Casein phosphopeptide and fluoride
CPP powder (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and sodium fluoride (Sigma, Saint Louis, MO, USA) used in this study were reagent-grade.
Two-fold serially diluted CPP solutions in the range of 0.15625–10% (w/v) and 2.5% CPP solution supplemented with 900 ppm fluoride were prepared. The pH of solutions was adjusted to 7.0 using NaOH, and the solutions were sterilized through a 0.22 μm polyethersulfone membrane (Merck Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, IRL). Heat-inactivated 2.5% CPP solution was prepared by autoclaving at 121°C for 15 min. All solutions were prepared freshly and used on the same day.
Hydroxyapatite (HA) disc (9.7 mm in diameter and 1.5 mm in thickness, Clarkson Chromatography Products, South Williamsport, PA, USA) was used as the substrate in this study. To visualize the live and dead bacteria cells on the substrate surface, the HA disc was polished using a precision lapping/polishing machine (Unipol-1502, Kejing Auto-Instrument Co., LTD, Shenyang, China) with #360, #600, and #1000 silicon carbide sandpapers under constant water cooling, until the disc reached a thickness of 0.3 mm. After polishing, the disc was sonicated in ultrapure water for 10 min to remove the debris. Before experiments, the HA disc was autoclaved at 121°C for 15 min.
Bacterial strain and culture conditions
S. mutans UA159 (ATCC 700610) purchased from Guangdong Microbial Culture Collection Center was used in this study. To prepare the inoculum, S. mutans was firstly recovered on a brain heart infusion (BHI, Difco, Detroit, MI, USA) agar plate supplemented with 5% sterile defibrinated sheep blood for 48 h at 37 °C under anaerobic conditions (5% CO2, 10% H2, 85% N2). A single colony was selected and inoculated into 10 mL of BHI broth and incubated anaerobically at 37 °C overnight. Bacteria was harvested by centrifugation (3000 rpm, 4 °C, 5 min), washed twice with sterile phosphate buffered saline (PBS), and finally re-suspended in BHI broth. The optical density at 600nm (OD600nm) was adjusted to 0.2 (corresponding to a concentration of approximately 2.0 × 108 cells/mL).
Saliva collection and preparation
Human whole unstimulated saliva was collected from healthy volunteers with informed consent, and the procedure was approved by the Ethical Review Committee of Sun Yat-Sen University (No. ERC--24). Saliva samples were collected and prepared according to a published method with some modification . Briefly, saliva was collected from eight volunteers who had not taken in anything but water for at least 2 h before saliva collection, were never smokers, and had not consumed any antibiotics or mouthwashes for at least 3 months. The saliva was spitted into a sterile and iced centrifuge tube until 10 mL of saliva was obtained, and this process usually took about 50–60 minutes. The saliva from eight donors was pooled. After centrifugation at 4000 rpm and 4 °C for 20 min, the supernatant was collected and filtered using a 0.22 μm polyethersulfone membrane. Clarified saliva samples were frozen quickly in liquid nitrogen, and stored at -80 °C. Saliva collection from the same donors was carried out with an interval of at least 30 days, until the present study was completed. Saliva samples were used within 6 months and thawed at room temperature prior to experiments.
Adhesive inhibitory concentration assay
The HA discs were individually placed into a 24-well plate (Costar, Corning, NY, USA). A volume of 1 mL of the clarified saliva was added into each well and incubated at 37 °C for 2 h. After rinsing twice with sterile PBS to obtain salivary pellicle-coated HA (s-HA) discs, the discs were randomly divided into eight groups (n = 6 per group): control group (PBS), and seven treated groups containing serially diluted concentrations of CPP. The s-HA disc was incubated in 1 mL of the corresponding solutions for 2 h at 37 °C, followed by two rinses with PBS. A volume of 1 mL of the bacteria suspension was seeded and incubated for 30 min and 2 h in a humidified atmosphere of 5% CO2 at 37 °C (three discs per group and incubation time). After rinsing twice with PBS to remove non-attached and loosely bound bacteria, the HA disc was transferred into an eppendorf tube containing 2 mL of PBS. Adherent S. mutans was detached by sonication for 10 min, followed by vortexing for a further 60 seconds. The sonicated and vortexed S. mutans suspension was serially diluted and 100 μL of the diluted solution was spread over a BHI agar plate and incubated for 48 h at 37 °C in a humidified atmosphere of 5% CO2. The number of adherent S. mutans was expressed as colony-forming units (CFU) per disc. The experiment was repeated three times in triplicate. The adhesion reduction percentage was calculated as follows: (CFU counts of the control group- CFU counts of the treated group)/ CFU counts of the control group× 100%.
Bacterial adhesion assay
CPP at 2.5% (w/v) was used for further experiments based on the ability to reduce the adhesion of S. mutans by approximately 50% . For the S. mutans adherence assay, s-HA disc was prepared as described above. The s-HA discs were randomly divided into four groups (n = 10 per group): (1) blank control (PBS); (2) negative control (heat-inactivated 2.5% CPP); (3) CPP (2.5% CPP); and (4) CPP + F (2.5% CPP supplemented with 900 ppm fluoride). Pellicle modification and inoculation of S. mutans were performed as described above (three discs per group and incubation time). The number of adherent S. mutans was expressed as CFU per disc. The experiment was repeated three times in triplicate.
Scanning electron microscopy (SEM) was used to visualize S. mutans adhesion on the HA surface. Two samples from each group and incubation time were prefixed in 2.5% glutaraldehyde at room temperature for at least 3 h. After washing four to six times using ultrapure water, samples were dehydrated using gradient concentrations of ethanol (30%, 50%, 70%, 80%, 85%, 90%, 95%, and 100%) for 15 min, and then substituted by tert butyl alcohol three times, freeze-dried, sputter-coated with gold, and examined by SEM (JSM-6330F, JEOL, Japan).
Bacteria LIVE/DEAD staining
Adhesion of S. mutans was also measured using a LIVE/DEAD BackLight Bacterial Viability Kit (L7012, Thermo Scientific, USA). The polished HA discs with a thickness of 0.3 mm were used. Pellicle formation and modification as well as cultivation of S. mutans were performed as described above (three HA discs per group and incubation time). After incubation for 30 min and 2 h, the HA disc was washed twice with 0.9% NaCl and then stained with 1 mL of LIVE/DEAD® BacLight™ solution at room temperature in the dark for 15 min. A volume of 1 mL of LIVE/DEAD® BacLight™ solution contained 997 μL of ultrapure water, 1.5 μL of propidium iodide (PI), and 1.5 μL of SYTO 9, which was prepared according to the manufacturer’s instructions. Dead bacterial cells with damaged membrane were finally stained in red color by PI, while live bacterial cells with intact cell membrane were stained in green color by SYTO 9. Samples were observed by a confocal laser scanning microscopy (CLSM, LSM 780, Zeiss, Oberkochen, BW, Germany) with a 20× water-immersion objective lens. Dual-channel scanning observations were performed through a green channel for SYTO 9 (excitation wavelength: 488 nm) and a red channel for PI (excitation wavelength: 543 nm). Four fields were randomly selected on each sample for scanning. Image analysis was performed with ImageJ software. The ratio of the area occupied by green or red fluorescence to the whole area on each visual field was measured. For each specimen, the ratio (%) of the area covered by total fluorescence of each color to the whole area was calculated. Subsequently, the proportion (%) of red to total fluorescence was measured. The experiment was replicated three times.
Ion quantified analysis
The total and free calcium and fluoride concentrations in 2.5% CPP and 2.5% CPP supplemented with 900 ppm fluoride solutions were quantified, and the corresponding bound ion concentrations were calculated. Before detecting the total ion concentrations, 1 mL of the initial solution was diluted with 19 mL of 1.0 M HNO3 and reacted for 24 h, followed by centrifugation at 1000g for 15 min at room temperature; the supernatant was collected to detect the total ion concentration . Before detection of free ion concentrations, macromolecular CPP with a molecular weight of approximately 3 kDa was filtered using a magnetically stirred ultrafiltration device (Amicon, Model 8200, 200 mL, Millipore) equipped with an ultrafiltration disc (1kDa NMWL, PLAC06210, Ultracel® regenerated cellulose, Millipore). According to the instructions, 50 mL of the initial solution was added into the device; equipped with a magnetic stirrer at 300 rpm, the ultrafiltration separation was driven using high-purity N2 (99.999%) and the pressure was maintained within 2.4 atm. CPP was filtered and the filtrate was collected to detect the free ion concentrations. Fluoride concentration was detected by ion chromatography (IC-1100, Thermo Fisher Scientific, USA), and calcium concentration was measured using inductively coupled plasma-optical emission spectroscopy (ICP-OES, ICP-OES 730, Agilent, USA). Calcium and fluoride concentrations were expressed as mmol/L (mM).
The hydrophobicity of the s-HA surface with and without modification was determined by measuring the water contact angle with a Contact Angle Meter (DMo-501, Kyowa Kogyo CO., LTD, Japan). Deionized water was used as the medium (2 μL per drop), and the sessile drop technique was employed. The right and left water contact angles for each droplet were measured at room temperature and averaged. For each sample, the measurement was repeated at three randomly selected regions. Finally, the result was expressed as degree (°).
Zeta potential analysis
The electrophoretic mobilities of pellicle coated ceramic hydroxyapatite powder (Clarkson Chromatography Products, Williamsport, PA, USA) with and without modification were measured by a Zeta Potential Analyzer (Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK) according to a published method . The ceramic hydroxyapatite powder is the raw material for the HA disc that was used in the current study. Both the ceramic hydroxyapatite powder and HA disc were purchased from the same company. Pellicle formation and modification were carried out as described above in an incubator shaker (50 rpm) at 37 °C. Five measurements of electrophoretic mobilities for each sample were averaged, and the zeta potential was calculated according to the Helmholtz-Smoluchowski formula.
For all statistical analyses, SPSS v.19.0 software (IBM, Armonk, NY, USA) was used. All values were expressed as the mean ± standard deviation (SD). The inter-group differences were estimated by one-way analysis of variance (ANOVA) followed by a Turkey post hoc multiple comparison test. The level of significance was set at p < 0.05.