Ryan A. Koppes

Multifunctional fibers for simultaneous optical, electrical and chemical interrogation of neural circuits in vivo

Brain function depends on simultaneous electrical, chemical and mechanical signaling at the cellular level. This multiplicity has confounded efforts to simultaneously measure or modulate these diverse signals in vivo. Here we present fiber probes that allow for simultaneous optical stimulation, neural recording and drug delivery in behaving mice with high resolution. These fibers are fabricated from polymers by means of a thermal drawing process that allows for the integration of multiple materials and interrogation modalities into neural probes. Mechanical, electrical, optical and microfluidic measurements revealed high flexibility and functionality of the probes under bending deformation. Long-term in vivo recordings, optogenetic stimulation, drug perturbation and analysis of tissue response confirmed that our probes can form stable brain-machine interfaces for at least 2 months. We expect that our multifunctional fibers will permit more detailed manipulation and analysis of neural circuits deep in the brain of behaving animals than achievable before.

Robust neurite extension following exogenous electrical stimulation within single walled carbon nanotube-composite hydrogels

UNLABELLED The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: (1) nanofillers influence neurite extension and (2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10-100-μg/mL) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-μg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. STATEMENT OF SIGNIFICANCE Novel biomedical devices delivering electrical stimulation are being developed to mitigate symptoms of Parkinsons, treat drug-resistant depression, control movement or enhance verve regeneration. Carbon nanotubes and other novel materials are being explored for novel nano-neuro devices based on their unique properties. Neuronal growth on carbon nanotubes has been studied in 2D since the early 2000s demonstrating increased outgrowth, synapse formation and network activity. In this work, single-walled carbon nanotubes were selected as one possible electrically-conductive material, dispersed within a 3D hydrogel containing primary neurons; extending previous 2D work to 3D to evaluate outgrowth within nanomaterial composites with electrical stimulation. This is the first study to our knowledge that stimulates neurons in 3D composite nanomaterial-laden hydrogels. Examination of electrically conductive biomaterials may serve to promote regrowth following injury or in long term stimulation.

Optogenetic control of nerve growth

Due to the limited regenerative ability of neural tissue, a diverse set of biochemical and biophysical cues for increasing nerve growth has been investigated, including neurotrophic factors, topography, and electrical stimulation. In this report, we explore optogenetic control of neurite growth as a cell-specific alternative to electrical stimulation. By investigating a broad range of optical stimulation parameters on dorsal root ganglia (DRGs) expressing channelrhodopsin 2 (ChR2), we identified conditions that enhance neurite outgrowth by three-fold as compared to unstimulated or wild-type (WT) controls. Furthermore, optogenetic stimulation of ChR2 expressing DRGs induces directional outgrowth in WT DRGs co-cultured within a 10 mm vicinity of the optically sensitive ganglia. This observed enhancement and polarization of neurite growth was accompanied by an increased expression of neural growth and brain derived neurotrophic factors (NGF, BDNF). This work highlights the potential for implementing optogenetics to drive nerve growth in specific cell populations.

The Mechanical Properties of Drosophila Jump Muscle Expressing Wild-Type and Embryonic Myosin Isoforms

Transgenic Drosophila are highly useful for structure-function studies of muscle proteins. However, our ability to mechanically analyze transgenically expressed mutant proteins in Drosophila muscles has been limited to the skinned indirect flight muscle preparation. We have developed a new muscle preparation using the Drosophila tergal depressor of the trochanter (TDT or jump) muscle that increases our experimental repertoire to include maximum shortening velocity (V(slack)), force-velocity curves and steady-state power generation; experiments not possible using indirect flight muscle fibers. When transgenically expressing its wild-type myosin isoform (Tr-WT) the TDT is equivalent to a very fast vertebrate muscle. TDT has a V(slack) equal to 6.1 +/- 0.3 ML/s at 15 degrees C, a steep tension-pCa curve, isometric tension of 37 +/- 3 mN/mm(2), and maximum power production at 26% of isometric tension. Transgenically expressing an embryonic myosin isoform in the TDT muscle increased isometric tension 1.4-fold, but decreased V(slack) 50% resulting in no significant difference in maximum power production compared to Tr-WT. Drosophila expressing embryonic myosin jumped <50% as far as Tr-WT that, along with comparisons to frog jump muscle studies, suggests fast muscle shortening velocity is relatively more important than high tension generation for Drosophila jumping.

Force enhancement in lengthening contractions of cat soleus muscle in situ: transient and steady-state aspects

Force enhancement (FE) associated with lengthening is a well‐accepted phenomenon of active skeletal muscle, but the underlying mechanism(s) remain unknown. Similar to force depression (FD) following active shortening, the mechanism of FE may be attributed, at least in part, to cross‐bridge kinetics. To examine this relationship, a post hoc analysis was performed on the transient force relaxation phase of previous in‐situ FE experiments in soleus muscle‐tendon units of anesthetized cats. For each muscle (n = 8), nine eccentric lengthenings (3 amplitudes, 3 velocities) were performed while tetanically stimulated (3T at 30 Hz, 3× α motorneuron, 35 ± 1°C). To determine transient aspects of FE, the period immediately following stretching was fit with an exponential decay function (R2 > 0.95). Statistical analyses revealed that total steady‐state FE (FESS) increased with stretching amplitude and applied mechanical work. A positive relationship was observed between the active FESS and rate of force decay (k), indicating that a kinetic mechanism may explain active FE. However, for all muscles and stretch conditions, there was no correlation between the total amount of FESS and rate of decay. Therefore, FE cannot be explained solely by an active FE mechanism involving the interaction of actin and myosin. Rather, these findings suggest a combination of underlying mechanisms, including a kinetic mechanism for active FE, contributions of a passive elastic element, and possibly an activatable passive component operating outside of actin–myosin cross‐bridging. Moreover, this transient analysis identifies that FE is not simply the opposite of FD, and its underlying mechanism(s) cannot simply be the opposite in nature.

The influence of specimen thickness and alignment on the material and failure properties of electrospun polycaprolactone nanofiber mats.

Electrospinning is a versatile fabrication technique that has been recently expanded to create nanofibrous structures that mimic ECM topography. Like many materials, electrospun constructs are typically characterized on a smaller scale, and scaled up for various applications. This established practice is based on the assumption that material properties, such as toughness, failure stress and strain, are intrinsic to the material, and thus will not be influenced by specimen geometry. However, we hypothesized that the material and failure properties of electrospun nanofiber mats vary with specimen thickness. To test this, we mechanically characterized polycaprolactone (PCL) nanofiber mats of three different thicknesses in response to constant rate elongation to failure. To identify if any observed thickness-dependence could be attributed to fiber alignment, such as the effects of fiber reorientation during elongation, these tests were performed in mats with either random or aligned nanofiber orientation. Contrary to our hypothesis, the failure strain was conserved across the different thicknesses, indicating similar maximal elongation for specimens of different thickness. However, in both the aligned and randomly oriented groups, the ultimate tensile stress, short-range modulus, yield modulus, and toughness all decreased with increasing mat thickness, thereby indicating that these are not intrinsic material properties. These findings have important implications in engineered scaffolds for fibrous and soft tissue applications (e.g., tendon, ligament, muscle, and skin), where such oversights could result in unwanted laxity or reduced resistance to failure.

Engineering Cellular Fibers for Musculoskeletal Soft Tissues Using Directed Self-Assembly

Engineering strategies guided by developmental biology may enhance and accelerate in vitro tissue formation for tissue engineering and regenerative medicine applications. In this study, we looked toward embryonic tendon development as a model system to guide our soft tissue engineering approach. To direct cellular self-assembly, we utilized laser micromachined, differentially adherent growth channels lined with fibronectin. The micromachined growth channels directed human dermal fibroblast cells to form single cellular fibers, without the need for a provisional three-dimensional extracellular matrix or scaffold to establish a fiber structure. Therefore, the resulting tissue structure and mechanical characteristics were determined solely by the cells. Due to the self-assembly nature of this approach, the growing fibers exhibit some key aspects of embryonic tendon development, such as high cellularity, the rapid formation (within 24 h) of a highly organized and aligned cellular structure, and the expression of cadherin-11 (indicating direct cell-to-cell adhesions). To provide a dynamic mechanical environment, we have also developed and characterized a method to apply precise cyclic tensile strain to the cellular fibers as they develop. After an initial period of cellular fiber formation (24 h postseeding), cyclic strain was applied for 48 h, in 8-h intervals, with tensile strain increasing from 0.7% to 1.0%, and at a frequency of 0.5 Hz. Dynamic loading dramatically increased cellular fiber mechanical properties with a nearly twofold increase in both the linear region stiffness and maximum load at failure, thereby demonstrating a mechanism for enhancing cellular fiber formation and mechanical properties. Tissue engineering strategies, designed to capture key aspects of embryonic development, may provide unique insight into accelerated maturation of engineered replacement tissue, and offer significant advances for regenerative medicine applications in tendon, ligament, and other fibrous soft tissues.

Restoring the sense of touch

Flexible circuitry mimics the way skin transduces pressure signals [Also see Report by Tee et al.] Amputation of damaged tissue is one of the oldest surgical techniques, reaching prevalence in the 16th century (1). Improved emergency medicine has allowed more individuals to survive traumatic injuries as amputees, but prosthetic limbs remain the only means to restore any degree of function to these patients. Inadequate tactile feedback is a leading shortcoming of prosthetic limbs, but for artificial hands, just a few sensors that relay grasp pressure back to the user can provide the functionality needed to enable delicate tasks (2). In addition to improved motor control, sensory stimulation could alleviate phantom limb pain, which affects ~80% of amputees (2). On page 314 of this issue, Tee et al. (3) report a Digital Tactile System (“DiTact”) based on a low-power flexible organic transistor circuit that transduces pressure stimuli into oscillating signals like those generated by skin mechanoreceptors. Mammalian skin is a multilayered viscoelastic material that can stretch up to ~125% from its resting dimensions without any apparent loss in sensitivity to external stimuli such as pressure or temperature. Replicating skin mechanical and functional properties remains an elusive engineering challenge. Meanwhile, the rapidly expanding field of flexible electronics has made substantial strides, and complex circuits can now be produced on soft substrates. Advances in microcontact printing, inkjet deposition, and organic electronics have delivered stretchable and flexible, wearable, and even epidermal sensors (4–6).

A new experimental model to study force depression: the Drosophila jump muscle

Force depression (FD) is a decrease in isometric force following active muscle shortening. Despite being well characterized experimentally, its underlying mechanism remains unknown. To develop a new, genetically manipulatable experimental model that would greatly improve our ability to study the underlying mechanism(s) of FD, we tested the Drosophila jump muscle for classical FD behavior. Steady-state force generation following active shortening decreased by 2, 8, and 11% of maximum isometric force with increasing shortening amplitudes of 5, 10, and 20% of optimal fiber length, and decreased by 11, 8, and 5% with increasing shortening velocities of 4, 20, and 200% of optimal fiber length per second. These steady-state FD (FDSS) characteristics of Drosophila jump muscle mimic those observed in mammalian skeletal muscle. A double exponential fit of transient force recovery following shortening identified two separate phases of force recovery: a rapid initial force redevelopment, and a slower recovery toward steady state. This analysis showed the slower rate of force redevelopment to be inversely proportional to the amount of FDSS, while the faster rate did not correlate with FDSS. This suggests that the mechanism behind the slower, most likely cross-bridge cycling rate, influences the amount of FDSS. Thus the jump muscle, when coupled with the genetic mutability of its sarcomere proteins, offers a unique and powerful experimental model to explore the underlying mechanism behind FD.

A new experimental model for force enhancement: steady-state and transient observations of the Drosophila jump muscle.

The increase in steady-state force after active lengthening in skeletal muscle, termed force enhancement (FE), has been observed for nearly one century. Although demonstrated experimentally at various structural levels, the underlying mechanism(s) remain unknown. We recently showed that the Drosophila jump muscle is an ideal model for investigating mechanisms behind muscle physiological properties, because its mechanical characteristics, tested thus far, duplicate those of fast mammalian skeletal muscles, and Drosophila has the advantage that it can be more easily genetically modified. To determine if Drosophila would be appropriate to investigate FE, we performed classic FE experiments on this muscle. Steady-state FE (FESS), following active lengthening, increased by 3, 7, and 12% of maximum isometric force, with increasing stretch amplitudes of 5, 10, and 20% of optimal fiber length (FLOPT), yet was similar for stretches across increasing stretch velocities of 4, 20, and 200% FLOPT/s. These FESS characteristics of the Drosophila jump muscle closely mimic those observed previously. Jump muscles also displayed typical transient FE characteristics. The transient force relaxation following active stretch was fit with a double exponential, yielding two phases of force relaxation: a fast initial relaxation of force, followed by a slower recovery toward steady state. Our analyses identified a negative correlation between the slow relaxation rate and FESS, indicating that there is likely an active component contributing to FE, in addition to a passive component. Herein, we have established the Drosophila jump muscle as a new and genetically powerful experimental model to investigate the underlying mechanism(s) of FE.