Fluoridated Enamel Crystals Bind Ameloblastin More Tightly than Control Crystals

The effectiveness of systemic fluoride as a preventative agent against dental caries is dose dependent. One unfortunate side-effect of systemic fluoride administration is that as the concentration increases above 1 ppm so does the incidence of dental fluorosis. The cellular mechanisms of dental fluorosis are not well understood, but it has been proposed that impaired removal of enamel matrix proteins, from enamel crystal surfaces may impede crystal growth, leading to hypoplastic defects in enamel. Objective: To describe and compare the binding capacity of fluorotic and non-fluorotic enamel crystals for recombinant ameloblastin, an important enamel matrix protein. Methods: Enamel crystals were obtained from rat mandibular incisors. Litter mate rats were raised either in the absence of fluoride (control), or with 75 ppm fluoride administered in the drinking water from day 21. Crystals were imaged on a Nanoscope IIIa Multimode Atomic Force Microscope (AFM) in fluid tapping mode. The crystals were then exposed to 5 uL of recombinant ameloblastin and immediately imaged. Finally, the crystals were washed in increasing concentrations of phosphate buffer, pH 7.4, and imaged. Roughness measurements (RMS) were obtained for both sets of crystals after each stage. Results: Roughness measurements of the surface of the crystals confirmed that fluoridated enamel bound ameloblastin more tightly. Control enamel returned to normal roughness after a 100mM phosphate buffer wash (RMS=0.56), while the fluoridated enamel roughness remained high (RMS=0.81). Fluorotic crystal roughness did not return to normal until a 200mM phosphate buffer wash was applied. Conclusion: Fluorotic enamel crystals have a higher affinity for ameloblastin than the control enamel crystals. Further work is being done to determine whether this increased affinity is due to an increased roughness of the enamel crystal itself, or due to an alteration of the surface charge of the crystal. Research supported by NIH Grant #12899.