Efficacy of Ca2+- or PO43−-conjugated mesoporous silica nanoparticles on dentinal tubule occlusion: an in-vitro assessment
Original Article

Efficacy of Ca2+- or PO43−-conjugated mesoporous silica nanoparticles on dentinal tubule occlusion: an in-vitro assessment

Tianjia Yu1, Yu Wang2, Qiang Cai2, Lin Wu1

1Department of Prosthodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang 110002, China; 2School of Materials Science & Engineering, Tsinghua University, Beijing 100084, China

Contributions: (I) Conception and design: All authors; (II) Administrative support: All authors; (III) Provision of study materials or patients: All authors; (IV) Collection and assembly of data: All authors; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

Correspondence to: Lin Wu. Department of Prosthodontics, China Medical University School of Stomatology, Shenyang 110002, China. Email: wulin13@163.com.

Background: Maintaining a long-term biological effect of dental materials on dentinal tubule occlusion is one of the great technical challenges in dental clinics. In addition to physical treatment, chemical treatment to produce insoluble precipitates to seal dentinal tubules has been used. As dentin is mostly composed of calcium and phosphate complexes, in this study, we have developed a novel tubule-occluding material [Ca2+/PO43−@mesoporous silica nanoparticles (MSNs)] by separately conjugating either Ca2+ or PO43− with MSNs.

Methods: The shape and structure of the MSNs were examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The surface morphology and chemical compositions of Ca2+@MSNs/PO43−@MSNs and Ca2+/PO43−@MSNs were examined using SEM and X-ray fluorescence (XRF). The element distribution of Ca2+/PO43−@MSNs was detected using energy dispersive spectrometer (EDS). The sustained release ability of Ca2+@MSNs/PO43−@MSNs was detected using inductively coupled plasma atomic emission spectrometry (ICP-AES). The efficacy of Ca2+/PO43−@MSNs on dentinal tubule sealing was evaluated using SEM, and the results were analyzed by Image-Pro software to determine the best water-powder ratio. We also compared the sealing efficacy between Ca2+/PO43−@MSNs and NovaMin, which is currently used in clinics, under the simulated conditions of oral acidic corrosion and mechanical friction.

Results: Ca2+/PO43−@MSNs are a new type of tubule-occluding material with sustained release properties. The ratio of Ca2+@MSNs: PO43−@MSNs: H2O =0.015 g: 0.015 g: 150 µL exhibited an excellent sealing effect on dentinal tubules as well as resistance to oral acid corrosion and daily oral friction.

Conclusions: The novel dental material Ca2+/PO43−@MSNs demonstrates potential long-term effectiveness in sealing dentinal tubules and reducing dentin sensitivity, which is one of the most important problems in dental clinics.

Keywords: Dentin hypersensitivity; mesoporous silica nanoparticles (MSNs); calcium phosphates, dentinal tubule

Submitted Oct 09, 2019. Accepted for publication Jan 02, 2020.

doi: 10.21037/atm.2020.01.98


Prosthesis is a widely accepted dental procedure to repair a larger area of tooth defect. However, the preparation of vital teeth often leads to dentin exposure, even very close to the pulp cavity. In addition, toxic materials released from bacteria and external temperature stimulus from temporary crown prostheses could result in dentin hypersensitivity as well as pulpitis under certain conditions (1-3). Cutting, caries, attrition, abrasion and erosion result in dentinal tubule exposure followed by dentin hypersensitivity, which is commonly seen in dental practice (4,5).

One of the main treatments for dentin hypersensitivity is dentin tubule occlusion (6). For this treatment, chemical therapy, dental material filling and laser therapy are the most commonly used methods in dental clinics (7,8), where chemical therapy is the most frequently used. Unfortunately, the effectiveness of these therapies is short term since physical friction of daily tooth brushing and chewing as well as acid corrosion of food and drinks could lead to dentinal tubule dilation and/or gradual loss of tubule-occluding materials (9). Thus, it is crucial to develop a novel material with excellent resistance to daily acid corrosion and friction for effective occlusion of dentinal tubules.

Mesoporous silica nanoparticles (MSNs) demonstrate many superior properties: stable mesh structure, high surface energy, large surface area, high penetration rates, high solubility and high reaction activity (10-14). Thus, MSNs can effectively diffuse into narrow dentinal tubules of 2–3 µm in diameter. In addition, MSNs are able to attach tightly onto the tooth surface due to their high binding affinity and basic OH groups (15). All these excellent properties make MSNs one of the best carriers (16). Considering the fact that the main component of human teeth is hydroxyapatite (HAP) (17), we proposed conjugating Ca2+ or PO43− with MSNs to form more biocompatible Ca2+@MSN and PO43−@MSN complexes. This MSN complex (Ca2+/PO43−@MSNs) is expected to deeply penetrate into the dentinal tubules, and a gradual release of Ca2+ and PO43− could form a stable phosphate calcium precipitate to seal the dentinal tubules (18). In this study, a Ca2+/PO43−@MSN complex was prepared. The features of the MSNs were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The surface morphology and chemical compositions of Ca2+@MSNs/PO43−@MSNs and Ca2+/PO43−@MSNs were examined by SEM and X-ray fluorescence (XRF). The element distribution of Ca2+/PO43−@MSNs was detected using energy dispersive spectrometer (EDS). The sustained release ability was detected by inductively coupled plasma atomic emission spectrometry (ICP-AES). The optimal water–powder ratio for sealing dentinal tubules was determined. Compared with that of NovaMin (NovaMin Technology, Wuhan, China), one of the most commonly used materials in current dental practice, the capability of Ca2+/PO43−@MSNs to resist acid corrosion and physical friction was evaluated.


Preparation of Ca2+- or PO43−-conjugated MSNs

MSNs were prepared following a previously reported protocol (18). In brief, tetraethyl orthosilicate (TEOS) was used as a raw material, and hexadecyl trimethyl ammonium bromide (CTAB) was used as a template to prepare SiO2 microspheres under weakly alkaline conditions. We reduced the diameter of the particles by increasing the amount of water (from 420 to 480 mL) and prolonging the reaction time (from 2 to 3 h). The shape and structure of the MSNs were examined using TEM (HT-7700, Hitachi, Japan) at 100 kV and SEM (S-4800, Hitachi, Japan) at 10 kV. To conjugate MSNs with either Ca2+ or PO43−, 1.5 g MSNs were mixed with 2.8 g anhydrous calcium chloride and 0.3 g oxalic acid or with 2.7 g phosphoric acid (30%). Deionized water was then added, and the mixture was stirred gently. Next, the mixture was then dried in a drying oven at 105 °C for 4 h, transferred into a muffler furnace and heated at 200 °C for 4 h. The mixtures were finally cooled to room temperature and ground into fine powders. The surface appearance of Ca2+@MSNs, PO43−@MSNs and Ca2+/PO43−@MSNs was examined with SEM at 10 kV. The element distribution of Ca2+/PO43−@MSNs was detected using EDS. The chemical compositions of Ca2+@MSNs/PO43−@MSNs were analyzed using XRF (XRF-1800, Shimadzu, Japan).

Detection of the sustained release of Ca ions and P ions in vitro

Ca2+@MSNs (100 mg) or PO43−@MSNs (100 mg) were dissolved in 10 mL Tris-buffered saline (TBS, 50 mM Tris-HCl, 150 mM NaCl, and 0.02% NaN3, pH 7.4) solution in a centrifuge tube at 37 °C. The tubes were centrifuged at 4,000 rpm for 10 min, and the supernatant was collected for analysis. The separate sampling points were set at 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h. The Ca and P ions in the sampled supernatants were determined using ICP-AES (IRIS Intrepid XSP, Thermo Fisher Scientific Inc, USA).

Preparation of dentin discs

Caries-free premolars, clinically extracted for orthodontic treatment, were collected. The study was approved by our institutional review board (IRB K2016011), and written informed consent was obtained from all subjects. Each tooth was sectioned 1 mm above the cementoenamel junction perpendicular to the long axis of the tooth using a low-speed, water-cooled diamond saw. The dentin discs (1.0±0.1 mm in thickness) were polished stepwise with sandpapers at mesh sizes of 400, 600, 800, 1,000 and 1,200. These polished discs were immersed in 6% citric acid solution for 2 min and cleaned twice by ultrasonication in distilled water. These cleaned discs were stored in artificial saliva at 4 °C for use.

Optimization of different water-powder ratios

Five groups of different water–powder ratios were prepared (Table 1). All dentin discs were examined under a light microscope to ensure the consistency of the samples. Five dentin discs were selected, and each disc was cut into five equal parts that were randomly assigned to five groups and coated with Ca2+/PO43−@MSNs of different water-powder ratios. After mixing Ca2+@MSNs/PO43−@MSNs with deionized water, the material was immediately brushed on the surface of the selected discs, and this brushing process was repeated 20 s later. These discs were immersed in a solution of 3% glutaraldehyde overnight, dehydrated with gradient concentrations of ethyl alcohol, and sprayed with gold in vacuum after drying for SEM observation. The sealing rate of dentinal tubules in all discs was calculated using Image-Pro Plus 6.0.

Table 1
Table 1 Five groups of different water-powder ratios
Full table

Evaluation of the sealing effect under simulated conditions of acid corrosion and physical friction

Six dentin discs were randomly divided into two groups: the Ca2+/PO43−@MSN group (discs coated with the optimal concentration of Ca2+/PO43−@MSNs: Ca2+@MSNs: PO43−@MSNs: H2O =0.015 g: 0.015 g: 150 µL) and the NovaMin group (discs coated with NovaMin). For NovaMin (SiO2, Na2O, CaO, and P2O5), the application procedure followed the manufacturer’s instructions. Each disc was cut into three equal parts for the untreated disc test, acid corrosion test, and friction test. For the untreated test, the discs were stored in artificial saliva. For the acid corrosion test, the dentin discs were soaked in fresh Coca-Cola (pH 2.5) for 10 min every day to mimic the oral acidic environment. For the friction test, all the dentin discs were brushed by the same operator using a Colgate soft bristle toothbrush (a kind of toothbrush commonly used worldwide) with the same strength as was used in daily brushing. The dentin discs were brushed twice a day for 3 min each to mimic the oral friction environment. The discs for acid corrosion test and friction test were stored in artificial saliva daily. All discs were examined by SEM after one week of treatment. The sealing rates of dentinal tubules from both the Ca2+/PO43−@MSN and NovaMin groups under each treatment condition were calculated using Image-Pro Plus 6.0.

Statistical analysis

SPSS 21.0 was used for statistical analysis. The data are expressed as the mean ± standard deviation. One-factor ANOVA was used for the statistical analysis of the data when testing the different water-powder ratios, and multiple comparisons were conducted by Tukey’s test. Student’s t-test was used to compare two independent groups of data in the evaluation of the sealing effect under the simulated conditions of acid corrosion and physical friction. The significance level was set at α=0.05.


Characterizations of MSNs and Ca2+/PO43−@MSNs

The TEM and SEM results showed that MSNs exhibited regular agglomeration and excellent dispersion ability, with even-sized particles and nearly spherical structure with a diameter of approximately 50 nm. The distribution of the inner core was also even and similar in size (Figure 1). SEM images (Figure 2) revealed that the surface of the Ca2+@MSNs/PO43−@MSNs was relatively rough [(A) Ca2+@MSNs; (B) PO43−@MSNs; (C) Ca2+/PO43−@MSNs]. Figure 2C showed a significant decrease in particle diameter due to the ion release. The EDS map (Figure 3) of Ca2+/PO43−@MSNs demonstrated that Ca, P, O elements were evenly distributed. The XRF results (Table 2) of the Ca2+@MSNs and PO43−@MSNs provided evidence that SiO2 and CaO as well as SiO2 and P2O5 were mainly present, respectively. The data showed that the loading rate was 38.976–41.227%.

Figure 1 TEM (A) and SEM (B) images of MSNs. MSNs exhibit even particle size and nearly spherical structure with a diameter of approximately 50 nm. The distribution of the inner core is also even and similar in size. TEM, transmission electron microscopy; SEM, scanning electron microscopy; MSNs, mesoporous silica nanoparticles.
Figure 2 SEM images of Ca2+@MSNs (A), PO43−@MSNs (B) and Ca2+/PO43−@MSNs (C). The surface of the Ca2+@MSNs/PO43−@MSNs was relatively rough. Ca2+/PO43−@MSNs showed a significant decrease in particle diameter due to the ion release. MSNs, mesoporous silica nanoparticles.
Figure 3 EDS elemental mapping of the Ca2+/PO43−@MSN complex. (A) Image of Ca2+/PO43−@MSNs, (B) EDS O elemental map, (C) EDS Ca elemental map, (D) EDS P elemental map. The elements of Ca, P, O were evenly distributed. EDS, energy dispersive spectrometer; MSNs, mesoporous silica nanoparticles.
Table 2
Table 2 The XRF results of Ca2+@MSNs and PO43−@MSNs
Full table

Release of Ca and P ions in vitro

Figure 4 shows that the release of Ca and P ions was rapid in the first 1 h and then gradually slowed until the end of the experiments. The Ca/P ratio of the new material at 72 h was 1.61, which is close to the Ca/P ratio of HAP.

Figure 4 In vitro release of cumulative Ca2+ and PO43− from Ca2+@MSNs/PO43−@MSNs in TBS. MSNs, mesoporous silica nanoparticles; TBS, Tris-buffered saline.

Effect of different water–powder ratios of Ca2+/PO43−@MSNs on dentinal tubule occlusion

Ca2+/PO43−@MSNs demonstrated an excellent effect on dentinal tubule occlusion at all five different concentrations but with variable efficacies depending on the concentration (Figure 5). Among the five groups, group 3 with Ca2+@MSNs: PO43−@MSNs: H2O =0.015 g: 0.015 g: 150 µL showed the best sealing effect, 97.6%.

Figure 5 (A) Sealing rate of Ca2+/PO43−@MSNs with five different water–powder ratios. (B) SEM image of the most dentinal tubules sealed by the Ca2+/PO43−@MSNs with the optimal water–powder ratios. Group 3, Ca2+@MSNs: PO43−@MSNs: H2O =0.015 g: 0.015 g: 150 µL, shows the best sealing ability. Significant differences are labeled as (*, P<0.05). MSNs, mesoporous silica nanoparticles.

Sealing effect under simulated conditions of acid corrosion and physical friction

As shown in Figure 6, although both Ca2+/PO43−@MSNs and NovaMin had different degrees of resistance to acid corrosion and friction, Ca2+/PO43−@MSNs exhibited better results than NovaMin. After acid etching treatment, we observed demineralization on the surface of the dentin discs. The dentinal tubules in the Ca2+/PO43−@MSN group remained well sealed, while most of the dentinal tubules in the NovaMin group were reopened. After friction treatment, the sealing rates of dentin discs in the Ca2+/PO43−@MSN and NovaMin groups were 88.6% and 71.7%, respectively.

Figure 6 Sealing effect of Ca2+/PO43−@MSNs and NovaMin under simulated oral conditions for seven days. (A) and (B) are dentinal tubules treated with Ca2+/PO43−@MSNs and NovaMin. The dentinal tubules are almost sealed with the Ca2+/PO43−@MSN group under the simulated conditions of the acid corrosion treatment (C) and friction treatment (E), while most dentinal tubules have been reopened with the NovaMin group under the same conditions (D and F). (G) The sealing rates of Ca2+/PO43−@MSNs are better than those of NovaMin. Significant differences are labeled as (*, P<0.05). MSNs, mesoporous silica nanoparticles.


The occlusion of dentinal tubules has become the preferred treatment for reducing dentin hypersensitivity, which is supported by the widely accepted hydrodynamics theory (19). Nanomaterials are promising for sealing dentinal tubules. One of the most commonly studied materials for such purposes is MSNs (18,20-22).

At present, most of the clinically applied tubule-occluding materials are immediate-release materials, generally resulting in the sealing materials accumulating only at the orifice of dentinal tubules. In comparison with other tubule-occluding materials, the Ca2+/PO43−@MSNs used in this experiment exhibit potential advantages of deeper and firmer sealing effects, which might be attributed to the following three reasons. First, the diameter of Ca2+@MSNs/PO43−@MSNs was much smaller than that of dentinal tubules, so the material could easily penetrate into the dentinal tubules. In our previous study, Ca2+ and PO43− were loaded into the pores of MSNs with diameters of 80 nm, with loading rates of 2.662–3.089% (18). In the current experiment, the diameter of MSNs was reduced to 50 nm for a deeper sealing effect, and Ca2+/PO43− was loaded on the surface of the particles and inside the pores, with an improved loading rate of 38.976–41.227%.

Second, owing to the sustained release capacity of the material, Ca2+ and PO43− were continuously released during the process of particle penetration for at least 72 h in this experiment, forming a stable calcium phosphate precipitate to continue consolidating the sealing effect. The initial pH of the composite is 4.7. Under this acidic condition, the dentin surface is likely to be slightly dissolved, and calcium ions from the dissolved dentin surface react with phosphate ions in the Ca2+/PO43−@MSNs to form precipitates and facilitate the sealing effect. At the same time, the dentin surface can also be roughened to strengthen the bonding strength with Ca2+/PO43−@MSNs. Therefore, proper acidity is desirable to improve the sealing effect. Clinically, 30% phosphoric acid (pH <1) is used to etch both enamel and dentin, so the acidity of Ca2+/PO43−@MSNs is acceptable for teeth. Moreover, MSNs had a certain difference in the loading of calcium and phosphorus because MSNs were negatively charged in aqueous solution (23). Compared with negatively charged phosphate ions, MSNs could load more positively charged calcium ions. Therefore, when the ions were released, the content of calcium ions would be more than that of phosphate ions. Under the experimental conditions, Ca2+/PO43−@MSNs had a Ca/P ratio of 1.61 at 72 h, which was close to that of HAP (1.67). The formed calcium and phosphorus compounds depositing on the wall of dentinal tubules might promote tooth remineralization (24,25), which would improve the reliability and effectiveness of the curative effect on dentin hypersensitivity.

Third, the mechanical sealing of Ca2+/PO43−@MSNs combined with the chemical sealing of calcium and phosphorus facilitated the sealing effect. From the XRF results in this study, we know that the proportion of MSNs is approximately 52.265–55.70%, which is lower than before, but the material contains much more calcium and phosphorus instead. In the dentinal tubules, calcium and phosphate ions are gradually released and form a precipitate that plays an important role in sealing dentin tubules together with the MSNs. The MSNs function by mechanical stacking, and the gaps between the MSN particles will be filled with the calcium-phosphate precipitate. Our previous study showed that although MSNs can seal dentinal tubules, their aggregates can most likely collapse after mechanically sealing the dentinal tubules if the bottoms of the dentinal tubules are not fully filled and consolidated (18). In the case of Ca2+/PO43−@MSNs, calcium phosphate precipitation caused by the release of calcium and phosphate ions played a key role in consolidating the sealing effect together with MSNs. Therefore, in the long run, calcium and phosphorus ions could enhance the stability of the sealing material in the dentinal tubules.

Although nanoscale MSNs can be immediately dissolved in deionized water, the water–powder ratios may influence the sealing effect. Insufficient sealing of the exposed dentinal tubules was observed when lower concentrations were used. At higher concentrations, the viscosity of suspensions increases and liquidity deterioration may occur, which prevents the diffusion of Ca2+/PO43−@MSNs into the dentinal tubules. For the viscosity of the solution is directly proportional to the concentration of the dispersion according to Einstein’s formula.

where µm is the viscosity of the suspension, φ is the volume fraction of the dispersed system in suspension, and µ is the viscosity of the continuous phase liquid. We found that Ca2+/PO43−@MSNs at the concentration of Ca2+@MSNs: PO43−@MSNs: H2O =0.015 g: 0.015 g: 150 µL displayed the best sealing effect on dentinal tubules. These results illustrate that it is critical to optimize the concentration to achieve an excellent sealing effect, and the optimized concentration provides a solid foundation for future clinical practice.

NovaMin, a common clinical tubule-occluding material, has chemical components and mechanical properties that are similar to those of Ca2+/PO43−@MSNs in terms of sealing dentinal tubules (26,27). Therefore, NovaMin was chosen as the control in this experiment. In comparison, Ca2+/PO43−@MSNs with the optimal water–powder ratio demonstrated a better sealing of the dentinal tubules and a better resistance to daily oral acid corrosion and tooth brushing. The ability to resist chemical corrosion and mechanical friction is an important parameter to evaluate the utility of sealing dentinal tubules. The major component of dentin is HAP, which can be partially dissolved under acidic conditions. It is a rational assumption that dental materials with chemical components similar to those of dentin could be decomposed by oral conditions (28). There is a possibility of dentinal tubule dilation and loss of the tubule-occluding material. As shown by the SEM images, the dentin surface started to demineralize or wear out under the conditions of simulated daily oral acid corrosion or tooth brushing. If the tubule-occluding materials are not embedded deeply and firmly enough in the dentinal tubules, they will wear out and dissolve, and even the sealed dentinal tubules will reopen.

The in vivo evaluation of this Ca2+/PO43−@MSN material is under investigation, and the sealing effect will be reported soon.


In summary, we have developed a novel dental material, Ca2+/PO43−@MSNs. The optimal concentration of this material (Ca2+@MSNs: PO43−@MSNs: H2O =0.015 g: 0.015 g: 150 µL) demonstrates a good effect on dentinal tubule sealing and can resist simulated oral acid corrosion and daily oral friction. These results suggest the potential efficacy of this material in long-term dentinal tubule sealing to reduce dentin supersensitivity and indicate its feasibility for clinical utilization.


Funding: None.


Conflicts of Interest: The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by an institutional ethics committee and followed the tenets of the Declaration of Helsinki (No. K2016011).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.


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Cite this article as: Yu T, Wang Y, Cai Q, Wu L. Efficacy of Ca2+- or PO43−-conjugated mesoporous silica nanoparticles on dentinal tubule occlusion: an in-vitro assessment. Ann Transl Med 2020;8(5):173. doi: 10.21037/atm.2020.01.98