Bioinspired injectable hydrogels for bone regeneration

Graphical abstract

Highlights

• •A unique bioinspired design perspective, offering fresh insights for injectable hydrogel development in bone/cartilage regeneration.
• •A comprehensive overview of injectable hydrogel applications across multiple bone disease models, surpassing existing reviews in both scope and depth.
• •Systematic categorization and elucidation of various bioinspired strategies for injectable hydrogels, exceeding current methodologies in precision and comprehensiveness.
• •Exploration of the integration of emerging technologies like artificial intelligence in injectable hydrogel research, paving the way for future advancements.
• •Injectable hydrogels with attractive potential for enhancing organoid applications; their synergistic integration marking the onset of a groundbreaking era in organoid-centric research and therapeutic strategies.

Abstract

The effective regeneration of bone/cartilage defects remains a significant clinical challenge, causing irreversible damage to millions annually. Conventional therapies such as autologous or artificial bone grafting often yield unsatisfactory outcomes, emphasizing the urgent need for innovative treatment methods. Biomaterial-based strategies, including hydrogels and active scaffolds, have shown potential in promoting bone/cartilage regeneration. Among them, injectable hydrogels have garnered substantial attention in recent years on account of their minimal invasiveness, shape adaptation, and controlled spatiotemporal release. This review systematically discusses the synthesis of injectable hydrogels, bioinspired approaches-covering microenvironment, structural, compositional, and bioactive component-inspired strategies-and their applications in various bone/cartilage disease models, highlighting bone/cartilage regeneration from an innovative perspective of bioinspired design. Taken together, bioinspired injectable hydrogels offer promising and feasible solutions for promoting bone/cartilage regeneration, ultimately laying the foundations for clinical applications. Furthermore, insights into further prospective directions for AI in injectable hydrogels screening and organoid construction are provided.

Introduction

As the global population ages rapidly [1], the incidence of bone-related diseases, often accompanied by significant comorbidities [2], has sharply increased [3]. While the aging population significantly contributes to this trend, other factors, including accidental trauma, bone tumors, and critical-sized bone defects (CSBD) [4], also play crucial roles in the over two million annual bone graft surgeries performed worldwide [5], imposing tremendous burdens on patients and public health resources [6]. Conditions such as bone infections, trauma, non-union fractures, and tumor resections [7], [8] can result in CSBD that do not heal spontaneously [9], [10]. Cartilage regeneration is further hindered by the tissue's lack of blood vessels, nerves, and lymphatic systems [11], [12], [13]. Traditional bone regeneration therapies, such as autologous, allogeneic, and synthetic bone grafts, face issues like immune rejection, limited donor supply, and risk of disease transmission [14], [15]. Similarly, conventional cartilage repair methods, including osteochondral autografts and microfracture surgery, often lead to joint impairment [16], low efficacy [17], and movement disorders [18]. Consequently, there is an urgent need for novel and effective therapies for bone/cartilage repair, as current treatments often fail to address diverse clinical needs.

Bone tissue engineering has emerged as a promising alternative, integrating biomaterials, stem cells, and bioactive factors into defect sites to promote bone/cartilage regeneration [19], [20], [21]. Hydrogels, known for their diverse applications in biomedicine [22], serve as an effective biomaterial in tissue engineering, drug delivery, and regenerative medicine [23], [24], [25]. These 3D porous polymer networks mimic the extracellular matrix (ECM), providing an ideal environment for cell growth and tissue regeneration while allowing the sustained release of bioactive factors [26], [27], [28]. Compared to traditional scaffolds, hydrogels offer dynamic properties, excellent biocompatibility, biodegradability, and morphological adaptability. Their soft texture reduces inflammation and enhances integration with surrounding tissues. However, conventional bulk hydrogels face limitations such as the need for large surgical incisions, inability to conform to irregular defect shapes, implant migration, and prolonged postoperative recovery [29], all of which increase infection risks [30] and patient discomfort. To address these limitations, injectable hydrogels have been developed, allowing for the minimally invasive filling of irregularly shaped defects [31]. This significantly reduces surgical trauma and pain. Once injected, the liquid gel solidifies in situ to form a 3D matrix that supports cell adhesion, proliferation, and differentiation, facilitating the development of new bone tissue [32], [33]. Given the complex and highly regulated nature of biological systems, injectable hydrogels that closely mimic the ECM’s structure and function can effectively replicate its physical and biochemical properties. For instance, Dong et al. [34] developed an injectable chitosan (CS)/silk fibroin (SF) hydrogel system capable of releasing stromal cell-derived factor-1 (SDF-1) and kartogenin (KGN)-loaded microspheres, which enhanced mesenchymal stem cell (MSC) recruitment and differentiation into chondrocytes, promoting articular cartilage regeneration. Bai et al. [35] introduced a self-strengthening injectable hydrogel using non-covalent and Diels-Alder dual crosslinking, which stimulated bone regeneration without requiring additional cells or growth factors.

In conclusion, bioinspired injectable hydrogels present a promising approach for bone/cartilage regeneration. Although previous reviews have addressed injectable hydrogels for these applications, the bioinspired design has not been comprehensively reviewed. This article systematically classifies the physicochemical crosslinking mechanisms in injectable hydrogels and elucidates bioinspired strategies, including those inspired by microenvironments, structures, compositions, and bioactive components. The practical applications of these hydrogels in bone/cartilage disease models are also summarized. Finally, future directions for integrating Artificial Intelligence (AI) in injectable hydrogel screening and its potential in constructing bone/cartilage organoids are explored.

Injectable hydrogels

Crosslinking techniques of injectable hydrogels

Crosslinking is a critical step in forming 3D scaffolds in injectable hydrogels, ensuring an interconnected network that provides mechanical stability as the material transitions from a sol to a gel state. The choice of gelation method plays a vital role in the injectable hydrogel's structural properties and application. Current gelation methods are classified into two categories: irreversible chemical crosslinking and reversible physical crosslinking [36] (Fig. 1). Crosslinking reactions used for in situ formation strengthen the mechanical properties of the scaffold by establishing solid systems in polymeric scaffolds through supramolecular interactions such as hydrogen, ionic, or covalent bonds. This enhances polymer rigidity, flexibility, and tensile strength while improving the dynamics between cells and the matrix and fortifying resistance to environmental stressors like temperature, chemicals, and enzymatic degradation.

Fig. 1.

Crosslinking techniques for synthesis of injectable hydrogel.

Chemical crosslinking

Chemical crosslinking methods are widely used to enhance the mechanical strength and degradation resistance of hydrogels. In chemically crosslinked hydrogels, polymer chains form covalent bonds, resulting in networks with greater mechanical stability but higher volume fluctuations compared to physically crosslinked systems [37]. Key chemical crosslinking methods include click chemistry, enzyme-mediated crosslinking, photo-crosslinking, and Michael addition.

Click chemistry

Click chemistry represents a spontaneous, swift, exceedingly selective, and efficient chemical reaction between two molecules under gentle conditions [38]. It characterized by the absence of byproducts, or water being the sole byproduct during synthesis [39], making it an ideal method for hydrogel synthesis with tailored chemical and physical properties [40]. Hubbell et al. [39] pioneered biohydrogels using click chemistry, employing Michael addition to fabricate hydrogels with high selectivity under physiological conditions [41]. Click chemistry encompasses copper-catalyzed CuAAC reaction [42], copper-free catalysis such as thiol-ene reactions between thiols and double bonds and strain-promoted azide-alkyne cycloaddition reaction [43]. Within the realm of click chemistry applications, diverse injectable hydrogels have been devised to cater to drug delivery and tissue engineering requirements [44], [45], [46], [47]. For instance, Piluso et al. [48] utilized CuAAC to develop hyaluronic acid (HA) hydrogels with tunable mechanical properties. However, copper toxicity limits its use in cell-based applications, prompting researchers like Fu et al. [49] to develop copper-free systems using strain-promoted azide-alkyne cycloaddition for HA-polyethylene glycol (PEG) hydrogels in mild biophysical environments, without the need for a catalyst or exposure to ultraviolet (UV) light [50], [51], [52].

Enzyme-Mediated crosslinking

Enzyme-mediated crosslinking is characterized by high reaction rates under physiological conditions, maintaining bioactivity during in situ hydrogel formation. This eliminates concerns about bioactivity loss associated with natural polymers that may be adversely affected by the harsh chemical conditions necessitated for conventional crosslinking [53]. In general, transglutaminases (protein glutamine gamma-glutamyl transferase), tyrosinase, horseradish peroxidase, lysyl oxidase, hydrogen peroxide (H₂O₂) along with phosphatases have gained application in fabricating diverse polymeric systems and hydrogels adopted for tissue engineering applications owing to their mild reaction conditions, efficient crosslinking, and outstanding cytocompatibility [54], [55]. Among these enzymes, horseradish peroxidase and H₂O₂ emerge as the predominant enzyme-mediated crosslinking agents through phenol partial carbon–oxygen/nitrogen bond or carbon–carbon bond oxidative coupling [56], [57]. Jin and colleagues generated an injectable hydrogel by co-crosslinking of dextran and tyramine derivatives of heparin employing H₂O₂ and horseradish peroxidase [58]. Broguiere et al. [59] employed bacterial Sortase A to modify hyaluronan, yielding injectable hydrogels with excellent biocompatibility and low in vivo inflammatory responses.

Photo-Crosslinking

In recent years, visible and near-UV photo-crosslinking have gained substantial attention as a prominent gelation process in the development of injectable hydrogels due to their ability to facilitate rapid in situ gelation [60], [61]. These methods are suitable for aqueous media, allowing for spatial and temporal manipulation of the crosslinking process while minimally affecting cell viability. Certain hydrogels can undergo in vivo and in vitro photopolymerization, where the interaction between photoinitiators and light induces free radical generation and subsequent polymerization. Qi and colleagues [62] developed injectable hydrogels from sericin (Ser) functionalized to sericin methacryloyl (SerMA) using UV light for crosslinking, which promoted chondrocyte proliferation and effectively formed artificial cartilage. Liu et al. [63] successfully prepared injectable hydrogels by utilizing amino gelatin (Gel) and aldehyde methacrylate sodium alginate (SA) that exhibited improved mechanical properties, remarkable biocompatibility characteristics and manageable degradation rate.

Michael addition

Michael addition, a reaction between nucleophiles and electrophiles, has been extensively employed in the development of injectable hydrogels due to its mild reaction conditions and controlled reaction [63], [64], [65], [66]. Typically, Michael addition denotes the addition of thiol-containing polymers to activated α,β-unsaturated carbonyl polymers under basic conditions [67]. Given that proteins inherently contain thiols in cysteine residues, hydrogel synthesis between proteins and vinyl-containing polymers becomes easily achievable. The advantages of Michael addition in biomedical applications include its high selectivity, absence of toxic reagents or byproducts, and compatibility with injectable systems [68]. Common biomaterials like HA, PEG, and CS are frequently used in injectable hydrogels designed for cartilage tissue engineering via Michael addition under physiological conditions [65], [69], [70], [64]. Jin and colleagues [69] designed HA injectable hydrogels through Michael addition reaction, which demonstrated significant promise for use in cartilage tissue engineering. Similarly, Fiorica and colleagues [71] formulated two HA injectable hydrogel variants with controllable swelling properties that incorporated articular chondrocytes. Liu et al. [72] developed hydrogels via Michael addition reaction between thiolated HA, thiolated Gel and acrylated PEG aiming at osteochondral regeneration in a rabbit model.

Physical crosslinking

A prevailing inclination in injectable hydrogel development involves the utilization of physical crosslinking techniques as chemical crosslinking often employ toxic crosslinkers that can negatively impact the encapsulated substances such as proteins and cells within the hydrogel, and removing or extracting unreacted crosslinkers prior to in vivo application is necessary. Different from chemical crosslinking, physical crosslinking overcomes these limitations by relying on intermolecular forces, such as hydrogen bonds, ionic bonds, hydrophobic interactions, London dispersion forces, van der Waals forces, and π-interactions, or physically self-crosslinking occurred such as host–guest interactions, stereo-complexation and complementary binding, all without requiring a crosslinker or catalyst [73]. This section focuses on ionic interactions, thermal gelation, and hydrogen bonding.

Ionic interaction

Ionic crosslinking, is one of the methods of crosslinking ionizable polymers using divalent and/or trivalent ions. A fundamental approach to crafting ionic crosslinked hydrogels involves the amalgamation of ionizable polymers with counter-ions and change the interactions between the polymer molecules by varying the ionic concentration of the solvent, temperature, or pH that the kinetics of the sol–gel transition achieves the injectable property of hydrogel [74], [75]. Commonly used naturally derived polymer polymer is alginate (Alg) [76], which initiates gelation with calcium ions (Ca²⁺) and other divalent ions. For example, Park et al. [77] prepared ionically crosslinkable injectable hydrogels which formed with hyaluronate-g-Alg in the presence of Ca²⁺ and provided a suitable 3D environment for chondrocyte transplantation, promoting chondrogenic differentiation and cartilage regeneration in a mouse model. Ishii and colleagues [78] prepared injectable hydrogels by using electrostatic interaction of polyion complex as a driving force for flower-type micelle formation.

An alternative approach to ionic crosslinking hydrogels involves the combination of two polyelectrolytes with opposite charges [79]. Commonly employed polyelectrolytes involve HA, polylysine, poly (glutamic acid), and SA, and gelation process is intricately influenced by factors such as the concentration of gelators, temperature, electric density and environmental pH [74], [80], [81]. Additionally, the immediate blending of two polyelectrolytes possessing opposite charge property in solution often results in vigorous interactions, leading to an inhomogeneous mixture with substantial aggregates. For achieving precise injection control, Wu and colleagues [82] chose a stable mixing system to fabricate injectable polyelectrolyte composites hydrogels by using CS and polyglutamate.

Thermal gelation

Thermally crosslinked hydrogels have received much attention, which have lower critical solution temperatures below body temperature [83]. Consequently, polymer solutions remain in a liquid state at room temperature or under refrigeration and experience thermogelation upon an increase in temperature within the body. Natural polymers, such as cellulose derivatives [36], the combination of CS and glycerol phosphate disodium salt [68], and Gel [84], demonstrate thermo-responsive behavior. Chenite and colleagues [85] showcased the transition to hydrogel formation occurs when CS solutions, brought to a physiological pH (∼7.2) with β-glycerol phosphate, were heated to 37 °C. PNIPAAm, derived from polyacrylic acid, stands out as a frequently employed temperature-sensitive polymer in research, appreciated for its swift phase transition occurring at a low critical solution temperature, approximately ∼ 32 °C [86], [87]. Ren and colleagues [88] utilized atom transfer radical polymerization to graft temperature-sensitive PNIPAAm onto Gel, yielding hydrogels exhibiting a sol-to-gel transition at physiological temperature. Meanwhile, Tan and colleagues [89] successfully designed an injectable hydrogel featuring a lower solution temperature of approximately 35 °C, characterized by non-cytotoxicity, effectively preserved the viability of encapsulated cells, thus rendering it highly suitable for delivering cells in applications related to cartilage tissue engineering.

Hydrogen bonding

The formation of injectable hydrogels can be accomplished through the utilization of hydrogen bonding among hydrogel precursors or polymers [90]. The weak nature of hydrogen bonds facilitates shear thinning during injection, with subsequent hydrogel recovery after the applied force diminishes. Examples include the application of hydrogen bonding for the creation of injectable poly (N-acryloyl glycinamide) [91], PVA-based [92], or methylcellulose/HA [93] hydrogels. Injectable hydrogels ameliorated by numerous hydrogen bonds exhibit commendable toughness, self-recovery, and recoverability, serving as a driving force and showcasing exceptional biocompatibility attributed to the absence of chemical crosslinkers. However, these hydrogels may lack stability under hydration due to hydrogen bond dissociation, primarily stemming from the dissociation of hydrogen bonding between polymer chains. Consequently, hydrogen bonding is often combined with covalent or noncovalent bonds to establish robust adhesion between hydrogel adhesives and tissues [94]. Basu et al. [95] prepared injectable hydrogels through a two-step physical crosslinking method, where initial network points were formed by hydrogen bonding between complementary DNA strands, and the resulting hydrogels showed thermal, elastic, and mechanical stability properties.

Advantages of injectable hydrogels for Bone/Cartilage regeneration

Injectable hydrogels are progressively gaining prominence in the realm of minimally invasive surgeries, a trend observed across diverse surgical disciplines, such as orthopedics, neurology, and cardiovascular interventions. These hydrogels, injected at specific sites with minimal surgical wounds [96], undergo in situ polymerization regulated by factors like pH and temperature, simplifying the implantation process [97] and reducing the risk of postoperative infection [98]. Advantages of in situ injection include significantly reduced patient compliance, minimized damage to surrounding tissues, decreased postoperative pain, and reduced scar size, all leading to substantial cost savings [99]. Injectable hydrogels, in contrast to preformed counterparts requiring in vitro crosslinking and subsequent surgical implantation, not only overcome the challenges of solid porous scaffolds but also exhibit high modularity, allowing for casting into diverse shapes, sizes, and forms. This grants them the capacity to suitably fill defects with irregular shapes [99], thereby overcoming the limitations of traditional methods, which may occasionally fall short of forming an ideal bone/cartilage shape in the damaged area.

Injectable hydrogels also possess 3D architectures with substantial porosity, closely resemble the native ECM in physical characteristics, which facilitates the encapsulation of a substantial cell population, offering an optimal site and a favorable natural microenvironment for cell attachment, specific differentiation, and proliferation [100]. Furthermore, they exhibit non-toxicity and biocompatibility, ensuring their safe and prolonged presence in vivo. Gelation occurs under mild physiological conditions, allowing successful encapsulation of cells, proteins, and peptides for tissue regeneration [101]. When combined with bioactive molecules, they can deliver chemical signals to promote cell proliferation and regeneration. While injectable hydrogels have distinct advantages, they also present certain limitations. Traditional bulk hydrogels have superior mechanical strength and pre-designed structural properties [102], which make them suitable for specific clinical applications requiring load-bearing support. Injectable hydrogels may exhibit lower initial mechanical strength compared to bulk hydrogels, and controlling gelation kinetics can be challenging, potentially affecting their performance in vivo.

Bioinspired strategies for injectable hydrogels

Microenvironment bioinspired strategies

The cellular microenvironment exerts a fundamental influence on dictating cell behavior and function across developmental, physiological, and pathophysiological contexts. Serving as both a structural scaffold for cellular habitation and a source of diverse biochemical and biophysical cues [103], [104], the bone microenvironment consists of three key components: physiological (e.g., neighboring cells like macrophages), chemical (e.g., oxygen, pH), and physical factors (e.g., mechanics, acoustics), which collaboratively impact osteoconduction, osteoinduction, osteointegration, and osteogenesis [105]. Physiologically, the microenvironment, containing immune and endothelial cells, fosters immunosuppression, maintaining hematopoietic stem cell homeostasis and supporting bone tissue function through the regulation of cellular energy metabolism. Recent studies suggest that M2 macrophages play a key role in improving the physiological environment at injury sites, transforming an inflammatory milieu into an anti-inflammatory one, thereby promoting osteointegration. Chemically, essential nutrients like oxygen, pH, and signaling molecules from various cell types are vital for cell survival and functionality. Li and colleagues [106] endeavored to increase oxygen supply to sites of bone defects, ameliorating the hypoxic environment and significantly augmenting osteogenic capacity. Physically, external stimuli can profoundly influence cell fate, offering novel insights for bone tissue engineering. Brady et al. [107] observed a notable difference between osteoblasts in stationary culture and those subjected to mechanical stimulation, highlighting that the paracrine factors released post-stimulation markedly improved the migration, proliferation, and osteogenic activity of osteoprogenitor cells. To sum up, the engineering of bone microenvironment stands as a prospective research focus for the bioinspired design of injectable hydrogels which, acting as bone ECM, emulate numerous biological processes and respond to various bone microenvironmental changes.

Physiological microenvironment

The immune system is a pivotal element in maintaining the homeostasis of bone regenerative microenvironment and determining the degree of bone/cartilage regeneration [108], [109], [110]. In multiple physiological contexts, macrophages are key players in the host immune response, which have a crucial function in tissue homeostasis hand in addition to influencing innate and adaptive immunity [111]. Macrophages in the innate immune system experience various polarization states; M1 macrophages display pro-inflammatory traits, while M2 macrophages show anti-inflammatory features. Inducing macrophage polarization towards the M2 phenotype is instrumental in creating a conducive osteogenic microenvironment, and is thus important to regulate the response of macrophages.

Li and colleagues [112] prepared injectable hydrogels incorporating a novel anti-oxidative and anti-inflammatory material referred to as TPCD, which was synthesized by covalently attaching tempol and phenylboronic acid pinacol ester onto β-cyclodextrin. In periodontitis rats, TPCD nanoparticles (NPs) demonstrated the ability to normalize the intracellular/extracellular inflammatory and oxidative microenvironments of ectodermal stem cells (EMSCs), leading to enhanced survival, proliferation, and osteogenic differentiation of EMSCs (Fig. 2A). Yao et al. [113] developed an injectable composite thermal hydrogel with highly hydrophilicity and flexible properties that closely resembles living tissue, providing an almost normal growth environment for chondrocytes thereby mitigating the inflammatory reaction of adjacent cells and tissues. Li and colleagues [114] formulated an injectable double-network hydrogel incorporating phenylboronic-acid-crosslinked poly (vinyl alcohol) and Gel colloids, designed for diabetic bone regeneration. After being injected into a diabetic rat model, the hydrogel loaded with IL-10 and bone morphogenetic protein 2 (BMP-2) could regulate macrophage polarization and generate an inflammation-suppressive microenvironment, thus promoting bone regeneration (Fig. 2B). Seo et al. [115] prepared an injectable hydrogel composed of polymeric NPs encapsulating triamcinolone acetonide (TCA), a model drug of nonsteroidal anti-inflammatory, which long-term released TCA after intra-articular injection, promoting cartilage regeneration in osteoarthritis (OA). It achieved this therapeutic effect by inhibiting matrix proteinase expressions in cartilage through the dual mechanism of decreasing the expression of pro-inflammatory cytokines while elevating the expression of anti-inflammatory cytokines. Yao et al. [116] fabricated injectable HA methacrylate hydrogel microspheres loaded with polyhedral oligomeric silsquioxane-diclofenac sodium NPs which has anti-inflammatory and chondro-differentiating properties. The final hydrogel promoted proliferation of chondrocytes and ameliorated the local tissue inflammatory microenvironment by continuously releasing diclofenac sodium.

Fig. 2.

Microenvironment bioinspired strategies of injectable hydrogels to promote bone/cartilage regeneration. A) Thermo-sensitive inflammation-resolving injectable hydrogels promoting bone regeneration in periodontitis via delivering TPCD and EMSCs. Reproduced and adapted with permission [112]. Copyright 2022, Wiley-VCH GmbH. B) IL-10 and BMP-2 loaded injectable composite hydrogel via immune regulation to promote diabetic bone regeneration. Reproduced and adapted with permission [114]. Copyright 2021, Wiley-VCH GmbH. C) The injectable SMS hydrogel releasing Ca²⁺ and Si²⁺ to induce new bone formation and angiogenesis. Reproduced and adapted with permission [117]. Copyright 2023, Elsevier. D) Dynamic stiffening injectable hydrogels significantly accelerating calvarial defect regeneration compared to the Low and High hydrogels. Reproduced and adapted with permission [118]. Copyright 2022, Elsevier.

Vascularization plays a vital role in bone regeneration, as evidenced by a well-established correlation between compromised blood supply and the unsuccessful healing of bone defects [119], [120]. Additionally, animal studies consistently reveal that neovascularization is engaged with fracture healing, and inhibition of angiogenesis suppresses the formation of both callus and periosteal woven bone [121]. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor for bone regeneration [122], which holds a pivotal position in various facets of skeletal development, influencing the chemo-attraction, differentiation, and survival of mesenchymal progenitor cells, including osteoblasts, chondrocytes, and osteoclasts [123], [124]. Kaigler et al. [125] prepared injectable Alg hydrogels, which, upon injection into cranial critical-sized defects in rat, released VEGF and promoted early-stage angiogenesis, ultimately contributing to enhanced bone regeneration in later stages. Wang et al. [126] fabricated an injectable nano-HA (nHA)/poly lactic-co-glycolic acid microspheres/CS hydrogel, verifying that this hydrogel, featuring a sustained release of BMP-2 and VEGF, significantly promoted vascularization and the formation of new bone in mandibular critical-sized defects in rabbits. 

Chemical microenvironment

Bone defects often coincide with local microvascular ruptures, posing a challenge to bone regeneration in hypoxic microenvironments resulting from interrupted blood supply [127], [128]. Low-oxygen level can inhibit the metabolic transformation and osteogenesis of bone marrow stem cells (BMSCs), which in turn significantly reduce the therapeutic effect of bone regeneration [129], [130]. Reactive oxygen species (ROS) are significant components of the chemical microenvironment and play a complex role in bone regeneration. While ROS can facilitate signaling pathways that promote osteogenesis, excessive levels can lead to oxidative stress, resulting in the demise of osteoblast precursor cells and fully differentiated osteoblasts. This disruption hampers the osteogenic differentiation of BMSCs and osteoblast precursor MC3T3-E1 cells [131], [132]. Hypoxia-induced increases in pro-inflammatory mediators, particularly ROS [106], have been shown by Huang and colleagues [133] to negatively impact mineralization and alkaline phosphatase activity, which are key markers of osteogenic differentiation. Therefore, maintaining a balanced ROS environment is essential for fostering bone regeneration. Strategies to ensure a reasonable oxygen supply and to scavenge excessive ROS are crucial for enhancing the therapeutic outcomes of bone regeneration interventions. Li et al. [134] prepared an adipose mesenchymal stem cells (ADSCs)-loaded injectable hydrogel, which could continuously and efficiently capture redundant ROS, protecting ADSCs from ROS-mediated death and bioactivity inhibition. Intra-articular injection of this hydrogel notably enhanced the formation of the cartilage matrix and facilitated the repair of cartilage damage in OA.

Studies have shown that Ca²⁺ plays a pivotal role in inducing the migration, proliferation, and osteogenic differentiation of osteoblasts through the activation of calcium-sensitive receptor-directed signaling pathways [135]. In the injectable SF/mesoporous bioglass/SA composite hydrogel system prepared by Zheng et al. [117], mesoporous bioglass, which is classified as an inorganic non-metallic material [136], exhibits a gradual release of Ca²⁺ and Si²⁺. In a rabbit model of maxillary sinus elevation, the hydrogel displayed responsive degradation in an acidic microenvironment, which led to the release of Ca²⁺ and Si²⁺, thereby fostering osteogenesis and angiogenesis in cells to address lacunar bone deficiencies in vivo (Fig. 2C). Additionally, Mg²⁺ assumes a versatile role in bone regeneration, participating in the migration and tube formation of vascular endothelial cells (VECs) while also promoting the osteogenic differentiation of stem cells [137]. Consequently, the incorporation of Ca²⁺ and Mg²⁺ into injectable hydrogels not only enhances their mechanical properties but also significantly augments their capacity for bone regeneration. Yu et al. [138] fabricated the injectable Ca²⁺/Mg²⁺-doped supramolecular hydrogel by introducing a novel physical crosslinking mechanism involving the robust chelation of the comonomer alendronate (ALN) with Ca²⁺/Mg²⁺. After injection into cranial defects in rats, the hydrogel effectively released Ca²⁺ and Mg²⁺, which supported the migration and infiltration of intrinsic cells, thereby facilitating new bone regeneration.

Physical microenvironment

Biophysical properties play a pivotal role in the tissue regeneration process [139]. Mechanical cues in the surrounding microenvironment, such as matrix stiffness, significantly influence bone and cartilage regeneration [118]. The stiffness of the ECM stiffens dynamically with changes in structure and composition, thereby influencing various cellular behaviors [140], [141], including attachment, proliferation, migration, differentiation and survival [142], [143]. Acellular bone matrix scaffolds with uniform microstructures but varying stiffness have been demonstrated to exhibit distinct osteoinductive capabilities for bone regeneration [144], [145]. For example, Huebsch et al. [146] demonstrated with injectable Alg hydrogels that varying bulk matrix stiffness distinctly influenced the osteodifferentiation of transplanted MSCs and the formation of new bone in a cranial defect model. Injectable hydrogels with adjustable stiffness can emulate dynamic mechanical microenvironment in response to environmental changes of native ECM for bone/cartilage regeneration. Chen et al. [118] presented an injectable hydrogel that could dynamically change stiffness to better simulate the in vivo mechanical microenvironment, which was injected into rat mandibular defects, promoting bone regeneration more effectively compared to the ones without mechanical manipulation (Fig. 2D). Mahajan et al. [147] developed an injectable SF/carboxymethyl cellulose/Gel triple-network hydrogel system that utilized the capability of SF to undergo dynamic transformation and stiffening over time, resembling the developmental process of biological tissues, consequently facilitating the regeneration of mature hyaline cartilage when ectopically implanted in mice. The hydrogel demonstrated an augmented chondrogenesis attributed to its capacity to offer dynamic mechanical niches to cells. Ghosh et al. [148] prepared an injectable Alg-peptide composite hydrogel for bone regeneration, which could imitate the nanofibrillary characteristics of the ECM and tailor its mechanical attributes to achieve the desired stiffness and strength, thereby inducing adhesion, proliferation, and osteogenic differentiation of MC3T3-E1 pre-osteoblasts.

Structure and composition bioinspired strategies

Structure bioinspired strategies

Structural characteristics of the ECM exert significant impacts on cellular behaviors and are intricately linked to the functionalities of bone and cartilage tissues. Specifically, the pores within the interstitial framework of ECM networks play a crucial role, with their size dictating the available space [105], thereby affecting cell attachment, migration, morphology and proliferation. In addition, porosity significantly influences the mechanical properties of the matrix, governs the delivery of nutrients and oxygen, and facilitates the removal of waste products [149], [150]. Notably, the inherent high porosity property facilitates efficient penetration of nutrients, growth factors, and oxygen [151], providing lower diffusion resistance for the exchange of nutrients and gases, which make tissue growth faster and better. Hence, a pivotal emphasis has been the development of materials that emulate the structures, properties, and functions of the native ECM, thereby facilitating the investigation of cells in vitro within a realistic and adaptable cell microenvironment. Through emulating bone ECM architecture, injectable hydrogels have 3D networks with high porosity, which provide a physical scaffold for cells in bone tissues and make a quantity of cells and proteins encapsulated in the mesh, providing good sites for cell adhesion, proliferation and specific differentiation [152]. Due to the crosslinked 3D network, hydrogels can absorb significant amounts of water while retaining their shape, facilitating the transfer of nutrients, oxygen, and metabolic products throughout the porous network. Li and colleagues [153] developed an injectable laponite-calcium@Gel hydrogel with a porous structure, which facilitated both cell migration and vascular infiltration, consequently promoting the effective regeneration of functional bone tissue (Fig. 3A). Wu et al. [154] fabricated an injectable stem cell-loaded homogenous porous hydrogel microspheres, which rapidly adsorbed murine BMSCs, preserving the viability and osteogenic potential in vitro for cancellous bone regeneration (Fig. 3B). Sa-Lima et al. [155] developed a CS-glycerol-phosphate-starch injectable hydrogel for encapsulating ADSCs that induced chondrocyte differentiation for cartilage regeneration.

Fig. 3.

Structure and composition bioinspired strategies of injectable hydrogels to promote bone/cartilage regeneration. A) The porous structure of the injectable hybrid hydrogel promoting cell recruitment and migration, facilitating bone regeneration. Reproduced and adapted with permission [153]. Copyright 2023, Wiley-VCH GmbH. B) The injectable cell-loaded hydrogel microspheres promoting osteogenesis. Reproduced and adapted with permission [154]. Copyright 2020, Elsevier. C) The injectable matrix metalloproteinase-responsive hydrogels with sustained release of DEX sodium phosphate to treat OA. Reproduced and adapted with permission [156]. Copyright 2022, Elsevier. D) The injectable hydrogel-biphasic calcium phosphate ceramic bi-layer scaffold improving the regeneration of condylar osteochondral defects. Reproduced and adapted with permission [157]. Copyright 2021, Elsevier. E) The injectable HA-HAP-fibroin hydrogel mimicking ECM for bone regeneration. Reproduced and adapted with permission [158]. Copyright 2021, Elsevier.

Composition bioinspired strategies

The ECM of bone primarily consists of organic and inorganic components, such as collagen (Col) and hydroxyapatite (HAP), both of which possess specific biological functions [159]. Col plays a crucial role in the sequential events resulting in the formation of new bone [160] and the integrin-mediated adhesion to type I Col (Col I) enhances the osteodifferentiation of human BMSCs (hBMSCs) [161]. HAP, a major inorganic component found in natural bones and teeth, is commonly used as an additive in the fabrication of bone-mimetic substitutes and polymers for bone tissue engineering [162], [163], [164]. HAP exhibits exceptional biocompatibility and establishes stable interfacial binding within bone tissue [165], [166], which involves the direct binding to newly formed bone, fostering osteoblast adhesion, promoting growth and differentiation, and facilitating the deposition of new bone through mechanisms distinct from those involving adjacent living bone. The incorporation of HAP renders the hydrogel similar to the natural bone ECM in terms of mechanical properties, structure, and composition, thereby promoting biocompatibility by preserving cellular viability and enhancing cellular proliferation. Mimicking the composition of bone/cartilage ECM represents a logical and practicable strategy [167]. Shi et al. [168] developed an injectable Gel methacryloyl (GelMA)-HAP-SN hydrogel, which exhibited exceptional potential for bone regeneration in vivo and in vitro. When injecting MSC-encapsulated hydrogels into cranial critical-sized defects in rats, an increased formation of new bone and vascular tissue was observed.

HA, the main component of the human ECM [169] is cytocompatible, low immunogenic and biodegradable. The oxidized HA, recognized as a highly biocompatible macromolecular crosslinking agent, can be synthesized through the periodate oxidation of HA [170]. Dexamethasone (DEX) sodium phosphate is commonly employed for intra-articular management of osteoarthrosis, offering symptomatic relief, mitigating inflammation, enhancing mobility, and posing minimal risk to the viability of chondrocytes [171]. Yi et al. [156] demonstrated that the Col-oxidized HA matrix metalloproteinase-responsive hydrogels loading DEX sodium phosphate could effectively alleviate the symptoms of OA continuously (Fig. 3C). Wang et al. [157] designed a self-crosslinking thiolated HA (HA-SH)/Col I injectable hydrogel and biphasic calcium phosphate ceramics for in situ condylar osteochondral regeneration. In vitro findings demonstrated that the hybrid hydrogel created a conducive microenvironment, enabling the simultaneous proliferation and chondrogenic matrix secretion of both rabbit BMSCs and chondrocytes, effectively facilitating the regeneration of subchondral and fibrocartilage bone (Fig. 3D). Arjama et al. [158] fabricated the injectable HA-HAP-SF hydrogels with higher mechanical strength, which successfully increased osteoblast cellular attachment, ameliorated osteogenic activity as well as promoted bone tissue regenerations (Fig. 3E). Shao et al. [172] developed the injectable composite hydrogel composed of poly (ethylene glycol)-poly (ε-caprolactone)-poly (ethylene glycol)/Col/nHA, designed to mimic the compositions of natural bone. This hydrogel guided the growth of osteoblasts and new vascular tissue into the inner part of the composite due to its excellent degradability, consequently fostering increased formation of bone tissue. Sun et al. [173] prepared a physical crosslinked thermo-sensitive CS/Col/β-glycerophosphate hydrogel, which exhibited the persistent capacity to promote osteogenesis in vivo when it was pre-loaded with osteodifferentiated dog-BMSCs in vitro, holding promise as both a vehicle for delivering cells and an injectable substitute for bone.

Bioactive components bioinspired strategies

In the pursuit of an effective strategy for in situ bone/cartilage regeneration, diverse tissue-engineering cells, active factors, functional drugs and nanocarriers have been developed [174], [175], [176], [177]. Hydrogels serve as effective delivery agents for cells or biological payloads such as growth factors, NPs, drugs, controlling release and targeted delivery [178]. The characteristics of hydrogels, which include immunoisolation coupled with facile diffusion of nutrients, oxygen, and metabolic products, make them exceptionally advantageous in cell transplantation. Furthermore, to achieve the targeted drug release, the management of delivery kinetics involves the control of hydrogel swelling degree, crosslinking extent and the biodegradation rate.

Cell-Derived nanocarriers

Exosomes (Exos), nanovesicles naturally secreted by cells, act as intercellular messengers with less safety issues, reduced toxicity and diminished concerns related to immunogenicity [179], [180], [181] that effectively reach the intended targets via direct injection, stimulating precursor cells, osteoclasts, osteoblasts, chondrocytes, and immune cells to promote bone/cartilage regeneration [182], [183], [184]. In this review, we summarize the Exos secreted by various types of cells, alongside their quintessential roles in the bone/cartilage regeneration (Table 1). Incorporating therapeutic agents into Exos employs two methods: passive and active encapsulation. During passive encapsulation, the target drug undergoes incubation with Exos. Conversely, active modification utilizes a mechanical shear force to alter the membranes of Exos, facilitating the diffusion of drug molecules during this process of membrane modification [185]. To date, Gel, CS, HA, as well as polypeptide-based hydrogels have found application in encapsulating Exos derived from various cell sources [186], [187]. The PEG/DNA hybrid injectable hydrogel loading stem cells from apical papilla (SCAP)-derived Exo was developed by Jing et al. [188], which released SCAP-Exo in a controlled manner upon injection into mandibular bone defect models of diabetic rats, resulting in substantial therapeutic effects on facilitating vascularized bone regeneration (Fig. 4A). Yang et al. [189] used injectable hydrogels for the sustained release of Exos derived from human umbilical cord MSC s, revealing that Exos encapsulated in an injectable hydrogel composed of ALG and HA could significantly enhance bone regeneration in a rat model of calvarial bone defects.

Table 1.

A summary of cell-derived nanocarriers in bone/cartilage regeneration.

Cell-derived nanocarriers	Roles in bone/cartilage regeneration	Ref.
Bone mesenchymal stem cell-derived Exos	Regeneration Specificity: Primarily tailored for bone defect regeneration. Clinician Accessibility: Enhanced ease of access for clinical practitioners. Osteogenic Potential: Possessing robust osteogenic capabilities. miRNA Role in BMSCs: Regulating osteoblast differentiation and bolstering bone regeneration.	[187], [206]
Human urine-derived stem cell Exos	Multifunctional Differentiation: Capable of suppressing osteolysis while promoting angiogenesis and osteogenesis.	[207]
Dental pulp stem cells-derived Exos	Enhancement of HUVECs: Significantly boosting the tube-forming ability of HUVECs and supporting angiogenic and osteogenic differentiation, alongside promoting pre-osteoblast migration.	[188]
Stem cells from apical papilla-derived Exos	Angiogenesis and Osteogenesis Enhancement: Facilitated by the high expression of miRNAs (miR-126-5p, miR-150-5p).	[188]
Exos derived from M2 macrophages	Osteogenic Stimulation: Promoting MSC differentiation and acting as immune regulator for fracture regeneration.	[208], [209]
Adipose mesenchymal stem cell-derived Exos	Cellular Regulation: Governing BMSCs, osteoblasts, osteoclasts, and VECs for chondrogenesis and osteogenesis. Immune Response and MSC Support: Modulating immune activity near bone and enhancing MSC viability and migration.	[210], [211]
Human umbilical cord mesenchymal stem cells-derived Exos	Immune and Tumorigenic Safety: Exhibiting minimal immune rejection and non-tumorigenicity in vivo. Regenerative Efficacy: Effectively enhancing new bone formation and increasing blood vessel density.	[212], [213]
Primary chondrocyte-derived Exos	Mitochondrial and Immune Function: Eliminating mitochondrial dysfunction and restoring immune regulation. Inflammation and Regeneration: Promoting M1 to M2 macrophage polarization, reducing excessive articular inflammation and enhancing cartilage regeneration.	[192], [214], [215]
Hypoxia-induced Exos derived from adipose-derived mesenchymal stem cells	Chondrocyte Support under Hypoxia: Increasing IL-1β-induced chondrocyte autophagy in hypoxic conditions. Autophagy and Proliferation: Enhancing chondrocyte autophagy, reducing apoptosis, and promoting chondrocyte activity and proliferation.	[216]

Fig. 4.

Bioactive components bioinspired strategies of injectable hydrogels to promote bone/cartilage regeneration. A) The synthesis and therapeutic principles for promoting diabetic vascularized bone regeneration of SCAP-Exo-loaded PEG/DNA hybrid hydrogel. Reproduced and adapted with permission [188]. Copyright 2022, American Chemical Society. B) The injectable WYRGRL-Exo-L@GelMA hydrogel inhibiting OA-related inflammation and promoting cartilage regeneration in OA. Reproduced and adapted with permission [191]. Copyright 2023, Springer Link. C) The injectable primary chondrocyte-derived Exos-loaded hydrogel inducing efficient polarization of M1 to M2 macrophages and alleviating OA. Reproduced and adapted with permission [192]. Copyright 2022, Springer Nature. D) The injectable Van/DFO/DEX liposome-hydrogel enhancing vascularized bone regeneration. Reproduced and adapted with permission [202]. Copyright 2022, Elsevier. E) The pre-osteoblasts MC3T3-E1 cells-encapsulated hydrogel regenerating calvarial defect. Reproduced and adapted with permission [217]. Copyright 2022, Elsevier. F) The injectable hydrogel delivering BMP-2 and VEGF to enhance vascularization and osteogenesis of rabbit mandibular bone defects. Reproduced and adapted with permission [126]. Copyright 2020, Elsevier.

Given the inherent drawbacks of BMSCs transplantation, Exo supplementation provides a potential therapeutic method for cartilage defects [190]. In a study by Wan et al. [191], photo-crosslinking spherical GelMA hydrogel was fabricated to encapsulate engineered Exos modified with the cartilage-affinity WYRGRL peptide for the treatment of OA. In a mouse model of OA, the hydrogel significantly suppressed inflammation associated with OA and downregulated the expression of immune-related genes, showing excellent therapeutic results in improving cartilage regeneration (Fig. 4B). Sang et al. [192] developed an injectable thermo-sensitive hydrogel loading Exos derived from primary chondrocytes, which could alleviate OA by sustainedly releasing Exos, facilitating positive regulation of chondrocyte migration, proliferation, and differentiation while also inducing the phenotypic transition of macrophages from M1 to M2 after intra-articular injection (Fig. 4C). Zhang et al. [193] prepared the AD/CS/ regenerated SF/Exo hydrogel, which, after injection into rat patellar grooves, released Exos and recruited BMSCs into both the hydrogel and neo-cartilage through the chemokine signaling pathway, thereby enhancing the in situ cartilage regeneration and promoting the ECM remodeling. In a study by Zhang et al. [194], injectable hydrogels based on PEG maleate citrate/β-TCP was fabricated to promote angiogenesis and osteogenesis in spinal fusion, which was achieved by optimizing the BMSC microenvironment and Exos secretion. Nevertheless, a significant challenge remains in realizing the efficient extraction of engineered bone-functionalized Exos, primarily attributed to constraints related to cell passages and the inherent difficulty in maintaining their totipotency [195].

Liposomes, recognized as super microsphere carriers [196], exhibit the capacity to encapsulate both hydrophilic and hydrophobic drugs within their lipid bilayer film and cavity, including protein drugs, antibody cytokines, water-soluble macromolecules, and water-soluble small molecules [197]. Ensuring uniform dispersion within hydrogels, liposomes form the groundwork for a collaborative drug delivery process [198], effectively mitigating burst release. The establishment of covalent [199] and non-covalent [200], [201] bonds between the liposome groups and hydrogel network serves to significantly enhance the mechanical properties of the composite hydrogel, all without the risk of interface separation. Consequently, the liposome-modified hydrogel emerges as an ideal solution, meeting the demands for a versatile and robust multi-drug delivery system. Xun and colleagues [202] developed injectable liposome–hydrogels containing vancomycin/deferoxamine (Van/DFO)/DEX, which was injected into a rabbit model undergoing maxillary sinus floor augmentation, releasing Van (with a powerful antibacterial action), DFO and DEX (with a role in fostering angiogenic sprouting and promoting osteoblastic differentiation) in a sequential and sustained manner that promoting regeneration of complex bone defects and the vascularized bone formation (Fig. 4D). Cheng et al. [203] developed an injectable 4-arm-PEG-thiol hydrogel incorporated with DFO loaded liposomes-calcium phosphate NPs with osteogenesis, angiogenesis and antibacterial effects. Injecting this hydrogel into a rat model of cranial critical-sized defects, the angiogenesis and osteogenesis were significantly enhanced. Liu et al. [204] developed an injectable PEG hydrogel incorporating BMP-2-loaded adhesive liposomes, a potent inducer of osteogenesis in vivo [205]. Upon the direct injection of the hydrogel into osteoporotic fracture sites and the surrounding bone marrow cavity in rats, the liposomes maintained the adhesive properties after releasing from the hydrogel and continued to adhere to the tissue surrounding the fracture, harnessing the optimal effects of hydrogel and drug, consequently, facilitating enhanced osteogenic differentiation and accelerated local bone reconstruction in rats with osteoporotic fractures.

Functional cells

Cell-based therapy for regenerating damaged bone/cartilage is gaining prominence, with effective cell delivery being a prerequisite for regenerative medicine centered on cell therapy [218]. Typically, cellular therapy approaches for bone regeneration involve adult stem cells, like MSCs, owing to their capacity to differentiate into specific lineage cells. MSCs not only are the key healing cells involved in natural bone regeneration [219], but also are immunoprivileged [220] and capable of secreting immunomodulatory factors and others with therapeutic functionality [221], [222] that make them excellent for promoting bone regeneration [223]. BMSCs were the first MSCs to be reported [224] and have been successfully used in tissue engineering and disease treatment [225], [226], [227], holding great promise in bone regeneration [228].

Chen et al. [217] prepared a tunable dual-crosslinked hydrogel with injectability, aiming for the implantation of non-viral CRISPR activation-engineered MC3T3-E1 preosteoblast cells, thereby activating the expression of VEGF-A genes and transforming growth factor β1 (TGF-β1) to promote osteogenesis (Fig. 4E). Kaigler et al. [125] demonstrated that the injection of VEGF-Alg hydrogel into a CSBD in the rat skull enhanced early angiogenesis and significantly promoted bone regeneration. Zhang et al. [229] fabricated an injectable hydrogel comprising Col-HA laden with BMSCs that exhibited excellent biocompatibility and facilitated chondrogenic differentiation of the BMSCs. This hydrogel system was shown to achieve good hyaline cartilage regeneration in vivo experiments. Wu et al. [154] constructed injectable hydrogel microspheres laden with stem cells, characterized by notable biocompatibility, high cell-loading capacity, and uniform size, which efficiently attracted BMSCs, exhibiting considerable osteogenic potential in vitro and contributing to bone remodeling in vivo. As a specific subset of hydrogels, cryogels are sponge-like, porous structures that gelating at subzero temperatures from a precursor hydrogel solution. HBMSCs, recognized as postnatal stem cells, possess multipotent capabilities and can differentiate into osteoblasts and chondrocytes. They are widely acknowledged as the premier cell source for various tissue engineering therapies and have achieved successful clinical applications [230], [231]. Yuan et al. [232] fabricated GelMA Cryogel Microspheres and mixed hBMSC-loaded Cryogel Microspheres-30 and HUVEC-loaded Cryogel Microspheres-30 at a 1:1 ratio, which were subsequently injected into nude mice, showing osteogenesis and angiogenesis.

Bioactive factors

Growth factors, which are soluble signaling molecules, regulate various cellular responses through specific binding to transmembrane receptors on target cells [213]. The incorporation of growth factors serves as a strategic approach of functionalizing injectable hydrogels, including angiogenic, osteogenic, and inflammatory factors. These hydrogels deliver the growth factors to injury sites, inducing signaling cascades that promote bone tissue regeneration [233], [234]. Local enrichment of these factors can effectively modulate and stimulate cellular proliferation, differentiation, recruitment, migration, and angiogenesis. BMPs, a member of the highly conserved TGF-β superfamily, are a class of transforming growth factors with osteoinductive activity that initiate the mesenchymal cells to undergo differentiation, leading to the formation of osteoblasts and chondrogenic cells [235]. BMP-2, BMP-4, and BMP-7, in particular, have been extensively investigated as pivotal osteogenic molecules, promoting de novo bone formation in ectopic and orthotopic sites, including critical-sized defect [236], [237], [238]. Hydrogels hold significant appeal for osteoinductive proteins [239], capable of initiating chain-level reactions within cells, inducing the differentiation of mesenchymal cells into chondrocytes, prompting the secretion of the cartilage matrix, followed by mineralization to facilitate bone formation. Li and colleagues [240] prepared an injectable thermo-responsive hydrogel with plasmid DNA-BMP2, which released plasmid DNA-BMP2 continuously, affecting the growth of osteocytes within the lacunae, enhancing trabecular thickness, and fostering the formation of trabecular bone. Yoon and colleagues [241] prepared an injectable hydrogel system based on glycol CS incorporating TGF-β1 and BMP-2 for continuous release that enhanced bone volume and density at sites of the bone defects.

Vascularization is crucial for facilitating the transport of growth factors, oxygen, nutrients, differentiation factors, and circulating cells, essential elements for bone formation and homeostasis [242], [243]. A local microvascular network is particularly supportive of osteogenic, chondrogenic, and MSCs, integral to the process of bone regeneration. Soluble molecules play a pivotal role in the regulation of angiogenesis, including VEGF, a potent angiogenic growth factor that emerges as a promising candidate for delivery in bone regeneration efforts [122], [244], [245]. Wang et al. [126] prepared injectable hydrogel comprising nHA/Poly lactic-co-glycolic acid/CS, featuring sustained release of VEGF and BMP-2, promoting vascularization and osteogenesis in rabbit mandibular CSBDs (Fig. 4F). Chen et al. [246] fabricated injectable hydrogels to encapsulate microspheres loaded with VEGF and VECs. Upon injection into the necrotic site of the femur head, the formation of new blood vessels was observed over weeks, accompanied by the concomitant formation of new bone.

Apoptotic bodies are extracellular vesicles released during the process of apoptosis [247]and exhibit a variety of biological activities. They contain a diverse array of cargo, including proteins, metabolites, and nucleic acids, which can be transferred to neighboring cells, thereby modulating cellular functions and signaling pathways[248]. This transfer plays a critical role in promoting healing and tissue regeneration. Additionally, Apoptotic bodies are involved in various biological processes such as inflammation, autoimmunity, and cancer, by regulating the activities of recipient cells[249], [250]. In the study by Ma et al. [251], apoptotic bodies derived from preosteoclasts enhance angiogenesis by delivering PDGF-BB to endothelial progenitor cells, which is crucial for bone tissue repair. Additionally, apoptotic bodies from mature osteoclasts utilize RANKL reverse signaling to promote osteogenic differentiation of MSCs, thereby directly enhancing bone formation. Future studies should, therefore, explore the integration of apoptotic bodies into injectable hydrogels with the aim of developing the next generation of therapeutic strategies for bone/cartilage repair.

Gene delivery systems

Tissue engineering has emerged as a promising approach for addressing large-scale bone defects [252], with gene delivery systems playing a pivotal role in modulating cellular functions at the genetic level. These systems facilitate the sustained expression of specific osteoinductive growth factors by host or transplanted cells at the defect site. Among the various gene delivery vehicles, injectable hydrogels have gained significant attention for their minimally invasive nature, efficacy, and controllability in delivering a range of genetic materials, including plasmid DNA, small interfering RNA, and the CRISPR/Cas9 system. Gan et al. [253] prepared a novel injectable hydrogel system that enables precise control over the release of microRNA-26a through UV irradiation, promoting osteogenic differentiation in human MSCs, and demonstrates its potential in bone defect repair through in vitro and in vivo experiments. Krebs et al [254]. have developed an injectable hydrogel containing calcium phosphate-DNA nanoparticles that can stably carry plasmid DNA encoding for BMP-2. When these hydrogels were injected subcutaneously in mice and mixed with MC3T3-E1 preosteoblast cells, they were able to sustain the release of DNA and induce the formation of bone tissue in as little as two and a half weeks. Sun et al. [255] developed an innovative injectable hydrogel system based on GelMA integrated with sticky end-loaded tetrahedral framework nucleic acids modified with miR29c. This system is designed to achieve sustained release of miR29c, which is essential for the osteogenic differentiation of BMSCs. It significantly upregulates osteogenic markers including ALP, RUNX2, and OSX in vitro and promotes rapid bone tissue formation in a CSBD mouse model, highlighting its therapeutic potential for the repair of bone defects.

Injectable hydrogels in Bone/Cartilage regeneration

CSBDs regeneration

Although the skeleton has a remarkable ability for spontaneous healing, most CSBDs caused by acute injury, severe infection, or degenerative diseases in clinical practice result in delayed union or nonunion [256]. Injectable hydrogels have shown great promise as bone defect fillers (15), which combining with stem cells offer high efficacy for regeneration of irregular bone injury. An injectable PEG hydrogel loaded with rat BMSCs and recombinant human BMP-2 was fabricated by Lao et al. [257], which, when injected into cranial defects in rats, could release recombinant human BMP-2 in a spatiotemporal manner and enhance both proliferation and osteogenic differentiation of rat BMSCs, thus resulting in rapid bone regeneration in CSBDs (Fig. 5A). Ingavle et al. [258] demonstrated that an injectable mineralized microsphere-loaded composite hydrogels loaded with MSCs promoted healing of sheep iliac crest CSBDs.

Fig. 5.

Injectable hydrogel for critical sized bone defects regeneration. A) The injectable hydrogel loading BMSCs and recombinant human BMP-2 facilitating regeneration of bone CSDs. Reproduced and adapted with permission [257]. Copyright 2023, Royal Society of Chemistry. B) The injectable DFO- gelatin microspheres hydrogel regenerating femoral CSDs. Reproduced and adapted with permission [256]. Copyright 2022, Elsevier. C) The injectable mucin-monetite hydrogel facilitating vascularization and the regeneration of bone CSDs in mice. Reproduced and adapted with permission [263]. Copyright 2023, Elsevier. D) The injectable hydrogel system promoting critical-sized bone formation in ovariectomized-rats [264]. Copyright 2021, Elsevier.

DFO is a small-molecule-based iron chelator and can accelerate vascularized bone healing [259]. H-type vessels represent a recently characterized subtype of blood vessels with pivotal roles in influencing bone regeneration [260]. Zeng et al. [256] successfully fabricated a DFO-gelatin microspheres injectable hydrogel whose sustained release of DFO induced the formation of functional H-type vessels that regenerated femoral critical-sized defects in rats (Fig. 5B). Monetite, an acidic calcium phosphate, has been shown to exhibit osteogenic capabilities in vitro and in vivo [261], [262]. In a study by Chen et al. [263], the monetite-mucin hydrogel with injectability was developed, which performed early bone immunomodulation to promote early angiogenesis and ultimately augmented fracture healing in a rat cranial CSBDs model (Fig. 5C). Calcitriol is universally acknowledged as a fat-soluble small molecule drug known for its reported anti-osteoporotic activity, promoting osteogenesis of osteoblasts. Hu et al. [264] created an injectable calcitriol/micelle/polydopamine modified nHAP hydrogel system with diverse sustained release effects of calcitriol. In the CSBDs model of ovariectomized rats, the hydrogel greatly enhanced the migration and proliferation of ovariectomized-BMSCs and the osteogenic differentiation process, effectively promoting bone regeneration at the site of bone defects (Fig. 5D).

Deng et al. [265] utilized an environmentally friendly approach to create an injectable hydrogel by combining nHAP with methylcellulose. The addition of nHAP contributed to increase protein absorption and osteoblasts adhesion, serving as nucleating sites for mineral deposition. Injecting the hydrogel into calvarial critical-sized defects, where it could serve as an effective carrier for BMSCs, resulting in osteogenic differentiation and bone regeneration. Patrick et al. [266] created nanoporous injectable microgels, which could support attachment, growth, and differentiation of BMSC, as well as create a conducive environment for osteogenesis and vascularization. In a mouse model with a full-thickness cranial critical-sized defect, the microgels facilitated the bridging of the critical-sized defect by MSCs, leading to the development of mineralized Col-rich osteoid bone and successful bone regeneration. SDF-1α, an important chemokine, additionally boosted the recruitment of BMSCs, augmenting bone regeneration within the critical-sized defects [267], [268], [269]. Mi et al. [270] prepared an injectable composite of SDF-1α/CS/carboxymethyl-CS NPs hydrogels that promoted the healing of calvarial critical-sized defects in rats via controlled in situ release of SDF-1α.

Osteoporosis (OP) treatment

OP is a systemic, metabolic, and progressive skeletal disorder, commonly associated with aging and primarily affecting the elderly and postmenopausal women [271]. The condition is characterized by the inability to maintain bone mass and the disruption of bone microarchitecture, resulting from an imbalance between bone formation and resorption [272], [273], which leads to a reduction in bone strength and an increased susceptibility to fractures. Most fractures tend to manifest in the later stages of life, a period characterized by heightened rates of bone loss and microarchitectural deterioration [274]. The intricate connection between aging processes of bone and development of osteoporosis has prompted a surge in comprehensive research, including basic, observational, translational, and clinical studies in recent years, all aimed at exploring the mechanisms that contribute to age-related bone loss and the occurrence of fragility fractures [275], [276].

Wu and colleagues [277] constructed an injectable mineralized hydrogel incorporating a zippered G4-Hemin DNAzyme to enhance osteogenic capacity under osteoporotic conditions. Injected into a skull defect model in osteoporotic rats, the hydrogel scavenged excessive ROS molecules, alleviating oxidative stress and promoting high-quality bone defect regeneration (Fig. 6A). Several drugs, including bisphosphonate (BP), calcitonin, and parathyroid hormone (PTH), have been clinically approved for inhibiting bone resorption and promoting bone formation. PTH, a peptide hormone secreted by the parathyroid gland, possesses a distinctive capability to activate osteoblasts, offering a clinical approach to treat osteoporosis through systemic intermittent administration [278]. Kuang et al. [279] designed an injectable poly (dimethylaminoethyl methacrylate-co-2-hydroxyethyl methacrylate) (DHCP)/PNAm-ICG-PTH (PIP) hydrogel loaded with PTH for near infrared-stimulated release. This hydrogel, when employed to restore bone defects in ovariectomized rats, exhibited increased functionality in osteoclasts and osteoblasts, as observed both in vivo and in vitro (Fig. 6B).

Fig. 6.

A) Injectable hydrogels for the treatment of OP. The injectable MDH promoting osteogenesis for osteoporosis via scavenging excess ROS and providing bone raw materials. Reproduced and adapted with permission [277]. Copyright 2023, Wiley-VCH GmbH. B) The injectable DHCP-PIP hydrogel facilitating new bone formation in osteoporosis rats [279]. Copyright 2021, Wiley-VCH GmbH. C) The injectable GelMA-BP-Mg hydrogel microsphere capturing Mg⁺ and facilitating regeneration of cancellous bone. Reproduced and adapted with permission [280]. Copyright 2021, American Chemical Society. D) The injectable hydrogel loading DEX and REX for the regeneration of osteoporotic bone defects. Reproduced and adapted with permission [281]. Copyright 2023, Wiley-VCH GmbH.

Zhao et al. [280] utilized the coordination reaction of metal ion ligand to construct a GelMA-BP-Mg hydrogel microsphere, which could capture and sustainably release Mg²⁺ to promote cancellous bone reconstruction and regeneration of osteoporotic bone defect in ovariectomized rats (Fig. 6C). Resveratrol, recognized for the potent anti-inflammatory properties, exhibits the capability to efficiently eliminate surplus free radicals at the injured site, direct macrophage polarization toward the M2 phenotype, and modulate immune responses. Concurrently, DEX has been proven to effectively stimulate osteogenic differentiation. Li et al. [281] prepared an injectable thermo-sensitive hydrogel based on poly (D, L-lactide)-poly (ethylene glycol)-poly (D, L-lactide) system, loaded with resveratrol and DEX to establish a microenvironment favorable for osteogenesis within bone defects affected by osteoporosis. The injection of this hydrogel into osteoporotic rats demonstrated a synergistic modulation of the bone damage microenvironment, mitigating inflammatory responses, removing excess intracellular ROS and promoting osteogenesis (Fig. 6D). ALN, the most commonly prescribed oral nitrogen-containing BP drug for addressing post-menopausal osteoporosis and osteoporosis induced by osteoporosis [282], induces osteoclast apoptosis through suppressing the resorptive function of mature osteoclasts. Li et al. [283] developed the injectable tetra-PEG hydrogels containing a prodrug of ALN modified with PEG. When injected into the femoral mid-region of osteoporotic rabbits, they released ALN in a slow and sustained way, increasing the mineral density of the femoral bone of rabbits thereby providing local reinforcement to cure bone osteoporosis effectively. Ye et al. [284] synthesized a thermos-responsive and injectable poloxamer 407/HA hydrogel incorporating MnO₂ NPs, which functioned as a protective carrier for BMSCs deliver. Upon injection into rabbits with osteoporosis and segmental bone defects, the hydrogel enhanced osteoporotic bone defects regeneration by reducing the accumulation of local ROS and provided an optimal microenvironment to support the osteogenic differentiation and bone regeneration of transplanted BMSCs.

Integrated osteochondral regeneration

Osteochondral unit integration has emerged as a promising strategy for cartilage defect repair [285] while continues to pose a significant challenge attributed to the intricate structure and composition of articular cartilage, coupled with its limited self-repair capabilities [286]. Additionally, treating surface of the chondral lesion lacks efficiency in the absence of support from an intact subchondral bed. Therefore, it is essential to develop a promising strategy to achieve osteochondral integration regeneration. In a study by Zhu et al. [287], a continuous stratified injectable scaffold was developed, encompassing both injectable SA/bioglass composite hydrogel and injectable SA/agarose thermo-sensitive hydrogel layer with components in both layers involving various bioactive materials and inoculated cells, mimicking the natural structure of osteochondral tissue. In a rat model, this construct improved hyaline cartilage formation, subchondral bone maturation, and integration with surrounding tissues (Fig. 7A). Liu et al. [288] prepared a cytomodulin-10-KGN GelMA injectable hydrogel for the regeneration of osteochondral defect. KGN is a potent and stable small molecule known for its capacity in cartilage regeneration and protection and Cytomodulin-10 demonstrates persistent regulation of chondrogenesis in BMSCs. In rat models, the hydrogel showed excellent regenerative effects in cartilage regeneration and contributed to the subchondral bone reconstruction (Fig. 7B). Research by Wu et al. [289] demonstrated that an injectable SF-MA hydrogel loading TGF-β3 along with a bilayer scaffold incorporating a cartilage layer resembling native cartilage and a BMP-2-loaded porous subchondral bone layer could achieve osteochondral regeneration. In rabbit model, the TGF-β3/SF-MA hydrogel played a crucial role in closing the marginal gap between the cartilage layer of the native cartilage and scaffold, expediting lateral integration and better promoting osteochondral regeneration (Fig. 7C).

Fig. 7.

Injectable hydrogels for integrated osteochondral regeneration. A) The injectable composite hydrogel enhancing the osteochondral regeneration. Reproduced and adapted with permission [287]. Copyright 2019, Elsevier. B) The injectable cytomodulin-KGN@GelMA composite hydrogel for the regeneration of the osteochondral defect. Reproduced and adapted with permission [288]. Copyright 2023, Elsevier. C) The injectable integral bilayer silk scaffold combined with SF-MA hydrogel in osteochondral regeneration. Reproduced and adapted with permission [289]. Copyright 2021, Elsevier.

Xu et al. [290] created the injectable supramolecular hydrogels laden with stem cells to enhance in situ osteochondral regeneration through the sustained co-release of hydrophilic and hydrophobic chondrogenic molecules. These resulting hydrogels enhanced the chondrogenic differentiation of encapsulated hBMSCs both in vivo and in vitro. Furthermore, the hydrogels loaded with MSCs, upon injection, resulted in the generation of high-quality neocartilage in a rat knee cartilage defect model. Radhakrishnan et al. [291] have fabricated a semi-interpenetrating network injectable hydrogel containing chondroitin sulfate NPs (a glycosaminoglycan that can impart chondrogenic potential) and nHAP for subchondral and cartilage layers with gradient interface. The injectable biomimetic hydrogel, featuring a gradient of nHAP and chondroitin sulfate, demonstrated enhanced tissue coverage in rabbit osteochondral defects that accelerated osteochondral tissue regeneration. And animals injected with the gradient nHAP-chondroitin sulfate hydrogel exhibited confined subchondral bone formation characterized by bone volume to total defect volume, higher bone mineral density, and trabecular thickness compared to other experimental groups. Ji et al. [292] prepared hydroxypropyl chitin injectable hydrogels encapsulating the dimethyloxallyl glycine (an inhibitor of HIF prolylhydroxylase, serving of promoting macrophage M2 polarization [293] and preventing the hypertrophy of chondrocytes [294]) and porous CS microspheres conjugated with KGN (a small drug molecule employed to stimulate chondrogenesis of MSCs [295] that initiate the endochondral ossification to facilitate reconstruct of the subchondral bone). Injecting the composite scaffold into osteochondral defect in rat bilateral knee joints could effectively modulate the microenvironment, achieve localized polarization of macrophages toward the M2 phenotype and enhance the regeneration of cartilage.

Arthritis cartilage regeneration

OA, a progressive joint disease, characterized by cartilage degradation, osteophyte formation, inflammation, and bone hyperplasia [296]. Among older adults, OA has risen as a predominant reason for chronic disability. Patients with OA not only endure persistent physical and physiological pain but also experience significant emotional and mental distress, impairing daily activities [297]. Han et al. [298] fabricated an injectable GelMA@DMA-MPC hydrogel microspheres with prolonged drug release and enhanced lubrication, which improved cartilage morphology and reduced cartilage lesion depth in OA rats (Fig. 8A). Lei et al. [299] formulated rapamycin-liposome-incorporating HA-based hydrogel microspheres, which alleviated joint damage and osteophyte formation while delaying OA progression in rat models. Zhou et al. [300] successfully created a self-healing, pH-responsive hydrogel composed of HA, platelet-rich plasma, and BSA-MnO2. Upon injection into OA rats, this hydrogel promoted chondrocyte proliferation and reduced oxidative stress, mitigating joint damage and slowing OA progression (Fig. 8B).

Fig. 8.

Injectable hydrogels for arthritis cartilage regeneration. A) The injectable GelMA@DMA-MPC hydrogel microspheres releasing drug to treat OA. Reproduced and adapted with permission [298]. Copyright 2021, Elsevier. B) A HA/platelet-rich plasma hydrogel containing BSA-MnO₂ nanoparticles efficiently alleviating OA. Reproduced and adapted with permission [300]. Copyright 2022, Elsevier. C) The injectable ARP hydrogel nano/microsphere releasing MTX/ARPL liposomes for RA. Reproduced and adapted with permission [301]. Copyright 2022, AAAS. D) The injectable MnCoO nanozyme hydrogels improving prosthetic interface osseointegration in RA treatment. Reproduced and adapted with permission [302]. Copyright 2022, Springer Nature.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects joints, leading to stiffness, pain, and inflammation of the synovial membrane, which progressively damages joint structures [303]. Conventional systemic therapies have limited effectiveness in providing localized symptom relief and often produce adverse effects. Injectable hydrogels with prolonged intra-articular drug release are thus a promising alternative [304]. Deoxyribonuclease I, a natural endonuclease that hydrolyzes extracellular deoxyribonucleic acid, has the potential to hinder or disrupt these traps, suggesting its utility in the management of RA [305]. An injectable carboxymethyl CS hydrogel crosslinked with deoxyribonuclease-functionalized oxidized HA was prepared by Wang et al [306], targeting neutrophil extracellular traps in a Col −induced arthritis mouse model. This hydrogel effectively reduced inflammation and alleviated RA symptoms without causing significant adverse effects.

Chen et al. [307] developed a hydrogel composed of F127-HA-poly (γ-glutamic acid), loaded with infliximab, for intra-articular injection in a RA rabbit model. This hydrogel reduced surface temperature and joint swelling, decreased inflammatory cytokines, and provided sustained relief from RA symptoms. Kumar et al. [308] prepared an enzyme-responsive hydrogel that encapsulated budesonide into the hydrophobic mesh and provided prolonged release of budesonide over an extended duration. Upon injection into rats with OA, the hydrogel enhanced Col synthesis to downregulate the expression of inflammatory mediators, contributing to cartilage regeneration. Simultaneously, it downregulated protein Matrix metalloproteinase-9 expression, a key inflammatory marker of RA and ameliorated the morphology of articular cartilage and the synovial space gap. Kim et al. [309] prepared HA- tyramine injectable hydrogels encapsulating DEX for RA treatment, which were intra-articular injected into arthritis rats and resulted in sustained release of DEX, successfully treating RA by the reduction in prostaglandin E2, interlukine-6, as well as four types of cytokine levels. The cartilage that healed after being treated with HA-tyramine hydrogels containing DEX was almost equivalent to the cartilage of normal SD rats. Concurrently, a new phospholipid-mimic artemisinin prodrug (ARP) has been documented as an alternative medical option to traditional phospholipids for the direct construction of liposomes. Du and colleagues [301] developed an injectable liposome-embedded ARP/methotrexate hydrogel nano/microsphere (MTX/ARPL@MS) for a localized RA lesion. Upon local injection into rats with arthritis, the MTX/ARPL was gradually released via imine hydrolysis, targeting RA synovial macrophages and fibroblasts concurrently that inhibiting RA inflammation and ameliorating the degree of arthropathy (Fig. 8C). Zhao and colleagues [302] developed injectable MnCoO nanozyme hydrogels as H₂O₂-driven oxygenerators to regulate stem cell behavior. In vitro and in vivo analyses have demonstrated that the hydrogel could successfully decompose the endogenous H₂O₂ to produce O₂, thereby assuaging the hypoxic and oxidative microenvironment of RA and fostering a conducive 3D microenvironment for the proliferation and osteogenesis of BMSCs (Fig. 8D).

Diabetic bone healing

Diabetes mellitus is linked to various bone health complications [310], such as heightened fracture risk, hindered bone healing, and diminished bone mineral density. Characterized by chronic hyperglycemia, inflammation [311], and metabolic irregularities, Diabetes mellitus leads to increased oxidative stress [312] and impaired osteogenic potential of bone-forming cells, disrupting the natural bone remodeling process. To address these challenges, injectable hydrogels present a targeted delivery system for drugs and bioactive molecules to diabetic bone defects, providing controlled and sustained release over an extended period, thus creating a more favorable environment for bone regeneration. For instance, lithium-modified bioglass hydrogels activate the GSK-3β/β-catenin pathway to boost bone formation [313], while phenylboronic acid-functionalized hydrogels provide an H₂O₂-responsive release of antibiotics and anti-inflammatory drugs, beneficial for treating periodontitis in diabetic rats [314]. Additionally, PDGF-BB nanocomposite hydrogels enhance the PI3K/AKT pathway, promoting angiogenesis and osteogenic differentiation [315]. Thermo-sensitive hydrogels, such as CS/GO/HEC/β-GP, mitigate inflammation, crucial for healing bone fractures in diabetic mice [316]. Furthermore, hydrogels like GelMA/Polyoxometalate can reduce oxidative stress, beneficial for bone healing, by scavenging reactive oxygen species [317]. These hydrogels often work by immune modulation and inflammation suppression. For example, MgH₂@PLGA/F-GM hydrogel facilitates immune regulation and reduces oxidative stress [318], while PEG/DNA hybrid hydrogels respond to MMP-9, activating the NOTCH pathway to increase angiogenesis and osteogenic differentiation [188]. PVA gelatin double network hydrogels, on the other hand, release IL-10 and BMP-2 to enhance M2 macrophage polarization, promoting immune regulation [319] (Table 2).

Table 2.

Injectable Hydrogels for Diabetic Bone Healing.

Hydrogel Type	Mechanisms	Targeted Sites	Effects	Ref.
Lithium modified bioglass- hydrogel	Sustained lithium ions release for activating the GSK-3β/β-catenin pathway; Increasing M2 macrophages	Femur bone defects in diabetic rats	Immune regulation	[313]
Phenylboronic acid-functionalized poly (ethylene imine)/Oxidized dextran hydrogel	Achieving H2O2-responsive drug release of doxycycline and metformin	Subgingival pocket of chronic periodontitis in diabetes rats	Inflammation suppression	[314]
PDGF-BB nanocomposite hydrogel	Release of PDGF-BB and beneficial ions; Improving the PI3K/AKT pathway	Calvarial defects in diabetic rats	Increasing angiogenesis, and osteogenic differentiation	[315]
CS/GO/HEC/β-GP thermosensitive hydrogel	Atstrin regulating the TNF-α-triggered pro-inflammatory response	Closed gravity-induced femur fracture in diabetic mice	Inflammation suppression	[316]
GelMA/ Polyoxometalate hydrogel	Scavenging ROS by slow release of polyoxometalate	Distal femoral defects in diabetic mice	Reducing oxidative stress	[317]
MgH2@PLGA/F-GM hydrogel	Sustained release of Mg2+ and H2 ions to modulate macrophage phenotype and reduce ROS	Skull defects in diabetic mice	Immune regulation, inflammation suppression, and reducing oxidative stress	[318]
PEG/DNA hybrid hydrogel	MMP-9-responsive activation; Activating the NOTCH pathway;	The mandibular bone defect in diabetic rats	Increasing angiogenesis and osteogenic differentiation	[188]
PVA gelatin double network hydrogel	Releasing IL-10 and BMP-2; Increasing M2 macrophages	Calvarial bone defects in diabetes mellitus rats	Immune regulation	[319]

Conclusion and perspective

Compared to traditional bulk hydrogels, bioinspired injectable hydrogels emulating multiple biological processes and properties of bone/cartilage, build a innovate bridge from biomaterials to bone/cartilage regeneration. In this review, we highlighted the synthesis and bioinspired strategies-specifically, microenvironmental, structural, and compositional strategies-as well as bioactive component-based approaches and further applications of injectable hydrogels for bone/cartilage regeneration.

Recently, parallel to the fast-paced developments in organoid technology, injectable hydrogels have promising applications as matrix gels in organoid construction [320]. Organoids are 3D structures originating from stem cells that can accurately emulate the structure and function of organs [321] and have been employed to investigate the evolution and activity of distinct organs, drug screening and disease modeling [322], [323]. Typically, organoid construction depends on Matrigel, an animal-derived ECM, which presents challenges including limited reproducibility, potential immunogenicity, and variability. Injectable synthetic and hybrid hydrogel matrices have emerged as viable alternatives, offering easy-to-modify functional features, cost-effectiveness, robust mechanical properties, and simplified purification and handling [324], [325], [326]. By closely mimicking the native structural and functional attributes of human organs, these synthetic matrices significantly enhance the quality and broaden the scope of potential applications for organoids [327].

The synthesis of injectable hydrogels is complex due to the need for precise control over their physical, chemical, and biological properties. This complexity arises from the requirement to tailor the hydrogels for specific applications, such as organoid engineering, where properties like biocompatibility, biodegradability, mechanical strength, and responsiveness to environmental stimuli are crucial. The integration of AI in this process can expedite the design and screening of the hydrogels, ensuring optimal performance for targeted applications. Machine learning, a subset of artificial intelligence, is adept at processing extensive datasets to identify optimal hydrogel synthesis methods, understand the intricate spatial structure of hydrogels, optimize cell culture conditions, determine key active inducers, and evaluate the impact of external stimuli [328]. Crucially, machine learning significantly influences hydrogel synthesis by analyzing how different polymer compositions affect stem cell behavior and organoid development. It excels in analyzing matrix gel imaging, identifying subtle spatial patterns, and predicting changes in cellular behavior within organoids, which is essential for their successful growth and development [320]. This integration of AI significantly enhances the refinement of spatial structures and organoid construction, ultimately enabling the development of more accurate models of human organ function and disease (Figure 9).

Fig. 9.

Advanced injectable hydrogels with AI-assisted construction and further application in organoid research.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Xuan Tang is currently a postgraduate student under the supervision of Prof. Jiacan Sui at the Institute of Translational Medicine, Shanghai University. She focuses on the research of bone biomaterials in bone/cartilage regeneration and their clinical applications.

Fengjin Zhou is an associate chief physician at the Department of Orthopaedics, Honghui Hospital, Xi’an Jiao Tong University. He received his M.S. degree from the Orthopedics Hospital of Shanghai Changzheng Hospital. He is a doctor of clinical medicine and graduated from the Department of Clinical Medicine of the Second Military Medical University under the supervision of Prof. Ni Bin. His research interests include the fixation and reconstruction of traumatic fractures, the diagnosis and treatment of osteoporotic fractures, and the role of biomaterials in facilitating fracture repair.

Sicheng Wang is a chief physician at orthopedic of Shanghai Zhongye Hospital. He obtained his bachelor's degree from Medical College of Shandong University in 2001 and obtained his master's degree from Medical College of Wuhan University of Science and Technology in 2015. Currently, he is standing member of orthopedics (Shanghai) Expert Committee of Geriatrics Branch of Chinese Gerontology and Geriatrics Society, and standing member of Osteoporosis Committee of Shanghai Society of Integrated Traditional Chinese and Western Medicine.His research mainly focuses on osteoporosis and osteoarthritis.

Guangchao Wang is an associate chief physician at the Orthopedics Department of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. He obtained his doctoral degree from the Second Military Medical University in 2017. His research mainly focuses on trauma treatment, bone and joint diseases, and orthopedic implant materials.

Long Bai is an associate professor at the Institute of Translational Medicine, Shanghai University. He obtained his doctoral degree from the Taiyuan University of Technology in 2018. Then, he worked as a postdoc at the East China University of Science and Technology from 2019 to 2022. His research mainly focuses on the construction strategy, data analysis, and application exploration of bone organoids.

Jiacan Su is the Principal scientist and director of Translational Medicine Research Institute, Shanghai University. He is also the chief orthopedic physician of the Department of Orthopedics at Shanghai Xinhua Hospital and the chief scientist of key projects of the Science and Technology Commission of Military Commission. His research focuses on the basic and clinical research of bone and joint degenerative diseases, the development and application of new biomaterials, and the development and transformation of bone/cartilage organoids.