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Currently, mitochondrial dysfunction caused by oxidative stress is a growing concern in degenerative diseases, notably intervertebral disc degeneration (IVDD). Dysregulation of the balance of mitochondrial quality control (MQC) has been considered the key contributor, while it's still challenging to effectively harmonize different MQC components in a simple and biologically safe way. Hydrogen gas (H2) is a promising mitochondrial therapeutic molecule due to its bio-reductivity and diffusibility across cellular membranes, yet its relationship with MQC regulation remains unknown. Herein, we propose a mitochondrial 'Birth-Death' coordinator achieved by an intelligent hydrogen nanogenerator (Fe@HP-OD), which can sustainably release H2 in response to the unique microenvironment in degenerated IVDs. Both in vitro and in vivo results prove alleviation of cellular oxidative stress and restoration of nucleus pulposus cells function, thereby facilitating successful IVD regeneration. Significantly, this study for the first time proposes the mitochondrial 'Birth-Death' coordination mechanism: 1) attenuation of overactivated mitochondrial 'Death' process (UPRmt and unselective mitophagy); and 2) activation of Adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway for mitochondrial 'Birth-Death' balance (mitochondrial biogenesis and controlled mitophagy). These pioneering findings can fill in the gaps in molecular mechanisms for H2 regulation on MQC homeostasis, and pave the way for future strategies towards restoring equilibrium of MQC system against degenerative diseases.

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Intervertebral disc degeneration (IVDD) may lead to an increase in extracellular matrix (ECM) stiffness, contributing to the progression of the disease. Melatonin reportedly mitigates IVDD; while its potential to attenuate elevated matrix stiffness-induced IVDD remains unexplored. Therefore, we aimed to investigate whether melatonin can alleviate the progression of IVDD triggered by increased matrix stiffness and elucidate its mechanisms. Nucleus pulposus (NP) tissues were collected from patients, and ECM stiffness, reactive oxygen species (ROS) levels, apoptosis rates, and p65 expression in these tissues with varying Pfirrmann scores were determined. In vitro experiments were conducted to investigate the effects of melatonin on the NP cells cultured on soft substrate with differing stiffness levels. Our findings revealed a positive correlation between ECM stiffness in human NP tissue and degree of IVDD. Additionally, phosphorylation of P65 exhibited a strong association with matrix stiffness. Enhanced levels of ROS and cellular apoptosis were observed within degenerated intervertebral discs. In vitro experiments demonstrated that melatonin significantly inhibited catabolism and apoptosis induced by stiff matrices, along with elevated ROS levels. Furthermore, we observed that melatonin inhibited NP cell catabolism and apoptosis by reducing the melatonin receptors mediated activation of the PI3K/AKT and NF-κB pathways. Also, we found that the reduction of ROS by melatonin can assist in inhibiting the activation of the NF-κB pathway. The outcomes of the in vivo experiments corroborated the results of the in vitro experiments. Collectively, melatonin can potentially alleviate high matrix stiffness-induced IVDD by reducing intracellular ROS levels and inhibiting the PI3K/AKT/NF-κB pathway.

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Low back pain significantly impacts individuals' quality of life, with intervertebral disc degeneration (IDD) being a primary contributor to this condition. Currently, IDD treatment primarily focuses on symptom management and does not achieve a definitive cure. The cartilage endplate (CEP), a crucial nutrient-supplying tissue of the intervertebral disc, plays a pivotal role in disc degeneration. This review examines the mechanisms underlying CEP degeneration, summarizing recent advancements in understanding the structure and function of CEP, the involvement of various signaling pathways, and the roles of cartilage endplate stem cells (CESCs) and exosomes (Exos) in this process. The aim of this review is to provide a comprehensive reference for future research on CEP. Despite progress in understanding the role of CEP in IDD, the mechanisms underlying CEP degeneration remain incompletely elucidated. Future research poses significant challenges, necessitating further investigations to elucidate the complexities of CEP.

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The NRF2 pathway is a metabolic- and redox-sensitive signaling axis in which the transcription factor controls the expression of a multitude of genes that enable cells to survive environmental stressors, such as oxidative stress, mainly by inducing the expression of cytoprotective genes. Basal NRF2 levels are maintained under normal physiological conditions, but when exposed to oxidative stress, cells activate the NRF2 pathway, which is crucial for supporting cell survival. Recently, the NRF2 pathway has been found to have novel functions in metabolic regulation and interplay with other signaling pathways, offering novel insights into the treatment of various diseases. Numerous studies have shown that targeting its pathway can effectively investigate the development and progression of age-related musculoskeletal diseases, such as sarcopenia, osteoporosis, osteoarthritis, and intervertebral disc degeneration. Appropriate regulation of the NRF2 pathway flux holds promise as a means to improve musculoskeletal function, thereby providing a new avenue for drug treatment of age-related musculoskeletal diseases in clinical settings. The review summarized an overview of the relationship between NRF2 and cellular processes such as oxidative stress, apoptosis, inflammation, mitochondrial dysfunction, ferroptosis, and autophagy, and explores the potential of targeted NRF2 regulation in the treatment of age-related musculoskeletal diseases.

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Background: Intervertebral disc degeneration (IDD) is a chronic disabling disease caused by degeneration of nucleus pulposus cells, decreased activity and the number of nucleus pulposus cells, decreased extracellular matrix, and infiltration of inflammatory factors, resulting in low back and leg pain. Recent studies have shown that non-surgical treatment is of great significance in reversing the progression of degenerative disc disease, and there are more relevant literature reports. However, there is no bibliometric analysis in this area. This study aimed to describe the knowledge structure and thematic trends of non-surgical treatment methods for IDD through bibliometrics. Methods: Articles and reviews on non-surgical treatment of disc degeneration from 1998 to 2022 were collected on the Web of Science. VOSviewer 1.6.18, CiteSpace 6.1.R3, R package "bibliometrix" and two online analysis platforms were used for bibliometric and visual literature analysis. Results: 961 articles were screened for inclusion, including 821 articles and 140 reviews. The analysis of our study shows that publications in the non-surgical treatment of disc degeneration are increasing annually, with publications coming mainly from North America and Asia, with China and the United States dominating. Huazhong Univ Sci & Technol and Wang K are the most prolific institutions and authors, respectively, and Le Maitre CL is the most co-cited author. However, there is less collaboration between institutions in different countries. Spine is both the most published and the most cited journal. According to the co-citation and co-occurrence analysis results, "mesenchymal stem cells," "exosomes," "medication," and "tissue engineering" are the current research hotspots in this field. Conclusions: This study employs bibliometric analysis to explore the knowledge structure and trends of non-surgical treatments for IDD from 2013 to 2022. Key research hotspots include mesenchymal stem cells, exosomes, medication, and tissue engineering. The number of publications, especially from China and the USA, has increased significantly, though international collaboration needs improvement. Influential contributors include Wang K and the journal Spine. These findings provide a comprehensive overview and highlight important future directions for the field.

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There is still a lack of high-level evidence regarding the causal relationship between smoking and intervertebral disc degenerative diseases. This study utilized data from genome wide analysis studies and conducted two-sample Mendelian randomization analyses across multiple heterogeneous datasets. We evaluated the causal relationships between smoking behavior, serum inflammatory factors, serum chemokines, and intervertebral disc degeneration. Sensitivity analysis was performed to examine data heterogeneity and the pleiotropy of causal effects. The results indicated that smokers were liable to develop intervertebral disc degeneration (OR 1.770; 95 % CI, 1.519-2.064; p = 2.992 × 10-13), and long-term smoking behavior increased the risk of intervertebral disc degeneration (OR 1.715; 95 % CI 1.475-1.994; P = 2.220 × 10-12). Additionally, a causal relationship was confirmed between serum IL-1ß level and intervertebral disc degeneration (OR 1.087; 95 % CI, 1.023-1.154; p = 0.007). The "smoking index" representing lifelong smoking habit was also found to be causally related to serum MCP-3 level(ß = 0.292; SE = 0.093; p = 0.002). All of the causality mentioned above remained stable in sensitivity tests. Based on the analysis results and fundamental medicine theories around macrophage-induced inflammation in degenerative intervertebral discs, we have constructed a new mechanism that long-term smoking could induce an increase in serum MCP-3 level, promoting the gathering and activation of monocyte macrophages. Furthermore, the recruited macrophages led to an increase in local IL-1ß within the intervertebral disc, ultimately exacerbating the process of intervertebral disc degeneration. What we have found is expected to accelerate the development of prevention and treatment of intervertebral disc degeneration.

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Intervertebral disc (IVD) degeneration damaging the extracellular matrix (ECM) of IVDs is the main cause of spine-associated disorders. Degenerative disc disease (DDD) is a multifaceted disorder, where environmental factors, inflammatory cytokines and catabolic enzymes act together. DDD starts typically due to imbalance between ECM biosynthesis and degradation within IVDs, especially through unbalanced degradation of aggrecan and collagen II in nucleus pulposus (NP). Current treatment approaches are primarily based on conservative or surgical therapies, which are insufficient for biological regeneration. The disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) and matrix metalloproteinases (MMPs) are the key proteolytic enzymes for degradation of aggrecan and collagens. Previously, high expression levels of ADAMTS4, ADAMTS5, MMP3 and MMP13, which are accompanied with low levels of aggrecan and collagen II, were demonstrated in degenerative human NP cells. Moreover, self-complementary adeno-associated virus type 6 (scAAV6) mediated inhibitions of ADAMTS4 and ADAMTS5 by RNA-interference (RNAi) could specifically enhance aggrecan level. Thus, MMPs are apparently the main degrading enzymes of collagen II in NP. Furthermore, scAAV6-mediated inhibitions of MMP3 and MMP13 have not yet been investigated. Therefore, we attempted to enhance the level of collagen II in degenerative NP cells by scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13. MRI was used to determine preoperative grading of IVD degeneration in patients. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting MMP3 or MMP13; and analysed by fluorescence microscopy, FACS, MTT assay, RT-qPCR, ELISA and western blotting. scAAV6-shRNRs have no impact on cell viability and proliferation, despite high transduction efficiencies (98.6%) and transduction units (1383 TU/Cell). Combined knockdown of MMP3 (92.8%) and MMP13 (90.9%) resulted in highest enhancement of collagen II (143.2%), whereby treatment effects were significant over 56 days (p < 0.001). Conclusively, scAAV6-RNAi-mediated inhibitions of MMP3 and MMP13 help to progress less immunogenic and enduring biological treatments in DDD.

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BACKGROUND: Intervertebral disc (IVD) degenerative disease is a multifactorial disease for which genetics plays an integral role. Several genes, and their variants, associated with the development and progression of IVD degenerative disease have been identified. While several studies have investigated these genes in Asian and European populations, no available evidence exists for the South African population. Therefore, this study aimed to investigate these parameters. METHODS AND RESULTS: Biological samples were collected in the form of buccal swabs from patients and DNA was extracted using a standard salt-lysis protocol. DNA purity and quantity was assessed by spectrophotometry, and subsequent genotyping was performed using the MassARRAY®System IPLEX extension reaction. For associations between variants and the presence of IVD degenerative disease, odds ratios (OR), confidence intervals (CI), chi-squared analysis and logistic regression was calculated. Age and sex were adjusted for, and Bonferroni's correction was applied. This study found statistically significant associations for five of the evaluated single nucleotide polymorphisms (SNPs) with IVD degenerative disease, whereby IL-1α rs1304037 and rs1800587, ADAMTs-5 rs162509, and MMP-3 rs632478 demonstrated increased odds of a positive diagnosis for IVD degenerative disease, while decreased odds of IVD degenerative disease were seen for GDF-5 rs143383. CONCLUSION: To the best of our knowledge, this study represents the first of its kind to investigate the association of gene variants associated with IVD degenerative disease within the South African population. This study has shown that 5 of these gene variants were significantly associated with the presence of IVD degenerative disease, reflecting their integral roles in development and possible progression of the disease.

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Diabetes mellitus (DM) is a well-documented risk factor of intervertebral disc degeneration (IVDD). The current study was aimed to clarify the effects and mechanisms of NADH: ubiquinone oxidoreductase subunit A3 (NDUFA3) in human nucleus pulposus cells (HNPCs) exposed to high glucose. NDUFA3 was overexpressed in HNPCs via lenti-virus transduction, which were co-treated with high glucose and rotenone (a mitochondrial complex I inhibitor) for 48 h. Cell activities were assessed for cell viability, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) ratio, oxygen consumption rate (OCR) and mitochondrial complexes I activities. High glucose decreased cell viability, increased apoptotic cells, increased ROS production, decreased MMP levels and OCR values in HNPCs in a dose-dependent manner. Rotenone co-treatment augmented the high glucose-induced injuries on cell viability, apoptosis, ROS production and mitochondrial function. NDUFA3 overexpression counteracted the high glucose-induced injuries in HNPCs. HDAC/H3K27ac mechanism was involved in regulating NDUFA3 transcription. NDUFA3 knockdown decreased cell viability and increased apoptotic cells, which were reversed by ROS scavenger N-acetylcysteine. HDAC/H3K27ac-mediated transcription of NDUFA3 protects HNPCs against high glucose-induced injuries through suppressing cell apoptosis, eliminating ROS, improving mitochondrial function and oxidative phosphorylation. This study sheds light on candidate therapeutic targets and deepens the understanding of molecular mechanisms behind DM-induced IVDD.

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STUDY DESIGN: Retrospective study. OBJECTIVE: The objective of this study is to investigate the association of waterpipe smoking with lumbar intervertebral disc degeneration (IVDD). METHODS: This is a retrospective chart review study. A total of 286 adults who underwent a lumbar magnetic resonance imaging (MRI) at a tertiary medical center were included and divided into three groups. Group 1 (n = 125) included non-smokers, group 2 (n = 80) smoked cigarettes only, and group 3 (n = 81) smoked waterpipe only. The intervertebral discs were graded using the Pfirmann disc degeneration grading system. RESULTS: The study showed higher lumbar disc degeneration scores for waterpipe and cigarette smokers compared to non-smokers at all spinal levels. Specifically, post hoc analysis showed that there was a significant difference at L1-L2 between cigarette smokers and non-smokers (P = 0.007) and between waterpipe smokers and non-smokers (P = 0.013), and a significant difference at L3-L4 and L4-L5 between non-smokers and cigarettes smokers (P < .001 and P = .029 respectively). CONCLUSION: Waterpipe smoking is associated with lumbar intervertebral disc degeneration.

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Intervertebral disc disease (IDD) is a debilitating spine condition that can be caused by intervertebral disc (IVD) damage which progresses towards IVD degeneration and dysfunction. Recently, human pluripotent stem cells (hPSCs) were recognized as a valuable resource for cell-based regenerative medicine in skeletal diseases. Therefore, adult somatic cells reprogrammed into human induced pluripotent stem cells (hiPSCs) represent an attractive cell source for the derivation of notochordal-like cells (NCs) as a first step towards the development of a regenerative therapy for IDD. Utilizing a differentiation method involving treatment with a four-factor cocktail targeting the BMP, FGF, retinoic acid, and Wnt signaling pathways, we differentiate CRISPR/Cas9-generated mCherry-reporter knock-in hiPSCs into notochordal-like cells. Comprehensive analysis of transcriptomic changes throughout the differentiation process identified regulation of histone methylation as a pivotal driver facilitating the differentiation of hiPSCs into notochordal-like cells. We further provide evidence that specific inhibition of histone demethylases KDM2A and KDM7A/B enhanced the lineage commitment of hiPSCs towards notochordal-like cells. Our results suggest that inhibition of KDMs could be leveraged to alter the epigenetic landscape of hiPSCs to control notochord-specific gene expression. Thus, our study highlights the importance of epigenetic regulators in stem cell-based regenerative approaches for the treatment of disc degeneration.

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Spinal tuberculosis usually involves intervertebral disc, pardiscal vertebra and has associated perivertebral collection. Involvement of vertebral body including the posterior element, noncontagious vertebral involvement and sparing of intervertebral discs is typical of metastasis. Index case, a 19 years old Nepali patient presented to our hospital with complaints of severe backache. Blood and urine investigations were normal except elevated erythrocyte sedimentation rate and C- reactive protein. A contrast enhanced computed tomography (CT) showed lytic lesions in clivus, C7, T10, and L3 vertebral bodies with involvement of right sided posterior elements of C7 and T10. Mild surrounding soft tissue was seen. Contrast enhanced MRI showed similar findings and also lesions in S1 and right sacral ala. Possibility of metastatic lesion was considered over tuberculosis. A CT guided biopsy from right pedicle of T10 vertebra confirmed tubercular etiology.

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Background: Intervertebral disc degeneration (IDD) can lead to disc herniation and spinal instability, sometimes requiring surgical intervention. Currently, estrogen has a potential protective effect on IDD, and estrogen is associated with an increased risk of some cancers, such as breast and endometrial cancer. Therefore, it is important to identify natural compounds that estrogen analogues treat IDD while reducing the risk of tumor development. Objective: This study aims to explore a natural metabolic treatment strategy by targeting CRISP2 with the natural compound Hesperidin to mimic the protective effects of estrogen on IDD and reduce the risk of tumor development. Methods: Microarray data from healthy volunteers and IDD patients were extracted from the Gene Expression Omnibus (GEO) database, and RNA sequencing and clinical data from various cancer types were analyzed. Differentially expressed genes (DEGs) were identified using the Bioconductor Limma package, followed by principal component analysis, volcano plot, and heatmap visualization. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, CIBERSORT and ssGSEA immune cell infiltration assessments, survival analysis, metabolite enrichment analysis, and molecular docking were performed. Hesperidin's interaction with CRISP2 was further validated through molecular docking and experimental studies. Results: Hesperidin significantly reduced the expression of CRISP2, iNOS, and COX2 in IDD models, decreased reactive oxygen species (ROS) and apoptosis, and diminished inflammatory markers. CIBERSORT and ssGSEA analyses revealed a correlation between CRISP2 and immune cell infiltration. Survival analysis demonstrated that CRISP2 expression levels were associated with patient survival across various cancer types. Hesperidin was found to mimic estrogen's effects on IDD and reduce tumor progression. Cell culture and experimental validation confirmed Hesperidin's protective effects on nucleus pulposus cells (NPCs). Conclusion: Hesperidin, as a potential natural metabolic regulator, not only has therapeutic effects on IDD but may also synergize with estrogen therapy to promote spinal health without increasing cancer risk. This study presents a new clinical approach for IDD treatment and lays the foundation for further drug development and experimental research.

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Introduction: Large clinical studies regarding cervical intervertebral disc extrusion (IVDE) in Dachshunds are lacking. This retrospective multicentric study therefore aims to describe the clinical features, magnetic resonance imaging (MRI) findings and outcomes of Dachshunds diagnosed with cervical IVDE. Methods: Medical records of Dachshunds with cervical IVDE were reviewed for signalment, onset of clinical signs, neurological examination, MRI features, treatment and outcome. Results: Eighty Dachshunds were included in the study, mostly ambulatory (55% grade 1 and 33% grade 2) and without nerve root signature (85% of cases) on presentation. Information on coat type was available for 56% of dogs; specifically, 41% were smooth-haired, 9% were long-haired and 6% were wire-haired Dachshunds. There were 29 (36%) neutered female, 27 (34%) male entire, 15 (19%) male neutered and 9 (11%) entire female dogs. The onset of clinical signs was most often >48 h (84%). The most common intervertebral disc space affected was C2-C3 (38%) and foraminal IVDEs were reported in 14% of dogs. A foraminal IVDE was diagnosed in only 25% of dogs presented with nerve root signatures. Most dogs (77.5%) were treated surgically. In this group, a higher body condition score on presentation and a higher mean spinal cord compression ratio calculated on MRI were directly and moderately associated with a longer hospitalization time (r = 0.490 p = 0.005 and r = 0.310 p = 0.012, respectively). The recovery time was longer in dogs with an onset of clinical signs <24 h or 24-48 h compared to those with an onset of clinical signs >48 h (3.1 ± 6.5 days versus 1.6 ± 6.2, p < 0.001) in both medically and surgically treated groups. Data about the outcome was available for 83% of dogs. Eighty percent of the entire population of dogs was considered to have completely returned to normal. There was no association between the therapeutic choice (surgical versus medical management) and the outcome of the dogs included in this study.

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BACKGROUND: Low back pain is the most prevalent and disabling condition worldwide, with a high recurrence rate in the general adult population. METHODS: A set of open-sourced trunk musculoskeletal models was used to investigate trunk flexion kinematics under different motor control strategies, including minimizing shearing or compressive loads at the L4/L5 or L5/S1 level. FINDINGS: A control strategy that minimizes the load on the lower lumbar intervertebral disc can result in two kinematic patterns-the "restricted lumbar spine" and the "overflexed lumbar spine"-in performing the trunk flexion task. The "restricted" pattern can reduce the overall load on the lower lumbar levels, whereas the "overflexed" pattern can reduce the shearing force only at the L4/L5 level and increase the compressive and shearing forces at the L5/S1 level and the compressive force at the L4/L5 level. INTERPRETATION: This study investigated the relationships between specific trunk kinematics in patients with low back pain and lumbar intervertebral loading via musculoskeletal modelling and simulation. The results provide insight into individualized treatment for patients with low back pain.

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PURPOSE: To use transabdominal ultrasound (US) to investigate the impact of posture and axial loading on the lumbar intervertebral disc (IVD) dimensions in healthy adults. METHOD: For this single-center, prospective cross-sectional study 54 healthy volunteers (mean age 23.76 ± 3, 26 men) underwent transabdominal US. Lumbar IVD dimensions (height, length, width) at the levels L3/4 and L4/5 were assessed in three test conditions: supine, standing, and standing position with additional axial load of 50 % body weight (standing+50%). Success rates for the longitudinal and transverse US acquisitions and IVD dimension measurements were determined. IVD dimensions were compared across test conditions using two-way repeated measures analysis of variance and post-hoc pairwise t-tests with Bonferroni correction. RESULTS: The success rate for longitudinal and transverse US acquisition was 100 %. The overall success rate for IVD dimension measurement was 96.4 %, it was highest for IVD height (99.2 %) and lowest for IVD length (93.3 %). IVD height at L4/5 decreased significantly from the supine to standing position (p < 0.05) and from the supine to standing+50% position (p < 0.01). IVD width at L3/4 increased significantly from the supine to standing+50% position (p < 0.05). No significant differences were found for IVD length. CONCLUSIONS: Transabdominal US is a feasible tool to investigate IVD dimensions at L3/4 and L4/5 in different postures and with axial loading. Posture and axial loading significantly influence IVD height and width, but not length.

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STUDY DESIGN: Double-center retrospective study. PURPOSE: Utilization trends in interventional treatment for lumbar disc herniation (LDH) have not yet been examined. Furthermore, limited information is currently available on motor recovery with condoliase therapy. Therefore, the present study investigated utilization trends in treatment for LDH and the effects of condoliase therapy on muscle weakness. METHODS: This retrospective, double-center study involved patients with leg pain caused by LDH who received interventional treatment between September 2017 and August 2022. LDH patients were divided into two groups: an operative treatment group and condoliase therapy group. The period between September 2017 and August 2022 was divided into 5 equal parts and changes in the percentage of intervention treatment were examined. Motor recovery was also assessed in the two groups. Patients receiving condoliase therapy were divided into two groups: an effective group and non-effective group. Sex, age, the body mass index, duration of symptoms, herniation level, neurological and radiographic findings, a visual analog scale for leg pain, and the Oswestry disability index were examined in the two groups. RESULTS: Subjects included 226 males and 115 females with a mean age of 49.2 years, mean BMI of 22.8, and mean duration of symptoms of 5.0 months. The utilization of condoliase therapy for LDH surpassed surgery in the third year after its introduction. In the fourth year, condoliase therapy became the main treatment for LDH. Lower limb muscle strength improved in 76 % of cases receiving condoliase therapy. CONCLUSIONS: Condoliase therapy has become an intermediate treatment before surgery in our institutions. Motor recovery in patients receiving condoliase therapy was not inferior to that after surgery; however, in cases with severe muscle weakness with manual muscle test ≤3, the improvement rate was approximately 60 %. These results will be useful for clinicians when providing informed consent and selecting condoliase therapy.

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METHODS: Single-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1ß. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action. RESULTS: CFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1ß, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3). CONCLUSION: The study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs.

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BACKGROUND: Limited supply of certain nutrients and deregulation of nucleus pulposus (NP) plays a key role in the pathogenesis of intervertebral disc degeneration (IVDD). However, whether nutrient deprivation-induced cell death, particularly disulfidptosis, contributes to the depletion of NP cells and the development of IVDD, is unknown. METHODS: RNA-seq, single-cell RNA-seq, and Genome-wide DNA methylation datasets of nucleus pulposus tissue were collected for bioinformatic analysis. Predictive models of disulfidptosis related genes in IVDD were constructed by machine learning and their differential expression was analyzed. In addition, we performed cell subsets identification analysis, cell-cell communications analysis, and functional enrichment analysis of key genes in the core subset based on single-cell RNA-seq data of NP tissues isolated from one normal sample and one IVDD sample. Finally, glucose deprivation-induced disulfidptosis in human NP cells (HNPCs) was verified by various cell death inhibitors and disulfidptosis-related molecular markers. RESULTS: We found the disulfidptosis signal was significantly activated in the IVDD group. Using single-cell RNA-seq analysis, we focused on the chondrocytes and found that disulfidptosis-related genes significantly highly expressed in the IVDD C4 chondrocyte subset, which was identified as a new disulfidptosis-associated cell subset. Correlation analysis revealed the negative correlation between SLC7A11 (driving gene of disulfidptosis) and the glucose transporter GLUTs (SLC2A1-4) family genes (suppressing genes of disulfidptosis) in the IVDD group. We also found obvious cell death in HNPC upon glucose starvation, while employment of various cell death inhibitors could not inhibit glucose starvation-induced death in HNPCs. Moreover, the accumulation of disulfide bonds in cytoskeletal proteins was indicated by slowed migration in non-reducible protein blotting experiments. 2-DG, a key disulfidptosis inhibitor, significantly rescued cell death caused by glucose starvation through lowering the NADP+/NADPH ratio. CONCLUSIONS: We validated the occurrence of disulfidptosis in HPNCs and identified a novel disulfidptosis-associated cell subset, followed by experimental verification of disulfidptosis in a glucose-limited context to mimic a fall in nutrient supply during the development disc degeneration. These findings provided new insights into the pathological mechanisms of IVDD and encourage us to explore potential therapeutic targets involved in the regulation of disulfidptosis for the prevention of intervertebral disc degeneration.

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PURPOSE: To compare the mechanical properties of human annulus fibrosus obtained by forceps versus bistoury and observe whether the measurement could be affected by forceps sampling method. METHODS: In this study, the mechanical properties of the the extracellular matrix (ECM) of human annulus fibrosus, including elastic modulus and stiffness, were investigated using atomic force microscope (AFM). Tissue was obtained from patients during operation using a bistoury or nucleus pulposus forceps. Tissues obtained with the nucleus pulposus forceps were considered as the forceps group and those obtained with a bistoury were considered as the bistoury group. RESULTS: There was no significant difference observed between the forceps and bistoury group according to histological staining. The elastic modulus of the forceps group was 0.41 ± 0.08 MPa, and that of bistoury group was 0.53 ± 0.13 MPa, and the difference between the two groups was statistically significant (p < 0.05). The stiffness of the forceps group was 0.024 ± 0.003 N/m, and that of the bistoury group was 0.037 ± 0.003 N/m, and the difference between the two groups was statistically significant (p < 0.05). CONCLUSION: The results indicate that the forceps sampling method has a substantial negative effect on the micromechanical properties of the ECM of the annulus fibrosus. Bistoury sampling method is recommended as the experimental subject for exploring the micromechanics mechanisms of cervical degenerative disease.