Enamel structure properties controlled by engineered proteins in transgenic mice.

Abstract

Amelogenin protein has regulatory effects on enamel biofabrication in mammalian tooth. Using teeth obtained from transgenic mice that express two separate protein-engineered versions of amelogenins, we made structure-nanomechanical properties correlations and showed 21% hardness and 24% elastic modulus degradation compared with the age-matched wildtype littermates. We attribute the inferior properties to the disorganization of the protein matrix resulting in defective mineral formation. Enamel is a bioceramic initiated by the biosynthesis of a complex mixture of proteins that undergoes self-assembly to produce a super molecular ensemble that controls the nucleation and habit of the crystalline mineral phase. Ultimately, the inorganic crystals grow to almost fully replace the organic phase. This biofabrication process occurs at physiologic conditions of pH, temperature, pressure, and ion concentration and results in the hardest tissue in the vertebrate body, with the largest and longest substituted-hydroxyapatite crystals known to biomineralizing systems. The most abundant protein of forming mammalian enamel, amelogenin, has been shown to have a significant regulatory effect on this complex process. In this work, we present the effect of protein engineering of amelogenin on the mechanical properties of the resultant mouse enamel. We have produced two types of transgenic animals that express separate versions of amelogenin proteins that lack the required self-assembly domains. The resultant matured enamel was extensively characterized for its mechanical properties at the nanoscale by means of nanoindentation and atomic force microscopy (AFM). These techniques have enabled us to probe the mechanical properties that are representative of a single enamel rod. Our nanoindentation measurements have revealed that the altered amelogenin with dysfunctional self-assembly properties resulted in a degradation by as much as 21% in hardness and 24% in elastic modulus compared with the age-matched wildtype littermates. Furthermore, the enamel formed by these defective proteins is found to display a decrease in indentation surface pile-up volume by up to 32%. We attribute these inferior mechanical properties for the enamel grown by the engineered amelogenins to result from the disorganization of the nanospheres formed in the protein matrix starting at the mineral nucleation stage with a consequential alteration to the fully grown mineral component. By engineering the properties of proteins that contribute to the nanoscale level of hierarchy in enamel biomineralization, it is possible to regulate the properties of the resulting bioceramic at the mesoscale level of the tissue.