Structural analysis of proteoglycans synthesized by mineralizing bone cells in vitro in the presence of fluoride

This study investigated the biochemical structure of proteoglycans synthesized during matrix maturation by mineralizing bone cells in vitro, in the presence and absence of fluoride. Bone cells were obtained from rat femur washes and cultured in alpha MEM media supplemented with fetal calf serum, ascorbic acid, beta-glycerophosphate and dexamethasone. Cells were characterized as osteoblast-like by the expression of alkaline phosphatase activity and the synthesis of collagen type I and osteocalcin. Fluoride, present in the culture media at concentrations of 10(-5) M or 10(-7) M, had negligible effect on cell viability. However, calcium deposition was increased in cell cultures incubated in the presence of fluoride. Proteoglycans were extracted from the extracellular matrix with 4 M guanidinium chloride and purified by anion exchange chromatography. Biochemical analysis identified the presence of the small leucine rich proteoglycan, decorin and biglycan, in addition to degradation products relating to the larger chondroitin sulphate protoeglycan, versican. Fluoride had little effect on the size or amino acid composition of the protein core, but resulted in significant alterations to the GAG chains, including a dramatic reduction in chain length, reduction in sulphation and decrease in the proportion of dermatan sulphate compared to chondroitin sulphate. The influence of fluoride on proteoglycan structure synthesized by mineralizing bone cells provides valuable information, indicating specific roles for dermatan sulphate and chondroitin sulphate proteoglycans. The results suggested that fluoride affected the post-translational assembly of the GAG chains which may be an influential factor in the mineralization process.