Actinobacillus actinomycetemcomitans lipopolysaccharide-mediated experimental bone loss model for aggressive periodontitis

Abstract

Background: Bacterial constituents, such as Gram-negative derived lipopolysaccharide (LPS), can initiate inflammatory bone loss through induction of host-derived inflammatory cytokines. The aim of this study was to establish a model of aggressive inflammatory alveolar bone loss in rats using LPS derived from the periodontal pathogen Actinobacillus actinomycetemcomitans.

Methods: Eighteen female Sprague-Dawley rats were divided into LPS test (N = 12) and saline control (N = 6) groups. All animals received injections to the palatal molar gingiva three times per week for 8 weeks. At 8 weeks, linear and volumetric alveolar bone loss was measured by micro-computed tomography (microCT). The prevalence of inflammatory infiltrate, proinflammatory cytokines, and osteoclasts was assessed from hematoxylin and eosin, immunohistochemical, or tartrate-resistant acid phosphatase (TRAP)-stained sections. Statistical analysis was performed.

Results: A. actinomycetemcomitans LPS induced severe bone loss over 8 weeks, whereas control groups were unchanged. Linear and volumetric analysis of maxillae by microCT indicated significant loss of bone with LPS administration. Histologic examination revealed increased inflammatory infiltrate, significantly increased immunostaining for interleukin IL-6 and -1beta and tumor necrosis factor-alpha, and more TRAP-positive osteoclasts in the LPS group compared to controls.

Conclusion: Oral injections of LPS derived from the periodontal pathogen A. actinomycetemcomitans can induce severe alveolar bone loss and proinflammatory cytokine production in rats by 8 weeks.

Figure 1

Characterization of A. actinomycetemcomitans LPS used in these studies. A) Purified A. actinomycetemcomitans LPS demonstrated characteristic laddering on a silver nitrate–stained polyacrylamide gel with indicated quantities of A. actinomycetemcomitans LPS. B) Absence of protein as indicated by Coomassie-stained gel with the same LPS. C) Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra showing singly charged anions representing the major lipid A structures present. The major peak corresponds to the hexa-acylated form (mass-to-charge ratio [m/z] at 1,827) with minor forms being present as well.

Figure 2

A. actinomycetemcomitans LPS induced more inflammatory cell infiltrate into the area adjacent to bone loss. Histologic appearance of representative control at low magnification (A), and at high magnification (B), enlarged box area from A shows normal structure with minimal inflammatory cells. A. actinomycetemcomitans LPS-injected rat periodontal tissues at low magnification (C), and at high magnification (D), enlarged box area from C shows significant inflammatory infiltrate is present.

Figure 3

μCT shows A. actinomycetemcomitans LPS induced significant linear bone loss. A) Reformatted μCT isoform display from 8-week A. actinomycetemcomitans LPS-injected rat maxillae exhibits dramatic palatal and interproximal bone loss. B) Linear bone loss as measured from the CEJ to the ABC. Significant bone loss (*P < 0.01) was observed between control (N = 6) and A. actinomycetemcomitans LPS (Aa LPS)-injected rats (N = 12).

Figure 4

A. actinomycetemcomitans LPS induced significant loss of proximal bone volume. A) Reformatted μCT isoform display showing volumetric ROI used for analysis. B) Analysis of μCT volumes were assessed using software. Data are presented as percentage bone within ROI. Significant bone loss (*P < 0.001) was observed between control (N = 6) and A. actinomycetemcomitans LPS (Aa LPS)-injected rats (N = 12).

Figure 5

Pronounced expression of proinflammatory cytokines was observed in A. actinomycetemcomitans LPS (Aa LPS)-injected animals. Histologic sections were immunostained and detected using Nova Red reagent for IL-6 (A), IL-1β (C), or TNF-α (E). B, D, and F) Immunohistochemical (IHC) scores from control animals and 8 weeks post-LPS injections. Significant differences were observed for IL-6 (*P = 0.0104) and TNF-α (*P = 0.0305) and approached significant differences between control and LPS-treated groups for IL-1β (P = 0.0671). (Original magnification × 60.)

Figure 6

A. actinomycetemcomitans LPS delivery enhanced osteoclastogenesis. A) Histologic sections were stained for TRAP. Arrows indicate TRAP-positive cells in A. actinomycetemcomitans LPS (Aa LPS)-injected rats. B) Stained cells that were TRAP-positive and had three or more nuclei were enumerated. LPS-injected animals (N = 12) exhibited significantly more TRAP-positive cells than control animals (N = 6; *P = 0.0023). (Scale bar = 1μm; original magnification × 60.)