Preparation Techniques of CS-Based Hydrogels

Hydrogels are generally fabricated via crosslinking the polymer network through physical and chemical strategies. Based on these two basic approaches, there are various preparation techniques for construction of hydrogel configurations. On the one hand, physical crosslinkers mainly include the hydrophobic-hydrophobic, host-guest, ionic or electrostatic, crystallization and stereo complex interactions, which can cause the formation of polymer network in mild conditions (Hoffman, 2012; Sivashanmugam et al., 2015; Fan et al., 2019; Zhu C.X. et al., 2019; Wang et al., 2020a). Physically crosslinked hydrogels possess great advantages in biological applications because of the absence of chemical crosslinkers that may bring about the unpredictable and potential toxicity for the tissues, but their reversible architectures, poor stability and low mechanics greatly limited the scope of applications (Zhang et al., 2013; Maitra and Shukla, 2014; Parhi, 2017; Li et al., 2018). On the other hand, chemical crosslinkers are constructed by the covalent linkage of polymer chains together within the network using high-effective synthetic methods, such as click chemistry, Schiff base reaction, free radical polymerization, etc. (Hennink and van Nostrum, 2012; Wang et al., 2013; Zhang J.F. et al., 2019). On account of the permanent and irreversible junctions among polymeric chains, chemically crosslinking hydrogels possess stable structures and excellent mechanics for tissue engineering fields. However, toxic chemical agents and difficult sterilization may produce adverse effects and certain insecurities.

Physico-Chemical Properties of CS-Based Injectable Hydrogels

In recent years, the application of injectable in-situ forming hydrogels in orthopedics has been widely investigated by many scientists (Gyawali et al., 2013). Unlike the prefabricated stents that require surgical implantation, injectable hydrogels can be in situ injected into the defect sites to fill in any geometric deformities using the minimally invasive surgery methods. Hydrogel is usually used for confronting bone defects in non-weight bearing parts or injured bone tissue through delivering and releasing the therapeutic biomolecules and agents once the stimulus response that triggers its physical and/or chemical properties has been changed. On account of numerous hydroxy and amine groups within the cationic CS framework, CS can be feasibly modified to improve its multifunction, such as antibacterial property, solubility, stimulus-response, adhesion and degradation behaviors (Lavanya et al., 2020). Therefore, CS-based injectable hydrogels possess more advantages on the tailor of topological structures, biodegradability behaviors, cytocompatibility and adhesive force, which can be widespread used as effective biopharmaceutical materials to promote bone regeneration (Liu et al., 2017; Shariatinia and Jalali, 2018).

Chitosan is usually combined with other natural-derived or synthetic biomaterials via the covalent and/or non-covalent bonds, yielding a varied of multifunctional hydrogels. Wherein, physical gelation is a typical approach for fabrication of CS-based hydrogels with good biocompatibility and gradual degradability to promote the cell-materials interactions and stimulate the proliferation and differentiation of osteoprogenitor cells (Berger et al., 2004). Therefore, development of CS-based injectable hydrogels would allow for the effective therapy for the bone regeneration, especially for the irregular defective sites of bone tissue. Based on this physical gelation of CS-based injectable hydrogel, environmentally responsive injectable hydrogels, such as pH, light and temperature, are widely used for repair of large bone defect, because an externally applied trigger for gelation can easily tailor sol-gel transition with the facile permeation into the defect sites and quick gelation in situ to fully seal the injury. Recently, Li et al. (2015) prepared a series of soluble UV-crosslinkable CS-based injectable hydrogels by modification of amine groups of CS with methacrylic anhydride in the absence of any activators. These methacryloyl groups improved the CS solubilization in water by impairing intra-/intermolecular hydrogen bonds and provided the crosslinkable CS derivatives. Xia et al. (2017) reported a photopolymerized injectable water-soluble maleilated chitosan (MCS)/poly(ethylene glycol) diacrylate (PEGDA) hydrogels, which had faster gelation rate, higher compressive strength than MCS hydrogel under UV radiation. Control of the MCS/PEGDA ratio could tailor the swelling behavior and mechanical properties of composite hydrogels that could promote the L929 cells attachment and tissue regeneration. Instead of UV light, Hu et al. (2012) prepared CS hydrogels crosslinked under visible light for tissue engineering. By tailoring three various blue light initiators (camphorquinone, fluorescein and riboflavin), methacrylate glycol CS can form the CS hydrogels under the visible light (400–500 nm at 500 mW cm^–2), displaying good mechanical properties and less damages to cells than energetic UV light. Further, diverse therapeutic agents or drugs can be encapsulated into these temperature and pH-responsive injectable hydrogels to form the multifunctional hydrogels in tissue engineering and drug delivery systems. For example, Qi et al. (2013) incorporated the bone morphogenetic proteins (BMPs) and mesenchymal stem cells (MSCs)/osteoprogenitors into CS-based injectable hydrogels. The thermo-sensitive CS/poly (vinyl alcohol) (PVA) composite hydrogel can offer the strength support and tailored degradation time, which induced the tissue repair via the space conduction and occupation of defect part. In addition, compared to the pure CS hydrogel, introduction of non-degradable PVA could obviously postpone the degradation and prolong the self-healing term of tissue within the defect center.

Besides, rheological properties also play vital roles in injectable CS-based hydrogels, especially for the gelation, firmness and durability of crosslinking network, which provide intriguing guidance on the effective selection of appropriate chitosan for wide application. Many studies on rheological properties of chitosan in solution have been conducted to characterize the effects various parameters such as polymer concentration, degree of deacetylation, concentrated chitosan, chitosan and vinyl polymers, temperature, shearing and storage time, and addition of other polymers/nanoparticles on the viscoelastic behaviors, which are closely relative to a peculiar mechanism of association with the double-chain strands connected network (Hwang and Shin, 2000; Chen et al., 2003; Martiínez et al., 2004; Lewandowska, 2009; El-hefian and Yahaya, 2010; Racine et al., 2017). Until now, injectable hydrogels are widely applied for tissue engineering and orthopedic applications. Unlike surgical implantation methods, pre-fabricated hydrogel can be injected into any geometries of defect bearing sites using minimally invasive procedures and quick in situ gelation through a sol-gel transition once exposure to a simulative change. In addition, injectable hydrogels can also be blended with other biomolecules, therapeutic agents and cells to form multifunctional carriers to repair the bone and dental tissues (Figure 2; Saravanan et al., 2019).

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FIGURE 2

Schematic representation of injectable hydrogels for treating bone loss at defective sites. Reproduced from Saravanan et al. (2019) with permission from Copyright 2019 Elsevier.

pH-Responsive CS-Based Injectable Hydrogels

Chitosan and its derivatives are a series of natural biopolymers with good biocompatibility, pH sensitivity, enzymatic biodegradability and polycationic attributes. Especially, tailor the protonation/deprotonation of –NH₂ group can obviously furnish CS-based injectable hydrogels with pH-responsive behaviors (Xu and Matysiak, 2017). Lower pH can protonate amine group to induce the electrostatic repulsion, and thus the polymer chains can easily expand and interact with water molecules to effectively improve the water-solubility. In contrast, higher pH can deprotonate the -NH₂ group and cause the collapse of globular structure, severely impairing the water-solubility of CS. In this case, the water solubility and swelling property of CS-based hydrogels is mainly relied on the its pKa value and external pH conditions (Rizwan et al., 2017). Therefore, CS-based injectable hydrogels possess the innate pH response that has been attracted considerable attentions in the bone regeneration process. In addition, incorporation of various polymers with CS can improve this pH responsiveness. Nevertheless, CS has poor solubility in alkaline and neutral solutions as well as the insufficient mechanical performance. To overcome these limitations, various physical or chemical modification strategies have been well-developed to make CS soluble without impairing its other properties.

For instance, Rogina et al. (2017) demonstrated a pH-responsive CS-hydroxyapatite hydrogel with a gelling agent of NaHCO₃. Tailoring of NaHCO₃ can achieve the quick gelation of CS-hydroxyapatite-based hydrogel within 4 min, which exhibited good viability for cell proliferation and differentiation as a potential cell carrier. Zhao et al. (2019) developed a composite CMCh-ACP hydrogel via combining the carboxymethyl chitosan (CMCh) and amorphous calcium phosphate (ACP). Incorporation of an acidifier of glucono δ-lactone could endow pH-responsive injectable hydrogels with good biocompatibility and effective cell adhesion/proliferation, which upregulated the expression of bone markers and enhanced the new bone regeneration (Figure 3).

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FIGURE 3

Schematic representation of pH-responsive CS-based hydrogels for bone tissue engineering. Reproduced from Zhao et al. (2019) with permission from Copyright 2019 American Chemical Society.

Thermo-Responsive CS-Based Injectable Hydrogels

Chitosan itself has not thermosensitive behaviors, and thermo-responsive CS-based hydrogels are generally fabricated via introducing the thermo-responsive polymers into CS-based hydrogels (Argüelles-monal et al., 2018). In views of simple regulation and easy utilization for in vitro and in vivo testing, temperature is recognized as a typical stimulus for the hydrogel system. In recent years, ideally thermo-responsive CS injectable hydrogels with the sol-gel transition at a physiological temperature have been developed in application of tissue engineering because of their temperature response, attractive moldable ability, tailorable rheological property, excellent biocompatibility and biodegradability to the cells and tissues, which can provide suitable manipulation and enhance cellular activity for bone and dental regenerations. In addition, encapsulation of diverse polymers (natural and synthetic polymers), bioactive molecules and nanoparticles can also expand the functional properties of CS injectable hydrogels for biomedical applications (Mohebbi et al., 2019; Zarrintaj et al., 2019; Bagheri et al., 2020; Khalili et al., 2020; Mahmodi et al., 2020; Nourbakhsh et al., 2020). For example, CS/β-glycerophosphate (GP) hydrogel is a famous scaffold for wide-range delivery of growth factors, cells, small drugs and nucleic acid. Since cationic CS chains can be connected with negatively charged GP molecules via the electrostatic attraction and hydrogen bonds, injectable CS/GP hydrogels can achieve structural optimization systems with a sol-gel transition temperature of 37°C for the bone regeneration (Figure 4; Dhivya et al., 2015; Saravanan et al., 2019).

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FIGURE 4

Schematic representation of thermal-responsive CS/β-GP hydrogels encapsulating with various growth factors, cells, drugs and nucleic acids for bone tissue engineering. Reproduced from Saravanan et al. (2019) with permission from Copyright 2019 Elsevier.

The gelation mechanism of this thermo-responsive CS/GP hydrogel has been detailedly elucidated as follows (Chenite et al., 2001). Firstly, on account of poor solubility of CS in neutral and basic solutions, CS solutions above pH 6.2 can result in the immediate formation of hydrated gel-like precipitate as well as the occurrence of phase separation of CS at pH >6 to form a hydrogel. Maintaining CS solution within a physiological pH (6.8–7.2) in the presence of GP polyol salt could generate a thermo-responsive injectable hydrogel, which kept a sol state at room temperature and transferred into a gel state at physiological temperature (cloud point of ≥37°C). Incorporation of GP components into CS solutions can reduce electrostatic repulsion between phosphate groups of GP and amino groups of CS, enhance CS-CS hydrophobic interactions and increase hydrogen bonding effects between CS chains during gel formation, and therefore the pH range can be modulated from 7.0 to 7.4 and the sol-gel transition temperature can be tailored at 37°C. The temperature dependence of CS/GP hydrogels has been investigated by the rheological studies. Owing to the hydrophobic interactions between CS chains and glycerol groups, CS chains aggregation can be prevented by the CS-water interactions at low temperature. Once upon heating, water molecules are partially removed from the glycerol moieties and CS chains can associate and aggregate into a gel formation. In this CS/GP gelling system, temperature rise can also tailor CS-CS hydrophobic interactions in addition to the regulation of electrostatic forces, because the orientation of dipolar water molecules surrounding CS polymer chains is effectively limited via increasing the vibrational and rotational energy of water molecules. These energized water molecules are removed around the CS polymeric chains, thereby causing the interconnection among dehydrated hydrophobic segments. At low temperatures, CS displays a helical structure due to the presence of intramolecular hydrogen bonds and the masking of physical connections. At high temperature, the decreasing number of intramolecular hydrogen bonds allows the CS molecules to unfold and facilitate the gelation. Thus, pH and temperature are significantly important factors for this thermo-responsive CS/GP injectable hydrogels.

Polymers Introducing the CS-Based Injectable Hydrogels

Incorporation of various polymers like naturally-derived (e.g., alginate, hyaluronic acid, collagen, starch, and silk fibroin) and synthetic macromolecules [e.g., poly(vinylalcohol), polyethylene glycol and polycaprolactone] into CS hydrogels can further improve their multifunctionality and thermo-responsive behaviors for effective bone repair. For example, Bagheri et al. (2019) prepared a kind of electroactive hydrogel based on chitosan–aniline oligomer and agarose, wherein the aniline oligomer can tailor the swelling, degradation rate, thermal and conductive properties of CS-based hydrogels. Owing to its conductivity, control of the electrical stimulus can manipulate the on-demand drug release that may promote the cell activity, growth and proliferation. Chen et al. (2013) prepared a biocompatible injectable thermo-responsive HA-CPN/BCP hydrogel based on a hyaluronic acid-g-chitosan-g-poly (N-isopropylacrylamide) containing biphasic calcium phosphate (BCP) ceramic microparticles. The HA-CPN/BCP possessed better biocompatibility with human fetal osteoblast cell attachment, proliferation and osteoblastic differentiation. In addition, higher mechanical strength and elasticity of these thermo-responsive injected hydrogels was benefited for calcium deposition, extracellular matrix mineralization and generation of ectopic bone tissue. Additionally, incorporation of synthetic polymers endows the CS hydrogels with multifunction to achieve the advanced regulation. Wu et al. (2018) constructed a thermo-sensitive NIPAAm-g-CS hydrogel with disulfide crosslinkers throughout the networks (Figure 5), and found that three types of cells exhibited excellent biocompatibility with favorable cell attachment, growth and proliferation.

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FIGURE 5

(A) Synthetic preparation of NIPAAm-g-CS. (B) Gelation mechanism of physical cross-linking (helix-coil structure) and chemical cross-linking (disulfide bonds). Reproduced from Wu et al. (2018) with permission from Copyright 2018 Elsevier.

CS-Based Hydrogels for Bone Regeneration

Bone tissues are highly functional connective that can constitute the human skeleton, provide the mechanical support, protect internal organs and participate in many physiological activities (Arvidson et al., 2011). Once it is injured, bone tissues can undergo the self-repair process by stimulating MSCs toward osteogenic differentiation and forming the neo-angiogenesis for small size of bone defect, but large-size defects should require bone grafts in the clinical procedures (Gómez-Barrena et al., 2015; Loi et al., 2016). On account of low immunogenicity, physiological inertia, osteoconductivity and osteogenesis, CS-based hydrogels can significantly promote cell proliferation and cell adhesion, presenting great application prospects in bone tissue regeneration (Jiang et al., 2019). In addition, to improve its mechanics and multiple function, CS-based hydrogels are always required to be mixed with other synthetic or natural polymers and bioactive pharmacological molecules to construct multifunctional biomaterials scaffolds. For example, Oudadesse et al. (2020) proposed a nBG-loaded hybrid CS-based hydrogel using freeze-gelation method. The surface area, porosity and mechanical properties of BGN/CH composite hydrogel could be tailored by altering the BGN proportion, which could favor the generation of apatite layer on the surface to promote the direct bone bonding with the implanted hydrogels. Zhang Y.H. et al. (2019) developed a biodegradable hybrid DN hydrogel via interspersing a methacrylated gelatin network into a nanocomposite hydrogel comprised of methacrylated chitosan and polyhedral oligomeric silsesquioxane. Based on its easy manipulation and excellent mechanics, hybrid DN hydrogels could preferentially guide the MSCs toward the osteogenic differentiation in vitro and accelerate new bone regeneration in situ using a rat of calvarial defects. These advantageous characteristics and attractive capacities endowed these functional DN hydrogels with great potentials for stem cell therapy and tissue engineering application. It is noted that tunable osteogenesis and degradation rates are important for repairing the bone defect. Huang et al. (2020) prepared a novel composite CaCO₃/MgO/CMC/BMP2 scaffold with high mechanical strength, mineralization ability, biomimetic activity and osteogenic differentiation. The sustainable release of Mg²⁺ and BMP2 played an important role in activating the phosphorylation of ERK1/2 and Akt pathways, which guided the osteogenesis formation using an in-situ rat calvarial defect model (Figure 6).

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FIGURE 6

Schematic preparation of CaCO₃/MgO/CMC/BMP2 scaffolds and their applications in vivo. Reproduced from Huang et al. (2020) with permission from Copyright 2020 Elsevier.