The immune microenvironment extensively participates in tumorigenesis as well as progression in osteosarcoma (OS). However, the landscape and dynamics of immune cells in OS are poorly characterized. By analyzing single-cell RNA sequencing (scRNA-seq) data, which characterize the transcription state at single-cell resolution, we produced an atlas of the immune microenvironment in OS. The results suggested that a cluster of regulatory dendritic cells (DCs) might shape the immunosuppressive microenvironment in OS by recruiting regulatory T cells. We also found that major histocompatibility complex class I (MHC-I) molecules were downregulated in cancer cells. The findings indicated a reduction in tumor immunogenicity in OS, which can be a potential mechanism of tumor immune escape. Of note, CD24 was identified as a novel “don’t eat me” signal that contributed to the immune evasion of OS cells. Altogether, our findings provide insights into the immune landscape of OS, suggesting that myeloid-targeted immunotherapy could be a promising approach to treat OS.

Traumatic brain injury (TBI) accelerates fracture healing, but the underlying mechanism remains largely unknown. Accumulating evidence indicates that the central nervous system (CNS) plays a pivotal role in regulating immune system and skeletal homeostasis. However, the impact of CNS injury on hematopoiesis commitment was overlooked. Here, we found that the dramatically elevated sympathetic tone accompanied with TBI-accelerated fracture healing; chemical sympathectomy blocks TBI-induced fracture healing. TBI-induced hypersensitivity of adrenergic signaling promotes the proliferation of bone marrow hematopoietic stem cells (HSCs) and swiftly skews HSCs toward anti-inflammation myeloid cells within 14 days, which favor fracture healing. Knockout of β3- or β2-adrenergic receptor (AR) eliminate TBI-mediated anti-inflammation macrophage expansion and TBI-accelerated fracture healing. RNA sequencing of bone marrow cells revealed that Adrb2 and Adrb3 maintain proliferation and commitment of immune cells. Importantly, flow cytometry confirmed that deletion of β2-AR inhibits M2 polarization of macrophages at 7th day and 14th day; and TBI-induced HSCs proliferation was impaired in β3-AR knockout mice. Moreover, β3- and β2-AR agonists synergistically promote infiltration of M2 macrophages in callus and accelerate bone healing process. Thus, we conclude that TBI accelerates bone formation during early stage of fracture healing process by shaping the anti-inflammation environment in the bone marrow. These results implicate that the adrenergic signals could serve as potential targets for fracture management.

Bupivacaine is frequently administered for diagnosing and controlling spine-related pain in interventional spine procedures. However, the potential cytotoxic effects of bupivacaine on intervertebral disc (IVD) cells and the underlying molecular mechanisms have not yet been fully established. Here, we showed that bupivacaine decreased the viability of rabbit IVD cells in a dose- and time-dependent manner. Moreover, the short-term cytotoxicity of bupivacaine in IVD cells was primarily due to cell necrosis, as assessed by Annexin V-propidium iodide staining and live/dead cell staining. Necrosis was verified by observations of swollen organelles, plasma membrane rupture, and cellular lysis under transmission electronic microscopy. Interestingly, our data indicated that bupivacaine-induced primary necrosis might involve the necroptosis pathway. The key finding of this study was that bupivacaine was able to induce lysosomal membrane permeabilization (LMP) with the release of cathepsins into the cytosol, as evidenced by LysoTracker Red staining, acridine orange staining, and cathepsin D immunofluorescence staining. Consistently, inhibitors of lysosomal cathepsins, CA074-Me and pepstatin A, significantly reduced bupivacaine-induced cell death. Finally, we found that bupivacaine resulted in an increase in intracellular reactive oxygen species (ROS) and that inhibition of ROS by N-acetyl-L-cysteine effectively blocked bupivacaine-induced LMP and cell death. In summary, the results of this in vitro study reveal a novel mechanism underlying bupivacaine-induced cell death involving ROS-mediated LMP. Our findings establish a basis for the further investigation of bupivacaine cytotoxicity in an in vivo system.

As a controllable biological process, regulated cell death (RCD) extensively participates in cellular homeostasis, organismal development, and the pathogenesis of diseases. This review addresses the research gaps by synthesising the findings on the complexity of RCD modes and their role in disc degeneration, and summarises the preclinical strategies to alleviate disc degeneration and promote disc repair by regulating RCD. Intervertebral disc degeneration (IDD) is the major source of chronic low back pain. As a controllable biological process, regulated cell death (RCD) extensively participates in the pathogenesis of IDD. Nevertheless, the initiation and progression of RCD remain unclear, and more importantly, the interaction between different RCD modes during IDD and therapy is far from well understood. Literature search was performed using “regulated cell death AND intervertebral disc degeneration” in PubMed, Embase, and Web of Science. Meanwhile, relevant findings have been reviewed and quoted. In this review, we discuss the inducing factors of IDD, various modes of RCD in intervertebral disc, the interactions between different RCD modes, as well as the obstacles to achieve disc regeneration. Meanwhile, the research gaps and perspective in studies that targeting RCD are also presented. Increasing evidence demonstrated the presence of different RCD modes in intervertebral disc during the progression of IDD. RCD in the resident disc cells is probably induced by the multiple factors such as abnormal mechanical loading, nutritional imbalance, inflammation microenvironment, circadian rhythm changes, withdraw of hormones, and other biomechanical factors. A better understanding of the fundamental mechanisms and the interactions between different RCD modes might contribute to the rescuing of disc degeneration and development of promising therapeutics. The Translational potential of this article. This review aims to demonstrate a better understanding of the fundamental mechanisms governing RCD, which might contribute to the rescuing of disc degeneration and to the development of promising therapeutics in a clinical setting.

Stem cell‐based therapy has been indicated to be beneficial for intervertebral disc regeneration. However, the underlying mechanisms have not been fully identified. The present study showed that bone marrow mesenchymal stem cells (BMSCs) donated mitochondria to adjacent nucleus pulposus cells (NPCs) in a coculture system. The mode of mitochondrial transfer between these cells was intercellular tunneling nanotube (TNT), which acted as a transportation expressway for mitochondria. NPCs acquired additional mitochondria from BMSCs in a concentration‐dependent manner after rotenone‐induced mitochondrial dysfunction in NPCs. Further research demonstrated that TNT‐mediated mitochondrial transfer rescued NPCs from mitochondrial dysfunction and apoptosis, which was indicated by the recovery of the mitochondrial respiratory chain, the increase in mitochondrial membrane potential, and the decreases in reactive oxygen species (ROS) levels and apoptosis rates. Furthermore, Miro1, a critical protein that regulates mitochondrial movement, was knocked down in BMSCs and significantly reduced mitochondrial transfer from BMSCs to NPCs. These results suggested that Miro1 depletion inhibited the rescue of NPCs with mitochondrial dysfunction. Taken together, our data shed light on a novel mechanism by which BMSCs rescue impaired NPCs, providing a concrete foundation to study the critical role of intercellular interactions in disc regeneration.