Cartilage calcification studied by proton nuclear magnetic resonance microscopy

A three-dimensional (3D) mineralizing culture system using hollow fiber bioreactors has been developed to study the early stages of endochondral ossification by proton nuclear magnetic resonance (NMR) microscopy. Chondrocytes harvested from the cephalic half of the sterna from 17-day-old chick embryos were terminally differentiated with 33 nM of retinoic acid for 1 week and mineralization was initiated by the addition of 1% beta-glycerophosphate to the culture medium. Histological sections taken after 6 weeks of development in culture confirmed calcification of the cartilage matrix formed in bioreactors. Calcium to phosphorus ratios (1.62-1.68) from X-ray microanalysis supported electron diffraction of thin tissue sections showing the presence of a poorly crystalline hydroxyapatite mineral phase in the cultures. After 4 weeks of culture, quantitative proton NMR images showed water proton magnetization transfer rate constants (km) were higher in premineralized cartilage compared with uncalcified cartilage, a result suggesting collagen enrichment of the matrix. Notably after 5 weeks mineral deposits formed in bioreactors principally in the collagen-enriched zones of the cartilage with increased km values. This caused marked reductions in water proton longitudinal (T1) and transverse (T2) relaxation times and water diffusion coefficients (D). These results support the hypothesis that mineralization proceeds in association with a collagen template. After 6 weeks of culture development, the water proton T2 values decreased by 13% and D increased by 7% in uncalcified areas, compared with the same regions of tissue examined 1 week earlier. These changes could be attributed to the formation of small mineral inclusions in the cartilage, possibly mediated by matrix vesicles, which may play an important role in cartilage calcification. In summary, NMR images acquired before and after the onset of mineralization of the same tissue provide unique insights into the matrix events leading to endochondral mineral formation.