Biodentine-induced remineralisation of dentine: Tetracycline labelling and two-photon fluorescence microscopy

Methods: Fourteen human dentine discs were totally demineralised and placed on top of cement blocks of Biodentine. Samples were stored in three different media: Phosphate buffered saline (PBS)/TC, water/TC, and PBS alone. Additionally, three dentine discs were stored separately in the first solution without the cement. Following 8 weeks storing at 37˚C, discs were collected, cleaned, sectioned, and polished; one half was examined using 2p fluorescence microscope (x40/1.3 NA objective lens, 800 nm excitation, 550 +/- 20 nm emission), while the other half was examined using a Raman spectrometer for chemical analysis (x50/ 0.75 NA objective lens, 785 nm laser, 1200 lines/mm diffraction grating). Fluorescence intensity and fluorescence lifetime were both measured for each image, and average values were calculated for each group.

Results: The highest fluorescence intensity was exhibited by the dentine samples stored with the cement in the PBS/TC solution. Dentine samples of this group have also shown the formation of a mineralization front, intra-tubular crystallisation, and small fluorescent globular structures in the inter-tubular matrix. Dentine stored in the other solutions exhibited much weaker fluorescence, with none of these features detected. Fluorescence life-time analysis indicated that the fluorescence origin detected in the samples stored in TC-containing solutions was Tetracycline. Raman spectra confirmed the formation of mineral deposits, composed of calcium phosphate and calcium carbonate, no mineral formation was detectable in the dentine stored in cement-free or PBS-free media.

Conclusions: Biodentine induced calcium-phosphate mineral formation within the dentine matrix in a phosphate rich media, which was selectively detectable using the Tetracycline labelling and 2p fluorescence microscope.