Abstract
Introduction:
Pulp capping is a viable treatment option for management of deep carious lesions in pulp preservation with reduced remaining thickness of dentin. Traditionally, calcium hydroxide (Ca(OH)2) and currently contemporary bioceramic pulp capping agents have been well researched in the dental domain and evidenced varying but promising success rates. However, there is a growing interest in exploring indigenous, innovative, alternative natural products derived from apiarian sources, which possess characteristics and properties which can potentiate predictable treatment outcome including antimicrobial properties. Hence, this study aims to compare the efficacy of indigenously developed apiarian products like royal jelly (RJ) with contemporary pulp capping agents in eradicating Streptococcus mutans biofilm, a predominant cariogenic microorganism.
Materials and Methods:
RJ was prepared at 4% concentration by dissolving 4 g of pure RJ in 100 mL dimethyl sulfoxide (DMSO). Ca(OH)2 was prepared by mixing 2 g of Dycal (Dentsply) in 100 mL of sterile water. Chlorhexidine (CHX) was used as a 2% gel formulation (Gluco-Chex, Cerkamed). DMSO was used as a solvent to ensure proper dispersion. Agar diffusion results: RJ (97.89%), CHX (72.95%), and Ca(OH)2 (78.19%). Biofilm assay: RJ (97.89%), CHX (72.95%), and Ca(OH)2 (78.19%). Evaluation of the antimicrobial activity was conducted with various tests such as zone of inhibition and biofilm viability assay. Data collection was done and statistical analysis was done using G*Power (P < 0.05).
Results:
Agar diffusion: RJ showed the highest antimicrobial effect (97.89%), followed by CHX (72.95%) and Ca(OH)2 (78.19%). Biofilm assay: 4% RJ exhibited the highest inhibition (97.89%), outperforming CHX (72.95%) and Ca(OH)2 (78.19%).
Conclusion:
The findings of this comparative evaluation support the potential of the innovation of indigenously developed apiarian pulp capping agent as an effective antimicrobial agent against S. mutans cariogenic biofilm. Further research is warranted to explore the optimal inhibitory concentration against dental biofilm and further evaluate their long-term clinical efficacy of this indigenous innovation and safety in dental practice as a pulp capping agent.
Keywords: Antimicrobial efficacy, apitherapy, biofilm inhibition, calcium hydroxide, pulp capping agent, royal jelly, Streptococcus mutans
INTRODUCTION
Streptococcus mutans is one of the principal causative pathogens that significantly contributes to the pathogenesis of human dental caries.[1,2,3,4,5,6]
The virulence factor that initiates dental caries is the presence of various adhesins which bind on the galactoside of the enamel pellicle, which in turn has a vital role to play in colonization of cariogenic biofilm.[1,2] Vital pulp therapy is the treatment which aims to preserve and maintain vital pulp tissue that has been compromised but not yet invaginated or violated the integrity of the pulp dentin complex violated by caries, trauma, and/or restorative procedures.[3,4,5]
Calcium hydroxide (Ca(OH)2) has inherent antibacterial properties by virtue of an alkaline pH which can aid in elimination of microbial penetration and subsequent pulpal tissue noxious irritation.[7] Ca(OH)2 has inherent limitations such as dissolution in oral fluids and presence of tunnel defects in the dentin bridge formation.[8] Chlorhexidine (CHX) is a commonly used dental antimicrobial and has demonstrated a therapeutic role as an oral disinfectant and a nonspecific matrix metalloproteinase (MMP) inhibitor with documented effect against MMP-2, MMP-8, and MMP-9 entrapped in dentin. Ca(OH)2 and CHX have been combined as a pulp capping agent to enhance the synergism action and potentiate the antimicrobial efficacy.[9] While CHX is primarily used in clinical dentistry for its strong antimicrobial and hemostatic properties, it lacks bioinductive capacity unlike materials such as mineral trioxide aggregate (MTA) or Biodentine. In vital pulp therapy, CHX is often used as a disinfectant to control bleeding and reduce microbial load prior to placement of a pulp capping material. In this study, CHX was included as a comparative benchmark for antimicrobial efficacy because it is a widely accepted and clinically validated agent used adjunctively in pulp capping procedures.[9,10] Its inclusion allows for a standardized comparison of the antimicrobial performance of the test materials (such as royal jelly [RJ]) against a known antimicrobial agent, even though CHX itself does not possess regenerative potential. The primary goal of this study was to evaluate antimicrobial and antibiofilm activity, for which CHX serves as a useful reference standard.
Propolis is a natural biocompatible apiarian derivative that has been widely researched in dentistry for its anti-inflammatory, antibacterial, and immunomodulatory properties.[11] Propolis is documented to be more efficacious than Ca(OH)2, MTA, Biodentine, and BioAggregate as it induces the formation of tubular dentin which is qualitatively more well organized.[12]
RJ is a natural secretion produced by the hypopharyngeal and mandibular glands of worker honeybees (Apis mellifera) and is used as the exclusive food source for queen larvae and adult queens. It is rich in bioactive compounds including major RJ proteins, 10-hydroxy-2-decenoic acid, royalisin (a peptide with antibacterial activity), flavonoids, phenolic acids, enzymes, vitamins (B-complex), and trace minerals.[13,14] These components contribute to its antibacterial, anti-inflammatory, antioxidant, and regenerative properties, making it a promising candidate for applications in pulp therapy. Prior studies have confirmed the biocompatibility and low cytotoxicity of RJ when used in dental applications.[13,14,15] Its antimicrobial action has been demonstrated against several oral pathogens, including S. mutans, further supporting its potential as a pulp capping material. Hence, it can be a viable therapeutic option for vital endodontics.[16,17,18] While several materials such as Ca(OH)2 and MTA have shown positive results in pulp capping procedures, there is a need for additional alternatives that may offer enhanced antimicrobial properties.[19,20,21]
Aim
This study was conceived to compare the antimicrobial effect of an indigenously developed innovative apiarian pulp capping agent, i.e. RJ and propolis, with Ca(OH)2, and CHX which has been incorporated in various pulp capping agents for its substantivity.[22]
MATERIALS AND METHODS
The study was conducted to evaluate the antimicrobial efficacy of indigenously developed apiarian products with Ca(OH)2 and CHX by agar diffusion method and to assess the biofilm inhibition by crystal violet assay. Study design included three groups with Group A: RJ was prepared at 4% concentration by dissolving 4 g of pure RJ in 100 mL dimethyl sulfoxide (DMSO)., Group B: CHX was used as a 2% gel formulation (Gluco-Chex, Cerkamed) for the experimental group and Group C (Control Group): Ca(OH)2 was used at a 2% concentration, in accordance with the abstract and experimental standardization. To prepare this, 2 g of Dycal (Dentsply) was mixed in 100 mL of sterile distilled water, forming a uniform 2% w/v solution. The mixture was stirred with a magnetic stirrer to ensure homogeneity before application.
RJ 50 g was obtained in pure form from Queen Bees Honey Pvt. Ltd, Chennai, India, and mixed with DMSO at 4% concentration. Ca(OH)2 was obtained from Dycal (Dentsply) which is used for pulp capping and CHX from Gluco-Chex (Cerkamed) 2% gel.
S. mutans clinical strain was acquired from Saveetha Microbiology Culture Centre, Chennai, India, and inoculated into brain–heart infusion broth. After incubation, the S. mutans strains were maintained in brain–heart infusion agar at 4° 20% glycerol at 80°. S. mutans ferments a variety of carbohydrates, produces large amounts of acid, with resultant decrease in pH, secretes extracellular polysaccharides, and leads to plaque or biofilm formation.
Antimicrobial assessment: Agar diffusion method
The antimicrobial activity was assessed using the agar well diffusion method as shown in Figure 1. Mueller-Hinton agar plates were inoculated with S. mutans using a sterile cotton swab to create a uniform lawn. Wells (6 mm diameter) were punched into the agar, and 100 μL of each test material (RJ, CHX, ad Ca(OH)2) was added to respective wells. Plates were incubated at 37°C for 24 h, and the zones of inhibition were measured in millimeters using a digital Vernier caliper. Results were expressed as inhibition percentages relative to the maximum inhibition observed.
Figure 1.
Antimicrobial assessment using agar diffusion method. (a) Zone of inhibition of Calcium Hydroxide and Chlorhexidine against E. faecalis and S. mutans. (b) Zone of inhibition of Royal Jelly extracts (aqueous, ethanolic, and raw) against S. mutans
Biofilm inhibition
Crystal violet staining was used to assess the ability of each agent to inhibit biofilm formation on microtiter plates. Biofilm presence was quantified using spectrophotometry at 570 nm. The samples after diluting to desired concentrations (0.1 ml to 0.003 ml) were added to the MHI broth in the wells of 96-well microplates as shown in Figure 2. Then, the samples were inoculated with 50 μL of the broth culture and incubated for 48 h at 37°C. After the incubation, the broth was aspirated from the wells using a sterile pipette and washed with phosphate-buffered saline (PBS) solution. Then, 150 μL of crystal violet (0.2%) was added to each well and allowed to stand for 15–20 min. This was followed by removal of the dye and washed with PBS to remove unbound and excess dye. Then, the dye was dissolved by adding 150 μL of glacial acetic acid (30%) in each well. Readings were taken using an ELISA plate reader at 570 nm, and the absorbance value was recorded. Data collection was done, and statistical analysis was performed using G*Power software version 3.1 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, North Rhine-Westphalia, Germany) (P < 0.05).
Figure 2.
Antibiofilm assessment by crystal violet stain
RESULTS
The highest antimicrobial efficacy was shown by RJ using agar diffusion method. Biofilm inhibition was shown highest by RJ. 4% RJ demonstrated the greatest zones of inhibition followed by 2% CHX, which showed more inhibition than Ca(OH)2.
DISCUSSION
Traditionally for over a century, Ca(OH)2 has been used as a pulp capping agent. Historically, the mechanism of action of Ca(OH)2 in inducing dentin bridge formation has not been well elucidated. Ca(OH)2 has inherent antibacterial properties by virtue of an alkaline pH which can aid in elimination of microbial penetration and subsequent pulpal tissue noxious irritation. Ca(OH)2 despite limitations has long been known to induce dentin bridge formation and promote pulp healing through stimulation of hard tissue deposition due to its alkaline pH and calcium ion release.[6] Although chlorhexidine (CHX) is primarily known for its antimicrobial properties, studies by Priyadarshini et al. have demonstrated that, when combined with calcium hydroxide (Ca(OH)2), it can enhance pulp healing by inhibiting matrix metalloproteinases (MMPs) and minimizing dentin matrix degradation thereby promoting a more favorable bioactive environment for regeneration.[9] Studies have shown that RJ stimulates regeneration, fibroblast proliferation, and collagen synthesis, making it a promising agent in regenerative dentistry. It contains royalisin and bioactive proteins that contribute to tissue repair and dentinogenic stimulation.[13,14,16] The antimicrobial and antibiofilm properties of pulp capping agents play a crucial role in achieving predictable outcomes, especially in vital pulp therapy, where the goal is to maintain the integrity of healthy pulp. Although carious dentin is clinically removed before applying the pulp capping material, studies have shown that microbial remnants, especially biofilm-forming S. mutans, may persist within the dentinal tubules or at the pulp–dentin interface.[23,24] If not adequately neutralized, these remnants can lead to pulpal irritation, inflammation, or failure of the capping procedure.
Therefore, testing for biofilm inhibition in vitro is justified as it provides predictive evidence of a material’s ability to suppress residual bacteria that may otherwise compromise healing. Materials with strong antibiofilm activity, like RJ, may enhance the clinical success of vital pulp capping, not only by reducing bacterial load but also through bioinductive potential by stimulating repair and dentinogenesis.[13,14] The antibacterial effect of a pulp capping agent is thus a crucial deciding factor in achieving predictable outcomes.[25] Bacterial elimination or suppression or entombment is essential to prevent further progression of infection and enhance and promote dentin bridge formation, which in turn will lead to the preservation of pulp vitality. Pulp capping agents containing antibacterial components have, over time, been associated with the emergence of resistant strains of Streptococcus mutans.[2,3] Pedrinha et al. observed that although Ca(OH)2 initially disrupts bacterial biofilm, over time, the changes it induces in the biofilm matrix and its physicochemical interactions may paradoxically support the viability and regrowth of bacterial cells particularly S. mutans within the polysaccharide-rich matrix.[26] Kaspar et al. demonstrated that S. mutans strains subjected to subinhibitory concentrations of CHX can develop spontaneous tolerance. This adaptation is accompanied by genetic mutations, including defects in genes associated with stress response, cell wall integrity, and antimicrobial resistance pathways, which may also reduce bacterial fitness.[10]
Biofilm plays a critical etiological role in the pathogenesis of dental caries and pulpal inflammation. Even though the carious dentin is clinically removed prior to pulp capping, complete eradication of microorganisms is often challenging. Residual bacteria and biofilms, especially those within dentinal tubules or near the pulp, can persist and compromise the success of pulp capping procedures.[23,24] Therefore, evaluating the antibiofilm activity of pulp capping agents is crucial to ensure that any residual infection is neutralized, reducing the risk of posttreatment inflammation or failure. This test provides insight into the ability of these materials to suppress or eliminate bacterial communities that might remain post excavation, thus supporting predictable pulp healing.
Hence, this study design was designed to compare and introduce innovative, indigenous material for pulp capping. One of the active components of apiarian products such as propolis and RJ and beeswax is caffeic acid phenethyl ester which is effective in stimulating collagen as well as inhibiting the inflammation and degeneration of dental pulp. The indigenous novel material which was selected was an apiarian product RJ. This study aimed to assess the antibacterial efficacy of indigenous RJ compared to contemporary pulp capping agents in inhibiting S. mutans biofilm formation. The results of our experiments reveal important insights into the potential applications of these agents in dental procedures.
The highest antimicrobial efficacy by agar diffusion method was shown by RJ followed by 2% CHX followed by Ca(OH)2 [Graph 1]. Biofilm inhibition when done under crystal violet assay proved 4% RJ demonstrated greatest zones of inhibition followed by 2% CHX, which showed more inhibition than Ca(OH)2. However, CHX and Ca(OH)2 performed more or less similarly. Thus, no significant difference is seen in Ca(OH)2 and CHX. Since RJ is an indigenous innovative product, more studies are needed for optimization of dose [Tables 1, 2 and Graph 2].
Graph 1.
Antimicrobial efficacy by agar diffusion method
Table 1.
Biofilm Inhibition shown at various concentrations in mg
| Concentration (mg) | Biofilm inhibition | ||
|---|---|---|---|
|
| |||
| RJ | CHX | Ca(OH)2 | |
| 1 | 44.00 | 33.07 | 28.68 |
| 0.5 | 56.00 | 43.09 | 33.01 |
| 0.25 | 71.79 | 50.90 | 38.90 |
| 0.125 | 85.68 | 63.13 | 48.72 |
| 0.062 | 95.16 | 69.14 | 52.65 |
| 0.031 | 97.89 | 72.95 | 78.19 |
CHX: Chlorhexidine, Ca(OH)2: Calcium hydroxide, RJ: Royal jelly
Table 2.
Biofilm inhibition at various concentrations (crystal violet assay)
| Concentration (mg/mL) | RJ | CHX | Ca(OH)2 |
|---|---|---|---|
| 1.000 | 44.00 | 33.07 | 28.68 |
| 0.500 | 56.00 | 43.09 | 33.01 |
| 0.250 | 71.79 | 50.90 | 38.90 |
| 0.125 | 85.68 | 63.13 | 48.72 |
| 0.062 | 95.16 | 69.14 | 52.65 |
| 0.031 | 97.89 | 72.95 | 78.19 |
CHX: Chlorhexidine, Ca(OH)2: Calcium hydroxide, RJ: Royal jelly
Graph 2.
Antibiofilm assessment by crystal violet stain
This study demonstrated the highest antimicrobial efficacy shown by RJ, followed by CHX and Ca(OH)2 against S. mutans. No significant difference in antimicrobial efficacy has been demonstrated between Ca(OH)2 and CHX, more studies need to be conducted with optimization of dose, especially that of RJ.
We found that both indigenous RJ and contemporary pulp capping agents exhibited antibacterial activity against S. mutans. Specifically, the inhibition zones observed in our experiments demonstrated the ability of these agents to impede biofilm formation, which is a critical factor in dental caries development.
The findings of the current study are consistent with previous research investigating the antibacterial properties of apiarian products, such as propolis, royal jelly as well as those of contemporary pulp capping agents.[5] Yalcin et al. reported similar antibacterial effects of RJ against oral pathogens, substantiating our results.[5] Additionally, our observations are consistent with the well-documented antibacterial properties of contemporary pulp capping agents, as described in studies by Aljandan et al.[1] and Poggio et al.[15]
The antimicrobial properties of RJ present a promising avenue for enhancing infection control in endodontics.[27] The antibacterial mechanisms of RJ and comparison with contemporary pulp capping agents warrant further investigation. RJ contains various bioactive compounds, such as royalisin, which have been associated with antimicrobial effects.[13]
Conventional pulp capping agents, on the other hand, often contain materials such as Ca(OH)2 or MTA, which are known for their alkaline pH and ability to induce tissue repair. These properties may contribute to their antibacterial effects by creating an unfavorable environment for bacterial growth.[4,5,6,8,12,28,29,30] These findings have significant clinical implications. Considering that RJ exhibits superior antimicrobial activity, it can be a viable therapeutic option to clinicians for vital pulpal procedures and contributing to predictable treatment outcomes and reduced dependency on synthetic chemical-based materials.[14,23,26]
The antibacterial efficacy of both indigenous RJ makes it a viable natural alternative, particularly in prevention or inhibition of dental caries and an alternative pulp capping agent. Dentists may consider these agents as viable alternatives or complementary treatment adjuncts, depending on patient-specific factors and clinical scenarios.
Future research directions should include long-term studies to assess the substantivity of the antibacterial effects. Cytotoxicity assessments to ensure patient safety should also be conducted especially of each constituent of RJ extract. Further clinical investigations should be conducted to assess the clinical efficacy and long-term outcomes of these agents in human applications. Future research should explore its compound’s specific interactions with S. mutans and their potential to disrupt biofilm formation. Further research can also encompass the inclusion of RJ extract with other restorative materials or intracanal medicaments and synergism may be evaluated. It should expand the scope to encompass a wider range of cariogenic and other oral pathogens.
It is important to acknowledge the limitations of our study, such as the use of in vitro assays that may not fully replicate the complexity of the oral environment. Moreover, the study focused on a single bacterial strain of S. mutans, while the oral microbiome comprises a plethora of species.[24] The study demonstrates that both indigenous RJ and contemporary pulp capping agents exhibit antibacterial efficacy against S. mutans biofilm formation. These findings open avenues for the development of novel dental treatments and the optimization of existing pulp capping techniques. The potential of RJ as a natural alternative warrants further exploration in clinical settings, with the ultimate goal of improving dental health and treatment outcomes.
CONCLUSION
Within the limitations of the study, it can be concluded that indigenously developed apiarian products are effective antimicrobial agents against S. mutans biofilm. Maximum antimicrobial efficacy was shown by RJ when compared between CHX and Ca(OH)2.
Further research is warranted to explore the inhibitory concentration and evaluate their long-term clinical efficacy and safety in dental practice as pulp capping agents.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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