Poster N10 - Developing Peripheral Nerve Recovery with Layer-by-Layer ECM Coatings and study of the Schwann Cell Response

Introduction: Peripheral nerve injuries (PNIs) frequently involve surgical intervention for repair, typically relying on autografts as the gold standard for nerve regeneration. However, autograft usage frequently leads to complications such as infections, scarring, and mobility loss at the donor site, along with the burden of multiple surgeries for patients. In response to these challenges, Nerve Guide Conduits (NGCs) have emerged as a promising alternative. Despite their potential, NGCs are constrained by the absence of crucial extracellular matrix (ECM) components present in autografts, limiting their efficacy, particularly for larger lesions. To overcome these limitations and bolster the regenerative capacity of NGCs, novel strategies integrating tissue engineering principles and advanced surface modification techniques are being pursued. Among these, layer-by-layer (LbL) assemblies present a versatile approach for engineering biomimetic coatings on NGC surfaces. Particularly noteworthy is the utilization of natural polymers such as Collagen (COL) and Heparin (HEP) via LbL technique, offering a promising avenue for augmenting NGC functionality and fostering nerve regeneration. This study aims to assess the efficacy and potential of (HEP/COL) coatings on NGC surfaces for promoting nerve repair bioactivity of human Schwann cells (hSCs), encompassing evaluations of coating impact on cell proliferation, stability and degradation kinetics, and applicability to commercial collagen-based NGCs.

Materials and

Methods: Polymeric multilayers were formed using the Layer-by-layer technique on a 96-well cell culture plate and Nerve Guide conduits. A polyanion solution (HEP) and a polycation solution (COL) at 1 mg/ml concentration in pH 5 sodium acetate buffer were alternated, totaling 6 bilayers of the polymer blend. Type I collagen sponges from cow tendon (donated by Integra Lifesciences Holdings Corporation, Añasco, PR) and sodium heparin from Celsius Laboratories, Inc. were used to prepare the solutions. Additionally, Nerve Growth Factor (NGF) was added to the culture medium at 10 ng/ml to investigate its effect on Schwann cells, with control groups lacking NGF and coatings. Fluorescence microscopy was employed to observe cell morphology and migration, while protein expression was assessed using a Luminex MAGPIX system after 3 and 6 days of cell culture. Proliferation was evaluated via a PrestoBlue viability assay. Finally, (HEP/COL) coatings were applied onto commercial collagen-based NGCs and cell viability and morphology, and coating stability studies were also carried out.

Results, Conclusions, and Discussions: In vitro cell culture assays were conducted to compare NGCs and Tissue culture plastic (TCP), both coated with (HEP/COL)6, to uncoated controls, with and without the supplementation of Nerve Growth Factor (NGF). Results demonstrated enhanced cell proliferation, adhesion, and migration on (HEP/COL)6 surfaces, attributed to the bioactive properties of the coatings and the presence of growth factors. Furthermore, systematic characterization employing techniques like IR Variable Angle Spectroscopic Ellipsometry (IR-VASE) highlighted the durability and sustained functionality of (HEP/COL)6 coatings under simulated physiological conditions, essential for their translational potential. This complete approach, integrating in vitro cell culture assays and morphological analyses, exhibited significant enhancements in human Schwann cell viability, adhesion, and migration on (HEP/COL)6, emphasizing the translational promise of this surface modification strategy.
Comprehensive strategies employed to advance peripheral nerve regeneration, particularly the utilization of (HEP/COL)6 on various surfaces, offer valuable understandings into the mechanisms governing nerve regeneration. These findings lay the groundwork for developing next-generation NGCs with enhanced regenerative capacities through the integration of tissue engineering principles and advanced surface modification techniques. This research represents a significant advancement in addressing the clinical challenges associated with peripheral nerve injuries.

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