Material properties and electrical stimulation regimens of polycaprolactone fumarate-polypyrrole scaffolds as potential conductive nerve conduits

Abstract

The mechanical and electrical properties of polycaprolactone fumarate-polypyrrole (PCLF-PPy) scaffolds were studied under physiological conditions to evaluate their ability to maintain the material properties necessary for application as conductive nerve conduits. PC12 cells cultured on PCLF-PPy scaffolds were stimulated with regimens of 10 μA of either a constant or a 20 Hz frequency current passed through the scaffolds for 1h per day. PC12 cellular morphologies were analyzed by fluorescence microscopy after 48 h. PCLF-PPy scaffolds exhibited excellent mechanical properties at 37 °C which would allow suturing and flexibility. The surface resistivity of the scaffolds was 2 kΩ and the scaffolds were electrically stable during the application of electrical stimulation (ES). In vitro studies showed significant increases in the percentage of neurite bearing cells, number of neurites per cell and neurite length in the presence of ES compared with no ES. Additionally, extending neurites were observed to align in the direction of the applied current. This study shows that electrically conductive PCLF-PPy scaffolds possess the material properties necessary for application as nerve conduits. Additionally, the capability to significantly enhance and direct neurite extension by passing an electrical current through PCLF-PPy scaffolds renders them even more promising as future therapeutic treatments for severe nerve injuries.

Figure 1

Schematic overview of the project’s main steps: fabrication of PCLF-PPy composite scaffolds, cell seeding, and application of ES treatment regimens.

Figure 2

Effect of thermal transition of the materials on mechanical properties of PCLF, PCLF-PPy_NSA and PCLF-PPy_DBSA scaffolds at room temperature and 37°C, measured by a) differential scanning calorimetry, (b) tensile test, (c) 3-point bending, and (d) suture pullout test. *denotes significant differences (p <0.01) in material properties between room temperature and 37°C. **denotes significant differences (p <0.01) between PCLF-PPy and PCLF at room temperature.

Figure 3

Surface resistivity (R_s) over the course of 100 h when different stimulation regimens at an intensity of 10 μA are applied on PCLF-PPy_DBSA samples (a) and PCLF-PPy_NSA samples (b) incubated in media. Figure 3c shows the temperature increase of the surrounding media when 1 mA of direct current is constantly applied for 8 h.

Figure 4

a) Percent weight loss of PCLF, PCLF-PPy_NSA and PCLF-PPy_DBSA samples incubated at 37°C in PBS for 34 weeks. b) Percent weight gain of scaffolds after 24h swelling in PBS at 37°C. No statistically significant difference was found between different scaffolds.

Figure 5

Protein adsorption on PCLF, PCLF-PPy_NSA and PCLF-PPy_DBSA scaffolds after incubation for 24 h in media with or without NGF. Samples with no media were used as control. *denotes significant differences (p <0.05) in the amount of protein adsorbed on scaffolds in media with NGF compared to scaffolds incubated in media without NGF.

Figure 6

Fluorescence microscopy of PC12 cells imaged at 10× and 40× magnification after undergoing different ES treatment regimens for 48 h.

Figure 7

Percentage of neurite bearing cells seen in fluorescent microscopy when ES treatment regimens of 20Hz and constant current are applied on the PCLF-PPy_NSA scaffolds. *denotes significant difference (p <0.01) from no ES.

Figure 8

Average number of neurites per cell (a) and distribution of neurites (b) of PC12 cells cultured on PCLF-PPy_NSA scaffolds under different ES regimens. Fluorescence microscopy of an exemplary image of a cell stimulated for 1 h/day with 10 μA 20 Hz ES bearing multiple neurites (c). *denotes significantly higher number of neurites per cell compared to no ES (p <0.01). **denotes significant differences between 20Hz frequency and constant ES (p <0.01).

Figure 9

a) Median neurite length of PC12 cells cultured on PCLF-PPy_NSA scaffolds under ES treatment regimens of 20Hz and constant current. Figure 9b,c show the neurite length distribution in terms of total neurite counts (b) and in relative values (c) for different ES treatments applied. *denotes significant difference compared to cells cultured in the absence of ES (p <0.01).

Figure 10

With the direction of the current defined as 0° the distribution of the neurite alignment is displayed for ES treatment regimens of 20Hz and constant current in terms of absolute neurite counts (a) and relative values (b).