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 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.
Tendons are among the most mechanically stressed tissues of the body, with a functional core of type-I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load-bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial-like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell-laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146 + tendon stem/progenitor cell and CD45 + F4/80 + bone-marrow derived macrophage activity within a tendon injury-like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone-marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80 + "tenophages" from the intrinsic tissue core.
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.
Background:Modic type 1 changes (MC1) are vertebral bone marrow (BM) edema that associate with non-specific low back pain (LBP). Two etiologies have been described. In the infectious etiology the anaerobic aerotolerant Cutibacterium acnes (C. acnes) invades damaged intervertebral discs (IVDs) resulting in disc infection and endplate damage, which leads to the evocation of an immune response. In the autoinflammatory etiology disc and endplate damage lead to the exposure of immune privileged disc cells and matrix to leukocytes, thereby evoking an immune response in the BM. Different etiologies require different treatment strategies. However, it is unknown if etiology-specific pathological mechanisms exist.Objectives:The aim of this study was to identify etiology-specific dysregulated pathways of MC1 and to perform in-depth analysis of immune cell populations of the autoinflammatory etiology.Methods:BM aspirates and biopsies were obtained from LBP patients with MC1 undergoing spinal fusion. Aspirates/biopsies were taken prior screw insertion through the pedicle screw trajectory. From each patient, a MC1 and an intra-patient control aspiration/biopsy from the adjacent vertebral level was taken. If C. acnes in IVDs adjacent to MC1 were detected by anaerobic bacterial culture, patients were assigned to the infectious, otherwise to the autoinflammatory etiology.Total RNA was isolated from aspirates and sequenced (Novaseq) (infectious n=3 + 3, autoinflammatory n=5 + 5). Genes were considered as differentially expressed (DEG) if p-value < 0.01 and log2fc > ± 0.5. Gene ontology (GO) enrichment was performed in R (GOseq), gene set enrichment analysis (GSEA) with GSEA software.Changes in cell populations of the autoinflammatory etiology were analyzed with single cell RNA sequencing (scRNAseq): Control and MC1 biopsies (n=1 + 1) were digested, CD45+CD66b- mononuclear cells isolated with fluorescence activated cell sorting (FACS), and 10000 cells were sequenced (10x Genomics). Seurat R toolkit was used for quality-control, clustering, and differential expression analysis.Transcriptomic changes (n=5 + 5) of CD45+CD66b+ neutrophils isolated with flow cytometry from aspirates were analyzed as for total bulk RNAseq. Neutrophil activation (n=3 + 3) was measured as CD66b+ expression with flow cytometry. CD66bhigh and CD66blow fractions in MC1 and control neutrophils were compared with paired t-test.Results:Comparing MC1 to control in total bulk RNAseq, 204 DEG in the autoinflammatory and 444 DEG in the infectious etiology were identified with only 67 shared genes (Fig. 1a). GO enrichment revealed “T-cell activation” (p = 2.50E-03) in the autoinflammatory and “complement activation, classical pathway” (p=1.1E-25) in the infectious etiology as top enriched upregulated biological processes (BP) (Fig 1b). ScRNAseq of autoinflammatory MC1 showed an overrepresentation of T-cells (p= 1.00E-34, OR=1.54) and myelocytes (neutrophil progenitor cells) (p=4.00E-05, OR=2.27) indicating an increased demand of these cells (Fig. 1c). Bulk RNAseq analysis of neutrophils from the autoinflammatory etiology revealed an activated, pro-inflammatory phenotype (Fig 1d), which was confirmed with more CD66bhigh neutrophils in MC1 (+11.13 ± 2.71%, p=0.02) (Fig. 1e).Figure 1.(a) Venn diagram of DEG from total bulk RNAseq (b) Top enriched upregulated BP of autoinflammatory (left) and infectious (right) etiology (c) Cell clustering of autoinflammatory MC1 BM (d) Enrichment of “inflammatory response” gene set in autoinflammatory MC1 neutrophils (e) Representative histogram of CD66b+ expression in MC1 and control neutrophils.Conclusion:Autoinflammatory and infectious etiologies of MC1 have different pathological mechanisms. T-cell and neutrophil activation seem to be important in the autoinflammatory etiology. This has clinical implication as it could be explored for diagnostic approaches to distinguish the two MC1 etiologies and supports developing targeted treatments for both etiologies.Disclosure of Interests:None declared
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 a role of CRP in the tissue pathophysiology of MC1 bone marrow. From thirteen MC1 patients undergoing spinal fusion at MC1 levels, vertebral bone marrow aspirates from MC1 and intra-patient control bone marrow were taken. Bone marrow CRP, 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 PCR. Increased CRP in MC1 bone marrow (mean difference: +0.22 mg CRP/g protein, 95% CI [-0.04, 0.47], p=0.088) correlated with blood CRP (r=0.69, p=0.018), with bone marrow IL-1 (=0.52, p=0.029) and IL-6 (=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.
FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with remarkable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 activation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of responsiveness to light activation. These results demonstrate unexpected cell type–specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.
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