Abstract
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.
Highlights
Low back pain, affecting up to 85% of the population and resulting in considerable socioeconomic consequences [1, 2], has been associated with intervertebral disc (IVD) degeneration [3]
Porcine notochordal cells (NCs)-conditioned medium (NCCM)-derived extracellular vesicle (EV) exerted anabolic effects comparable to NCCM-derived proteins, while unfractionated NCCM was more potent in human chondrocyte-like cell (CLC)
EVqEV: NCCM-derived EVs obtained after size exclusion chromatography (SEC), EVUC: NCCM-derived EVs obtained after size-exclusion chromatography (SEC) and subsequent 100,000g ultracentrifugation (UC), PqEV: NCCM-derived proteins obtained after SEC, PUC: NCCMderived proteins obtained after SEC and subsequent 100,000g UC
Summary
Low back pain, affecting up to 85% of the population and resulting in considerable socioeconomic consequences [1, 2], has been associated with intervertebral disc (IVD) degeneration [3]. Since dogs experience back pain and IVD degeneration with similar characteristics as humans, they are considered a suitable animal model for human IVD degeneration [4, 5]. GAGs indirectly attract water, and in this way the IVD provides a shock absorption function for the spine. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the NP. When the IVD degenerates, the CLCs are not able to maintain healthy tissue anymore. The CLCs become depleted, the GAG and water content decreases and collagen type II is replaced by collagen type I, resulting in a more fibrous tissue. The avascular IVD shows inadequate repair, and a vicious circle develops in which the IVD experiences increased vulnerability to damage by physiologic loading [7]
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