Effects of bioactive glass with and without mesoporous structures on desensitization in dentinal tubule occlusion

Applied Surface Science 283 (2013) 833–842

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Effects of bioactive glass with and without mesoporous structures on desensitization in dentinal tubule occlusion Wen-Cheng Chen a,1 , Jung-Chang Kung b,1 , Cheng-Hwei Chen c , Yu-Cheng Hsiao d , Chi-Jen Shih d,∗ , Chi-Sheng Chien e,f,g,∗ a Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan b Department of Family Dentistry, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan c School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan d Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan e Department of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan f Department of Orthopaedics, Chi Mei Foundation Hospital, Tainan, Taiwan g Department of Electrical Engineering, Southern Taiwan University of Science and Technology, Tainan, Taiwan

a r t i c l e

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Article history: Received 9 March 2013 Received in revised form 5 July 2013 Accepted 5 July 2013 Available online 13 July 2013 Keywords: Mesoporous bioactive glass Dentin hypersensitivity Dentinal tubules occlusion Apatite

a b s t r a c t Bioactive glass (BG) is a potential material for treating dentin hypersensitivity due to its high ability of dissolution. In this study, conventional BG and BG with well-ordered mesopore structures (MBG) were applied for dentinal tubule occlusion. We used X-ray diffractometer (XRD), scanning electronic microscope (SEM), and Fourier transform infrared (FTIR) to investigate the physiochemical properties and the dentinal tubule occlusion ability of BG and MBG groups. The results showed that the major crystallite phase of MBG and BG agents was monocalcium phosphate monohydrate. MBG pastes, mixed with 30 and 40 wt% phosphoric acid hardening solutions, had the ability to create a penetration depth greater than 50 ␮m. These results showed that BG with mesoporous structures turned the pastes mixed with suitable phosphoric acid solution into a material with great ability for occluding dentinal tubules; it has a short reaction time and good operability, and these agents have better potential for the treatment of dentin hypersensitivity than BG without mesoporous structures. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The treatment of dentin hypersensitivity is one of the major challenges in dental practice, especially for a long-term outcome of restoration. Brossman proposed the ideal method to treat dentin hypersensitivity includes the following: non-sensitive to dentin, painless, easy operation, efficient, long-lasting, colorless, and continual results [1]. In keeping hydrodynamic theory in mind [2–4], dentinal tubule occlusion by reducing permeability of the dentin to external stimulations is the current, most widely used method to treat hypersensitivity [5]. Dentin is the basis of a tooth. The number of dentinal tubules within the dentin surface area increases near the pulp chamber, which starts from 15,000 to 20,000 tubules/mm2 at the enamel

∗ Corresponding authors at: Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan. Tel.: +886 7 3121101x2367; fax: +886 7 3210683. E-mail addresses: [email protected] (C.-J. Shih), [email protected] (C.-S. Chien). 1 These authors contributed equally to this work. 0169-4332/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2013.07.027

and dentin interface and reaches 45000–65000 tubules/mm2 at the pulp chamber [6]. Due to the continual mineralization of the intratubular dentin, the thickness of the intratubular dentin near the enamel will be thicker, causing surface dentinal tubules to be smaller in diameter [7]. When the dentin thickness is 3 mm, external stimuli will not transfer easily to the pulp chamber cavity. However, when this thickness is less than 0.3 mm, the pulp chamber caivity becomes extremely sensitive to outside stimulus [8]. Successful endodontic therapy appears to depend on complete sealing of the root canal and prevention of the micro leakage that may occur through cervically exposed dentinal tubules [9]. The external sealing of the exposed dentinal tubules appear to be partial and short term cover; end with re-exposing of the dentinal tubules [10]. The main ingredient of bioactive glass (BG) is SiO2 –CaO–Na2 O–P2 O5 . In the past, BG has been used to maintain ridge shape and integrity after teeth extraction in dentistry as an endosseous ridge maintenance implant, which is mainly composed of 45% SiO2 –24.5% CaO–24.5% Na2 O–6% P2 O5 (wt%) [11,12]. In addition, scholars have proposed that when BG powder contacts water, sodium ions are released and increase the pH to a base.

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Under these circumstances, the calcium ions and phosphate ions that infiltrate dentinal tubules form apatite to occlude the dentin [13,14]. Back in 2000 and 2001, Professor Lin attempted to use BG and CO2 laser to treat dentin hypersensitivity with success. In 2007, he also used mesoporous silica containing calcium oxide to treat hypersensitivity, proving that mesoporous silica and dentin form a strong bond to each other. [15,16]. Because the success rate of hypersensitivity treatment is related to how lasting the biomaterial and the teeth bond to each other [17], BG is a great option for desensitization with an occluding rate of 90%. However, biomaterials of this type do not hold high strength and the smear layer produced is easily smeared off by teeth brushing [18]. Mesoporous bioactive glass (MBG) is produced by using an evaporation-induced self-assembly method, EISA [19,20]. During the organic solvent evaporation process, the concentration of the three-block high molecular polymer gradually rises, inducing the silicon to self-assemble with the surfactant to create micelles. Later on, the process involves assembly into a liquid-crystalline mesoporous phase structure. Lastly, high temperature heat processing was performed to remove surfactants to reveal a mold template and form a highly patterned MBG material [21,22]. Recently, MBG has been proposed as a highly potential material to be used in bone implant as the mesoporous structure offers a high specific surface area with mesopores ranging within 2–50 nm distributed throughout the structural matrix. This material is capable of fast reactions, dissolution, and re-precipitation of bone-like apatite materials. Currently, both BG and MBG have both been applied in dentistry research and MBG has been known to exhibit better biocompatibility qualities compared to BG. MBG also contains more pores to act as drug delivery carriers. However, there is a lack of empirical study comparing the occlusion rate of BG and MBG in dentinal tubules. Our previous study showed that MBG and phosphoric acid (PA) reacting with each other creates a large amount of crystal [22]. We hypothesized that a novel MBG material containing high specific areas with a mesoporous distribution structure inside the matrix mixed with phosphoric acid can have a more positive impact on dentinal tubule occlusion and significantly reduce dentin permeability compared to BG without mesopores inside.

2. Materials and methods 2.1. Sample preparations BG, the control group with varied agents, was fabricated without F127 agent. The testing group, BG with mesoporous structures, was fabricated using a typical synthesis of MBG. F127 (7.0 g), tetraethyl orthosilicate (TEOS, 6.7 g), Ca(NO3 )2 ·4H2 O (1.4 g), triethyl phosphate (TEP, 0.73 g; 80:15:5 Si:Ca:P molar ratio), and 0.5 M HCl (1.0 g) were dissolved in ethanol (60 g) and stirred at room temperature for 1 day [21]. After this, polyurethane foam was completely immersed in the sol and compressed. To force the sol to migrate into the pores of the foam, the sponge body tissues were uniformly coated with the appropriate sol while the pores remained open. The raw porous scaffold bodies were fully dried and then thermally treated at constant heating rates (10 ◦ C/min) to calcination temperatures in the range of 600 ◦ C for 2 h. After cooling, the powders were ground up, sieved through #325 meshes and then subjected to characterization. The two groups of BG and MBG powders were mixed with the luting/hardening solutions of de-ionized water, phosphate buffered saline (PBS), 20, 30 and 40 wt% PA solutions. Nomenclature of groups with different preparation procedures for testing in this study are listed in Table 1.

Table 1 Nomenclature of BG and MBG groups mixed with varied hardening/lubricated agents for testing in this study. Tested sample prepared procedures

Abbreviation of groups

BG and MBG were respective mixed with de-ionized water BG and MBG were respective mixed with phosphate buffered saline (PBS) agent BG and MBG were respective mixed with 20, 30 and 40 wt% phosphoric acid (PA) agents

BG/W and MBG/W BG/PBS and MBG/PBS

BG/20PA, BG/30PA, BG/40PA and MBG/20PA, MBG/30PA, MBG/40PA

2.2. Re-mineralized ability evaluation to penetrate into the depth of dentinal tubule BG only and BG with mesopores in structures (BG and MBG, respectively) were mixed with the re-mineralized/hardening agents at the ratio of powder-to-solution ratios of 1 g/2 mL. The dentin samples were prepared from the caries-free human molars extracted for surgical reasons from healthy patients. Teeth were obtained after approval by the Institutional Review Board of Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Dentin sample preparations were referenced and modified from the study of Gandolfi et al. [23]. The procedures are shown in Fig. 1. Briefly, the upper dentin surface of each 1 mm thickness sample was sand polished with 800-grit SiC paper for 1 min followed by ultrasonic cleaning for 10 min as a standard flat dentin surfaces with opened dentinal tubes. The produced paste were spread on the dentin surface samples and incubated at 37 ◦ C at 100% relative humidity, to simulate natural environment of the oral cavity. Occlusion times were set at 5–10 min and were immediately removed and washed with large quantity of de-ionized water for 20 s and submerged into anhydrous ethanol to stop any reactions. After being dried for 24 h, the samples were mechanically split open for analysis of osmosis efficiency. Substrates were split along the ridge and plated with gold to determine the degree of occlusion and crystallization in the dentinal tubules of each group. The occlusion rate and penetration depth of the dentin tubules are calculated from scanning electron microscope (SEM) images. A total of 20 dentinal tubules from 3 different sets of SEM images were used for sampling. The occlusion rate is defined as the ratio of dentinal tubules with crystallization to total number of dentinal tubules. The penetration depth is defined as the average length of the crystallization of all the dentinal tubules in ␮m.

2.3. Textural characterization X-ray diffraction (XRD, Rigaku D-max IIIV, Tokyo, Japan) at a scanning speed of 4◦ /min within the 2 range of 10–80◦ and Fourier-transform infrared spectroscopy (FTIR) analyses (Thermo NICOLET 6700, MA, US) were performed. To study the remineralized topographies and the composition of the elements on cross-section topographies, the samples were examined using a field emission SEM (Hitachi S-3000N, Hitachi, Tokyo, Japan). Nitrogen adsorption and desorption isotherms were measured at 77 K on a Quantachrome Autosorb 1 sorption analyzer. All samples were purged for 12 h at 150 ◦ C under high vacuum in the degas port of the adsorption analyzer. The specific surface areas of the samples were measured using the BET method (ASAP 2010, Micromeritics, USA) with nitrogen as an absorbent. The textures of the MBG specimens were observed using a high-resolution TEM (Philips Tecnai G2 F20) with an electron beam accelerating energy of 200 kV.

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Fig. 1. Sample prepared procedures and the SEM image of the dentin surface and the dentinal tubules after etching with 37% phosphoric acid to remove the original smear layer.

2.4. Cytotoxicity of MBGs First, the sample powders were sterilized under high pressure at 121 ◦ C for 20 min in an autoclave. Then, 0.1, 0.5, and 1 g of powder were incubated in 10 mL of culture medium at 37 ◦ C and 5% CO2 . After 7 days of incubation, the media were centrifuged at 2000 rpm for 5 min and the supernatant were used as sample extracts. The cytotoxicity of the MBGs was tested by filtration and culture of the fibroblast cells (NIH 3T3, abbreviated 3T3). The cells were provided by the National Institute of Health (NIH) in Taiwan. The 3T3 cells were derived from newborn mouse fibroblasts and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen Taiwan Ltd., MD) containing 10% bovine serum (BS) (Biological Industries, Haemek, Israel). An XTT Cell Viability Assay Kit provided a simple method to count live cells using an absorbance reader. NIH-3T3 cells were plated at a seeding density of 1 × 104 cells/well into 96-well microliter plate for 24 h. Culture media were then exchanged to media extract (100 ␮L/well) for another 24 h. The cells’ metabolic abilities were measured at an early stage time point of 24 h. After the cultured time, the cells on the sample surface were washed with phosphate-buffered saline (PBS) and transferred to 100 ␮L of culture medium with a 50 ␮L XTT kit and were incubated for another 4 h. The reaction medium was then measured spectrophotometrically at 490 nm using an ELISA microplate reader UVM-340 (ASYS Hitech GmbH, Eugendorf, Austria). Finally, the cell numbers were determined from a plot of absorbance (OD values) versus the 3T3 cells after adjustment via XTT assays. Each experiment was performed five times (n = 5).

Institute Inc., Cary, NC, USA). In all cases, the results were considered to be significantly different when p < 0.05. 3. Results 3.1. Nitrogen adsorption-desorption and texture properties of MBG and BG The specific surface area versus the mesoporous volume of the BG and MBG samples is shown in Fig. 2. The MBG samples showed larger specific surface areas (roughly three times larger) compared to the non-porous BG. The main reason is obviously due to the

2.5. Statistical analyses The statistical analyses of the results were analyzed using one-way ANOVA to investigate the significant group comparisons between different populations using the JMP 9.0 software (SAS

Fig. 2. Nitrogen adsorption/desorption isotherm and the BET surface areas and pore volumes of MBG and BG original powders.

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Fig. 3. Typical TEM images of the MBG specimens (a); the pore and the wall are indicated by white triangles in (b).

porous structure, evidently shown in the nitrogen absorption curve from Fig. 2. The formation of a hysteresis loop also signifies the presence of mesoporous structures in the MBG samples [22]. The hysteresis loop is missing from the BG curve, resulting with 1/5 the pore volume of MBG. Typical TEM images of the MBG specimens as shown in Fig. 3.

Fig. 4. The XRD patterns of MBG (a) and BG (b) mixed with varied hardening solutions. Symbols in figures indicated as following: original powders of MBG and BG (a), MBG and BG reacted with W (b), PBS (c), 20%PA (d), 30%PA (e), and 40%PA (f) respectively.

3.2. Setting properties of MBG and BG As shown in Fig. 4a, MBG mixed with de-ionized water (MBG/W) and PBS (MBG/PBS) for 5 min showed very similar XRD patterns except for a difference in the new crystalline structure of CaSiO3 (29.4◦ ) that appeared. However, when mixed with phosphoric acid (d–f indicated in Fig. 4), two diffraction peaks in two theta value of 22.9◦ and 24.1◦ appeared. After matching with JCPD card and consulting the literature, these peaks correspond with the triclinic crystal structure of monocalcium phosphate monohydrate (MCPM, Ca(H2 PO4 )2 ·H2 O) [24]. XRD patterns in Fig. 4b show that in contrast to MBG groups, BG/W and BG/PBS groups do not contain crystallization after reaction and patterns are similar to original BG powders. On the other hand, BG/20, 30, and 40%PA groups exhibit both 22.9◦ and 24.1◦ MCPM diffraction peaks. FTIR spectra in Fig. 5a and b reveal the different patterns of the powders. MBG and BG powders showed C O asymmetric stretching bands 1650 cm−1 , 875 cm−1 C O group asymmetric bending vibration, 1000–1250 cm−1 Si O Si asymmetric stretching band, 800 cm−1 Si O Si symmetric stretching band, and the 475 cm−1 band Si O bending vibration band. MBG, BG/W and MBG, BG/PBS groups were similar to MBG and BG original powders. Both reveal functional groups of Si O and CO3 2− . When MBG and BG was

mixed with 20, 30, and 40%PA, a few extra peaks showed up compared to original MBG and BG powders. Specifically, the P O band that stretches asymmetrically from 700 to 1130 cm−1 , the Si O Si asymmetrical stretching and vibration double absorbance, the 960 cm−1 peak of P O stretching vibration, and finally the 570 cm−1 peak from the P O symmetrical stretching vibrational absorption. 3.3. Effects of MBG and BG pastes on penetrate in the depth of dentinal tubule Due to clinical concerns, there is a need to shorten and reduce the time to occlude dentinal tubules. To assess the reaction times, 5 min MBG reactions and 10 min BG reactions were used as standards. The assessment of short term MBG and BG to understand occlusion efficiency and the penetration depths and occlusion rates are listed in Table 2. 3.3.1. Reacted topographies of MBG and BG pastes The results of morphologies and occlusive ratios are respectively shown in Fig. 6a and Table 2, with regards to the surface occlusion performance. The pores present on the external surfaces

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Table 2 The percentage of tubule occlusion and the penetration depth of MBG and BG based occlusive agents for 5 and 10 min mixing with hardening solution (n = 20). Occlusive agents

MBG mixed with varied hardening agents

BG mixed with varied hardening agents

Concentrations of phosphate (wt%)

20PA

30PA

40PA

20PA

30PA

40PA

Percentage of tubule occlusion (%) Average of penetration depth (␮m) Standard deviation of penetration depth (␮m)

25 61.5 4.6

40 61.1 13.1

55 62.5 17.1

50 62.6 10.0

33 30.7 2.5

47 47.1 7.2

of MBG/W and MBG/PBS decreased respective in diameter from 2–4 to 1–2 ␮m. MBG/20PA surface occlusion is much better than MBG/W or MBG/PBS groups since the exposed dentinal tubules were less and diameters decreased to around 1 ␮m. The occlusion rate was around 25%, with the exception of MBG/30PA, which exposed a small portion of dentinal tubules on the exterior. Thus, initial inspections reveal that the crystals contained three dimensional fused (gelled) structures with an occlusion rate of around 40%. As for MBG/40PA, the surface of the substrates was completely covered and almost no pores were present on the external surface. Furthermore, there is obvious crystallite fusion unlike MBG/W and MBG/PBS groups. The simple crystallization covered the dentinal tubules at an occlusion rate of 55%. BG/W covered substrates

revealed only a small randomly distributed portion of crystalline as does BG/PBS groups. However, BG/20PA had more complete coverage and also exposed less pores as shown in Fig. 6a where most of the substrate surface has been covered by crystallization (50% occlusion rate) and fused together. BG/30PA dentin substrate was covered by many small crystallites with an occlusion ate of 33%. In contrast, BG/40PA surface crystallite fusion occurred much more pronounced with an occlusion rate of 47%, compared to BG/20 and 30%PA. 3.3.2. MBG and BG pastes on penetrate in the depth of dentinal tubule From the cross-sectional view of the dentinal tubules in Fig. 6b, the MBG/W and MBG/PBS groups are revealed that they cannot penetrate into the tubules and only form a surface as a 5–10 ␮m barrier. MBG/20PA, on the other hand, infiltrates the dentin tubules with an average depth of 61.5 ␮m. MBG/30PA penetrates to a depth of an average 61 ␮m while also forming some incomplete three dimensional fused structured crystals. MBG/40PA penetrates to an average depth of 62.5 ␮m. MBG, after mixed with phosphoric acid at three different ratios (MBG/20, 30 and 40%PA), all formed stabilized fusions with the substrate at a thickness of approximately 15–20 ␮m with the 40PA mixed group the thickest. The interior dentin tubules penetration of BG/W and BG/PBS groups were identical to MBG groups, the paste cannot enter to occlude the tubules. However, BG/20PA cross sectional view shows that part of the tubules contains crystalline precipitation at an average depth of 62.6 ␮m. Although BG/30 and 40%PA groups also developed precipitates in the dentin tubules, the depths that were penetrated were shallower at 30.7 and 47.1 ␮m, respectively. BG/W and BG/PBS did not form bonds between the protective barriers and the substrate itself with thicknesses of 5 and 10 ␮m. When mixed with phosphoric acid, BG/20, 30, and 40%PA fusion layers were about 10 ␮m thick. Although this layer seemed to seal the dentin tubules, results were not optimal. Most of the dentin tubules morphologies were exposed. Both dentin substrate surface coverage and penetration ability were shown most effective when MBG/40PA was used (Table 2). 3.4. Setting morphologies of MBG and BG

Fig. 5. FTIR spectra of MBG (a) and BG (b) mixed with varied hardening solutions. Symbols in figures indicated as following: original powders of MBG and BG (a), MBG and BG reacted with W (b), PBS (c), 20%PA (d), 30%PA (e), and 40%PA (f) respectively.

The setting morphologies of MBG and BG with varied hardening solutions were analyzed by SEM, shown in Fig. 7. As indications, MBG/20PA powdered particulate surface coexists with columnar and plate crystallized morphologies. Most of the crystallites were fine and small pieces of crystals were generally found nested next to large ones. The columnar crystals were only 0.5–1.5 ␮m in diameter and at least 7 ␮m long. In contrast, MBG/30 and 40%PA surfaces only contained plate crystallizations that were larger than 10 ␮m, respectively. Combining the XRD data shows that the formation upon the powdered particulate surfaces of MBG reacted with 20, 30, and 40%PA were indeed crystallites of MCPM (Fig. 4). In the mixing of BG with PA, BG/20PA formed small amounts of crystals at a larger geometric size up to around 10 ␮m. Most particulates maintained the original material’s morphology while only a small portion formed crystallites. The groups of BG reacted with 30 and 40%PA formed crystallites mainly in the shape of plates ranging

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from 7 to 15 ␮m in feature size. Microstructures of BG/40PA group reveal tiny micro-sized crystallites continue to grow, indicating a faster crystallization rate than BG/30PA. Taking into account of the above observations, the higher the phosphoric acid concentration, the more adequate phosphate ions are available. The readily available phosphate ions contribute to larger crystalline formation and faster crystallization.

3.5. Cytotoxicity of MBG mixed with 30 wt% phosphoric acid hardening solution Cell toxicity tests were analyzed with an XTT assay to determine cell viability. MBG/30PA was chosen as the occlusive agent cytotoxicity testing group because clinically available teeth etching paste is 37% phosphoric acid and has been proven to be safe.

Fig. 6. (a) Topographies occluded effects on dentinal tubules of MBG and BG mixed with varied hardening solutions: (a) MBG and BG original powders, (b) MBG/W, (c) MBG/PBS, (d) MBG/20PA, (e) MBG/30PA, and (f) MBG/40PA. (b) Cross-sectional morphologies of penetrated depths in dentinal tubules of MBG and BG mixed with varied hardening solutions: (a) MBG and BG original powders, (b) MBG/W, (c) MBG/PBS, (d) MBG/20PA, (e) MBG/30PA, and (f) MBG/40PA.

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Fig. 6. (Continued ).

Furthermore, the occluding ability MBG/40PA is only marginally better than MBG/30PA. However, MBG/30PA was still chosen as a safety precaution on account of a lower phosphoric acid ratio, even though 45S5-BG mixed with 50% PA has been suggested to treat dentin hypersensitivity and incipient enamel caries [25]. The direct exposure to phosphoric acid results in only 3% survival of cells in Fig. 8, signifying the toxicity of phosphoric acid.

As for MBG/30PA, the cell survival rate of 10, 50, and 100 mg/mL of MBG/30PA paste were declined, respectively. Although the survival rates of MBG/30PA treated cells were not optimal, they are comparatively less toxic than phosphoric acid with a much higher cell survival rate. This proves that the mixture of MBG with phosphoric acid significantly decreases the calcium ion concentration toxicity of the occlusive agent against cells.

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Fig. 7. Setting morphologies of MBG and BG mixed with varied concentrations of 20, 30 and 40 wt% phosphoric acid hardening solutions at a magnification of 3000×.

4. Discussions 4.1. Effects of BG with and without meso-pores distribution on dentinal occlusions and penetrated depths Sodium fluoride (NaF) is a commonly used desensitizing agent that not only prevents dental carries, but also increases defense against decalcification under acidic environments [26,27]. In addition, the particles that accumulate in the dentinal tubules [28] also have a desensitizing effect. However, Pashley discovered in

Fig. 8. The cell viability of NIH-3T3 cell cultured in the media for 1 day that were previously immersed with different amounts of MBG/30PA for 7 days (n = 5, p < 0.05).

1986 that the calcium fluoride (CaF2 ) crystals are extremely small (0.5 ␮m). Therefore, unless a large quantity is formed, the tiny crystals are ineffective at occluding the dentinal tubules [29]. Calcium hydroxide (Ca(OH)2 ) has a long history in treating hypersensitivity because it is osteoinductive and provides calcium ions to allow remineralization of the dentin. The mechanisms involve a physical barrier provided by the calcium hydroxide when used as a periodontal paste, which physically reduces stimulations to improve hypersensitivity [30,31]. However, the re-mineralization time of calcium hydroxide is extremely short and is easily washed away. Although the early desensitization effects are extremely effective, this effect wears off as time goes on. This phenomenon happens at an even faster pace as modern dietary consumption often includes acidic foods such as coke and vinegar, which are potent at wiping away the smear layers [17,29,32]. BG is mainly composed of silicon dioxide as a base with calcium, phosphor, and sodium additions. The molecular composition of BG is SiO2 –CaO–Na2 O–P2 O5 . Due to its high biocompatibility, the BG material is suitable for the body’s environment. Currently, there are two types of BG depending on synthesis method: meltderived BG or sol–gel BG. The use of melting derives requires the addition of sodium to stabilize the reaction. Although this method is relatively simple for mass production, crystal diameters vary greatly. On the other hand, the sol–gel method creates much more uniform crystals, but mass production using this method is difficult. The present study uses the sol–gel method to fabricate the MBG. In 2010, Banerjee et al. performed clinical trials and discovered that the addition of phosphoric acid in BG served as an occlusive agent that effectively reduces hypersensitivity in patients [33]. Furthermore in 2011, Mitchell et al. suggested that nanosized BG decreases fluid dynamics in the dentinal tubules, which

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is also very effective against hypersensitivity [18]. In the present study, the crystallization formation rate of BG treated substrates had the tendency to increase as the concentration of PA and phosphate ions increase (Fig. 7). This leads to better occlusion effects of the paste, and increasingly decreases the number of exposed dentinal tubules (Fig. 6). By observing three substrates mixed with PA, we believe that the crystallization initiates from around the particles due to the supersaturated ions effect, causing tiny crystallites of MCPM to heterogeneous nucleation. Eventually, crystal growth occurs and closely packed crystals fused together to form larger crystals. Between BG and MBG groups, the crystallite fusion rate is much slower in the BG group due to its inability to re-mineralize crystallites (Fig. 7). This phenomenon is most obvious in the groups of MBG/30 and 40%PA (Fig. 6a and b) where crystallite fusion can be seen on the MBG/30PA dentinal specimen surface topographies compared with BG/30PA where only separate crystals are formed without any bridging or fusion between the particles. For this material to be used clinically in the future, the dentin should grow in a heterogeneous nucleation fashion radiating plate crystallization on the dentinal tubule area that can be observed in Fig. 7. Additionally, the smear layers that form on the dentinal surfaces will continue to form nucleation and crystalline growth. Thus, crystallization forms a loosely fit protective layer after fusion protection layers continued to form. Due to dental saliva, food intake, water rinsing, and tooth brushing, these actions will affect the effective time of the paste against hypersensitivity. The occlusive agent of MBG mixed with phosphate solution not only forms a fusion crystallite as a protective layer, but also re-mineralizes to ensure lasting effects against previously mentioned stimuli. From cross sectional views of the dentinal tubules, although BG without mesopores penetrates into the tubes, the proportion is too low to effectively occlude the tubules. Furthermore, the fusion crystallite did not completely fuse together and the dentinal tubules are visible (Fig. 6b). In terms of the effect of anti-hypersensitivity, any clinical usage of this would prove to be ineffective. 4.2. Setting morphologies The XRD patterns between MBG groups (MBG/W and MBG/PBS) and BG original powders show a difference in crystallite formation of CaSiO3 due to higher specific surface areas of MBG (Fig. 2). When MBG/20, 30, and 40%PA were mixed, the calcium and phosphorus ions released to rapidly form MCPM. As the PA concentration increases, the crystallization slowly changes from a columnar shape into plates or blades morphology with patterns showing increase in crystal size (Fig. 7). The groups of BG resulted in the same plate shaped calcium dihydrogen phosphate crystal formation while 20%PA mixed group yielded non-plate shaped crystals when BG mixed with 30 and 40%PA. Unlike BG groups, MBG groups developed large quantities of crystals mainly due to the porous structure in the MBG that holds a high specific surface area. As a result, the high contact surface area for the PA to react and release of ions for more sites to produce embryonic nucleus yields the phase of MCPM, thus proves the effects of a porous structure in crystallization growth. In this study, MBG products do not form hydroxyapatite [21], which is different compared with other studies that do form this product. This is probably due to the use of the hardening solution as well as the reaction time specified for the reaction. As Mneimne et al. have shown in their research, BG that do not react with dentin formed stable apatite at 6 h (high phosphate concentration) to 3 days (low phosphate concentration) in buffer solution [34]. This study focuses on the crystallization morphology and occlusion rate at the short term reaction time. Thus the reaction product phases are CaSiO3 and MCPM instead of apatite. If the reaction time is extended to increase ion diffusion, such as incubation in hanks buffered solution, then MBG and BG functional groups such

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as Si OH could induce apatite formation in the dentinal tubules [21].

5. Conclusions MBG mixed with phosphoric acid formed crystals with more uniform distribution compared to BG without mesopores. This is mainly due to the high specific areas offered by the mesopores, allowing quick release of the ions. MBG mixed with 30 and 40% phosphoric acid results in a homogeneous, smooth, and fused surface. This effectively protects the crystallite precipitate against external forces or stimuli, proving that MBG reacting with higher concentrations of phosphate ions will result in faster crystallization growth. From our research, future clinical applications of MBG mixed with high ion concentrations from phosphoric acid will provide an acidic environment to catalyze the reaction. The acidic environment could allow etching of the peritubular tubules and also form crystals to occlude the dentinal tubules by forming 10–20 ␮m smear layers. This can efficiently enhance dentinal tubule occlusion. In terms of dentinal tubule infiltration, MBG surface covering rate is far superior compared to BG groups. The crystallization precipitate within the tubules requires a lower phosphoric acid concentration, showing that MBG is far better at occluding dentinal tubules. In the future, occluding dentinal tubules will be safer using MBG as opposed to BG. Furthermore, it is less toxic and offers fewer stimuli and will be even more effective against treating hypersensitivity when paired with CO2 laser treatment.

Acknowledgments The Authors gratefully appreciate the supported by Grants from the Kaohsiung Medical University Hospital (KMUH100-0M51, KMUH101-1M68, KMUH101-1M69) and Grants from the Kaohsiung Medical University Research Foundation (KMU-M110008, KMU-M102006). The authors also acknowledge the supported by Grants from the Chi Mei Foundation Hospital of Taiwan for the financial support provided under 102CM-KMU-13.

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