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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 115-121

Effectiveness of electrospun Ocimum sanctum nanofibers as an adjunct to scaling and root planning in the management of chronic periodontitis: A randomized controlled clinical trial


Department of Periodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Chennai, Tamil Nadu, India

Date of Submission20-Jul-2020
Date of Decision21-Dec-2020
Date of Acceptance05-Jan-2021
Date of Web Publication17-Apr-2021

Correspondence Address:
Dr. Gurumoorthy Kaarthikeyan
Department of Periodontics, Saveetha Dental College & Hospitals, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, 162, Poonamalle High Road, Tamil Nadu.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_245_20

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  Abstract 

Aim: Research based on periodontal therapy is focused on the use of natural products rather than chemical agents. Ocimum sanctum has been known to possess various medicinal properties. There is only a limited literature available on the therapeutic effectiveness of O. sanctum for the treatment of periodontal disease. Local drug delivery systems allowed for the direct delivery to the diseased site thereby bypassing systemic circulation and hence attaining maximum concentration at the site that requires the drug. The aim of the study was to analyze the effect of O. sanctum on the anti-inflammatory efficiency [interleukin 1-β (IL-1β)] in patients with periodontitis. Materials and Methods: A randomized, controlled, clinical trial was conducted with a sample size of 15 patients to compare the clinical parameters and IL-1β levels in subjects who underwent nonsurgical periodontal therapy along with local application of O. sanctum fibers and those subjects who had undergone non-surgical periodontal therapy alone. Results: From the present study, a statistically significant difference could not be observed in the IL-1β levels or in the clinical parameters between the test and the control groups at the end of 1 month. There was a significant difference in clinical attachment-level gain between groups (P = 0.046). Conclusion: Ocimum sanctum nanofibers provided benefits in terms of reduction in IL-1β levels. Our study concludes that O. sanctum delivers additional benefit when used as an adjuvant in the treatment of periodontitis.

Keywords: Electrospinning, interleukin-1, nanofibers, Ocimum sanctum, periodontitis


How to cite this article:
George PM, Jayakumar ND, Kaarthikeyan G. Effectiveness of electrospun Ocimum sanctum nanofibers as an adjunct to scaling and root planning in the management of chronic periodontitis: A randomized controlled clinical trial. J Int Oral Health 2021;13:115-21

How to cite this URL:
George PM, Jayakumar ND, Kaarthikeyan G. Effectiveness of electrospun Ocimum sanctum nanofibers as an adjunct to scaling and root planning in the management of chronic periodontitis: A randomized controlled clinical trial. J Int Oral Health [serial online] 2021 [cited 2021 Jun 17];13:115-21. Available from: https://www.jioh.org/text.asp?2021/13/2/115/313840


  Introduction Top


Chronic periodontitis is a disease of polymicrobial origin, which results in inflammation within the underlying supporting structures of the teeth, thereby leading to periodontal pocket formation, attachment loss, and bone loss. Periodontal disease is widely accepted as being caused by bacteria associated with dental plaque and recently viruses have also shown to play a role in periodontal destruction.[1],[2] The main aim of periodontal therapy is to remove plaque and calculus, thereby aiding in the restoration of the lost form and function. Mechanical therapy may fail to eliminate all the pathogens within the gingival tissues and in tooth structures inaccessible to periodontal instrumentation.

To overcome the limitations of conventional treatment, chemotherapeutic agents such as antibiotics and antiseptics have been used for the treatment of periodontal disease.[3] The drawback of systemic antibiotics is that they require the administration of large doses to obtain optimal concentrations at the site of the disease or within the gingival crevicular fluid (GCF). Such large doses lead to the development of drug-resistant strains of bacteria. To avoid the shortcomings of systemic administration, local drug delivery systems containing antibiotic or antiseptic agents were introduced.[4]

Local drug delivery systems allowed for the direct delivery to the diseased site, thereby bypassing systemic circulation and hence attaining maximum concentration at the site that requires the drug. Initial efforts included flushing of the periodontal pocket directly with antimicrobial solutions.[5] These treatments, however, have been ineffective as adjunct therapies, most likely due to the inability to maintain sufficient concentration of the agent in the pocket for adequate durations. Thus, the critical factor in the use of a locally delivered anti-microbial agent in periodontal treatment may be exposure of the microflora for sufficient duration to ensure the efficacious result and also its ability to deliver higher concentrations of the drug directly to the depth of the pocket at microbicidal concentrations.

Research is focused on the use of natural products rather than chemical agents. Medicinal plants have been used as a traditional treatment agent for numerous human diseases for ages in many parts of the world. Tulsi, commonly known as Holy Basil and botanically as Ocimum sanctum, belongs to the family Lamiaceae. Tulsi has been described as two types—Vanya (wild) and Gramya (homegrown). Both are known to have identical properties but the former has darker leaves. The chemical composition of tulsi is known to be highly complex, containing many nutrients and other biologically active compounds.

Ocimum sanctum has been known to possess various medicinal properties. It is known for its properties such as anti-inflammatory, anti-microbial, anti-oxidant, and anti-cancer.[6] The pharmacological and nutritional properties of the whole herb in natural form, as used in traditional medicine, results from the synergistic interaction of many different active phytochemicals. Consequently, the overall effects of tulsi cannot be fully duplicated with isolated compounds or extracts. The best known active components that have been identified and extracted are eugenol and ursolic acid.[7]

For a long time, bacteria were thought to be the sole causative factor for periodontal disease, but it is the subsequent inflammatory response that develops which leads to the destruction of the underlying supporting structures. As there is only a limited literature available on the therapeutic effectiveness of O. sanctum for the treatment of periodontal disease and hence it is worthwhile to analyze the anti-inflammatory efficiency of O. sanctum in patients with periodontitis. Thus, the aim of this study was to analyze the efficiency of locally delivered electrospun O. sanctum nanofibers as an adjuvant with nonsurgical periodontal therapy in the management of chronic periodontitis.


  Materials and Methods Top


Sampling criteria

The sample size was calculated using G power 3.1 software based on the mean and standard deviation obtained from a similar study done by Gupta et al.,[8] with a power calculation of 95% and the α error at 0.05. Study participants were recruited from the outpatient department of Saveetha Dental College and Hospitals based on the inclusion and exclusion criteria for periodontitis.

The inclusion criteria include those patients diagnosed clinically as having chronic periodontitis according to the American Academy of periodontics criteria 1999 with a periodontal probing depth of ≥5 mm and clinical attachment loss in more than 30% of sites along with radiographic evidence of bone loss. The mean age of the subjects was 43.67 ± 5.36.

The exclusion criteria include that the patients with a history of any systemic diseases like diabetes mellitus, cardiac problems, etc. Patients with a previous history of hospitalization/surgery in the past 1 year, pregnant or lactating women, patients with a history of antibiotic, anti-inflammatory drug intake in the past 6 months were excluded from the study.

Material preparation

Ocimum sanctum (Tulsi) extracts preparation

The leaves of O. sanctum were separated and shade dried for 1 week. Once the leaves were completely dry, it was ground into a fine powder. Fifty grams of this powder was mixed with 500mL of methanol (Sigma–Aldrich) and was stirred until it was completely dissolved. A rotary evaporator (BUCHI rotavapor R-200) under reduced pressure was used to completely remove the solvent (methanol). This led to obtaining a crude extract. The crude extract was suspended in organic solvents (hexane and methanol) and was filtered using Whatman No.41 filter paper to remove the particles. The particle-free extract obtained was evaporated to remove the under reduced pressure to obtain dry crude extract. This procedure was repeated twice to obtain dry crude extract.[9]

MIC determination by broth dilution

Equipment and materials involved were 96-well plate (Thermo Scientific), Nutrient broth (HIMEDIA M002), Nutrient Agar/Agar powder (HIMEDIA RM026), Resazurin dye (Sigma–Aldrich), Staphylococcus aureus (MTCC 737).

Staphylococcus aureus bacterial strain was incubated into Muller Hinton broth and incubated for around 5 h at 35ºC in a shaker water bath until turbidity of 0.5 McFarland units appeared. The final inoculum was adjusted to 5×105 cfu/mL. The tubes contained two-fold serial dilutions of the extract and Chlorhexidine (control). Each dilution was seeded with the test organism to the standard concentration (5×105 cfu/mL). The final dilution showing no noticeable growth (turbidity) was ascertained as the MIC. The end-points of colorimetric MIC are interpreted as the lowest sample concentration that remained blue (indicating no growth) or the first dilution that changed from blue to slightly purple (equivalent to growth inhibition). In this experiment, tulsi extract showed inhibition at 10 mg/mL concentration against Staphylococcus aureus.

Preparation of spinning solutions

Polyvinyl acetate (HIMEDIA)/tulsi solutions were prepared by dissolving tulsi in 10% (w/v) aqueous PVA solution. Solutions were prepared with tulsi at 1%, 5%, 10%, 15%, 20% wt. with respect to PVA content and were placed in a sonicator for 5min and then stirred for 8 h continuously at room temperature.[9]

Electrospinning process

By varying spinning parameters, the resultant fibers were analyzed under SEM to identify those that yielded the ideal fibers. The spinning parameters that resulted in ideal smooth and homogenous fibers were when the syringe containing the spinning solution was fixed horizontally in the HOLMARC’S appliance, the applied voltage set at 13kV, tip-to-collector distance of 12cm, the flow of the solution was at 500 µL/h, tip diameter of the needle was 12 mm, the volume of the solution was 2.5mL and the total duration of spinning took 5 h. SEM (Quanta 200 FEG) was used to examine the fiber morphology. The images obtained showed samples were homogenous and free of heterogeneities or artifacts. 10 wt% fibers seem to have a smooth surface with no visible beading.

Study method

This is a randomized controlled clinical trial to compare the clinical parameters and interleukin 1-β (IL-1β) levels in subjects who underwent nonsurgical periodontal therapy along with a local application of O. sanctum fibers and those subjects who had undergone nonsurgical periodontal therapy alone. Randomization was performed by computer-generated random numbers.

Study groups

  1. Group 1: Control group (n = 15): Subjects who underwent Scaling and Root planning alone.


  2. Group 2: Test group (n = 15): Scaling and root planning followed by the placement of O. sanctum fiber.


In both the groups, after the proper recording of the case sheet, thorough supragingival scaling was done with ultrasonic scalars and instructions for proper oral hygiene maintenance were given. The subjects were recalled after a week to evaluate the tissue response and oral hygiene maintenance. Then a thorough subgingival scaling and root planning were carried out. The patients were again recalled after a week for re-evaluation and in the test groups, the O. sanctum nanofibers were packed in the persistent pockets of more than 3 mm.

The O. sanctum electrospun fibers were packed into the pockets after careful isolation and drying of the sites. The fibers were packed gently from gingival margins to the depth of the pocket without inducing any bleeding. The patients were instructed with proper oral hygiene maintenance.

Observational parameters

Parameters were assessed before and after nonsurgical periodontal therapy. Nonsurgical therapy included scaling and root planning followed by oral hygiene instructions. Scaling was done using ultrasonic scalars. Root planning was performed using Gracey Curettes. The clinical parameters observed include gingival index,[10] sulcus bleeding index,[11] probing pocket depth, clinical attachment level, and the biochemical marker estimated pre- and post-treatment was GCF IL-1β level.

Human IL-1β ELISA assay

Total levels of IL-1β were determined using a preheated ELISA plate according to the manufacturer’s instructions (RayBiotech, Inc., Georgia). Briefly, 50 µL of standard solutions or samples were added in duplicate to the appropriate wells and incubated for 2 h at room temperature while shaking at 200rpm and plates were washed for four times with 1× buffer. One hundred microliters of human IL-1β detection solution was added to each well and incubated at room temperature for 1 h and the plates were washed again. One hundred microliters of Avidin-HRP D was added and incubated at room temperature for 30min. The color changes from blue to yellow and the absorbance was read at 450 nm (BIO reader™) within 30min.

Statistical analysis

SPSS 20 was used for the assessment of the results. Student’s t-test was used to analyze the statistical significance in the IL-1β levels, Bleeding index, gingival index, probing depth, and clinical attachment level between group 1 (SRP) and group 2 (SRP+ O. sanctum). P < 0.05 was considered statistically significant.


  Results Top


On intragroup comparison of a gingival index at the end of 1 month, group 1 showed a mean reduction of 0.47 (P = 0.00* [Throughout the article, the symbol asterisk indicates significance.]) and reduction of 0.42 in group 2 (P = 0.00*). On intergroup comparison, the baseline values of a gingival index (P = 0.29) showed no significant difference. At the end of 1 month, the mean difference observed between the groups was 0.09 (P = 0.35), thus showing no statistically significant difference [Table 1]. On intragroup comparison of bleeding index, at the end of 1 month, group 1 showed a mean reduction of 0.65 (P = 0.00*) and a reduction of 0.77 in group 2 (P = 0.02*). On intergroup comparison, the baseline values of the bleeding index (P = 0.75) showed no significant difference [Table 2]. At the end of 1 month, the mean difference observed between the groups was 0.04 (P = 0.55).
Table 1: Comparison of the mean gingival index between groups 1 and 2 using Student’s t-test

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Table 2: Comparison of the mean bleeding index scores between groups 1 and 2 using Student’s t-test

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Similar results were obtained for probing pocket depth with no statistically significant difference between the groups (P = 0.276) [Table 3]. On intragroup comparison of CAL, at the end of 1 month, group 1 showed a mean reduction of 1.2 (P = 0.00*) and a reduction of 1.4 in group 2 (P = 0.00*). On intergroup comparison, the baseline values of CAL (P = 0.47) showed no significant difference. At the end of 1 month, the mean difference observed between the groups was 1.01 (P = 0.046) [Table 4]. When compared between the groups, there was a nearing significance in the clinical attachment level with P = 0.046. This is a significant finding with O. sanctum showed good clinical attachment level improvement compared with nonsurgical therapy alone group. As far as the pro-inflammatory mediator interleukin 1-β levels in GCF were considered, there was a significant reduction in the level of IL-1β in the test group but there was no significant difference between the test and control groups [Table 5].
Table 3: Comparison of the mean probing depth between groups 1 and 2 using Student t-test

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Table 4: Comparison of the clinical attachment level between groups 1 and 2 using Student’s t-test

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Table 5: Comparison of GCF levels of IL-1β between groups using Student’s t-test

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  Discussion Top


Sustained-release local drug delivery systems showed promising results in the management of periodontitis due to higher drug concentrations achieved at target sites, thereby reducing the side effects. Agents incorporating tetracycline, doxycycline is the most commonly used agents in the treatment of periodontitis. The focus has been shifted to natural agents like tulsi, turmeric, neem, aloe vera which are known to have various medicinal properties. Many natural products (herbs) have shown promising results in the management of inflammatory diseases and shown good results even in the in vitro research of carcinomas.[12],[13] Administering a natural agent such as tulsi with anti-microbial and anti-inflammatory properties through an effective delivery system would be helpful in the treatment of periodontitis. Several delivery systems are available in the market such as strips, gels, injectables, fibers, etc.

Electrospinning, a versatile and cost-effective method for nanoscale fiber production, has been receiving great attention in recent years. They have various properties such as nanoporous structure, large surface to volume ratio, flexibility for physical or chemical modification. They allow for the incorporation of the drug into the system while fabricating the fiber. The extremely small diameter of the electrospun nanofiber, high surface area, and interconnected fibers make them desirable for various applications.[14] In the present study, O. sanctum was chosen as the natural agent to be incorporated into PVA to produce nanofibers that could be used within the periodontal pocket. Tulsi, known to possess several therapeutic activities such as antimicrobial, anti-inflammatory, anti-oxidant as claimed by various available literature, led the study in the direction of this plant. The antibacterial agents present in O. Sanctum, such as Eugenol, Carvacrol, Linalool, Caryophyllene, Ursolic acid, and methyl carvicol, can form high-molecular-weight complexes with soluble proteins in saliva which cause bacterial lysis and prevent bacterial colonization. The essential oils that tulsi yields are said to be responsible for its medicinal properties.[6] The leaves of tulsi contain 0.7% volatile oil comprising about 71% eugenol and 20% methyl eugenol. Antibacterial efficacy of O. sanctum against periodontal pathogens such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia was shown in an in vitro study by Mallikarjun et al.[15] In another in vitro study to assess the anti-microbial property of tulsi against Aggregatibacter actinomycetemcomitans, results showed inhibition at 6% concentration.[16]

The MIC of O. sanctum against Streptococcus mutans was 0.125 mg as seen in the study done by Mistry et al.[17] The periodontopathogens are mostly facultative anaerobes with more virulence than S. mutans, hence increasing the concentration of O. sanctum was thought to be ideal. The concentration incorporated was 10 mg/mL. In an animal study to assess the effect of fixed oil of O. sanctum on experimentally induced arthritis at varying concentrations of 1.0, 2, and 3mL/kg, improvement in the condition was observed with the use of O. sanctum.[18]

In a study done to compare O. sanctum mouth rinse and chlorhexidine based on the plaque score and gingival index by Gupta et al.[8] results showed that there was a significant reduction in plaque and gingival index at the end of 15 and 30 days in both O. sanctum and chlorhexidine groups, whereas no significant difference was observed between the groups treated with chlorhexidine and O. sanctum. The baseline gingival index value (mean and SD) for the O. sanctum group was 2.23 ± 0.43 and, at the end of 30 days, it was 1.35 ± 0.36.[9] The present study also showed a reduction from the baseline value of 1.45 ± 0.32 to 1.03 ± 0.17. Even though there was a reduction in both the groups from the baseline, no significant difference was observed between the groups in both the studies.

In a randomized controlled study, to compare the efficiency of 4% O. sanctum and 0.2% chlorhexidine irrigation as an adjunct to scaling and root planning, a statistically significant difference was observed between baseline and at the end of 30 days. No significant difference was found between the groups in plaque index, gingival index, probing pocket depth, and clinical attachment levels. The results obtained in the present study are in line with the above study, except there was an improvement in clinical attachment level in the present study.[19]

In an animal study done to analyze the efficiency and anti-inflammatory potential of 2% O. sanctum gel on experimental periodontitis, wherein the anti-inflammatory property was assessed by the carrageenan-induced paw edema method. The change in the periodontal status was assessed based on gingival index and Probing pocket depth. Anti-inflammatory activity of tulsi was better than the control (plain gel) but not statistically significant, whereas the periodontal parameters, that is, gingival index and probing pocket depth, showed a statistically significant difference between the groups.[20] The present study also showed similar results.

Evaluating inflammatory mediator levels is more sensitive in predicting disease activity. Increased levels of proinflammatory mediators like cytokines, endothelin, tumor necrosis factor in the saliva, and GCF of patients affected by periodontal diseases are a hallmark of the initiation and maintenance of the disease.[21],[22],[23] Among the various inflammatory mediators, IL-1β plays a major role in the inflammatory process of the periodontal.[24] The present study aimed at evaluating IL-1β levels with and without the use of O. sanctum. The ability of O. sanctum extract to inhibit periodontal pathogens could be the possible mechanism that led to the reduction in the inflammatory component and thus the proinflammatory mediator–IL-1β levels got reduced post-treatment in the test group.

Electrospinning of tulsi at the desired concentration could be pursued as seen from the available literature. The study done by Reise et al.[25] showed that metronidazole could be incorporated into poly(l-lactide-co-d/l-lactide) by electrospinning for the treatment of localized periodontitis. Sun et al.[26] did a study by incorporating curcumin in PVA by electrospinning at 5%, 10%, 15%, and 20% concentrations. Various nonantimicrobial drugs are also validated against many periodontal pathogens.[27]

The constituents of the GCF are one of the early, biochemical indicators of tissue changes within the periodontium. It is one of the early and objective methods of diagnosing periodontal disease. The association between IL-1β with periodontal disease is well established. There was a significant correlation between the level of IL-1β and the severity of the disease. In the present study, on intragroup comparison of IL-1β levels, at the end of 1 month, group 1 showed a mean reduction of 3.63 (P = 0.00*) and a reduction of 5.41 in group 2 (P = 0.01*). On intergroup comparison, the baseline values of IL-1β (P > 0.05) showed no significant difference. At the end of 1 month, the mean difference observed between the groups was 0.17 (P = 0.92). The significant reduction of IL-1β in the test group can be attributed to the anti-inflammatory property of O. sanctum. It is known to contain eugenol which can block the cyclooxygenase pathway. Another compound, linoleic acid that is found in the fixed oils of O. sanctum, has the ability to block both cyclooxygenase and lipooxygenase pathways of the arachidonic acid metabolism.[6] The intergroup difference after 30 days failed to show any statistical significance which may be due to the concentration of tulsi that was incorporated. Targeting periodontal pathogens that are more virulent may need an increased concentration to be able to inhibit their activity.


  Conclusion Top


Within the limitations of this study, it can be stated that, on comparison within the group, O. sanctum nanofibers provided benefits in terms of reduction in IL-1β levels and significant gain in the clinical attachment level. Further studies need to be carried out with different concentrations of O. sanctum and a larger sample size to expedite the role of O. sanctum in the management of periodontitis.

Acknowledgment

None.

Financial support and sponsorship

It is a self-funded study and we did not receive funding from any agency or organization.

Conflict of interest

There are no conflicts of interest.

Author contributions

Priyanka Mariam George - designing the study, data collection, and execution of the study. N. D. Jayakumar - manuscript preparation and statistical analysis. G. Kaarthikeyan - study design, execution of the study, and literature search.

Ethical policy and institutional review board statement

The study was registered in the clinical trials registry (CTRI/2018/07/014961). The ethical clearance was obtained from the institutional ethical committee. Written informed consent was obtained from all participants before including them in the population.

Patient declaration of consent

Not applicable.

Data availability statement

Data are available on valid request by contacting the corresponding author mail.

 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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