On the use of Parylene C polymer as substrate for peripheral nerve electrodes

Abstract

Parylene C is a highly flexible polymer used in several biomedical implants. Since previous studies have reported valuable biocompatible and manufacturing characteristics for brain and intraneural implants, we tested its suitability as a substrate for peripheral nerve electrodes. We evaluated 1-year-aged in vitro samples, where no chemical differences were observed and only a slight deviation on Young's modulus was found. The foreign body reaction (FBR) to longitudinal Parylene C devices implanted in the rat sciatic nerve for 8 months was characterized. After 2 weeks, a capsule was formed around the device, which continued increasing up to 16 and 32 weeks. Histological analyses revealed two cell types implicated in the FBR: macrophages, in contact with the device, and fibroblasts, localized in the outermost zone after 8 weeks. Molecular analysis of implanted nerves comparing Parylene C and polyimide devices revealed a peak of inflammatory cytokines after 1 day of implant, returning to low levels thereafter. Only an increase of CCL2 and CCL3 was found at chronic time-points for both materials. Although no molecular differences in the FBR to both polymers were found, the thick tissue capsule formed around Parylene C puts some concern on its use as a scaffold for intraneural electrodes.

Figure 1

(A) Tensile testing samples, according to DIN EN ISO 527-3. (B) Young’s modulus of aged Parylene C samples as box plots. Depiction of 10 samples per measurement set. Maximum length of whiskers is 1.5 times the interquartile range (IQR). ANOVA: F(6,60) = 3.04, p-value = 0.01. Significance (Tukey): *p < 0.05. (C) Strain at tensile strength of aged Parylene C samples as box plots. Depiction of 10 samples per measurement set. Maximum length of whiskers is 1.5 times IQR ANOVA: F(6,60) = 7.39, p-value = 6.1*10⁻⁶. Significance (Tukey): *p < 0.05, **p < 0.001.

Figure 2

FTIR spectra of fresh, aged, explanted and intentionally oxidized Parylene C samples. The absorption is presented in arbitrary units. Only oxidized Parylene C exhibits an additional peak.

Figure 3

Progression of tissue capsule formation around the Parylene C device implanted in the nerve. Representative light microscopy images of (A) sham nerves after 1 day and 2 weeks in which no tissue deposition can be appreciated, (B) Implanted nerves with progressive tissue capsule around the device at 1 day, 2, 8 and 32 weeks. (C) Increase in tissue capsule thickness along time. Scale bar = 50 µm. *p < 0.05 vs 2w. ^#p < 0.05 vs 16w.

Figure 4

Detailed evaluation of tissue capsule by TEM. (A–F) Representative images of the tissue capsule at 1 and 2 days, 2, 8, 16 and 32 weeks postimplant. Scale bar = 10 µm. At (A) 1 day, (B) 2 days and (C) 2 weeks only amoeboid cells (arrows) were seen in the vicinity of the device. From (D) week 8 two different zones can be distinguished in the capsule (dotted line), with spindle-shaped cells localized at the periphery of the capsule (arrowheads). After (E) 16 and (F) 32 weeks post-implant, the capsule was mainly formed by spindle-shaped cells and collagen fibers (insets, G–L). Insets show the progressive change from (G–H) amoeboid cells at early time points to (I–L) spindle-shaped cells, together with an increase in (K–L) collagen deposition from 8 weeks. Scale bar = 2 µm.

Figure 5

Iba1 labeling of infiltrating macrophages in the tibial nerve. Representative images of (A) implanted and (B) sham nerves at different time-points. Scale bar = 100 µm. (C) Number of iba1 positive cells in the whole tibial nerve along time. *p < 0.05 vs sham animals. ^#p < 0.05 vs 2w implanted.

Figure 6

Cellular composition of the capsule. Representative confocal microscopy images labeling against Iba1 and CD90 markers. Only iba1+ macrophages were observed from (A) 1 day to (E) 4 weeks post-implant. From (E) 8 to (F) 16 weeks, CD90+ fibroblasts were located in the periphery of the capsule and by (G) week 32w, CD90+ fibroblasts occupied most of the area of the capsule. Iba1 in red, CD90 in green, DAPI in blue. Scale bar = 5 µm.

Figure 7

Cytokines levels obtained by multiplex analysis. Heatmaps showing the changes in several proteins at different time points in (A) Parylene C and (B) polyimide implanted nerves versus sham nerves. Results are expressed as the mean of the ratio between each group and sham values. (C) Heatmap representing the changes in Parylene C implanted nerves with respect to polyimide implanted nerves. Results expressed as the mean of the ratio between Parylene C and polyimide values. Crossed out squares mean no protein detected.