In Vitro Calcification of Immature Bovine Articular Cartilage: Formation of a Functional Zone of Calcified Cartilage

INTRODUCTION: The zone of calcified cartilage (ZCC) anchors articular cartilage (AC) to subchondral bone through a layer of intermediate stiffness. The regulation and functional consequences of cartilage calcification may vary with depth from the articular surface. The hypothesis of this study was that the in vitro calcification of immature AC occurs selectively in the deep region and is associated with a local increase in stiffness. METHODS: AC and growth plate cartilage (GPC) from calves were incubated in DMEM, 1% fetal bovine serum, 100 µg/mL ascorbate, and ±10 mM β-glycerophosphate (βGP) for up to 3 weeks. To assess the time course and effects of cell viability and βGP, full-depth strips of AC and GPC were analyzed by histology, indentation, and (45)Ca(++) uptake. To assess the effect of tissue zone, disks harvested from surface and deep zone AC and from reserve and hypertrophic zone of GPC were incubated independently and analyzed by compression and for (45)Ca(++) uptake and biochemical components. RESULTS: The deep ~20% of immature AC calcified within 3 weeks, with calcification dependent on cell viability and βGP. Mineral was deposited continuously around cells in AC but only between cell columns in GPC. The deep zone of AC exhibited a compressive modulus of 0.53 MPa after βGP-induced calcification, ~4-fold stiffer than AC incubated without βGP. CONCLUSIONS: Cartilage explants exhibit inherent zone-specific calcification processes, resulting in an increase in stiffness associated with cartilage calcification. Such properties may be useful for engineering a biomimetic ZCC tissue to integrate cartilaginous tissue to bone, thereby forming a mechanically functional osteochondral unit.