Microanatomical changes and biomolecular expression at the PDL-entheses during experimental tooth movement

Abstract

The novel aspect of this study was to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL)-space within a mechanically activated periodontal complex. The PDL is unique as it is the only ligament with both innervation and vascularization. Maxillary molars in 6-week-old male C57BL/6 mice (N = 5) were experimentally translated for 2 weeks using an elastic spacer. Contralateral teeth were used as controls. Mechanical testing of the periodontal complex of a mouse in situ and imaging using X-ray micro-computed tomography (micro-XCT) illustrated deformations within blood vessels (BV) of the PDL. PDL-bone and PDL-cementum entheses at the widened and narrowed PDL-spaces following experimental tooth movement (ETM) illustrated osterix (OSX), bone sialoprotein (BSP), cluster of differentiation 146 (CD146), and protein gene product 9.5 (PGP9.5), indicating active remodeling at these sites. PGP9.5 positive nerve bundles (NBs) were co-localized with multinucleated cells (MCs), Howship's resorption lacunae, and CD146 positive BVs. Association between nerves and MC was complemented by visualizing the proximity of osmium tetroxide stained NBs with the ultrastructure of MCs by performing scanning transmission electron microscopy. Spatial association of NB with BV, and NB with MC, provided insights into the plausible co-activation of NBs to initiate osteoclastic activity. Resorption of mineral occurred as an attempt to restore PDL-space of the load-bearing complex, specifically at the PDL-entheses. Mapping of anatomy-specific structural elements and their association with regenerative molecules by correlating light and electron micrographs provided insights into the use of these extracellular matrix molecules as plausible targets for pharmacological interventions related to tooth movement. Within the realm of tissue regeneration, modulation of load can reverse naturally occurring mineral formation to experimentally induced resorption, and naturally occurring mineral resorption to experimentally induced formation at the enthesial sites to permit tooth translation.

Keywords: biomolecules; enthesis; orthodontics; periodontal ligament; tooth movement.

Figure 1.. Temporal tooth movement and rotation:

Global tooth movement and rotation of molars M1-M3 following experimental tooth movement (ETM) after T=0 (IA, IB, IC) and T=2weeks (IID, IIE, IIF) of dam placement. Experimental and contra-lateral control hemi-maxillae were overlaid and registered (IA)&(IID), with the centers of rotation of each tooth highlighted in white (control - CTRL) and orange (experimental - EXP) (see left hand corner legend in IA and IID). Arrows connect the centers of rotation of the CTRL and EXP, displaying the angle of torque (IA) & (IID). Global tooth movement of M1-M3 are shown as a color map on tooth-root surfaces (IB)&(IIE). Displacement frequency graphs demonstrate relative tooth displacement trends for T=0 and T=2 weeks (IC)&(IIF).

Figure 2.. Changes in PDL-space over time:

PDL-space of the first molar subjected to experimental tooth movement (ETM) for T=0 in a representative 8 week old mouse (IA, IB), and for T=2 (2 weeks of placement of elastic dam in 6 week old mouse, and euthanized at 8 weeks of age) (IIC, IID) are shown as a color map on tooth-root surfaces. A Gaussian fit is applied to the graphed PDL-spaces and significant peaks were labeled (IA, IIC). The PDL-space is color-mapped (IB, IID). Gaussian-fit curves and graphed PDL-spaces are overlaid for experimental sides of the first molar for both T=0 and T=2 (IIIE). Differences in PDL-space of experimental sides of the first molar for both T=0 and T=2 weeks are shown by side by side color maps on the tooth-root surface (IIIF). See Supplemental Figure 2 for details on average PDL-space changes.

Figure 3.. Correlating anatomical location with site-specific biological expressions within the periodontal complex:

(A) PDL-space after 2 weeks of experimental tooth movement (ETM) is color-mapped on the surfaces of the root of first maxillary molar (M1). A transverse virtual section containing change in PDL-space for roots one, two and three (R1, R2, R3) as indicated by colored tethers (pseudo ligament) is shown in detail in (B). A corresponding H&E stained transverse section, with bony growths indicated by asterisks, is shown for histological reference. The anatomy-specific immunolocalization of proteins (C) is from locations within the white rectangles in (B). White rectangles correspond to narrowed and widened zone (NZ, WZ). (C) Immunohistochemistry on serial transverse sections illustrate (left to right) cluster of differentiation 146 (CD146), protein gene product 9.5 (PGP), bone sialoprotein (BSP), osterix (OSX). Top row shows protein expressions within corresponding WZ, and bottom row shows the corresponding NZ results. BV: blood vessel, N: nerves, R1: first root, PDL: periodontal ligament, OB, OC, AB. All images were taken at 40X magnification and displayed at the same scale (top left image scale bar is 50 μm).

Figure 4.. Visualization of blood vessels within a loaded periodontal complex, and the association of blood vessels with nerves and cells:

The dentoalveolar complex is shown in (a), and under load, the change in periodontal space as indicated by narrowed (−ve) and widened (+ve) regions (scale bar) is shown in (b). Data from micro-XCT illustrated deformed blood vessel (BV) within a loaded periodontal complex (white arrows, c, d). Multiscale mapping and co-localization of blood vessels, nerves and multi-nucleated cells: CLEM image (e) illustrates localization of PGP9.5 (brown stain) within the periodontal complex containing resorption pits (Howship’s lacunae, HL) and multinucleated cells (f, MC, red arrows). Spatial association of nerves with blood vessels is shown in (g) and (h) SEM micrographs. At a higher resolution using STEM technique, osmium tetroxide stained nerve bundles (NB) in association with multinucleated cell (MC) including the ultrastructure of NB is shown in (j) and (k). Note: CLEM: Correlative Light and Electron Microscopy, SEM: Scanning Election Microscopy, STEM: Scanning Transmission Election Microscopy