ABSTRACT
The developing enamel matrix is a highly dynamic system mainly composed of the full-length amelogenin and its proteolytic cleavage products. In this study, size, zeta-potential, and the isoelectric points of nanoparticles of the recombinant full-length human amelogenin (rH174) and two proteolytic products (rH163 and rH146) were analyzed by dynamic light-scattering and electrokinetic measurements. We tested the hypothesis that zeta-potential may be used as a control parameter in directing the self-assembly of amelogenins. Extensive aggregation of amelogenin molecules with the particle size reaching about one micron occurred at a mildly acidic to neutral pH, and coincided with the red shift of the internal fluorescence. Zeta-potential was between +/- 15 mV in the same pH range, indicating that amelogenin aggregation occurred when surface potentials were minimal. This suggests that electrostatic interactions may be another crucial factor, aside from hydrophobic interaction, in the aggregation and hierarchical assembly of spherical particles of amelogenins into supramolecular structures of a higher order.
Subject(s)
Amelogenin/chemistry , Humans , Hydrogen-Ion Concentration , Hydrolysis , Isoelectric Focusing , Kinetics , Membrane Potentials , Nanospheres , Particle Size , Recombinant Proteins , Scattering, Radiation , Surface PropertiesABSTRACT
Dental enamel forms through a protein-controlled mineralization and enzymatic degradation with a nanoscale precision that new engineering technologies may be able to mimic. Recombinant full-length human amelogenin (rH174) and a matrix-metalloprotease (MMP-20) were employed in a pH-stat titration system that enabled a continuous supply of calcium and phosphate ions over several days, mimicking the initial stages of matrix processing and crystallization in enamel in-vitro. Effects on the self-assembly and crystal growth from a saturated aqueous solution containing 0.4 mg/ml rH174 and MMP-20 with the weight ratio of 1:1000 with respect to rH174 were investigated. A transition from nanospheres to fibrous amelogenin assemblies was facilitated under conditions that involved an interaction between rH174 and the proteolytic cleavage products. Despite continuous titration, the levels of calcium exhibited a consistent trend of decreasing, thereby indicating its possible role in the protein self-assembly. This study suggests that mimicking enamel formation in-vitro requires the synergy between the aspects of matrix self-assembly, proteolysis and crystallization.
