Electromechanical Stimulation Research Articles

A Tympanic Piezo-Bioreactor Modulates Ion Channel-Associated Mechanosignaling to Stabilize Phenotype and Promote Tenogenesis in Human Tendon-Derived Cells.

Preserving the function of human tendon-derived cells (hTDCs) during cell expansion is a significant challenge in regenerative medicine. In this study, a non-genetic approach is introduced to control the differentiation of hTDCs using a newly developed tympanic bioreactor. The system mimics the functionality of the human tympanic membrane, employing a piezoelectrically tuned acoustic diaphragm made of polyvinylidene fluoride-co-trifluoroethylene and boron nitride nanotubes. The diaphragm is vibrationally actuated to deliver targeted electromechanical stimulation to hTDCs. The results demonstrate that the system effectively maintains the tendon-specific phenotype of hTDCs, even under conditions that typically induce nonspecific differentiation, such as osteogenesis. This stabilization is achieved by modulating integrin-mediated mechanosignaling via ion channel-regulated calcium activity, potentially by TREK-1 and PIEZO1, yet targeted studies are required for confirmation. Finally, the system sustains the activation of key differentiation pathways (bone morphogenetic protein, BMP) while downregulating osteogenesis-associated (mitogen-ctivated protein kinase, MAPK and wingless integrated, WNT) pathways and upregulating Focal Adhesion Kinase (FAK) signaling. This approach offers a finely tunable, dose-dependent control over hTDC differentiation, presenting significant potential for non-genetic approaches in cell therapy, tendon tissue engineering, and the regeneration of other mechanosensitive tissues.

Piezoelectric biomaterials with embedded ionic liquids for improved orthopedic interfaces through osseointegration and antibacterial dual characteristics

Orthopedic implant failures, primarily attributed to aseptic loosening and implant site infections, pose significant challenges to patient recovery and lead to revision surgeries. Combining piezoelectric materials with ionic liquids as interfaces for orthopedic implants presents an innovative approach to addressing both issues simultaneously. In this study, films of poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) incorporated with 1-ethyl-3-methylimidazolium hydrogen sulfate ([Emim][HSO4]) ionic liquid were developed. These films exhibited strong antibacterial properties, effectively reducing biofilm formation, thereby addressing implant-related infections. Furthermore, stem cell-based differentiation assays exposed the potential of the composite materials to induce osteogenesis. Interestingly, our findings also revealed the upregulation of calcium channel expression as a result of electromechanical stimulation, pointing to a mechanistic basis for the observed biological effects. This work highlights the potential of piezoelectric materials with ionic liquids to improve the longevity and biocompatibility of orthopedic implants. Offering dual-functionality for infection prevention and bone integration, these advancements hold significant potential for advancing orthopedic implant technologies and improving patient outcomes.

Molecular Insights into the Low Complexity Secreted Venom of Calliactis polypus.

Sea anemones are venomous animals that rely on their venom for prey capture, defense against predators, and intraspecific competition. Currently, comprehensive molecular and evolutionary analyses of the toxin repertoire for sea anemones are limited by a lack of proteomic data for most species. In this study, proteo-transcriptomic analysis was used to expand our knowledge of the proteinaceous components of sea anemone venom by determining the secreted venom proteome of Calliactis polypus. Electromechanical stimulation was used to obtain the secreted venom of C. polypus. We identified a low complexity proteome that was dominated by toxins with similarity to known neurotoxins, as well as six novel toxin candidates. The novel putative toxin candidates were found to be taxonomically restricted to species from the superfamily Metridioidea. Furthermore, the secreted venom of C. polypus had only three putative toxins in common with the venom of acontia from the same species and little similarity with the secreted venom of closely related species. Overall, this demonstrates that regionalized and lineage-specific variability in toxin abundance is common among sea anemone species. Moreover, the limited complexity of the toxin repertoire found in C. polypus supports the idea that peptide neurotoxins make up the dominant toxin arsenal found in the venom of sea anemones.

An electromechanical stimulation regulating model with flexoelectric effect of piezoelectric laminated micro-beam for cell bionic culture

Cell bionic culture requires the construction of cell growth microenvironments. In this paper, mechanical force and electrical stimulations are applied to the cells cultured on the surface of the piezoelectric laminated micro-beam driven by an excitation voltage. Based on the extended dielectric theory, the electromechanical microenvironment regulating model of the current piezoelectric laminated micro-beam is established. The variational principle is used to obtain the governing equations and boundary conditions. The differential quadrature method and the iterative method are used to solve two boundary value problems for cantilever beams and simply supported beams. In two cases, the mechanical force and electrical stimulations applied to the cells are analyzed in detail and the microscale effect is investigated. This study is meaningful for improving the quality of cell culture and promoting the cross-integration of mechanics and biomedicine.

Biomechanical response of ultrathin slices of hypertrophic cardiomyopathy tissue to myosin modulator mavacamten

Hypertrophic cardiomyopathy (HCM) is the most common inherited myocardial disorder of the heart, but effective treatment options remain limited. Mavacamten, a direct myosin modulator, has been presented as novel pharmacological therapy for HCM. The aim of this study was to analyze the biomechanical response of HCM tissue to Mavacamten using living myocardial slices (LMS). LMS (n = 58) from patients with HCM (n = 10) were cultured under electromechanical stimulation, and Verapamil and Mavacamten were administered on consecutive days to evaluate their effects on cardiac biomechanics. Mavacamten and Verapamil reduced contractile force and dF/dt and increased time-to-relaxation in a similar manner. Yet, the time-to-peak of the cardiac contraction was prolonged after administration of Mavacamten (221.0 ms (208.8 – 236.3) vs. 237.7 (221.0 – 254.7), p = 0.004). In addition, Mavacamten prolonged the functional refractory period (FRP) (330 ms (304 − 351) vs. 355 ms (313 − 370), p = 0.023) and better preserved twitch force with increasing stimulation frequencies, compared to Verapamil. As such, Mavacamten reduced (hyper-)contractility and prolonged contraction duration of HCM LMS, suggesting a reduction in cardiac wall stress. Also, Mavacamten might protect against the development of ventricular tachyarrhythmias due to prolongation of the FRP, and improve toleration of tachycardia due to better preservation of twitch force at tachycardiac stimulation frequencies.

MyoLoop: Design, development and validation of a standalone bioreactor for pathophysiological electromechanical in vitro cardiac studies.

Mechanical load is one of the main determinants of cardiac structure and function. Mechanical load is studied in vitro using cardiac preparations together with loading protocols (e.g., auxotonic, isometric). However, such studies are often limited by reductionist models and poorly simulated mechanical load profiles. This hinders the physiological relevance of findings. Living myocardial slices have been used to study load in vitro. Living myocardial slices (LMS) are 300-μm-thick intact organotypic preparations obtained from explanted animal or human hearts. They have preserved cellular populations and the functional, structural, metabolic and molecular profile of the tissue from which they are prepared. Using a three-element Windkessel (3EWK) model we previously showed that LMSs can be cultured while performing cardiac work loops with different preload and afterload. Under such conditions, LMSs remodel as a function of the mechanical load applied to them (physiological load, pressure or volume overload). These studies were conducted in commercially available length actuators that had to be extensively modified for culture experiments. In this paper, we demonstrate the design, development and validation of a novel device, MyoLoop. MyoLoop is a bioreactor that can pace, thermoregulate, acquire and process data, and chronically load LMSs and other cardiac tissues in vitro. In MyoLoop, load is parametrised using a 3EWK model, which can be used to recreate physiological and pathological work loops and the remodelling response to these. We believe MyoLoop is the next frontier in basic cardiovascular research enabling reductionist but physiologically relevant in vitro mechanical studies.

Surface-Modified Piezoelectric Copolymer Poly(vinylidene fluoride-trifluoroethylene) Supporting Physiological Extracellular Matrixes to Enhance Mesenchymal Stem Cell Adhesion for Nanoscale Mechanical Stimulation.

There is an unmet clinical need to provide viable bone grafts for clinical use. Autologous bone, one of the most commonly transplanted tissues, is often used but is associated with donor site morbidity. Tissue engineering strategies to differentiate an autologous cell source, such as mesenchymal stromal cells (MSCs), into a potential bone-graft material could help to fulfill clinical demand. However, osteogenesis of MSCs can typically require long culture periods that are impractical in a clinical setting and can lead to significant cost. Investigation into strategies that optimize cell production is essential. Here, we use the piezoelectric copolymer poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), functionalized with a poly(ethyl acrylate) (PEA) coating that drives fibronectin network formation, to enhance MSC adhesion and to present growth factors in the solid phase. Dynamic electrical cues are then incorporated, via a nanovibrational bioreactor, and the MSC response to electromechanical stimulation is investigated.

Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

Sarcomeric network analysis of ex vivo cultivated human atrial appendage tissue using super-resolution microscopy

Investigating native human cardiac tissue with preserved 3D macro- and microarchitecture is fundamental for clinical and basic research. Unfortunately, the low accessibility of the human myocardium continues to limit scientific progress. To overcome this issue, utilizing atrial appendages of the human heart may become highly beneficial. Atrial appendages are often removed during open-heart surgery and can be preserved ex vivo as living tissue with varying durability depending on the culture method. In this study, we prepared living thin myocardial slices from left atrial appendages that were cultured using an air-liquid interface system for overall 10 days. Metabolic activity of the cultured slices was assessed using a conventional methyl thiazolyl tetrazolium (MTT) assay. To monitor the structural integrity of cardiomyocytes within the tissue, we implemented our recently described super-resolution microscopy approach that allows both qualitative and quantitative in-depth evaluation of sarcomere network based on parameters such as overall sarcomere content, filament size and orientation. Additionally, expression of mRNAs coding for key structural and functional proteins was analyzed by real-time reverse transcription polymerase chain reaction (qRT-PCR). Our findings demonstrate highly significant disassembly of contractile apparatus represented by degradation of alpha -actinin filaments detected after three days in culture, while metabolic activity was constantly rising and remained high for up to seven days. However, gene expression of crucial cardiac markers strongly decreased after the first day in culture indicating an early destructive response to ex vivo conditions. Therefore, we suggest static cultivation of living myocardial slices derived from left atrial appendage and prepared according to our protocol only for short-termed experiments (e.g. medicinal drug testing), while introduction of electro-mechanical stimulation protocols may offer the possibility for long-term integrity of such constructs.

Combining rotary wet-spinning biofabrication and electro-mechanical stimulation for thein vitroproduction of functional myo-substitutes.

In this work, we present an innovative, high-throughput rotary wet-spinning biofabrication method for manufacturing cellularized constructs composed of highly-aligned hydrogel fibers. The platform is supported by an innovative microfluidic printing head (MPH) bearing a crosslinking bath microtank with a co-axial nozzle placed at the bottom of it for the immediate gelation of extruded core/shell fibers. After a thorough characterization and optimization of the new MPH and the fiber deposition parameters, we demonstrate the suitability of the proposed system for thein vitroengineering of functional myo-substitutes. The samples produced through the described approach were first characterizedin vitroand then used as a substrate to ascertain the effects of electro-mechanical stimulation on myogenic maturation. Of note, we found a characteristic gene expression modulation of fast (MyH1), intermediate (MyH2), and slow (MyH7) twitching myosin heavy chain isoforms, depending on the applied stimulation protocol. This feature should be further investigated in the future to biofabricate engineered myo-substitutes with specific functionalities.

Regulation of TiO2 @PVDF piezoelectric nanofiber membranes on osteogenic differentiation of mesenchymal stem cells

Bioactive piezoelectric materials with similar properties as natural bone tissue with piezoelectricity have been explored for bone repair. Polyvinylidene fluoride (PVDF) is among the most studied piezoelectric polymers and has been manifested to promote osteogenic differentiation. Unfortunately, in the practical application of bone repair, bioactive materials as implants have faced poor electrical signal stability and poor modulation of bone regeneration, which have seriously hindered their further development. Here, we fabricated a composite piezoelectric fibrous membrane with titanium dioxide (TiO2) nanoparticles and PVDF (TiO2 @PVDF) for cellular osteogenic differentiation. The expression quantity of the gene osteocalcin (OCN) with the piezoelectric fibrous membrane was 49 times and 6 times higher than the expression quantity of OCN with the control group and with the pure polyvinylidene fluoride (PVDF), respectively. Moreover, the membrane with 0.3 wt% TiO2 (P-0.3TN) generated high surface potential and significantly promoted cell adhesion and proliferation through electromechanical stimulation early in osteogenic differentiation. Alkaline phosphatase (ALP) activity was increased up to 2-fold by stimulation of electrical and mechanical signals through piezoelectric fibers, effectively inducing osteogenic differentiation at the early stage. The proposed mechanism of regulating osteogenic differentiation behavior by the surface potential of TiO2 @PVDF piezoelectric fiber composite membrane provides guidance for designing electroactive materials to promote osteogenic differentiation and bone regeneration and remodeling.

Combining Piezoelectric Stimulation and Extracellular Vesicles for Cartilage Regeneration

Numerous patients experience articular cartilage defects (ACDs), which are characterized by progressive cartilage degradation and often lead to osteoarthritis (OA). Consequently, 44.7% of OA patients suffer from dyskinesia or disability. Current clinical drug treatments offer limited effectiveness in fully curing the disease. In this study, we propose a collaborative approach that combines physical and biological cues to promote cartilage regeneration. A biodegradable piezoelectric poly (l-lactic acid) (PLLA) nanofiber scaffold facilitates in situ, battery-free electrical stimulation under natural joint loading, while extracellular vesicles (EVs) serve as communication mediators between cells and promote cell proliferation, migration, and secretion of type II collagen. In this combined approach, EVs attached to PLLA are gradually released by localized piezoelectric electrical stimulation and taken up by chondrocytes. This process results in the organization of type II collagen along the PLLA fiber surface, ultimately forming cartilage lacunae that facilitate the residence of new chondrocytes. As an outcome, a significant round cartilage defect (diameter: 3 mm and depth: 1 mm) in the PLLA/EVs group (rat and knee) was rapidly restored within six weeks. In contrast, individual EVs and PLLA groups demonstrated considerably weaker cartilage regeneration capabilities. This research suggests that the synergistic effect of electromechanical stimulation and EVs-based biological cues is a crucial intervention method for treating OA.

PO-01-142 ACUTE ATRIAL DILATION INCREASES REFRACTORY PERIOD AND CONDUCTION ABNORMALITIES: EVIDENCE FROM IN-VIVO MAPPING AND IN-VITRO LIVING ATRIAL SLICES

Atrial dilation shows strong clinical association with atrial fibrillation (AF) and its proarrhythmic nature is confirmed by various studies. However, previous (animal) models reproducing acute dilation yielded in conflicting effects on the functional refractory period (FRP). Shortcomings of previous studies included lack of in-human measurements, high-density mapping, atrial conduction characterization and titration of dilation. To evaluate the effect of acute atrial dilation on human atrial refractoriness and conduction with a two-way approach of in-vivo high-density mapping and in-vitro living atrial slices. Epicardial mapping with a 192-electrode array (inter-electrode distance 2.0 mm) was performed in 4 patients undergoing mitral valve surgery, without history of right atrial (RA) dilation and AF. Mapping was performed on the mid RA during baseline and overloaded conditions (echocardiographic RA diameter increase 10-20%) by positioning patients in Trendelenburg. Atrial FRP and conduction were evaluated with programmed extrastimulation, consisting of a S1-sequence with cycle length (CL) of 400ms followed by S2 stimuli of 300ms with decrements of 20ms. In addition, 23 living myocardial slices (LMS) (thickness: 300μm) were produced from RA left-over material from cardiac surgeries and cultured under electromechanical stimulation (diastolic preload 1mN, electrical stimulation 1.0Hz). These LMS were overloaded with 10–40% extra uniaxial lengthening for evaluation of FRP and early aftercontractions (EAC). FRP significantly increased during acute dilation both in in-vitro LMS (211±49 vs. 271±34ms, p=0.01) and during in-vivo electrophysiological mapping (255±30 vs. 280±23ms, p=0.042). FRP further increased with increasing dilation and EAC were more present in dilated LMS (Table 1). During acute dilation, S2 pacing (CL: 300ms) resulted in an increase in total activation time (TAT) (31 (29–39) vs. 67 (49–82) ms), conduction block (0.6 (0.0–3.0) vs. 12.9 (8.4-16.7)%) and total length of conduction block (2 (0–9) vs. 48 (21–72) mm). Moreover, the S2 stimulus with shortest coupling interval showed even more abnormalities in atrial conduction during dilation compared to baseline (Table 1). This first in-human study showed an unambiguous increase in atrial FRP and frequency-dependent conduction abnormalities during acute atrial dilation. These observations provide novel insights in the enhanced vulnerability for the development of AF in patients with dilated atria.Tabled 1Table 1In-vivo: EP mappingBaselineDilationRA Ø (cm)4.6 ± 1.05.6 ± 1.2CVP (mmHg)8 ± 418 ± 8FRP (ms)255 ± 30280 ± 23S1 400 msTAT (ms)34 (32 – 41)44 (40 – 52)CV (cm/s)85.5 (81.1 – 86.1)80.9 (77.7 – 83.8)CB (%)1.5 (1.1 – 2.1)3.1 (1.7 – 4.4)Length CB (mm)7 (5 – 9)16 (9 – 22)S2 300 msTAT (ms)31 (29 – 39)67 (49 – 82)CV (cm/s)82.1 (75.3 – 86.0)68.9 (61.6 – 79.8)CB (%)0.6 (0.0 – 3.0)11.7 (5.6 – 16.7)Length CB (mm)2 (0 – 9)48 (21 – 72)S2 with shortest coupling intervalTAT (ms)49 (41 – 61)82 (75 – 84)CV (cm/s)72.2 (67.0 – 78.3)66.7 (61.6 – 73.2)CB (%)6.0 (2.9 – 11.3)12.9 (8.4 – 16.7)Length CB (mm)30 (11 – 49)53 (33 – 72)In-vitro: LMSBaselineDilationAcute 20% (n=10)FRP (ms)211 ± 49271 ± 34EAC (%)0%50%Titration (n=13)10%20%30%40%FRP (ms)241 ± 46263 ± 68278 ± 63297 ± 75330 ± 79 Open table in a new tab

Label-free quantitative SWATH-MS proteomic analysis of adult myocardial slices in vitro after biomimetic electromechanical stimulation

A special in vitro model maintained with ultrathin cardiac slices with a preserved architecture, multi-cellularity, and physiology of the heart tissue was used. In our experiments, we performed label-free quantitative SWATH-MS proteomic analysis of the adult myocardial slices in vitro after biomimetic electromechanical stimulation. Rat myocardial slices were stretched to sarcomere lengths (SL) within the physiological range of 1.8–2.2 μm. Electromechanically stimulated slices were compared with slices cultured without electromechanical stimulation (unloaded and nonstimulated-TW) on a liquid–air interface and with fresh myocardial slices (0 h-C). Quantitative (relative) proteomic analyses were performed using a label-free SWATH-MS technique on a high-resolution microLC-MS/MS TripleTOF 5600+ system (SCIEX). The acquired MS/MS spectra from the DDA LC–MS/MS analyses of the rat heart samples were searched against the UniProt Rattus norvegicus database (version of 15.05.2018) using the Paragon algorithm incorporated into ProteinPilot 4.5 (SCIEX) software. The highest number of differential proteins was observed in the TW group—121 when compared to the C group. In the 1.8 and 2.2 groups, 79 and 52 proteins present at a significantly different concentration from the control samples were found, respectively. A substantial fraction of these proteins were common for two or more comparisons, resulting in a list of 169 significant proteins for at least one of the comparisons. This study found the most prominent changes in the proteomic pattern related to mitochondrial respiration, energy metabolism, and muscle contraction in the slices that were stretched and fresh myocardial slices cultured without electromechanical stimulation.

Static response of small scale functionally graded piezoelectric nanobeams

ABSTRACT In this paper, we tackle the static response of power-law functionally graded piezoelectric materials (FGPM) subjected to electro-mechanical stimulation. Effects of material composition, nonlocal parameter, slenderness ratio and electrical voltage on different characteristics of FGPM nanobeams are addressed. This study corroborates that the power exponent, nonlocal parameter and slenderness ratio significant impacts on reaction force of various configurations of FGPM nanobeams.

Bright and uniform light emission from stretchable, dual-channel energy conversion systems: Simultaneous harnessing of electrical and mechanical excitations

Recently, significant progress has been made in the development of new techniques for the fabrication of mechanically durable, bright, and deformable electroluminescent devices, leading to the emergence of various technologies, such as soft robots, actuators, flexible/stretchable/wearable electronics, and self-healable devices. However, these devices mostly possess coplanar structures, wherein the internally generated light must be transmitted through at least one of the electrodes, and require a thin emissive layer (EML), causing low brightness and less applicability in soft devices. This is particularly challenging in the case of stretchable electroluminescent devices, which require electrodes exhibiting both high transmittance and low resistance even in the stretchable state because thin EMLs have low tolerance to external mechanical deformations. Herein, we report in-plane electric-field-driven, stretchable alternating-current electroluminescent devices with high brightness by utilizing a thick EML comprising multiple parallelly patterned silver nanowires embedded in a zinc-sulfide-embedded polydimethylsiloxane layer. Since the device is driven by an internal in-plane electric field, it can utilize a thick EML without using planar electrodes. At an electric field of 8 V/μm, the device showed 3.8 times higher electroluminescence luminance than a thin coplanar-structured device and achieved a maximum brightness of 1324 cd/m2 (at 9.12 V/μm), suggesting that the electric field expands throughout the thick EML. Furthermore, the device exhibited strong mechanoluminescence and good durability of dual-channel luminescence under simultaneous electromechanical stimulation. We believe that our results represent a breakthrough in electroluminescence and mechanoluminescence research and provide important insights into the development of sustainable and stretchable devices with high brightness.

Wafer‐Scale Electroactive Nanoporous Silicon: Large and Fully Reversible Electrochemo‐Mechanical Actuation in Aqueous Electrolytes (Adv. Mater. 1/2022)

Electro-Actuation In article number 2105923, Manuel Brinker and Patrick Huber report laser cantilever bending experiments that show large and reversible electrosorption-induced deformation of wafer-scale nanoporous silicon in aqueous electrolytes (isotonic saline solution) at exceptionally small operation voltages. This paves the way for on-chip integrated actuation and stress generation, e.g., in (bio-)medical environments, as is illustrated by electro-mechanical stimulation of living cells.

Viscosity-aided electromechanical poration of cells for transfecting molecules.

Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. Here, we demonstrate electromechanical poration using a core-shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.

Effect of Electrical and Electromechanical Stimulation on PC12 Cell Proliferation and Axon Outgrowth.

Peripheral nerve injuries have become a common clinical disease with poor prognosis and complicated treatments. The development of tissue engineering pointed a promising direction to produce nerve conduits for nerve regeneration. Electrical and mechanical stimulations have been incorporated with tissue engineering, since such external stimulations could promote nerve cell proliferation, migration and differentiation. However, the combination of electrical and mechanical stimulations (electromechanical stimulation) and its effects on neuron proliferation and axon outgrowth have been rarely investigated. Herein, silver nanowires (AgNWs) embedded polydimethylsiloxane (PDMS) electrodes were developed to study the effects of electromechanical stimulation on rat pheochromocytoma cells (PC12 cells) behaviors. AgNWs/PDMS electrodes demonstrated good biocompatibility and established a stable electric field during mechanical stretching. PC12 cells showed enhanced proliferation rate and axon outgrowth under electrical stimulation alone, and the cell number significantly increased with higher electrical stimulation intensity. The involvement of mechanical stretching in electrical stimulation reduced the cell proliferation rate and axon outgrowth, compared with the case of electrical stimulation alone. Interestingly, the cellular axons outgrowth was found to depend on the stretching direction, where the axons prefer to align perpendicularly to the stretch direction. These results suggested that AgNWs/PDMS electrodes provide an in vitro platform to investigate the effects of electromechanical stimulation on nerve cell behaviors and can be potentially used for nerve regeneration in the future.

MilliPillar: A Platform for the Generation and Real-Time Assessment of Human Engineered Cardiac Tissues.

Engineered cardiac tissues derived from human induced pluripotent stem cells (iPSCs) are increasingly used for drug discovery, pharmacology and in models of development and disease. While there are numerous platforms to engineer cardiac tissues, they often require expensive and nonconventional equipment and utilize complex video-processing algorithms. As a result, only specialized academic laboratories have been able to harness this technology. In addition, methodologies and tissue features have been challenging to reproduce between different groups and models. Here, we describe a facile technology (milliPillar) that covers the entire pipeline required for studies of engineered cardiac tissues. We include methodologies for (i) platform fabrication, (ii) cardiac tissue generation, (iii) electrical stimulation, (iv) automated real-time data acquisition, and (v) advanced video analyses. We validate these methodologies and demonstrate the versatility of the platform by showcasing the fabrication of tissues in different hydrogel materials and using cardiomyocytes derived from different iPSC lines in combination with different types of stromal cells. We also validate the long-term culture of tissues within the platform and provide protocols for automated analysis of force generation and calcium flux using both brightfield and fluorescence imaging. Lastly, we demonstrate the compatibility of the milliPillar platform with electromechanical stimulation to enhance cardiac tissue function. We expect that this resource will provide a valuable and user-friendly tool for the generation and real-time assessment of engineered human cardiac tissues for basic and translational studies.