© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
J Periodont Res 2016; 51: 26–37 All rights reserved
JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12274
Effect of the probiotic Saccharomyces cerevisiae on ligature-induced periodontitis in rats Garcia VG, Knoll LR, Longo M, Novaes VCN, Assem NZ, Ervolino E, de Toledo BEC, Theodoro LH Effect of the probiotic Saccharomyces cerevisiae on ligature-induced periodontitis in rats. J Periodont Res 2016; 51: 26–37. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
V. G. Garcia1,2, L. R. Knoll2, M. Longo1, V. C. N. Novaes1, N. Z. Assem1, E. Ervolino3, B. E. C. de Toledo2, L. H. Theodoro1 1 Group of Research and Study on Laser in Dentistry (GEPLO), Division of Periodontics, Department of Surgery and Integrated Clinic, University Estadual Paulista (UNESP), Aracßatuba, Brazil, 2Master Course, Barretos Dental School, University Center of the Educational Foundation of Barretos (UNIFEB), Barretos, Brazil and 3Department of Basic Science, University Estadual Paulista (UNESP), Aracßatuba, Brazil
Background and Objective: This study assessed the effects of the local use of Saccharomyces cerevisiae as monotherapy and as an adjuvant to the mechanical treatment of ligature-induced periodontitis in rats. Material and Methods: Periodontitis was induced in 72 rats via the installation of a ligature around the mandibular first molar. After 7 d, the ligature was removed and the rats were placed in one of the following groups: no treatment (C; n = 18); scaling and root planing (SRP; n = 18); local irrigation with probiotics (PRO; n = 18); and SRP followed by local irrigation with probiotics (SRP/ PRO; n = 18). Six rats from each group were killed at 7, 15 and 30 d. The histological characteristics, alveolar bone loss (ABL) and immunolabeling of tumor necrosis factor alpha (TNF-a), interleukin-1beta (IL-1b), interleukin-10 (IL-10) and TRAP on the furcation area of the first molar were assessed. Results: The PRO group showed features of acceleration of the tissue-repair process during the entire experiment. On day 15, there was less ABL in the SRP/PRO group compared with the C group. There were fewer TRAP-positive cells in the SRP and SRP/PRO groups at 30 d. There was less immunostaining for TNF-a in the PRO and SRP/PRO groups and less immunostaining for IL-1b in the PRO group. However, there was more immunostaining for IL-10 in the PRO group on day 15. Conclusion: Local use of the probiotic did not result in any adverse effects on periodontal tissues. When used as monotherapy or as an adjuvant, the probiotic was effective at controlling periodontitis in rats.
Periodontitis is a chronic infectious disease, initiated by plaque that accumulates on the dental surface and on the gingival margin and which is characterized by the presence of persistent inflammation, soft-tissue disruption and bone destruction (1,2). For the past three decades, researchers have argued that several factors should be
considered in the etiology of periodontal disease, such as the presence of pathogenic bacterial specimens, the susceptibility of the host and the reduction or absence of beneficial microorganisms (3–5). The microbial ecosystem of the oral cavity includes approximately 700 species of bacteria (6). Approximately
Leticia Helena Theodoro, DDS, MS, PhD, Faculdade de Odontologia de Aracßatuba Bonifa cio, 1193, Centro, UNESP, Rua Jose 16015-050 Aracßatuba, Brazil Tel: (55) 18 – 36363239 Fax: (005518) 36363333 e-mail: [email protected] (authorized to publish) Key words: alveolar bone loss; periodontitis;
probiotics; rats Accepted for publication March 03, 2015
500 species colonize the subgingival crevice (7). In this environment, there are also probiotic lactobacilli that represent approximately 1% of oral bacteria (8). In sites affected by chronic periodontal disease, lactobacilli are also present and exhibit inhibition of periodontopathogens (9).
Use of a probiotic to treat periodontitis Some bacterial species are considered to be periodontal pathogens, including Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Porphyromonas gingivalis (10). However, according to the literature, the presence of pathogens is required, but not sufficient, for the development of periodontal disease. Periodontitis is caused by the host response to bacteria or occurs as a consequence of the invasion of soft tissue by bacterial products (11). The contamination of root cementum and dentinal tubules of radicular dentin can act as a reservoir for periodontopathogens (12). The infected and inflamed periodontal tissue can act as a locus of infection, inasmuch as proinflammatory cytokines released can spread systematically (13). Mechanical control of the bacterial biofilm and calculus constitutes the treatment method that is most indicated for treating periodontitis (14). However, this therapy is not completely effective in bacterial elimination in some conditions, mainly when bacteria are located in the soft tissue or radicular dentin and in areas inaccessible to periodontal instruments, such furcation areas and radicular depressions (12,15). Adjuvant methods to mechanical periodontal treatment have been proposed (13,16–19). Recent research has demonstrated the possibility of clinical use of probiotic lactobacilli in subgingival microbiota (20,21), in gingival inflammation (22–27) and in periodontitis (28–31). Probiotics can act in different ways, as follows: to modulate host defense; to stimulate the production of antimicrobial substances; and to compete with periodontopathogenic bacteria (32). Saccharomyces cerevisiae is a yeast biotherapeutic agent that may possess probiotic properties (33). The S. cerevisiae cell wall consists of mannoproteins, b-glucans and a small amount of chitin, which become cross-linked in a variety of ways (34). Beta-glucans are polysaccharides with a backbone that comprises b-1,3-linked D-glucose molecules (b-1,3- D-glucan) and b-1, 6-linked side chains of varying lengths (35,36). The anti-infective effect of
b-glucans is based on their ability to activate leukocytes by stimulating their phagocytic activity and the production of inflammatory cytokines (37). A few experimental studies have evaluated the effect of topically or systemically administered probiotics (38–43) in periodontal disease in animals. One study evaluated the systemic effect of purified immunomodulatory water-soluble b-1,3/1,6 glucan, isolated from the cell wall of S. cerevisiae on the progression of ligatureinduced periodontitis (44). The results of these studies demonstrated satisfactory effects from the use of probiotics in periodontal disease control, although the results were not conclusive (38–44). This outcome is probably a result of the diversity of methodologies used, the type of probiotic microorganisms and the different protocols studied, which emphasizes the need for studies capable of establishing parameters for clinical practice. The aim of the present research was to evaluate the local use of S. cerevisiae as monotherapy and as an adjuvant to scaling and root planing in experimental periodontitis in rats.
Material and methods Animals
This study was conducted on 72 adult male Wistar rats (3 mo of age) that weighed 200–300 g. The rats were kept in plastic cages with access to food and water ad libitum. The rats were allowed to acclimate to the laboratory environment for a period of 3 wks. All protocols described in this study were approved by the Institutional Review Board of Aracßatuba Dental School, S~ ao Paulo State University, Aracßatuba, SP, Brazil (no. 00262/2012). Experimental periodontal disease protocol
For all procedures, the rats were anesthetized with ketamine (70 mg/kg; Vitaset, Fort Dodge, IA, USA) and xylazine (6 mg/kg; Coopazine, Coopers, S~ao Paulo, SP, Brazil), which
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were administered via intramuscular injection. One left-mandibular first molar from each rat in all groups was selected to receive a cotton ligature in a submarginal position to induce experimental periodontitis (45). The ligatures were removed after 7 d. In the rats treated with scaling and root planing, the left molars were subjected to scaling and root planing with manual#1–2 mini-five curettes (Hu-Friedy Co. Inc., Chicago, IL, USA) through 10 distal–mesial traction movements in the buccal and lingual aspects. The furcation and interproximal areas were scaled with the same curettes using cervico–occlusal traction movements (45). All scaling and root planing procedures were performed by the same experienced operator (M.L.). Rats in each group were randomly assigned, using a computer-generated table, to different treatments. Groups containing 18 rats each were defined according to the following treatments applied locally to the left-mandibular first molars: no-treatment control (C); scaling and root planing and irrigation with 0.6 mL of physiological saline solution (SRP); irrigation with 0.6 mL of probiotic solution at 0, 48 and 96 h (PRO); and scaling and root planing and irrigation with 0.6 mL of probiotic solution at 0, 48 and 96 h (SRP/PRO) (Fig. 1). PRO treatment
Probiotic solution (S. cerevisiae – FloraxÒ; Hebron Farmac^eutica, Caruaru, PE, Brazil) was applied immediately after ligature removal in the PRO group and following scaling and root planing in the SRP/PRO group and 48 and 96 h later. The probiotic solution (60 million cells) was slowly poured into the periodontal pocket around the left-mandibular first molar using a syringe (1 mL) and an insulin needle (13 mm 9 0.45 mm) without a bevel. Experimental periods
Six rats from each group were killed on days 7, 15 and 30 after periodontitis treatment by overdose of thiopental (150 mg/kg; Crist alia, Ltd, Itapira,
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Garcia et al. sues. The area analyzed was the entire furcation region of the first molar with ligature-induced periodontitis. Histometric analysis
Fig. 1. Flowchart showing the design of the study. EP, experimental periodontitis; SRP, scaling and root planing.
SP, Brazil). Their jaws were removed and fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 48 h. Laboratory procedures
The specimens were demineralized in a solution of 10% EDTA (Sigma Chemical Co., St Louis, MO, USA) and were subjected to conventional histological processing, including paraffin embedding. Serial paraffin sections (4 lm) were obtained in the mesial–distal direction. Some sections were stained with hematoxylin and eosin and some sections were processed for immunohistochemical analyses. For the immunohistochemical method, the sections were deparaffinized in xylene and hydrated in a decreasing ethanol series. Antigen retrieval was achieved by immersing the histological slides in buffer solution (Diva DecloakerÒ; Biocare Medical, Concord, CA, USA) in a pressurized Decloaking ChamberÒ (Biocare Medical) at 95°C for 10 min. The slides were rinsed with 0.1 M phosphate-buffered saline (pH 7.4) at the end of each of these stages. Then, the histological sections were immersed in 3% hydrogen peroxide for 1 h to block endogenous peroxidase activity and in 1% bovine serum albumin for 12 h to block nonspecific sites. Histological slides containing samples from all experimental groups were divided into four batches, and each batch was incubated with one of the following primary antibodies: goat anti-tumor necrosis factor alpha (TNF-a) (1 : 100 dilution; goat antiTNF-a – SC 1348; Santa Cruz Biotechnology, Santa Cruz, CA, USA);
rabbit anti-interleukin 1 beta (IL-1b) (1 : 100 dilution; rabbit anti-IL-1b – SC 7884; Santa Cruz Biotechnology); goat anti-interleukin-10 (IL-10) (1 : 100 dilution; goat anti-IL-10 – SC 1783; Santa Cruz Biotechnology); and goat anti-TRAP (1 : 100; goat antiTRAP – SC 30833; Santa Cruz Biotechnology). The primary antibodies were diluted in phosphate-buffered saline containing 0.1% Triton X-100 (Sigma Chemical Co., added to the sections) and placed in a moist chamber for 24 h. The histological sections were incubated with a biotinylated secondary antibody for 2 h and then with streptavidin–horseradish peroxidase conjugate for 1 h (Universal Dako Labeled Streptavidin-Biotin KitÒ; Dako Laboratories, Carpinteria, CA, USA). The reaction was developed using the chromogen 3,30 -diaminobenzidine tetrahydrochloride (DAB chromogen KitÒ; Dako Laboratories) and the slides were counterstained with Harris hematoxylin. The negative control consisted of specimens subjected to the aforementioned procedures but without the primary antibodies. Histopathological analysis
The sections stained with hematoxylin and eosin were analyzed by a certified histologist (E.E.) who was blinded to the treatment groups, in which the following parameters were examined: nature and extension of the inflammatory process; presence of necrotic tissue; presence, extension and nature of resorption of bone, cementum and dentin; state of the vasculature; pattern extracellular matrix structure in periodontal tissue; and types of cells and distribution in periodontal tis-
The same sections that were stained with hematoxylin and eosin were used to assess the inter-radicular bone levels. One trained examiner (E.E.), who was blinded to the treatments, selected the sections for histometric analysis. Five equidistant sections from each specimen were selected and imaged using a digital camera coupled to a light microscope (AxioStar Plus; Carl Zeiss MicroImaging Gmb, Gottingen, Germany). Another calibrated examiner (M.L.), who was also blinded to the treatments, conducted the histometric analysis. At 509 magnification, the outer surface of the cementum and the boundary of the alveolar bone crest in the furcation region was circumvented; from this, the area of alveolar bone loss (ABL) (mm2) was determined using an image-analysis system (Axiovision 4.8.2; Carl Zeiss MicroImaging GmbH). ABL was measured in the furcation region of each section by the same examiner three times, on different days, to reduce variations in the data. The mean values were averaged and statistically compared. Immunohistochemical analysis
A treatment-blinded, trained examiner (V.C.N.N.) selected the sections for the immunohistochemical analyses. A certified histologist who was blinded to the treatments (E.E.) conducted the data analysis. The values or scores for each section were measured three times by the same examiner on different days to reduce variations in the data. For TNF-a, IL-1b and IL-10, semi-quantitative immunolabeling analysis was performed. Three histologic sections from each rat were used, and the criteria for establishing the immunoreactivity pattern were adapted from those given in Garcia et al. 2014: score 0: total absence of immunoreactivity (IR); score 1 (low IR): few immunoreactive cells (≤ 10 cells per
Use of a probiotic to treat periodontitis area) and weak labeling in the extracellular matrix; score 2 (moderate IR): a moderate number of immunoreactive cells (11–20 cells per area) and moderate labeling in the extracellular matrix; score 3 (high IR): a large number of immunoreactive cells, ≥ 21 cells per area, and strong labeling in the extracellular matrix (19). The entire area of the furcation region was analyzed at 4009 magnification (Leica Microsystems, Wetzlar, Germany). For TRAP-positive multinucleated osteoclasts, quantitative immunolabeling analysis was performed. Five equidistant sections from each rat were used, and TRAP-positive multinucleated osteoclasts were quantified. A 1600 lm 9 1200 lm area of the central part of the inter-radicular septum was analyzed at 4009 magnification. The coronal limit was the bone crest, which apically spanned a distance of 1200 lm. The values were expressed as the numbers of TRAPpositive multinucleated osteoclasts (19) Intra-examiner reproducibility
Before the histometric and immunohistochemical analyses were performed, the examiner was trained by double measurements of 24 specimens with a 1-week interval. Additionally, Pearson’s correlation coefficient revealed a very high correlation (0.95) between the two sets of measurements for both histometric and immunohistochemical analyses. Statistical analysis
With a sample size of 6 (p < 0.05), the power of the study was 85%. Data were analyzed using BIOESTAT 5.3 software (Bioestat, Mamiraua Institute, Tefe, AM, Brazil). The normality of the histometric (ABL) and immunohistochemical (TRAP, TNFa, IL-1b and IL-10) data were analyzed using the Shapiro–Wilk test. Intra- and intergroup analyses were performed using ANOVA (p < 0.05). When ANOVA detected a significant difference, multiple comparisons were performed using the Bonferroni test
(p < 0.05), Tukey’s test was used for analyzing the TRAP data (p < 0.05), and the Kruskal–Wallis test was used for analyzing the TNFa-, IL-1b and IL-10 data (p < 0.05).
Results Histological analysis
In the C group, 7 d after ligature removal, an intense inflammatory infiltrate, predominantly of neutrophils, was present in all connective tissue of the furcation region. All specimens of this group presented necrotic spicules of bone covered by inflammatory cells in the furcation region. The intraradicular septum presented a very irregular contour and with thin bone trabeculae, which contained many active osteoclasts (Fig. 2A, B). At 15 and 30 d, the inflammation was still very intense, although compared with the results at 7 d there was a gradual reduction of the volume occupied by inflammatory infiltrate, composed mainly of neutrophils and mononucleated cells. The connective tissue near the inter-radicular septum was less inflamed, especially at 30 d. The inter-radicular septum presented thin bone trabeculae, with an irregular external contour and very active osteoclasts. In this group, it was evident that the inflammatory response was becoming chronic and not as an effective repair process (Figs 3A, B, 4A, B). In the SRP and SRP/PRO groups, the histopathological characteristics were very similar. In these groups, at 7 d, connective tissue with intense inflammatory infiltrate, composed mainly of polymorphonuclear neutrophils, prevailed in the furcation region. Next to the inter-radicular septum, less inflamed connective tissue was observed, composed of high numbers of fibroblasts and blood vessels, interlaced with a delicate net of collagen fibers. The bone tissue of the inter-radicular septum was composed of bone trabeculae of external irregular contour because of the presence of several lacunae of resorption and fully active osteoclasts. Some specimens presented small spicules of necrotic
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bone in the furcation region (Fig. 2C, D, G, H). At 15 d, the histopathological characteristics were similar to those mentioned above. However, there was a reduction of the volume occupied by inflammatory infiltrate, especially in the SRP/PRO group (Fig. 3C, D, G, H). At 30 d, there was an even greater reduction of volume occupied by inflammatory cells (mainly lymphocytes and macrophages, although a few neutrophils were also present). The connective tissue located near the bone of the interradicular septum showed a moderate amount of fibroblasts and collagen fibers. The bone tissue was still composed of delicate bone trabeculae, but with a less irregular surface contour (Fig. 4C, D, G, H). In the PRO group, at 7 d, the inflammatory infiltrate, composed mainly of neutrophils, was smaller than that previously described (C, SRP and SRP/PRO groups). The connective tissue near the inter-radicular septum contained moderate numbers of fibroblasts, few inflammatory cells and large numbers of collagen fibers and blood vessels. The inter-radicular septum showed trabeculae with more regular external morphology, full of fully active osteoblasts and with less active areas of bone resorption than other groups (Fig. 2E, F). At 15 d, an even smaller volume of inflammatory infiltrate and a clear organization of connective tissue were observed, which started to present with greater numbers of collagen fibers and moderate numbers of fibroblasts. Some specimens presented new bone tissue in the inter-radicular septum (Fig. 3E, F). At 30 d, only a very small amount of inflammatory infiltrate was present in the dense connective tissue, composed of a great amount of collagen fibers and few fibroblasts. The interradicular septum was composed, in part, of new bone, the surface of which was covered with osteoblasts actively synthesizing new bone (Fig. 4E, F). When comparing this group with SRP and SRP/PRO groups, it was clear that there was an acceleration in the tissue-repair process of the PRO group at all experimental time points, indicating that the
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Fig. 2. Photomicrographs of the longitudinal sections of the furcation region of the mandibular first molar showing the area of alveolar bone loss and inflammatory infiltrate observed at 7 d in control (C) (A, B), scaling and root planing (SRP) (C, D), local irrigation with probiotics (PRO) (E, F) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, H) groups. ab, alveolar bone; nb, necrotic bone; *, inflammatory infiltrate. Hematoxylin and eosin staining. Original magnification: A, C, E and G, 950; B, D, F and H, 9250. Scale bars: A, C, E and G, 500 lm; B, D, F and H, 100 lm.
therapy trauma.
did
not
provoke
tissue
Histometric assessment
The results of histometric assessment are presented in Fig. 5. A significant reduction in ABL was noted in the SRP/PRO group at 15 d compared with the findings in the C group at the same study time point (p < 0.05). Immunohistochemical assessment
The immunohistochemical method used for detection of TNF-a, IL-1b, IL-10 and TRAP yielded high specificity in the detection of these proteins, as evidenced by the total absence of staining in the negative control. Immunoreactive cells and extracellular matrix stained a brown-
ish color. Immunostaining was predominantly observed for TNF-a and IL-1b in inflammatory cells and extracellular matrix, for IL-10 in osteoblasts and for TRAP in osteoclasts.
group (p < 0.05) at all time points. At day 15 only, the SRP/PRO group exhibited lower immunolabeling (score 1) compared with the C group (p < 0.05) (Fig. 6B,H–K).
TNF-a— In the C and SRP groups, moderate immunolabeling (score 2) was observed at all time points. At day 15, the SRP/PRO group showed low immunolabeling (score 1) compared with the C group (p < 0.05). At 30 d, the PRO and SRP/PRO groups showed low immunolabeling (both score 1) compared with the C group (p < 0.05) (Fig. 6A, D–G).
IL-10— In the C and SRP groups, moderate immunolabeling (score 2) was observed at all time points, except at days 7 and 15 in the SRP group, for which low immunostaining (score 1) was observed. In the PRO and SRP/PRO groups, high immunolabeling (score 3) was observed at all time points. At day 15, the SRP/ PRO group showed greater immunolabeling (score 3) than the SRP group (p < 0.05) (Fig. 6C, L–O).
IL-1b— In the C and SRP groups, moderate immunolabeling (score 2) was observed at all time points. The PRO group showed lower immunolabeling (score 1) compared with the C
TRAP— The highest number of TRAP-positive multinucleated osteoclasts was detected at day 7 in all
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Fig. 3. Photomicrographs of the longitudinal sections of the furcation region of the mandibular first molar showing the area of alveolar bone loss and inflammatory infiltrate observed at 15 d in control C (A, B), scaling and root planing (SRP) (C, D), local irrigation with probiotics (PRO) (E, F) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, H) groups. ab, alveolar bone; nb, necrotic bone; *, inflammatory infiltrate. Hematoxylin and eosin staining. Original magnification: A, C, E and G, 950; B, D, F and H, 9250. Scale bars: A, C, E and G, 500 lm; B, D, F and H, 100 lm.
experimental groups. Smaller amounts of TRAP-positive multinucleated osteoclasts were observed at day 7 in the SRP (p < 0.05), PRO (p < 0.01) and SRP/PRO (p < 0.01) groups compared with the C group. Compared with the C group, there were fewer TRAP-positive multinucleated osteoclasts in the SRP (p < 0.05) and SRP/ PRO (p < 0.01) groups at day 30 (Fig. 7A–I).
Discussion The use of animals in research has increased, mainly because of the difficulty of studying the host response to therapeutic conditions and disease pathogenesis in humans. Rats (Rattus norvegicus) are most often used in studies of experimental periodontitis because they are easy to manipulate,
cost effective and the macroscopic and microscopic anatomy of their periodontium are similar to those of humans (46,47). This experimental model has commonly been used in studies by our group (18,19,45,48). A recent study (49) evaluated the host response in several models of the induction of experimental periodontitis. The authors demonstrated that the model of periodontopathogenic bacteria (P. gingivalis) injection and ligature placement constitute the most representative models of periodontal disease in humans (49). Still, according to these authors, only the ligature model promotes significant alveolar bone loss after 7 d, maintaining it during the study period (15 and 30 d). Histopathological analysis in this study demonstrated a greater magni-
tude of the inflammatory response and severe tissue breakdown in the furcation region when rats received no type of treatment after ligature removal, and it was evident that ligature installation was effective in the development of experimental disease. This finding confirms the report of Graves et al. (50), which observed that ligature favors plaque accumulation, epithelium ulceration and invasion of periodontal tissue by bacteria. Cytokines induced by the host response to bacterial products stimulate alterations in the epithelium and provoke an inflammatory reaction in periodontal tissue (11). Bacterial products in periodontal tissue induce the host response, which is able to promote inflammation, loss of attachment and alveolar bone loss in a 7-d period (51,52).
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Fig. 4. Photomicrographs of the longitudinal sections of the furcation region of the mandibular first molar showing the area of alveolar bone loss and inflammatory infiltrate observed at 30 d in control (C) (A, B), scaling and root planing (SRP) (C, D), local irrigation with probiotics (PRO) (E, F) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, H) groups. ab, alveolar bone; nb, necrotic bone; *, inflammatory infiltrate. Hematoxylin and eosin staining. Original magnification: A, C, E and G, 950; B, D, F and H, 9250. Scale bars: A, C, E and G, 500 lm; B, D, F and H, 100 lm.
Fig. 5. Graph showing the mean and standard deviation of the histometric data for alveolar bone loss in the furcation regions of the left-mandibular first molars, according to groups and time points. *, significant difference compared with the control group (C) at the same time point (p < 0.05), analyzed using ANOVA and the Bonferroni test.
The results of the present study showed that in an area of experimental periodontitis that was not treated (group C), the inflammatory process was exacerbated even more, even though the periodontitis was chronic
and all treatments were highly effective, as it accelerated the process of local tissue repair. Only the conventional mechanical treatment of scaling and root planing (SRP group) showed similar results
when this procedure was associated with probiotic use (SRP/PRO group), although some of the parameters evaluated had a better response in rats of the SRP/PRO group. However, therapy with isolated probiotics (PRO group) presented promising results, emphasizing a reduction in both the magnitude of the local inflammatory response and tissue destruction, resulting in acceleration of the tissue-repair process compared with other groups at all experimental time points. These observations can be justified because treatment with probiotics caused less tissue trauma than scaling and root planing (51). The attenuation of the inflammatory process observed in the PRO group favored the earlier onset of the tissuerepair process. Another hypothesis for these findings considers the effect of
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Fig. 6. Immunolabeling of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-10 (IL-10) in the furcation region of the left-mandibular first molar in the different experimental groups. (A–C) Graphs showing the distribution of scores of TNF-a (A), IL1b (B) and IL-10 (C) immunolabeling, according to group, treatment and time point. *, significant difference compared with the control (C) group at the same time point (p < 0.05); #, significant difference compared with the scaling and root planing (SRP) group at the same time point (p < 0.05). (D–O) Photomicrographs showing TNF-a (D–G), IL-1b (H–K) and IL-10 (L–O) immunolabeling at the furcation region of the mandibular first molar at day 15 in the C (D, H, L), SRP (E, I, M), local irrigation with probiotics (PRO) (F, J, N) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, K, O) groups. ab, alveolar bone. Arrowheads: immunoreactive cells. Harris’s hematoxylin counterstaining. Original magnification: 9400. Scale bars: 60 lm.
probiotics on local immunity, modulating systemic immune function (44) besides production of chemicals that inhibit oral bacteria or act on plaque formation by competing and intervening with bacteria or becoming involved in the metabolism of substrates (53). These results can also be justified because specimens treated with S. cerevisiae were demonstrated to have increased levels of anti-inflammatory cytokines (IL-10) and reduced levels of proinflammatory cytokines
(TNF-a and IL-1b). In accordance with the present study, Ryan et al. (54) demonstrated a lower level of expression of IL-6 in liver tissue of the group supplemented with S. cerevisiae. Beta-glucan from S. cerevisiae induces the expression of immunoregulatory cytokines (IL-10, TGF-B1 and IL-2) in bone marrow-derived dendritic cells (55). Another study demonstrated that treatment with different strains of S. cerevisiae
decreased expression of the proinflammatory cytokine IL-1a in Escherichia coli-infected tissue (33). Beta-glucan might increase the concentration of TGF-b1 in gingival crevicular fluid in chronic periodontitis, thereby augmenting the periodontal healing potential (56). The histometric results demonstrated less ABL in the furcation area only in rats of the SRP/PRO group compared with rats that received no treatment in the 15-d period of evalu-
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Fig. 7. Immunolabeling for TRAP in the furcation region of the left-mandibular first molar in the different experimental groups. (A) Graph showing mean and standard deviation of the number of TRAP-positive cells, according to group, treatment and time point. *, significant difference compared with the C group at the same time point (p < 0.05). (B–I) Photomicrographs showing TRAP-positive osteoclasts (black arrowheads) at the furcation region of the mandibular first molar at day 7 in the control (C) (B, C), scaling and root planing (SRP) (D, E), local irrigation with probiotics (PRO) (F, G) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (H, I) groups. ab, alveolar bone. Harris’s hematoxylin counterstaining. Original magnification: B, D, F and H, 9160; C, E, G, I, 91000. Scale bars: B, D, F and H, 120 lm; C, E, G and I, 40 lm.
ation. These findings present a possible adjuvant effect of probiotic use in nonsurgical periodontal therapy as a result of the effect of b-glucan on decreasing bacterial counts (57). Betaglucan can exert protective effects in proinflammatory conditions (58). One study demonstrated the enhanced experimental periodontal disease resistance induced by treatment with soluble b-1,3/1,6-glucan (44). In addition, smaller numbers of TRAP-positive multinucleated osteoclasts were observed at 7 d in the SRP, PRO and SRP/PRO groups compared with the C group. In comparison with the C group, there were fewer TRAP-positive multinucleated osteoclasts in the SRP and SRP/PRO groups at 30 d. These results showed less osteoclast activity in all treated groups at the initial time point (7 d) and in rats in the SRP and SRP/PRO groups at 30 d. These results show the importance of SRP in controlling osteoclast activity in the long term. It must be considered that the results observed in specimens of groups that received mechanical treatment were probably caused by different factors, such as the inefficiency of the SRP procedure, the number of SRP sessions and the force of instrumentation.
Use of a probiotic to treat periodontitis Reports have documented that one session of SRP is not sufficient to maintain healthy subgingival microbes (59), that the instrumentation force and the number of movements during instrumentation are important factors and must be considered (60) and that periodontal pathogens are still present after mechanical instrumentation (61). However, these results showed the importance of SRP. The results obtained in rats of groups treated with probiotic were more promising than the results in rats of other groups. The lowest degree of inflammation, patterns of structuring of connective and bone tissues and presence of alveolar bone neoformation were greater evident at 15 and 30 d. These observations demonstrated acceleration of the tissuerepair process in the PRO group compared with other groups. These results can also be justified because glucan enhances wound healing by increasing macrophage infiltration into the wound, stimulating tissue granulation, collagen biosynthesis (62) and re-epithelialization, and altering the tensile strength (63). Our findings are corroborated by previous research that verifies the beneficial effects of probiotic use in periodontal treatment in animals (41–43) and supports the beneficial clinical results of probiotic use on different conditions, such as reduction in the numbers of periodontopathogens (9,20,21) and inflammatory cytokines (25), in the degree of gingival inflammation (24,26) and of chronic periodontitis (28–31). It also draws attention to its possible use in locations where the scaling and root planing procedure cannot be performed, such as hospital clinics. It is important to emphasize that no specimens in the groups in which probiotic S. cerevisiae was used (PRO and SRP/PRO) presented histological characteristics that could prevent its use, which shows the possibility of its use in the clinical practice of periodontal treatment. A previous investigation evaluated the structural aspects of polysaccharides that influence the host immune response (57). An important consider-
ation is that b-glucan derived from S. cerevisiae can exert beneficial effects by modulating immunological parameters in addition to affecting the microbiota. Considering that this topic has only recently been highlighted in dental literature, new experimental studies in animals and clinical trials in humans must be performed, especially considering the number of probiotics that have been discussed in the literature.
Conclusion It can be assumed that the local use of S. cerevisiae had no adverse effects on periodontal tissues and, when used as monotherapy in the treatment of ligature-induced periodontitis, it had the same effectiveness as SRP. In addition, the use of probiotic S. cerevisiae as an adjuvant to SRP showed promising results for the treatment of experimental periodontitis.
Acknowledgements This study was financially supported by the “Pro-Reitoria de Pesquisa da UNESP” and “FUNDUNESP” (Process no. 0468/011/14-PROPe/CDC), S~ao Paulo, SP. Brazil. The authors report no conflicts of interest related to this study.
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J Periodont Res 2016; 51: 26–37 All rights reserved
JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12274
Effect of the probiotic Saccharomyces cerevisiae on ligature-induced periodontitis in rats Garcia VG, Knoll LR, Longo M, Novaes VCN, Assem NZ, Ervolino E, de Toledo BEC, Theodoro LH Effect of the probiotic Saccharomyces cerevisiae on ligature-induced periodontitis in rats. J Periodont Res 2016; 51: 26–37. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
V. G. Garcia1,2, L. R. Knoll2, M. Longo1, V. C. N. Novaes1, N. Z. Assem1, E. Ervolino3, B. E. C. de Toledo2, L. H. Theodoro1 1 Group of Research and Study on Laser in Dentistry (GEPLO), Division of Periodontics, Department of Surgery and Integrated Clinic, University Estadual Paulista (UNESP), Aracßatuba, Brazil, 2Master Course, Barretos Dental School, University Center of the Educational Foundation of Barretos (UNIFEB), Barretos, Brazil and 3Department of Basic Science, University Estadual Paulista (UNESP), Aracßatuba, Brazil
Background and Objective: This study assessed the effects of the local use of Saccharomyces cerevisiae as monotherapy and as an adjuvant to the mechanical treatment of ligature-induced periodontitis in rats. Material and Methods: Periodontitis was induced in 72 rats via the installation of a ligature around the mandibular first molar. After 7 d, the ligature was removed and the rats were placed in one of the following groups: no treatment (C; n = 18); scaling and root planing (SRP; n = 18); local irrigation with probiotics (PRO; n = 18); and SRP followed by local irrigation with probiotics (SRP/ PRO; n = 18). Six rats from each group were killed at 7, 15 and 30 d. The histological characteristics, alveolar bone loss (ABL) and immunolabeling of tumor necrosis factor alpha (TNF-a), interleukin-1beta (IL-1b), interleukin-10 (IL-10) and TRAP on the furcation area of the first molar were assessed. Results: The PRO group showed features of acceleration of the tissue-repair process during the entire experiment. On day 15, there was less ABL in the SRP/PRO group compared with the C group. There were fewer TRAP-positive cells in the SRP and SRP/PRO groups at 30 d. There was less immunostaining for TNF-a in the PRO and SRP/PRO groups and less immunostaining for IL-1b in the PRO group. However, there was more immunostaining for IL-10 in the PRO group on day 15. Conclusion: Local use of the probiotic did not result in any adverse effects on periodontal tissues. When used as monotherapy or as an adjuvant, the probiotic was effective at controlling periodontitis in rats.
Periodontitis is a chronic infectious disease, initiated by plaque that accumulates on the dental surface and on the gingival margin and which is characterized by the presence of persistent inflammation, soft-tissue disruption and bone destruction (1,2). For the past three decades, researchers have argued that several factors should be
considered in the etiology of periodontal disease, such as the presence of pathogenic bacterial specimens, the susceptibility of the host and the reduction or absence of beneficial microorganisms (3–5). The microbial ecosystem of the oral cavity includes approximately 700 species of bacteria (6). Approximately
Leticia Helena Theodoro, DDS, MS, PhD, Faculdade de Odontologia de Aracßatuba Bonifa cio, 1193, Centro, UNESP, Rua Jose 16015-050 Aracßatuba, Brazil Tel: (55) 18 – 36363239 Fax: (005518) 36363333 e-mail: [email protected] (authorized to publish) Key words: alveolar bone loss; periodontitis;
probiotics; rats Accepted for publication March 03, 2015
500 species colonize the subgingival crevice (7). In this environment, there are also probiotic lactobacilli that represent approximately 1% of oral bacteria (8). In sites affected by chronic periodontal disease, lactobacilli are also present and exhibit inhibition of periodontopathogens (9).
Use of a probiotic to treat periodontitis Some bacterial species are considered to be periodontal pathogens, including Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Porphyromonas gingivalis (10). However, according to the literature, the presence of pathogens is required, but not sufficient, for the development of periodontal disease. Periodontitis is caused by the host response to bacteria or occurs as a consequence of the invasion of soft tissue by bacterial products (11). The contamination of root cementum and dentinal tubules of radicular dentin can act as a reservoir for periodontopathogens (12). The infected and inflamed periodontal tissue can act as a locus of infection, inasmuch as proinflammatory cytokines released can spread systematically (13). Mechanical control of the bacterial biofilm and calculus constitutes the treatment method that is most indicated for treating periodontitis (14). However, this therapy is not completely effective in bacterial elimination in some conditions, mainly when bacteria are located in the soft tissue or radicular dentin and in areas inaccessible to periodontal instruments, such furcation areas and radicular depressions (12,15). Adjuvant methods to mechanical periodontal treatment have been proposed (13,16–19). Recent research has demonstrated the possibility of clinical use of probiotic lactobacilli in subgingival microbiota (20,21), in gingival inflammation (22–27) and in periodontitis (28–31). Probiotics can act in different ways, as follows: to modulate host defense; to stimulate the production of antimicrobial substances; and to compete with periodontopathogenic bacteria (32). Saccharomyces cerevisiae is a yeast biotherapeutic agent that may possess probiotic properties (33). The S. cerevisiae cell wall consists of mannoproteins, b-glucans and a small amount of chitin, which become cross-linked in a variety of ways (34). Beta-glucans are polysaccharides with a backbone that comprises b-1,3-linked D-glucose molecules (b-1,3- D-glucan) and b-1, 6-linked side chains of varying lengths (35,36). The anti-infective effect of
b-glucans is based on their ability to activate leukocytes by stimulating their phagocytic activity and the production of inflammatory cytokines (37). A few experimental studies have evaluated the effect of topically or systemically administered probiotics (38–43) in periodontal disease in animals. One study evaluated the systemic effect of purified immunomodulatory water-soluble b-1,3/1,6 glucan, isolated from the cell wall of S. cerevisiae on the progression of ligatureinduced periodontitis (44). The results of these studies demonstrated satisfactory effects from the use of probiotics in periodontal disease control, although the results were not conclusive (38–44). This outcome is probably a result of the diversity of methodologies used, the type of probiotic microorganisms and the different protocols studied, which emphasizes the need for studies capable of establishing parameters for clinical practice. The aim of the present research was to evaluate the local use of S. cerevisiae as monotherapy and as an adjuvant to scaling and root planing in experimental periodontitis in rats.
Material and methods Animals
This study was conducted on 72 adult male Wistar rats (3 mo of age) that weighed 200–300 g. The rats were kept in plastic cages with access to food and water ad libitum. The rats were allowed to acclimate to the laboratory environment for a period of 3 wks. All protocols described in this study were approved by the Institutional Review Board of Aracßatuba Dental School, S~ ao Paulo State University, Aracßatuba, SP, Brazil (no. 00262/2012). Experimental periodontal disease protocol
For all procedures, the rats were anesthetized with ketamine (70 mg/kg; Vitaset, Fort Dodge, IA, USA) and xylazine (6 mg/kg; Coopazine, Coopers, S~ao Paulo, SP, Brazil), which
27
were administered via intramuscular injection. One left-mandibular first molar from each rat in all groups was selected to receive a cotton ligature in a submarginal position to induce experimental periodontitis (45). The ligatures were removed after 7 d. In the rats treated with scaling and root planing, the left molars were subjected to scaling and root planing with manual#1–2 mini-five curettes (Hu-Friedy Co. Inc., Chicago, IL, USA) through 10 distal–mesial traction movements in the buccal and lingual aspects. The furcation and interproximal areas were scaled with the same curettes using cervico–occlusal traction movements (45). All scaling and root planing procedures were performed by the same experienced operator (M.L.). Rats in each group were randomly assigned, using a computer-generated table, to different treatments. Groups containing 18 rats each were defined according to the following treatments applied locally to the left-mandibular first molars: no-treatment control (C); scaling and root planing and irrigation with 0.6 mL of physiological saline solution (SRP); irrigation with 0.6 mL of probiotic solution at 0, 48 and 96 h (PRO); and scaling and root planing and irrigation with 0.6 mL of probiotic solution at 0, 48 and 96 h (SRP/PRO) (Fig. 1). PRO treatment
Probiotic solution (S. cerevisiae – FloraxÒ; Hebron Farmac^eutica, Caruaru, PE, Brazil) was applied immediately after ligature removal in the PRO group and following scaling and root planing in the SRP/PRO group and 48 and 96 h later. The probiotic solution (60 million cells) was slowly poured into the periodontal pocket around the left-mandibular first molar using a syringe (1 mL) and an insulin needle (13 mm 9 0.45 mm) without a bevel. Experimental periods
Six rats from each group were killed on days 7, 15 and 30 after periodontitis treatment by overdose of thiopental (150 mg/kg; Crist alia, Ltd, Itapira,
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Garcia et al. sues. The area analyzed was the entire furcation region of the first molar with ligature-induced periodontitis. Histometric analysis
Fig. 1. Flowchart showing the design of the study. EP, experimental periodontitis; SRP, scaling and root planing.
SP, Brazil). Their jaws were removed and fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 48 h. Laboratory procedures
The specimens were demineralized in a solution of 10% EDTA (Sigma Chemical Co., St Louis, MO, USA) and were subjected to conventional histological processing, including paraffin embedding. Serial paraffin sections (4 lm) were obtained in the mesial–distal direction. Some sections were stained with hematoxylin and eosin and some sections were processed for immunohistochemical analyses. For the immunohistochemical method, the sections were deparaffinized in xylene and hydrated in a decreasing ethanol series. Antigen retrieval was achieved by immersing the histological slides in buffer solution (Diva DecloakerÒ; Biocare Medical, Concord, CA, USA) in a pressurized Decloaking ChamberÒ (Biocare Medical) at 95°C for 10 min. The slides were rinsed with 0.1 M phosphate-buffered saline (pH 7.4) at the end of each of these stages. Then, the histological sections were immersed in 3% hydrogen peroxide for 1 h to block endogenous peroxidase activity and in 1% bovine serum albumin for 12 h to block nonspecific sites. Histological slides containing samples from all experimental groups were divided into four batches, and each batch was incubated with one of the following primary antibodies: goat anti-tumor necrosis factor alpha (TNF-a) (1 : 100 dilution; goat antiTNF-a – SC 1348; Santa Cruz Biotechnology, Santa Cruz, CA, USA);
rabbit anti-interleukin 1 beta (IL-1b) (1 : 100 dilution; rabbit anti-IL-1b – SC 7884; Santa Cruz Biotechnology); goat anti-interleukin-10 (IL-10) (1 : 100 dilution; goat anti-IL-10 – SC 1783; Santa Cruz Biotechnology); and goat anti-TRAP (1 : 100; goat antiTRAP – SC 30833; Santa Cruz Biotechnology). The primary antibodies were diluted in phosphate-buffered saline containing 0.1% Triton X-100 (Sigma Chemical Co., added to the sections) and placed in a moist chamber for 24 h. The histological sections were incubated with a biotinylated secondary antibody for 2 h and then with streptavidin–horseradish peroxidase conjugate for 1 h (Universal Dako Labeled Streptavidin-Biotin KitÒ; Dako Laboratories, Carpinteria, CA, USA). The reaction was developed using the chromogen 3,30 -diaminobenzidine tetrahydrochloride (DAB chromogen KitÒ; Dako Laboratories) and the slides were counterstained with Harris hematoxylin. The negative control consisted of specimens subjected to the aforementioned procedures but without the primary antibodies. Histopathological analysis
The sections stained with hematoxylin and eosin were analyzed by a certified histologist (E.E.) who was blinded to the treatment groups, in which the following parameters were examined: nature and extension of the inflammatory process; presence of necrotic tissue; presence, extension and nature of resorption of bone, cementum and dentin; state of the vasculature; pattern extracellular matrix structure in periodontal tissue; and types of cells and distribution in periodontal tis-
The same sections that were stained with hematoxylin and eosin were used to assess the inter-radicular bone levels. One trained examiner (E.E.), who was blinded to the treatments, selected the sections for histometric analysis. Five equidistant sections from each specimen were selected and imaged using a digital camera coupled to a light microscope (AxioStar Plus; Carl Zeiss MicroImaging Gmb, Gottingen, Germany). Another calibrated examiner (M.L.), who was also blinded to the treatments, conducted the histometric analysis. At 509 magnification, the outer surface of the cementum and the boundary of the alveolar bone crest in the furcation region was circumvented; from this, the area of alveolar bone loss (ABL) (mm2) was determined using an image-analysis system (Axiovision 4.8.2; Carl Zeiss MicroImaging GmbH). ABL was measured in the furcation region of each section by the same examiner three times, on different days, to reduce variations in the data. The mean values were averaged and statistically compared. Immunohistochemical analysis
A treatment-blinded, trained examiner (V.C.N.N.) selected the sections for the immunohistochemical analyses. A certified histologist who was blinded to the treatments (E.E.) conducted the data analysis. The values or scores for each section were measured three times by the same examiner on different days to reduce variations in the data. For TNF-a, IL-1b and IL-10, semi-quantitative immunolabeling analysis was performed. Three histologic sections from each rat were used, and the criteria for establishing the immunoreactivity pattern were adapted from those given in Garcia et al. 2014: score 0: total absence of immunoreactivity (IR); score 1 (low IR): few immunoreactive cells (≤ 10 cells per
Use of a probiotic to treat periodontitis area) and weak labeling in the extracellular matrix; score 2 (moderate IR): a moderate number of immunoreactive cells (11–20 cells per area) and moderate labeling in the extracellular matrix; score 3 (high IR): a large number of immunoreactive cells, ≥ 21 cells per area, and strong labeling in the extracellular matrix (19). The entire area of the furcation region was analyzed at 4009 magnification (Leica Microsystems, Wetzlar, Germany). For TRAP-positive multinucleated osteoclasts, quantitative immunolabeling analysis was performed. Five equidistant sections from each rat were used, and TRAP-positive multinucleated osteoclasts were quantified. A 1600 lm 9 1200 lm area of the central part of the inter-radicular septum was analyzed at 4009 magnification. The coronal limit was the bone crest, which apically spanned a distance of 1200 lm. The values were expressed as the numbers of TRAPpositive multinucleated osteoclasts (19) Intra-examiner reproducibility
Before the histometric and immunohistochemical analyses were performed, the examiner was trained by double measurements of 24 specimens with a 1-week interval. Additionally, Pearson’s correlation coefficient revealed a very high correlation (0.95) between the two sets of measurements for both histometric and immunohistochemical analyses. Statistical analysis
With a sample size of 6 (p < 0.05), the power of the study was 85%. Data were analyzed using BIOESTAT 5.3 software (Bioestat, Mamiraua Institute, Tefe, AM, Brazil). The normality of the histometric (ABL) and immunohistochemical (TRAP, TNFa, IL-1b and IL-10) data were analyzed using the Shapiro–Wilk test. Intra- and intergroup analyses were performed using ANOVA (p < 0.05). When ANOVA detected a significant difference, multiple comparisons were performed using the Bonferroni test
(p < 0.05), Tukey’s test was used for analyzing the TRAP data (p < 0.05), and the Kruskal–Wallis test was used for analyzing the TNFa-, IL-1b and IL-10 data (p < 0.05).
Results Histological analysis
In the C group, 7 d after ligature removal, an intense inflammatory infiltrate, predominantly of neutrophils, was present in all connective tissue of the furcation region. All specimens of this group presented necrotic spicules of bone covered by inflammatory cells in the furcation region. The intraradicular septum presented a very irregular contour and with thin bone trabeculae, which contained many active osteoclasts (Fig. 2A, B). At 15 and 30 d, the inflammation was still very intense, although compared with the results at 7 d there was a gradual reduction of the volume occupied by inflammatory infiltrate, composed mainly of neutrophils and mononucleated cells. The connective tissue near the inter-radicular septum was less inflamed, especially at 30 d. The inter-radicular septum presented thin bone trabeculae, with an irregular external contour and very active osteoclasts. In this group, it was evident that the inflammatory response was becoming chronic and not as an effective repair process (Figs 3A, B, 4A, B). In the SRP and SRP/PRO groups, the histopathological characteristics were very similar. In these groups, at 7 d, connective tissue with intense inflammatory infiltrate, composed mainly of polymorphonuclear neutrophils, prevailed in the furcation region. Next to the inter-radicular septum, less inflamed connective tissue was observed, composed of high numbers of fibroblasts and blood vessels, interlaced with a delicate net of collagen fibers. The bone tissue of the inter-radicular septum was composed of bone trabeculae of external irregular contour because of the presence of several lacunae of resorption and fully active osteoclasts. Some specimens presented small spicules of necrotic
29
bone in the furcation region (Fig. 2C, D, G, H). At 15 d, the histopathological characteristics were similar to those mentioned above. However, there was a reduction of the volume occupied by inflammatory infiltrate, especially in the SRP/PRO group (Fig. 3C, D, G, H). At 30 d, there was an even greater reduction of volume occupied by inflammatory cells (mainly lymphocytes and macrophages, although a few neutrophils were also present). The connective tissue located near the bone of the interradicular septum showed a moderate amount of fibroblasts and collagen fibers. The bone tissue was still composed of delicate bone trabeculae, but with a less irregular surface contour (Fig. 4C, D, G, H). In the PRO group, at 7 d, the inflammatory infiltrate, composed mainly of neutrophils, was smaller than that previously described (C, SRP and SRP/PRO groups). The connective tissue near the inter-radicular septum contained moderate numbers of fibroblasts, few inflammatory cells and large numbers of collagen fibers and blood vessels. The inter-radicular septum showed trabeculae with more regular external morphology, full of fully active osteoblasts and with less active areas of bone resorption than other groups (Fig. 2E, F). At 15 d, an even smaller volume of inflammatory infiltrate and a clear organization of connective tissue were observed, which started to present with greater numbers of collagen fibers and moderate numbers of fibroblasts. Some specimens presented new bone tissue in the inter-radicular septum (Fig. 3E, F). At 30 d, only a very small amount of inflammatory infiltrate was present in the dense connective tissue, composed of a great amount of collagen fibers and few fibroblasts. The interradicular septum was composed, in part, of new bone, the surface of which was covered with osteoblasts actively synthesizing new bone (Fig. 4E, F). When comparing this group with SRP and SRP/PRO groups, it was clear that there was an acceleration in the tissue-repair process of the PRO group at all experimental time points, indicating that the
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A
C
E
G
B
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Fig. 2. Photomicrographs of the longitudinal sections of the furcation region of the mandibular first molar showing the area of alveolar bone loss and inflammatory infiltrate observed at 7 d in control (C) (A, B), scaling and root planing (SRP) (C, D), local irrigation with probiotics (PRO) (E, F) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, H) groups. ab, alveolar bone; nb, necrotic bone; *, inflammatory infiltrate. Hematoxylin and eosin staining. Original magnification: A, C, E and G, 950; B, D, F and H, 9250. Scale bars: A, C, E and G, 500 lm; B, D, F and H, 100 lm.
therapy trauma.
did
not
provoke
tissue
Histometric assessment
The results of histometric assessment are presented in Fig. 5. A significant reduction in ABL was noted in the SRP/PRO group at 15 d compared with the findings in the C group at the same study time point (p < 0.05). Immunohistochemical assessment
The immunohistochemical method used for detection of TNF-a, IL-1b, IL-10 and TRAP yielded high specificity in the detection of these proteins, as evidenced by the total absence of staining in the negative control. Immunoreactive cells and extracellular matrix stained a brown-
ish color. Immunostaining was predominantly observed for TNF-a and IL-1b in inflammatory cells and extracellular matrix, for IL-10 in osteoblasts and for TRAP in osteoclasts.
group (p < 0.05) at all time points. At day 15 only, the SRP/PRO group exhibited lower immunolabeling (score 1) compared with the C group (p < 0.05) (Fig. 6B,H–K).
TNF-a— In the C and SRP groups, moderate immunolabeling (score 2) was observed at all time points. At day 15, the SRP/PRO group showed low immunolabeling (score 1) compared with the C group (p < 0.05). At 30 d, the PRO and SRP/PRO groups showed low immunolabeling (both score 1) compared with the C group (p < 0.05) (Fig. 6A, D–G).
IL-10— In the C and SRP groups, moderate immunolabeling (score 2) was observed at all time points, except at days 7 and 15 in the SRP group, for which low immunostaining (score 1) was observed. In the PRO and SRP/PRO groups, high immunolabeling (score 3) was observed at all time points. At day 15, the SRP/ PRO group showed greater immunolabeling (score 3) than the SRP group (p < 0.05) (Fig. 6C, L–O).
IL-1b— In the C and SRP groups, moderate immunolabeling (score 2) was observed at all time points. The PRO group showed lower immunolabeling (score 1) compared with the C
TRAP— The highest number of TRAP-positive multinucleated osteoclasts was detected at day 7 in all
Use of a probiotic to treat periodontitis A
C
E
G
B
D
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Fig. 3. Photomicrographs of the longitudinal sections of the furcation region of the mandibular first molar showing the area of alveolar bone loss and inflammatory infiltrate observed at 15 d in control C (A, B), scaling and root planing (SRP) (C, D), local irrigation with probiotics (PRO) (E, F) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, H) groups. ab, alveolar bone; nb, necrotic bone; *, inflammatory infiltrate. Hematoxylin and eosin staining. Original magnification: A, C, E and G, 950; B, D, F and H, 9250. Scale bars: A, C, E and G, 500 lm; B, D, F and H, 100 lm.
experimental groups. Smaller amounts of TRAP-positive multinucleated osteoclasts were observed at day 7 in the SRP (p < 0.05), PRO (p < 0.01) and SRP/PRO (p < 0.01) groups compared with the C group. Compared with the C group, there were fewer TRAP-positive multinucleated osteoclasts in the SRP (p < 0.05) and SRP/ PRO (p < 0.01) groups at day 30 (Fig. 7A–I).
Discussion The use of animals in research has increased, mainly because of the difficulty of studying the host response to therapeutic conditions and disease pathogenesis in humans. Rats (Rattus norvegicus) are most often used in studies of experimental periodontitis because they are easy to manipulate,
cost effective and the macroscopic and microscopic anatomy of their periodontium are similar to those of humans (46,47). This experimental model has commonly been used in studies by our group (18,19,45,48). A recent study (49) evaluated the host response in several models of the induction of experimental periodontitis. The authors demonstrated that the model of periodontopathogenic bacteria (P. gingivalis) injection and ligature placement constitute the most representative models of periodontal disease in humans (49). Still, according to these authors, only the ligature model promotes significant alveolar bone loss after 7 d, maintaining it during the study period (15 and 30 d). Histopathological analysis in this study demonstrated a greater magni-
tude of the inflammatory response and severe tissue breakdown in the furcation region when rats received no type of treatment after ligature removal, and it was evident that ligature installation was effective in the development of experimental disease. This finding confirms the report of Graves et al. (50), which observed that ligature favors plaque accumulation, epithelium ulceration and invasion of periodontal tissue by bacteria. Cytokines induced by the host response to bacterial products stimulate alterations in the epithelium and provoke an inflammatory reaction in periodontal tissue (11). Bacterial products in periodontal tissue induce the host response, which is able to promote inflammation, loss of attachment and alveolar bone loss in a 7-d period (51,52).
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A
C
E
G
B
D
F
H
Fig. 4. Photomicrographs of the longitudinal sections of the furcation region of the mandibular first molar showing the area of alveolar bone loss and inflammatory infiltrate observed at 30 d in control (C) (A, B), scaling and root planing (SRP) (C, D), local irrigation with probiotics (PRO) (E, F) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, H) groups. ab, alveolar bone; nb, necrotic bone; *, inflammatory infiltrate. Hematoxylin and eosin staining. Original magnification: A, C, E and G, 950; B, D, F and H, 9250. Scale bars: A, C, E and G, 500 lm; B, D, F and H, 100 lm.
Fig. 5. Graph showing the mean and standard deviation of the histometric data for alveolar bone loss in the furcation regions of the left-mandibular first molars, according to groups and time points. *, significant difference compared with the control group (C) at the same time point (p < 0.05), analyzed using ANOVA and the Bonferroni test.
The results of the present study showed that in an area of experimental periodontitis that was not treated (group C), the inflammatory process was exacerbated even more, even though the periodontitis was chronic
and all treatments were highly effective, as it accelerated the process of local tissue repair. Only the conventional mechanical treatment of scaling and root planing (SRP group) showed similar results
when this procedure was associated with probiotic use (SRP/PRO group), although some of the parameters evaluated had a better response in rats of the SRP/PRO group. However, therapy with isolated probiotics (PRO group) presented promising results, emphasizing a reduction in both the magnitude of the local inflammatory response and tissue destruction, resulting in acceleration of the tissue-repair process compared with other groups at all experimental time points. These observations can be justified because treatment with probiotics caused less tissue trauma than scaling and root planing (51). The attenuation of the inflammatory process observed in the PRO group favored the earlier onset of the tissuerepair process. Another hypothesis for these findings considers the effect of
Use of a probiotic to treat periodontitis A
B
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C
D
E
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J
K
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O
Fig. 6. Immunolabeling of tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-10 (IL-10) in the furcation region of the left-mandibular first molar in the different experimental groups. (A–C) Graphs showing the distribution of scores of TNF-a (A), IL1b (B) and IL-10 (C) immunolabeling, according to group, treatment and time point. *, significant difference compared with the control (C) group at the same time point (p < 0.05); #, significant difference compared with the scaling and root planing (SRP) group at the same time point (p < 0.05). (D–O) Photomicrographs showing TNF-a (D–G), IL-1b (H–K) and IL-10 (L–O) immunolabeling at the furcation region of the mandibular first molar at day 15 in the C (D, H, L), SRP (E, I, M), local irrigation with probiotics (PRO) (F, J, N) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (G, K, O) groups. ab, alveolar bone. Arrowheads: immunoreactive cells. Harris’s hematoxylin counterstaining. Original magnification: 9400. Scale bars: 60 lm.
probiotics on local immunity, modulating systemic immune function (44) besides production of chemicals that inhibit oral bacteria or act on plaque formation by competing and intervening with bacteria or becoming involved in the metabolism of substrates (53). These results can also be justified because specimens treated with S. cerevisiae were demonstrated to have increased levels of anti-inflammatory cytokines (IL-10) and reduced levels of proinflammatory cytokines
(TNF-a and IL-1b). In accordance with the present study, Ryan et al. (54) demonstrated a lower level of expression of IL-6 in liver tissue of the group supplemented with S. cerevisiae. Beta-glucan from S. cerevisiae induces the expression of immunoregulatory cytokines (IL-10, TGF-B1 and IL-2) in bone marrow-derived dendritic cells (55). Another study demonstrated that treatment with different strains of S. cerevisiae
decreased expression of the proinflammatory cytokine IL-1a in Escherichia coli-infected tissue (33). Beta-glucan might increase the concentration of TGF-b1 in gingival crevicular fluid in chronic periodontitis, thereby augmenting the periodontal healing potential (56). The histometric results demonstrated less ABL in the furcation area only in rats of the SRP/PRO group compared with rats that received no treatment in the 15-d period of evalu-
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B
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Fig. 7. Immunolabeling for TRAP in the furcation region of the left-mandibular first molar in the different experimental groups. (A) Graph showing mean and standard deviation of the number of TRAP-positive cells, according to group, treatment and time point. *, significant difference compared with the C group at the same time point (p < 0.05). (B–I) Photomicrographs showing TRAP-positive osteoclasts (black arrowheads) at the furcation region of the mandibular first molar at day 7 in the control (C) (B, C), scaling and root planing (SRP) (D, E), local irrigation with probiotics (PRO) (F, G) and scaling and root planing followed by local irrigation with probiotics (SRP/PRO) (H, I) groups. ab, alveolar bone. Harris’s hematoxylin counterstaining. Original magnification: B, D, F and H, 9160; C, E, G, I, 91000. Scale bars: B, D, F and H, 120 lm; C, E, G and I, 40 lm.
ation. These findings present a possible adjuvant effect of probiotic use in nonsurgical periodontal therapy as a result of the effect of b-glucan on decreasing bacterial counts (57). Betaglucan can exert protective effects in proinflammatory conditions (58). One study demonstrated the enhanced experimental periodontal disease resistance induced by treatment with soluble b-1,3/1,6-glucan (44). In addition, smaller numbers of TRAP-positive multinucleated osteoclasts were observed at 7 d in the SRP, PRO and SRP/PRO groups compared with the C group. In comparison with the C group, there were fewer TRAP-positive multinucleated osteoclasts in the SRP and SRP/PRO groups at 30 d. These results showed less osteoclast activity in all treated groups at the initial time point (7 d) and in rats in the SRP and SRP/PRO groups at 30 d. These results show the importance of SRP in controlling osteoclast activity in the long term. It must be considered that the results observed in specimens of groups that received mechanical treatment were probably caused by different factors, such as the inefficiency of the SRP procedure, the number of SRP sessions and the force of instrumentation.
Use of a probiotic to treat periodontitis Reports have documented that one session of SRP is not sufficient to maintain healthy subgingival microbes (59), that the instrumentation force and the number of movements during instrumentation are important factors and must be considered (60) and that periodontal pathogens are still present after mechanical instrumentation (61). However, these results showed the importance of SRP. The results obtained in rats of groups treated with probiotic were more promising than the results in rats of other groups. The lowest degree of inflammation, patterns of structuring of connective and bone tissues and presence of alveolar bone neoformation were greater evident at 15 and 30 d. These observations demonstrated acceleration of the tissuerepair process in the PRO group compared with other groups. These results can also be justified because glucan enhances wound healing by increasing macrophage infiltration into the wound, stimulating tissue granulation, collagen biosynthesis (62) and re-epithelialization, and altering the tensile strength (63). Our findings are corroborated by previous research that verifies the beneficial effects of probiotic use in periodontal treatment in animals (41–43) and supports the beneficial clinical results of probiotic use on different conditions, such as reduction in the numbers of periodontopathogens (9,20,21) and inflammatory cytokines (25), in the degree of gingival inflammation (24,26) and of chronic periodontitis (28–31). It also draws attention to its possible use in locations where the scaling and root planing procedure cannot be performed, such as hospital clinics. It is important to emphasize that no specimens in the groups in which probiotic S. cerevisiae was used (PRO and SRP/PRO) presented histological characteristics that could prevent its use, which shows the possibility of its use in the clinical practice of periodontal treatment. A previous investigation evaluated the structural aspects of polysaccharides that influence the host immune response (57). An important consider-
ation is that b-glucan derived from S. cerevisiae can exert beneficial effects by modulating immunological parameters in addition to affecting the microbiota. Considering that this topic has only recently been highlighted in dental literature, new experimental studies in animals and clinical trials in humans must be performed, especially considering the number of probiotics that have been discussed in the literature.
Conclusion It can be assumed that the local use of S. cerevisiae had no adverse effects on periodontal tissues and, when used as monotherapy in the treatment of ligature-induced periodontitis, it had the same effectiveness as SRP. In addition, the use of probiotic S. cerevisiae as an adjuvant to SRP showed promising results for the treatment of experimental periodontitis.
Acknowledgements This study was financially supported by the “Pro-Reitoria de Pesquisa da UNESP” and “FUNDUNESP” (Process no. 0468/011/14-PROPe/CDC), S~ao Paulo, SP. Brazil. The authors report no conflicts of interest related to this study.
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