Annulus fibrosus cells can induce mineralization: an in vitro study

Background contextThere is still no consensus as to whether the calcification observed in degenerate intervertebral discs (IVDs) is a cause or a consequence of disc degeneration.PurposeTo investigate the mineralization potential of healthy (independent of other associated changes) annulus fibrosus (AF) cells under controlled in vitro conditions.Study design/settingIn vitro study to investigate the mineralization potential of the AF cells.MethodsAnnulus fibrosus cells, isolated from bovine IVDs, were grown in monolayer. The effect of cell density, culture time, age of cell source, and passage on the percentage of AF cells with alkaline phosphatase activity (ALPa) was evaluated. Gene expression of mineralization-associated markers was determined. Cells were immunostained for Type I, II, and X collagens. To study mineralization potential, AF cells and AF cells that were sorted into two populations, high (top 5%±1%) or low (bottom 5%±1%) ALPa expressors, were grown in the presence of β-glycerophosphate for 2 weeks.ResultsThe percentage of AF cells that express ALPa changes with time in culture and seeding density for primary immature and mature cell sources but not for passaged cells. Gene expression of ALP, matrix metallopeptidase-13 (MMP-13), osteopontin, and runt-related transcription factor 2 was upregulated by Day 7. Under mineralization-inducing conditions, high ALPa expressors and unsorted AF cells formed von Kossa-positive nodules, composed of hydroxyapatite as determined by electron diffraction analysis. Low ALPa expressors had significantly fewer von Kossa-positive nodules (p<.01) compared with high ALPa expressors. Cells showed colocalization of Type I collagen and ALPa. No Type II collagen was detected suggesting that these were AF cells and not chondrocytes.ConclusionsAnnulus fibrosus cells have mineralizing capability and form hydroxyapatite crystalline deposits when cultured under appropriate conditions. This system could be used to investigate mineralization mechanisms in the AF during pathological calcification and at the AF-bone interface in disc degeneration.