Wen H B, Moradian-Oldak J, Leung W, Bringas P, Fincham A G
School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 1st Floor, Los Angeles, California 90033, USA.
J Struct Biol. 1999 Jun 1;126(1):42-51. doi: 10.1006/jsbi.1999.4086.
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.
釉原蛋白凝胶基质的热可逆转变（透明<-->不透明）已为人所知约三十年，现在通过微观结构研究得以阐明。从猪发育中的牙釉质基质中提取的混合釉原蛋白制剂（含有“25K”，7.4%；“23K”，10.7%；“20K”，49.5%；以及较小的肽段，32.4%）溶解于稀甲酸中，并用氢氧化钠溶液将pH值调至6.8使其再沉淀。通过在微量离心管中沉淀沉淀物在体外形成釉原蛋白凝胶。凝胶在4℃和24℃下用卡尔诺夫斯基固定剂固定，发现其能保持相应的透明（4℃）和不透明（24℃）状态。采用扫描电子显微镜、原子力显微镜和透射电子显微镜对固定的透明和不透明凝胶进行微观结构表征。观察到釉原蛋白凝胶基质具有准球形釉原蛋白纳米球及其聚集体的分级结构。直径为8 - 20nm的纳米球组装形成直径为40 - 70nm的小球形聚集体，这些聚集体进一步聚集形成直径为70 - 300nm的大球形聚集体。在透明凝胶中，大多数大聚集体小于150nm，纳米球和聚集体均匀分散，使得它们之间的流体分布均匀。然而，在不透明凝胶中，观察到许多直径为0.3至7微米的球形充满流体的空间，大多数大聚集体的直径为150 - 200nm。这些空间可能是由于温度升高时纳米球和/或聚集体之间增强的疏水相互作用导致的。与低温（4℃）制剂相比，不透明（24℃）凝胶的高不透明度显然源于观察到的众多充满流体的空间。这些观察结果表明，纳米球和不同级别的釉原蛋白聚集体之间的疏水相互作用对于确定牙釉质基质的结构完整性很重要。
