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.

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.

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.

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.

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.

The roles of autophagy in osteogenic differentiation in rat ligamentum fibroblasts: Evidence and possible implications.

Autophagy, a macromolecular degradation process, plays a pivotal role in cell differentiation and survival. This study was designed to investigate the role of autophagy in the osteogenic differentiation in ligamentum fibroblasts. Rat ligamentum fibroblasts were isolated from the posterior longitudinal ligament and cultured in osteogenic induction medium. Ultrastructural analysis, immunofluorescence assay, western blot, flow cytometry, and lysosomal activity assessment were performed to determine the presence and activity of autophagy in the cells. The mineralization deposit and osteogenic gene expressions were evaluated to classify the association between autophagy activity and the bone formation ability of the spinal ligament cells. The influence of leptin and endothelin-1 on the autophagy activity was also evaluated. Our study demonstrated that autophagy was present and increased in the ligament cells under osteogenic induction. Inhibition of autophagy with either pharmacologic inhibitors (Bafilomycin A and 3-methyladenine) or Belcin1 (BECN1) knocking down weakened the mineralization capacity, decreased the gene expressions of COL1A1, osteocalcin (Ocn), and runt-related transcription factor 2 (Runx2) in the ligamentum fibroblasts and increased cell apoptosis. The Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-BECN1 autophagic pathway was activated in the osteogenic differentiating ligamentum fibroblasts. Leptin significantly increased the autophagy activity in the ligament cells under osteogenic induction. These discoveries might improve our understanding for the mechanism of ossification of the posterior longitudinal ligament (OPLL) and provide new approaches on the prevention and treatment of this not uncommon disease.

Methylation-mediated down-regulation of microRNA-497-195 cluster confers osteogenic differentiation in ossification of the posterior longitudinal ligament of the spine via ADORA2A.

Aberrant expression of microRNAs (miRNAs) has been associated with spinal ossification of the posterior longitudinal ligament (OPLL). Our initial bioinformatic analysis identified differentially expressed ADORA2A in OPLL and its regulatory miRNAs miR-497 and miR-195. Hence, this study was conducted to clarify the functional relevance of miR-497-195 cluster in OPLL, which may implicate in Adenosine A2A (ADORA2A). PLL tissues were collected from OPLL and non-OPLL patients, followed by quantification of miR-497, miR-195 and ADORA2A expression. The expression of miR-497, miR-195 and/or ADORA2A was altered in posterior longitudinal ligament (PLL) cells, which then were stimulated with cyclic mechanical stress (CMS). We validated that ADORA2A was expressed highly, while miR-497 and miR-195 were down-regulated in PLL tissues of OPLL patients. miR-195 and miR-497 expression in CMS-treated PLL cells was restored by a demethylation reagent 5-aza-2'-deoxycytidine (AZA). Moreover, expression of miR-195 and miR-497 was decreased by promoting promoter CpG island methylation. ADORA2A was verified as the target of miR-195 and miR-497. Overexpression of miR-195 and miR-497 diminished expression of osteogenic factors in PLL cells by inactivating the cAMP/PKA signaling pathway via down-regulation of ADORA2A. Collectively, miR-497-195 cluster augments osteogenic differentiation of PLL cells by inhibiting ADORA2A-dependent cAMP/PKA signaling pathway.

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.

Radiographic analysis of the correlation between ossification of the nuchal ligament and sagittal alignment and segmental stability of the cervical spine in patients with cervical spondylotic myelopathy.

BACKGROUND: Ossification of the nuchal ligament (ONL) caused by chronic injury to the nuchal ligament (NL) is very common in instability-related cervical disorders. PURPOSE: To determine possible correlations between ONL, sagittal alignment, and segmental stability of the cervical spine. MATERIAL AND METHODS: Seventy-three patients with cervical spondylotic myelopathy (CSM) and ONL (ONL group) and 118 patients with CSM only (control group) were recruited. Radiographic data included the characteristics of ONL, sagittal alignment and segmental stability, and ossification of the posterior longitudinal ligament (OPLL). We performed comparisons in terms of radiographic parameters between the ONL and control groups. The correlations between ONL size, cervical sagittal alignment, and segmental stability were analyzed. Multivariate logistic regression was used to identify the independent risk factors of the development of ONL. RESULTS: C2-C7 sagittal vertical axis (SVA), T1 slope (T1S), T1S minus cervical lordosis (T1S-CL) on the lateral plain, angular displacement (AD), and horizontal displacement (HD) on the dynamic radiograph increased significantly in the ONL group compared with the control group. The size of ONL significantly correlated with C2-C7 SVA, T1S, AD, and HD. The incidence of ONL was higher in patients with OPLL and segmental instability. Cervical instability, sagittal malalignment, and OPLL were independent predictors of the development of ONL through multivariate analysis. CONCLUSION: Patients with ONL are more likely to have abnormal sagittal alignment and instability of the cervical spine. Thus, increased awareness and appreciation of this often-overlooked radiographic finding is warranted during diagnosis and treatment of instability-related cervical pathologies and injuries.

Serum oxidative stress influences neurological recovery after surgery to treat acutely worsening symptoms of compression myelopathy: a cross-sectional human study.

BACKGROUND: Recent reports indicate that oxidative stress induced by reactive oxygen species is associated with the pathobiology of neurodegenerative disorders that involve neuronal cell apoptosis. Here we conducted a cross-sectional study to evaluate serum levels of oxidative stress in cervical compression myelopathy. METHODS: Thirty-six serum samples were collected preoperatively from patients treated for acutely worsening compression myelopathy (AM) and chronic compression myelopathy (CM). Serum levels of oxidative stress markers were evaluated by measuring derivatives of reactive oxygen metabolites (ROM), which reflect concentrations of hydroperoxides. ROM in healthy individuals range from 250 to 300 (U. CARR), whereas ROM >340-400 and > 400 define moderate and severe levels of oxidative stress, respectively. Difference of ROM by the cause of disorders whether cervical spondylotic myelopathy (CSM) or cervical ossification of longitudinal ligament (OPLL), correlations between ROM and patient age, body mass index (BMI), history of smoking, existence of diabetes were examined. Neurological evaluations according to Japanese Orthopaedic Association (JOA) scores were performed and correlated with ROM. RESULTS: ROM increased to 349.5 ± 54.8, representing a moderate oxidative stress, in CM samples. ROM increased to 409.2 ± 77.9 in AM samples, reflecting severe oxidative stress which were significantly higher than for CM samples (p < 0.05). There was no significant difference by the cause of disorders (CSM or OPLL). ROM were significantly increased in AM serum samples from female patients versus AM male and CM patients (p < 0.05). There were no correlations between ROM and age, BMI, history of smoking, and existence of diabetes. A negative correlation between ROM and recovery rate of JOA score (R2 = 0.454, p = 0.047) was observed in the AM group. CONCLUSIONS: Although moderate oxidative stress was present in patients with CM, levels of oxidative stress increased in severity in patients with AM. These results suggest that postsurgical neurological recovery is influenced by severe oxidative stress in AM.

Epidemiology of ossification of the spinal ligaments and associated factors in the Chinese population: a cross-sectional study of 2000 consecutive individuals.

BACKGROUND: The epidemiology and cause of ossification of the spinal ligaments (OSL) remains obscure. To date, there is no study that comprehensively evaluates the prevalence, distribution, and concomitance of each type of OSL by CT among general Chinese population. We therefore aimed to comprehensively investigate epidemiological characteristics of OSL using whole spine CT in the Chinese population and examine the factors that correlate with the presence of OSL. METHODS: Ossification of the posterior longitudinal ligament (OPLL), ligamentum flavum (OLF), anterior longitudinal ligament (OALL), nuchal ligament (ONL), and diffuse idiopathic skeletal hyperostosis (DISH) were evaluated from the subjects who underwent PET/CT for the purpose of cancer screening in our hospital. Prevalence, distribution, and concomitance of OSL were reviewed. Logistic regression analysis was performed to identify the risk factors of OSL. RESULTS: A total of 2000 subjects (1335 men and 665 women) were included. The prevalence rate of cervical OPLL (C-OPLL) was 4.1%, thoracic OPLL (T-OPLL) 2.25%, lumbar OPLL (L-OPLL) 0.8%, thoracic OLF (T-OLF) 37.65%, lumbar OLF (L-OLF) 1.45%, ONL 31.5%, DISH 3.85%. The most commonly involved level was C5 for C-OPLL, T1 for T-OPLL, T10 for T-OLF, and T8/9 for OALL. 21% of subjects with C-OPLL had T-OPLL, 44% of C-OPLL had T-OLF, 38% of T-OPLL had C-OPLL, 53% of T-OPLL had T-OLF, 44% of L-OPLL had T-OPLL, and 56% of L-OPLL had T-OLF. The average age of OSL-positive subjects was significantly higher than that of OSL-negative subjects. The results of the multiple regression analysis revealed that males had a strong association with DISH (odds ratio, 3.15; 95% confidence interval, 1.27-7.78; P = 0.013). CONCLUSION: The prevalence of OSL in the Chinese was revealed. Tandem ossification is not uncommon in people with OSL. There is a high incidence of multiple-regional OPLL in the whole spine. Approximately half of the subjects with OPLL coexist with T-OLF. For patients with clinical symptoms induced by OPLL, thorough evaluation of whole spine using CT is recommended.

Distribution of ossified spinal lesions in patients with severe ossification of the posterior longitudinal ligament and prediction of ossification at each segment based on the cervical OP index classification: a multicenter study (JOSL CT study).

BACKGROUND: In patients with ossification of the posterior longitudinal ligament (OPLL) in the cervical spine, it is well known that the thoracic ossified lesions often coexist with the cervical lesions and can cause severe myelopathy. However, the prevalence of OPLL at each level of the thoracic and lumbar spinal segments is unknown. The aims of this study were to investigate how often OPLL occurs at each level in the thoracolumbar spine in patients with a radiological diagnosis of cervical OPLL and to identify the spinal levels most likely to develop ossification. METHODS: Data were collected from 20 institutions in Japan. Three hundred and twenty-two patients with a diagnosis of cervical OPLL were included. The OPLL index (OP index), defined as the sum of the vertebral body and intervertebral disc levels where OPLL is present, was used to determine disease severity. An OP index ≥20 was defined as severe OPLL. The prevalence of OPLL at each level of the thoracic and lumbar spinal segments was calculated. RESULTS: Women were more likely to have ossified lesions in the thoracolumbar spine than men. Severe OPLL was significantly more common in women than in men (20% vs. 4.5%). For thoracic vertebral OPLL, the most frequently affected was the T1 segment in both men and women, followed by the T1/2 and T3/4 intervertebral levels in men and women, respectively. Ossified lesions were frequently seen at the intervertebral and vertebral levels around the cervicothoracic and thoracolumbar junctions in men with severe OPLL, whereas OPLL was more diffusely distributed in the thoracic spine in women with severe OPLL. CONCLUSION: Thoracolumbar OPLL occurred most often at T1 in men and at T3/4 in women. In severe OPLL cases, although ossified lesions were frequently seen at the intervertebral and vertebral levels around the cervicothoracic and thoracolumbar junctions in men, OPLL could be observed more diffusely in the thoracic spine in women.

Wnt signaling pathway correlates with ossification of the spinal ligament: A microRNA array and immunohistochemical study.

BACKGROUND: Ossification of the posterior longitudinal ligament or the ligamentum flavum parallels endochondral ossification. Cell differentiation at the ossification front is known to be important during this process, although the factors regulating its initiation and progression are still unclear. The purpose of this study was to identify factors important for the regulation of chondrocyte/osteoblast differentiation during spinal ossification. METHODS: Ligamentum flavum tissue was isolated from 25 patients who underwent decompressive surgery for cervical ossification of the posterior longitudinal ligament. Tissue sections were used for in vitro culture to obtain primary cells through migration methods. To identify microRNAs associated with ossification of the posterior longitudinal ligament, cultured cells were prepared from the ligamentous tissue (n = 4; continuous type) or from control ligamentous samples harvested from patients with cervical spondylosis without spinal ossification, and analyzed using a microRNA array. The ligamentous sections were also examined by immunohistochemistry for the expression of candidate microRNA target genes. RESULTS: The microRNA array identified 177 factors; 12 of which were expressed at significantly different levels in patients with ossification of the posterior longitudinal ligament compared to those in control patients. The hsa-miR-487b-3p was down-regulated in patients with ossification of the posterior longitudinal ligament, which met the false discovery rate of <0.05. This microRNA was predicted to regulate the expression of genes involved in Wnt signaling. Furthermore, immunohistochemistry of Wnt signaling proteins, including Wnt 3a, LRP5/6, and beta-catenin, revealed positive expression in mesenchymal cells and/or premature chondrocytes at the ossification front. CONCLUSION: Our results suggested that down-regulation of miR-487b-3p plays an important role in the initiation of Wnt signaling during the ossification process. Wnt signaling may regulate both chondrocyte and osteoblast differentiation and the specification of endochondral ossification in the pathogenesis of ossification of the posterior longitudinal ligament or the ligamentum flavum.

Ossification of the posterior ligament is mediated by osterix via inhibition of the ß-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 ß-catenin, and these effects were offset by Osx knockdown. Activation of ß-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/ß-catenin signaling pathway, providing a basis for further research to identify potential targets for the treatment of OPLL.

Connexin 43 Affects Osteogenic Differentiation of the Posterior Longitudinal Ligament Cells via Regulation of ERK Activity by Stabilizing Runx2 in Ossification.

AIMS: Connexin 43 is one of the most potent gap junction proteins related to osteoblast differentiation and bone formation. We hypothesized that Connexin 43 is a significant factor in osteogenic differentiation in the posterior longitudinal ligament through the regulation of extracellular signal-regulated kinases (ERK) activity by converging on Runt-related transcription factor 2 (Runx2) activity. In this study, we mapped the activity of Connexin 43 to ERK and Runx2 by extracting longitudinal ligament cell for culture and silencing Connexin expression in addition to dexamethasone treatment in vitro. METHODS: qRT-PCR, Western Blot, and Runx2-responsive Luciferase Reporter Assay were performed to detect the activity of ERK, Runx2 and the expression levels of osseous genes under Connexin 43 modification. RESULTS: Downregulation of Connexin 43 resulted in suppression of dexamethasone-induced osteogenic differentiation, inhibition of the ERK and Runx2 activity, and reduction of osseous gene expression. CONCLUSION: these data support that Connexin 43 significantly regulates osteogenic differentiation in the cells from posterior longitudinal ligament by altering the activity of ERK, and subsequently causing the modification of Runx2.

Connexin 43 promotes ossification of the posterior longitudinal ligament through activation of the ERK1/2 and p38 MAPK pathways.

Although cervical ossification of the posterior longitudinal ligament (OPLL) is one of the most common spinal diseases, the pathogenic mechanism is still not fully understood. Abnormal mechanical stress distribution is believed to be one of the main causes of OPLL. We have previously found that mechanical stress can up-regulate connexin 43 (Cx43) expression in ligament fibroblasts; this transduces mechanical signals to promote osteoblastic differentiation. In the present study, in order to explore further the intracellular mechanisms of Cx43-induced osteoblast differentiation of ligament fibroblasts, we investigate the potential roles of the osteogenic signaling pathway components ERK1/2, p38 MAPK and JNK in Cx43-mediated mechanical signal transduction. We first confirm higher Cx43 levels in both in vivo ligament tissue from OPLL patients and in vitro cultured OPLL cells. We find that ERK1/2, p38 MAPK and the JNK pathway are all activated both in vivo and in vitro. The activation of these signals was dependent upon Cx43, as its knock-down resulted in diminished mechanical effects and reduced signaling. Moreover, its knock-down almost reversed the osteogenic effect of mechanical stress on ligament fibroblasts and the blocking of the ERK1/2 and p38 MAPK pathways but not the JNK pathway, partly diminished this effect. Therefore, Cx43, which is up-regulated by mechanical stress, seems to function partly via the activation of ERK1/2 and p38 MAPK signals to promote the osteoblastic differentiation of ligament fibroblasts.

Ossification of the Posterior Longitudinal Ligament: Imaging Findings in the Era of Cross-Sectional Imaging.

Imaging appearance and classification systems of ossification of the posterior longitudinal ligament (OPLL) on computed tomography and magnetic resonance imaging will be reviewed. Computed tomography evaluation most accurately demonstrates OPLL length and thickness, whereas magnetic resonance imaging has the advantage of demonstrating abnormal signal in the cord. Neurologic symptoms are most common in the cervical spine and are related to the degree of spinal stenosis and presence of cord edema. Surgical treatment usually involves cases of cervical OPLL and includes anterior or posterior decompression.

Implications of different patterns of "double-layer sign" in cervical ossification of the posterior longitudinal ligament.

PURPOSE: To make a preliminary classification of double-layer sign according to the morphological characteristics of the ossified and central hypodense mass and clarify implications of different patterns of "double-layer sign". METHODS: The 268 patients of cervical ossification of the posterior longitudinal ligament (OPLL) who underwent anterior corpectomy were retrospectively analyzed from January 2009 to January 2014. All these patients were performed cervical plain X-rays, CT and MRI. The double-layer sign was observed on axial bone window of CT images. According to the morphological characteristics of the ossified and central hypodense mass, this sign was classified into three types: type A was crescent shape, type B was short-straight shape and type C was long-straight shape. Type A was named when the central hypodense mass traced an arc and the OPLL is much more extensive than the dural ossification (DO). It belonged to type B when the central hypodense mass traced a short-straight line, less than or equal to half of the base width of the vertebrae. The OPLL may be extensive or equal to the DO. Type C was defined when it was more than half of the base width, presenting with a long-straight line and DO is much more extensive than OPLL. Intraoperative findings including dural mater ossification and adhesion, postoperative CSF leakage and outcome were all studied. Two spinal surgeons with rich experiences read the CT images according to this classification method and verified its consistency. RESULTS: Ninety-two patients were found in association with DO during the anterior decompression procedure, meanwhile the double-layer sign could be seen on axial bone window of CT imaging. In these 92 patients, there were 51 patients presenting with type A, and the ossified mass was completely resected with ossified dura mater reserved. Only two patients presented with dural defect and postoperative CSF leakage. Thirty-five patients was classified as type B, of which six accompanied by CSF leakage. All the other six patients of type C presented with CSF leakage after operation. There was a significant correlation between the occurence of CSF leakage and pattern of double-layer sign, but not gender, age, duration of symptoms, extent of OPLL and occupying rate. The Kappa value between the two surgeons was 0.82, showing a good consistency of the method. CONCLUSIONS: OPLL patients with double-layer sign of type C is almost inevitably followed by CSF leakage after anterior decompression. For type A and B, occurrence of CSF leakage is not as high as we thought before. Pattern of double-layer sign should be a considered factor when anterior or posterior approach is chosen.

Factors associated with intramedullary MRI abnormalities in patients with ossification of the posterior longitudinal ligament.

STUDY DESIGN: A retrospective clinical study of 113 patients with ossification of the posterior longitudinal ligament (OPLL), who underwent either anterior or posterior surgery between 2006 and 2009. OBJECTIVE: To evaluate the risk factors affecting the intramedullary spinal cord changes in signal intensity on magnetic resonance imaging (MRI) for the patients with OPLL. SUMMARY OF BACKGROUND DATA: The relationship between the intramedullary spinal cord changes in signal intensity on MRI and neurological deficits, as well as the surgical outcomes, has been described. To obtain better prognosis, early surgery should be conducted in patients with OPLL who have potential abilities to develop intramedullary spinal cord changes in signal intensity on MRI. Various factors may be affecting the development of intramedullary spinal cord changes in signal intensity on MRI. MATERIALS AND METHODS: The clinical and radiographic data of 113 patients with OPLL who underwent either anterior or posterior surgery between 2006 and 2009 were reviewed. Age, sex, complication, mean occupying ratio of OPLL (the greatest thickness of OPLL divided by the anteroposterior diameter of the bony spinal canal), duration of symptoms, type of OPLL, preoperative Japanese Orthopedic Association (JOA) score, and range of motion of the cervical spine were collected. Logistic regression analysis was used. RESULTS: Changes in the intramedullary signal intensity on MRI were observed in 33 of the 113 patients. Statistical results show that duration of symptoms, occupying ratio of OPLL, preoperative JOA score, kyphosis, and instability of the cervical spine are the relevant risk factors for intramedullary spinal cord changes in signal intensity on MRI, with regression coefficients of 2.437, 0.953, -1.952, 2.093, and 1.516, respectively. For patients with OPLL, the longer the duration of the symptoms, or the higher occupying ratio of OPLL, or the lower preoperative JOA score, the greater the likelihood of intramedullary spinal cord changes in signal intensity on MRI. CONCLUSIONS: As intramedullary spinal cord changes in signal intensity on MRI indicated severe damage to spinal cord and poor prognosis as we described before, early surgery is suggested for patients with OPLL who manifest one of the following factors: prolonged symptoms, high occupying ratio, low preoperative JOA score, kyphosis, or instability of the cervical spine. These factors are closely related to the intramedullary spinal cord changes in signal intensity on MRI.

Down-regulated expression of vimentin induced by mechanical stress in fibroblasts derived from patients with ossification of the posterior longitudinal ligament.

PURPOSE: The aim of this study was to investigate the potential role of vimentin in the signal transduction pathways initiated by mechanical stimulation that contribute to ossification of the posterior longitudinal ligament of the spine (OPLL). METHODS: We investigated the effects of in vitro cyclic stretch on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The expression levels of the osteoblast-specific genes encoding osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) were assessed by semi-quantitative reverse transcription-polymerase chain reaction. Vimentin protein expression in OPLL cells was detected by Western blotting. Small interfering RNA (siRNA) interference targeting vimentin was performed in OPLL cells induced by mechanical stress, and the expression levels of OCN, ALP and COL I were assessed. RESULTS: In response to mechanical stretch, the expression levels of OCN, ALP, and COL I were increased in OPLL cells, whereas no change was observed in non-OPLL cells. Furthermore, knockdown of vimentin protein expression by siRNA resulted in an increase in the mRNA expression levels of OCN, ALP, and COL I. CONCLUSION: The down-regulation of vimentin induced by mechanical stress plays an important role in the progression of OPLL through the induction of osteogenic differentiation in OPLL cells.