Abstract
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).
(a) GAG content as a percentage of the samples wet weight, bars indicate p < 0.05.
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