File:The-Adaptive-Nature-of-the-Bone-Periodontal-Ligament-Cementum-Complex-in-a-Ligature-Induced-876316.f1.ogv

English: Supplementary materials include a set of supplemental movies and 3 supplemental figures. Supplemental movies include specimens rotated relative to the crossed polarizer and analyzer to illustrate varying birefringence in collagen of the PDL from controls and ligated groups. Supplemental Figure 1 includes a single micrograph illustrating birefringence of PDL taken from control and ligated specimens across time points, respectively. Supplemental Figure 2 illustrates a merged fluorescence image of various immunolabeled biomolecules investigated in this study. Supplemental Figure 3 summarizes the observed changes in biomolecular expressions as a function of anatomical location within the complex. Supplemental Figure 4 presents a hypothesis illustrating the mechaniobioliogical-induced bone and cementum adaptation and, as a result, an overall change in organ-level biomechanics. Details pertaining to movies and figures can be found within respective captions.

Supplemental Movies. Movies showing rotation of PSR stained control and ligated complexes relative to the crossed polarizer and analyzer. 360 degrees rotation of each distal and mesial complex is shown in five degree increments. (A) 4 day control distal complex, (B) 4 day control mesial complex, (C) 4 day ligated distal complex, (D) 4 day ligated mesial complex, (E) 8 day control distal complex, (F) 8 day control mesial complex, (G) 8 day ligated complex, (H) 15 day control distal complex, (I) 15 day control mesial complex, (J) 15 day ligated distal complex, (K) 15 day ligated mesial complex. Supplemental Figure 1. Micrographs illustrating collagen birefringence differences between control and ligated bone-PDL-cementum complexes. Distal (red) and mesial (green) complexes are delineated for each control and ligated sections stained with PSR at 4, 8, and 15 days. These images at 4x magnification act as anatomical references for the supplementary movies. Alveolar bone (AB), dentin (Den), periodontal ligament (PDL), secondary cementum (SC), transseptal fibers (TF). Supplemental Figure 2. Merged image of RANKL, FN, and DAPI immunofluorescence signals. An overlay of panels representing immunofluorescence for RANKL (red), FBN (green), and DAPI (blue). (A1) 4 day control mesial complex, (A2) 4 day control distal complex, (B1) 8 day ligated mesial complex, (B2) 8 day ligated distal complex, (C1) 15 day ligated mesial complex, (C2) 15 day ligated distal complex. White arrows indicate osteoclast-like cells in resorption pits at the PDL-bone interface and the alveolar bone crest. Alveolar bone (AB), dentin (D), secondary cementum (SC), new cementum (N), and vasculature (V). Supplemental Figure 3. Models annotating (3A) fundamental changes in organ level biomechanics, and (3B) correlates between observed morphological changes to spatial biochemical expressions emphasizing morphological adaptations. (3A) TOP PANEL: (1) The healthy fibrous joint exhibits alveolar bone crest (ABC) height and PDL structure for normal attachment support. As such, it

undergoes limited, but controlled vertical displacement, torsion, and distal tooth rotation, during occlusal loading. This causes coronal compression at the distal complex of the fibrous joint. (2) Compression-induced resorption of mineralized tissues in coronal regions of the joint is shown with orange arrows. Within physiological limits, the functional PDL-space is maintained through load-induced strains and subsequent mechanobiological activity at soft-hard tissue interfaces and bulk PDL. This results in no net change to the functional PDL-space. BOTTOM PANEL: (3) Loss of coronal attachment through inflammation-induced resorption of the ABC and PDL degradation is highlighted. Loss of attachment support causes increased vertical tooth displacement and whole body rotation. Resorption of interdental and interradicular AB lowers the fulcrum, altering the whole body rotation of the tooth. Organ-level biomechanics prompts increased compressive loads at the distal coronal complex, along with increased tensile loads at the mesial coronal and mesial apical PDL-spaces and distal PDL-secondary cementum interface. (4) Perpetuating function (mastication) causes a shift in strains at soft-hard tissue interfaces, promoting net mineralized tissue formation (blue arrows, green line) in areas experiencing tensile strains and net mineralized tissue resorption (red arrows) in areas experiencing compressive strains. The net mineral-forming and -resorbing events cause large scale morphological changes in the bone-PDL-tooth complex that ultimately potentiates the joint towards disease progression and subsequent failure. (3B) Schematic drawing shows changes in the expression pattern of target biomolecules, including RANKL (red), TNF-β (brown), FN (green), TRAP (red), PSR (orange), and PDL-space (gray), of ligated groups compared to control groups.