Effects of the probiotic Bifidobacterium animalis subsp. lactis on the non-surgical treatment of periodontitis. A histomorphometric, microtomographic and immunohistochemical study in rats

Abstract

Lactobacillus probiotics have been investigated in periodontitis. However, the effects of the genus Bifidobacterium on periodontitis are hardly known. This study evaluated the effects of the probiotic (PROB) Bifidobacterium animalis subsp. lactis (B. lactis) HN019 as an adjunct to scaling and root planing (SRP) in rats with experimental periodontitis (EP). At baseline, 32 rats were assigned to 4 groups: C (control), PROB, EP-SRP and EP-SRP-PROB. In groups EP-SRP and EP-SRP-PROB, the mandibular first molars of the animals received a ligature. At day 14, the ligatures were removed and SRP was performed. Animals of groups PROB and EP-SRP-PROB were orally administered with 10 mL/day of 109 colony forming units of B. lactis HN019 for 15 days, starting at day 14. Animals were euthanized at day 29. Histomorphometric, microtomographic and immunohistochemical analyses were performed. Microbiological effects of B. lactis on biofilm were also evaluated. Data were statistically analyzed (ANOVA, Tukey; Kruskal-Wallis, Dunn's; Two-tailed t-test; p<0.05). Group EP-SRP-PROB presented reduced alveolar bone resorption and attachment loss when compared with Group EP-SRP (p<0.05). Group EP-SRP-PROB showed significantly fewer osteoclasts, increased expression of anti-inflammatory cytokines and reduced expression of proinflammatory cytokines compared with Group EP-SRP (p<0.05). B. lactis promoted a higher ratio between aerobic and anaerobic bacteria in biofilm samples (p<0.05). B. lactis HN019 may have a role in the treatment of EP in rats, as an adjunct to SRP.

Fig 1. Microtomographic analysis.

Three-dimensional rendered reconstructions of the microtomographic sections of groups C (A, E), PROB (B, F), EP-SRP (C, G) and EP-SRP-PROB (D, H). Buccal view (A-D). Internal surface view, sagittal section (E-H). Pixel size = 7.96 μ.

Fig 2. Histomorphometric analysis of periodontal tissues.

Means and standard deviations of ANB (A; furcation area) and AL (B; interproximal area), with comparisons among groups. Photomicrographs of periodontal tissues in the furcation (C-F) and interproximal areas (G-J) of mandibular first molars: Group C (C and G); Group PROB (D and H); Group EP-SRP (E and I); Group EP-SRP-PROB (F and J). Abbreviations and symbols: ab = alveolar bone; ct = connective tissue; pdl = periodontal ligament; ANB = area of no bone; AL = attachment loss; FM = first molar; SM = second molar; black arrows = cementoenamel junction; white arrows = epithelial attachment; * = p<0.05; ** = p<0.01; *** = p<0.001. Scale bars: C-J = 200 μm. (Hematoxylin and Eosin stain).

Fig 3. Immunohistochemical analyses–TRAP.

Means and standard deviations of the number of TRAP-positive multinucleated cells (A), with comparisons among groups. Photomicrographs demonstrating immunolabeling for TRAP (B-E) in the furcation regions of mandibular first molars: Group C (B); Group PROB (C); Group EP-SRP (D); Group EP-SRP-PROB (E). Abbreviations and symbols: ab = alveolar bone; ct = connective tissue; pdl = periodontal ligament; black arrowhead = TRAP-positive multinucleated cell; * = p<0.05; ** = p<0.01; *** = p<0.001. Scale bars: B-E = 20 μm. (Hematoxylin counterstaining).

Fig 4. Immunohistochemical analyses—IL-1β and CINC.

Medians, interquartile range and maximum and minimum values of the immunolabeling scores for IL-1β (A) and CINC (B), with comparisons among groups. Photomicrographs showing immunolabeling for IL-1β (C-F) and CINC (G-J) in the furcation regions of mandibular first molars: Group C (C and G); Group PROB (D and H); Group EP-SRP (E and I); Group EP-SRP-PROB (F and J). Abbreviations: ab = alveolar bone; ct = connective tissue; pdl = periodontal ligament; * = p<0.05. Scale bars: C-J = 80 μm. (Hematoxylin counterstaining).

Fig 5. Immunohistochemical analyses—IL-10 and TGF-β1.

Medians, interquartile range and maximum and minimum values of the immunolabeling scores for IL-10 (A) and TGF-β1 (B), with comparisons among groups. Photomicrographs showing immunolabeling for IL-10 (C-F) and TGF-β1 (G-J) in the furcation regions of mandibular first molars: Group C (C and G); Group PROB (D and H); Group EP-SRP (E and I); Group EP-SRP-PROB (F and J). Abbreviations: ab = alveolar bone; ct = connective tissue; pdl = periodontal ligament; * = p<0.05. Scale bars: C-J = 80 μm. (Hematoxylin counterstaining).

Fig 6. Histomorphometric analysis of small intestine.

Mean values and standard deviations of VH (A) and CD (B) in intestinal sections, with comparisons among groups. Photomicrographs of small intestine (duodenum sections): Group C (C); Group EP-SRP (D); Group PROB (E); Group EP-SRP-PROB (F). Abbreviations and symbols: LC = crypt of Lieberkühn; VH = villous height; CD = crypt depth; black arrowhead = calciform cells; * = Significant difference (p<0.05) when compared with Groups C, EP-SRP and EP-SRP-PROB; ^† = Significant difference (p<0.05) between Groups EP-SRP and EP-SRP-PROB; ^‡ = Significant difference (p<0.05) between Groups C and EP-SRP-PROB; ^§ = Significant difference (p<0.05) between Groups C and EP-SRP. Scale bars: C-F = 50 μm. (Hematoxylin and Eosin stain).

Fig 7. Effects of B. lactis HN019 on ligature-associated microbiota.

Means and standard deviations of the ratios between aerobic and anaerobic bacteria in groups Intervention and Placebo. * = Significant difference (p<0.05) when compared with Group Placebo.