Proteins are polymers with clearly defined sequences, and their chemical properties at the molecular level (crosslinking density and number of bioactive ligands) can be well controlled (Krishna and Kiick, 2010). Protein-based hydrogels can be composed entirely of proteins or mixed networks of proteins and other polymers. Protein-based hydrogel components are attractive because of their structural designability, specific biological functions, and stimuli responsiveness (Schloss et al., 2016). Compared with other polymers, the multifunctionality of proteins endows protein hydrogels with a variety of biofunctions. Collagen hydrogels are natural materials with excellent biocompatibility, adhesiveness, and biodegradability (Gao et al., 2018). Yang et al. (2021) designed a collagen hydrogel loaded with the small-molecule drugs LDN193189, SB431542, CHIR99021, and P7C3-A20. In vitro and in vivo experiments verified that this combined collagen hydrogel delivery system effectively promoted the differentiation of endogenous neural stem cells (NSCs) into neurons and inhibited their differentiation into astrocytes. Zhou et al. (2020) used gelatin methacryloyl (GelMA) hydrogel as a carrier to load bone mesenchymal stem cells (BMSCs) and neural stem cells (NSCs) and implanted them into animals. GelMA hydrogel is commonly used for drug delivery in vivo and is a photosensitive hydrogel composed of a processed natural ECM. The mechanical properties of GelMA can be changed by adjusting the hydrogel concentration, crosslinking degree, and gel time to simulate the softness of the spinal cord (Nichol et al., 2010). Owing to the structural properties of three-dimensional GelMA hydrogel, it has good permeability to oxygen and nutrients (Chen et al., 2019). GelMA hydrogels can also promote cell survival, proliferation, and migration, and NSCs differentiate into neurons, reducing the formation of astrocytes (Fan et al., 2018). Fan et al. (2022) directly loaded bone marrow stem cell-derived exosomes (BMSC-exosomes) into GelMA hydrogel scaffolds, and as the scaffolds were implanted into animals, they could enhance local NSC recruitment and promote neuronal and axonal regeneration, resulting in significant functional recovery in the early period in an SCI mouse model. Fibrin is a common raw material for protein-based hydrogels. Sudhadevi et al. (2021) synthesized a fibrin hydrogel loaded with neural progenitor cells (NPCs). Fibrin hydrogel is composed of a fibrinogen solution mixed with thrombin. The porosity, pore size, and fiber chain thickness were tuned by varying the amount of thrombin. Different thrombin contents also affect the morphology of NPCs and cell survival in vitro. In addition, fibrin hydrogel as a flexible scaffold can induce neural stem/progenitor cells (NSPCs) to differentiate into dopaminergic/noradrenergic neurons and synaptic protein networks (Bento et al., 2017). King et al. (2010) examined four biomaterials that can be injected into an injury site and gel in situ: collagen, viscous fibronectin, fibrin, and fibrin + fibronectin (FB/FN). The in vivo experiments were divided into four groups and injected into the knife-cut cavity in the rat spinal cord. At 1 week, all four materials showed good integration with the host spinal cord and supported some degree of axonal ingrowth. But at 4 weeks, the FB/FN mixture showed the best combination with the host spinal cord tissue and supported the robust growth of axons, which demonstrated that the mixture of fibrin and fibronectin could enhance the supporting properties of the scaffold.

Polysaccharides exist in organisms and can be obtained from renewable sources. Polysaccharide-based hydrogels can retain large amounts of water, are usually non-toxic and biocompatible, and possess special physical and chemical properties (Coviello et al., 2007). Kushchayev et al. (2016) demonstrated that hyaluronic acid (HA) hydrogel had a neuroprotective effect on the spinal cord by decreasing the magnitude of SSCI. It can reduce disorganized scar tissue and retain neurons near and above the lesion. Wen et al. (2016) fabricated a novel scaffold composed of an anti-Nogo receptor antibody (antiNgR)-modified HA hydrogel and poly(lactic-co-glycolic acid) (PLGA) microspheres containing BDNF and VEGF. They showed that the implants and host tissues could be well integrated and that inflammation and glial hyperplasia were inhibited. In particular, a large number of new blood vessels and regenerated nerve fibers were found inside and around the implants. Chitosan is prepared by the N-deacetylation of chitin, which is a natural polysaccharide obtained from the exoskeleton of crustaceans or the cell wall of fungi. Chitosan has excellent biocompatibility, biological activity, biodegradability, and high mechanical strength and is widely used in the field of tissue engineering (Othman et al., 2021; Yu et al., 2021; Liu et al., 2022). For example, Chedly et al. (2017) prepared physical chitosan microhydrogels as scaffolds for SSCI restoration and axon regeneration. Animal experiments showed that chitosan scaffolds could promote the regeneration of axons, and growing axons were myelinated or ensheathed by endogenous Schwann cells that migrated into the lesion site. Moreover, the chitosan hydrogel could regulate the inflammatory response at the injury site and improve the local microenvironment. Mahya et al. (2021) used chitosan and alginate as raw materials to crosslink and synthesize composite hydrogels containing berberine-loaded chitosan nanoparticles. The mixing of chitosan nanoparticles and alginate can regulate the swelling and degradation of hydrogels, thereby providing a suitable microenvironment for spinal cord tissue engineering. Huang et al. (2021) designed a thermosensitive quaternary ammonium chloride chitosan/β-Glycerophosphate (HACC/β-GP) hydrogel scaffold combined with BMSCs. BMSCs were transfected with adenovirus containing GDNF gene. GDNF can promote the differentiation of BMSCs into neurons (Zhu et al., 2015), whereas HACC can provide a non-toxic microenvironment that supports cell adsorption and growth. Heparin is another raw material commonly used for polysaccharide hydrogels. Heparin-based hydrogels are widely used in various applications, including implantation, tissue engineering, biosensors, and drug-controlled release, owing to the 3D constructs of hydrogels (He et al., 2019a). Albashari et al. (2020) reported a injectable heparin hydrogel loaded with bFGF and dental pulp stem cells (DPSCs). This team injected it into spinal cord defects of Sprague-Dawley rats. Animal experiments have shown that the system could regulate the inflammatory response, accelerate nerve regeneration, and inhibit the proliferation and activation of microglia/macrophages. Another common raw material for polysaccharide hydrogels is alginate, which can be prepared into hydrogels for cell culture (Andersen et al., 2015; Neves et al., 2020). Liu et al. (2017) transplanted Schwann cell-seeded alginate capillary hydrogels into a spinal cord lesion site. With adeno-associated viral 5 (AAV5) expressing BDNF injected and activated caudally, the number of regenerating axons significantly increased.

The cellular matrix comprises all components of the tissue, except cells, including a homogeneous matrix (proteoglycans and glycoproteins) and filamentous collagen fibers. It removes antigen components that can cause immune rejection after the acellular processing of allogeneic tissues while completely retaining the three-dimensional structure of the ECM and some growth factors that play an important role in cell differentiation, such as FGF2, transforming growth factor β (TGF-β), and VEGF (Badylak et al., 2011). The treated ECM material has excellent mechanical properties and histocompatibility that can support and connect cells in vivo, without immune rejection (Liang et al., 2020). Simultaneously, its 3D spatial structure and cytokines are conducive to cell adhesion and growth. Xu et al. (2021) compared the biological functions of a decellularized tissue matrix hydrogel derived from the spinal cord (DSCM-gel) and a decellularized matrix hydrogel derived from the peripheral nerve (DNM-gel). The results showed that the hydrogel processed by DSCM had a nano-fiber structure simulating the ECM and a relatively larger pore size. DSCM-gel provides a regenerative 3D microenvironment that enhances the survival, proliferation, and migration of NSCs/NSPCs), and is followed by a unique ability to promote neuronal differentiation and synapse formation in NSPCs.

Natural hydrogels can be loaded with drugs, cytokines, miRNAs, stem cells, and stem cell-derived exosomes for local delivery to treat SSCI. Most functional additives are loaded physically. However, they have different requirements in different situations (Chai et al., 2017). For drug loading, no substances affecting drug activity should be added during the preparation of hydrogels, and the temperature should also be within the appropriate range (Li and Mooney, 2016). Moreover, there are some limitations in delivering incompatible hydrophobic drugs. In order to address these problems, different strategies have been employed to modify the hydrogel system, including the introduction of hydrophobic domains, the addition of nanoparticles, and inclusion of cyclodextrin (CD) groups (Gu et al., 2017). For cytokine loading, a suitable temperature is required during the loading process, which cannot be inactivated. Therefore, there are certain requirements for the hydrogel type and preparation method (Schirmer et al., 2016). Stem cells and stem cell-derived exosomes have high requirements for the microenvironment of hydrogels because the survival of stem cells and exosomes requires sufficient nutritional support (Khayambashi et al., 2021). Stem cells and exosomes can be prepared in suspension together with the raw materials of the hydrogel to form an injectable hydrogel. They can also be co-cultured with hydrogel scaffolds for a certain period. After cell growth, differentiation, and adhesion to the hydrogel scaffold, it was implanted into the body to achieve spinal cord delivery (Albashari et al., 2020; Huang et al., 2021). Stem cell-derived exosomes have characteristics of stem cells, such as directional migration homing ability and low immunogenicity. Studies have shown that stem cell-derived exosomes can effectively transport bioactive substances, such as mRNA, microRNA, and protein, and have important biological functions, such as reducing apoptosis, reducing the inflammatory response, promoting angiogenesis, inhibiting fibrosis, and improving tissue repair potential (Khalatbary, 2021). Some recent studies on natural hydrogel-loaded different types of stem cells and substances that assist nerve cell differentiation and growth are summarized in Table 1 (Li et al., 2016; Thompson et al., 2018; He et al., 2019a; Yao et al., 2021; Chen et al., 2022a). During SSCI, endogenous NSCs can grow and differentiate, however, most differentiate into astrocytes rather than neurons without intervention. Hydrogels and drugs have the potential to recruit endogenous NSCs [(Lau and Wang, 2011; Chen et al., 2022a; Zabarsky et al., 2022), thus some researchers have focused on promoting the differentiation of endogenous NSCs into neurons by loading drugs or bioactive substances on hydrogels. Some drugs and bioactive substances used by researchers in recent years and their biological functions are listed in Table 2 (Zhao et al., 2016; Wang et al., 2017a; Xu et al., 2018; He et al., 2019b; Han et al., 2020; Nazemi et al., 2020; Raspa et al., 2021; Zhang et al., 2021; Gao et al., 2022; Shen et al., 2022; Xu et al., 2022).

Chen et al. (2022b) designed a hydrogel based on PVA and mixed it with molybdenum disulfide/graphene oxide (MoS2/GO). Studies have shown that compound hydrogels containing 10% (w/v) PVA exhibit the closest strength to the spinal cord and good adhesion. Hiraizumi et al. (1995) used PVA hydrogel membranes after spinal surgery at L5 in 30 adult cats. In vivo experiments demonstrated that PVA hydrogel membranes prevented the migration of inflammatory cells, thereby reducing the formation of intraspinal scar tissue and adhesion reactions. Other beneficial features of PVA include extreme resilience and low friction, eliminating the mechanical response to the spinal cord.

Nisbet et al. (2008) developed a PCL scaffold by electrospinning. PCL scaffolds were modified with ethylenediamine (ED), which demonstrated that electrospun PCL can direct the differentiation of NSCs towards a specific lineage. Similarly, Yen et al. (2019) prepared novel electrospun PCL/type I collagen nanofiber conduits for SSCI repair. In vivo experiments showed that the nanofiber conduit had the potential to repair sciatic nerve defects. It was also observed that PCL/type I collagen nanofiber conduits with excellent biocompatibility.

Another synthetic polymer, PLGA, was used by Zheng et al. (2021) to fabricate thermoreversible hydrogels. When PLGA is copolymerized with PEG, it can acquire a solid-liquid transition at physiological temperature (Wang et al., 2017b). PLGA/PEG block thermoreversible gelling polymers are widely studied hydrogels for drug delivery owing to their biodegradability and excellent biocompatibility (Chen et al., 2017). Meanwhile, baricitinib could be mixed into the PLGA-PEG-PLGA solution and injected into the site of SCI. The results showed that the hydrogel solution could gel in the body, disintegrate within 72 h, and release the drug.

Similar to the aforementioned PLGA-PEG-PLGA hydrogel, Bonnet et al. (2020) designed a poly (N-isopropyl acrylamide) (PNIPAAm)-polyethylene glycol (PEG) hydrogel. It has been demonstrated that PNIPAAm can form a hydrogel with thermosensitive and solid-liquid conversion properties (Klouda et al., 2011), and its universal conversion temperature is 32°C; however, when PNIPAAm is copolymerized with a hydrophilic polymer such as PEG, its solid-liquid conversion temperature can be increased to 37°C. In addition, their mechanical and viscoelastic properties can be tailored (Vernengo et al., 2008). Experiments showed that PNIPAAm-g-PEG improved sensory and motor recovery, and supported axonal regeneration.

It is usually used as a matrix for preparing hydrogels because it has the following advantages. First, in the acute phase of SCI, it can resist nerve fiber degeneration, reduce inflammation, inhibit vacuole, and scar formation, and protect the nerve membrane. Second, PEG coupling polymers can not only promote angiogenesis but also transport drugs or bioactive molecules to the injured site. Third, PEG hydrogels can be used as supporting substrates for stem cell growth after injury to induce cell migration, proliferation, and differentiation (Kong et al., 2017). Typically, PEG must be combined with other polymers to form a hydrogel system. Zhang et al. (2020a) designed a conductive hydrogel by combining graphene oxide and PEG. Hydrogels have good recoverability and injectability, allowing drugs to be injected in situ into the site of SCI and realizing injury repair.

Acrylic acids and their derivatives, such as polyacrylamide, can be used to fabricate hydrogel delivery systems. Dong et al. (2020) used polyacrylamide as a raw material to synthesize a conducting polymer hydrogel (CPH). In vitro experiments showed that near-infrared light irradiation enhanced the conductivity of the CPH, thereby promoting the conduction of bioelectrical signals. When the CPH is mechanically elongated, it still has high conductive durability, which can accommodate the unexpected strain of nerve tissue in motion. Wang et al. (2021b) prepared an F127-polycitrate-polyethyleneimine hydrogel (FE) loaded with extracellular vesicles for SSCI treatment. This delivery system was shown to inhibit fibrosis scar formation, reduce inflammation, promote myelin and axon regeneration, and synergistically induce effective spinal cord treatment. F127 is a copolymer of polycitrate-polyethyleneimine, which is rapidly crosslinked and solidified into a gel by ultraviolet (UV) and visible light in the presence of a photoinitiator. F127 has excellent thermo-induced gel characteristics and good biosafety, and polycitrate-polyethyleneimine can be loaded with extracellular vesicles through electrostatic interactions. The team prepared thermosensitive and injectable hydrogels based on the characteristics of these two materials, which greatly improved the possibility of their practical application in the treatment of SSCI.

Hydrogels with regularly oriented structures can act as scaffolds to support cell growth and guide axon regeneration. Yao et al. (2020) prepared an aligned fibrin hydrogel (AFG) as a cell growth scaffold to guide nerve cell growth. Stem cells were transplanted into the AFG, and the scaffolds were implanted into animals. AFG scaffolds can induce host nerve cells to migrate to hydrogels, which can replace the scaffold structure to form aligned cell fibers and create a bioactive environment for nerve fiber regeneration in vivo. It was worth mentioning that AFG has low elasticity which can better fit the soft tissue of the spinal cord. More importantly, the structure of the AFG directional arrangement was conducive to the directional growth of axons (Zhang et al., 2017; Yao et al., 2018) (Figure 5A). Yao et al. (2016) designed an AFG loaded with human umbilical mesenchymal stem cells (hUMSCs), and scanning electron microscopy (SEM) images showed that the AFG had regularly aligned internal structures at different magnifications (Figure 5B). In vitro experiments verified that hUMSCs can grow and differentiate into oriented fibrin hydrogels and form new directional axons. Four weeks after implantation of this loading system into rodents, it was found that stem cells could grow and differentiate normally in hydrogel scaffolds, and the newly formed axons grew along the fiber arrangement of the scaffolds. Subsequently, the team tested the mechanical characteristics of AFG in vitro (Cao et al., 2020). The results showed that the AFG could be stretched, knotted, twined, and clipped without breaking (Figure 5C). They further transplanted the AFG loaded with hUMSCs into the semi-resected spinal cord site of the canine. Animal experiments verified that with the help of hydrogel scaffolds, the motor functions of canines were greatly improved in SSCI. From rodents to large animals, the team took a big step in the application of hydrogels in SSCI.

The spinal cord plays an important role in electrical signal transduction in nerve cells. Hence, the reconstruction of electrical conductivity at the SCI site is beneficial for the recovery of spinal cord function (Shu et al., 2019). Zhou et al. (2018) designed a soft, highly conductive, biocompatible CPH based on plant-derived polyphenol, tannic acid (TA), and doping-conducting polypyrrole (PPy) chains. The resulting hydrogels exhibited excellent electronic conductivity (0.05–0.18 S/cm). In vitro, high-conductivity CPH inhibits the development of astrocytes and accelerates the differentiation of NSCs into neurons. In vivo, CPH has relatively high conductivity, which can activate endogenous NSC neurogenesis in the lesion area, leading to significant recovery of motor function. Similarly, Yang et al. (2022) synthesized an agarose/gelatin/polypyrrole (Aga/Gel/PPy, AGP3) hydrogel for SSCI treatment. PPy, as a conductive material, was doped into the hydrogel to impart high conductivity. In vivo studies have shown that AGP3 hydrogel provides a biocompatible microenvironment for promoting endogenous neurogenesis rather than glial fibrosis, leading to significant functional recovery. RNA sequencing analysis further showed that the AGP3 hydrogel regulated the expression of neurogenesis-related genes through the intracellular Ca²⁺ signaling pathway.

The cell adhesion ability of Arg-Gly-Asp (RGD) peptides is related to the integrins on the cell membrane. Integrins are the main family of cell surface receptors. RGD peptides have been shown to play a central role in adhesion-mediated cell migration required for tissue construction during development and repair. Recent studies have found that around 11 types of integrins can specifically bind to RGD peptides, which are antagonist peptides of integrin receptors (Liu, 2009; Vedaraman et al., 2021). Woerly et al. (2001) designed a poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) hydrogel containing RGD peptides. RGD peptides were connected to the hydrogels via chemical grafting. Experiments have indicated that the PHPMA-RGD hydrogel can reproduce some features of the ECM chemical environment. Similarly, laminin-derived peptides have cell-binding sites that can achieve specific binding. Laminin-derived peptides can chemically react with HA chains to modify the HA hydrogels. For example, Li et al. (2019) reported that laminin-derived peptides PPFLMLLKGSTR could tether HA chains through a reaction between the amino groups of the peptide and the partial aldehyde groups of aldehyde-modified HA. In addition, apart from the RGD peptides and laminin-derived peptides mentioned above, there are some ECM molecules such as collagen, fibronectin, vitronectin, and osteopontin (Onak et al., 2018; Tsiklin et al., 2022) that have corresponding cell binding sites. These peptides can play an important role in cell adhesion when modified into hydrogels.

Moreover, it is important to endow hydrogels with injectability. Injectability means that the hydrogel can be injected through a small surgical wound and solidified in vivo, which is of great significance for future clinical use. Zhang et al. (2022) developed a facile in-situ synthetic strategy for injectable lysine-containing peptide-functionalized hydrogels for the treatment of SSCI. This injectable hydrogel can fill irregular cavities to inhibit glial scar formation and significantly suppress inflammatory responses, thereby further promoting nerve regeneration. Wang et al. (2018) prepared a gelatin-based hydrogel, which was modified using polymer fibers with a shape memory function. The modified hydrogel could recover and maintain the microstructure after injection to a specified position, providing support and guidance for the differentiation of motor neurons. Hong et al. (2017) synthesized amphiphilic injectable poly (organophosphazene) hydrogels and implanted them in animals. In vivo experiments demonstrated that the hydrogel system could reconstruct the ECM and improve the microenvironment of SSCI. It contained a hydrophobic group and a hydrophilic group, which gave the hydrogel temperature sensitivity and achieved a temperature-dependent sol-gel transition behavior. The injectability of hydrogels is a promising strategy for in vivo implantation in SSCI (Caron et al., 2016; Fuhrmann et al., 2016; Tukmachev et al., 2016; Alizadeh et al., 2020).

Generally, a hydrogel, as a delivery system, possesses more than one function. Yuan et al. (2021) designed a physical dynamic cell-adaptable neurogenic hydrogel (CaNeu hydrogel). The CaNeu hydrogel network stabilized by the reversible ‘host-guest’ complexes was formed by the UV-mediated polymerization of the acryloyl group in acetone washed β-Cyclodextrin (Ac-β-CD). Hydrogels have improved physical properties and biological functions, including self-healing properties, mechanical elastic simulation of neural tissue structure and mechanical properties, injectability under gel state, shape remodeling ability, and supporting cell migration ability. The CaNeu hydrogel supported the expansion of adipose-derived stem cells (ADSCs) through the mechanical transmission signal of YAP. The dynamic network of the CaNeu hydrogel provided a permeable ECM for cell migration and growth, thereby promoting axon growth and eventually leading to improved coordination of motor-evoked potential, hind limb strength, and complete spinal cord transection in rats. Luo et al. (2022) prepared natural ECM biopolymer (chondroitin sulfate and gelatin)-based hydrogels for SSCI treatment. This hydrogel delivery system containing polypyrrole, which imparted electroconductive properties, mechanical strength (∼928 Pa), and conductive properties (4.49 mS/cm) are similar to those of natural spinal cord tissues. In addition, the hydrogels exhibited shear-thinning and self-healing abilities, which allowed them to be effectively injected into the injury site and to fill the spinal cord defect segment to accelerate tissue repair in SSCI. Functionalization or chemical modification endows the hydrogels with specific functions. These changes in the characteristics of hydrogels greatly improve their prospects for application in the spinal cord.