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Heterotopic ossification (HO) is pathological bone formation characterized by ossification within muscle, tendons, or other soft tissues. However, the cells of origin and mechanisms involved in the pathogenesis of HO remain elusive. Here we show that deletion of suppressor of fused (Sufu) in cathepsin K-Cre-expressing (Ctsk-Cre-expressing) cells resulted in spontaneous and progressive ligament, tendon, and periarticular ossification. Lineage tracing studies and cell functional analysis demonstrated that Ctsk-Cre could label a subpopulation of tendon-derived progenitor cells (TDPCs) marked by the tendon marker Scleraxis (Scx). Ctsk+Scx+ TDPCs are enriched for tendon stem cell markers and show the highest self-renewal capacity and differentiation potential. Sufu deficiency caused enhanced chondrogenic and osteogenic differentiation of Ctsk-Cre-expressing tendon-derived cells via upregulation of Hedgehog (Hh) signaling. Furthermore, pharmacological intervention in Hh signaling using JQ1 suppressed the development of HO. Thus, our results show that Ctsk-Cre labels a subpopulation of TDPCs contributing to HO and that their cell-fate changes are driven by activation of Hh signaling.

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization. Runx2 is essential for tuning the generation of bone from skeletal stem cells (SSCs). Here, the authors demonstrate that the CK2/HAUSP pathway stabilizes RUNX2 protein thereby regulating the commitment of SSCs to osteoprogenitors as well as their subsequent maturation, and that inhibition of this pathway can block heterotopic ossification.

In a new study, Yingzi Yang and her colleagues show that a careful balance between Wnt and Hedgehog signaling is required to maintain proper differentiation of osteogenic precursor cells. Upon mutation of GNAS , this balance is disturbed and severe bone disease develops, including either heterotopic ossification or fibrous dysplasia. Heterotopic ossification, the pathologic formation of extraskeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gα s restricts bone formation to the skeleton by inhibiting Hedgehog signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia, a human disease caused by null mutations in GNAS , which encodes Gα s , Hedgehog signaling is upregulated in ectopic osteoblasts and progenitor cells. In animal models, we show that genetically-mediated ectopic Hedgehog signaling is sufficient to induce heterotopic ossification, whereas inhibition of this signaling pathway by genetic or pharmacological means strongly reduces the severity of this condition. As our previous work has shown that GNAS gain-of-function mutations upregulate WNT–β-catenin signaling in osteoblast progenitor cells, resulting in their defective differentiation and fibrous dysplasia, we identify Gα s as a key regulator of proper osteoblast differentiation through its maintenance of a balance between the Wnt–β-catenin and Hedgehog pathways. Also, given the results here of the pharmacological studies in our mouse model, we propose that Hedgehog inhibitors currently used in the clinic for other conditions, such as cancer, may possibly be repurposed for treating heterotopic ossification and other diseases caused by GNAS inactivation.

Heterotopic ossification (HO) occurs as a common complication after injury, while its risk factor and mechanism remain unclear, which restricts the development of pharmacological treatment. Clinical research suggests that diabetes mellitus (DM) patients are prone to developing HO in the tendon, but solid evidence and mechanical research are still needed. Here, we combined the clinical samples and the DM mice model to identify that disordered glycolipid metabolism aggravates the senescence of tendon-derived stem cells (TSCs) and promotes osteogenic differentiation. Then, combining the RNA-seq results of the aging tendon, we detected the abnormally activated autocrine CXCL13-CXCR5 axis in TSCs cultured in a high fat, high glucose (HFHG) environment and also in the aged tendon. Genetic inhibition of CXCL13 successfully alleviated HO formation in DM mice, providing a potential therapeutic target for suppressing HO formation in DM patients after trauma or surgery.

Neutrophils are emerging as an important player in skeletal muscle injury and repair. Neutrophils accumulate in injured tissue, thus releasing inflammatory factors, proteases and neutrophil extracellular traps (NETs) to clear muscle debris and pathogens when skeletal muscle is damaged. During the process of muscle repair, neutrophils can promote self-renewal and angiogenesis in satellite cells. When neutrophils are abnormally overactivated, neutrophils cause collagen deposition, functional impairment of satellite cells, and damage to the skeletal muscle vascular endothelium. Heterotopic ossification (HO) refers to abnormal bone formation in soft tissue. Skeletal muscle injury is one of the main causes of traumatic HO (tHO). Neutrophils play a pivotal role in activating BMPs and TGF-β signals, thus promoting the differentiation of mesenchymal stem cells and progenitor cells into osteoblasts or osteoclasts to facilitate HO. Furthermore, NETs are specifically localized at the site of HO, thereby accelerating the formation of HO. Additionally, the overactivation of neutrophils contributes to the disruption of immune homeostasis to trigger HO. An understanding of the diverse roles of neutrophils will not only provide more information on the pathogenesis of skeletal muscle injury for repair and HO but also provides a foundation for the development of more efficacious treatment modalities for HO. Neutrophils in Muscle Injury: Implications for Heterotopic Ossification Skeletal muscle, the body’s most common tissue, often gets injured and lacks highly effective treatments. This review investigates the complex relationship between skeletal muscle and neutrophils during injury and healing. Researchers study how neutrophils can both worsen muscle damage and assist in tissue repair. It looks at how neutrophil activity affects muscle repair, explaining the processes of inflammation, tissue regeneration, and the factors causing heterotopic ossification. It also emphasizes the significance of controlling neutrophil activity for effective muscle healing and avoiding complications. Key findings show that neutrophils are crucial in both harming and repairing skeletal muscle. Overactive neutrophils can cause extended inflammation, hindering the healing process, while controlled activity aids tissue regeneration. The researchers suggest that focusing on neutrophil activity could be a promising method for treating muscle injuries and preventing heterotopic ossification. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Heterotopic ossification (HO) refers to an abnormal process characterized by the aberrant development of bone within soft tissues, leading to significant impairments in patients’ mobility and overall quality of life. Macrophages, as a crucial element of the immune system, are instrumental in the different stages of heterotopic ossification through their dynamic polarization state (pro-inflammatory M1 and anti-inflammatory M2 phenotypes) and secretion of different cytokines. This review explores novel mechanisms of M1 and M2 macrophage-mediated heterotopic ossification, emphasizing the involvement of the inflammatory microenvironment, osteogenic factors, and osteogenic signaling pathways. In addition, we explore promising therapeutic strategies targeting macrophage polarization and function, including agents that modulate the inflammatory microenvironment, such as IL-1 inhibitors, parovastatin, and metformin, as well as agents that affect macrophage osteogenic signaling, such as TGF-βRII-Fc, Galunisertib, and Ruxolitinib. A more comprehensive understanding of these mechanisms may open up new avenues for developing novel approaches to reducing HO in high-risk patients.

Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.

ObjectivesThe aim of this study was to identify the role of tenascin-C (TNC) in entheseal new bone formation and to explore the underlying molecular mechanism.MethodsLigament tissue samples were obtained from patients with ankylosing spondylitis (AS) during surgery. Collagen antibody-induced arthritis and DBA/1 models were established to observe entheseal new bone formation. TNC expression was determined by immunohistochemistry staining. Systemic inhibition or genetic ablation of TNC was performed in animal models. Mechanical properties of extracellular matrix (ECM) were measured by atomic force microscopy. Downstream pathway of TNC was analysed by RNA sequencing and confirmed with pharmacological modulation both in vitro and in vivo. Cellular source of TNC was analysed by single-cell RNA sequencing (scRNA-seq) and confirmed by immunofluorescence staining.ResultsTNC was aberrantly upregulated in ligament and entheseal tissues from patients with AS and animal models. TNC inhibition significantly suppressed entheseal new bone formation. Functional assays revealed that TNC promoted new bone formation by enhancing chondrogenic differentiation during endochondral ossification. Mechanistically, TNC suppressed the adhesion force of ECM, resulting in the activation of downstream Hippo/yes-associated protein signalling, which in turn increased the expression of chondrogenic genes. scRNA-seq and immunofluorescence staining further revealed that TNC was majorly secreted by fibroblast-specific protein-1 (FSP1)+fibroblasts in the entheseal inflammatory microenvironment.ConclusionInflammation-induced aberrant expression of TNC by FSP1+fibroblasts promotes entheseal new bone formation by suppressing ECM adhesion forces and activating Hippo signalling.

Ectopic ossification often involves the transformation of soft tissue into bone. In this new study, Paul Yu et al. show that inflammation is a key step in disease progression and that a small molecule inhibitor of the disease gene’s protein product is therapeutic, thus offering a potential treatment for this devastating condition. Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues 1 , 2 , 3 , 4 and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2) 5 . Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6 ), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling.

Heterotopic ossification (HO) is a pathological condition characterized by the formation of bone within soft tissues. The development of HO is a result of abnormal activation of the bone formation programs, where multiple signalling pathways, including Wnt/β‐catenin, BMP and hedgehog signalling, are involved. The Wnt/β‐catenin signalling pathway, a conserved pathway essential for various fundamental activities, has been found to play a significant role in pathological bone formation processes. It regulates angiogenesis, chondrocyte hypertrophy and osteoblast differentiation during the development of HO. More importantly, the crosstalk between Wnt signalling and other factors including BMP, Hedgehog signalling, YAP may contribute in a HO‐favourable manner. Moreover, several miRNAs may also be involved in HO formation via the regulation of Wnt signalling. This review aims to summarize the role of Wnt/β‐catenin signalling in the pathogenesis of HO, its interactions with related molecules, and potential preventive and therapeutic measures targeting Wnt/β‐catenin signalling.