Antibacterial and physical properties of calcium-phosphate and calcium-fluoride nanocomposites with chlorhexidine

Abstract

Objectives

Previous studies have developed calcium phosphate and fluoride releasing composites. Other studies have incorporated chlorhexidine (CHX) particles into dental composites. However, CHX has not been incorporated in calcium phosphate and fluoride composites. The objectives of this study were to develop nanocomposites containing amorphous calcium phosphate (ACP) or calcium fluoride (CaF₂) nanoparticles and CHX particles, and investigate S. mutans biofilm formation and lactic acid production for the first time.

Methods

Chlorhexidine was frozen via liquid nitrogen and ground to obtain a particle size of 0.62 µm. Four nanocomposites were fabricated with fillers of: Nano ACP; nano ACP+10% CHX; nano CaF₂; nano CaF₂+10% CHX. Three commercial materials were tested as controls: A resin-modified glass ionomer, and two composites. S. mutans live/dead assay, colony-forming unit (CFU) counts, biofilm metabolic activity, and lactic acid were measured.

Results

Adding CHX fillers to ACP and CaF₂ nanocomposites greatly increased their antimicrobial capability. ACP and CaF₂ nanocomposites with CHX that were inoculated with S. mutans had a growth medium pH > 6.5 after 3 d, while the control commercial composites had a cariogenic pH of 4.2. Nanocomposites with CHX reduced the biofilm metabolic activity by 10–20 folds and reduced the acid production, compared to the controls. CFU on nanocomposites with CHX were three orders of magnitude less than that on commercial composite. Mechanical properties of nanocomposites with CHX matched a commercial composite without fluoride.

Significance

The novel calcium phosphate and fluoride nanocomposites could be rendered antibacterial with CHX to greatly reduce biofilm formation, acid production, CFU and metabolic activity. The antimicrobial and remineralizing nanocomposites with good mechanical properties may be promising for a wide range of tooth restorations with anti-caries capabilities.

1. Introduction

Nearly 200 million dental restorations are placed annually in the USA [1]. Resin composites are increasingly used for dental caries restorations because of their esthetics and direct-filling capability [2–8]. Remarkable progress has led to esthetic composite restoratives with less removal of tooth structures, enhanced load-bearing properties, and improved clinical performance [9–15]. However, secondary caries at the restoration margins is identified as a main limitation to the longevity of the restorations [16–18]. The replacement of existing restorations accounts for 50% to 70% of all restorations performed [19,20]. Replacement dentistry costs $5 billion annually in the U. S. alone [21]. Dental caries is a dietary carbohydrate-modified bacterial infectious disease, and is one of the most common bacterial infections in humans [22–24]. The basic mechanism of caries is demineralization of dental tissue (enamel/dentin) via acid generated by bacterial biofilms (dental plaque) [25–27]. Acidogenic bacterial growth and biofilm formation in the presence of fermentable carbohydrates are known to be responsible for caries. However, resin composites do not hinder bacteria colonization and plaque formation. On the contrary, previous studies have shown that composites allow more accumulation of plaque on their surfaces than other restoratives [28–30].

Efforts are underway to develop novel antibacterial composites to reduce caries. One approach involves the incorporation of antibacterial monomers to decrease the viability of bacteria such as Streptococcus mutans (S. mutans) [4,29,31]. In one study, a polymerizable bactericidal monomer, 12-methacryloyloxydodecylpyridinium bromide (MDPB), was immobilized in the resin which reduced bacteria growth via contact inhibition [4]. In another, ionic liquid dimethacrylate monomers that contained quaternary ammoniums groups were used to develop antimicrobial resins, which reduced bacterial colonization [32]. Other studies incorporated chlorhexidine (CHX) particles as fillers into dental resin composites, which resulted in CHX release and reduced bacteria growth [33–35]. CHX particles were also mixed into glass ionomer cements [36], thus combining antimicrobial activity with fluoride ions (F) [37].

Another promising class of composites consists of resins filled with calcium phosphate (CaP) particles of about 1 µm to 55 µm in sizes [38–40]. These composites released supersaturating levels of calcium (Ca) and phosphate (PO₄) ions and remineralized tooth lesions in vitro [39,40]. Recently, CaP nanoparticles of about 100 nm in size were synthesized via a spray-drying technique for the first time [41,42]. Composites containing CaP nanoparticles with high specific surface areas were found to release high levels of Ca and PO₄ while possessing mechanical properties nearly two-fold those of previous CaP composites [41,42]. Nanocomposites containing CaF₂ nanoparticles were also developed that released fluoride (F) ions matching that of a resin-modified glass ionomer [43,44]. The mechanical properties of the CaF₂ nanocomposite were much higher than that of resin-modified glass ionomer, and matched those of commercial composites with little F release [43,44]. However, there has been no report of CaP and CaF₂ nanocomposites containing CHX to achieve the triple benefits of remineralization, antibacterial, and load-bearing capabilities.

Therefore, the objectives of this study were to combine CHX with CaP or CaF₂ nanoparticles into the nanocomposites, and to determine the mechanical and antibacterial properties. It was hypothesized that: (1) incorporating CHX into CaP and CaF₂ nanocomposites will impart a potent antibacterial capability to diminish S. mutans biofilm viability and therefore reduce the acid production; and (2) the mechanical properties of the CaP and CaF₂ nanocomposites containing CHX will match those of commercial composites that have no ion release or antibacterial capability.

2. Materials and methods

2.1. Fabrication of CaP and CaF₂ nanocomposites containing CHX

Nanoparticles of amorphous calcium phosphate (ACP), Ca₃(PO₄)₂, were synthesized via a spray-drying technique [45]. ACP is an important compound because it is a precursor that can convert to apatite, similar to the minerals in tooth enamel and dentin. A spraying solution was prepared by dissolving calcium carbonate (CaCO₃, Fisher, Fair Lawn, NJ) and dicalcium phosphate anhydrous (CaHPO₄) (J. T. Baker, Phillipsburg, NJ) into an acetic acid solution. This solution was sprayed through a nozzle into a heated chamber [46]. The water and volatile acid were evaporated and expelled into an exhaust-hood. The dried particles were collected by an electrostatic precipitator [45]. Transmission electron microscopy (TEM, 3010-HREM, JEOL, Peabody, MA) was used to examine the ACP particles.

CaF₂ nanoparticles were synthesized using the same spray-drying apparatus, except that a two-liquid nozzle was employed [43,47]. This allowed two solutions to be mixed during atomization: Ca(OH)₂ and NH₄F. The two solutions were atomized leading to the formation of CaF₂ nanoparticles: Ca(OH)₂ + NH₄F → CaF₂ + NH₄OH. The NH₄OH was removed as NH₃ and H₂O vapors. TEM was used to examine the CaF₂ particles.

Chlorhexidine diacetate (Sigma, St. Louis, MO) was frozen via liquid nitrogen, and then ground in a mortar and pestle to obtain a fine particle size. The particles were sputter coated with gold and examined in a scanning electron microscope (SEM, FEI Quanta 200, Hillsboro, OR). These particles are referred to as CHX. The sizes of 100 random CHX particles were measured via SEM in this study.

Barium aluminosilicate glass particles with a mean diameter of 1.4 µm (Caulk/Dentsply, Milford, DE) were used as a co-filler and silanized with 4% (all mass fractions) 3-methacryloxypropyltrimethoxysilane and 2% n-propylamine. For ACP nanocomposite, a resin of Bis-GMA (bisphenol glycidyl dimethacrylate) and TEGDMA (triethylene glycol dimethacrylate) at 1:1 ratio was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate [45]. For the CaF₂ nanocomposite, because the paste was relatively opaque, a two-part chemically-activated resin was used. The initiator resin consisted of 48.975% Bis-GMA, 48.975% TEGDMA, 0.05% 2,6-di-tert-butyl-4-methylphenol, and 2% benzoyl-peroxide. The accelerator resin consisted of 49.5% Bis-GMA, 49.5% TEGDMA, and 1.0% N,N-dihydroxyethyl-p-toluidine [44].

Four nanocomposites were made with the following filler mass fractions: (1) 30% nano ACP + 35% glass (referred to as “NanoACP”); (2) 30% nano ACP + 25% glass + 10% CHX (referred to as “NanoACP+CHX”); (3) 30% nano CaF₂ + 35% glass (“NanoCaF₂”); (4) 30% nano CaF₂ + 25% glass + 10% CHX (“NanoCaF₂+CHX”).

The glass and nanoparticle filler levels were selected following previous studies [44,48]. The CHX filler level was based on previous studies which ranged from 0.5% to 33% [33–36]. The total filler mass fraction of 65% yielded a cohesive paste. The paste was placed into rectangular molds of (2 × 2 × 25) mm for mechanical testing, and disk molds of 9 mm in diameter and 2 mm in thickness for biofilm experiments. NanoCaF₂ specimens were self-cured. NanoACP specimens were photo-cured (Triad 2000, Dentsply, York, PA) for 1 min on each side.

Three commercial materials were tested as controls. A resin-modified glass ionomer (Vitremer, 3M, St. Paul, MN), referred to as “RMGI”, consisted of fluoroaluminosilicate glass, and a light-sensitive, aqueous polyalkenoic acid. Indications include Class III, V and root-caries restoration, Class I and II in primary teeth, and core-buildup. A powder/liquid mass ratio of 2.5/1 was used according to the manufacturer. A composite with nanofillers of 40–200 nm and a low level of F release was used (Heliomolar, Ivoclar, Amherst, NY), and is referred to as “CompositeF”. The fillers were silica and ytterbium-trifluoride with a filler level of 66.7%. Heliomolar is indicated for Class I, II, III, IV and V restorations. Renamel (Cosmedent, Chicago, IL) served as a non-releasing control, and is referred to as “CompositeNoF”. It consisted of nanofillers of 20–40 nm with 60% fillers in a multifunctional methacrylate ester resin [49]. Renamel is indicated for Class III, IV, and V restorations. Specimens were photo-cured in the same manner as the ACP nanocomposite.

2.2. Flexural testing

Flexural strength and elastic modulus were measured using a three-point flexural test with a 10 mm span at a crosshead-speed of 1 mm/min on a computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC). Flexural strength (S) was calculated by: S = 3P_maxL/(2bh²), where P_max is the fracture load, L is span, b is specimen width and h is specimen thickness. Elastic modulus (E) was calculated by: E = (P/d)(L³/[4bh³]), where load P divided by displacement d is the slope of the load-displacement curve in the linear elastic region.

2.3. CHX release measurement

CHX release was measured for NanoACP+CHX and NanoCaF₂+CHX composites. A physiological-like buffer solution with pH of 7 (133 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES) was prepared. Following previous studies [41–44], three specimens of (2 × 2 × 12) mm were immersed in 50 mL solution. The CHX concentrations were measured at days 1, 2, 3, 7, 14, 21, and 28. At each time period, aliquots of 200 µL were removed and replaced by fresh solution. A series of CHX reference solutions was prepared and a standard curve was constructed. The absorbance at 255 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA).

2.4. S. mutans inoculation and pH measurement

The use of S. mutans (ATCC 700610, UA159, American Type Culture, Manassas, VA) was approved by the University of Maryland. S. mutans is a cariogenic, aerotolerant anaerobic bacterium and the primary causative agent of dental caries [22]. Brain heart infusion (BHI) broth (BD, Franklin Lakes, NJ) supplemented with 0.2% sucrose is termed “growth medium”. Fifteen µL of stock bacteria was added into 15 mL of growth medium and incubated at 37 °C with 5% CO₂ for 16 h. During this culture, the S. mutans were suspended in the BHI broth. Then, this S. mutans culture was diluted 10-fold in growth medium to form the inoculation medium [50].

The composite disks were sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC). Each disk was placed in a well of a 24-well plate, and 1.5 mL of the inoculation medium was added to each well. The samples were incubated at 5% CO₂ and 37 °C for 24 h to form the initial biofilms on the disk. At 24 h, each disk with biofilm was transferred to a new 24-well plate containing 1.5 mL of fresh growth medium. The pH of the medium was measured via a pH meter (Accumet Excel XL25, Fisher, Pittsburgh, PA) from 24 h to 48 h. pH measurements were not collected during the first 24 h of culture, because the non-adherent bacteria in the growth medium could contribute to the pH changes. In this way, the measured pH was solely related to the biofilm on the composite, and there was no contribution from planktonic bacteria in the media. At 48 h, each disk was transferred to a new 24-well plate containing 1.5 mL of fresh growth medium. The pH of the medium was measured again from 48 h to 72 h.

2.5. Live/dead assay

Each disk was placed in a well of a 24-well plate, inoculated with 1.5 mL of inoculation medium, and cultured for 1 d (initial biofilm), or 3 d (mature biofilm). The growth medium was changed every 24 h, by transferring the disks to a new 24-well plate with fresh growth medium. After 1 d or 3 d, the biofilms on the disks were stained using the BacLight live/dead bacterial viability kit (Molecular Probes, Eugene, OR). Live bacteria were stained with Syto 9 to produce green fluorescence, and bacteria with compromised membranes were stained with propidium iodide to produce red fluorescence. The stained disks were imaged using laser scanning confocal microscopy (TCS SP5, Leica, Germany). A minimum of three x–y images were collected at random locations on each disk. At each time point, a minimum of three disks were evaluated for each material yielding a minimum of 9 images for each sample.

2.6. Lactic acid production and viable cell counts

After 3 d, mature biofilms were formed on the disks. Each disk was rinsed in cysteine peptone water (CPW) to remove loose bacteria, and placed in a new 24-well plate. Then, 1.5 mL of buffered peptone water (BPW) supplemented with 0.2% sucrose was added to each well. The reason for using the BPW media was that the mature biofilm would remain stable during this 3 h culture for the acid production assay. In addition, BPW has a relatively high buffer capacity, so the pH should not become significantly acidic, as a low pH could hinder bacterial acid production. The samples were incubated at 5% CO₂ and 37 °C for 3 h to allow the biofilms to produce acid. After 3 h, the BPW solutions were stored for lactate analysis. Lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method [51]. The microplate reader was used to measure the absorbance at 340 nm (optical density OD₃₄₀) for the collected BPW solutions. Standard curves were prepared using a standard lactic acid (Supelco Analytical, Bellefonte, PA).

After treatment for lactic acid production, colony-forming unit (CFU) counts were used to quantify the total number of viable bacteria present on each disk. When biofilms are properly dispersed and diluted, each viable bacterium results in a single, countable colony on an agar plate. The disks were transferred into tubes with 2 mL CPW. The biofilms were harvested by sonication (3510R-MTH, Branson, Danbury, CT) for 3 minutes, and then vortexing at maximum speed for 20 s using a vortex mixer (Fisher, Pittsburgh, PA), thus removing and dispersing the biofilms from the sample disks. The bacterial suspensions were serially diluted, spread onto BHI agar plates, and incubated for 3 d at 5% CO₂ and 37 °C. The number of colonies that grew were counted and used, along with the dilution factor, to calculate total CFUs on each composite disk.

2.7. MTT metabolic assay

Disks were placed in a 24-well plate, inoculated with 1.5 mL of the inoculation medium, and cultured for 1 d or 3 d. Each disk was then transferred to a new 24-well plate for the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan [52]. One mL of MTT dye (0.5 mg/mL MTT in PBS) was added to each well and incubated at 37 °C in 5% CO₂ for 1 h. During this process, metabolically active bacteria metabolized the MTT and reduced it to purple formazan inside the living cells. After 1 h, the disks were transferred to a new 24-well plate, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min with gentle mixing at room temperature in the dark. After brief mixing via pipetting, 200 µL of the DMSO solution from each well was transferred to a 96-well plate, and the absorbance at 540 nm (OD₅₄₀) was measured via the microplate reader. A higher absorbance indicates a higher formazan concentration, which in turn indicates more metabolic activity in the biofilm present on the composite disk.

One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison test was used to compare the data at a p-value of 0.05. Each standard deviation (sd) serves as the estimate for the standard uncertainty associated with a particular measurement.

3. Results

Fig. 1A plots the histogram of CHX particle size distribution. Based on 100 particles measured via SEM, the particle size ranged from approximately 0.1 µm to 5 µm, with (mean ± sd; n = 100) of (0.62 ± 0.48) µm. The CHX release from the ACP nanocomposite and CaF₂ nanocomposite is plotted in (B) (mean ± sd; n = 3). The CHX release from the ACP nanocomposite was similar to that of the CaF₂ nanocomposite (p > 0.1).

Figure 1.

(A) CHX particle size distribution, based on the measurement via SEM of 100 random particles. (B) CHX release from ACP and CaF₂ nanocomposites. Each value is the mean of three measurements with the error bar showing one standard deviation (mean ± sd; n = 3), with specimens immersed in a physiological solution at pH 7 from 1 d to 28 d.

Fig. 2 shows TEM images of nanoparticles and composite mechanical properties: (A) Example of smaller ACP nanoparticles, (B) example of ACP clusters, (C) CaF₂ nanoparticles, (D) flexural strength and (E) elastic modulus after 1 d and 28 d of immersion. In (A), arrows indicate individual ACP particles that overlapped a larger ACP particle. In (B), arrows indicate individual ACP particles near a cluster. The cluster appeared to contain numerous small particles, which likely had stuck to form the cluster in the spray-drying chamber before they were completely dried. In general, the individual ACP particles had sizes of the order of 10 nm, and the clusters had sizes of about 100–300 nm. Measurement of 100 random particles yielded an average size of 37 nm for the individual ACP particles, and an average size of 225 nm for the ACP clusters. Similar features were observed for CaF₂ in (C), and measurement of 100 random particles yielded an average size of 20 nm for the individual CaF₂ particles, and an average size of 306 nm for CaF₂ clusters.

Figure 2.

TEM micrographs of the spray-dried nanoparticles as well as the composite mechanical properties. (A) Small ACP nanoparticles, (B) ACP cluster, (C) CaF₂ nanoparticles, (D) flexural strength, and (E) elastic modulus, after 1 d and 28 d of immersion. Each value is mean ± sd; n = 6. CompositeF is Heliomolar. CompositeNoF is Renamel. RMGI is Vitremer. NanoACP composite and NanoCaF₂ composite had no CHX. NanoACP+CHX and NanoCaF₂+CHX contained 10% CHX particles by mass.

In Fig. 2D, CompositeNoF, NanoACP, NanoCaF₂, and NanoCaF₂+CHX had a significant decrease in strength from 1 d to 28 d (p < 0.05). CompositeF, CompositeNoF, NanoACP, and NanoCaF₂ had strengths similar to each other at 28 d (p > 0.1). The strengths of NanoACP+CHX and NanoCaF₂+CHX at 28 d were 2-fold that of RMGI (p < 0.05). In 2E, elastic moduli in general were similar to each other for the different materials at 1 d and 28 d.

The pH of biofilm medium is plotted in Fig. 3. (A) pH measurements were started at 24 h, when the medium was changed, and then collected hourly. (B) Then the pH was measured again after the medium change at 48 h. In (A), for NanoCaF₂+CHX and NanoACP+CHX, the pH remained at 6.5 or higher. For all other materials, the pH decreased with time, reaching 4.7 for RMGI, 4.6 for NanoCaF₂, and 4.2 for the other composites at 48 h. Comparing (A) with (B) shows a similar trend and similar end pH values, indicating that the effect of inhibiting bacteria growth and acid production for the CHX composites was maintained over 3 d.

Figure 3.

The pH of the culture medium with biofilm on the composite disk. Each value is mean ± sd; n = 6. During the first 24 h after inoculation, an initial biofilm was established on the composite disk. At 24 h, the disk was transferred to a new well with new medium, and the pH measurement was started. The plot in (A) shows the pH from 24 h to 48 h. At 48 h, a new culture medium was used (because the medium was changed daily), and the pH is plotted in (B) from 48 h to 72 h. The initial pH was 7.2 for each new medium.

Images of biofilms stained with the live/dead stain are shown in Fig. 4. The live bacteria appear green, and the compromised bacteria appear red. In some areas, the live and compromised (likely dead) bacteria are closely associated and/or colocalized, hence the red color was mingled with green to yield the yellow and orange colors. At 1 d, the biofilms were predominantly viable in (A) and (C), whereas in (B), biofilm surface coverage was slightly patchy and there was some cell death. In (D), the biofilms grown on the NanoACP+CHX also had patchy surface coverage and had increased numbers of dead bacteria relative to (A) to (C). The biofilm structure and viability for NanoCaF₂+CHX at 1 d (not shown) had similar features to those in (D), with even more cell death evident by more yellow in the images.

Figure 4.

Representative live/dead images: Early biofilms (1 d) are shown for (A) CompositeF, (B) RMGI, (C) NanoACP, and (D) NanoACP+CHX. Mature biofilms (3 d) are shown for the same materials in (E) to (H). Live bacteria appear green. Membrane-compromised bacteria appear red, and, when mingled with live bacteria, appear yellow/orange. At 1 d, bacteria were predominantly alive in (A) to (C), with some cell death in (B). (D) NanoACP+CHX had increased dead bacteria relative to (A) to (C). At 3 d, live bacteria formed mature biofilms in (E) to (G) with slightly increased death in (F). (H) NanoACP+CHX had significant amounts of dead bacteria. Images of CompositeNoF and NanoCaF₂, which had qualitatively similar features to the other materials without CHX, were omitted to save space. Likewise, images for NanoCaF₂+CHX were not shown, as their biofilms had similar features to biofilms on the NanoACP+CHX.

At 3 d, S. mutans had formed a mature biofilm in Fig. 4E to 4G, where the bacteria were primarily alive, with RMGI having slightly more dead bacteria. Biofilms on CompositeNoF and NanoCaF₂ (not shown) were similar to those in (E) and (G). NanoACP+CHX in (H) had significantly more dead bacteria. NanoCaF₂+CHX had similar images to (H), with increased cell death evident by mostly yellow and red staining and very little green present.

Fig. 5 plots results on (A) lactic acid production, and (B) CFU counts. Biofilm on CompositeNoF produced the most acid, closely followed by that of nano ACP. Between F-releasing materials, biofilm on CompositeF had the most acid, while biofilms on NanoCaF₂ and RMGI had similarly lower acid production. Acid production on composites with CHX was nearly 10-fold less than that on CompositeNoF. In (B), the CFU counts were ≈ 10⁹ per disk for CompositeNoF, and ≈ 10⁸ for all other materials without CHX. CFU counts for nanocomposites with CHX were reduced 1,000-fold (to ≈ 10⁶) from those of CompositeNoF.

Figure 5.

Quantitative response of S. mutans biofilms on the composite disks: (A) Lactic acid production, and (B) bacteria colony-forming units (CFU) on the disks at 3 d. In each plot, dissimilar letters indicate values (mean ± sd; n = 6) that are different from each other (p < 0.05). Values with the same letters are not significantly different (p > 0.1).

The MTT results are plotted in Fig. 6 for (A) 1 d, and (B) 3 d. In each plot, values (mean ± sd; n = 6) with dissimilar letters are significantly different (p < 0.05). In (A), CompositeNoF had the highest absorbance. The two nanocomposites containing CHX had absorbance 10-fold less than that of RMGI, and 20-fold less than that of CompositeNoF. A similar trend was maintained at 3 d in (B), although the absorbance was 1.5 to 2 fold higher than that at 1 d.

Figure 6.

Results of the MTT metabolic activity assay for S. mutans on composite disks at: (A) 1 d, and (B) 3 d. A higher absorbance indicates an overall higher metabolically active biofilm on the composite disk that metabolized the MTT tetrazole. In each plot, values (mean ± sd; n = 6) with dissimilar letters are significantly different from each other (p < 0.05). Values with the same letters are not significantly different (p > 0.1).

4. Discussion

The present study investigated the effects of novel nanocomposites containing ACP or CaF₂ nanoparticles and CHX on biofilm formation, viability, acid production, and metabolic activity for the first time. An important approach to the inhibition of demineralization and the promotion of remineralization was the development of CaP composites [38–41]. Previous studies showed that the remineralization of tooth lesions was greatly promoted by increasing the solution calcium and phosphate ion concentrations [39,40]. Composites containing CaP particles released Ca and PO₄ ions to supersaturating levels with respect to tooth mineral, and were shown to protect the teeth from demineralization, and even regenerate lost tooth mineral in vitro [39,40]. A recent study showed that the new nano ACP composite released Ca and PO₄ ions at concentrations matching those of traditional CaP composites known to remineralize tooth lesions, while having much higher mechanical properties [45]. Another study demonstrated that the new nano CaF₂ composite released F ions at similar amounts to a commercial resin-modified glass ionomer, while having mechanical properties equivalent to a commercial composite without F release [44]. Previous studies indicated that the release of Ca, PO₄ and F ions could lead to the remineralization of the tooth structure [24,25,39,40,53]. In the present study, the composites not only could release Ca, PO₄ and F ions [43–45], but also increased the pH (Fig. 3) which could have an additional effect on the remineralization of the tooth structure. Further studies are needed to measure the mineral contents of tooth structures with the use of the antibacterial composites containing ACP and CaF₂ nanoparticles.

CHX particles have been incorporated into glass ionomer materials [36,37] and dental polymeric composites to render the filling materials antibacterial [33,35]. The present study showed that the CHX release was relatively high in the first week and then plateaued after 2 weeks, which is similar to previous studies [36]. The percentage of CHX released from the ACP and CaF₂ nanocomposites during one month of immersion was relatively small (about 2%), which is comparable to previous studies. Such a small amount of CHX release, while having an antibacterial effect near the surface of the tooth cavity restoration locally, is expected to have a negligible systemic effect. In previous studies, the CHX released from a glass ionomer cement was about 3–5% after 240 d [36]. Another study showed that the percentage of CHX released from a dental composite was about 10% after 4 months of immersion in a pH 6 solution; when the solution pH was reduced to 4, the CHX release increased to 50% in 4 months due to polymer degradation [35]. The present study showed that even a small amount of the CHX release from the nanocomposites greatly reduced acidogenic bacterial CFU counts, biofilm viability, biofilm formation, biofilm metabolic activity, and therefore lactic acid production. The 2% release indicates a large reservoir of CHX remaining in the composite. A previous study estimated that if the CHX reservoir in the resin was fully utilized, the release could last 6–10 years [35].

Most previous studies [33,34,36,37] did not mention attempts to obtain fine CHX particles by grinding the as-received particles, which were about 40 µm in diameter for the CHX diacetate from Sigma. A particle size of 40 µm is much larger than the glass fillers in dental composites, which are typically about 1 µm or less. One study reported the grinding of the as-received CHX, yielding a particle size of 13.5 µm [35]. Preliminary studies failed to obtain smaller CHX particles, until the use of liquid nitrogen to chill the CHX prior to grinding. The lower temperature appeared to embrittle and help shatter the particles, yielding an average particle size of 0.62 µm in the present study. A small CHX particle size could improve the polishability and mechanical properties of the composite. Previous studies on CHX composites did not report their mechanical properties [33,35]. In the present study, after 28 d immersion, the strength for NanoACP+CHX was approximately 80% that without CHX. A similar 20% strength loss occurred for NanoCaF₂. However, the strength of the nanocomposites containing CHX was only slightly lower than the commercial composite without CHX. The strength of the nanocomposites containing CHX was twice that of a commercial resin-modified glass ionomer.

Previous studies have shown that bacteria will colonize surfaces and form biofilms, which are heterogeneous structures consisting of cell clusters embedded in an extracellular matrix [54]. Acidogenic bacteria in dental plaque, such as S. mutans, metabolize carbohydrates to acids and can result in a local plaque pH drop to 4.5 or 4 after a sucrose rinse. Acids cause demineralization of the tooth structure beneath the biofilm. Studies have shown that there is a critical pH of about 5.5, below which demineralization dominates, leading to a net enamel mineral dissolution [55]. Therefore, it would be highly desirable for the local pH at the tooth surface to remain greater than 5.5 in order to inhibit secondary caries at the restoration-tooth interface. The NanoACP+CHX and NanoCaF₂+CHX likely slowed down or eliminated the bacterial growth, reducing the acid production by the bacteria, thereby yielding a pH of 6.5 or higher. In contrast, the two commercial composites had pH below 4.5. It should be noted that although the biofilms likely had a dominant effect on the pH of the media, the materials such as the resin-modified glass ionomer and the NanoACP composite could also affect the pH in the absence of a biofilm [48]. Hence, the measured pH resulted from contributions from the material and the biofilm. The results of this study demonstrated that NanoACP+CHX and NanoCaF₂+CHX composites with S. mutans biofilms in the presence of sucrose were able to maintain the pH at a safe level to inhibit tooth mineral dissolution.

Another potential benefit of nanocomposites containing CHX is the ability to reduce biofilm acid production, resulting in a near neutral pH. The biofilm surrounding a tooth caries likely has a low pH, and may therefore have a high proportion of acidogenic, aciduric (acid-tolerant) bacteria and a low proportion of other benign bacteria that are less acid-tolerant [22,56]. Restorations that release CHX can potentially kill all the bacteria in the vicinity. Eventually, the CHX release will be exhausted, and new biofilms will form. Because the pH has been close to neutral during the CHX release, the new biofilm could have a less pathogenic composition as compared to the acidogenic biofilm that likely would have regrown, had there been no CHX treatment. Indeed, without the CHX release, the repeated acidification in the plaque would likely have continued, resulting in even more predominance of acidogenic and aciduric bacteria such as S. mutans [23]. Compared to the commercial composites that had a pH of 4.2 in the biofilm medium, the pH in the biofilm medium of the new CHX-releasing nanocomposites was greater than 6.5. Thus, these new composites may be able to promote recolonization of the area with benign bacteria and a normal oral flora (with < 1% acidogenic bacteria [25]). This effect may help prevent the dominance of cariogenic bacteria and hence help inhibit dental caries.

It is interesting to note that the NanoCaF₂+CHX had a higher pH than that of NanoACP+CHX. The mechanism for this is likely that the F ion release helped reduce the acid production of the bacteria, via the inhibition of metabolic pathways such as the fermentation pathway for lactic acid production [54]. A previous study used a constant depth film fermentor (CDFF) model and showed that while F treatment had little effect on S. mutans viability, it did reduce the acid production of the bacteria [53]. This notion is also supported by the higher pH of RMGI and NanoCaF₂ than the pH of CompositeNoF. These results are corroborated by the F-containing materials having lower lactic acid production and CFU, than the commercial CompositeNoF. Therefore, the following two points should be noted: (1) While the release of Ca and PO₄ ions are beneficial in remineralization, the incorporation of CHX was needed in NanoACP to maintain a safe (non-demineralizing) pH of 6.5; (2) the additional F release of NanoCaF₂+CHX was beneficial in further reducing the lactic acid production of bacteria.

Compared to the CompositeNoF, the biofilm acid production on NanoACP+CHX and NanoCaF₂+CHX was reduced by 10 fold. The metabolic activity, related to the bacteria metabolism, was reduced by 10 to 20 fold. The flexural strength and elastic modulus of the nanocomposites with CHX were not significantly different from those of CompositeNoF after 28 d of immersion. According to the manufacturer, CompositeNoF (Renamel) is indicated for Class III, IV, and V restorations. This suggests that the new nanocomposites containing fine CHX particles may also be suitable for these applications. Further study is needed to improve and optimize the ACP and CaF₂ nanocomposites, and to systematically investigate their mechanical and physical properties as well as anti-caries capabilities.

5. Conclusion

The present study developed novel nanocomposites containing ACP and CaF₂ nanoparticles and CHX particles and determined their effects on S. mutans biofilm formation, acid production, CFU, and metabolic activity for the first time. Incorporating CHX into the ACP and CaF₂ nanocomposites imparted a potent antibacterial capability. The S. mutans biofilm-coated ACP and CaF₂ nanocomposites containing CHX maintained a growth medium pH at a safe level of above 6.5, while that of commercial composites had a cariogenic pH of 4.2, a level known to cause tooth lesions. The new nanocomposites reduced the biofilm acid production and metabolic activity by 10–20 times, compared to a commercial composite. Mechanical properties of the new nanocomposites matched those of a commercial composite without fluoride. These novel ACP and CaF₂ nanocomposites have the mechanical properties to be used in restorations where the commercial control composites are used, and could potentially inhibit biofilm formation, lactic acid production and caries. Further studies are needed to optimize the nanocomposites and investigate the anti-caries capabilities.