Hippocampal neurons respond uniquely to topographies of various sizes and shapes

Abstract

A number of studies have investigated the behavior of neurons on microfabricated topography for the purpose of developing interfaces for use in neural engineering applications. However, there have been few studies simultaneously exploring the effects of topographies having various feature sizes and shapes on axon growth and polarization in the first 24 h. Accordingly, here we investigated the effects of arrays of lines (ridge grooves) and holes of microscale (approximately 2 microm) and nanoscale (approximately 300 nm) dimensions, patterned in quartz (SiO2), on the (1) adhesion, (2) axon establishment (polarization), (3) axon length, (4) axon alignment and (5) cell morphology of rat embryonic hippocampal neurons, to study the response of the neurons to feature dimension and geometry. Neurons were analyzed using optical and scanning electron microscopy. The topographies were found to have a negligible effect on cell attachment but to cause a marked increase in axon polarization, occurring more frequently on sub-microscale features than on microscale features. Neurons were observed to form longer axons on lines than on holes and smooth surfaces; axons were either aligned parallel or perpendicular to the line features. An analysis of cell morphology indicated that the surface features impacted the morphologies of the soma, axon and growth cone. The results suggest that incorporating microscale and sub-microscale topographies on biomaterial surfaces may enhance the biomaterials' ability to modulate nerve development and regeneration.

Figure 1

Quartz surfaces patterned with arrays of structures to form topographies. (A), (B) Grooves (lines) of 300 nm width with a spacing of 1 μm (1.3 μm pitch); (C), (D) holes with a 300 nm diameter with horizontal and vertical spacings of 1 μm (1.3 μm pitch); (E), (F) grooves (lines) of 2 μm width with a spacing of 1 μm (3 μm pitch); (G), (H) holes with a 2 μm diameter with horizontal and vertical spacings of 1 μm (3 μm pitch). All topographies consist of structures with 400–500 nm depth.

Figure 2

Adhesion of neurons to topography and smooth surface calculated by dividing the total number of cells sampled (polarized and unpolarized) by the surface area. Differences in adhesion on topography were similar to the smooth surface according to statistical analysis. Error bars = SD.

Figure 3

(A) Fraction of neurons polarized (%) relative to the total cell count on each topography and smooth surface. % polarization was statistically greater on the topographies than the smooth surface; however, differences among the topographies did not affect polarization to a statistical degree. (B) Mean axon length (μm) of polarized neurons on the topography and smooth surface. Error bars = SD. ^*Statistically significant, where p ≤ 0.05, †p ≤ 0.10 (not deemed significant, but consistent with statistical trends). At least 100 neurons (n = 100) were analyzed on each topography (except for 300 nm holes, n = 73).

Figure 4

(A) Histogram showing the percentage of axons having an alignment, θ, of 0–30° (parallel), 30–60° (unaligned) and 60–90° (perpendicular) on the 2 μm and 300 nm lines. (Inset) Axon orientation angle θ. Average θ for all angles 0–90° for the 2 μm and 300 nm lines was 19.1° (n = 45) and 28.9° (n = 73), respectively (p ≤ 0.05). (B) Average axon length for axons of various alignments. (C) Axon alignment plotted against the axon length for the data represented in (A). Error bars = SD. ^*p ≤ 0.05.

Figure 5

SEM and optical images showing the influence of the line width on the orientation of elongated axons seeded on arrays of microfabricated lines (line topographies). (A), (B) Parallel alignment on the 2 μm lines; (C)–(E) perpendicular alignment on the 300 nm lines.

Figure 6

Soma and axon orientation and morphology based on topography. The images in the right column show the enlarged areas indicated in the white boxes shown in the juxtaposing images in the left column. Sample cells on (A), (B) 2 μm lines, (C), (D) 2 μm holes, (E)–(H) 300 nm lines, (I), (J) 300 nm holes and (K), (L) smooth surface.