Microstructures of an amelogenin gel matrix.

The thermo-reversible transition (clear <--> opaque) of the amelogenin gel matrix, which has been known for some three decades, has now been clarified by microstructural investigations. A mixed amelogenin preparation extracted from porcine developing enamel matrix (containing "25K," 7.4%; "23K," 10.7%; "20K," 49.5%; and smaller peptides, 32.4%) was dissolved in dilute formic acid and reprecipitated by adjusting the pH to 6.8 with NaOH solution. Amelogenin gels were formed in vitro by sedimenting the precipitate in microcentrifuge tubes. The gels were fixed with Karnovsky fixative at 4 and 24 degrees C, which was found to preserve their corresponding clear (4 degrees C) and opaque (24 degrees C) states. Scanning electron microscopy, atomic force microscopy, and transmission electron microscopy were employed for the microstructural characterization of the fixed clear and opaque gels. The amelogenin gel matrix was observed to possess a hierarchical structure of quasi-spherical amelogenin nanospheres and their assemblies. The nanospheres of diameters 8-20 nm assemble to form small spherical assemblies of diameters 40-70 nm that further aggregated to form large spherical assemblies of 70-300 nm in diameter. In the clear gel, most of the large assemblies are smaller than 150 nm, and the nanospheres and assemblies are uniformly dispersed, allowing an even fluid distribution among them. In the opaque gel, however, numerous spherical fluid-filled spaces ranging from 0.3 to 7 microm in diameter were observed with the majority of the large assemblies sized 150-200 nm in diameter. These spaces presumably result from enhanced hydrophobic interactions among nanospheres and/or assemblies as the temperature increased. The high opacity of the opaque (24 degrees C) gel apparently arises from the presence of the numerous fluid-filled spaces observed compared to the low-temperature (4 degrees C) preparation. These observations suggest that the hydrophobic interactions among nanospheres and different orders of amelogenin assemblies are important in determining the structural integrity of the dental enamel matrix.