The role of the bone in complex regional pain syndrome 1-A systematic review

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

Development of a standardized histopathology scoring system for intervertebral disc degeneration in rat models: An initiative of the ORS spine section

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

Should Degenerated Intervertebral Discs of Patients with Modic Type 1 Changes Be Treated with Mesenchymal Stem Cells?

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

Do Cells of Intervertebral Discs with type 1 Modic Change on MRI show a History of Impaired Spinal Fusion?

INTRODUCTION: Type 1 Modic changes (MC1) in the spine on magnetic resonance imaging are believed to be related to changes in the crosstalk of the intervertebral disc (IVD) with the neighbouring bone-marrow of the vertebrae and to contribute to low back pain [1]. Here, we hypothesized that cells of the MC1 discs possess a specific transcriptome that is di!erent to relatively healthy non-degenerative cells coming from trauma. METHODS:13 IVDs were sampled with written consent from seven patients, four IVDs from three trauma (“control”) and nine IVDs from four chronic low back pain patients diagnosed with MC1. Total RNA was extracted and purified by silica columns from passage 0 cells that were expanded in normoxia in low glucose Dulbecco Modified Eagle Medium (DMEM) and 10% fetal calf serum after mild enzymatic overnight digestion with pronase/collagenase 2. From each IVD, nucleus pulposus cells (NPC) and annulus fibrosus cells (AFC) were sequenced, thus 26 samples. High-quality total RNA was prepared for sequencing using the Illumina TruSeq Stranded mRNA Library Prep assay. The samples were processed at the Functional Genomics Center of the University of Zürich and were run on an Illumina sequencer. Routine bioinformatic analyses were conducted. RESULTS: The di!erential expression analysis (DEGs) of the NGS data showed that 811 genes were di!erently either up or down-regulated comparing the two groups in NPC, 240 in AFC and 83 genes in both NPC and AFC when comparing four control IVDs compared to the nine MC1 discs. We found among MC1 and control cells that genes were di!erentially expressed involved in the matrix synthesis (e.g. COL9A3 -4.04 times down in MC1 (P = 0.00016)). In bone metabolism (e.g. SPP1 = secreted phosphoprotein 1 or better known as Osteopontin and SP7, aka Osterix) among many others (Fig. 1). We detected DEGs related to matrix production between MC1 and the “control” group: COL2A1 P = 0.0003 for NPC and 0.226 for AFC, respectively), SOX9 (P = 0.004 for NPC, and P < 0.0001 for AFC, respectively). Pathway overrepresentation analysis (PRA) revealed that inflammatory pathways and pathways in skeletal system development and ossification and cartilage development were significantly down-regulated in MC1

Bone marrow stromal cells in Modic type 1 changes promote neurite outgrowth

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

Protocol for Biospecimen Collection and Analysis within the BACPAC Research Program

The Biospecimen Collection and Processing Working Group of the 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

Intervertebral disc cell chondroptosis elicits neutrophil response in Staphylococcus aureus spondylodiscitis

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

CD90-positive stromal cells associate with inflammatory and fibrotic changes in modic changes

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. Keywords: Bone marrow oedema; Fibrosis; Inflammation; Low back pain; Modic changes

Intervertebral disc cell chondroptosis elicits neutrophil response in Staphylococcus aureus spondylodiscitis

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

Intervertebral disc microbiome in Modic changes: Lack of result replication underscores the need for a consensus in low-biomass microbiome analysis

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