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Figure 1. In vitro experimental set-up for the nucleation of apatite by amelogenins and the 32-kDa enamelin. (A) Sodium dodecyl sulfate-polyacrylamide gel (15% acrylamide) electrophoresis (SDS-PAGE) patterns of extracted amelogenins and the 32-kDa enamelin. Lane 1: Gibco BRL protein molecular-weight standard containing proteins with 43-k, 29-k, 18-k, 14-k, and 5-k molecular weights. Lane 2: amelogenins extracted from the extracellular enamel matrix of developing pig mandibular molars by the dissociative technique as described in MATERIALS & METHODS. The extract was characterized to be a mixture of secreted amelogenin (25 K, 7.4%) and its processed products (23 K, 10.7%; 20 K [the major band], 49.5%; 14–18 kDa, 32.4%) (Wen et al., 1999). Lane 3: The 32-KDa enamelin with an apparent molecular weight of 32 kDa. (B) Schematic representation for the experimental set-up used for monitoring nucleation of apatite crystals. Induction time was determined based on the difference in calcium uptake by the gel between the control (without phosphate) and the samples (with phosphate) as described in Fig. 3 .
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, sample) and without (
, control) phosphate ions. Comparisons between the sample and the control for each measurement were made by Student’s t test, and statistically significant differences were defined at p < 0.05. Induction time was defined as the time when the comparison between the sample (phosphate-containing gel) and the control for each measurement was statistically significant. Values are based on ratio percentages of 45Ca radioactivity (counts per min) at time t to that measured at time zero as the function of time. The data (13 points) shown are the mean ± SD of 3 separate experiments. The variability ranged between SD = 0.4–1.9 for control and SD = 0.3–2.3 for sample.