Bioinspired Peptide-Decorated Tannic Acid for in Situ

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Bioinspired peptide-decorated tannic acid for in situ remineralization of tooth enamel: in vitro and in vivo evaluation Xiao Yang, Bo Yang, Libang He, Ruiqi Li, Yixue Liao, Shuhui Zhang, Yinxin Yang, Xinyuan Xu, Dongyue Zhang, Hong Tan, Jiyao Li, and Jianshu Li ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00623 • Publication Date (Web): 06 Oct 2017 Downloaded from http://pubs.acs.org on October 8, 2017

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ACS Biomaterials Science & Engineering

Bioinspired peptide-decorated tannic acid for in situ remineralization of tooth enamel: in vitro and in vivo evaluation Xiao Yang†, #, Bo Yang†, #, Libang He§, #, Ruiqi Li†, Yixue Liao†, Shuhui Zhang†, Yinxin Yang†, Xinyuan Xu†, Dongyue Zhang†, Hong Tan†, Jiyao Li§ and Jianshu Li†,*

AFFILIATIONS †

Department of Biomedical Polymers and Artificial Organs, College of Polymer Science and

Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, NO.24, 1st Section of South Yihuan Road, Chengdu 610065, P. R. China. §

State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University,

NO.14, 3rd Section of Ren Min Nan Road, Chengdu 610065, P. R. China. *

Corresponding author: E-mail: [email protected] (JS. Li)

#

These authors contributed equally to this work.

ABSTRACT: Tooth enamel can be eroded by the local cariogenic bacteria in plaque or the non-bacterial factors in oral environment. The damage is irreversible in most situations. In order to restore the etched human tooth enamel in situ, a salivary acquired pellicle (SAP) bioinspired tannic acid (SAP-TA) is synthesized. Statherin is one of the SAP proteins which can selectively adsorb onto enamel surface. Peptide sequence DDDEEKC is a bioinspired sequence of statherin and has the adsorption capacity on hydroxyapatite (HAP). TA has an abundant polyphenol groups which can grasp the Ca2+ in the saliva to induce the regeneration of HAP crystal. Hence, SAP1 ACS Paragon Plus Environment


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TA not only enhances the binding force at the interface of remineralization, but also mimics the biomineralization process of tooth enamel. Besides, ferric ion can coordinate with SAP-TA to form a compact coating which increases the adsorbed amounts of SAP-TA on tooth enamel. Compared with SAP-TA alone, the etched enamels treated with SAP-TA/Fe (III) has a better remineralization effect and mechanical properties (surface microhardness recovery > 80% and binding force of 64.85 N) when being incubated in artificial saliva for two weeks. Moreover, in vivo remineralization performance is evaluated in a classical rat caries model. The polarizing microscope and Micro-CT results show that SAP-TA/Fe (III) has a good effect on the remineralization process in real oral environment, indicating that it is a promising repair material of in situ remineralization of enamel. KEYWORDS: polyphenol, salivary acquired pellicle, effective adsorption, biomineralization, dental restoration.

INTRODUCTION Human tooth enamel is composed of inorganic components (96%) and organic components (4%). Hydroxyapatite (HAP) is the main component of tooth, which is nanorod and has highly organized hierarchical structures.1 Amelogenin, which is secreted by ameloblasts, is the major protein to form around 90% of the organic matrix components in the process of enamel biomineralization.2-5 Tooth enamel is known to be the hardest human tissue.6-7 However, the tooth enamel can be eroded by 2 ACS Paragon Plus Environment

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local cariogenic bacteria in plaque (caries), or non-bacterial factor (acidic beverages, mechanical force) in oral environment.8 For the purpose of restoring the etched tooth enamel, researchers have developed numerous of restorative materials, such as filling the hole with resin, metal and ceramics. Biomineralization, as a strategy to repair etched tooth enamel has been studied for decades. Biomineralization is a mineralization process which is modulated by organic matrix, for instance, the formation of tooth enamel was regulated by amelogenin. In order to mimic this natural process, scientists have developed quite a few of natural or synthetic materials to induce the regeneration tooth enamel. For examples, amelogenin and recombinant amelogenins were directly utilized to induce the generation of mineral layers containing arranged needle-like fluoridated hydroxyapatite crystals on the surface of etched enamel.9-10 Snead et al. developed a self-assembling bioactive matrix which is peptide amphiphiles to trigger the formation of dental enamel in vivo.11 Dendrimer is also an ideal analogue of proteins due to its tunable three-dimensional nanostructures, narrowed molecular weight distribution and easily modified peripherial groups. Thus, dendrimer-based materials have been widely used to mimic the proteins’ function to induce HAP mineralization.12 For example, poly (amido amine) (PAMAM) dendrimer has been modified into carboxylterminated PAMAM,1 alendronate (ALN) conjugated PAMAM,8 and phosphateterminated PAMAM13 to induce needle-like HAP crystal on the surface of etched enamel.14

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Tannic acid (TA) is a type of natural polyphenols, which is widely existed in tea and wine.15 Because of its polyphenolic structure, it has interesting functions such as antioxidation16-17 and antibacterial property.18 Recently, Caruso et al. reported a new application of TA by preparing a coordination complexes of TA with Fe (III) ions as a functional coating of various interfaces.19 After that, this system was utilized to repair dentin in vitro as it can not only efficiently occlude dentinal tubule. Moreover, it can induce the remineralization of HAP by pyrogallol-mediated Ca2+ adsorption in the tubules during incubation in artificial saliva.20 However, although it is an efficient and quick method to restore dentin, it has no effect on enamel because TA/Fe (III) complex is predominantly deposited on the dentin surface20-21 and TA cannot specially adsorb on HAP. It is known that oral bacteria could form biofilms on hard tooth surface so that they could adhere to teeth instead of being swallowed by human in oral environment.23 In the initial stage of building a biofilm, some oral bacteria adhere to the surface of teeth by recognition of the receptors in the salivary acquired pellicle (SAP), which is a proteins / glycoproteins layer tightly coated on the teeth surface.22-23 The specific adsorption of salivary macromolecules on enamel surfaces is important in the beginning of the SAP formation.24 For example, statherin is one of the SAP proteins which can selectively adsorb onto enamel surface.25 It contains 43 aminoacid with a high degree of structural and charge asymmetry. It has been previously revealed that the density of negative charge, 1-15 sequence, as well as the helical conformation at N-terminal region of statherin are all key factors for its absorption on 4 ACS Paragon Plus Environment

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HAP.26 Further, the first six residues DpSpSEEK is proved to form an α-helix and adsorb on HAP.27 Meanwhile, it was found that DDDEEK, in which phosphoserines are substituted by Asp residues, has the comparable HAP adsorption capacity as that of DpSpSEEK.26 The SAP inspired strategy of constructing specific peptide-decorated chemicals may be general to prepare biomaterials or coatings with strong interface binding force.28-29 In this work, we synthesized a SAP-bioinspired polyphenols, i.e., DDDEEKC modified tannic acid, which was named as SAP-TA (Scheme 1). In this molecular structure, cysteine is designed to link DDDEEK and tannic acid. Then we evaluate its adsorption capability on HAP and explore its remineralization behavior on acidetched human tooth enamel. Moreover, its in vivo remineralization behavior was directly performed on the dental enamel of Spraguee Dawley (SD) rats.

EXPERIMENTAL SECTION Materials Tannic acid (TA), acryloyl chloride, triethylamine, dimethylformamide (DMF), dimethylsulfoxide (DMSO), , dimethylphenylphosphine, fluorochrome rhodamine B (RB), ethyl dimethylaminopropyl carbodiimide (EDC) were purchased from Sigma (HPLC degree). Hydroxyapatite (HA) powder and slice were obtained from National Engineering Research Center for Biomaterials, Sichuan University (medical grade, M30, the diameter is 8 mm and the thickness is 2 mm). Peptide sequence (Fmoc) DDDEEK (Fmoc) C (95% of purity) was purchased from Shanghai Biotech 5 ACS Paragon Plus Environment


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Bioscience & Technology Company. Human tooth samples were prepared according to standard procedures for preparation at the Hospital of Stomatology, and handled with permission of the university. HCl, NH3•H2O were obtained from Tianjin Bodi Chemical Holding Company.

Synthetic route of acrylated TA 1.7 g (1 mM) of TA and 140 µL of triethylamine were dissolved in 30 mL of dimethylformamide (DMF) in three-necked flask. The solution was stirred vigorously and cooled to 0 °C in an ice bath. Then 82 µL of acryloyl chloride was added dropwise to the TA solution (0 °C). The reaction was carried out for 24 h at room temperature. After reaction, the system was dialyzed (MWCO 2000) against deionized water to remove the impurity. Finally, the product was obtained by lyophilization.30

Synthesis of peptide sequences (Fmoc) DDDEEK (Fmoc) C-terminated TA (Fmoc-SAP-TA) 40 mg of (Fmoc) DDDEEK (Fmoc) C and 20 µL of dimethylphenylphosphine were dissolved in 10 mL of deionized water. Then the solution of acrylated TA (3.61 mg/mL) in deionized water was dropwisely introduced to the mixture. The reaction was proceeded for 24 h (room temperature). After reaction, the system was dialyzed (MWCO 2000) against deionized water to remove the impurity, then it was freezedried to obtain the Fmoc-SAP-TA powder. 6 ACS Paragon Plus Environment

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Synthesis of SAP-TA Fmoc-SAP-TA powder was dissolved in mixed solvent (40 mL), which contained 4 mol/L NaOH, C4H8O2, NH3•H2O, and (the volume ratio was 1:9:30). The solution was reacted at 25 oC for 24 h and then the sample was further purified by dialyzing in ultrapure water for three days. Finally, the final sample was obtained by a freeze-dryer. Cell toxicity assay MTT assays were carried out to measure the cytotoxicity of the SAP-TA. MG63 cells were cultured in Dulbecco’s modified eagle medium (DMEM), 10% heatinactivated fetal bovine serum (FBS), 100 units/mL of penicillin and 100 mg/mL of streptomycin at 37 °C, with 5% CO2 and 95% relative humidity. The cells were put in a 96-well microtiter plate with the density of 104 cells/well and incubated in DMEM/well (100 µL) for 24 h. The media was refreshed with new culture media that contains serial dilutions of SAP-TA. The cells were incubated for a predetermined time of 24 h. Then, sterile-filtered MTT stock solution (20 µL, in 5 mg/mL PBS) was added. The unreacted dye was cleaned by 5 h aspiration. The formed formazan crystals were dissolved in 150 µL/well DMSO. The absorbance was measured using a microplate reader (Spectra Plus, Tecan, Zurich, Switzerland) at 570 nm. The cell viability (%) was calculated as 100*([A]test-[A]zero)/([A]control-[A]zero), where[A]control, [A]zero and[A]test is the absorbance values of the wells without SAP-TA (negative



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control), without cells (zero set) and with SAP-TA, respectively. The absorbance was the average data calculated from six wells.

Preparation of human tooth enamel The teeth collected from and approved by West China Hospital of Stomatology were stored at 4 °C in saturated thymol aqueous solution. A diamond-coated band saw was applied to cut root from crowns and to cut sections longitudinally, leading to approximately 5*5 mm2 square plate. Thes samples were ground flat and polished with water-cooled carborundum discs. All the surfaces were protected with acrylic resin except the polished enamel surface. The sample surfaces were partly painted with two layers of acid-resistant nail varnish, leaving only an exposed window of 3*4 mm2. The samples were stored at 4 °C in PBS.

The preparation of etched enamel samples The prepared enamel samples were clean with ultrasonic washer for 30 minutes before etched. Then each of samples was immersed in 37 wt % phosphoric acid for 45 s. PBS was used to wash the etched samples. After washed, those samples were sonicated for 5 minutes and stored in PBS (4 °C).

Adsorbability of SAP-TA on HAP powder


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To explore the adsorbability of SAP-TA, 50 mg HAP powder was added to SAPTA aqueous solutions at different concentration (from 0.25 to 3.75 mg/mL). These solutions were stirred at room temperature for 24 h. After that, they were centrifuged at 10000 r/min for 4 minutes to get supernatant. The supernate was analyzed by UV (MAPADA 1800PC, China) absorbance analysis. The extent of adsorption of SAPTA on HAP was counted by the difference value of SAP-TA. The process is as follows: Firstly, we construct a standard curve for concentration and absorbance of SAT-TA or TA. After adsorbing, we test the absorbance of remained SAP-TA or TA in solution. According to the standard curve, we calculate the concentration of SAPTA or TA in solution. The adsorbing capacity was calculated according to the following formula: The adsorbing capacity= (Cbefore-Cafter)×V×M

(Equation 1)

Cbefore is the initial concentration of dissociative SAP-TA or TA in solution. Cafter is the post-adsorption concentration of dissociative SAP-TA or TA in solution.

The adsorption capability of SAP-TA on HAP slice With the purpose of observing the adsorption capability of SAP-TA on hydroxyapatite (HAP) slice, we utilized fluorochrome rhodamine B (RB) to label SAP-TA. The procedure was report previously.31-3213.7 mg of RB was dissolved in 10 mL of DMSO and then 4.4 mg of EDC and 3.3 mg of SAP-TA was added to the mixture. The reactions were stirring at room temperature for 3 days in the dark conditions, then dialyzed against deionized water for 3 days. After that, the conjugate 9 ACS Paragon Plus Environment


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was lyophilized. And equimolar TA were treated following the same method. Bare HAP slices were soaked in RB-labelled SAP-TA and RB-labelled TA for 24 h and rinsed with DMSO and HCl, separately. The absorbed slices were dried in vacuum oven and observed by confocal laser scanning microscope (CLSM) (ZEISSLSM700, Germany).

The coating of SAP-TA and SAP-TA/Fe (III) on HAP slice 300 µL of 6.25 mg/mL of SAP-TA and 96 µL of 2.782 mg/mL of FeCl3•6H2O were added to the 1.5 mL Eppendorf tube. Then the pH is adjusted by 1 M NaOH to pH=8. Tris buffer was used to prepare the above solutions and added to the mixture to increase the volume to 500 µL. 100 µl of 3.75 mg/mL SAP-TA solution and SAPTA/Fe (III) solution were dropwise added on the surface of HAP slices for four times and dried at 25 oC separately. The morphologies of SAP-TA on HA surface were measured by scanning electron microscope (SEM) (Quanta 250, USA). The EDS spectra was performed using a HITACHI S3400.

The adsorbability of SAP-TA on human enamel samples Apart from the materials, the method was the same to section 2.9. RB-labelled SAP-TA solution was dropwise added to the enamel samples surface and etched enamel surface instead of HAP slices and dried in vacuum (1 day). After that, the samples were washed with DMSO 3 times and dried in vacuum drier for another 1


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day. Then adsorbed enamel samples were dried in vacuum oven and measured by CLSM.

Remineralization in artificial saliva 100 µL of 3.75 mg/mL SAP-TA solution and SAP-TA/Fe (III) solution were dipped on the surface of the etched enamel, respectively. After 30 minutes, these samples are washed by PBS buffer. The samples were incubated in 5 mL of artificial saliva 6 and placed in a 37 °C water bath for one day, one week and two weeks. The treatment was repeated and the artificial saliva was refreshed every day.

Structure and composition of remineralized crystal After remineralization, the new crystal on the enamel were observed and analyzed by SEM (Quanta 250, USA), atomic force microscope (AFM) (BRUKER MultiMode 8) and X-ray diffractometer (XRD) (Ultima IV, Japan). The cross-section was also observed by SEM.

Characterization of mechanical property of new crystal on tooth enamel Knoop microhardness testing machine (Duramin-1/-2; Struers, Copenhafen, Denmark) were applied to measure the knoop microhardness of the tooth enamel surface under a 50 g load for 10 s and each sample was tested 5 times. We took the mean value of 5 measurements as the microhardness of each sample.



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Scratch tests were characterized by a coating adhesion scratch device under the condition of that diamond stylus R was 0.2 mm, scratching speed was 3 mm/min and loading rate was 80 N/min.

Animal experiments The in vivo experiments were permitted by the Animal Research Committee of the University. Male Spraguee Dawley (SD) rats were used to carry out the animal experiment, which were Specific pathogen-free, 21-day-old male rats. They were weaned and weighed at the age of 21 days. On the next 3 days, they were provided distilled and deionized water containing streptomycin (0.05% w/v). Then oral swabs dipped in their mouth were streaked onto Mitis salivarius agar (Difco) and cultured anaerobically at 37 oC to confirm there is no endogenous S. mutans infection. After confirmation, the pups were continuously infected with S. mutans ATCC27157 for 4 days. After that, oral swabs from their mouth were used to confirm the infection, which has a similar method to the above. The rats were randomly and averagely divided into three groups. Their molars were treated with distilled and deionized water (DDW), SAP-TA aqueous solution (3.75 mg/mL) and SAP-TA/Fe (III) (3.75 mg/mL) separately with the aid of cotton swabs for twice a day. After a 3consecutive-week treatment, they were sacrificed by decapitation. Their maxillae and mandibles were dissected and immersed in 4% Paraformaldehyde fixed solution.


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During the infection and treatment period, these rats were weighed weekly and fed a cariogenic diet (Diet 2000, which contains 28% sucrose and 28% glucose) and provided 10 wt % sucrose aqueous solution. In order to evaluate the side effect of SAP-TA (III), SAP-TA (III) treated maxillae and mandibles were sagittally dissected and the lateral half of the maxillae and mandibles were placed in 4% paraformaldehyde solution for 24 h. Subsequently, 5-µm longitudinal sections were cut with a Leica SM2500S microtome. The sections were stained with hematoxylin–eosin and were observed by optical microscope. For the polarized light microscopy (PLM) observation, their maxillae and mandibles were hemisected and embedded in polymethylmethacrylate (PMMA). Then the samples were cut along the mesiodistal sagittal plane using a diamondcoated band saw and the thickness of sections were reduced into approximately 100 µm by hand-grind. After that, slices were observed by polarized light microscopy (Olympus, CX31P-OC-1). The halves of the maxillae were scanned at a 10-µm isotropic voxel resolution by an Inveon micro-CT system (µCT50, Scanco medical AG, Switzerland) to get their 3dimensional images under an operating voltage of 80 kV and 500 µA of current. Six HAP disks were scanned as reference phantoms to get a mineral density (MD) calibration curve. Then the MD of enamel was calculated using the curve-fitting equation.



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RESULTS Characterizations of synthetic materials The whole synthetic process is presented in Figure S1. In the FTIR spectra of acrylated TA (Figure S2), there appears a new peak of 1652.73 cm-1 in contrast of the FTIR spectra of TA, because of the C=C stretch vibration in acrylated TA. When the peptide sequence (Fmoc) DDDEEK (Fmoc) C is grafted to acrylated TA, the characteristic 1401.10 cm-1 peak appeared, which is contributed by C-N stretching vibration of amido link among Fmoc-SAP-TA. The 1H NMR data (Figure S3) also shows the same result: the chemical shifts at about 6-7 ppm demonstrate that acryloyl group is introduced to TA. When the peptide sequence (Fmoc) DDDEEK (Fmoc) C is linked to acrylated TA, the chemical shifts of acryloyl group disappears. The chemical shifts at approximately 1-2 ppm is the methyl protons adjacent to the carbonyl and 6-8 ppm arise is and the protons of secondary amide. Because there are no characteristic peaks appearance after deprotection of FmocSAP-TA, the Matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) is utilized to characterize Fmoc-SAP-TA and SAPTA. The data (Figure S4, a) of Fmoc-SAP-TA shows a peak at m/z of 3076.079 [M + Na+]. The molecular mass of TA would increase by 1350.93 g/mol after the introduction of each (Fmoc) DDDEEK (Fmoc) C, there are one (Fmoc) DDDEEK (Fmoc) C in each TA molecule. After deprotection, the data (Figure S4, b) of SAPTA shows a peak at m/z of 2673.706 [M + R4NX], which indicates that the Fmoc groups have been removed from SAP-TA. 14 ACS Paragon Plus Environment

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The cytotoxicity of SAP-TA and SAP-TA/Fe (III) were evaluated using MG63 cell by MTT assay. The MG63 cell viability remains 80-100% within the SAP-TA and SAP-TA/Fe (III) concentration from 2 to 256 µg/mL, which indicates SAP-TA has a low cytotoxicity (Figure 1). Therefore, SAP-TA is suitable for further biomedical applications.

Figure 1. Cytotoxicity assay of SAP-TA and SAP-TA/Fe (III) using MG63 cells by MTT method (The concentration from left to right is 2, 4, 8, 16, 32, 64, 128 and 256 µg/mL). Date are expressed as mean + SD, n=6.

The adsorption capability of SAP-TA to HAP To verify the adsorbability of SAP-TA to HAP, HAP slices were soaked in the solution of RB-labelled SAP-TA, RB-labelled TA and RB, separately. After dried, the samples were observed by CLSM. Figure S5 (C, E) shows the red fluorescence distribution on both samples’ surface, but there was no fluorescence on RB coated surface (Figure S5A). It demonstrates that SAP-TA and TA can effectively adsorb on HAP slice and RB have no adsorption of HAP slices. The fluorescence of slice soaked in RB-labelled SAP-TA solution is stronger than that in RB-labelled TA solution. The 15 ACS Paragon Plus Environment


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slice soaked in RB-labelled TA could also show a weak fluorescence because RBlabelled TA can be deposited on the surface of HAP slice due to oxidation.15 In order to verify the assumption, the three samples were rinsed with HCl. After that, the fluorescence intensity of the slice soaked in RB-labelled TA become weaker while the intensity of the slice soaked in RB-labelled SAP-TA is still strong (Figure S5, D, F). This phenomenon indicates that SAP-TA can tightly adsorb on the surface of HAP slice and this adsorption is acid-resistant, which is significant for oral clinic applications.33 The ATR-IR data (Figure 2) of SAP-TA treated HAP slice also indicate that SAP-TA adsorbed on the surface of HAP slice. The characteristic peak of SAP-TA (amide vibration: 3514.42 cm-1, amide carbonyl: 1636.9 cm-1) can be obviously observed. The HAP slice treated by SAP-TA is washed sufficiently by deionized water. After washing, the characteristic peak of SAP-TA can still be clearly observed. All above appearance could explain that SAP-TA has a tightly affinity with the surface of HAP slice and cannot be washed off by ultrapure water. To quantify its adsorbability on HAP, SAP-TA and TA aqueous solutions were made up with a series of concentrations and 50 mg HAP powder was added with adequately stirring and then centrifuged. The extent of adsorption of SAP-TA on HAP was measured by the difference value of SAP-TA (or TA) to obtain the adsorption isotherm (Figure S6), which reveals that the saturated amount of SAP-TA on HAP powder (50 mg) is 2.95 mg at the concentration of 3.25mg/mL. After nonlinear data fitting, the isotherm accords with the Freundlich model (R2 > 0.95) that the SAP-TA 16 ACS Paragon Plus Environment

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is monolayer adsorption on HA slices. Therefore, 3.25 mg/mL was regarded as the work concentration for reminerlization investigations. Meanwhile, TA show little adsorption to HAP powder. This result is in correspondence to the result of fluorescence on HAP discs.

Figure 2. ATR-IR spectra of blank HAP slice, SAP-TA coated HAP and the one after being washed with deionized water.

The adsorption capability of SAP-TA to human tooth enamel Since SAP-TA is designed to repair demineralized enamel, we try to illustrate the different adsorption behaviors of SAP-TA on normal and acid-etched enamel samples. Normal enamel sample is the human enamel sample which was polished ground flat using water-cooled carborundum discs and these surfaces were protected with acrylic resin except the polished enamel surface, but without acid-etched. Further experiment is carried out to test the adsorbability of SAP-TA to human tooth enamel samples. RB and RB-labelled SAP-TA were dropwise added to the surface of normal 17 ACS Paragon Plus Environment


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enamel model and etched enamel model, separately, and the samples were rinsed with DMSO. Figure 3 demonstrates the results observed by CLSM. As can be seen, without treated and RB treated group shows no fluorescence dispersion while the RBlabelled SAP-TA group presents an uneven and sporadically fluorescence dispersion on normal enamel sample (Figure 3, A, C, E). After rising by DMSO, without treated and RB treated group still shows no fluorescence dispersion (Figure 3, B, D). The fluorescence dispersion on RB-labelled SAP-TA etched enamel sample is much stronger and well-distributed (Figure 3F), which demonstrated that RB have no specific adsorption to human tooth enamel. The adsorbability of RB-labelled SAP-TA on the control sample are less than that on etched one because the etched enamel has more specific surface area thus it can provide additional binding site for SAP-TA than the control sample.

Figure 3. CLSM images of normal tooth enamel (A), acid-etched tooth enamel (B), RB treated normal tooth enamel (C), RB treated acid-etched tooth enamel (D), RBlabelled SAP-TA coated normal enamel model (E) and RB-labelled SAP-TA coated acid-etched tooth enamel model (F), n=6. 18 ACS Paragon Plus Environment

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The SEM morphology of SAP-TA/Fe (III) and SAP-TA In order to observe the morphology of SAP-TA on HAP slices, the SAP-TA and SAP-TA/Fe (III) treated HAP slices were analyzed by SEM. The surface of bare HAP is porous and rough (Figure 4, a). While after SAP-TA treatment, the porous structure disappear and the surface become smoother (Figure 4, b), which illustrates that SAPTA can specifically adsorb on the surface of HAP slice. Compared with SAP-TA treating HAP slice, the surface of HAP slice treated by SAP-TA/Fe (III) appears some aggregates (Figure 4, c). As analyzed by EDS, the ingredients of the coating contain C, N, O, Fe, thus we think they are the aggregates of SAP-TA/Fe (III) (Figure S8). This phenomenon is because that ferric ion could coordinate with SAP-TA to form a compact coating by interact with TA.19 Remineralization of enamel in vitro The acid-etched tooth enamel samples treated with DDDEEKC (A group), TA (B group), without treatment (C group), SAP-TA (D group) and SAP-TA/Fe (III) (E group) were incubated in artificial saliva for predetermined time intervals of one day, one week and two weeks. After being incubated, the morphologies of these samples were observed by SEM. As shown in Figure S7 (c and d), the surface of tooth enamel sample become asperous and the enamel prism structure is exposed to air after acidetching as compared with the original enamel (Figure S7, a and b). As shown in Figure 5, all the samples have formed new mineral layer on their surfaces after being soaked in artificial saliva, but their morphologies are different for different treatments 19 ACS Paragon Plus Environment


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and incubating time. After be incubating in artificial saliva for 1 day, DDDEEKC group (Figure 5, A1), SAP-TA group (Figure 5, D1) and SAP-TA/Fe (III) complex group (Figure 5, E1) have more newly generated mineral than the control group (Figure 5, B1, C1) and their enamel prism structure is sheltered compared with the TA and without treated group. The regenerated HA also shows different morphology: as for the TA group and the group without treatment, the crystals distributed randomly on the surface of initial crystal. However, the crystals of SAP-TA and SAP-TA/Fe (III) treated sample appear ordered nanorod structure. While the morphology of TA coated HAP slices are quite same to the without treatment group. It indicated that TA has no specific adsorption to enamel surface. The reminelization capacity is SAP-TA (III)>SAP-TA>DDDEEKC>TA. Among them, the SAP-TA/Fe (III) treated samples’ crystals aggregate much denser than that of SAP-TA group. But there still are some spaces between crystals on the surface of these samples.

Figure 4. SEM images of the bare HAP slice surface (A), SAP-TA treated (B) and SAP-TA/Fe (III) (C) treated HAP slice surface, n=6.


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Figure 5. SEM images of the surface of acid-etched tooth enamel treated with DDDEEK (A group), TA (B group), without treatment (C group), SAP-TA (D group) and SAP-TA/Fe (III) (E group), after being incubated in artificial saliva for one day (A1, B1, C1, D1, E1), one week (A2, B2, C2, D2, E2) and two weeks (A3, B3, C3, D3, E3). The inserts are enlarged details. n=6, pH=6.8. As the incubating time goes on, there were increased regenerated crystals on the surface of all samples. At the end of 2 weeks, the surface of untreated sample was covered by irregular regenerated crystals. However, the surfaces of SAP-TA and SAP-TA/Fe (III) complex samples become smooth, appearring more ordered nanorod structure than the TA group and the group without treatment. Moreover, the nanorod crystals distribute densely on the SAP-TA/Fe (III) complex surface compared with SAP-TA only, which demonstrates that SAP-TA/Fe (III) coating has a better regulating growth ability of new HAP crystal on the acid-etched surface. Moreover, SEM photos of the cross-section of acid-etched enamel without treatment (A group) and SAP-TA (B group) treated and SAP-TA (Ⅲ) treated sample (C group) also show different morphology (Figure 6). As for B, C group, the new regrown crystals on the etched enamel are uniform and oriented bundles of nanorod 21 ACS Paragon Plus Environment


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HAP and the thickness of layer is more than 6 and 10 µm. However, the layer of A group (2 µm) is thinner than of B group. This should be attributed to the in situ biomineralization effect of SAP-TA.

Figure 6. SEM images of the cross-sections of acid-etched enamel without treatment (A), SAP-TA treated sample (B) and SAP-TA (Ⅲ) treated sample (C) for two weeks, n=6, pH=6.8.

Figure 7. AFM data of the intact enamel (a), the acid-etched enamel without treatment (control) (b), SAP-TA treated (c) or SAP-TA/Fe (III) treated sample (d) 22 ACS Paragon Plus Environment

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treatment after being incubated in artificial saliva for 2 weeks. Ra shows the mean roughness, n=6. AFM is also applied to estimate the average mean roughness (Ra) of the samples (Figure 7). The Ra was calculated from 10*10 µm squares area on the surface of all samples. The polished intact enamel has a Ra of 58 ± 4 nm. After the acid-etched enamel (without coating treatment) was incubated in artificial saliva, the Ra was 421 ± 23 nm. The roughness has a significant increase because the acid destroy the orderly structure of enamel. But the Ra of SAP-TA and SAP-TA/Fe (III) treated acid-enamel surface were 222 ± 22 nm and 166 ± 12 nm, respectively. When the acid-etched enamel sample was soaked in artificial saliva, the surface of etched enamel became rough and porous due to the random deposition of mineral layer. However, after the treatment of SAP-TA and SAP/Fe (III), the new regenerated HAP crystals regularly distributed on the etched enamel surface. Hence, Ra of between SAP-TA and SAPTA/Fe (III) treated model is lower than that of non-treatment sample. Moreover, SAPTA/Fe (III) treated samples shows a smoother surface than SAP-TA only. This is due to the dense stack of regrown crystals on etched enamel by regulating of SAP-TA/Fe (III). In order to analyse the phase and orientation of the newly formed HA crystal, XRD was applied to measure the samples (soaked for two weeks). In Figure 8, the diffraction peaks (002) at 2θ=25.8 (According to Braggs law, d=0.345nm), (211) at 2θ=31.8 (According to Braggs law, d=0.281nm) and (300) at 2θ= 32.8 (According to Braggs law, d=0.273nm) are characteristic peaks of HAP, which are appeared in all samples. It shows that the SAP-TA could control the growth of the regenerated 23 ACS Paragon Plus Environment


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mineral to form HAP. To investigate the orientation degree of renewed crystals, the ratio of diffraction intensity of axis (002) to direction (211) is used.8, 10 The ratios are 1.21, 2.03, 1.46, 1.17 and 1.12 for the intact enamel, acid-etched one, TA treated sample, without treated sample, SAP-TA treated and SAP-TA/Fe (III) treated samples, respectively, which shows that with the help of SAP-TA, the acid-etched enamel could keep the orientation in the biomineralization process.

Figure 8. XRD patterns of intact tooth enamel, acid-etched tooth enamel, etched enamel treated with SAP-TA or with SAP-TA/Fe (III), or without treatment (control) after being incubated in simulated saliva for two weeks, n=6.

The sample mechanical properties were studied by Knoop microhardness method and the scratch measurement. The microhardness value of original and acid-etched tooth enamel are about 529 and 248, respectively (Figure 9). After being incubated in artificial saliva, the control group shows a little surface microhardness recoveries (%SMHR), while the SAP-TA/Fe (III) and SAP-TA group exhibit an effective


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recovery. Especially, SAP-TA/Fe (III) group has a significant %SMHR values of 80% after 2 weeks’ incubation in artificial saliva. This data illustrates that the dense mineral layer of SAP-TA/Fe (III) treated model is positive correlation to the microhardness improvement compared with the SAP-TA group. In the scratch test (Figure 10), the adhesion force of regenerated HAP crystal on the original enamel surface is following this order: SAP-TA/Fe (III) treated group (64.85 N) ≈ SAP-TA treated group (62.95 N) >> control group (0.1 N), which means that SAP-TA could improve the binding force of newly generated HAP layer on original enamel surface.

Figure 9. Surface microhardness (SMH) of original enamel, demineralized enamel, SAP-TA treated sample, and SAP-TA/Fe (III) treated sample after being incubated in simulated saliva for one / two weeks, comparing with the microhardness data of the original enamel and demineralized enamel, n=6.



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Figure 10. Acoustic emission signal in the scratch test. The acid-etched enamel (a), SAP-TA treated (b) and SAP-TA/Fe (III) treated sample (c) were tested after being soaked in simulated saliva for 2 weeks. n=6. Remineralization of enamel in vivo During the experiment period, the body weight data was similar among all three rat groups (Figure 11) while no rats died or other oral mucosal disease was observed, which indicated that both SAP-TA and SAP-TA/Fe (III) did not cause obviously toxicity and had no side effect (Figure S9).

Figure 11. Weight changes of Sprague-Dawley rats during the infection and 26 ACS Paragon Plus Environment

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subsequent treatments with DDW, SAP-TA or SAP-TA/Fe (III). All data are mean ± SD for 6 animals in each treatment.

The subsurface remineralization appeared as bright layer in PLM images.34 As shown in Figure 12, there were few bright layers on the surface of molars in DDW treated group (Figure 12a) while significant bright layers could be found in SAP-TA and SAP-TA/Fe (III) treated groups, which suggested that SAP-TA and SAP-TA/Fe (III) had a good effect on the remineralization process.

Figure 12. Typical polarized light micrographs of molars from SD rats infected with Streptococcus mutans and then treated with (a) distilled and deionized water (DDW), (b) SAP-TA or (c) SAP-TA/Fe (III). White arrow indicates the typical impaired enamel while red arrows indicate the remineralization layer on the initial lesions.

Micro-CT was a useful tool for qualitative and quantitative evaluation of dental hard tissue. It could be used to calculate the Mineral density (MD) of enamel. The groups treated with SAP-TA or SAP-TA/Fe (III) had a higher MD than that of treated by DDW (1000 mg/mL) (Figure 13), which suggested that SAP-TA and SAP-TA/Fe (III) provide an enhanced remineralization effect.35 Moreover, the group treated SAPTA/Fe (III) had the highest MD value about 1150 mg/mL (Figure 13), indicating the 27 ACS Paragon Plus Environment


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addition of ferric ion resulted in an enhanced remineralization effect.

Figure 13. Mineral density of the first molar enamels treated with DDW, SAP-TA aqueous solution (3.75 mg/mL) and SAP-TA/Fe (III) (3.75 mg/mL) separately. Date are expressed as mean + SD, n=6.

DISCUSSION By quantifying the adsorption capability of SAP-TA to HAP (Figure S6), the amount of SAP-TA adsorbed on HAP linearly increase with the increase of concentration until 3.25 mg/mL and the saturated amount of SAP-TA on 50 mg of HAP is 2.95 mg while the concentration of SAP-TA is 3.75 mg/mL. This adsorption behavior should be put down to the specific adsorption between DDDEEKC and HAP.26 On the basis of this result, we choose the concentration of 3.75 mg/mL of SAP-TA to proceed the further experiment and it is confirmed to be appropriate for the remineralization in the next step. In vitro remineralization, the TA and without treated group shows few degrees of restorative effect and the regenerated crystal are randomly deposited on the acid-


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etched enamel surface with irregular shape. This mineralization (no mediator) should arise from the electrostatic interaction between the free ions in artificial saliva (free calcium and phosphate ions) and the charged domains on the enamel.36 On the other hand, the SAP-TA and SAP-TA/Fe (III) treated samples present obvious restoration and there are uniform nanorod crystals on the acid-etched enamel surface. The formation of nanorod HAP crystal is caused by the regulation of TA as its pyrogallol groups could grasp the Ca2+ in the simulated saliva.20 SAP-TA and SAP-TA/Fe (III) treated samples induced a large mineral density of nanorod HAP minerals on the sample surface. This is because that SAP-TA molecule could adsorb on the acidetched enamel surface with the help of peptide sequence DDDEEKC and then the pyrogallol groups of SAP-TA can coordinate with Fe (III) to generate a stable crosslinked coating in the mixture solution of SAP-TA/Fe (III).19-20,

37-38

The interface

property of SAP-TA and SAP-TA/Fe (III) on HAP slice also supports this conclusion that they can form dense film on HAP slice. For the clinic application, the acidic oral environment has very little influence on the coordination of SAP-TA with Fe (III). The coordination of SAP-TA with Fe (III) mainly depends on the hydroxyl groups in the TA moiety. At low pH, most of the hydroxyl groups were protonated, resulting in rapid destabilization of cross-links. With the increase of pH value, the hydroxyl groups were deprotonated and coordinated with Fe (III). Generally, TA and Fe (III) formed mono-complex at pH < 2, tris-complex at pH > 7 and bis-complex when the pH was in the range of 3 to 7.19 In our experiment, the solution of SAP-TA/Fe (III) was prepared in alkaline environment (pH=8) and artificial saliva was neutral 29 ACS Paragon Plus Environment


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(pH=7.02). SAP-TA easily coordinated with Fe (III) under these conditions. Although the pH of normal oral environment was about 6.6, SAP-TA still could coordinate with Fe (III) to form coatings and enhance the remineralization effect of this system. Further mechanical property experiments indicate that SAP-TA and SAP-TA/Fe (III) treated groups have superior microhardness and stronger binding force at the interface of remineralization as compared with control group. That is due to the tight interaction between peptide sequence DDDEEKC and the surface of human tooth enamel.26 With the specific adsorption on HAP and coordination between SAP-TA and Fe (III), SAP-TA/Fe (III) could tightly and densely adsorb on HAP. Meanwhile, TA moiety of SAP-TA can be the act as the template of biomineralization. So it can show a good biomineralization behavior in the artificial saliva in the whole 2 weeks period. In the in vivo remineralization process, the rats are treated with different solution (DDW, SAP-TA, and SAP-TA (Ⅲ)).The weight increase of rats are quite same, it demonstrating that SAP-TA and SAP-TA/Fe (Ⅲ) have no side effect (Figure S9) and is more safety than fluoride treatment.39 Compared with DDW treated rats molar, the surface of SAP-TA and SAP-TA (Ⅲ) treated molar have an intensified polarized light signal. It demonstrated that SAP-TA and SAP-TA (Ⅲ) can induce the formation of hydroxyapatite crystal. While, there are seldom polarized light signal on DDW treated rats molar since DDW cannot induce new mineralized layer.40 We hypothesized that mineral density is related to the mineralization degree. The DDW treated rats molar have a low mineral density because of its low mineralization degree ability.41 With the 30 ACS Paragon Plus Environment

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mineralization degree increase, the mineral density shows an increasing trend. According to the in vitro experiment, SAP-TA ( Ⅲ ) have a better mineralization degree than that of SAP-TA. Hence, SAP-TA treated rats molar have a higher mineral density as shown in Figure 13.

CONCLUSION In this work, for the purpose of specific adsorption and remineralization of tooth enamel in situ and in vivo, SAP-TA with low cytotoxicity is successfully synthesized. Several experiments were carried out to investigate the selective absorption on HAP and the results shows that SAP-TA could have a tightly affinity with human enamel (2.95 mg / 50 mg HAP) and can resist washing by HCl and deionized water. After incubated in artificial salivary, SAP-TA and SAP-TA/Fe (III) could induce the remineralization of HAP in situ. The regenerated crystals are uniform nanorod-like HAP, with a surface microhardness recovery of larger than 80% and a strong adhesion force of 64.85 N. Moreover, animal experiment also shows that SAP-TA and SAPTA/Fe (III) could effectively induce the remineralization of HAP in oral cavity of SD rats. Hence, the DDDEEKC peptide-decorated natural polyphenol can be used as a promising biomaterial for enamel remineralization.

ASSOCIATED CONTENT Supporting Information 31 ACS Paragon Plus Environment


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The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXXXXXXX FTIR; 1H NMR; Time of Flight Mass Spectrometry of chemicals; CLSM images; Adsorption isotherm; SEM images; EDS data; Histological images.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (JS. Li) Author Contributions # These authors contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGE The authors thank the finanical supports from National Natural Science Foundation of China (51573110) and National Key Research and Development Plan of China (2016YFC1100404).


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Bioinspired peptide-decorated tannic acid for in situ remineralization of tooth enamel: in vitro and in vivo evaluation Xiao Yang, Bo Yang, Libang He, Ruiqi Li, Yixue Liao, Shuhui Zhang, Yinxin Yang, Xinyuan Xu, Dongyue Zhang, Hong Tan, Jiyao Li and Jianshu Li


Bioinspired Peptide-Decorated Tannic Acid for in Situ

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