To evaluate the effects of 8% arginine-containing toothpaste on the dental plaque of no caries (NC) and high caries (HC) individuals in situ .
6 no caries (DMFT = 0) and 6 high caries (DMFT ≥ 6) individuals wearing a self-developed in situ dental plaque acquisition device were involved in a randomized double-blinded crossover study for 6 weeks: including lead-in (1 week), arginine-free (2 weeks), washout (1 week) and arginine-active (2 weeks) stages. The in situ plaque samples were collected at the endpoint of arginine-free and arginine-active stages and subjected to lactic acid production, metabolic activity, live/dead bacteria ratio and total biofilm biomass detections.
The arginine-containing dentifrice reduced lactic acid production significantly in both the NC and HC groups, while the inhibitory abilities in the HC group were stronger than that in the NC group. In addition, the arginine-containing dentifrice didn’t significantly decrease the metabolic activity, live/dead bacteria ratio and total biofilm biomass in either the NC or the HC group.
Arginine-containing toothpaste can significantly reduce the lactic acid production from the in situ plaques to a low level without changing the metabolic activity, live/dead bacteria ratio and total biofilm biomass through a critical clinical randomized double-blinded crossover study.
Arginine is a potential ecological prevention and control agent for dental caries. Meanwhile, the in situ model is an easy and pragmatic way to evaluate oral hygiene products (clinical trial registration: ChiCTR-INR-16010226).
Dental caries is one of the most common infectious and chronic diseases in human oral cavity. The loss of the oral microbial balance caused the increase of glycolytic acid production such as lactic acid from fermentable carbohydrates is considered as the major cariogenic agent. The increase of aciogenic/aciduric oral bacteria can not only cause the demineralization of the tooth by leading to a drop of pH value, but also provides a competitive acidic growth environment over other commensal species . Arginine, an alkali-generating substrate, can inhibit tooth demineralization by neutralizing glycolytic acid, which is a promising strategy for dental caries prevention through an exogenous source from oral care products .
Recently, our group demonstrated that arginine may augment ecological benefits by enriching alkali-generating S. sanguinis and prevent the overgrowth of the periodontal pathogen P. gingivalis in multi-species biofilms in vitro . Meanwhile, accumulating conventional clinical trial data have shown that arginine-containing toothpaste significantly increased ecological benefits, rendering it less susceptible to cariogenic challenges . Those studies mostly focused on the control of multispecies biofilms cultured in saliva in vitro , which may not be representative of dental plaque under real clinical conditions. However, according to previous studies in vivo , the 3-dimensional structure of the dental plaque was crashed, when bacterial samples were scaled from the tooth surface or dorsum of the tongue . To get whole clinical dental plaques from oral cavity and access the in situ dental plaque characters, a self-developed in situ dental plaque acquisition device was developed in our lab and was designed to clinically reproduce the oral environment of individuals with different caries statuses and to obtain the integrated biofilm without the distorting of the 3-dimensional structure during formation, collection or analysis processes.
The aim of the current study was to clinically evaluate the effects of arginine-containing toothpaste on plaque samples collected from the self-developed in situ dental plaque acquisition device by the analysis of acid production, metabolic activity and biomass change.
Materials and methods
Registration and ethical aspects
This in situ , double-blinded, single-center, randomized controlled crossover study was authorized by the Institutional Review Board, West China Hospital of Stomatology, Sichuan University, Chengdu, China (WCHSIRB-ST-2014-085) and the Chinese Clinical Trial Registry (registration no. ChiCTR-INR-16010226). The manuscript was prepared in compliance with the CONSORT checklist. A principal investigator was responsible for adherence to the study protocol, two investigators performed all clinical and technical procedures. An external monitor followed the study (initiation, follow-up, and close-out). Written informed consent was obtained from all the participants in the study. Participants had the right to withdraw from the study at any time and for any reason without prejudice.
A total of 12 adult volunteers (mean age, 22.5 ± 2.6 yrs; 6 females and 6 males) were recruited from West China Hospital of Stomatology at Sichuan University (Table S1). The following exclusion criteria were employed: smoker or former smoker, presence of any systemic disease that could alter the production or composition of the saliva, treatment with antibiotics, steroids or any medication known to cause dry mouth in the last 3 months, having any known allergy to previously used oral hygiene products or dental materials, and presence of dental prostheses or orthodontic devices that might affect the oral environment. Participants were categorized into 2 groups. The no caries (NC) group consisted of 6 individuals with no clinical evidence of caries experience [decayed, missing and filled teeth (DMFT = 0)]. The high caries (HC) group consisted of 6 individuals with decayed, missing and filled teeth (DMFT ≥ 6). In this study, 10 participants (5 NC and 5 HC) completed all the study visits. 2 participants were excluded from the study due to self-reported lack of compliance.
This in situ , double-blinded, randomized controlled crossover study was carried out for 6 weeks, including a 1-week lead-in period, 2-week arginine-free phase, 1-week washout period and 2-week arginine-active phase. In each phase, participants were randomly assigned to use either arginine-free (arginine-free phase) or arginine-containing (arginine-active phase) toothpaste. The arginine-free toothpaste was Colgate ® Total ® Advanced Toothpaste (containing 1450 ppm F − as sodium fluoride). The arginine-containing toothpaste was Colgate ® Sensitive Pro-Relief™ Toothpaste (containing 8% arginine and 1450 ppm F − as sodium fluoride). Dental plaque samples were collected at the endpoint of both phase 1 (first 2-week treatment) and phase 2 (second 2-week treatment). Participants who used arginine-free toothpaste in phase 1 used arginine-containing toothpaste in phase 2 and vice versa ( Fig. 1 ). During the lead-in and washout period, participants brushed their teeth using Colgate ® Total ® Advanced Toothpaste (containing 1450 ppm F − as sodium fluoride).
Preparation of the palatal device and specimen
The independently designed in situ model , was made with six hydroxyapatite (HA) slabs recessed into a palatal appliance. A schematic illustration and an intraoral view of the in situ model is presented in Fig. 1 . Every site had a 1-mm uniform gap covered by plastic mesh to allow for free contact of the saliva with the specimen’s surface to form the plaque biofilms and to protect it from a mechanical disturbance. HA slabs (4 mm × 4 mm × 2 mm, BAM, National Engineering Research Center for Biomaterials, Chengdu, China) were stored in 0.1% thymol solution (pH = 7.0) at 4 °C, and randomly assigned to each site, fixed with silicone rubber. New slabs were inserted into the appliance before each stage. To minimize the contact between the tongue and the specimens, the sites were positioned posterior to the incisive papillae. Participants had an appliance try-in appointment where the necessary adjustments were made before each phase.
Intervention and sample collection
Participants performed oral hygiene with the provided toothbrush and toothpaste habitually. The frequency of tooth brushing was twice a day for 3 min. The recommended amount of dentifrice was approximately 1 g or 2 cm in length. Participants should wear the appliances for longer than 20 h per day. They could only remove the device while eating their main meals and brushing, and the appliance should have been rinsed off food debris and stored in PBS. Participants were also required to eat and drink as usual and keep a record of their daily food intake during phase 1 to guide them to eat similar food during phase 2. They were not permitted to use any other source of fluoride throughout the whole study period.
Participants were asked to refrain from brushing and flossing their teeth, eating or drinking anything other than water for 8 h prior to each collection visit, which was performed between 8 and 11 a.m. . The biofilms from the in situ dental plaque acquisition device of each phase were transferred to sterile 24-well plates and analyzed immediately or, if necessary, snap-frozen and stored at −80 °C until further analysis . There were 6 blocks available for analysis from each in situ model appliance; 2 of 6 specimens were used for lactic acid measurement and MTT assay, 2 were used for SEM imaging and 2 were used for live/dead staining, respectively.
Randomization and blinding
The study products and test materials were provided as coded packages labeled with participant number and study period. The order of treatments for participants was randomly allocated by using a computer-generated randomization list. The toothpastes were provided in similar plain-labelled containers. None of the participants, investigators, or monitor knew the type of toothpaste until all procedures were finalized, and the close-out visit of the monitor had been finished satisfactorily.
Lactic acid measurement
Specimens with 14-day biofilms were transferred to individual wells of 24-well plates with 1.5 mL of buffered peptone water (BPW, Sigma-Aldrich, St. Louis, MO) supplemented with 0.2% sucrose in each well. During acid production assay, BPW medium was used to maintain the stability of the mature biofilm due to its relatively high buffer capacity. After incubation at 5% CO 2 and 37 °C for 3 h, lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method . The absorbance at 340 nm (optical density OD340) was measured via a microplate reader (SpectraMax, Molecular Devices, Sunnyvale, CA, USA) for the collected BPW solutions. Standard curves were prepared using a lactic acid standard (Supelco, Bellefonte, PA, USA).
MTT metabolic assay
For MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) metabolic assay, specimens with 14-day biofilms were rinsed in phosphate buffer solution (PBS) to remove loose bacteria and were placed in 24-well plates containing buffered peptone water (BPW) supplemented with 0.2% sucrose. After 3 h of incubation, the BPW solutions were stored for lactate acid analysis, and each specimen was transferred to a new 24-well plate with 1 mL of MTT dye (0.5 mg/mL of MTT in PBS) in each well for the MTT assay described previously . A microplate reader (SpectraMax, Molecular Devices, Sunnyvale, CA) was used to measure absorbance at the 540 nm (optical density OD540).
For the scanning electron microscopy (SEM), specimens with 14-day biofilms were washed twice with PBS, fixed with 2.5% glutaraldehyde overnight, and serially dehydrated with ethanol (50%, 60%, 70%, 80%, 90%, 95%, and 100%). Then, the dehydrated samples were sputter-coated with gold and examined at × 10,000 magnification (FEI, Hillsboro, OR, USA).
For live/dead imaging, the biofilms were stained according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Briefly, the biofilms were stained with 2.5 μM of Syto 9 (Molecular Probes, Invitrogen) and propidium iodide (Molecular Probes) for 15 min. The labeled biofilms were imaged using a DMIRE2 confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with a 60 × oil immersion objective lens. The channels were set as follows: excitation/emission maxima 480/500 nm for Syto 9 stain, 490/635 nm for propidium iodide stain .
All three-dimensional reconstructions of the in situ biofilms and the quantification of live/dead biomass were performed using Imaris 7.0.0 (Bitplane, Zürich, Switzerland). Quantification of the live/dead bacteria ratio was performed using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA).
The lactic acid measurement and MTT metabolic assay were performed in triplicate. The washout period was designed long enough to rule out a carryover effect and was checked in a preliminary test to improve statistical power. We measured the residual effects of arginine-containing toothpaste on acid production, metabolic activity and biomass change within in situ biofilms. SPSS statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. P < 0.01 were regarded as being statistically significant. In Table S2, S3, S8, S9, the normality of results was measured by a Shapiro-Wilk test and Boxplot graphs, and unpaired t -test or the Wilcoxon rank sum test was used to measure the significant difference between groups, depending on the results distribution. In Table 1 , the general linear model identified an interaction effect between caries status and treatment methods. In Table S10, Fisher’s LSD test was used for simple effect analysis.
|Lactic acid production||MTT||Total biofilm biomass||Live/dead bacterial ratio|
|Caries status a||277.6||0.000**||199.4||0.000**||85.9||0.000**||0.8||0.380|
|Caries status* Treatment e||123.8||0.000**||2.4||0.150||0.2||0.660||13.8||0.003**|