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Introduction: Modic changes (MC) are bone marrow lesions of vertebral bones, which can be detected with magnetic resonance imaging (MRI) adjacent to degenerated intervertebral discs. Defined by their appearance on T1 and T2 weighted images, there are three interconvertible types: MC1, MC2, and MC3. The inter-observer variability of the MRI diagnosis is high, therefore a diagnostic serum biomarker complementing the MRI to facilitate diagnosis and follow-up would be of great value., Methods: We used a highly sensitive and reproducible proteomics approach: DIA/SWATH-MS to find serum biomarkers in a subset of the Northern Finland Birth Cohort 1966. Separately, we measured a panel of factors involved in inflammation and angiogenesis to confirm some potential biomarkers published before with an ELISA-based method called V-Plex., Results: We found neither an association between the serum concentrations of the proteins detected with DIA/SWATH-MS with the presence of MC, nor a correlation with the size of the MC lesions. We did not find any association between the factors measured with the V-Plex and the presence of MC or their size., Conclusion: Altogether, our study suggests that a robust and generally usable biomarker to facilitate the diagnosis of MC cannot readily be found in serum., Competing Interests: All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 The Author(s). JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Introduction: The emerging field of the disc microbiome challenges traditional views of disc sterility, which opens new avenues for novel clinical insights. However, the lack of methodological consensus in disc microbiome studies introduces discrepancies. The aims of this study were to (1) compare the disc microbiome of non-Modic (nonMC), Modic type 1 change (MC1), and MC2 discs to findings from prior disc microbiome studies, and (2) investigate if discrepancies to prior studies can be explained with bioinformatic variations., Methods: Sequencing of 16S rRNA in 70 discs (24 nonMC, 25 MC1, and 21 MC2) for microbiome profiling. The experimental setup included buffer contamination controls and was performed under aseptic conditions. Methodology and results were contrasted with previous disc microbiome studies. Critical bioinformatic steps that were different in our best-practice approach and previous disc microbiome studies (taxonomic lineage assignment, prevalence cut-off) were varied and their effect on results were compared., Results: There was limited overlap of results with a previous study on MC disc microbiome. No bacterial genera were shared using the same bioinformatic parameters. Taxonomic lineage assignment using "amplicon sequencing variants" was more sensitive and detected 48 genera compared to 22 with "operational taxonomic units" (previous study). Increasing filter cut-off from 4% to 50% (previous study) reduced genera from 48 to 4 genera. Despite these differences, both studies observed dysbiosis with an increased abundance of gram-negative bacteria in MC discs as well as a lower beta-diversity. Cutibacterium was persistently detected in all groups independent of the bioinformatic approach, emphasizing its prevalence., Conclusion: There is dysbiosis in MC discs. Bioinformatic parameters impact results yet cannot explain the different findings from this and a previous study. Therefore, discrepancies are likely caused by different sample preparations or true biologic differences. Harmonized protocols are required to advance understanding of the disc microbiome and its clinical implications., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Disc degeneration and vertebral endplate bone marrow lesions called Modic changes are prevalent spinal pathologies found in chronic low back pain patients. Their pathomechanisms are complex and not fully understood. Recent studies have revealed that complement system proteins and interactors are dysregulated in disc degeneration and Modic changes. The complement system is part of the innate immune system and plays a critical role in tissue homeostasis. However, its dysregulation has also been associated with various pathological conditions such as rheumatoid arthritis and osteoarthritis. Here, we review the evidence for the involvement of the complement system in intervertebral disc degeneration and Modic changes. We found that only a handful of studies reported on complement factors in Modic changes and disc degeneration. Therefore, the level of evidence for the involvement of the complement system is currently low. Nevertheless, the complement system is tightly intertwined with processes known to occur during disc degeneration and Modic changes, such as increased cell death, autoantibody production, bacterial defense processes, neutrophil activation, and osteoclast formation, indicating a contribution of the complement system to these spinal pathologies. Based on these mechanisms, we propose a model how the complement system could contribute to the vicious cycle of tissue damage and chronic inflammation in disc degeneration and Modic changes. With this review, we aim to highlight a currently understudied but potentially important inflammatory pathomechanism of disc degeneration and Modic changes that may be a novel therapeutic target., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

The cartilaginous endplates (CEP) are key components of the intervertebral disc (IVD) necessary for sustaining the nutrition of the disc while distributing mechanical loads and preventing the disc from bulging into the adjacent vertebral body. The size, shape, and composition of the CEP are essential in maintaining its function, and degeneration of the CEP is considered a contributor to early IVD degeneration. In addition, the CEP is implicated in Modic changes, which are often associated with low back pain. This review aims to tackle the current knowledge of the CEP regarding its structure, composition, permeability, and mechanical role in a healthy disc, how they change with degeneration, and how they connect to IVD degeneration and low back pain. Additionally, the authors suggest a standardized naming convention regarding the CEP and bony endplate and suggest avoiding the term vertebral endplate. Currently, there is limited data on the CEP itself as reported data is often a combination of CEP and bony endplate, or the CEP is considered as articular cartilage. However, it is clear the CEP is a unique tissue type that differs from articular cartilage, bony endplate, and other IVD tissues. Thus, future research should investigate the CEP separately to fully understand its role in healthy and degenerated IVDs. Further, most IVD regeneration therapies in development failed to address, or even considered the CEP, despite its key role in nutrition and mechanical stability within the IVD. Thus, the CEP should be considered and potentially targeted for future sustainable treatments., Competing Interests: Benjamin Gantenbein and Christine Le Maitre are editorial board members of JOR Spine and co‐author of this article. They were excluded from editorial decision‐making related to the acceptance of this article for publication in the journal. All other authors have no conflicts of interest to declare in relation to this article., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources., Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated., Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture., Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide., Competing Interests: The authors have no relevant conflicts of interest to declare in relation to this article., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Vertebral endplate signal intensity changes visualized by magnetic resonance imaging termed Modic changes (MC) are highly prevalent in low back pain patients. Interconvertibility between the three MC subtypes (MC1, MC2, MC3) suggests different pathological stages. Histologically, granulation tissue, fibrosis, and bone marrow edema are signs of inflammation in MC1 and MC2. However, different inflammatory infiltrates and amount of fatty marrow suggest distinct inflammatory processes in MC2., Aims: The aims of this study were to investigate (i) the degree of bony (BEP) and cartilage endplate (CEP) degeneration in MC2, (ii) to identify inflammatory MC2 pathomechanisms, and (iii) to show that these marrow changes correlate with severity of endplate degeneration., Methods: Pairs of axial biopsies ( n  = 58) spanning the entire vertebral body including both CEPs were collected from human cadaveric vertebrae with MC2. From one biopsy, the bone marrow directly adjacent to the CEP was analyzed with mass spectrometry. Differentially expressed proteins (DEPs) between MC2 and control were identified and bioinformatic enrichment analysis was performed. The other biopsy was processed for paraffin histology and BEP/CEP degenerations were scored. Endplate scores were correlated with DEPs., Results: Endplates from MC2 were significantly more degenerated. Proteomic analysis revealed an activated complement system, increased expression of extracellular matrix proteins, angiogenic, and neurogenic factors in MC2 marrow. Endplate scores correlated with upregulated complement and neurogenic proteins., Discussion: The inflammatory pathomechanisms in MC2 comprises activation of the complement system. Concurrent inflammation, fibrosis, angiogenesis, and neurogenesis indicate that MC2 is a chronic inflammation. Correlation of endplate damage with complement and neurogenic proteins suggest that complement system activation and neoinnervation may be linked to endplate damage. The endplate-near marrow is the pathomechanistic site, because MC2 occur at locations with more endplate degeneration., Conclusion: MC2 are fibroinflammatory changes with complement system involvement which occur adjacent to damaged endplates., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions., Aims: This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models., Materials & Methods: This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter- and intra-rater reliability., Results: A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP-AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely-accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 μm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories., Conclusion: The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Pro-inflammatory cytokines are recognized contributors to intervertebral disc (IVD) degeneration and discogenic pain. We have recently reported the anti-inflammatory effect of pulsed electromagnetic fields (PEMF) on IVD cells in vitro. Whether these potentially therapeutic effects are sufficiently potent to influence disc health in vivo has not been demonstrated. We report here the effect of PEMF on acute inflammation arising from a rat-tail IVD injury model. Disc degeneration was induced by percutaneously stabbing the Co6-7, Co7-8, and Co8-9 levels using a 20-gauge needle. Seventy-two (72) rats were divided into three groups: sham control, needle stab, needle stab+PEMF. Treated rats were exposed to PEMF immediately following surgery and for either 4 or 7 days (4 hr/d). Stab and PEMF effects were evaluated by measuring inflammatory cytokine gene expression (RT-PCR) and protein levels (ELISA assay), anabolic and catabolic gene expression (RT-PCR), and histologic changes. We observed in untreated animals that at day 7 after injury, inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor α, and IL-1β) were significantly increased at both gene and protein levels ( P  < .05). Similarly, catabolic factors (MMP [metalloproteinases]-2, MMP-13 and the transcriptional factor NF-kβ gene expression) were significantly increased ( P  < .05). At day 7, PEMF treatment significantly inhibited inflammatory cytokine gene and protein expression induced by needle stab injury ( P  < .05). At day 4, PEMF downregulated FGF-1 and upregulated MMP-2 compared to the stab-only group. These data demonstrate that previously reported anti-inflammatory effects of PEMF on disc cells carry over to the in vivo situation, suggesting potential therapeutic benefits. Though we observed an inhibitory effect of PEMF on acute inflammatory cytokine expression, a consistent effect was not observed for acute changes in disc histology and anabolic and catabolic factor expression. Therefore, these findings should be further investigated in studies of longer duration following needle-stab injury., (© 2019 The Authors. JOR Spine published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)