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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.

Microstructures of non-unions of human humeral shaft fractures were investigated by using scanning electron microscopy, transmission electron microscopy, and X-ray microdiffraction. The non-union has a trabeculae structural framework similar to woven bone. Among the trabeculae are cavities that are subdivided into small chambers by thin plates of collagen fibrils. Some chambers are filled with variously shaped mineralized particles several micrometers in size. The collagen fibrils in both the trabeculae and the thin plates were only slightly mineralized by hydroxyapatite. Vesicles loaded with noncrystalline calcium phosphate (NCP) were observed in most mineralized particles, and brushite crystals with special morphology were seen to be embedded in some particles in irregular shapes. X-ray microdiffraction results indicated that the mineral phases in the non-unions were mainly NCP in addition to small amounts of hydroxyapatite and brushite. NCP deposition and insufficient mineralization of the collagen fibrils may be two important microstructural features of the non-unions of human humeral shaft fractures different from normally repaired bone callus.

Microstructures of the early external callus after diaphyseal fractures of human long bone were investigated by using scanning electron microscopy, X-ray diffraction, and transmission electron microscopy. It was found that the main structural framework of the human early callus consists of disordered, mineralized collagen fibrils with a small fraction of regions of ordered collagen fibrils. X-ray diffraction analyses show that hydroxyapatite containing some carbonate impurity has been the dominant crystalline phase in the human early callus. In addition, a small amount of brushite phase was detected. Selected area diffraction analyses indicated that hydroxyapatite microcrystals were embedded in microfibrils with a diameter of 4.5 nm and well-banded fibrils, whereas brushite particles of 15-20 nm in an irregular shape were located in the noncollagenous organic matter around the nonmineralized, ordered collagen fibrils. The spatial distribution of the brushite particles in the human early callus was for the first time determined. The brushite particles probably serve as the reservoir of calcium and phosphate ions for subsequent mineralized periods rather than the precursor of hydroxyapatite.

In order to understand further the mechanism of bone fracture repair, and thus to innovate better operative treatment for bone fracture and to design new implant materials for bone repair, microstructures of external periosteal callus (EPC) of repaired femoral fracture in both children and adults were investigated by using a scanning electron microscope, transmission electron microscopy, and an X-ray microdiffractometer. The repair time after the fractures in children and adults is on average 155 and 370 days, respectively. Collagen fibrils making up children's EPC (CEPC) are underdeveloped and insufficiently mineralized by hydroxyapatite (HA), while those from adults' EPC (AEPC) are similar to normal bone. A lot of particles loaded by brushite (DCPD) minerals were found among the collagen fibrils of CEPC. The main mineral phases in CEPC consist of DCPD and HA, while only HA exists in AEPC. Deposition of DCPD minerals could have compensated for the insufficient mineralization of the collagen fibrils of CEPC, thereby making fractured bone repair more rapidly in children than in adults.