Tea tree oil in inhibiting oral cariogenic bacterial growth an in vivo study for managing dental caries

Abstract

Dental caries is considered a major health burden, and preventive strategies are needed to improve oral health. It is suggested that natural essential oils possess anti-plaque formation properties and exhibit strong antimicrobial activity; however, in vivo studies to support these concepts are scarce. We evaluated the effects of tea tree oil (TTO) on caries initiation and progression in vivo to generate supportive data for clinical studies in patients at high risk of caries. We first assessed TTO in vitro against Streptococcus mutans and Streptococcus sobrinus, two of the most common oral bacteria associated with dental caries development, using bacterial growth assays, biofilm formation, and adhesion assays. TTO efficacy on caries initiation and caries lesion progression was then evaluated in vivo, where complex biofilms are formed on dental enamel. Our results showed that TTO demonstrates strong antimicrobial efficacy by inhibiting bacterial growth and biofilm formation while preventing bacterial adhesion on human teeth. In vivo, TTO application reduced the number and depth of carious lesions. Specifically, the number of caries lesions was lower in the TTO-treated group compared to the control group (13 vs. 19 lesions), and the lesion area was significantly smaller in the TTO-treated group compared to the untreated group (p = 0.003). TTO did not affect the extent of reparative dentin formation. The clinical relevance is primarily for individuals who have difficulties brushing their teeth or those at high risk of developing dental caries, serving as an adjunct to preventive dentistry.

Introduction

Dental caries counts among the most widespread chronic diseases, characterized by localized destruction of dental hard tissues¹. Dental plaque is a microbial biofilm that forms on the tooth surface and contains a complex structure of microorganisms. The metabolic activity of the bacteria in this biofilm is directly linked with the dental caries process^2,3. Various microbial species have been isolated from caries lesions, such as the Streptococcus salivarius, Lactobacilli, and Veillonella species. There is a strong association between caries initiation and progression with acidogenic and aciduric Gram-positive bacterial species, most commonly Streptococcus mutans and Streptococcus sobrinus^4,5. The growth of acidogenic/aciduric bacteria disrupts the balance of plaque biofilms and leads to initiating caries lesions⁶. Interactions between acid-producing bacteria and host factors play a crucial role in caries formation. Sucrose-dependent and sucrose-independent attachment methods are used by S. mutans⁷.

Tea tree oil (TTO) is an essential oil extracted from steaming the leaves of Melaleuca alternifolia. It is generally accepted that essential oils possess bioactive properties, including antimicrobial properties⁸. Therapeutic properties of the TTO, including antimicrobial activity, were reported in the early 20th century⁹. Several studies indicated that TTO is very effective in vitro against a broad range of bacteria, including oral bacteria^10,11. Moreover, in 2002, Groppo et al., showed that the total number of S. mutans and total oral bacteria were reduced when 0.2% TTO mouthwash was used¹².

Over the past few decades, dentistry has been moving toward preventive and noninvasive treatments for dental caries¹³. Caries prevention strategies include controlling the dental biofilm with mechanical and chemical methods^14,15. For instance, chlorhexidine (CHX), a widely used antimicrobial agent, is often preferred as an antiplaque solution¹⁶. With the widespread use of these approaches in caries prevention, it is essential to note that these methods have some limitations and side effects. Excessive or abusive use of mechanical approaches in caries prevention, such as dental brushing and flossing, have a potentially harmful impact on both dental and gingival tissues¹⁷. Moreover, the use of mechanical dental caries prevention approaches is also difficult for older people and those suffering from systemic diseases associated with limited mobility, such as arthritis¹⁸. Furthermore, using chemoprophylactic agents for caries prevention has its limitations and side effects¹⁹. By examining the impact of TTO on key bacteria implicated in dental caries, such as S. mutans and S. sobrinus, we may be able to overcome these limitations and add new intervention methods to the field of preventive dentistry. Based on the antimicrobial activity of TTO, its essential oil compounds have the potential to inhibit the ability of microorganisms to form biofilms and, consequently, the development of caries. The present study aims to evaluate the efficacy of TTO in limiting bacteria growth and the formation of microbial biofilm. Results of this study could set the basis for novel interventional approaches in the field of preventive dentistry and lay the foundation for the development of a new caries prevention therapy.

Methods

Bacterial strains and evaluation of anti-cariogenic potential

Essential oil from M. alternifolia (tea tree oil; Sigma-Aldrich, W390208-K) and chlorhexidine (CHX) (Sigma-Aldrich, C9394) were compared and evaluated in the present study.

The anti-cariogenic therapeutic potential was assessed against two bacterial strains, S. mutans (ATCC700610) and S. sobrinus (ATCC27351). The bacteria were cultured on Brain Heart Infusion (BHI) Sgar (BD™ Difco™) at 37℃ under microaerophilic conditions (5% CO₂) for 24–48 h. For the bacterial growth and biofilm formation assays, each culture was prepared to a suspension containing bacteria at a concentration of 1 × 10⁶ CFU/mL in BHI broth with the addition of 5% sucrose. The total bacterial count was assessed after plating bacteria on BHI agar and culturing them overnight at 37℃ with 5% CO₂.

Bacterial growth assay

The antimicrobial properties of the treatments were evaluated by assessing their ability to inhibit planktonic growth. Furthermore, the Minimum Bactericidal Concentration (MBC) was determined as previously described by Mah (2014), with minor modifications²⁰. Liquid cultures were prepared in 96-well plates. Each well was inoculated with 100 µL of bacterial suspension at 1 × 10⁶ CFU/mL in BHI broth with 5% sucrose and 100 µL of the respective treatment in serial dilutions (TTO and CHX concentrations ranged from 1.56 to 50%), with the serial dilutions performed in duplicate. Bacterial cultures in BHI broth supplemented with 5% sucrose, without the presence of any treatment, were prepared to serve as a positive control for bacterial growth. All cultures were then incubated for 24 h at 37℃ with 5% CO₂. At the end of the incubation period, 5 µL aliquots of the liquid cultures were sub-cultured from the 96-well plates on BHI agar plates and incubated for 24 h at 37℃ in a 5% CO₂ aerobic atmosphere. After that, colony counting was performed to quantify bacterial growth. MBC was defined as the lowest dilution able to completely inhibit bacterial growth.

Biofilm formation assay

The efficiency of the evaluated treatments to inhibit biofilm formation developed by each bacterial strain was assessed with a biofilm formation assay²¹. Briefly, bacterial cultures containing 100 µL of the respective strain at a final concentration of 1 × 10⁶ CFU/mL in BHI broth with 5% sucrose were combined with 100 µL of the respective treatment in serial dilutions in 96-well plates (TTO and CHX concentrations ranged from 0.1 to 50%). Additional cultures containing solely bacteria in BHI broth with 5% sucrose, without being exposed to any treatment, served as a positive control for the biofilm formation. The 96-well plates were incubated for 48 h at 37℃ in the presence of 5% CO₂. Once the incubation period was completed, the supernatant was removed, and each well was rinsed three times with phosphate-buffered saline (PBS) (Hyclone, SH3025802). After that, 96% (v/v) ethanol solution was added to fix the biofilm that had been bound at the bottom of each well. The plates were air-dried and were then stained with 0.1% crystal violet for 20 min at room temperature. Crystal violet was aspirated, and the plates were washed and dried, followed by the addition of 30% (v/v) glacial acetic acid solution to each well. The plates were then incubated for 20 min at 37℃. After that, biofilm formation was assessed spectrophotometrically by measuring the optical density (OD_600nm) in a microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo Scientific).

Adhesion assay on human teeth

To investigate TTO efficacy in preventing oral bacterial adhesion on teeth, we conducted adhesion assays using previously discarded, anonymous, non-identifiable human teeth (waste material, College of Dentistry, University of Saskatchewan). All teeth were permanent molars and were macroscopically intact, without any visible signs of carious lesions, wear, or hard tissue defects. Upon collection, the teeth were immediately stored in 10% neutral buffered formalin for disinfection. Over the course of one month, the teeth underwent repeated washes with sterile 1× PBS to remove residual fixative and debris. Following the washing phase, the teeth were sterilized by autoclaving at 121 °C for 15 min at 15 psi to ensure complete microbial decontamination²². The teeth were randomly divided into three groups: a treatment group, and a positive and a negative control, respectively. A tooth and the respective treatment were placed into each well of a 12-well plate. The lowest concentration able to completely inhibit planktonic growth as well as biofilm production was used. A bacterial suspension containing overnight cultures of S. mutans and S. sobrinus (each strain at a final concentration of 1 × 10⁶ CFU/mL in BHI broth with the addition of 5% sucrose) was also added into the corresponding wells. The plates were incubated for 4 days at 37℃ and 5% CO₂. After the first 48 h of incubation, the medium was carefully aspirated and replaced with a fresh one consisting of fresh bacteria in BHI broth with 5% sucrose and the respective treatment. The plates were then incubated for additional 48 h. Once the incubation period was over, the teeth were transferred to a fresh BHI medium and vortexed in it. Subsequently, 100 µL of this medium was plated on BHI agar plates and incubated for 24 h at 37℃. After that, bacterial colonies counting was performed to determine bacterial growth.

Cell viability

To assess the cytotoxicity of TTO, cell viability was evaluated using the CCK-8 assay, as previously described by Vu et al., with minor modifications²³. Briefly, Human Gingival Fibroblast Cells (HGFs) were cultured in a 96-well plate and incubated for 24 h at 37 °C with 5% CO2. After that, the HGFs were treated with TTO (12.5%). The CCK-8 Cell Viability Assay Kit (CK04-01, Dojindo) was used to evaluate the cell viability following the manufacturer’s instructions. After incubation with CCK-8 solution for 2 h at 37 °C, we measured the absorbance at 450 nm using a microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo Scientific).

In vivo treatment preparation

A solution of 25% TTO was prepared in distilled water. Upon application to rat’s teeth, the concentration of the prepared solutions was decreased to half because of the presence of a bacterium suspension. The final concentration for TTO was 12.5%.

In vivo rat model for dental caries

To assess the effect of TTO treatment on tertiary dentine formation, we used the rat model. The animal experiment protocol (#20210032) was reviewed and approved by the University Animal Care Committee (UACC) at the University of Saskatchewan. All procedures were conducted in strict accordance with the guidelines and regulations approved by the UACC. Furthermore, all experimental procedures involving animals complied with the ARRIVE 2.0 guidelines²⁴. Sixteen Sprague-Dawley rats (8 males and 8 females) were used in this study. Rats were randomly divided into 2 groups. Each group had 8 rats (4 males and 4 females). The sample size was based on previously published rat caries models^25,26. All rats were fed a high cariogenic diet 2000 and 5% sucrose water; food and drinking were provided ad libitum as previously published²⁵. A bacteria suspension containing S. mutans and S. sobrinus was directly applied to tooth surfaces in all groups. Group 1 was the control group; rats in this group were only given bacteria suspension without any treatment. Group 2 was treated with TTO. The treatment was applied twice a week using a micro-brush on the teeth surface immediately after applying the bacteria mixture. Upon completion of 12 weeks, humane euthanasia was performed under anesthesia with isoflurane, and then the maxillae and mandibles were collected and fixed with 70% ethanol and analyzed by micro-computed tomography (micro-CT).

Micro-CT

The collected samples were scanned with micro-CT (SkyScan 1172; Bruker, MA) at 60 kVp and 165 µA, 0.5 mm aluminium filter, 100 ms exposure, 4-frame averaging, 0.4 degree rotation step (through 180 degrees) and 12 μm pixel size. Images were reconstructed (12 μm isotropic voxels) with NRecon version 1.6.4.6 (Bruker) and visualized with ImageJ²⁷. Each of the two dentists independently evaluated the micro-CT scans. The initial detection and scoring of caries lesions were performed visually by carefully examining the micro-CT images. Following visual identification, the area of each caries lesion was quantitatively measured using ImageJ software on the micro-CT slice showing the deepest point of the lesion. A total of 96 molars were evaluated per each group (N = 8). During data analysis, micro-CT images were coded with unique, anonymized identifiers, preventing the analysts from knowing the treatment groups. This blinding procedure helped reduce subjective bias in measuring caries lesions and other outcomes.

Hematoxylin and Eosin (H&E) staining

H&E staining was performed using 5 μm thick paraffin-embedded molar sections to assess the impact of TTO treatment on tertiary dentine formation. Briefly, after deparaffination and rehydrating, the tissue sections were immersed in hematoxylin for 2 min, washed with tap water, quickly dipped in acid alcohol (0.5% hydrochloric acid in 95% ethanol), rinsed in tap water, immersed in saturated aqueous lithium carbonate for 5 s, washed with tap water and immersed in eosin for 1 min. Finally, sections were dehydrated, mounted with Permount, and photographed using an Aperio Virtual Microscopy System (Leica Biosystems Inc.).

Statistical analysis

The treated and untreated groups were compared using the Mann-Whitney U test and the student’s unpaired t-test after testing for normally distributed and equal variance in the data. P-values were considered significant when < 0.05. Values are presented as the mean ± standard error. Analysis was performed with SPSS^® v28 software (IBM, Armonk, NY, USA).

Results

Effect on bacterial growth and MBC determination

TTO shows antimicrobial efficacy against both bacterial species. As illustrated in Fig. 1 (A and B), TTO is able to completely inhibit the planktonic growth of S. mutans and S. sobrinus at a concentration of 12.5%. The control treatment with CHX at the same concentration of 12.5% also exhibits complete inhibition of the growth of S. mutans and S. sobrinus (Fig. 1C and D).

Fig. 1.

Effects of Tea Tree Oil (TTO) and Chlorhexidine (CHX) on Bacterial Growth. The mean (± SE) of the number of colonies of S. mutans and S. sobrinus in different concentrations of treatments (0–50%), n = 2/group. TTO completely inhibits the growth of S. mutans and S. sobrinus at a concentration of 12.5% (A and B, respectively). At a concentration of 12.5%, CHX effectively suppresses the growth of S. mutans and S. sobrinus (C and D, respectively).

Effect on biofilm formation

Both treatment (TTO) and control (CHX) effectively inhibit biofilm formation. More specifically, TTO completely inhibits the ability of both S. mutans and S. sobrinus to form biofilms at the concentration of 12.5% (Fig. 2A and B). At the same concentration of 12.5%, CHX exhibits similar inhibitory effects on biofilm formation by S. mutans and S. sobrinus (Fig. 2C and D).

Fig. 2.

Effects of Tea Tree Oil (TTO) and Chlorhexidine (CHX) on in vitro Biofilm Production by S. mutans and S. sobrinus. The mean (± SE) of the number of OD values in different percentages of TTO concentration (0–50%) and CHX concentration (0–50%), n = 2/group. At a concentration of 12.5%, TTO completely inhibits biofilm formation by S. mutans and S. sobrinus (A and B). CHX exhibits similar efficacy, completely inhibiting biofilm formation by S. mutans and S. sobrinus at a concentration of 12.5% (C and D).

Effect on bacterial adhesion to tooth surfaces

No bacterial colonies were grown in the treatment group, indicating that TTO treatment is able to completely inhibit the adhesion of S. mutans and S. sobrinus on the surfaces of human teeth (Fig. 3A). Bacterial colonies were observed only in the positive control group that was not exposed to treatment.

Fig. 3.

Effects of Tea Tree Oil (TTO) on Bacterial Adhesion and its Cytotoxicity. (A) Mean (± SE) number of bacterial colonies after treatment with TTO (n = 3), TTO completely inhibits the adhesion of both S. mutans and S. sobrinus on human teeth surfaces. (B) Mean (± SE) viability of Human Gingival Fibroblasts after a 2-hour treatment with TTO (n = 3), no significant cytotoxic effects of TTO treatment are observed. * indicates a statistically significant difference (p-value < 0.05) as assessed by Student’s unpaired t-test.

Cytotoxicity effects

Our findings indicate that there were no substantial differences in cell viability among the HGFs treated with TTO at 12.5% concentration, as illustrated in Fig. 3B. This consistent cell viability across treatments suggests the absence of significant cytotoxic effects on our treatment.

Effect of TTO on dental caries formation in rat caries model

Caries lesions are found in all groups, and carious lesions are detected on both occlusal and interproximal surfaces. Caries lesions are found in 7 out of 8 rats within the control group (group 1, which received bacteria suspension without treatment), and in 7 out of 8 rats in group 2 (which received bacteria suspension and TTO treatment). Upon analysis of micro-CT images, the influence of treatments on the incidence and progression of caries lesions was evaluated. We found a reduction in the number of caries lesions within the group treated with TTO compared to the control group (13 vs. 19 caries lesions, respectively). The percentage of carious molars in group treated with TTO was less than in the control group but was not statistically significant (p-value = 0.43) (Fig. 4A). Additionally, we found that caries lesions were more severe in group 1 compared to the treated group. More specifically, caries lesions in the control group extensively extend into the dentine layer showing a deep destruction of the enamel layer. In contrast, in the group treated with TTO, caries lesions detected are mostly superficial being limited to enamel and dentino–enamel junctions (Fig. 4B). Further quantitative analysis of lesion size using the Mann-Whitney U test reveals a statistically significant reduction in caries lesion area in the TTO-treated group compared to the untreated group (p = 0.003) (Fig. 4C). This suggests that TTO treatment is effective in limiting the extent and progression of caries lesions.

Fig. 4.

Effects of Tea Tree Oil (TTO) on the Number and Depth of Dental Caries and Tertiary Dentine Formation: An In Vivo Study. (A) The percentage of molar with caries number in different groups (± SE). Each group contains eight rats (4 males and 4 females). Comparison between the TTO-treated and control groups shows a decrease in caries occurrence in the TTO group, although not statistically significant (p-value = 0.43). (B) Micro-CT analysis of molars with caries reveals that the untreated group exhibits more extensive and invasive caries lesions compared to the treated group. (C) Mean of caries lesion area (mm² ± SE) in the TTO-treated and untreated groups. A significant reduction in lesion area was observed in the TTO group compared to the control (p-value = 0.003, Mann-Whitney U test). (D) H&E staining shows increase in tertiary dentin (TD) deposition in carious molars treated with TTO compared to untreated. Scale bar: 100 μm. ** indicates a statistically significant difference (p-value < 0.01) as assessed by Mann–Whitney U test.

Tertiary dentine deposition

Microscopic evaluation of H&E-stained sections reveals a marginally higher rate of tertiary dentin deposition within carious molars in the group subjected to TTO treatment compared to untreated counterparts (Fig. 4D). Quantitative analysis using ImageJ showed that TTO-treated molars exhibited approximately 1.5 times more tertiary dentin deposition compared to controls. However, it is important to emphasize that this difference was not statistically significant, indicating that TTO treatment did not exert any obvious effect on tertiary dentine formation, as shown in the representative image in Fig. 4D.

Discussion

In this study, we assessed the antibacterial properties of TTO and demonstrated that it inhibited the planktonic growth of S. mutans and S. sobrinus, and, in the process, TTO inhibited the bacteria’s ability to form biofilm. Moreover, we found that TTO inhibited the adhesion of S. mutans and S. sobrinus to the surfaces of the extracted human teeth. Most importantly, our in vivo findings revealed a reduction in the presence of carious teeth and an inhibition of deep caries lesions. However, TTO treatment did not induce any significant effects on reactive dentinogenesis as response to dental caries progression suggesting that its mechanisms of action are mostly through bacteria growth inhibition.

A range of preventive strategies, including mechanical methods and chemoprophylactic agents, are available to prevent dental caries^14,15. These preventive strategies are critically important in patients with combined risk factors resulting in higher caries prevalence, such as in patients with brushing difficulties, low saliva levels (xerostomia), and higher bacteria plaque adhesion. However, traditional preventive methods and agents come with drawbacks such as tooth staining, toxicity, and xerostomia^19,28. Consequently, there has been an increasing interest in investigating the potential of natural products to inhibit oral bacteria growth and prevent dental caries. In the current study, we demonstrated that TTO exhibited inhibitory effects on the planktonic growth of S. mutans and S. sobrinus, as well as on their ability to form biofilm. The results of this study corroborate the growing body of evidence highlighting TTO as a potent antimicrobial agent against cariogenic bacteria. The observed inhibition of oral bacterial growth and adhesion in vitro aligns with previous studies demonstrating TTO’s efficacy against oral bacteria, key pathogens implicated in biofilm formation and enamel demineralization^11,12. Such antimicrobial activity is critical for caries prevention, as biofilms are the primary drivers of acid production and enamel degradation³. Moreover, the reduction in caries lesions observed in vivo in the current study further underscores TTO’s clinical relevance. This aligns with a study showing that oral mouthwashes containing TTO may limit the accumulation of dental plaque²⁹ supporting the need for additional clinical studies and larger population dental caries prevention strategies.

Our in vivo results showed that TTO contributed to a decrease in the frequency and severity of dental caries, suggesting its potential efficacy in caries prevention, although the reduction in the percentage of carious molars was not statistically significant. It is important to note that the lack of statistical significance in the percentage of carious molars may be attributed to the relatively small sample size in our study. A larger cohort would increase statistical power and may better clarify the effects of TTO treatment on caries incidence. Additionally, quantitative analysis indicated a significant reduction in caries lesion size in the TTO-treated group, further supporting its effectiveness in limiting caries progression. These findings align well with prior studies utilizing various natural products, such as green tea and citrus lemon oil, which have demonstrated efficacy in inhibiting oral bacteria growth and in preventing dental caries^30,31. Notably, a recent double-blinded clinical trial comparing TTO and CHX mouthwashes in gingivitis treatment reported comparable reductions in gingival inflammation and plaque scores between the two groups, with TTO demonstrating a favorable safety profile and fewer adverse effects, such as taste alteration or mucosal irritation, compared to CHX³². This parallels our findings, further supporting TTO’s role as a viable natural alternative or complement to conventional antimicrobial agents. Our findings, thus, present a promising alternative to conventional approaches, exhibiting the efficacy of TTO towards caries prevention while potentially mitigating risks associated with long-term use of synthetic agents like CHX.

The current study revealed that the TTO inhibited the ability of S. mutans and S. sobrinus to form a biofilm. It is universally accepted that dental biofilm significantly contributes to the initiation of dental caries. The initial stage in the formation of dental biofilms involves bacterial attachment and colonization on tooth surfaces^33,34. A dental biofilm consists of diverse microorganisms enclosed within an extracellular matrix of host and microbial polymers adhering to the tooth surface³⁵. The metabolic activity of bacteria within dental biofilms directly influences the initiation and progression of caries processes in dental tissues³. Multiple studies have indicated that inhibiting dental biofilm formation could potentially prevent dental caries^36,37. The demonstrated efficacy of TTO in inhibiting biofilm formation by S. mutans and S. sobrinus, as observed in the current study, may play a significant role in preventing the initiation and progression of dental caries. Utilizing TTO alone or combined with other caries-preventing agents could offer a promising approach to caries prevention. Future in vivo studies assessing the preventive effects of TTO in clinical settings could provide additional insights into its efficacy for caries prevention. Moreover, our study was focused on the effect of TTO on S. mutans and S. sobrinus. Since many types of bacteria are involved in the initiation and progression of dental caries, further studies are needed to conclude on the efficacy of TTO against additional types of microorganisms involved in the dental biofilm and caries process. Furthermore, TTO effectiveness could potentially be further enhanced through combination with other anti-caries agents, a strategy that warrants further investigation in future studies.

Chlorhexidine is well-known as an antimicrobial agent and is considered the gold standard of antiplaque agents^16,38. CHX has demonstrated significant efficacy in reducing S. mutans and S. sobrinus^39,40. Our findings demonstrate that TTO exhibits comparable efficacy to CHX in inhibiting the growth of S. mutans and S. sobrinus at a concentration of 12.5%. Additionally, we observed that TTO inhibits the ability of S. mutans and S. sobrinus to form biofilm at a same concentration as CHX. Studies have reported that CHX usage can exhibit several adverse effects, including taste alteration, oral discomfort, tooth discoloration and xerostomia^19,41. The efficacy of TTO in inhibiting S. mutans and S. sobrinus growth, as well as inhibiting their ability to form biofilms, as demonstrated in the current study, presents a promising alternative natural product to CHX. Although our current data suggest that TTO treatment did not exhibit any toxic effects on HGF, further investigations are warranted to evaluate the long-term effects of TTO usage and to explore its potential advantages and any associated risks on both oral and systemic health.

Tertiary dentin, recognized as a reactionary response to dental tissue insult by caries or injury, assumes a pivotal role in dentin structure restoration by safeguarding the pulp against external or pathological influences^42,43. This form of dentinogenesis is triggered by mild stimuli generated by caries, prompting odontoblasts to initiate tertiary dentin formation⁴⁴. Moreover, the slow progression of carious lesions plays a crucial role in the enhanced formation of tertiary dentin⁴⁵. The presence of treatment substances may provide additional benefits in facilitating this process. However, notably, our study found no evidence that TTO promotes tertiary dentin formation, a process critical for pulp repair in advanced caries. This contrasts with bioactive agents such as calcium hydroxide, which directly stimulate odontoblast activity and dentin regeneration^46,47. The slower progression and less invasive caries lesions in the group treated with TTO, as demonstrated in the current study, suggest that TTO treatment might help stimulate defence mechanisms and mitigate early-stage caries by reducing bacterial load. In the present study, microscopic examination demonstrated a slightly increased rate of tertiary dentin deposition in carious molars subject to TTO treatment relative to untreated molars. However, the absence of significant regenerative effects in our results reinforces the conclusion that TTO’s therapeutic value lies in its antimicrobial, rather than reparative, properties. This distinction is clinically significant, emphasizing that while TTO may address bacterial adhesion and proliferation, it does not effectively resolve structural tooth damage, necessitating combined therapies for comprehensive caries management. Further research is needed into the mechanisms by which TTO treatment influences tertiary dentin formation. These future studies will provide further evidence on potential TTO clinical applications.

Limitation

Although our study clearly demonstrates the antimicrobial effect of TTO, including its proven ability to significantly reduce both the number and size of caries lesions in vivo, some limitations should be acknowledged. First, the investigation focused mainly on S. mutans and S. sobrinus, despite the polymicrobial nature of dental biofilms and the complex interactions among diverse oral microorganisms. This may limit the generalizability of our findings to the broader oral microbiome. Additionally, the relatively small sample size may affect the statistical power and robustness of our conclusions. Future studies should include a wider range of oral bacteria and larger sample sizes to better reflect clinical conditions and further validate the efficacy of TTO.

Conclusion

In conclusion, our study highlights the potential of a natural product, TTO, in inhibiting the growth of oral bacteria and preventing dental caries. TTO demonstrated inhibitory effects on both planktonic growth and biofilm formation of S. mutans and S. sobrinus, suggesting its ability to act as a preventive agent. The antibacterial efficacy of TTO holds thus promise for potential caries prevention and lays a solid foundation for innovative approaches in preventive dentistry for patients with high-risk caries and/or limited access to dental care.

Abbreviations

TTO

Tea Tree Oil

CHX

Chlorhexidine

S. mutans

Streptococcus mutans

S. sobrinus

Streptococcus sobrinus

MBC

Minimum Bactericidal Concentration

HGFs

Human Gingival Fibroblast Cells

Author contributions

I.H.: Performed Experiments, Data Analysis and Interpretation, Visualization, Writing – Original Draft; Z.D.: Performed Experiments, Data Analysis and Interpretation, Visualization, Writing – Original Draft; L.L.: Performed Experiments, Visualization, Writing – Original Draft; R.S.: Performed Experiments, Microcomputed topography analysis, Writing – Review & Editing; D.C.: Microcomputed topography analysis, Writing – Review & Editing; K.A.: Conceptualization, Supervision, Writing – Review & Editing; G.K.: Resources, Supervision, Writing – Review & Editing; S.P.: Supervision, Validation, Writing – Review & Editing; P.P.: Conceptualization, Funding Acquisition, Project Administration, Supervision, Validation, Writing – Review & Editing.

Funding

This work was supported by Institute of International Education, Greek diaspora fellowship program (P.P.); Centennial Enhancement Chair funds, University of Saskatchewan (P.P.); Start-up funds, university of Saskatchewan (P.P.)

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.