The fate of notochord cells during disc development and aging is still a subject of debate. Cells with the typical notochordal morphology disappear from the disc within the first decade of life. However, the pure morphologic differentiation of notochordal from non-notochordal disc cells can be difficult, prompting the use of cellular markers. Previous reports on these notochordal cell markers only explored the occurrence in young age groups without considering changes during disc degeneration. The aim of this study, therefore, was to investigate presence, localization, and abundance of cells expressing notochordal cell markers in human lumbar discs during disc development and degeneration. Based on pilot studies, cytokeratins CK-8, -18 and -19 as well as Galectin-3 were chosen from a broad panel of potential notochordal cell markers and used for immunohistochemical staining of 30 human lumbar autopsy samples (0-86 years) and 38 human surgical disc samples (26-69 years). In the autopsy group, 80% of fetal to adolescent discs (0-17 years) and 100% of young adult discs (18-30 years) contained many cells with positive labeling. These cells were strongly clustered and nearly exclusively located in areas with granular changes (or other matrix defects), showing predominantly a chondrocytic morphology as well as (in a much lesser extent) a fibrocytic phenotype. In mature discs (31-60 years) and elderly discs (≥ 60 years) only 25 and 22-33%, respectively, contained few stained nuclear cells, mostly associated with matrix defects. In the surgical group, only 16% of samples from young adults (≤ 47 years) exhibited positively labeled cells whereas mature to old surgical discs (>47 years) contained no labeled cells. This is the first study describing the presence and temporo-spatial localization of cells expressing notochordal cell markers in human lumbar intervertebral discs of all ages and variable degree of disc degeneration. Our findings indicate that cells with a (immunohistochemically) notochord-like phenotype are present in a considerable fraction of adult lumbar intervertebral discs. The presence of these cells is associated with distinct features of (early) age-related disc degeneration, particularly with granular matrix changes.

While there is consensus in the literature that blood vessels are confined to the outer anulus fibrosus of normal adult intervertebral disc, debate continues whether there is a vascular in-growths into inner parts of the intervertebral disc during degeneration. We therefore tested the hypothesis that vascular in-growth is not a distinct feature of disc degeneration. The specific endothelial cell marker CD 31 (PECAM) was used to immunohistochemically investigate 42 paraffin-embedded complete mid-sagittal human intervertebral disc sections of various ages (0-86 years) and varying extent of histomorphological degeneration. Additionally, 20 surgical disc samples from individuals (26-69 years) were included in this study. In discs of fetal to infantile age, blood vessels perforated the cartilaginous end plate and extended into the inner and outer anulus fibrosus, but not into the nucleus pulposus. In adolescents and adults, no blood vessels were seen except for the outer zone of the anulus fibrosus adjacent to the insertion to ligaments. The cartilaginous end plate remained free of vessels, except for areas with circumscribed destruction of the end plate. In advanced disc degeneration, no vessels were observed except for those few cases with complete, scar-like disc destruction. However, some rim lesions and occasionally major clefts were surrounded by a small network of capillary blood vessels extending into deeper zones of the anulus fibrosus. A subsequent morphometric analysis, revealed slightly "deeper" blood vessel extension in juvenile/adolescent discs when compared to young, mature and senile adult individuals with significantly "deeper" extension in the posterior than anterior anulus. The analysis of the surgical specimens showed that only sparse capillary blood vessels which did not extend into the nucleus pulposus even in major disc disruption. Our results show that vascular invasion deeper than the periphery was not observed during disc degeneration, which supports the hypothesis that vascular in-growth is not a distinct feature of disc degeneration.