Conducting polymer-based granular hydrogels for injectable 3D cell scaffolds

Abstract

Injectable 3D cell scaffolds possessing both electrical conductivity and native tissue-level softness would provide a platform to leverage electric fields to manipulate stem cell behavior. Granular hydrogels, which combine jamming-induced elasticity with repeatable injectability, are versatile materials to easily encapsulate cells to form injectable 3D niches. In this work, we demonstrate that electrically conductive granular hydrogels can be fabricated via a simple method involving fragmentation of a bulk hydrogel made from the conducting polymer PEDOT:PSS. These granular conductors exhibit excellent shear-thinning and self-healing behavior, as well as record-high electrical conductivity for an injectable 3D scaffold material (~10 S m^-1). Their granular microstructure also enables them to easily encapsulate induced pluripotent stem cell (iPSC)-derived neural progenitor cells, which were viable for at least 5 days within the injectable gel matrices. Finally, we demonstrate gel biocompatibility with minimal observed inflammatory response when injected into a rodent brain.

Keywords: 3D cell scaffolds; conductive hydrogels; injectable hydrogels.

Figure 1.

Fabrication of granular conductive hydrogels. (A) Granular conductive gels are fabricated via a simple three-step process. First, a colloidal gel is formed by mixing PEDOT:PSS solution with concentrated PBS buffer. Second, the colloidal gel is immersed in acetic acid to induce PEDOT aggregation to increase electrical conductivity. Finally, the granular gel is formed by fragmenting the bulk conductive gel with a stir bar. (B) The granular nature of the resultant gel enables the material to flow in response to applied pressure, since microgel particles are not covalently linked and can move past one another. After removal of applied pressure, the particles return to a jammed configuration, with elasticity and conductivity that emerge due to the junctions between jammed particles. (C) Optical microscopy of the granular gel dispersed in water shows that the particles are highly heterogeneous, though with an approximate length scale that is smaller than the inner diameter of a standard 20 gauge needle (603 μm). (Scale bar = 200 μm) (D) Scanning electron microscopy of the lyophilized granular gel confirms the porosity of the hydrogel is retained even after fragmentation. (Scale bar = 50 μm)

Figure 2.

Rheological properties of granular conductive hydrogels. (A) Fragmentation of the bulk PEDOT:PSS hydrogel results in a slight decrease in storage modulus (blue, dashed line) compared to the bulk gel (blue, solid line), indicating that bond breakage is required to obtain the desired granular structure. Nevertheless, the tan delta of the fragmented gel (gray, dashed line) remains similar and less than 1, indicative of the elasticity that is maintained by the jammed microparticles. (B) The granular gel exhibits clear shear-thinning behavior in its viscosity (dark blue) and shear stress (light blue) profiles, with a power law index of 0.0501, as well as a dynamic yield stress of 1.9 kPa. (C) In an oscillatory shear amplitude sweep, the granular gel exhibits a crossover from solid-like (G’, dark blue > G”, light blue) to liquid-like (G” > G’) behavior at a strain of 15%. The peak in the loss modulus at this point is consistent with a jamming-induced elastic-to-viscous transition. (D) The granular gel is able to quickly and reversibly alternate between liquid-like behavior (G”, light blue > G’, dark blue) at high strains (500%) and solid-like (G’ > G”) behavior at low strains (1%). (E) The gel can be injected through a 20 gauge needle and (F) quickly reforms its solid-like elasticity after injection.

Figure 3.

Electrical properties of granular conductive hydrogels. (A) The impedance of the gel after fragmentation (dashed blue) is slightly higher than before (solid blue). However, the increase is minimal, especially compared to an ionically conductive control of PBS solution (pink). (B) The phase angle of both the fragmented (dashed blue) and bulk (solid blue) conductive gels is near zero, compared to the highly negative phase angle of 1x PBS (pink) at low frequencies. (C) Chronoamperometric measurement of current with the application of a DC voltage of 0.1V confirms that the granular gel (blue) is electrically conductive, with a conductivity of 10.8 S m⁻¹. By comparison, a colloidal PEDOT:PSS gel without acid treatment (pink) draws a small current that rapidly drops off over time. (D) Demonstrating that the improved impedance is not just an interfacial effect, the impedance of the granular gel increases with increasing gap size, consistent with a typical resistor. (E) The resistance across a granular gel sample loaded between two parallel plates (grey squares) tracks its rheological characteristics with the application of high and low strain. This corroborates our hypothesis that the jammed granular microstructure gives rise to macroscopic electrical conductivity in addition to elasticity. (F) Using the granular gel to complete a circuit to light an LED.

Figure 4.

Cytocompatibility of granular conductive hydrogels. (A) Live/dead assay showing the viability of NPCs within the gel on day 2. (B) Percentage viability of cells encapsulated within the gel matrices determined based on the fluorescence in live/dead assay. The cells within the gel matrices are viable without any significant different between the two groups when analyzed using the t- test. (C) alamarBlue assay illustrates the viability of cells encapsulated within the gel. There was no significant difference between the cells encapsulated within the gel compared to the positive control (cells alone). Negative control is the cells treated with cell lysis solution for 1 hour on day 1 prior to the assay. Cells encapsulated within PEDOT:PSS gel were at least 90% viable compared to the positive control on day 5. Analyzed using a mixed-effects model, followed by Tukey’s HSD post hoc test with * P < 0.05, **P<0.01, ***P<0.001. Values represent the mean of independent experiments (N = 4), error bars are S.D.

Figure 5.

Inflammatory response post-granular hydrogel injection in rats. (A) Fluorescent images of GFAP (astrocytes) and Iba1 (microglia) staining in rat brain tissue closer to the injection site. The injectable gels are well-formed within the brain tissue and labelled as gel. Percent area under (B) GFAP and (C) Iba1 fluorescence at injection and peri-injection site are quantified. The data demonstrates that the GFAP and Iba1 fluorescence near both the gels were comparable to that of the control–needle tract without any significant difference between the groups when analyzed through nested 1-way ANOVA.