Formation of articular cartilage-like tissue in vitro with a calcifying zone requires elevated levels of phosphate

Articular cartilage (AC) deep zone (DZ) merges with a zone of calcified cartilage (ZCC) which is responsible for anchoring AC to bone. However, regulators of ZCC formation are poorly understood. As inorganic phosphate (P_i) modulates growth plate chondrocyte differentiation and is a component of hydroxyapatite, we hypothesize that increased P_i is required for formation of a localized ZCC by DZ chondrocytes (DZC).

Calf metacarpophalangeal joint DZC were grown in 3D culture for up to 7 weeks with or without P_i-containing compounds. In vitro-formed tissues and native AC were evaluated histologically, immunohistochemically, biochemically, and by electron microscopy and diffraction.

P_i levels and tissue non-specific alkaline phosphatase (TNAP) activities showed depth-dependent distribution in native DZ. With β-glycerophosphate (βGP) treatment, both TNAP_low and TNAP_high DZC formed tissue with a lower mineralized zone, however, TNAP_low DZC revealed delayed mineralization and P_i accumulation when compared to TNAP_high DZC. In the absence of βGP, no mineral accumulated. Both control and βGP-treated TNAP_low and TNAP_high tissues contained differentially distributed TNAP activity, ColX, and MMP13. Separation of upper and lower aspects of βGP-treated in vitro-formed tissues revealed greater P_i content in the lower, mineralizing region than in the upper, non-mineralized region. Chemical inhibition of P_i generation and transport prevented mineralization. P_i-treated DZC resulted in biphasic tissues with a lower, mineralized, high-P_i region, and an upper, non-mineralized, low-P_i region.

This work establishes P_i as an important regulator of DZC mineralization and ZCC formation and suggests that P_i may regulate the phenotype of a DZC subpopulation.