Initial experiments were designed to test the effects of two diphosphonates on acid dissolution of mature human enamel. Polished facets of enamel were treated with 10 mmol/L of EHDP or Cl2MDP (pH 7.4) for five min, H20-washed, acid-etched with 0.01 N lactic acid (pH 5.4) for 15 min, and examined in the scanning electron microscope. Controls were run with distilled H2O substituted for diphosphonates. In addition, the experimental window was outlined with lacquer, thus providing a reference to the original polished enamel surface. Results showed that EHDP markedly reduced enamel dissolution, with less than 1 μm of enamel removed, while Cl2MDP was similar to controls, with approximately 4-5 μm of enamel dissolved. It was decided to initiate further experiments with the objective of determining effects of diphosphonates on developing enamel during the secretory stage in rat molars. Five-day-old animals were injected s.c. with one of the following: EHDP, ADP, Cl2MDP, or NaCl (0.9% control). The diphosphonate dosages were 2.5, 5, 10, and 20 mg P/kg. Rat incisors were also examined in initial experiments on older rats (21-day) following daily s.c. injections of EHDP. Twenty-four hr after the injections, all rats were killed by perfusion fixation and routinely prepared with and without decalcification for light and transmission electron microscopy. Light microscopic examination of developing maxillary first molars frequently revealed subameloblastic cyst formation, which was used as an indicator of severe cellular alteration of overlying secretory ameloblasts. By this criterion, the EHDP- and ADP-treated rats showed more severe cellular effects at lower dosages (2.5 and 5 mg P/kg) than did the Cl2MDP-treated rats. In addition, when undecalcified sections were examined in the transmission electron microscope, disturbances in crystal formation were observed. Since both the cyst formation and the disturbed enamel beneath the cyst had many similarities to previous results reported on the effects of high doses of fluoride, the majority of results observed may be due to a non-specific cellular toxicity. However, some crystal growth effects appear to be specific for diphosphonates, as indicated by preliminary results with daily injections of EHDP. In the rat incisor, a definitive banding that shows both inhibition and return to more normal development within a 24-hour cycle has been detected. In the ultrastructure examination of undecalcified sections, the reaction to EHDP is clearly defined as bands of normal crystal size, electron density, and orientation alternating with disturbed bands of small crystal size and decreased electron density. The bands are approximately 2-3 μm in width.
