KLF5 promotes the ossification process of ligamentum flavum by transcriptionally activating CX43.

BACKGROUND: Ossification of ligamentum flavum (OLF) is a prevalent degenerative spinal disease, typically causing severe neurological dysfunction. Kruppel-like factor 5 (KLF5) plays an essential role in the regulation of skeletal development. However, the mechanism KLF5 plays in OLF remains unclear, necessitating further investigative studies. METHODS: qRT-PCR, immunofluorescent staining and western blot were used to measure the expression of KLF5. Alkaline Phosphatase (ALP) staining, Alizarin red staining (ARS), and the expression of Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcin (OCN) were used to evaluate the osteogenic differentiation. Luciferase activity assay and ChIP-PCR were performed to investigate the molecular mechanisms. RESULTS: KLF5 was significantly upregulated in OLF fibroblasts in contrast to normal ligamentum flavum (LF) fibroblasts. Silencing KLF5 diminished osteogenic markers and mineralized nodules, while its overexpression had the opposite effect, confirming KLF5's role in promoting ossification. Moreover, KLF5 promotes the ossification of LF by activating the transcription of Connexin 43 (CX43), and overexpressing CX43 could reverse the suppressive impact of KLF5 knockdown on OLF fibroblasts' osteogenesis. CONCLUSION: KLF5 promotes the OLF by transcriptionally activating CX43. This finding contributes significantly to our understanding of OLF and may provide new therapeutic targets.

"Bone in the penis" or fasciitis ossificans of the penis - a first time description of a pseudo-tumor at an extraordinary site.

BACKGROUND: Fasciitis ossificans is a rare subtype of nodular fasciitis, a benign soft tissue tumor with reactive characteristics. Due to its rapid growth, it is often misdiagnosed as a malignant tumor. While fasciitis ossificans commonly originates from the subcutaneous tissue and can appear throughout the body, it may also arise from extraordinary sites. CASE PRESENTATION: We report the first-ever documented case of fasciitis ossificans arising from the penis in a male patient who presented with a tumor on the glans penis. The tumor was surgically resected due to suspicion of penile cancer. Initial histopathological analysis led to a misdiagnosis of squamous cell carcinoma. However, pathological consultation ultimately confirmed the diagnosis of fasciitis ossificans of the penis originating from the glans penis by demonstrating ossification. CONCLUSION: This case underscores the importance of considering fasciitis ossificans in the differential diagnosis of soft tissue tumors, even in unusual locations such as penile soft tissue.

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.

A rare case of extensive neurogenic heterotopic ossification: a case report.

INTRODUCTION: Neurogenic Heterotopic ossification (NHO) is a potential sequalae and a detrimental complication following neurological insult. It is characterized by formation of localized gradually progressive, peri-articular lamellar bone formation in extra-skeletal tissues. We would like to report a rare case of heterotopic ossification involving all 4 limbs, in which we tried to restore joint mobility to improve his functional status so that he could perform his daily tasks. CASE PRESENTATION: We present a case of a 33-year-old bed ridden male, diagnosed with NHO involving all 4 limbs (bilateral hip, right knee, right shoulder, left elbow). The patient had a crippled posture, significant pain and impaired range of motion hampering movement of all four limbs which prevented him from lying down supine, sitting, walking and performing activities of daily living. After three surgeries, the patient achieved wheelchair mobilization and upright posture with the assistance of calipers. CONCLUSION: The management of NHO requires a multidisciplinary approach involving orthopaedic surgeons, neurologists & rehabilitation specialists. Prognosis of NHO depends on factors such as extent of ossification, underlying neurological condition & patients overall health.

The HIF-1α/PLOD2 axis integrates extracellular matrix organization and cell metabolism leading to aberrant musculoskeletal repair.

While hypoxic signaling has been shown to play a role in many cellular processes, its role in metabolism-linked extracellular matrix (ECM) organization and downstream processes of cell fate after musculoskeletal injury remains to be determined. Heterotopic ossification (HO) is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues. Hypoxia and hypoxia-inducible factor 1α (HIF-1α) activation have been shown to promote HO. However, the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells (MPCs) contributes to pathologic bone formation remain to be elucidated. Here, we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate. Using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analyses of the HO site, we found that collagen ECM organization is the most highly up-regulated biological process in MPCs. Zeugopod mesenchymal cell-specific deletion of Hif1α (Hoxa11-CreERT2; Hif1afl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreERT2; Hif1afl/fl mice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO. Importantly, our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion. From a translational aspect, a pan-LOX inhibitor significantly decreased HO. A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism. This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate. These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.

Intersections of Fibrodysplasia Ossificans Progressiva and Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating pathology where ectopic bone develops in areas of soft tissue. HO can develop as a consequence of traumatic insult or as a result of dysregulated osteogenic signaling, as in the case of the orphan disease fibrodysplasia ossificans progressiva (FOP). Traumatic HO (tHO) formation is mediated by the complex interplay of signaling between progenitor, inflammatory, and nerve cells, among others, making it a challenging process to understand. Research into the pathogenesis of genetically mediated HO (gHO) in FOP has established a pathway involving uninhibited activin-like kinase 2 receptor (ALK2) signaling that leads to downstream osteogenesis. Current methods of diagnosis and treatment lag behind pre-mature HO detection and progressive HO accumulation, resulting in irreversible decreases in range of motion and chronic pain for patients. As such, it is necessary to draw on advancements made in the study of tHO and gHO to better diagnose, comprehend, prevent, and treat both.

Hedgehog Signaling Controls Chondrogenesis and Ectopic Bone Formation via the Yap-Ihh Axis.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by abnormal bone formation due to ACVR1 gene mutations. The identification of the molecular mechanisms underlying the ectopic bone formation and expansion in FOP is critical for the effective treatment or prevention of HO. Here we find that Hh signaling activation is required for the aberrant ectopic bone formation in FOP. We show that the expression of Indian hedgehog (Ihh), a Hh ligand, as well as downstream Hh signaling, was increased in ectopic bone lesions in Acvr1R206H; ScxCre mice. Pharmacological treatment with an Ihh-neutralizing monoclonal antibody dramatically reduced chondrogenesis and ectopic bone formation. Moreover, we find that the activation of Yap in the FOP mouse model and the genetic deletion of Yap halted ectopic bone formation and decreased Ihh expression. Our mechanistic studies showed that Yap and Smad1 directly bind to the Ihh promoter and coordinate to induce chondrogenesis by promoting Ihh expression. Therefore, the Yap activation in FOP lesions promoted ectopic bone formation and expansion in both cell-autonomous and non-cell-autonomous manners. These results uncovered the crucial role of the Yap-Ihh axis in FOP pathogenesis, suggesting the inhibition of Ihh or Yap as a potential therapeutic strategy to prevent and reduce HO.

Immunologic Aspects in Fibrodysplasia Ossificans Progressiva.

BACKGROUND: Inflammation is a major driver of heterotopic ossification (HO), a condition of abnormal bone growth in a site that is not normally mineralized. PURPOSE OF REVIEW: This review will examine recent findings on the roles of inflammation and the immune system in fibrodysplasia ossificans progressiva (FOP). FOP is a genetic condition of aggressive and progressive HO formation. We also examine how inflammation may be a valuable target for the treatment of HO. Rationale/Recent findings: Multiple lines of evidence indicate a key role for the immune system in driving FOP pathogenesis. Critical cell types include macrophages, mast cells, and adaptive immune cells, working through hypoxia signaling pathways, stem cell differentiation signaling pathways, vascular regulatory pathways, and inflammatory cytokines. In addition, recent clinical reports suggest a potential role for immune modulators in the management of FOP. FUTURE PERSPECTIVES: The central role of inflammatory mediators in HO suggests that the immune system may be a common target for blocking HO in both FOP and non-genetic forms of HO. Future research focusing on the identification of novel inflammatory targets will help support the testing of potential therapies for FOP and other related conditions.

Heterotopic bone formation in a case of clear cell renal cell carcinoma: A case report.

Renal cell carcinoma (RCC) with heterotopic formation has been reported very rarely. We report this rare entity in a 33-year-old female patient who came to the out-patient department after complaining of pain in the lumbar region of the left side for 2 years. A computed tomography scan showed a heterogeneously enhancing lesion originating from the posterior cortex of the left kidney in the upper pole. It had many chunky calcification foci and was treated with left robotic partial nephrectomy. Histo-pathological examination revealed clear cell RCC with the heterotopic bone formation with a tumor size measuring 5 × 4 × 2.5 cm; the tumor was limited to the kidney, and the tumor resection margin were free of tumor, WHO/ISUP Grade 2. The pathological stage (AJCC 8th edition PTNM) was p T1b p NX p MX. The prognostic implications regarding calcification are poorly addressed in the literature. Patients suffering from osseous metaplasia are often in their early stages of the disease and have a favorable prognosis.

Magnetic resonance imaging of femoral nerve injury in the setting of anterior approach total hip arthroplasty.

PURPOSE: To illustrate MRI findings in patients with femoral neuropathy following anterior approach total hip arthroplasty (THA). METHODS: This was a retrospective review of patients who underwent MRI for femoral neuropathy following anterior approach THA between January 1, 2010, and July 1, 2022. Included patients had no preexisting neurologic condition. Clinical and diagnostic data were collected. MRIs were reviewed in consensus by 2 musculoskeletal radiologists. RESULTS: A total of 115 patient records were reviewed, 17 of which were included in the final analysis (mean age 64 years; 11 females). Study subjects presented with weakness with hip flexion and knee extension and pain and numbness in the femoral nerve distribution. In 7 subjects, the femoral nerve appeared normal. In 5 subjects, the femoral nerve was hyperintense on fluid-sensitive fat-suppressed imaging. In 4 patients, mass effect on the femoral nerve by either ill-defined soft tissue edema (n = 2), seroma (n = 1), or heterotopic ossification (n = 1) was detected. Only 1 patient had a nerve transection. Patients were imaged at a median time of 8 months (range: 1 day to 11 years) following arthroplasty placement. Clinical follow-up was available in 8 patients, of whom half had complete symptomatic resolution and half had partial improvement at a mean follow-up time of 39.3 months (SD 51.1). Of these 8, 1 underwent revision arthroplasty, 1 had removal of hardware, and another had excision of heterotopic ossification. CONCLUSION: MRI provides a means to directly evaluate the femoral nerve following anterior approach THA in both the immediate and chronic postoperative periods.

Trauma patient heterotopic ossification diagnosis is associated with increased hospital length of stay.

BACKGROUND: Traumatic heterotopic ossification (tHO) refers to the development of extra-skeletal bone in muscle and soft tissues following tissue insult secondary to surgery or trauma. This presents a persistent clinical concern associated with significant patient morbidity and expense to diagnose and treat. Traumatic HO is a substantial barrier to rehabilitation for trauma-injured patients. As such, the development of tHO after burn and other trauma is hypothesised to prolong inpatient length of stay (LOS) and thus increase health care costs. OBJECTIVE: To investigate the association between an inpatient tHO diagnosis and hospital LOS in trauma patients. METHODS: A retrospective audit of trauma patients over a 14-year period was completed using data from four WA hospitals. Burn and neurological trauma patients diagnosed with tHO as an inpatient (tHO+) and control subjects (tHO-), matched (1:3) by age, gender, and injury severity factors, were identified using medical diagnostic codes. Data relating to patient and injury-related determinants of LOS from tHO+ and tHO- subjects were analysed to model the association of tHO on total hospital length of stay. RESULTS: 188 identified patients were hospitalised due to traumatic injury; 47 patients with tHO following burn injury (n = 17), spinal cord injury (n = 13) and traumatic brain injury (n = 17), and 141 control patients. Those who developed tHO during hospitalisation had a significantly higher median LOS than matched trauma patients who did not develop tHO (142 days vs. 61 days). Multivariate regression analyses identified the following independent predictive factors of a prolonged hospital LOS: tHO diagnosis, mechanical ventilation hours, injury to the hip region and thigh area, other ossification disorder, pressure injury, admission to intensive care unit and deep vein thrombosis. Trauma patients diagnosed with tHO during their hospital admission stayed 1.6 times longer than trauma patients matched for injury severity without a tHO diagnosis (IRR 1.56, 95% CI 1.35-1.79, p<0.001). CONCLUSION: Traumatic heterotopic ossification is an independent explanatory factor for increased hospital LOS in patients following burns, spinal cord, and traumatic brain injury. Early diagnosis may assist in reducing the impact of tHO on acute hospital stay after trauma.

iMSC-mediated delivery of ACVR2B-Fc fusion protein reduces heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease caused by a gain-of-function mutation in ACVR1, which is a bone morphogenetic protein (BMP) type I receptor. Moreover, it causes progressive heterotopic ossification (HO) in connective tissues. Using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and mouse models, we elucidated the underlying mechanisms of FOP pathogenesis and identified a candidate drug for FOP. METHODS: In the current study, healthy mesenchymal stem/stromal cells derived from iPSCs (iMSCs) expressing ACVR2B-Fc (iMSCACVR2B-Fc), which is a neutralizing receptobody, were constructed. Furthermore, patient-derived iMSCs and FOP mouse model (ACVR1R206H, female) were used to confirm the inhibitory function of ACVR2B-Fc fusion protein secreted by iMSCACVR2B-Fc on BMP signaling pathways and HO development, respectively. RESULTS: We found that secreted ACVR2B-Fc attenuated BMP signaling initiated by Activin-A and BMP-9 in both iMSCs and FOP-iMSCs in vitro. Transplantation of ACVR2B-Fc-expressing iMSCs reduced primary HO in a transgenic mouse model of FOP. Notably, a local injection of ACVR2B-Fc-expressing iMSCs and not an intraperitoneal injection improved the treadmill performance, suggesting compound effects of ACVR2B-Fc and iMSCs. CONCLUSIONS: These results offer a new perspective for treating FOP through stem cell therapy.

Heterotopic Ossification of Bilateral Hips Post-COVID-19 and Prolonged Immobilization: A Case Report.

CASE: A 43-year-old healthy man developed hip pain post-coronavirus disease 2019 (COVID-19) immobilization. Imaging confirmed bilateral bridging heterotopic ossification (HO) of the hips, Brooker Class IV. Bilateral HO caused functional arthrodesis (45° flexion: -20° internal rotation). Bilateral HO resection resulted in almost full mobility at 1-year follow-up (90° flexion; 30° internal rotation). CONCLUSION: Many cases of HO after immobilization for COVID-19 have been reported, but as far as we know, this is the first case report describing surgical intervention as an adequate treatment option for severe restricted mobility caused by HO due to COVID-19-induced prolonged immobilization. Caution and preoperative 3D planning are recommended of HO formation near neurovascular structures.

Arthroscopic debridement improves range of motion for heterotopic ossification after total knee replacement: a retrospective cohort study.

The presence of heterotopic ossification (HO) after primary total knee replacement (TKR) is rare and associated with limited mobility and stiffness of the knee. This study aimed to identify if the arthroscopic debridement after TKR could decrease HO and improve the function and range of motion. Thirty HO patients after TKR were retrospectively separated into 2 cohorts. 15 patients of group A accepted the arthroscopic debridement, while 15 patients of group B only had non-operative treatment, mainly including oral nonsteroidal anti-inflammatory drugs (NSAIDs) and rehabilitative treatment. Visual analog scale (VAS) scores, knee society knee scores (KSS), range of motion (knee flexion and knee extension) were obtained before treatment and at 1 month, 3 months, and 6 months after treatment. Radiography of after-treatment was also evaluated to assess the changes in HO. There were 3 males and 27 females with a mean age of 67.4 ± 0.8 years in group A and 68.2 ± 1.3 in group B. The onset time of HO was 3-6 months. The maximum size of the ossification was < 2 cm in 23 knees, 2 cm < heterotopic bone < 5 cm in 6 knees and > 5 cm in 1 knee. The size of HO decreased gradually in all knees by X-ray film at the last follow-up. There were no significant differences in VAS scores after replacement between two groups (p > 0.05). The average range of motion preoperatively in group A was - 15.2-90.6°, which postoperatively increased to - 4.2-110.0°. Meanwhile, the KSS scores and average range of motion of the group A were better than those of the group B at each follow-up time after treatment. Arthroscopic debridement can decrease HO seen from postoperative X-rays, improve the function and range of motion, as well as the pain remission between two groups are comparable. Consequently, arthroscopic resection of HO after TKR is recommended as soon as there is aggravating joint stiffness.

Healing an ischial tuberosity pressure ulcer in a patient with neurogenic heterotopic ossification: a case report.

Neurogenic heterotopic ossification (NHO) is widely recognised as an aberrant bone formation in soft tissue following central nervous system injury. It is most frequently associated with pain and limited movement, especially in the hip. However, it may be neglected in patients with paraplegia with a pressure ulcer (PU). We report the case of an 18-year-old male patient who presented with a hard-to-heal ischial tuberosity PU and who had undergone three operations at other hospitals during the previous six months, which had failed to repair the PU. There was a history of paraplegia as a consequence of spinal cord injury two years previously. Computed tomography and three-dimensional reconstruction showed massive heterotopic ossification (HO) in the wound bed and around the right hip. Histological findings were consistent with a diagnosis of HO. The HO around the wound was completely excised, negative pressure wound therapy was used to promote granulation, and a gluteus maximus musculocutaneous flap was used to cover the wound. We conclude that for patients with paraplegia, with a hard-to-heal PU, it should be determined whether it is associated with NHO. Surgical resection of HO surrounding the wound and improving the microcirculation are critical for repair and reconstruction of these PUs.

Role of the NF-kB signalling pathway in heterotopic ossification: biological and therapeutic significance.

Heterotopic ossification (HO) is a pathological process in which ectopic bone develops in soft tissues within the skeletal system. Endochondral ossification can be divided into the following types of acquired and inherited ossification: traumatic HO (tHO) and fibrodysplasia ossificans progressiva (FOP). Nuclear transcription factor kappa B (NF-κB) signalling is essential during HO. NF-κB signalling can drive initial inflammation through interactions with the NOD-like receptor protein 3 (NLRP3) inflammasome, Sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK). In the chondrogenesis stage, NF-κB signalling can promote chondrogenesis through interactions with mechanistic target of rapamycin (mTOR), phosphatidylinositol-3-kinase (PI3K)/AKT (protein kinase B, PKB) and other molecules, including R-spondin 2 (Rspo2) and SRY-box 9 (Sox9). NF-κB expression can modulate osteoblast differentiation by upregulating secreted protein acidic and rich in cysteine (SPARC) and interacting with mTOR signalling, bone morphogenetic protein (BMP) signalling or integrin-mediated signalling under stretch stimulation in the final osteogenic stage. In FOP, mutated ACVR1-induced NF-κB signalling exacerbates inflammation in macrophages and can promote chondrogenesis and osteogenesis in mesenchymal stem cells (MSCs) through interactions with smad signalling and mTOR signalling. This review summarizes the molecular mechanism of NF-κB signalling during HO and highlights potential therapeutics for treating HO.

Sex as a Critical Variable in Basic and Pre-Clinical Studies of Fibrodysplasia Ossificans Progressiva.

Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.

The HIF-1α and mTOR Pathways Amplify Heterotopic Ossification.

Fibrodysplasia ossificans progressiva (FOP; MIM# 135100) is an ultra-rare congenital disorder caused by gain-of-function point mutations in the Activin receptor A type I (ACVR1, also known as ALK2) gene. FOP is characterized by episodic heterotopic ossification (HO) in skeletal muscles, tendons, ligaments, or other soft tissues that progressively causes irreversible loss of mobility. FOP mutations cause mild ligand-independent constitutive activation as well as ligand-dependent bone morphogenetic protein (BMP) pathway hypersensitivity of mutant ACVR1. BMP signaling is also a key pathway for mediating acquired HO. However, HO is a highly complex biological process involving multiple interacting signaling pathways. Among them, the hypoxia-inducible factor (HIF) and mechanistic target of rapamycin (mTOR) pathways are intimately involved in both genetic and acquired HO formation. HIF-1α inhibition or mTOR inhibition reduces HO formation in mouse models of FOP or acquired HO in part by de-amplifying the BMP pathway signaling. Here, we review the recent progress on the mechanisms of the HIF-1α and mTOR pathways in the amplification of HO lesions and discuss the future directions and strategies to translate the targeting of HIF-1α and the mTOR pathways into clinical interventions for FOP and other forms of HO.

3D-Printed Model in Preoperative Planning of Sciatic Nerve Decompression Because of Heterotopic Ossification: A Case Report.

CASE: A 31-year-old patient presented with an encapsulated sciatic nerve secondary to extensive hip heterotopic ossification (HO), which prevented visualization of a safe osteotomy site to avoid nerve damage. The 3D-printed model demonstrated an easily identifiable osseous reference point along the inferior aspect of the heterotopic mass, allowing for a vertical osteotomy to be safely performed. CONCLUSION: HO is associated with loss of normal anatomic topography. The current case report illustrates the use of a 3D-printed model to identify pertinent anatomic landmarks required for safe decompression of an encapsulated sciatic nerve within the anatomic region of the hip.