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

Superiority of nucleus pulposus cell- versus mesenchymal stromal cell-derived extracellular vesicles in attenuating disc degeneration and alleviating pain (#PP-1a)

Luca Ambrosio 1 2 , Jordy Schol 1 , Clara Ruiz-Fernandez 1 3 , Shota Tamagawa 1 4 , Hazuki Soma 1 , Veronica Tilotta 2 , Giuseppina Di Giacomo 2 , Claudia Cicione 2 , Shunya Nakayama 5 , Kosuke Kamiya 5 , Rocco Papalia 2 , Masato Sato 1 , Gianluca Vadalà 2 , Masahiko Watanabe 1 , Vincenzo Denaro 2 , Daisuke Sakai 1
  1. Department of Orthopaedic Surgery, Tokai University School of Medicine, Isehara, Japan
  2. Department of Orthopaedic and Trauma Surgery, Campus Bio-Medico University of Rome, Rome, Italy
  3. Neuroendocrine Interactions in Rheumatology and Inflammatory Diseases, Santiago University Clinical Hospital, Santiago de Compostela, Spain
  4. Department of Medicine for Orthopaedics and Motor Organ, Juntendo University Graduate School of Medicine, Tokyo, Japan
  5. Department of Hematological Malignancy, Tokai University School of Medicine, Isehara, Japan

Introduction: Despite promising results in attempting intervertebral disc regeneration, intradiscal cell transplantation is affected by several drawbacks, including poor viability in the harsh disc environment, low cost-effectiveness, and immunogenic/tumorigenic concerns1. Recently, the development of cell-free approaches is gaining momentum in the field, with a particular regard towards extracellular vesicles (EVs)2. Nucleus pulposus cell (NPC) progenitors characterized by Tie2 expression have shown a higher chondrogenic differentiation potential compared to bone marrow-derived mesenchymal stromal cells (BM-MSCs)3. This study aimed to investigate the putative regenerative effects of EVs isolated from Tie2-overexpressing NPC progenitors on degenerative NPCs in vitro and in an in vivo rat model of intervertebral disc degeneration (IDD). We hypothesized that such EVs would exert a higher anabolic and anticatabolic effect compared to EVs derived from BM-MSCs and differentiated NPCs characterized by a low Tie2 expression.

Methods: NPCs were isolated from young donors (n=6) and underwent an optimized culture protocol to maximize Tie2 expression (NPCTie2+)4 or a standard culture protocol (NPCSTD). EVs were extracted from NPCTie2+ and NPCSTD and characterized according to the ISEV guidelines5. NPCs isolated from surgical specimens of patients with IDD (n=6) were treated with either NPCTie2+-EVs or NPCSTD-EVs, with or without 10 ng/mL interleukin (IL)-1β. Cell proliferation and viability were assessed with the CCK-8 assay. Cell senescence was investigated with β-galactosidase staining. EV uptake was assessed with PKH26 staining of EVs under confocal microscopy. IDD was induced by annular puncture of 3 caudal discs in 16 Sprague Dawley rats aged between 10 and 12 weeks. Animals were randomly assigned to sham injection, NPCTie2+-derived EV, NPCSTD-derived EV, or BM-MSC-derived EV intradiscal injection (n=4 per group) and sacrificed at 12 weeks. Behavioral tests (Von Frey and open field test), radiographic disc height index (DHI) measurements, evaluation of serum pain biomarkers, and histological analyses (hematoxylin/eosin, Safranin-O/Fast-Green, and Picrosirius-red staining) were performed to assess the outcomes of injected EVs.

Results: NPC-derived-EVs exhibited the typical exosomal morphology and were efficiently internalized by degenerative NPCs, enhancing cell proliferation, and reducing senescence, especially in samples treated with NPCsTie2+-EVs (Fig. 1). In vivo, a single injection of NPC-derived-EVs preserved DHI and notably reduced mechanical hypersensitivity. Discs treated with NPC-derived EVs showed significantly lower Thompson scores and attenuated tissue degenerative changes at histological analyses compared to the sham group and samples treated with BM-MSC-derived EVs (Fig. 2). BM-MSC-derived-EVs showed marginal improvements over sham controls across all measured outcomes.

Discussion: Our results underscore the regenerative potential of young NPC-derived EVs, particularly NPCTie2+-EVs, surpassing MSC-derived counterparts. The use of committed progenitors naturally residing within the nucleus pulposus may optimize EV regenerative properties and constitute the basis for a new therapy for IDD. Further in vitro characterization and applications in larger animals are warranted to confirm and expand the preliminary knowledge base provided by this study. These findings raise questions about the validity of MSCs as both EV sources and cellular therapeutics against IDD. The study emphasizes the critical influence of cell type, source, and culture conditions in EV-based therapeutics.

 

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