Introduction

Flowable resin composites have increased in popularity for restoring cavities due to their prolific properties and ease of handling characteristics. With such characteristics, the flowable composite materials have gained a wide range of usage for clinical applications, including preventive dental restoration, restoring cervical lesions, deciduous teeth, cavity lining, and pit and fissure sealing [1, 2]. However, the unfavorable property such as higher polymerization shrinkage has been reported to form gaps and voids, which eventually disturbs bonded interface [1,2,3]. In such scenario, complications such as secondary caries, fracture, sensitivity, and discolorations have been found [4] limiting the longevity of composites restorations.

In addition, among dental restorative materials, the resin-based composite has a higher tendency to accumulate more bacteria and dental plaque [5,6,7]. Surface roughness and free energy of the material are linked to the higher bacterial accumulation and bacterial enzymes formation, leading to marginal failure of the resin bonded restoration [8, 9]. Nevertheless, restorations typically fail within a span of 6–10 years, accounting for 60 to 70% of the clinician’s time and cost [9, 10]. Therefore, the approaches of formulating novel flowable composite materials to have superior antibiofilm properties are crucial in restorative dentistry to enhance their clinical longevity.

Studies [10,11,12] on metal nanoparticles such as silver and zinc oxide have been performed to enhance the durability of the resin-based flowable composite restorations due to their antibacterial properties. However, the antimicrobial efficacy of those metal nanoparticles was found to be short-lived [13, 14]. Therefore, for clinical applicability, designing flowable composites having a smart-drug delivery system, for long-term release is necessitated that aims to reduce incidence of secondary caries, antimicrobial resistance, and thus enhance the bonded restorations.

Chlorhexidine (CHX) is considered as the “gold standard” antibiotic/antiplaque agent against oral pathogenic microbiomes including S. mutans and is widely used to control oral infections [15,16,17]. Despite being a potent antibiotic/antiplaque agent, the incorporation of CHX in free form within resin-matrix has been reported to reduce the mechanical properties of the resin-based restorative materials [16]. CHX is insoluble with resin monomers, it forms aggregation and forms voids inside the resin matrix after dissolution, which further increases the incidence of bacterial accumulation [18].

Mesoporous silica nanoparticles (MSN), on the other hand, have shown a massive interest in the field of dentistry as a nanocarrier, due to their characteristic properties including biocompatibility, large pore volume, and surface area. The hollow structure provides a reservoir to encapsulate different types of molecules controlling release for more advance targeted treatment [19,20,21]. Yan et al. developed a glass ionomer cement using CHX and MSN as a drug and a nanocarrier, respectively. The resulting composition showed a potent antibiofilm properties [22]. Inspired from this, the main aim of this study was to modify the flowable resin composite by incorporating chlorhexidine-loaded mesoporous silica nanoparticles (CHX-MSN) and investigate the antibiofilm and mechanical properties of newly formed nanocomposites. The null hypothesis of this study is that modified flowable composite with synthesized nanoparticles will not show bactericidal properties.

Materials and methods

Formulation of CHX loaded MSN nanoparticles

Mesoporous silica nanoparticles were synthesized according to previously described protocol with slight modification [21]. Briefly, 1 g of N-cetyltrimethylammonium bromide (CTAB; Sigma-Aldrich, Australia), a 7 ml of mesitylene (TMB; Sigma-Aldrich, Australia) used as a porosity template were added into a container contained 450 ml of distilled water and 3 ml of sodium hydroxide (2 N). The obtained mixture was kept stirring for 4 h at 80 °C. After that, 5 ml of tetraethyl orthosilicate (TEOS; Sigma-Aldrich, Australia) was added and stirred further for 2 h. The resultant precipitate was centrifuged and washed thrice with ethanol, followed by overnight oven drying at 60 °C. The dried powder was then calcinated at 550 °C for 5 h.

To encapsulate CHX into the synthesized MSN nanoparticles, 10 mg of base CHX (digluconate, M.W: 505.45 g/mol, Sigma-Aldrich, Australia) was dissolved in 1 ml of absolute ethanol and mixed with 50 mg of synthesized MSN nanoparticles sonicated for 30 min and stirred for 24 h at room temperature. The formed CHX-MSN mixture was centrifuged, vacuum oven dried, and stored at room temperature until further use.

Drug loading and encapsulation efficiency

Supernatant was used to determine the drug loading content. The amount of loaded CHX was analyzed by UV–Vis spectrophotometer (UV-1900i, Shimadzu, Japan) at 289 nm wavelength. The percentage of drug loading content and encapsulation efficiency were measured using the following formulas (1) and (2):

$$Encapsulation\;efficiency\;(\%) = initial\; wt.\;of\;CHX - free\;non-entrapped\;CHX\;/\;initial\;wt.\;of\;CHX\times\;100$$
(1)
$$Drug\;loading\;content\;(\%) = initial\;wt.\;of\;CHX-encapsulated\;CHX\;/\;total\;wt.\;of\;CHX\;-\;MSN\;\times\;100$$
(2)

Characterization of the formulated CHX-MSN nanoparticles

Morphological characterizations

The morphological features of the MSN nanoparticles were investigated under Scanning electron microscope (SEM; Verios XHR; Thermo Fisher Scientific, USA). The synthesized nanoparticles were sprinkled on the SEM stub and coated with platinum and the images were acquired at an accelerating voltage of 5.0 kV. The particle size was calculated using Image J software.

Further confirmation of MSN and CHX-MSN nanoparticles was done by Transmission electron microscope (TEM; JEOL-200, Japan) at 200 kV coupled with energy-dispersive X-ray spectroscopy (EDS) for elemental analysis. For TEM analysis, the synthesized nanoparticles were suspended into pure ethanol and a drop of the suspension was loaded onto the copper mesh grid carbon-coated (200 mesh; Emgrid, Australia) and allowed to air dry at room temperature. The TEM images of the dried specimens were obtained with a Gatan OneView camera and analyzed using ImageJ software.

Fourier-transform infrared spectroscopy

The spectral analysis of the synthesized nanoparticles was performed using Fourier-transform infrared spectrometer (FTIR; IR Spirit, Shimadzu, Japan) coupled with attenuated total reflectance (ATR). The chlorhexidine-loaded (CHX-MSN) and the unloaded chlorhexidine (MSN) mesoporous silica nanoparticles, respectively, were placed onto the diamond crystal and compressed with an anvil. The spectrum was then acquired in the range of 400–4000 cm−1, at a resolution of 4 cm−1 and analyzed using IR LabSolutions software.

Formulation of the modified experimental flowable composites

The unmodified commercially available flowable resin composite (FRC; 3 M ™ Filtek ™, Australia) was used as the control. The experimental groups were set at three different concentrations by incorporating CHX-MSN nanoparticles into the commercial FRC (3 M ™ Filtek ™, Australia) at 1%, 5%, and 10% (w/w), mixed homogenously with a spatula on a glass slab. It was then transferred into a mould (Ø 10 mm, H 2 mm) to ensure consistency in the dimensions and light cured with 315–500 nm LED (Bluephase 20i LED, Ivoclar, Vivadent, USA) for 40 s, with an intensity of 1200 mW cm2.

Specimens ageing

The specimens from the control and experimental groups (1% CHX-MSN, 5% CHX-MSN, and 10% CHX-MSN) were aged in artificial saliva at 37 ℃, and then evaluated the long-term effect on the antibiofilm and mechanical properties. For sustainable antibiofilm properties, specimens were evaluated after 1 month of storage in artificial saliva. The artificial saliva was prepared following a previously described protocol: 0.103 g of calcium chloride anhydrous, 0.544 g of potassium dihydrogen phosphate, 0.019 g of magnesium chloride hexahydrate, 2.24 g of potassium chloride, 2 g of sodium azide and 4.77 g of hydroxyethyl-piperazineethanesulfonic acid were dissolved into 1 L of distilled water, and pH were adjusted to 7.0 [22]. The artificial saliva for all the groups was replenished every 2 weeks.

Characterization of modified experimental flowable resin-composites

Degree of conversion

The degree of conversion (DC) of the control and the experimental specimens was measured using FTIR spectroscopy. Each specimen (Ø 10 mm, H 2 mm) was placed on top of diamond crystal, and pressure was exerted with an anvil to make contact between the sample and diamond crystals. Multiple sites of each specimen were measured in the wavelength ranging from 1500 to 1700 cm−1 at the resolution of 4 cm−1 (n = 5). DC was determined using the baseline approach of the aliphatic (C = C) absorbance peak at 1638 cm−1 and aromatic (C–C) reference peak at 1607 cm−1 and calculated using following formula [23]:

$$\%\;DC = [1\;-\;(Caliphatic/Caromatic)/(Ualiphatic/Uaromatic)]\;\times\;100$$
(3)

where Caliphatic and Caromatic are the peak absorption of the polymerized resin at 1638 cm−1 and 1607 cm−1 respectively, while Ualiphatic and Uaromatic are the peak absorption of the unpolymerized resin at 1638 cm−1 and 1607 cm−1 respectively.

Evaluation of CHX release profile from the experimental composites

To evaluate the CHX release profile from each of experimental group (n = 5), the specimens were immersed in 5 ml of phosphate buffered saline (PBS; Sigma-Aldrich, Australia) at pH 7.4 and stored in an incubator at 37 °C for different time-points (1, 5, 10, 15, 20, 25, and 30 days). At each time point, 2 ml of the solution was withdrawn and replaced with PBS. The extract aliquots were analyzed to determine CHX release by using UV–Vis spectrophotometer (UV-1900i, Shimadzu, Japan) at the wavelength of 289 nm. The cumulative release profile of CHX from each experimental specimen was plotted.

Cytotoxicity evaluation of the experimental composites

The biocompatibility of all the specimens was assessed using human oral fibroblast cells (HOrF; ScienCell Research Laboratories, USA) by indirect method as described in Aati et al. [24]. The viability of the cell against control and experimental groups (n = 9/group) was assessed by acid phosphatase (APH) assay. Briefly, baseline and aged (1 M and 3 M) specimens (Ø 10 mm, H 2 mm) of the control and experimental groups were sterilized under ultraviolet light and then immersed in the Fibroblast Medium (FM; ScienCell Research Laboratories, USA) at 37 °C for 24 h to obtained extracts of each group. The HOrF cells (5th passage), were culture in FM supplemented with 2% fetal bovine serum, 1% fibroblast growth supplement and 1% antibiotics (penicillin/streptomycin) and placed in an incubator under 5% CO2 at 37 °C. The cultured cells were then seeded at a density of 1 × 104 cells/ml in sterilized 96-well plate and incubated for 24 h under 5% CO2 at 37 °C. Later, the cell culture medium was discarded and 200 µl of resin extracts were added. After incubation for 24 h, the supernatant was discarded and 150 µl of APH buffer was added in each well and plates were incubated under 5% CO2 at 37 °C for 1.5 h, followed by addition of 20 µl of 1 M of NaOH and incubated for 10 min. Then the optical density (OD) values were measured using microplate reader (Sunrise™, Tecan, Switzerland) at the wavelength of 405 nm. The percentage of cell viability was calculated using the following formula:

$$Cell\;viability\;(\%)\;=\;OD\;of\;the\;test\;group\;/\;OD\;of\;the\;control\;group\;\times\;100$$
(4)

Evaluation of antibiofilm properties of the experimental flowable composite

Biofilm formation

The biofilm of S. mutans strain ATCC 700610 (Invitro technologies, Australia) was formed on a substrate as previously described with slight modification [25]. Initially, 1–2 colonies from the brain heart infusion (BHI) agar plate were transferred to a falcon tube containing 10 ml of BHI broth (Sigma-Aldrich, Australia) and incubated at 37 °C for 24 h anaerobically, followed by centrifugation of the bacterial culture at 3000 rpm for 15 min to obtain the pellet. The supernatant was discarded, and the cells were resuspended in PBS and adjusted to 0.5 MacFarland (108 CFU/ml) cell concentration using McFarland densitometer (DEN-1, Fisher biotec, Australia). The baseline and aged specimens (Ø 10 mm, H 2 mm) were placed in a 24-well plate sterilized under ultraviolet light and inoculated with 100 µl of S. mutans in a BHI suspension plus 900 µl of BHI broth supplemented with 1% sucrose. The plate was then incubated at 37 °C for 7 days to obtain biofilm and maintained by replacing media every 48 h. Later, the specimens were washed thrice with PBS to remove non-adherent bacterial cells before evaluations.

Crystal violet assay

Specimens of each group coated with biofilm were evaluated using crystal violet assay as previously described with slight modifications (n = 5) [26]. The biofilm attached specimens of each group were placed on 24 well plates and 500 µl of aqueous 0.1% crystal violet (CV; Sigma-Aldrich, Australia) were added into each well and incubated for 15 min. The CV solution was then gently aspirated and washed thrice with PBS, followed by solubilizing with 30% acetic acid. Finally, the CV was quantified by measuring the absorbance at a wavelength of 590 nm using microtiter plate reader (Sunrise™, Tecan, Switzerland).

MTT assay

The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide kit (MTT; Sigma-Aldrich, Australia) was used to investigate the bacterial viability of the control and experimental group (n = 5). The biofilm coated disc from each group were placed into 24 well plate, followed by addition of 500 µl of MTT reagent. The plates were then incubated for 4 h at 37 °C. The reagent was aspirated, then replaced with 500 µl of solubilizing solution and incubated overnight at 37 °C. The absorbance of the supernatant was than measured with a micro plate reader (Sunrise™, Tecan, Switzerland) at 570 nm.

Confocal laser scanning microscopy

The microscopic evaluation of the specimens from each group coated with biofilm was performed using confocal laser microscopy (CLSM; Nikon A1Si Confocal Microscope, Japan). The biofilm coated specimens (n = 5) were then stained by the LIVE/DEAD BacLight bacterial viability kit stain (Invitrogen, USA) for 15 min. The kit included propidium iodide and SYTO-9 which stains dead bacteria and live bacteria, respectively. Each specimen was gently washed with PBS to remove excess stains and fixed with 4% paraformaldehyde and then imaged using CLSM.

Evaluation of mechanical properties of the experimental flowable composite

Microhardness

The surface microhardness of the control and experimental specimens was performed using Vickers microhardness tester (VH, Duramin-40, Australia). The microhardness of the specimens from each group was evaluated immediately at 24 h (n = 5). The specimens were subjected to a load of 200 g and a dwell time of 20 s using diamond intender with a pyramid shaped and a specified angle [27]. On each specimen’s surface, six areas were randomly selected and tested. After initial measurement, the samples were immediately stored in artificial saliva to simulate oral physiological environment and placed in an incubator at 37 °C for 3 months. After 3 months, the specimens were tested again with the same parameters as used for initial measurement. The outcome was reported as the average of each specimen’s indented locations.

Flexural strength

The specimens from each group (n = 10) were tested according to ISO 4049 and evaluate at two-time points (baseline and aged). Specimens were fixed on a 3-point fixture with a 20-mm supporting distance (ElectroPuls™ E3000, Instron, UK), a load was applied until fracture occurred at a crosshead speed of 1 mm/min. The flexural strength (FS) was calculated using the formula [28]:

$$FS = 3 \times F \times L \div 2 \times b \times h2$$
(5)

where F is the failure load (N), L is the distance (20 mm) between the supports, b is the width (mm) and h is the height of the specimen (mm).

Statistical analysis

The normal distribution of data was investigated through the Shapiro–Wilk test. Data were shown as means and standard deviations. The statistical package for the social sciences (SPSS) software (version 23.0, USA) was used to analyze data and the significant effects of variables were determined by one-way ANOVA followed by Tukey’s post hoc for pairwise comparison at a p-value < 0.05 to determine level of significance difference.

Results

Characterization of synthesized nanoparticles

Figure 1a shows formulated MSN nanoparticles with spherical-shaped particles with the particle size of around 100 nm. TEM images of MSN revealed nanoparticles with typical spherical, or elliptical-shaped particles and honeycomb like hexagonal shaped mesopores, confirming MSN nanoparticle formation, as shown in Fig. 1b. After CHX loading, the honeycomb-shaped mesopores of MSN became less clearly defined, as shown in Fig. 1c. To further validate the CHX-loading into the MSN nanoparticles, EDS was performed. The presence of Si, O2, Cl, and N in CHX-loaded MSN nanoparticles shown by EDS spectrum confides the CHX-loading into the MSN nanoparticles, as shown in Fig. 1e.

Fig. 1
figure 1

Shows SEM images (a) of synthesized MSN; TEM (b, c) images of synthesized MSN and CHX loaded MSN (CHX-MSN) nanoparticles; (d, e) TEM-EDS elemental analysis of the MSN and CHX-MSN, respectively. The orange arrow indicates honeycomb appearance of synthesized mesoporous silica nanoparticles and red arrow represents presence of Cl and N, confirming successful synthesis of CHX-MSN nanoparticles. (f, g) Schematic representation of mesoporous silica (MSN) and CHX loaded mesoporous silica nanoparticles (CHX-MSN), respectively. The blue circle represents CHX

Full size image

The representative FTIR spectra of CHX-MSN and MSN are shown in Fig. 2. The distinctive peaks at 455 cm−1, 794 cm−1, and 1050 cm−1, belongs to Si–O-Si bending, symmetrical stretching, and asymmetrical stretching, respectively, confirming the formation of MSN nanoparticles in the final product [29, 30].The pure CHX spectrum displayed a prominent peak at 1084 cm−1, 1240 cm−1, and 1668 cm−1 [31].The distinctive narrow vibrational band at 1668 cm−1 and 1598 cm−1 assigned to aromatic C = N bending and -NH stretching of CHX [32] can be observed in CHX loaded MSN formulations, as shown in Fig. 2. After confirming the successful synthesis of CHX-MSN nanoparticles, the drug loading capacity of the CHX-MSN nanoparticles was determined. The result revealed that the maximum amount of CHX loaded in MSN nanoparticles was 177 µg/mg, with an encapsulation efficiency of 65.54%.

Fig. 2
figure 2

Represents the FTIR spectra of the chlorhexidine (CHX), formulated mesoporous silica nanoparticles (MSN), and CHX loaded mesoporous silica nanoparticles

Full size image

Release profile of CHX-MSN experimental modified flowable composite

Figure 3 depicts the concentration of the cumulative release of CHX from each experimental group modified with different concentrations of CHX-MSN. All the experimental groups (1%, 5%, and 10% CHX-MSN) showed rapid release of CHX on day 1 and 5, followed by a decrease in the CHX release behavior and was relatively stable until day 30 for all the experimental groups. Compared to the cumulative release amount of CHX of all the experimental groups, the trend of higher CHX release was observed when resin was doped with higher concentration (10%) CHX-MSN.

Fig. 3
figure 3

The cumulative in vitro CHX releasing behavior from the experimental modified flowable resin-composites (1%, 5%, and 10% CHX-MSN groups) after storing in PBS solution at 37 ℃ at different time points

Full size image

Degree of conversion evaluation

The values of degree of conversion (DC) of the control and the experimental specimens are demonstrated in Fig. 4. When compared to the control group, the DC values of the experimental specimens doped with the CHX-MSN at the lowest concentration (1%) were nearly identical. The experimental group incorporated with 5% CHX-MSN exhibited no significant change when compared to the control group (p > 0.05). However, the addition of 10% CHX-MSN into the resin significantly reduced the DC values (p < 0.05).

Fig. 4
figure 4

Demonstrates the degree of conversion of the control and experimental modified groups; a significant decrease in DC was observed with specimens doped with 10% CHX-MSN. Dissimilar letters indicate significant difference of (p < 0.05)

Full size image

Cytotoxicity evaluation

Figure 5 illustrates that the cell viability of each group increased after ageing compared to the baseline, indicating the biocompatibility of the experimental specimens. The highest cell viability percentage for three-time points was demonstrated by specimens doped with 5% CHX-MSN. At 1 month, although 10% CHX-MSN demonstrated a statistically significant dropped in cell viability than 5% CHX-MSN (p < 0.05), its value was still greater than 80%. Moreover, at 3 months of ageing 5% CHX-MSN displayed significantly higher cell viability compared to the control and experimental group (1% and 10% CHX-MSN). Overall, the viability of cell exposed to the extracts of experimental groups for three-time points was greater than 80% suggesting low cytotoxicity profile of the experimental specimens according to ISO standards 10993–5 [33].

Fig. 5
figure 5

Shows the viability of human oral fibroblast cells after being exposed to the extracts of control and experimental modified groups (baseline and aged) by APH assay at different time points i.e., baseline, 1 month (1 M), and 3 months (3 M). Dissimilar letters indicate significant difference of p < 0.05. (*) represents significant difference within group

Full size image

Antimicrobial evaluation

The antibiofilm efficacy of the experimental groups with different concentrations of CHX-MSN nanoparticles against S. mutans is presented in Fig. 6. The highest crystal violet absorbance was demonstrated by the control compared to all the experimental groups (Fig. 6b) in both time-points, indicating significantly higher bacterial adhesion on the surface of the control group for both time-points. The experimental group with 10% CHX-MSN showed the lowest absorbance value suggesting reduction of biofilm biomass by almost half compared to the control group for both times. To further validate the antibiofilm potential of the experimental groups with respect to bacterial viability, MTT assay was performed. For both time points, the relative microbial viability significantly decreased with the increase in the concentration of CHX-MSN nanoparticles (p < 0.05) (Fig. 6c). The highest antibiofilm characteristic was observed in experimental group doped with 10% CHX-MSN, indicating the lowest metabolic activity of the adhered biofilm on the resin specimens. Compared to baseline, although the antimicrobial capacity of all the experimental groups has decreased, 5% and 10% CHX-MSN groups presented significantly low biofilm viability when compared to the control (p < 0.05) (Fig. 6c).

Fig. 6
figure 6

Shows the antibiofilm efficacy of the experimental groups against S. mutans. (a) Schematic illustration of formation of biofilm on experimental modified flowable resin-composite specimens. (b) Crystal violet assay showing the total biomass of S. mutans on the surface of control and experimental specimens; (c) relative viability of S. mutans biofilm. Dissimilar letter indicates significant difference at p < 0.05. (*) represents statistical significance. (d–g) CLSM images showing the distribution of live and dead bacterial cells of the control and experimental groups. The live cell (Green stain — live) was observed in the control specimens with almost no dead bacterial cells indicates no inherent antibiofilm efficacy (d), while all the experimental specimens showed dead cells (Red stain — dead) in varying degree; (e) 1%, (f) 5%, and (g) 10% of CHX-MSN addition. Scale bar represents 100 µm

Full size image

For additional confirmation, qualitative analysis using CLSM imaging was conducted to investigate the viability and the adherence of S. mutans biofilm on the specimens of the control and the experimental groups (Fig. 6d–g). The thick green biomass of the S. mutans biofilms with almost no red colonies were observed in the control group (Fig. 6d). Within experimental groups, 1% CHX-MSN showed mixture of red and green colonies (Fig. 6e), 5% CHX-MSN illustrated more red colonies with some surviving green colonies of bacteria (Fig. 6f), and the highest bacterial cell death (red stain) was observed with the experimental group doped with 10% CHX-MSN (Fig. 6g). Consistent results were observed with both qualitative and quantitative analysis.

Mechanical characterizations

Figure 7a depicts that at baseline, there was a significant increase in Vickers microhardness value of experimental group with 10% CHX-MSN. However, after ageing, the group with 5% CHX-MSN showed the highest microhardness value compared to the control. Furthermore, before and after ageing, the significant changes in the microhardness values were shown by the group loaded with 10% CHX-MSN (p < 0.05).

Fig. 7
figure 7

Illustrates the mean and standard deviation of control and experimental modified flowable resin composite doped with (1%, 5%, and 10% CHX-MSN) for (A) microhardness and (B) flexural strength. Dissimilar letter indicates significant difference at (p < 0.05). (*) represent statistical significance within the group at different time points (i.e., baseline and aged)

Full size image

Figure 7b shows the flexural strength (FS) for all groups at baseline and after 3 months of incubation in the artificial saliva. At baseline, with the increase in concentration of nanoparticles, the FS values of experimental groups significantly decreased (p < 0.05). Notably, after ageing, the FS of the control group reduced significantly (p < 0.05) and no significant difference between the control and the experimental groups (1 and 5%) was observed (Fig. 7b). Interestingly, the experimental modified group doped with 5% CHX-MSN showed no significant changes in the FS values between two time-points (p > 0.05).

Discussion

The present study showed the development of modified flowable restorative resin nanocomposites with enhanced antimicrobial properties against S. mutans bacterial biofilms. The CLSM imaging, crystal violet, and MTT assay demonstrated that flowable resin-composite doped with CHX-MSN nanoparticles inhibited S. mutans biofilm and that it’s antibiofilm efficacy increased with the rise in the concentration of CHX-MSN nanoparticles. The surface microhardness property of the experimental flowable resin-composite was further significantly enhanced when CHX-MSN was incorporated at the concentration of 5% even after 3 months of ageing.

Mesoporous silica nanoparticles (MSN) were synthesized with an addition of a pore-expanded agent (mesitylene) as previously described [21]. The TEM analysis revealed that MSN structure had spherical or elliptical shape with characteristic highly regular honeycomb porosity (Fig. 1b). The distinct structure of mesopores, however changed and became less defined after CHX loading (Fig. 1c), implying CHX adsorption on the MSN surface and into the mesopores [19]. The successful formulation of CHX-loaded MSN nanoparticles was further verified by FTIR where in it displayed distinct sharp peaks at 794 cm−1, which were assigned to MSN and the characteristic peaks at 1250 cm−1 ascribed to CHX, and distinctive peaks between 1084 cm−1 and 1240 cm−1 were allocated to CHX-loaded MSN nanoparticles (Fig. 2) [29,30,31,32].

To monitor the behavior of drug release from the modified flowable composite doped with different concentration of CHX-MSN at various time-points, the specimens were immersed into PBS solutions having pH of 7.4. The idea behind studying at pH 7.4 is to mimic the pH of normal body fluids (blood and saliva). Figure 3 showed a short initial burst release of CHX followed by a sustainable continuous steady release even until a month. Furthermore, the releasing pattern was dependent on the amount of drug loaded into the flowable resin composite. Similar, sustainable dose-depended CHX releasing behavior was demonstrated by Yan et al. [21] when CHX-MSN nanoparticles were incorporated into glass ionomer cement. The aforementioned sustainable release kinetic mechanism may be attributed to electrostatic interaction developed between negatively charged MSN surface because of the dissociation of surface –OH groups and positively charged CHX molecules [34].

The inhibition of biofilm formation with sustainable drug release and antibacterial action against S. mutans could introduce the potential application of the experimental modified flowable resin composites in restorative dentistry. Because S. mutans produces higher organic acid and esterase enzyme, making them potent contributor for biodegradation of resin composite by hydrolyzing resin monomers [35, 36]. More specifically, the total biomass of bacterial biofilm was significantly reduced by the addition of CHX-MSN into experimental specimens even after 30 days (Fig. 6b). This observation indicates a lower number of bacteria being attached on the surface of specimens doped with CHX-MSN. Since crystal violet assay may also stain the extracellular matrix in addition to the bacterial cell wall and therefore measures combine live and dead bacteria [37]; thus, MTT assay and CLSM imaging was performed to determine the actual viable bacterial population on the surface of the specimens. The CLSM displayed a potent antibiofilm efficacy of the experimental groups compared to the control (Fig. 6d–g). The significant bacterial cell death was observed with highest concentrations (5% and 10% CHX-MSN), which is consistent with the previous study [21]. Furthermore, a statistically significant decrease in bacterial viability was apparent with the modified composite with 5% and 10% CHX-MSN even after ageing for 1 month when compared to the control (Fig. 6c). This entails firstly the long-term antibiofilm capacity of the experimental composite, this result was consistent with previous study [21]. Secondly, the cationic molecule CHX is a potent antibacterial drug against S. mutans because of its higher affinity towards negatively charged bacterial cell wall, consequently, interferes with osmosis followed by infiltration into the cytoplasm by rupturing the cell wall and causing cell death [38, 39].

The degree of conversion should not be interfered with the addition of CHX-MSN nanoparticles as it has a significant effect on the physio-mechanical, biological properties, and durability of the cured composited resins [23]. The degree of conversion values (Fig. 4) showed no significant difference between the control and the experimental groups doped with 1% and 5% CHX-MSN. However, experimental group doped with 10% CHX-MSN significantly reduced when compared with the control. The decrease in the DC values of experimental group with highest concentration of CHX-MSN could be attributed to the agglomeration of nanoparticles which may interfere with the polymerization process and hinder penetration of photons [40]. Cytotoxicity was performed using human oral fibroblast cells utilizing APH assay to determine biocompatibility of the modified experimental specimens. As it is well documented that an excessive concentration of CHX can trigger cell death, leading to unwarranted side effects [41]. The result showed that 10% CHX-MSN aged (1 M and 3 M) specimens had significant decrease in cell viability in comparison to the control and low concentration (1% and 5%) modified specimens (Fig. 5). The reduction in viability with 10% experimental group might be associated with degree of conversion findings (Fig. 4). As at higher concentration of CHX-MSN in resin resulted in significant decrease in value of degree of conversion leaving unpolymerized monomers consequently causing toxic response [42, 43]. Nevertheless, the percentage of cell viability demonstrated by 10% CHX-MSN is still above 80%, indicating slight cytotoxicity according to ISO standards 10,993–5 [33]. Moreover, the cell viability of each group increased after ageing compared to baseline, indicating that the cell proliferated on the process of culture and hence biocompatibility.

To be clinically acceptable, resin composites should exhibit sufficient mechanical properties to withstand occlusal stress along with antibacterial properties. The result from the present study showed that the addition of CHX-MSN into the resin composite significantly increased in the surface microhardness values compared to the control (Fig. 7a), indicating enhanced resistance against abrasion. This could be due to the smaller size of nanoparticles, which have a larger surface area and hence have a large contact area with resin, resulting in better dispersion of filler [44]. Interestingly, among the experimental groups, 5% CHX-MSN group showed enhanced microhardness values in both the time points (Fig. 7a). One possible explanation for the enhanced microhardness value of the 5% CHX-MSN group is the lesser aggregation caused by the uniform dispersion of nanoparticles. This consistent distribution of nanoparticles inhibits filler loss from the composite resin, increasing its stability, as evidenced by the hardness values after ageing. Higher concentrations, on the other hand, can cause agglomeration, which can interfere with curing [40] and eventually result in loss of filler from the poorly cured composite resin, resulting in diminished hardness with age. On the other hand, the resin loaded with CHX-MSN had unfavorable effect on its flexural properties, i.e., the higher the concentration of nanoparticles in the resin, the lower the flexural strength (Fig. 7b), which was consistent with the previous study [11]. One of the plausible explanations may be that the higher concentration of nanoparticles forms large agglomeration, which contribute to structural defects and flaws, which in turn favor stress concentration and mechanical failure even under early-stage flexural loading. However, when comparing baseline and aged samples, the experimental group with 5% CHX-MSN showed an insignificant change compared to the control, indicating the stability of the materials when exposed to oral fluids for long duration.

The limitation of this study is associated with agglomerations of the nanoparticles with their concentration. This is one of the important factors to influence the physico-mechanical properties of the resin-based flowable composite. Therefore, further optimization should be performed in regard to viscosity and flowability, to ensure efficient dispersal of nanoparticles into the resin. Moreover, although the present study showed mechanical stability with 5% CHX-MSN over time in an artificial saliva compared to the control. However, the value of flexural strength decreased with increase in concentration, therefore, more in vitro mechanical study should be conducted.

Conclusion

The present study shows that flowable resin-composites modified with drug-loaded nanoparticles (CHX-MSN) displayed significant enhancement in antibiofilm properties against S. mutans. In addition, the modified composites possessed acceptable biocompatibility without adversely affecting mechanical properties and degree of conversion up to 5% addition of CHX-MSN nanoparticles. This study introduced a protocol to develop resin-based flowable dental composite material having an inherent superior antibacterial property against dental cariogenic biofilms aiming to enhance the clinical longevity of dental restorations.