Dec. 15, 1998 Researchers at the University of Southern California's School of Dentistry are closing in on making tooth enamel, the hardest substance found in vertebrates. They have identified tiny spheres that regulate the formation and organization of tooth enamel by controlling the substance's crystalline growth.
Called nanospheres because they are only 20 nanometers in diameter, these structures are formed by a naturally occurring family of tooth-specific proteins known as amelogenins. These spheres are also a component of the synthetic amelogenin first cloned at the USC School of Dentistry's Center for Craniofacial Molecular Biology (CCMB) four years ago.
"More than 98% of tooth enamel consists of carbonated calcium hydroxy-apatite," says CCMB research professor A.G. Fincham, Ph.D. "Essentially, your teeth are made of rock."
For two decades, CCMB researchers have been studying tooth enamel with the goal of one day replacing mercury-based gold and silver fillings with restorations of man-made material identical or similar to natural tooth enamel. "Beyond that, the same principles that nature uses to make enamel might also be applied to create novel synthetic materials," Dr. Fincham says.
Tooth enamel begins to form in the human embryo when a specialized layer of cells, called ameloblasts, in the embryonic tooth bud secretes amelogenin proteins. The amelogenins self- assemble to form the extracellular matrix within which the inorganic crystals of mineral start to form. "The earliest enamel crystals form in extremely long, thin ribbons and are rather beautifully parallel," Fincham notes.
CCMB researchers first saw the spheres in 1994. "Magnified in an electron microscope, they looked like tiny ping pong balls among the long ribbons of crystal," Fincham reports.
A more powerful atomic force microscope recently revealed that the spheres are uniformly 18 to 20 nanometers in diameter. (A nanometer is a billionth of a meter, and a 20-nanometer- diameter sphere is roughly 1/500th the size of a red blood cell.)
Chemically, the mineral crystals in tooth enamel are a calcium hydroxy-apatite formed from calcium and phosphate ions, which are transported into the nanosphere matrix by ameloblast cells.
"At first," Fincham explains, "the elongated apatite crystals will grow solely on their end faces, becoming ever longer. With the nanospheres acting as spacers, these early crystals build a scaffold on which mature enamel can eventually form. After enzymes have broken down the amelogenin proteins, the crystals start to grow on all of their faces. They thicken, clump together and create mature enamel."
Apatite crystals grown in the lab by traditional methods are about 100 times smaller than the crystals nature makes. They grow haphazardly, and the resulting material is considerably weaker than natural enamel.
Four years ago, the CCMB researchers took the gene for an amelogenin protein from a mouse, placed it in a bacterial cell, and then used the bacterial reproductive process to produce an identical recombinant amelogenin protein. This recombinant amelogenin protein, which the researchers can now produce in quantity, has since been shown to self-assemble to make nanosphere structures identical to those seen in the mouse and other animals, including humans.
"The structure of the amelogenin enamel protein is virtually the same in all vertebrates, from wallabies to humans, suggesting it has a very specialized function," says Fincham. "That function is to spontaneously self-assemble into a matrix with nanospheres -- a matrix that controls the microarchitecture of the developing enamel, both the three-dimensional spacing between the initial mineral crystals and the later crystal growth."
Currently, CCMB researchers are growing apatite crystals within synthetic matrices made from recombinant amelogenin protein.
"We get very long, straight structures," Fincham reports. "The crystals grow only on their end faces, although the material is unremarkable. We can't make enamel yet, but we can see how nature does it. The nanospheres clearly have a capacity to regulate the way crystals grow."
Other researchers working on aspects of the enamel mineral/enamel protein project include: Janet Moradian-Oldak, Ph.D.; Maggie Zeichner-David, Ph.D.; Malcolm Snead, Ph.D.; Michael Paine, Ph.D.; Hai Bo Wen, Ph.D.; and Raji Ravindranath, Ph.D.
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