Physically and Chemically Crosslinked Hyaluronic Acid-Based Hydrogels Differentially Promote Axonal Outgrowth from Neural Tissue Cultures

Abstract

Our aim was to investigate axonal outgrowth from different tissue models on soft biomaterials based on hyaluronic acid (HA). We hypothesized that HA-based hydrogels differentially promote axonal outgrowth from different neural tissues. Spinal cord sliced cultures (SCSCs) and dorsal root ganglion cultures (DRGCs) were maintained on a collagen gel, a physically crosslinked HA-based hydrogel (Healon 5^®) and a novel chemically crosslinked HA-based hydrogel, with or without the presence of neurotrophic factors (NF). Time-lapse microscopy was performed after two, five and eight days, where axonal outgrowth was assessed by automated image analysis. Neuroprotection was investigated by PCR. Outgrowth was observed in all groups; however, in the collagen group, it was scarce. At the middle timepoint, outgrowth from SCSCs was superior in both HA-based groups compared to collagen, regardless of the presence of NF. In DRGCs, the outgrowth in Healon 5^® with NF was significantly higher compared to the rest of the groups. PCR revealed upregulation of NeuN gene expression in the HA-based groups compared to controls after excitotoxic injury. The differences in neurite outgrowth from the two different tissue models suggest that axons differentially respond to the two types of biomaterials.

Keywords: HA-based hydrogel; axonal outgrowth; dorsal root ganglion culture; neuroprotection; spinal cord slice culture.

Figure 1

Axonal outgrowth from SCSCs and DRGCs at 5 days. Axonal outgrowth from SCSCs and DRGCs was observed in both crosslinked and non-crosslinked HA-based hydrogels while outgrowth in collagen gel was scarce((A,D): collagen, (B,E): non-crosslinked, (C,F): crosslinked). Cell migration was evident in cultures maintained in the non-cross-linked HA-based hydrogel.

Figure 2

Quantitative analysis of axonal outgrowth from SCSCs using automated analysis with NeuriteSegmentation. Images (A,C,E) show the time lapse development of outgrowth parameters in SCSCs in the absence of NM. Images (B,D,F) show the respective parameters in the presence of NM. (* indicates signifigance p ≤ 0.05).

Figure 3

Quantitative analysis of axonal outgrowth from DRGCs using automated analysis with NeuriteSegmentation. Images (A,C,E) show the time lapse development of outgrowth parameters in DRGCs in the absence of NM. Images (B,D,F) show the respective parameters in the presence of NM. (* indicates signifigance p ≤ 0.05).

Figure 4

Gene expression analysis of NeuN and IL-1β from SCSCs using the ΔCt method. (* indicates signifigance p ≤ 0.05).