Progress and prospects of nanocomposite hydrogels in bone tissue engineering

Abstract Hydrogels are hydrophilic polymers with high flexibility and elasticity, and are widely used in the field of bone tissue engineering. However, their low toughness and insufficient mechanical strength have limited their applications. Nanocomposite hydrogels are flexible and porous as well as possess excellent biocompatibility and mechanical strength, thus exhibiting a significant potential to be used in the field of bone tissue engineering. Nanocomposite hydrogels are synthesized by chemically or physically crosslinking nanomaterials and hydrogels, and their physical, chemical, and biological properties can be enhanced by changing the properties and compositions of the nanomaterials. Both nanomaterials and hydrogel polymers can be customized, and therefore, nanocomposite hydrogels can be developed for bone repair under different conditions. In this review, we first introduce the synthesis methods and properties of nanocomposite hydrogels, and then summarize the applications and progress of nanocomposite hydrogels in bone tissue engineering. Graphical Abstract Nanocomposite hydrogels are used in bone tissue engineering. Adapted from Huang et al. (2019) [121]


Introduction
Hydrogels, crosslinked three-dimensional polymers composed mainly of water, are widely used in bone tissue engineering [1]. Hydrogels can mimic natural tissues, providing a suitable microenvironment for cells [2][3][4]. They also possess good biocompatibility, and hence, they are excellent for bone tissue engineering applications [5,6]. However, the properties of conventional hydrogels are dependent on the polymer content and crosslink density, which significantly limit their mechanical strength and internal structure [7][8][9][10]. To overcome these limitations, nanomaterials are used for the modification of hydrogels, and the resulting nanocomposite hydrogels exhibit improved mechanical strength and biological properties [7,11]. For example, Julien E. Gautrot et al. used electrospun PLA fibers and carboxymethyl cellulose (CMC) to prepare nanocomposite hydrogels, and its storage modulus increased by 3 times compared with pure hydrogels [12]. Irmina Samba et al. prepared nanocomposite hydrogels containing PLGA nanoparticles with a more uniform pore structure than pure hydrogels [13]. Nanomaterials and hydrogels can be crosslinked physically or chemically to form nanocomposite hydrogels, and different synthesis and combination methods (natural polymers/nanomaterials) can be used to alter the properties of the final composite hydrogels [14]. In this review, we first introduce the synthesis methods for nanocomposite hydrogels and their properties, and then summarise the applications and progress of nanocomposite hydrogels in bone tissue engineering.
The main synthesis methods for nanocomposite hydrogels are chemical (covalent bonding) and physical (non-covalent bonding) crosslinking [19,20]. In the chemical method, organic crosslinking agents are used to achieve irreversible covalent bonding between nanomaterials and polymers, whereas the physical method involves reversible bonding through hydrogen bonding and ionic interactions [13,21]. Nanocomposite hydrogels formed via physical crosslinking can be decomposed by changing physical conditions such as pH and temperature, whereas those formed via chemical crosslinking have better mechanical properties, can withstand greater mechanical stress, and are difficult to decompose [22][23][24][25]. In addition, nanomaterials can be used as crosslinkers instead of organic crosslinkers to crosslink hydrogels as polymers, and the resulting products have greater mechanical toughness [11,26].
Nanocomposite hydrogels are generally synthesised from inorganic nanomaterials with a variety of properties and synthetic/natural polymers [7]. The properties of the resulting nanocomposite hydrogels are dependent on the composition of the nanomaterials and polymers, the proportional concentration of nanomaterials and polymers, and the size and degree of dispersion of the nanomaterials in the hydrogel [11]. Numerous studies have shown that as long as a small number of nanomaterials (2%$10%) is added, the properties of the prepared nanocomposite hydrogels can be significantly improved [27][28][29][30]. The combination of nanomaterials and hydrogel polymers directly affects the properties of nanocomposite hydrogels. Therefore, controlling the dispersion degree of nanomaterials in the hydrogel and its mutual combination with the molecular chains of the hydrogel is the key to the preparation of nanocomposite hydrogels. According to the dispersion method of nanomaterials in hydrogels, the current preparation methods include the blending method, grafting method, in situ precipitation method and freeze-thaw method [14,31].

The blending method
The blending method is the simplest method for preparing nanocomposite hydrogels, which directly mixes polymer monomers, crosslinking agents and nanomaterials, and initiates polymerization to form nanocomposite hydrogels [32]. Filippi et al prepared magnetic responsive nanocomposite hydrogels from iron oxide nanoparticles (average particle size 15 nm) and polyethylene glycol (PEG) hydrogel polymers by co-blending, which have good biocompatibility and high elastic modulus, while it acts as a promoter of osteogenesis and angiogenesis in response to external magnetic field stimulation [33]. The preparation process of the blending method is simple, and the nanocomposite hydrogel with uniform particle size can be obtained by controlling the stirring speed, material concentration and preparation period during mixing [34]. However, when the nanocomposite hydrogel is prepared by the blending method, the nanoparticles are prone to agglomeration, which makes the structural enhancement effect of the prepared product not obvious. For example, Tong et al prepared nanocomposite hydrogels by blending carbon nanotubes and polyvinyl alcohol hydrogel polymers [35]. The swelling properties of the composite hydrogels were significantly enhanced, but the mechanical properties were not significantly improved. The agglomeration phenomenon generated during the blending method weakens the direct interaction between the nanoparticles and the hydrogel polymer, resulting in the improvement of the mechanical properties of the prepared nanocomposite hydrogel is not obvious, which is a disadvantage that the blending method needs to overcome [36,37].

The Grafting-Onto method
The grafting method can effectively improve the agglomeration phenomenon of inorganic nanomaterials in hydrogel by modifying the surface of the nanomaterials to form a covalent bond between the inorganic nanomaterials and the hydrogel network [21,38]. At the same time, due to the enhanced interaction between nanoparticles and hydrogel polymers, the prepared nanocomposite hydrogel has stronger mechanical properties, and the surface of the nanomaterial is mainly modified by grafting method for polyacrylamide (PAM) hydrogels. Messing et al used methacrylic acid groups to modify the surface of magnetic nanoparticles, and then added the modified magnetic nanoparticles to the polyacrylamide hydrogel network to prepare nanocomposite hydrogels, which shows relatively high mechanical properties, including tensile strength and fracture toughness [39]. Another similar study synthesized a nanocomposite hydrogel by grafting method, and its nano iron oxide particles were fused with 3-(trimethoxysilyl) propyl methacrylate and polydimethylsiloxane (PDMS) was used to modify the surface of the hydrogel [40]. The mechanical properties of this nanocomposite hydrogel are greatly improved, and at the same time, it can maintain stability under fatigue load, which is very suitable for the application of bone tissue engineering [41]. Therefore, the advantage of the grafting method is that after the surface of the nanomaterial is modified, the nanoparticles and the hydrogel polymer are combined in a covalent bond, which greatly improves the mechanical strength and stability of the nanocomposite hydrogel [7,42,43].

In Situ precipitation
In situ precipitation is to uniformly disperse inorganic nanoparticles in hydrogel polymer monomers, and then polymerize the monomers under certain temperature conditions to form nanocomposite hydrogels [44,45]. This method can prepare nanocomposite hydrogels with good dispersion effect, can maintain the excellent properties of nanocomposite particles, and at the same time make the mutual bonding between nanoparticles and hydrogels more stable [46,47]. For example, Wang et al. synthesized magnetically responsive nanocomposite hydrogels by in situ precipitation. The main preparation process is to mix the magnetic nanoparticles and chitosan hydrogel, and then the mixture is polymerized in NaOH solution, in which chitosan and iron ions can chelate, this interaction makes the magnetic nanoparticles evenly dispersed in the hydrogel, and finally obtain a nanocomposite hydrogel with good magnetic response and mechanical properties [28]. Meanwhile, the mechanical properties of nanocomposite hydrogels can be enhanced by increasing the concentration of magnetic nanoparticles (0 to 15 wt%). In addition, in situ precipitation can be triggered by appropriate heating, Yu et al. added an ultrasonically treated graphene aqueous suspension to an acrylamide monomer, and an in situ free radical polymerization reaction occurred under 25 C water bath conditions, and a graphene nanocomposite hydrogel was formed after 48 h. In situ precipitation can ensure that the nanoparticles are evenly distributed in the hydrogel and maintain the characteristics of the nanoparticles, so the mechanical strength of the nanocomposite hydrogel can be prepared, the disadvantage is that the alkaline solution used may affect the ability of the hydrogel network cells to load, and the inorganic nanoparticles must be modified before the polymerization reaction [36,37,48].

Freeze-thaw method
The freeze-thaw method is a method for preparing nanocomposite hydrogels by physical cross-linking. The method is mainly to repeatedly thaw the hydrogel mixed solution at low temperature and room temperature [35,49]. In this process, polymer aggregation and non-aggregation areas are formed internally when the hydrogel mixture solidifies, and when thawing, the polymer aggregation areas will undergo physical crosslinking to form a crosslinking network [50]. Huang et al. prepared a magnetic nanocomposite hydrogel with good mechanical properties and biocompatibility by freeze-thaw method. During the preparation process, they mixed PVA, nanohydroxyapatite (n-HA) and Fe2O3 in a certain proportion, mixed evenly at 60 C, frozen at À20 C for 16 h, thawed at room temperature for 4 h, and repeated freeze-thawed 7 times to finally obtain a nanocomposite hydrogel [51]. Studies have reported that the number of freeze-thaws affects the mechanical strength and magnetization strength of magnetic nanocomposite hydrogels. The disadvantage of the freeze-thaw method is that the physical crosslinking is reversible, so the structure of the prepared hydrogel crosslinking network is not very strong, and it is easy to decompose quickly due to external influences.
3. Classification of nanocomposite hydrogels for bone tissue engineering applications

Clay-based nanocomposite hydrogel
Clay minerals are a class of inorganic layered nanomaterials consisting of two main units: tetrahedral (T) and octahedral (O) sheets [42]. Nanoclay is a type of ultrathin (1 nm) polar nanomaterial [52,53] with excellent biocompatibility [54], and its degradation products (e.g. magnesium and silicates) are non-toxic, resorbable, and capable of promoting cellular osteogenesis [55]. The addition of nanoclay materials (dispersed phase) to the crosslinked network of the hydrogel (continuous phase) improves the mechanical properties and bioadhesion of the hydrogel, and the resulting nanocomposite hydrogel retains its previous inherent processing properties [56]. These properties provide a theoretical basis for the application of clay-based nanocomposite hydrogels in bone tissue engineering ( Figure 1) [57].
According to the chemical composition and morphology of the nanoparticles, nanoclays can be divided into several categories, such as montmoril- The main nanoclay used in bone tissue engineering is Laponite, which consists of nanosheets with a high aspect ratio, width close to 25 nm, and thickness of 1-2 nm [58]. Laponite has a negative surface charge due to the presence of surface hydroxyl groups. Owing to this, it is prone to electrostatic interactions with positively charged polymers. These interactions allow Laponite to be uniformly dispersed in the hydrogel, making the resulting nanocomposite hydrogel highly stable [59,60].
Several studies have reported that Laponite composite hydrogels can promote osteogenesis through multiple pathways. For example, its degradation product (magnesium ions) can promote osteogenesis by activating the hypoxia-inducible factor-1 (HIF-1a) and peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a) signalling pathways [61], and direct the differentiation of gel-encapsulated cells (e.g. endothelial cells and mesenchymal stem cells) [55,62]. In addition to the interaction with cells, some studies have suggested that nanoclay is internally 'bioactive' and can initiate biomineralisation to enhance calcium phosphate (CaP) deposition and thus promote osteogenesis [63][64][65][66]. Owing to the size and shape of clay nanoparticles, theoretically, they can be endocytosed by cells (optimal particle size for endocytosis is 25-30 nm). The clay nanoparticles interact with cells after endocytosis to exert their osteogenesis-promoting effects, but the exact mechanism is not clear [54,67,68]. In addition to the biological function of promoting osteogenesis, nanosilicates can also combine with various substances for bone repair, such as DNA Multifunctional DNA nanostructures have been created for drug delivery and therapy [69]. The surface of a nanosilicate (Laponite XLS) has an anisotropic charge distribution and can interact with the phosphate groups on the DNA backbone through non-covalent bonds, which not only enhances the mechanical strength of the hydrogel but also loads the DNA for slow release. Based on this property, a research team has developed an injectable DNA-nSi nanocomposite hydrogel with a porous structure and shear-thinning property ( Figure 2) [70].(The shear-thinning nanocomposite hydrogels can be easily injected using a fine 22-gauge needle, and because of their ability to reassemble after shear stress is removed, gelation occurs almost immediately after injection. The injectable nature allows it to be used in minimally invasive procedures, reducing the risk of blood loss and infection, and it can be used for filling irregular bone defects. Nanoclay has the advantages of high surface reactivity and extensive interactions with polymers, cells, and minerals. The nanocomposite hydrogels made from nanoclay have significantly enhanced mechanical strength while maintaining the physical properties of the hydrogel (the nanoclay acts as a physical crosslinking agent) [66,[71][72][73]. The results of Haraguchi K's research show that the elongation at break of nanoclay composite hydrogels is 10 times that of pure hydrogels [74]. Another study reported that the tensile modulus, elongation, and tensile strength of PNIPAAM/Laponite XLG nanocomposite hydrogels were as high as 453 kPa, 1,000%, and 1.1 MPa, respectively. Its breaking energy is more than 1,000 times that of the classic PNIPAAM hydrogel [75] Clay-based nanocomposite hydrogels have significant potential for bone tissue engineering; multiple studies have been conducted to test different combinations of formulations for improving the performance of these hydrogels, while the research on enhancing hydrogel's bioactivity and osteogenesis on stem cells has also progressed.

Ceramic-based nanocomposite hydrogel
Ceramics are inorganic materials that are mixtures with the main components of silica and silicates (e.g. aluminium silicate, calcium silicate). Nanoceramic materials have grains and grain boundaries, and their bonding is at the nanometre level. This gives them higher strength, toughness, superplasticity, and electromagnetic properties than ordinary ceramic materials [76]. The addition of nanoceramics can enhance the mechanical properties of hydrogels, thereby increasing the effectiveness of hydrogels for bone repair [26,77,78]. The most widely used inorganic phosphate-based ceramics for bone tissue engineering are calcium phosphates, including hydroxyapatite (HA), b-tricalcium phosphate (b-TCP), and their mixtures-bidirectional calcium phosphate (BCP) [79][80][81]. The inorganic component of bone is mainly composed of HA; hence, nanohydroxyapatite (nHAp) composite hydrogels have good biocompatibility with bone tissues. Meanwhile, nHAp composite hydrogels promote osteogenesis and vascularisation as well as possess high mechanical strength [82][83][84][85]. C Giordano et al. found that the nHA composite hydrogel maintains excellent swelling properties, its maximum deformation in the swollen state increases (from about 0.7% to 19%) and the elastic modulus decreases (from 2027.51 MPa to 19.92 MPa), which also meets the mechanical properties of cancellous bone [86]. In another study, nHA composite hydrogels exhibited excellent mechanical properties. Its tensile stress at break ranges from 0.21 to 0.86 MPa, the tensile strain at break can reach 30 mm/mm, and its compressive strength can reach 35.8 MPa [87]. He et al. reported a polyacrylamide/nHAp hydrogel; with nHAp as a crosslinking agent that binds to the polymer through interactions such as hydrogen bonding, the nanocomposite hydrogel has better mechanical properties than ordinary hydrogels. This hydrogel also has interconnected homogeneous pores that allow better adhesion of mesenchymal stem cells to the scaffold, whereas nHAp promotes osteogenic differentiation of cells [88]. Similarly, Gaharwar et al. proposed a photocrosslinked elastic nanocomposite hydrogel composed of nHAp and gelatin methacrylate (GelMA) (Figure 3), which exhibited better mechanical properties and cell adhesion, and promoted mesenchymal stem cells differentiation towards osteogenesis, the use of this nanocomposite hydrogel results in more cartilage tissue production, which promotes more collagen II production compared to pure hydrogels [89,90]. In another study, Jeong et al. also demonstrated the significant enhancing effect of nHAp on the mechanical strength of hydrogels while improving cell adhesion function and promoting osteogenic differentiation of mesenchymal stem cells. In the study, the osteogenesis-related indicators (alkaline phosphatase, bone morphogenetic protein 2, and osteocalcin) of mesenchymal stem cells loaded on the heat-responsive poly(ethylene glycol)poly(l-alanine-co-l-phenylalanine) (PEG-PAF)/calcium phosphate hydrogels were highly expressed [91]. In addition to directly promoting osteogenesis, nHAp can also regulate the bone immune microenvironment and enhance the activity of vascular endothelial cells, acting as an indirect promoter for osteogenesis [92][93][94].
Both b-TCP and BCP have also been used to synthesise nanocomposite hydrogels, which are superior to hydrogels synthesised from nHAp in terms of certain properties. Nanocomposite hydrogels formed from natural hydrogels and BCP not only displayed improved mechanical strength and cell adhesion and proliferation but also overcame the drawback of slow degradation of nHAp, Song et al. found that Young's modulus of the BCP nanocomposite hydrogel is more than 2.5 times higher than that of the pure hydrogel, and it has a good interconnected porous structure with a pore size of 65.3 mm [95]. In addition to natural hydrogels, another study reported poly(glycolylated poly(glycerol sebacate)) compounded with b-TCP nanoparticles as a hydrogel. The advantage of this composite hydrogel is that PEG and b-TCP synergistically enhanced osteoconduction and promoted mineralisation, its maximum tensile strength is about 9.58 MPa, which is 1.5 times that of the pure hydrogel group [96].
In addition to the above-mentioned nanoceramic materials, other ceramic-based materials, such as alumina, bioactive glass, and titanium dioxide, with higher elasticity are used in bone tissue engineering. The hydrogels prepared from these ceramic-based materials and different polymers exhibit immunomodulatory, antibacterial, superabrasive, and selfhealing properties [16,[97][98][99][100][101][102]. Strontium, an alkaline earth metal, is a novel nanomaterial that promotes osteogenesis and has a significant potential for applications in the field of bone tissue engineering. Strontium-based nanocomposite hydrogels are expected to replace the current ceramic-based nanocomposite hydrogels; however, it has only been reported in limited studies so far.

Iron oxide-based nanocomposite hydrogel
Iron oxide nanoparticles are biocompatible and can adhere to cells, while the composite hydrogels based on iron oxide nanoparticles have magnetic and thermal responsive properties [103][104][105]. Iron oxide nanocomposite hydrogel as a magnetic biomaterial has been proven to promote osteogenesis [106]. The possible mechanism is that the contact between cells and magnetic materials generates nanoscale forces similar to mechanical forces, which stretches cell membranes and activates the associated channels and receptors [107]. In addition to the effect of intrinsic magnetic field on cells, applied magnetic field interference influences cell behaviour (cell proliferation, migration direction, and differentiation) [108][109][110][111][112]. Besides the effect of magnetic field, studies have found that iron oxide nanoparticles exhibit a peroxidase-like ability in a cytoplasmic environment to activate the p38 mitogen-activated protein kinase (MAPK) signalling pathway to accelerate cell cycle progression and promote cell proliferation [113,114]. So far, many iron oxide-based nanocomposite hydrogels have been developed for bone tissue engineering applications. These nanocomposite hydrogels have excellent mechanical strength and magnetic-responsive properties that can be tuned by the type, content, and size of the iron oxide particles. For example, in a chitosan hydrogel with iron oxide nanoparticles, the mechanical properties of the hydrogel improved with an increase in the content of the incorporated magnetic particles. The strength and elastic modulus of the iron oxide nanocomposite hydrogel were increased by 416% and 265% compared with the pure hydrogel group. Under the action of a low-frequency alternating magnetic field (LAMF), the drug in the nanocomposite hydrogel was switched from passive release to pulsed release, and the cumulative and partial drug release was increased by 67.2% and 31.9% compared with the pure hydrogel group [28]. In another study, it was found that larger iron oxide nanoparticles could effectively avoid aggregation, better crosslink with the hydrogel, and improve the mechanical strength of the composite. The addition of iron nanoparticles increased the mechanical stiffness of the hydrogel by 10 times and the toughness by 20 times, while the mechanical stiffness of the composite hydrogel was tuned by changing the concentration and size of the iron nanoparticles (0.2KPa$200KPa) [107,115]. The addition of iron oxide nanoparticles not only enhanced the mechanical strength of the composite hydrogels but also added more suitable cell adhesion sites. For example, in a mechanically rigid collagen/iron oxide (Fe 3 O 4 ) NPs hydrogel, the iron oxide nanoparticles acted as the crosslinking agent and substantially enhanced the hydrogel strength and increased the number of cell adhesion sites (Figure 4) [115]. Externally applied magnetic fields can also alter the properties of iron oxide nanocomposite hydrogels by causing vibrational and rotational displacements of magnetic particles. This results in microscopiclevel changes in the shape of the composite and the appearance of increased micropores on the surface of the hydrogel that are favourable for cell growth. Based on this phenomenon, iron oxide nanoparticles were added into hydrogels containing arginine glycyl aspartate (RGD) to dynamically regulate cellmatrix interactions through an applied magnetic field ( Figure 5), promoting cell proliferation and differentiation, and ultimately realising tissue regeneration [116]. The strength of the magnetic field also influences the proliferation and differentiation functions of the cells. It has been reported that an applied magnetic field of moderate strength (from 1 mT to 1 T) can effectively promote mesenchymal stem cell proliferation and osteogenic differentiation [43,107]. In addition to directly promoting osteogenesis, iron oxide nanocomposite hydrogels can indirectly promote bone regeneration by enhancing blood vessel formation. The composite hydrogel formed by adding iron oxide nanoparticles to the PEG hydrogel containing stromal vascular component cells of human adipose tissues can promote bone tissue formation as well as blood vessel angiogenesis in tissues. The mechanism behind this may be that the magnetic drive stimulates the function of progenitor

Black phosphorus (BP)-based nanocomposite hydrogel
Black phosphorus (BP) is a nanomaterial composed of a single element, phosphorus, with stable chemical properties and excellent biocompatibility [117]. During bone remodelling, bone mineralisation induced by proper calcium and phosphorus metabolism is required in addition to the involvement of osteoblasts and osteoclasts [118,119]. BP is highly homologous to the inorganic components of bones [120]. Its degradation products are nontoxic phosphates, which can promote calcium deposition in bone tissues and facilitate bone repair. The compressive modulus of this nanocomposite hydrogel is more than 2 times that of the pure hydrogel group, and it can also form more surface mineralization to promote bone formation [121]. Based on this feature, researchers constructed a BP/PEA/GelMA composite hydrogel system for bone tissue engineering, which was prepared by photo-crosslinking GelMA, BP nanosheets (BPN), and cationic arginine-based unsaturated polyesteramide. BPNs were encapsulated inside the hydrogel, which not only improved the mechanical strength of the hydrogel but also released phosphorus ions upon degradation to promote bone defect repair. Its compressive modulus is 3-4 times higher than that of the pure hydrogel. In vitro and in vivo experiments have shown that it can form more mineralization than pure hydrogel, and the final bone repair effect is better [121]. In another study, researchers constructed a BPN composite double network (DN) hydrogel, with the ductile network comprising PHEA, PDMA, or PAM and the brittle network comprising chitosan-methacrylate (ChiMA), alginate-methacrylate (AlgMA), or gelatin-methacrylate (GelMA). BPNs were compounded into various double network hydrogels in different combinations. Under weak alkaline conditions, the composite hydrogel could induce accelerated CaP nanocrystal formation via BPNs to mimic biomineralisation, thereby promoting bone formation [122]. In addition, BPNs have excellent light absorption in the near-infrared (NIR) region, which enables stable and durable photo-controlled regulation of the release of loaded active substances as well as photothermal conversion and thermal therapy at the composite hydrogel sites [123]. Local thermal therapy has been confirmed to enhance the expression of alkaline phosphatase (ALP) and heat shock protein (HSP), thereby accelerating bone regeneration [124][125][126]. Based on this property, researchers constructed a heat-responsive BPN/PRP/chitosan hydrogel system to treat bone defects caused by rheumatoid arthritis (RA). The BPN in this composite hydrogel generates local heat in the NIR region while producing reactive oxygen species (ROS), which are delivered to diseased joints to treat abnormal synovial hyperplasia. The composite hydrogel can also precisely control the release of BP degradation products (phosphorus ions), providing sufficient raw material for bone regeneration and facilitating the repair of bone defects caused by RA [127].
Owing to their high specific surface area, BPNs are excellent drug carriers(its drug loading efficiency can reach 950%), and drugs are adsorbed on BPNs through non-chemical bonding(this property preserves drug activity and purity); controlled drug release can be achieved under NIR light irradiation due to the photothermal effect of BPNs [128]. Based on this property, researchers developed PLGA hydrogel microspheres combined with SrCl 2 and BPNs for bone regeneration. The composite exhibited good light absorption and photothermal effects under NIR light, and its temperature increases by 24 C after 10 min of NIR light irradiation. The BPN-induced photothermal conversion generated heat at the nanocomposite hydrogel sites to break the hydrogen bonds in the network. After turning off the light irradiation, the heat dissipated, the nanocomposite hydrogel cooled down gradually, and the broken hydrogen bonds reappeared in the nanocomposite hydrogel [129]. Therefore, we conclude that the BP-based nanocomposite hydrogels possess multiple functions, including stronger mechanical properties, promotion of bone formation, stronger drug loading properties, and photothermal properties. It shows great potential in bone tissue engineering, especially in repairing bone defects caused by bone tumors

Carbon-based nanocomposite hydrogel
Owing to their excellent optical, mechanical, and electrical properties, carbon-based nanomaterials, such as graphene and carbon nanotubes (CNTs), have been used to prepare nanocomposite hydrogels with unique properties. After the addition of CNTs, the elastic modulus of GelMA hydrogel increased from 15KPa to 60KPa and the compressive modulus increased from 10KPa to 30KPa similar to the compressive modulus of bone matrix (25 À 40 kPa). At the same time, the nanocomposite hydrogel maintains a highly microporous structure. However, the elongation at break of the GelMA hydrogel (72%) is higher than that of the CNT-incorporated hydrogel (58%) ( Figure 6) [130,131]. Compounding modified carbon-based nanomaterials (e.g. adding polar groups to improve their water solubility) with hydrogels, the mechanical strength of hydrogels can be substantially enhanced and the synthesised nanocomposite hydrogels can be equipped with other properties (e.g. electrical properties, optical properties, and elasticity) [132,133]. Although CNTs have many successful applications in biomedical engineering, some studies have reported that the use of carbon nanotubes may induce increased cytotoxicity [134,135].
Graphene is a novel material in which carbon atoms connected by sp 2 hybridisation are tightly packed into a monolayer two-dimensional honeycomb lattice structure. Its derivatives include graphene oxide (GO), carboxyl graphene (CXYG), reduced graphene oxide (rGO), and graphene quantum dots (GQDs) [136]. GO contains hydroxyl and carboxyl groups that make it hydrophilic and enable it to disperse uniformly in hydrogels. GO exhibits good biocompatibility and promotes cell proliferation and differentiation, in vitro studies confirmed that GO can promote the differentiation of hMSCs towards osteogenesis [137]. It provides a microenvironment for cell proliferation while controlling cell differentiation; moreover, its broad surface can provide interaction sites for proteins and cytokines, enriching osteogenesis-related factors and minerals. In addition, GO also has excellent antibacterial properties; graphene family materials can act as electron acceptors and cause non-dependent oxidation of superoxide anions in bacteria to kill bacteria [138][139][140]. Based on these properties of GO, its complexation with hydrogels has led to the synthesis of several nanocomposite hydrogels for bone tissue engineering applications. For example, scaffolds with dual functions of inducing bone regeneration and preventing bacterial infection were fabricated by depositing GO on a polydopamine (PDA)-modified titanium scaffold to form a GO/Ti porous scaffold and then assembling gelatin microspheres (GelMS) encapsulated with 2(bone morphogenetic protein) (BMP2) and vancomycin (Van) on the scaffold, the common stent released 76.9% of the loaded drug in 5 days, while the nanocomposite hydrogel stent released 63.4% of the loaded drug in 21 days [141]. Moreover, an alginate/GO composite as a 3 D printing ink for repairing bone defects has been reported. This nanocomposite hydrogel improved cell printing performance and increased cell survival rate and differentiation capability. The GO-added hydrogels all exhibited shear-thinning properties, and the shear moduli of alginate/GO-0.5 and alginate/GO-1.0 at 1 Hz were 1.19 and 1.23 times higher than those of alginate/GO-0, respectively [142]. Similarly, nanocomposite hydrogel scaffolds (GO/ PDLLA) have been proposed to promote cartilage regeneration by releasing TGF-b3. Many studies have incorporated transforming growth factor-b (TGF-b) into scaffolds to promote cartilage repair, but TGF-b has limited utility due to its short functional half-life and/or rapid clearance. TGF-b loaded by GO/PDLLA hydrogel scaffolds can be released stably for 4 weeks, and the compressive modulus of GO/PDLLA hydrogel is about 1.32 to 1.45 times higher than that of control PDLLA hydrogel [143]. GO can also enhance the mechanical strength of hydrogels. It has been reported that the addition of graphene to sericin methacryloyl (SerMA) hydrogels (SMH) can improve the compression modulus of the composite. Specifically, the compression modulus of the composite hydrogel increased by 17 kPa upon increasing the content of GO from 0.02% to 0.04% (the compressive modulus is 17 kPa for SMH, 34 kPa for SMH/GO-0.01%, and 43 kPa for SMH/GO-0.02%), and the SMH/GO hydrogel exhibited a superior therapeutic effect in a rat cranial defect model (Figure 7) [144]. GO-reinforced regenerated cellulose/polyvinyl alcohol (GO-RCE/ PVA) ternary hydrogels were prepared in another study, and it was confirmed that the elongation at break and tensile strength of the composite hydrogels significantly increased on increasing the GO content. With the addition of 1.0 wt% GO, the tensile strength increased from 0.52 MPa to 0.73 MPa (40.4% increase) and the elongation at break increased from 103% to 238%. With the addition of 0.8 wt% GO, the swelling ratio of the GO-RCE/PVA ternary hydrogel increased from 150% to 310% [145].

Summary
Bone is a highly organized composite material consisting of 50-70% inorganic components (mainly  Mechanical properties: The compressive modulus of the outermost nanosilicate composite hydrogel is 6.5 ± 1.0 kPa 3 D printing properties: The printed tubular scaffold is divided into three layers, the innermost layer degrades after 12 days to form 500 lm channels (to promote blood vessel ingrowth); the outermost layer is loaded with mesenchymal stem cells to promote osteogenesis. Biocompatibility: After 7 days of culture, HUVECs and hMSCs survived at 94% and 77% and maintained proliferation in the printed scaffolds. Drug Loading and Release Performance: The composite scaffolds released almost four times the amount of VEGF that physically mixed GelMA þ VEGF [155] Clay-based nanocomposite hydrogel Silicate Nanoparticlesþ Gelatin þ DNA Injectable properties：Their shear-thinning properties are maintained after drug loading, confirming their potential as injectable drug delivery systems.
Mechanical properties: Self-supporting structure can be formed after injection, yield stress increases from 29.45 ± 3.75 to 73.98 ± 9.42 Pa Drug Loading and Release Performance: Gelatin hydrogels released approximately 80% of the loaded drug within 2 days, and nanocomposite hydrogels released 50% of the loaded drug within 5.5 days Biocompatibility: After 72 h in vitro, all hydrogeltreated groups showed more than 85% cell viability [70] Ceramic-based nanocomposite hydrogel Calcium phosphate Nanoparticles þ collagen þ dexamethasone Mechanical properties: It has a porous structure similar to that of pure collagen hydrogel scaffolds, but Young's modulus of BCP/collagen composite scaffolds is at least 2.5 times higher than that of collagen scaffolds Porous structure: The diameter of the spherical large hole is 405 mm$450mm, and the diameter of the small hole is 65.3 mm ± 23.5 mm Biocompatibility: MSCs were able to adhere and proliferate better in BCP scaffolds, with more [95] (continued) (continued) (continued) The ALP activity of alg/GO hydrogel was 2.5 times higher than that of the control group. [142] Carbon-based nanocomposite hydrogel Graphene oxide (GO)þ Ti scaffolds þ gelatin microspheres (GelMS) Mechanical properties:The fixation rate of GO/Ti scaffold was 60%, which was higher than that of pure Ti scaffold (30%) Porous structure: a 3 D interconnected porous structure with a pore size of 100 to 200 mm Drug Loading and Release Performance: GelMS GO/ Ti scaffolds exhibit sustained-release and dual-release [141] (continued) hydroxyapatite (HAp), 20-40% organic components (mainly type I collagen), 5-10% Water, and 3% lipid. Macroscopically, bone can be divided into the outer hard cortical bone and inner spongy cancellous bone [146]. Bone tissue engineering aims to develop new bone repair strategies to regenerate and repair damaged or diseased bone tissue, replacing more traditional bone grafting methods (such as autografts or allografts). Materials used for bone tissue engineering should possess some basic properties. Such as biocompatibility: the materials used should allow cell adhesion, proliferation, and differentiation without cytotoxicity and excessive immune response during implantation [147]. For example, osteoconductivity and osteoinductivity refer to the properties of materials that can promote the precipitation of bone on non-material surfaces and the differentiation of mesenchymal stem cells, respectively [148]. The implanted biomaterial should have sufficient mechanical strength to support the bone tissue. For example, the elastic modulus of cortical bone is 14 $ 20 GPa, the compressive strength is 130 $ 225 MPa, and the elastic modulus of cancellous bone is 1 $ 2 GPa, the compressive strength is 7 $ 10 MPa, and the compressive modulus of cartilage is 1.9 $ 15 MPa [149][150][151]. The interconnected porous structure of the material is also a key factor in the design of scaffolds for bone repair, which allows native cell migration and proliferation. In the structure of bone tissue, cancellous bone is highly porous (30-90% porosity) and cortical bone has only 5-30% porosity, so most metabolic activity occurs in cancellous bone. Typically, materials with pore sizes above 200 mm can promote new bone formation and vascularization [55]. The geometry of the porous structure has a profound effect on both the mechanical and biological responses of bone tissue, and 3 D printing technology enables the production of customized bone tissue engineering scaffolds that can tune its porosity, mechanical properties, and structure. For 3 D printing strategies, printable bioinks are the key to success. Several aspects should be considered for printable bioinks, such as rheological behavior (viscosity and shear thinning), surface tension, swelling, gelling kinetics, and mechanical properties of the material. Another important aspect is the cross-linking (physical, chemical or enzymatic) of the bioink during processing and post-processing steps to maintain the biomechanical stability of the printed product structure [152].
Nanocomposite hydrogels represent a new generation of advanced biomaterials that are widely used in bone tissue engineering. Bone tissue engineering aims to repair bone defects caused by various reasons and faces diverse challenges. Nanocomposite hydrogels have good biocompatibility and can mimic the cellular matrix environment; cells can grow and proliferate well in the scaffold; and various cytokines can flow in the porous three-dimensional structure. Moreover, nanocomposite hydrogels can gradually degrade and get absorbed in the process of bone repair, providing space for bone regeneration. Furthermore, its degradation products are non-toxic and can even promote osteogenesis (e.g. the degradation products are calcium and phosphorus ions). The scaffold material used for bone repair should also have a certain amount of mechanical strength. By adding hard inorganic nanomaterials, the mechanical strength of nanocomposite hydrogels can be enhanced and adjusted by changing the formula. Moreover, multi-layered bionic structures can also be constructed for different load-bearing stages. Vascular coupling is important in bone repair; the porous structure of nanocomposite hydrogels can facilitate oxygen exchange and vascular endothelial cell growth, and the addition of appropriate nanomaterials and encapsulation of pro-vascular factors can facilitate the angiogenesis of blood vessels. Bone repair is more difficult in infected and inflammatory environments. To address this problem, the addition of nanomaterials with antimicrobial and anti-inflammatory capabilities can equip the hydrogels with antimicrobial and anti-inflammatory effects in bone regeneration to promote osteogenesis. The incorporation of various nanomaterials can endow the composite hydrogels with stimulus-responsive properties, such as heat and magnetic responses, which can further enhance bone repair performance while providing a new strategy for controlled drug release. We summarize the properties of various types of nanocomposite hydrogels in Table 1.
In general, the nanocomposite hydrogels used for bone tissue engineering should be based on various factors of bone repair. By reasonable design and matching of nanomaterials and polymers, nanocomposite hydrogels have been developed with multiple adjustable functions. These functions include mechanical strength, elasticity, surface topography, controlled biodegradability, wettability, self-healing property, and stimulus responsiveness. Moreover, the cell loading capacity of nanocomposite hydrogels is an important factor. The effect of nanomaterials on the fate of mesenchymal stem cells should be explored, as it is an important factor for the construction of suitable bioactive materials.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The authors acknowledge the funding provided by the National Natural Science Foundation of China (81972069).

Notes on contributors
Chengcheng Du holds a bachelor's degree in clinical medicine from Nanchang University and a bachelor's degree in biomedicine from Queen Mary University of London and is currently studying for a master's degree in orthopaedics at Chongqing Medical University. His research focuses on bone defect repair with mesenchymal stem cells combined with nanocomposite hydrogels.
Wei Huang is the director and professor of the First Affiliated Hospital of Chongqing Medical University and the head of the Orthopaedic Research Institute of Chongqing Medical University. His research focuses on bone defect repair and cartilage regeneration. Currently developed antibacterial bone repair materials, nano-bone filling materials and porous tantalum have achieved clinical transformation, and he is working on the development of biomaterials for cartilage repair.