Abstract
Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGKCre, Rosa26tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 μg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.
Highlights
Central nervous system (CNS) lesions are a leading cause of disability worldwide due to their limited regeneration potential [1,2] Numerous three-dimensional (3D) neural cell culture systems within biomaterials, mimicking the CNS extracellular environment, are being developed for drug screening studies [3,4] and as scaffolds in tissue engineering approaches [5,6]
Porosity analysis based on the pore area and the total scaffold area ratio was obtained from confocal acquisitions using fluorescein isothiocyanate (FITC)-Dextran in the formulations
The goal of this study was to determine the potential of tailored macroporous polysaccharide-based scaffolds for the 3D culture and manipulation of cortical neurons isolated from embryonic mouse cortex
Summary
Central nervous system (CNS) lesions are a leading cause of disability worldwide due to their limited regeneration potential [1,2] Numerous three-dimensional (3D) neural cell culture systems within biomaterials, mimicking the CNS extracellular environment, are being developed for drug screening studies [3,4] and as scaffolds in tissue engineering approaches [5,6]. Hydrogels are 3D hydrated porous polymer networks whose physicochemical properties can be tailored with interconnected pores that can allow for extensive neurite extension before they degrade [8,9,10]. Natural polymers such as collagen [11], chitosan [12], gelatin, hyaluronic acid [13], alginate [14] and heparan sulfate [15] have retained some interest as scaffolding biomaterial for neural tissue engineering because of their superior biological compatibility compared with synthetic scaffolds [7,10,16]. Dextran is a polysaccharide synthesized from sucrose by bacteria, which is composed of glucose units joined mostly by α1,6-glycosidic bonds, with α1,2-, α1,3- or α1,4side chains, which provides structural stability thanks to its high molecular weight [18,19,20]
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