Development of Epigallocatechin-3-gallate-Encapsulated

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Development of epigallocatechin-3-gallate-encapsulated nanohydroxyapatite/ mesoporous silica for therapeutic management of dentin surface Jian Yu, Hongye Yang, Kang Li, Hongyu Ren, Jinmei Lei, and Cui Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06597 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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ACS Applied Materials & Interfaces

Development of Epigallocatechin-3-gallate-Encapsulated Nanohydroxyapatite/Mesoporous Silica for Therapeutic Management of Dentin Surface

Jian Yu,†,§ Hongye Yang,†,§ Kang Li,† Hongyu Ren,† Jinmei Lei,† Cui Huang†,*

†

The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST)

& Key Laboratory for Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China. * Corresponding author at: The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, #237 Luoyu Road, Hongshan District, Wuhan 430079, China. Tel.: +86 27 87686130.

Fax.: +86 27 87873260.

E-mail address: [email protected] (Cui Huang). §

These authors contributed equally to this work and are co-first authors.

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ABSTRACT: In dental clinic, unsatisfactory management of the dentin surface after dentin exposure often leads to the occurrence of dentin hypersensitivity and caries. Current approaches can occlude the tubules on the dentin surface to relieve dentin hypersensitivity, however, the blocked tubules are generally weak in combating daily tooth erosion and abrasion. Moreover, cariogenic bacteria, such as Streptococcus mutans (S. mutans), produce biofilm on the dentin surface, causing caries and compromising the tubules’ sealing efficacy. To overcome this problem, the

present

study

focused

epigallocatechin-3-gallate-encapsulated

on

establishing

a

versatile

nanohydroxyapatite/mesoporous

silica

biomaterial, nanoparticle

(EGCG@nHAp@MSN), for therapeutic management of the dentin surface. The effectiveness of the biomaterial on dentinal tubule occlusion including resistances against acid and abrasion, was evaluated by field-emission scanning electron microscopy (FESEM) and dentin permeability measurement. The inhibitory capability of the biomaterial on S. mutans biofilm formation was investigated

by

confocal

laser

scanning

microscopy

(CLSM),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming units (CFU) counts, and FESEM. Results demonstrated for the first time that the use of EGCG@nHAp@MSN on the dentin surface was capable of effectively occluding dentinal tubules, reducing dentin permeability, and achieving favorable acid- and abrasion-resistant stability. Furthermore, EGCG@nHAp@MSN held the capability to continuously release EGCG, Ca, and P, and significantly inhibit the formation and growth of S. mutans biofilm on the dentin surface. Thus, the development of EGCG@nHAp@MSN bridges the gap between multifunctional concept and dental clinical practice, and is promising in providing dentists a therapeutic strategy for the 2


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management of the dentin surface to counter dentin hypersensitivity and caries.

Keywords: dentin hypersensitivity, mesoporous silica nanoparticles, nanohydroxyapatite, epigallocatechin-3-gallate, caries, Streptococcus mutans, biofilm

1. INTRODUCTION As a global challenge in dental clinic, dentin hypersensitivity is characterized by a transient, sharp pain originating from the exposure of dentin.1,2 Based on the most widely embraced hydrodynamic hypothesis, the occlusion of tubules on the dentin surface is considered to be a reliable strategy for avoiding fluid movement and reducing dentin permeability, thereby alleviating dentin hypersensitivity.3,4 Therefore, various of approaches have been developed to obstruct dentinal tubules, including the application of fluorides, oxalates, calcium phosphates, bioglass, adhesives, and laser treatment.5–9 Although dentin permeability can be reduced to some extent, the sealed tubules on the dentin surface are generally weak in combating daily adverse conditions in a complicated oral environment (e.g., tooth erosion and abrasion).10,11

Because the principle components of dentin are apatite crystals, it is reasonable to assume that the tubules should be occluded using analogous mineral compounds with respect to biomimetics. When compared with current tubule-occluding approaches including the application of fluorides, oxalates, etc, the principle components of nanohydroxyapatite (nHAp) more closely resemble those of normal dentin. Furthermore, nHAp can promote the deposition of calcium and phosphate on

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demineralized teeth for mineralization.12 nHAp has long been employed in blocking dentinal tubules,13,14 however, it dissolves slowly when confronted with an acid challenge.15 On account of the stable framework, large surface area, and excellent chemical and thermal performance, mesoporous silica nanoparticles (MSN) have been widely utilized in biomedical fields as ideal nanocarriers for drug and gene delivery over the past decades.16,17 Numerous researches have demonstrated that MSN plays a significant role in tubule obstruction.18 For these reasons, developing an nHAp-loaded MSN composite (nHAp@MSN) would be promising. MSN may protect nHAp to a certain degree from being dissolved, thus achieving satisfactory remineralization effects; and the unique acid resistance and superior mechanical strength of MSN can be expected to produce a powerful and functional surface to tackle complicated oral conditions.19,20

Owing to the existing of open tubules on the dentin surface and lower mineralization degree than the enamel, exposed dentin tends to be more susceptible to dental caries.21 As one of the most widespread oral infectious diseases, dental caries is principally derived from the cariogenic bacteria such as S. mutans, which participate in the formation and progression of biofilms, and the subsequent cariogenesis.22 Although contemporary desensitizing therapy methods can cut off the pathway of S. mutans invading into the tubules, the local acidic microenvironment induced by bacterial metabolism might hamper the efficacy and longevity of such therapies.23 Hence, a desensitizing strategy that also includes an effective inhibition of S. mutans biofilm on the dentin surface may be more promising.

The past decades have witnessed the use of varied antimicrobials being doped into

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desensitizing agents to inhibit cariogenic bacteria from forming biofilms, such as triclosan, chlorhexidine, stannous fluoride, and zinc salts.24–27 However, the application of these additives remains limited due to the possibility of drug resistance, tooth staining, instability, and cytotoxicity.28–32 Recently, epigallocatechin-3-gallate (EGCG), a natural extract derived from green tea, has attracted great attention because of its versatile function in antimicrobial, antioxidant, anti-inflammatory, and anticancer applications.33 EGCG possesses high biological activity and low toxicity, and it can suppress the expression of matrix metalloproteinases (MMP-2 and MMP-9) to inhibit the degradation of dentin collagen.34 Previous studies have also reported the inhibitory effects of EGCG on biofilm formation of S. mutans.35,36 Considering these advantages, EGCG might serve as a safe, stable, and effective antimicrobial to be encapsulated into nHAp@MSN, thus inhibiting the formation of S. mutans biofilm on the dentin surface by means of EGCG release and delivery.

To maximize the strength of nHAP, MSN, and EGCG in enabling the dentin surface to combat dentin hypersensitivity and caries, a facile integration based on the properties of each component is necessary. In our opinion, an EGCG-encapsulated nHAp@MSN (EGCG@nHAp@MSN) assembly might be the most optimal. It is speculated that EGCG@nHAp@MSN would be a desirable, versatile biomaterial to manage the dentin surface, which can not only treat dentin hypersensitivity by blocking the dentinal tubules and resisting acid and abrasion challenge, but also prevent caries by inhibiting the formation of S. mutans biofilm. However, no information is yet available on this topic to the best of our knowledge.

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Therefore, the objective of this study was to fabricate EGCG@nHAp@MSN biomaterial and to explore its potential application in treating dentin hypersensitivity and preventing caries. The null hypothesis was that EGCG@nHAp@MSN could achieve therapeutic management of the dentin surface by occluding the dentinal tubules, resisting acid and abrasion challenge, and inhibiting the formation of S. mutans biofilm.

2. MATERIALS AND METHODS 2.1. Chemicals and Reagents Absolute ethanol, ethylenediamine tetraacetic acid (EDTA), calcium nitrate tetrahydrate [Ca(NO3)2·4H2O], citric acid monohydrate, agar, sucrose, diammonium hydrogen phosphate [(NH4)2HPO4], and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). MSN, EGCG, and MTT assay kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). Brain Heart Infusion (BHI) broth was purchased from Difco, Becton, Dickinson and Co. (Sparks, MD, USA). Alpha-modified essential medium (α-MEM) and fetal bovine serum (FBS) were obtained from HyClone (Logan, UT, USA). Penicillin and streptomycin were obtained from Amresco LLC (Solon, OH, USA). All chemicals and reagents were used as received without further purification.

2.2. Synthesis and Characterization of EGCG@nHAp@MSN Biomaterial The nHAp@MSN composite was synthesized via a homogeneous precipitation technique based on

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our previous study.37 Briefly, Ca(NO3)2·4H2O and (NH4)2HPO4 aqueous solutions with Ca/P molar ratio of 1.67 were prepared and used as sources of Ca2+ and PO43− of nHAp, respectively. Then, Ca(NO3)2·4H2O aqueous solution was added dropwise to the aqueous suspension of MSN, followed by the dropwise addition of (NH4)2HPO4 aqueous solution. After centrifugation, vacuum drying, and calcination, the powder of nHAp@MSN was obtained. Then, 20 mg of pure EGCG was dissolved in 10 mL of absolute ethanol. Dried nHAp@MSN (100 mg) was dispersed into the EGCG solution (2 mg/mL) with magnetic stirring at room temperature for 2 h, followed by vigorous shaking for 72 h in darkness. This process promoted the EGCG molecules to infiltrate into the inner pores within nHAp@MSN to reach maximum loading. The mixture was then centrifuged, triple-washed with ethanol, filtered and vacuum-dried to obtain EGCG@nHAp@MSN, and stored in darkness at 4 °C until use.

Ultrastructural features of nHAp@MSN and EGCG@nHAp@MSN were examined by transmission electron microscopy (TEM) (JEM-2100, JEOL, Tokyo, Japan). The encapsulating capacity of EGCG and thermal stability of nanoparticles were determined by thermogravimetric analysis (TGA) using a thermogravimetric analyzer (STA449F3, NETZSCH, Selb, Germany) at a heating rate of 10 °C/min from 25 °C to 1000 °C under a nitrogen atmosphere.

2.3. Detection of EGCG, Ca, and P Ions Release in vitro To eliminate the influence of Ca and P ions existing in phosphate buffered saline (PBS) or simulated body fluid (SBF), Tris-buffered saline (TBS) solution was employed to assess the actual release profile of Ca and P ions, as well as EGCG from EGCG@nHAp@MSN in vitro. 7



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EGCG@nHAp@MSN (100 mg) was immersed in 10 mL of TBS [50 mM Tris-HCl, 150 mM NaCl, 0.02% (w/v) NaN3, pH 7.4] and shaken at 37 °C while being protected from light.38 The release profiles of EGCG, Ca, and P ions were detected at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, and 96 h, respectively. The suspension of EGCG@nHAp@MSN was centrifuged at 4000 rpm for 5 min at each time interval, and a 1 mL aliquot of the supernatant was collected then replaced by an equal volume of fresh TBS solution. These aliquots were used to analyze the concentration of EGCG using an UV-Vis spectrophotometer (UV-2401 PC, Shimadzu Co., Tokyo, Japan) at a detection wavelength of 325 nm. Meanwhile, the aliquots were also employed to determine the concentrations of Ca and P ions by an inductively coupled plasma-atomic emission spectrometer (ICP-AES) (IRIS Intrepid II XSP, Thermo Fisher Scientific Inc., Waltham, MA, USA).

2.4. Dentin Specimen Preparation Freshly extracted, non-carious human third molars were collected after informed consents from the donors were achieved based on a protocol approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University. These teeth were cleaned and stored in 1% (w/v) chloramine T solution at 4 °C for no more than one month before use. Each tooth was sectioned parallel to the occlusal plane below the enamel-dentinal junction by utilizing a low-speed, water-cooled diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) to produce dentin disks (1.0 mm ± 0.1 mm thickness). To eliminate the influence of dentin thickness on dentin permeability, all disks were polished to uniform thickness of 1.0 mm, and unqualified disks were excluded.

2.5. Experimental Design 8


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The occlusal surface of the prepared dentin disks was ground with water-irrigated 600-grit SiC polishing paper for 60 s to yield a standardized smear layer. Subsequently, each disk was immersed into a solution of EDTA (0.5 M, pH 7.4) for 2 min to remove the sear layer, thus simulating a sensitive tooth model.10 Thirty-two EDTA-etched dentin specimens were randomly assigned into two groups (n = 16 each group) as follows:

Group 1 (control): No treatment was applied.

Group 2 (EGCG@nHAp@MSN-treated): A slurry of EGCG@nHAp@MSN (prepared by mixing EGCG@nHAp@MSN powder with deionized water in a powder/liquid ratio of 10 mg/100 µL) was applied twice to the occlusal surfaces of the dentin specimens for 30 s at a low speed with a rotary cup.

After different treatments, all specimens were stored in artificial saliva (0.7 mM CaCl2, 0.2 mM MgCl2·6H2O, 4.0 mM KH2PO4, 30 mM KCl, 20 mM HEPES buffer, 0.3 mM NaN3, pH 7.4) at 37 °C for 24 h.39 Subsequently, the specimens in each group were randomly selected and equally assigned into two subgroups (n = 8 disks). Specimens in one of the subgroup was challenged by immersing the disks in a 6% (w/v) citric acid solution (pH 1.5) for 1 min to test the resistance to dietary acid erosion. Specimens in the other subgroup was challenged by mechanical brushing for 3 min using a toothbrush (360° Total Advanced, Colgate-Palmolive Co., New York, NY, USA) with soft bristles to test the resistance to daily abrasion. The toothbrush was applied to the occlusal surface of each dentin specimen at an inclination of approximately 90° under a constant load (150 g) measured by an electronic top-loading balance (EL202, Mettler-Toledo, Shanghai, China) for 150 9



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strokes per min.

2.6. Dentin Permeability Measurement All dentin specimens mentioned above were used for the measurement of dentin permeability. The permeability was evaluated through a fluid infiltration apparatus in a modified split-chamber unit which was linked to a water container at a simulated pulpal pressure of 20 cm deionized water.10,40,41 Each dentin specimen was tightly attached between a pair of ‘‘O’’ rubber rings, which were linked to a plexiglass block of 2 × 2 × 0.5 cm3 (length × width × depth) perforated by an 18-gauge steel tube, providing an available dentin surface area (0.38 cm2) for fluid infiltration (Figure 1). The movement of an air bubble (in mm) produced within a glass micro-capillary tube (horizontally placed between the split-chamber unit and the pressure reservoir) of 25 µL was monitored and transformed into volume flow (µL·min−1) across each dentin specimen. The hydraulic conductance (Lp) value (µL·min−1·cm H2O−1·cm−2) was then calculated by dividing the fluid flow (µL·min−1) by the hydrostatic pressure (20 cm H2O) and the available surface area (cm2).

Figure 1. Schematic illustration of dentin permeability measurement using a fluid infiltration apparatus in a modified split-chamber unit working under 20 cm H2O pressure. 10


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The dentin permeability of each dentin specimen was represented as a percentage (Lp%) of the maximum permeability (obtained after EDTA application and identified as a value of 100% permeability) of the same specimen. The Lp value was recorded at four treatment time points, namely, after EDTA application, desensitizing treatment application, exposure to 6% citric acid challenge, or exposure to mechanical brushing. The dentin permeability data were showed as mean ± standard deviation and statistically analyzed by an IBM SPSS 20.0 software (Armonk, NY, USA). Two-factor repeated measurements ANOVA was employed to analyze the data, considering the treatment (group) as the main effect and the treatment time as the repeated measurement. One-factor repeated measurement ANOVA was utilized for pairwise comparisons within each group, and Tukey’s test was used to perform post hoc multiple comparisons (α = 0.05).

2.7. Morphology Observation of Tubule Occlusion For the morphology observation of tubule occlusion on dentin surface, three dentin specimens were prepared for each treatment group. For each specimen, a groove was sectioned on the pulpal surface to the occlusal surface with half of the disk’s thickness (0.5 mm) by using the Isomet diamond saw (Figure 2). Then, each specimen, which was pinched between the forefinger and thumb, was split longitudinally into two halves by exerting pressure on the distal and medial surfaces to examine the cross and longitudinal sections of the dentin. The splitting operation should be performed such that the occlusal surface would not be influenced, and longitudinal observation should focus on the tubules which were close to the occlusal surface rather than the pulpal surface. All dentin specimens were desiccated, Au−Pd alloy sputter coated, and observed by FESEM at 5

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kV. Micrographs at the magnifications of 2500×, 5000×, and 7500× were captured.

Figure 2. Schematic summarizing the process of the specimen preparation for morphology observation.

2.8. Evaluation of S. mutans Biofilms Formation 2.8.1. Bacterial Culture and Biofilm Preparation

S. mutans (ATCC 25175), obtained from the State Key Laboratory for Oral Biomedicine Ministry of Education, School of Stomatology, Wuhan University (China), was cultured in BHI broth anaerobically at 37 °C for 24 h. Then, the bacterial suspension was adjusted to a concentration of 107 colony forming units (CFU)/mL prior to use. The inoculation medium was achieved by diluting the bacterial culture medium with fresh BHI broth containing 1% (w/v) sucrose.

Twenty EDTA-etched dentin specimens were prepared, disinfected under UV light for 2 h for each side, and then randomly assigned into two groups (the control group and the EGCG@nHAp@MSN-treated group, n = 10 each group) in a same procedure as mentioned in Section 2.4 and 2.5. Each specimen was placed in a well of a 24-well plate and added with 1 mL of inoculation medium. After cultivation at 37 °C under anaerobic conditions for 24 h for biofilm formation, the biofilm-coated specimens were triple-washed with sterile PBS to wash away non-adherent bacteria and transferred to a new 24-well plate. 12


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2.8.2. CLSM analysis

One biofilm-coated specimen randomly chosen from each group was dyed with a LIVE/DEAD Bacterial Viability Kit (Molecular Probes, Invitrogen, Eugene, OR, USA). Live and dead microorganisms were dyed with SYTO-9 and propidium iodide to yield green and red fluorescence, respectively.42 The biofilm-coated specimens were examined by CLSM (Fluoview FV1200, Olympus, Tokyo, Japan) at 40× magnification. For each specimen, two characteristic stacks (Z-stack) of each biofilm image were achieved at a Z-step of 2 µm, starting from the bottom (contacting with the treated surface) to the top of the biofilm. An Imaris 7.2.3 software (Bitplane, Zurich, Switzerland) was applied to analyze confocal images obtained from each stack. The live/dead bacteria biomass distributions from the first 10 layers (20 µm thickness) of each Z-stack were analyzed to evaluate the biofilm inhibition efficacy of different treatments on dentin surfaces.

2.8.3. MTT Assay

Three biofilm-coated specimens randomly selected from each group were utilized to detect the metabolic activity of S. mutans biofilms using an MTT assay. Briefly, 1 mL of MTT solution (0.5 mg/mL) was added into each well of the 24-well plate containing the specimen and cultivated at 37 °C under conditions of 5% CO2 for 4 h. After incubation, the supernatant in each well was pipetted out, and 1 mL of DMSO was added to dissolve the purple/blue formazan. After gently shaken for 20 min in darkness, the absorbance was recorded with a PowerWave XS2 microplate reader (BioTek Instruments Inc., Winooski, VT, USA) at 570 nm. Four readings were accomplished for each biofilm-coated specimen in each group (N = 12). The data of MTT assay 13



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were statistically analyzed by Student’s t-test (α = 0.05).

2.8.4. CFU Counts

Three biofilm-coated specimens randomly selected from each group were transferred into a microtube including 1 ml of sterile PBS. After vortex mixed for 2 min to detach the coated biofilm,43,44 ten-fold serial dilutions were conducted. Then, 40 mL aliquots of each dilution were plated onto BHI agar plates, and the plates were anaerobically cultivated at 37 °C for 24 h. After that, the CFUs of each specimen were manually calculated. Three replicates were implemented for each biofilm-coated specimen in each group (N = 9). The data of CFU counts were statistically analyzed by Student’s t-test (α = 0.05).

2.8.5. FESEM Examination

The remaining three biofilm-coated specimens in each group were employed to examine the S. mutans adhesion and biofilm morphology on dentin surfaces subjected to different treatments. Each specimen was fixed with 2.5% glutaraldehyde in PBS at 4 °C for 4 h, and then triple-rinsed with PBS. After graded ethanol dehydration (30%, 50%, 70%, 80%, 90%, and 100%) and desiccation overnight, the specimens were sputter coated with Au−Pd alloy and inspected by FESEM at 5 kV.

2.9. Cytotoxicity Test Human dental pulp stem cells (hDPSCs) derived from healthy pulp tissues of premolars for orthodontic extraction after achieving the donors’ informed consent were employed to investigate the cytotoxicity of EGCG@nHAp@MSN. The hDPSCs were seeded at 5,000 cells per well in

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96-well plates and cultivated at 37 °C under conditions of 5% CO2 for 24 h in α-MEM containing 10% FBS and 1% Penicillin/Streptomycin until 80−85% confluence was achieved. These cells were exposed to a series of concentrations of EGCG@nHAp@MSN (0, 10, 20, 40, 80, 160, 320, 640, 1280 µg/mL). After cultivation for 24 h, each well was added with 10 µL of MTT solution (5 mg/mL) and cultured at 37 °C for another 4 h in darkness. Then, the supernatant was pipetted out, and DMSO was added to dissolve the MTT formazan produced by mitochondrial succinic dehydrogenase. The absorbance was monitored with the microplate reader at 490 nm. The results were expressed as relative mitochondrial succinic dehydrogenase activity (%) of a control group (0 µg/mL). This assay was implemented in sextuplicate. One-factor ANOVA was used to statistically analyze the data, and Tukey’s test was used to perform post hoc multiple comparisons (α = 0.05).

3. RESULTS 3.1. Characterization TEM image in Figure 3A shows that nHAp@MSN contained well-ordered nanopores and channel framework (red box), and the MSN were enveloped by the nHAp crystals. TEM image in Figure 3B shows that the characteristic nanopores and channel framework were obscured (red box) after the introduction of EGCG, indicating the EGCG molecules were largely adsorbed into the internal pores of MSN. The nHAp crystals remained extensively loaded on the surface of the MSN. Furthermore, TGA result in Figure 3C shows that the respective weight loss of nHAp@MSN and EGCG@nHAp@MSN was 6.12% and 17.41% from 25 °C to 1000 °C. The loading efficiency of 15



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EGCG was calculated to be 11.29%, suggesting that a significant amount of EGCG was capable to be encapsulated into the MSN.

Figure 3. TEM pictures of (A) nHAp@MSN and (B) EGCG@nHAp@MSN. Pointers indicate that the HAp nanocrystals were loaded on the MSN. Red box in (A) revealing well-ordered nanopores and channel framework and in (B) revealing obscured nanopores and channel framework. (C) TGA curves of nHAp@MSN and EGCG@nHAp@MSN. In vitro release profiles of (D) EGCG and (E) cumulative Ca and P ions from EGCG@nHAp@MSN in TBS at 37 °C (pH 7.4) for 96 h. (F) Ca/P ratio of the released minerals from EGCG@nHAp@MSN in TBS at 37 °C (pH 7.4) for 96 h.

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3.2. Release Profiles of EGCG, Ca, and P Ions Release profiles of EGCG, Ca, and P ions from EGCG@nHAp@MSN were studied in vitro. Figure 3D shows EGCG release, which occurred in a two-step pattern, with an initial rapid release in the first 8 h and a relatively slow, continuous release over 96 h. According to the standard curve (y = 0.0036x + 0.0124; R2 = 0.9992), the release percentage of EGCG reached approximately 55% after 96 h. Figure 3E shows the cumulative release of Ca and P ions, which presented as an initial stage of rapid release then gradually slowed down until the end of the observation time at 96 h. As shown in Figure 3F, the Ca/P ratio of the released minerals during 96 h was around 1.67, which corresponds to the typical Ca/P ratio of HAp.45

3.3. Dentin Permeability Measurements Table 1 and 2 summarize the dentin permeability produced by the control group and the EGCG@nHAp@MSN group at each designated treatment time point. Both tables show that a two-way repeated measurements ANOVA showed a significant main effect for time (p < 0.001), group (p < 0.001), and time × group interactions (p < 0.001). After the desensitizing treatment by EGCG@nHAp@MSN, the dentin permeability decreased significantly (p < 0.001), and the EGCG@nHAp@MSN group showed a significantly lower Lp% value than that of the control group (p < 0.001). After citric acid challenge, the Lp% value of the control group increased significantly (p < 0.001), and although that of the EGCG@nHAp@MSN-treated group increased as well (p < 0.05), it was statistically lower than that of the control group (p < 0.001) (Table 1). After mechanical brushing, the Lp% value of the EGCG@nHAp@MSN-treated group did not 17



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increase when compared with that before mechanical brushing (p > 0.05), and the group manifested significantly lower Lp% value than that of the control group (p < 0.001) (Table 2).

Table 1. Dentin permeability data at different time points (after EDTA application, desensitizing treatment, and citric acid challenge)

EDTA

Desensitizing

Citric acid

application

treatment

challenge

Control (n = 8)

100 ± 0 A,1

100.0 ± 8.7 A,1

145.7 ± 13.6 B,1

EGCG@nHAp@MSN (n = 8)

100 ± 0 a,1

14.9 ± 8. 3 b, 2

24.2 ± 10.5 c, 2

Groups

Notes: The values of dentin permeability are expressed as percentage (Lp%) and shown as means ± SD. The Lp% after EDTA application represents the maximum permeability (100%). For each row, different superscript letters indicate significant differences (p < 0.05). For each column, different superscript numbers indicate significant differences (p < 0.05).

Table 2. Dentin permeability data at different time points (after EDTA application, desensitizing treatment, and mechanical brushing)

EDTA

Desensitizing

Mechanical

application

treatment

brushing

Control (n = 8)

100 ± 0 A,1

101.9 ± 8.0 A,1

106.0 ± 9.7 A,1

EGCG@nHAp@MSN (n = 8)

100 ± 0 a,1

16.3 ± 10.0 b, 2

20.1 ± 11.4 b, 2

Groups

Notes: The values of dentin permeability are expressed as percentage (Lp%) and shown as means ± SD. The Lp% after EDTA application represents the maximum permeability (100%). For each row, different superscript letters indicate significant differences (p < 0.05). For each column, different superscript

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numbers indicate significant differences (p < 0.05).

3.4. FESEM Examination of Tubule-Occluding Effects Figure 4 shows the cross and longitudinal sectional morphology of tubule occlusion of all the groups. After the desensitizing treatment, the control group (Figure 4A1–A3) showed a smear-free layer on the dentin surfaces, and all the dentinal tubules were open after being etched with EDTA for 2 min. The EGCG@nHAp@MSN-treated group (Figure 4B1–B3) showed completely occluded dentinal tubules, and the intratubular EGCG@nHAp@MSN precipitates were firmly associated with the tubular inwall with the penetration depth of approximately 10 µm. After citric acid challenge, the control group (Figure 4A4–A6) showed a smooth surface, and the diameters of patent tubules were expanded. The EGCG@nHAp@MSN-treated group (Figure 4B4–B6) showed that most of the tubules remained sealed, and the underlying intratubular precipitates remained tightly incorporated with the tubular inwall. After mechanical brushing, the control group (Figure 4A7–A9) showed that the patent dentinal tubules were visible, and some debris was left on the intertubular dentin. The EGCG@nHAp@MSN-treated group (Figure 4B7–B9) showed that tubule orifices were scarcely open, and the infiltration depth of the crystals remained unchanged.

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Figure 4. Representative FESEM images of the cross and longitudinal sectional examination of tubule occlusion. Graphs of (A1–A3) Group 1 (control) revealing patent tubules (pentagrams); graphs of

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(B1–B3) Group 2 (EGCG@nHAp@MSN-treated) revealing completely blocked tubules, and the precipitates firmly combined with the tubular inwall (box). Graphs of (A4–A6) Group 1 revealing patent tubules with expanded diameters (pentagrams); graphs of (B4–B6) Group 2 revealing that most of the tubules were obstructed, while the underlying deposits were well-retained (box). Graphs of (A7–A9) Group 1 revealing open tubules (pentagram) with some debris left on the intertubular dentin; graphs of (B7–B9) Group 2 revealing hardly any tubule orifices were open, and the infiltration depth of the precipitates was almost unchanged (box).

3.5. Evaluation of the Inhibition of S. mutans Biofilm Formation Figure 5A and C respectively shows stacked confocal images of S. mutans biofilm grown on the surfaces of each dentin specimen obtained from the control and the EGCG@nHAp@MSN-treated groups. The corresponding relative distribution of the live/dead bacteria on each layer is summarized on the right of each CLSM image as line plots (Figure 5B and D). The total biomass of live/dead bacteria of the control group appeared to be more significant than that of the EGCG@nHAp@MSN-treated group. Furthermore, the control group showed a potential growth of a considerably thicker bacterial biofilm more than the first 10 layers (20 µm of thickness) of each Z-stack, while the EGCG@nHAp@MSN-preteated group showed an apparently thinner biofilm with less than 20 µm of thickness. The result of the MTT assay (Figure 5E) indicates that the bacteria belonging to the EGCG@nHAp@MSN-treated group showed a significantly lower cellular metabolic activity than those belonging to the control group (p < 0.01). The result of the CFU counts (Figure 5F) suggests that the EGCG@nHAp@MSN-treated group showed significantly fewer bacterial colonies on future culturing compared to the control group (p < 0.05).

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Figure 5. CLSM images (3D overlay projections) of representative live/dead stained S. mutans biofilm (live bacteria, green; dead bacteria, red) in the (A and B) control group and (C and D) EGCG@nHAp@MSN-treated group after 24 h of incubation on the dentin surfaces. The line plots on the right of each CLSM image indicate the corresponding biomass distribution of total live/dead bacteria (Z-step = 2 µm). (E) MTT assay of S. mutans biofilm after 24 h of incubation on the dentin surfaces. Data are expressed as mean ± SD (N = 12). * denotes p < 0.01. (F) CFU counts of S. mutans biofilm after 24 h of incubation on the dentin surfaces. Data are expressed as mean ± SD (N = 9). # denotes p < 0.05. 22


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Representative FESEM pictures of S. mutans biofilm grown on dentin surfaces are shown in Figure 6. In the control group (A and B), dense and thick bacterial biofilm was observed on the dentin surface. In the EGCG@nHAp@MSN-treated group (C and D), the orifices of the tubules were almost absolutely occluded by EGCG@nHAp@MSN, and there were fewer bacteria growing on the dentin surface than those in the control group.

Figure 6. Representative FESEM images of S. mutans biofilm formation on the dentin surfaces after cultivation for 24 h in the (A and B) control group and (C and D) EGCG@nHAp@MSN-treated group. (B) and (D) are high-magnification images of (A) and (C). Pointers in (A) indicate the patent dentinal tubules, and pointers in (C) indicate the dentinal tubules occluded by EGCG@nHAp@MSN.

3.6. Cytotoxicity Test 23



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The relative mitochondrial succinic dehydrogenase activity of hDPSCs exposed to a series of concentrations of EGCG@nHAp@MSN (0–1280 µg/mL) is shown in Figure 7. A statistically significant difference (p < 0.01) was noted between the 640 µg/mL group and 0 µg/mL group, and the 1280 µg/mL group and 0 µg/mL group. However, even at the highest concentration of 1280 µg/mL, EGCG@nHAp@MSN triggered less than 20% of hDPSCs death.

Figure 7. Relative mitochondrial succinic dehydrogenase activities of hDPSCs after incubation with EGCG@nHAp@MSN for 24 h. Data are expressed as mean ± SD (n = 6). The same letter denotes no statistical difference (p > 0.05).

4. DISCUSSION In this study, the EGCG@nHAp@MSN biomaterial was synthesized. Our results demonstrated that the application of EGCG@nHAp@MSN on the dentin surface efficiently occluded the dentinal tubules, reduced dentin permeability, achieved excellent acid- and abrasion-resistant stability, and inhibited the formation and growth of S. mutans biofilm. These findings indicate that EGCG@nHAp@MSN exhibits great potential in providing a therapeutic management of the dentin

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surface for treating dentin hypersensitivity and preventing caries, thereby confirming our null hypothesis.

EGCG, the most active component of green tea polyphenols, has been extensively used as a natural antimicrobial owing to its excellent antibacterial capability and low toxicity.33,35 MSN and nHAp have been proven to be practical in obstructing dentinal tubules because of their specific properties.13,18 To successfully manage the dentin surface for combating dentin hypersensitivity and inhibiting S. mutans biofilm inhibition, we encapsulated MSN with nHAp and EGCG to develop a versatile biomaterial in this study. TEM images and TGA analysis provided direct evidence for the successful encapsulation of EGCG and nHAp into MSN, leading to the establishment of EGCG@nHAp@MSN.

The

cumulative

release

profile

of

Ca

and

P

ions

from

EGCG@nHAp@MSN reflected an initial stage of rapid release, and then gradually slowed down until 96 h (Figure 3E). The Ca/P ratio of the released minerals was analogous to HAp (Figure 3F), suggesting that EGCG@nHAp@MSN likely acts as a reservoir of calcium and phosphate to facilitate crystal deposition and growth for tooth remineralization.12,46 Furthermore, a sustained release of EGCG was observed over 96 h, which may play an crucial role in the inhibition of S. mutans biofilm formation. Thus, EGCG@nHAp@MSN’s efficacy in occluding the dentinal tubules and inhibiting S. mutans biofilm formation were investigated to confirm the potential application of EGCG@nHAp@MSN as a versatile biomaterial for therapeutic management of the dentin surface.

The dentin permeability after EDTA application, desensitizing treatment, acid challenge, and mechanical brushing were evaluated with a modified fluid infiltration apparatus, and further

25



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morphological alterations concerning tubule occlusion were examine using FESEM. After the desensitizing treatment, the tubules in the control group exposed (Figure 4A1–A3), while the tubules in the EGCG@nHAp@MSN group were completely obstructed, and the precipitates were firmly incorporated with the tubular inwall (Figure 4B1–B3). The latter phenomenon may be attributable to several reasons. First, TEM image showed that the average diameter of the EGCG@nHAp@MSN was around 300 nm (Figure 3), and the material was in a slurry state when applied to the dentin surface during the desensitizing procedure. Thus, it may easily infiltrate into the tubules. Second, continuous release of Ca2+ and PO43– from EGCG@nHAp@MSN would likely yield a local supersaturation to facilitate mineral formation in the tubules.12,46 Third, the remarkable blocking ability might also profit from the favorable adhesion effects between the dentin surface and HAp nanoparticles. Since nHAp are of small particle sizes, the large atomic numbers on its surface would offer high surface energy that readily binds to other atoms. Fourth, the interspace between MSNs inside the tubules was occupied and complemented by the loaded nHAp, which is why a close adherence to the tubular wall was observed.37

The results of dentin permeability measurement are consistent with the FESEM observation. The dentin specimens treated with EGCG@nHAp@MSN decreased dentin permeability significantly, and the EGCG@nHAp@MSN-treated group manifested significantly lower Lp% value compared to the control group (Table 1 and 2), which confirming the efficacy of EGCG@nHAp@MSN on sealing the dentinal tubules and reducing dentin permeability. The reduction of dentin permeability (approximately 85%) via the application of EGCG@nHAp@MSN

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in this study was higher than that obtained via the application of a bioactive glass-ceramic toothpaste (nearly 51%) or a Sensodyne Repair toothpaste (nearly 43%), as reported by Zhong et al.47 or the application of a Novamin-containing toothpaste (almost 76%), as reported by Wang et al.48 Considering dentin permeability, the application of EGCG@nHAp@MSN achieved the most effective tubule-occluding ability. Different treatment methods may affect dentin permeability differently. Specifically, in the present study, EGCG@nHAp@MSN slurry was polished twice on the surfaces of the dentin specimens for 30 s. The specimens were brushed for 20 s with toothpaste then maintained for 4 min, according to Zhong et al. and were brushed for 2 min with toothpaste, according to Wang et al. Actually, the dentin permeability values are highly variable and could be potentially influenced by many factors, such as dentin thickness, surface area, test temperature, post-extraction time, measurement apparatus, and the operator who produces the specimens and performs the test.49–51 Therefore, the parameters and instruments for dentin permeability measurement should be reasonably selected and standardized so that published data can be compared and analyzed. Furthermore, since the presence of the smear layer and demineralization may affect the initial hydraulic conductance,52,53 it is reasonable to recommend that both of them should be taken into account and measured to establish the real effect of the new materials.

A crucial standard for evaluating the capability of the new desensitizers on tubule occlusion is the acid-resistant stability. Because citric acid is the most common acidic component of fruit juices and soft drinks in daily diet, six percent (w/v) citric acid solution was used in this study to simulate dietary erosion in oral cavity and examine the acid-resistance of EGCG@[email protected],54 After

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exposure to acid attack, the diameters of patent tubule orifices in the control group were expanded (Figure 4A4–A6), and the group exhibited the highest Lp% value (Table 1), which is identical with previously published investigations.10,54 This feature is primarily due to the fact that the mineralization degree of the intertubular dentin is lower than the peritubular dentin, thus the latter is much more vulnerable to acidic conditions that may eventually lead to the increase of dentin permeability.55 However, most of the orifices remained sealed in the EGCG@nHAp@MSN-treated group, while the underlying deposits were firmly associated with the tubular inwall (Figure 4B4–B6). Although the dentin permeability of the EGCG@nHAp@MSN-treated group increased after acid challenge, it was statistically lower than that of the control group (Table 1). These findings highlight the desirable acid-resistant stability of EGCG@nHAp@MSN. The possible mechanism underlying such stability mainly originates from the resistance of MSN to acid challenge;19 and the favorable occlusion inside the tubules by EGCG@nHAp@MSN could defend the deposits from being attacked by erosion. Despite the fact that acid challenge dissolves loose deposits, Ca2+ and PO43– released from the underlying nHAp inside the tubules might promote crystals deposition for remineralization.46 In addition, EGCG may inactivate the dentin-derived MMPs for reducing the solubilization of dentin collagen during the remineralization period to prevent dentin erosion.56

For appraising the tubule-occluding effectiveness of desensitizing agents, abrasion-resistant stability is another important criterion. The desensitizing treatment can generate a crystal layer on the dentin surface and obstruct the tubule orifices, whereas the crystal layer or obstructed orifices

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may be readily removed or be open again by daily mechanical brushing.11,54 After exposure to mechanical brushing, the EGCG@nHAp@MSN-treated group showed not only essentially sealed tubule orifices and well-maintained infiltration depth (Figure 4B7–B9), but also a significantly lower Lp% value than that of the control group. More importantly, the mechanical brushing treatment for the EGCG@nHAp@MSN-treated group did not increase dentin permeability (Table 2). These findings highlight the strong abrasion-resistant stability of EGCG@nHAp@MSN. In our opinion, the probable mechanism underlying such anti-abrasion action is as follows: firstly, the MSN is of superior mechanical property and has been utilized extensively in improving mechanical strength.20 Secondly, the tightly occluded dentinal tubules and well-maintained infiltration depth together considerably reduce the possibility of being brushed away, and the remineralization effect of nHAp is also involved in this process. Thirdly, the EGCG holds the inhibitory activity against MMPs and might play a vital role in preventing the degradation of the collagen matrix and in maintaining the organic layer on the abrasion surface.57

Considering the above discussion, it is reasonable to believe that EGCG@nHAp@MSN can efficiently block the tubules, reduce dentin permeability, and provide excellent acid- and abrasion-resistant stability. Theoretically, larger the proportion of tubules obstructed by EGCG@nHAp@MSN, higher is the acid- and abrasion-resistant stability. These attributes will apparently strengthen the durability and stability of desensitizing agents on tubule occlusion for preventing fluid movements by different aspects, thereby suggesting a promising strategy to overcome dentin hypersensitivity and providing an ideal alternative for clinical dentists.

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Dental caries, which is chiefly caused by biofilm formation of cariogenic bacteria, is universally acknowledged as one of the most widespread dental problems. Among the cariogenic bacteria, S. mutans is deemed to play a important role in the initial development of dental caries since it can adhere onto tooth surfaces and metabolize carbohydrates to produce acids, thus triggering tooth demineralization.58 Owing to the patent dentinal tubules and lower mineralization degree compared to enamel, the cariogenic process on the dentin surface is usually very quick.21 Besides, the stability and functionality of the desensitizers might be affected by the local low pH microenvironment induced by bacteria metabolism after the application of desensitizing treatment.23 Therefore, it is of great importance for desensitizing agents to prevent the formation of S. mutans biofilm on dentin surface to prevent caries. In this study, the confocal images indicated that treatment with EGCG@nHAp@MSN effectively inhibited the growth of S. mutans biomass compared with that of the control (Figure 5A–D). The MTT assay and CFU counts are supplementary to the CLSM data, among which the MTT assay quantified the intracellular metabolic activities of the biofilm-forming and planktonic bacteria, showing a decline in the metabolisms of S. mutans (Figure 5E). The CFU counts quantified the total number of viable bacteria on the dentin surfaces, revealing a reduction in the number of viable S. mutans colonies (Figure 5F). FESEM observation confirmed that there were fewer bacteria growing on the EGCG@nHAp@MSN-treated dentin surface (Figure 6C and D). Taken together, these results verified that the application of EGCG@nHAp@MSN could efficiently inhibit the formation and growth of S. mutans biofilm on the dentin surface. We deduce that the outstanding inhibitory effect is derived from the EGCG, as an initial burst release of EGCG was followed by a relatively slow 30


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release

over

96

h;

furthermore,

after

nearly

55%

of

EGCG

was

released

from

EGCG@nHAp@MSN, the remaining EGCG might be released continuously, and the inhibitory effects would be maintained for a relatively long time. The relevant mechanism of action of EGCG has been already elucidated by several researchers. Xu et al suppose that EGCG suppresses the specific virulence factors (e.g., lactate dehydrogenase) of cariogenic bacteria, and affect the acid production of S. mutans, thereby inhibiting the cariogenicity.59 Moreover, EGCG is able to suppress the expression of GTF B, C, and D genes to interfere in the adhesion of S. mutans on tooth surface, and it may decrease the cariogenicity of cariogenic foods to provide an anti-caries function.60,61 The findings confirm that EGCG@nHAp@MSN is capable of inhibiting the formation and growth of S. mutans biofilm on the dentin surface, thus preventing caries.

Cytotoxicity assay is indispensable in evaluating the safety of the application of biomaterials to human tissues. EGCG@nHAp@MSN should be nontoxic or negligibly toxic before it may be employed for future use in vivo. Results illustrated that EGCG@nHAp@MSN showed low cytotoxicity in vitro in the range of concentrations measured, and even at the highest concentration (1280 µg/mL), it caused less than 20% of hDPSCs death (Figure 7). Hence, EGCG@nHAp@MSN is highly biocompatible and maybe appropriate for in vivo applications.

To the best of our knowledge, this is the first endeavor to date to establish a therapeutic dentin surface by applying EGCG@nHAp@MSN biomaterial for treating dentin hypersensitivity and inhibiting S. mutans biofilm formation in vitro. The principal mechanism of action of EGCG@nHAp@MSN is presented in Figure 8. It is anticipated that our promising experimental

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fruits could ultimately be transformed into clinical benefits, therefore further in vitro and in vivo studies are definitely required to investigate the long-term functioning of EGCG@nHAp@MSN.

Figure 8. Schematic illustrating the mechanism of EGCG@nHAp@MSN for therapeutic management of the dentin surface. (A) represents exposed dentinal tubules, S. mutans biofilm forms on the dentin surface and infiltrates into the dentinal tubules to induce caries; when exposed to stimulus, such as cold or acid, the pulp nerve would become sensitive, thereby triggering dentin hypersensitivity. (B) represents tightly occluded dentinal tubules by the application of EGCG@nHAp@MSN, EGCG@nHAp@MSN not only cuts off the pathway of stimulus or S. mutans invading into the tubules but also effectively inhibits the formation of S. mutans biofilm on the dentin surface. In this regard, caries can be prevented, and the sensitive pulp nerve can be recovered, thereby treating dentin hypersensitivity.

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5. CONCLUSION To sum up, EGCG@nHAp@MSN biomaterial was successfully developed. The application of EGCG@nHAp@MSN on the dentin surface could efficiently occlude the dentinal tubules, reduce dentin permeability, provided favorable acid- and abrasion-resistant stability. Furthermore, EGCG@nHAp@MSN was capable of sustainedly releasing EGCG, Ca, and P, and markedly inhibiting the formation and growth of S. mutans biofilm on the dentin surface. Therefore, EGCG@nHAp@MSN exhibits tremendous potential to serve as a novel, biocompatible, and versatile biomaterial to therapeutically manage the dentin surface for combating dentin hypersensitivity and caries.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Cui Huang).

Author Contributions §

Jian Yu and Hongye Yang contributed equally to this work and are co-first authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No.

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81371191, 81571012), the Natural Science Foundation of Hubei Province of China (No. 2015CFA072), and the Fundamental Research Funds for the Central Universities (2042017kf0102).

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Table of Contents Graphic

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Development of Epigallocatechin-3-gallate-Encapsulated

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