Does dentine mineral change with anatomical location, microscopic site and patient age?

Abstract

Objective: To determine the effect of patient age (young or mature), anatomical location (shallow/deep and central/peripheral) and microscopic site (intertubular/peritubular) on dentine mineral density, distribution and composition.

Methods: Extracted posterior teeth from young (aged 19-20 years, N = 4) and mature (aged 54-77 years, N = 4) subjects were prepared to shallow and deep slices. The dentine surface elemental composition was investigated in a SEM using Backscattered Electron (BSE) micrographs, Energy Dispersive X-ray Spectroscopy, and Integrated Mineral Analysis. Qualitative comparisons and quantitative measures using machine learning were used to analyse the BSE images. Quantitative outcomes were compared using quantile or linear regression models with bootstrapping to account for the multiple measures per sample. Subsequently, a Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM) was used to mill large area (100 µm) cross-sections to investigate morphology through the dentine tubules using high resolution secondary electron micrographs.

Results: With age, dentine mineral composition remains stable, but density changes with anatomical location and microscopic site. Microscopically, accessory tubules spread into intertubular dentine (ITD) from the main tubule lumens. Within the lumens, mineral deposits form calcospherites in the young that eventually coalesce in mature tubules and branches. The mineral occlusion in mature dentine increases overall ITD density to reflect peritubular dentine (PTD) infiltrate. The ITD observed in micrographs remained consistent for age and observation plane to suggest tubule deposition affects overall dentine density. Mineral density depends on the relative distribution of PTD to ITD that varies with anatomical location.

Significance: Adhesive materials may interact differently within a tooth as well as in different age groups.

Keywords: Age; Apatite; BSE; BSE, Backscatter Electron; Ca, Calcium; Cl, Chloride; DEJ, Dentine-enamel junction; DT, Dentine Tubule; Dentine; EPMA, Electron Probe Microanalyser; Ga, Gallium; H, Hydrogen; Human; ITD, Intertubular Dentine; Intertubular dentine; LA-ICP-MS, Laser Ablation Induction Coupled Plasma Mass Spectroscopy; Mg, Magnesium; Mineral; Na, Sodium; O, Oxygen; Odontoblasts; P, Phosporus; PTD, Peritubular Dentine; Peritubular dentine; SEM, Scanning Electron Microscope; SEM-EDS; SEM-EDS, Scanning Electron Microscope Energy Dispersive X-ray Spectroscopy; TEM, Transmission Electron Microscope; TIMA, Integrated Mineral Analysis; XE PFIB-SEM, Xenon Plasma Focussed Ion Beam Scanning Electron Microscope; Xe PFIB-SEM; β-TCMP, Magnesium-whitlockite.

Graphical abstract

Fig. 1

A. Sound molar teeth were collected and immediately stored in Phosphate Buffered Saline. Teeth were hemi-sectioned to locate the dentine-enamel junction (DEJ) and pulp roof and prepared to shallow (0.5–0.75 mm below the DEJ) or deep (0.5–0.75 mm above the roof of the pulp) sites. B. A shallow and deep section was prepared from each tooth, dried in ambient conditions before resin embedding. C. Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy identified elemental composition of peritubular and intertubular dentine. D. Backscattered Electron Micrographs analysed for percentage black, white & grey as well as relative grey intensity. E. Integrated Mineral Analysis of a young and mature tooth dentine was performed F. Xenon Plasm Focussed Ion Beam was used to mill dentine G. i. Depicts the sub-regional classification of central and peripheral dentine ii. The microscopic sites analysed in this study include dentine tubules, peritubular and intertubular dentine.

Fig. 2

Scanning electron micrographs of xenon plasma focussed ion beam (Xe PFIB-SEM) milling of tooth surface to create a cross-section of dentine tubules. A. Manipulator needle attached to a single crystal silicon mask (100 µm × 50 µm × 50 µm) held over the surface of a polished tooth slice to mill a flat surface using focussed xenon ions. B. Figure ‘a’ magnified to show the surface of the tooth and the cross-section after removal of the mask. C. The surface of the dentine from a tooth slice before embedding and polishing. D. The ‘top’ surface view of the polished tooth after milling. E. The ‘top’ surface of the polished surface after milling at the edge of the mask. The left hand side of this micrograph shows the pillaring effect from the xenon beam if a mask is not used. Scale bars = A. 100 µm, B. 20 µm, C-E. 5 µm.

Fig. 3

Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe-PFIB SEM) micrographs of dentine tubules (DT) from shallow central dentine from a A. mature person in transverse plane, a B. young person in longitudinal plane and a C. young person in an oblique plane. Micrographs reveal the location of the peritubular dentine (PTD) with the tips of the double-ended arrows demarcating PTD thickness, nanoporous intertubular dentine (ITD) with smaller tubules in cross-section (circles). A white arrow delineates the ITD from the PTD and a black arrow reveals the intersection between the PTD wall and the internal mineral architecture that has detached due to dehydration and resulting shrinkage. A. In the mature dentine cross-section, a black asterisk differentiates the original DT margin from internal calcifications and the unfilled tubule centre (white asterisk). B. The young dentine tubule in longitudinal cross-section depicts a white triangle where debris blocks the tubule entrance. Internally, a white circle identifies round deposits that line the lower tubule wall and a white square is located where material has formed against the superior wall. The PTD wall cross-section (white star) remains intact. C. The DT from a young person in oblique cross-section with smaller accessory tubules (white bracket) appears to originate from the main tubule.

Fig. 4

Scanning electron microscopy backscattered electron micrographs of polished dentine surfaces in the transverse plain from A. young 18-year-old and B. mature 63-year-old. Micrographs are shown for i. shallow peripheral ii. shallow central iii. deep peripheral and iv. deep central locations. Av. and Bv. are magnified versions of Aii. and Bii. respectively. Note the major dentine tubule branches in longitudinal views (white arrows) and transverse views (circles). The intact tubules in transverse view are visible (triangle) and intertubular dentine weave more prominent. Dentine tubules with dehydration cracks traversing through lie below a. Note the micrographs in Panel B are relatively lighter in intensity with more grey/white regions compared with Panel A. Scale bar = 5 µm.

Fig. 5

Summary of major findings for Scanning Electron Microscopy backscattered electron micrograph analysis and Energy Dispersive X-ray results for young and mature dentine at shallow and deep, central and peripheral locations.

Fig. 6

Panel of elemental and phase maps from Scanning Electron Microscopy Integrated Mineral Analysis (TIMA) deep central dentine surfaces in the transverse plane from a A. young and B. mature person. Maps are shown for i. Apatite ii. Calcium iii. and Phosphorus. Below each Calcium and Phosphorus map is a scale that indicates the number of x-rays identified at each pixel (2500 x-rays were collected at every pixel). Scale bar = 50 µm.