Elsevier

Carbohydrate Polymers

Volume 99, 2 January 2014, Pages 483-490
Carbohydrate Polymers

Comparative evaluation of chitosan, cellulose acetate, and polyethersulfone nanofiber scaffolds for neural differentiation

https://doi.org/10.1016/j.carbpol.2013.08.050Get rights and content

Highlights

Abstract

Based on accumulating evidence that the 3D topography and the chemical features of a growth surface influence neuronal differentiation, we combined these two features by evaluating the cytotoxicity, proliferation, and differentiation of the rat PC12 line and human neural stem cells (hNSCs) on chitosan (CS), cellulose acetate (CA), and polyethersulfone (PES)-derived electrospun nanofibers that had similar diameters, centered in the 200–500 nm range. None of the nanofibrous materials were cytotoxic compared to 2D (e.g., flat surface) controls; however, proliferation generally was inhibited on the nanofibrous scaffolds although to a lesser extent on the polysaccharide-derived materials compared to PES. In an exception to the trend toward slower growth on the 3D substrates, hNSCs differentiated on the CS nanofibers proliferated faster than the 2D controls and both cell types showed enhanced indication of neuronal differentiation on the CS scaffolds. Together, these results demonstrate beneficial attributes of CS for neural tissue engineering when this polysaccharide is used in the context of the defined 3D topography found in electrospun nanofibers.

Introduction

Several features position polysaccharides as attractive biomaterials for use in tissue engineering, stem cell research, and regenerative medicine. Three factors in particular have contributed to the growing potential of sugar-based materials for these applications. The first is the long-standing recognition that the oligosaccharide components of glycoproteins and glycolipids ubiquitously displayed on the surfaces of mammalian cells are essential for the function of these molecules; for example they play many roles at the nexus of cell adhesion and signaling (Hakomori, 2002). The second factor has been the realization that extracellar polysaccharides, which are major structural components of the extracellular matrix (ECM), also play critical roles in modulating signaling (Du & Yarema, 2010). For example the ECM can spatially sequester signaling molecules, which can enhance their ability to engage signaling pathways to promote and guide nerve regeneration (Krick, Tammia, Martin, Höke, & Mao, 2011) as well as direct asymmetric cell division (Hayes, Tudor, Nowell, Caterson, & Hughes, 2008). The third factor is the ongoing need for new materials with specific and controllable biological activity, a high degree of safety, and biodegradability for use in tissue engineering and regenerative medicine (Tirtsa Ehrenfreund-Kleinman & Domb, 2006); polysaccharides meet all of these criteria.

Chitosan, an abundant natural polysaccharide, has many attractive qualities for tissue engineering because of its biocompatibility, biodegradability, non-toxicity, and its pH dependent solubility facilitating its processing into micro- and nano-scaffolds (Kim et al., 2008a). The chemical structure of chitosan is similar to glycosaminoglycans such as hyaluronic acid present in mammalian ECM insofar as both contain amino-sugar residues, and its hydrophilicity enhances its interaction with growth factors, cellular receptors, and adhesion proteins (Kumar, 2000). Previous studies showed that chitosan is biocompatible with neural cells (Freier, Koh, Kazazian, & Shoichet, 2005) with its surface amines and its low equilibrium water content contributing to the viability, survival, and integration of neural precursor cells during prolonged periods of cell culture (Scanga, Goraltchouk, Nussaiba, Shoichet, & Morshead, 2010). Chitosan scaffolds can also enhance cell proliferation (Prabhakaran et al., 2008) and accelerate repair in the peripheral nervous system (Pfister, Papaloïzos, Merkle, & Gander, 2007). Similarly, Ao et al. demonstrated the excellent affinity of multimicrotubule chitosan-containing conduits for nerve cells (Ao et al., 2006), and most recently, chitosan-based nerve grafts have successfully repaired the 50 mm nerve defects in rhesus monkeys after 12 months of implantation (Hu et al., 2013).

In addition to the chemical composition of a biomaterial, surface properties such as topography, surface charge, and protein adsorption influence cell behavior by providing appropriate signals for cell growth, differentiation and subsequent tissue formation (Lim & Mao, 2009). In this regard, electrospun nanofibers have gained considerable interest because their architecture is similar to the naturally-occurring protein fibrils in the extracellular environment and each individual nano-scale fiber has a high surface to volume and aspect ratio allowing for a high level of surface area contact of the scaffold with the cell. Because scaffolds consisting of electrospun fibers mimic the extracellular matrix (ECM), they have been used in recent years to enhance nerve regeneration both in vitro and in vivo. For example, electrospun nanofibers fabricated from poly-L-lactic acid (PLLA) and poly-ɛ-caprolactone (PCL) have been used successfully as scaffolding materials for nerve tissue engineering (Ghasemi-Mobarakeh et al., 2010, Koh et al., 2008). Furthermore, tuning the fiber dimension and pattern of PLLA fibers regulates the viability, proliferation, and neurite outgrowth of neonatal mouse cerebellum C17.2 cells (He et al., 2010). Hurtado et al. investigated the repair of a completely transected spinal cord with a 3-mm defect in a rat model (Hurtado et al., 2011) by filling the spinal cord gap with cells enclosed in PLLA electrospun fiber conduits four weeks after surgery. Kim et al. carried out both in vitro and in vivo studies on the impacts of aligned electrospun poly(acrylonitrile-co-methylacrylate) fibers on neuron outgrowth and Schwann cell migration for the repair of a 17-mm nerve gap in a rat peripheral nerve injury model (Kim, Haftel, Kumar, & Bellamkond, 2008).

As described above, chitosan as a material and electro-spun nanofibers as a scaffold design strategy hold promise for neural tissue engineering. However, to date these two approaches have not been systematically combined by investigating electrospun chitosan nanofibers as scaffolds for nerve cell growth and differentiation. To fill this void, the present study explores the electrospinning of two polysaccharides, chitosan (CS) and cellulose acetate (CA), to obtain the nanofibrous scaffolds with similar structures. We predict that unique features of CS, specifically that it is the only natural cationic polysaccharide with a more hydrophilic and positively charged surface than CA, will provide enhanced benefits for biocompatibility, viability, and differentiation of nerve cells. The results obtained from nanofibrous scaffolds constructed from these two sugar-based materials will be compared with structurally-similar polyethersulfone electrospun (PES) fibers as well as with cells grown under standard, widely used 2D conditions (e.g., on collagen- and laminin-coated tissue culture plastic for rat PC12 and human neural stem cells, hNSCs, respectively). The resulting findings verified that a combination of the chemical properties of CS together with nanofiber topography constituted highly compatible scaffolds for nerve tissue regeneration.

Section snippets

Materials

All chemicals were purchased from Sigma–Aldrich unless otherwise stated. Polyethersulfone granules (Mw = 55,000) was purchased from Goodfellow Cambridge Limited. Cellulose acetate (CA, Mv = 30 kDa and DS = 2.45) and chitosan (CS, Mv = 405 kDa and DDA = 82.5%) were obtained from Sigma–Aldrich. Rat pheochromocytoma (PC12) cells and human neural stem cells (hNSC) were purchased from the American Type Culture Collection (ATCC) and EMD Millipore, respectively.

Electrospinning of polyethersulfone (PES), cellulose acetate (CA), and chitosan (CS) fibers

By adopting and optimizing methodology from the

Characterization of fiber diameters

We constructed electrospun PES, CA, and CS nanofibrous scaffold based on results from the Mao group, which previously optimized fiber diameter to influence neural cell differentiation and proliferation (Christopherson, Song, & Mao, 2009). As shown from the SEM micrographs presented in Fig. 1, bead-free and continuous PES, CA, and CS electrospun fiber substrates with a similar range of diameters (394 ± 143 nm, 360 ± 123 nm, and 334 ± 166 nm, respectively) were prepared using the optimized protocols

Conclusions

This study demonstrated that a combination of the chemical features and the topography of electrospun nanofibers influence the fate of neuronal precursor cells. From a chemical point of view, many polysaccharide candidates for construction of electrospun nanofibers suffer from limitations. For example, cellulose (e.g., CA used in this study) is an excellent structural material (indeed, is one of the most abundant structural biomacromolecules used throughout nature and industry) but lacks amino

Acknowledgment

Funding for this study was provided by the National Institute for Biomedical Imaging and Bioengineering (EB 005692).

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