A Convolutional Neural Network for Automated Detection of Cervical Ossification of the Posterior Longitudinal Ligament using Magnetic Resonance Imaging.

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: We aimed to develop and validate a convolutional neural network (CNN) model to distinguish between cervical ossification of posterior longitudinal ligament (OPLL) and multilevel degenerative spinal stenosis using Magnetic Resonance Imaging (MRI) and to compare the diagnostic ability with spine surgeons. SUMMARY OF BACKGROUND DATA: Some artificial intelligence models have been applied in spinal image analysis and many of promising results were obtained; however, there was still no study attempted to develop a deep learning model in detecting cervical OPLL using MRI images. MATERIALS AND METHODS: In this retrospective study, 272 cervical OPLL and 412 degenerative patients underwent surgical treatment were enrolled and divided into the training (513 cases) and test dataset (171 cases). CNN models applying ResNet architecture with 34, 50, and 101 layers of residual blocks were constructed and trained with the sagittal MRI images from the training dataset. To evaluate the performance of CNN, the receiver operating characteristic curves of 3 ResNet models were plotted and the area under the curve were calculated on the test dataset. The accuracy, sensitivity, and specificity of the diagnosis by the CNN were calculated and compared with 3 senior spine surgeons. RESULTS: The diagnostic accuracies of our ResNet34, ResNet50, and ResNet101 models were 92.98%, 95.32%, and 97.66%, respectively; the area under the curve of receiver operating characteristic curves of these models were 0.914, 0.942, and 0.971, respectively. The accuracies and specificities of ResNet50 and ResNet101 models were significantly higher than all spine surgeons; for the sensitivity, ResNet101 model achieved better values than that of the 2 surgeons. CONCLUSION: The performance of our ResNet model in differentiating cervical OPLL from degenerative spinal stenosis using MRI is promising, better results were achieved with more layers of residual blocks applied.

Anterior direct decompression significantly relieves spinal cord high signal in patients with ossification of the posterior longitudinal ligament: a case-control study.

BACKGROUND: In patients with cervical spondylotic myelopathy caused by ossification of the posterior longitudinal ligament, high cord signal (HCS) is frequently observed. However, limited research has investigated the variations in HCS improvement resulting from different surgical approaches. This study aims to explore the potential relationship between the choice of surgical approach and the postoperative improvement of intramedullary high signal in ossification of the posterior longitudinal ligament (OPLL) patients. METHODS: We extensively reviewed the patients' medical records, based on which demographic information such as gender, age, and body mass index (BMI) were recorded, and assessed the severity of the patients' neurological status preoperatively and postoperatively by using the Japanese Orthopedic Association score (JOAs), focusing on consecutive preoperative and postoperative Magnetic resonance imaging (MRI) T2WI measurements, to study the statistical correlation between the improvement of HCS and the choice of surgical approach. RESULTS: There were no significant differences in demographic, imaging parameters, and clinical symptoms between patients undergoing anterior and posterior surgery (p > 0.05, Table 1). However, both improvement in JOAs (Recovery2) and improvement in HCS (CR2) were significantly better in the anterior surgery group two years after surgery (p < 0.05, Table 1). Multifactorial logistic regression analysis revealed that posterior surgery and higher preoperative signal change ratio (SCR) were identified as risk factors for poor HCS improvement at the two-year postoperative period (p < 0.05, Table 2). Table 1 Differences in demographic, imaging parameters, and clinical symptoms in patients with anterior and posterior approach Anterior approach Posterior approach P-Values Demographic data  Sex (male/female) 10/12 6/17 0.175  Age 58.59 ± 5.68 61.43 ± 9.04 0.215  Hypertension 14/8 14/9 0.848  Diabetes 16/6 19/4 0.425  BMI 25.58 ± 4.72 26.95 ± 4.58 0.331  Smoking history 19/3 16/7 0.175 Preoperative measured imaging parameters  Preoperative SCR 1.615 ± 0.369 1.668 ± 0.356 0.623  CR1 0.106 ± 0.125 0.011 ± 0.246 0.08  CNR 0.33 ± 0.073 0.368 ± 0.096 0.15  C2-7 Cobb angle 8.977 ± 10.818 13.862 ± 13.191 0.182  SVA 15.212 ± 8.024 17.46 ± 8.91 0.38  mK-line INT 3.694 ± 3.291 4.527 ± 2.227 0.323 Imaging follow-up  6 months postoperative SCR 1.45 ± 0.44 1.63 ± 0.397 0.149  2 years postoperative SCR 1.26 ± 0.19 1.65 ± 0.35 0.000**  CR2 0.219 ± 0.14 - 0.012 ± 0.237 0.000** Clinical symptoms  Preoperative JOAs 10.64 ± 1.59 10.83 ± 1.47 0.679  6 months postoperative JOAs 11.82 ± 1.37 11.65 ± 1.4 0.69  2 years postoperative JOAs 14.18 ± 1.01 12.52 ± 2.06 0.001**  Recovery1 0.181 ± 0.109 0.128 ± 0.154 0.189  Recovery2 0.536 ± 0.178 0.278 ± 0.307 0.001** *, statistical significance (p < 0.05). **, statistical significance (p < 0.01) BMI = body mass index. SCR = the signal change ratio between the localized high signal and normal spinal cord signal at the C7-T1 levels. CR1 = the regression of high cord signals at 6 months postoperatively (i.e., CR1 = (Preoperative SCR-SCR at 6 months postoperatively)/ Preoperative SCR). CR2 = the regression of high cord signal at 2 years postoperatively (i.e., CR2 = (Preoperative SCR-SCR at 2 years postoperatively)/ Preoperative SCR). CNR = canal narrowing ratio. SVA = sagittal vertical axis. mK-line INT = modified K-line interval. JOAs = Japanese Orthopedic Association score. Recovery1 = degree of JOAs recovery at 6 months postoperatively (i.e., Recover1 = (JOAs at 6 months postoperatively-Preoperative JOAs)/ (17- Preoperative JOAs)). Recovery2 = degree of JOAs recovery at 2 years postoperatively (i.e., Recover2 = (JOAs at 2 years postoperatively-Preoperative JOAs)/ (17-Preoperative JOAs)) Table 2 Linear regression analyses for lower CR2 values 95% CI P value Uni-variable analyses Demographic data  Sex (male/female) - 0.01 0.221 0.924  Age - 0.015 0.003 0.195  Hypertension - 0.071 0.204 0.334  Diabetes - 0.195 0.135 0.716  BMI - 0.375 0.422 0.905  Smoking history - 0.249 0.077 0.295  Surgical approach - 0.349 - 0.113 0.000# Preoperative measured imaging parameters  C2-7 Cobb angle - 0.009 0.002 0.185  SVA - 0.008 0.008 0.995  mK-line INT - 0.043 0.005 0.122  Preoperative SCR 0.092 0.445 0.004#  CR1 0.156 0.784 0.004#  CNR - 0.76 0.844 0.918 Multi-variable analyses  Surgical approach - 0.321 - 0.118 0.000**  Preoperative SCR 0.127 0.41 0.000**  CR1 - 0.018 0.501 0.067 #, variables that achieved a significance level of p < 0.1 in the univariate analysis *statistical significance (p < 0.05). **statistical significance (p < 0.01) BMI = body mass index. SCR = the signal change ratio between the localized high signal and normal spinal cord signal at the C7-T1 levels. CR1 = the regression of high cord signals at 6 months postoperatively (i.e., CR1 = (Preoperative SCR-SCR at 6 months postoperatively)/ Preoperative SCR). CR2 = the regression of high cord signal at 2 years postoperatively (i.e., CR2 = (Preoperative SCR-SCR at 2 years postoperatively)/ Preoperative SCR). CNR = canal narrowing ratio. SVA = sagittal vertical axis. mK-line INT = modified K-line interval CONCLUSIONS: For patients with OPLL-induced cervical spondylotic myelopathy and intramedullary high signal, anterior removal of the ossified posterior longitudinal ligament and direct decompression offer a greater potential for regression of intramedullary high signal. At the same time, this anterior surgical strategy improves clinical neurologic function better than indirect decompression in the posterior approach.

Prevalence and Characteristics of Cervical Ossified Posterior Longitudinal Ligament in the Jewish Population.

BACKGROUND: Numerous studies have demonstrated an association between ethnic identity and the prevalence rate of cervical ossified posterior longitudinal ligament (C-OPLL). To date, its prevalence rate in the Jewish population has not been determined. The aim of this historical prospective study is to evaluate the prevalence and characteristics of C-OPLL in the Jewish population. METHODS: We performed a retrospective evaluation of imaging studies of all adult patients who underwent both cervical computed tomography and magnetic resonance imaging for all clinical indications within a span of 36 months between January 2017 and July 2020 at a single tertiary referral hospital located in central Israel. Identified C-OPLL carriers were interviewed by telephone. All the patients provided informed consent and then were questioned for current symptoms and demographics, including religion, Jewish ethnic identity, birthplace, parental birthplace and ethnic identity, and family history of spinal disorders. RESULTS: Overall, 440 participants were radiographically evaluated. The prevalence of C-OPLL in the Jewish population was 7.5% (33 of 440). The mean age of the C-OPLL carriers was 65.8 years. All the C-OPLL carriers were symptomatic at analysis. The carriers had an increased proportion with a Sephardic Jewish ethnic identity (65.4%), with a significantly high rate of homogeneous parental Jewish identity (92.4%), suggesting a prominent genetic contribution to the development of this condition. CONCLUSIONS: The prevalence of C-OPLL in the Jewish population in central Israel was 7.5%. This rate is significantly higher than that in other previously studied populations. To the best of our knowledge, this is the first study to identify the Jewish population as experiencing an increased prevalence of C-OPLL.

Genetic insights into ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is an intractable disease leading to severe neurological deficits. Its etiology and pathogenesis are primarily unknown. The relationship between OPLL and comorbidities, especially type 2 diabetes (T2D) and high body mass index (BMI), has been the focus of attention; however, no trait has been proven to have a causal relationship. We conducted a meta-analysis of genome-wide association studies (GWASs) using 22,016 Japanese individuals and identified 14 significant loci, 8 of which were previously unreported. We then conducted a gene-based association analysis and a transcriptome-wide Mendelian randomization approach and identified three candidate genes for each. Partitioning heritability enrichment analyses observed significant enrichment of the polygenic signals in the active enhancers of the connective/bone cell group, especially H3K27ac in chondrogenic differentiation cells, as well as the immune/hematopoietic cell group. Single-cell RNA sequencing of Achilles tendon cells from a mouse Achilles tendon ossification model confirmed the expression of genes in GWAS and post-GWAS analyses in mesenchymal and immune cells. Genetic correlations with 96 complex traits showed positive correlations with T2D and BMI and a negative correlation with cerebral aneurysm. Mendelian randomization analysis demonstrated a significant causal effect of increased BMI and high bone mineral density on OPLL. We evaluated the clinical images in detail and classified OPLL into cervical, thoracic, and the other types. GWAS subanalyses identified subtype-specific signals. A polygenic risk score for BMI demonstrated that the effect of BMI was particularly strong in thoracic OPLL. Our study provides genetic insight into the etiology and pathogenesis of OPLL and is expected to serve as a basis for future treatment development.

Modified ACDF Technique for the Treatment of Centrum Focal Ossification of the Posterior Longitudinal Ligament: A Case Report.

BACKGROUND: Anterior cervical discectomy fusion (ACDF) is a surgical procedure used to treat cervical spondylosis with anterior spinal cord compression. However, there are limitations to traditional ACDF and posterior indirect decompression when the anterior source lesion is in the center of the cervical vertebra. CASE PRESENTATION: On June 8, 2022, our department treated a patient with cervical spondylotic myelopathy-whose high posterior longitudinal ligament (OPLL) occupied the central position of the vertebral body-with modified ACDF. The preoperative surgical plan was designed based on the relevant imaging data and assay index. Also, the visual analogue scale (VAS), Japanese Orthopaedic Association (JOA) scores, and imaging parameters of neck pain were recorded and compared. Postoperative imaging data showed that cervical curvature was recovered and spinal canal compression was relieved. The VAS score for neck pain decreased from 7 preoperatively to 1.5 at the last follow-up, while the JOA score increased from 10 preoperatively to 29 at the last follow-up. The volume of the spinal canal was restored. Simultaneously, the patient's extremity muscle strength improved and muscle tension decreased. CONCLUSIONS: Modified ACDF may be an effective surgical method for resolving spinal cord compression in a specific location when bone mineral density is good. We can effectively avoid iatrogenic nerve injury and symptom recurrence by removing the vertebral body and the lesion directly.

Regulatory Mechanism between Ferritin and Mitochondrial Reactive Oxygen Species in Spinal Ligament-Derived Cells from Ossification of Posterior Longitudinal Ligament Patient.

Primary spinal ligament-derived cells (SLDCs) from cervical herniated nucleus pulposus tissue (control, Ctrl) and ossification of the posterior longitudinal ligament (OPLL) tissue of surgical patients were analyzed for pathogenesis elucidation. Here, we found that decreased levels of ferritin and increased levels of alkaline phosphatase (ALP), a bone formation marker, provoked osteogenesis in SLDCs in OPLL. SLDCs from the Ctrl and OPLL groups satisfied the definition of mesenchymal stem/stromal cells. RNA sequencing revealed that oxidative phosphorylation and the citric acid cycle pathway were upregulated in the OPLL group. SLDCs in the OPLL group showed increased mitochondrial mass, increased mitochondrial reactive oxygen species (ROS) production, decreased levels of ROS scavengers including ferritin. ROS and ferritin levels were upregulated and downregulated in a time-dependent manner, and both types of molecules repressed ALP. Osteogenesis was mitigated by apoferritin addition. We propose that enhancing ferritin levels might alleviate osteogenesis in OPLL.

Usefulness of K-line in predicting prognosis of laminoplasty for cervical spondylotic myelopathy.

BACKGROUND: K-line is widely recognized as a useful index for evaluating cervical alignment and the size of the cervical ossification at the posterior longitudinal ligament (OPLL). The purpose of this study was to investigate whether the K-line could be a useful clinical tool for predicting the prognosis of laminoplasty (LP) for cervical spondylotic myelopathy (CSM). METHODS: Adult CSM patients scheduled for cervical LP were recruited for this study. C2-7 angle, local kyphosis angle, and K-line was evaluated by T2-weighted sagittal magnetic resonance imaging (MRI). Clinical findings were evaluated by the JOA score and the recovery rate. Clinical and radiological findings were evaluated preoperation and final follow-up. Patients were grouped into K-line ( +) and K-line (-). Patients with Kline (-) were further divided into two sub-groups: disc type (anterior cord compression due to disc protrusion with kyphosis) and osseous type (due to osseous structure such as osteophyte). RESULTS: Sixty-eight patients were included in the analysis. The recovery rate of K-line (-) group (n = 11,19.4%) was significantly worse than that of K-line ( +) group (n = 57, 50.6%, p<0.05). Among 11 K-line (-) patients, 7 were disc type and 4 were osseous type. Over the period of follow-up, the disc type K-line (-) patients changed to K-line ( +) and showed significantly better recovery rate (27.6%) compared to the osseous type K-line (-) group (5.0%, p < 0.05). CONCLUSION: The present of this study indicate that K-line may have a predictive value for clinical outcome in patients undergoing LP for CSM. K-line (-) of osseous type was worse than k-line (-) of disc type.

Uniaxial cyclic stretch enhances osteogenic differentiation of OPLL-derived primary cells via YAP-Wnt/ß-catenin axis.

The pathogenesis of posterior longitudinal ligament ossification (OPLL) remains inadequately understood. Mechanical stimulation is one of the important pathogenic factors in OPLL. As one of the mechanical stimulation transduction signals, the yes-associated protein (YAP) interacts with the Wnt/ß-catenin signalling pathway, which plays an important role in osteogenic differentiation. This study aimed to demonstrate the role of YAP-Wnt/ß-catenin axis in cell differentiation induced by mechanical stress. Primary cells extracted from posterior longitudinal ligament tissues from OPLL or non-OPLL patients were subjected to sinusoidal uniaxial cyclic stretch (5 %, 0.5 Hz, 3 d). The expression of runt-related transcription factor 2, collagen I, osterix, osteocalcin and alkaline phosphatase were compared between the static and the experimental groups. In addition, the cytoskeleton was detected using phalloidin staining while YAP phosphorylation states and nuclear location were identified using immunofluorescence. The results showed that mechanical stretching loading increased the expression of osteogenic genes and proteins in the OPLL group, while it had no significant effect on the control group. When OPLL cells were stretched, YAP exhibited an obvious nuclear translocation and the Wnt/ß-catenin pathway was activated. Knocking down YAP or ß-catenin could weaken the impact upon osteogenic differentiation induced by mechanical stimulation. YAP-mediated mechanical stimulation promoted osteogenic differentiation of OPLL cells through Wnt/ß-catenin pathway and this progress was independent of the Hippo pathway.

Molecular and Genetic Mechanisms of Spinal Stenosis Formation: Systematic Review.

Spinal stenosis (SS) is a multifactorial polyetiological condition characterized by the narrowing of the spinal canal. This condition is a common source of pain among people over 50 years old. We perform a systematic review of molecular and genetic mechanisms that cause SS. The five main mechanisms of SS were found to be ossification of the posterior longitudinal ligament (OPLL), hypertrophy and ossification of the ligamentum flavum (HLF/OLF), facet joint (FJ) osteoarthritis, herniation of the intervertebral disc (IVD), and achondroplasia. FJ osteoarthritis, OPLL, and HLF/OLFLF/OLF have all been associated with an over-abundance of transforming growth factor beta and genes related to this phenomenon. OPLL has also been associated with increased bone morphogenetic protein 2. FJ osteoarthritis is additionally associated with Wnt/ß-catenin signaling and genes. IVD herniation is associated with collagen type I alpha 1 and 2 gene mutations and subsequent protein dysregulation. Finally, achondroplasia is associated with fibroblast growth factor receptor 3 gene mutations and fibroblast growth factor signaling. Although most publications lack data on a direct relationship between the mutation and SS formation, it is clear that genetics has a direct impact on the formation of any pathology, including SS. Further studies are necessary to understand the genetic and molecular changes associated with SS.

Exosomal miR-140-5p inhibits osteogenesis by targeting IGF1R and regulating the mTOR pathway in ossification of the posterior longitudinal ligament.

BACKGROUND: Ossification of the posterior longitudinal ligament (OPLL) is a disabling disease whose pathogenesis is still unclear, and there are no effective cures or prevention methods. Exosomal miRNA plays an important role in the osteogenesis of ectopic bone. Therefore, we focused on the downregulation of miR-140-5p in OPLL cell-derived exosomes to explore the mechanism by which exosomal miR-140-5p inhibits osteogenesis in OPLL. RESULTS: Exosomes were isolated by differential centrifugation and identified by transmission electron microscopy, nanoparticle tracking analysis, and exosomal markers. Exosomal RNA was extracted to perform miRNA sequencing and disclose the differentially expressed miRNAs, among which miR-140-5p was significantly downregulated. Confocal microscopy was used to trace the exosomal miR-140-5p delivered from OPLL cells to human mesenchymal stem cells (hMSCs). In vitro, we verified that exosomal miR-140-5p inhibited the osteoblast differentiation of hMSCs by targeting IGF1R and suppressing the phosphorylation of the IRS1/PI3K/Akt/mTOR pathway. In vivo, we verified that exosomal miR-140-5p inhibited ectopic bone formation in mice as assessed by micro-CT and immunohistochemistry. CONCLUSIONS: We found that exosomal miR-140-5p could inhibit the osteogenic differentiation of hMSCs by targeting IGF1R and regulating the mTOR pathway, prompting a further potential means of drug treatment and a possible target for molecular therapy of OPLL.

Coexistence of flavum ligament ossification with diffuse idiopathic skeletal hyperostosis in the cervical spine: Review of literature and technical note starting from a rare case.

BACKGROUND: Cervical flavum ligament ossification (C-OLF) is very rare source of myeloradiculopathy. Less than 100 cases have been reported in modern English literature up to 2020. Association between C-OLF and Diffuse Idiopathic Skeletal Hyperostosis (DISH) at cervical level has never been described. METHODS: In this article we performed a systematic review about epidemiology, physiopathology, clinical and surgical management of C-OLF. Moreover, we research its possible association with other cervical spine ligament ossification and in particular with anterior longitudinal ligament ossification. We report a case of 73 years-old woman experiencing mild cervical myeloradiculopathy caused by C6-C7 C-OLF compression and coexistence of DISH at cervico-thoracic level. A brief technical note about intraoperative management of C-OLF has also been described. RESULT: Our research found 81 previous reported case of C-OLF. The coexistence of Posterior longitudinal ligament ossification has been reported in 21.3% of C-OLF case. Conversely, we reported the first case describing the association between DISH and C-OLF. Posterior surgical decompression is the only useful treatment providing good long-term functional outcome. Instrumentation should be tailored according to pre-operative findings. CONCLUSIONS: C-OLF is a rare source of myeloradiculopathy and it may coexists with DISH probably due to alteration in the cervical mechanical stress and tendency of bone formation in patients harboring coexistent ligament ossifications. According to our result, skip en-bloc microsurgical laminectomy is safe and less invasive method to avoid complication and to provide optimal cervical spinal cord and nerve decompression avoiding CSF-leak.

Small extracellular vesicle-mediated miR-320e transmission promotes osteogenesis in OPLL by targeting TAK1.

Ossification of the posterior longitudinal ligament (OPLL) is an emerging spinal disease caused by heterotopic ossification of the posterior longitudinal ligament. The pathological mechanism is poorly understood, which hinders the development of nonsurgical treatments. Here, we set out to explore the function and mechanism of small extracellular vesicles (sEVs) in OPLL. Global miRNA sequencings are performed on sEVs derived from ligament cells of normal and OPLL patients, and we have showed that miR-320e is abundantly expressed in OPLL-derived sEVs compare to other sEVs. Treatment with either sEVs or miR-320e significantly promote the osteoblastic differentiation of normal longitudinal ligament cells and mesenchymal stem cells and inhibit the osteoclastic differentiation of monocytes. Through a mechanistic study, we find that TAK1 is a downstream target of miR-320e, and we further validate these findings in vivo using OPLL model mice. Together, our data demonstrate that OPLL ligament cells secrete ossification-promoting sEVs that contribute to the development of ossification through the miR-320e/TAK1 axis.

Clinical feasibility of MR-generated synthetic CT images of the cervical spine: Diagnostic performance for detection of OPLL and comparison of CT number.

ABSTRACT: We aimed to determine the incremental value of magnetic resonance generated synthetic computed tomography (MRCT), evaluate cervical ossification of the posterior longitudinal ligament (OPLL), and compare the computed tomography (CT) numbers between MRCT and conventional CT.Twenty-two patients who underwent magnetic resonance imaging (MRI) with MRCT protocols and CT were enrolled. MRCT images were generated from 3D-T2-weighted imaging, 3D-pointwise-encoding time reduction with radial acquisition, 3D-T1-Dixon, and 3D-time-of-flight sequences. Two radiologists independently evaluated the presence of OPLL at each cervical spine level during sessions 1 (MRI alone) and 2 (MRI + MRCT). CT was the reference standard for the presence of OPLL. One reader measured the mean CT number of the vertebral body and spinous process at each cervical spine level in the MRCT and CT images.Sensitivity for the detection of OPLL was markedly higher in session 2 (MRI + MRCT) than in session 1 (MRI alone), as measured by both readers (47% vs. 90%, reader 1; 63% vs. 93%, reader 2). The mean CT number of MRCT and CT showed a moderate to strong positive correlation (ρ = .42-.72, P < .001).The combined use of MRCT and MRI showed improved sensitivity for the evaluation of cervical OPLL. The mean CT number of MRCT and CT showed a positive correlation.

Bone marrow mesenchymal stem cell-derived extracellular vesicles containing miR-497-5p inhibit RSPO2 and accelerate OPLL.

AIMS: Muscle and adipose tissue-derived mesenchymal stem cells presented high osteogenic potentials, which modulate osteoblast function through releasing extracellular vesicles (EVs) containing miRNAs. Herein, this study evaluated the function of bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) delivering miR-497-5p in ossification of the posterior longitudinal ligament (OPLL). MAIN METHODS: The expression level of miR-497-5p was validated in ossified posterior longitudinal ligament (PLL) tissues and BMSC-EVs. The uptake of BMSC-EVs by ligament fibroblasts was observed by immunofluorescence. miR-497-5p was overexpressed or downregulated to assess its role in osteogenic differentiation of ligament fibroblasts. Further, an OPLL rat model was established to substantiate the effect of BMSC-EVs enriched with miR-497-5p on OPLL. KEY FINDINGS: Ossified PLL tissues presented with high miR-497-5p expression. PLL fibroblasts were identified to endocytose BMSC-EVs. BMSC-EVs could upregulate miR-497-5p and shuttle it to ligament fibroblasts to accelerate the osteogenic differentiation. miR-497-5p targeted and inversely regulated RSPO2. Then, RSPO2 overexpression activated Wnt/ß-catenin pathway and repressed the osteogenic differentiation of ligament fibroblasts. In vivo experiments further showed that miR-497-5p-containing BMSC-EVs enhanced OPLL through diminishing RSPO2 and inactivating Wnt/ß-catenin pathway. SIGNIFICANCE: BMSC-EVs could deliver miR-497-5p to ligament fibroblasts and modulate RSPO2-mediated Wnt/ß-catenin pathway, thereby accelerating OPLL.

Potential Link between Ossification of Nuchal Ligament and the Risk of Cervical Ossification of Posterior Longitudinal Ligament: Evidence and Clinical Implication from a Meta-Analysis of 8429 Participants.

OBJECTIVE: The aim of the present paper was to evaluate the strength and the magnitude of the association between ossification of the nuchal ligament (ONL) and the risk of cervical ossification of the posterior longitudinal ligament (COPLL) and to determine whether there is a direct association or whether COPLL is a consequence of shared risk factors. METHODS: Medline, Web of Science, Cochrane Library, and Embase databases were searched for studies evaluating the association of COPLL-ONL published before July 2020. Eligible studies were selected based on certain inclusion and exclusion criteria. Two investigators independently conducted the quality assessment and extracted the data, including study designs, countries, patients' age, gender, body mass index (BMI), and the risk of COPLL between individuals with and without ONL. A meta-analysis of homogenous data, a sensitivity analysis, a publication bias assessment, and a subgroup analysis were performed using Stata 12.0 software. RESULTS: A total of 10 cohort studies involving 8429 participants were incorporated into this analysis. Pooled results demonstrated a statistically significant association between the presence of ONL and the increased COPLL risk (odds ratio [OR] 3.84; 95% confidence interval [CI] 2.68-5.52, P < 0.001). Furthermore, subgroup analyses indicated that this association was independent of study design (6.36-fold in case-control studies vs 3.22-fold in cross-sectional studies), sex (6.33-fold in male-female ratio >2.5 vs 2.91-fold in male-female ratio <2.5), age (4.28-fold in age ≥55 years vs 3.45-fold in age <55 years), and BMI (3.88-fold in BMI ≥ 25 kg/m2 vs 2.43-fold in BMI < 25 kg/m2 ), which also indicated that obese, older male patients with ONL had a higher risk of OPLL. Moreover, combined two articles revealed that patients with larger-type ONL had a significantly higher risk of long-segment COPLL compared with controls (OR 1.86; 95% CI 1.41-2.47, P < 0.001). CONCLUSION: This is the first meta-analysis to demonstrate a strong and steady association between ONL and higher risk of COPLL. This association was independent of sex, age, and BMI. Considering that ONL is generally asymptomatic and easily detectable on X-ray, our findings implied that ONL might serve as an early warning sign of the onset of COPLL and provide clinicians an opportunity for early detection and early intervention.

A Novel Scoring System for Ossification of Posterior Longitudinal Ligament of the Cervical Spine Based on Magnetic Resonance Imaging-CSFM Scoring System.

OBJECTIVE: To establish a new scoring system to assess spinal cord compression of ossification of posterior longitudinal ligament (OPLL) of the cervical spine. METHODS: Literature review and expert advice were used to determine variables of the novel CSFM scoring system. The CSFM score included 4 variables: curvature of spinal cord (C), increased signal intensity of spinal cord (S), cerebrospinal fluid imaging (F), and cross-section morphology of spinal cord (M). From June 2015 to June 2018, clinical and imaging data of 387 patients with cervical OPLL were retrospectively analyzed. The 4 variables were measured and recorded. Different scores were assigned based on analysis of the relationship between the variables and the Japanese Orthopaedic Association score. Two spine surgeons scored the patients according to the CSFM score and analyzed the internal consistency and reliability of the CSFM score. RESULTS: The CSFM scoring system consisted of 4 variables, each of which was divided into 4 grades. Each variable was assigned a score of 0-3 according to different grades. The total possible score was 12, and the minimum score was 0. A higher score indicated more severe spinal cord compression. CONCLUSIONS: The CSFM scoring system can effectively reflect the degree of spinal cord compression for cervical OPLL.

Enhanced Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in Ossification of the Posterior Longitudinal Ligament Through Activation of the BMP2-Smad1/5/8 Pathway.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic OPLL. To date, the specific molecular pathogenesis of OPLL has not been clearly elucidated. In this study, bone marrow-derived mesenchymal stem cells obtained from healthy donors (HD-MSCs) and patients with OPLL (OPLL-MSCs) were cultured in osteogenic differentiation medium for 21 days. The osteogenic differentiation capacity was determined by alizarin red S (ARS) and alkaline phosphatase (ALP) assays. Gene expression levels of osteoblastic markers were measured by quantitative reverse transcription-polymerase chain reaction. Protein levels of related genes and the activation of related signaling pathways were measured by western blotting. LDN193189 was used to inhibit the Smad1/5/8 pathway, and small interfering RNA was used to regulate BMP2 expression. Our results showed that the OPLL-MSCs had stronger ARS staining and ALP activity and higher expression of RUNX2, Osterix, and OCN than the HD-MSCs. During osteogenic differentiation, the Smad1/5/8 pathway was overactivated in the OPLL-MSCs, and LDN193189 inhibition reversed the enhanced osteogenic ability of these cells. Besides, BMP2 was upregulated in the OPLL-MSCs. After BMP2 knockdown, the abnormal osteogenic differentiation of OPLL-MSCs was rescued. Thus, abnormal activation of the BMP2-Smad1/5/8 pathway induces enhanced osteogenic differentiation of OPLL-MSCs compared with HD-MSCs. These findings reveal a mechanism of pathological osteogenesis in OPLL and provide a new perspective on inhibiting pathological osteogenesis by regulating BMP2.

Segmental motion at the peak of the ossification foci is independent risk factor except for mal-alignment and thick ossification foci for poor outcome after laminoplasty for cervical ossification of the posterior longitudinal ligament: analyses in patients with positive K-line, lordotic alignment, and lower canal occupying ratio.

PURPOSE: To elucidate the independent preoperative factors that have a significant impact on poor surgical outcome after laminoplasty for K-line (+) ossification of the posterior longitudinal ligament (OPLL). Analyses in K-line (+) patient population can exclude the influence by mal-alignment and thick OPLL, both of which are well known two major factors that have significant impact on clinical outcome. METHODS: The present study included 72 patients (50 male and 22 female) who underwent laminoplasty for K-line (+) cervical OPLL and were followed-up for at least 1 year. Recovery of Japanese Orthopedic Association score (JOA score) for cervical myelopathy was used as the measure of clinical outcome. For radiographic assessment, the type of OPLL, the maximum OPLL occupation ratio, the C2-C7 angle, and the segmental range of motion at the peak of OPLL (segmental ROM) were assessed. To elucidate the factors that are significantly associated with a poor clinical outcome after laminoplasty for K-line (+) OPLL, statistical analyses were conducted. RESULTS: The mean preoperative JOA score was 8.9 points and improved to 12.8 points after surgery. The recovery of JOA score was 47 ± 35%. Stepwise logistic regression following univariate analyses revealed that preoperative segmental ROM at the peak of OPLL is an independent factor associated with a poor outcome (p = 0.04, odds ratio = 1.15). CONCLUSIONS: Large preoperative segmental ROM at the peak of the OPLL is an independent factor that has significant impact on poor surgical outcome after laminoplasty for K-line (+) OPLL.

Expression Analysis of Susceptibility Genes for Ossification of the Posterior Longitudinal Ligament of the Cervical Spine in Human OPLL-related Tissues and a Spinal Hyperostotic Mouse (ttw/ttw).

STUDY DESIGN: Immunohistochemical and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. OBJECTIVE: The aim of this study was to analyze the expression of five susceptibility genes (RSPO2, HAO1, CCDC91, RHPH9, and STK38L) for human ossification of the posterior longitudinal ligaments (OPLL) identified in a genome-wide association study. SUMMARY OF BACKGROUND DATA: Detailed expression and functional studies for the five susceptibility genes are needed to aid in clarification of the etiology and pathogenesis of OPLL. METHODS: Immunostaining, cell culture, and real-time RT-PCR were performed on ossified ligament samples collected during anterior cervical decompression for symptomatic OPLL (n = 39 patients) and on control non-OPLL samples (n = 8 patients). Immunohistochemical analysis in spinal hyperostotic mice (ttw/ttw) (n = 25) was also performed. The sample sections were stained for RSPO2, HAO1, CCDC91, RHPH9, STK38L, Runx2, Sox9, and CD90. The mRNA expression levels of the five susceptibility genes were also analyzed in cultured human OPLL and non-OPLL cells subjected to cyclic tensile strain. RESULTS: Immunoreactivity for RSPO2 and Sox9 was evident in proliferating chondrocytes in human OPLL tissues and ttw/ttw mice. Application of cyclic tensile strain to cultured human OPLL cells resulted in increases in mRNA levels for RSPO2, HAO1, and CCDC91. However, individual differences in expression in human OPLL-related samples were seen. HAO1-positive cells were detected only in 3- to 6-week-old ttw/ttw mice that did not simultaneously express RSPO2-positive samples. CONCLUSION: Among the five susceptibility genes, RSPO2, HAO1, and CCDC91 might be contributory factors in progression of OPLL. RSPO2 may be involved in endochondral ossification, especially in mixed or continuous type OPLL, HAO1 may be an initiation factor for OPLL that is rarely seen in mature human OPLL samples, and CCDC91 may be associated with progression of ossification caused by mechanical stress. These findings provide important insights into the pathogenesis and therapeutic targets for OPLL. LEVEL OF EVIDENCE: N/A.

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/ß-catenin signaling, as indicated by stabilized ß-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.