Mechanical Characterization and Shape Optimization of Fascicle-Like 3D Skeletal Muscle Tissues Contracted with Electrical and Optical Stimuli.
ABSTRACT: In this study, we present a quantitative approach to construct effective 3D muscle tissues through shape optimization and load impedance matching with electrical and optical stimulation. We have constructed long, thin, fascicle-like skeletal muscle tissue and optimized its form factor through mechanical characterization. A new apparatus was designed and built, which allowed us to measure force-displacement characteristics with diverse load stiffnesses. We have found that (1) there is an optimal form factor that maximizes the muscle stress, (2) the energy transmitted to the load can be maximized with matched load stiffness, and (3) optical stimulation using channelrhodopsin2 in the muscle tissue can generate a twitch force as large as its electrical counterpart for well-developed muscle tissue. Using our tissue construct method, we found that an optimal initial diameter of 500??m outperformed tissues using 250??m by more than 60% and tissues using 760??m by 105%. Using optimal load stiffness, our tissues have generated 12 pJ of energy per twitch at a peak generated stress of 1.28?kPa. Additionally, the difference in optically stimulated twitch performance versus electrically stimulated is a function of how well the overall tissue performs, with average or better performing strips having less than 10% difference. The unique mechanical characterization method used is generalizable to diverse load conditions and will be used to match load impedance to muscle tissue impedance for a wide variety of applications.
Project description:Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3?V/mm amplitude, 4?ms width, and 1?Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.
Project description:Integrative approaches to studying the coupled dynamics of skeletal muscles with their loads while under neural control have focused largely on questions pertaining to the postural and dynamical stability of animals and humans. Prior studies have focused on how the central nervous system actively modulates muscle mechanical impedance to generate and stabilize motion and posture. However, the question of whether muscle impedance properties can be neurally modulated to create favorable mechanical energetics, particularly in the context of periodic tasks, remains open. Through muscle stiffness tuning, we hypothesize that a pair of antagonist muscles acting against a common load may produce significantly more power synergistically than individually when impedance matching conditions are met between muscle and load. Since neurally modulated muscle stiffness contributes to the coupled muscle-load stiffness, we further anticipate that power-optimal oscillation frequencies will occur at frequencies greater than the natural frequency of the load. These hypotheses were evaluated computationally by applying optimal control methods to a bilinear muscle model, and also evaluated through in vitro measurements on frog Plantaris longus muscles acting individually and in pairs upon a mass-spring-damper load. We find a 7-fold increase in mechanical power when antagonist muscles act synergistically compared to individually at a frequency higher than the load natural frequency. These observed behaviors are interpreted in the context of resonance tuning and the engineering notion of impedance matching. These findings suggest that the central nervous system can adopt strategies to harness inherent muscle impedance in relation to external loads to attain favorable mechanical energetics.
Project description:A biopsy needle with electrical impedance sensor array based on stainless steel microelectrodes (EIS needle) was developed for real-time four electrode measurement and multi-spot sensing of tissues during the biopsy process. The sensor performance was characterized by using saline solutions with various concentrations, which proved accurate, stable and reliable electrical impedance measurement. The capability of impedance-based tissue sensing was verified by the conductivity measurement of agarose hydrogel based phantom mimicking cancer tissue. Furthermore, multi-spot impedance sensing during needle insertion was demonstrated using porcine meat with muscle and fat layers, which exhibited a clear discrimination between different types of tissues. Also, the electrical impedance difference between normal and fatty livers of mouse model was measured by the EIS needle. We could successfully demonstrate that the EIS needle can provide localized and accurate characterization of biological tissues at the needle tip.
Project description:Dystrophic muscle is particularly susceptible to eccentric contraction-induced injury. We tested the hypothesis that electrical impedance myography (EIM) can detect injury induced by maximal-force lengthening contractions.We induced injury in the quadriceps of wild-type (WT) and dystrophic (mdx) mice with eccentric contractions using an established model.mdx quadriceps had significantly greater losses in peak twitch and tetany compared with losses in WT quadriceps. Injured muscle showed a significant increase in EIM characteristic frequency in both WT (177?±?7.7%) and mdx (167?±?7.8%) quadriceps. EIM also revealed decreased extracellular resistance for both WT and mdx quadriceps after injury.Our results show overall agreement between muscle function and EIM measurements of injured muscle, indicating that EIM is a viable tool to assess injury in dystrophic muscle. Muscle Nerve 56: E85-E94, 2017.
Project description:<h4>Background</h4>The structural elements of the vascular wall, namely, extracellular matrix and smooth muscle cells (SMCs), contribute to the overall stiffness of the vessel. In this study, we examined the crosslinking-dependent and crosslinking-independent roles of tissue transglutaminase (TG2) in vascular function and stiffness.<h4>Methods and results</h4>SMCs were isolated from the aortae of TG2-/- and wild-type (WT) mice. Cell adhesion was examined by using electrical cell-substrate impedance sensing and PicoGreen assay. Cell motility was examined using a Boyden chamber assay. Cell proliferation was examined by electrical cell-substrate impedance sensing and EdU incorporation assays. Cell micromechanics were studied using magnetic torsion cytometry and spontaneous nanobead tracer motions. Aortic mechanics were examined by tensile testing. Vasoreactivity was studied by wire myography. SMCs from TG2-/- mice had delayed adhesion, reduced motility, and accelerated de-adhesion and proliferation rates compared with those from WT. TG2-/- SMCs were stiffer and displayed fewer cytoskeletal remodeling events than WT. Collagen assembly was delayed in TG2-/- SMCs and recovered with adenoviral transduction of TG2. Aortic rings from TG2-/- mice were less stiff than those from WT; stiffness was partly recovered by incubation with guinea pig liver TG2 independent of crosslinking function. TG2-/- rings showed augmented response to phenylephrine-mediated vasoconstriction when compared with WT. In human coronary arteries, vascular media and plaque, high abundance of fibronectin expression, and colocalization with TG2 were observed.<h4>Conclusions</h4>TG2 modulates vascular function/tone by altering SMC contractility independent of its crosslinking function and contributes to vascular stiffness by regulating SMC proliferation and matrix remodeling.
Project description:Development of tissue-engineered hearts for treatment of myocardial infarction or biologic pacemakers has been hindered by the production of mostly arrhythmic or in-synergistic constructs. Electrical stimulation (ES) of these constructs has been shown to produce tissues with greater twitch force and better adrenergic response. To further our understanding of the mechanisms underlying the effect of ES, we fabricated a bioreactor capable of delivering continuous or intermittent waveforms of various types to multiple constructs simultaneously. In this study, we examined the effect of an intermittent biphasic square wave on our artificial heart muscle (AHM) composed of neonatal rat cardiac cells and fibrin gel. Twitch forces, spontaneous contraction rates, biopotentials, gene expression profiles, and histologic observations were examined for the ES protocol over a 12 day culture period. We demonstrate improved consistency between samples for twitch force and contraction rate, and higher normalized twitch force amplitudes for electrically stimulated AHMs. Improvements in electrophysiology within the AHM were noted by higher conduction velocities and lower latency in electrical response for electrically stimulated AHMs. Genes expressing key electrophysiologic and structural markers peaked at days 6 and 8 of culture, only a few days after the initiation of ES. These results may be used for optimization strategies to establish protocols for producing AHMs capable of replacing damaged heart tissue in either a contractile or electrophysiologic capacity. Optimized AHMs can lead to alternative treatments to heart failure and alleviate the limited donor supply crisis.
Project description:Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death that often goes undetected in the general population. HCM is also prevalent in patients with cardio-facio-cutaneous syndrome (CFCS), which is a genetic disorder characterized by aberrant signaling in the RAS/MAPK signaling cascade. Understanding the mechanisms of HCM development in such RASopathies may lead to novel therapeutic strategies, but relevant experimental models of the human condition are lacking. Therefore, the objective of this study was to develop the first 3D human engineered cardiac tissue (hECT) model of HCM. The hECTs were created using human cardiomyocytes obtained by directed differentiation of induced pluripotent stem cells derived from a patient with CFCS due to an activating BRAF mutation. The mutant myocytes were directly conjugated at a 3:1 ratio with a stromal cell population to create a tissue of defined composition. Compared to healthy patient control hECTs, BRAF-hECTs displayed a hypertrophic phenotype by culture day 6, with significantly increased tissue size, twitch force, and atrial natriuretic peptide (ANP) gene expression. Twitch characteristics reflected increased contraction and relaxation rates and shorter twitch duration in BRAF-hECTs, which also had a significantly higher maximum capture rate and lower excitation threshold during electrical pacing, consistent with a more arrhythmogenic substrate. By culture day 11, twitch force was no longer different between BRAF and wild-type hECTs, revealing a temporal aspect of disease modeling with tissue engineering. Principal component analysis identified diastolic force as a key factor that changed from day 6 to day 11, supported by a higher passive stiffness in day 11 BRAF-hECTs. In summary, human engineered cardiac tissues created from BRAF mutant cells recapitulated, for the first time, key aspects of the HCM phenotype, offering a new in vitro model for studying intrinsic mechanisms and screening new therapeutic approaches for this lethal form of heart disease.
Project description:We introduce a novel method to form 3D biomimetic tissues from a droplet of a cell-extracellular matrix (ECM) mixture on a sensor stage and to quantify tissue force and stiffness as a function of time under optical microscopes. This method exploits advances in micro-nano fabrication and capillarity for self-assembly and self-alignment of tissues on the stage. It allows simultaneous investigation of the microstructure of the tissue in situ while its mechanical response is quantified, thus linking tissue biophysics with physiology and revealing structural-functional properties of 3D tissues. We demonstrate the functionality of the stage by studying the mechanical behavior of different cell-collagen mixtures under mechanical, chemical and electrical stimulation. This includes force evolution in cell-free collagen during curing, myotubes differentiated from muscle cell-collagen/Matrigel ECM subjected to electrical stimulation, and fibroblast-collagen tissue subjected to cancer cell conditioned media (CM) and a Rho-kinase inhibitor, Y27632. Muscle contraction decreases with increasing frequency of electrical stimulation, and fibroblasts respond to CM by increasing contractility for a short time and completely relax in the presence of Y27632 but restore force with Y27632 washout.
Project description:A model of myocardial electromechanics is suggested. It combines modified and simplified versions of previously published models of cardiac electrophysiology, excitation-contraction coupling, and mechanics. The mechano-calcium and mechano-electrical feedbacks, including the strain-dependence of the propagation velocity of the action potential, are also accounted for. The model reproduces changes in the twitch amplitude and Ca2+-transients upon changes in muscle strain including the slow response. The model also reproduces the Bowditch effect and changes in the twitch amplitude and duration upon changes in the interstimulus interval, including accelerated relaxation at high stimulation frequency. Special efforts were taken to reduce the stiffness of the differential equations of the model. As a result, the equations can be integrated numerically with a relatively high time step making the model suitable for multiscale simulation of the human heart and allowing one to study the impact of myocardial mechanics on arrhythmias.
Project description:Most striated muscles generate steady contractile tension when activated, but some preparations, notably cardiac myocytes and slow-twitch fibers, may show spontaneous oscillatory contractions (SPOC) at low levels of activation. We have provided what we believe is new evidence that SPOC is a property of the contractile system at low actin-myosin affinity, whether caused by a thin-filament regulatory system or by other means. We present a quantitative single-sarcomere model for isotonic SPOC in skeletal muscle with three basic ingredients: i), actin and myosin filaments initially in partial overlap, ii), stretch activation by length-dependent changes in the lattice spacing, and iii), viscoelastic passive tension. Modeling examples are given for slow-twitch and fast-twitch fibers, with periods of 10 s and 4 s respectively. Isotonic SPOC occurs in a narrow domain of parameter values, with small minimum and maximum values for actin-myosin affinity, a minimum amount of passive tension, and a maximum transient response rate that explains why SPOC is favored in slow-twitch fibers. The model also predicts the contractile, relaxed and SPOC phases as a function of phosphate and ADP levels. The single-sarcomere model can also be applied to a whole fiber under auxotonic and fixed-end conditions if the remaining sarcomeres are treated as a viscoelastic load. Here the model predicts an upper limit for the load stiffness that leads to SPOC; this limit lies above the equivalent loads expected from the rest of the fiber.