Peptide-Based Bioinspired Approach to Reconstructing Multi-layered Aprismatic Enamel

Objectives: To characterize amelogenin-inspired peptides and test their potential to a) assemble into an organized scaffold and b) to reconstruct an organized layer of synthetic aprismatic enamel in situ via a bottom-up mineralization strategy.
Methods: Two amelogenin-inspired peptides (P26 and P32) were rationally designed based on the functional domains of native amelogenin. Full-length recombinant amelogenin (rP172) was used for comparison. Human third molars were longitudinally sectioned into 2mm slices and immersed in a demineralizing solution (pH 4.6) at 37°C for 2 hours. Peptides were applied on days 1 and 3 and the coated slices (n=5) were incubated in artificial saliva for 7 days. The structure and assembly of the synthetic peptides at physiological pH were characterized using CD and TEM. In vitro mineralization with and without the peptides was monitored under in situ Raman spectroscopy and TEM. Crystal morphology, orientation, composition and mechanical performance of the remineralized layers were studied using SEM, XRD, EDS and nanoindentation tests.
Results: Peptides P26 and P32 displayed a random-coil conformation with the formation of dispersed, nanospherical assemblies (~23nm) similar to rP172. In vitro mineralization experiments indicated that peptides accelerated crystal nucleation when compared to the control or rP172, transiently stabilized octacalcium phosphate (OCP) and controlled the size of apatitic crystals. SEM and XRD images revealed that repeated peptide applications to tooth slices reconstituted multi-layer, oriented enamel-like apatite, forming a seamless interface with underlying native enamel. There was a ~2-fold increase in the hardness and modulus of peptide-treated samples when compared to demineralized enamel.
Conclusions: Application of amelogenin-inspired peptides with active domains promoted the formation of durable enamel-like apatite layers. Functional domains of native proteins may offer an effective strategy for designing peptides for hard tissue engineering.