Effects of MMP-20 on amelogenin self-assembly and crystal growth

During enamel maturation proteases eliminate most of the extracellular matrix proteins and facilitate the formation of a hard tissue containing 96 wt.% mineral with a unique architecture. Enamel matrix metalloproteinase-20 (MMP-20) is the first protease in this process to cleave amelogenins. Objective: The purpose of this study was to mimic amelogenin interactions with mineralizing solutions in the presence of MMP-20 in vitro. Method: A pH-stat titration system was used to continuously supply calcium and phosphate ions to a mineralizing solution at constant pH over a period of up to seven days. Mineralizing solutions at pH 7.40 contained 1.6 mM Ca²⁺, 1.0 mM PO4^3- ions and 0.4 mg/ml recombinant human amelogenin, rH174. MMP-20 was added to the solutions initially or after 2 days of experimental run. The effect on amelogenin self-assembly and the crystal growth was evaluated on FAP glass-ceramic substrates immersed in the solutions. Results: Titration rates decreased from 8 to 4 mL/h when rH174 was added to the mineralizing solution and additionally decreased upon the introduction of MMP-20, indicating a reduction in nucleation and crystal growth rates when the protein is present. In the absence of MMP-20, amelogenin formed nanospheres that transited into nanostrings after three days, as observed by AFM. When MMP-20 was added, larger agglomerates of protein formed beside nanospheres. With time a network of nanostrings (d = 30 nm) developed and covered the entire glass-ceramic surface. Circular assemblies of nanospheres were frequently observed. None of the samples, however, showed significant apatite crystal growth on FAP substrates.

Conclusion: MMP-20 reduces the tendency for crystal formation in the mineralizing solution containing 0.4 mg/ml amelogenin, although appears to promote hierarchical self-assembly.

Support: by NIH/NIDCR R01-DE017529 and R01-DE015821.