|Year : 2021 | Volume
| Issue : 6 | Page : 525-531
The adjunctive effect of probiotics to nonsurgical treatment of chronic periodontitis: A randomized controlled clinical trial
Ghadeer Khalil Mohamed El-bagoory1, Hoda Mohamed El-guindy1, Malak Yousef Mohamed Shoukheba1, Enas Arafa El-zamarany2
1 Department of Periodontology, Oral Medicine, Oral Diagnosis and Radiology, Faculty of Dentistry, Tanta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||19-Feb-2021|
|Date of Decision||28-Jun-2021|
|Date of Acceptance||11-Jul-2021|
|Date of Web Publication||01-Nov-2021|
Ghadeer Khalil Mohamed El-bagoory
Faculty of Dentistry, Tanta University, Tanta
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Probiotics catch more attention as adjunctive periodontal therapy. This study was conducted to assess the benefit of locally delivered Lactobacillus reuteri (L. reuteri) probiotic as an adjunctive to scaling and root planing (SRP) in the treatment of chronic periodontitis clinically and microbiologically. Materials and Methods: Bacterial cultures and clinical evaluation were recorded in 20 sites of chronic periodontitis in 12 patients and followed up at 3 and 6 months from the start of intervention using clinical attachment level, probing pocket depth, plaque index (PI), bleeding on probing, and microbiologically for Porphyromonas gingivalis (P. gingivalis) load. Patients meeting the inclusion criteria were scheduled within 1 week for two sessions of SRP. After SRP, oral hygiene measures were reassured, and sites were divided randomly into two groups 10 sites each. Group I received SRP only, while Group II received SRP and subgingival delivery of 1 ml of probiotic L. reuteri suspension at baseline and 1, 2, and 4 weeks using a blunt syringe. A periodontal pack was applied after the placement of the drug. Results: We found noticeable variation between the two groups in all evaluation aspects at 3 and 6-month follow-up periods except PI at 6 months in which there was no significant difference between both groups. Conclusions: The results proved the antimicrobial benefit of L. reuteri probiotic as a promising adjunctive therapy in improving periodontal parameters. However, further long-term studies with large sample size are needed to evaluate the extent of the added value of L. reuteri suspension.
Keywords: Lactobacillus reuteri, periodontitis, polymerase chain reaction, probiotics, root planing
|How to cite this article:|
El-bagoory GK, El-guindy HM, Shoukheba MY, El-zamarany EA. The adjunctive effect of probiotics to nonsurgical treatment of chronic periodontitis: A randomized controlled clinical trial. J Indian Soc Periodontol 2021;25:525-31
|How to cite this URL:|
El-bagoory GK, El-guindy HM, Shoukheba MY, El-zamarany EA. The adjunctive effect of probiotics to nonsurgical treatment of chronic periodontitis: A randomized controlled clinical trial. J Indian Soc Periodontol [serial online] 2021 [cited 2021 Dec 4];25:525-31. Available from: https://www.jisponline.com/text.asp?2021/25/6/525/329735
| Introduction|| |
The goal of periodontal treatment is to alter the microbial etiology, arresting the disease progression and resolve inflammation. Several treatment modalities are available to achieve these goals and they can be broadly classified into nonsurgical and surgical treatment.
Many studies described the effectiveness of scaling and root planing (SRP) as a nonsurgical treatment modality., However, recolonization of pathogenic bacteria toward pretreatment levels occurs within weeks.
Different therapeutic approaches have been established to inhibit bacterial growth and colonization such as antibiotics and LASER beam therapy.,, Recently, probiotics were suggested as a potential treatment by inhibiting pathogenic bacterial colonization and overgrowth by competition over nutrition, decreasing adhesion, and producing antimicrobial substances and immunity modulation.
Probiotics are live bacteria that include a large spectrum of bacteria such as Lactobacillus spp. and Bifidobacterium spp., which are the most famous types.
Lactobacillus reuteri (L. reuteri) withdrew attention. Its modes of action include beneficial alteration of local ecology, antibiotic substance production, and host-immunity modulation.
Many studies describe the ability of Lactobacilli to inhibit periodontal pathogen's growth., Several trials proved the synergistic effect of L. reuteri and SRP in CP treatment.,
Hence, the goal of this research is to determine the added benefit of L. reuteri probiotic to SRP in treating CP cases clinically and microbiologically.
| Materials and Methods|| |
This blinded interventional prospective study (randomized controlled trials [RCT]) was done in agreement with principles guidelines declared in Helsinki experimentation on human subjects and after the approval of the ethics committee. This research was done in the Periodontology Department, Faculty of Dentistry.
A total of 20 sites limited to posterior teeth from 12 systemically healthy patients of both sexes (three males and nine females) enrolled in the study after fulfilling inclusion criteria which were
- Systemically healthy patients of both sexes between 35 and 55 years, suffering moderate chronic periodontitis according to Armitage criteria
- Having at minimum two teeth with one proximal site with (clinical attachment level [CAL]) of 3–4 mm, and (probing pocket depth [PPD]) of 5–6 mm
- Horizontal bone erosion by panoramic radiography [Figure 1]
- Patients who are compliant, with no history of missed clinic appointments.
The following criteria were also excluded:
- Previous use of systemic or local antibiotic, anti-inflammatory drugs, probiotics, or periodontal therapy in the previous 6 months
- Patients who are lactose intolerant were excluded.
All participants gave consent after a clear explanation of the goal, nature, and health-care benefits in this study was given to all subjects. The study protocol was performed between May 2018 and February 2019.
The sample size considered a standard deviation of 0.61 mm and a difference between the test and control group of 0.82 mm for the primary outcome variable which was PPD, based on Vivekananda et al. It was calculated that 10 sites were needed to provide 80% power with an α of 0.05 using StatsDirect (StatsDirect, Cheshire, UK) program.
A thorough gingival assessment was performed and complete medical and dental history was taken. Periodontal probing was done after fabrication of a customized-made acrylic stent to mark the probe depth at every evaluation point [Figure 2]. The primary outcome variable was PPD and CAL. The secondary outcome variables were bleeding on probing (BOP) and plaque index (PI).
|Figure 2: Measurement of probing pocket depth using UNC-15 probe through a customized made acrylic stent with groove|
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A calibration exercise was performed to determine intra-examiner reproducibility. Six patients not enrolled in the study who exhibited at least 7 teeth with a Pocket depth (PD) of ≥5 mm on at least one aspect of each tooth were evaluated by the examiner on two separate visits 48 h apart. The PD and CAL were measured. Calibration was accepted if the measurements at baseline and 48 h were consistent (in mm) in ≥90% of the measurements.
Investigator GH K screened the patients and randomly assigned them to test and control groups and performed the treatment procedures for all participants. Investigator M. Sh recorded the clinical parameters and she was masked to the randomization for the extent of the study.
Oral hygiene instructions including regular tooth brushing twice daily for a minimum of 2 min by modified bass brushing technique and interdental flossing was explained to the patients. Randomization of the sites was accomplished through sealed envelopes into two groups ten each by a masked supervisor.
Patients were scheduled within 1 week for two SRP appointments, performed by scalers and hand instrumentation. Group I had SRP alone. Group II patients had SRP and subgingival delivery of 1 ml of probiotic suspension using a blunt syringe (consists of L. reuteri DSM 17938 [1 × 108 CFU]); sunflower oil, medium-chain triglyceride oil, and anti-caking agent (silicon dioxide) (BioGaia, Lund, Sweden) were applied to these sites at baseline (after SRP) and 1, 2, and 4 weeks [Figure 3] and [Figure 4]. Periodontal pack was applied after drug application [Figure 5].
|Figure 4: Application of Lactobacillus reuteri probiotic suspension using a blunt syringe|
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Patients were advised not to harsh food, avoid dental manipulation near the treated areas, and not to use any interdental aids for 24 h. No antiplaque agents, systemic antibiotics, anti-inflammatory drugs, or any other type of probiotics were allowed during the period of the study.
The gingival crevicular fluid (GCF) was collected initially, at 3–6 months using a paper point. The supragingival plaque was removed with sterile cotton pellets. Two sterilized paper points (size 35) were applied to the maximum depth of the periodontal pocket and kept for 10 s [Figure 6], then they were placed in one ml phosphate-buffered saline in an Eppendorf tube.
|Figure 6: Gingival crevicular fluid sample collection using sterile paper point size 35|
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Samples were dispersed using a vortex mixer and preserved at −20°C until analysis. The frozen samples were sent to the central laboratory on dry ice and kept at − 80°C at once. After defrosting, 400 μl of each sample was centrifuged at 13,000 g. The gained pellet was dispersed in 200 μl InstaGene. DNA was retrieved with QIAamp DNA Mini Kit following the manufacturer's instructions. Five microliiter of the purified DNA was used for the quantification of P. gingivalis. The steps were done according to the manufactures' instructions (Primer design, UK).
The data were analyzed using version 19 (SPSS) created by IBM, Illinois, Chicago, USA. For numerical values, the range, mean, and standard deviations were calculated. The differences between the two mean values were tested using Mann–Whitney (Z) test. For comparison of series of observations measured during follow-up, Friedman Chi-square (X2) was used, and when found significant, pairwise comparison by Mann–Whitney (Z) test was used. Data were considered statistically significant when P > 0.05.
| Results|| |
The patient demographics showed no difference in both groups. No subjects were lost or excluded over the following 6 months. The performed treatment was well tolerated by all the patients without any side effects [Table 1] and [Figure 7] flowchart].
|Table 1: Comparison between the two studied groups according to biographic data|
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No difference was observed between the two groups at baseline regarding the clinical and bacteriological parameters as evident by their mean baseline values P > 0.05.
For clinical results, Group II exhibited a statistically significant decrease in the mean PI and BOP scores at 3–6 months as compared to their mean baseline value P < 0.05. Group I recorded statistically significant reductions of the mean PI score at 3–6 months as compared to their mean baseline value P < 0.05. However, Group I recorded no difference in BOP score at 3 and 6 months in comparison to baseline value P > 0.05 [Table 2]. PI intergroup results showed a statistically significant difference between the two groups at 3-month follow-up period in favor of Group II (P < 0.05) and no difference between the two studied groups after 6 months P > 0.05, While for the BOP intergroup, interpretation recorded a statistically significant difference between them at 3 and 6 months (P < 0.05) follow-up periods in favor of Group II [Table 2].
|Table 2: Comparison of plaque index and bleeding on probing percentage between the studied groups at different periods of follow-up|
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Intragroup results showed a statistically significant reduction in the mean PPD and CAL scores at 3–6 months (P < 0.05) in comparison to the mean baseline for both groups. Intergroup results recorded a slight statistically significant difference in PPD reduction CAL gain in favor of Group II at 3 months (P < 0.05), whereas we found a highly statistically significant difference at 6 months in favor of Group II (P = 0.001) [Table 3].
|Table 3: Comparison of pocket depth, loss of attachment, and polymerase chain reaction between the studied groups at different periods of follow-up|
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For the microbiological results, Group I showed no statistically significant reduction in the P. gingivalis load at 3 months as compared to the mean baseline value (P > 0.05), while at 6 months, there was a statistically significant increase in P. gingivalis load (P < 0.05), whereas Group II showed a statistically significant reduction in P. gingivalis load at 3–6 months (P < 0.05) as compared to the mean baseline value. Intergroup results showed a slight statistically significant difference in P. gingivalis load at 3 months in favor of Group II P < 0.05, whereas there was a highly statistically significant difference at 6 months in favor of Group II (P < 0.05) [Table 3].
By correlating clinical and bacteriological results, Spearman rank test showed a statistically significant positive correlation between PPD, CAL, and the presence of P. gingivalis at 3–6 months (P < 0.05) in both the groups [Table 4].
|Table 4: Correlation between pocket depth, loss of attachment and polymerase chain reaction results|
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| Discussion|| |
This RCT assessed the benefit of L. reuteri probiotic suspension to SRP on clinical and microbiological parameters in chronic periodontitis patients. A parallel study design was selected to overcome the shortcoming of the split-mouth design as it is prone to the “carry-across” effect of the intervention especially when it cannot be localized.
Topical application of L. reuteri probiotic suspension was allowed to increase its concentration at the pocket and the direct contact with the pathogens. Moreover, the periodontal dressing was applied to remove the washout effect of the GCF and to prolong its contact time with the inflamed sites.
The clinical results were confirmed microbiologically by PCR technique to quantify the level of P. gingivalis in GCF at baseline and 3 and 6 months of treatment as P. gingivalis is known to be the main pathogen that can create dysbiosis between the host and dental plaque. The probiotic application was found to be well tolerated by all patients. No side effects were reported during the study period.
Regarding PI, the more favorable results obtained in Group II can be explained as the plaque reduction brought about by SRP was enhanced by the antiplaque effect of L. reuteri through direct interactions within dental plaque due to the competition of probiotics with the colonizing bacteria on the binding sites and nutrients leading to disruption of the colonizing pathogens. However, the rebound that occurred in Groups II at 6 months evaluation period reflects the best effect of probiotic that was maintained for more than 3 months.
Our results agreed with various studies on different probiotic supplements.,, and with the recent review which stated that probiotics prevent quorum sensing and the survival of biofilm pathogens, interfere with biofilm integrity, and lead to biofilm elimination. In contrary to our results, Iniesta et al. showed no difference in PI or GI at 8 weeks between interventional and control groups.
For BOP, the effect of SRP as a standard means of controlling inflammation seemed to be enhanced by the anti-inflammatory and host immune-modulatory effects of probiotics. Our results agreed with Twetman et al., who studied the benefit of a chewing gum containing two strains of L. reuteri and found that the pro-inflammatory cytokines such as interleukin (IL)-1 β, tumor necrosis factor α, and IL-8 in GCF were decreased by this probiotic therapy. However, there are opposite results, which showed that probiotics are ineffective regarding the PI and GI scores.,, This can be explained by the difference in the study design, patient population, probiotic bacteria, and method of introducing probiotics. SRP was not done in the majority of these studies and it is difficult for therapeutic agents to penetrate biofilms unless they are mechanically disrupted.
At 3-month follow-up period, there was a slight statistically significant difference in PPD and CAL reduction in favor of group II, whereas there was a highly statistically significant difference at 6-month follow-up period in favor of Group II. These results suggested that the effect of probiotics could be maintained for 6 months.
The favorable effect of Group II is explained by the antimicrobial and anti-inflammatory actions of L. reuteri probiotic on a wide variety of cells to modulate the host immune response. The introduction of L. reuteri into the oral microbiome will shift the microbiota from pathological to commensal making the oral environment favorable for periodontal health. The more CAL gain in Group II in comparison to Group I could be a result of the bone growth promoting properties of L. reuteri Similarly, the results of the systematic review of Ikram et al. suggested that the adjunctive use of probiotics could result in superior benefits in CAL gain in CP. Results of the present study were comparable to another study which showed better clinical outcomes to the groups treated with probiotics compared to tetracycline fibers.
The favorable microbiological results in Group II can be explained by the antimicrobial property of L. reuteri as it produces reuterin, three-hydroxypropionaldehyde, within biofilms., that produces oxidative stress in pathogenic organisms which accounts for its anti-pathogenic effect.
In addition, it can be explained by microbial antagonism of the Lactobacilli strain in the test product toward P. gingivalis as described by Van Essche et al., who proved that lactic acid inhibited the growth of other bacterial strains by producing a huge amount of organic acids.
Correlating clinical and microbiological results indicates that P. gingivalis is inseparable from CP and there is a positive correlation between them. These results agreed with Kulkarni et al., who quantified P. gingivalis in CP cases and normal individuals by PCR and found a positive correlation between P. gingivalis and severity of the chronic sequel of periodontitis.
It is well known that local drug delivery in periodontal disease has shown promising results; hence, the local administration of probiotics containing L. reuteri may have a better result. However, the favorable effect was shown to be short.
The main limitation of the present study is the relatively small number of participants. Nevertheless, this study could serve as a basis for future studies conducted in larger cohorts. Further studies are required to evaluate how the process of periodontal colonization works over time, substantively of probiotics, and to determine the frequency and course for optimal dose.
| Conclusion|| |
Our study shows that local application of L. reuteri in combination with SRP gives more favorable results than SRP alone in cases with chronic periodontitis.
The authors would like to thank our department colleagues and the staff nurses for their help.
Financial support and sponsorship
This research received no external funding (self-funding) except for the PCR kit supplied by our institution.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Klokkevold PR, Newman MG, Takei HH. Carranza's Clinical Periodontology. St. Louis, Missouri: Elsevier Saunders; 2015.
Greenstein G, Caton J. Periodontal disease activity: A critical assessment. J Periodontol 1990;61:543-52.
Yashima A, Gomi K, Maeda N, Arai T. One-stage full-mouth versus partial-mouth scaling and root planing during the effective half-life of systemically administered azithromycin. J Periodontol 2009;80:1406-13.
Taba M, Kinney J, Kim AS, Giannobile WV. Diagnostic biomarkers for oral and periodontal diseases. Dent Clin 2005;49:551-71.
Harper DS, Robinson PJ. Correlation of histometric, microbial, and clinical indicators of periodontal disease status before and after root planing. J Clin Periodontol 1987;14:190-6.
Quirynen M, Teughels W, De Soete M, van Steenberghe D. Topical antiseptics and antibiotics in the initial therapy of chronic adult periodontitis: Microbiological aspects. Periodontol 2000 2002;28:72-90.
Herrera D, Alonso B, León R, Roldán S, Sanz M. Antimicrobial therapy in periodontitis: The use of systemic antimicrobials against the subgingival biofilm. J Clin Periodontol 2008;35:45-66.
Yilmaz S, Kut B, Gursoy H, Eren-Kuru B, Noyan U, Kadir T. Er: YAG laser versus systemic metronidazole as an adjunct to nonsurgical periodontal therapy: A clinical and microbiological study. Photomed Laser Surg 2012;30:325-30.
Ikram S, Hassan N, Baig S, Borges KJJ, Raffat MA, Akram Z. Effect of local probiotic (Lactobacillus reuteri
) vs systemic antibiotic therapy as an adjunct to non-surgical periodontal treatment in chronic periodontitis. J Invest Clin Dent 2019;10:e12393.
Mu Q, Tavella VJ, Luo XM. Role of Lactobacillus reuteri
in human health and diseases. Front Microbiol 2018;9:757.
Song D, Liu X. Role of probiotics containing L. reuteri in adjunct to SRP for management of patients with CP: A meta-analysis. Eur Rev Med Pharmacol Sci 2020;24:4495-505.
Vives-Soler A, Chimenos-Küstner E. Effect of probiotics as a complement to non-surgical periodontal therapy in CP: A systematic review. Med Oral Patol Oral Cir Bucal 2020;25:e161-7.
İnce G, Gürsoy H, İpçi ŞD, Cakar G, Emekli-Alturfan E, Yılmaz S. Clinical and biochemical evaluation of lozenges containing Lactobacillus reuteri
as an adjunct to non-surgical periodontal therapy in chronic periodontitis. J Periodontol 2015;86:746-54.
Tekce M, Ince G, Gursoy H, Dirikan Ipci S, Cakar G, Kadir T, et al.
Clinical and microbiological effects of probiotic lozenges in the treatment of chronic periodontitis: A 1-year follow-up study. J Clin Periodontol 2015;42:363-72.
Penala S, Kalakonda B, Pathakota KR, Jayakumar A, Koppolu P, Lakshmi BV, et al.
Efficacy of local use of probiotics as an adjunct to SRP in CP and halitosis: A randomized controlled trial. J Res Pharm Pract 2016;5:86-93.
] [Full text]
Armitage GC. Classifying periodontal diseases – A long-standing dilemma. Periodontol 2000 2002;30:9-23.
Vivekananda MR, Vandana KL, Bhat KG. Effect of the probiotic Lactobacilli reuteri
(Prodentis) in the management of periodontal disease: A preliminary randomized clinical trial. J Oral Microbiol 2010;2:10-2.
Ramfjord SP. The periodontal disease index (PDI). J Periodontol 1967;38:l602-10.
Ainamo J, Bay I. Problems and proposals for recording gingivitis and plaque. Int Dent J 1975;25:229-35.
Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condtion. Acta Odontol Scand 1964;22:121-35.
Andrade R, Espinoza M, Gómez EM, Espinoza JR, Cruz E. Intra- and inter-examiner reproducibility of manual probing depth. Braz Oral Res 2012;26:57-63.
Tokede O, Jadotte Y, Tounsi A. Efficacy of ozone as an adjunctive anti-microbial in the non-surgical treatment of chronic and aggressive periodontitis-part 2: Review findings and meta-analysis. Int J Dent Sci Res 2016;4:28-34.
Hajishengallis G, Darveau RP, Curtis MA. The keystone-pathogen hypothesis. Nat Rev Microbiol 2012;10:717-25.
Barzegari A, Kheyrolahzadeh K, Hosseiniyan Khatibi SM, Sharifi S, Memar MY, Zununi Vahed S. The battle of probiotics and their derivatives against biofilms. Infect Drug Resist 2020;13:659-72.
Mani A, Saini R, Saini S. Efficacy of oral probiotics as an adjunct to scaling and root planing in nonsurgical treatment outcome of generalized chronic periodontitis patients: A clinico-microbiological study. Int J Exp Dent Sci. 2017;6:6-13.
Iniesta M, Herrera D, Montero E, Zurbriggen M, Matos AR, Marín MJ, et al.
Probiotic effects of orally administered lactobacillus reuteri
-containing tablets on the subgingival and salivary microbiota in patients with gingivitis. A randomized clinical trial. J Clin Periodontol 2012;39:736-44.
Twetman S, Derawi B, Keller M, Ekstrand K, Yucel-Lindberg T, Stecksen-Blicks C. Short-term effect of chewing gums containing probiotic Lactobacillus reuteri
on the levels of inflammatory mediators in gingival crevicular fluid. Acta Odontol Scand 2009;67:19-24.
Hallström H, Lindgren S, Yucel-Lindberg T, Dahlén G, Renvert S, Twetman S. Effect of probiotic lozenges on inflammatory reactions and oral biofilm during experimental gingivitis. Acta Odontol Scand 2013;71:828-33.
Staab B, Eick S, Knöfler G, Jentsch H. The influence of a probiotic milk drink on the development of gingivitis: A pilot study. J Clin Periodontol 2009;36:850-6.
Teughels W, Loozen G, Quirynen M. Do probiotics offer opportunities to manipulate the periodontal oral microbiota? J Clin Periodontol 2011;38 Suppl 11:159-77.
Teughels W, Durukan A, Ozcelik O, Pauwels M, Quirynen M, Haytac MC. Clinical and microbiological effects of Lactobacillus reuteri
probiotics in the treatment of chronic periodontitis: A randomized placebo-controlled study. J Clin Periodontol 2013;40:1025-35.
McCabe LR, Parameswaran N. Advances in probiotic regulation of bone and mineral metabolism. Calcif Tissue Int 2018;102:480-8.
Ikram S, Hassan N, Raffat MA, Mirza S, Akram Z. Systematic review and meta-analysis of double-blind, placebo-controlled, randomized clinical trials using probiotics in chronic periodontitis. J Invest Clinic Dent 2018;9:e12338.
Boyeena L, Koduganti RR, Panthula VR, Jammula SP. Comparison of efficacy of probiotics versus tetracycline fibers as adjuvants to scaling and root planing. J Indian Soc Periodontol 2019;23:539-44.
] [Full text]
Jones SE, Versalovic J. Probiotic Lactobacillus reuteri
biofilms produce antimicrobial and anti-inflammatory factors. BMC Microbiol 2009;9:35.
Schaefer L, Auchtung TA, Hermans KE, Whitehead D, Borhan B, Britton RA. The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups. Microbiology (Reading) 2010;156:1589-99.
van Essche M, Loozen G, Godts C, Boon N, Pauwels M, Quirynen M, et al.
Bacterial antagonism against periodontopathogens. J Periodontol 2013;84:801-11.
Kulkarni PG, Gosavi S, Haricharan PB, Malgikar S, Mudrakola DP, Turagam N, et al.
Molecular detection of Porphyromonas gingivalis
in chronic periodontitis patients. J Contemp Dent Pract 2018;19:992-6.
Da Rocha HA, Silva CF, Santiago FL, Martins LG, Dias PC, De Magalhães D. Local drug delivery systems in the treatment of periodontitis: A literature review. J Int Acad Periodontol 2015;17:82-90.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4]