Effects of Probiotic Lactobacilli plantarum in Treatment of Experimentally Induced Periodontal Disease in Rabbits

Abstract

The aim of this study was to evaluate the therapeutic effects of probiotic Lactobacillus plantarum in experimentally induced periodontal disease in rabbits. The incisor teeth of 24 rabbits were scaled under general anesthesia. Two weeks later, silk ligatures were placed at the gingival margin of the incisor teeth to induce periodontal disease. After confirming the presence of periodontal disease by periodontal probing four weeks later, incisor mucogingival flaps were created and gingival pocket lavage and debridement was performed. The rabbits were randomly divided into four groups. Group 1: Control; Group 2: Microencapsulated form of the probiotic; Group 3: Planktonic form of the probiotic; and Group 4: Biofilm form of the probiotic. The rabbits were euthanized eight weeks later, and gingival connective tissue and epithelium were resected for histopathological and histomorphometric evaluation. The results showed that the rate of epithelial regeneration was lower and bone regeneration was significantly higher in the treatment groups compared to the Control group. The highest level of bone regeneration was in Group 2 (Microencapsulated probiotic). There was no significant difference in bone regeneration observed between the biofilm and planktonic probiotic groups. This study showed that applying the probiotic Lactobacillus plantarum in microencapsulated form improved bone regeneration in experimentally induced periodontal disease in rabbits.

Keywords

periodontal probiotic microencapsulated rabbit

Introduction

Periodontal disease is one of the most common oral diseases seen in cats and dogs, after the age of three years.¹ Eighty percent of dogs and 70% of cats show some degree of the disease.² The initial stage is termed gingivitis, which includes gingival inflammation, hyperemia, discoloration, halitosis, and sometimes hemorrhage. The second stage is periodontitis, which occurs when infection and inflammation of the gingiva progress to involve the cementum, periodontal ligament, and alveolar bone. Periodontitis can result in tooth loss and other serious health problems. As the severity of the soft tissue change progresses, alveolar bone is resorbed. Alveolar bone loss may result in the periodontal pocket formation or gingival recession and root exposure.³ The goal of treatment is to salvage or remove the teeth, combined with ongoing oral hygiene to maintain oral health.¹ The use of probiotics in the management of oral disease in humans has been recently considered, but there are few studies on the use of probiotics for periodontal disease in dogs and cats.⁴

The origin of the word “probiotic” is derived from two Greek words which are “pro” and “biotic” meaning “for life,” which is the opposite of the word “antibiotic.”⁵ Probiotics have the ability to inhibit pathogens. They also produce several antimicrobial agents such as biosurfactants, various organic acids, and bacteriocins.^6,7 Probiotics are living, distinct substances that, when administered to humans or animals, improve health conditions by affecting the body's microbial flora.^6,8 An important feature of probiotic bacteria is the ability to inhibit the proliferation of pathogens and thus their pathogenicity. Bacteria that produce lactic acid occupy the target sites for pathogenic bacteria and prevent the binding of pathogens, reducing their effect. In addition, these bacteria produce a variety of antimicrobial agents and bacteriocins, such as cyclic antimicrobial peptides, antibiotics, and organic acids such as acetic acid and lactic acid.^6,9 The most common probiotic microorganisms are bacteria and fungi. Some of these microorganisms are strains of Bifidobacterium and Lactobacilli, although strains of Escherichia coli, Enterococcus, and Streptococcus are also used for this purpose.¹⁰ Among the yeasts, Saccharomyces cerevisiae and S. boulardii can be used as probiotics. Most Bifidobacterium and Lactobacillus bacteria have been diagnosed as harmless.¹¹ Bacteria producing lactic acid, except Streptococcus and Enterococcus, are sometimes pathogenic to humans and animals.⁷ Under normal conditions, the predominant oral flora is made up of gram-positive bacteria such as Streptococcus, Enterococcus, and Staphylococcus.¹²

In order to increase the viability of probiotics, the microencapsulated technique using different hydrocolloid coatings (carrageenan, chitosan, alginate, xanthan, gelatin, etc.) is used. Sodium alginate is most widely used in microencapsulated probiotics.^13,14 It has also been reported that mixing alginate and glycerol in microencapsulated probiotics greatly increases the possibility of survival of these bacteria under freezing conditions.¹⁵ In addition to proper protection of probiotic bacteria against adverse environmental factors, what is important and less considered in most studies is intense and targeted release of probiotics in various organs.¹⁵ This study aims at evaluating the therapeutic effects of the microencapsulated probiotic Lactobacillus plantarum in experimentally induced periodontal disease in rabbits. This study was approved by an institutional animal care and use committee, Ethic code: IR.UM.REC.1400.119.

Materials and Methods

Microencapsulation of Probiotic Bacteria

Microencapsulation of probiotic bacteria was performed by an external gelation technique. Two grams of sodium alginate^a with medium viscosity was dissolved in 100 mL of autoclaved distilled water^b using a magnetic stirrer^c. It was stored overnight at 4 °C to allow the alginate efficiently to absorb the water. The next day, 18 g of sterile sodium alginate solution was mixed with 1 g of bacterial suspension. Alginate and bacterial suspensions were slowly added using a sterile pipette in 100 g of rapeseed liquid vegetable oil containing 5 g/L of Tween 80^d. This was stirred by a magnetic stirrer at 900 rpm and then dispersed evenly for 20 min. Next, the gelatinization process was started by adding 32 mL of emulsion containing calcium ions (prepared by dissolving 60 g of rapeseed liquid vegetable oil, 5 g/L of Tween 80, and 62 mg calcium chloride^e. Stirring continued for 20 min to form alginate clusters, and then for an additional 30 min until gelatinization occurred. At completion, a two-phase system remained; the upper phase of which was oil, and the lower phase a sodium alginate granule dissolved in calcium chloride solution. The oil phase was separated by centrifugation at 500 g at 25 °C for 5 min. The grains were washed using 0.1% peptone water solution^f and were centrifuged under the above conditions. The grains were then allowed to harden completely in the refrigerator at 4 °C for 10 h.

The resulting alginate grains were then used directly.¹⁶ In order to coat the probiotic bacteria, microencapsulated Lactobacillus plantarum (PTCC1745) was prepared with alginate grains according to the above method. Then, the prepared microcapsules were filtered by Whatman filter paper No. 4^g, followed by adding an additional 15 g and mixing with 100 mL of alginate solution (half weight/volume) of medium viscosity and stirred at 500 rpm for 20 min until the grains were scattered.

Afterward, the grains were collected and stirred in 75 g of rapeseed oil for 20 min to form cross-linking of external calcium ions with the surrounding layer of alginate. To break the emulsion, 45 mL of the previously prepared 0.05 M calcium chloride solution was added and transferred to a separatory funnel. The microencapsulated grains intertwined with calcium were collected and stored in 0.1% peptone solution^h at 4 °C for subsequent tests. The procedure described in this section was repeated for re-microencapsulation single-layer grains.¹⁶

Production of Biofilm

To prepare the biofilm, 0.1 mL of the bacterial suspension of Lactobacilli was inoculated with 0.5 McFarland turbidity into De Man Rogosa and Sharpe mediumⁱ (MRS) containing a 1 cm catheter and the samples were heated at 37 °C. After heating and biofilm formation, the catheter containing the biofilm was gently washed three times with phosphate-buffered saline^j (PBS) so that the biofilm cells attached to the catheter surface remained. After disposing of the medium from each tube, 1 mL of PBS was added, and each tube was placed on a shaker for 5 min to remove the surviving biofilm cells from the surface of the catheter to be used in the next steps.

Production of Planktonic Form

The vial containing the lyophilized bacteria was broken under aseptic conditions using a diamond blade. 1 g of lyophilized bacteria was transferred to 10 mL of MRS broth previously sterilized and placed for 48 h at 37 °C under anaerobic conditions in an anaerobic jar in the presence of pack gas and was incubated. At the end of the incubation period, 100 μL of activated bacteria was transferred to MRS agar medium and incubated again under the above-mentioned conditions. After the emergence of colonies, several were removed using a sterile annular swab and mixed in sterile distilled water. Then, using a UV–Vis spectrophotometer^k, it was adjusted to a concentration of 109 Colony Forming Units (CFU)/mL at a wavelength of 600 nm with light absorption of 0.921 nm.

Inducing Periodontal Disease

In the study groups, experimental periodontal disease was induced by silk ligation at the incisors. Rabbits were anesthetized by intramuscular (IM) injection of acepromazine^l (0.75 mg/kg) and xylazine^m (3 mg/kg) mixed in the same syringe, followed by IM ketamineⁿ (10-20 mg/kg). Ultrasonic scaling of the incisor teeth was performed two weeks before the onset of the experimental disease.

Silk sutures^o (4-0) were placed between the tooth and the periodontium to mechanically widen the periodontal sulcus and accelerate plaque accumulation.¹⁷ To stabilize the ligature during the study period and not require multiple anesthetic episodes, a 4-0 silk suture was placed on the free gingiva. The ligature was checked weekly to ensure it remained in place.

Experiment Grouping

In this study, 24 rabbits^p with the same age, weight range, and experimental periodontal disease were used. After confirmation of the presence of periodontal disease at four weeks by periodontal examination, the ligatures were removed. The animals were then randomly divided into four 6-member groups.

Group 1. Rabbits were treated with gel.

Group 2. Rabbits were treated with a microencapsulated form of Lactobacillus plantarum.

Group 3. Rabbits were treated with a planktonic form of Lactobacillus plantarum.

Group 4. Rabbits were treated with a biofilm form of Lactobacillus plantarum.

In the three treatment groups in which probiotics were used, mucogingival flaps around incisor teeth were initially created and lavage and debridement of the gingival pockets was performed. Probiotics were placed in the desired location in treatment groups as mentioned above. Patients were kept in appropriate cages under controlled environmental and nutritional conditions for eight weeks. According to previous studies, the minimum concentration of probiotics used in the management of the periodontal disease is 1 × 10⁸ CFU.¹⁸ Before using probiotics in treatment groups, the appropriate concentration was determined in the laboratory.¹⁸

Eight weeks post-treatment, rabbits were euthanized ethically for pathological examination. To euthanize the rabbits, a mixture of acepromazine (1 mg/kg) and xylazine (5 mg/kg) was injected IM to ensure deep anesthesia. This was followed by IM ketamine (35 mg/kg) and pentobarbital^q (150 mg/kg) intravenously. Sampling was performed for histopathological and histomorphometric evaluation of gingival tissue and junctional epithelium. The extent of epithelial healing was also examined. Mandibles of sacrificed animals were removed for histopathological analysis. Afterward, specimens were fixed in 10% buffered formalin for five days and decalcified using 10% ethylenediamine tetra acetic acid^r (EDTA, pH 7.2) for the next four weeks. Then, routine histopathological processing was carried out for all the decalcified samples. Tissues were cut serially into 4 μm sections and stained with hematoxylin and eosin^s for histopathological evaluation by a pathologist blinded to the groups.¹⁹

Histomorphometrical Analysis

0.1% Alizarin Red Sigma^t-stained sections obtained from each of the four groups were evaluated for morpho-metrical measurement of newly formed bone surface area under 20× magnification using an optical microscope^u equipped with a digital camera^v. The microsope was connected to a computer for use of image software. Consecutive images from five randomly chosen slides were analyzed for morpho-metrical measurement of the newly engineered bone surface area, quantified according to the previously described formula.²⁰

Data Analysis

Statistical software^w was used for quantitative data analysis. Parametric and non-parametric data were determined firstly by the Kolmogorov–Smirnov test, followed by the Kruskal–Wallis test to analyze non-parametric data. In all tests, the results with P < .05 were considered significant.

Results

Histomorphometrical Analysis of Epithelial Tissue

In Group 1 (Control), regeneration of junctional epithelial tissue, lack of bone tissue regeneration, and hyperemia of the surrounding connective tissues were observed (Figure 1A and B). In Group 2 (microencapsulated form), junctional epithelial tissue regeneration and more marked bone regeneration were observed. The alveolar bone crest was well differentiated, and inflammation and hyperemia were not observed. In this group, large bone marrow spaces, indicative of bone, were being generated (Figure 2A and B). In Group 3 (planktonic form), regeneration of junctional epithelium and regeneration of bone in islands with intermediate areas of fibrous tissue was present. Inflammation and hyperemia were not observed (Figure 3A and B). In Group 4 (biofilm form), there was significant regeneration of junctional epithelium with some bone regeneration between the treatment groups. Inflammation and moderate inflammatory cell infiltration were observed (Figure 4). In general, the results of histological examinations indicated better healing in the treatment groups compared to the Control group.

Epithelial Tissue Regeneration

The results of epithelial tissue regeneration are shown in Table 1. The percentage of epithelial tissue regeneration in the Control group was significantly higher than that of the other groups (P < .05). Among the treatment groups, the highest rate of epithelial tissue healing was in Group 4 (biofilm form), which was significantly higher than for Group 2 (microencapsulated form) or Group 3 (planktonic form) (P < .05). No statistical significance was observed between groups 2 (microencapsulated form) and 3 (planktonic form) (P > .05).

Table 1.

Results of Epithelial Tissue Regeneration.

Group	Control	Microencapsulated	Planktonic	Biofilm
Average Percentage	3.48 ± 77.91	3.03 ± 20.20	7.30 ± .21.00	2.00 ± 40.00
P-value	.01	.00	.00	.01

The results are expressed in terms of mean ± standard error, with a difference under 0.05 being significant.

Bone Regeneration

The results of the percentage of bone surface area to total tissue are shown in Table 2. The percentage of bone surface area to entire tissue in the Control group was significantly lower than that of the other groups (P < .05). Among the treatment groups, the highest amount of bone was in Group 2 (microencapsulated form), which was significantly higher than in groups 3 (planktonic form) and 4 (biofilm form) (P < .05). No statistical significance was observed between these latter groups (P > .05).

Table 2.

Results of Bone Surface Area in Proportion to Entire Tissue Percentage.

Group	Control	Microencapsulated	Planktonic	Biofilm
Average Percentage	0	7.13 ± 48.20	4.21 ± 31.03	2.00 ± 17.24
P-value	.00	.00	.00	.01

The results are expressed in terms of mean ± standard error, with a difference under 0.05 being significant.

Discussion

Periodontal disease affects the structures and tissues supporting teeth. It is the most common oral disease found in small animals. The use of probiotics in the management of oral disease in humans has been considered, but their use in controlling canine and feline periodontal disease has received limited attention.⁴ Therefore, very few studies using probiotics to control periodontal disease in dogs and cats have been published. In the present study, the therapeutic effect of the probiotic Lactobacillus plantarum in experimentally induced periodontal disease in rabbits was evaluated.

Studies of probiotics have revealed their effect on cell healing and differentiation. It has been found that the mechanism of action of probiotics is in the stimulation of the acquired and innate immune systems. Probiotics affect the immune system at various levels, such as by increasing the levels of cytokines and immunoglobulins, increasing the proliferation of mononuclear cells, activation of macrophages, enhancement of natural killer cell activity, and immune stimulation against pathogenic bacteria and protozoa.²¹ It has been shown that all bacterial cells increase the proliferation of immune cells and induce the production of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin-6.²² Also, probiotics affect the proliferation of lymphocytes and production of cytokines by T cells. Most importantly, probiotics positively affect the immune system without causing harmful inflammatory responses.²³ Histopathological results of this study indicated that Lactobacillus plantarum significantly enhances the healing process in periodontal disease in rabbits.

During this study, no infection was observed in the experimental groups. This could be because probiotics prevent wound infections caused by antimicrobial activity, which includes the secretion of antimicrobial peptides, inhibition of bacterial invasion, and inhibition of pathogenic bacterial adhesion to epithelial cells.²³ The ability to stimulate the synthesis of fibroblasts and macrophages as well as inhibit inflammation are prominent features that have led to the use of probiotic bacteria in dairy products.²⁴ Some probiotic bacteria secrete a large amount of exopolysaccharides which may improve wound healing and promote faster closure due to adhesions and better conditions for epithelial cell movement.²⁵ Perhaps the mechanism of action of Lactobacillus plantarum in this study was to stimulate inflammation and cell division (fibroblasts) for the production of collagen, which probably resulted from bacterial secretions and the ability to stimulate the production of growth factors. Previous studies have also shown the beneficial effects of probiotics in tissue healing.

In this study, the beneficial and significant effect of Lactobacillus plantarum probiotic use for bone healing was also observed. It has recently been shown that probiotics can increase osteoprotegerin expression.²⁶ The positive effect observed in this study may be related to this matter. Osteoprotegerin is a TNF-α induced compound produced by osteoblasts, mesenchymal stem cells, endothelial cells, adipocytes, bone marrow mesenchymal cells, and osteocytes. This protein ultimately reduces the production and differentiation of osteoclasts; and consequently, reduces bone resorption through its receptors.²⁷ This protein acts by binding to its receptor called receptor activator of nuclear factor kappa-B ligand (RANKL) and by competing with the receptor activator of nuclear factor kappa B (RANK) which is a surface receptor expressed on osteoclasts and dendritic cells. RANK is a vital cytokine for differentiation, activity, and survival of osteoclasts. Osteoprotegerin inversely inhibits the binding of RANK and RANKL, preventing osteoporosis and other diseases such as rheumatoid arthritis and bone metastasis.²⁷

Several studies indicate that probiotics such as Lactobacillus brevis and Lactobacillus rhamnosus have the potential to improve health.⁷ It has been shown that probiotics can prevent nitric oxide production, thus providing an anti-inflammatory effect, and thereby increase alveolar bone surface in periodontal disease,^28,29 improve clinical and microbiological parameters in patients with chronic periodontal disease, and reduce tooth decay in children.^30,31 Further studies are needed to determine the precise histochemical and molecular aspects of the healing process in periodontal disease after the application of probiotics. The therapeutic properties of other types of probiotics in periodontal disease should also be investigated and their regenerative effects compared. Probiotics have also been used to modulate the microbiota as a treatment for various bone disorders, such as osteoporosis, and thereby improve bone health.³²

Conclusion

This study found that the application of the probiotic Lactobacilli plantarum in microencapsulated, biofilm, and planktonic forms enhances the regeneration of bone tissue and improves the regeneration of periodontal tissue in rabbits, with the best improvement attained by the microencapsulated form. However, long-term evaluation of the therapeutic effects of probiotics on periodontal tissues in other species, including dogs and cats, is recommended.

Materials

Sodium alginate, Merck KGA, Germany

Distilled water, autoclaved water, Tehran, Iran

Magnetic stirrer, Dynalon Labware, USA

Tween 80, Sigma-Aldrich, Missouri, United States

Calcium Chloride, Sigma-Aldrich, Missouri, United States

Peptone Water Solution, Sigma-Aldrich, Missouri, United States

Paper, MilliporeSigma, Sigma-Aldrich, Missouri, United States

Peptone Solution, Sigma-Aldrich, Missouri, United States

MRSAgar,Sigma-Aldrich, Merck KGaA, Darmstadt, Germany

Phosphate-Buffered Saline, PBS, HyClone, Utah, United States

Spectrometer, Mecasys, Korea

Acepromazine, Alfasan, Netherlands

Xylazine, Alfasan, Netherlands

Ketamine, Alfasan, Netherlands

Silk sutures, Supa medical devices, Iran

Razi research institute Mashhad, Iran

Pentobarbitol, Sigma-Aldrich, United States

EDTA, Sigma-Aldrich, United States

H&E, Abcam, Cambridge, United Kingdom

Alizan – red, Sigma-Aldrich, United States

Microscope, Zeiss, Germany

Camera, Olympus, Japan

Software, SPSS Statistics 23.0, IBM, NY, USA

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Yeganeh Mehrani

Hossien Kazemi Mehrjerdi

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