With continued progress of wearable sensor technology and drug-screening in-vitro models, there is a need for more advanced biomaterials and scaffolds to enhance electrical performance for stimulation and recording.1 Poly(3,4-ethylenedioxythiophene):poly-styrenesulfonate (PEDOT:PSS) is an electroconductive polymer often applied within biosensors and more recently as scaffold in tissue engineering.2 In this context, its conductive properties are hypothesized to enhance the effect of electrical stimulation; known to play a potent role in differentiation of progenitor stem cell sources into cardiomyocytes and in the maturation of cardiac engineered organoids.3
In this project, PEDOT:PSS was engineered into tunable, aligned, three-dimensional (3D) porous sponge-like structures. Scaffolds were functionalised via a crystallisation treatment to enhance their properties for tissue engineering. Afterward, we conceptualized and fabricated a bioelectric pacing bioreactor to deliver electrical stimulation to 3D scaffolds and an ad-hoc rig for in-vitro contraction-tracking, validated in-vitro using C3H10, primary rat cardiomyocytes and induced pluripotent stem cell derived cardiomyocytes.
Materials and methods
PEDOT:PSS was covalently crosslinked using glycidoxypropyl-trimethoxysilane (GOPS) and fabricated via lyophilisation. Crystallisation was achieved with incubation in pure sulphuric acid to improve conduction networks and remove excess PSS. Morphology of constructs was evaluated qualitatively using scanning electron microscopy (SEM) and quantitatively through image analysis of scaffold microtomed sections. Ethanol intrusion provided quantification of the overall porosity. Mechanical properties were determined using a Zwick-Roell uniaxial testing apparatus, while electrical features were simultaneously obtained from a Keithley sourcemeter. Via X-ray diffraction (XRD) it was possible to confirm the crystallisation of PEDOT:PSS. In-vitro studies determined the material biocompatibility and effectiveness of custom designed bioreactor. Viability, proliferation via DNA quantitation, metabolism and cell orientation were chosen as performance indicators. Bioreactor designs were generated with Solidworks® and rapid-prototyped with either Prusa-i3 or Formlabs SLA printers. Matlab® was adopted for the writing of scripts and the analysis of datasets, such as piezoresistivity, pore size, stress-relaxation, cell-directionality.
Results & Discussion
Controlled freeze-drying parameters achieved highly porous structures with either isotropic or aligned architectures. Crystallised scaffolds exhibited 1000-fold higher conductivity compared to untreated ones, while preserving stiffness and biocompatibility in a range matching to induce myogenic differentiation.4 We designed and prototyped both a pacing bioreactor that can fit standard 6-well plate and a chip with flexible anchorage for tracking of contraction (R3S), that is reusable and easy to manufacture. A 7-day study applying electrical pacing to C3H10 cells, showed that pacing does not decrease cells viability, and that it also promotes metabolism and alignment of cells, synergistically with the aligned topography of the scaffolds. Studies on primary rat cardiomyocytes and induced pluripotent stem cell derived cardiomyocytes further corroborated the use of these scaffolds for in vitro models.
Overall, PEDOT:PSS scaffolds provided an asset for the production of versatile platforms for tissue engineering applications.
1. Nezakati T, et al. Chemical Reviews. 118(14). 6766-6843. 2018.
2. Guex AG, et al. Acta Biomater. 62(91-101. 2017.
3. Solazzo M, et al. APL Bioengineering. 3(4). 041501. 2019.
4. Solazzo M, et al. Biomater Sci. 9(12). 4317-4328. 2021.