3D anisotropic photocatalytic architectures as bioactive nerve guidance conduits for peripheral neural regeneration

Abstract

Great research efforts have been invested in developing nerve guidance conduits (NGCs), which can direct axons advance and guide peripheral neural regeneration. Here, three different aspects of NGC design, namely anisotropy, photocatalytic stimulation and self-assembly at implantation site, were unitedly addressed. Firstly, melt electrowriting (MEW) was used to print anisotropic, microfibrous PCL architectures. Specifically, by tailoring the fiber spacing ratio between two arms of the grid patterns (1-1, 1–2, 1–3), preferential neurite extension of PC 12 cells along the long arm direction was achieved. Such anisotropic neurites guidance was further strengthened when the intersection angles were reduced from 90° to 30°. Secondly, functionalization of PCL micropatterns with graphene oxide and graphitic carbon nitride (g-C₃N₄), a visible-light photocatalyst, may enable optoelectronic conversion and wireless neural stimulation. As a result, photocatalytic stimulation further enhanced neurite extension length under visible light irradiation. Last but not the least, NGC were successfully obtained either by manually rolling or self-assembly using a thermo-responsive bi-layer system. Interestingly, the anisotropic micropattern design dictated the self-assembly process, and an underlying mechanism was proposed. With a synergy of three unique design parameters, the herein presented NGCs may possess great potential for repairing peripheral nerve injuries.

Introduction

In the clinic, autografts are commonly used for repairing larger nerve injuries (e.g. >20 mm in human), despite their limitations by the shortage of donor nerves, mismatch in size and modality between donor nerve and injured sites etc. [[1], [2], [3], [4]] Great research efforts have been invested in developing nerve guidance conduits (NGCs) as synthetic alternatives, which can direct axons advance and guide nerve growth along conduit axis [[5], [6], [7], [8], [9]]. For this purpose, an intriguing trend concerns introducing anisotropic guiding cues in the NGCs for enhancing therapeutic effects in injured peripheral nerves. Anisotropic distribution of well-defined biophysical (e.g. surface topography [[10], [11], [12], [13], [14], [15], [16], [17]] and mechanical properties [[18], [19], [20], [21], [22], [23]] of growth permissive scaffolds), biochemical (e.g. extracellular matrix (ECM) proteins or peptides [24], growth factor gradients [25,26]), or biological cues (e.g. supportive Schwann cells [6,27]) could enable more efficient regeneration.

Topographical cues have been demonstrated to induce specific response of neuronal cells. Neurons grown on substrates with a regular geometry or clear edge, such as aligned fibrous scaffolds [[10], [11], [12], [13]] or micropatterned graphene surfaces [[14], [15], [16], [17]], prefer to extend geometrically ordered neurites. The introduction of such anisotropic guidance within NGCs, through the application of well-defined architectures, would promote the bridging in the injured nerve gap.

On the other hand, communication between neurons relying on action potential in the cell membrane [[28], [29], [30]], and a variety of related cellular behavior (e.g. migration, proliferation, axonal outgrowth, differentiation) can be influenced by endogenous electrical signals or external electrical stimuli [28,[31], [32], [33], [34]]. Electrical stimulation was regarded as an important type of non-chemical cues for promoting neural regeneration. As an alternative for direct electrical stimjulation, optoelectronic conversion triggered by photocatalytic systems, such as reduced graphene oxide (rGO)/titanium dioxide (TiO₂) [35], or rGO/graphtic carbon nitride (g-C₃N₄) [36], enabled a remote, spatiotemporal, indirect electrical stimulation. The latter could avoid potential harmful effect from the UV light source as g-C₃N₄ works in visible light, while rGO plays an important role in the separation of photogenerated charge carriers [36,37].

Melt electrowriting (MEW) is an emerging additive manufacturing technology for producing low range micrometer scale polymeric filaments with precisely controlled deposition in 3D [[38], [39], [40], [41], [42], [43], [44], [45]]. Though relatively young, MEW has demonstrated a momentum for tissue engineering, just to name a few, guiding cell growth of cardiac progenitor cells [46], formation of capillary-like networks from HUVECs [47], and influencing neuronal differentiation and cell extension of human iPSC-derived neural progenitors [48]. Polycaprolactone (PCL) has been widely demonstrated as a biocompatible material for biomedical applications. Being a thermoplastic with low melting point at ~60 °C, PCL is a good candidate for MEW [38,39,41].

Herein, three different yet important aspects of NGCs for peripheral neural regeneration were unitedly addressed (Fig. 1). Firstly, MEW PCL microarchitectures with anisotropic, mechanically stable grid patterns, were obtained to provide anisotropic guidance for PC12 cells, an electroactive neural model cell line [[49], [50], [51]]. After optimizing the topographical pattern, a photocatalytic scaffold was achieved by sequentially decorating the PCL architectures with graphene oxide (GO) nanosheets and g-C₃N₄ nanoparticles via electrostatic interactions. Scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy were used to characterize the modifications. The neurite outgrowth on the PCL-GO-C₃N₄ scaffolds under monochromatic light irradiation (450 nm) was studied. Finally, based on the anisotropic micropatterned structures, NGCs assembly for implantation were explored.