In-situ forming thermosensitive hydroxypropyl chitin-based hydrogel crosslinked by Diels-Alder reaction for three dimensional cell culture
Graphical abstract
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.
Section snippets
Materials
Furfuryl glycidyl ether (FGE) was purchased from Acros Organics (New Jersey, USA). Chitin was obtained from Golden-Shell Biochemical (Zhejiang, China), for which the viscosity-average molecular weight (Mη) was determined to be 7.56 × 105 Da based on the Mark-Houwink equation (Li et al., 2010), and its degree of acetylation was 0.97 using nuclear magnetic resonance (NMR) analysis according to our previous work (Liu, Yang, Zhang, Zhuo, & Jiang, 2016). Propylene oxide and biological grade lysozyme
Synthesis and characterization of furyl-modified hydroxypropyl chitin and maleimide-terminated PEG
The thermoresponsive hydroxypropyl chitin (HPCH) was synthesized homogeneously as reported in the previous work (Yuan et al., 2018). The furyl-containing HPCH conjugates (FGE-HPCHs) were prepared in one-step by coupling furfuryl glycidyl ether (FGE) to HPCH chains through etherification reaction in 1 mol/L NaOH solution (Scheme 1a). Different molar substitution (MS) degrees of FGE samples were obtained through changing the feed ratio (molar ratio of FGE to HPCH unit). The structure of the
Conclusions
Novel water soluble and thermosensitive furyl-modified hydroxypropyl chitin (FGE-HPCH) conjugates were successfully synthesized by further functionalization of the hydroxypropyl chitin with furfuryl glycidyl ether. The dually crosslinked hydrogel was prepared in-situ in the presence of maleimide-terminated PEG under mild physiological conditions combining the thermosensitivity and Diels-Alder reaction. As expected, the dually crosslinked hydrogel gelled rapidly at 37℃ and its mechanical
Declarations of interest
None.
Acknowledgements
This research was financially supported by the National Natural Science Foundation of China (21674083, 21875168 and 51533006) and the Natural Science Foundation of Jiangsu Province of China (BK20151249).
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