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 (CaF 2 ) nanoparticles and CHX particles, and investigate Streptococcus mutans biofilm formation and lactic acid production for the first time.
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 CaF 2 ; nano CaF 2 + 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.
Adding CHX fillers to ACP and CaF 2 nanocomposites greatly increased their antimicrobial capability. ACP and CaF 2 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.
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.
Nearly 200 million dental restorations are placed annually in the USA . Resin composites are increasingly used for dental caries restorations because of their esthetics and direct-filling capability . Remarkable progress has led to esthetic composite restoratives with less removal of tooth structures, enhanced load-bearing properties, and improved clinical performance . However, secondary caries at the restoration margins is identified as a main limitation to the longevity of the restorations . The replacement of existing restorations accounts for 50–70% of all restorations performed . Replacement dentistry costs $5 billion annually in the U.S. alone . Dental caries is a dietary carbohydrate-modified bacterial infectious disease, and is one of the most common bacterial infections in humans . The basic mechanism of caries is demineralization of dental tissue (enamel/dentin) via acid generated by bacterial biofilms (dental plaque) . 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 .
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 ) . In one study, a polymerizable bactericidal monomer, 12-methacryloyloxydodecylpyridinium bromide (MDPB), was immobilized in the resin which reduced bacteria growth via contact inhibition . In another, ionic liquid dimethacrylate monomers that contained quaternary ammoniums groups were used to develop antimicrobial resins, which reduced bacterial colonization . Other studies incorporated chlorhexidine (CHX) particles as fillers into dental resin composites, which resulted in CHX release and reduced bacteria growth . CHX particles were also mixed into glass ionomer cements , thus combining antimicrobial activity with fluoride ions (F) .
Another promising class of composites consists of resins filled with calcium phosphate (CaP) particles of about 1–55 μm in sizes . These composites released supersaturating levels of calcium (Ca) and phosphate (PO 4 ) ions and remineralized tooth lesions in vitro . Recently, CaP nanoparticles of about 100 nm in size were synthesized via a spray-drying technique for the first time . Composites containing CaP nanoparticles with high specific surface areas were found to release high levels of Ca and PO 4 while possessing mechanical properties nearly two-fold those of previous CaP composites . Nanocomposites containing CaF 2 nanoparticles were also developed that released fluoride (F) ions matching that of a resin-modified glass ionomer . The mechanical properties of the CaF 2 nanocomposite were much higher than that of resin-modified glass ionomer, and matched those of commercial composites with little F release . However, there has been no report of CaP and CaF 2 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 CaF 2 nanoparticles into the nanocomposites, and to determine the mechanical and antibacterial properties. It was hypothesized that: (1) incorporating CHX into CaP and CaF 2 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 CaF 2 nanocomposites containing CHX will match those of commercial composites that have no ion release or antibacterial capability.
Materials and methods
Fabrication of CaP and CaF 2 nanocomposites containing CHX
Nanoparticles of amorphous calcium phosphate (ACP), Ca 3 (PO 4 ) 2 , were synthesized via a spray-drying technique . 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 (CaCO 3 , Fisher, Fair Lawn, NJ) and dicalcium phosphate anhydrous (CaHPO 4 ) (J.T. Baker, Phillipsburg, NJ) into an acetic acid solution. This solution was sprayed through a nozzle into a heated chamber . The water and volatile acid were evaporated and expelled into an exhaust-hood. The dried particles were collected by an electrostatic precipitator . Transmission electron microscopy (TEM, 3010-HREM, JEOL, Peabody, MA) was used to examine the ACP particles.
CaF 2 nanoparticles were synthesized using the same spray-drying apparatus, except that a two-liquid nozzle was employed . This allowed two solutions to be mixed during atomization: Ca(OH) 2 and NH 4 F. The two solutions were atomized leading to the formation of CaF 2 nanoparticles: Ca(OH) 2 + NH 4 F → CaF 2 + NH 4 OH. The NH 4 OH was removed as NH 3 and H 2 O vapors. TEM was used to examine the CaF 2 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 . For the CaF 2 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 .
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 CaF 2 + 35% glass (“NanoCaF 2” ); (4) 30% nano CaF 2 + 25% glass + 10% CHX (“NanoCaF 2 + CHX”).
The glass and nanoparticle filler levels were selected following previous studies . The CHX filler level was based on previous studies which ranged from 0.5% to 33% . The total filler mass fraction of 65% yielded a cohesive paste. The paste was placed into rectangular molds of 2 mm × 2 mm × 25 mm for mechanical testing, and disk molds of 9 mm in diameter and 2 mm in thickness for biofilm experiments. NanoCaF 2 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 . Renamel is indicated for Class III, IV, and V restorations. Specimens were photo-cured in the same manner as the ACP nanocomposite.
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 = 3 P max L /(2 bh 2 ), 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 )( L 3 /[4 bh 3 ]), where load P divided by displacement d is the slope of the load–displacement curve in the linear elastic region.
CHX release measurement
CHX release was measured for NanoACP + CHX and NanoCaF 2 + 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 , three specimens of 2 mm × 2 mm × 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).
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 . 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% CO 2 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 .
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% CO 2 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.
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.
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% CO 2 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 . The microplate reader was used to measure the absorbance at 340 nm (optical density OD 340 ) 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 min, 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% CO 2 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.
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 . One mL of MTT dye (0.5 mg/mL MTT in PBS) was added to each well and incubated at 37 °C in 5% CO 2 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 (OD 540 ) 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.