Title: Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration

Authors: FC Bach, SAH de Vries, FM Riemers, J Boere, FWM van Heel, M van Doeselaar, SS Goerdaya, PGJ Nikkels, K Benz, LB Creemers, AF Maarten Altelaar, BP Meij, K Ito, MA Tryfonidou

Address: Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 108, 3584 CM Utrecht, the Netherlands

E-mail: m.a.tryfonidou at uu.nl

Key Words: Intervertebral disc degeneration, nucleus pulposus, notochordal cells, notochordal cell-conditioned medium, proteomics, extracellular vesicles, regenerative medicine, canine, porcine, human.

Publication date: August 30th 2016

Abstract: During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

Article download: Pages 163-180 (PDF file)
DOI: 10.22203/eCM.v032a11