Microstructures of an amelogenin gel matrix

The thermo-reversible transition (clear <-> opaque) of the amelogenin gel m atrix, 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 formi c acid and reprecipitated by adjusting the pH to 6.8 with NaOH solution. Am elogenin gels were formed in vitro by sedimenting the precipitate in microc entrifuge tubes. The gels were fixed with Karnovsky fixative at 4 and 24 de grees C, which was found to preserve their corresponding clear (4 degrees C ) and opaque (24 degrees C) states. Scanning electron microscopy, atomic fo rce microscopy, and transmission electron microscopy were employed for the microstructural characterization of the fixed clear and opaque gels. The am elogenin gel matrix was observed to possess a hierarchical structure of qua si-spherical amelogenin nanospheres and their assemblies. The nanospheres o f diameters 8-20 nm assemble to form small spherical assemblies of diameter s 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 sma ller than 150 nm, and the nanospheres and assemblies are uniformly disperse d, allowing an even fluid distribution among them. In the opaque gel, howev er, numerous spherical fluid-filled spaces ranging from 0.3 to 7 mu m in di ameter were observed with the majority of the large assemblies sized 150-20 0 nm in diameter. These spaces presumably result from enhanced hydrophobic interactions among nanospheres and/or assemblies as the temperature increas ed. The high opacity of the opaque (24 degrees C) gel apparently arises fro m 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 ame logenin assemblies are important in determining the structural integrity of the dental enamel matrix. (C) 1999 Academic Press.