Introduction: Degenerative disc disease is the leading cause of disability and back pain. However the molecular signals that guide the normal development, and those involved in the degeneration of disc are not well defined. Nucleus pulposus (NP) cells of the disc are known to originate from embryonic notochord that act during embryogenesis as an important signaling center in patterning of neural crest and somites.
Objectives: We hypothesize that NP cells continue to be an active signaling center during the postnatal stages, and are required for the maintenance of the annulus fibrosus (AF) and endplate (EP) cells.
Methods: 1–4 days old mice discs were cultured in DMEM Ham/F-12 medium containing fetal calf serum (FCS) or insuline-transferrin-sodium-selenite (ITS) supplement at 37°C in 5% CO2 for 2, 4, 6 and 8 days on Collagen IV coated inserts, and snap frozen at the end of the culture. Cryosections were collected at 8μm thickness and immunostaining was carried out using specific antibodies.
Results: NP cell markers like Brachyury, sonic hedgehog and cytokeratin 19 were expressed at all the time points. The absence of FCS did not affect the expression. All the components of the disc responded to BMP and TGFβ signaling, analyzed using antibodies against the activated cytoplasmic messengers: phospho-Smad 1/5/8 and phospho-Smad 2/3 respectively. Changes in the matrix assembly were monitored by assaying for the collagen I and II accumulation in the AF & EP cells.
Conclusions: Results show that the NP cells continue to express their characteristic molecular markers, and the disc continues to grow in vitro even in the absence of the serum.
Significance: Understanding the molecular mechanism of disc growth will help design experiments to delineate what goes wrong during degeneration of the disc.