Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture
Authors: de Vries Stefan , van Doeselaar Marina , Meij Björn , Tryfonidou Marianna , Ito Keita 
Published: May 2015

Introduction

Notochordal cell-conditioned medium (NCCM) has shown to stimulate matrix production by nucleus pulposus cells (NPCs) in alginate bead cultures.1,2 This cell culture method, however, provides a markedly different environment than the nucleus pulposus (NP) tissue, in which the bioactive factors should exert their effect. Therefore, the main objective of this study was to test the stimulatory effect of NCCM in an NP explant culture system.

Materials and Methods

NCCM was generated by incubating porcine NC-rich NP tissue in plain high glucose DMEM (hgDMEM) at 37°C, 5% CO2 and 5% O2 for 4 days. The medium was filtered and the filtrate was resuspended in fresh hgDMEM. NP tissue samples were isolated from the caudal IVDs of 2-year old cows ( n = 5), excluding the annulus fibrosus (AF) tissue. The NP tissues were cultured, according to a previously established method,3 in a fiber jacket, constraining the tissue and thereby acting as an AF. Samples were cultured in base medium (BM: hgDMEM supplemented with pen/strep, ITS, ascorbic acid, proline, bovine serum albumin, and sodium pyruvate), NCCM, supplemented with the same factors as BM, or positive control medium (Pos, BM supplemented with 1 µg/mL Link-N). After 4 weeks, NP tissue explants were assayed for water, GAG, DNA, and hydroxyproline content, histology with safranin O/fast green staining and gene expression of collagen type 1 and 2, aggrecan, and SOX9.

Results

The GAG content of tissue explants treated with NCCM significantly increased ( p < 0.05) compared with day 0 and BM ( Fig. 1a ). GAG content in the positive control group tended to increase compared with BM ( p = 0.086). This was confirmed by safranin O/fast green staining, showing a more intense red staining for the NCCM culture group ( Fig. 1b ). No significant differences were observed between groups regarding water, DNA, and hydroxyproline content. Gene expression results did not confirm the finding of the biochemical content assays. The relative expression of collagen type 1 and 2, aggrecan and SOX9 did not differ between groups, and only minor changes were observed compared with day 0. NCCM may have an initial and a transient stimulatory effect on transcriptional level, which could explain why the stimulatory effect of NCCM after 4 weeks of culture was not observed at the gene level. Nevertheless, we cannot exclude that GAGs originating from the NCCM may have been incorporated into the NP tissue explant during culture.

Conclusion

These results confirm the stimulatory effect of NCCM in an NP tissue explant culture. Moreover, NCCM appears to be at least as potent as Link-N, of which the stimulatory effect on NP tissue has been previously established in vivo.4 This study, therefore provides further evidence for the promising role of NCCM in the treatment of IVD regeneration. Identification of the bioactive factors in NCCM will be helpful to further develop a treatment method and achieve maximal anabolic effects.

Acknowledgment

This work was supported by AOSpine International through an AOSpine Research Network grant (SRN2011_11). [Figure: see text] References Potier E, de Vries S, van Doeselaar M, Ito K. Potential application of notochordal cells for intervertebral disc regeneration: an in vitro assessment. Eur Cell Mater 2014;28:68–80, discussion 80–81 Abbott RD, Purmessur D, Monsey RD, Iatridis JC. Regenerative potential of TGFb3 + Dex and notochordal cell conditioned media on degenerated human intervertebral disc cells. J Orthop Res 2012;30(3):482–488 van Dijk BG, Potier E, Ito K. Long-term culture of bovine nucleus pulposus explants in a native environment. Spine J 2013;13(4):454–463 Mwale F, Masuda K, Pichika R, et al. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011;13(4):R120