In-situ forming thermosensitive hydroxypropyl chitin-based hydrogel crosslinked by Diels-Alder reaction for three dimensional cell culture

Highlights

• Novel thermosensitive and degradable furyl-modified hydroxypropyl chitin synthesized.

• Injectable and dually crosslinked hydrogel formed from thermogelation and DA reaction.

• The dually crosslinked hydrogel showed improved strength than the physical thermogel.

• This chitin-modified hydrogel with good biocompatibility useful for 3D cell culture.

Abstract

Injectable thermosensitive hydrogels crosslinked physically have been extensively studied as scaffolds in biomedical field, however, their low gel stability with weak strength limits their potential applications. Here, a novel thermosensitive furyl-modified hydroxypropyl chitin polymer was synthesized homogeneously in aqueous solution for developing injectable and dually crosslinked degradable hydrogel by integration of Diels−Alder click reaction using crosslinker maleimide-terminated PEG under physiological conditions. The dually crosslinked chitin-modified hydrogel showed much higher mechanical strength and slower biodegradation rate in contrast with the solely physically crosslinked thermosensitive hydroxypropyl chitin hydrogel. Cells encapsulated in the hydrogel displayed sustainable proliferation and good ability of self-assembling to form multicellular spheroids. In vivo investigation of the thermosensitive chitin-based dually crosslinked hydrogel showed favorable injectability, in-situ thermogelation and good biocompatibility. Thus the injectable, biodegradable and biocompatible dually crosslinked chitin-based hydrogel holds great potential for being applied in three dimensional cell culture and tissue repair.

Introduction

Hydrogels are widely investigated for biomedical applications, such as cell culture, regenerative tissue engineering and drug delivery due to their similar structure as natural extracellular matrixes (Jiang, Chen, Deng, Suuronen, & Zhong, 2014; Owen, Fisher, Tam, Nimmo, & Shoichet, 2013; Tong & Yang, 2018; Zhao et al., 2017). Hydrogels are composed of three dimensional (3D) hydrophilic networks having high water content, and are benefit for the transportation of oxygen, nutrients and other water-soluble metabolites, providing an adaptive environment for cell growth. The in-situ forming hydrogels can encapsulate therapeutic molecules and/or cells easily and be injected into body in minimally invasive procedure, which provides the most attractive materials for injection (Ressler et al., 2018; Xu, Gao et al., 2018, 2018b). In-situ forming thermosensitive hydrogels are promising carriers for cell delivery and tissue regeneration because of their good capability of filling complex tissue defects, by non-invasive injection (Abandansari et al., 2018; Bermejo-Velasco, Dou, Heerschap, Ossipov, & Hilborn, 2018; Chen et al., 2016; He, Sui et al., 2017). However, the physical hydrogels are often degraded and eroded fast due to their weak mechanical properties, which restricts their potential applications. On the other hand, the chemical hydrogels crosslink too quickly, which restricts to make the gels homogeneously and conveniently, or too slowly, leading to the polymer precursor solutions diluted and leached away from the injection site. Therefore, combining both methods in dual gelation can provide an easy injection with the formation of strong network (Bian et al., 2017; Boere et al., 2015; Ghanian, Mirzadeh, & Baharvand, 2018; Gregoritza, Messmann, Abstiens, Brandl, & Goepferich, 2017; Yuan, Bi, Huang, Zhuo, & Jiang, 2018). In this way, thermosensitive crosslinking induces an immediate gelation to stabilize the network after injection, while at the same time, chemical crosslinking improves the mechanical property.

The metal-free “click” reactions are gaining more and more attention for the development of in-situ crosslinking hydrogels (Huang & Jiang, 2017; Wang et al., 2017). Among them, Diels–Alder (DA) reactions between maleimide and furyl groups are particularly helpful, because of their high efficiency, no catalysts required, no toxic side products formed, and processing under physiological conditions (Guaresti, García–Astrain, Aguirresarobe, Eceiza, & Gabilondo, 2018; Meng & Edgar, 2016). For example, the DA reaction has been introduced for the preparation of degradable polysaccharide-based crosslinking hydrogels (Bai et al., 2017; Shao, Wang, Chang, Xu, & Yang, 2017; Yu, Cao, Du, Wang, & Chen, 2015). Thus these hydrogels show promising potential for 3D cell culture and tissue engineering. However, the relatively long gel times of these hydrogels is deemed to be a major limitation for injectable hydrogels due to the slow DA reaction (Yu et al., 2014). Therefore, many efforts have been made to promote gelation rate based on DA reactions, such as, utilizing hydrophobic association (Gregoritza, Messmann, Goepferich, & Brandl, 2016) or introducing additional branches (Kirchhof et al., 2015).

Chitin and its derivatives have widely been studied to prepare new functional biomaterials, due to their inherent biocompatibility, biodegradability, antimicrobial properties and bioactivity (Couto, Hong, & Mano, 2009; Kang, Bi, Zhuo, & Jiang, 2017; Liu, Liu et al., 2016; Yuan et al., 2018). It is worthwhile pointing out that the chitin-based biomaterials with higher degree of acetylation (DA) could be degraded more rapidly than chitosan-based biomaterials (DA of chitin is above 50% and DA of chitosan is below 50%) under in vivo condition (Ren, Yi, Wang, & Ma, 2005; Tomihata & Ikada, 1997). In our previous work, the novel thermoresponsive hydroxypropyl chitin (HPCH) was synthesized in homogeneous “green” solvent aqueous sodium hydroxide/urea solution through the etherification of the chitin side hydroxyl groups with propylene oxide (Jiang et al., 2017; Yuan et al., 2018). The HPCH aqueous solution can be injected directly into the desired sites easily due to its fluidity at low temperature, which forms hydrogel in-situ rapidly under physiological conditions. Hydrogels prepared by this HPCH material can be degraded by lysozyme and have good in vivo biocompatibility (Jiang et al., 2017; Yuan et al., 2018). In addition, it can promote 3D cell growth and be applied to tissue engineering.

Here, we hypothesized that a combination of fast in-situ thermosensitive crosslinking with slow DA reaction would solve the problems of slow gelation and weak mechanical strength for hydrogels without using any initiator or catalyst under physiological conditions. Therefore, we developed a novel injectable dually crosslinked hydrogel combining thermal gelation of thermosensitive HPCH and the DA crosslinked reaction. First, thermosensitive furyl-modified hydroxypropyl chitin (FGE-HPCH) was synthesized. Then dually crosslinked hydrogels were prepared based on FGE-HPCH polymer in the presence of maleimide-terminated PEGs (MAL-PEG-MAL) under physiological conditions. The mechanical property, stability and biodegradation behavior of the resulted thermosensitive FGE-HPCH based dually crosslinked hydrogel were studied. In addition, the cytotoxicity and biocompatibility of the dually crosslinked hydrogel was also investigated.