Background: The nucleus pulposus is a constituent structure of the human intervertebral disc, and its degeneration can cause intervertebral disc degeneration (IDD). However, the cellular and molecular mechanisms involved remain elusive.Methods: Through bioinformatics analysis, the single-cell transcriptome sequencing expression profiles of human normal nucleus pulposus (NNP) cells and human degenerative nucleus pulposus (DNP) cells were compared to clarify the transcriptome differential expression profiles of human NNP and DNP. The single-cell sequencing results of the two samples were analyzed using bioinformatics methods to compare the differences in histiocytosis between human NNP and DNP, map the histiocytes of NNP and DNP, perform cell differentiation trajectories for the cell populations of interest and predict cell function, and explore their heterogeneity by pathway analysis and Gene Ontology analysis.Results: Nine cell types were identified, which were chondrocyte 1, chondrocyte 2, chondrocyte 3, chondrocyte 4, chondrocyte 5, endothelial, macrophage, neutrophil, and T cells. Analysis of the proportion of chondrocytes in different tissues revealed that chondrocyte 1 accounted for a higher proportion of NNP cells and highly expressed COL2A1 compared with DNP cells; chondrocyte 2, chondrocyte 3, chondrocyte 4, and chondrocyte 5 accounted for a higher proportion of DNP cells compared with NNP cells. Among them, chondrocyte 2 was an inhibitory calcified chondrocyte with high expression of MGP, chondrocytes 3 were fibrochondrocytes with high expression of COL1A1, chondrocytes 4 were chondrocytes that highly express pain inflammatory genes such as PTGES, and chondrocytes 5 were calcified chondrocytes with high expression of FN1 (chondrocytes 4 and chondrocytes 5 were found for the first time in a study of single-cell transcriptome sequencing of disc tissue). Cell trajectory analysis revealed that chondrocyte 1 was at the beginning of the trajectory and chondrocyte 3 was at the end of the trajectory, while chondrocyte 5 appeared first in the trajectory relative to chondrocyte 2 and chondrocyte 4.Conclusion: After functional identification of the specifically expressed genes in five chondrocytes, it was found that chondrocyte 1 was a chondrocyte with high expression of COL2A1, COL9A2, COL11A2, and CHRDL2 in a high proportion of NNP cells, and chondrocyte 3 was a fibrochondrocyte with high expression of COL1A1, COL6A3, COL1A2, COL3A1, AQP1, and COL15A1 in an increased proportion during nucleus pulposus cell degeneration. Through cell trajectory analysis, it was found that chondrocytes 5 specifically expressing FN1, SESN2, and GDF15 may be the key cells leading to degeneration of nucleus pulposus cells. Chondrocytes 2 expressing MGP, MT1G, and GPX3 may play a role in reversing calcification and degeneration, and chondrocytes 4 expressing PTGES, TREM1, and TIMP1 may play a role in disc degeneration pain and inflammation.

We first investigated the difference in microRNA expression between normal NP cells and degenerative NP cells using gene chip. We have found that the expression of ERK1/2 was decreased with overexpression of miR-155 in normal nucleus pulposus cell. Expression of ERK1/2 was increased with inhibition of miR-155. Overexpression or inhibition of miR-155 had no effects on the expression level of mRNA ERK1/2 in nucleus pulposus cell, which showed that miR-155 affected the expression of pERK1/2 after transcription of ERK1/2 mRNA indicating that ERK1/2 was a new target protein regulated by miR-155. In the degeneration of intervertebral disc, inhibited miR-155 decreased the expressions of extracellular main matrix collagen II and glycosaminoglycan and increased expression of ERK1/2. Taken together, our data suggested that miR-155 was the identified miRNA which regulated NP cells degenerated through directly targeting ERK1/2.

Background: Single-cell RNA sequencing (scRNA-seq) enables specific analysis of cell populations at single-cell resolution; however, there is still a lack of single-cell-level studies to characterize the dynamic and complex interactions between osteoporotic vertebral compression fractures (OVCFs) and Kümmell’s disease (KD) in the osteoimmune microenvironment. In this study, we used scRNA-seq analysis to investigate the osteoimmune microenvironment and cellular composition in OVCFs and KD.Methods: ScRNA-seq was used to perform analysis of fractured vertebral bone tissues from one OVCF and one KD patients, and a total of 8,741 single cells were captured for single-cell transcriptomic analysis. The cellularity of human vertebral bone tissue was further analyzed using uniform manifold approximation and projection. Pseudo-time analysis and gene enrichment analysis revealed the biological function of cell fate and its counterparts. CellphoneDB was used to identify the interactions between bone cells and immune cells in the osteoimmune microenvironment of human vertebral bone tissue and their potential functions.Results: A cellular profile of the osteoimmune microenvironment of human vertebral bone tissue was established, including mesenchymal stem cells (MSCs), pericytes, myofibroblasts, fibroblasts, chondrocytes, endothelial cells (ECs), granulocytes, monocytes, T cells, B cells, plasma cells, mast cells, and early erythrocytes. MSCs play an immunoregulatory function and mediate osteogenic differentiation and cell proliferation. The differentiation trajectory of osteoclasts in human vertebral bone tissue was also revealed. In addition, ECs actively participate in inflammatory infiltration and coupling with bone cells. T and B cells actively participate in regulating bone homeostasis. Finally, by identifying the interaction of ligand–receptor pairs, we found that immune cells and osteoclasts have bidirectional regulatory characteristics, have the effects of regulating bone resorption by osteoclasts and promoting bone formation, and are essential for bone homeostasis. It is also highlighted that CD8-TEM cells and osteoclasts might crosstalk via CD160–TNFRSF14 ligand–receptor interaction.Conclusion: Our analysis reveals a differential landscape of molecular pathways, population composition, and cell–cell interactions during OVCF development into KD. OVCFs exhibit a higher osteogenic differentiation capacity, owing to abundant immune cells. Conversely, KD results in greater bone resorption than bone formation due to depletion of MSCs and a relatively suppressed immune system, and this immune imbalance eventually leads to vertebral avascular necrosis. The site of action between immune cells and osteoclasts is expected to be a new therapeutic target, and these results may accelerate mechanistic and functional studies of osteoimmune cell types and specific gene action in vertebral avascular necrosis and pathological bone loss diseases, paving the way for drug discovery.

We first applied moderate fluid shear stress to nucleus pulposus cells. The correlation of AP-1 with type II collagen, proteoglycan, Cytokeratin 8 protein, MAP-1, MAP-2, and MAP-4 and the correlation of AP-1 with IL-1β, TNF-α, IL-6, IL-8, MIP-1, MCP-1, and NO were detected. Our results document that moderate fluid shear stress could activate the FAK-MEK5-ERK5-cFos-AP1 signaling pathway. AP1 could downregulate the construct factors of cytoskeleton such as type II collagen, proteoglycan, Cytokeratin 8 protein, MAP-1, MAP-2, and MAP-4 in nucleus pulposus cell after the fluid shear stress was loaded. AP1 could upregulate the inflammatory factors such as IL-1β, TNF-α, IL-6, IL-8, MIP-1, MCP-1, and NO in nucleus pulposus cell after the fluid shear stress was loaded. Taken together, our data suggested that moderate fluid shear stress may play an important role in the cytoskeleton of nucleus pulposus and surrounding inflammatory mediators by activating the FAK-MEK5-ERK5-cFos-AP1 signaling pathway, thereby affecting cell degeneration.

Background: Giant cell tumor of bone (GCTB) presents considerable complexity in tumor microenvironment (TME) because of its intricate intercellular heterogeneity and the presence of an immunosuppressive milieu. In order to understand the complex gene expression patterns and cell interactions in GCTB, we carried out a thorough investigation using single‐cell RNA sequencing (scRNA‐seq). Methods: We examined scRNA‐seq data from 7091 cells that were collected after surgical removal of GCTB. Following the initial quality control process, 10 separate groups of cells were distinguished, which consisted of dendritic cells, endothelial cells, macrophage cells, mast cells, monocyte cells, neutrophil cells, tumor cells, osteoclast cells, pericyte cells, and T cells. Additional analysis uncovered distinct categories within tumor‐associated macrophages (TAMs), CD8+ T cells, and CD4+ T cells. The differentiation mechanisms of TAMs, CD8+ T cells, and CD4+ T cells were explored using pseudo‐time trajectory analysis. The CellPhoneDB study revealed the interactions between various cell types within the TME of GCTB. Results: TAMs have been identified as the main infiltrating cells in GCTB. These TAMs exhibit several subtypes that are characterized by specific marker genes and functional states. The identification of several subgroups within CD8+ T cells that are involved in regulating immunological checkpoints underscores the difficulties encountered when attempting to employ immune checkpoint blockade therapy for GCTB. T cell exhaustion poses a major barrier to the efficacy of antitumor immune responses. Research suggests a strong correlation between TAMs and exhausted T cells (Texs) in the TME. The high number of regulatory T cells (Tregs) highlights the immunosuppressive nature of the immunological environment in GCTB. Significant interactions have been observed between TAMs and tumor cells, highlighting their crucial involvement in immune evasion strategies. Conclusion: This scRNA‐seq study provides a general overview of the different cellular compositions and immune interactions within GCTB. The identified subtypes and communication networks provide valuable information about the immunosuppressive environment of GCTB, laying the foundation for prospective therapeutic approaches targeting specific cell types or interactions.

Osteolytic bone diseases are commonly presented with enhanced osteoclast formation and bone resorption. Sesquiterpene lactone natural compounds have been found to possess anti-inflammatory and immune-modulation effects. Here, we identified three germacrane sesquiterpenes using computer-based virtual screening for the structural similarity with sesquiterpene lactone, parthenolide. We showed that natural germacrane sesquiterpene compounds A, B, and C inhibit osteoclast formation and bone resorption in a dose-dependent manner, with relative potency compound A > compound C > compound B based on their equimolar concentrations. Mechanistic studies by Luciferase reporter gene assay and Western blot analysis showed that germacrane sesquiterpene compound A inhibits RANKL-induced activation of NF-κB and IκBα degradation. This study reveals that natural germacrane sesquiterpene compounds are inhibitors for osteoclast formation and bone resorption, and provides evidence that naturally-occurring compounds might be beneficial as alternative medicine for the prevention and treatment of osteolysis.