The in vivo study was conducted in the Department of Conservative Dentistry and Endodontics in Kalinga Institute of Dental Sciences, Bhubaneswar, Odisha, India, during August 2016 and December 2016. Patients reporting to the department suitable for ART, irrespective of sex, age, race and socio-economic status were included in this study.

Inclusion criteria consisted of occlusal or occluso-proximal lesion suitable for ART such as deep dentinal lesions without involving pulp as diagnosed on radiograph, without pain, abscess, swelling or adjacent soft tissue lesions.

Forty patients were divided into four groups consisting of ten patients each. The groups were as described in the [Table/Fig-1].

Commercially available 2% chlorhexidine gel (Consepsis, Ultradent) in syringe form was used in this study.

For preparing 1% TTO, 1 ml of commercially available pure TTO (Mother Herbs Private Limited, New Delhi) was mixed with 0.5% polyoxyethylene sorbitan monolaurate-Tween 80, an emulsifying agent (with no antimicrobial activity) and 1 ml of distilled water. After mixing it well, distilled water was again added to make it a solution of 100 ml. As TTO is sparingly soluble in water, an emulsifying agent was added to enhance its solubility [1]. This preparation was stored in sterile syringes for easy application.

The leaves of Aloe vera plant were washed with distilled water, cut opened and fresh pulp was collected in a sterile container. Slurry was formed with the help of mortar and pestle and stored in sterile syringes for easy application [14].

Distilled water was put in syringes for ease in application and for maintaining the uniformity in test agents application.

A baseline sample of the carious lesion was obtained using a sterile spoon excavator from the center of the lesion, after isolation with a rubber dam prior to excavation of caries in each sample.

After complete excavation of caries, a second dentin sample for microbial evaluation was collected from the hard dentin using another sterile spoon excavator. The teeth in each group were then disinfected with the test agent according to the group. Approximately 1 ml of the disinfectant test agent was syringed out into the cavity for one minute and then the cavity was washed with distilled water and air dried. Another dentin sample after disinfection was then collected for microbial analysis using a spoon excavator from the same place. After collection of the third sample, the tooth was restored with glass ionomer cement (GC Fuji II®).

Thus, dentinal samples were collected three times from each carious tooth, viz., baseline- before excavation of caries, after hand excavation of caries and after disinfection of the cavity. These samples were subjected to microbiological evaluation for TVC [15,16].

The samples collected were immediately transferred to brain heart infusion broth and was incubated overnight for microbial growth in it. The samples were homogenized in a tube shaker for three minutes, and 25 μl aliquots of this solution were placed onto the plate surfaces containing blood agar, with a micropipette. Each culture plate was inoculated with samples collected in three phases from a single tooth. Subsequently, the cultures were incubated at 37ºC for 24 hrs, after which a visual assessment of the total number of viable bacterial colonies were counted and subjected to statistical analysis [15,17].

The data obtained were analysed using analysis of variance (ANOVA) to test the significance. Bonferroni post-hoc test was used for pair-wise comparison between the means when ANOVA test was significant. The level of significance was set at p≤0.05. Statistical analysis was carried out using the SPSS 14.0 software (Statistical Package for Scientific Studies Inc. Chicago, IL, USA.).

The first test group (2% CHX) showed a mean TVC pre-excavation, post-excavation and post-disinfection of 131.5±9.107, 69.2±5.959, 3.1±1.197 respectively. Similarly second test group (1% TTO) showed values of 135.7±6.8, 69.4±6.72, 13.1±1.729 and third test group (Aloe vera) values of 130.8±9.151, 67.5±6.399, 16.9±0.994 respectively as shown in [Table/Fig-2].

The results of present study showed that there was a statistically significant reduction in TVC when compared between pre and postexcavation in all the groups (p<0.05) and postexcavation and post-disinfection in all the test groups (p<0.05) (except control group) using Analysis of Variance (ANOVA)

Pair-wise comparison of viable bacterial colony using Bonferroni post-hoc test was done for each study group at different phases viz., pre-excavation, post-excavation and post-disinfection which showed statistically significant difference in the number of bacterial colonies between each phase in all the three study groups (p<0.05) [Table/Fig-3].

An important and clinically relevant finding of the present study is that there was a statistically significant reduction in the bacterial counts after cavity disinfection in all the three groups as compared to the control group (distilled water). (p<0.05) as shown in [Table/Fig-4].

A pair-wise comparison of mean bacterial colony counts in post disinfection procedure showed maximum reduction of TVC with 2% CHX followed by 1% TTO and Aloe vera in which the difference in results were statistically significant.

Both TTO and Aloe vera showed reduced bacterial count post disinfection, but 1% TTO was found to be better than Aloe vera although the difference was not statistically significant.(p≥0.05) [Table/Fig-5].

Traditional restorative dentistry propagated early operative intervention to remove diseased tissue and bacteria. Modern dentistry, however, emphasises on arresting the caries progression and restoring the tooth with minimum tissue destruction. Minimal invasive dentistry is based on this axiom.

A drawback in restorative dentistry is the occurrence of secondary caries [18,19] that has compelled us to practice a more extensive form in the past. Minimally Invasive Dentistry (MID) also displays the same drawback. Our study, in accordance with other studies has also shown incomplete bacterial elimination with excavation only [20]. Several antimicrobial agents have been tried and tested to eliminate bacteria underneath the restorations, and are now being used as cavity disinfectants [21]. With the advent of cavity disinfectants, there is an upsurge in the use of MID in the recent years.

Varied concentrations of CHX; 0.2% [22], 0.12% [23-25], 0.06% [23] have been tested and shown to act effectively against S. mutans. A major disadvantage of this agent is that it adversely affects the micro-tensile [26] and shears bond strength [27] of composite resins as well as increases the microleakage [26,28]. Thus, there is the need for an alternate agent which could overcome these difficulties as well as serve the purpose of a cavity disinfectant.

Two antimicrobial herbal agents, TTO and Aloe vera, were used in our study to compare and illustrate their efficacy as cavity disinfectants.

Tea tree oil is the volatile essential oil obtained primarily from the Australian native plant, Melaleuca alternifolia. It consists of terpene hydrocarbons, mainly monoterpenes, sesquiterpenes and their associated alcohols which are volatile, aromatic hydrocarbons [29-33].

The terpinen-4-ol, α-terpineol and 1, 8-cineole, are the antimicrobial components in TTO which cause leakage of 260 nm-light absorbing material and render cells susceptible to sodium chloride [34]. Thus, tea tree oil shows antimicrobial effect through cell lysis and the loss of membrane integrity which leads to leakage of ions and the inhibition of respiration [29].

Aloe barbadensis Mill (A. vera) is a short succulent herb filled with a clear viscous gel which has potent antibacterial, antifungal, and antiviral properties [35,36]. The therapeutic use of Aloe vera has been known to us since inception.

The antimicrobial activity of Aloe vera is attributed to its constituent compounds, the most important being the anthraquinones [37]. Aloe vera gel has a number of components such as aloin, aloe emodin, aloetic acid, anthracene, aloe mannan, aloeride, antranol, chrysophanic acid, resistanol, and saponin [38]. Aloin and aloe emodin possess strong antibacterial and antiviral activities. They inhibit protein synthesis from bacterial cells, thus explaining their antimicrobial activity [39]. It is noteworthy that some compounds like anthraquinones and saponin present in Aloe vera gel have direct antibacterial activities while some other components, such as acemannan, have been considered to exert indirect bactericidal activity through stimulation of phagocytosis [40].

Many studies on TTO have advocated its antibacterial property. Studies conducted by Takarada K et al., Groppo FC et al., showed antibacterial activity of TTO on S. mutans [41,42]. Filoche SK et al., have shown that combination of TTO and CHX mouthwash as a better antibacterial agent for oral pathogens [43]. Our study also, in accordance with other studies has shown TTO to be an effective antibacterial when used as a cavity disinfectant. Another in vitro study showed significant reduction in S. mutans count when tooth brushes were soaked for 12 hrs in 0.2% TTO and also showed more reduction in salivary S. mutans counts when compared with 0.12% CHX mouthwash [1,42]. However, in the present study 2% CHX was found to be more effective than 1% TTO. This could be due to the difference in the concentration of CHX and TTO used and the time duration it was applied for. Most bacteria are susceptible to TTO at concentrations of 1.0% or less, however, for organisms like Enterococcus faecalis, and Pseudomonas aeruginosa the MICs was found to be 2% [29,44,45]. Although TTO has shown its antibacterial efficacy against various oral microfloras, there is no literature available on its use as a cavity disinfectant.

It was also shown in various studies that mouth rinses and dentifrices containing Aloe vera have shown a prodigious reduction in gingivitis and plaque accumulation after its use. Gupta RK et al., in his study observed Aloe vera mouth rinse to be equally effective as 0.2% CHX [46], but in our study 2% CHX was found to have better antibacterial efficacy than pure Aloe vera gel. This could again be attributed to the difference in the concentration and type of Aloe vera preparation used. Mohammadmehdi Fani M et al., have reported mean MIC values for Aloe vera gel against clinical isolates of S. mutans to be 12.5 μg/ml [47]. A study by Prabhakar R et al., has shown Aloe vera to be a more effective cavity disinfectant when compared to propolis [15].

In our study, although TTO showed more reduction of TVC than Aloe vera, the difference was statistically insignificant.

Limitations of this study were that neither specific bacteria were isolated from the pre excavated dentin samples nor the test agents were tested against any specific strain which could provide a better understanding of the pathogens and the effect of the test agents on them in isolation.

The present study concluded that only excavation cannot eliminate all the cariogenic bacteria. Natural antibacterial agents like tea tree oil and Aloe vera could be effectively used as cavity disinfectants which will help in minimizing secondary caries and rendering a long term restorative success. However, further scientifically sound clinical research and studies should be carried out to broaden our understanding of various antimicrobial agents, particularly natural agents, in the prevention of dental caries.