INTRODUCTION: Notochordal cells (NCs) get lost in the disc nucleus by aging and degeneration. Two hypotheses exist: they might be subsequently replaced by chondrocytes from the endplate or annulus, or they might differentiate into mature disc cells resembling chondrocyte‐like cells. To gain further insight in the possible dedifferentiation of NCs into chondrocyte‐like cells, we analyzed expression of potential NC markers in immature pig tail discs, rich of NCs.
METHODS: For analysis, we chose cytokeratin‐8 (Cytk‐8), which is an accepted marker for NCs, and carbonic‐anhydrase III (CAIII) as a potential NC marker, as we could selectively determine CAIII in notochordal tissue by mass spectrometry. Using young pig tails, heterogenous notochordal shaped cells were isolated from the nucleus whereas uniform chondrogenic shaped small cells were isolated from the inner annulus, which was verified by fluorescence‐activated‐cell‐sorting‐analysis. For Cytk‐8 protein‐expression, we performed Western Blot of freshly isolated cells and Immunohistochemistry of pig‐tail slides. We performed Cytk‐8 and CAIII semiquantitative RT‐PCR; first, freshly isolated cells were analyzed, then cells after 3D alginate bead culture for 3, 7 and 14d.
RESULTS: Cytk‐8 protein was only detectable in the nucleus by both Western Blot and Immunohistochemistry (95% Cytk‐8 positive cells). Gene expression was detectable in both nucleus and annulus but was expressed much higher in the nucleus (Cytk‐8: 5X (p=0.0006); CAIII: 3X (p=0.04)). After cell culture, gene‐expression was maintained, though reduced compared with freshly isolated nucleus cells. Morphologically, nucleus cells lost their large inclusions and obtained a more chondrocyte phenotype.
DISCUSSION: The unique molecular pattern of NCs was maintained in cell culture, though significant morphological changes occurred. Hence, these markers may be attractive to detect notochordal origin in discs that lost notochordal morphology.