Calcif Tissue Int (1993) 52:392-398 Calcified Tissue International 9 1993 Springer-Verlag New York Inc. The Influence of Fluoride on Proteoglycan Structure Using a Rat Odontoblast In Vitro System R. J. Waddington, G. Embery, and R. C. Hall Department of Basic Dental Science, Dental School, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XY, UK Received September 23, 1992, and in revised form November 16, 1992 Summary. Using an in vitro rat incisor odontoblast system, the effect of fluoride on proteoglycans was investigated at both the metabolic and structural level. Incisors were re- moved from 4-week-old rats, split longitudinally, and the pulps removed. Teeth were incubated at 37~ 5% CO2 in Eagle's Minimum Essential Medium containing 35S-sulfate for 7 hours in the presence of 0 raM, 3 mM, or 6 mM sodium fluoride. Teeth were demineralized in EDTA, proteoglycan was extracted from the residue with 4 M guanidinium chlo- ride, and further purified by anion exchange chromatogra- phy. Uptake of radiolabel was monitored by liquid scintilla- tion counting. The resultant products were examined by cel- lulose acetate electrophoresis, SDS-PAGE, chondroitinase digestion, and amino acid analysis. Differential effects of fluoride were observed in both metabolism and biochemical characterization of proteoglycans following incubation at the two concentrations. Fluoride decreased uptake of the radi- olabel but led to an accumulation of glycosaminoglycan within the proteoglycan of the matrix. Chondroitin sulfate was the predominant glycosaminoglycan identified, with the additional presence of dermatan sulfate and heparan sulfate identified. Dermatan sulfate levels increased in 3 raM- treated teeth. Fluoride-treated proteoglycans had a reduced molecular weight (200-90K to 180-79K); this reduction is pri- marily a result of smaller glycosaminoglycan chains, with limited reduction in the size of the core protein of 6 raM- treated teeth occurring. Such alterations in the biochemical metabolism and hence structure and function of proteogly- can may be implicated in the hypomineralization seen in fluorosis. Key words: Fluoride - Proteoglycans - Odontoblasts. Fluoride administered at low concentrations is known to be a potent stimulator of bone formation and is often used as a therapeutic agent (10-5 M serum concentrations) in diseases such as osteoporosis. However, the prolonged ingestion of excessively high amounts of fluoride leads to both skeletal and dental fluorosis, characterized by hypomineralization of calcified tissues. Both collagenous and noncollagenous com- ponents are known to undergo structural alteration during fluorosis. However, the precise mechanisms of fluoride tox- icity with respect to biochemical changes of the organic ma- trix within the mineralized tissues, including synthetic events, have not been fully elucidated. The organic matrix of all connective tissues, both miner- alized and nonmineralized, can be generally described as a Offprint requests to: R. J. Waddington collagenous fibrous network embedded in a ground sub- stance, an important component of which are proteoglycans. These nonfibrous macromolecules are composed of a central protein core, the amino acid composition of which are char- acteristically enriched in aspartic acid, glutamic acid, serine, and glycine. Attached to this protein core are highly anionic glycosaminoglycan (GAG) chains, covalently bound via the serine residue. The nature of the proteoglycan is such that it is considered to play possible roles in hydration, compress- ibility, collagen fibril formation, and mineralization. Chon- droitin 4-sulfate has been identified as the predominant GAG present in mineralized tissues studied from various sources [1-3]. Rahemtulla et al. [4] have characterized three chon- droitin sulfate proteoglycan populations from rat incisor: a chondroitin 4-sulfate and a chondroitin 4- and 6- sulfate iso- mer associated with the predentine and the mineralized ma- trix of dentine. Studies on the effect of fluoride on proteoglycan present in developing rat incisor dentine in vivo following ingestion of 112 ppm fluoride over a 10-week period have shown a reduction in the molecular size of proteoglycan compared with nonfluorotic teeth [5]. Further, Jha and Susheela [6, 7] have observed an alteration in the GAG composition of can- cellous bone after prolonged fluoride ingestion, demonstrat- ing the increased presence of dermatan sulfate, whereas in nonfluorotic bone, negligible to trace amounts were evident. The same group have also reported an increased appearance of dermatan sulfate in fluorosed human teeth [8]. Fluoride has been shown to inhibit the uptake of 35S-sulfate into sul- fated GAG in vitro by odontoblasts of rat incisor teeth [3], but no information on the nature of the resultant products has been established. Biochemical changes due to the effect of fluoride on the collagenous components of mineralized tissues have also been reported. Susheela et al. [9] have identified changes in the ratio of proline and lysine with their respective hydroxylate moieties on comparing fluorotic and nonfluorotic teeth. Such changes, together with the bio- chemical changes witnessed in the proteoglycan components of fluorotic tissues may be partly responsible for the irregu- lar mineralization leading to reduced bone strength. Using an in vitro rat incisor odontoblast system, the present study was carried out to examine the effect of various concentrations of fluoride within the culture medium on the metabolism and biochemical structure of proteoglycan within the mineralized matrix. Materials and Methods Preparation of Teeth and Incubation Procedure The procedure used for the isolation and exposure of the incisor