Fluoride alters bone matrix protein expression within mineralising bone cells

Bone associated glycoproteins are important in directing matrix assembly in the formation of osteoid and its subsequent mineralisation to form bone. Fluoride is known to alter mineralisation within bone, although the mechanism for its action is unclear. Objective: The aim of this study was to investigate the influence of fluoride on the temporal expression of bone associated proteins during the culture of bone cells whilst depositing a mineralised matrix. Methods: Bone cell cultures were established from femur bone marrow washes of male Wistar rats and maintained in a-MEM, foetal calf serum, ascorbic acid, b-glycerophosphate, and dexamethasone. 5 days post isolation, cultures were reseeded to 10⁴ cells/cm² and supplemented with 0, 10^-7M, or 10^-5M sodium fluoride. von Kossa staining indicated mineral deposition was initiated at day 8. Expression of the bone associated proteins was examined 2-13 days post reseeding by immunohistochemical localisation and Western blot analysis of extracted proteins using rabbit polyclonal antibodies specific for osteonectin, osteopontin, osteocalcin and bone sialoprotein. Results: Expression of the bone associated proteins was detectable during the mineralisation phase in the development of the bone cell culture. During this phase supplementation with low concentrations of fluoride (10^-7M) caused a relative decrease in levels of bone sialoprotein and osteopontin and an increase in osteonectin. No difference was seen in osteocalcin expression. The high concentration of fluoride (10^-5M), on the other hand, was associated with increased levels of osteopontin and osteocalcin, a small decrease in bone sialoprotein, but no difference with osteonectin. Conclusion: The results highlight the changing profile of bone matrix proteins during the formation mineralised bone and indicate the potential for fluoride to influence the temporal expression of bone matrix proteins, thereby offering a potential mechanism by which fluoride alters mineralisation. (Supported by the Wolfson Foundation)