Functional Remineralization of Dentin Lesions Using Polymer-Induced Liquid-Precursor Process

It was hypothesized that applying the polymer-induced liquid-precursor (PILP) system to artificial lesions would result in time-dependent functional remineralization of carious dentin lesions that restores the mechanical properties of demineralized dentin matrix. 140 µm deep artificial caries lesions were remineralized via the PILP process for 7–28 days at 37°C to determine temporal remineralization characteristics. Poly-L-aspartic acid (27 KDa) was used as the polymeric process-directing agent and was added to the remineralization solution at a calcium-to-phosphate ratio of 2.14 (mol/mol). Nanomechanical properties of hydrated artificial lesions had a low reduced elastic modulus (ER = 0.2 GPa) region extending about 70 μm into the lesion, with a sloped region to about 140 μm where values reached normal dentin (18–20 GPa). After 7 days specimens recovered mechanical properties in the sloped region by 51% compared to the artificial lesion. Between 7–14 days, recovery of the outer portion of the lesion continued to a level of about 10 GPa with 74% improvement. 28 days of PILP mineralization resulted in 91% improvement of ER compared to the artificial lesion. These differences were statistically significant as determined from change-point diagrams. Mineral profiles determined by micro x-ray computed tomography were shallower than those determined by nanoindentation, and showed similar changes over time, but full mineral recovery occurred after 14 days in both the outer and sloped portions of the lesion. Scanning electron microscopy and energy dispersive x-ray analysis showed similar morphologies that were distinct from normal dentin with a clear line of demarcation between the outer and sloped portions of the lesion. Transmission electron microscopy and selected area electron diffraction showed that the starting lesions contained some residual mineral in the outer portions, which exhibited poor crystallinity. During remineralization, intrafibrillar mineral increased and crystallinity improved with intrafibrillar mineral exhibiting the orientation found in normal dentin or bone

Typical 2d reconstructions showing x-ray attenuation with depth in the lesions at different remineralization times.

<p>a) control lesion (0 time) showing protected portion at the left, the severely demineralized region which gradually blends with normal dentin. Widened dentin tubules were apparent at the depth of the lesion. b) 7 day remineralization treatment seems more complete in the deeper part of the lesion. c) 14 day remineralization shows some unevenness in the remineralization, but much of the lesion appeared fully mineralized. d) 28 day remineralization was similar to 14 day specimens. Note there is no apparent shrinkage in any of the specimens as the imaging was done in fully hydrated tissue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038852#pone.0038852.s001" target="_blank">supplemental Figure 1</a>). Dotted line shows approximate lesion depth, magnification bar = 100 µm. A movie at 10X, demonstrating widened tubules at the lesion site is also provided as supplemental information.</p

Schematic diagram showing specimen preparation and analyses.

<p>a) dentin block covered with nail varnish with exposed window for lesion formation showing subsequent sections 1–3 used for nanoindentation, MicroXCT™ and microscopy. b) Schematic of lesion section such as that labeled 1 in a). c) nanoindentations at 4 µm intervals made through lesion into normal dentin. d) regions of interest used from a thinned region of lesion marked 2 in a) that was used to determine X-ray attenuation from MicroXCT™ tomograms at intervals of ∼ 785 nm (not drawn to scale).</p

Average shrinkage measurement at three time points relative to control

<p>Average shrinkage measurement at three time points relative to control</p

Analysis of the areas under the curves from the data of Figures 2, 3, and 7 with percent change from lesion (%).

*<p>Significantly different from control; + Significantly different from lesion, (p<0.05, n = 4, mean ± S.E.M).</p

TEM of unstained lesion, 7 and 14 day specimens with corresponding SAED patterns showing intrafibrillar mineral within collagen fibrils after remineralization.

<p>Diffraction was consistent with apatite with its long axis along the axis of the collagen fibrils. Inset of lesion shows lower magnification of the specimen appearance and the inset at 14 days shows intrafibrillar crystallites at higher magnification.</p

Reduced elastic modulus values increased with remineralization time as shown by statistical change-point diagrams with 95% CLs.

<p>Artificial lesion profiles (grey) had a low modulus region that extended nearly 80 µm, followed by a sloped region that extended to the depth of the lesion at 140–150 µm, Seven day PILP remineralization (green) showed significantly increased properties with normal dentin values starting at about 100 µm from the surface. These trends continued with time at 14 days (blue) and 28 days (red) of remineralization. Little additional change was observed between the 14 and 28 day treatments that had restored normal properties from about 85 µm inward. In the outer portion modulus values were restored to about half of normal levels. Average control values (open circles, squares, triangles, and diamonds) are shown without error bars for clarity.</p

a) SEM showing marked structural differences in the outer (bottom) and gradient zones of remineralization.

<p>b) EDS analysis showed little difference at 14 days. Figs. 7a and 7b show outer zone mineral which seemed to cover the region and fill most of the tubule lumens. In the gradient zone the mineral formed a lip around each tubule lumen.</p