Thermo-irreversible glycol chitosan/hyaluronic acid blend hydrogel for injectable tissue engineering

Highlights

• A blend thermogel (AcHA/HGC) was successfully prepared by mixing of HGC and AcHA.

• The AcHA/HGC hydrogel underwent a unique thermo-irreversible sol-gel transition.

• The AcHA/HGC exhibited the time-dependent improvement in mechanical strength.

• The AcHA/HGC showed good biocompatibility and more effective cartilage formation.

Abstract

Thermogels that undergo temperature-dependent sol-gel transition have recently attracted attention as a promising biomaterial for injectable tissue engineering. However, conventional thermogels usually suffer from poor physical properties and low cell binding affinity, limiting their practical applications. Here, a simple approach for developing a new thermogel with enhanced physical properties and cell binding affinity is proposed. This thermogel (AcHA/HGC) was obtained by simple blending of a new class of polysaccharide-based thermogel, N-hexanoyl glycol chitosan (HGC), with a polysaccharide possessing good cell binding affinity, acetylated hyaluronic acid (AcHA). Gelation of AcHA/HGC was initially triggered by the thermosensitive response of HGC and gradually intensified by additional physical crosslinking mechanisms between HGC and AcHA, resulting in thermo-irreversible gelation. Compared to the thermos-reversible HGC hydrogel, the thermo-irreversible AcHA/HGC hydrogel exhibited enhanced physical stability, mechanical properties, cell binding affinity, and tissue compatibility. These results suggest that our thermo-irreversible hydrogel is a promising biomaterial for injectable tissue engineering.

Introduction

Tissue engineering has emerged as an alternative therapy to treat damaged tissue and organs by implanting a scaffold incorporating the patient’s own cells (Feig, Tran, Lee, & Bao, 2018; Lee & Mooney, 2001; Vlierberghe, Dubruel, & Schacht, 2011; Wang & Heilshorn, 2015). In this case, the biomaterials for scaffolds should fulfill the following important requirements (O’Brien, 2011; Sun et al., 2012): (i) be biocompatible without being toxic to cells or tissues, (ii) biodegrade at a rate similar to the rate of new tissue formation to avoid unnecessary repeated surgery, and (iii) have adequate mechanical strength to maintain structural stability and (iv) an interconnected porous structure to facilitate cellular migration and the diffusion of nutrients and oxygen into the scaffolds during treatment. Consideration of all of the above properties highlights the need for an ideal biomaterial with tunable physical properties and good tissue compatibility to aid the recovery of dysfunctional tissue (Lutolf & Hubbell, 2005; Madl, Heilshorn, & Blau, 2018).

Hydrogels are fascinating biomaterials for building extracellular matrix (ECM) that mimics a scaffold because of their high water content, biocompatibility, and biodegradability; appropriate mechanical properties; and interconnected pore structure, which enable these materials to consistently meet the requirements for use as scaffolds in tissue engineering (Hoffman, 2012; Hunt, Chen, Veen, & Bryan, 2014; Li, Rodrigues, & Tomas, 2012). In particular, stimuli-responsive hydrogels that exhibit phase transitions in response to changes in the surrounding environment, such as changes in temperature, pH, light, electric field and ionic strength, have received substantial attention due to their tunable and in situ gelation properties (Konieczynska et al., 2016; Roy, Cambre, & Sumerlin, 2010). Especially, thermogelling hydrogels (thermogels) that demonstrate a thermo-reversible sol-gel transition allow bioactive molecules or cells to be simply loaded in a sol state and directly injected into the desired sites. The rheological behavior and viscoelastic properties of thermogels are important features for successful application of injectable thermogels because the injection through clinical catheters may strongly change their physical stability and properties (Giordano et al., 2011). Injectable thermogels should have low storage modulus (G′) at room temperature and demonstrate a sufficiently high storage modulus with fast thermogelation after injection, enabling easy encapsulation of cells and minimally invasive administration (Chung & Park, 2009; Li, Yan, Yang, Chen, & Zeng, 2015; Shu, Liu, Palumbo, Luo, & Prestwich, 2004). However, conventional thermogels usually suffer from low physical stability and poor mechanical properties due to the inherent characteristics of physical hydrogels that are constructed by only weak thermally induced hydrophobic interactions (Bhattarai, Gunn, & Zhang, 2010; Yang, Yeom, Hwang, Hoffman, & Hahn, 2014). In addition, their slow gelation kinetics may often cause significant mass or cell loss of administered thermogels, especially at an initial stage. To overcome these limitations, dual crosslinking systems that have both physical and chemical crosslinking mechanisms have been demonstrated to enhance the physicochemical properties of thermogels. However, chemical crosslinking often requires additional chemical or crosslinking agents that may have undesired effects on surrounding cells or tissues, and such crosslinking may not occur to a sufficient extent or in a timely manner under physiological conditions (Han et al., 2018; Li, Xu et al., 2015; Reddy, Reddy, & Jiang, 2015). On the other hand, other physical crosslinking mechanisms, such as ionic interactions and hydrogen bonding, can also be introduced to reinforce the thermogels (Qin et al., 2018; Zhang, Chen, Deng, Ngai, & Wang, 2017). Although the ionic interactions between polyanions and polycations have been popularly adopted for the formation of ionically complexed hydrogels with good mechanical properties (Daemi et al., 2016; Deng et al., 2014), they are not suitable for conventional thermogels that have typical non-ionic block copolymer structures (PEG-PPG-PEG and PEG-PLGA-PEG).

Recently, N-acyl glycol chitosans (AGCs) have been investigated by our group as a new class of polysaccharide-based thermogelling hydrogels and demonstrated many promising properties, such as fast gelation kinetics and good physical stability, for various biomedical applications such as injectable drug delivery, cell encapsulation, and 3D cell culture (Cho, Cho et al., 2016; Cho, Li et al., 2016; Cho, Park et al., 2016; Li et al., 2018). AGCs have been evaluated as useful thermogelling biomaterials, but their performance as injectable cell scaffolds has been limited due to their poor cell binding affinity and insufficient physical properties. To overcome the current limitations of AGC thermogels for injectable tissue engineering applications, a new blend thermogel system with enhanced cell binding affinity and tunable mechanical properties is suggested in this study. A typical polysaccharide with good cell binding affinity, hyaluronic acid (HA), which is an essential component of the ECM, is utilized as a building block for preparing blend thermogels with AGCs because HA has not only promising properties, such as biodegradability and biocompatibility, as a useful biomaterial but also carboxyl functional groups available for ionic interactions and hydrogen bonding (Jeong et al., 2017; Larrañeta et al., 2018). HA itself is also known to have a short turnover rate and limited mechanical properties, so additional chemical or physical modifications for many practical applications are possible (Fraser, Laurent, & Laurent, 1997; Prestwich, Marecak, Marecek, Vercruysse, & Ziebell, 1998; Schanté, Zuber, Herlin, & Vandamme, 2011). As illustrated in Scheme 1, the aim of this study was to develop a new thermo-irreversible hydrogel system constructed by blending AGC and HA, which produce a gel with improved characteristics, especially the cell binding affinity and tunable mechanical properties. Herein, we present a blend thermogel model using hexanoyl glycol chitosan (HGC) as a thermogelling AGC and acetylated HA (AcHA). AcHA was used instead of HA due to its amphiphilicity that may increase the miscibility with HGC and better anti-inflammatory activity compared to free HA form. (Saturnino et al., 2014). The ratio of AcHA to HGC was modulated to effectively compensate for the deficiencies of the individual hydrogel component. The AcHA/HGC hydrogels with different compositions were prepared and their physicochemical and biological properties were evaluated in terms of the thermo-irreversible sol-gel transition, mechanical properties, biodegradability, in vitro cytotoxicity, and tissue compatibility of the gel for potential application as a useful injectable scaffold system for cartilage regeneration.