Human nucleus pulposus cell cultures and disc degeneration grading systems: comment on the article by Le Maitre et al.

To the Editor: In the recent article by Le Maitre et al, the authors compare the integrin mechanotransduction pathways in degenerated human nucleus pulposus (NP) cells with those in normal human NP cells, after cells were subjected to dynamic compression loading (1). NP cells exhibiting various grades of degeneration were loaded into a three-dimensional culture system and were suspended in alginate, in which NP cells exhibit spherically shaped morphology and a chondrocyte-like phenotype (2). Dynamic compression loading, as used in the study by Le Maitre and colleagues, simulates the physiologic daily load of the spine in order to determine the underlying mechanisms leading to disc degeneration. Notwithstanding the progress that has been made in understanding disc cell biology in the past decades, investigations into universal human NP cell lines and their application in disc cell research are still in the earliest stages. The reasons for this might include both the inherently low NP cellularity in human tissue, with NP cells making up only 1% of the total volume (3), and limited tissue specimens (4). Hitherto, only one human NP cell line, HNPSV-1 (derived from a 19-year-old man with a lumbar burst fracture), has been investigated (5), but the cell line is in monolayer culture and, consequently, loses the NP cell phenotype (6). Besides, HNPSV-1 is not commercially available. Nevertheless, NP cells in threedimensional scaffolds (e.g., alginate in the study by Le Maitre et al) can be redifferentiated (7). The histology of NP cells in such scaffolds, however, is different from that in vivo. For example, the capsule around NP cells in vivo, which is a distinct pericellular matrix or “nest” (8), is lost in cultures using alginate (9). Nevertheless, among studies, there are variations in the conditions of monolayer cultures of human NP cells (1,5,10,11), including variations in diverse digestion medium and duration. These scenarios reflect the earliest stages of investigations into NP cells in human intervertebral discs. In our own preliminary study, human NP cell culture conditions were consistent with those described by both Le Maitre et al (1) and Sakai et al (5), but differed with regard to the process of digestion with collagenase overnight, as described in other studies (10,11). Furthermore, we found that cell clusters, as one of the hallmarks of degeneration, diminished gradually in a monolayer culture within 48 hours of initial culture (Figure 1). Additionally, Le Maitre and colleagues analyzed and graded discs for the degree of degeneration according to the classification system based on histologic results proposed by Sive et al (12). A variety of systems for classifying disc degeneration have been described previously, including systems using gross macroscopy (13), histology (12), and magnetic resonance imaging (MRI) (14,15). However, there has been neither a universally accepted classification system nor a comparative study of the various existing systems, factors which might limit the application of research into disc biology and potential degenerative mechanisms. In our opinion, the classification system using MRI proposed by Pfirrmann et al (14) appears to be simple and accurate. In comparison, the classification system using histology is relatively complicated and demanding with regard to specimens. Briefly, in such a system, specimens should be intact and should have similar NP and annulus fibrosus tissue, with subsequent slides at the parasagittal planes. Given the fact that discs undergo degenerative changes earlier in life than other tissue (16), the “normal” human disc described in the study by Le Maitre and colleagues is normal only to a certain extent. In fact, age-related alterations in human discs occur in the first decade of life, as early as 2 years of age (16). Age-related morphologic changes include cleft, cluster, and nest formations (8,17), as well as an increased number of necrotic NP cells (8). As a consequence, studies using grading systems to investigate degeneration should exclude age as a factor by selecting age-matched cases. In the study by Le Maitre et al, normal and degenerated tissue for NP cell cultures was obtained from controls (mean age 48 years, range 18–79 years) and patients (mean age 51 years, range 35–75 years), respectively, who were relatively inconsistently matched for age (1). In summary, the study by Le Maitre and colleagues contributes greatly to an understanding of mechanotransduction mechanisms in human NP cells. However, research on Figure 1. Cell cluster formation of human nucleus pulposus cells in a monolayer culture within 48 hours of initial culture. Cells were obtained from a 37-year-old man with disc degeneration classified as grade IV according to the grading system established by Pfirrmann et al (14). Cell cluster formation diminished gradually in culture (original magnification 400).