Notochord cells regulate intervertebral disc chondrocyte proteoglycan production and cell proliferation

Non-chondrodystrophic dog notochord cell conditioned medium was used to evaluate chondrocyte proteoglycan production and cell proliferation.

To evaluate the responsiveness of bovine disc-derived chondrocytes to notochord-cell conditioned medium with respect to proteoglycan and cell proliferation. In addition, to examine phenotypic changes of notochord cells cultured in monolayered as compared to 3-dimensional culture.

Non-chondrodystrophic dogs maintain their intervertebral disc notochord cells into adulthood and are protected from having degenerative disc disease develop. The chondrodystrophic breeds such as beagles do not preserve these cells and have disc disease develop much earlier in life. The role of the notochord cell within the disc nucleus is poorly understood.

Canine notochord cells were cultured within alginate beads in serum-deficient conditions using Dulbecco modified Eagle medium to produce notochord cell conditioned medium (NCCM). NCCM was used to stimulate bovine disc chondrocytes from which we evaluated proteoglycan production and cell proliferation as compared to chondrocytes grown in DMEM alone. In addition, parallel cultures of notochord cells were seeded within alginate beads as well as in monolayer and cultured in order to examine for differences in phenotype between the 2 culture conditions.

The morphologic aspects of the intervertebral disc between the species differed markedly. A dose- dependent relationship was seen between proteoglycan production and NCCM concentration across various concentrations of NCCM in repeated experiments. Although there was a 4-fold increase in cell proliferation under all NCCM concentrations, this increase in cell proliferation was not dose dependent in the concentrations tested. Unlike chondrocytes, notochord cells do not adhere to tissue culture plate (monolayer) until at least day 4-6, do not markedly alter their phenotype, and rapidly assume masses of cells while floating within tissue culture medium.

The biology of the disc-derived chondrocyte is profoundly affected by NCCM in that various concentrations of NCCM activate proteoglycan production in a dose-dependent fashion. However, in the doses tested in our study, cell proliferation was increased but in a nondose-dependent fashion. Notochord cells retain their phenotype even in monolayer and through the development of floating intimately associated masses of cells suggest the development and maintenance of cell-cell interaction. These masses of cells are retained even after 6 days in culture when they do attach to the tissue plate surface. The persistence of notochord cells in non-chondrodystrophic dog species suggests that these in vitro studies may mirror the milieu of the disc in vivo, in which the notochord cell may play a key role in disc homeostasis.