A novel application of nanohydroxyapatite/mesoporous silica biocomposite on treating dentin hypersensitivity: An in vitrostudy

Abstract

Objectives

To fabricate a nanohydroxyapatite/mesoporous silica nanoparticle (nHAp@MSN) biocomposite and investigate its effectiveness on dentinal tubule occlusion, acid-resistant stability, and microtensile bond strength (MTBS).

Methods

The nHAp@MSN biocomposite was synthesized and characterized by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, N 2 adsorption–desorption isotherms, field-emission scanning electron microscopy (FESEM), and high-resolution transmission electron microscopy (HRTEM). Thirty-two simulated sensitive dentin discs were prepared and randomly divided into four groups according to the following treatments (n = 8 each): Group 1, no treatment; Group 2, NovaMin, 15 s × 2; Group 3, MSN, 15 s × 2; Group 4, nHAp@MSN, 15 s × 2. Then, four discs per group were post-treated with 6 wt.% citric acid challenge to test their acid-resistant stability. The effects on dentinal tubule occlusion were observed by FESEM. A self-etch adhesive (G-Bond) was applied to evaluate the MTBS. The cytotoxicity was detected using the Cell Counting Kit-8 (CCK-8) assay.

Results

Results revealed that the nHAp@MSN biocomposite was successfully fabricated. nHAp@MSN could effectively occlude the dentinal tubules, and the intratubular crystals were tightly associated with the tubular wall. After citric acid attack, nHAp@MSN exhibited the highest acid-resistant stability among the four groups. Moreover, no significant difference in MTBS was noted among the four groups ( P > 0.05). CCK-8 assay identified that nHAp@MSN induced no more than 20% cell death even at the highest concentration of 640 μg/mL.

Conclusions

The application of the nHAp@MSN biocomposite resulted in efficient dentinal tubule occlusion, acid-resistant stability, and did not compromise immediate bond strength between dentin and self-etch adhesive system.

Clinical significance

The nHAp@MSN biocomposite indicates enormous potential as a new strategy for relieving dentin hypersensitivity.

Introduction

As a relatively common dental complaint, dentin hypersensitivity (DH) is principally derived from the exposure of dentin owing to enamel loss ( e.g ., abrasion, attrition, or acid erosion) and/or gingival recession caused by periodontal surgery or periodontitis, leading to a transient, sharp pain and discomfort . Several hypotheses have been proposed in the past decades to illuminate the mechanism of DH. Currently, the hydrodynamic theory is the most extensively recognized.

Based on the theory, a potential strategy for the management of DH is to effectively occlude the dentinal tubules to prevent fluid flow, thus relieving clinical symptoms . Essentially, the past decades have witnessed the application of various approaches to provide tubule obstruction for treating DH, including the use of potassium oxalate , sodium fluoride , dentin adhesives , laser treatment , and calcium-containing pastes . Although some success was achieved, the tubules sealed by traditional therapies were superficial with limited infiltration depth and could be readily re-exposed when dietary acid attack is encountered . This phenomenon would then result in the short-lived effects of the treatment and the relapse of sensitivity.

Hence, developing a desirable biomaterial for treating DH is imperative, not only to efficiently occlude the dentinal tubules but also maintain long-term stability, especially when confronted with dietary acid challenge . Furthermore, in clinical practice, sensitive teeth are often accompanied by the defects of hard tissue. Consequently, subsequent resin restoration may become necessary, and desensitizing treatment should also cause no detrimental effect to the bond strength .

Over the past decades, mesoporous silica nanoparticles (MSN) have been widely employed in biomedical fields because of their stable network structure, large surface area, adsorption performance, and thermal and chemical stability . MSN may offer significant advantages as an ideal medium for drug, gene, and functional nanoparticle loading . Chiang et al. demonstrated that CaCO 3 -containing MSN mixed with 30% H 3 PO 4 deeply permeated dentinal tubules . Ishikawa et al. suggested that the supersaturation of Ca 2+ and PO 4 3− ions could promote the formation of calcium phosphate minerals that can penetrate more deeply into tubules . In addition, numerous reports have shown that nano-hydroxyapatite (nHAp) holds the potential to act as a Ca 2+ and PO 4 3− reservoir to maintain the supersaturated state for tooth mineralization . These studies highlighted the capability of nHAp for dentinal tubule occlusion, as well as its capacity to facilitate crystal deposition and growth on demineralized teeth. Considering the unique properties of MSN and the mineralizing potential of nHAp, a nHAp/MSN (nHAp@MSN) biocomposite would be highly desirable. However, to the best of our knowledge, few reports are available on nHAp@MSN with regard to DH research.

Therefore, a new type of nHAp@MSN biocomposite was designed in the present study. The objectives of this study were (1) to fabricate and characterize the nHAp@MSN hybrid material and (2) to test the null hypothesis that the application of nHAp@MSN can effectively occlude the dentinal tubules, maintain acid-resistant stability, and cause no adverse effect on microtensile bond strength.

Materials and methods

Synthesis of nHAp@MSN biocomposite

A commercial product of mesoporous silica nanoparticles (MSN) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The nHAp@MSN biocomposite was fabricated following the reported techniques with slight modification. Briefly, 8 mM Ca(NO 3 ) 2 ·4H 2 O and 4.8 mM (NH 4 ) 2 HPO 4 aqueous solutions were prepared, respectively. 0.48 g dried MSN was dispersed into 20 mL deionized water ultrasonically and then added drop-wise to the solution containing Ca(NO 3 ) 2 to enable the latter to infiltrate into the mesopores. After vigorous stirring for 12 h at room temperature, the mixture was centrifuged to obtain a white precipitate. The precipitate was triple-washed with deionized water and ethanol, followed by dissolving in 40 mL deionized water. The solution containing (NH 4 ) 2 HPO 4 was added drop-wise to the above mixture under constant stirring for 30 h. After aging for 24 h under ambient conditions, the mixture was centrifuged to retrieve another white precipitate. The precipitate was triple-washed with deionized water and ethanol, filtered, air-dried overnight at 80 °C and calcined at 550 °C for 6 h to obtain the final powdery product.

Materials characterization

The MSN and the white powdery product were characterized as fellows. X-ray diffraction (XRD) analysis was applied to determine the crystalline phase on an X-ray diffractometer (X’Pert PRO, PANalytical, Almelo, The Netherlands) using Cu-Kα radiation at 40 kV and 40 mA. Wide-angle XRD patterns were recorded in the 2 θ range from 10° to 70° with a scanning speed of 4°/min, and small-angle XRD patterns were scanned from 0.5° to 10° with the speed of 1.2°/min. The typical functional groups were examined by Fourier transform infrared spectroscopy (FT-IR) using a Nicolet 5700 spectrometer (Thermo Scientific Inc., Madison, WI, USA), and the spectra were recorded from 4000 to 400 cm −1 at 4 cm −1 of resolution. N 2 adsorption–desorption isotherms were collected on an ASAP 2020 M gas adsorption analyzer (Micromeritics, Atlanta, GA, USA) at 77 K. Specific surface area and pore size distribution were calculated by BET and BJH method, respectively. The overall morphology of the samples was analyzed by a field-emission scanning electron microscopy (FESEM) (Sigma, Zeiss, Germany) and a high-resolution transmission electron microscopy (HRTEM) (JEM-2100, JEOL, Japan).

Specimen preparation and experimental design

Thirty-two extracted caries-free human third molars were collected after obtaining the donors’ informed consent under a protocol approved by the Ethics Committee for Human Studies of the School & Hospital of Stomatology, Wuhan University, China. The teeth were stored in 0.5 wt.% thymol at 4 °C and used within 3 months. Dentin discs with 1 mm thick were prepared by sectioning perpendicular to the long axis of the teeth below the enamel-dentinal junction by a low-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) under water cooling. The discs were wet-ground with 600-, 800-, 1000-grit silicon carbide (SiC) polishing papers for 60 s, respectively, then the dentinal tubules of the discs were opened by soaking in a 1 wt.% citric acid solution for 20 s to simulate a sensitive tooth model , followed by rinsing thoroughly with water-spray. The specimens were randomly divided into four groups (n = 8 discs) as fellows:

Group 1: No treatment (control).

Group 2: A calcium sodium phosphosilicate-containing desensitizing paste NovaMin (NovaMin Company Ltd., Wuhan, China) of 100 mg was applied to the dentin surfaces with a rotary cup at a low speed for 15 s, then repeated for another 15 s for a total of 30 s.

Group 3 and Group 4: The slurry of the MSN and nHAp@MSN prepared by a powder/liquid (deionized water) ratio of 100 mg/200 μL were applied to the dentin surfaces in the same procedure as described in Group 2, respectively.

Subsequently, four discs randomly selected from each group were ready for post-treatment. The chosen discs were soaked in a 6 wt.% citric acid solution (pH = 1.5) for 1 min then rinsed with deionized water, so as to determine the resistance to strong acid conditions after desensitizing treatments (n = 4 discs).

FESEM evaluation of tubule occlusion

After mentioned above, a groove was prepared from the pulpal to the enamel surface of each dentin disc, then each disc was sectioned longitudinally into halves for the top and longitudinal surface examination, respectively. These specimens were rinsed with water-spray, dehydrated, sputter-coated with Au-Pd alloy, and the morphological alterations of dentinal tubule occlusion on exposed dentin after different treatments were examined using FESEM at 5 kV. Micrographs at ×2000, ×5000, ×10,000 magnifications were taken from two central sites of each disc, Image J (NIH, Frederick, MD, USA) was employed to compute the area ratios of the occluded dentinal tubules (the area of occluded tubules/the total tubules area) using ×2000 images (n = 8).

Microtensile bond strength (MTBS) test

Additional twenty teeth were sectioned parallel to the occlusal surface to expose the mid-coronal dentin surface by the Isomet saw under water cooling. The exposed surfaces were ground with 600-grit SiC and soaked in 1 wt.% citric acid solution to simulate the sensitive tooth model, then the teeth were randomly divided into four groups (n = 5 teeth) and each group was treated as mentioned in 2.3, followed by thoroughly rinsed with water-spray for 30 s and air-dried. A commercial self-etch adhesive G-Bond (GC, Tokyo, Japan) containing 4-MET was applied to the pretreated dentin surfaces following the manufacturers’ instructions to evaluate the effects of different desensitizing procedures on immediate MTBS. A resin composite (Charisma, Heraeus Kulzer, Hanau, Germany) build-ups were constructed in 4 mm increments, and each increment was polymerized for 20 s.

After stored in deionized water for 24 h at 37 °C, the bonded teeth were sectioned perpendicular to the bonding interfaces to produce 0.9 mm × 0.9 mm beams, and eight beams were obtained from each tooth. In MTBS test, each beam (40 beams for each group) was fixed with a cyanoacrylate adhesive to an MTBS tester (Bisco, Schaumburg, IL, USA), setting the tensile force at a cross-head speed of 1 mm/min until failure. The cross-sectional interface area of each beam was measured using a digital caliper, and the MTBS values (MPa) were then calculated.

Cell culture and cytotoxicity assay

Human dental pulp cells (HDPCs) were isolated from healthy human dental pulp tissues of premolars for orthodontic extraction with informed consents. The extracted pulp tissues were harvested and minced and then transferred into plastic flasks containing α-modified essential medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone), 100 units/mL penicillin and 100 mg/mL streptomycin. The tissues were cultured in a humidified atmosphere of 5% CO 2 at 37 °C. The medium was replaced every 2 days, and the third passage cells were used. In vitro cytotoxicity of nHAp@MSN was identified by a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. The HDPCs were seeded in 96-well plates at a density of 5000 cells per well and incubated under the condition of 5% CO 2 at 37 °C for 24 h. The cells were exposed to a series of increasingly concentrated nHAp@MSN (0, 10, 20, 40, 80, 160, 320, 640 μg/mL) for another 24 h. Then, 10 μL of CCK-8 solution was added into each well and incubated for 2 h. The optical density was monitored by a microplate reader (PowerWave XS2, BioTek Instruments Inc., Winooski, VT, USA) at 450 nm. The results were expressed as the relative cell viability (%) with respect to a control group only with the culture medium. The experiment was performed in sextuplicate.

Statistical analysis

Statistical analysis was performed by SPSS 19.0 (SPSS Inc., Chicago, IL, USA). The values of the occluded area ratio in FESEM study and the data of MTBS and CCK-8 assay were analyzed using one-way ANOVA, respectively. Multiple comparisons were conducted by Tukey’s test and the significance level was set at α = 0.05.

Materials and methods

Synthesis of nHAp@MSN biocomposite

A commercial product of mesoporous silica nanoparticles (MSN) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The nHAp@MSN biocomposite was fabricated following the reported techniques with slight modification. Briefly, 8 mM Ca(NO 3 ) 2 ·4H 2 O and 4.8 mM (NH 4 ) 2 HPO 4 aqueous solutions were prepared, respectively. 0.48 g dried MSN was dispersed into 20 mL deionized water ultrasonically and then added drop-wise to the solution containing Ca(NO 3 ) 2 to enable the latter to infiltrate into the mesopores. After vigorous stirring for 12 h at room temperature, the mixture was centrifuged to obtain a white precipitate. The precipitate was triple-washed with deionized water and ethanol, followed by dissolving in 40 mL deionized water. The solution containing (NH 4 ) 2 HPO 4 was added drop-wise to the above mixture under constant stirring for 30 h. After aging for 24 h under ambient conditions, the mixture was centrifuged to retrieve another white precipitate. The precipitate was triple-washed with deionized water and ethanol, filtered, air-dried overnight at 80 °C and calcined at 550 °C for 6 h to obtain the final powdery product.

Materials characterization

The MSN and the white powdery product were characterized as fellows. X-ray diffraction (XRD) analysis was applied to determine the crystalline phase on an X-ray diffractometer (X’Pert PRO, PANalytical, Almelo, The Netherlands) using Cu-Kα radiation at 40 kV and 40 mA. Wide-angle XRD patterns were recorded in the 2 θ range from 10° to 70° with a scanning speed of 4°/min, and small-angle XRD patterns were scanned from 0.5° to 10° with the speed of 1.2°/min. The typical functional groups were examined by Fourier transform infrared spectroscopy (FT-IR) using a Nicolet 5700 spectrometer (Thermo Scientific Inc., Madison, WI, USA), and the spectra were recorded from 4000 to 400 cm −1 at 4 cm −1 of resolution. N 2 adsorption–desorption isotherms were collected on an ASAP 2020 M gas adsorption analyzer (Micromeritics, Atlanta, GA, USA) at 77 K. Specific surface area and pore size distribution were calculated by BET and BJH method, respectively. The overall morphology of the samples was analyzed by a field-emission scanning electron microscopy (FESEM) (Sigma, Zeiss, Germany) and a high-resolution transmission electron microscopy (HRTEM) (JEM-2100, JEOL, Japan).

Specimen preparation and experimental design

Thirty-two extracted caries-free human third molars were collected after obtaining the donors’ informed consent under a protocol approved by the Ethics Committee for Human Studies of the School & Hospital of Stomatology, Wuhan University, China. The teeth were stored in 0.5 wt.% thymol at 4 °C and used within 3 months. Dentin discs with 1 mm thick were prepared by sectioning perpendicular to the long axis of the teeth below the enamel-dentinal junction by a low-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) under water cooling. The discs were wet-ground with 600-, 800-, 1000-grit silicon carbide (SiC) polishing papers for 60 s, respectively, then the dentinal tubules of the discs were opened by soaking in a 1 wt.% citric acid solution for 20 s to simulate a sensitive tooth model , followed by rinsing thoroughly with water-spray. The specimens were randomly divided into four groups (n = 8 discs) as fellows:

Group 1: No treatment (control).

Group 2: A calcium sodium phosphosilicate-containing desensitizing paste NovaMin (NovaMin Company Ltd., Wuhan, China) of 100 mg was applied to the dentin surfaces with a rotary cup at a low speed for 15 s, then repeated for another 15 s for a total of 30 s.

Group 3 and Group 4: The slurry of the MSN and nHAp@MSN prepared by a powder/liquid (deionized water) ratio of 100 mg/200 μL were applied to the dentin surfaces in the same procedure as described in Group 2, respectively.

Subsequently, four discs randomly selected from each group were ready for post-treatment. The chosen discs were soaked in a 6 wt.% citric acid solution (pH = 1.5) for 1 min then rinsed with deionized water, so as to determine the resistance to strong acid conditions after desensitizing treatments (n = 4 discs).

FESEM evaluation of tubule occlusion

After mentioned above, a groove was prepared from the pulpal to the enamel surface of each dentin disc, then each disc was sectioned longitudinally into halves for the top and longitudinal surface examination, respectively. These specimens were rinsed with water-spray, dehydrated, sputter-coated with Au-Pd alloy, and the morphological alterations of dentinal tubule occlusion on exposed dentin after different treatments were examined using FESEM at 5 kV. Micrographs at ×2000, ×5000, ×10,000 magnifications were taken from two central sites of each disc, Image J (NIH, Frederick, MD, USA) was employed to compute the area ratios of the occluded dentinal tubules (the area of occluded tubules/the total tubules area) using ×2000 images (n = 8).

Microtensile bond strength (MTBS) test

Additional twenty teeth were sectioned parallel to the occlusal surface to expose the mid-coronal dentin surface by the Isomet saw under water cooling. The exposed surfaces were ground with 600-grit SiC and soaked in 1 wt.% citric acid solution to simulate the sensitive tooth model, then the teeth were randomly divided into four groups (n = 5 teeth) and each group was treated as mentioned in 2.3, followed by thoroughly rinsed with water-spray for 30 s and air-dried. A commercial self-etch adhesive G-Bond (GC, Tokyo, Japan) containing 4-MET was applied to the pretreated dentin surfaces following the manufacturers’ instructions to evaluate the effects of different desensitizing procedures on immediate MTBS. A resin composite (Charisma, Heraeus Kulzer, Hanau, Germany) build-ups were constructed in 4 mm increments, and each increment was polymerized for 20 s.

After stored in deionized water for 24 h at 37 °C, the bonded teeth were sectioned perpendicular to the bonding interfaces to produce 0.9 mm × 0.9 mm beams, and eight beams were obtained from each tooth. In MTBS test, each beam (40 beams for each group) was fixed with a cyanoacrylate adhesive to an MTBS tester (Bisco, Schaumburg, IL, USA), setting the tensile force at a cross-head speed of 1 mm/min until failure. The cross-sectional interface area of each beam was measured using a digital caliper, and the MTBS values (MPa) were then calculated.

Cell culture and cytotoxicity assay

Human dental pulp cells (HDPCs) were isolated from healthy human dental pulp tissues of premolars for orthodontic extraction with informed consents. The extracted pulp tissues were harvested and minced and then transferred into plastic flasks containing α-modified essential medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone), 100 units/mL penicillin and 100 mg/mL streptomycin. The tissues were cultured in a humidified atmosphere of 5% CO 2 at 37 °C. The medium was replaced every 2 days, and the third passage cells were used. In vitro cytotoxicity of nHAp@MSN was identified by a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. The HDPCs were seeded in 96-well plates at a density of 5000 cells per well and incubated under the condition of 5% CO 2 at 37 °C for 24 h. The cells were exposed to a series of increasingly concentrated nHAp@MSN (0, 10, 20, 40, 80, 160, 320, 640 μg/mL) for another 24 h. Then, 10 μL of CCK-8 solution was added into each well and incubated for 2 h. The optical density was monitored by a microplate reader (PowerWave XS2, BioTek Instruments Inc., Winooski, VT, USA) at 450 nm. The results were expressed as the relative cell viability (%) with respect to a control group only with the culture medium. The experiment was performed in sextuplicate.

Statistical analysis

Statistical analysis was performed by SPSS 19.0 (SPSS Inc., Chicago, IL, USA). The values of the occluded area ratio in FESEM study and the data of MTBS and CCK-8 assay were analyzed using one-way ANOVA, respectively. Multiple comparisons were conducted by Tukey’s test and the significance level was set at α = 0.05.

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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on A novel application of nanohydroxyapatite/mesoporous silica biocomposite on treating dentin hypersensitivity: An in vitrostudy
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