Design and fabrication of a chitosan hydrogel with gradient structures via a step-by-step cross-linking process

Highlights

• Gradient chitosan hydrogels were fabricated by a step-by-step gelation.

• Ionic-dependent solubility of chitosan played a key role.

• Structures and properties of hydrogels were tuned by the gelling conditions.

Abstract

The development of scaffolds to mimic the gradient structure of natural tissue is an important consideration for effective tissue engineering. In the present study, a physical cross-linking chitosan hydrogel with gradient structures was fabricated via a step-by-step cross-linking process using sodium tripolyphosphate and sodium hydroxide as sequential cross-linkers. Chitosan hydrogels with different structures (single, double, and triple layers) were prepared by modifying the gelling process. The properties of the hydrogels were further adjusted by varying the gelling conditions, such as gelling time, pH, and composition of the crosslinking solution. Slight cytotoxicity was showed in MTT assay for hydrogels with uncross-linking chitosan solution and non-cytotoxicity was showed for other hydrogels. The results suggest that step-by-step cross-linking represents a practicable method to fabricate scaffolds with gradient structures.

Introduction

The developing field of tissue engineering aims to regenerate damaged tissues by combining cells from the body with highly porous scaffold biomaterials, which act as templates for tissue regeneration (O’Brien, 2011). One of the challenges in the development of such scaffolds is to adequately mimic the gradient structure of natural tissue such as bone, cartilage, tooth, skin, and vessels (Levingstone, Matsiko, Dickson, O’Brien, & Gleeson, 2014). Systems with gradients in material composition, bioactive signals, or substrate modulus have been widely explored for different applications including protein delivery systems, directing neuronal growth, investigating cell-microenvironment interactions, enhancing cell proliferation, and for interfacial tissue engineering (Chatterjee, Sun, Chow, Young, & Simon, 2010; Mohan et al., 2011; Seidi, Ramalingam, Elloumi-Hannachi, Ostrovidov, & Khademhosseini, 2011).

In particular, hydrogels, which comprise insoluble hydrophilic polymer networks formed through the gelling of water-soluble polymers, have found widespread application in tissue engineering as their aqueous, structured environment may partially mimic the natural extracellular matrix (Geckil, Xu, Zhang, Moon, & Demirci, 2010; Slaughter, Khurshid, Fisher, Khademhosseini, & Peppas, 2009). Hydrogels also represent attractive scaffolds for controlling cell function, as a variety of technologies and chemistries have been developed for tailoring their biochemical and physical properties (Burdick, Chung, & Jia, 2005). Generally, the methods used to modulate hydrogel properties typically result in isotropic properties because the gels are created from homogeneously mixed solutions. Alternatively, hydrogels with gradients in biosignals, composition, and structure have been fabricated in recent years by freezing-thawing (Kim et al., 2015), gradient polymerization (Karpiak, Ner, & Almutairi, 2012; Tan et al., 2015), 3D printing (Sobral, Caridade, Rui, Mano, & Rui, 2011), and microfluidics (Mahadik, Wheeler, Skertich, Kenis, & Harley, 2014; Pedron, Becka, & Harley, 2015) methodologies. However, these systems often employ long and sophisticated fabrication procedures that necessitate expertise and expensive equipment (Jeon, Alt, Linderman, & Alsberg, 2013; Karpiak et al., 2012).

Chitosan, a compound derived from the partial deacetylation of natural chitin, consists of a linear, semi-crystalline polysaccharide that exhibits many desirable intrinsic properties such as biocompatibility, biodegradability, and sterilization, which make it an outstanding candidate for biomedical applications (Bhattarai, Gunn, & Zhang, 2010; Domard, 2011, Sashiwa and Aiba, 2004; Silva, Juenet, Meddahi-Pellé, & Letourneur, 2015; Zhang et al., 2012). Accordingly, chitosan hydrogels including thermosensitive hydrogels and micro/nanogels, represent the most widely used biomaterials for drug delivery, gene delivery, and tissue engineering (Abd-Allah, Kamel, & Sammour, 2016; Hui, Ling, Pei, Li, & Xi, 2015). Notably, the primary aliphatic amines (pKa = 6.3) of chitosan may be protonated under acidic conditions, which renders the molecule fully soluble (Bhattarai et al., 2010). Consequently, the chitosan solution forms entangle physical gels via secondary interactions (Van der Waals interactions and hydrogen bonds) resulting from the increase in pH (Berger, Reist, Mayer, Felt, & Gurny, 2004; Domard, 2011, Ho et al., 2004, Hsieh et al., 2007, Silva et al., 2015). Multilayered bulk hydrogels are fabricated by exploiting the pH-dependent solubility of chitosan through periodic neutralization of the chitosan solution with NaOH (Dash, Chiellini, Ottenbrite, & Chiellini, 2011; Ladet, David, & Domard, 2008; Montembault, Viton, & Domard, 2005). Furthermore, tripolyphosphate (TPP) has often been used as an ionic gelling agent to prepare chitosan beads, gels, nanoparticles, and films owing to its non-toxic properties and gelling ability and stability in acid environments (Anitha et al., 2011; Jin, Zeng, Liu, & He, 2013; Rampino, Borgogna, Blasi, Bellich & Cesàro, 2013). Together, for example, these techniques have allowed the successful preparation of biomimetic multi-layered hollow chitosan-TPP hydrogel rods in NaOH solution by semipermeable membrane (Nie, Wang, Zhang, & Hu, 2015).

As a result, fabrication of physical chitosan hydrogels with the same gradient structure as human tissues was one of important research topic. By now, more literatures focused on the preparation methods on nano/micro gels, fibers and films of chitosan (Agnihotri, Mallikarjuna, & Aminabhavi, 2004). The methods for bulk chitosan hydrogels such as lyophilization and neutralization were difficult to control the gradient structure (Levengood & Zhang, 2014). The current study aimed to fabricate a chitosan-based physical gradient hydrogel via a step-by-step gelation by TPP and NaOH in sequence with assistant of NaCl. The influence of the preparation conditions on hydrogel structure and properties were further examined.