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

OBJECTIVE

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 (Pi) modulates growth plate chondrocyte differentiation and is a component of hydroxyapatite, we hypothesize that increased Pi is required for formation of a localized ZCC by DZ chondrocytes (DZC).

DESIGN

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

RESULTS

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

CONCLUSIONS

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