Biocompatibility and characterization of a peptide amphiphile hydrogel for applications in peripheral nerve regeneration

Abstract

Peripheral nerve injury is a debilitating condition for which new bioengineering solutions are needed. Autografting, the gold standard in treatment, involves sacrifice of a healthy nerve and results in loss of sensation or function at the donor site. One alternative solution to autografting is to use a nerve guide conduit designed to physically guide the nerve as it regenerates across the injury gap. Such conduits are effective for short gap injuries, but fail to surpass autografting in long gap injuries. One strategy to enhance regeneration inside conduits in long gap injuries is to fill the guide conduits with a hydrogel to mimic the native extracellular matrix found in peripheral nerves. In this work, a peptide amphiphile (PA)-based hydrogel was optimized for peripheral nerve repair. Hydrogels consisting of the PA C16GSH were compared with a commercially available collagen gel. Schwann cells, a cell type important in the peripheral nerve regenerative cascade, were able to spread, proliferate, and migrate better on C16GSH gels in vitro when compared with cells seeded on collagen gels. Moreover, C16GSH gels were implanted subcutaneously in a murine model and were found to be biocompatible, degrade over time, and support angiogenesis without causing inflammation or a foreign body immune response. Taken together, these results help optimize and instruct the development of a new synthetic hydrogel as a luminal filler for conduit-mediated peripheral nerve repair.

FIG. 1.

Chemical structure of the peptide amphiphile (PA), C₁₆GSH.

FIG. 2.

Images of Schwann cells grown on 2D hydrogel surfaces of C₁₆GSH (A–D) or collagen (E) for 18 h. Schwann cells spread most on gels C₁₆GSH of 0.1 wt% (D) corresponding to 0.92 kPa stiffness. Observed spreading was less on more concentrated C₁₆GSH gels of 1 wt% (A), 0.5 wt% (B), 0.2 wt% (C), and collagen (E).

FIG. 3.

Scanning electron micrographs of Schwann cells grown on 2D surfaces of 0.1 wt% C₁₆GSH (A) and collagen (B) gels after 24 h. Conditions of 0.1 wt% C₁₆GSH and collagen have roughly equivalent stiffness, 0.92 and 1.07 kPa, respectively.

FIG. 4.

Proliferation of Schwann cells on collagen and C₁₆GSH gels after 48 h (mean±SD, *p<0.01, ANOVA, Dunnett's test). At all concentrations, cells proliferate more on C₁₆GSH gels than on collagen.

FIG. 5.

Representative images of Schwann cells migrating through microchannels filled with collagen (A) and 0.2 wt% (B) or 0.05 wt% (C) C₁₆GSH hydrogels. Cells have migrated from the inlet area (white arc) on the left toward the right over a period of 6 days without additional growth factors.

FIG. 6.

Migration of Schwann cells through three-dimensional (3D) michrochannels of collagen or C₁₆GSH gels. Calculated number of cells entering the microchannel from the entrance port for each gel, as calculated using ImageJ software. (mean±SD, *p<0.01, ANOVA, Dunnett's test).

FIG. 7.

Histological images of 1 wt% C₁₆GSH hydrogels implanted subcutaneously at days 3, 10, and 30. Dashed outlines indicate the gel implant area. Over a 30-day period, gels are infiltrated by cells and degrade (hematoxylin and eosin [H&E] staining). The presence of macrophages is confirmed with F4/80 staining at days 3 and 10 and is significantly reduced by day 30.

FIG. 8.

Day 10 histological images of 1 wt% C₁₆GSH implanted gels showing spontaneous development of blood vessels within the gel (black arrows). Scale bar is 75 μm.

FIG. 9.

Systemic antibody production as measured by total IgG ELISA. No statistical difference was measured between treatment groups within each time point (ANOVA with Tukey's post hoc correction).