Electroactive Tissue Scaffolds with Aligned Pores as Instructive Platforms for Biomimetic Tissue Engineering

Abstract

Tissues in the body are hierarchically structured composite materials with tissue-specific chemical and topographical properties. Here we report the preparation of tissue scaffolds with macroscopic pores generated via the dissolution of a sacrificial supramolecular polymer-based crystal template (urea) from a biodegradable polymer-based scaffold (polycaprolactone, PCL). Furthermore, we report a method of aligning the supramolecular polymer-based crystals within the PCL, and that the dissolution of the sacrificial urea yields scaffolds with macroscopic pores that are aligned over long, clinically-relevant distances (i.e., centimeter scale). The pores act as topographical cues to which rat Schwann cells respond by aligning with the long axis of the pores. Generation of an interpenetrating network of polypyrrole (PPy) and poly(styrene sulfonate) (PSS) in the scaffolds yields electroactive tissue scaffolds that allow the electrical stimulation of Schwann cells cultured on the scaffolds which increases the production of nerve growth factor (NGF).

Keywords: electroactive polymers; microfabrication; nerve guide; peripheral nerve; plastic electronics; topography.

Figure 1

Hydrogen bond-mediated self-assembly of urea.

Figure 2

(A) Illustration of the experimental setup used to produce the hard acrylic master template with grooves with widths and heights of 2 mm. (B) Examples of the hard acrylic master templates produced: (left) grooves with widths and heights of 2 mm; (center) grooves with widths and heights of 1 mm; (right) grooves with widths and heights of 0.5 mm. (C) Examples of the flexible PDMS templates produced: (left) grooves with widths and heights of 2 mm; (right) grooves with widths and heights of 1 mm. (D) Illustration of the experimental setup using the flexible, grooved PDMS template covered with a glass slide, which facilitates controlled solvent evaporation and thereby preferential alignment of urea crystals within the grooves.

Figure 3

Experimental setup for electrical stimulation of electroactive PCL-based tissue scaffolds (Not to scale). (CE) counter electrode. (CT) copper tape. (PCL) electroactive PCL-based tissue scaffolds. (PCW) polycarbonate well. (RE) reference electrode. (WE) working electrode.

Figure 4

Scanning electron microscope images of sections of PCL-based tissue scaffolds with aligned pores. (A) Millimeter and micrometer scale topography of non-electroactive scaffolds, scale bar represents 500 µm. (B) Micrometer and nanometer scale topography of non-electroactive scaffolds, scale bar represents 10 µm. (C) Millimeter and micrometer scale topography of electroactive scaffolds, scale bar represents 500 µm. (D) Micrometer and nanometer scale topography of electroactive scaffolds showing evidence of increased nanometer scale surface roughness due to the presence of an interpenetrating network of PPy and PSS interwoven within the PCL matrix, scale bar represents 10 µm.

Figure 5

(A and B) FTIR spectra of PCL-based tissue scaffolds with aligned pores: (A) non-electroactive scaffolds, (B) electroactive scaffolds. Peaks observed at ca. 1543 and ca. 1480 cm⁻¹ are characteristic of the antisymmetric and symmetric ring stretching modes of pyrrole [65,78]. (C and D) XPS spectra of PCL-based tissue scaffolds with aligned pores: (C) non-electroactive scaffolds, (D) electroactive scaffolds. Peaks at ca. 400 eV (N 1s) and ca. 168 eV (S 2p) are characteristic of PPy and PSS, respectively [65,73,79].

Figure 6

In vitro degradation profiles of the PCL-based tissue scaffolds in PBS. (A) Non-electroactive scaffolds: black circles in the absence of cholesterol esterase; grey circles in the presence of cholesterol esterase. (B) Electroactive scaffolds: black circles in the absence of cholesterol esterase; grey circles in the presence of cholesterol esterase. Error bars represent standard deviations.

Figure 7

Cells respond to the topography of the PCL-based tissue scaffolds substrates and align on the substrates. (A) Schwann cells on non-electroactive scaffolds (scale bar represents 50 µm); (B) Schwann cells on electroactive scaffolds without electrical stimulation (scale bar represents 50 µm).

Figure 8

Concentration of Schwann cell-produced NGF in the culture medium. Black circles) commercially available tissue-culture treated Corning^® Costar^® tissue culture plates. (Red circles) non-electroactive PCL-based tissue scaffolds. (Yellow circles) electroactive PCL-based tissue scaffolds without electrical stimulation. (Blue circles) electroactive PCL-based tissue scaffolds with electrical stimulation. Error bars represent standard deviations.