Measuring Electric Currents from Piezoelectric Hydrogel Substrates for Excitable Cells

Introduction: Piezoelectric materials are materials that produce a voltage when placed under mechanical stress. Biocompatible piezoelectric materials are a growing area of interest for biomedical engineering applications for their potential rehabilitative and regenerative properties for electrically excitable cells such as nervous and muscle cells. By further developing these materials, it may be possible to use them to better address issues such as volumetric muscle loss and nerve damage. For researchers to produce these piezoelectric devices, they must be able to robustly validate these materials as reliable forms of treatment. To accomplish this, electronic systems that can reliably measure the electrical properties of piezoelectric materials are needed. However, electronic systems used for detecting the small currents produced by these materials are too big for practical use and could be unreliable in their detection of the currents. This work proposes a portable design for a device to measure small electric currents produced by piezoelectric hydrogels undergoing mechanical deformation. We further developed a protocol for the synthesis of an interpenetrating network of polymers to form poly(vinylidene fluoride)-poly(ethylene glycol diacrylate) (PVDF-PEGDA) piezoelectric hydrogels to be used as a substrate for electrically excitable cells. The developed set-up was tested with C2C12 myoblast cells cultured on the piezoelectric hydrogels.

Materials and

Methods: To prepare PVDF-PEGDA gels that are 20% PVDF by weight (Fig. 1A), PVDF was first solubilized in dimethyl sulfoxide (DMSO) to make a 5% w/v solution. PEGDA dissolved in DMSO (13.3%w/v) was added to the PVDF solution in a ratio of 2:3 (10% w/v PVDF-PEGDA final) and mixed under Argon gas. A photoinitiator (Irgacure 2959, 1% w/v) was then added to the solution before the precursor solution was irradiated with UV light to polymerize the gel. The gel was then soaked in DI water for 24 h with 3 water exchanges to remove the DMSO. C2C12 myoblast cells (200 cells/L) were seeded on the gels and cultured in DMEM/F12 media containing 10% fetal bovine serum (FBS) and 1% Penicillin/streptomycin at 5% CO2 and 37oC for 24 hours. To apply mechanical stimulation to the gels, gels were cut into 1.8 cm (about 0.71 in) x 1.8 cm squares and glued into a PDMS gasket using Sylgard 184. The gasket was then stretched to 5% strain using an IonOptix C-Pace EM culture stimulator. After gels were glued to the PDMS gasket, copper wire electrodes were used to detect voltage produced by the gels undergoing mechanical stimulation. Electrical current was measured using a custom device comprised of National Instruments Data Acquisition (DAQ) box and a non-inverting amplifier.

Results, Conclusions, and Discussions: PVDF-PEGDA gels were successfully made in a robust, reproducible process (Fig. 1A). The gels became opaque when soaked in water, compared to fully transparent PEGDA gels, confirming PVDF incorporation within the gels (Fig. 1B, C). C2C12 cells seeded on top of the PVDF-PEGDA gels showed poor adhesion and low spreading, with only a few cells extending cell protrusions (Fig. 2). Cells showed poor adhesion and no spreading on the control PEGDA gels as well. This was attributed to the hydrophilic and inert nature of PEGDA. To address this, current work is focused on covalent methods for coating the gels with adhesive ligands and including interstitial elements to stabilize the polarized phase of PVDF to promote cell attachment. We were able to successfully ‘glue’ the gels to the chambers of the IonOptix C-Pace EM culture stimulator and mechanically stimulate them. Future experiments will include mechanically deforming cell-seeded gels to observe the effects of applying piezoelectric stimulation on muscle cell proliferation and morphology. We then designed a device to measure the electrical currents produced by the gels upon mechanical stimulation (Fig. 3). Due to the expected small magnitude of the voltages produced by the gels, a non-inverting amplifier was implemented and was found to have a gain of 23.1 (Fig. 3C). A difficulty faced with our design was the mechanical mismatch between the soft gels and stiff electrodes, where wire electrodes placed in the gels tore them easily, compromising the direct contact between the electrodes and the gel. This will be addressed in the future by replacing the wire electrodes with planar electrodes placed on the sides of the gel. Overall, this work demonstrates the successful synthesis of a piezoelectric hydrogel with potential for affecting growth and proliferation of electrically excitable cells and proposes a design for measuring the electric properties of the gels. Future work will be aimed at studying the effects of using piezoelectric hydrogels to grow electrically excitable cells and characterizing the piezoelectric properties of PVDF-PEGDA hydrogels.

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