Phosphoric acid

After demineralization, the enamel was pretreated as follows and fissure sealant was applied:
Group 1 (control): The enamel was etched with phosphoric acid 35% (3 M, ESPE, St. Paul, MN, USA) for 20 s, rinsed and dried under a weak air stream. Then an unfilled fissure sealant (FS) (Clinpro, 3 M ESPE, St. Paul, Minn, USA) was applied and cured with a halogen light curing unit (Coltolux, Coltene, Whaledent, Altstaetten, Switzerland) at a power density of 550 mW/cm² for 40 s.
Group 2 (nano-HA 0.15%): After the enamel was etched as described above for group 1, each sample was immersed in 5 mL of a solution that contained 0.15% nano-HA in a closed glass vial with continuous slow speed rotation (4 rmp) for 5 min to ensure that the nanoparticles remained in suspension and to avoid precipitation [24 (link)]. Then each tooth was dried under an air stream and the sealant was applied.
Group 3 (nano-HA 0.03%): The procedures were similar to group 2, except that the solution contained nano-HA at 0.03% concentration.
Group 4 (nano-HA 0.15% + SHMP 0.05%): The solution powder contained nano-HA 0.15% and SHMP 0.05%, which were mixed together before the solvent was added. The other procedures were similar to groups 2 and 3.
Group 5 (nano-HA 0.03% + SHMP 0.01%): The procedures were similar to group 4, except that the solution contained nano-HA at 0.03% and SHMP at 0.01%.

A total of 30 dentin discs were randomly assigned to five groups (n = 6/group) according to biofilm formation and surface treatment as follows:(1)

Group C (control): no biofilm formation;

(2)

Group BF: biofilm formation and no surface treatment;

(3)

Group BF-E: biofilm formation and treatment with etching using 37% phosphoric acid (3M ESPE, St. Paul, MN, USA) gel for 15 s and rinsing with distilled water for 30 s;

(4)

Group BF-EC: biofilm formation and treatment with etching using 37% phosphoric acid gel for 15 s, soaking in chlorhexidine for 5 min after drying, and rinsing with distilled water for 30 s;

(5)

Group BF-RE: biofilm formation and prophylaxis using a rubber cup and plain pumice for 30 s, followed by etching using 37% phosphoric acid gel for 15 s, and rinsing with distilled water for 30 s.

Half of the samples in each group (n = 3) were observed with confocal laser scanning microscopy (LSM800, Carl Zeiss, Oberkochen, Germany) and scanning electron microscopy (SEM, S-4700, Hitachi, Tokyo, Japan) to quantitatively and qualitatively assess the biofilm, respectively.

Before the start of the adhesive procedure, the surfaces of the glass-fiber posts were treated with 35% phosphoric acid (3M ESPE, St. Paul, MN, EUA) for 60 seconds. They were then washed and air dried. The post surfaces were silanized for 60 seconds (Ângelus) and gently dried with an air jet. Finally, on the basis of the treatment to be performed in the intraradicular dentin, the adhesive system used on the dentin surface was also applied on the post surface if required. Thereafter, the post was not manipulated further to prevent contamination.