A study of canine notochord cell-derived soluble factors that enhance proteoglycan production by intervertebral disc chondrocytes

Introduction. There is no biological explanation for the disparity that exists between individuals who do or do not develop degenerative disc disease in the absence of trauma. The canine species is a case in point with respect to factors that may have a genetic link. Nonchondrodystrophic (NCD) dogs maintain their notochord cells for many years and are not known to develop degenerative disc disease until much later in life, whereas other purebred species such as beagles (chondrodystrophic breeds) do not preserve these cells and develop disc disease much earlier. The purpose of this study was to evaluate the responsiveness of bovine disc-derived chondrocytes to notochord-cell conditioned medium (NCCM), to develop a preliminary profile of the soluble proteins produced by notochord cells, identify gene expression of the important aggregating proteoglycans aggrecan and versican as well as the enzyme hyaluronic acid synthase-2 and to identify the components of NCCM. Hypothesis. Intervertebral disc integrity is dependent upon factors produced by notochord cells within the nucleus of NCD dogs that interact with chondrocyte disc matrix. Results. A dose dependent relationship exists between proteoglycan production and NCCM concentration that is statistically significant at P = 0.018. 3H thymidine incorporation demonstrated four-fold increase in cell proliferation under all NCCM concentrations that was statistically significant at all levels tested at the P = .01 level or better but was not dose dependent. Aggrecan, Versican and Hyaluronic acid synthase-2 are all up regulated by NCCM with aggrecan by far being the most potently stimulated gene examined. NCCM was found to contain aggrecan, ubiquitin, Cu/Zn superoxide dysmutase, fibronectin and connective tissue growth factor precursor. Methods. Canine notochord cells (obtained from NCD dogs) were cultured within alginate beads in serum-deficient conditions (DMEM) to produce notochord cell conditioned medium (NCCM). Proteoglycans were extracted with Guanidine HCl after first culturing the bovine disc chondrocytes with NCCM. Column chromatography and subsequent scintillation counting were used after the NCCM cultured chondrocytes were exposed to overnight 35S sulfate. 3H thymidine incorporation was used to assay cell proliferation in parallel cultures. NCCM was evaluated with SDS-PAGE and MS/MS Mass Spectroscopy. Semi-quantitative RT-PCR was used to evaluate gene expression of aggrecan, versican and the enzyme hyaluronic acid synthase-2. Discussion. Canine notochord cells secrete soluble factors that up regulate proteoglycan production by bovine disc-derived chondrocytes including the important aggregating proteoglycans aggrecan and versican as well as the enzyme hyaluronic acid synthase-2. Though the increased proteoglycan production is dose-dependent cell proliferation is not. Cell proliferation was statistically significantly increased in all doses when cultured with NCCM indicating a strong mitogenic capacity of the components of NCCM, however the lack of dose-dependency suggests a proteoglycan-specific anabolic role for these soluble factors.