Preparation of the nano-HA solution
Effect of hydroxyapatite nanoparticles on enamel remineralization and estimation of fissure sealant bond strength to remineralized tooth surfaces: an in vitro study
Abstract
Background
The management of noncavitated caries lesions before sealant therapy is a clinical challenge when the tooth needs sealant application. Sealing noncavitated carious lesions in pits and fissures may lead to failure of the fissure sealant (FS) due to incomplete sealing. Therefore the use of remineralizing agents such as nanoparticles has been suggested. This study investigated the ability of hydroxyapatite nanoparticles (nano-HA) to remineralize enamel, and their effect on sealant microleakage and shear bond strength (SBS).
Methods
A total of 192 third molars were demineralized and pretreated with two concentrations of nano-HA with and without sodium hexametaphosphate (SHMP), followed by phosphoric acid etching and resin FS application. The study groups were 1) etching + FS, 2) etching + nano-HA 0.15% + FS, 3) etching + nano-HA 0.03% + FS, 4) etching + mixture of nano-HA 0.15% and SHMP 0.05% + FS, 5) etching + mixture of nano-HA 0.03% + SHMP 0.01% + FS. The laboratory tests included microleakage in 50 teeth, scanning electron microscopy (SEM) evaluation in 10 samples, and SBS in 100 samples. Enamel remineralization changes were evaluated in 32 teeth with energy-dispersive X-ray spectroscopy (EDS) and field emission scanning electron microscope (FESEM).
Results
Nano-HA enhanced the SBS to remineralized enamel in a large percentage of nanoparticles. Mean SBS in group 2 was significantly greater than in groups 1, 3 and 4 (all P < 0.05). SBS was related to nano-HA concentration: nano-HA 0.15% yielded greater SBS (16.8 ± 2.7) than the 0.03% concentration (14.2 ± 2.1). However, its effect on microleakage was not significant. Nano-HA with or without SHMP led to enhanced enamel remineralization; however, the Calcium (Ca)/Phosphate (P) weight percent values did not differ significantly between the groups (P > 0.05). SEM images showed that SHMP did not affect sealant penetration into the deeper parts of fissures. FESEM images showed that adding SHMP led to increased nanoparticle dispersal on the tooth surface and less cluster formation.
Conclusions
The ultraconservative approach (combining nano-HA 0.15% and SHMP) and FS may be considered a minimal intervention in dentistry to seal demineralized enamel pits and fissures.
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Background
Pit and fissure sealing is an accepted method to prevent dental caries or arrest the progression of noncavitated carious lesions in the deep parts of occlusal surfaces. The number of bacteria and their production are reduced after pits and fissures are sealed [1]. However, some studies have recommended using a pit and fissure sealant on teeth with noncavitated lesions [2, 3]. In some cases, practitioners may be uncertain about sealing noncavitated carious lesions or incipient caries such as lesions in the deepest parts of pits and fissures. In addition, some dentists have concerns about applying the sealant on decalcified enamel in the deep parts of occlusal grooves or lateral walls of fissures, which are not detectable by visual examination [4, 5]. Several methods are available for enamel pretreatment prior to sealant therapy, e.g. preparation with burrs, air abrasion or laser [6, 7]. Some researchers have reported the use of the resin infiltration technique as a microinvasive approach to preserve demineralized enamel. A low-viscous resin infiltrant combined with a flowable composite resin has been used to seal the porous occlusal subsurface in initial caries lesions. This technique increased marginal adaption and internal integrity compared to the use of a conventional flowable composite as a fissure sealant [8].
Other methods use the benefits of remineralizing agents such as pit and fissure sealants containing fluoride, amorphous calcium phosphate or nanoparticles [2, 9,10,11,12,13,14,15,16]. With the introduction of nanotechnology, some researchers have tested the use of nanoparticles in restorative and preventive dentistry [17,18,19]. One type of nanoparticle used in dentistry is nanohydroxyapatite (nano-HA). Nano-HA was considered promising because of its similarity to the bone and mineral structure of teeth, biocompatibility, and bioactivity [19, 20]. The particles are “similar in morphology and crystal structure to dental apatite” [21]. The remineralization characteristics of nano-HA particles have been reported in studies in which nanoparticles were added to a glass ionomer or other restorative materials [20, 22]. Toothpastes containing nano-HA showed greater remineralization effects in dentin compared to amine fluoride toothpastes [23].
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To improve nano-HA infiltration and deagglomerate nano-HA particles, the addition of the deflocculant agent sodium hexametaphosphate (SHMP) to nano-HA has been recommended [24]. This results in more efficient hydroxyapatite crystal remineralization by providing a smaller particle size. Sodium hexametaphosphate is widely used as a food additive (E number E452i), water softener, and dispersing agent to break down clay and other soil types [25]. In the present study the effects of nano-HA with and without SHMP were compared.
Some studies have investigated commercial fissure sealants with nanoparticles added to sealant materials. These studies focused on the mechanical and chemical properties of the sealants as well as their antibacterial and remineralization characteristics [13,14,15,16]. However, no data are available on the use of nano-HA with pit and fissure sealants in demineralized enamel. The aim of the present study was to investigate the efficacy of nano-HA applied prior to the pit and fissure sealant to remineralize artificial enamel caries lesions. Our hypothesis (H0) was that two concentrations of nano-HA would be similar to conventional fissure sealant in their ability to infiltrate demineralized enamel without influencing the sealant’s sealing ability or bond strength of the sealant to tooth surface. The null hypothesis was tested against an alternative hypothesis (HA) that differences would be found. We compared microleakage and shear bond strength (SBS) after treatment with nano-HA. In addition, we studied the changes in enamel mineral composition and nano-HA particle microstructure with energy dispersive X-ray spectroscopy (EDS) and field emission scanning electron microscopy (FESEM). Scanning electron microscopy (SEM) was used to observe the sealant–enamel interface.
Methods
The research protocol was approved by the Human Ethics Review Committee of the School of Dentistry, Shiraz University of Medical Sciences. A total of 192 sound extracted third molars were cleaned and disinfected by immersion in 0.1% chloramine T solution for 4 weeks. The aim of the study was explained to patients who decided to have their third molar extracted, and their informed consent in writing was obtained. The teeth were observed under a stereomicroscope (Motic K, Wetzlar, Germany) at 20× to rule out those with defects, cracks or caries.
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Early caries lesions
Briefly, for microleakage and SEM assessment, each tooth was randomly selected and demineralized by immersion in 15 mL demineralization solution at 37 °C for 96 h. For SBS, EDS and FESEM studies, the specimens were demineralized followed by polishing and sonication of the buccal and lingual enamel surfaces. The demineralization solution contained 6 μM methyl hydroxydiphosphonate, 50 mM lactic acid solution, 3 mM calcium chloride dihydrate and 3 mM potassium dihydrogen phosphate, and the final pH was adjusted to 4.5 [26]. The solution was replaced with freshly made solution after 48 h. After 96 h, each sample was washed with deionized water for 20 s, air dried and subjected to different conditions as detailed below.
Preparation of the nano-HA solution
Before starting tooth treatments, nano-HA particle size (nano-HA, Merck, nGimat, Darmstadt, Germany) was measured with a nanoparticle size analyzer (Horiba Ltd., Kyoto, Japan). The average diameter of the particles was recorded as 10.67 nm. Then two types of nano-HA were prepared, one with and one without SHMP (Merck, nGimat, Darmstadt, Germany).
The solution contained distilled water and ≥ 99.5% acetone (Acetone, Merck, nGimat, Darmstadt, Germany) as a solvent at a 1:1 ratio. The nano-HA and SHMP powder was measured and mixed at a 3:1 ratio and added to the solution. The prepared solutions contained nano-HA 0.15% or 0.03% (w/v) and SHMP at 0.05% or 0.01% (w/v). The amounts of powder and solvent needed to produce suitable concentrations of nano-HA and SHMP for testing were determined according to previous research [24] and a pilot study carried out in our laboratory.
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Experimental groups
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]. 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%.
Microleakage assessment
A total of 60 selected demineralized teeth were randomly divided into 5 groups of 12 teeth each and pretreated as explained above. After the sealant was applied and cured, the teeth were aged in a thermocycling bath at temperatures between 5 °C and 55 °C for 1000 cycles with a dwell time of 30 s and a 15 s transit time between baths. Two samples in each group were randomly selected for SEM evaluation.
Next the root apices were sealed and each tooth surface was covered with two layers of nail polish, leaving a 1-mm margin around the sealant. Then the samples were immersed in 0.5% basic fuchsin dye (Merck, Darmstadt, Germany) solution for 24 h, rinsed and sectioned buccolingually across the sealant area with a diamond saw (Mecatome, Presi, Eybens, France) to obtain two halves of the sealant. Two dentists who were trained prior to the study observed the sectioned teeth under a calibrated digital microscope (Dino Lite, Taipei, Taiwan) at 50× magnification. The proportion of microleakage was calculated by dividing the total distance of dye penetration (in mm) by the total length of the enamel sealant interface (in mm) [27].
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