Fluoride Inhibits Longitudinal Bone Growth by Acting Directly at the Growth Plate in Cultured Neonatal Rat Metatarsal Bones

Abstract

Excessive intake of fluoride inhibits bone growth in both humans and animals. It is unknown whether fluoride acts directly on the growth plate to inhibit longitudinal bone growth, and its mechanism of action has not been elucidated. In this study, we used an organ culture system and SW1353 cells to evaluate the effects of fluoride on endochondral ossification. Neonatal rat metatarsal bones were dissected and cultured with or without fluoride for 7 days. The total length and width of the metatarsal rudiments and the length of the calcification zone were measured. Chondrocyte proliferation, differentiation, and apoptosis were analyzed by immunohistochemistry and TUNEL assay in sectioned bones. The apoptosis was detected by flow cytometry, and the expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3 were detected by western blotting in SW1353 cells. Linear measurements demonstrated that fluoride induced a biphasic effect on longitudinal bone growth in organ culture, with a significant growth inhibition at a high concentration (10−4 M) and a stimulatory action at low concentration (10−6 M) of fluoride. Histomorphometrical analysis of growth plate from fluoride-exposed metatarsal rudiments showed a significant reduction in the height of the proliferative and hypertrophic chondrocyte zones. Analysis of the Col2α1 and Col10α1 expression by immunohistochemistry revealed fluoride-suppressed metatarsal growth plate chondrocyte proliferation and differentiation. In addition, fluoride increased the number of apoptotic chondrocytes in the metatarsal growth plate. Western blotting showed an up-regulated expression of Caspase-3 and Bax and down-regulated expression of anti-apoptotic protein Bcl-2 after treatment with 5 × 10−4 M fluoride in SW1353 cells. Our findings indicated that fluoride inhibited longitudinal bone growth by acting directly at the growth plate in cultured neonatal rat metatarsal bones. Such growth inhibition was mediated by suppressing proliferation and differentiation, increasing apoptosis of resting chondrocytes and causing premature cell senescence in the growth plate.