The adaptive nature of the bone-periodontal ligament-cementum complex in a ligature-induced periodontitis rat model

Abstract

The novel aspect of this study involves illustrating significant adaptation of a functionally loaded bone-PDL-cementum complex in a ligature-induced periodontitis rat model. Following 4, 8, and 15 days of ligation, proinflammatory cytokines (TNF- α and RANKL), a mineral resorption indicator (TRAP), and a cell migration and adhesion molecule for tissue regeneration (fibronectin) within the complex were localized and correlated with changes in PDL-space (functional space). At 4 days of ligation, the functional space of the distal complex was widened compared to controls and was positively correlated with an increased expression of TNF- α. At 8 and 15 days, the number of RANKL(+) cells decreased near the mesial alveolar bone crest (ABC) but increased at the distal ABC. TRAP(+) cells on both sides of the complex significantly increased at 8 days. A gradual change in fibronectin expression from the distal PDL-secondary cementum interfaces through precementum layers was observed when compared to increased and abrupt changes at the mesial PDL-cementum and PDL-bone interfaces in ligated and control groups. Based on our results, we hypothesize that compromised strain fields can be created in a diseased periodontium, which in response to prolonged function can significantly alter the original bone and apical cementum formations.

Figure 1

In vivo rat ligature model for the induction of acute periodontitis. (a) Photograph illustrates lipopolysaccharide (LPS) soaked 4–0 braided silk threads in the diastemata flanking left/right second maxillary molars. Controls were flossed in the same interproximal regions. (b) Schematic illustrates the targeted regions of the fibrous joint within the study. (c) 3D tomogram illustrates the lingual-sagittal, mid-sagittal, and buccal-sagittal 2D virtual sections through a second maxillary molar used for morphometrics. Anatomical landmarks used to measure alveolar bone crest recession (CEJ-ABC) are indicated. (d) 2D virtual section illustrates anatomical landmarks to measure interradicular distance and PDL width. Division of the bone-PDL-cementum complex into coronal, middle, and apical sections for PDL-space measurements is also illustrated.

Figure 2

Identification of RANKL using immunofluorescence. (a) Representative micrographs illustrate immunofluorescence of antibodies against RANKL at 4-day control and 8- and 15-day ligated groups. Note RANKL expression around the vasculature (V) and endosteal spaces (asterisks). Multinucleated osteoclast-like cells were also observed at the PDL-bone interface (white arrows). (b) Higher magnification micrographs show RANKL immunofluorescence in local regions of the complex at 15 days of ligation. (c) Schematic of rat periodontal tissue (mesiodistal section) with gray boxes (125 μm × 125 μm) that indicate target areas used to count RANKL(+) cells. (d, e) Bar graphs illustrate RANKL(+) cell count within specified target areas between control and ligated groups on mesial (d) and distal (e) sides. *Statistically significant difference at 95% confidence interval was observed. Junctional epithelium (JE), epithelium (E), periodontal ligament (PDL), alveolar bone (AB), alveolar bone crest (ABC), dentin (D), and secondary cementum (SC).

Figure 3

Alveolar bone resorption through TRAP(+) osteoclast identification. (a) Mesiodistal histological sections illustrate TRAP(+) cells on distal surfaces. The relative height of the alveolar bone crest (ABC) in relation to the cementoenamel junction (CEJ) is shown to decrease with duration of ligation. (b) Magnified images of 3A show alveolar bone crest (ABC), coronal, and apical regions of distal surfaces across time points and between control and ligated complexes (i–ix). The number of multinucleated osteoclasts (OC) located along the bone perimeter was counted in coronal and apical root segments on mesial (x) and distal (xi) sides. *Statistically significant difference at 95% confidence interval was observed. Alveolar bone (AB), periodontal ligament (PDL), dentin (D), and secondary cementum (SC).

Figure 4

Immunohistochemical staining for identification of TNF-α. (a) Representative light micrograph of the complex at 15 days of ligation illustrates localization of TNF-α. (b) Representative images illustrate immunohistochemical localization of TNF-α at subepithelial connective tissue, coronal and apical PDL spaces, and interradicular PDL regions, according to experimental conditions. For example, note TNF-α expression at the distal coronal PDL-bone interface (black arrow heads). Connective tissue (CT), alveolar bone (AB), periodontal ligament (PDL), dentin (D), and secondary cementum (SC).

Figure 5

Morphometric comparisons of control and ligated bone-PDL-cementum complexes, including changes in collagen birefringence. (a) Comparisons of measurements in mesial and distal regions of the CEJ-ABC (i), the interradicular region (ii), and the PDL-space (iii, iv) between control and ligated complexes at 4, 8, and 15 days. Individual graphs were used to compare PDL-space measurements between coronal and apical anatomical locations within mesial (iii) and distal (iv) complexes. (b) The differences between average ligated and average control PDL-space measurements are plotted for each aforementioned anatomical location across time. (c) Histological sections show the distal complex stained with PSR and visualized under polarized light microscopy. Alveolar bone (AB), periodontal ligament (PDL), secondary cementum (SC), and transseptal fibers (TF). Symbols within plots indicate statistically significant differences at 95% confidence interval. ^&,†,Ψ,¥Significant difference between control and ligated groups. ^η,‡,κSignificant difference over time. ^#,+Significant difference between control and ligated groups. *Significant difference over time. ^{A,Β,C,D,E,F,G,H}Significant difference between control and ligated groups. ^▲,∆Significant difference over time.

Figure 6

Line profiles and micrographs of immunolabeled fibronectin (FN). Representative micrographs illustrate FN immunofluorescence in apical regions of control and ligated complexes at 4, 8, and 15 days. The intensity of FN expression was measured along the anatomical locations indicated by the 300 μm long bar. For the mesial complex, the x-axis of the 300 μm long profile corresponds to alveolar bone (AB), periodontal ligament (PDL), secondary cementum (SC), and dentin (D). Note that the direction for the distal complex is reversed. (a1) 4-day control mesial complex, (a2) 4-day control distal complex, (a3) 4-day ligated mesial complex, (a4) 4-day ligated distal complex, (b1) 8-day control mesial complex, (b2) 8-day control distal complex, (b3) 8-day ligated mesial complex, (b4) 8-day ligated distal complex, (c1) 15-day control mesial complex, (c2) 15-day control distal complex, (c3) 15-day ligated mesial complex, and (c4) 15-day ligated distal complex. Dentin (D), secondary cementum (SC), periodontal ligament (PDL), alveolar bone (AB), new bone (NB).