Homocysteine decreases chondrocyte-mediated matrix mineralization in differentiating chick limb-bud mesenchymal cell micro-mass cultures

Abstract

The differentiating chick limb-bud mesenchymal cell micro-mass culture system has been used as a model for monitoring the effects of matrix modification on cell-mediated calcification. In this study, we show that treating these micro-mass cultures with homocysteine (Hcys) impairs cartilage calcification. Cultures were treated from day 2 to day 7 with two nonphysiological concentrations of Hcys equivalent to 100× and 1000× avian serum levels (0.36 and 3.6 mmol/L), and from days 9–13 with one tenth the concentration. Mineralization assays were done at days 16, 19, and 21, and matrix and cell properties were examined between days 5 and 21. Mineral accretion, based on differential 45Ca uptake (mineralizing minus control cultures), was significantly reduced in the high-Hcys-concentration group, and slightly reduced in the low-Hcys-concentration group. Electron microscopy at culture day 21 showed that the collagen matrix was less abundant and its banding pattern less obvious in the Hcys-treated groups than in the untreated cultures. Pyridinoline (Pyr) and deoxypyridinoline (d-Pyr) contents were not detectable in day 21 cultures with either 0.36 or 3.6 mmol/L homocysteine, whereas values in mineralizing and nonmineralizing controls ranged from 0.06 to 0.08 and 0.03 to 0.06 (moles/mole collagen) for Pyr and d-Pyr, respectively. Fourier transform infrared (FTIR) imaging also indicated a decreased content of pyridinoline cross-links. Hcys caused other matrix changes as well. Whereas at culture day 5 there was no significant difference in the number of chondrocyte nodules formed, by day 11 the proteoglycan content (measured by Alcian blue dye binding at 595 nm) was significantly reduced in both mineralizing and control cultures in the high- and low-Hcys groups. In contrast, there were no detectable differences in type X collagen and alkaline phosphatase staining in the mineralizing cultures with or without Hcys supplements. Because vital dye stains and electron microscopy studies indicated that cells in the control and experimental groups did not differ in terms of viability, the observed differences cannot be attributed to toxicity. Thus, Hcys treatment, which causes matrix disorganization, decreases the ability of the matrix to support mineralization.