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 Table of Contents  
Year : 2021  |  Volume : 13  |  Issue : 3  |  Page : 298-305

Ozonated olive oil gel versus nano-silver mouthwash in root biomodification: A scanning electron microscopic study

1 Department of Oral Medicine, Periodontology, Oral Diagnosis and Radiology, Faculty of Dentistry, Tanta University, Cairo, Egypt
2 Department of Oral Biology, Faculty of Dentistry, Tanta University, Cairo, Egypt

Date of Submission21-Dec-2020
Date of Decision04-Jan-2021
Date of Acceptance03-May-2021
Date of Web Publication18-Jun-2021

Correspondence Address:
Dr. Sarah Yasser A Ali
Department of Oral Biology, Faculty of Dentistry, Tanta University, Algeish Street, Tanta City, Al gharbeyah.
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jioh.jioh_355_20

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Aim: This in vitro scanning electron microscopic (SEM) study was planned to assess and compare the effect of ozone gel versus nano-silver mouthwash when used as a root bio-modifier on the surface of the periodontally affected extracted roots. Materials and Methods: Twenty single-rooted teeth were selected for this in vitro cross-sectional study. The teeth were decoronated and split longitudinally to form 40 radicular halves. They were randomly allocated to four groups of 10 fragments each. Then, the other groups were scaled. Group 1: left untreated (non-scaled) (Negative control group). Group 2: treated with saline (Positive control group). Group 3: treated with ozone gel 20:25 µgm/ml concentration. Group 4: treated with nano-silver mouthwash. The specimens were prepared to undergo scanning electron microscopy and elemental analysis (SEM-EDX) to determine the percentage of calcium (Ca) atom. Further, the acquired photomicrographs were evaluated for the presence of calculus, smear layer, and product residues and they were analyzed using Fisher Exact test followed by Dunn’s test. SEM-EDX were analyzed by ANOVA test followed by Tukey’s post hoc test for a pairwise comparison between groups. Results: The nano-silver mouthwash treated group showed the absence of the smear layer and calculus and had significantly higher residue levels than ozone-treated specimens (P = 0.001), whereas the ozone group showed that the smear layer was not completely removed with the absence of remnant of the material. The negative control group scored the highest level in the presence of calculus compared with other examined groups (P = 0.001). Interestingly, the nano-silver treated group showed a significant decrease in Ca levels compared with all other experimental groups. Conclusion: The nano-silver mouthwash removed the smear layer effectively and provided notable substantivity, as evidenced by the presence of the material remnant after application. Moreover, it decreased Ca level significantly compared with all other experimental groups, which is expected to increase cellular attachment and proliferation. Thus, using the nano-silver mouthwash as a root bio-modifier could yield better results in periodontal reattachment.

Keywords: Nano Silver, Ozone, Periodontitis, Root Bio-modification, SEM

How to cite this article:
Shoukheba MY, Yasser A Ali S, Gamal SM. Ozonated olive oil gel versus nano-silver mouthwash in root biomodification: A scanning electron microscopic study. J Int Oral Health 2021;13:298-305

How to cite this URL:
Shoukheba MY, Yasser A Ali S, Gamal SM. Ozonated olive oil gel versus nano-silver mouthwash in root biomodification: A scanning electron microscopic study. J Int Oral Health [serial online] 2021 [cited 2021 Oct 26];13:298-305. Available from:

  Introduction Top

Periodontitis resulted in exposure of the cementum of the roots to the environment in the periodontal pocket and to the pathogenic microorganisms and its toxins.[1] This initiates inflammation, and substantial physical, chemical, and histological alterations in the root surfaces exposed to this unsuitable environment.[2] These cause damage to the periodontal tissue structures, such as loss of collagen fiber integrity, disturbance in mineral density and composition, which become non-biocompatible for growth of the periodontal ligament cells and, consequently, affect the process of regeneration.[3]

Further, scaling and root planing results in the precipitation of a smear layer on the root surface, creating a physical barrier that inhibits new attachment, and acts as a substrate for bacterial growth on the root surfaces.[4],[5] Hence, to achieve considerable amount of periodontal regeneration, the diseased root surface should be devoid of local and cytotoxic substances.[6] Thus, removal of this smear layer as an adjunct to root surface debridement for exposure of the dentinal collagen fibers is mandatory and known as root biomodification.[7] This latter procedure encourages cementogenesis, improving fibrin linkage, reinforcing connective tissue attachment, and promoting fibroblast migration.

Various agents have been used in root biomodification, such as citric acid, ethylene diamine tetra-acetic acid (EDTA), tetracycline hydrochloride (HCl), hydrogen peroxide, fibronectin, enamel matrix proteins, recombinant platelet-derived growth factor, and dentin bonding conditioner.[8]

The old protocol of root treatment was mostly focused on the use of acids such as citric acid and tetracycline hydrochloride.[9] The application of low pH acting agents has many harmful effects on the nearby vital periodontal tissues, which may inhibit healing and regeneration.[10] Studies have shown that EDTA, a neutral pH chelating agent, is better than other acidic agents as it maintains the integrity of the periodontal tissues after root surface treatment and biomodification.[7]

The use of ozone in dentistry is gaining its place in everyday dental practice. Ozone therapy is widely used as an adjunctive therapy and is proven to be a new therapeutic modality, with great benefits to dental patients.[11] Ozone is a naturally occurring gas that is formed from combining three oxygen atoms. It is a potent oxidizing agent with a great antimicrobial effect against periodontal pathogens, and it has the capacity to work as a metabolic and host immune modulator.[12]

Ozone has been used mainly for sterilizing early carious cavities, avulsed teeth, root canals, and periodontal pockets. It has also been utilized to enhance epithelial wound healing, such as ulcerations and herpetic lesions. Ozone can be administered either topically or systemically, in both gaseous and aqueous forms.[13] Moreover, ozone showed in vitro biocompatibility with human oral epithelial cells, gingival fibroblast cells, cementoblasts, and periodontal cells, suggesting its efficiency against oral infectious diseases such as periodontal disease and peri-implantitis.[14]

In addition, ozone can effectively terminate the root sensitivity problem. Its desensitizing effect on dentine lasts longer than other conventional methods.[15] On the other hand, the use of nanotechnology that has affected all aspects of science and dentistry is no exception. Nanotechnology is the science of manipulating matter at the nanometer scale. It permits control over the material up to levels invisible to the naked eyes.[16] Nano-silver mouthwash has been shown to have antibacterial activity due to the small size of the silver nanoparticles that increase the surface area and permit more contact with the bacterial cells. Also, nanoparticles can penetrate bacteria and other microorganisms and destroy them.[17] Surprisingly, many studies have reported that the nano-silver has a higher antimicrobial effect than chlorhexidine.[18],[19] Thus, nano-silver particles are considered one of the most important metallic nanoscale materials with magnificent antimicrobial properties against many types of microorganisms, from which the oral bacteria emerge. Studies have stated that the usage of nano-silver in periodontal treatment has the potential to fight the dental biofilm and to decrease the incidence of periodontal disease.[20] However, León et al.[21] reported that there is no sufficient information regarding the antimicrobial capability of nano-silver against dental biofilms accompanying periodontal diseases.

The present study was conducted to evaluate the efficacy of two novel materials, namely, ozonated olive oil gel against nano-silver mouthwash when used as root bio-modifiers on the surface of the periodontally affected extracted roots in vitro by a scanning electron microscope.

  Materials and Methods Top

Setting and design

The present in vitro cross-sectional study was carried out in the Department of Oral Medicine, Periodontology, Oral diagnosis and Radiology Department, Faculty of Dentistry, Tanta University from October 2020 to November 2020. Sample size was calculated by G Power (version, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) depending on the primary outcome. The required sample size per group was 10 according to these assumptions, effect size was 0.5, margin of error was 5%, power was 80%, and the number of the studied groups was 4. The procedures were in accordance with the ethical standards of the Research Ethics Committee of Faculty of Dentistry, Tanta University and with the Helsinki Declaration.

Twenty single-rooted human teeth from patients with advanced periodontal disease were used in this study. The inclusion criteria were: untreatable, hopeless single-rooted teeth that have clinical attachment loss >5 mm. The exclusion criteria were: badly decayed teeth with periapical infection, teeth with enamel pearls or any cemental anomalies, root caries, and recent periodontal therapy.

Study method

After extraction, the teeth were washed with saline to eliminate adherent soft tissues. Using a high-speed cylindrical bur, two parallel grooves were made on the proximal root surfaces of the teeth: one at the cemento-enamel junction (CEJ) and the second one 5 mm apically from the first groove. To adapt a smear layer, the zone located between the two grooves was scaled and planed using Gracey curettes 5/6 (Hu-Friedy, Chicago, IL, USA) by the same investigator. The roots were then decoronated at the level of the first groove and separated longitudinally into two identical halves, to obtain 40 specimens. The specimens taken from the same root were allocated to different treatment groups and marked by a number. Then, they were well maintained in a mixture of anhydrous glycerol/absolute alcohol 1:1 until the time of treatment to maintain the tissues, as advised by Wen et al. (1992).[22]

The specimens were randomly allocated to four groups comprising 10 specimens each as follows:

  • Group 1: served as a negative control group without scaling and root planning.

  • Group 2: was treated with saline for 3min after scaling and root planing. (Positive control group).

  • Group 3: was treated with ozonated olive oil gel with a concentration of 20:25 µgm/ml (Pharmoxid Arznei GmbHandCo.KG, Nordring 8, D-76473 Iffezheim) for 3min after scaling and root planing.

  • Group 4: was treated with nano-silver mouthwash with a concentration of 0.02mg/ml (Nanogist co., Korea) for 3min after scaling and root planing.

  • Scanning electron microscopic analysis (SEM) and elemental analysis (EDX)

    SEM and EDX were carried out in the department of science, Alexandria University. Preparation of samples for SEM was done as follows: first, rinsing in buffer solution and next, washing with distilled water. Then, dehydration was conducted in a series of concentrations from 70% to 90% of aqueous ethanol solution. Specimens were exposed to air drying with a blower and preserved overnight in silica gel. Then, they were mounted on conductive carbon tape pasted on the standard specimen mounting SEM stubs. They were coated with a thin layer of gold powder by a sputter coating device for 1min. The specimens were examined using JSM 5200 LV SEM and photographed by Orion 6.60 software package containing camera drivers.

    During sample analysis, the electron beam parameters were kept constant and maintained at a 08-tilt angle to maintain standardization. The microscope accelerating voltage was kept between 15kV and 20kV at high vacuum. The photomicrographs were obtained at 500 and 1000 magnification.

    The SEM micrographs were analyzed based on three parameters: presence of calculus: 1 (where 1 indicated the presence of a great amount on dentin, whereas 2 indicated the absence of calculus); presence of smear layer (where 1 indicated the presence of a great amount of smear layer and smear plug, whereas 2 indicated the absence of a smear layer and smear plug); and presence of the product residues (where 1 indicated the presence of a great amount of product residues, whereas 2 indicated no product residues left for use).[23] Nominal data were represented by score distribution frequency.

    The SEM-EDX mapping analysis of the root surfaces measures the concentration and distribution of elements, such as Ca. For the point analysis to measure the concentrations of Ca, two points on the root surface were randomly selected from each specimen and the mean of the percentage of Ca atom was calculated. The analysis was done under the same conditions of voltage, current, working distance, and pressurization.

    Statistical analysis

    The data of the present study were collected, tabulated, and analyzed using Statistical Program for Social Science (SPSS) version 20.0. Data of the presence of calculus, smear layer, and product residues were analyzed by Fisher Exact test and then Dunn’s test. However, SEM-EDX were analyzed by using ANOVA test followed by Tukey’s post hoc test for a pairwise comparison between groups.

      Results Top

    The SEM analysis

    The scores of the presence of calculus, smear layer, and the residue of the products for all groups are shown in [Table 1]. The SEM analysis showed that in all samples of the negative control group, the root cementum showed a rough irregular surface with cracks and it was covered by debris and calculus [Figure 1]. The scaled group showed no residual calculus, whereas the root surface was irregular and covered with a smear layer [Figure 2]. The SEM of the ozone-treated group showed that the smear layer was not effectively removed and there were no residual particles of the used material [Figure 3]. The nano-silver mouthwash treated group showed absence of a smear layer with the presence of residual particles in all specimens [Figure 4]. A significant difference among groups was observed by Fisher Exact test (P = 0.001). Dunn’s test showed that the silver nanoparticles group had a significantly lower smear layer level than other experimental groups [Table 1]. Dunn’s test also revealed that the silver nanoparticles group had significantly higher residue levels than ozone-treated specimens (P = 0.001). The presence of calculus results revealed that the negative control group had the highest significant level of the presence of calculus than other examined groups (P = 0.001).
    Table 1: Parameter scores evaluated by SEM analysis for all groups

    Click here to view
    Figure 1: SEM image of the negative control group showing calculus and debris covering almost the entire root surface (Original magnifications ×1000)

    Click here to view
    Figure 2: SEM image of the positive control scaled group showing no residual calculus, the presence of debris, and a smear layer covering the entire root surface (Original magnifications ×1000)

    Click here to view
    Figure 3: SEM image of the ozone treated group showing the presence of a smear layer covering the entire root surface; no residual remnants are evident (Original magnifications ×1000)

    Click here to view
    Figure 4: SEM image of the nano-silver treated group showing complete removal of the smear layer with the presence of residual particles (Original magnifications ×1000)

    Click here to view

    SEM-EDX results

    The SEM-EDX mapping analysis of the root surfaces measures the concentration and distribution of elements, such as Ca. The mapping analysis in [Table 2] and [Table 3] shows that there was a significant difference between the experimental groups in the percentage of Ca atom. There was a significant decrease in the percentage of Ca atom in both ozonated and silver nanoparticle groups compared with the control. However, in comparison to the scaled/saline group, only the silver nanoparticles group showed a significant decrease in the percentage of Ca atom. Interestingly, there was a significant difference between the silver nanoparticles group and the ozonated olive oil group [Figure 5].
    Table 2: Comparison between Means ± S.D of Ca atom% of all groups using ANOVA test

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    Table 3: Tukey’s post hoc test for pairwise comparison between groups

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    Figure 5: Comparison between Means of Ca atom% of all group

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      Discussion Top

    In comparison with classical medicine modalities such as antibiotics and disinfectants, nanotechnology and ozone therapy allow us to take a minimally invasive and conservative approach to dental treatment. Periodontal therapy aims at modifying the periodontally affected root surface into a biologically compatible surface that allows re-attachment with both the epithelial and connective tissue components of the periodontium.[23] This is done primarily by the mechanical removal of dental plaque and calculus. In our study, we removed only the superficial diseased cementum layer without deliberately exposing dentin. Recent studies reported that endotoxins were not located within the cementum; rather, they were only loosely attached to the cementum surface,[24],[25] and most of the endotoxins were related to the bacterial biofilm.[26] Moreover, the preservation of cementum on the root surface was crucial for soft tissue re-attachment as it acts as a source of growth factors that are important for periodontal regeneration.[27] Subsequent to the scaling and root planing process, a smear layer is formed on the treated root surfaces. This smear layer is composed of organic and inorganic debris that interferes with periodontal healing, as it acts as a physical barrier between periodontal tissues and the blood clot from one side and the root surface from the other side. This barrier delays the formation of periodontal re-attachment. Microscopically, the smear layer contains the remaining calculus, bacterial-derived products, and cementum.[27],[28]

    To increase the potential of periodontal regeneration, further chemical treatment on the instrumented root surface is needed to remove the smear layer. Several agents were used to decontaminate, demineralize, and remove this layer from the root surface, thus exposing collagen fibers of dentin and cementum to facilitate re-attachment between the root surface and the periodontal ligaments.[29] This re-attachment is led by blood clot formation, fibrin linkage, more fibronectin binding sites, and enhanced mesenchymal cell adhesion, chemotaxis, and growth. These modifying chemicals include EDTA, phosphoric acid, and tetracycline.[4],[ 30],[ 31]

    The present study aimed at comparing the efficacy of using ozonated olive oil gel versus nano-silver mouthwash in the root surface biomodification of periodontally affected human teeth. Results of the present study found that in all scaled specimens, no remaining calculus was found; however, the root surfaces were covered by a smear layer except in the nano-silver treated group.

    Ozone has multiple beneficial effects, including its antimicrobial activity and oxidation of bacterial biomolecules and microbial toxins. It has been implicated in periodontal diseases due to its healing and regenerative properties. Patel et al. (2013) found that ozone removes the smear layer, thus opening the dentinal tubules to allow deep penetration of Ca and fluorine ions into them. This process reduces root dentin hypersensitivity.[32] However, unlike the previous findings, we found that ozone did not effectively remove the smear layer formed on the root surface. This was in correspondence with Habashneh et al., who concluded that irrigation with ozonated water as an adjunctive therapy to scaling and root planing produced no statistically significant benefit compared with scaling and root planing plus distilled water irrigation.[33]

    Silver nanoparticles are well known for their antimicrobial and anti-inflammatory effects[34] and high penetration capacity due to their small size.[35] Moreover, they enhance fibroblast proliferation and maturation.[36] Also, it was found that silver nanoparticles eliminate bacterial biofilms on the root surface,[20] and this is in connection with our findings indicating that silver nanoparticles successfully removed the smear layer. This can be explained by the nature of the ionic liquid, imidazole, used as a coating agent and a stabilizer in the silver nanoparticle synthesis. Further, the charge distribution dissimilarity on the cationic part of these particles and the root surface might also be a contributing factor to our findings.[37],[38] It is worth noting that remnants of the nanoparticles were found on the treated root surfaces. This provides active ingredient distribution over an extended time, thus exerting prolonged benefit from the material (substantivity) and reducing the frequency of their administration. On the other hand, there were no remnants of the ozonated gel after its application.

    This finding is in agreement with that of Espinosa-Cristóbal et al.,[21] who found that nano-silver particles have good substantivity and antimicrobial inhibitory effect against various oral biofilms isolated from subjects with active dental caries and active periodontal disease.

    In this study, we were also concerned with elemental or mineral changes on the root cementum surface that can be attributed to chronic periodontitis and the relationship between using different agents and the mineral content of the cementum surface. The SEM–EDX analysis was found to be the most suitable technique for elemental analysis. We found that the negative control group scored the highest percentage of Ca atom. This is in concomitance with other studies indicating that the diseased surface cementum has a relative increase of 7–10% in the Ca content as compared with the deeper layers[39] and healthy cementum.[40] Also, Eide et al.[41] observed that diseased cementum, in periodontal diseases, has a highly mineralized surface coating derived from saliva and inflammatory exudates within periodontal pockets. The present study found that merely scaling and root planing has no effect on the Ca level, due to the presence of the smear layer, which is highly mineralized, and was found to interfere with cellular attachment.[42] Furthermore, using ozonated olive oil gel failed to effectively remove the smear layer and yielded a nonsignificant decrease in Ca levels, compared with the scaled group. Interestingly, we found that the nano-silver treated group showed a significant decrease in Ca levels compared with all other experimental groups. The present results were in harmony with those of Kurt et al.,43 who found that the level of Ca concentration on dentin surfaces was negatively correlated with the number of attached dental stem cells to these surfaces. Furthermore, it was found that reducing the Ca content on the dentin surface increases cellular proliferation.[44] Therefore, using silver nanoparticles is expected to increase cellular attachment and proliferation; however, more studies are needed to address this point.

    According to the results of the present study, it can be concluded that the nano-silver mouthwash demonstrated good antimicrobial and substantivity properties; thus, it can be considered more beneficial than ozonated olive oil as a root bio-modifier. However, further research is still needed to conduct more scientific studies to compare nano-silver mouthwash with the present conventional therapeutic modalities.


    Not applicable.

    Financial support and sponsorship


    Conflicts of interest

    There are no conflicts of interest.

    Authors’ contributions

    All authors contributed toward data collection, article writing and revising.

    Ethical policy and Institutional Review board statement

    Ethical approval for the study was taken at December 2020 from the Research Ethics Committee of the Faculty of Dentistry, Tanta University.

    Patient declaration of consent: (If In vivo Study / Case reports)

    Not applicable.

    Data availability statement

    Raw data can be sent on request from the corresponding author.

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

      [Table 1], [Table 2], [Table 3]


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