Diabetes aggravates periodontitis by limiting repair through enhanced inflammation

Abstract

Periodontitis is the most common lytic bone disease and one of the first clinical manifestations of diabetes. Diabetes increases the risk of periodontitis. The aim of the present study was to examine mechanisms by which diabetes aggravates periodontitis. Ligature-induced periodontitis was examined in Goto-Kakizaki rats with type 2 diabetes. A tumor necrosis factor (TNF)-specific-inhibitor, pegsunercept, was applied to diabetic rats after the onset of periodontal disease. Interferon-γ (IFN-γ), TNF-α, interleukin-1 β (IL-1β), fibroblast growth factor-2 (FGF-2), transforming growth factor beta-1 (TGFβ-1), bone morphogenetic protein-2 (BMP-2), and BMP-6 were measured by real-time RT-PCR, and histological sections were examined for leukocyte infiltration and several parameters related to bone resorption and formation. Inflammation was prolonged in diabetic rats and was reversed by the TNF inhibitor, which reduced cytokine mRNA levels, leukocyte infiltration, and osteoclasts. In contrast, new bone and osteoid formation and osteoblast numbers were increased significantly vs. untreated diabetic animals. TNF inhibition in diabetic animals also reduced apoptosis, increased proliferation of bone-lining cells, and increased mRNA levels of FGF-2, TGFβ-1, BMP-2, and BMP-6. Thus, diabetes prolongs inflammation and osteoclastogenesis in periodontitis and through TNF limits the normal reparative process by negatively modulating factors that regulate bone.

Figure 1.

Diabetes prolongs an inflammatory infiltrate. Destructive periodontitis was initiated in rats by placement of ligatures around the molar teeth, which facilitates bacterial invasion of gingiva. Resolution of periodontal inflammation was induced by removal of ligatures. TNF was inhibited in diabetic rats by injection with pegsunercept when ligatures were removed and every 4 d thereafter (diab+inh group). Control rats received vehicle alone. Rats were euthanized before placing the ligature (0 d), simultaneously with ligature removal (7 d), and after ligature removal (11, 15, and 22 d). Numbers of PMN cells (A) and mononuclear cells (B) and depth of PMN cells (C) were examined in the gingival connective tissue from the epithelium adjacent to the tooth to distance of 1 mm toward alveolar bone, assessed in H&E-stained sections. *P < 0.05 vs. normoglycemic (normal) group; ⁺P < 0.05 vs. diabetic (diab) group.

Figure 2.

Diabetes increases osteoclast formation, which is due to prolonged inflammation. Induction and resolution of periodontal disease and application of TNF inhibitor were performed as in Fig. 1. Osteoclasts were counted in TRAP-stained sections as described in Materials and Methods. *P < 0.05 vs. normoglycemic group; ⁺P < 0.05 vs. diabetic group.

Figure 3.

Diabetes prolongs mRNA levels of inflammatory cytokines. Induction and resolution of periodontitis and application of TNF inhibitor were performed as in Fig. 1. IFN-γ (A), TNF-α (B), and IL-1β (C) mRNA levels were measured in RNA extracted from rat periodontium by RT-PCR. ⁺P < 0.05 vs. diabetic group.

Figure 4.

Diabetes reduces periodontal bone formation and osteoblast numbers, and this decrease is reversed in diabetic animals by TNF inhibition. New osteoid formation (A) and bone formation (B) were measured in histological sections; osteoblasts were counted per millimeter of bone length (C) in H&E-stained sections. Osteoblast counts were confirmed using osteocalcin-specific immunostaining. *P < 0.05 vs. normoglycemic group; ⁺P < 0.05 vs. diabetic group.

Figure 5.

Diabetic rats have increased apoptosis and decreased proliferation of bone-lining cells, which is reversed by TNF inhibition. Induction and resolution of periodontal disease and application of TNF inhibitor were performed as in Fig. 1. A) Apoptotic bone-lining cells in histological sections were measured by the TUNEL assay. B) Number of PCNA-immunopositive cells was measured by immunohistochemistry. *P < 0.05 vs. normoglycemic group; ⁺P < 0.05 vs. diabetic group.

Figure 6.

Diabetes reduces growth factor mRNA levels during resolution of periodontal inflammation, which is reversed by TNF inhibition. Induction and resolution of periodontal disease and application of TNF inhibitor were performed as in Fig. 1. RNA was isolated from periodontal tissue, and mRNA levels of TGFβ (A), BMP-2 (B), BMP-6 (C), and FGF-2 (D) were measured by real-time PCR. ⁺P < 0.05 vs. diabetic group.

Figure 7.

TNF inhibition enhances BMP-2 and FGF-2 production in the periodontium in diabetic specimens. Induction and resolution of periodontal disease and application of TNF inhibitor were performed as in Fig. 1. Numbers of BMP-2- and FGF-2-expressing cells in proximity to bone (within the periodontal ligament) were measured by immunohistochemistry using antibodies specific for BMP2 (A) and FGF-2 (B). *P < 0.05 vs. normoglycemic group; ⁺P < 0.05 vs. diabetic group.