Notochordal-cell derived extracellular vesicles exert regenerative effects on canine and human nucleus pulposus cells
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Frances Bach1, Sten Libregts2, Laura Creemers3, Björn Meij1, Keita Ito3,4, Marca Wauben2 and Marianna Tryfonidou1
1Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
2Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
3Department of Orthopaedics, University Medical Centre Utrecht, Utrecht, The Netherlands
4Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
Marianna Tryfonidou, email: firstname.lastname@example.org
Keywords: extracellular vesicles, intervertebral disc, notochordal cell, regeneration, nucleus pulposus
Received: July 08, 2017 Accepted: August 26, 2017 Published: October 04, 2017
During intervertebral disc ageing, chondrocyte-like cells (CLCs) replace notochordal cells (NCs). NCs have been shown to induce regenerative effects in CLCs. Since vesicles released by NCs may be responsible for these effects, we characterized NC-derived extracellular vesicles (EVs) and determined their effect on CLCs.
EVs were purified from porcine NC-conditioned medium (NCCM) through size exclusion chromatography, ultracentrifugation or density gradient centrifugation. Additionally, the EVs were quantitatively analyzed by high-resolution flow cytometry. The effect of NCCM-derived EVs was studied on canine and human CLC micro-aggregates in vitro and compared with NCCM-derived proteins and unfractionated NCCM.
Porcine NCCM contained a considerable amount of EVs. NCCM-derived EVs induced GAG deposition in canine CLCs to a comparable level as NCCM-derived proteins and unfractionated NCCM, and increased the DNA and glycosaminoglycan (GAG) content of human micro-aggregates, although to a lesser extent than unfractionated NCCM. The biological EV effects were not considerably influenced by ultracentrifugation compared with size exclusion-based purification. Upon ultracentrifugation, interfering GAGs, but not collagens, were lost. Nonetheless, collagen type I or II supplemented to CLCs in a concentration as present in NCCM induced no anabolic effects.
Porcine NCCM-derived EVs exerted anabolic effects comparable to NCCM-derived proteins, while unfractionated NCCM was more potent in human CLCs. GAGs and collagens appeared not to mediate the regenerative EV effects. Thus, NC-derived EVs have regenerative potential, and their effects may be influenced by the proteins present in NCCM. The optimal combination of NC-secreted factors needs to be determined to fully exploit the regenerative potential of NC-based technology.
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