Self-Assembling Peptide Nano-Apatite Hybrid Material for Dentine Mineralisation

Dentine sensitivity continues to be a problem for patients who experience painful symptoms associated with the condition and there is a clear clinical need for the development of new treatments to manage the problem. Currently available treatments often require frequent application or do not produce the required alleviation of symptoms. The aim of this thesis was to establish if self-assembling peptides (SAPs) combined with nanohydroxyapatite seed crystals could be an effective treatment for the occlusion of human dentine tubules and so potentially alleviate the symptoms of dentine sensitivity.
Nanohydroxyapatite (nanoHA) seed crystals were produced via hydrothermal synthesis and characterised using x-ray diffraction, dynamic light scattering, scanning electron microscopy and transmission electron microscopy. This confirmed the presence of highly crystalline rod shaped crystals with a median size of 45 nm (longest axis). A steady state in vitro nucleation and crystal growth assay (IVNCG) was used to determine the minimum nanoHA seed crystal density in agarose required to generate a total phosphate mass (indicative of quantity of mineral formed) comparable to that produced by the poly-L-glutamic acid positive control after 5 days exposure to buffers containing calcium and phosphate ions at physiological concentrations, pH and temperature. A transition in crystal morphology and composition, reflecting a shift from an OCP-like towards a HA-like chemistry and appearance, was seen on increasing nanoHA seeding density from 20 µg/mL to 30 µg/mL. SAPs (P11-13 and P11-14, complementary pair) were then combined with nanoHA seed crystals to produce a novel hybrid material. Its ability to support HA crystal growth was assessed using the IVNCG, without success, due to precipitation of the hybrid material. A novel precipitation assay was developed and used alongside a 1H NMR study to determine the cause of the observed precipitation. Precipitation was found to be associated with interaction and aggregation of the P11-13 peptide with nanoHA in the presence of calcium ions at the same concentration as those used in the IVNCG assay, possibly through calcium bridging.
SAP/nanoHA hybrid material and P11-4 were applied to human dentine samples and their ability to reduce dentine permeability assessed using a fluid filtration method after initial application and following 7 days incubation in artificial saliva (AS). Surprisingly, following treatment with the hybrid material, dentine permeability increased after 7 days in AS, possibly due to P11-13 interaction with the dentine tubule wall (as suggested by the NMR experiments) resulting in compression of the non-slip layer. In contrast, P11-4 treated dentine showed a decrease in permeability after 7 days incubation in AS which was statistically insignificant compared to the positive control (I Bond universal, Kulzer)(p>0.05). The reduction in fluid flow was attributed to the formation of a 10 µm thick layer of crystals, believed to be HA, present 20 µm down the tubule and seen in the SEM analysis.
In conclusion the P11-13/14 self-assembling peptides combined with nanoHA treatment produced no evidence of human dentine tubule occlusion and actually resulted in an increase in fluid flow through dentine. P11-4 treatment, in contrast, resulted in reduced dentine permeability and should be considered a candidate for further development as a dentine sensitivity treatment.