INTRODUCTION:

Dental caries is a polymicrobial biofilm disease driven by the diet and microbiota-matrix interactions that occur in hard structures¹. Among various bacteria groups, Streptococcus mutans is naturally found in the oral cavity, which is considered a renowned member of the mutans streptococci group and is widely accepted to be a major etiological factor behind the cause of dental caries². However, based on the ecological-based hypothesis, dysbiosis of acidogens is to be the aetiology of caries rather than a specific microbial agent³.The fact that most supporters of the ecological plaque hypothesis still concede some level of specificity or repeatability to the species associated with decaying surfaces adds to S. mutans function in the multifactorial caries process⁴.

Many attempts have been made to eliminate mutans streptococci from the oral flora with the help of various antibiotics therapy. However, excessive use can cause undesirable side effects⁵.During the last decades, composites have become the most commonly used restorative material, because of their desirable properties compared to other indirect restorations.⁶However, multiple clinical studies have found that composite restorations have a shorter lifespan and a higher failure rate than amalgam because polymerization reactions are coupled with volumetric shrinkage, which causes internal contraction strains at the interface^7,8,9.

Over the years researchers focused to produce the best material to prevent the development of caries associated with bacterial flora. There has been a conflicting result concerning various materials. In this study, more focus is given to the influence of Vitamin C as a material to prevent bacterial flora associated with dental caries attributed to its antioxidant effects^10,11.

It has been demonstrated that there is a link between increased caries activity and lower vitamin C levels in the saliva¹². Vitamin C was found to be just as effective as chlorhexidine at inhibiting microbial growth in the mouth¹³. Furthermore, research on Bacillus subtillis, a model organism for biofilm production, discovered that vitamin C inhibits quorum sensing and other biofilm development pathways¹⁴.

Over the past decade, various controversies remained about the use of natural materials and chemical agents in the field of dentistry. Therefore the purpose of the study was to evaluate the effects of Vitamin C against secondary caries formation around margins of class V composite restorations by in vitro bacterial caries model.

MATERIALS AND METHODS:

In the present study, 30 extracted mandibular premolars were collected from the Department of Oral and Maxillofacial Surgery. These extracted teeth are placed in a 0.1% solution of Thymol for 4 weeks. The teeth were then washed and cleaned to make them debris-free. The samples were sectioned 4 mm below the cementoenamel junction and then mesiodistally in the long axis of the tooth to obtain a rectangular buccal block. Class V cavities were made at the level of the CEJ using a flat-end carbide bur in a handpiecethat permits water cooling.¹⁵

Total samples were divided into three groups (n=10). (figure 1)

Group I- Application of 2% 20 mM HEPES primer. (Sigma Aldrich, Bangalore)

Group II - Application of 2% chlorhexidine primer.(Septodont, India)

Group III - Application of ascorbic acid ( Sigma Aldrich, Bangalore)

In the case of Group I, the walls of the cavity were etched with 37% phosphoric acid(ultradent, Bangalore) for 15 seconds before rinsing for 30 seconds with distilled water. Specimens were dried using an absorbent tissue, then primed for one minute with 2% 20mM HEPES buffer, then rinsed and dried with an absorbent tissue, before applying dentin bonding agent (3M single bond universal adhesive) to the preparation surfaces.

After was the removal of excess solvent. It was then light-cured(IvoclarVivadent blue phase) for 20 seconds. Preparations were filled with Universal composite (Filtek Z250 XT) material and light-cured for forty seconds each¹⁶. The restorations were then polished with an abrasive disc using a slow-speed micromotor straight handpiece right after the final curing. Group II and Group III followed the same restorative procedure, except for the change in the primer used¹⁷.

Group II (CHX primer), primed with 2% chlorhexidine solution (30 seconds) and dried and Group III (Ascorbic Acid primer), primed with an ascorbic solution for one minute followed by rinsing with distilled water for 30 seconds and finally it was blotted dry¹⁸.

Bacterial Caries Model Induction:

Approximately 1 mm away from the restoration margins, the cosmetic nail varnish was applied and then air-dried. Followed by disinfection of the specimens in 70% ethanol ( Sigma Aldrich, Bangalore) for 20 minutes before being rinsed twice with sterile phosphate-buffered saline (PBS) ( Sigma Aldrich, Bangalore) and stored overnight in sterile PBS at 4^oC¹⁹.

Streptococcus mutans (25175 ATCC) were aerobically cultivated on the Brain Heart Infusion agar before being injected into BHI broth and incubated at 370C for 18-20 hours. The cells were then washed twice with PBS and suspended in a fresh medium supplemented with 1% sucrose (BHIS) and spectrophotometrically standardised to 1 108 cells/mL (vita zahnfabrik Germany). The S. mutans suspension in BHI broth was used to inoculate the specimens with 1% sucrose for 4 hours at 37°C, further, the media was transferred to BHI for 20 hrs that doesnot contain sucrose²⁰.

Following each media change, PBS buffer was used to rinse the wells gently. After fourdays, rinsing was done followed by sectioning along the axis of the tooth.

Fluorescent Microscopic Analysis:

The dye application using 0.1mMRhodamine B solution (Therma Fisher Scientific, Bangalore) was performed after immersing the specimens in distilled water for an hour²¹.After some time, specimens were cleaned for one minute under running water and dried with an absorbent tissue. Then the samples were examined using a fluorescent microscope with a digital camera (Jenoptik laser optic system GmbH). For all specimens, the same microscope settings were employed. Images were evaluated and measured using the system's built-in scale.

Statistical analysis:

The collected data were statisticallyanalysed using Statistical Package for Social Sciences [SPSS] for Windows, Version 22.0. Released 2013. Armonk, NY: IBM Corp. Descriptive Statistics included expression of the distance from restoration and lesion depth in each study group using Mean and SD. In inferential statistics,the Kruskal Wallis test followed by the Mann-Whitney U test was used to compare the mean distance from restoration and Lesion Depth in the Dentinal and Enamel region between three study groups. The level of significance [P-Value] was set at P<0.05.

RESULTS:

This study was conducted to assess the protective effect of Vitamin- C primer against secondary caries development around enamel and dentine margins of class V restoration, using an in vitro bacterial caries model. The lesion depth and distance at which the initial lesion was observed from the restoration were measured using a fluorescent microscope(Figure 1).

A comparison of the mean distance from restoration in dentinal and enamel region between three study groups showed that the distance at which initial lesion is seen for group III for enamel was 899µm and dentin was 739µm when compared to the other group I ( 580µm and 729 µm) and Group II ( 519µm and 711µm) which is statistically significant that indicates the protective effect of vitamin C on inhibiting secondary caries in enamel and dentin (Graph 1)(Graph 2) (Table1) (Table 2).

Table 4: Multiple comparisons of mean difference in the Lesion depth between 3 groups using Mann Whitney Test

Region		G1 Vs G2	G1 Vs G3	G2 Vs G3
Dentin	P-Value	0.71	0.004*	<0.001*

Table 5: Comparison of mean Lesion Depth in Enamel region between Group 1 and Group 2 using Mann Whitney U Test

Region	Group	N	Mean	SD	Mean Diff	Z	P-Value
Enamel	Group 1	10	4.205	2.284	1.762	-1.663	0.10
Enamel	Group 2	10	2.443	0.800	1.762	-1.663	0.10

Note: Group 3 - No Lesion Penetration was observed in Group 3 [Vitamin C] in the Enamel region

Graph 3: Comparison of mean Lesion Depth in Dentinal region between 3 study groups

A comparison of mean lesion depth in the enamel region between three study groups stated that there was no progression of caries in group III in the enamel region. Lesion depth was less in group II when compared to group I (Graph 4).

Graph 4: Comparison of mean Lesion Depth in Enamel region between 2 study groups

Hence from the results obtained by comparing the lesion depth and distance at which the initial lesion formed, it can be noted that Vitamin C had a positive response in inhibiting secondary caries formation. Regardless of the material used for restoration, secondary caries cannot be completely avoided. The current findings confirm that the dentin-resin interface seems to be more susceptible to caries progression around the restoration margin than the enamel-resin interface.

DISCUSSION:

The most important causative factor for failure of bonded restorations is secondary caries²² which can cause micro-leakage and gap formation that will weaken the resin-dentin interface further providing a pathway for invasion of various oral plaque biofilms.²³ Secondary caries promoted by the acid production from bacteria including Streptococcus mutans is another major reason for dental resin composite restoration replacement²⁴.

Few probable causes that favour adhesion of oral streptococci in composite resin is hydrophobicity and lack of surface smoothnesswhich contributes to the demineralization adjacent to both dentin and enamel margins²⁵. The bond between the tooth-resin interface impacts the progression of dental caries below the restorative margin along with microleakage providing a second point of entry for bacteria. Thus, enhancement of bond durability by inhibition of bacterial ingress are essential issues to be considered for decreasing the formation of secondary caries.

During the process of dental decay, large numbers of free radicals are produced. Neutralisation of these free radicalsis brought about by antioxidants such as vitamin C and thus reducing its capability to cause further damage²⁶. Recently ZehdiEydou et al ²⁷ studied the effect of vitamin C on S. mutans and found vitamin C has a deleterious effect on S. mutans growth and biofilm formation. Vitamin C is also known to have both bactericidal and bacteriostatic effects²⁸.

Hence,this study was done to evaluate the effect of ascorbic acid against S. Mutans which contributes to secondary caries progression in and around tooth margins using an in vitro bacterial caries model of class V restorations,

The results of this study showed the ascorbic acid primer has a protective effect on secondary caries progression adjacent to the dentine resin interface. This is similar to the study by ZehdiEydou et al,²⁷ who also explained that vitamin C inhibited growth and biofilm formation of S. mutans.

Furthermore, as dentin is relatively porous, the ascorbic acid could diffuse further and protect dentin beyond the interface²⁹ Ascorbic acid is also known to increase the bond strength of resin composites by inhibition of MMPS thereby providing optimal use with resin composites ³⁰.

Another material that was used in the study was 2% chlorhexidine primer. It is a cationic molecule, which acts by binding to the bacterial wall. And depending on the concentration in which it is used, it can have either a bacteriostatic or a bacteriocidal action³¹.The finding of the study showed chlorhexidine was less effective to inhibit secondary caries when compared to ascorbic acid. This could be explained based on the low permanent binding mechanism of chlorhexidine in the dentin structure.

Russell AD et al,³² explained that CHX had a reduced permanent binding mechanism to the dentin structure and the residual amount of CHX was too low to exhibit bactericidal activity because it is only bacteriostatic at low doses. Other studies by Velumurugan et al,³³ and Usha C et al,³⁴ also concluded that CHX was able to inhibit S. Mutans. In addition to the anti-cariogenic activity CHX diacetate in concentrations as low as 0.2% hasbeen shown to improve the long-term stability of resin-dentine interfaces, without jeopardizing the mechanical properties of the adhesives³⁵.

Another agent assessed was HEPESbuffer which had no noteworthy effect on inhibiting the formation of secondary caries around the margins of resin composite restorations.HEPES acts like water in which its dissociation reduces as the temperature decreases. Which, at low temperatures makes it a better buffering agent for maintaining the enzyme structure and function. This is supported by a study by Zhou W et al.,³⁶ who explains the effect of various buffer solutions in maintaining enzyme structure.

Our study's shortcoming, as with any in vitro investigation, is that extrapolating results to the actual oral environment, where several other factors may be confounding, must be done with caution. Furthermore, just one species of cariogenic bacteria was used in this study's bacterial caries model (S. mutans). The current study approach, on the other hand, yields important and promising results that pave the way for future research.

CONCLUSION:

Regardless of the priming solution's composition, secondary caries occur in enamel and dentin. Antioxidants like ascorbic acid are important for maintaining tooth integrity as well as acting as a non-enzymatic antioxidant defence system. The findings suggested that including ascorbic acid into the resin composite restorative technique inhibits the development of secondary caries, particularly near vulnerable root dentine borders.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 27.10.2021 Modified on 26.05.2022

Research J. Pharm. and Tech 2023; 16(7):3245-3250.

DOI: 10.52711/0974-360X.2023.00533

Region	Groups	N	Mean	SD	Min	H	P-Value
Dentin	Group 1	10	580.30	95.39	437	8. 508	0.01*
	Group 2	10	519.60	172.10	312
	Group 3	10	739.20	152.62	410
Enamel	Group 1	10	729.95	66.20	616	18. 797	<0.001*
	Group 2	10	711.35	32.89	675
	Group 3	10	899.71	68.52	792

Region	Groups	N	Mean	SD	Min	Max	H	P-Value
Dentin	Group 1	10	6.912	3.200	2.78	11.32	14.650	0.001*
	Group 2	10	6.339	2.233	4.2	11.19
	Group 3	10	2.773	1.013	1.25	4.3