Oral Presentation 51st International Society for the Study of the Lumbar Spine Annual Meeting 2025

Extracellular vesicles derived from primed MSCs modulate intervertebral disc metabolism in C6-deficient mouse spine cultures (114778)

Graciosa Quelhas Teixeira 1 , David Arff 1 , Julia Kräutler 1 , Mubashir Ahmad 1 , Melanie Haffner-Luntzer 1 , Anita Ignatius 1 , Cornelia Neidlinger-Wilke 1
  1. Institute of Orthopaedic Research and Biomechanics, Ulm University, Ulm, BADEN-WüRTTEMBERG, Germany

INTRODUCTION

The terminal complement complex (TCC), a key component of innate immunity, promotes cell lysis andinflammation, and is involved in intervertebral disc (IVD) degeneration.1 This study investigated the role of complement activation in IVD degeneration and its therapeutic modulation. Specifically, the therapeutic potential of extracellular vesicles (EVs) derived from interleukin (IL)-1β-primed mesenchymal stem cells was investigated, because previous studies have shown that the secretome of MSCs influences complement regulation, inflammation, and matrix metabolism in IVD cells.2 This research focuses on the effects of IL-1β-primed MSC-derived EVs in mouse spine cultures deficient in C6,3 a TCC component.

METHODS

MSCs from C57BL/6J mice were cultured with or without 1 ng/mL IL-1β for 48h (n=3), and EVs were isolated from their secretome by ultracentrifugation. Control- and IL-1β-EV were characterized for morphology and size. Secretome and EVs were analysed by proteomics. Mouse lumbar spines from both C57BL/6J wild-type and C6-deficient mice (n=4-6, with ethical approval) were then cultured with either i) mouse serum alone (MS), or including ii) IL-1β, or iii) IL-1β+IL-1β-EV. After two days, gene expression of anti-apoptotic (Bcl2), complement regulatory (Cd46, Cd55, Cd59), inflammation (Il6), and matrix metabolism (Mmp3, Col1a1, Col2a1) markers was measured. Disc degeneration was assessed after 14 days through histological staining and modified Thompson scale.4 Statistical analysis was conducted using Kruskal-Wallis or one-way ANOVA test (significance, p<0.05).

RESULTS

Control- and IL-1β-EV displayed typical size distribution (diameter: 105±53 nm,  polydispersity index: 0.30). Proteomic analysis indicated distinct protein profiles in IL-1β-Sec and IL-1β-EV (Fig. 1A), with 36 proteins shared between IL-1β-EV and IL-1β-Sec (Fig. 1B). IL-1β-EV were enriched in pathways related to inflammatory response regulation, complement system processes, and extracellular matrix organization (Fig. 1C,D). In IVDs from wild-type mice, IL-1β stimulation upregulated pro-inflammatory and catabolic markers Il6and Mmp3 (p<0.05, Fig. 2), while IL-1β-EV treatment upregulated anti-apoptotic (Bcl2) and complement regulatory (Cd55) markers compared to the control and IL-1β groups (p<0.05). IL-1β increased degeneration scores in the annulus fibrosus and in the nucleus pulposus (p<0.05), but these scores remained similar to control levels with IL-1β-EV treatment. In IVDs from C6-deficient mice, IL-1β upregulated Il6 and Mmp3(p<0.05), while IL-1β-EV treatment mitigated these effects, reducing the inflammatory response without significantly altering degeneration scores.

DISCUSSION

This organ culture model enabled the investigation of IVD degeneration ex vivo while incorporating TCC deficiency to examine complement-mediated inflammation's role in tissue degradation and repair. IL-1β priming notably altered the molecular composition of MSC-derived EVs, with enrichment in processes and pathways associated with complement regulation. In C6-deficient spine cultures, IL-1β-EVs reducel local inflammation and tissue degeneration, suggesting that TCC may counteract the beneficial effects of EVs. These findings indicate the therapeutic potential of IL-1β-EVs as a novel treatment for spinal disorders associated with complement dysregulation and validate the mouse spine culture as a promising research model prior to in vivo studies. Funding. Deutsche Wirbelsäulenstiftung

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  2. Neidlinger-Wilke C, Ekkerlein A, Goncalves RM, Ferreira JR, Ignatius A, Wilke HJ, Teixeira GQ. Mesenchymal stem cell secretome decreases the inflammatory response in annulus fibrosus organ cultures. Eur Cell Mater. 2021;42:1-19.
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