Number of Dental Stem Cells on Root Canal Dentin after Application of Triple Antibiotic Paste or Calcium Hydroxide: An In Vitro Study.

OBJECTIVE: The aim of this study was to evaluate the viability of stem cells from exfoliated and deciduous teeth (SHED) on dentin surface treated with triple antibiotic paste or calcium hydroxide. MATERIALS AND METHODS: Nine single-rooted extracted premolars were prepared appropriately and divided into three groups. In group A, the root canals were left empty, a triple antibiotic paste was placed in the root canals of group B, and calcium hydroxide was placed in the root canals of group C. After 1 week, the intracanal medicaments were removed, and stem cells were seeded on the treated surface of the specimens for 1 more week. The cells were stained and then observed under confocal microscope over the entire surface of each test material. Counting of the cells was made by Image J (3D) software, as well as manually. STATISTICAL ANALYSIS: To investigate any statistically significant differences between the experimental groups, statistical tests including Kruskal-Wallis and Mann-Whitney U-test were performed. Significance level was set to P < 0.05, and all analyses were performed with SPSS IBM program, v. 21. RESULTS: Groups B and C showed statistically significantly higher number of cells compared to Group A, whereas cells developed in a substrate of calcium hydroxide residues appeared in majority with distinct cores and widened unlike other groups. CONCLUSIONS: The effect of calcium hydroxide manifested better results regarding the number of stems cells on root canal surfaces.

A comparative evaluation of two working length determination methods.

The purpose of this study was to explore the level of agreement between the radiographic and the electronic apex locator (EAL) working length assessment methods, amongst teeth with different diagnoses. Working length measurements along with pulpal and periapical diagnoses data were collected from the Patient Registry Database of our Institution. The null hypothesis of this study was that pulp status does not affect the level of agreement between the two methods. The degree of agreement was assessed using the Bland-Altman method, followed by a Kruskal-Wallis test (a = 5%) that would allow the null hypothesis to be accepted. The EAL measurements agree in general with those provided by a 1.5 mm file placed coronal to the radiographic apex amid teeth with different pulpal diagnoses. Within the limitations of this study, we suggest that this setting (i.e. the distance) may be applied to all cases of endodontically treated teeth, despite the differences in pulpal diagnosis.

Assessment of cell viability in four novel endodontic sealers.

OBJECTIVE: The aim of this study was to evaluate the viability of human periodontal ligament (PDL) cells on MTA-Fillapex, GuttaFlow 2, TotalFill Sealer, and BioRoot™ RCS in comparison to conventional epoxy resin-based (AH Plus) and zinc oxide-eugenol-based (Roth's 801) sealers. MATERIALS AND METHODS: Sealers were divided into two groups, and five coverslips for each material per group were prepared. In the first group, PDLs were added immediately after the preparation of sealers (Fresh Group), and in the second, PDLs were added after 24 h. PDLs were cultured for 72 h and afterward, counted using standard hematocytometry. A Mann-Whitney U-test and Kruskal-Wallis test were used for the statistical analysis. The level of significance was set at 5%. Furthermore, cell morphology was assessed by confocal microscopy. RESULTS: The number of viable cells for the 24 h-set groups was higher than the freshly mixed in all sealers except Roth's 801. In both groups, GuttaFlow 2 presented the highest number of viable cells. In a descending order of cells' survival, TotalFill, BioRoot, and MTA-Fillapex are following and the conventional sealers, AH Plus and Roth's 801, seem not to exhibit the biological properties of the others. Cells grown on GuttaFlow 2, TotalFill, and BioRoot were observed to be well-formed. In contrast, MTA-Fillapex exhibited untypical morphology. No cells were detected on the surfaces of AH Plus, as well as Roth's 801. CONCLUSIONS: All novel sealers presented increased cell viability in comparison to conventional sealers. GuttaFlow 2 exhibited the highest cell viability.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers.

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

Different Patterns of Restoration Provision Between Initial Endodontic Treatment and Retreatment: A Retrospective Clinical Study.

PURPOSE: The aim of this study was to investigate the relationship between type of endodontic treatment and choice of definitive restoration and to show the prevalence of endodontic treatment options according to patient age and type of tooth. MATERIALS AND METHODS: Data were collected from the archive system of the School of Dentistry, National and Kapodistrian University of Athens in Athens, Greece. The sample included endodontically treated teeth being restored definitively at the time of data collection. RESULTS: Statistically significant difference was found regarding the type of restoration between initial endodontic treatments and retreatments (P < .001). CONCLUSION: Endodontic retreatment seemed to have a significant effect on the choice of definitive restoration of the tooth.

Interaction of dental pulp stem cells with Biodentine and MTA after exposure to different environments.

OBJECTIVE:: The aim of the present study was to evaluate and compare the cytotoxic effects of Biodentine and MTA on dental pulp stem cells (DPSCs) and to assess cell viability and adherence after material exposure to an acidic environment. MATERIAL AND METHODS:: DPSCs were cultured either alone or in contact with either: Biodentine; MTA set for 1 hour; or MTA set for 24 hours. After 4 and 7 days, cell viability was measured using the MTT assay. Biodentine and MTA were also prepared and packed into standardized bovine dentin disks and divided into three groups according to the storage media (n=6/group): freshly mixed materials without storage medium (Group A); materials stored in saline (Group B); materials stored in citric acid buffered at pH 5.4 (Group C). After 24 hours, DPSCs were introduced in the wells and cell adherence, viability, and cellular morphology were observed via confocal microscopy after three days of culture. Cell viability was analyzed using repeated-measures analysis of variance test with Tukey's post hoc tests (α=0.05). RESULTS:: Biodentine expressed significantly higher cell viability compared with all other groups after 4 days, with no differences after 7 days. Notably, cell viability was significantly greater in 24-hour set MTA compared with 1-hour set MTA and control groups after 7 days. Material exposure to an acidic environment showed an increase in cell adherence and viability in both groups. CONCLUSIONS:: Biodentine induced a significantly accelerated cell proliferation compared with MTA. Setting of these materials in the presence of citric acid enhanced DPSC viability and adherence.

Interaction of dental pulp stem cells with Biodentine and MTA after exposure to different environments

ABSTRACT Objective: The aim of the present study was to evaluate and compare the cytotoxic effects of Biodentine and MTA on dental pulp stem cells (DPSCs) and to assess cell viability and adherence after material exposure to an acidic environment. Material and Methods: DPSCs were cultured either alone or in contact with either: Biodentine; MTA set for 1 hour; or MTA set for 24 hours. After 4 and 7 days, cell viability was measured using the MTT assay. Biodentine and MTA were also prepared and packed into standardized bovine dentin disks and divided into three groups according to the storage media (n=6/group): freshly mixed materials without storage medium (Group A); materials stored in saline (Group B); materials stored in citric acid buffered at pH 5.4 (Group C). After 24 hours, DPSCs were introduced in the wells and cell adherence, viability, and cellular morphology were observed via confocal microscopy after three days of culture. Cell viability was analyzed using repeated-measures analysis of variance test with Tukey's post hoc tests (α=0.05). Results: Biodentine expressed significantly higher cell viability compared with all other groups after 4 days, with no differences after 7 days. Notably, cell viability was significantly greater in 24-hour set MTA compared with 1-hour set MTA and control groups after 7 days. Material exposure to an acidic environment showed an increase in cell adherence and viability in both groups. Conclusions: Biodentine induced a significantly accelerated cell proliferation compared with MTA. Setting of these materials in the presence of citric acid enhanced DPSC viability and adherence.

Primary Inadequate Management of Dental Trauma.

Tooth fractures are common complications due to trauma in the oral cavity. Tooth fragments and foreign bodies may be embedded in soft tissues as a result of dentofacial trauma and go unnoticed in emergency situations. The inadequate management of such cases may lead to complications, such as foreign-body reaction and scarring. This report describes two cases with dental fragments embedded in the lower lip, which went unnoticed until the patients presented later for completely different treatments and emphasizes the importance of clinical and radiographic examination of soft tissues, even in cases that present late for dental trauma management.

Comparative evaluation of sealing ability and microstructure of MTA and Biodentine after exposure to different environments.

OBJECTIVES: The aim of this study was to evaluate the sealing ability and morphological microstructure of Biodentine in comparison to ProRoot mineral trioxide aggregate (MTA) after storage in an acidic environment. MATERIALS AND METHODS: Biodentine and ProRoot MTA were prepared and packed into the canal lumen of dentin disks. Twenty specimens of each material were further randomly divided into two groups according to the storage media: group A: materials with saline as storage medium; group B: materials with citric acid buffered at pH 5.4 as storage medium. The sealing ability was evaluated at 1, 3, 6, and 24 h and 1 or 3 months, using a fluid transport model for quantitative analysis of endodontic microleakage. The morphological microstructures of the materials were also evaluated using scanning electron microscopy. RESULTS: During the first 24 h, MTA showed greater fluid transport values than Biodentine in both environments. At the 3-month measurement, when the materials were stored in saline, MTA showed greater ability to prevent fluid movement than Biodentine (p < 0.0001). However, when the materials were stored in an acidic environment, no statistical significant difference was found after 3 months. After storage in saline, both materials showed an uneven crystalline surface with similar hexagonal crystals. The microstructure of Biodentine changed after exposure to citric acid, showing a relatively smooth surface with more spheroidal crystals. CONCLUSIONS: The exposure to an acidic environment, within the limits of this study, seems to result in morphological changes of Biodentine in a different manner than MTA. MTA shows good ability to prevent fluid movement over time, in both environments. The ability of Biodentine to prevent fluid movement over time was enhanced in the acidic environment. CLINICAL RELEVANCE: The findings of the present study could imply that both materials are indicated for use in an acidic environment.

Evolving Trends in Endodontic Research: An Assessment of Published Articles in 2 Leading Endodontic Journals.

INTRODUCTION: The aim of this study was to evaluate and analyze the evolving trends in endodontic research in 2 leading endodontic journals (ie, Journal of Endodontics and International Endodontic Journal) in articles published from January 2009 to December 2013. The differences in content between this period and a 10-year earlier period from January 1999 to December 2003 were also evaluated. METHODS: Each journal's content was accessed through the web edition. For each article, the following parameters were recorded: number of authors, article type, number of affiliations, field of study, source of article, and geographic origin. The recorded data were analyzed using both descriptive and analytic statistics. RESULTS: During 2009-2013 (second period), the mean number of authors per article increased significantly compared with 1999-2003 (first period). The main volume of the literature in both periods and journals was original research articles. The number of published reviews increased significantly from the first to the second study period in contrast to case reports/clinical articles, which presented a significant decrease. "Endodontic materials" was the most prevalent thematic category in both study periods. The number of published articles related to "biology" and "chemical preparation and disinfection" increased significantly from the first to the second study period. On the contrary, the number of articles regarding "obturation and microleakage" presented a considerable decrease at the same time. The United States was the leading country in the number of publications in the first period followed by Brazil. In the second period, this rank was reversed with Brazil becoming the leading country followed by the United States. CONCLUSIONS: In the last 15 years, the progress of the specialty of endodontology was apparent as shown through the trends and shifts in research orientation in published articles in the 2 leading endodontic journals. The results of the present reviewing process encourage both journals to publish well-designed research projects with a high degree of difficulty, thus contributing to a more evidence-based approach of endodontology.

Comparison of Bacterial Community Composition of Primary and Persistent Endodontic Infections Using Pyrosequencing.

INTRODUCTION: Elucidating the microbial ecology of endodontic infections (EIs) is a necessary step in developing effective intracanal antimicrobials. The aim of the present study was to investigate the bacterial composition of symptomatic and asymptomatic primary and persistent infections in a Greek population using high-throughput sequencing methods. METHODS: 16S amplicon pyrosequencing of 48 root canal bacterial samples was conducted, and sequencing data were analyzed using an oral microbiome-specific and a generic (Greengenes) database. Bacterial abundance and diversity were examined by EI type (primary or persistent), and statistical analysis was performed by using non-parametric and parametric tests accounting for clustered data. RESULTS: Bacteroidetes was the most abundant phylum in both infection groups. Significant, albeit weak associations of bacterial diversity were found, as measured by UniFrac distances with infection type (analyses of similarity, R = 0.087, P = .005) and symptoms (analyses of similarity, R = 0.055, P = .047). Persistent infections were significantly enriched for Proteobacteria and Tenericutes compared with primary ones; at the genus level, significant differences were noted for 14 taxa, including increased enrichment of persistent infections for Lactobacillus, Streptococcus, and Sphingomonas. More but less abundant phyla were identified using the Greengenes database; among those, Cyanobacteria (0.018%) and Acidobacteria (0.007%) were significantly enriched among persistent infections. Persistent infections showed higher phylogenetic diversity (PD) (asymptomatic: PD = 9.2, standard error [SE] = 1.3; symptomatic: PD = 8.2, SE = 0.7) compared with primary infections (asymptomatic: PD = 5.9, SE = 0.8; symptomatic: PD = 7.4, SE = 1.0). CONCLUSIONS: The present study revealed a high bacterial diversity of EI and suggests that persistent infections may have more diverse bacterial communities than primary infections.

A quantitative and diametral analysis of human dentinal tubules at pulp chamber ceiling and floor under scanning electron microscopy.

The purposes of this study are (i) to evaluate and compare the dentinal tubule density, tubule diameter and percentage area of dentin occupied by tubules at the pulp chamber ceiling and floor; and (ii) to evaluate the effects of age on the number and dimensions of tubule openings. Twelve intact, human mandibular third molars were recruited. Six teeth belonged to patients up to 30 years of age and six teeth belonged to patients over 50 years. Scanning electron microscopic evaluations were made at two different locations: the pulp chamber ceiling and floor. The pulp chamber ceiling presented higher tubule density (P < 0.0001) and greater percentage area of dentin occupied by tubule openings (P < 0.0001) compared to the pulp chamber floor. Also, younger age group had greater tubule diameter (P < 0.0001), higher tubule density (P < 0.0001) and greater percentage area of dentin occupied by tubule openings (P < 0.0001) compared to older age group. Pulp chamber ceiling presents higher tubule density and greater area of exposed tubules. In younger people, the pulp chamber floor presents considerably high tubule diameter. The number and dimensions of dentinal tubule openings significantly decrease with age.

Regenerative endodontic therapy: a data analysis of clinical protocols.

INTRODUCTION: The aim of the present study was to systematically analyze the protocols that have been used in regenerative endodontic therapy and to detect any variations in clinical procedures. METHODS: An electronic search was executed in PubMed using appropriate Medical Subject Heading terms covering the period from January 1993 to May 2014. Additional publications from hand searching and the reference section of each relevant article enriched the article list. The relevance of each article was initially evaluated by scanning all titles and corresponding abstracts. The definite inclusion of each article in the study was determined by using specific criteria applied independently by 3 reviewers. RESULTS: Sixty relevant publications were finally included. The canal walls were not mechanically instrumented in 68% of the clinical articles. Sodium hypochlorite was included in 97% of the clinical studies either as the only irrigant or in combination with other irrigants. Antibiotic combination paste was used as the intracanal medicament in 80% of the clinical articles. Sodium hypochlorite, chlorhexidine, and EDTA were used in the final irrigation protocol in 75%, 4%, and 13% of the clinical studies, respectively. Neither the creation of a blood clot nor the use of platelet-rich plasma/platelet-rich fibrin was described in 13% of the clinical articles. Mineral trioxide aggregate was used as an intracanal coronal barrier in 85% of the relevant clinical studies. CONCLUSIONS: The variability of the clinical protocols applied during regenerative enododontic procedures is considerably high. A thorough analysis of regenerative protocols may constitute an additional source to provide useful clinical considerations for REPs.

Re-establishing apical patency after obturation with Gutta-percha and two novel calcium silicate-based sealers.

OBJECTIVE: Aim of the present study was to evaluate the retreatability and reestablishment of apical patency of two calcium silicate-based sealers, TotalFill BC Sealer (BCS) and mineral trioxide aggregate Fillapex (MTA F), versus AH Plus, when used in combination with Gutta-percha (GP). MATERIALS AND METHODS: The canals of 54 single-rooted anterior teeth were instrumented and filled with GP/AH Plus (Group A), GP/MTA F (Group B), or GP/BCS (Group C) using continuous wave obturation technique. The groups were subdivided into subgroups with the master-GP cone placed to the working length (WL) or intentionally 2 mm short. The retreatment procedures were performed using ultrasonics, chloroform, rotary, and hand files. The ability to establish the patency and reach WL was determined as well as the time taken to reach WL was calculated in minutes. Furthermore, the samples were observed under a dental, optical microscope, after vertically splitting them. RESULTS: The WL and patency were reestablished in 100% of specimens in all groups. The Mann-Whitney U-test indicated that there was a significant difference in the amount of time required to reach WL between the groups (P < 0.05) with group GP/BCS short of the WL showing the most amount of time to be retreated. CONCLUSION: The novel calcium silicate-based sealers are negotiable under simple root canal anatomy. However, the conventional retreatment techniques are not able to fully remove them.

Levels of evidence for the outcome of regenerative endodontic therapy.

INTRODUCTION: The aim of this systematic review was to assign levels of evidence (LOEs) to existing clinical articles related to the outcome of regenerative endodontic therapy and to evaluate the clinical and radiographic outcomes of this treatment modality. METHODS: Electronic search was executed in PubMed, Scopus, and Cochrane databases by using appropriate Medical Subject Headings terms covering the period from January 1993 to December 2013. Additional publications from hand-searching and reference section of each relevant article enriched the article list. The LOE of each article was assessed according to guidelines provided by the Oxford Centre of Evidence-Based Medicine. Quality assessment of the observational studies was executed by using the Newcastle-Ottawa scale. RESULTS: Fifty-one relevant publications were included in this review. There were 2 high-level cohort studies (LOE 2), 8 case series (LOE 4), and 41 case reports (LOE 5). The vast majority of the treated teeth in those publications showed resolution of clinical signs, symptoms, and periapical radiolucencies at follow-up period. Furthermore, the majority of treated teeth presented further increase in root length and root wall thickness and apical closure at the follow-up period. However, because of lack of sufficient high-level evidence it was not possible to answer totally the review question and determine definitely the outcome of regenerative endodontic therapy. CONCLUSIONS: The lack of adequate high-level studies that could possibly strengthen the satisfactory current data and allow practicing more evidence-based dentistry constitutes a significant knowledge gap in the endodontic literature. However, the current best available evidence undeniably allows clinicians to provide this treatment modality safely to patients.

Estimation of radiographic angles and distances in coronal part of mandibular molars: A study of panoramic radiographs using EMAGO software.

OBJECTIVE: This study was designed to investigate radiographically the effects of age and external irritating stimuli on the morphology and thickness of the pulp chamber ceiling and floor in mandibular molars. MATERIALS AND METHODS: A total of 234 panoramic radiographs were recruited and 494 mandibular molars were examined in this study. The sample was divided into six age groups: Group 1, <20 years; Group 2, 20-29 years; Group 3, 30-39 years; Group 4, 40-49 years; Group 5, 50-59 years and Group 6, >60 years. Each group was subdivided into two subgroups with regard to the tooth condition (intact or non-intact teeth). Four distances, two angles and two ratios were measured, in order to estimate the dimensions of the pulp chamber and the thickness of the pulp chamber ceiling and floor. RESULTS: The thickness of the pulp chamber ceiling and floor increased significantly from Group 1 to 6. The pulp chamber floor angle presented progressive sharpness from Group 1 to 6 whereas the pulp chamber ceiling angle presented progressive bluntness from Group 1 to 5 and sharpness from Group 5 to 6. Significant differences were identified in the thickness of the pulp chamber ceiling, the thickness of the pulp chamber floor and the ceiling-floor distance between intact and non-intact teeth. CONCLUSION: Based on those results, there are differences in the location of secondary dentin formation between the two pulp chamber walls. Age is related to diminished pulp chamber size. The increase rate of the pulp chamber ceiling thickness is similar to that of the pulp chamber floor thickness. Furthermore, external irritating stimuli have an effect on the pulp chamber dimensional changes.

Device and materials for in vitro evaluation of forces developed to teeth and periodontal structures during dental practices.

This study aimed at providing a gauge device (Ekontak et al Gauge K-Device) in order to analyze the forces applied to teeth and periodontal tissues during dental practices in vitro. This force gauge device can be used in the investigation of the possible defect generation to tooth structures when overloaded forces are applied during dental procedures in vitro. Ekontak et al Gauge K-Device consists of three units: the specimen's holder, a high-performance digital force gauge, and the support frame. The holder was fabricated by an Al alloy providing a steady detachable attachment between the specimens and the force gauge's pin connector. The clinical simulation was achieved with the use of a proper silicone material, selected to provide similar elastic behavior with the human periodontal ligament and to join the teeth inside a solid matrix of an acrylic resin. The digital force gauge is a high-speed collection and recording (1000 Hz) product coupled with data recording software. The forces developed to 15 specimens' root canals during lateral condensation and vertical compaction of cold gutta-percha obturation procedures were monitored, saved as graphs, CSV, and excel files and presented over time. The forces developed during vertical compaction (mean maximum force per obturation circle = 13.22 N) were more excessive than those during lateral condensation (mean maximum force per obturation circle = 10.14 N). In conclusion, Ekontak et al Gauge K-Device is provided as a modern gauge device, capable of performing clinical simulation in vitro, under the terms of its protocol.

Apical barrier formation after incomplete orthograde MTA apical plug placement in teeth with open apex--report of two cases.

Two cases are reported in which incomplete placement of 4 mm mineral trioxide aggregate (MTA) plug was performed unintentionally at the apical third of permanent immature teeth with open apex and apical periodontitis. As confirmed radiographically, there were gaps between MTA and dentinal walls along the MTA-dentin interface. After setting of MTA was confirmed, endodontic treatment was completed and access was sealed with composite resin. At 6 to 16 months follow-up examinations, formation of dentin in contact with the MTA surface, as well as apical closure and periapical healing were ideied radiographically for both cases. The results of these cases showed that apical barrier formation and complete periapical healing is possible despite the incomplete apical placement of the MTA plug. This might be due to the biological properties of the MTA. Even so, an incomplete three-dimensional placement of the filling material is not advocated.

Influence of dentin from the root canal walls and the pulp chamber floor on the pH of intracanal medicaments.

INTRODUCTION: The objective of this study was to evaluate the alterations in the pH of calcium hydroxide (Ca[OH]2), 2% chlorhexidine (CHX) gel, and their combination after adding dentin powder from the root canal walls and the pulp chamber floor. METHODS: Ca(OH)2 paste, 2% CHX gel, and their combination were examined. Dentin powder was obtained from the root canal walls and the pulp chamber floor of bovine teeth and added as 1.8% to the volume of the medications. The pH values of the mixtures were assessed immediately after preparation, after 24 hours, and on days 7 and 14. Data were analyzed statistically by analysis of variance and the Student's t test (P = .05). RESULTS: pH values of Ca(OH)2 were always above 12 in the presence or absence of dentin powder during the 14 days of the study. A significant decrease in pH values of Ca(OH)2 was observed after 14 days when dentin from the root canal wall was added (P < .001). The pH values of CHX gel were significantly increased by the presence of dentin (P = .001). The t test showed a significant difference in the increase of the pH values when the different dentin types were added (P = .002). The pH of the Ca(OH)2 and CHX gel combination was not influenced by dentin powder from the root canal walls, and a reduction was observed after 14 days, exactly like in the control group. The pH values of the combination did not alter after 14 days when pulp chamber floor dentin was added. CONCLUSIONS: The different dentin types variably affect the pH of Ca(OH)2 paste, 2% CHX gel, and their combination in the studied periods.