Injectable hydrogels attract a great deal of attention due to their in situ formation in vivo, which minimizes the risk of major trauma during the surgical procedure as compared to traditional hydrogel scaffolds. Click chemistry is widely used in the preparation of injectable hydrogels, although some of them require photoinitiators or catalysts that may be cytotoxic and impair the proliferation of encapsulated cells. In the present study, an injectable dual crosslinking hydrogel was designed by a thiol–ene click reaction and conversion between sulfhydryl and disulfide bonds at a neutral pH. The sulfhydryl group and acrylate group were successfully grafted onto HA and characterized using FT-IR and ¹H-NMR. The gelation time, morphology, injection force, swelling, degradation, mechanical properties and drug release behavior of the hydrogel varied with the molecular weight of the hydrogel (M_w = 0.1, 1.0, 2.0 MDa). MTT, FDA/PI staining and scanning electron microscopy (SEM) showed that the encapsulated L929 proliferated well in different molecular weight hydrogels. It was also observed that in a low molecular weight (0.1 MDa) hydrogel system, the cells were better distributed and secreted more extracellular matrix. Subcutaneous injection in mice also demonstrated that hydrogels could support the proliferation of encapsulated L929 cells in vivo, and no significant infiltration of peripheral cells into the interior of the material was observed. These results indicate that this injectable hyaluronan-based hydrogel is an excellent cell protectant and exhibits good biocompatibility, with potential for biomedical applications such as cell delivery and postoperative anti-adhesion.

Translation of mRNA to protein is tightly regulated by transfer RNAs (tRNAs), which are subject to various chemical modifications that maintain structure, stability, and function. Deficiency of tRNA N⁷-methylguanosine (m⁷G) modification in patients causes a type of primordial dwarfism, but the underlying mechanism remains unknown. Here we report that the loss of m⁷G rewires cellular metabolism, leading to the pathogenesis of primordial dwarfism. Conditional deletion of the catalytic enzyme Mettl1 or missense mutation of the scaffold protein Wdr4 severely impaired endochondral bone formation and bone mass accrual. Mechanistically, Mettl1 knockout decreased abundance of m⁷G-modified tRNAs and inhibited translation of mRNAs relating to cytoskeleton and Rho GTPase signaling. Meanwhile, Mettl1 knockout enhanced cellular energy metabolism despite incompetent proliferation and osteogenic commitment. Further exploration revealed that impairment of Rho GTPase signaling upregulated the level of branched-chain amino acid transaminase 1 (BCAT1) that rewired cell metabolism and restricted intracellular α-ketoglutarate (αKG). Supplementation of αKG ameliorated the skeletal defect of Mettl1-deficient mice. In addition to the selective translation of metabolism-related mRNAs, we further revealed that Mettl1 knockout globally regulated translation via integrated stress response (ISR) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Restoring translation by targeting either ISR or mTORC1 aggravated bone defects of Mettl1-deficient mice. Overall, our study unveils a critical role of m⁷G tRNA modification in bone development by regulation of cellular metabolism and indicates suspension of translation initiation as a quality control mechanism in response to tRNA dysregulation.

Trauma rapidly mobilizes the immune response of surrounding tissues and activates regeneration program. Manipulating immune response to promote tissue regeneration shows a broad application prospect. However, the understanding of bone healing dynamics at cellular level remains limited. Here, we characterize the landscape of immune cells after alveolar bone injury and reveal a pivotal role of infiltrating natural killer T (NKT) cells. We observe a rapid increase in NKT cells after injury, which inhibit osteogenic differentiation of mesenchymal stem cells (MSCs) and impair alveolar bone healing. Cxcl2 is up-regulated in NKT cells after injury. Systemic administration of CXCL2-neutralizing antibody or genetic deletion of Cxcl2 improves the bone healing process. In addition, we fabricate a gelatin-based porous hydrogel to deliver NK1.1 depletion antibody, which successfully promotes alveolar bone healing. In summary, our study highlights the importance of NKT cells in the early stage of bone healing and provides a potential therapeutic strategy for accelerating bone regeneration.

The integrated stress response (ISR), regulated by general control nonderepressible 2 (GCN2), is essential for maintaining tissue homeostasis, yet its role in periodontitis remains poorly understood. Here, through transcriptomic analysis and immunohistochemistry of gingival biopsies from patients and a ligature-induced mouse periodontitis model, we demonstrate that GCN2-mediated ISR is activated in both human and mouse periodontitis and mainly functioned in macrophages. Using Gcn2 ^-/- mice, we show that Gcn2 deletion exacerbates gingival inflammation and bone loss in experimental periodontitis. Mechanistically, bulk RNA-seq and in vitro assays revealed that the loss of GCN2 impairs autophagy and leads to overactivation of the NLRP3/CASPASE1 inflammasome pathway. Notably, local administration of halofuginone, a GCN2 activator, mitigates oral inflammation and tissue destruction in a GCN2-dependent manner. In summary, our work highlights the protective role of the GCN2-mediated ISR in oral mucosa and indicates GCN2 as a promising therapeutic target for periodontitis.