Mechanical Characterization and Shape Optimization of Fascicle-Like 3D Skeletal Muscle Tissues Contracted with Electrical and Optical Stimuli

Here we present a quantitative approach to constructing 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 their 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 a) there is an optimal form factor that maximizes the muscle stress, b) the energy transmitted to the load can be maximized with matched load stiffness, and c) optical stimulation using channelrhodopsin2 in the muscle tissue can generate twitch force as large as its electrical counterpart for well developed muscle tissue. Using our tissue construct method we found an optimal initial diameter of 500 microns outperformed tissues using 250 microns by more than 60% and tissues using 760 microns by 105%. Using an optimal load stiffness, our tissues have generated 12 pJ of energy at a peak generated stress of 1.28 kPa. Additionally, the difference in optically stimulated twitch performance vs. 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.National Science Foundation (U.S.) (Grant No. CBET-0939511)Singapore-MIT Alliance for Research and Technology (BioSyM IRG)National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular System

Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions.National Science Foundation (U.S.). Office of Emerging Frontiers in Research and Innovation (grant #0735997

Formation and optogenetic control of engineered 3D skeletal muscle bioactuators

Densely arrayed skeletal myotubes are activated individually and as a group using precise optical stimulation with high spatiotemporal resolution. Skeletal muscle myoblasts are genetically encoded to express a light-activated cation channel, Channelrhodopsin-2, which allows for spatiotemporal coordination of a multitude of skeletal myotubes that contract in response to pulsed blue light. Furthermore, ensembles of mature, functional 3D muscle microtissues have been formed from the optogenetically encoded myoblasts using a high-throughput device. The device, called “skeletal muscle on a chip”, not only provides the myoblasts with controlled stress and constraints necessary for muscle alignment, fusion and maturation, but also facilitates the measurement of forces and characterization of the muscle tissue. We measured the specific static and dynamic stresses generated by the microtissues and characterized the morphology and alignment of the myotubes within the constructs. The device allows testing of the effect of a wide range of parameters (cell source, matrix composition, microtissue geometry, auxotonic load, growth factors and exercise) on the maturation, structure and function of the engineered muscle tissues in a combinatorial manner. Our studies integrate tools from optogenetics and microelectromechanical systems (MEMS) technology with skeletal muscle tissue engineering to open up opportunities to generate soft robots actuated by a multitude of spatiotemporally coordinated 3D skeletal muscle microtissues.National Science Foundation (U.S.) (Science and Technology Center—Emergent Behaviors of Integrated Cellular Systems (EBICS) grant No. CBET-0939511)National Institutes of Health (U.S.) (EB00262)National Science Foundation (U.S.) (GM74048)National Science Foundation (U.S.) (HL90747)National Institute for Biomedical Imaging and Bioengineering (U.S.) (RESBIO, Integrapted Technologies for Polymeric Biomaterial)University of Pennsylvania. Center for Engineering Cells and RegenerationSingapore-MIT Alliance for Research and Technolog

Investigational chemotherapy and novel pharmacokinetic mechanisms for the treatment of breast cancer brain metastases

In women, breast cancer is the most common cancer diagnosis and second most common cause of cancer death. More than half of breast cancer patients will develop metastases to the bone, liver, lung, or brain. Breast cancer brain metastases (BCBM) confers a poor prognosis, as current therapeutic options of surgery, radiation, and chemotherapy rarely significantly extend life and are considered palliative. Within the realm of chemotherapy, the last decade has seen an explosion of novel chemotherapeutics involving targeting agents and unique dosage forms. We provide a historical overview of BCBM chemotherapy, review the mechanisms of new agents such as poly-ADP ribose polymerase inhibitors, cyclin-dependent kinase 4/6 inhibitors, phosphatidyl inositol 3-kinaseinhibitors, estrogen pathway antagonists for hormone-receptor positive BCBM; tyrosine kinase inhibitors, antibodies, and conjugates for HER2+ BCBM; repurposed cytotoxic chemotherapy for triple negative BCBM; and the utilization of these new agents and formulations in ongoing clinical trials. The mechanisms of novel dosage formulations such as nanoparticles, liposomes, pegylation, the concepts of enhanced permeation and retention, and drugs utilizing these concepts involved in clinical trials are also discussed. These new treatments provide a promising outlook in the treatment of BCBM

Dynamic Modeling of Cell Migration and Spreading Behaviors on Fibronectin Coated Planar Substrates and Micropatterned Geometries

An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling, actin motor activity, and lamellipodia protrusion is developed for predicting cell spreading and migration behaviors. This work is motivated by two experimental works: (1) cell migration on 2-D substrates under various fibronectin concentrations and (2) cell spreading on 2-D micropatterned geometries. These works suggest (1) cell migration speed takes a maximum at a particular ligand density (~1140 molecules/µm2) and (2) that strong traction forces at the corners of the patterns may exist due to combined effects exerted by actin stress fibers (SFs). The integrative model of this paper successfully reproduced these experimental results and indicates the mechanism of cell migration and spreading. In this paper, the mechanical structure of the cell is modeled as having two elastic membranes: an outer cell membrane and an inner nuclear membrane. The two elastic membranes are connected by SFs, which are extended from focal adhesions on the cortical surface to the nuclear membrane. In addition, the model also includes ventral SFs bridging two focal adhesions on the cell surface. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bond to ligands on the ECM surface, activate SFs, and form focal adhesions. The relationship between the cell migration speed and fibronectin concentration agrees with existing experimental data for Chinese hamster ovary (CHO) cell migrations on fibronectin coated surfaces. In addition, the integrated model is validated by showing persistent high stress concentrations at sharp geometrically patterned edges. This model will be used as a predictive model to assist in design and data processing of upcoming microfluidic cell migration assays

Nonlinear, large-strain PZT actuators using controlled structural buckling

Buckling is a highly nonlinear and singular phenomenon in thin beams, and is usually an undesired characteristic that must be prevented from occurring in engineered systems. Buckling, however, can be a useful mechanism for gaining extremely large displacement amplification, since a tiny displacement in the axial direction of the beam may lead to a large defection in the middle of the beam. This paper presents a novel large-strain piezoelectric actuator exploiting the buckling of a structure with imbedded PZT stacks. Although the free displacement of a PZT stack is only 0.1% of the stack length, the buckling mechanism, controlled with an effective algorithm and strategically placed redirecting stiffness, can produce a large bi-polar displacement that is approximately 150 times larger than the original PZT displacement. Furthermore, the structural buckling produces a pronounced nonlinearity in output impedance; the effective stiffness viewed from the output port varies as a function of output displacement, which can be a useful property for those applications where actuator stiffness needs to vary

Dynamic Performance of Nonlinear 100X Displacement Amplification Piezoelectric Actuator

The dynamic frequency response of a nonlinear piezoelectric amplification mechanism capable of over 150 fold displacement amplification is presented. Research to amplify the displacement of piezoelectric actuators has included flexure based approaches that utilize geometric configuration, and has included frequency based approaches that utilize resonance and small steps to contribute to a full motion. The dynamic operation of the actuator presented here utilizes the benefits from both of these methods. The geometry allows for great displacement amplification, and operation within a specific frequency band allows for the exploitation of a nonlinear singularity. The actuator has three distinct modes of dynamic operation, one of which achieves significant displacement by utilizing momentum to pass through a singular configuration. A nonlinear model, linear models for each frequency response mode, and multiple prototypes are presented. This actuator operating in the high displacement frequency band is promising as an input actuator for stepping mechanisms. Copyright © 2010 by ASME

A PZT Array Actuator Using Buckling Strain Amplification and Preload Mechanisms

Displacement amplification mechanisms have been a topic of research for piezoelectric actuators for decades to overcome their significantly small strain, but still utilize their high power density, force, and efficiency This paper further analyzes a nonlinear buckling mechanism to improve its efficiency, defined as the ratio of mechanical work output of the buckling actuator to the mechanical work output of the PZT actuator, as well as, employing two methods, preload and loading conditions, that improve its work output per cycle. This is accomplished by running a numerical analysis of the geometry of the flexure joints in the buckling mechanism which found a maximum mechanical efficiency of 48%. The preload is applied using shape memory alloy wire to exploit the low stiffness of the super elastic regime; which in turn allows a larger work output due to a loading condition supplied by a novel gear design. Finally a prototype was fabricated to provide a baseline of comparison against these concepts. Topics: Actuators, Buckling, Mechanism

Towards the Development of Optogenetically-Controlled Skeletal Muscle Actuators

Engineered skeletal muscle tissue has the potential to be used as dual use actuator and stress-bearing material providing numerous degrees of freedom and with significant active stress generation. To exploit the potential features, however, technologies must be established to generate mature muscle strips that can be controlled with high fidelity. Here, we present a method for creating mature 3-D skeletal muscle tissues that contract in response to optical activation stimuli. The muscle strips are fascicle-like, consisting of several mm-long multinucleate muscle cells bundled together. We have found that applying a tension to the fascicle-like muscle tissue promotes maturation of the muscle. The fascicle-like muscle tissue is controlled with high spatiotemporal resolution based on optogenetic coding. The mouse myoblasts C2C12 were transfected with Channelrhodopsin-2 to enable light (∼470 nm) to control muscle contraction. The 3D muscle tissue not only twitches in response to an impulse light beam, but also exhibits a type of tetanus, a prolonged contraction of continuous stimuli, for the first time. In the following, the materials and culturing method used for 3D muscle generation is presented, followed by experimental results of muscle constructs and optogenetic control of the 3D muscle tissue.National Science Foundation (U.S.)National Science Foundation (U.S.). Science and Technology Center. Emergent Behaviors of Integrated Cellular Systems (grant no. NSF STC-0902396)Singapore-MIT Alliance. BioSystems and Micromechanics (BioSyM) Inter-Disciplinary Research Grou

Distributed Live Muscle Actuators Controlled by Optical Stimuli

A multi degree of freedom skeletal muscle system stimulated via optical control is presented. These millimeter-scale, optically excitable 3D skeletal muscle bio-actuators are created by culturing genetically modified precursory muscle cells that are activated with light: optogenetics. These muscle bio-actuators are networked together to create a distributed muscle system. Muscle systems can manipulate loads having no fixed joint. These types of loads include shoulders, the mouth, and the jaw. Topics: Actuators , Muscl