Ossification Of Spinal Ligaments Research Articles

Coagulation, Vascular Morphology, and Vasculogenesis in Spinal Ligament Ossification Model Mice.

In vivo studies of the vascular system in ossification of the posterior longitudinal ligament (OPLL) model mice. The aim of this study was to investigate blood coagulability, vascular morphology, and vasculogenesis capability, known as venous thromboembolism (VTE) risk factors in the ossification model, tiptoe walking (ttw) mice. Patients with OPLL are more likely to develop VTE after spinal cord injury. Capillary mesh invasion of spinal ligaments precedes spinal ligament ossification in ttw mice. Investigation on vascular systems of ttw mice may contribute to clarifying its pathology. Coagulability of blood samples from ttw and C57BL/6 (WT) mice were evaluated at 8, 16, and 24 weeks of age. Vascular morphology was assessed from a Hematoxylin-Eosin stained section by measuring vessel area. A tube formation assay was performed with endothelial cells isolated from the aorta to assess vasculogenesis. Prothrombin time was significantly shorter in ttw mice than in WT at 8 and 16 weeks. Fibrinogen had a greater increase in ttw mice than in WT at 16 weeks. The vascular area and vascular wall area were significantly smaller in ttw mice than in WT at all timepoints. The ratio of vascular wall area to vascular area was significantly smaller in ttw mice than in WT at 24 weeks. The endothelial cells from ttw mice formed significantly higher numbers of total branching points than WT cells. Ossification model mice had impaired blood coagulation and vascular morphology and high capacity for vasculogenesis. With regard to the pathogenesis of VTE, ttw mice harbor an environment that promotes the development of VTE.Level of Evidence: N/A.

Diffuse idiopathic skeletal hyperostosis (DISH) – A case report

Diffuse idiopathic skeletal hyperostosis is a non inflammatory systemic disease affecting axial skeleton which results in ossification and calcification of spinal ligaments and entheses. The pathophysiology of the disease is poorly understood. The closest differential diagnosis for DISH remains ankylosing spondylitis. Further research is necessary to treat the disease and to improve the prognosis and quality of life of patients affected with DISH. This article reports a patient with DISH and its management in a tertiary care hospital. Keywords: Forestier’s disease, Diffuse idiopathic skeletal hyperostosis, Calcification, Ossification, Anterior longitudinal ligament, Candle wax, Ankylosing spondylitis, Syndesmophytes.

ADAM10 promotes the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway.

Ligamentum flavum hypertrophy (LFH) is an important cause of spinal canal stenosis and posterior longitudinal ligament ossification. Although a number of studies have focused on the mechanisms responsible for LFH, the cellular mechanisms remain poorly understood. The aim of the present study was to investigate the roles of differentially expressed genes (DEGs) in LFH, elucidate the mechanisms responsible for LFH and provide a potential therapeutic target for further studies. The GSE113212 dataset was downloaded from the Gene Expression Omnibus (GEO) database. The microarray data were analyzed and DEGs were obtained. Bioinformatics methods, such as Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein-protein interaction (PPI) network analyses were used to obtain the key genes and signaling pathways. In addition, cells derived from hypertrophied ligamentum flavum were cultured, and the key genes and signaling pathways in ligamentum cells were identified through in vitro cell biology and molecular biology experiments. A total of 2,123 genes were screened as DEGs. Among these DEGs, 1,384 genes were upregulated and 739 genes were downregulated. The KEGG pathway analysis revealed that the DEGs were mainly enriched in the PI3K/AKT signaling pathway, and the PPI network analysis screened A disintegrin and metalloproteinase 10 (ADAM10) as a key gene. In vitro experimental verification revealed that ADAM10 promoted the proliferation of ligamentum flavum cells and led to the hypertrophy of the ligamentum by activating the PI3K/AKT pathway. On the whole, the in vitro experimental results suggested that ADAM10 promoted the proliferation of ligamentum flavum cells by activating the PI3K/AKT pathway, which may represent a pathogenic mechanism of LFH. The findings of the present study may provide a basis and direction for further studies on the cellular mechanisms of LFH and present a potential novel therapeutic target and clinical approach.

P1446DIFFUSE IDIOPATHIC SKELETAL HYPEROSTOSIS IN PATIENTS UNDERGOING MAINTENANCE HEMODIALYSIS

Abstract Background and Aims Diffuse idiopathic skeletal hyperostosis (DISH) is an idiopathic degenerative disease characterized by ossification of spinal ligaments and entheses, especially in the anterior longitudinal ligament. The ankylosed spine is prone to fracture with minor trauma and may result in spinal cord injury. It is usually asymptomatic and often diagnosed incidentally by computed tomography or other imaging methods. Several reports have revealed DISH as a risk factor for stroke, cardiovascular disease, and metabolic disorders, such as diabetes mellitus, hyperinsulinemia, obesity, dyslipidemia, and hyperuricemia. However, there are few reports of DISH in patients with end stage kidney disease (ESKD) requiring hemodialysis. Epidemiology, etiology and risk factors of DISH in patients with ESKD are unknown. We aimed to examine the prevalence and predictors of DISH in maintenance hemodialysis patients. Method This was a case-control study using patients who received maintenance hemodialysis in our hospital in December 2018. The prevalence of DISH at enrollment was evaluated according to Resnick and Niwayama criteria by computed tomography (CT). The criteria is defined as follows: (1) Flowing ossification along the anterolateral aspect of at least four contiguous vertebral bodies, (2) Relative preservation of intervertebral disc height in the involved segment, (3) Absence of apophyseal joint bony ankylosis and sacroiliac joint erosion. We excluded patients who could not receive chest CT or with a history of thoracic spine surgery. Fisher exact tests were used to evaluate the relationships between categorical variables, and Kruskal-Wallis or Mann-Whitney U tests were used to evaluate continuous variables. We performed logistic regression analyses to identify variables that had a statistically significant association with DISH, as measured by the estimated odds ratio (OR) with the 95% confidence interval (CI). Results A total of 395 consecutive patients were treated with maintenance hemodialysis in our hospital in December 2018. After excluding 4 patients who could not receive chest CT and 2 patients with a history of thoracic spine surgery, the remaining 389 patients were analyzed. The median age was 70 years, and 61.2% of patients were men. One hundred and twenty-three (31.6%) patients were diagnosed with DISH. We assigned them to the DISH group and other 266 patients to the control group. The patients with DISH were significantly more likely to have advanced age, male sex, high body weight, high bone mineral density, prior vertebral fracture, and diabetic nephropathy. Multivariate analysis showed that having DISH was significantly associated with advanced age (OR 1.06, 95%CI 1.03-1.09, P<0.001), high bone mineral density (OR1.02, 95%CI 1.00-1.03, P=0.03), prior vertebral fracture (OR3.22, 95%CI 1.23-8.41, P=0.017), diabetic nephropathy (OR 2.00, 1.03-3.90, P=0.041). Conclusion The prevalence of DISH in maintenance hemodialysis patients was high. Advanced age, male sex, high body weight, high bone mineral density, prior vertebral fracture, and diabetic nephropathy were identified as a risk factor for DISH. Physicians involved in hemodialysis treatment should be aware of the existence of DISH as one of the comorbidities in the elderly ESKD patients.

Functional characterization of a unique mutant of ALK2, p.K400E, that is associated with a skeletal disorder, diffuse idiopathic skeletal hyperostosis.

Bone morphogenetic protein (BMP) signaling regulates the physiological and pathological development of skeletal tissues. Activin receptor-like kinase 2 (ALK2) is a BMP type I transmembrane serine/threonine kinase receptor. Recently, a p.K400E mutation was found in ALK2 in a patient with diffuse idiopathic skeletal hyperostosis (DISH), which is a disorder characterized by calcification and ossification of spinal ligaments and entheses. We report here the functional characterization of ALK2 p.K400E in vitro. Cells overexpressing ALK2 p.K400E activated BMP signaling in response to osteogenic BMP ligands. However, ALK2 p.K400E was not activated by a nonosteogenic ligand, Activin A. BMP signaling through ALK2 p.K400E was further enhanced by the coexpression of a BMP type II receptor. The type II receptor increased the phosphorylation level of ALK2 p.K400E, suggesting that ALK2 p.K400E is a hypersensitive mutant to the BMP type II receptor kinases. Our findings suggest that pathological calcification and ossification in DISH are caused by overactivated BMP signaling through ALK2 p.K400E enhanced by type II receptors in response to osteogenic BMPs rather than Activin A.

Effects of Aging on Impairment Ratings: Part 4: Musculoskeletal Issues

Abstract This article is the fourth of five in a series on the effects of age-related changes in impairment evaluations as defined by the AMA Guides to the Evaluation of Permanent Impairment (AMA Guides), Fifth and Sixth Editions. The present article addresses the musculoskeletal system and differs from the first three articles, which focused on apportionment of an impairment rating between aging and other causes. The medical literature supports the notion that age-related osteoarthritis (OA) changes in the hand and digits frequently are associated with injury and/or repetitive motion. Thus, apportionment is indicated, but deciding which came first, the imaging abnormality or the injury, requires consummate skill on behalf of the rating physician. OA also occurs in the knees and hips of older individuals. Diffuse idiopathic skeletal hyperostosis (DISH) is a noninflammatory disorder characterized by calcification and ossification of spinal ligaments and entheses and is unique, in the authors’ opinion, because of a positive correlation between aging and back pain caused by this condition. The article also addresses the association—or the lack thereof—between pathology and aging, as well as degenerative changes and symptoms, to facilitate causation analysis. For a fuller discussion of causation analysis for the spine, readers can consult the AMA Guides to the Evaluation of Disease and Injury Causation, Second Edition.

Progress in animal models of ossification of spinal ligaments

Ossification of spinal ligament (OSL) is a common clinical disease associated with many factors while its etiology is unknown. It is characterized by heterotopic ossification of spinal ligament, which leaded to narrowing of spinal canal diameter, followed by spinal cord and nerve root lesions in varying degrees, including ossification of posterior longitudinal ligament (OPLL) and ossification of flavum ligament (OLF). At present, surgical intervention of ossification of spinal ligament is the only effective treatment method, while the clinical effect is not very satisfactory. Therefore, understanding the etiology and pathogenesis of the ossification of the spinal ligament has important clinical significance for the prevention and treatment of ossification of the ligament. The pathogenesis of ossification of spinal ligament is complex. An ideal animal model is of great significance in studying its etiology and pathogenesis. In this paper, the modeling methods of various animal models of spinal ligament ossification in recent years were reviewed, which could provide reference for the selection of animal models for future experiments.

Clinical characteristics of patients with thoracic myelopathy caused by ossification of the posterior longitudinal ligament.

Although ossification of the posterior longitudinal ligament (OPLL) commonly develops in the cervical spine, it also occurs, albeit less commonly, in the thoracic spine. However, data are scarce regarding the characteristics of patients with thoracic OPLL. In the current study, we performed a cross-sectional study on a total of 133 patients with OPLL to clarify the clinical characteristics of patients with thoracic OPLL compared with those of patients with cervical OPLL. The subjects were divided into four groups according to the main region of OPLL and treatment type: C-OPLL-C, cervical OPLL treated conservatively; C-OPLL-S, cervical OPLL treated via surgery; T-OPLL-C, thoracic OPLL treated conservatively; and T-OPLL-S, thoracic OPLL treated via surgery. Symptoms developed at an earlier age in the T-OPLL-S group than in the C-OPLL groups. Current body mass index (BMI), maximum lifetime BMI, and BMI at the age of 20years were significantly higher in the T-OPLL-S group than in the C-OPLL groups. Yearly weight gain from the age of 20years to the age at which maximum body weight was attained was significantly greater in the T-OPLL-S group than in the C-OPLL groups. The T-OPLL group showed a higher rate of co-existence of OPLL at other regions or ossification of the ligamentum flavum compared to the C-OPLL groups. Our findings demonstrate that severe obesity, early-onset of symptoms, and diffuse ossification of spinal ligaments are distinct features of patients with myelopathy caused by thoracic OPLL.

Molecular characterization of known and novel ACVR1 variants in phenotypes of aberrant ossification.

Diffuse idiopathic skeletal hyperostosis (DISH) is a disorder principally characterized by calcification and ossification of spinal ligaments and entheses. Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disabling disorder characterized by progressive ossification of skeletal muscle, fascia, tendons, and ligaments. These conditions manifest phenotypic overlap in the ossification of tendons and ligaments. We describe herein a patient with DISH, exhibiting heterotopic ossification of the posterior longitudinal ligament where clinical whole exome sequencing identified a variant within ACVR1, a gene implicated in FOP. This variant, p.K400E, is a novel variant, not identified previously, and occurs in a highly conserved region across orthologs. We used sequence-based predicative algorithms, molecular modeling, and molecular dynamics simulations, to test the potential for p.K400E to alter the structure and dynamics of ACVR1. We applied the same modeling and simulation methods to established FOP variants, to identify the detailed effects that they have on the ACVR1 protein, as well as to act as positive controls against which the effects of p.K400E could be evaluated. Our in silico molecular analyses support p.K400E as altering the behavior of ACVR1. In addition, functional testing to measure the effect of this variant on BMP-pSMAD 1/5/8 target genes was carried out which revealed this variant to cause increased ID1 and Msx2 expression compared with the wild-type receptor. This analysis supports the potential for the variant of uncertain significance to contribute to the patient's phenotype.

Facilitation of Chemotaxis Activity of Mesenchymal Stem Cells via Stromal Cell-Derived Factor-1 and Its Receptor May Promote Ectopic Ossification of Human Spinal Ligaments.

Mesenchymal stem cells (MSCs) have been used to elucidate the pathogenesis of numerous diseases. Our recent study showed that MSCs may conduce to the ossification of spinal ligaments. Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) regulate MSC migration. Moreover, their expression is elevated in sites of damage and remodeling in pathologic states. We explored the possible role of the SDF-1/CXCR4 axis in the chemotactic behavior of MSCs in the ossification of spinal ligaments. Specimens of thoracic vertebra ossified ligamentum flavum (OLF) and non-OLF plaques were received from patients in whom we had performed spine surgery. Paraffin-embedded tissue sections were prepared for immunohistochemical staining. Cultured MSCs from the ligamentum flavum were prepared for in vitro analyses. We observed SDF-1 and CXCR4 localization immunohistochemically in the perivascular area and collagenous matrix of ligaments and in chondrocytes near the ossification front of OLF. And then, immunohistochemical staining showed a close relationship between MSCs and the SDF-1/CXCR4 axis. In the in vitro analyses, expression of the SDF-1/CXCR4 and the migratory capacity of MSCs in OLF were remarkably higher compared with non-OLF MSCs. Furthermore, the migration of MSCs was upregulated by SDF-1 and downregulated by treatment with AMD3100 (C28H54N88HCl), a specific antagonist for CXCR4. All in vitro test data showed a significant difference in MSCs from OLF compared with non-OLF MSCs. Our results reveal that the SDF-1/CXCR4 axis may contribute to an MSC-mediated increase in the ossification process, indicating that the SDF-1/CXCR4 axis may become a potential target for a novel therapeutic strategy for ossification of spinal ligaments.

Angiopoietin-2 promotes osteogenic differentiation of thoracic ligamentum flavum cells via modulating the Notch signaling pathway.

Thoracic ossification of the ligamentum flavum (TOLF) is heterotopic ossification of spinal ligaments, which may cause serious thoracic spinal canal stenosis and myelopathy. However, the underlying etiology remains inadequately understood. In this study, the ossification patterns of TOLF were analyzed by micro-computer tomography (micro-CT). The expression profile of genes associated with angiogenesis was analyzed in thoracic ligamentum flavum cells at sites of different patterns of ossification using RNA sequencing. Significant differences in the expression profile of several genes were identified from Gene Ontology (GO) analysis. Angiopoietin-2 (ANGPT2) was significantly up-regulated in primary thoracic ligamentum flavum cells during osteogenic differentiation. To address the effect of ANGPT2 on Notch signaling and osteogenesis, ANGPT2 stimulation increased the expression of Notch2 and osteogenic markers of primary thoracic ligamentum flavum cells of immature ossification, while inhibition of ANGPT2 exhibited opposite effect on Notch pathway and osteogenesis of cells of mature ossification. These findings provide the first evidence for positive regulation of ANGPT2 on osteogenic differentiation in human thoracic ligamentum flavum cells via modulating the Notch signaling pathway.

Dysphagia Due to Diffuse Idiopathic Skeletal Hyperostosis

Diffuse Idiopathic Skeletal Hyperostosis (DISH) affects up to 20% of the elderly population, with a greater prevalence in men. It is characterized by the non-inflammatory ossification of spinal ligaments and soft tissues. Most patients are asymptomatic, but the developing osteophytes may cause esophageal compression and DISH should be considered in evaluation of dysphagia. Our case illustrates the importance of maintaining a high index of suspicion, and the difficulty in resolving dysphagia secondary to DISH. A 68-year-old man referred for persistent dysphagia reported solid dysphagia up to 85% of the time with occasional regurgitation. He complained of no odynophagia, dyspepsia, or heartburn. Physical exam was grossly normal. The patient had a history of hepatic steatosis, well controlled gastroesophageal reflux disease, diabetes mellitus, hyperlipidemia, and hypertension. An esophagram revealed large anterior bridging osteophytes from C3-C7, which were effacing portions of the hypopharynx and upper esophagus. The osseous findings were consistent with a diagnosis of DISH. Of note, a CT scan from five years ago had identified osteophytes in the thoracic and lumbar spine. Esophagogastroduodenoscopy (EGD) was remarkable for severe extrinsic stenosis of the upper third of the esophagus. Dilation was performed without subsequent symptom improvement. The patient was counseled regarding possible surgical options but declined further intervention. Over the next eight months the patient continued to report dysphagia prompting two more upper endoscopies with dilation, each failing to provide symptom resolution. A repeat esophagram revealed 40-50% narrowing of the cervical esophagus of about 10 cm in length. Aspiration of barium contrast was also noted. Chest computed tomography with contrast revealed only a small hiatal hernia and hypertrophic spondylosis of the dorsal spine. This patient with DISH experienced dysphagia secondary to osseous esophageal compression. Esophageal dilation is not an effective treatment for dysphagia secondary to DISH. Surgical resection of the osteophytes may be considered, although sufficient studies are lacking regarding outcomes of surgical management. In conclusion, DISH is surprisingly common and should be considered more often in the evaluation of dysphagia in elderly patients. This disorder will be encountered with increasing frequency as the geriatric population expands.2973_A Figure 1. CT showing anterior osteophyte formation (arrows) with narrowing of the oropharynx (Image from similar case)2973_B Figure 2. Barium swallow radiogram showing obstruction opposite C5 (arrow) and DISH bridging C4-C6 (Image from similar case)

Co-existence of ossification of the nuchal ligament is associated with severity of ossification in the whole spine in patients with cervical ossification of the posterior longitudinal ligament -A multi-center CT study-

BackgroundPrevious studies have shown that patients with cervical ossification of the posterior longitudinal ligament (OPLL) often have co-existing ossification of the nuchal ligament (ONL). However, no studies have focused on ONL and its relevance to the severity of OPLL or ossification of other spinal ligaments, such as anterior longitudinal ligament (OALL), ligamentum flavum (OLF), and supraspinous/interspinous ligament (OSIL). MethodsIn this multicenter study, we investigated ossification of the spinal ligaments in the whole spine computed tomography (CT) images of 233 cervical OPLL patients. The severity of ossification was evaluated using ossification index for each spinal ligament, calculated as the sum of the level of ossification. We compared the severity of ossification in each spinal ligament between patients with ONL and those without ONL. Furthermore, we investigated how the number of segments, where ONL exists, affects the severity of ossification in each spinal ligament. ResultsOne hundred thirty patients (55.8%) had co-existing ONL in the cervical OPLL patients included in this study. The ONL (+) group included more male and aged patients. The cervical ossification indexes of OPLL and OALL were higher in ONL (+) patients than in ONL (−) patients. The thoracolumbar ossification indexes of OALL and OSIL were also higher in ONL (+) patients. Logistic regression analysis revealed that age, gender and cervical OA-index were independent factors correlating to the existence of ONL. In the cervical spine, both the ossification indexes of OALL and OPLL increased as the levels of ONL increased. Similarly, in the thoracolumbar spine, both the ossification indexes of OALL and OSIL were increased as the levels of cervical ONL increased. In the multiple regression analysis, cervical OA-index and thoracolumbar OSI-index showed significant correlation with the number of ONL levels. ConclusionsCo-existence of ONL in cervical OPLL patients was associated with the severity of spinal hyperostosis especially in cervical OPLL, OALL, thoracolumbar OALL and OSIL.

Skeletal evidence of Diffuse Idiopathic Skeletal Hyperostosis (DISH) in a collective burial from Byzantine Greece

The paper reports on a collective burial from a 13th c. AD cist grave in Attica, Greece. The grave was located inside a basilica and held the remains of at least ten adults. Bone representation analysis showed secondary manipulation of previous deceased including long bone selection for reburial in the same grave and/or bones transported from a different burial place. Paleopathological analysis used macroscopy and radiology, and revealed several lesions on the axial and appendicular skeleton expressed mainly by spinal ligament ossification and multiple peripheral enthesopathies. Individuation of the remains pointed to a middle-aged male with DISH, a condition often correlated to high social rank. Byzantine period is marked by increasing development and prosperity in Greece including among others the creation of many local monastic centers. Although the precise social and professional profile of these individuals cannot be revealed, the combined investigation of skeletal and archaeological evidence suggests that the grave gathered the remains of individuals belonging to an upper class social group.

MiR-490-3p inhibits osteogenic differentiation in thoracic ligamentum flavum cells by targeting FOXO1.

Thoracic ossification of the ligamentum flavum (TOLF) is a rare heterotopic ossification of spinal ligaments, which is the major cause of thoracic spinal canal stenosis and myelopathy. In this study, the roles of miR-490-3p and forkhead box O1 (FOXO1) in osteogenesis of human thoracic ligamentum flavum cells were investigated. MiR-490-3p was found to be down-regulated during osteogenic differentiation of thoracic ligamentum flavum cells, while their overexpression inhibited osteogenic differentiation. In addition, the analysis of target prediction and dual luciferase reporter assays supported that miR-490-3p directly targeted FOXO1 and suppressed the expression of FOXO1. Moreover, FOXO1 knockdown was displayed to attenuate the effect of miR-490-3p inhibition. ChIP assays showed that miR-490-3p negatively regulated the interaction of FOXO1 and RUNX2. These findings suggest that miR-490-3p performs an inhibitory role in osteogenic differentiation of thoracic ligamentum flavum cells by potentially targeting FOXO1.

Drug therapy targeting pyrophosphate slows the ossification of spinal ligaments in twy mice.

The lack of an effective drug therapy against ossification of spinal ligament (OSL) warrants investigation into the therapeutic target of this disease. An endogenous inhibitor of biomineralization, pyrophosphate (PPi) is a potential therapy for ectopic ossification; however, exogenous PPi is rapidly hydrolyzed by tissue non-specific alkaline phosphatase (TNAP) present in body fluids. In this study, we examined whether a drug therapy targeting PPi is efficacious for the treatment of OSL using the Enpp1ttw/ttw (twy) mouse model. Twenty male twy mice were randomized into four groups: (i) vehicle (Control); (ii) alkaline phosphatase inhibitor levamisole (5 mg/kg/day sc continuously); (iii) levamisole + exogenous PPi (160 µmol/kg/day sc continuously); and (iv) nuclear retinoic acid receptor-γ (RARγ) agonist (6 µg/kg sc daily). The RARγ agonist, which is a proven inhibitor of ectopic endochondral ossification, was used as a positive control. Treatments commenced when the mice were 5 weeks of age and continued for 4 weeks. Longitudinal micro-computed tomography and postmortem histological analysis were performed. Administration of levamisole alone and in combination with PPi increased serum PPi concentration by 17% and 52%, respectively, compared to that in vehicle-treated mice. The development of OSL in twy mice was suppressed by levamisole + PPi and RARγ agonist treatments, but not by levamisole alone. The levamisole + PPi therapy did not cause osteoporosis, whereas RARγ agonist-treated mice developed osteoporosis. Treatment of twy mice with levamisole in combination with exogenous PPi increased serum PPi level, which slowed the progression of OSL without producing adverse effect on bone. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1256-1261, 2018.

MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1.

Ossification of the ligamentum flavum (OLF) is a disease of heterotopic ossification in spinal ligaments. The key of the OLF pathogenesis is the differentiation of fibroblasts into osteoblasts. In this study, we explored the role of miR-615-3p in the osteogenic differentiation of human LF cells. The expression of miR-615-3p was detected during the osteogenic differentiation of hFOB1.19 human osteoblasts, human BMSCs, and human LF cells. The qPCR results showed that miR-615-3p was being decreased during the osteogenic differentiation of these cell lineages. Then, both gain- and loss-function experiments, respectively performed by single-strand miR-615-3p mimic and antagomir, revealed that miR-615-3p negatively regulated the osteogenesis of hLF cells, manifested by a lighter staining degree with Alizarin Red and a decreased level of osteogenic marker genes, including alkaline phosphatase (ALP), RUNX2, osterix (ostx), osteocalcin (OCN), and osteopontin (OPN). Subsequently, our data on bioinformatic analysis, 3'-UTR luciferase activity assay, and protein level detection indicated that miR-615-3p directly targeted and suppressed the expression of FOXO1 and GDF5. Furthermore, knockdown of either FOXO1 or GDF5 could inhibit the osteogenic differentiation of hLF cells, which displayed a similar effect with the miR-615-3p mimic. In conclusion, miR-615-3p negatively regulates the osteogenic differentiation of hLF cells through post-transcriptionally suppressing osteogenic regulators GDF5 and FOXO1. It can be regarded as a potential target for human OLF therapy.

Prevalence, Concomitance, and Distribution of Ossification of the Spinal Ligaments: Results of Whole Spine CT Scans in 1500 Japanese Patients.

Cross-sectional study. To investigate the prevalence, concomitance, and distribution of various types of ossification of the spinal ligaments in healthy subjects using computed tomography (CT). CT has better diagnostic accuracy for ossification of the spinal ligaments compared to plain radiography. Currently there is no study that examines the prevalence of ossification of the spinal ligaments using whole spine CT scans. One thousand five hundred Japanese patients (888 men and 612 women) who underwent positron emission tomography and CT (PETCT) in a private health check center between 2006 and 2013 were included. This PETCT was performed on self-paying participants as a preventive cancer screen. Existence of ossification of the posterior longitudinal ligament (OPLL), ligamentum flavum (OLF), anterior longitudinal ligament (OALL), diffuse idiopathic skeletal hyperostosis (DISH), and nuchal ligament (ONL) was examined. The prevalence of spinal ligament ossifications was found to be 6.3% in cervical OPLL (8.3% in men and 3.4% in women), 23% in ONL (33% in men and 8.8% in women), 1.6% in thoracic OPLL (1.4% in men and 2.0% in women), 12% in thoracic OLF (15% in men and 7.7% in women), 37% in thoracolumbar OALL (45% in men and 26% in women), and 12% in DISH (16% in men and 6.2% in women). Thirteen percent of patients with cervical OPLL had thoracic OPLL, 34% of cervical OPLL had thoracic OLF, 45% of cervical OPLL had ONL, and 36% of cervical OPLL had DISH. The most common level was C5 for cervical OPLL, T1/2 for thoracic OPLL, T11 for thoracic OLF, and T8/9 for OALL. Accurate prevalence of various types of ossification of the spinal ligaments evaluated by CT was revealed. High concomitance was observed in each classification of spinal ligament ossification. 3.

Ossification of the posterior ligament is mediated by osterix via inhibition of the β-catenin signaling pathway

Ossification of the posterior longitudinal ligament (OPLL) involves ectopic calcification of the spinal ligament preferentially at the cervical spine. OPLL is associated with different diseases and occurs by endochondral ossification, which is associated with the activity of different transcription factors. However, the pathogenesis of OPLL remains unclear. Here, we investigated the role of osterix (Osx), a transcription factor that functions downstream of Runx2 and is an important regulator of osteogenesis, in the process of OPLL in a dexamethasone (Dex)-induced model of spinal ligament ossification. Our results showed that Osx is upregulated in patients with OPLL and during the ossification of ligament cells in parallel with the upregulation of osteogenic markers including osteocalcin (OCN), alkaline phosphatase (ALP) and collagen-1 (Col-1). Dex-induced ossification of ligament cells was associated with the downregulation and inactivation of β-catenin, and these effects were offset by Osx knockdown. Activation of β-catenin signaling abolished the effect of Dex on ossification and the upregulation of osteogenic markers. Taken together, our results suggest that OPLL is mediated by Osx via a mechanism involving the Wnt/β-catenin signaling pathway, providing a basis for further research to identify potential targets for the treatment of OPLL.

MiR-132-3p Regulates the Osteogenic Differentiation of Thoracic Ligamentum Flavum Cells by Inhibiting Multiple Osteogenesis-Related Genes.

Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.