Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION: A new promising treatment for intervertebral disc (IVD) degeneration-related low back pain is represented by extracellular vesicles (EVs), cell-derived membrane-bound vesicles that carry bioactive molecules and deliver them to recipient cells. EVs derived from notochordal cells (NCs) residing within the IVD core of young pigs have been shown to induce proliferation of degenerate human and dog nucleus pulposus cells (NPCs) and limited healthy extracellular matrix production (1). Considering that NCs maintain disc homeostasis, we hypothesize that pro-degenerative stimuli induce NCs to produce more potent regenerative EVs, in the attempt to counterbalance the micro-environment stress.

METHODS: NC-rich explants were isolated from one porcine spine and cultured at 37⁰C, 5% O₂ for 4 days in low glucose (1 gr/L) DMEM, 7.1 pH, 450 mOsm medium (healthy, H-EVs), or 6.8 pH, 350 mOsm medium (pro-degenerate, D-EVs). The effect of culture media on NP explants was tested qualitatively via (immuno)stainings for matrix deposition and NCs phenotype and quantitatively measuring inflammatory factors (PGE2 and CCL2) production and proteoglycans release into the medium. NC-EVs were isolated from the conditioned media via differential centrifugation, followed by Size Exclusion Chromatography. The particle size and NC-EVs concentration were determined via Nanoparticle Tracking Analysis. The regenerative potential of H-EV and D-EV, including their respective EV-depleted controls, was tested on NPC pellets using histological and biochemical assays.

RESULTS: Histological analysis showed a slight reduction of matrix content within the NP explant after 4 days of culturing in both conditions compared to the baseline (Fig. 1). However, the proteoglycans amount released from the NP explants into the conditioned media, as well as the inflammatory factors production, were comparable among both conditions. Additionally, the NC-markers KRT8+18+19 expression was conserved in both conditions upon NP explant culture (Fig. 1). Comparable average number and size of D-EVs (3*10¹⁰ particles/gram tissue; 199 nm) and H-EVs (2.8*10¹⁰ particles/gram tissue; 195 nm) were isolated from the conditioned media. Histological and biochemical analysis showed comparable GAG content and DNA levels in NPC pellets treated with D-EVs and H-EVs, and their respective EV-depleted controls. Nonetheless, the total GAG produced (pellet content + released in medium) by NPCs treated with D-EVs was 2.8-fold higher compared to H-EVs. Furthermore, both H- and D-EVs induced higher total GAG production than the respective EV-depleted controls (3.4-fold and 14.5-fold, respectively).

CONCLUSIONS: Even though the prodegenerative stimulus on NC-rich NP explants did not seem to distinctly affect matrix quality, tissue inflammation, cell viability and phenotype, and resulted in conditioned media with comparable particle concentration, this pro-degenerative environment promoted the regenerative effect of NC-EVs compared to those secreted under healthy conditions. This preliminary study showed an EV-specific positive effect on healthy matrix production by NPCs derived from degenerate discs. These data suggest that the difference between the D-EV and H-EV anabolic effects on NPCs resides within the EV cargo, rather than the particle number. Therefore, follow-up work focuses on replicating the work and expanding on proteomic and RNA-sequencing studies, aiming to decode the differential EVs content composition.