Assessment of chlorohexidine-coated cotton filler addition on resin-based fissure sealants’ mechanical, water sorption, and antibacterial properties

Abstract

The occlusal surfaces of posterior teeth are susceptible to caries formation due to the presence of pits and fissures. The bacteria present in the oral cavity and food particles are accumulated on these complex structures. To prevent and protect the tooth structure, different additives were added to pits and fissures sealants to improve adhesion with tooth structure, enhance the mechanical properties, and prevent bacterial growth. In this regard, cotton was coated with chlorohexidine (CHX) and incorporated in a resin-based sealant to improve its mechanical properties (compressive and flexural strength), mass change, and bacterial properties. The mean compressive strength values of CHX coated cotton modified RBS samples S1, S2 & S3 were significantly higher than the control group. The mean flexural strength value of group S1 was significantly higher as compared to the C, S2 & S3. Disc diffusion methods showed a significantly higher value of (p-value = 0.003) antibacterial potential of groups S2 and S3 as compared to group C. The mean percent mass change values of S1, S2 & S3 were higher than the control group. Hence the addition of CHX-coated cotton fillers in RBS improved its properties.

Keywords:

• Cotton cellulose
• chlorhexidine
• pits and fissures sealants
• mechanical properties
• water sorption
• antibacterial properties

1. Introduction

Dental caries, the most common intraoral disease, affects most of the population. It is considered the most prevailing oral health problem (Yadav & Kumar, 2019; Fei et al., 2020). About 44% of people are suffering from untreated caries in their primary and permanent teeth (Simonsen, 2011). The occlusal pits and fissures are the most susceptible areas for caries formation and plaque accumulation (Kervanto-Seppälä et al., 2009). The primary prevention includes plaque control, topical fluoride application, and use of pits and fissure sealant. The sealant acts as a physical barrier by blocking nutrients from the bacteria (Ahovuo-Saloranta et al., 2020). Generally, sealants are categorized into three main groups: glass ionomer cement (GIC) based sealants, resin-based sealants (RBS), and polyacid-modified sealants (Naaman et al., 2017).

Dental resin based sealant (RBS) has superior mechanical properties as compared to other sealants (Lynch et al., 2014). Resin based sealant (RBS) contains Bis-GMA, methyl methacrylate (MMA) or triethylene glycol, micro-particles of silica, and sodium monofluorophosphate (Morphis et al., 2000). The use of RBS is a recommended procedure for the prevention and control of caries. Ahuvuo-Saloranta et al.in 2008, proved that there is a 78% decrease in occlusal caries up to two years after the use of fissure sealants when compared to unsealed occlusal surfaces (Ahovuo-Saloranta et al., 2004). Resin-based fissure sealants (RBS) have several challenges such as compressive strength, flexural strength, plaque accumulation, scraping of materials with chewing, polymerization shrinkage, water sorption, discoloration, sealing ability, and marginal leakage which provide a pathway for bacteria causing recurrent caries (Lin et al., 2007)

Due to these shortcomings, there is demand for the development of novel materials in prophylactic dentistry, especially for the enhancement of mechanical properties and antibacterial activity against S. mutans (Kalia et al., 2008). Different researchers have added various fillers to enhance mechanical properties and decrease bacterial activity in RBS. Sodium-monofluorophosphate helps to prevent demineralization due to fluoride release (Awah et al., 2016). Cellulose acetate derived from bamboo cellulose (B-CA) was used to enhance the mechanical properties (Cai et al., 2016). Muhammad et al in 2020 added Hydroxyapatite (HP) to resin based restorative materials and it improves mechanical properties (Sabir et al., 2020). Jirun Sun et al in 2011 added titanium dioxide (TiO2) to improve mechanical properties and photo activity (Sun et al., 2011). Silver nanoparticles helped to inhibit biofilm growth (Agnihotri et al., 2014). Synthetic (nylon-6) and natural (chitosan) polymers have been added as fillers in resin based fissure sealants (Hamilton et al., 2015). Titanium dioxide nanoparticles (TiO2 NPs) have been used to enhance the antibacterial properties of restorative materials but due to the inconsistent deposition of TiO2 NPs, their mechanical properties were not improved (Klapdohr & Moszner, 2005). Cotton cellulose material has many advantages, they are cheap, easily available, renewable, environment friendly, nontoxic, biocompatible, biodegradable inside the human body, and possesses substantial mechanical strength as compared to other materials (Singh et al., 2020). They are also used in biomedical engineering (Zulkifli et al., 2019). Due to superb properties for bone tissue engineering surgical cotton microfibers were used as primary material for 3D scaffold fabrication. Cotton cellulose microparticles, used as active filler in dental material, are obtained from raw cotton by sulphuric acid hydrolysis (Ibrahim et al., 2015).

In the oral cavity various pathogens especially S.mutans plays a major role in caries formation. To decrease S.mutans count in saliva different materials such as zinc oxide nanoparticles, silver ions, titanium dioxide, chlorhexidine, etc. are incorporated in dental resin based composite (Yadav et al., 2022). The antimicrobial effect of silver dioxide (Ag₂O) is short, with no changes in mechanical properties but causes discoloration (Davari et al., 2022).

Chlorhexidine in different concentrations has an antibacterial activity but they are slowly released from the polymer in the oral cavity leaving spaces/holes and thus decreasing the mechanical properties leading to the dislodgement of material (Takahashi et al., 2006).

Since the development of pit and fissure sealants, they had shortcomings like lower mechanical properties and no antibacterial properties. This fails restoration which leads to the formation of new caries lesions called secondary or recurrent caries under the existing restoration. To prevent the formation of recurrent caries, there is a need for materials that have both properties, i.e., strong mechanical properties and antibacterial activity against oral pathogens. It is important to emphasize that several attempts were made to improve the mechanical properties and antibacterial activity. But none of them developed such a material that has both properties at the same time. So in this study, an attempt was made to develop pit and fissure sealants by incorporating CHX coated cotton fillers containing cellulose and antibacterial extract to improve the mechanical as well as antibacterial properties. Cotton cellulose and in general cellulose having a good tensile strength of 7.5–7.7 GPa and Young’s modulus of 110–220 GPa was coated with chlorhexidine (having good antibacterial properties) and added in commercial pits and fissure sealant. The cotton cellulose and chlorhexidine-coated cotton cellulose were characterized with FTIR and SEM analysis for confirmation of cellulose matrix and coating with chlorhexidine. The modified fissure sealant was evaluated for mechanical properties (flexural strength, compressive strength), water sorption, and antibacterial properties against S. mutants.

2. Materials and methods

Materials used in this study has been listed in Table 1.

Table 1. List of materials.

Materials	Brand Name	CAS-NO
Pits and fissure sealants	SDI Conseal F	NIL
Chlorhexidine powder 99.9%	Sigma Aldrich	CAS-NO: 55-56-1
Cotton Fibers	Pure cotton from local market	NIL
Sodium hydroxide pellets	DAEJUNG, Korea	CAS NO: 1310-73-2
Concentrated sulphuric acid	DAEJUNG, Korea	CAS NO: 7664-93-9
Streptococcus Mutans	CLARKE	ATCC25175
Mueller Hinton Agar	OXOID, KSFE, Malaysia	CMO 337

2.1. Settings and design

This was an in vitro experimental study. A total of 80 pits and fissure sealant samples were divided into three experimental groups (S1, S2, and S3) and one control group (C). The experimental group samples were prepared by incorporating three concentrations (1, 2, and 3%) of chlorhexadine coated cotton fillers into the commercial pits and fissure sealants.

2.2. Preparation of cotton cellulose powder

5g of natural cotton was cut into small pieces, washed, and dried at ambient room temperature (25 ± 1 °C) for 48 hrs. Acid hydrolysis of cotton cellulose was done as described by An and Le Chi (2023) with slight modification. A 40% solution of sulphuric acid was prepared by adding 40 ml of sulphuric acid dropwise into 60 ml of distilled water in a beaker. The cotton fibers were transferred into a sulfuric acid solution and magnetic stirrer for 3 hrs at 250 rpm keeping the temperature at 40 °C. The hydrolyzed cotton solution was centrifuged at 500 rpm for 25 minutes. After centrifugation, sediment was transferred into the beaker containing 150 ml of distilled water to stop the exothermic reaction. After cooling add 1% sodium hydroxide (NaOH) dropwise to make it pH neutral. The neutral solution was centrifuged and dried in an oven at a temperature of 45 °C for 6 hrs. The dried cotton cellulose powder was treated by a ball milling machine and the fine powder was sealed in an air-tight bag.

2.3. Preparation of chlorhexidine (CHX) coated cotton powder

A 3% chlorhexidine solution was made by adding 3 gm of chlorhexidine powder into 100 ml of distilled water. Put the solution on a magnetic stirrer at 200 rpm for 1 hr at room temperature for complete mixing. The cotton powder was then added into the chlorhexidine solution, and stirred on a magnetic stirrer at 200 rpm for 24 hrs, to achieve maximum adsorption of chlorhexidine onto the cotton powder. Centrifuge the solution at 12000 rpm for 20 minutes. Dried in an oven at 40 °C for 6 hrs and stored in an airtight bag.

2.4. Preparation of experimental resin based fissure sealant (RBS)

Samples were divided into control and experimental groups. The control group specimens were prepared from resin based fissure sealant (RBS) (Conseal F™) in recommended size molds, and cured through a light curing unit for 35 seconds according to the manufacturer’s instructions. The experimental group samples were prepared by adding chlorhexidine-coated cotton filler to the RBS in proportions of (1, 2, and 3%) by weight. The samples were mixed by hand spatula for 5 minutes followed by magnetic stirring for about 20 minutes at 100 rpm. These modified pits and fissure sealants of different concentrations were stored in dark bottles to prevent premature polymerization. Each bottle was properly labeled.

2.5. Characterization

2.5.1. Scanning electron microscope (SEM)

The SEM of cotton cellulose powder and chlorhexidine-coated cotton powder was performed using a scanning electron microscope (JSM-IT-100). The samples were gold coated in gold sputter (QUORUM) and images were taken in a range of 700X to 10000 X at a voltage of 20 keV.

2.5.2. Fourier transform infrared spectroscopy (FTIR) of cotton powder

Fourier transform infrared spectroscopy of cotton cellulose powder and chlorhexidine-coated cotton powder with FTIR coupled with Attenuated Total Reflectance (ATR) mode as an accessory (IR Tracer 100 Shimadzu). The spectra were collected over the region 4000-650cm^-1 at a resolution of 4 cm^-1 and an average of 32 scans.

2.6. Evaluation of mechanical properties

To make clean and clear samples, the mold was cleaned using ethanol to remove any contamination or debris present in it. One side of the mold was covered with a Mylar strip and then placed on a glass slab. The experimental materials, CHX-coated cotton cellulose filler modified RBS were inserted in a preformed Teflon mold using a plastic instrument with great care. To prevent the oxygen inhibition layer another Mylar strip was placed on the top of the material in the mold. The samples were cured using a light curing unit (Woodpacker™ curing light) on both sides for 35 seconds. Samples were then removed from the mold. The specimens were polished to be refined by using a 500-grit abrasive (silicon carbide) paper in a moist environment.

2.6.1. Flexural strength

For measuring the flexural strength of the prepared material, the procedure cited in ISO standard 6874: 2015 dentistry polymer-based pits and fissure sealants was used. Teflon molds in the dimensions of 25 mm in length, 2 mm in width, and 2 mm in height were used for the preparation of the samples. Before measuring the flexural strength of the specimen, the samples were kept in distilled water at 37 °C for 24 hours in a drying oven (Memmert UNB-500). The specimens were then presented to the universal testing machine at a load cell of 50 KN at a cross-head speed of 2 mm/min. The flexural strength (FS) was analyzed using the following equation. (1) $$\text{FS}=\text{3PL}/\text{2bd2}$$(1)

FS is the flexural strength in MPa, P stands for load at fracture (N), L is the length of span, b is the width and d is the thickness of the specimens in millimeters (Osorio et al., 2007; Hojati et al., 2013).

2.6.2. Compressive strength

For measuring the compressive strength of the prepared material, CHX coated cotton cellulose fillers modified RBS, the rules mentioned in ISO standard 6874: 2015 dentistry polymer-based pits, and fissure sealants were used. The mold in the dimensions of 4 mm in diameter and 6 mm in height was used for the preparation of the samples. Before measuring the compressive strength of the specimen, the samples were kept in distilled water at 37 °C for 36 hours in a drying oven (Memmert UNB-500). The specimens were then launched to the universal testing machine at a load cell of 50 KN at a cross-head speed of 2 mm/min. The compressive strength (FS) was analyzed using the following equation.

(2) $$\text{Compressive Strength}\left(\text{CS}\right)=\text{F}/\text{A}$$(2)

2.7. Water sorption test

For measuring water sorption of the CHX coated cotton cellulose-modified RBS the protocol mentioned in ISO standard 6874: 2015 dentistry polymer-based pits and fissure sealants was used. The samples were prepared in a disc-shaped metal mold with a dimension of 2 mm in height and 4 mm in diameter. After curing the samples were then kept in a desiccator at 37 °C for 48 hours, noted dry mass as m₁, on a digital balance. For measuring water sorption, the samples were then kept in distilled water taken in an Eppendorf tube at 25 ± 1 °C and measured wet mass as m₂, at different time intervals (first after 24 hours then 3, 7, and 10 days). Weighing was carried out using an analytical balance (Shimadzu electronic balance) with 0.2 mg accuracy. The following equation was used for measuring water sorption (WS). (3) $$w_{\mathit{\text{sp}}}=\frac{m_2-m_1}{m_1}x\mathrm{ }100$$(3)

Where m₁ is the dry mass of the sample and m₂ is the wet mass of a specimen.

2.8. Anti-bacterial test

2.8.1. Disc diffusion method

To assess the antibacterial activity of CHX coated cotton cellulose fillers modified RBS, disc diffusion tests were performed. For this purpose, a preformed metal mold of about 6 mm in diameter and 2 mm in height was used for preparing the samples. The prepared samples were then disinfected and sterilized using Ultraviolet (UV) light: 256 micrometers for 60 minutes (Model no 54-O-432, ESCO private limited) on both sides (Farrugia et al., 2015). About 38 grams of Muller Hinton agar (OXOID, CMO 337) was measured using digital balance and then mixed in 100 ml of distilled water to make a solution. The solution was put in an autoclave to sterilize it (Model WAC-60). The agar was poured into petri dishes in the safety cabinet. The control group C and experimental groups (S1, S2 & S3) were then placed in Muller Hinton agar plates which were injected with 200 micro-liter bacterial solution containing S. mutans (ATCC 25175). Plates were kept warm at 37°Cabout for 48 hrs. and the zone of inhibition around the sample disc was measured in millimeters with the help of Vernier caliper (Al-Mosawi & Al-Badr, 2017).

3. Results

3.1. Scanning electron microscope (SEM) of natural cotton and CHX coated cotton powder

SEM of natural cotton powder showed irregularly shaped particles of different sizes which were in the range of 10-20 micrometers Figure 1. SEM of Chlorhexidine (CHX) coated cotton fillers showed irregularly shaped particles of different sizes with luminous appearance as compared to the pure cotton powder Figure 2. The particles were densely packed, showing that CHX was properly adsorbed.

Figure 1. (a) SEM of cotton powder at 700x (b) SEM of cotton powder at 1200x.

Figure 2. (a) SEM of CHX coated cotton powder at 750x (b) SEM of CHX coated cotton powder 1000x.

3.2. Fourier transform infrared spectroscopy (FTIR) cotton powder

FTIR spectrum for CHX powder presents a large band related to N-H stretch at 3220 cm^-1. The region between 1400 cm^-1 and 1750 cm^-1 relates to the CHX-coated cotton cellulose. Bands around 1640 cm^-1 and 1580 cm^-1 refer to C = N and N-H stretching vibration, respectively. The chloride bonded to the aromatic ring of the CHX was observed at 1095 cm^-1 as shown in Figure 3.

Figure 3. (a) Cotton, CHX, and CHX coated cotton powder.

3.3. Statistical analysis

The results of each test were analyzed with one-way ANOVA followed by Post Hoc Tukey’s test using GraphPad Prism 8.0.1 at a significance level of P < 0.05.

3.4. Flexural strength

The highest flexural strength was found in group S1 (125.9 ± 6.1 MPa) and the lowest flexural strength was found in S3 (64.7 ± 8.2 MPa), which showed that at low concentration flexural strength was increased. Similarly, for group C it was (117.5 ± 11.5 MPa) and for S2 it was (105.8 ± 5.5 MPa) as shown in Figure 4.

Figure 4. Flexural strength comparisons between Control group (C) and experimental groups (S1, S2, S3). The calculated mean flexural strength values with standard deviation error bars are expressed.

3.5. Compressive strength

The highest compressive strength was found in group S1 (416.9 ± 14.7 MPa). The lowest compressive strength was found in control group C (139.3 ± 14.9 MPa). This result indicates that there was a statistically significant difference between group C and S1 as shown in Figure 5.

Figure 5. Compressive strength comparisons between Control group (C) and experimental groups (S1, S2, S3). The calculated mean compressive strength values with standard deviation error bars are expressed.

3.6. Water sorption

The results for water sorption are shown in Figure 6. One-way ANOVA revealed significant differences in water sorption (P < 0.05) among the different groups. Water sorption showed an decreasing trend with an increasing proportion of fillers.

Figure 6. Graphical presentation of mean percent mass change (%) of group C and experimental groups (S1. S2, S3), after 1 day., 3 days, 7 days, and 10 days. The calculated mean % mass change values along with standard deviation error bars are expressed for group C and experimental groups (S1, S2, S3).

3.7. Anti-bacterial study

For measuring the antibacterial activities of S. mutans strain disc diffusion method was performed for both the control group C and experimental groups (S1, S2, and S3) containing 1, 2, and 3% of CHX coated cotton cellulose fillers modified resin-based sealant (RBS). There was no zone of inhibition for the control group C. Similarly, group S1 showed slight sensitivity. However, the group S2 and S3 were sensitive to the media. Group S1 showed a 1 ± 0.6 mm zone of inhibition, S2 showed a 5 ± 1.2 mm zone of inhibition and S3 showed an 8 ± 2.3 mm inhibition zone. Statistical analysis showed significant differences (p < 0.003) between group C and groups S2 and S3, Figure 7.

Figure 7. Graphical presentation of the mean zone of inhibition (mm) with standard deviation error bars for control group C and experimental groups S1, S2 &S3 after 24 hrs.

4. Discussion

The addition of antibacterial agents into resin based sealant (RBS) has sought to form a material with enhanced therapeutic properties to prevent dental caries. The present review mapped the studies to evaluate dental resin based sealants containing antibacterial agents.

4.1. Scanning electron microscope (SEM) of pure cotton and CHX coated cotton powder

In this study, the surface characteristics of pure cotton powder and as well as CHX coated cotton powder were performed through SEM study. The SEM images of cotton powder showed irregular particle structures arranged in a complex manner. Besides this, they were highly smooth and had varied sizes. This shows a typical structure of cellulose. The results of this study closely resemble the study conducted by Hassabo et al and Farahbakhsh et al in 2015., in which SEM of cotton powder was performed (Farahbakhsh et al., 2015; Hassabo et al., 2015). The SEM images of CHX coated cotton powder showed that CHX particles are properly adsorbed onto the cotton powder and give irregularly shaped particles of different sizes that were densely packed, and with a luminous appearance.

4.2. Fourier transform infrared spectroscopy (FTIR)

FTIR spectroscopy of non-invasive method was used to detect and recognize the chemical bonds and functional groups present in a specimen. It can measure both the absorbance as well as emission infrared spectrum which is recorded between wavenumber 4000 and 400 cm^-1. This technique is used to analyze the powder of CHX coated cotton fillers because it contains complex molecules and structural components of both the cotton and Chlorhexidine powder. So in this study, it was used to detect any possible molecular interactions between cotton cellulose and Chlorhexidine powder. The characteristic peaks obtained from FTIR data for cotton powder were similar to the previous studies reported (Yue et al., 2013). FTIR data shown in Chapter 4, indicates that all the groups in the study uniformly showed the peak at 4000-400 cm^-1 representing OH stretching vibration of the hydroxyl group of the cotton cellulose.

4.3. Mechanical properties

For improving the mechanical properties of dental resin based fissure sealant different fillers were incorporated by many researchers, which were obtained from natural and synthetic resources (Yadav et al., 2022). In this study, cotton cellulose fillers were used to enhance the mechanical properties (compressive and flexural strength) while CHX was added to enhance the anti-bacterial properties of commercial resin based sealants (RBS). The cotton cellulose fillers obtained from natural cotton contain approximately 79% cellulose and 13.7% lignin (Reddy & Yang, 2009).

In this study, maximum flexural strength was shown by group S1 which kept on decreasing with increasing concentration of cotton cellulose fillers, resulting in minimum flexural strength showed by S3. The values of flexural strength measured in this study were high as compared to the study conducted by Chung et al in 2004 which might be related to testing methods adopted or the composition of materials (Chung et al., 2004). Increasing the number of fillers decreases the quantity of normal constituents of resin based fissure sealants. All experimental groups showed a similar trend in the increase of flexural strength (high with a lower concentration of CHX coated cotton fillers and then decreased with an increased quantity of fillers). The cotton fillers usually increase the strength but the decrease in strength might be due to the slow release of CHX (Zhang et al., 2014). According to the literature, when CHX was used as an antibacterial agent in dental restorative material, it inhibited biofilm growth but due to its slow release, it leaves voids which results in decreased mechanical strength (Aydin Sevinç & Hanley, 2010). The results of the current study closely resembled a study done by Nishimura et al., 2019, they added cellulose nanofibers in Glass Ionomer Cement and concluded, an improved compressive and flexural strength (Nishimura et al., 2019). Similarly, the inclusion of a tiny amount of cellulose nanocrystals CNCs (0.2–0.4 wt. %) enhanced the mechanical properties of the material (Lai et al., 2022).

The compressive strength results showed the lowest strength in control group C (0% filler) which showed a mean value of (153.26 ± 56 MPa) and the highest compressive strength was found in group S1 (1% filler) which showed a mean value of (436.88 ± 45 MPa). As the concentration of CHX coated cotton filler was increased the compressive strength showed the reverse pattern. The compressive strength was decreased by increasing the concentration of filler content. Initially, the filler content increased the strength but as the filler fraction was increased, there was a decrease in strength which might be related to the mixing technique because there was a chance of air bubbles incorporation into the material during mixing. Similar finding was reported by Yadav and Kumar, (2020) which showed increase in compressive strength of restorative material by adding silanized silica up to 6% and subsequenty decreased to by adding 8%. These results closely resemble the result of a study conducted by Chung and Greener (1990). This decrease might be due to the fact, that the main constitutes of fissure sealants were lowered or it may be due to the improper mixing of filler content in commercial pits and fissure sealant material (Yadav et al., 2023).

In addition, the cotton cellulose fibers are opaque and durable while the CHX salt is transparent so in this case the cotton cellulose fibers did not allow light to pass through a transparent pit and fissure sealant which reduces the degree of conversion and consequently mechanical properties. Mechanical properties were influenced by the lengths of fibers, the ability of fibers to transfer force, and the fiber-resin interface. The surface energy and adhesion with the resin matrix were decreased by increasing the concentration of cotton cellulose fiber consequently affecting the mechanical properties of commercial pits and fissure sealant (Ranganathan et al., 2015). During the mixing of fibers in pit and fissure sealants care was taken to mix them uniformly but with an increasing concentration of cotton cellulose fibers, the varied distribution of fillers in the pit and fissure sealant cannot be ruled out (Nishimura et al., 2019). Secondly, cotton cellulose powder is hydrophilic and is less resistant to moisture when comes in direct contact with oral fluids (Halder et al., 2005). Before measuring the mechanical properties (i-e compressive & flexural strength) all specimens were kept in distilled water for about 24 hours at a temperature of 37 °C and in that case, the absorbed water acted as a plasticizer, therefore this fact cannot be ruled out, which in turn causes a decrease in mechanical properties. In this study, the cotton cellulose powder was prepared by acid hydrolysis and then passed through the ball milling and sieving process in the laboratory, to make a fine powder, which decreased its mechanical properties. According to the literature, no previous study was found in which natural cotton cellulose fibers were used in combination with Chlorhexidine and were added in pit and sealants. So the results of the current study cannot be compared.

4.4. Water sorption

Water plays a significant role in the chemical degradation of resin based material, which results in hydrolytic reaction and swelling of material. Therefore water sorption property of material is of great interest to the researchers (Saini et al., 2023). The resin matrix is responsible for water uptake in dental material and it is a diffusion-controlled process.

Water sorption is mainly related to solubility and composition of resin based sealant (RBS) in an aqueous condition (Mccabe & Rusby, 2004). However, there are numerous factors that affect water sorption such as the presence of filler content (Osorio et al., 2007). In this study in the case of fillers (CHX coated cotton cellulose) modified fissure sealant, the lowest water sorption values were obtained. Results showed a decreasing trend with an increasing mass fraction of filler. However, commercial pit and fissure sealants without incorporation of filler content showed the highest water sorption values. It was most likely because of the presence of hydrophilic filler content, which does cause water sorption.

4.5. Anti-bacterial properties

In this study, the disc diffusion method was used to evaluate the anti-bacterial properties of the control group C and experimental groups S1, S2, and S3 containing different concentrations of CHX coated cotton cellulose fillers. The results showed no zone of inhibition for control group C (0% filler). However, a minimum zone of inhibition of 1 ± 0.6 mm for experimental group S1 (1% fillers) was observed. Similarly, S2 and S3 showed 5 ± 1.2 mm and 8 ± 2.3 mm zone of inhibition respectively. However one of the major problems with incorporating antimicrobials or antibacterial agents into the monomer phase is its adverse effect on the mechanical properties of resin based fissure sealant. This result was similar to the result of the Cheng et al study conducted in 2012, which showed that Nano-CHX particles could be effective against S. mutans biofilm growth (Cheng et al., 2012). However one of the drawbacks of CHX was that it might decrease the mechanical properties (flexural strength) of fissure sealant by increasing the content of fillers, which was shown by Leikin et al in their study in 2008., he added 1% Chlorhexidine gluconate in composite material resulting in a reduction of tensile and compressive strengths (Leikin & Paloucek, 2008). The reason for this phenomenon might be related to the disturbance of monomers during curing or the interference of binding of the filler and matrix phases by the incorporated agent. Moreover, it is clear that the release of Chlorhexidine particles produces a porous structure in the material and the mechanical properties of composites containing soluble antimicrobial agents decrease over time (Imazato, 2003).

5. Conclusion

In this study, the addition of CHX coated cotton filler into pit and fissure sealants enhanced mechanical, water sorption, and antibacterial properties relative to commercial pit and fissure sealants without fillers. The addition of fillers increased the compressive and flexural strength of commercial resin based fissure sealant. By increasing the proportion of CHX cotton cellulose filler, the mean percent mass change declined after 10 days of immersion in water. Similarly, due to the presence of CHX, the antibacterial property against streptococcus mutants was increased. In conclusion, this research rejected the null hypothesis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work has been supported Department of Dental Materials, Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Khyber Pakhtunkhwa, Pakistan.