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This study aimed to develop a method allowing high-dimensional and technically uniform screening of surface markers on cells of hematopoietic origin. High-dimensional screening of cell phenotypes is primarily the domain of single-cell RNA sequencing (RNAseq), which allows simultaneous analysis of the expression of thousands of genes in several thousands of cells. However, rare cell populations can often substantially impact tissue homeostasis or disease pathogenesis, and dysregulation of rare populations can easily be missed when only a few thousand cells are analyzed. With the presented methodological approach, it is possible to screen hundreds of markers on millions of cells in a technically uniform manner and thus identify and characterize changes in rare populations. We utilize the highly expressed markers CD45 on immune cells and CD71 on erythroid progenitors to create unique fluorescent barcodes on each of the 25 samples. Double-barcoded samples are co-stained with a broad immunophenotyping panel. The panel is designed in such a way that allows the addition of PE-labelled antibody, which was used for screening purposes. Multiplexed samples are divided into hundreds of aliquots and co-stained, each aliquot with a different PE-labelled antibody. Utilizing a broad immunophenotyping panel and machine-learning algorithms, we can predict the co-expression of hundreds of screened markers with a high degree of precision. This technique is suitable for screening immune cells in bone marrow from different locations, blood specimens, or any tissue with a substantial presence of immune cells, such as tumors or inflamed tissue areas in autoimmune conditions. It represents an approach that can significantly improve our ability to recognize dysregulated immune cell populations and, if needed, precisely target subsequent experiments covering lower cell counts such as RNAseq., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The vertebral cartilage endplate (CEP), crucial for intervertebral disc health, is prone to degeneration linked to chronic low back pain, disc degeneration, and Modic changes (MC). While it is known that disc cells express toll-like receptors (TLRs) that recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), it is unclear if CEP cells (CEPCs) share this trait. The CEP has a higher cell density than the disc, making CEPCs an important contributor. This study aimed to identify TLRs on CEPCs and their role in pro-inflammatory and catabolic gene expression., Methods: Gene expression of TLR1-10 was measured in human CEPs and expanded CEPCs using quantitative polymerase chain reaction. Additionally, surface TLR expression was measured in CEPs grouped into non-MC and MC. CEPCs were stimulated with tumor necrosis factor alpha, interleukin 1 beta, small-molecule TLR agonists, or the 30 kDa N-terminal fibronectin fragment. TLR2 signaling was inhibited with TL2-C29, and TLR2 protein expression was measured with flow cytometry., Results: Ex vivo analysis found all 10 TLRs expressed, while cultured CEPCs lost TLR8 and TLR9 expression. TLR2 expression was significantly increased in MC1 CEPCs, and its expression increased significantly after pro-inflammatory stimulation. Stimulation of the TLR2/6 heterodimer upregulated TLR2 protein expression. The TLR2/1 and TLR2/6 ligands upregulated pro-inflammatory genes and matrix metalloproteases (MMP1, MMP3, and MMP13), and TLR2 inhibition inhibited their upregulation. Endplate resorptive capacity of TLR2 activation was confirmed in a CEP explant model., Conclusions: The expression of TLR1-10 in CEPCs suggests that the CEP is susceptible to PAMP and DAMP stimulation. Enhanced TLR2 expression in MC1, and generally in CEPCs under inflammatory conditions, has pro-inflammatory and pro-catabolic effects, suggesting a potential role in disc degeneration and MC.

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.)

Background: The multimodal properties of mesenchymal stromal cells (MSCs), particularly their ability to modulate immune responses is of high interest in translational research. Pro-inflammatory, hypoxic, and 3D culture priming are promising and often used strategies to improve the immunosuppressive potency of MSCs, but the underlying mechanisms are not well understood. Therefore, the aims of this study were (i) to compare the effects of pro-inflammatory, hypoxic, and 3D culture priming on the in vitro immunosuppressive potential of MSCs, (ii) to assess if immunosuppressive priming effects are temporally preserved under standard and translationally relevant culture conditions, and (iii) to investigate if the three priming strategies engage the same immunosuppressive mechanisms., Methods: Functional in vitro T cell suppressive potency measurements were conducted to assess the impact of pro-inflammatory, hypoxic, and 3D culture priming on the immunosuppressive potential of human bone marrow-derived MSCs. Primed MSCs were either cultured under standard cell culture conditions or translationally relevant culture conditions, and their transcriptomic adaptations were monitored over time. Next-generation sequencing was performed to assess if different priming strategies activate distinct immunosuppressive mechanisms., Results: (i) Pro-inflammatory, hypoxic, and 3D culture priming induced profound transcriptomic changes in MSCs resulting in a significantly enhanced T cell suppressive potential of pro-inflammatory and 3D culture primed MSCs. (ii) Priming effects rapidly faded under standard cell culture conditions but were partially preserved under translationally relevant conditions. Interestingly, continuous 3D culture priming of MSCs maintained the immunosuppressive potency of MSCs. (iii) Next-generation sequencing revealed that priming strategy-specific differentially expressed genes are involved in the T cell suppressive capacity of MSCs, indicating that different priming strategies engage distinct immunosuppressive mechanisms., Conclusion: Priming can be a useful approach to improve the immunosuppressive potency of MSCs. However, future studies involving primed MSCs should carefully consider the significant impact of translationally relevant conditions on the preservation of priming effects. Continuous 3D culture could act as a functionalized formulation, supporting the administration of MSC spheroids for a sustainably improved immunosuppressive potency., (© 2024. The Author(s).)

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 pain in patients with Modic type 1 changes (MC1) is often due to vertebral body endplate pain, which is linked to abnormal neurite outgrowth in the vertebral body and adjacent endplate. The aim of this study was to understand the role of MC1 bone marrow stromal cells (BMSCs) in neurite outgrowth. BMSCs can produce neurotrophic factors, which have been shown to be pro-fibrotic in MC1, and expand in the perivascular space where sensory vertebral nerves are located. The study involved the exploration of the BMSC transcriptome in MC1, co-culture of MC1 BMSCs with the neuroblastoma cell line SH-SY5Y, analysis of supernatant cytokines, and analysis of gene expression changes in co-cultured SH-SY5Y. Transcriptomic analysis revealed upregulated brain-derived neurotrophic factor (BDNF) signaling-related pathways. Co-cultures of MC1 BMSCs with SH-SY5Y cells resulted in increased neurite sprouting compared to co-cultures with control BMSCs. The concentration of BDNF and other cytokines supporting neuron growth was increased in MC1 vs. control BMSC co-culture supernatants. Taken together, these findings show that MC1 BMSCs provide strong pro-neurotrophic cues to nearby neurons and could be a relevant disease-modifying treatment target., Competing Interests: SD was employed by Aclarion. The remaining 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., (Copyright © 2023 Mengis, Herger, Heggli, Devan, Spirig, Laux, Brunner, Farshad, Distler and Dudli.)

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.)

The Biospecimen Collection and Processing Working Group of the National Institutes of Health (NIH) HEAL Initiative BACPAC Research Program was charged with identifying molecular biomarkers of interest to chronic low back pain (cLBP). Having identified biomarkers of interest, the Working Group worked with the New York University Grossman School of Medicine, Center for Biospecimen Research and Development-funded by the Early Phase Pain Investigation Clinical Network Data Coordinating Center-to harmonize consortium-wide and site-specific efforts for biospecimen collection and analysis. Biospecimen collected are saliva, blood (whole, plasma, serum), urine, stool, and spine tissue (paraspinal muscle, ligamentum flavum, vertebral bone, facet cartilage, disc endplate, annulus fibrosus, or nucleus pulposus). The omics data acquisition and analyses derived from the biospecimen include genomics and epigenetics from DNA, proteomics from protein, transcriptomics from RNA, and microbiomics from 16S rRNA. These analyses contribute to the overarching goal of BACPAC to phenotype cLBP and will guide future efforts for precision medicine treatment., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Academy of Pain Medicine.)

Objective: The aim of this systematic review was to appraise and analyse the knowledge on bone-related biochemical and histological biomarkers in complex regional pain syndrome 1 (CRPS 1)., Database: A total of 7 studies were included in the analysis (biochemical analyses n  = 3, animal study n  = 1, histological examination n  = 3)., Results: Two studies were classified as having a low risk of bias and five studies with a moderate risk of bias. Biochemical analysis indicated an increased bone turnover with increased bone resorption (elevated urinary levels of deoxypyridinoline) and bone formation (increased serum levels of calcitonin, osteoprotegerin and alkaline phosphatase). The animal study reported an increased signalling of proinflammatory tumour necrosis factor 4 weeks postfracture, which did, however, not contribute to local bone loss. Histological examination from biopsies revealed thinning and resorption of cortical bone, rarefication and reduction in trabecular bone and vascular modification in the bone marrow in acute CRPS 1, and replacement of the bone marrow by dystrophic vessels in chronic CRPS 1., Conclusion: The limited data reviewed revealed certain potential bone-related biomarkers in CRPS. Biomarkers hold the potential to identify patients who may benefit from treatments that influence bone turnover. Thus, this review identifies important areas for future research in CRPS1 patients., (© 2023 The Authors. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation - EFIC ®.)

Modic type 1 changes (MC1) are vertebral bone marrow lesions and associate with low back pain. Increased serum C-reactive protein (CRP) has inconsistently been associated with MC1. We aimed to provide evidence for the role of CRP in the tissue pathophysiology of MC1 bone marrow. From 13 MC1 patients undergoing spinal fusion at MC1 levels, vertebral bone marrow aspirates from MC1 and intrapatient control bone marrow were taken. Bone marrow CRP, interleukin (IL)-1, and IL-6 were measured with enzyme-linked immunosorbent assays; lactate dehydrogenase (LDH) was measured with a colorimetric assay. CRP, IL-1, and IL-6 were compared between MC1 and control bone marrow. Bone marrow CRP was correlated with blood CRP and with bone marrow IL-1, IL-6, and LDH. CRP expression by marrow cells was measured with a polymerase chain reaction. Increased CRP in MC1 bone marrow (mean difference: +0.22 mg CRP/g, 95% confidence interval [CI] [-0.04, 0.47], p = 0.088) correlated with blood CRP (r = 0.69, p = 0.018), with bone marrow IL-1β (ρ = 0.52, p = 0.029) and IL-6 (ρ = 0.51, p = 0.031). Marrow cells did not express CRP. Increased LDH in MC1 bone marrow (143.1%, 95% CI [110.7%, 175.4%], p = 0.014) indicated necrosis. A blood CRP threshold of 3.2 mg/L detected with 100% accuracy increased CRP in MC1 bone marrow. In conclusion, the association of CRP with inflammatory and necrotic changes in MC1 bone marrow provides evidence for a pathophysiological role of CRP in MC1 bone marrow., (© 2022 The Authors. Journal of Orthopaedic Research ® 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.)

Extensive extracellular matrix production and increased cell-matrix adhesion by bone marrow stromal cells (BMSCs) are hallmarks of fibrotic alterations in the vertebral bone marrow known as Modic type 1 changes (MC1). MC1 are associated with non-specific chronic low-back pain. To identify treatment targets for MC1, in vitro studies using patient BMSCs are important to reveal pathological mechanisms. For the culture of BMSCs, fibroblast growth factor 2 (FGF2) is widely used. However, FGF2 has been shown to suppress matrix synthesis in various stromal cell populations. The aim of the present study was to investigate whether FGF2 affected the in vitro study of the fibrotic pathomechanisms of MC1-derived BMSCs. Transcriptomic changes and changes in cell-matrix adhesion of MC1-derived BMSCs were compared to intra-patient control BMSCs in response to FGF2. RNA sequencing and quantitative real-time polymerase chain reaction revealed that pro-fibrotic genes and pathways were not detectable in MC1-derived BMSCs when cultured in the presence of FGF2. In addition, significantly increased cell-matrix adhesion of MC1-derived BMSCs was abolished in the presence of FGF2. In conclusion, the data demonstrated that FGF2 overrides key pro-fibrotic features of MC1 BMSCs in vitro. Usage of FGF2-supplemented media in studies of fibrotic mechanisms should be critically evaluated as it could override normally dominant biological and biophysical cues.

To understand the pathophysiology of spondylodiscitis due to Staphylococcus aureus , an emerging infectious disease of the intervertebral disc (IVD) and vertebral body with a high complication rate, we combined clinical insights and experimental approaches. Clinical data and histological material of nine patients suffering from S. aureus spondylodiscitis were retrospectively collected at a single center. To mirror the clinical findings experimentally, we developed a novel porcine ex vivo model mimicking acute S. aureus spondylodiscitis and assessed the interaction between S. aureus and IVD cells within their native environment. In addition, the inflammatory features underlying this interaction were assessed in primary human IVD cells. Finally, mirroring the clinical findings, we assessed primary human neutrophils for their ability to respond to secreted inflammatory modulators of IVD cells upon the S. aureus challenge. Acute S. aureus spondylodiscitis in patients was characterized by tissue necrosis and neutrophil infiltration. Additionally, the presence of empty IVD cells' lacunae was observed. This was mirrored in the ex vivo porcine model, where S. aureus induced extensive IVD cell death, leading to empty lacunae. Concomitant engagement of the apoptotic and pyroptotic cell death pathways was observed in primary human IVD cells, resulting in cytokine release. Among the released cytokines, functionally intact neutrophil-priming as well as broad pro- and anti-inflammatory cytokines which are known for their involvement in IVD degeneration were found. In patients as well as ex vivo in a novel porcine model, S. aureus IVD infection caused IVD cell death, resulting in empty lacunae, which was accompanied by the release of inflammatory markers and recruitment of neutrophils. These findings offer valuable insights into the important role of inflammatory IVD cell death during spondylodiscitis and potential future therapeutic approaches., Competing Interests: The 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., (Copyright © 2022 Schweizer, Andreoni, Acevedo, Scheier, Heggli, Maggio, Eberhard, Brugger, Dudli and Zinkernagel.)

Objective: Modic changes (MC) are vertebral bone marrow lesions seen on magnetic resonance images, that associate with disc degeneration and low back pain (LBP). Few studies described MC histopathology qualitatively based on a few patient samples. CD90-positive bone marrow stromal cells were shown to be pro-fibrotic in MC. We aimed to provide the first semi-quantitative histomorphometric analysis of MC bone marrow. We hypothesized a role of CD90-positive cells in MC pathomechanisms., Design: Human biopsies from Modic type 1 changes (MC1, n ​= ​8), Modic type 2 changes (MC2, n ​= ​6), and control biopsies (MC0, n ​= ​8) from adjacent vertebrae were obtained from 14 LBP patients during lumbar spinal fusion. Biopsies were processed for histology/immunohistochemistry. Inflammatory changes (oedema, inflammatory infiltrates), fibrotic changes (connective tissue, type I and III collagen, fibronectin, α-smooth muscle actin), and amount of bone marrow stromal cells (CD90, CD105) were scored. Scores for MC0, MC1, and MC2 were compared with non-parametric tests. Pairwise correlations, hierarchical clustering, and principal component analysis of histological readouts were calculated to identify most important histomorphometric MC characteristics., Results: Compared to MC0, MC1 had more connective tissue, oedema, inflammatory infiltrates, and CD90 + cells. MC2 compared to MC0 had more oedema and CD90 + cells. Scores of CD90 correlated and clustered with inflammatory and fibrotic changes. Amount of connective tissue correlated with LBP., Conclusion: Accumulation of CD90 + cells is a major characteristic of MC in patients undergoing lumbar spinal fusion and associates with inflammatory and fibrotic changes. Therefore, CD90 + cells may play an important role in the inflammatory-fibrotic pathomechanisms of MC., Competing Interests: None of the authors have a conflict of interest., (© 2022 The Authors.)

Low back pain (LBP) has been among the leading causes of disability for the past 30 years. This highlights the need for improvement in LBP management. Many clinical trials focus on developing treatments against degenerative disc disease (DDD). The multifactorial etiology of DDD and associated risk factors lead to a heterogeneous patient population. It comes as no surprise that the outcomes of clinical trials on intradiscal mesenchymal stem cell (MSC) injections for patients with DDD are inconsistent. Intradiscal MSC injections have demonstrated substantial pain relief and significant disability-related improvements, yet they have failed to regenerate the intervertebral disc (IVD). Increasing evidence suggests that the positive outcomes in clinical trials might be attributed to the immunomodulatory potential of MSCs rather than to their regenerative properties. Therefore, patient stratification for inflammatory DDD phenotypes may (i) better serve the mechanisms of action of MSCs and (ii) increase the treatment effect. Modic type 1 changes-pathologic inflammatory, fibrotic changes in the vertebral bone marrow-are frequently observed adjacent to degenerated IVDs in chronic LBP patients and represent a clinically distinct subpopulation of patients with DDD. This review discusses whether degenerated IVDs of patients with Modic type 1 changes should be treated with an intradiscal MSC injection.

(1) Background: Three-dimensional (3D) collagen I-based skin models are commonly used in drug development and substance testing but have major drawbacks such as batch-to-batch variations and ethical concerns. Recently, synthetic nanofibrous scaffolds created by electrospinning have received increasing interest as potential alternatives due to their morphological similarities to native collagen fibrils in size and orientation. The overall objective of this proof-of-concept study was to demonstrate the suitability of two synthetic polymers in creating electrospun scaffolds for 3D skin cell models. (2) Methods: Electrospun nanofiber mats were produced with (i) poly(acrylonitrile-co-methyl acrylate) (P(AN-MA)) and (ii) a blend of pullulan (Pul), poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) (Pul/PVA/PAA) and characterized by scanning electron microscopy (SEM) and diffuse reflectance infrared Fourier transform (DRIFT) spectra. Primary skin fibroblasts and keratinocytes were seeded onto the nanofiber mats and analyzed for phenotypic characteristics (phalloidin staining), viability (Presto Blue HS assay), proliferation (Ki-67 staining), distribution (H/E staining), responsiveness to biological stimuli (qPCR), and formation of skin-like structures (H/E staining). (3) Results: P(AN-MA) mats were more loosely packed than the Pul/PVA/PAA mats, concomitant with larger fiber diameter (340 nm ± 120 nm vs. 250 nm ± 120 nm, p < 0.0001). After sterilization and exposure to cell culture media for 28 days, P(AN-MA) mats showed significant adsorption of fetal calf serum (FCS) from the media into the fibers (DRIFT spectra) and increased fiber diameter (590 nm ± 290 nm, p < 0.0001). Skin fibroblasts were viable over time on both nanofiber mats, but suitable cell infiltration only occurred in the P(AN-MA) nanofiber mats. On P(AN-MA) mats, fibroblasts showed their characteristic spindle-like shape, produced a dermis-like structure, and responded well to TGFβ stimulation, with a significant increase in the mRNA expression of PAI1 , COL1A1 , and αSMA (all p < 0.05). Primary keratinocytes seeded on top of the dermis equivalent proliferated and formed a stratified epidermis-like structure. (4) Conclusion: P(AN-MA) and Pul/PVA/PAA are both biocompatible materials suitable for nanofiber mat production. P(AN-MA) mats hold greater potential as future 3D skin models due to enhanced cell compatibility (i.e., adsorption of FCS proteins), cell infiltration (i.e., increased pore size due to swelling behavior), and cell phenotype preservation. Thus, our proof-of-concept study shows an easy and robust process of producing electrospun scaffolds for 3D skin cell models made of P(AN-MA) nanofibers without the need for bioactive molecule attachments.

Modic type 1 changes (MC1) are painful vertebral bone marrow lesions frequently found in patients suffering from chronic low-back pain. Marrow fibrosis is a hallmark of MC1. Bone marrow stromal cells (BMSCs) are key players in other fibrotic bone marrow pathologies, yet their role in MC1 is unknown. The present study aimed to characterise MC1 BMSCs and hypothesised a pro-fibrotic role of BMSCs in MC1. BMSCs were isolated from patients undergoing lumbar spinal fusion from MC1 and adjacent control vertebrae. Frequency of colony-forming unit fibroblast (CFU-F), expression of stem cell surface markers, differentiation capacity, transcriptome, matrix adhesion, cell contractility as well as expression of pro-collagen type I alpha 1, α-smooth muscle actin, integrins and focal adhesion kinase (FAK) were compared. More CFU-F and increased expression of C-X-C-motif-chemokine 12 were found in MC1 BMSCs, possibly indicating overrepresentation of a perisinusoidal BMSC population. RNA sequencing analysis showed enrichment in extracellular matrix proteins and fibrosis-related signalling genes. Increases in pro-collagen type I alpha 1 expression, cell adhesion, cell contractility and phosphorylation of FAK provided further evidence for their pro-fibrotic phenotype. Moreover, a leptin receptor high expressing (LEPRhigh) BMSC population was identified that differentiated under transforming growth factor beta 1 stimulation into myofibroblasts in MC1 but not in control BMSCs. In conclusion, pro-fibrotic changes in MC1 BMSCs and a LEPRhigh MC1 BMSC subpopulation susceptible to myofibroblast differentiation were found. Fibrosis is a hallmark of MC1 and a potential therapeutic target. A causal link between the pro-fibrotic phenotype and clinical characteristics needs to be demonstrated.

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.)

Purpose: Lumbar Modic change (MC) can serve as a diagnostic marker as well as an independent source of chronic low back pain (CLBP). This study aimed to test for the existence of serum biomarkers in CLBP patients with MC., Methods: Age- and sex-matched CLBP patients with confirmed MC on lumbar MRI (n = 40) and pain-free controls (n = 40) were assessed. MC was classified into M1, predominating M1, predominating M2 and M2. MC volumes were calculated. Fasting blood samples were assessed for inflammatory mediators, signalling molecules, growth factors and bone turnover markers. Serum concentrations of 46 biomarkers were measured., Results: Median concentrations of interleukin (IL)-15 (p < 0.001), IL-8 (p < 0.001), tumour necrosis factor (TNF)-alpha (p < 0.001), Eotaxin-1 (p < 0.05), Eotaxin-3 (p < 0.001), monocyte chemotactic protein (MCP)-1 (p < 0.05), macrophage inflammatory protein (MIP)-1alpha (p < 0.01), TEK receptor tyrosine kinase (Tie)-2 (p < 0.001), vascular cell adhesion molecule (VCAM)-1 (p < 0.001), RANTES (p < 0.001), C telopeptide of type I collagen (CTX)-1 (p < 0.001), vascular endothelial growth factor (VEGF)-C (p < 0.001), VEGF-D (p < 0.05), fms-related tyrosine kinase (Flt)-1 (p < 0.01) and intercellular adhesion molecule (ICAM)-1 (p < 0.01) were significantly higher among controls. IL-1sRII (23.2 vs. 15.5 ng/ml, p < 0.001) and hepatocyte growth factor (HGF)-1 (169 vs. 105 pg/ml, p < 0.01) concentrations were significantly higher among patients. Type or volume of MC was not associated with biomarker concentrations., Conclusions: This is the first study to assess the blood serum biomarker profile in individuals with CLBP with MC. Several biomarkers were suppressed, while two markers (IL-1sRII and HGF) were elevated among MC patients, irrespective of MC type or size, with CLBP compared with asymptomatic controls.

Previously, we proposed the hypothesis that similarities in the inflammatory response observed in acne vulgaris and degenerative disc disease (DDD), especially the central role of interleukin (IL)-1β, may be further evidence of the role of the anaerobic bacterium Cutibacterium (previously Propionibacterium ) acnes in the underlying aetiology of disc degeneration. To investigate this, we examined the upregulation of IL-1β, and other known IL-1β-induced inflammatory markers and neurotrophic factors, from nucleus-pulposus-derived disc cells infected in vitro with C. acnes for up to 48 h. Upon infection, significant upregulation of IL-1β, alongside IL-6, IL-8, chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 4 (CCL4), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), was observed with cells isolated from the degenerative discs of eight patients versus non-infected controls. Expression levels did, however, depend on gene target, multiplicity and period of infection and, notably, donor response. Pre-treatment of cells with clindamycin prior to infection significantly reduced the production of pro-inflammatory mediators. This study confirms that C. acnes can stimulate the expression of IL-1β and other host molecules previously associated with pathological changes in disc tissue, including neo-innervation. While still controversial, the role of C. acnes in DDD remains biologically credible, and its ability to cause disease likely reflects a combination of factors, particularly individualised response to infection.

Vertebral endplate bone marrow lesions, visualized on magnetic resonance imaging (MRI) as Modic changes (MC), are associated with chronic low back pain (cLBP). Since guidelines recommend against routine spinal MRI for cLBP in primary care, MC may be underdiagnosed. Serum biomarkers for MC would allow early diagnosis, inform clinical care decisions, and supplement treatment monitoring. We aimed to discover biomarkers in the blood serum that correlate with MC pathophysiological processes. For this single-site cross-sectional study, we recruited 54 subjects with 38 cLBP patients and 16 volunteers without a history of LBP. All subjects completed an Oswestry Disability Index (ODI) questionnaire and 10-cm Visual Analog Score (VAS) for LBP (VASback) and leg pain. Lumbar T1-weighted and fat-saturated T2-weighted MRI were acquired at 3T and used for MC classification in each endplate. Blood serum was collected on the day of MRI. Biomarkers related to disc resorption and bone marrow fibrosis were analyzed with enzyme-linked immune-absorbent assays. The concentration of biomarkers between no MC and any type of MC (AnyMC), MC1, and MC2 were compared. The Area Under the Curve (AUC) of the Receiver Operating Characteristics were calculated for each biomarker and for bivariable biomarker models. We found that biomarkers related to type III and type IV collagen degradation and formation tended to correlate with the presence of MC ( p = 0.060-0.088). The bivariable model with the highest AUC was PRO-C3 + C4M and had a moderate diagnostic value for AnyMC in cLBP patients (AUC = 0.73, specificity = 78.9%, sensitivity = 73.7%). In conclusion, serum biomarkers related to the formation and degradation of type III and type IV collagen, which are key molecules in bone marrow fibrosis, correlated with MC presence. Bone marrow fibrosis may be an important pathophysiological process in MC that should be targeted in larger biomarker and treatment studies., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The aim of the current study was to compare changes in serum biomarkers, including inflammatory mediators, signaling molecules, growth factors and markers of bone turnover after a single intravenous infusion of 5 mg zoledronic acid (ZA, a long-acting bisphosphonate; n = 20) or placebo ( n = 20) among patients with Modic changes (MC) and chronic low back pain in a randomized controlled design. The MCs were classified into M1, predominating M1, predominating M2, and M2. We measured the serum concentrations of 39 biomarkers at baseline, and one month and one year after treatment. After Benjamini-Hochberg (B-H) correction, we observed significant differences in three biomarkers over one year: Interferon-γ-inducible protein (IP10) had risen in the ZA group (p = 0.005), whereas alkaline phosphatase (AFOS) and intact procollagen I N-terminal propeptide (iPINP) had significantly decreased in the ZA group, but had not changed in the placebo group (p < 0.001 for both). Change in iPINP correlated with change in the volume of all MC and M1 lesions. ZA downregulated bone turnover markers as expected and, surprisingly, increased the chemokine IP-10 relative to placebo treatment. This adds to our knowledge of the effects of ZA on MC and the biomarkers that signal this process., Competing Interests: The authors declare no conflict of interest.

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.)

Study Design: Research., Objective: The goal of this study was to investigate whether Propionibacteria acnes infection of the intervertebral disc can be detected noninvasively by nuclear magnetic resonance (NMR) spectroscopy., Summary of Background Data: Microbiological studies of surgical samples suggest that a significant subpopulation of back pain patients may have occult disc infection with P. acnes bacteria. This hypothesis is further supported by a double-blind clinical trial showing that back pain patients with Modic type 1 changes may respond to antibiotic treatment. Because significant side effects are associated with antibiotic treatment, there is a need for a noninvasive method to detect whether specific discs in back pain patients are infected with P acnes bacteria., Methods: P. acnes bacteria were obtained from human patients. NMR detection of a propionic acid (PA) in the bacteria extracts was conducted on 500 MHz high-resolution spectrometer, whereas in vivo NMR spectroscopy of an isolated bovine disk tissue infected with P. acnes was conducted on 7 T magnetic resonance imaging scanner., Results: NMR spectra of P. acnes metabolites revealed a distinct NMR signal with identical chemical shits (1.05 and 2.18 ppm) as PA (a primary P. acne metabolite). The 1.05 ppm signal does not overlap with other bacteria metabolites, and its intensity increases linearly with P. acnes concentration. Bovine disks injected with P. acnes bacteria revealed a very distinct NMR signal at 1.05 ppm, which linearly increased with P. acnes concentration., Conclusion: The 1.05 ppm NMR signal from PA can be used as a marker of P. acnes infection of discs. This signal does not overlap with other disc metabolites and linearly depends on P. acnes concentration. Consequently, NMR spectroscopy may provide a noninvasive method to detect disc infection in the clinical setting., Level of Evidence: N/A.

Purpose: Intervertebral disc with Propionibacterium acnes (P. acnes) is suggested to be an etiology of Modic type I changes in the adjacent bone marrow. However it is unknown if disc cells can respond to P. acnes and if bone marrow cells respond to bacterial and disc metabolites draining from infected discs., Methods: Human disc cells (n = 10) were co-cultured with 10- and 100-fold excess of P. acnes over disc cells for 3 h and 24 h. Lipopolysaccharide was used as positive control. Expression of IL1, IL6, IL8, and CCL2 by disc cells was quantified by quantitative PCR. Lipase activity was measured in culture supernatants (n = 6). Human vertebral bone marrow mononuclear cells (BMNCs) (n = 2) were cultured in conditioned media from disc cell/P. acnes co-cultures and expression of IL1, IL6, IL8, and CCL2 was measured after 24 h., Results: All disc cells responded to lipopolysaccharide but only 6/10 responded to P. acnes with increased cytokine expression. Cytokine increase was time- but not P. acnes concentration-dependent. Disc cell responsiveness was associated with the presence of lumbar Modic changes in the donor. Lipase activity was increased independent of disc cell responsiveness. BMNCs responded with inflammatory activity only when cultured in supernatants from responsive disc cell lines., Conclusion: Disc cell responsiveness to P. acnes associates with the presence of lumbar Modic changes. Furthermore, bone marrow cells had an inflammatory response to the cocktail of disc cytokines and P. acnes metabolites. These data indicate that low virulent P. acnes infection of the disc is a potential exacerbating factor to Modic changes.

Background Context: Modic changes (MCs) are magnetic resonance imaging (MRI) evidence of inflammatory and fibrotic vertebral bone marrow lesions that associate with adjacent disc degeneration and end plate damage. Although MC etiology is uncertain, historical data suggest a linkage to an autoimmune response of bone marrow triggered by the nucleus pulposus (NP)., Purpose: The aim of this study was to test whether bone marrow has an autoimmune response to NP cells that is amplified by an inflammatory milieu and ultimately leads to MC development in vivo. We hypothesized that an inflammatory co-stimulus is required for bone marrow/NP crosstalk to stimulate MC., Study Design: This is an in-vitro cell co-culture study plus in-vivo experiments in rat caudal vertebrae., Methods: In in-vitro study, bone marrow mononuclear cells (BMNCs) and NP cells (NPCs) from rats were co-cultured with and without interleukin (IL)-1α stimulation. Cell viability (n=3) of BMNCs and NPCs and gene expression (n=7) were analyzed. In in-vivo study, proinflammatory lipopolysaccharide (LPS) and control disc nucleus surrogates (NP micromass pellets) were generated in vitro from rat NPCs and implanted into rat tail vertebrae, and the response was compared with sham surgery (n=12 each). Tissue changes were investigated with T1w and T2w MRI (7T), histology, and immunohistochemistry (tumor necrosis factor, CD3) 1 (n=6) and 2 weeks (n=6) after implantation., Results: BMNC/NPC co-culture significantly increased lymphocyte viability (42%-69%, p<.05) and reduced NPC viability (96%-88%, p<.001), indicating immunogenicity of NPC. However, IL-1α was required to cause significant transcriptional upregulation of IL-1, IL-6, IL-10, and tropomyosin receptor kinase A. Therefore, an inflammatory activation is required to amplify the immune response. Immunogenicity of the NP was corroborated in vivo by CD3 cell accumulation around LPS and control disc surrogates at Day 7. However, only the LPS disc surrogate group demonstrated infiltration of CD3 cells at Day 14. Furthermore, end plate defects (p<.05, LPS: n=4/6, Ctrl: n=0/6, sham: n=0/6) and MC1-like MRI changes (T2w hyperintensity, p<.05) were only seen with LPS disc surrogates., Conclusions: NPCs are immunogenic but cannot trigger MC without an additional proinflammatory stimulus. Our data suggest that MC requires end plate defects that allow marrow/NPC co-mingling plus an adjacent inflammatory "MC disc" that can amplify the immune response., (Copyright © 2017 Elsevier Inc. All rights reserved.)