In this study, we identified two distinct cell populations within the bovine NP based on the expression of the intermediate filament protein CK8. We found by proteomic tools (Figure ), by immunostaining isolated cells (Figure ), and by immunostaining tissue sections (Figure ) that CK8 was exclusively present in the NP and was not seen in the AF or in articular cartilage. However, we also found that only approximately 10% of the NP cells were positive for CK8 (Figure ). These CK8-positive cells were not uniformly distributed throughout the tissue but were grouped in small clusters in isolated regions of the matrix which appeared more gelatinous than the surrounding matrix of the nucleus (Figure ); these clusters were found, independently of stages of maturity, in all discs examined (Figure ).
The apparent co-existence of distinct cell populations in the bovine disc raises a number of questions. First, as notochordal disc cells are known to express CK8 [19
], are the CK8-positive cells remnants of the original notochordal population of the disc? There are several indications that support this idea. Intermediate filaments such as CK8 are often used to classify the origin of a tissue as differentiated cells usually express only one intermediate filament type; tissues are classified as epithelial when expressing CK8 [36
] or as mesenchymal when they express vimentin [33
]. Intermediate filaments from two different families can be expressed simultaneously, however, in tumors or in development [37
]. Disc notochordal cells, epithelial in origin [19
], are CK8-positive as expected but are also positive for vimentin [19
] as we saw in the porcine discs (Figures and ). All bovine NP and also all AF cells were positive for vimentin (Figure ) as reported previously [35
]. Thus, like disc notochordal cells [19
], the CK8 subpopulation of the bovine NP is also vimentin-positive (Figures and ), supporting the idea that these cells could have originated from the original population of notochordal cells. This hypothesis is strengthened by the organization of the bovine CK8-positive cells in clusters and by the more gelatinous texture of the surrounding matrix (Figures and ), both features of the notochordal nucleus. However, notochordal cells of the NP have a morphology very different from that of chondrocyte-like nucleus cells as the former are markedly greater in diameter and contain large vacuoles [20
]; indeed, notochordal cells of the disc are identified mostly by their morphological features [31
]. Thus, if the CK8-positive cells are notochordal-like, they would have had to undergo a large size reduction since we found that the size distributions of CK8-positive and -negative cell populations were very similar (Figure ). Such loss of size of NP cells has been seen experimentally; the diameter of porcine notochordal cells decreased markedly over 15 days in culture with loss of vacuoles and approached that of chondrocyte-like NP cells [40
]. In addition, rabbit notochordal cells have been shown to differentiate toward 'chondrocyte-like' cells when maintained in culture [41
]. Thus, on balance, it seems likely that the CK8-positive cells are remnants of the original cell population of the bovine disc. The question, however, could be resolved definitively by gene or proteomic profiling of the two cell populations; at present, only gene profiles from rat notochordal cells and mature chondrodystrophoid canine disc tissue are available [14
Second, if, as we suggest above, the bovine disc contains a notochordal-like cell population, does the adult human disc do so too? The NP of bovine discs is reported to be similar to that of human discs in regard both to matrix composition and to cell phenotype [17
]; thus, if notochordal remnants are present in bovine discs, it is possible that they are also present in adult human discs. If, as in bovine discs, the majority of any remaining CK8-positive cells shrink and lose vacuoles (Figure ), only the small fraction of larger cells would be identifiable morphologically as notochordal cells; indeed, some few cells resembling notochordal cells have been reported in adult discs [43
]. Immunohistochemical studies have, however, identified cytokeratin-positive cells and also the co-expression of CK8 and vimentin in adult human NP [45
], but whether all cells or only a subpopulation was immunopositive was not stated in these reports.
If, as these various reports suggest, cells from the original notochordal population are retained in discs regarded as non-notochordal, do these cells have any functional significance? Notochordal cell-conditioned medium or co-culture of notochordal and adult NP cells has been found to stimulate matrix production by adult NP cells; in addition, notochordal cells are reported to retard disc degeneration when inserted into damaged rabbit discs [47
]. These results could be explained by the finding that notochordal cells secrete growth factors that stimulate production of extracellular matrix [21
]. It has also been suggested that notochordal cell remnants could serve as a stem cell population [20
]; indeed, in preliminary experiments, we find that the CK8 population proliferates faster than the CK8-negative population in vitro
(data not shown). Functionally distinct non-chondrocytic subpopulations have been identified within the NP of adult human discs [52
], indicating the possibility that some notochordal cells survive and remain active. Could it be that the presence of these resident notochordal cells and the growth factors they secrete help in the maintenance of a healthy disc, as suggested by Aguiar and colleagues [47
] in a study on dog discs, and that these cells are thus essential for disc homeostasis?
Finally, does the presence of CK8-positive cells provide any information on the origin of the chondrocyte-like cells of the adult human or bovine disc? Studies in rabbits suggest that notochordal cells die off to be replaced by mesenchymal cells originating in the inner annulus or cartilage end-plate [32
]. However, it has also been suggested that notochordal cells could differentiate into the adult disc nucleus cell phenotype [20
] since chordomas, which arise from embryonic notochordal remnants [13
], show chondrogenic potential and can differentiate into cartilage-type cells expressing collagen II and aggrecan [53
]. Perhaps the possible differentiation pathway from notochordal to mature NP discs should be revisited.