Development Of Ossification Of The Posterior Longitudinal Ligament Research Articles

Hyperlipidemia, Obesity, and Diabetes, and Risk of Ossification of the Posterior Longitudinal Ligament

Several risk factors of ossification of the posterior longitudinal ligament (OPLL) have been established, including diabetes and obesity. However, the relationship between hyperlipidemia (HLD) and OPLL is incompletely understood. PearlDiver was queried to identify adults with (+) and without (-) HLD, diabetes, and obesity. Comparative analyses were performed on demographics, comorbidities, and OPLL rates before and after matching for age, sex, and comorbidities. Stepwise logistic regression modeling assessing the relationship between HLD and OPLL with the addition of predictor variables was also performed. In total, 31,677 cervical OPLL patients, as well as 170,467 HLD+ and 118,665 HLD-, 168,985 Diabetes+ and 137,966 Diabetes-, and 150,363 Obesity+ and 142,553 Obesity- patients, were examined. Mean age ranged 43.44-59.46years, 54.94-63.12% were females, and mean Charlson Comorbidity Index ranged from 0.06 from 1.53, all higher in those with the comorbidity. Before matching, OPLL rates were higher in those with HLD (HLD+=0.05% vs. HLD-=0.03%, P= 0.005), diabetes (Diabetes+=0.06% vs. Diabetes-=0.02%, P < 0.001), and obesity (Obesity+=0.05% vs. Obesity-=0.02%, P= 0.001). However, after matching by age, sex, and Charlson Comorbidity Index, the associations between the studied comorbidities and OPLL were attenuated (all P > 0.05). Stepwise regression modeling revealed an association between HLD and cervical OPLL that was most impacted by the addition of age (OR=1.95, R2= 0.029 to OR=1.38, R2= 0.075) and obesity (OR=1.21, R2= 0.086 to OR=1.07, R2= 0.111) into the model. Cervical OPLL rates were higher in patients with HLD even after accounting for demographics and comorbidities. HLD may be an independent risk factor for OPLL development.

MRI-based vertebral bone quality score in cervical ossification of the posterior longitudinal ligament: a comparison with cervical spondylotic myelopathy using propensity score matching

BACKGROUNDBone mineral density plays a key role in the assessment of operative instrumentation complications and clinical outcomes. The MRI-based vertebral bone quality (VBQ) score has been introduced as a novel marker of bone quality. However, few studies have investigated the relationship between VBQ score and patients associated with cervical ossification of the posterior longitudinal ligament (OPLL). PURPOSEThe aims of the study were (1) to reveal bone mineral density between cervical OPLL and cervical spondylotic myelopathy (CSM) group by VBQ score, (2) to compare the VBQ score of cervical OPLL between male and female group, (3) to explore the relationship between segmental VBQ scores associated with OPLL. STUDY DESIGNRetrospective cohort study. PATIENT SAMPLEConsecutive series of 425 patients at a single academic institution. OUTCOME MEASURESMRI based measurements of C2–C7 VBQ scores. METHODSPreoperative noncontrast T1-weighted MRIs of the cervical spine was used to measure the VBQ score. The VBQ score was defined as the mean value of the signal intensity of the vertebrae divided by that of the cerebrospinal fluid (CSF) space at the cisterna magna. Patients with cervical OPLL and CSM were matched based on age, sex, body mass index (BMI), comorbidity, medication history, diet habit, smoking, alcohol consumption via propensity score matching (PSM). Normality of each VBQ score was tested by the Shapiro-Wilk test. Wilcoxon's rank-sum test was used to compare matched cohorts. Kruskal-Wallis test was performed to compare the VBQ scores between segments. Multivariate logistic regression analysis was used to evaluate factors associated with the development of cervical OPLL. RESULTSA total of 425 patients were assessed. For final analysis, 135 paired patients were compared between the cervical OPLL and CSM groups, and 22 paired patients were compared between male and female group associated with cervical OPLL. There were no statistically significant differences in age, sex, BMI, comorbidity, medication history, diet habit, smoking, alcohol between the matched cohorts. OPLL group was associated with lower VBQ score compared with CSM group at C3, while there were no differences in VBQ score for the other levels between the two groups. There were no differences between male and female group associated with OPLL in C2–C7 VBQ scores. VBQ scores of cervical OPLL are variable between segments, with significantly lower scores at C6, C7 compared with C1–C5. Multivariate logistic regression analysis showed that BMI was correlated with the development of OPLL (regression coefficient, 0.162; 95% confidence interval, 0.010–0.037). Additional risk factors included hypertension, calcium supple history and smoking. CONCLUSIONSThis study demonstrates that cervical OPLL is associated with lower VBQ score at C3, with no differences for the other levels when compared with CSM derived from measurements on MRI. No differences were found between male and female group associated with OPLL in C2–C7 VBQ scores. Cervical OPLL were found to have smaller VBQ score at C6, C7 compared with C1–C5. Our findings provide new insight for bone density assessment in cervical OPLL patient.

Dyslipidemia as a novel risk for the development of symptomatic ossification of the posterior longitudinal ligament

BACKGROUND CONTEXTObesity and visceral fat have been implicated as potential factors in the pathogenesis of the ossification of the posterior longitudinal ligament (OPLL); the details of the factors involved in OPLL remain unclear. PURPOSEWe aimed to determine the association between dyslipidemia and symptomatic OPLL. STUDY DESIGNSingle institution cross-sectional study. PATIENT SAMPLEData were collected from Japanese patients with OPLL (n=92) who underwent whole-spine computed tomography scanning. Control data (n=246) without any spinal ligament ossification were collected from 627 Japanese participants who underwent physical examination. OUTCOME MEASURESBaseline information and lipid parameters, including triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) from fasting blood samples were collected to assess the comorbidity of dyslipidemia. METHODSPatient data were collected from 2020 to 2022. Patients with dyslipidemia were defined as those who were taking medication for dyslipidemia and who met one of the following criteria: TG ≥150 mg/dL, LDL-C ≥140 mg/dL, and/or HDL-C <40 mg/dL. The factors associated with OPLL development were evaluated using multivariate logistic regression analysis. RESULTSThe comorbidity of dyslipidemia in the OPLL group was more than twice that in the control group (71.7% and 35.4%, respectively). The mean body mass index (BMI) of the OPLL group was significantly higher than that of the control group (27.2 kg/m2 and 23.0 kg/m2). Multivariate logistic regression analysis revealed that dyslipidemia was associated with the development of OPLL (regression coefficient, 0.80; 95% confidence interval, 0.11–1.50). Additional risk factors included age, BMI, and diabetes mellitus. CONCLUSIONSWe demonstrated a novel association between dyslipidemia and symptomatic OPLL development using serum data. This suggests that visceral fat obesity or abnormal lipid metabolism are associated with the mechanisms of onset and exacerbation of OPLL as well as focal mechanical irritation due to being overweight.

Increased Cellular Expression of Interleukin-6 in Patients With Ossification of the Posterior Longitudinal Ligament.

We performed histologic, immunohistochemical, immunoblot examination and suspension array analyses of cytokine expression in cultured cells derived from human cervical ossification of the posterior longitudinal ligament (OPLL). To determine the roles of interleukin-6 (IL-6) during the maturation of osteoblasts and chondrocytes associated with the development of OPLL. Ectopic OPLL affects ~3% of the general population, with a higher incidence in Asian ethnic groups. Alterations in cytokine profiles may influence osteoblast differentiation, but the mechanisms and signaling pathways associated with the ossification process remain unclear. Samples were collected from 14 patients with OPLL who had undergone spinal surgery and seven with cervical spondylotic myelopathy without OPLL. Tissue sections were used for histologic and immunohistochemical studies, and primary cells from ligamentum samples were used for cytokine array and immunoblotting. A suspension array was used to measure the concentrations of 27 inflammatory cytokines or growth factors. Suspension array and immunoblot analyses revealed significantly elevated levels of IL-6 in OPLL patients. Alterations in IL-6 concentrations were found to alter the expression of the genes Sox9 , Runx2 , and SIRT1 . In addition, immunohistochemical analysis revealed that these factors are present in mesenchymal cells within the degenerative portion of the ligament matrix that is adjacent to the ossification front. IL-6 plays a profound role in the osteoblast differentiation process along with the induction of chondrocyte hypertrophy and cell apoptosis in the early stages of ossification in OPLL. These changes in cytokine profiles are essential factors for regulation of the ectopic ossified plaque in OPLL.

Investigation of the biomarkers involved in ectopic ossification: The shared mechanism in ossification of the spinal ligament.

Background: Ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF) are multifactor diseases characterized by progressively ectopic ossification in the spinal ligament. However, the shared ossification mechanism of OPLL and OLF remains to be elucidated. The study aims to investigate the common biomarkers related to ectopic ossification and the potential molecular regulatory mechanism. Methods: Microarray and RNA-seq datasets were obtained from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) from OPLL and OLF were identified to construct the protein-protein interaction (PPI) network. Furthermore, the hub intersection genes were screened and the diagnostic performance was assessed in the external OLF and OPLL cohorts. We also depicted the landscape of immune cell infiltration and m6A modification meanwhile further estimating the relationship with BMP4. Results: A total of nine up-regulated DEGs and 11 down-regulated DEGs were identified to construct the PPI networks. The integrative bioinformatic analysis defined five hub genes (BMP4, ADAMTS4, HBEGF, IL11, and HAS2) as the common risk biomarkers. Among them, BMP4 was the core target. ROC analysis demonstrated a high diagnostic value of the hub genes. Moreover, activated B cells were recognized as shared differential immune infiltrating cells and significantly associated with BMP4 in OPLL and OLF. Meanwhile, a strong correlation was detected between the expression pattern of the m6A regulator METTL3 and BMP4. Conclusion: This study first identified BMP4 as the shared core biomarker in the development of OPLL and OLF. Activated B cells and m6A writer METTL3 might be involved in the osteogenesis process mediated by BMP4. Our findings provide insights into the pathogenesis in the ossification of the spinal ligament and unveil the potential therapeutic targets.

Nature or nurture: a latent ossification of the posterior longitudinal ligament after atlantoaxial fusion. Illustrative case.

The natural history of ossification of the posterior longitudinal ligament (OPLL) remains poorly understood and multiple etiologies have been reported. However, most have focused on the characteristics of the patient rather than alternation of mechanical stress after spinal fusion. This report describes, for the first time, a de novo OPLL found at the subaxial cervical spine 7 years after an atlantoaxial fusion surgery. A 57-year-old female initially required atlantoaxial arthrodesis for os odontoideum and stenosis that caused myelopathy. The posterior fusion surgery went smoothly without complications and the patient had good recovery of neurological functions. There was no associated instability, trauma, or reoperations during the follow-up. Seven years later, the patient presented with slight neck pain and a newly developed OPLL at C3-4 caudal to the C1-2 fusion construct. Conflicting with the conventional concept that OPLL is common in elderly men with genetic or hormonal factors, or associated spondyloarthropathies, OPLL could develop in women even after solid C1-2 fusion. The adjacent subaxial cervical spine is not free of risks for subsequent development of OPLL and cervical spondylotic myelopathy. This case illustration extends the scope of etiologies of OPLL within the present literature.

Hao1 Is Not a Pathogenic Factor for Ectopic Ossifications but Functions to Regulate the TCA Cycle In Vivo.

Ossification of the posterior longitudinal ligament (OPLL), a disease characterized by the ectopic ossification of a spinal ligament, promotes neurological disorders associated with spinal canal stenosis. While blocking ectopic ossification is mandatory to prevent OPLL development and progression, the mechanisms underlying the condition remain unknown. Here we show that expression of hydroxyacid oxidase 1 (Hao1), a gene identified in a previous genome-wide association study (GWAS) as an OPLL-associated candidate gene, specifically and significantly decreased in fibroblasts during osteoblast differentiation. We then newly established Hao1-deficient mice by generating Hao1-flox mice and crossing them with CAG-Cre mice to yield global Hao1-knockout (CAG-Cre/Hao1flox/flox; Hao1 KO) animals. Hao1 KO mice were born normally and exhibited no obvious phenotypes, including growth retardation. Moreover, Hao1 KO mice did not exhibit ectopic ossification or calcification. However, urinary levels of some metabolites of the tricarboxylic acid (TCA) cycle were significantly lower in Hao1 KO compared to control mice based on comprehensive metabolomic analysis. Our data indicate that Hao1 loss does not promote ectopic ossification, but rather that Hao1 functions to regulate the TCA cycle in vivo.

New Development and Progression of Ossification of the Posterior Longitudinal Ligament (OPLL) after Cervical Disc Arthroplasty: A Case Report

Purpose: To report an extremely rare case of new development and progression of ossification of posterior longitudinal ligament (OPLL) after cervical disc arthroplasty (CDA). Background: To our knowledge, new development and progression of ossification of OPLL after CDA have not been reported. Methods: A 44-year-old female with cervical disc herniation at C5-6 presented with radiculopathy. The patient had no evidence of preexisting OPLL on plain radiographs and magnetic resonance imaging. She underwent CDR at C5-6 and her symptoms were significantly improved after surgery. Results: New development of OPLL at C5-7-T1 was identified for the first time at 6 years after CDA at C5-6, and progression of OPLL was observed at 3 &amp; 13 years’ follow-up. Due to new development and progression of OPLL, there was no segmental motion at C5-6 with CDA. The patient continued to follow up without further surgery because there was no deterioration in clinical symptoms. Conclusion: Our study demonstrated an extremely rare case of new development and progression of OPLL after CDA with a long term follow-up. Our case suggests potential clues for discovering the complex pathological mechanism of OPLL. Keywords: Ossification of the posterior longitudinal ligament, New development, Progression, Cervical disc arthroplasty

Cytokine Profile From the Ligamentum Flavum in Patients with Ossification of the Posterior Longitudinal Ligament in the Cervical Spine.

Histological, immunohistochemical, and suspension array analyses of cytokine expression in human cervical ossification of the posterior longitudinal ligament (OPLL). The aim of this study was to determine whether changes in the cytokine profile reflect the maturation of chondrocytes and osteoblasts are associated with OPLL development. OPLL progresses gradually over a prolonged period and may lead to serious spinal cord complications. However, treatment methods only include conservative therapy for neurological symptoms or surgical decompression, whereas preventive therapy for OPLL remains nonexistent. Ligamentous samples were harvested from 24 patients with OPLL who underwent spinal surgery, and five control samples from cervical spondylotic myelo/radiculopathy patients without OPLL. Tissue sections were used for immunohistochemical studies and primary cells were cultured from the ligamentous samples for cytokine profiling. Using a suspension array system, concentrations of 27 inflammatory cytokines or growth factors were measured to generate the cytokine profiles. Suspension array and immunoblot analysis revealed significant increments in the levels of interleukin (IL)-6, IL-1α, basic fibroblast growth factor, and RANTES in patients with OPLL. Immunohistochemical analysis further revealed that these factors were present in mesenchymal cells within the degenerative portion of the ligamentous matrix. Our findings suggest that specific changes in the cytokine profile during ossification promote osteoblast differentiation, thereby providing new insights into OPLL pathogenesis. Moreover, this work supports the development of a new therapeutic method for preventing OPLL progression by regulating the cytokine profiles.Level of Evidence: 3.

Hsa-circ-0007292 promotes the osteogenic differentiation of posterior longitudinal ligament cells via regulating SATB2 by sponging miR-508-3p.

Ossification of the posterior longitudinal ligament (OPLL) is a disorder with multiple pathogenic mechanisms and leads to different degrees of neurological symptoms. Recent studies have revealed that non-coding RNA (ncRNA), including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), could influence the development of OPLL. Nevertheless, the molecular mechanisms linking circular RNAs (circRNAs) and the progression of OPLL is still unknown. The current research explored the expression profiles of OPLL-related circRNAs by microarray analysis, and applied qRT-PCR to validate the results. Subsequently, we confirmed the upregulation of hsa_circ_0007292 in OPLL cells by qRT-PCR and validated the circular characteristic of hsa_circ_0007292 by Sanger sequencing. Fluorescence in situ hybridization (FISH) unveiled that hsa_circ_0007292 was predominantly located in the cytoplasm. Functionally, gain-of-function and loss-of-function experiments showed that hsa_circ_0007292 promoted the osteogenic differentiation of OPLL cells. Mechanistically, the interaction of hsa_circ_0007292 and miR-508-3p was predicted and validated by bioinformatics analysis, dual-luciferase reporter assays, and Ago2 RNA immunoprecipitation (RIP). Similarly, we validated the correlation between miR-508-3p and SATB2. Furthermore, rescue experiments were performed to prove that hsa_circ_0007292 acted as a sponge for miR-508-3p, and SATB2 was revealed to be the target gene of miR-508-3p. In conclusion, our research shows that hsa_circ_0007292 regulates OPLL progression by the miR-508-3p/SATB2 pathway. Our results indicate that hsa_circ_0007292 can be used as a promising therapeutic target for patients with OPLL.

Unveiling the genetic variation of severe continuous/mixed-type ossification of the posterior longitudinal ligament by whole-exome sequencing and bioinformatic analysis

Ossification of the posterior longitudinal ligament (OPLL) in the cervical spine is known as a rare, complex genetic disease, its complexity being partly because OPLL is diagnosed by radiological findings regardless of clinical or genetic evaluations. Although many genes associated with susceptibility have been reported, the exact causative genes are still unknown. We performed an analysis using next-generation sequencing and including only patients with a clear involved phenotype. This was a case control study. A total of 74 patients with severe OPLL and 26 healthy controls were included. Causal single-nucleotide variant (SNV), gene-wise variant burden (GVB), and related pathway METHOD: We consecutively included the severe OPLL patients with continuous-/mixed-type and an occupying ratio of ≥ 40%, and performed whole-exome sequencing (WES) and bioinformatic analysis. Then, a validation test was performed for candidate variations. Participants were divided into 4 groups (rapidly-growing OPLL, growing rate ≥ 2.5%/y; slow-growing, < 2.5%/y; uncertain; and control). WES was performed on samples from 74 patients with OPLL (rapidly-growing, 33 patients; slow-growing, 37; and uncertain, 4) with 26 healthy controls. Analysis of 100 participants identified a newly implicated SNV and 4candidate genes based on GVB. The GVB of CYP4B1 showed a more deleterious score in the OPLL than the control group. Comparison between the rapidly growing OPLL and control groups revealed seven newly identified SNVs. We found significant association for 2 rare missense variants; rs121502220 (odds ratio [OR]=infinite; minor allele frequency [MAF]=0.034) in NLRP1 and rs13980628 (OR= infinite; MAF=0.032) in SSH2. The 3 genes are associated with inflammation control and arthritis, and SSH2 and NLRP1 are also related to vitamin D modulation. Identification of unique variants in novel genes such as CYP4B1 gene may induce the development of OPLL. In subgroup analysis, NLRP1 and SSH2 genes coding inflammation molecules may related with rapidly-growing OPLL.

Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

BackgroundThe molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remain to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy-associated gene expression was correlated with the expression of osteogenic differentiation genes.MethodsExpression of autophagy-associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy-associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy-associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts.ResultsIn clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001), and collagen type 1 (COL 1) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy-associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP, and COL 1 by 63.2% (P < 0.01), 52% (P < 0.01), and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively.ConclusionsBeclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.

Dkk1 acts as a negative regulator in the osteogenic differentiation of the posterior longitudinal ligament cells

Ossification of the posterior longitudinal ligament (OPLL) is a spinal disorder characterized by progressive ectopic bone formation in the PLL of the spine. Dickkopf-1 (Dkk1) is a secreted inhibitor of the Wnt pathway that negatively regulates bone formation during skeletal development. However, whether Dkk1 impacts the pathogenesis of OPLL has not been reported. This study is to investigate the role of Dkk1 in the development of OPLL. Our results show that the serum levels of Dkk1 are decreased in OPLL patients compared with non-OPLL controls. The expression of Dkk1 is also reduced in OPLL ligament cells. Downregulation of Dkk1 in ligament cells is associated with activation of the Wnt/β-catenin signaling, as indicated by stabilized β-catenin and increased T-cell factor-dependent transcriptional activity. Functionally, Dkk1 exerts a growth-inhibitory effect by repressing proliferation but promoting apoptosis of ligament cells. Dkk1 also suppresses bone morphogenetic protein 2-induced entire osteogenic differentiation of ligament cells, and this suppression is mediated via its inhibition of the Wnt pathway. Our results demonstrate for the first time that Dkk1 acts as an important negative regulator in the ossification of the PLL. Targeting the Wnt pathway using Dkk1 may represent a potential therapeutic strategy for the treatment of OPLL.

MicroRNA-181 regulates the development of Ossification of Posterior longitudinal ligament via Epigenetic Modulation by targeting PBX1.

Objectives: Ossification of the posterior longitudinal ligament (OPLL) presents as the development of heterotopic ossification in the posterior longitudinal ligament of the spine. The etiology of OPLL is genetically linked, as shown by its high prevalence in Asian populations. However, the molecular mechanism of the disease remains obscure. In this study, we explored the function and mechanism of OPLL-specific microRNAs.Methods: The expression levels of the ossification-related OPLL-specific miR-181 family were measured in normal or OPLL ligament tissues. The effect of miR-181a on the ossification of normal or pathogenic ligament cells was tested using real-time polymerase chain reaction (PCR), Western blot, alizarin red staining and alkaline phosphatase (ALP) staining. The candidate targets of miR-181 were screened using a dual luciferase reporter assay and functional analysis. The link between miR-181a and its target PBX1 was investigated using chromatin immunoprecipitation, followed by real-time PCR detection. Histological and immunohistochemical analysis as well as micro-CT scanning were used to evaluate the effects of miR-181 and its antagonist using both tip-toe-walking OPLL mice and in vivo bone formation assays.Results: Using bioinformatic analysis, we found that miR-181a-5p is predicted to play important roles in the development of OPLL. Overexpression of miR-181a-5p significantly increased the expression of ossification-related genes, staining level of alizarin red and ALP activity, while the inhibition of miR-181a-5p by treatment with an antagomir had the opposite effects. Functional analysis identified PBX1 as a direct target of miR-181a-5p, and we determined that PBX1 was responsible for miR-181a-5p's osteogenic phenotype. By chromatin immunoprecipitation assay, we found that miR-181a-5p controls ligament cell ossification by regulating PBX1-mediated modulation of histone methylation and acetylation levels in the promoter region of osteogenesis-related genes. Additionally, using an in vivo model, we confirmed that miR-181a-5p can substantially increase the bone formation ability of posterior ligament cells and cause increased osteophyte formation in the cervical spine of tip-toe-walking mice.Conclusions: Our data unveiled the mechanism by which the miR-181a-5p/PBX1 axis functions in the development of OPLL, and it revealed the therapeutic effects of the miR-181a-5p antagomir in preventing OPLL development both in vivo and in vitro. Our work is the first to demonstrate that microRNA perturbation could modulate the development of OPLL through epigenetic regulation.

Long non-coding RNA MALAT1 functions as miR-1 sponge to regulate Connexin 43-mediated ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is the major cause for several deteriorate bone and joint diseases. Its development is a highly organized dynamic process as modulated by various physiological and pathophysiological factors. Both long non-coding RNAs (lncRNAs) and small non-coding RNAs (miRNAs) have been postulated to involve into almost all the biological conditions. Here, we applied high through-put transcriptome screening to unveil lncRNAs highly regulated under OPLL condition. siRNA assay in combination with western blot and quantitative PCR deciphered the lncRNA and miRNA functions in OPLL and their underlying mechanism. Here we identified an lncRNA, named Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) engaged into the development of OPLL by indirectly targeting Connexin 43 (Cx43) gene. As previously reported, Cx43 is one of the main proteins contributing to OPLL partially through enhancing inflammatory signaling. On top of that, we provided another regulatory layer that MALAT1 served as the upstream effector governing the transcription of Cx43 gene. Perturbation of MALAT1 significantly inhibited Cx43 expression, inflammation, and osteogenesis. Mechanistically, in silico analysis and experimental validation both confirmed that microRNA-1 (miR-1) was the mediator connecting MALAT1-Cx43 axis: overexpression of miR-1 diminished Cx43 expression and OPLL process; meanwhile, MALAT1 acted as miR-1 sponge to inhibit its suppressive transcription effect on downstream ossification related genes. Knock-down of MALAT1 released sequestered miR-1, which repressed Cx43 expression and associated OPLL. Likewise, induced OPLL caused by overexpression of MALAT1 can be ameliorated by enhanced miR-1 function, knock-down of Cx43 or inhibition of inflammation. More importantly, further validation using patient ligament samples from non-OPLL and OPLL individuals identified MALAT1-miR-1-Cx43 regulatory axis. Collectively, we found a novel mechanism through lncRNA-miRNA interaction that provides more insights into understanding the development of OPLL.

Ossification of the Posterior Longitudinal Ligament: Analysis of the Role of Craniovertebral and Spinal Instability.

This paper reviews an experience of surgically treating ossification of the posterior longitudinal ligament (OPLL) with fixation of the involved spinal segments alone, without resorting to any bony or soft tissue decompression or attempts at direct resection of the OPLL. While in the early part of the experience, stabilization of only the involved subaxial cervical spinal segments was done, in the later part of the experience, atlantoaxial fixation was included in the multisegmental spinal fixation construct. This treatment is based on the understanding that spinal instability that includes atlantoaxial instability forms the nodal point of the pathogenesis and development of OPLL, and maturation of the presenting clinical symptoms. Twenty-nine patients were treated in this series. There were 28 males and one female, and their ages ranged from 28 to 75years (average 57years). All patients presented with symptoms of neck pain, and progressive and disabling myelopathy-related quadriparesis. In the early part of the series (from 2012 to 2014), 14 patients underwent multilevel subaxial cervical spinal fixation by a transarticular technique of facetal fixation. After November 2014, atlantoaxial lateral mass fixation was included in the fixation construct in the subsequent 15 patients. Clinical assessments were done using a visual analogue scale (VAS), the Japanese Orthopaedic Association (JOA) scale and Goel's clinical grading scale. All patients' clinical symptoms improved in the immediate postoperative period, and the improvement was sustained and progressive in 28 patients. Atlantoaxial and subaxial spinal instability seems to be the nodal pathogenetic factor in OPLL. Only stabilization of spinal segments that includes the atlantoaxial joint can provide a safe, simple and rational form of treatment.

Progression of Ossification of the Posterior Longitudinal Ligament after Cervical Total Disc Replacement.

ObjectiveTotal disc replacement (TDR) is frequently performed anterior approaching method for the patients diagnosed with cervical disc herniation. This study aimed to assess the degree of ossification of the posterior longitudinal ligament (OPLL) progression after cervical TDR.MethodsTwenty-two male soldiers who underwent cervical TDR surgery from 2009 to 2016 and were followed-up for more than 12 months were enrolled. The enrolled patients were classified as; 1) patients with pre-existing OPLL and without; and 2) patient showing progression of OPLL or not.ResultsTwenty-two men were included in the analysis. The mean follow-up period from the surgery was 41.4 months (range, 12–114 months). The mean age of all patients was 40.7 years (range, 31–52 years). TDR-only was used in 7 cases, and the hybrid surgery (TDR+ACDF) was used in 15 cases. The incidence of progression or newly development of OPLL was significantly higher in pre-existing OPLL group (p=0.01). In 11 cases showing the progression of the OPLL, the mean size of OPLL progression was 4.16 mm (range, 0.34–18.87 mm) in the longitudinal height and 1.57 mm (range, 0.54–3.91 mm) in thickness.ConclusionThe progression of OPLL after cervical TDR was more frequent in patients with pre-existing OPLL than in patients without OPLL. Even though TDR is a major alternative to the treatment of cervical lesions to preserve vertebral segmental motion, careful attention should be paid to whether TDR should be used in patients with OPLL and this should be fully explained to the patient.

Ossification of the posterior longitudinal ligament in the cervical spine: a review.

Ossification of the posterior longitudinal ligament (OPLL) is a rare pathologic process of lamellar bone deposition that can result in spinal cord compression. While multiple genetic and environmental factors have been related to the development of OPLL, the pathophysiology remains poorly understood. Asymptomatic patients may be managed conservatively and patients with radiculopathy or myelopathy should be considered for surgical decompression. Multiple studies have demonstrated the morphology and size of the OPLL as well as the cervical alignment have significant implications for the appropriate surgical approach and technique. In this review, we aim to address all the available literature on the etiology, history, presentation, and management of OPLL in an effort to better understand OPLL and give our recommendations for the treatment of patients presenting with OPLL.

The microRNA-10a/ID3/RUNX2 axis modulates the development of Ossification of Posterior Longitudinal Ligament

Ossification of the posterior longitudinal ligament (OPLL) presents as pathological heterotopic ossification of the spinal ligaments. However, its underlying molecular mechanism is still unclear. Our previous findings suggested that altered microRNA regulatory network are critical for the development of OPLL. Here, we set out to unveiling the detailed mechanism of those altered OPLL-specific microRNAs. We screened a set of differentially expressed OPLL-specific microRNAs from the previous sequencing data and showed that microRNA-10a actively modulates the ossification of posterior ligament cells in vitro. Using a tissue-engineered scaffold grown from 4-week-old BALB/c homozygous nude mice, we found that altered microRNA-10a expression in posterior ligament cells indeed affected the heterotopic bone formation in vivo. Furthermore, computational analysis showed that the negative ossification regulator ID3 is a functional target gene of microRNA-10a, and its expression was also significantly altered during microRNA-10a modulation both in vitro and in vivo. Also, we have demonstrated that the ossification promoting function of microRNA-10a requires ID3, as ID3 actively inhibits RUNX2. Thus, we identified a critical role for highly altered OPLL-specific microRNA-10a in regulating the development of OPLL by modulating the ID3/RUNX2 axis.

Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification.

Ossification of the posterior longitudinal ligament (OPLL), a spinal ligament, reduces the range of motion in limbs. No treatment is currently available for OPLL, which is why therapies are urgently needed. OPLL occurs in obesity, is more common in men, and has an onset after 40 years of age. The mechanisms underlying OPLL remain unclear. In this study, we performed a serum proteomic analysis in both OPLL patients and healthy subjects to identify factors potentially involved in the development of OPLL, and found reduced levels of a protein that might underlie the pathology of OPLL. We isolated the protein, determined its amino acid sequence, and identified it as chemokine (C-X-C motif) ligand 7 (CXCL7). Based on these proteomics findings, we generated a CXCL7 knockout mouse model to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossification in the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7 deficiency in OPLL, we searched for single nucleotide polymorphisms and altered DNA exons, but no abnormalities were found. Although miR-340 levels were found to be high in an miRNA array, they were insufficient to reduce CXCL7 levels. Ubiquitin C-terminal hydrolase1 (UCHL1) was found to be overexpressed in CXCL7-null mice and in the sera of patients with OPLL, and was correlated with OPLL severity. Post-translational modifications of proteins with ubiquitin and ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) enzymes, are thought to control a wide range of cellular processes, and alterations in the ubiquitin–proteasome system have been associated with several degenerative disorders. In addition, the OPLL tissue of CXCL7-null mouse and its primary cells expressed the antibody to ubiquitin (linkage-specific K48). Our data clearly show decreased CXCL7 levels in patients with OPLL, and that OPLL developed in mice lacking CXCL7. Tumor necrosis factor receptor-associated factor (TRAF)6 expression was decreased in CXCL7-null mouse primary cells. Furthermore, K48 polyubiquitination was found in posterior longitudinal ligament ossified tissue and primary cells from CXCL7-null mice. We performed a phosphoproteomics analysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase (ME3K)15, ubiquitin protein ligase E3C (UBE3C) and protein kinase C (PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7 deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitination in the onset of OPLL. In conclusion, CXCL7 levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7 levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL.