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BackgroundDegeneration of intervertebral disc is a major cause of lower back pain and neck pain. Studies have tried to unveil the regulatory network using either transcriptomic or proteomic analysis. However, neither have fully elucidated the exact mechanism of degeneration process. Since post-transcriptional regulation may affect gene expression by modulating the translational process of mRNA to protein product, a combined transcriptomic and proteomic study may provide more insight into the key regulatory network of Intervertebral disc degeneration.MethodsIn order to obtain the proteomic and transcriptomic data, we performed label-free proteome analysis on freshly isolated nucleus pulposus cells and obtained transcriptome profiling data from the Gene Expression Omnibus repository. To identify the key regulatory network of intervertebral disc degeneration in nucleus pulposus cells, we performed bioinformatic analyses and established a protein-RNA interacting network. To validate the candidate genes, we performed in vitro experimentation and immunochemistry labeling to identify their potential function during nucleus pulposus degeneration.ResultsThe label-free proteome analysis identified altogether 656 proteins, and 503 of which were differentially expressed between nucleus pulposus cells from degenerated or normal disc cells. Using the existing nucleus pulposus transcriptomic profiling data, we integrated the proteomic and transcriptomic data of nucleus pulposus cells, and established a protein-RNA interacting network to show the combined regulatory network of intervertebral disc degeneration. In the network, we found 9 genes showed significant changes, and 6 of which (CHI3L1, KRT19, COL6A2, DPT, TNFAIP6 and COL11A2) showed concordant changes in both protein and mRNA level. Further functional analysis showed these candidates can significantly affect the degeneration of the nucleus pulposus cell when altering their expression.ConclusionsThis study is the first to use combined analysis of proteomic and transcriptomic profiling data to identify novel regulatory network of nucleus pulposus cells in intervertebral disc degeneration. Our established protein-RNA interacting network demonstrated novel regulatory mechanisms and key genes that may play vital roles in the pathogenesis of intervertebral disc degeneration.

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.

BACKGROUND: The surgical outcomes of individual patient with ossification of the posterior longitudinal ligament (OPLL) can vary depending on various patient-related factors. Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ) is a well-developed tool for outcome measurement and considers both disease-specific and general health aspects. This study aimed to investigate the reliability, validity, and responsiveness of the JOACMEQ in patients with OPLL in mainland China and to compare post-operative outcomes of OPLL patients between mainland China and Japan. METHODS: This multicenter trial was performed between July 2009 and June 2019. The procedure for the JOACMEQ translation followed Beaton’s guidelines. All patients enrolled were diagnosed with OPLL and had completed the JOACMEQ, the modified Japanese Orthopaedic Association (mJOA) scale, and the 36-Item Short Form Health Survey (SF-36) before and after surgery. The reliability (Cronbach’s α and Pearson’s correlation), construct validity (factor analysis), concurrent validity (Spearman’s correlation with SF-36) and responsiveness (effect sizes) of JOACMEQ were evaluated. A mixed-model analytic approach was used to analyze differences in postoperative outcomes between the 2 countries. RESULTS: Ninety-one patients from mainland China and ninety-one patients from Japan were recruited. JOACMEQ showed satisfactory internal consistency (Cronbach’s α=0.75). In test-retest reliability evaluation, except for the bladder function domain, the JOACMEQ domains had good test-retest reliability (0.89–0.96). In factor analysis, most of the items (19/24) were well clustered. Regarding clinical validity, all 5 domains were found to have moderate correlations with the physical component summary (PCS) of SF-36 (r=0.25–0.50), and the bladder function and quality of life domains also had moderate correlations (r=0.25–0.50) with the mental component summary (MCS) of SF-36. JOACMEQ showed a variable responsiveness in different domains (effect size =0.17–0.84; standardized response means =0.15–0.85). Regarding postoperative improvements in the JOACMEQ score, mixed-model analysis revealed a significant difference in the quality of life domain between Chinese and Japanese patients (16.0±18.7 vs. 7.8±17.7, P

Study Design A retrospective observational study. Objective To describe a novel outcome indication system, the posterior compression score (PCS), and investigate its clinical value in cervical ossification of the posterior longitudinal ligament (OPLL) patients treated with laminoplasty. Methods A total of 282 OPLL patients who underwent laminoplasty from January 2013 to December 2018 were reviewed. The patients were divided into high-score (HS) or low-score (LS) groups based on whether the PCS was over 8. Propensity score matching analysis with a caliper of .1 was used to attenuate the potential selection bias. Clinical measurements, including the Japanese Orthopedic Association (JOA) score, visual analog scale (VAS), neck disability index (NDI), and radiological measurements, including C2–C7 lordotic angle and range of motion (ROM), were compared between the groups. Results The mean follow-up period was 29.87 ± 9.17 months. There were no significant differences between the two groups regarding patients’ baseline demographical and clinical characteristics after propensity score matching. No significant differences were found in the operative time, blood loss, postoperative VAS score for neck and arm pain, postoperative C2–C7 lordotic angle, or postoperative ROM ( P > .05). However, the postoperative JOA score and recovery rate were significantly higher in the HS group than in the LS group, while the postoperative NDI was significantly lower in the HS group ( P < .05). Conclusion OPLL patients with higher PCS scores displayed better clinical outcomes. The novel PCS system is suggested to be a reliable scoring system for surgical outcome evaluation in patients with cervical OPLL.

The three-grade classification of increased signal intensity (ISI) on T2-weighted magnetic resonance imaging (MRI) is used extensively in patients with cervical compressive myelopathy (CCM). However, the efficacy and value in the prediction of this classification are still unclear and no systematic review and meta-analysis have been conducted on this topic. The objective of this study is to investigate the efficacy and value in prediction of the three-grade classification of ISI on the severity of myelopathy and surgical outcomes. Randomized or non-randomized controlled studies using three-grade classification of ISI (grade 0, none; grade 1, light or obscure; and grade 2, intense or bright) in patients with CCM were sought in the following databases: PubMed, Embase, and Cochrane Library. The pooled Japanese Orthopedic Association (JOA)/modified JOA (mJOA) score, neuro-functional recovery rate, C2-C7 lordotic angle, and range of motion (ROM) were calculated. A total of 8 studies containing 1101 patients were included in this review. Patients in grade 0 had the highest preoperative and postoperative JOA/mJOA score and recovery rate, while those parameters for patients in grade 2 were the lowest. Nevertheless, no statistically significant difference was found regarding the preoperative C2-C7 lordotic angle and ROM among three grades. Our meta-analysis suggests that the three-grade classification of ISI on T2-weighted MRI can reflect the severity of myelopathy and surgical outcomes in patients with CCM. The higher ISI grade indicates more severe myelopathy and surgical outcomes. Overall, the three-grade classification of ISI is instructive and should be used universally.

To investigate whether different quantitative measurements of increased signal intensity (ISI) on T2-weighted magnetic resonance imaging are useful for predicting surgical outcome in patients with cervical spondylotic myelopathy (CSM).We retrospectively reviewed 108 patients with ISI who underwent surgery for CSM. Clinical outcome was evaluated via the Japanese Orthopaedic Association (JOA) score and the recovery rate. Patients were divided into 2 groups based on the JOA recovery rate: good (≥50%) and fair (50%). The quantitative measurements of ISI included the maximum vertical length and the signal change ratio (SCR). C2-C7 sagittal alignment, C2-C7 sagittal vertical axis, C2-C7 range of motion, maximum spinal cord compression, and maximum canal compromise also were assessed.There were no significant differences in cervical sagittal alignment parameters between the groups. Multivariate analysis showed that a longer duration of symptoms, a lower preoperative JOA score, a longer preoperative ISI length, and a greater preoperative SCR were negatively associated with clinical outcomes. Receiver operating characteristic curve analysis showed that the best cutoff values of ISI length and SCR as negative predictors of surgical outcome were 15.50 mm and 1.56, respectively, and the areas under the receiver operating characteristic curve of preoperative ISI length, SCR that discriminate recovery rate of JOA score (≥50%,50%) were 0.8507, 0.8422, respectively, and was 0.8903 for a combination of the 2.Duration of symptoms, preoperative JOA score, preoperative ISI length, and SCR can reflect surgical outcome in patients with CSM; however, cervical sagittal alignment may not affect surgical outcome. Combining ISI length and SCR to depict ISI on T2-weighted magnetic resonance imaging is optimal and accurate. Patients with ISI length15.50 mm and SCR1.56 have fair surgical recovery.

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.

A reader alerted the journal to duplicated images in Fig. 2C and Fig. 4D of Development (2019) 146, [dev175547][1] ([doi:10.1242/dev.175547][2]). In the top panel of Fig. 4D, the bottom part of Stage I and the top part of Stage II-III images show an overlap. This is permissible since

Loss of chondroitin sulfate (CS) has been reported to play a key role during intervertebral disc degeneration (IDD). However, the detailed mechanism of CS and its synthases have not been elucidated. Since CS is mainly synthesized by chondroitin synthases 3 (Chsy3), here, the Chsy3 knockout mice are generated by using CRISPR-Cas9 and semi-cloning technology to study its mechanism during IDD. We find that CS and Chsy3 expression are decreased during IDD both in human and mice nucleus pulposus (NP) tissue, and knockout of Chsy3 shows that spontaneous IDD phenotype resembles that of human samples in the Chsy3-/- mice. Taking advantage of RNA-Seq data, we confirm increased catabolic and decreased anabolic changes in Chsy3-/- NP cells. By using bioinformatic analysis and validation, we find that Hippo signaling pathway is significantly downregulated, and the activation of Yap1 is mainly affected in Chsy3-/- NP cells. Furthermore, functional analyses have shown that Chsy3 could regulate NP cell degeneration by Actin tension mediated activation of Yap1, which is independent of Hippo/Lats signaling. In summary, our findings reveal a novel mechanism that depletion of CS-related Chsy3 can cause spontaneous intervertebral disc degeneration by mediating Yap activation through CS-related actin-tension in NP cells.

Intervertebral disc degeneration (IDD) is the main cause of low back pain and the mechanism of which is far from fully revealed. Although inflammation directed nucleus pulposus (NP) extracellular matrix metabolism dysregulation is known to be the main cause of the degeneration process, few is known about the protective factors. Using high-throughput label-free proteomics, we found that inflammation-related autocrine factor Chitinase-3-like protein 1 (CHI3L1, or YKL-40) is highly expressed in the NP cells during degeneration. Immunohistochemical analysis show that the expression of CHI3L1 is NP tissue specific, and increase significantly during degeneration. Overexpression of CHI3L1 significantly decrease the catabolism, and increase the anabolism of extracellular matrix. Knockdown of CHI3L1 using siRNAs show the opposite results, which imply that the protective role of CHI3L1 in IDD. Using high-throughput RNA sequencing and functional analyses, we find that AKT3 expression and its phosphorylation is mainly regulated by CHI3L1. And lastly, the mechanism of which is also validated using human and mouse degenerated NP tissues. In summary, our findings show that the inflammation-related autocrine factor CHI3L1 is NP specific, and it protects IDD by promoting the AKT3 signaling, which may serve as a potential therapeutic target in intervertebral disc degeneration.

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2 and its RING domain, a histone writer, can ubiquitinate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitinate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

To compare the clinical and radiologic outcomes of 3 anterior surgical techniques for the treatment of 3-level cervical spondylotic myelopathy (CSM) and the evolution of intramedullary T2-weighted increased signal intensity (ISI).A total of 98 consecutive patients (61 males, 37 females) with 3-level CSM who underwent anterior cervical surgery between January 2006 and January 2016 were retrospectively enrolled. Based on different anterior reconstructive techniques, the patients were divided into 3 groups: anterior cervical discectomy and fusion (ACDF), anterior cervical corpectomy and fusion (ACCF), and hybrid decompression and fusion (HDF) groups. The Japanese Orthopaedic Association score and its recovery rate were used to evaluate the clinical outcomes. The cervical alignment and range of motion were used to assess radiologic outcomes. In addition, the signal change ratio and length of ISI were used to measure the ISI.No statistical differences in preoperative factors were found among the 3 groups (P0.05). Although the HDF group had intermediate surgery time and blood loss compared with other groups (P0.05), it achieved greater postoperative Japanese Orthopaedic Association score and recovery rate than other groups (P0.05). The postoperative C2-C7 lordotic angle and postoperative range of motion in the HDF group were similar to that in the ACDF group (P0.05), both greater than that in the ACCF group (P0.05). The incidence of complications in the HDF group was close to the ACDF group (P0.05), and both groups were lower than that in the ACCF group (P0.05). In addition, the postoperative signal change ratio in the HDF group was lower than in other groups (P0.05). The postoperative length of ISI in the HDF group was similar to the ACCF group (P0.05), which was both shorter than that in the ACDF group (P0.05).For patients with 3-level CSM with ISI on T2-weighted MRI, HDF can be considered as the optimal technique that achieves better clinical and radiologic outcomes than the ACDF or ACCF procedure. HDF also has a better postoperative regression of ISI compared with the ACDF or ACCF procedure, which may potentially be an important indicator for improving surgical outcomes.

To investigate preoperative factors affecting the presence of intramedullary increased signal intensity (ISI) on T2-weighted magnetic resonance imaging (MRI) in patients with cervical spondylotic myelopathy (CSM) and the impact of ISI on clinical manifestations.Eighty-nine patients with CSM were retrospectively reviewed from January 2013 to December 2016 in our hospital. Based on the presence or absence of ISI on axial and sagittal T2-weighted MRI, patients were divided into ISI group (48 cases) and non-ISI group (41 cases). Factors such as age, sex, body mass index (BMI), duration of symptoms, clinical symptoms and signs, number and distribution of spinal cord compression levels, preoperative Japanese Orthopedic Association (JOA) score, preoperative C2-C7 lordotic angle, preoperative C2-C7 range of motion (ROM), maximal canal compromise (MCC) and maximal spinal cord compression (MSCC) were initially compared using univariate analysis. Factors with significant result in univariate analysis were included in multivariate logistic regression analysis. Receiver operating characteristic (ROC) curve and the area under the curve (AUC) were applied to evaluate the reliability of multivariate logistic regression model.Univariate analysis showed that the number of spinal cord compression levels, preoperative JOA score, MCC and MSCC might be related to the presence of ISI (P 0.05). Furthermore, multivariate logistic regression analysis revealed that the number of spinal cord compression levels (OR = 0.203, P 0.05), preoperative JOA score (OR = 4.274, P 0.05) and MSCC (OR = 0.250, P 0.05) were independent preoperative risk factors associated with the presence of ISI, yielding an AUC of 0.9558. Patients with ISI showed a trend of increasing clinical symptoms and signs, and also exhibited statistically significantly increased frequencies of clumsy hands, lower limb spasticity, impairment of gait, broad-based, unstable gait, weakness and motor deficits (P 0.05).Multilevel spinal cord compression, lower preoperative JOA score and greater MSCC are independent preoperative risk factors related to the presence of ISI on T2-weighted MRI in patients with CSM. Patients with ISI tend to have more clinical symptoms and signs, especially in lower limb manifestations and motor deficits.

To investigate clinical and radiologic results of anterior cervical discectomy and fusion for cervical spondylotic myelopathy in elderly patients with T2-weighted increased signal intensity (ISI), focusing specifically on the quantitative analysis of ISI.We retrospectively reviewed 88 patients with cervical spondylotic myelopathy with ISI who underwent anterior cervical discectomy and fusion with a minimum 1-year follow-up. Patients were divided into 2 groups: patients older than 65 (elderly group, 36 patients) or younger (young group, 52 patients). The Japanese Orthopaedic Association (JOA) score was used to evaluate the neurologic status. The signal change ratio (SCR) was defined as the grayscale of ISI region divided by that at C7-T1 disc level. The C2-C7 sagittal alignment, range of motion, SCR, and ISI length were measured.There was no statistically significant difference between the 2 groups in C2-C7 sagittal alignment and range of motion. However, the JOA score at 1-year follow-up and recovery rate in elderly group were significantly lower than in young group (P0.001). SCR and ISI length were significantly greater in elderly group than in young group, whereas their changes were significantly lower in elderly group (P0.05). Multivariate logistic regression analysis showed that an older age, a lower preoperative JOA score, a greater preoperative SCR, and a longer preoperative ISI length at 1-year follow-up were negatively correlated with the clinical outcomes in the elderly group (P0.05).Compared with young patients with ISI, the elderly patients had a lower preoperative JOA score, a greater preoperative SCR, and a longer preoperative ISI length, indicating poor surgical outcomes.