated bioactive moieties. This study demonstrates a stereolithographic 3D printing approach for designing and fabricating large arrays of biphasic poly (ethylene glycol) diacrylate (PEGDA) gel particles. The fabrication parameters governing the physical and biochemical properties of multi-layered particles are thoroughly investigated, yielding a readily tunable approach to manufacturing customizable arrays of multifunctional particles. The advantage in spatially organizing functional epitopes is examined by loading superparamagnetic iron oxide nanoparticles (SPIONs) and bovine serum albumin (BSA) in separate layers of biphasic PEGDA gel particles and examining SPION-induced magnetic resonance (MR) contrast and BSA-release kinetics. Particles with spatial segregation of functional moieties have demonstrably higher MR contrast and BSA release. Overall, this study will contribute significant knowledge to the preparation of multifunctional particles for use as biomedical tools.


Subject(s)
Hydrogels/chemistry , Particle Size , Polyethylene Glycols/chemistry , Printing, Three-Dimensional , Drug Delivery Systems , Equipment Design , Microscopy, Confocal , Nanoparticles/chemistry , Serum Albumin, Bovine
17.
Proc Natl Acad Sci U S A ; 113(13): 3497-502, 2016 Mar 29.
Article in English | MEDLINE | ID: mdl-26976577

ABSTRACT

Complex biological systems sense, process, and respond to their surroundings in real time. The ability of such systems to adapt their behavioral response to suit a range of dynamic environmental signals motivates the use of biological materials for other engineering applications. As a step toward forward engineering biological machines (bio-bots) capable of nonnatural functional behaviors, we created a modular light-controlled skeletal muscle-powered bioactuator that can generate up to 300 µN (0.56 kPa) of active tension force in response to a noninvasive optical stimulus. When coupled to a 3D printed flexible bio-bot skeleton, these actuators drive directional locomotion (310 µm/s or 1.3 body lengths/min) and 2D rotational steering (2°/s) in a precisely targeted and controllable manner. The muscle actuators dynamically adapt to their surroundings by adjusting performance in response to "exercise" training stimuli. This demonstration sets the stage for developing multicellular bio-integrated machines and systems for a range of applications.


Subject(s)
Muscle, Skeletal/physiology , Optogenetics/methods , Animals , Cell Line , Equipment Design , Finite Element Analysis , Locomotion , Mice , Muscle Contraction/physiology , Optogenetics/instrumentation , Printing, Three-Dimensional , Robotics/instrumentation , Robotics/methods , Time-Lapse Imaging , Tissue Engineering/instrumentation , Tissue Engineering/methods
18.
Adv Healthc Mater ; 5(5): 610-9, 2016 Mar 09.
Article in English | MEDLINE | ID: mdl-26696464

ABSTRACT

To gain a quantitative understanding of the way cells sense, process, and respond to dynamic environmental signals in real-time requires developing in vitro model systems that accurately replicate the 3D structure and function of native tissue. A high-resolution projection stereolithography apparatus (µSLA) capable of multimaterial and grayscale 3D patterning of cells and biomaterials at <5 µm resolution is presented. Murine cells (fibroblasts, myoblasts, endothelial, and bone marrow stromal cells) encapsulated within photosensitive hydrogels using the µSLA remain viable up to two weeks after fabrication. Harnessing the high-resolution fabrication capabilities of this machine, sub-millimeter scale angiogenic cell-encapsulating patches designed to promote targeted growth of neovasculature are printed, as assessed in vitro via enzyme-linked immunosorbent assay (ELISA) and in ovo via a chick chorioallantoic membrane assay (CAM). This application establishes the µSLA as an enabling technology that is widely adaptable to any application that requires high-resolution patterning of cells and cells signals. By providing an efficient and robust method of engineering microscale tissues with encapsulated cells, this apparatus has a range of applications including fundamental studies of extracellular matrix interactions, high throughput drug testing of physiologically relevant substitutes for native tissue, and programmable tissue engineering for applications in regenerative medicine.


Subject(s)
Bioprinting/methods , Neovascularization, Physiologic , Animals , Cell Survival/drug effects , Cells, Cultured , Chickens , Chorioallantoic Membrane/drug effects , Chorioallantoic Membrane/metabolism , Hydrogel, Polyethylene Glycol Dimethacrylate/pharmacology , Imaging, Three-Dimensional , Mice , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor A/metabolism
19.
Biotechnol Bioeng ; 112(4): 777-87, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25384798

ABSTRACT

In vitro models that recapitulate the liver's structural and functional complexity could prolong hepatocellular viability and function to improve platforms for drug toxicity studies and understanding liver pathophysiology. Here, stereolithography (SLA) was employed to fabricate hydrogel scaffolds with open channels designed for post-seeding and perfused culture of primary hepatocytes that form 3D structures in a bioreactor. Photopolymerizable polyethylene glycol-based hydrogels were fabricated coupled to chemically activated, commercially available filters (polycarbonate and polyvinylidene fluoride) using a chemistry that permitted cell viability, and was robust enough to withstand perfused culture of up to 1 µL/s for at least 7 days. SLA energy dose, photoinitiator concentrations, and pretreatment conditions were screened to determine conditions that maximized cell viability and hydrogel bonding to the filter. Multiple open channel geometries were readily achieved, and included ellipses and rectangles. Rectangular open channels employed for subsequent studies had final dimensions on the order of 350 µm by 850 µm. Cell seeding densities and flow rates that promoted cell viability were determined. Perfused culture of primary hepatocytes in hydrogel scaffolds in the presence of soluble epidermal growth factor (EGF) prolonged the maintenance of albumin production throughout the 7-day culture relative to 2D controls. This technique of bonding hydrogel scaffolds can be employed to fabricate soft scaffolds for a number of bioreactor configurations and applications.


Subject(s)
Hepatocytes/physiology , Hydrogel, Polyethylene Glycol Dimethacrylate , Organ Culture Techniques/methods , Tissue Scaffolds , Bioreactors , Cell Survival , Epidermal Growth Factor/metabolism , Humans
20.
Proc Natl Acad Sci U S A ; 111(28): 10125-30, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24982152

ABSTRACT

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼ 156 µm s(-1), which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design.


Subject(s)
Biomimetics , Bioprinting , Locomotion , Muscle, Skeletal , Animals , Cell Line , Collagen Type I/chemistry , Insulin-Like Growth Factor I/chemistry , Mice
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