2.1

Specimen preparation

Forty-five extracted non-carious human third molars were collected from young patients (20–28 years old) after their informed consent had been obtained under a protocol reviewed and approved by the Human Assurance Committee of the University of São Paulo, São Paulo, Brazil. Teeth were stored in 0.9% NaCl containing 0.02% sodium azide at 4 °C for less than 1 month. After organic debris/calculus removal, teeth were sectioned at the cementum–enamel junction to remove the roots. The pulp tissue was scraped off with sterile spoon excavators. Enamel and cementum were completely removed from the crown segment with high-speed diamond burs under copious water cooling. Dentin disks (0.5 ± 0.02 mm thick and 6.0 ± 0.01 mm diameter) were obtained from the mid-coronal portion of each tooth using a slow speed diamond saw (Labcut 1010, Extec Corp., Enfield, CT, USA) under water cooling followed by the use of a diamond-incrusted coring drill with a 6 mm internal diameter (Continental Diamond Tool Corp., New Haven, IN, USA). Dentin disks were ultra-sonicated in distilled-water bath for 15 min. Then, they were pre-dried in a sealed desiccator containing fresh silica gel at 37 °C, until a constant dry mass was obtained in analytical balance (Mettler Toledo Inc., Columbus, OH, USA) (i.e. 0.057 ± 0.009 g). Afterwards, they were individually immersed in distilled water at 37 °C for 1 week, gently wiped with absorbent paper and weighed in an analytical balance for determination of their wet mass (i.e. 0.062 ± 0.01 g). The inner, exposed surface of dentin disks (i.e. those that previously were in close contact with the pulp chamber) was completely covered with two layers of a nail varnish (Risqué, Niasi S/A, São Paulo, SP, Brazil), left to dry for 30 min (at 25 °C, 56% relative humidity) and had their dry mass re-measured (i.e. 0.063 ± 0.008 g).

Specimens were assigned for one of the following groups ( n = 15): (1) dentin disks were kept mineralized (MD); (2) the outer, uncovered dentin surface of dentin disks was partially demineralized with liquid 37% phosphoric acid for 15 s and thoroughly rinsed with distilled water for 60 s (PDD); (3) dentin disks were totally demineralized with liquid 10% phosphoric acid for 12 h and washed for 24 h (TDD). Although a pilot study showed that the selected nail varnish is apparently acid-resistant (data not shown), care was taken to avoid direct contact between the varnish-covered dentin surface and the phosphoric acid solution. Disks of synthetic hydroxyapatite were also prepared . One of the surfaces of these disks was completely covered with two layers of the same nail varnish while the other was kept exposed, as previously described for the dentin disks. Additionally, films of nail varnish (0.5 ± 0.01 mm thick and 6.0 ± 0.01 mm diameter) were also prepared.

2.2

Treatment of specimen surface

The exposed, dried, uncovered surface of each substrate (i.e. MD, PDD, TDD and HA) was treated with: (1) 10 μL of distilled water (controls); (2) 10 μL of 0.2% CHX (3.2 mM) or (3) 10 μL of 2% CHX (32.0 mM). The test solutions were applied uniformly on the specimens’ surface using a micro-syringe and they were left undisturbed for 30 s. No excess solution was removed from the specimens’ surface. With a pair of small forceps, the specimens were carefully transferred to 2-mL plastic centrifuge tubes containing 1 mL of PBS (pH 6.8) and incubated at 37 °C. Film-like specimens of nail varnish were also individually incubated under same conditions.

2.3

Analysis of eluates

The 1 mL PBS contents of each tube containing any eluted CHX were spectrophotometrically analyzed in quartz cuvettes at 260 nm after the following periods of incubation: 0.5 h, 1 h, 3, 6, 24, 168, 672 and 1344 h. The eluates were never discarded so that after measuring their absorbance values, at each determined period, the eluates were returned to the same plastic centrifuge tubes for additional incubation of specimens. The cumulative concentration of CHX in the eluates was estimated as a function of their UV-absorbances plotted against a CHX standard curve. Substantivity of CHX to tested substrates was expressed (in percentage) based on the CHX-applied dose. In theory, if CHX was not able to bind to the tested substrates, one should expect to detect the entire amount of applied CHX (10 μL of 0.2% or 2%) to be released into 1000 μL of PBS. This theoretical amount is 32 nmol (in 1000 μL) for specimens treated with 0.2% CHX and 320 nmol (in 1000 μL) for specimens treated with 2% CHX.

Data were analyzed by a three-way repeated measures ANOVA with substrate (MD, PDD, TDD and HA), specimen treatment mode (without, with 0.2% CHX or with 2% CHX) and time of incubation (0.5–1344 h) as main factors, and with the eluate as factor of repetition. Post hoc multiple comparisons were performed using Holm-Sidak test. Statistical significance was preset at α = 0.05.

2.1

Specimen preparation

Forty-five extracted non-carious human third molars were collected from young patients (20–28 years old) after their informed consent had been obtained under a protocol reviewed and approved by the Human Assurance Committee of the University of São Paulo, São Paulo, Brazil. Teeth were stored in 0.9% NaCl containing 0.02% sodium azide at 4 °C for less than 1 month. After organic debris/calculus removal, teeth were sectioned at the cementum–enamel junction to remove the roots. The pulp tissue was scraped off with sterile spoon excavators. Enamel and cementum were completely removed from the crown segment with high-speed diamond burs under copious water cooling. Dentin disks (0.5 ± 0.02 mm thick and 6.0 ± 0.01 mm diameter) were obtained from the mid-coronal portion of each tooth using a slow speed diamond saw (Labcut 1010, Extec Corp., Enfield, CT, USA) under water cooling followed by the use of a diamond-incrusted coring drill with a 6 mm internal diameter (Continental Diamond Tool Corp., New Haven, IN, USA). Dentin disks were ultra-sonicated in distilled-water bath for 15 min. Then, they were pre-dried in a sealed desiccator containing fresh silica gel at 37 °C, until a constant dry mass was obtained in analytical balance (Mettler Toledo Inc., Columbus, OH, USA) (i.e. 0.057 ± 0.009 g). Afterwards, they were individually immersed in distilled water at 37 °C for 1 week, gently wiped with absorbent paper and weighed in an analytical balance for determination of their wet mass (i.e. 0.062 ± 0.01 g). The inner, exposed surface of dentin disks (i.e. those that previously were in close contact with the pulp chamber) was completely covered with two layers of a nail varnish (Risqué, Niasi S/A, São Paulo, SP, Brazil), left to dry for 30 min (at 25 °C, 56% relative humidity) and had their dry mass re-measured (i.e. 0.063 ± 0.008 g).

Specimens were assigned for one of the following groups ( n = 15): (1) dentin disks were kept mineralized (MD); (2) the outer, uncovered dentin surface of dentin disks was partially demineralized with liquid 37% phosphoric acid for 15 s and thoroughly rinsed with distilled water for 60 s (PDD); (3) dentin disks were totally demineralized with liquid 10% phosphoric acid for 12 h and washed for 24 h (TDD). Although a pilot study showed that the selected nail varnish is apparently acid-resistant (data not shown), care was taken to avoid direct contact between the varnish-covered dentin surface and the phosphoric acid solution. Disks of synthetic hydroxyapatite were also prepared . One of the surfaces of these disks was completely covered with two layers of the same nail varnish while the other was kept exposed, as previously described for the dentin disks. Additionally, films of nail varnish (0.5 ± 0.01 mm thick and 6.0 ± 0.01 mm diameter) were also prepared.

2.2

Treatment of specimen surface

The exposed, dried, uncovered surface of each substrate (i.e. MD, PDD, TDD and HA) was treated with: (1) 10 μL of distilled water (controls); (2) 10 μL of 0.2% CHX (3.2 mM) or (3) 10 μL of 2% CHX (32.0 mM). The test solutions were applied uniformly on the specimens’ surface using a micro-syringe and they were left undisturbed for 30 s. No excess solution was removed from the specimens’ surface. With a pair of small forceps, the specimens were carefully transferred to 2-mL plastic centrifuge tubes containing 1 mL of PBS (pH 6.8) and incubated at 37 °C. Film-like specimens of nail varnish were also individually incubated under same conditions.

2.3

Analysis of eluates

The 1 mL PBS contents of each tube containing any eluted CHX were spectrophotometrically analyzed in quartz cuvettes at 260 nm after the following periods of incubation: 0.5 h, 1 h, 3, 6, 24, 168, 672 and 1344 h. The eluates were never discarded so that after measuring their absorbance values, at each determined period, the eluates were returned to the same plastic centrifuge tubes for additional incubation of specimens. The cumulative concentration of CHX in the eluates was estimated as a function of their UV-absorbances plotted against a CHX standard curve. Substantivity of CHX to tested substrates was expressed (in percentage) based on the CHX-applied dose. In theory, if CHX was not able to bind to the tested substrates, one should expect to detect the entire amount of applied CHX (10 μL of 0.2% or 2%) to be released into 1000 μL of PBS. This theoretical amount is 32 nmol (in 1000 μL) for specimens treated with 0.2% CHX and 320 nmol (in 1000 μL) for specimens treated with 2% CHX.

Data were analyzed by a three-way repeated measures ANOVA with substrate (MD, PDD, TDD and HA), specimen treatment mode (without, with 0.2% CHX or with 2% CHX) and time of incubation (0.5–1344 h) as main factors, and with the eluate as factor of repetition. Post hoc multiple comparisons were performed using Holm-Sidak test. Statistical significance was preset at α = 0.05.