Critical-sized bone defects are usually caused by trauma, fracture, infection, congenital malformation, osteosarcoma, or other degenerative diseases, and the repair of this defect remains a great challenge that will not spontaneously recover without intervention. The present therapeutic options, such as autografts and allografts, have their limits because of the scarcity of donor bones and the risk of immunological rejection. Additionally, acute injuries are often accompanied by the ascent of reactive oxygen species (ROS) produced by the inflammatory immune cells around the defect area, causing oxidative stress, leading to the damage of proteins and DNA structure, and finally inducing the apoptosis of cells and a delay in bone healing [[1], [2], [3]]. Alternatively, the development of biomaterials has provided a promising way to deal with critical-sized bone defects [4,5]. An increasing number of recently developed biomaterials have ROS-scavenging properties as an essential design in bone regeneration [3,6] or other inflammation related diseases [7]. In addition, irregular bone defects are very common in clinical practice, and minimally invasive treatment and implantation are favored by surgeons. In this setting, injectable biomaterials are extremely appealing. Therefore, the ideal biomaterials for bone regeneration should possess biosafety, good biocompatibility, biodegradability, suitable mechanical properties [8], ROS-scavenging, injectable, and osteogenic promotive properties.

Researchers have employed advanced fabrication technologies to develop various biomaterials or scaffolds with remarkable properties, aiming to repair and regenerate critical-sized bone defects. The commonly investigated scaffolds, such as bioceramics [9], porous polymeric or metallic scaffolds [10], bioactive glass scaffolds [11], calcium phosphate scaffolds [12], micro- or nanofibers [13], hydrogels [14], and other composite scaffolds-based biomaterials [15,16], have been developed for bone regeneration. These reported biomaterials provided good paradigms and demonstrated promising results in treating critical-sized bone defects. Furthermore, a thorough understanding of the process of repairing defective bone tissue should not overlook the intrinsic attributes of biomaterials, including their ability to scavenge ROS. Among these options, hydrogels, especially natural polymer-based hydrogels such as chitosan [17] and gelatin [18], showed great promise in tissue regeneration, such as in the repair of non-load-bearing bone defects, as this hydrogel possesses biosafety, biocompatibility, biodegradability, biomimetic structure, and components for the extracellular matrix. However, the mechanical property of gelatin-based hydrogels is low, and this hydrogel remains challenged by insufficient osteogenic-promotive performance and a lack of ROS-scavenging properties, which hinder potential further applications. Recently, researchers have reported various advanced studies aimed at addressing these issues with gelatin-based hydrogels. The commonly used strategies to improve these properties included incorporating nanoparticles, such as polydopamine-functionalized nanohydroxyapatite [19], mesoporous bioactive glass nanoparticles [20], nanofibers [21], nanosilver-incorporated halloysite nanotubes [22], increasing the degree of crosslinking of hydrogels [23], such as double network structures [24], nanoparticulate-reinforced cross-linked [19], and other combinations of bioactive components in gelatin [25,26]. Additionally, grafting functional groups or components to the matrix is considered an effective strategy to endow the targeted biomaterials with appealing properties [27]. Alternatively, gallic acid-grafted gelatin (GGA) showed antioxidant, anti-inflammatory, and ROS-scavenging properties [28], as gallic acid (GA) contains phenolic hydroxyl groups that can easily consume overexpressed reactive oxygen. GA is a natural antioxidant polyphenol, and it has three phenolic hydroxyl groups and one carboxyl group. The carboxyl group can combine with the amino group in gelatin to synthesize GGA polymers. GA is widely found in nature and has numerous biological activities, such as anticancer, antibacterial, anti-inflammatory, and others; thus, GA is widely used in biomedical applications [29]. In bone regeneration, GA, as an important component, is grafted onto matrix materials to improve the osteogenic promoting property [30]. GGA has been frequently used as matrix materials for biomedical applications or tissue engineering [31], especially in the case of ROS scavenging, anti-bacterial [32], and anti-inflammation, such as in osteoarthritis treatment [33], neural repair [28], wound healing [34], and others [35]. However, as per our review of the literature, we have found a few research works that have focused on GGA-based hydrogels for bone regeneration.

2D nanomaterials such as graphene oxide [36,37], nano-clay [38], and nanolayered 2D Ti3C2 MXenes have shown great potential for tissue engineering due to their attractive properties in drug loading, controlled release, and biological activities. Further modifications of these 2D nanomaterials can improve their mechanical and biological properties [39]. Ti3C2 MXene possesses unique planar structures, conductivity, biocompatibility, antibacterial property, biodegradability, photothermal effect, and biological imaging. Numerous biomedical fields, including biosensing [40], soft robots [41], antibacterial [42], photothermal therapy [43], neural modulation [44], bone tissue engineering [45], cancer therapy, and regenerative medicine [46], have explored these properties of MXene extensively for potential usage. MXene nanosheets were added to matrix materials in the field of bone tissue engineering to make them better at killing bacteria, promoting bone growth, or regulating immune cell behavior. As of now, there are only a few studies on the incorporation of MXene nanosheets into various kinds of matrix materials, such as polymeric membrane [47], nanocomposite membrane [45], and alginate methacrylate [48]. Researchers have used MXene nanosheets as additive materials in coatings [49] and incorporated them into natural polysaccharide composites for bone regeneration [50]. A recent study confirmed that MXene nanosheets can regulate the osteogenic differentiation of adipose-derived stem cells by activating the ERK signaling pathway [51]. The Ti-contained components released from MXene in the physiological environment (with the presence of oxygen) could promote new bone growth [52]. These research outcomes suggested that incorporation of MXene nanosheets into GGA-based hydrogel systems might have a positive effect on the osteogenic promotive and ROS-scavenging properties of this hydrogel in bone regeneration. Moreover, GGA possessed the ROS-scavenging property, and the phenolic hydroxyl groups in GGA can chelate with Ti in MXene, which may enhance the mechanical property of this hydrogel. To our best knowledge, there has been no such hydrogel system incorporating MXene nanosheets to explore the multifunctional properties in the regeneration of bone defects.

In this study, we have used the modified layer delamination method to synthesize MXene nanosheets. We incorporated MXene into the GGA aqueous pre-gel solution in the presence of the TG enzyme to develop an injectable GM nanocomposite hydrogel. We investigated and optimized the gelatin time of this hydrogel by adding varying quantities of TG enzymes. Further, we have investigated the swelling, biodegradable, mechanical, ROS-scavenging to OH- and DPPH radicals, and osteogenic inducing properties of GM hydrogels. Finally, we tested the in vivo osteogenic ability of the GM hydrogels by implanting them in critical-sized calvarial defects in rats for 4 and 12 weeks.