Author ORCID Identifier

https://orcid.org/0000-0001-7084-8216

Date of Award

17-8-2025

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.S.Anuradha

Keywords

Peripheral Nerve Injury, Biomimetic, Multifilament Nerve Conduit, Anatomic Equivalance, 3D Printing

Abstract

Long segmental defects in peripheral nerves impair sensory and motor function by disrupting neural impulse transmission. End-to-end autologous grafting with proper fascicular complementation has demonstrated functional recovery per the Medical Research Council Classification (MRCC). However, obtaining donor nerve tissue exceeding 30 mm remains a significant challenge. Artificial hollow conduits serve as an alternative but are limited to defects smaller than 30 mm due to inadequate innervation across the lumen.

Multichannel nerve guidance conduits (mNGCs) have emerged as a promising solution by enhancing fascicular complementation similar to autografts. This study presents a two-step approach for fabricating nerve conduits with precise fascicular organization. A customized 3D-printed multifilament extrusion system enables the rapid creation of perineurium-like structures, encapsulated within a biomimetic epineurial sheath via dip-coating. Pectin-based multifilament conduits, designed with tunable filament numbers (4, 6, 8, and 10), were fabricated using a 3D wet writing process to closely mimic native nerve fascicles.

To address the limited structural integrity of ionically crosslinked pectin, methacrylate groups were introduced to the hydroxyl functionalities of pectin via aldehyde-mediated chemistry, enabling covalent network formation through photo-induced crosslinking. This chemical modification significantly enhances the mechanical stability and durability of the resulting conduits. However, native pectin lacks intrinsic bioactive cues necessary for effective cellular adhesion.

To overcome this limitation, keratin extracted from human hair was covalently conjugated to the methylene functional groups of the methacrylated pectin (PecMA) via thiol-ene click chemistry. The keratin conjugated pectin methacrylate (Keratin-PecMA) provides biologically active motifs for cellular attachment while ensuring its stable integration into the polymer backbone, thereby preventing leaching during fabrication or physiological implantation.

The bio-ink’s dual crosslinkable properties and template-free fabrication allowed the rapid formation of structurally stable, cell-laden conduits. Scanning electron microscopy (SEM) and micro-Computed Tomography (Micro-CT) reconstructions confirmed that the acellular multifilament conduits closely resembled native rat and goat sciatic nerves, replicating epi-, peri-, and endoneurial features.

In vitro studies demonstrated that the cellular multifilament conduits supported cytocompatibility and enhanced Neurofilament Heavy chain (NF200) expression in PC12 cells and S100 expression in RSC96 cells. Ex vivo micro-CT imaging of anastomosed decellularized goat sciatic nerve with an 8-filament conduit using Vetbond® revealed precise fascicular alignment. In vivo, Keratin-PecMA multifilament conduits remained non-irritant for 8 weeks in a rat subcutaneous pouch model, confirming biocompatibility. In a rat sciatic nerve defect model, the conduits improved sciatic function index (SFI) and reduced foot slips 8 weeks post-implantation.

Proper fascicular alignment facilitated greater nerve fibre orientation, with Keratin-PecMA conduits supporting larger fibre diameters than PecMA conduits, emphasising keratin’s role in nerve regeneration. Immunohistochemical analysis further revealed enhanced neuronal and Schwann cell marker expression in Keratin-PecMA conduits. Additionally, the comparable muscle mass ratio between Keratin-PecMA and autograft groups indicated effective end-organ innervation. These findings suggest that customizable Keratin-PecMA multifilament conduits could serve as a viable alternative to autografts.

Download

Share

COinS