Heterotopic Ossification Progression Research Articles

Eupalinolide A attenuates trauma-induced heterotopic ossification of tendon in mice by promoting YAP degradation through TOLLIP-mediated selective autophagy

BackgroundInhibiting the aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs) is an effective strategy for treating traumatic heterotopic ossification (HO) in tendons. PurposeThis study aimed to investigate whether eupalinolide A (EA) could prevent tendon HO progression by suppressing the osteogenic differentiation of TDSCs. MethodsThe effects of EA on osteogenic differentiation and key signaling pathways in TDSCs were examined in vitro to assess its therapeutic potential and elucidate the underlying molecular mechanisms. Furthermore, the therapeutic efficacy of EA was evaluated in a mouse model of trauma-induced tendon HO via local injection therapy. ResultsEA significantly inhibited the osteogenic differentiation of TDSCs by targeting YAP in vitro. Specifically, EA facilitated the recruitment of E3 ubiquitin ligase HECW1, which mediated K27-linked polyubiquitination of YAP, leading to its degradation via the TOLLIP-mediated selective autophagy pathway. In vivo, EA mitigated trauma-induced tendon HO by inhibiting the YAP pathway. ConclusionsEA could be a potential therapeutic agent for treating traumatic tendon HO. The therapeutic target HECW1 involved in YAP degradation via autophagy presents a new therapeutic avenue to attenuate the progression of traumatic tendon HO.

Molecular Developmental Biology of Fibrodysplasia Ossificans Progressiva: Measuring the Giant by Its Toe.

When a genetic disease is characterized by the abnormal activation of normal molecular pathways and cellular events, it is illuminating to critically examine the places and times of these activities both in health and disease. Therefore, because heterotopic ossification (HO) in fibrodysplasia ossificans progressiva (FOP) is by far the disease's most prominent symptom, attention is also directed toward the pathways and processes of bone formation during skeletal development. FOP is recognizable by effects of the causative mutation on skeletal development even before HO manifests, specifically in the malformation of the great toes. This signature skeletal phenotype is the most highly penetrant, but is only one among several skeletal abnormalities associated with FOP. Patients may present clinically with joint malformation and ankylosis, particularly in the cervical spine and costovertebral joints, as well as characteristic facial features and a litany of less common, non-skeletal symptoms, all stemming from missense mutations in the ACVR1 gene. In the same way that studying the genetic cause of HO advanced our understanding of HO initiation and progression, insight into the roles of ACVR1 signaling during tissue development, particularly in the musculoskeletal system, can be gained from examining altered skeletal development in individuals with FOP. This review will detail what is known about the molecular mechanisms of developmental phenotypes in FOP and the early role of ACVR1 in skeletal patterning and growth, as well as highlight how better understanding these processes may serve to advance patient care, assessments of patient outcomes, and the fields of bone and joint biology.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Calcified apoptotic vesicles from PROCR+ fibroblasts initiate heterotopic ossification.

Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we found that calcified apoptotic vesicles (apoVs) led to increased calcification and stiffness of tendon extracellular matrix (ECM), which initiated M2 macrophage polarization and HO progression. Specifically, single-cell transcriptome analyses of different stages of HO revealed that calcified apoVs were primarily secreted by a PROCR+ fibroblast population. In addition, calcified apoVs enriched calcium by annexin channels, absorbed to collagen I via electrostatic interaction, and aggregated to produce calcifying nodules in the ECM, leading to tendon calcification and stiffening. More importantly, apoV-releasing inhibition or macrophage deletion both successfully reversed HO development. Thus, we are the first to identify calcified apoVs from PROCR+ fibroblasts as the initiating factor of HO, and might serve as the therapeutic target for inhibiting pathological calcification.

Incidence and risk factors of heterotopic ossification after cervical Baguera C disc arthroplasty.

This retrospective study was designed to evaluate the incidence and predisposing factors of heterotopic ossification (HO) after cervical disc arthroplasty (CDA) with a specific implant at 1 and 2 levels, and to investigate the biomechanical effects related to HO. The study goal was to identify ways to reduce the likelihood of HO formation after surgery. The study included patients who underwent only 1- or 2-level CDA with the Baguera C disc between November 2014 and December 2021 at a single medical center. All patients were operated on by the same neurosurgeon. The surgical indication included 1-level or 2-level disc herniation between C3 and C7 with radiculopathy, myelopathy, or both, with minimal spondylosis. The various factors were assessed by evaluating plain radiographs and cervical CT scans. The presence of HO was evaluated at different intervals postsurgery, and HO severity was graded using the McAfee classification. Of 107 patients who underwent CDA, 47 (43.9%) had HO at 63 of 171 levels (36.8%). Most cases with HO were grade 1, and no grade 4 was observed. Statistically significant risk factors for HO were the length of endplate coverage ratio and inferior anterior residual exposed endplate (AREE); sex, age, implant height and width, shell angle, and pre- and postoperative functional spinal unit (FSU) angle were not significant. More AREE and greater kyphotic postoperative FSU angle in the flexion position were significant factors differentiating HO grades 0 and 1 from grades 2 and 3. Furthermore, the non-HO group showed a trend of higher range of motion at any postoperative time compared to the HO group, especially at 1 month after surgery. The HO incidence after CDA was correlated with the residual length of endplate coverage and inferior AREE. Additionally, the AREE and kyphotic postoperative FSU angle in the flexion position were associated with HO grade progression. Patients with HO also showed a trend of lower range of motion at 1 month after surgery. Using an adequately sized implant and encouraging neck motion may help prevent HO development and progression.

Study methodology and insights from the palovarotene clinical development program in fibrodysplasia ossificans progressiva

BackgroundThe design of clinical trials in rare diseases is often complicated by a lack of real-world translational knowledge. Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by skeletal malformations and progressive heterotopic ossification (HO). Palovarotene is a selective retinoic acid receptor gamma agonist. Here, we describe the methodology of three studies in the palovarotene clinical development program in FOP and discuss insights that could inform future research, including endpoint suitability and the impact of trial design.MethodsPVO-1A-001 (NCT02322255) was a prospective, protocol-specified, longitudinal FOP natural history study (NHS). PVO-1A-201 (NCT02190747) was a randomized, double-blind, placebo-controlled phase II trial; PVO-1A-202 (NCT02279095) was its open-label extension. Trial designs, including treatment regimens and imaging assessments, were refined between PVO-1A-201 and PVO-‍1A-202, and within PVO-1A-202, based on emerging data as the studies progressed. Palovarotene doses were administered using a flare-up treatment regimen (higher dose for 2/4 weeks, followed by lower dose for 4/≥8 weeks; from flare-up onset), with or without accompanying chronic (daily) treatment. Flare-up and disease progression outcomes were assessed, including incidence and volume of new HO during flare-ups and/or annually, as well as other clinical, patient-reported, and exploratory outcomes. Safety was monitored throughout all studies.ResultsOverall, 114 and 58 individuals with FOP were enrolled in the NHS and phase II trials, respectively. Results of the NHS and PVO-1A-201 were published in 2022; complete results of PVO-1A-202 will be publicly available in due course. Together the studies yielded important information on endpoint suitability, including that low-dose whole-body computed tomography was the optimum imaging modality for assessing HO progression annually and that long study durations are needed to detect substantial changes in functional and patient-reported outcomes.ConclusionsA flexible clinical development program is necessary for underexplored rare diseases to overcome the many challenges faced. Here, the NHS provided a longitudinal evaluation of FOP progression and interventional trials were based on emerging data. The studies described informed the design and endpoints implemented in the phase III MOVE trial (NCT03312634) and provide a foundation for future clinical trial development.Trial registrationNCT02322255 (registered 23/12/2014); NCT02190747 (registered 15/07/2014); NCT02279095 (registered 30/10/2014).

Macrophage-Derived Extracellular DNA Initiates Heterotopic Ossification.

Heterotopic ossification (HO) severely affects people's lives; however, its pathological mechanism remains poorly understood. Although extracellular DNA (ecDNA) has been shown to play important roles in pathological calcification, its effects in HO development and progression remain unknown. The in vivo rat Achilles tendon injury model and in vitro collagen I calcification model were used to evaluate the effects of ecDNA in the ectopic calcifications and the main cell types involved in those pathological process. Histology, immunofluorescent staining, reverse transcriptase-polymerase chain reaction analysis and micro-computed tomography were used to identify the distribution of macrophage-derived ecDNA and elucidate their roles in HO. The results showed that the amount of ecDNA and ectopic calcification increased significantly and exhibited a strong correlation in the injured tendons of HO model compared with those of the controls, which was accompanied by a significantly increased number of M2 macrophages in the injured tendon. During in vitro co-culture experiments, M2 macrophages calcified the reconstituted type I collagen and ectopic bone collected from the injured tendons of HO rats, while those effects were inhibited by deoxyribonuclease. More importantly, deoxyribonuclease reversed the pathological calcification in the injured rat tendon HO model. The present study showed that ecDNA from M2 macrophages initiates pathological calcification in HO, and the elimination of ecDNA might be developed into a clinical strategy to prevent ectopic mineralization diseases. The use of deoxyribonuclease for the targeted degradation of ecDNA at affected tissue sites provides a potential solution to treat diseases associated with ectopic mineralization.

Inhibition of NF-κB Signaling-Mediated Crosstalk Between Macrophages and Preosteoblasts by Metformin Alleviates Trauma-Induced Heterotopic Ossification.

Heterotopic ossification (HO) is a pathological condition that occurs in soft tissues following severe trauma. The exact pathogenesis of HO remains unclear. Studies have shown that inflammation predisposes patients to the development of HO and triggers ectopic bone formation. Macrophages are crucial mediators of inflammation and are involved in HO development. The present study investigated the inhibitory effect and underlying mechanism of metformin on macrophage infiltration and traumatic HO in mice. Our results found that abundant levels of macrophages were recruited to the injury site during early HO progression and that early administration of metformin prevented traumatic HO in mice. Furthermore, we found that metformin attenuated macrophage infiltration and the NF-κB signaling pathway in injured tissue. The monocyte-to-macrophage transition in vitro was suppressed by metformin and this event was mediated by AMPK. Finally, we showed that inflammatory mediator's regulation by macrophages targeted preosteoblasts, leading to elevated BMP signaling, and osteogenic differentiation and driving HO formation, and this effect was blocked after the activation of AMPK in macrophages. Collectively, our study suggests that metformin prevents traumatic HO by inhibiting of NF-κB signaling in macrophages and subsequently attenuating BMP signaling and osteogenic differentiation in preosteoblasts. Therefore, metformin may serve as a therapeutic drug for traumatic HO by targeting NF-κB signaling in macrophages.

Galunisertib attenuates progression of trauma-induced heterotopic ossification via blockage of Smad2/3 signaling in mice

Heterotopic ossification (HO) is the formation of bony tissues in the extraskeletal system. To date, no effective therapy has been developed for the treatment of HO, although increasing evidences have shown that inhibition of TGF-β signaling has potential as a new therapeutic approach for attenuating HO progression. Results from previous clinical trials have demonstrated that patients with malignant tumors exhibit excellent tolerability to Galunisertib, a TGF-β receptor I kinase inhibitor. However, its therapeutic potential in preventing HO and inhibitory effect on osteogenesis remain unclear. In this study, we demonstrated that intragastrical administration of Galunisertib, at a concentration as low as 10 mg/kg, was not only fairly effective in preventing HO development in a dose-dependent manner, but also generated a non-toxic response in a novel Achilles tendon puncture-induced traumatic HO model in mice. Moreover, Galunisertib treatment in the early phases of HO development, including the inflammatory and chondrogenic period, resulted in better therapeutic effects instead of eliminating already formed bony tissues. Mechanistically, Galunisertib suppressed the osteogenic differentiation capacity of tendon-derived stem cells (TDSCs) by interfering with the Smad2/3 signaling pathway, blocking the phosphorylation of Smad2/3 translocated from cytoplasm into the nucleus to regulate the expression of both osteogenesis-related transcription factors and related proteins. Results from in vivo experiments further validated Galunisertib's effect on HO attenuation, by intercepting the TGF-β/Smad2/3 signaling pathway. In conclusion, our findings demonstrated Galunisertib's potential as a prophylactic drug for the treatment of traumatic HO or other related diseases triggered by over-expressed TGF-β.

Pharmacological activation of SIRT1 by metformin prevented trauma-induced heterotopic ossification through inhibiting macrophage mediated inflammation

Trauma-induced heterotopic ossification (HO) is the aberrant extra-skeletal bone formation that severely incapacitates patient's daily life. Inflammation is the first stage of this progression, becoming an appealing target of early therapeutic intervention. Metformin, a widely used antidiabetic drug, also poses the therapeutic potential to modulate various inflammatory-related diseases. Therefore, this study aimed to investigate the preventive effect of metformin on trauma-induced HO progression, and unveil the underlying molecular mechanisms. A murine burn/tenotomy model was established to mimic trauma-induced HO in vivo. The anti-inflammation and anti-ossification effects of metformin were evaluated by histological staining and micro-CT. The inhibitory effects of metformin on macrophages activation in vitro were examined by ELISA and qRT-PCR. The underlying molecular mechanisms were further explored by immunofluorescence staining and western-blotting in vivo. Increased macrophages infiltration and inflammatory responses were found at early stage during HO progression. However, metformin dose-dependently attenuated the macrophage-mediated inflammatory responses both in vivo and vitro, which might account for the inhibitory effect of metformin on chondrogenesis and HO formation after trauma. Furthermore, elevated SIRT1 expression and decreased NF-κB p65 acetylation were found in the beneficial effects of metformin. Moreover, similar preventive effects were also found in SRT1720 HCI, a specific SIRT1 activator, while were remarkably reversed after the administration of EX527 (a specific SIRT1 inhibitor) with metformin. Taken together, our results provide a novel evidence that metformin can effectively attenuate trauma-induced HO by mitigating macrophage inflammatory responses through inhibiting NF-κB signaling via SIRT1-dependent mechanisms, which favors future therapeutic investigations for trauma-related disease.

Inhibition of IL-17 prevents the progression of traumatic heterotopic ossification.

Traumatic heterotopic ossification (HO) is the abnormal formation of bone in soft tissues as a consequence of injury. However, the pathological mechanisms leading to traumatic HO remain unknown. Here, we report that aberrant expression of IL‐17 promotes traumatic HO formation by activating β‐catenin signalling in mouse model. We found that elevated IL‐17 and β‐catenin levels are correlated with a high degree of HO formation in specimens from patients and HO animals. We also show that IL‐17 initiates and promotes HO progression in mice. Local injection of an IL‐17 neutralizing antibody attenuates ectopic bone formation in a traumatic mouse model. IL‐17 enhances the osteoblastic differentiation of mesenchymal stem cells (MSCs) by activating β‐catenin signalling. Moreover, inhibition of IL‐17R or β‐catenin signalling by neutralizing antibodies or drugs prevents the osteogenic differentiation of isolated MSCs and decreases HO formation in mouse models. Together, our study identifies a novel role for active IL‐17 as the inducer and promoter of ectopic bone formation and suggests that IL‐17 inhibition might be a potential therapeutic target in traumatic HO.

Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult.

Heterotopic ossification (HO) is one of the most intractable disorders following musculoskeletal injury and is characterized by the ectopic presence of bone tissue in the soft tissue leading to severe loss of function in the extremities. Recent studies have indicated that immune cell infiltration and inflammation are involved in aberrant bone formation. In this study, we found increased monocyte/macrophage and mast cell accumulation during early HO progression. Macrophage depletion by clodronate liposomes and mast cell stabilization by cromolyn sodium significantly impeded HO formation. Therefore, we proposed that the dietary phytochemical quercetin could also suppress immune cell recruitment and related inflammatory responses to prevent HO. As expected, quercetin inhibited the monocyte-to-macrophage transition, macrophage polarization, and mast cell activation in vitro in a dose-dependent manner. Using a murine burn/tenotomy model, we also demonstrated that quercetin attenuated inflammatory responses and HO in vivo. Furthermore, elevated SIRT1 and decreased acetylated NFκB p65 expression were responsible for the mechanism of quercetin, and the beneficial effects of quercetin were reversed by the SIRT1 antagonist EX527 and mimicked by the SIRT agonist SRT1720. The findings in this study suggest that targeting monocyte/macrophage and mast cell activities may represent an attractive approach for therapeutic intervention of HO and that quercetin may serve as a promising therapeutic candidate for the treatment of trauma-induced HO by modulating SIRT1/NFκB signaling.

The TrkA Antagonist AR786 Prevents Heterotopic Ossification

Soft tissue trauma can result in the pathologic formation of cartilage and bone outside of the skeleton, termed heterotopic ossification (HO). Therapeutic options for treatment of HO are lacking. We recently recognized the role of sensory nerve fibers in trauma-induced HO through nerve growth factor (NGF) and tropomyosin receptor kinase A (TrkA) signaling whereby the deletion of NGF-TrkA signaling impairs axonal invasion and blunts osteochondral differentiation after HO induction in a mouse model. Given this observation, we hypothesized that utilization of a small molecule antagonist for TrkA in clinical development, AR786, would demonstrate therapeutic benefit in the prevention of HO. Association of NGF and innervation with human HO of the posterior longitudinal ligament (HOPLL) was analyzed by microarray and immunohistochemistry. In a mouse model, trauma-induced HO was generated by the combination of cutaneous burn and Achilles tenotomy in 10 week old C57BL/6J mice, which causes HO over a well characterized 9 week time period. NGF expression was examined by single cell sequencing, immunohistochemistry, and western blot. Dorsal root ganglion (DRG) from the innervating lumbar tracts were examined for pTrkA expression by immunohistochemistry. The phenotypic consequences of TrkA inhibition by AR786 on HO formation was next assessed (Vehicle, n=5 and AR786, n=5). Nerve innervation within the injury site was visualized using immunohistochemistry stained with Beta III Tubulin (TUBB3) and confocal microscopy. Abnormal chondrogenesis was assessed by cartilage histomorphometry, and immunohistochemical detection (SOX9 and COL2). Data were presented as Means ± SD. Results showed that NGF and associated downstream NGF signaling were increased among HOPLL patient cells. NGF immunoreactivity was present in perivascular cells adjacent to HO, fibroblastic cells, and bone-associated cells. Increased nerve fibers were present surrounding HO and axonal sprouting was confirmed by neural antigen S100 and neurofilament SM31 in human HO tissue sections. During HO progression in mice, NGF, proNGF, and pTrkA were upregulated following HO induction. Nerve frequency was significantly reduced with AR786 treated animals compared to vehicle control in uninjured (p<0.001, Vehicle; 548.2 ± 118.0 vs. AR786; 104.2 ± 47.9, µm3) and 3-weeks post-injury (p<0.001, Vehicle; 3255.6 ± 1405.1 vs. AR786; 462.8 ± 201.1, µm3). Consequently, reduced axonal ingrowth significantly delays in ectopic cartilage formation (p<0.01, Vehicle; 0.33 ± 0.07 vs. AR786; 0.07 ± 0.06, mm2). In conclusion, NGF-TrkA signaling mediates crucial functions in human and rodent HO, and inhibition of TrkA-expressing neural inputs by AR786 abrogates HO progression. These findings suggest that NGF-TrkA signaling inhibition can be a potential therapeutic use in HO patients as negative regulator of HO disease progression.

Genetic and acquired heterotopic ossification are driven by a self‐amplifying positive feedback loop of Hedgehog signaling

Heterotopic ossification (HO), which refers to extra-skeletal pathological bone formation, occurs as a common complication after injury or in genetic disorders. However, limited by poor understanding of the underlying cellular and molecular mechanisms, there is currently no effective treatment and surgical eviction often results in recurrence. We have found that in both genetic and injury-induced HO, activated ectopicexpression of a hedgehog ligand drives HO progression by forming a positive feedback loop with Yap activation that results in non-cell-autonomous and self-propagation of osteoblast differentiation of wild type cells. In mouse models of progressive osseous heteroplasia (POH), a human disease caused by null mutations in GNASthat encodes Gαs, we found previously that Hedgehog signaling was activated, which is both necessary and sufficient to induce HO. We recently further found that progressively expanded ectopic bone was formed by progressively recruited wild type cells. Mechanistically, the Gnas-/-mesenchymal cells differentiate into osteoblasts and recruit wild type cells to form bone by activatingexpression of Hedgehog ligands. Secreted Hedgehog further induces Yap activation that directly activating more Hedgehogligand expression in surrounding wild type cells. Activated Yap and Hedgehog signaling induces osteoblast differentiation and this self-propagating positive feedback loop is both necessary and sufficient for ectopic bone formation and expansion and can act independently of Gαs. Importantly, Hedgehog activation was also found in fibrodysplasia ossificans progressive (FOP), Achilles tendon puncture (ATP)-induced HO mouse models and in human HO samples. Genetic removal of Hedgehog and Yap abolished HO not only in POH, but also in FOP and acquired HO mouse models. We therefore identify a Hedgehog and Yap-driven, self- amplifying and self-propagating osteoblast differentiation as a common cellular and molecular mechanism underlying HO initiation and expansion. Our work highlights the importance of rare genetic disease studies in identifying a shared core pathological mechanism underlying a class of diseases. Our results further suggest that the Hedgehog pathway is a promising new therapeutic target for HO without affecting the normal bone.

Genetic and acquired heterotopic ossification are driven by a self‐amplifying positive feedback loop of Hedgehog signaling

Heterotopic ossification (HO), which refers to extra‐skeletal pathological bone formation, occurs as a common complication after injury or in genetic disorders. However, limited by poor understanding of the underlying cellular and molecular mechanisms, there is currently no effective treatment and surgical eviction often results in recurrence. We have found that in both genetic and injury‐induced HO, activated ectopic expression of a hedgehog ligand drives HO progression by forming a positive feedback loop that results in non‐cell‐autonomous and self‐propagation of osteoblast differentiation of wild type cells. In mouse models of progressive osseous heteroplasia (POH), a human disease caused by null mutations in GNAS that encodes Gαs, we found previously that Hedgehog signaling was activated, which is both necessary and sufficient to induce HO. We recently further found that progressively expanded ectopic bone was formed by progressively recruited wild type cells. Mechanistically, the Gnas−/− mesenchymal cells differentiate into osteoblasts and recruit wild type cells to form bone by activating expression of Hedgehog ligands. Secreted Hedgehog further induces Hedgehog expression and osteoblast differentiation in surrounding wild type cells. This self‐propagating positive feedback loop is both necessary and sufficient for ectopic bone formation and expansion and can act independently of Gαs. Importantly, Hedgehog activation was also found in fibrodysplasia ossificans progressive (FOP), Achilles tendon puncture (ATP)‐induced HO mouse models and in human HO samples. Genetic removal of Hedgehog abolished HO not only in POH, but also in FOP and aquired HO mouse models. We therefore identify a Hedgehog‐driven, self‐amplifying osteoblast differentiation as a common cellular and molecular mechanism underlying HO initiation and expansion. Our work highlights the importance of rare genetic disease studies in identifying a shared core pathological mechanism underlying a class of diseases. Our results further suggest that the Hedgehog pathway is a promising new therapeutic target for HO without affecting the normal bone.Support or Funding InformationR01DE025866 from NIDCR, R01AR070877 from NIAMS

Hypofractionated Radiation Therapy for Progressive Heterotopic Ossification: The Relationship between Dose and Efficacy

We sought to evaluate treatment outcomes after hypofractionated short-course radiation therapy (RT) for progressive heterotopic ossification (HO). Nine consecutive patients were treated between January 2016 and December 2018. None had received previous preventive RT. RT was performed to prevent further HO progression with one of the following dose-fractionation regimens: 7 Gy × 2 fractions, 9 Gy × 1 fraction, 6 Gy × 2 fractions, 6 Gy × 3 fractions, 8 Gy × 2 fractions, or 7 Gy × 3 fractions. All patients were male, with a median age of 30 years (range, 16-55). Eight patients presented with grade III Brooker classification. The most commonly involved site was the hip, followed by femur and knee. With a median assessment time of 7.1 (range, 5.1-23.1) months, eight patients achieved decreased HO, with five showing a dramatic (≥50%) reduction. Among the five excellent responders, a 21 Gy with 7 Gy per fraction schedule was used in four patients. HO status was maintained at the final median follow-up of 11.6 months (range, 6.2-36.1), and Brooker's grade was improved in three patients. Nosignificant RT-related toxicity was noted. The current RT scheme was effective for the management of progressive HO. It is speculated that higher RT doses could result in the superior efficacy of progressive HO.

Celecoxib cannot inhibit the progression of initiated traumatic heterotopic ossification

Heterotopic ossification (HO) is a recognized sequela after trauma and arthroplasty. The purpose of this study was to evaluate the therapeutic effect of celecoxib on HO. We hypothesized that celecoxib may inhibit the progression of initiated HO. We performed a retrospective review of 37 patients who underwent elbow joint surgery between January 2014 and June 2018. Seventeen patients were prescribed orally administered celecoxib (200 mg/dose, twice daily) for 2 months after the diagnosis of HO, whereas the remaining 20 patients were administered celecoxib for 1 month starting immediately after surgery. HO progression was evaluated by plain radiographs. By use of an Achilles tendon puncture-induced HO mouse model, the curative effect of celecoxib was illustrated at different HO progression stages. The mice were assigned to 1 of 4 groups: sham group, vehicle group, group receiving celecoxib on day 1, and group receiving celecoxib in week 6. Achilles tendons were analyzed by micro-computed tomography and histochemistry after 12 weeks. Celecoxib did not inhibit the progression of initiated HO in the patients in whom HO was diagnosed, whereas those who received celecoxib after surgery had lower morbidity. Achilles tendon puncture effectively induced typical HO in mice. The ectopic bone volume was significantly reduced in the day 1 celecoxib group compared with the vehicle group; however, the difference was not statistically significant in the week 6 celecoxib group. Administration of celecoxib starting immediately after surgery can significantly inhibit the formation of HO. Once HO is visible on plain radiographs or micro-computed tomography, celecoxib cannot effectively attenuate further progression of HO in humans and mice.

Tamoxifen Inhibits the Progression of Trauma-Induced Heterotopic Ossification in Mice.

BackgroundHeterotopic ossification (HO) is a kind of abnormal mineralized bone which usually occurs in muscle, tendon, or ligament. There are currently no effective drugs for the treatment and prevention of HO. Developing effective drugs that can inhibit HO is of profound significance and would provide new strategies for clinical treatment of this disease. The present investigation evaluated the inhibitory effect of tamoxifen against HO.Material/MethodsUsing an Achilles tendon trauma-induced HO female mice model, we screened different doses of tamoxifen (1, 3, and 9 mg/kg) in mice to determine the optimal dosage on the inhibition of the HO formation. The curative effect of tamoxifen was also illustrated at different HO progression stages including inflammation, chondrogenesis, osteogenesis, and HO maturation.ResultsHeterotopic bone was formed with typical endochondral ossification in Achilles tendons 6 weeks after surgery and continued to enlarge up to 12 weeks. The formation of HO was significantly inhibited with the treatment of tamoxifen at the dosage of 9 mg/kg, whereas 1 mg/kg and 3 mg/kg did not reduce HO bone volume remarkably. The progression of HO was both attenuated by tamoxifen from Day 1 and Week 4 post-surgery, whereas no inhibitory effect was shown at the osteogenesis and maturation stages treated with tamoxifen.ConclusionsTamoxifen exerts an inhibitory effect on the heterotopic bone progression at inflammation and chondrogenesis stages, with the TGF-β signaling pathway suppressed following the increase in estrogen receptor α activity.

Evolution of heterotopic bone in fibrodysplasia ossificans progressiva: An [18F]NaF PET/CT study.

Fibrodysplasia ossificans progressiva (FOP) is a rare, autosomal dominant disorder characterized by heterotopic ossification (HO) in muscles, ligaments and tendons. Flare-ups often precede the formation of HO, resulting in immobilization of joints. Due to progression of the disease without signs of a flare-up, co-existence of a chronic progression of HO has been postulated, but conclusive evidence is lacking. Recently, it has been shown that [18F]NaF PET/CT is able to identify early ossifying disease activity during flare-ups. Therefore, the purpose of the present study was to assess whether [18F]NaF PET/CT might also be able to identify the possible presence of chronic progressive HO in FOP. A total of thirteen [18F]NaF PET/CT scans from five FOP patients were analysed. Scans were acquired over a period of 0.5 to 2 years. Volumes of HO and standardized uptake values (SUV) were obtained based on manual segmentation of CT images. SUVpeak values, defined as the average SUV value of a 1 mL sphere containing the hottest voxel pixels, were obtained. Two out of five patients experienced ≥1 active clinical flare-ups at the time of the [18F]NaF PET/CT scan. In addition, in four out of five patients, serial scans showed radiological progression of HO (3 to 8 cm3), as assessed by CT volume, in the absence of a clinical flare-up. This volumetric increase was present in 6/47 (12.8%) of identified HO structures and, in all cases, was accompanied by increased [18F]NaF uptake, with SUVpeak ranging from 8.4 to 17.9. In conclusion, HO may progress without signs of a flare-up. [18F]NaF PET/CT is able to identify these asymptomatic, but progressive HO lesions, thereby demonstrating the presence of chronic activity in FOP. Consequently, future drugs should not only target new HO formation, but also this chronic HO progression.

Inhibition of overactive TGF-\u03b2 attenuates progression of heterotopic ossification in mice

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-β initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-β that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-β in tendon induces spontaneous HO, whereas systemic injection of a TGF-β neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-β type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-β as inducers and promoters of ectopic bone formation, and suggest that TGF-β might be a therapeutic target in HO.