Bone tissue is a highly dynamic and complex structure, which is constantly updated and iterated via the balance of the osteoblasts and osteoclasts (Rodolfi et al., 2023). The structure of bone primarily includes the periosteum, sclerotin, and bone marrow (Abdalla and Pendegrass, 2023). The periosteum is made up of fibrous connective tissue, which is rich in nerves and blood vessels and plays a vital role in bone nutrition, regeneration, and sensation (Caffarelli et al., 2023). Sclerotin is a hierarchical and anisotropic structure, which is mainly composed of dense cortical bone and spongy cancellous bone (Delawan et al., 2023; Tang et al., 2023). The dense cortical bone possesses high mechanical strength and encompasses the cancellous bone on the periphery, which possesses an excellent stabilizing and supporting function. The sponge-like cancellous bone consists of countless flaky trabeculated bones, which are laid out inside the bone. Bone marrow is present in the spongy cancellous bone space and the bone marrow cavity of long bones and is composed of various types of cells and reticulated connective tissue (Caffarelli et al., 2023; Cao et al., 2024). Thus, the bone tissue can be precisely divided into multiple levels for investigation. For example, at the micron level, the cortical bone is arranged along the long axis of the bone. An osteon is the functional basic unit of the long bone, consisting of lamellar bones arranged in concentric circles. Nerve fibers and blood vessels pass through the bones to form the Havers system. The cancellous bone, an anisotropic arrangement of rod-like trabecular bones, forms a honeycomb-like network. Observing the microscopic structure, the main component of ECM is collagen fibers, which have a diameter of approximately 35–60 nm. The collagen fibers are formed by the self-assembly of unique triple-helix biomolecules (Yin and Li, 2006; Wegst et al., 2015; Zhu et al., 2021). Hydroxyapatite crystals with superior anisotropic mechanical properties play a vital role during controlled bio-mineralization (Figure 2A).

Cartilage is a powerful gliding tissue that covers the moving extremities of the bone skeleton. However, cartilage regeneration is challenging due to its limited ability to self-heal, owing to complex structures and components and a lack of blood vessels (Zhang et al., 2023). Cartilage primarily consists of chondrocytes and ECM (mainly composed of water and type II collagen fibers) (Sun L. et al., 2023). Furthermore, the cartilage defects may initiate the development of osteoarthritis (OA). One work demonstrated bottom-up primary degeneration from subchondral bone during cartilage erosion in OA with aging (Wang H. et al., 2024). The special relationship between subchondral bone and cartilage via various molecular signaling has been widely reported (Wu Z. et al., 2023; Ouyang et al., 2023). For example, the subchondral bone builds a bridge between cartilage and bone to provide both mechanical and nutritional support for cartilage and maintain the stability of the cartilage microenvironment, which is the bony layer below the hyaline cartilage and cement line (Luo et al., 2024). Anatomically, the subchondral bone consists of the subchondral plate and the subchondral bone trabeculae. The subchondral plate is a dense and crisscrossed poly-porous calcified plate. The subchondral bone trabeculae possess a spongy-like cancellous structure behind the subchondral plate, which contributes to continuous bone regeneration and remodeling and maintaining the physiological homeostasis microenvironment of the cartilage (Su et al., 2023; Yu et al., 2023; Zhao et al., 2024) (Figure 2B).

Photo-sensitive hydrogels have the ability to achieve a liquid-gel transition when exposed to special wavelengths of light stimuli (Patel et al., 2022). They usually include polymeric systems and functional photoreceptive parts, in which the physicochemical properties are altered (liquid-gel) after light exposure. They have multiple biomedical applications and mainly utilize visible light. Photo-responsiveness is an attractive stimulus method because of its advantages (e.g., light is non-invasive and allows remote control of materials without byproducts). In addition, the light could be precisely modulated by irradiation parameters (e.g., light intensity, irradiation time, input power) to regulate the physical properties of the gels.

However, most natural biomaterials fail to respond to the trigger condition due to the absence of photo-responsive functional groups (Ding et al., 2015). Fortunately, these in situ response systems are achieved by introducing functional groups through photochemical addition reactions or activation of functional groups via the release of photo-caging groups and photoisomerization processes. Some bioactive materials are modified via methacryloyl (MA) or unsaturated bond groups (C=C or clickable C≡C) to respond to light triggers (e.g., gelatin methacryloyl (GelMA) (Zhang et al., 2016; Byambaa et al., 2017; Sun X. M. et al., 2018), hyaluronic acid- butyramide (HA-NB) (Zhang et al., 2016), polyethylene glycol (PEG) (Burke et al., 2019), etc.). For example, Qiao et al. (2020) designed a novel injectable in situ osteogenic polypeptide hydrogel system (GelMA-c-OGP) via ultraviolet radiation co-crosslinking with photo-cross-linkable osteogenic growth peptides (OGP). The in situ hydrogel system accelerates the bone regeneration process of osteogenic precursor cells by reinforcing the expression of osteogenic-related genes and increasing the precipitation of calcium in osteoblasts (Qiao et al., 2020) (Figure 4A). The injectable hydrogel precursor converts to solid hydrogel with UV in situ irradiation, which also can reinforce the mechanical properties (e.g., stress-strain, shear force.) of the defective bone, avoid burst release osteogenic drugs, and achieve timed release throughout the bone-healing term. Similar work was reported by Montazerian, H. to enhance the toughness, stretchability, and adhesive properties of GelMA hydrogel. They conjugated the polydopamine to the GelMA to improve the cell migration capacity and enhance bone regeneration (Montazerian et al., 2021). Furthermore, researchers have recently focused on RNA treatment due to its high effectiveness. For example, Gan et al. (2021) utilized the cholesterol-modified non-coding microRNA chol-miR-26a to promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Chol-miR-26a was conjugated to an injectable poly(ethylene glycol) (PEG) hydrogel by an ultraviolet (UV)-cleavable ester bond. The injectable PEG hydrogel was formed by a copper-free click reaction between the terminal azide groups of 8-armed PEG and dibenzocyclooctyne-biofunctionalized PEG, while UV-cleavable chol-miR-26a was simultaneously conjugated via a Michael addition reaction. Upon UV irradiation, Gel-c-miR-26a (ML^Caged) not only released chol-c-miR-26a selectively but also exhibited a significantly improved efficacy in bone regeneration compared to the hydrogel without UV irradiation and UV-uncleavable ML^Control (Gan et al., 2021) (Figure 4B).

In addition to traditional bone regeneration, the UV-mediated in situ hydrogel is combined with an autologous bone grafting technique to improve osseointegration and bone fusion. For example, Wu et al. (Wu H. et al., 2023) initiated a new method to mend the gaps between osteochondral plugs after mosaicplasty. This approach employs an injectable photo-sensitive hydrogel (BSN-GelMA), which is fabricated by GelMA loaded with bioactive supramolecular nanofibers to mimic IGF-1(IGF-1bsn) that can bind to IGF-1 receptors and improve the bioavailability, due to their high stability and tissue retention. The result demonstrated that the BSN-GelMA could realize seamless osteochondral integration in the gap region between plugs of osteochondral defects after mosaicplasty because they can prevent cartilage degeneration and promote graft survival. These bio-interactive materials offer a way to bridge the gap and enhance osteochondral integration after mosaicplasty (Figure 4C). Recently, UV-mediated in situ hydrogel became an ideal strategy for improving cartilage regeneration (Huang et al., 2018; Xue et al., 2022a). Articular cartilage has limited self-regenerative capacity, and the therapeutic methods for cartilage defects are still dissatisfactory. To overcome this problem, Hu et al. (2020) fabricated a GelMA/nanoclay hydrogel medium for sustaining the release of extracellular vesicles that come from human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (hUC-MSCs-sEVs) to improve the cartilage regeneration, which exhibited outstanding mechanical property. The results revealed that the GelMA/nanoclay hydrogel containing hUC-MSCs-sEVs stimulated chondrogenesis and healed cartilage damage by improving the expression of collagen II and glycosaminoglycan (GAG) (Figure 4D).

Although photo-sensitive hydrogels possess high sensitivity and are widely applied in research, there are several shortcomings that need to be considered in clinical application. Due to the limited ability of ultraviolet light to penetrate tissue, photoactivation must be directed in situ after gel injection, which is difficult in clinical practice. Moreover, there is a need for additional chemical reactions to graft special functional groups due to the most natural biomaterials do not have triggers for photoactivation. This complicates the preparation of photo-sensitive hydrogels and makes industrial production more difficult. Therefore, the production and preparation of photo-responsive gel precursors with high safety can improve the clinical practice of UV-stimulated in situ hydrogels.

Near-infrared (NIR) is an electromagnetic wave between visible light (VI.S) and mid-infrared light (MIR) and has a wavelength in the range of 780–2,526 nm as defined by the American Association for Testing and Materials Testing (ASTM) (Zhang et al., 2024c). The NIR spectrum belongs to the frequency multiplier and main frequency absorption spectrum of the molecular vibration spectrum due to the non-resonance of the molecular vibration (the molecular vibration is generated when the molecular vibration transitions from the ground state to the high energy level). The NIR possesses a strong penetration ability because of its special physical properties (Wang K. et al., 2024). In addition, NIR, without being strongly absorbed by body fluid, presents better tissue transparency than other wavelengths and is an ideal candidate for designing light-responsive in situ hydrogels (Raza et al., 2019). Thus, many injectable hydrogel systems have been explored to construct an in situ hydrogel in niduses via an NIR light trigger due to its noninvasive nature, high spatial resolution, temporal control, and convenience (Abueva et al., 2020) (Figure 5).

The photothermal effect is the primary mechanism of NIR-responsive in situ hydrogels. Precisely, it refers to the release of vibrational energy (heat) while the nanoparticle (NP) photosensitizer is excited by a specific band of light in the precursor (the conversion of light to heat) and then forms a hydrogel via the tune temperature (Kim and Lee, 2018). The NIR light was applied as an “on/off” trigger to remotely heat and activate thermosensitive in situ hydrogels. Similar systems are used for on-demand drug delivery and synergistic photothermal chemotherapy. For example, Liu et al. (2019) designed an injectable, thermosensitive photothermal-network hydrogel (PNT-gel) through host-guest self-assembly of photothermal conjugated polymers and ɑ-cyclodextrin. The conjugated polymer backbones can directly convert incident light into heat, endowing the PNT-gel with high photothermal conversion efficiency (g = 52.6%) and enhancing photothermal stability. Meanwhile, the mild host-guest assembly enables shear-thinning injectability and the photothermally driven and reversible gel-sol conversion of the hydrogel.

Many nanoparticles with photothermal conversion properties (including nanostructures of Au, Ag, Pt, Pd, graphene oxide, carbon nanotubes, CuS, MoS₂, and PDA) have been developed to work as multifunctional nanoplatforms to realize photothermal therapy in tissue engineering (Jiang et al., 2019). Lee et al. (2021) designed a novel type of NIR-triggered in situ gelation system based on the defect-rich 2D MoS₂ nano-assemblies and thiol-functionalized thermos-responsive poly (N-isopropyl acrylamide-co-acrylamide-co-2-mercaptoethylacrylamide) (PNAM). In this process, the dynamic polymer–nanomaterial interactions activate under the NIR radiation without a photoinitiator due to the photothermal characteristics of MoS₂ and the intrinsic phase transition ability of the PNAM thermos-responsive polymer. Specifically, this phase transition behavior of PNAM stems from dynamic changes in the hydrophilic properties of the lateral radicals. Thus, as the temperature increases, the hydrophobic interaction of MoS₂ nanoparticles increases with the PNAM chain. In addition, the MoS₂ nanoparticles generate heat under near-infrared irradiation to attract vulcanized PNAM chains to wrap themselves, while the defects of MoS₂ nanoparticles react rapidly with the thiol side groups to form covalent bonds (Figure 5A). Consequently, the remotely controlled on-demand release of drugs is achieved via a photothermal-induced gel-sol transition (Wu et al., 2017; Meng et al., 2018; Wang et al., 2020) (Figure 5B).

The photothermal effect of the incorporated photothermal agents not only induced the on-demand drug release through the gel-sol phase transition of thermosensitive hydrogel but also induced proliferative differentiation of cells via hyperthermia (Shen et al., 2021). Here, Zheng et al. (2020) described an NIR strategy for controlling activated cell processes (3D cell diffusion and angiogenesis) by embedding up-conversion nanoparticles (UCNPs) into the hydrogel, which is modified with a photo-activated cell adhesion motif. The UCNPs can convert NIR light (974 nm) into localized UV emission and activate photochemical reactions on demand. This light regulation is spatially controllable and dose-dependent and can be performed at different time points in cell culture without causing significant photodamage to the cells. Human umbilical vein endothelial cells (HUVEC) cells embedded in this hydrogel can form a network of blood vessels at predefined geometries determined by the irradiation pattern (Figure 6C). There are two main mechanisms of in situ hydrogel gel formation induced by NIR. The first is to realize photothermal conversion through a photothermal factor, and the other is to convert near-infrared light into ultraviolet light by UCNPs and then induce in situ gelling. However, the NIR utilization rate of both types of gelatinization is very low.

Ultrasound is a potentially valuable trigger for hydrogels because of the mechanical pressure waves that oscillate at high frequencies (≥20 kHz) and produce a range of thermal and nonthermal effects (Wiklund et al., 2012). The thermal effects refer to the transition from acoustic energy to thermal energy, inducing a rise in temperature in tissues (Gao et al., 2005). The nonthermal effects, also named cavitation effects, refer to the tiny gas bubbles generated by ultrasound vibration (Manouras and Vamvakaki, 2017).

Ultrasound can trigger reactions in polymers, which has been reported since the 1930s (Price, 2003). This system enables noninvasive, spatiotemporally controlled modulation and morphological properties using focused ultrasound in the body. For example, Wu S. et al. (2023) designed and prepared a novel ROS-responsive controlled-release in situ drug nanohydrogel for articular cartilage repair (Figure 6). Ultrasonic-cleavable ketothiol (TK)-based nanoliposomes are loaded into cartilage drugs (KGNs), thrombin, and sonics (PpIX). Partial rupture of liposomes under ultrasound stimulation is accompanied by enzymatic reactions of fibrinogen and thrombin to achieve in situ gelation for filling tissue defects. In addition, the controllable release of KGN drugs and the appropriate amount of ROS production were achieved by regulating the ultrasound conditions. More importantly, the appropriate amount of ROS and the continuous release of KGN in the in situ nanocomposite hydrogel microenvironment effectively promoted the differentiation of BMSCs in the direction of cartilage formation by stimulating the Smad5/mTOR signaling pathway, thereby accelerating the repair of articular cartilage defects. Current research also focuses on inducing in situ gelatinization through cascade reactions. The development of ultrasound in situ hydrogels is slow due to the lack of ultrasound responsiveness of natural gel materials. There is still a long distance to clinical application.

Magnetic hydrogels (MHGs) have extensive tissue engineering and biomedical applications and possess the advantages of quick response, biocompatibility, tunable mechanical properties, porosity, and internal morphology (Asadi et al., 2024; Liu et al., 2024). MHGs can be fabricated by blending exogenous additives (e.g., paramagnetic, ferromagnetic) in the polymeric matrix and placing the matrix in a magnetic field (static or oscillating) (Zhou K. et al., 2024; Zhang K. et al., 2024).

The most common and straightforward method for making magnetic composite hydrogels is blending with magnetically responsive nanoparticles. For example, Fe₃O₄ is one of the most common magnetic nanoparticles (MNPs) mixed with a polymer for the preparation of magnetic hydrogels. The Fe₃O₄ magnetic nanocomposite gel can be adjusted by applying a magnetic field (Yan et al., 2022). In addition, the type of hydrogel networks and MNPs (the content, size, distribution), as well as the interactions between MNPs and polymer networks, can make a significant impact on the physicochemical properties and magnetic responsiveness of magnetic hydrogels (Thoniyot et al., 2015). Magnetite and maghemite have been prepared and loaded into dextran hydrogel to produce a magneto-sensitive composite hydrogel system by photopolymerization (Brunsen et al., 2012). The in situ synthesis of MNP-based composite hydrogels is more accurate in terms of particle size and hydrogel architecture (Farzaneh et al., 2021). Gao et al. (2014) developed a magnetic hydrogel composed of anion networks of poly (2-acrylamide-2-methylpropane sulfonic acid) (PAMPS) and anisotropic nanocrystals of Fe₃O₄. The Fe₃O₄ nanocrystals were prepared in situ in the PAMPS networks, where negatively loaded SO₃ facilitated the adsorption and uniform distribution of iron ions in the hydrogel network. They found that the size and shape of Fe₃O₄ nanocrystals can be adjusted by modifying the crosslinking density of hydrogel without applying stringent experimental conditions (Gao et al., 2014). Although the magnetic response in situ hydrogel has achieved excellent results, it still faces great challenges in the clinical transformation stage due to the complex composition and the need to introduce magnetic response factors. Therefore, simplifying the composition of magnetic response in situ hydrogels and increasing their usability will be conducive to their use in tissue engineering.

Amongst several stimuli-responsive in situ hydrogel systems, the thermal stimulation in situ hydrogel is one of the most widely investigated classes because they have a lower critical solution temperature (LCST) and can flexibly modify their physical characteristic (e.g., changes in volume) in response to alters in various temperatures (Ma et al., 2019; Li X. et al., 2021; Wang J. et al., 2021; Nguyen et al., 2021). Thermo-gel behavior combines different thermosensitive mechanisms, such as the precipitation of water-insoluble polymers above LCST and then the aggregation of thermo-gelling (Zhang K. et al., 2021). Precisely, the hydrogel precursor is liquid when kept at temperatures lower than the critical solution temperature and transforms into solid-phase gel at body physiological temperature (Zhang et al., 2019). This smart strategy can form an in situ hydrogel and offer the possibility of injecting those systems and conforming to any irregular defects (El-Husseiny et al., 2022).

Many kinds of thermo-responsive hydrogels have been reported to improve the regeneration of bone tissue. Temperature-responsive hydrogels usually possess distinctive thermoplastic characteristics owing to hydrophobic moieties (e.g., propyl, ethyl, methyl group, etc.) (Sood et al., 2016). For example, poly (N-isopropyl acrylamide) (PNIPAm) gel has an obvious coil–globule transition at 32 °C (LCST); the compound is hydrophilic below this temperature and hydrophobic above it. Thus, these injectable precursors show a solution state at ambient temperature; the gel form is produced in situ at physiologic temperature (Karimi et al., 2016). Many thermo-responsive hydrogels based on PNIPAms have been designed and applied in tissue engineering, including N-isopropyl acrylamide hydrogel crosslinked with di(ethylene glycol) divinyl ether poly (NIPAAm-co-DEGDVE) (Werzer et al., 2019) and polymer poly [(propylene sulfide)-block-(N,N-dimethyl acrylamide)-block-(N-isopropyl acrylamide)] (PPS-b-PDMA-b-PNIPAAM) (Gupta et al., 2014).

Poloxamers are another biodegradable biomaterial that has been widely applied to thermo-responsive in situ hydrogel systems in tissue engineering due to the suitable sol-gel transition temperature (Liu et al., 2020; Zhang T. et al., 2021). For example, Madry et al. (2020) designed an injectable and thermosensitive hydrogel based on poly(ethylene oxide) (PEO)–poly(propylene oxide)(PPO)–PEO poloxamers, capable of controlling the release of a therapeutic recombinant adeno-associated virus (rAAV) vector overexpressing the chondrogenic Sox9 transcription factor in full-thickness chondral defects. This work indicated that the rAAV-FLAG-hsox9/PEO–PPO–PEO hydrogel significantly improves cartilage repair with a collagen fiber orientation similar to the normal cartilage and protects the subchondral bone plate from early bone loss in a minipig model (Madry et al., 2020) (Figure 7A).

Several natural biomaterials (e.g., cellulose and gelatin) and artificial synthetic polymers (e.g., poly (N-isopropyl acrylamide) (PNIPAAm), poloxamer, and polyfluorene 127, etc.) are exploited to fabricate thermosensitive hydrogels which suggest promising applications for bone tissue engineering (Chatterjee et al., 2018). For example, Ren et al. (2015) developed a thermosensitive hydrogel by gelatin grafted with PNIPAAm (Gel–PNIPAAm). These injectable hydrogel precursors exhibit an excellent biocompatible, sol-to-gel transformation property at physiological temperature and an excellent fill-ability for complex and irregular bone defects. The result demonstrated that this gel–PNIPAAm thermosensitive in situ hydrogel can induce bone differentiation and improve bone regeneration compared to the control in a cranial damage model.

As recently reported, several natural polysaccharides (e.g., agarose, amylose, carrageenan, etc.) possess thermo-gelling properties, but they failed to form gels at body temperature (Zhang B. et al., 2024; Elizalde-Cárdenas et al., 2024; Riviello et al., 2024). Fortunately, when xyloglucan is partially degalactosylated (Deg-XG), it will achieve sol-to-gel at a physiological temperature of concentration over 2 wt% (Chandramouli et al., 2012). For example, E.M and colleagues presented a simple strategy based on the synergic combination of forming in situ hydrogels and spheroids of adipose stem cells (SASCs) with great potential for minimally invasive regenerative interventions aimed to treat bone and cartilage defects. They loaded SASCs in aqueous dispersions of partially Deg-XG and either a chondroinductive or an osteoinductive medium to induce bone and cartilage regeneration. The dispersions rapidly set into hydrogels when the temperature was brought to 37°C. The physicochemical and mechanical properties of the hydrogels are controlled by polymer concentration (Muscolino et al., 2021) (Figure 7B). Similar results were reported by Dispenza et al. (2017). They designed the same injectable thermosensitive hydrogel (Deg-XG loading with the growth factor FGF-18) to promote cartilage reconstruction at gelation at 37°C.

Thermosensitive hydrogels were also extensively applied in the effective treatment of osteoarthritis by local injection to replace oral administration. For example, Seo et al. (2022) designed an injectable polymeric nanoparticle hydrogel system with poly(organophosphazenes), which is loaded with triamcinolone acetate (TCA, nonsteroidal anti-inflammatory drugs) for achieving a long-term anti-inflammatory effect and treat osteoarthritis. The TePN precursor turned into a solid hydrogel after intra-articular injection, and the formed 3D TePN hydrogel released TePNs for months in an in vivo microenvironment. Long-term release of TCA treats OA by inhibiting matrix proteinase (MMP) expressions in the cartilages via decreased pro-inflammatory and increased anti-inflammatory cytokine expressions (Figure 7C).

Insufficient vascularization is still a great challenge in the regeneration of critical-sized bone tissue defects (Takaoka et al., 2024; Yuan et al., 2024). The formation of the H-type vessel plays a key role in the progress. To address this problem, Zeng et al. (2022) designed an injectable thermosensitive demethoxyamine (DFO) gelatin microsphere (GMs) hydrogel complex to stimulate the production of functional H-type blood vessels. The results showed that the DFO-GMs thermosensitive hydrogel complex could stimulate the production of functional H-type blood vessels and effectively promote the proliferation, formation, and migration of HUVECs in vitro. Moreover, this thermosensitive hydrogel can expand the distribution range of H-type blood vessels in the defect area and increase the native bone tissue, which was confirmed via fluorescence immunostaining and radioactivity examination in vivo (Figure 7D).

The thermo-responsive hydrogels possess great potential to adjust the threshold response levels and allow on-demand triggers due to excellent biocompatibility, mild trigger conditions, no toxicity, etc. More studies should examine the integration of thermo-responsive hydrogels with multi-gradient temperature triggers. In addition, thermo-responsive hydrogel systems are generally regarded as crucial triggers in designing advanced external stimuli (e.g., NIR, magnetic, US, etc.) for smart, responsive platforms.

Biocatalytic reactions of enzyme complexes are especially important in naturally occurring multicellular organisms (Bleier et al., 2015). Furthermore, the enzymes catalyze the recognition of the 3D substrate structures that are adapted to the enzymes for binding. The “substrate specificity” closely regulates enzymatic activity without adverse side effects. In recent years, the enzyme medium stimulus-responsive in situ hydrogels have played an increasing role in tissue repair due to the mild reaction mechanism. Most enzymes involved in crosslinking also catalyze natural reactions in our body (Jin et al., 2011) (Figure 8). Furthermore, the enzymatic reactions are catalyzed with a neutral-pH microenvironment in an aqueous medium at moderate temperatures, which means that they can also be utilized to develop in situ hydrogels. Smart enzymatic reaction systems can be designed not only to create native extracellular matrices (ECM) but also to construct degradable biomaterials (Hubbell, 2003). These events capture, in substance, one of the most significant biological features of the ECM, which is remodeling. In addition to degradability, it is essential to adapt the gelation rate to applications such as drug administration and tissue regeneration strategies. A controlled gelation rate is essential to prevent diffusion of the precursors, ensure localized drug delivery, obtain a suitable cell distribution, and properly integrate the gel with the surrounding tissues (mainly for irregular-shape filling applications) (Teixeira et al., 2012). The enzyme stimulus-responsive in situ hydrogel has raised much attention in bone tissue engineering (e.g., OA, bone defects, fracture, etc.) (Zhou S. et al., 2024; Vidal et al., 2024). In this contribution, the enzymatically crosslinked gels are reviewed in the context of regenerative strategy applications.

Compared with physical crosslinking, the in situ hydrogels produced by chemical crosslinking possess higher stability in the physiological state and showed excellent mechanical properties, gel stiffness, and slower degradation rates due to the covalent linking of the interpolymer chain (Chai et al., 2024). In addition, they exhibit flexible biodegradation and mechanical properties by adjusting hydrogel precursors and providing longer-lasting spatial support for slow regeneration processes (Pranantyo et al., 2024). Usually, those chemical reactions promote covalent linkage in hydrogels through chemical crosslinking, such as click chemistry (e.g., Diels–Alder reaction, Michael addition reaction, or thiol–Michael addition reaction). In the assembly of hydrophilic polymers, those deterministic groups, such as COOH-, OH-, and NH₂-, are utilized to construct a hydrogel system via a covalent bond between amine-carboxylate and a Schiff alkali or isocyanate-OH/NH₂ (Liu H. et al., 2023; Wang X. et al., 2024; Qu et al., 2024).

Metal ions are an indispensable microelement in regulating whole-body homeostasis (Zhang et al., 2024b). They can be elaborately designed by linking with polymer chains to transform in situ functional hydrogels to improve bone tissue regeneration (Zhang P. et al., 2024). Ions can recognize biomaterial monomers, promote crosslinking between molecules, and enhance the stability of the structure. Therefore, the ion-triggered biomaterials that form in situ hydrogels have received extensive research attention (Ramírez et al., 2024). Alginate, a naturally derived polysaccharide, has been regarded as an ideal hydrogel biomaterial for bone tissue engineering because of its biocompatibility, tunable mechanism properties, low immunogenicity, and ease of gelation by metal ions (Li et al., 2024). It is generally believed that this crosslinking mechanism is the interaction of two carboxyl groups on the adjacent polymer chain with divalent cations to form an ion bridge or chelate with ions via the hydroxyl and carboxyl groups on polymer chains. Thus, alginate-based hydrogel systems can be flexibly manufactured to achieve metal ions response (e.g., Ca²⁺, Si²⁺, etc.) (Chen et al., 2024; Li et al., 2024; Zhong et al., 2024). Usually, alginate-based hydrogels are prepared by contacting alginate precursors with CaCl₂ aqueous solutions. However, the gelling rate is too fast and difficult to control when using CaCl₂ as a crosslinker. Thus, various crosslinking retardation agents have been applied to tune the gelation rate to form an injectable in situ alginate-based hydrogel (Nandi et al., 2008). For instance, Jung et al. (2018) prepared in situ gel alginate (ALG)/HA hydrogel system with a controlled gelatin rate by blending CaSO₄ and Na₂HPO₄ as crosslinking retardation agents. The ALG/HA hydrogels possess a controlled gelation rate in vivo. It is possible to stabilize and control the release of bioactive molecules (BMP-2 immobilized in the hydrogel for 5 weeks) for bone regeneration. The results of cell culture and animal studies on micropigs revealed that osteogenesis differentiation of hBMSCs improved with the increase of HA components in BMP-2-stimulated ALG hydrogels. In addition, hBMSCs/BMP-2 loaded in ALG/HA hydrogels can extensively enhance bone regeneration by synergistic action compared to single ALG/HA hydrogels.

Metal-chelating hydrogels are physical hydrogels that are completely crosslinked by complexes between ligand-modified polymers and metal ions. The mechanical properties of these hydrogels depend on the density and kinetics of the metal coordination interaction (Raymond et al., 2015). For example, HA and the catechol compound gallic acid (GA) have iron coordination activity. The conjugates of HA and GA (HA-GA) immediately form the hydrogel in the presence of the Fe³⁺ ion. After subcutaneous injection into mice, HA-GA and Fe³⁺ ions form an in situ hydrogel and remain at the injection site for at least 8 days (Ko et al., 2019). The ions in the ion crosslinking can replenish the trace elements of the human body. However, the amount of ions in the gel is much higher than the normal level of ions in the human body. Therefore, the safety of the tissues surrounding the gel must be thoroughly evaluated and studied.