Dentin remineralization at the bonded interface would protect it from external risk factors, therefore, would enhance the longevity of restoration and combat secondary caries. Dental biofilm, as one of the critical biological factors in caries formation, should not be neglected in the assessment of caries preventive agents. In this work, the remineralization effectiveness of demineralized human dentin in a multi-species dental biofilm environment via an adhesive containing nanoparticles of amorphous calcium phosphate (NACP) and dimethylaminohexadecyl methacrylate (DMAHDM) was investigated.
Dentin demineralization was promoted by subjecting samples to a three-species acidic biofilm containing Streptococcus mutans , Streptococcus sanguinis, Streptococcus gordonii for 24 h. Samples were divided into a control group, a DMAHDM adhesive group, an NACP group, and an NACP + DMAHDM adhesive group. A bonded model containing a control-bonded group, a DMAHDM-bonded group, an NACP-bonded group, and an NACP + DMAHDM-bonded group was also included in this study. All samples were subjected to a remineralization protocol consisting of 4-h exposure per 24-h period in brain heart infusion broth plus 1% sucrose (BHIS) followed by immersion in artificial saliva for the remaining period. The pH of BHIS after 4-h immersion was measured every other day. After 14 days, the biofilm was assessed for colony-forming unit (CFU) count, lactic acid production, live/dead staining, and calcium and phosphate content. The mineral changes in the demineralized dentin samples were analyzed by transverse microradiography.
The in vitro experiment results showed that the NACP + DMAHDM adhesive effectively achieved acid neutralization, decreased biofilm colony-forming unit (CFU) count, decreased biofilm lactic acid production, and increased biofilm calcium and phosphate content. The NACP + DMAHDM adhesive group had higher remineralization value than the NACP or DMAHDM alone adhesive group.
The NACP + DMAHDM adhesive was effective in remineralizing dentin lesion in a biofilm model. It is promising to use NACP + DMAHDM adhesive to protect bonded interface, inhibit secondary caries, and prolong the longevity of restoration.
Dental caries remains to be one of the major oral diseases challenging global public health . Adhesives and resin composites are widely used for restoring dental caries due to their esthetics and direct-filling ability . However, the dentin-resin bonded interface is considered to be the weak link in the restoration . The failure of adhesion may induce marginal discoloration, loss of retention of composite restoration, and secondary caries . Regardless of different techniques used in resin bonding procedures, a structure composed of demineralized collagen fibrils reinforced by resin matrix is formed, namely hybrid layer (HL). Dentin bonding relies on the integrity of HL . Nevertheless, exposed collagen fibrils that have failed to be reinforced by resin matrix can be destroyed by enzymes or acids produced by oral bacteria, leading to HL degradation and bonding failure . Thus, protecting HL from the attack by external risk factors is key to strengthening dentin-resin bonds. Because minerals have great HL protecting effectiveness, remineralization of HL in resin-sparse regions is an effective method to maintain the tight dentin-resin bonds . However, natural remineralization could be too weak to protect HL when faced with severe bacterial acid challenge . Therefore, exploring remineralization strategies could be helpful to dentin-resin bond preservation, which could prolong the longevity of restorations.
An effective strategy for remineralization is adding calcium phosphate (CaP) filler particles to adhesive resins . Previous studies have developed adhesive containing nanoparticles of amorphous calcium phosphate (NACP) , which could neutralize acids and raise fluid pH , and could release large amounts of calcium (Ca) and phosphate (P) ion “smartly”, there was an increase in the Ca and P ion release at a cariogenic pH . Both of those could be beneficial for remineralization. Due to the small particle size, NACP could easily flow with adhesive into dentinal tubules, forming resin tags . NACP-containing adhesive achieved dentin remineralization after an artificial saliva/lactic acid cycle . Poly(amido amine) dendrimer combined with NACP adhesive achieved dentin remineralization in a lactic acid environment .
Acquired pellicle, as the early form of dental biofilm, can be formed within 20 min after cleaning and polishing tooth surface. Dental biofilm is difficult to remove completely at the tooth-composite bond. Dental biofilm plays a critical biological role in caries formation. Cariogenic bacteria in dental biofilm produce acids, challenging the effectiveness of anti-caries agents. Thus, investigating remineralization ability in cariogenic biofilm-challenged environment corresponds to practical condition in clinic, which should not be neglected when studying caries preventive agents . To date, there has been no report on NACP adhesive for dentin remineralization in a biofilm environment, except our previous study, in which NACP adhesive was shown to have the ability to remineralize dentin demineralized lesion in a Streptococcus mutans ( S. mutans ) single-species biofilm environment . However, the ecology of dental caries is rather complex and dental biofilm contains more than S. mutans alone. Multi-species biofilm corresponds to practical dental biofilm better than S. mutans single-species biofilm. In addition, fewer bacteria are supposed to produce less acid, which could be beneficial for remineralization. However, whether incorporating antibacterial agent could improve dentin remineralization effect of NACP adhesive remains unknown. It would be necessary to evaluate the dentin remineralization effectiveness of NACP adhesive after incorporating antibacterial agent in a multi-species biofilm-challenged environment.
Dimethylaminohexadecyl methacrylate (DMAHDM) is a kind of quaternary ammonium salt with an alkyl chain length of 16, which has a strong antibacterial effect. It can cause cytoplasmic leakage through binding to bacterial cell membrane . DMAHDM was shown to have strong antibacterial and antibiofilm effects. Minimum inhibitory concentration (mg/mL) with S. mutans for DMAHDM was 0.0006 . CFU for DMAHDM adhesive was 4 log lower than the control group . The total microorganisms, total streptococci and S. mutans on DMAHDM containing adhesive were almost 4 orders of magnitude less than those of the control group . Therefore, DMAHDM was chosen to incorporate into NACP adhesive in present study.
Accordingly, the aim of this study was to assess the effects of NACP + DMAHDM adhesive on dentin remineralization in a multi-species biofilm-challenged environment for the first time. It was hypothesized that (1) NACP + DMAHDM adhesive would increase Ca and P ion concentrations in biofilm, neutralize bacterial acids, kill bacteria, and achieve remineralization of dentin lesions; and (2) NACP + DMAHDM adhesive would achieve greater remineralization effects on dentin demineralized lesions than NACP or DMAHDM adhesive alone in the biofilm-challenged environment.
Materials and methods
Fabrication of NACP and DMAHDM
Nanoparticles of ACP (Ca 3 [PO 4 ] 2 ), referred to as NACP, were fabricated via a spray-drying technique as reported previously . In brief, calcium carbonate and dicalcium phosphate were dissolved into an acetic acid solution to get Ca and P ionic concentrations of 8 and 5.333 mmol/L, respectively. The Ca/P molar ratio was 1.5, which was consistent with that of ACP. The solution was subsequently sprayed into a heated chamber, then an electrostatic precipitator was used to collect the dried particles, obtaining NACP with a mean particle size of 116 nm .
DMAHDM was fabricated using a modified Menschutkin reaction . Briefly, 10 mmol 1-bromohexadecane (BHD, TCI America, Portland, OR, USA) and 10 mmol 2-(dimethy-lamino) ethyl methacrylate (DMAEMA, Sigma, St. Louis, MO) were added into 3 g ethanol in a 20 mL scintillation vial . The vial was stirred at 70 °C for 24 h. The sample was subsequently put under vacuum to remove the solvent, any impurities and unreacted components, yielding DMAHDM as solid at room temperature .
Fabrication of adhesive samples
Scotchbond Multi-Purpose (SBMP, 3M, St. Paul, MN, USA) adhesive was used to test the effects of incorporation of NACP and DMAHDM in a biofilm-challenged environment, following previous studies . The SBMP adhesive included 60–70% Bisphenol A diglycidyl methacrylate (Bis-GMA) and 30–40% 2-hydroxyethyl methacrylate (HEMA), according to the manufacturer information. NACP was mixed into SBMP adhesive at mass fraction of 40%, and DMAHDM was mixed into the adhesive at mass fraction of 5%. A previous study demonstrated that raising NACP filler level from 10% to 40% significantly raised the Ca and P ion release . Similarly, raising DMAHDM filler level also raised the antibacterial ability , and mixing 40% NACP and 5% DMAHDM into SBMP adhesive did not undermine the dentin bond strength . Hence, SBMP adhesive containing 40% NACP or/and 5% DMAHDM was used in current study.
Thus, three kinds of SBMP adhesive were fabricated as follows: (1) SBMP + 5% DMAHDM, referred as SBMP-DMAHDM; (2) SBMP + 40% NACP, referred as SBMP-NACP; (3) SBMP + 40% NACP + 5% DMAHDM, referred as SBMP-NACP-DMAHDM. These three kinds of SBMP adhesive were placed into a rectangular mold (2 × 2 × 12 mm) as previously described , and light-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side. The SBMP bars were sterilized in ethylene oxide sterilizer (Anprolene AN 74i, Andersen, Haw River, NC, USA) before use.
Preparation of dentin specimens
Extracted caries-free human molars were collected at West China Hospital of Stomatology, Sichuan University, after being approved by the Institutional Review Board. The cleaned molars were stored in 0.5% thymol at 4 °C, which were stored no more than four weeks before use.
Each molar was cut at the cement-enamel junction by a low-speed water cooled diamond saw (Minitom, Struers, Copenhagen, Denmark). Enamel on the dentin block surface was removed using 120-grit carbide polishing papers under running water, and then dentin disks of 3.5 ± 0.5 mm thickness were obtained. The dentin samples were subsequently protected with acrylic resin. The dentin sample surfaces were polished with 800, 1200 and 2400-grit carbide polishing papers under running water, which were then ultrasonicated with an ultrasonic cleaner (FS20, Fisher Scientific Co., Pittsburgh, PA, USA) in distilled water for 20 min to remove the smear layer produced during the polishing process. Dentin hardness of all the prepared samples was examined by a Vickers hardness tester (MMT-X7A, Matsuzawa, Japan) with a diamond indenter under a 25-gf load for 10 s . Five indentations were made for each dentin specimen, and only those in the range from 0.6 to 0.8 GPa were collected for the following study . The collected dentin sample surfaces were partly painted by two layers of acid-resistant nail varnish, leaving an exposed 4 × 4 mm 2 window.
To mimic the real oral environment when adhesive is applied to dentin, a bonded model was also used in present study . For the bonded model in our current study, a dentin block was bonded with composite (Z250XT, 3M, USA) on a lateral face using SBMP, SBMP-DMAHDM, SBMP-NACP, SBMP-NACP-DMAHDM adhesive. The following embedding and polishing procedures were the same as previously described. The tooth samples were then stored in Ca-free phosphate buffered saline (PBS) at 4 °C before use. Before the following experiment. The dentin specimens were sterilized in ethylene oxide sterilizer (Anprolene AN 74i, Andersen) before the next experiment procedures.
Artificial caries biofilm formation
The multi-species biofilm including S. mutans UA159, Streptococcus sanguinis ( S. sanguinis ) ATCC 10556, Streptococcus gordonii ( S. gordonii ) DL1, which were provided by the State Key Laboratory of Oral Diseases (Sichuan University, Chengdu, China). Firstly, the bacteria were cultured overnight in brain-heart infusion broth (BHI; Difco, Sparks, MD, USA) at 37 °C anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ). The resulting bacterial suspensions were then mixed to obtain an inoculum containing a defined microbial population including 10 7 CFU/mL of each species above in BHIS for the following experiment .
The sterile dentin samples were placed in 24-well plates containing 2 mL of microbial suspension as previously mentioned. The samples were incubated at 37 °C anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ) for 24 h to induce a demineralization lesion. After 24 h, demineralized dentin lesions beneath the multi-species biofilms were formed, which had been demonstrated via transverse microradiography (TMR) analysis during the pre-experiment ( Fig. 6 A). The microbial supernatant was then removed, the biofilm formed on the dentin samples was also removed with pipette tips under aseptic conditions except for the exposed dentin area .
A dentin specimen with multi-species biofilm was put into a 5-mL Eppendorf tube under the aseptic conditions. These samples were then randomly divided into four groups and treated as described in the next paragraph.
Each biofilm-coated dentin sample underwent the entire remineralization/demineralization protocol without any treatment, to serve as a negative control .
Each biofilm-coated dentin sample was placed in contact with three SBMP-DMAHDM bars . Three bars were applied because when immersed in 1-mL solution, there would be a sample volume/solution volume ratio of 0.14/1, as was done in a previous study .
Three SBMP-NACP bars were placed on the dentin sample as described in (2) .
Three SBMP-NACP-DMAHDM adhesive bars were placed on a dentin sample as described in (2).
These four groups are denoted by Control, DMAHDM, NACP and NACP + DMAHDM. Twelve specimens were examined for each group ( n = 12). As for the bonded model, four groups with dentin blocks bonding composites using SBMP/SBMP-DMAHDM/SBMP-NACP/SBMP-NACP-DMAHDM adhesive are denoted by Control-bonded, DMAHDM-bonded, NACP-bonded and NACP + DMAHDM-bonded, respectively. Six dentin blocks were included for each bonded model group ( n = 6). A 5-mL Eppendorf tube was used to store each specimen, which was immersed in 1 mL solution cycle as described in the next paragraph.
An artificial saliva solution was prepared by dissolving 1.5 mmol/L CaCl 2 , 0.9 mmol/L KH 2 PO 4 , 130 mmol/L KCl, and 20 mmol/L 4-(2-hydroxyethyl-)-1-piperazineethanesulfonic acid (HEPES), adjusting pH to 7.0 with potassium hydroxide (1 mmol/L) . It was filtered by cylinder membrane filter to remove any bacteria before use. Each day, each biofilm-coated sample of the aforementioned eight groups was immersed in 1 mL of fresh artificial saliva for 20 h, and in sterilized BHIS for the other 4 h at 37 °C anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ), following a previous study . The 4-h immersion in BHIS presented a degree of correlation with oral conditions with respect to the accumulated food cycling in a 24-h period orally . The everyday medium change was performed under aseptic conditions and the samples were rinsed using sterile 0.9% normal saline at every medium refreshment point, in order to remove any unattached bacterial cell. This cyclic treatment was repeated for 14 days (d). Fig. 1 shows the entire experimental process.
At 1, 3, 5, 7, 10 and 14 d, the pH of BHIS broth for each sample after 4 h immersion treatment was measured by Orion Dual Star, pH/ISE Benchtop (Thermo Scientific, Waltham, MA, USA) .
Lactic acid measurement
Each sample was removed out of the Eppendorf tube at the end of cyclic solution treatment, rinsed by cysteine peptone water and transferred to a 24-well plate. Buffered peptone water (BPW) with 0.2% sucrose was added to each well and kept for 3 h at 37 °C anaerobically. An enzymatic method was used to analyze the lactic acid concentration of BPW . The absorbance of collected BPW solution for each sample was measured at 340 nm via a microplate reader (Multiskan Go, Thermo Scientific). The lactic acid concentration was calculated according to the standard curve of the standard lactic acid (Supelco Analytical, Bellefonte, PA, USA).
Colony-forming unit counts of biofilm
Biofilm was scraped/resuspended from the bottom of the exposed dentin area into 1.5 mL Eppendorf tubes with 1 mL sterile PBS using pipette tips and repeated pipetting . Serially diluted solution samples were then inoculated onto BHI agar plates. After a 2-day incubation, the colony numbers were counted.
Live/dead bacteria staining of biofilms
Dentin samples with biofilms were rinsed with sterile 0.9% normal saline to remove any non-adherent bacteria, and then stained using Baclight Live/dead bacterial viability kit (Molecular Probes, Eugene, OR, USA). Live bacteria were stained by SYTO 9 to produce a green fluorescence, and dead bacteria were stained by propidium iodide. Confocal imaging was performed using an Olympus FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan) with 60× oil immersion objective lens. The image collection gates were set at 495–515 nm for SYTO 9 and 565–665 nm for propidium iodide. The 3-dimensional reconstructions were performed with Imaris 7.2.3 (Bitplane, Zürich, Switzerland) .
Biofilm Ca and P content measurement
Biofilm was removed and transferred into Eppendorf tubes. 0.3 mL 0.5 M HCl per 10 mg biofilm wet weight was added into each tube to extract the Ca and P ions from the collected biofilms. The tubes were then shaken at 30 rpm for 3 h at room temperature . Ca and P ion concentrations were measured using a spectrophotometric method (Multiskan Go, Thermo Scientific) with known standards and calibration curves, as per previous studies ..
Dentin specimens were cut into sections approximately 300 μm thick using a diamond-coated band saw, vertically to the windows that were exposed to the cyclic immersion treatment. Similarly, the bonded dentin samples were cut vertically to the adhesion interface into sections about 300 μm thick. The sections were then polished into slices of approximately 100 μm thick, measured by a digital micrometer (Mitu-toyo, Tokyo, Japan). Slices were fixed on Plexiglass slides in a TMR sample holder (Inspektor Research Systems BV, Amsterdam, Netherlands). The slices were microradiographed alongside an aluminum calibration step-wedge with a monochromatic CuK X-ray source (Philips, Eindhoven, Netherlands) operated at 20 kV and 20 mA, and an exposure time of 25 s . Lesion depth, mineral loss and mineral content at selected depths were examined by imaging software (Transversal Microradiography Software 2006, Inspektor Research Systems BV). Six dentin slices were analyzed from each sample, five traces within areas exposed to cyclic immersion treatment were measured on each slice .
Remineralization in dentin lesion that occurred during the cyclic immersion treatment is calculated as: Remineralization R = (M Before − M After )/M Before , where M Before is the mineral loss value of dentin disk before remineralization protocol, and M After is the mineral loss value of the lesion after remineralization protocol .
Statistical analyses were performed with SPSS software version 21.0 (SPSS, Inc., IBM, Chicago, IL, USA). Kolmogorov–Smirnov test was used to test the normal distribution of data. One-way analysis of variance was conducted to examine the significant effects of the variables. Student–Newman–Keuls multiple comparison tests were conducted at a value of P = 0.05.
The dentin specimens with biofilm of four groups (Control, DMAHDM, NACP and NACP + DMAHDM) were immersed in artificial saliva for 20 h, and in BHIS for 4 h, every day during the remineralization/demineralization cycle. The BHIS after 4-h immersion for each sample was collected, of which the pH is plotted in Fig. 2 (mean ± SD). For the Control group, the pH decreased to approximately 4.5. For the NACP group, NACP achieved acid-neutralization function, and the pH was close to 6.0, which showed a slight decrease at the end of the cycle. DMAHDM achieved antibacterial function, bacterial acids were reduced, and the pH was approximately 6.5 for the DMAHDM group. NACP + DMAHDM achieved the best acid-neutralization effect, and the pH was close to 7.0 for the NACP + DMAHDM group, which showed a slight decrease at the end of the cycle. During the whole cycle, the pH values of BHIS after 4-h immersion for the four groups were NACP + DMAHDM > DMAHDM > NACP > Control ( P < 0.05).