Optimizing the Pore Structure of 3D Conducting Polymer Scaffolds for Bioelectronic Devices

The integration of 3D tissue-engineered microporous scaffolds based on conductive polymers (CPs) into bioelectronic devices have led to the possibility of dynamic monitoring of biological phenomena through electrical measurements. The 3D structure and morphology of the porous scaffolds mimics the topographical features of human physiology, while the electrically conductive nature of the polymers allows for electrical measurements for label-free monitoring and live-sensing of cells. 3D scaffolds based on the polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) prepared by the freeze drying process have been the focus of many studies and well integrated into bioelectronic devices such as electrodes[1] and organic electrochemical transistors[2].<br/>The freeze-drying process involves freezing the aqueous dispersion of the conducting polymer, followed by the sublimation of the ice crystals by lowering the pressure in the system, leaving behind a porous network of the conducting polymer. This porous network is representative of the physiological tissue structure, making the ice nucleation and crystallization a crucial component of the scaffold fabrication process. A deeper understanding of the freeze drying process and a higher degree of control on the freezing parameters will help establish the correlation between the material properties of the scaffolds and the biomimetic tissue engineering requirements for optimum cell culture platforms.<br/>Three different freeze drying protocols were employed, with a pre-cooling treatment used before the freeze-drying process. Two different pre-cooling rates, 0.5 °C/min and 1 °C/min along with a control protocol with no treatment were designed to prepare different scaffold types. Within each protocol, the freezing rate was varied between 0.2 °C/min and 1 °C/min and the thermal profile of the aqueous dispersion was recorded during the entire fabrication process.<br/>The influence of the different protocols and freezing rates on the pore size and structure was examined by scanning electron microscopy and micro-computed tomography. Pores of circular and elongated ellipses were observed by SEM and a pore size distribution (the longest dimension of the ellipsoid) between 80 µm and 220 µm was measured. It was observed that a higher cooling rate generated larger pores with more elongated pore morphology as well as a wider pore size distribution. The pre-cooling treatment with 0.5°C/min cooling rate was most useful in generating scaffolds with a more homogenous pore structure and a pore size of approximately 100 µm.<br/>The different scaffolds were incorporated into electronic devices such as transmembrane electrodes to characterize the electrical properties and evaluate the reproducibility of the scaffolds prior to cell culture. No significant change in the electrochemical impedance spectra was observed despite the large difference in porosity of the different scaffolds. In addition, the different scaffolds were used to host and monitor biological cells like fibroblasts and endothelial cells. Cell growth and proliferation in the different scaffolds was also monitored by immunofluorescence microscopy. It was observed that a pre-cooling protocol and a freezing rate of 0.5°C/min helps to fabricate scaffolds with an optimized, uniform and homogenous pore morphology. This design protocol is highly beneficial in reducing the random artifacts of the nucleation process and generating scaffolds highly conducive to cell growth and survival and to create a versatile bioelectronic platform for studying in-vitro cell models.<br/>References:<br/>[1] C. Pitsalidis, D. van Niekerk, C.M. Moysidou, A.J. Boys, A. Withers, R. Vallet, R.M. Owens, Sci. Adv. 8 (2022) eabo4761.<br/>[2] C. Pitsalidis, M.P. Ferro, D. Iandolo, L. Tzounis, R.M. Owens, Sci. Adv. 4 (2018) eaat4253.