Characterization of the Mineralization Process in Cultures of Rabbit Growth Plate Chondrocytes

Abstract

The present study was undertaken to investigate the mineralization process in chondrocyte cultures. Chondrocytes were isolated from the growth plate of ribs of 4-week-old rabbits. The nature and properties of mineral crystals precipitated in chondrocyte cultures were compared with those of crystals formed in the hypertrophic zone and bone of rabbit rib growth plates in vivo. The chondrocytes were maintained at high density on type II collagen-coated dishes in Eagle's medium, α-modification, with 10% fetal bovine serum and 50 μ/ml of ascorbic acid. These cells differentiated into hypertrophic cells 10 days after seeding and produced alkaline phosphatase and 1,25-dihydroxyvitamin D2 receptors on Days 30-70 at levels as high as those in the lower hypertrophic zone in vivo. Mineralization was initiated between Days 20 and 30 and advanced progressively throughout the culture period. However, mineralization was suppressed by the addition of parathyroid hormone (2 × 10-8M) or by the presence of fibroblasts. Examination by electron microscopy and Fourier transform infrared (FTIR) spectroscopy verified that mineralized nodules formed in vitro were composed of small apatite crystals. Importantly, FTIR spectral features of the apatite crystals (e.g., the prominent PO4 hands at 1125 and 1052 cm-1) were similar to those of cartilage apatites formed in vivo and differed markedly from those of carbonated bone apatites. These results suggest that growth plate chondrocytes cultured on collagen-coated dishes are an appropriate model for studies on cartilage mineralization.