Osteoarthritis (OA) poses a significant challenge in orthopedics. Inflammatory pathways are regarded as central mechanisms in the onset and progression of OA. Growing evidence suggests that senescence acts as a mediator in inflammation-induced OA. Given the lack of effective treatments for OA, there is an urgent need for a clearer understanding of its pathogenesis. In this review, we systematically summarize the cross-talk between cellular senescence and inflammation in OA. We begin by focusing on the mechanisms and hallmarks of cellular senescence, summarizing evidence that supports the relationship between cellular senescence and inflammation. We then discuss the mechanisms of interaction between cellular senescence and inflammation, including senescence-associated secretory phenotypes (SASP) and the effects of pro- and anti-inflammatory interventions on cellular senescence. Additionally, we focus on various types of cellular senescence in OA, including senescence in cartilage, subchondral bone, synovium, infrapatellar fat pad, stem cells, and immune cells, elucidating their mechanisms and impacts on OA. Finally, we highlight the potential of therapies targeting senescent cells in OA as a strategy for promoting cartilage regeneration.

Intervertebral disc degeneration (IVDD), a disease associated with ageing, is characterised by a notable increase in senescent nucleus pulposus cells (NPCs) as IVDD progresses. However, the specific mechanisms that regulate the senescence of NPCs remain unknown. In this study, we observed impaired autophagy in IVDD-NPCs, which contributed to the upregulation of NPCs senescence and the senescence-associated secretory phenotype (SASP). The dysregulated SASP disrupted NPCs viability and initiated extracellular matrix degradation. Conversely, the restoration of autophagy reversed the senescence phenotype by inhibiting GATA binding protein 4 (GATA4). Moreover, we made the novel observation that a cross-talk between histone H3 lysine 4 trimethylation (H3K4me3) modification and N6-methyladenosine(m⁶A)-methylated modification regulates autophagy in IVDD-NPCs. Mechanistically, lysine methyltransferase 2A (KMT2A) promoted the expression of methyltransferase-like 3 (METTL3) through H3K4me3 modification, whereas METTL3-mediated m⁶A modification reduced the expression of autophagy-associated 4a (ATG4a) by attenuating its RNA stability, leading to autophagy damage in NPCs. Silencing KMT2A and METTL3 enhanced autophagic flux and suppressed SASP expression in IVDD-NPCs. Therefore, targeting the H3K4me3-regulated METTL3/ATG4a/GATA4 axis may represent a promising new therapeutic strategy for IVDD.

Recent genome-wide association studies (GWAS) identified 518 significant loci associated with bone mineral density (BMD), including variants at the RUNX1 locus (rs13046645, rs2834676, and rs2834694). However, their regulatory impact on RUNX1 expression and bone formation remained unclear. This study utilized human induced pluripotent stem cells (iPSCs) differentiated into osteoblasts to investigate these variants’ regulatory roles. CRISPR/Cas9 was employed to generate mutant (Δ) iPSC lines lacking these loci at the RUNX1 locus. Deletion lines (Δ1 and Δ2) were created in iPSCs to assess the effects of removing regions containing these loci. Deletion lines exhibited enhanced osteogenic potential, with increased expression of osteogenic marker genes and Alizarin Red staining. Circularized chromosome conformation capture (4C-Seq) was utilized to analyze interactions between BMD-associated loci and the RUNX1 promoter during osteogenesis. Analysis revealed altered chromatin interactions with multiple gene promoters including RUNX1 isoform, as well as SETD4, a histone methyltransferase, indicating their regulatory influence. Interestingly, both deletion lines notably stimulated the expression of the long isoform of RUNX1, with more modest effects on the shorter isoform. Consistent upregulation of SETD4 and other predicted targets within the Δ2 deletion suggested its removal removed a regulatory hub constraining expression of multiple genes at this locus. In vivo experiments using a bone defect model in mice demonstrated increased bone regeneration with homozygous deletion of the Δ2 region. These findings indicate that BMD-associated variants within the RUNX1 locus regulate multiple effector genes involved in osteoblast commitment, providing valuable insights into genetic regulation of bone density and potential therapeutic targets.

Emerging evidence suggests a significant role of gut microbiome in bone health. Aging is well recognized as a crucial factor influencing the gut microbiome. In this study, we investigated whether age-dependent microbial change contributes to age-related bone loss in CB6F1 mice. The bone phenotype of 24-month-old germ-free (GF) mice was indistinguishable compared to their littermates colonized by fecal transplant at 1-month-old. Moreover, bone loss from 3 to 24-month-old was comparable between GF and specific pathogen-free (SPF) mice. Thus, GF mice were not protected from age-related bone loss. 16S rRNA gene sequencing of fecal samples from 3-month and 24-month-old SPF males indicated an age-dependent microbial shift with an alteration in energy and nutrient metabolism potential. An integrative analysis of 16S predicted metagenome function and LC-MS fecal metabolome revealed an enrichment of protein and amino acid biosynthesis pathways in aged mice. Microbial S-adenosyl methionine metabolism was increased in the aged mice, which has previously been associated with the host aging process. Collectively, aging caused microbial taxonomic and functional alteration in mice. To demonstrate the functional importance of young and old microbiome to bone, we colonized GF mice with fecal microbiome from 3-month or 24-month-old SPF donor mice for 1 and 8 months. The effect of microbial colonization on bone phenotypes was independent of the microbiome donors’ age. In conclusion, our study indicates age-related bone loss occurs independent of gut microbiome.

Osteoporosis (OP) is a common and fracture-prone skeletal disease characterized by deteriorated trabecular microstructure and pathologically involving various forms of regulated bone cell death. However, the exact role, cellular nature and regulatory mechanisms of ferroptosis in OP are not fully understood. Here, we reported that OP femurs from ovariectomized (Ovx) mice exhibited pronounced iron deposition, ferroptosis, and transcriptional suppression of a key anti-ferroptotic factor GPX4 (glutathione peroxidase 4). GPX4 suppression was accompanied by hypermethylation of the Gpx4 promoter and an increase in DNA methyltransferases DNMT1/3a/3b and was transcriptionally promoted by repressive KLF5 and the transcriptional corepressors NCoR and SnoN. Conversely, DNMT inhibition with SGI-1027 reversed promoter hypermethylation, GPX4 suppression and ferroptotic osteoporosis. In cultured primary bone cells, ferric ammonium citrate (FAC) mimicking iron loading similarly induced GPX4 suppression and ferroptosis in osteoblasts but not in osteoclasts, which were rescued by siRNA-mediated individual knockdown of DNMT 1/3a/3b. Intriguingly, SGI-1027 alleviated the ferroptotic changes caused by FAC, but not by a GPX4 inactivator RSL3. More importantly, we generated a strain of osteoblast-specific Gpx4 haplo-deficient mice Gpx4 ^Ob+/− that developed spontaneous and more severe ferroptotic OP alterations after Ovx operation, and showed that GPX4 inactivation by RSL3 or semi-knockout in osteoblasts largely abolished the anti-ferroptotic and osteoprotective effects of SGI-1027. Taken together, our data suggest that GPX4 epigenetic suppression caused by DNMT aberration and the resulting osteoblastic ferroptosis contribute significantly to OP pathogenesis, and that the strategies preserving GPX4 by DNMT intervention are potentially effective to treat OP and related bone disorders.

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss.

Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

The concept and development of bone/cartilage organoids are rapidly gaining momentum, providing opportunities for both fundamental and translational research in bone biology. Bone/cartilage organoids, essentially miniature bone/cartilage tissues grown in vitro, enable the study of complex cellular interactions, biological processes, and disease pathology in a representative and controlled environment. This review provides a comprehensive and up-to-date overview of the field, focusing on the strategies for bone/cartilage organoid construction strategies, progresses in the research, and potential applications. We delve into the significance of selecting appropriate cells, matrix gels, cytokines/inducers, and construction techniques. Moreover, we explore the role of bone/cartilage organoids in advancing our understanding of bone/cartilage reconstruction, disease modeling, drug screening, disease prevention, and treatment strategies. While acknowledging the potential of these organoids, we discuss the inherent challenges and limitations in the field and propose potential solutions, including the use of bioprinting for organoid induction, AI for improved screening processes, and the exploration of assembloids for more complex, multicellular bone/cartilage organoids models. We believe that with continuous refinement and standardization, bone/cartilage organoids can profoundly impact patient-specific therapeutic interventions and lead the way in regenerative medicine.

The human skeleton is a multifunctional organ made up of multiple cell types working in concert to maintain bone and mineral homeostasis and to perform critical mechanical and endocrine functions. From the beginning steps of chondrogenesis that prefigures most of the skeleton, to the rapid bone accrual during skeletal growth, followed by bone remodeling of the mature skeleton, cell differentiation is integral to skeletal health. While growth factors and nuclear proteins that influence skeletal cell differentiation have been extensively studied, the role of cellular metabolism is just beginning to be uncovered. Besides energy production, metabolic pathways have been shown to exert epigenetic regulation via key metabolites to influence cell fate in both cancerous and normal tissues. In this review, we will assess the role of growth factors and transcription factors in reprogramming cellular metabolism to meet the energetic and biosynthetic needs of chondrocytes, osteoblasts, or osteoclasts. We will also summarize the emerging evidence linking metabolic changes to epigenetic modifications during skeletal cell differentiation.

Bone and joint-related diseases, including osteoarthritis (OA), rheumatoid arthritis (RA), and bone tumors, pose significant health challenges due to their debilitating effects on the musculoskeletal system. 14-3-3 proteins, a family of conserved regulatory molecules, play a critical role in the pathology of these diseases. This review discusses the intricate structure and multifunctionality of 14-3-3 proteins, their regulation of signaling pathways, and their interactions with other proteins. We underscore the significance of 14-3-3 proteins in the regulation of osteoblasts, osteoclasts, chondrocytes, and bone remodeling, all key factors in the maintenance and dysfunction of bone and joint systems. Specific focus is directed toward elucidating the contribution of 14-3-3 proteins in the pathology of OA, RA, and bone malignancies, where dysregulated 14-3-3-mediated signaling cascades have been implicated in the disease processes. This review illuminates how the perturbation of 14-3-3 protein interactions can lead to the pathological manifestations observed in these disorders, including joint destruction and osteolytic activity. We highlight cutting-edge research that positions 14-3-3 proteins as potential biomarkers for disease progression and as innovative therapeutic targets, offering new avenues for disease intervention and management.

Cellular communication network factor 2 (CCN2) is a secreted extracellular matrix-associated protein, and its aberrantly increased expression has been implicated in a diversity of diseases involving pathological processes of fibrosis, chronic inflammation, or tissue injury, which has promoted the evaluation of CCN2 as therapeutic targets for multiple disorders. However, human phenotypes associated with CCN2 deficiency have remained enigmatic; variants in CCN2 have not yet been associated with a human phenotype. Here, we collected families diagnosed with spondyloepimetaphyseal dysplasia (SEMD), and screened candidate pathogenic genes for families without known genetic causes using next-generation sequencing. We identified a monoallelic variant in signal peptide of CCN2 (NM_001901.2: c.65 G > C [p.Arg22Pro]) as the cause of SEMD in 14 subjects presenting with different degree of short stature, premature osteoarthritis, and osteoporosis. Affected subjects showed decreased serum CCN2 levels. Cell lines harboring the variant displayed decreased amount of CCN2 proteins in culture medium and an increased intracellular retention, indicating impaired protein secretion. And the variant weakened the stimulation effect of CCN2 on osteogenesis of bone marrow mesenchymal stem cells. Zebrafish ccn2a knockout model and osteoblast lineage-specific Ccn2-deficient mice (Ccn2^fl/fl;Prx1^Cre) partially recapitulated the phenotypes including low bone mass observed in affected subjects. Pathological mechanism implicated in the skeletal abnormality in Ccn2^fl/fl;Prx1^Cre mice involved decreased bone formation, increased bone resorption, and abnormal growth plate formation. Collectively, our study indicate that monoallelic variants in CCN2 lead to a human inherited skeletal dysplasia, and highlight the critical role of CCN2 in osteogenesis in human.

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1–34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11⁺ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

Myeloid cells are pivotal in the inflammatory and remodeling phases of fracture repair. Here, we investigate the effect of periostin expressed by myeloid cells on bone regeneration in a monocortical tibial defect (MTD) model. In this study, we show that periostin is expressed by periosteal myeloid cells, primarily the M2 macrophages during bone regeneration. Knockout of periostin in myeloid cells reduces cortical bone thickness, disrupts trabecular bone connectivity, impairs repair impairment, and hinders M2 macrophage polarization. Mechanical stimulation is a regulator of periostin in macrophages. By activating transforming growth factor-β (TGF-β), it increases periostin expression in macrophages and induces M2 polarization. This mechanosensitive effect also reverses the delayed bone repair induced by periostin deficiency in myeloid cells by strengthening the angiogenesis-osteogenesis coupling. In addition, transplantation of mechanically conditioned macrophages into the periosteum over a bone defect results in substantially enhanced repair, confirming the critical role of macrophage-secreted periostin in bone repair. In summary, our findings suggest that mechanical stimulation regulates periostin expression and promotes M2 macrophage polarization, highlighting the potential of mechanically conditioned macrophages as a therapeutic strategy for enhancing bone repair.

Patients diagnosed with advanced osteosarcoma, often in the form of lung metastases, have abysmal five-year overall survival rates. The complexity of the osteosarcoma immune tumor microenvironment has been implicated in clinical trial failures of various immunotherapies. The purpose of this exploratory study was to spatially characterize the immune tumor microenvironment of metastatic osteosarcoma lung specimens. Knowledge of the coordinating cellular networks within these tissues could then lead to improved outcomes when utilizing immunotherapy for treatment of this disease. Importantly, various cell types, interactions, and cellular neighborhoods were associated with five-year survival status. Of note, increases in cellular interactions between T lymphocytes, positive for programmed cell death protein 1, and myeloid-derived suppressor cells were observed in the 5-year deceased cohort. Additionally, cellular neighborhood analysis identified an Immune-Cold Parenchyma cellular neighborhood, also associated with worse 5-year survival. Finally, the Osteosarcoma Spatial Score, which approximates effector immune activity in the immune tumor microenvironment through the spatial proximity of immune and tumor cells, was increased within 5-year survivors, suggesting improved effector signaling in this patient cohort. Ultimately, these data represent a robust spatial multiplexed immunofluorescence analysis of the metastatic osteosarcoma immune tumor microenvironment. Various communication networks, and their association with survival, were described. In the future, identification of these networks may suggest the use of specific, combinatory immunotherapeutic strategies for improved anti-tumor immune responses and outcomes in osteosarcoma.

Osteopetrosis is an inherited metabolic disease, characterized by increased bone density and narrow marrow cavity. Patients with severe osteopetrosis exhibit abnormal bone brittleness, anemia, and infection complications, which commonly cause death within the first decade of life. Pathologically, osteopetrosis impairs not only the skeletal system, but also the hemopoietic and immune systems during development, while the underlying osteoimmunological mechanisms remain unclear. Osteoclastic mutations are regarded as the major causes of osteopetrosis, while osteoclast non-autonomous theories have been proposed in recent years with unclear underlying mechanisms. Retinoic acid (RA), the metabolite of Vitamin A, is an essential requirement for skeletal and hematopoietic development, through the activation of retinoic acid signaling. RA can relieve osteopetrosis symptoms in some animal models, while its effect on bone health is still controversial and the underlying mechanisms remain unclear. In this study, we constructed an osteoblast-specific inhibitory retinoic acid signaling mouse model and surprisingly found it mimicked the symptoms of osteopetrosis found in clinical cases: dwarfism, increased imperfectly-formed trabecular bone deposition with a reduced marrow cavity, thin cortical bone with a brittle skeleton, and hematopoietic and immune dysfunction. Micro-CT, the three-point bending test, and histological analysis drew a landscape of poor bone quality. Single-cell RNA sequencing (scRNA-seq) of the femur and RNA-seq of osteoblasts uncovered an atlas of pathological skeletal metabolism dysfunction in the mutant mice showing that osteogenesis was impaired in a cell-autonomous manner and osteoclastogenesis was impaired via osteoblast-osteoclast crosstalk. Moreover, scRNA-seq of bone marrow and flow cytometry of peripheral blood, spleen, and bone marrow uncovered pathology in the hematopoietic and immune systems in the mutant mice, mimicking human osteopetrosis. Results showed that hematopoietic progenitors and B lymphocyte differentiation were affected and the osteoblast-dominated cell crosstalk was impaired, which may result from transcriptional impairment of the ligands Pdgfd and Sema4d. In summary, we uncovered previously unreported pathogenesis of osteopetrosis-like disorder in mice with skeletal, hematopoietic, and immune system dysfunction, which was induced by the inhibition of retinoic acid signaling in osteoblasts, and sheds new insights into a potential treatment for osteopetrosis.

Hachemi et al. report the immune cell atlas of bone repair revealing macrophages as pro-fibrotic regulators and a therapeutic target for musculoskeletal regeneration. Genetic depletion or pharmacological inhibition of macrophages improves bone healing in musculoskeletal trauma.