Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats

Abstract

Background and objective: Theaflavins (TFs), the major polyphenol in black tea, have the ability to reduce inflammation and bone resorption. The aim of this study was to evaluate the effects of TFs on experimental periodontitis in rats.

Material and methods: Thirty rats were divided into five groups: Control (glycerol application without ligation), Ligature (glycerol application with ligation), TF1 (1 mg/mL TF application with ligation), TF10 (10 mg/mL TF application with ligation), and TF100 (100 mg/mL TF application with ligation). To induce experimental periodontitis, ligatures were placed around maxillary first molars bilaterally. After ligature placement, 100 μL glycerol or TFs were topically applied to the rats daily, and rats were euthanized 7 days after ligature placement. Micro-computed tomography was used to measure bone resorption in the left side of the maxilla, and quantitative polymerase chain reaction was used to measure the expression of interleukin (IL)-6, growth-regulated gene product/cytokine-induced neutrophil chemoattractant (Gro/Cinc-1, rat equivalent of IL-8), matrix metalloproteinase-9 (Mmp-9), receptor activator of nuclear factor-kappa Β ligand (Rankl), osteoprotegerin (Opg), and the Rankl/Opg ratio in gingival tissue. With tissue from the right side of the maxilla, hematoxylin and eosin staining was used for histological analysis, immunohistochemical staining for leukocyte common antigen (CD45) was used to assess inflammation, and tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclasts.

Results: The TF10 and TF100 groups, but not the TF1 group, had significant inhibition of alveolar bone loss, reduction in inflammatory cell infiltration in the periodontium, and significantly reduced numbers of CD45-positive cells and TRAP-positive osteoclasts compared with the Ligature group. Correspondingly, the TF10 and TF100 groups had significantly downregulated gene expression of IL-6, Gro/Cinc-1(IL-8), Mmp-9, and Rankl, but not of Opg. Consequently, Rankl/Opg expression was significantly increased in the Ligation group but was attenuated in the TF10 and TF100 groups.

Conclusion: The results of this study suggest that topical application of TFs may reduce inflammation and bone resorption in experimental periodontitis. Therefore, TFs have therapeutic potential in the treatment of periodontal disease.

Keywords: cytokines; experimental periodontitis; periodontal disease; theaflavin.

Figure 1

Experimental groups. Control, glycerol application without ligation; Ligature, glycerol application with ligation; TF1, 1 mg/mL TF application with ligation; TF10, 10 mg/mL TF application with ligation; and TF100, 100 mg/mL TF application with ligation

Figure 2

Reconstructed 3D micro‐CT images. A, The arrows show the distance from the palatal CEJ to the ABC as a marker of alveolar bone height; the length of three areas (mesial, central, and distal sites) of the maxillary M1 was measured, and the mean value was determined as experimental data. B, A cube region was selected as the ROI to evaluate the ratio of the bone volume fraction (BV/TV)

Figure 3

Schematic illustration of the target regions of CD45‐positive and TRAP‐positive multinucleated cells of rat periodontal tissue. The four square fields (100 × 100 μm) of connective tissue adjacent to the JE indicate target areas that were used to count CD45‐positive cells. TRAP‐positive multinucleated cells were calculated on the linear surface of the alveolar bone. The segment from the top of the alveolar bone crest to the starting point of the curve was designated as the horizontal segment (Line X), and that from the top of the alveolar bone crest to the apex along the periodontal ligament was designated as the vertical segment (Line Y). Black arrow, cemento‐enamel junction. ABC, alveolar bone crest

Figure 4

Effects of TFs on alveolar bone loss. A, Reconstructed 3D micro‐CT images show the palatal view of the maxilla (scale bar = 3000 μm). B, The CEJ‐ABC distance data are expressed as mean ± SD. C, The BV/TV ratio in the interradicular regions of the first molars is expressed as mean ± SD. ^# P < 0.05, ^## P < 0.001 compared with the Control group; *P < 0.05, **P < 0.001 compared with the Ligature group. Control, glycerol application without ligation; Ligature, glycerol application with ligation; TF1, 1 mg/mL TF application with ligation; TF10, 10 mg/mL TF application with ligation; and TF100, 100 mg/mL TF application with ligation

Figure 5

Histological evaluation of inflammatory infiltration by H&E staining. H&E staining of specimens from the Control group (A, F), Ligature group (B, G), TF1 group (C, H), TF10 group (D, I), and TF100 group (E, J). Higher magnification micrographs show that the TF10 (I) and TF100 (J) groups had reduced inflammatory cell infiltration and less cementum exposed than the Ligature (G) and TF1 (H) groups. Control, glycerol application without ligation; Ligature, glycerol application with ligation; TF1, 1 mg/mL TF application with ligation; TF10, 10 mg/mL TF application with ligation; and TF100, 100 mg/mL TF application with ligation. Black arrows, cemento‐enamel junction (CEJ); white arrows, junctional epithelium (JE). Scale bar = 200 μm

Figure 6

Histological evaluation of inflammatory infiltration by immunohistochemical staining. CD45 (green) staining of specimens from the Control group (A), Ligature group (B), TF1 group (C), TF10 group (D), and TF100 group (E). Nuclear counterstaining by DAPI (blue) in fluorescent images. Higher magnification micrographs in the upper right corner show that the TF10 (D) and TF100 (E) groups had reduced CD45‐positive cell infiltration compared with the Ligature (B) and TF1 (C) groups. Control, glycerol application without ligation; Ligature, glycerol application with ligation; TF1, 1 mg/mL TF application with ligation; TF10, 10 mg/mL TF application with ligation; and TF100, 100 mg/mL TF application with ligation. Scale bar = 200 μm

Figure 7

Histological evaluation of osteoclasts. Osteoclasts were stained red by TRAP staining. Specimens from the Control group (A, F), Ligature group (B, G), TF1 group (C, H), TF10 group (D, I), and TF100 group (E, J) are shown. Higher magnification micrographs show that the Control group (F) had few TRAP‐positive cells on the Line Y segment. The TF10 (I) and TF100 (J) groups had fewer TRAP‐positive cells than the Ligature (G) and TF1 (H) groups, especially on the Line X segment. Control, glycerol application without ligation; Ligature, glycerol application with ligation; TF1, 1 mg/mL TF application with ligation; TF10, 10 mg/mL TF application with ligation; and TF100, 100 mg/mL TF application with ligation. ABC, alveolar bone crest. Scale bar = 200 μm

Figure 8

Effects of TFs on the mRNA expression of (A) IL‐6, (B) Gro/Cinc‐1 (IL‐8), (C) Mmp‐9, (D) Rankl, and (E) Opg (F) and the Rankl/Opg expression ratio in the gingival tissue of rats, as determined by qPCR. The data were normalized to the housekeeping gene, Gapdh. Data are expressed as mean ± SD. ^# P < 0.05, ^## P < 0.001 compared with the Control group; *P < 0.05, **P < 0.001 compared with the Ligature group. IL, interleukin; Gro/Cinc‐1, rat equivalent of IL‐8; Mmp, matrix metalloproteinase; Rankl, receptor activator of nuclear factor‐kappa B ligand; Opg, osteoprotegerin. Control, glycerol application without ligation; Ligature, glycerol application with ligation; TF1, 1 mg/mL TF application with ligation; TF10, 10 mg/mL TF application with ligation; and TF100, 100 mg/mL TF application with ligation