Analysis of proteoglycan synthesis, distribution and assembly of notochordal cells and small nucleus pulposus cells embedded in alginate beads and cultured in presence of [35S]-Na2SO4.Objective.
To determine whether the degeneration of the nucleus pulposus of the intervertebral disc is associated with a change in the cell phenotype.Summary of Background Data.
The loss of the notochordal cell from the nucleus pulposus is associated with ageing and disc degeneration. The reduction in their numbers after birth in humans and in the chondrodystrophoid dog has been suggested to result from cell death and replacement or differentiation by chondrocytes. The almost total disappearance of the notochordal cells in the nucleus pulposus correlates with early degenerative changes in the disc and a concomitant reduction in proteoglycan content, increased collagen, and loss of water content. The basic mechanism of this accelerated degeneration with ageing is poorly understood.Methods.
Nucleus pulposus and anulus fibrosus cells were isolated from the lumbar intervertebral discs of chondrodystrophoid and nonchondrodystrophoid dogs. The cells from the nucleus pulposus were further separated by size into notochordal cells and small nucleus pulposus cells. Cells were embedded in alginate beads and cultured in the presence of [35S]-Na2SO4 to measure proteoglycan size, rate of synthesis, and distribution into the pericellular and intercellular compartments.Results.
Large notochordal cells in the nucleus pulposus of chondrodystrophoid dogs formed 13% of the cell population in young dogs and fell to 0.4% in adults, whereas they were the predominant cell type in the nonchondrodystrophoid dogs at all ages. These cells were capable of 1.5-fold greater rate of synthesis of proteoglycans than the small nucleus pulpous cells. Proteoglycans secreted by the large cells were evenly distributed between the pericellular and intercellular compartments,whereas the small cells distributed 3-fold more proteoglycan into the intercellular phase. By size exclusion chromatography, the proteoglycans synthesized by the small cells of the chondrodystrophoid dogs formed large-size aggregates (Kav = 0.1) within the pericellular region, which then moved to the intercellular region over 5 to 10 days. In contrast, proteoglycans secreted by the notochordal cells were capable of rapid migration to the intercellular phase before assembly into large-sized aggregates. The ability to form aggregates was independent of age of the animal.Conclusions.
Our model shows that a change in intervertebral disc cell phenotype correlates with the grade of disc degeneration and that the notochordal cells synthesize proteoglycans, which exhibit delayed aggregation than those synthesized by the small nucleus pulposus cells. This implies that the cell type composition of the nucleus pulposus of the chondrodystrophoid and nonchondrodystrophoid dogs produces an extracellular matrix that is assembled in a distinct manner, which may affect tissue integrity.