Raman spectroscopy-multivariate analysis related to morphological surface features on nanomaterials applied for dentin coverage

Journal Pre-proof Raman spectroscopy-multivariate analysis related to morphological surface features on nanomaterials applied for dentin coverage

Luís Eduardo Silva Soares, Sídnei Nahórny, Vivian de Faria Braga, Fernanda Roberta Marciano, Tanmoy T. Bhattacharjee, Anderson Oliveira Lobo PII:

S1386-1425(19)31208-9

DOI:

https://doi.org/10.1016/j.saa.2019.117818

Reference:

SAA 117818

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date:

31 May 2019

Revised date:

28 October 2019

Accepted date:

18 November 2019

Please cite this article as: L.E.S. Soares, S. Nahórny, V. de Faria Braga, et al., Raman spectroscopy-multivariate analysis related to morphological surface features on nanomaterials applied for dentin coverage, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2019), https://doi.org/10.1016/j.saa.2019.117818

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© 2019 Published by Elsevier.

Journal Pre-proof Raman spectroscopy-multivariate analysis related to morphological surface features on nanomaterials applied for dentin coverage Luís Eduardo Silva Soares1*, Sídnei Nahórny1, Vivian de Faria Braga1, Fernanda Roberta Marciano2, Tanmoy T. Bhattacharjee3, Anderson Oliveira Lobo4 1

Laboratory of Dentistry and Applied Materials (LDAM), Research and Development Institute (IP&D),

Universidade do Vale do Paraíba, São José dos Campos, São Paulo, Brazil. Deparment of Physics, UFPI - Federal University of Piauí, 64049-550 Teresina – PI, Brazil.

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Sir John Walsh Research Center, 310 Great King Street, Dunedin 9016, New Zealand.

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LIMAV - Interdisciplinary Laboratory for Advanced Materials, Department of Materials Engineering, UFPI

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- Federal University of Piauí, 64049-550 Teresina – PI, Brazil.

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*Corresponding author

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Prof. Dr. Luís Eduardo Silva Soares. Universidade do Vale do Paraíba, UNIVAP, Instituto de Pesquisa e Desenvolvimento, IP&D, Laboratório de Odontologia e Materiais Aplicados, LOMA. Av. Shishima Hifumi,

Funding information

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39471165; E-mail: [email protected]

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2911, Urbanova, CEP 12244-000, São Jose dos Campos, SP, Brazil. Phone: +55 12 39471165; Fax: +55 12

The authors received financial support from Fundação de Amparo à Pesquisa do Estado de São

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Paulo, FAPESP (Grant numbers 01/14384-8, 2011/17877-7, 2011/20345-7, 2013/11192-8, 2014/02163-7, 2014/21587-2, 2015/09697-0 and 2016/00575-1) and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (Grant numbers 202439/2012-7 and 474090/2013-2).

Declarations of interest: none

Compliance with ethical standards The approval of the ethic committee of the Universidade do Vale do Paraíba, Univap, Brazil for the study was obtained (CEUA protocol n° 04/2012).

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ABSTRACT

Raman spectroscopy and scanning electron microscopy (SEM) were used to investigate the effect of coating materials and acidulated phosphate fluoride gel (APF) treatment on dentin before and after erosion-abrasion cycles. A multi-walled carbon nanotube/graphene oxide hybrid carbon-based material

(MWCNTO-GO),

nanohydroxyapatite

(nHAp),

or

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combined

composite

(nHAp/MWCNTO-GO) were used as a coating. Seventy root dentin fragments obtained from 40 bovine teeth were prepared and divided into groups (n = 10): negative control, artificial saliva - C,

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positive control – APF; nHAp; MWCNTO-GO; APF_nHAp; APF_MWCNTO-GO and

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APF_nHAp/MWCNTO-GO. All samples were subjected to cycles of demineralization (orange juice, pH ~3.7, room temperature, 1 min) followed by remineralization (saliva, 37ºC, 1h). The

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remineralization procedures were followed by tooth brushing (150 strokes). The above cycle was repeated 3/day for 5 days. The previous APF treatment of dentin allowed a better affinity of nHAp

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and MWCNTO-GO with the inorganic and organic portion of dentin, respectively. This interaction

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indicates the formation of a protective layer for the dentin surface and for the collagen giving possible protection against erosion. SEM micrographs illustrated the formation of a protective layer after application of the biomaterials and that it was partially or totally removed after the erosion and

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abrasion. Raman spectroscopy combined with multivariate analysis could distinguish samples with respect to treatment efficacy. The APF_nHAP/MWCNT-GO composite has shown to be a promising

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material since it has binding characteristics both to the inorganic and organic portion of the dentin and reduced solubility. Mineral-to-matrix ratio (MMR) parameter analysis confirmed the binding capability of MWCNTO-GO-based materials to dentin.

Keywords: Graphene oxide; Carbon nanotubes oxide; Nanohydroxyapatite; Dentin erosion; Raman spectroscopy; scanning electron microscopy.

Highlights

    

Dentin was modified by carbon nanotubes and nanohydroxyapatite composites Raman spectroscopy and multivariate analysis allows a prompt non-contact diagnosis. SEM revealed the interaction between biomaterials and dentin. Fluoride allowed a better affinity of composites with dentin. Biomaterials formed a layer which was partially or totally removed after erosion. 2

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1. Introduction

Dental erosion is the loss of tooth minerals as a consequence of chemical dissolution by acids, in the absence of microorganisms [1, 2]. There are intrinsic (acids resulting from eating disorders or gastric reflux) or extrinsic sources of acids (acidic drinks and food). In the last decades, the total amount and frequency of consumption of acid-containing products have increased because of changes in lifestyles [2-4]. The erosion lesion develops by a chemical process resulting in the dissolution of the inorganic phase of the teeth, thus changing mechanical properties of the tooth

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substrates as hardness. The modifications in the chemical and mechanical properties of the teeth are

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potentialized by the action of successive abrasive challenges through brushing [5, 6]. Erosive demineralization of dentin also occurs beneath the tissue surface, but in contrast to

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enamel erosion, demineralization leaves intact a surface layer of insoluble collagenous matrix. As erosion proceeds, the thickness of the demineralized layer, and thus the diffusion distance between

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the surface and the demineralization front, increases so the net rate of mineral loss decreases with time [7-9].

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The development of methods to avoid the erosion or treat the lesions is necessary due to the increase in the incidence of this lesion. The exposure of the tooth to fluoride or other

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protective/remineralizing agents is one of the options to perform the prevention of the erosive process and, consequently, arrest the erosive lesions. Considering the aspects of dental erosion lesion

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and treatment options, the efficacy of amine fluoride (AmF) [10], sodium fluoride (NaF) [1, 11-13] and acidulated phosphate fluoride (APF) [14, 15] against tooth erosion has been studied extensively as they are the most popular and used fluorides for hard tissue prevention. An important factor to note is that previous studies demonstrated a limited anti-erosive effect produced by fluoride [16, 17], probably due to the high dissolution of calcium fluoride remained on tooth surface subjected to erosive episodes [1, 11]. Conventional hygiene measures like fluoridated toothpaste resulted in a limited efficacy in the prevention of erosive tooth wear compared to the application of high-concentrated fluoride products, and this was more evident in situations at high-frequency application [18]. Due to these difficulties, research with new materials is done in the search for more effective alternatives. Hydroxyapatite (HA) based materials, may be able to provide calcium and phosphate ions with a similar composition to the main constituent of the inorganic phase of the tooth, for reducing tooth demineralization and/or improving tooth remineralization. The use of material with

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Journal Pre-proof nanoparticles HA (nHAp) is another option of surface treatment since it may penetrate tooth porosities resulting in a protective layer on the tooth surface [19-22]. Recently, the carbon nanotubes (CNTs) has been used in association with the hydroxyapatite (HAp) to increase the mechanical properties of this brittle material, because it has adequate mechanical properties, thus giving strength to the composite [23]. However, there are few reports related to the in-situ grown carbon nanotubes reinforced HAp (CNTs-HAp) coating [24]. Our group synthesized for the first time multi-walled carbon nanotube/graphene oxide hybrid carbon-base material (MWCNTO-GO, also called as Graphene Nanoribbons) [25]. Recently, our group evaluated the in vitro cytotoxicity and in vivo biocompatibility and potential to induce the

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osteogenesis process of MWCNTO-GO in bone defect in rats [26-28]. MWCNTO-GO composites

specific gene [27, 29] and induced bone formation [28].

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were non-toxic neither in vitro nor in vivo, besides induced the osteogenesis process upregulating

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Nahórny et al. [30] reported the effectiveness of nHAp/MWCNTO-GO in presence or absence of fluoride, as a protective coating for dentin erosion. The nHAp/MWCNTO-GO composite

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was stable in an erosive condition. Our previous study reported that the association of this composite with a previous fluoride application resulted in a thin and acid-resistant film deposition in dentin

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surface. The next step will be evaluate the stability of the nHAp/MWCNT-GO composite and do adherence of the formed layer after erosion-abrasion episodes.

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Thus, this in vitro study aimed to verify the effect of different coatings materials on dentin composition and morphology after erosion-abrasion on previously demineralized dentin. This

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condition of demineralized dentin was created to simulate clinical exposure of dentin tubules in patients. Furthermore, the study also aimed at applying Multivariate analysis of Raman spectra as a rapid content prediction modeling for dentin erosion. The hypothesis tested was that different coating materials, affect the molecular and surface features of root dentin and that Multivariate analysis could discriminate coatings.

2. Materials and Methods

2.1. Sample preparation and Experimental design Ethical approval for this study was granted by the local Ethics Committee (CEUA protocol n° 04/2012, Ethics Committee of the Universidade do Vale do Paraíba, Univap, Brazil). Sample preparation followed the protocol of our group previously tested [14, 30-36]. Briefly, forty nondamaged permanent bovine incisors were selected, and then, they were cleaned and stored in a 0.1% 4

Journal Pre-proof aqueous thymol solution [37]. The samples of root dentin were obtained after separation of the crown from the root through the cement-enamel junction (labio-lingually and perpendicularly to the long axis). A low-speed water-cooled diamond (Isomet1000 – Buehler, Lake Bluff, IL, USA) at a speed of ∼ 400 rpm with a mass of ∼ 100 g was used to separate roots from the crowns. The obtained segment of root dentin was then sectioned into two parts obtaining thus two slabs per root (8 mm long, 8 mm wide, and 6 mm thick). Then, all the 80 slabs were ground and polished to produce parallel surfaces (buccal and lingual sites) with wet 600 and 1200-grit silicon carbide paper (Arotec, Cotia, SP, Brazil) at 150 rpm. After polishing, the specimens were cleaned in an ultrasonic device

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with de-ionized water for 10 min. (Maxiclean 1450, Merse, Campinas, SP, Brazil) [37].

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2.2. Baseline inorganic composition analysis and sample selection

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The dentin slabs had their inorganic composition tested by micro energy-dispersive X-ray fluorescence spectrometry (µ-EDXRF, model µ-EDX 1300, Shimadzu, Kyoto, Japan), in order to

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standardize the samples selected for the study (Table 1), as previously reported [38]. Surface area mappings (n = 80) evaluated the Ca and P weight percentages (wt %) of dentin samples. The maps

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covered a selected central area of 40 × 30 points (steps of 20 μm, 5 s per point, 15 kV). In order to select the 70 samples with more homogeneous inorganic content, 10 samples that had values below

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or above the average of the Ca wt% (27.8 ± 0.6 %) were excluded. Statistical analysis of the results was performed by the Tukey-Kramer multiple comparison test using GraphPad Prism (GraphPad

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Software, San Diego CA, USA). After that, root dentin specimens were randomly allocated in seven groups (n=70) of 10 specimens each (Table 1).

2.3. Coating of root dentin using different nanomaterials The dentin blocks were pre-treated by an etching with 37% phosphoric acid (CONDAC 37, FGM, Joinville, SC, Brazil) for 1 min and cleaned with deionized water (10 times) according to a previous tested protocol [30]. This severe etching procedure prepared the dentin to the treatment with MWCNTOs because the etching exposed the superficial collagen fibers of dentin and allowed the interaction of the biomaterial with the exposed collagen fibers, as previously reported [24, 30]. Then, the samples were sonicated in deionized water for 10 min. The groups with designation APF (Table 1, APF, APF_nHap, APF_MWCNTO-GO, and APF_ nHAp/MWCNTO-GO) received one previous application of 1.23% of Acidulated Phosphate Fluoride gel (APF) for 1 min. (DFL, DFL Indústria e Comércio S.A., Rio de Janeiro, RJ, Brazil). Meanwhile, the other groups remained untreated. 5

Journal Pre-proof After this, the dentin slabs were dipped in a colloidal solution of 7.2 µg L-1 of biomaterials (nHAp, MWCNTO-GO, and nHAp/MWCNTO-GO composite) in distilled water. The solution was ultra-sonicated (35 W) for 5 min before dentin dipped. Next, each slab was individually stored in plastic vials (2 ml volume) and treated with 2 ml of dispersed solution (according to group division). After 1 h of mixing at 75 rpm and 36.6 °C using a shaker (CT-713RT, Cientec, Piracicaba, SP, Brazil), the tooth slabs were removed. The solution was renewed and the mixing time repeated [30]. To remove the unabsorbed MWCNTO-GO coating onto the dentin surface, the samples were washed 3 times with deionized water in the sequence [30].

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2.4. De-remineralization and cycles

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Samples were subjected to three daily consecutively de- and remineralization (DE-RE)

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cycles. The cycles were repeated for five days, with samples remained stored in artificial saliva at night [39].

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Demineralization was performed by immersing each sample in 0.3 mL of orange juice (Minute Maid Mais Laranja Caseira, 67% natural juice, pH ~3.67) and statically incubating it for 1

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min. Next, the samples were washed with deionized water for 10 s and immersed in 0.3 mL of artificial saliva (1.5 mmol/l Ca(NO3)2 H2O; 0.9 mmol/l Na2HPO4 2H2O; 150 mmol/l KCl; 0.1 mol/l

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H2NC(CH2OH)3 (TRIS); 0.05 mg ml−1 NaF, pH 7.0) for 60 min, 37ºC. Between each daily cycle,

2.5. Abrasion

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the abrasion was performed [39].

For a simulated abrasion, specimens were mounted individually in an automatic brushing machine (MSEt, 1500 W, Marcelo Nucci ME, Sao Carlos, SP, Brazil) under a 200-g load at 4.5 movements per second and 37 ± 0.5ºC and subjected to 150 cycles of the abrasive challenge. During the toothbrushing, the samples were bathed by a 1:3 w/w slurry of commercially available fluoride toothpaste and water (Oral-B 1-2-3, 1450 ppm F as NaF; Procter & Gamble do Brasil S/A, Queimados, RJ, Brazil). In each cycle, the samples were brushed using 2 ml of the slurry. Freshly mixed toothpaste slurries were prepared and applied in each cycle. The cycling was repeated three times per day for five days at room temperature. Samples were individually stored overnight in artificial saliva at 37ºC [39].

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2.6. Raman spectroscopy data acquisition Raman Spectroscopy analyzed the dentin slabs after application of materials (1st) and after erosion-abrasion (2nd) (Table 1). Raman spectra of samples were obtained by a 3510 Skin Composition Analyzer (River Diagnostics, Rotterdam, The Netherlands) confocal spectrometer with an inverted microscope using the near infrared laser (785 nm) [40]. The laser power at the sample was 20 mW. Each spectrum was obtained with 3 integrations of 10 sec. each (30 sec. per spectra). Three spectra/sample were collected in the fingerprint region (800–1800 cm−1). The area of the laser

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spot on the samples was 1μm diameter.

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2.7. Data Analysis

The processing of the Raman spectra consisted of first averaging the 3 points per sample

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region, obtaining a mean spectrum for each sample. The normalization adjustment was based on the CH2 wag Raman band at 1450 cm-1, as minimal changes in this band was observed in the data and

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according to previous studies [30, 41-43]. This band was also chosen for it low sensitivity to

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molecular orientation compared to amide I [44]. Then the fluorescence subtraction and the baseline corrections were performed. These processes were carried out in the OPUS software (Bruker Optik

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GmbH, Ettlingen, Germany). The areas of the Raman peaks were calculated using a Gaussian fit for the peaks in the Microcal Origin 5.0 software (Microcal Software, Inc., Northampton, MA, USA). The changes in the organic and inorganic components of dentin were evaluated by comparing the

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integrated areas of Raman peaks at 960, 1071 and 1662 cm-1 with respect to the peak at 1452 cm-1 between the treatment groups. The apparent gradient in mineral content was assessed as the relationship between the ratio of peak areas at 1070 cm-1 (carbonate) to 960 cm-1 (phosphate) [45]. The parameter mineral-to-matrix ratio (MMR) was calculated as the ratio of mineral specific Raman peak area (phosphate and carbonate peaks) to the area of amide I peak at 1662 cm-1 [42].

2.8 Statistical data analysis The integrated area of the phosphate, carbonate and collagen peaks obtained by Raman spectroscopy measurements were statistically analyzed using GraphPad Prism (version 5.01 for Windows, GraphPad Software, San Diego, CA). A one-way ANOVA with Dunnett's posttest was performed to compare the control (negative and positive) and other treatments. To evaluate significant statistical differences among the biomaterial groups (not including the negative and

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Journal Pre-proof positive controls), Tukey's multiple comparisons test was used. The data were statistically analyzed in two moments: after application of materials’ and after abrasion-erosion cycles.

2.9. Scanning electron microscopy (SEM) analyses After application of materials’ (n = 7) and after erosion (n = 7), the dentin samples were investigated by SEM (Table 1). The samples (n = 14) were dehydrated using a graded series of ethanol (50%, 70%, 90%, and 100%) for 10 min at each step. After that, the samples received a thin layer of gold (10 nm) and were examined using a scanning electron microscope (SEM, EVO-MA10,

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Carl ZeissVR STM, Oberkochen, BW, Germany) with an acceleration voltage of 20 kV [30].

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3. Results and discussion

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3.1. Raman spectroscopy

Raman spectra for negative control (saliva), positive control (APF) and treatments

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(biomaterials) before and after erosion-abrasion and remineralization cycles are presented in Figure 1 and 2 (A-C). Vibrations for both inorganic (phosphate and carbonate) and organic (collagen)

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components can be observed. These match the spectra of teeth reported earlier [30, 44]. The band centered at 960 cm-1 is attributed to the inorganic component P-O symmetric stretch (Figs. 1A, 2A).

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The band at 1070 cm-1 is associated with B-type carbonate (v1 CO32-) vibration modes (Figs. 1B, 2B). The band at 1666 cm-1 is related to collagen (Figs. 1C, 2C).

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After the application of materials’ (1st Raman spectroscopy analysis), in the case of the MWCNT-GO and APF_MWCNT-GO groups, there was great difficulty in obtaining the Raman spectra due to the very gray coloration of the sample surface. The spectra acquisition was normal in all groups, except for the MWCNTO-GO and APF_MWCNTO-GO groups, after the application of materials. In this specific case, the very greyish color of the sample surface affected the measurements, saturating the spectrum, especially in samples of MWCNTO-GO group were this material adhere to the surface. In the images obtained on the dentin surface of the MWCNT-GO group (Fig. 3A), a surface covered by lines of concentration of nanotubes and less gray areas with lower concentration was observed. In the spectrum, it is possible to verify that the phosphate peak is not present due to the prevalence of the MWCNT-GO peaks (MWCNT-GO D bands: at 1333 cm-1, black arrow and 1586 cm-1, blue arrow) (Fig. 3B) [46]. Thus, the material color’s influenced in the fluorescence background where the presence of gray material in the dentin surface became very difficult to collect the Raman spectrum with a poor signal to noise ratio.

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Journal Pre-proof In the dentin images of the APF_MWCNT-GO group, the deposition of carbon nanotubes on the surface is not very evident (Fig. 3C). However, unlike the MWCNT-GO group, the presence of fluoride interferes with nanotubes adhesion, and the phosphate peak is more evident (Fig. 3D, red arrow) but is still difficult to calculate area peak. The MWCNT-GO D bands [46] are also present in the spectra of this group (Fig. 3D, black and blue arrows). Peaks related to carbonate and collagen were not visualized in both groups (Figs. 3B and D), thus making it impossible to calculate the peak area. MWCNTO-GO pure material was used as a comparative material to the hybrid nHAp/MWCNTO-GO composite. The pure material has a grayish color and the composite a very

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white color. Due to the aesthetic characteristic, the composite material became the best choice of

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material to apply in patients, however, there was the need to use the pure material to compare. The association with the previous application of APF was also tested for both materials due to the routine

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application of fluoride by dentists in the clinic.

Although the 785 nm laser line used for this study allows for excellent signal-to-noise of

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hydroxyapatite content of dentin at relatively low laser power, the influence of MWCNTO-GO layer on dentin was a problem, as it introduced a high fluorescence background under 785 nm excitation.

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This experimental limitation caused by the sample color resulted in a low resolution in spectra and then the peak area calculation and statistical analysis for these two specific groups were

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not possible. After erosion-abrasion cycles, the dentin samples of these two groups become lighter and Raman spectra collection was performed normally, resulting in spectra with adequate

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visualization of inorganic and organic peaks and then allowing thus the peak area calculation. Faced with these difficulties, in the 1st Raman spectroscopy analysis, the peak area calculations were impossible due to this limitation of the technique. Hence, all statistical comparisons of peak areas were performed only between groups C, APF, nHAp, APF_nHAp and APF_nHAp/MWCNT-GO. In the case of this hybrid group (APF_nHAp/MWCNT-GO), the spectra could be obtained because the material has a slightly grayish coloration (light gray-white), which did not interfere in obtaining the Raman spectra (Fig. 1A-C). After application of materials’, the APF_nHAp treatment resulted in a significant reduction of the phosphate content than in the negative control group (p <0.05) (Fig. 1D). These changes in the phosphate component are directly noted in the mean Raman spectra (Fig 1A). The integrated area of the phosphate peak after APF_nHAp treatment was statistically significantly lower than in positive control APF and nHAP (p<0.05) (Fig. 1D). This reduction occurred due to the interaction of the nHAp with the dentin forming a surface coverage layer. The previous application of phosphoric acid and fluoride improved the deposition of the material that formed a thicker layer and, therefore, 9

Journal Pre-proof resulted in a lower exposure of the phosphate content of the dentin, with consequent lower relative peak area (Figs. 1A, 1D). The treatments APF, APF_nHAp, and APF_nHAp/MWCNTO-GO resulted in a statistically significant lower peak area of the carbonate content in comparison to the negative control group (p <0.05) (Fig. 1E). The integrated areas of the carbonate peak after nHAp and APF_nHAp treatments were statistically significantly higher than in positive control APF (p<0.01 and p<0.05, respectively) (Fig. 1E). These changes in the carbonate component are directly detectable in the mean Raman spectra with a reduction in the band intensity (Fig. 1B). A reduction in the intensity of amide I peak after APF_nHAp and APF_nHAp/MWCNTO-

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GO treatments is noted directly in the mean Raman spectra (Fig. 1C). The treatments nHAp,

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APF_nHAp and APF_nHAp/MWCNTO-GO resulted in a significant reduction in the peak area of the organic component than in the negative control and positive control groups (p <0.001) (Fig. 1F).

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The 1070/960 cm-1 ratio analysis (Fig. 4A) showed that APF and APF_nHAp/MWCNTO-GO treatments reduced the gradient in mineral content with statistical significance (p<0.001 and p<0.05,

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respectively) compared to the negative control. The apparent relative mineral concentration decreased after treatment with APF (positive control) in comparison to the nHAp (p<0.001),

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APF_nHAp (p<0.001) and APF_nHAp/MWCNTO-GO (p<0.05) treatments. The treatment APF_ MWCNTO-GO resulted in lower 1070/960 cm-1 ratio than in APF_nHAp (p<0.05) (Fig. 4A).

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After brushing (2nd Raman spectroscopy analysis), in the comparisons between the negative control group and treated groups, no statistically significant differences were observed for the

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phosphate and carbonate peaks (p> 0.05) (Fig. 2D, 2E). MWCNTO-GO group showed lower peak area for phosphate content than in positive control (p<0.01), nHAp (p<0.05) and APF_nHAp (p<0.01) (Fig. 2A). MWCNTO-GO group showed lower peak area for carbonate content than in nHAp (p<0.05) (Fig. 2E).

Regarding collagen, significant statistical differences were observed the between negative control group and MWCNT-GO (p <0.001) and APF_MWCNT-GO (p <0.001) with lower peak areas than in C (Fig. 2F). The same differences were observed concerning the positive control group (Fig. 2F). Statistical comparisons among dentin groups treated with biomaterials revealed significant differences among treatments (p<0.05) (Fig 2F). The treatments MWCNTO-GO and APF_ MWCNTO-GO resulted in lower peak area for collagen than in nHAP (p<0.001). The MWCNTOGO also showed statistically significant lower peak area for collagen component than in APF_nHAP (p<0.001), APF_ MWCNTO-GO (p<0.05) and APF_nHAp/ MWCNTO-GO (p<0.001). Peak area was lower in APF_ MWCNTO-GO than in APF_nHAp (p<0.001) and APF_nHAp/ MWCNTO-GO

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Journal Pre-proof (p<0.001) (Fig. 2F). Those results indicated that the MWCNT-GO binds to the dentin collagen and thus the intensity/area of this peak was reduced. After brushing-erosion, the 1070/960 cm-1 ratio was not affected by the treatments (p>0.05) (Fig. 4B). Based on the carbonate to phosphate (1070/960 cm-1) ratios generated via Raman spectra (Figs. 4A, 4B) the treatments APF and APF_nHAp/ MWCNTO-GO showed lower ratios than the other treatments. This result pointed out that in these two treatments probably there was less ion substitution than in the other treatments. Teraoka et al. [47] reported that the strength of a single HA crystal after immersion in water was reported to significantly decrease by 23–43 % in proportion to

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carbonate content. Consequently, in the two groups with lower carbonate to phosphate ratios the

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dentin surface may have had a more stable or thicker layer of deposits than in the other treatment groups. It is significant to perform the carbonate to phosphate ratio analysis because carbonate in the

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apatite structure is responsible for a decrease in the degree of crystallinity, weakening the bonds and increasing the mineral solubility [48].

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Figure 5 shows the MMR of bovine dentin after application of materials’ (Figs. 5A, 5C) and after brushing-erosion (Figs. 5B, 5D) estimated by using ratios between phosphate (Figs. 5A, 5B)

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and carbonate (Figs. 5C, 5D) to the amide I. In the case of the phosphate/amide I ratio, nHAP resulted in higher ratio than APF after application of materials’ (Fig. 5A). This result indicated that

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the coverage of dentin surface by nHAP after treatment was more effective than in APF. After brushing-erosion, the phosphate/amide I ratio was not affected by the treatments (p>0.05) (Fig. 5B).

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In the case of the carbonate/amide I ratio (Figs. 5C, 5D), different approaches of analysis can be done. First, the APF treatment resulted in significant statistical lower ratios than in the negative control (p<0.001, Fig. 5C). Thus, the MMR parameter indicated that the APF treatment reduced carbonate content and exposed the collagen fibers. Second, the nHAp, APF_nHAp and APF_nHAp/MWCNTO-GO resulted in significant statistical higher ratios than in the positive control (p<0.01, Fig. 5C). Again the MMR parameter showed that the dentin surface treated with these three materials resulted in a balance between carbonate content and collagen exposure in comparison to the APF treatment. Third, the APF_nHAp/MWCNTO-GO treatment resulted in significant statistical lower ratios than in the nHAp (p<0.05, Fig. 5C). This result indicated a slightly better balance between carbonate/amide I in nHAp treatment than in APF_nHAp/MWCNTO-GO. After brushingerosion, the carbonate/amide I ratio was statistically significant higher in MWCNTO-GO treatment than in negative control (p<0.001) (Fig. 5D). This result shows the balance between carbonate and collagen levels in MMR parameter. In this case, the MWCNTO-GO material has an affinity with dentin collagen and this treatment remained covering the dentin even after brushing-erosion. 11

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3.2. Multivariate analysis of Raman spectra The area under the phosphate, carbonate and protein peaks give an idea of the effect of coating on teeth before and after erosion-abrasion cycles and help evaluate the efficacy of coatings/treatments. However, it may be convenient and more objective, to apply multivariate analysis for a quick distinction of groups – the assumption being, if two groups show separation, significant chemical change has occurred. This allows a quick overview of changes in groups or lack of the same. Figure 6 (A) shows distinct cluster for the negative control (G1). Positive control (G2) and nHAp (G3) form a second separate cluster, and APF_nHAp (G5) and APF_nHAp/MWCNTO-

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GO (G7) form the third cluster. Based on SEM and phosphate/carbonate/protein area analysis,

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negative control shows no coverage, explaining why it forms a distinct cluster. Positive control shows partial teeth coverage, and demineralization, while nHAp shows coverage in spots by the

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respective treatment substance used, and hence form a second overlapping cluster. APF_nHAp and APF_nHAp/MWCNTO-GO, on the other hand, shows thick and effective coverage, explaining them

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forming a third distinctive cluster. This suggests that multivariate analysis could rapidly identify some parameters of treatment efficacy. This is also seen in PCA of groups after brushing, where

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treatments containing fluoride (positive control G2), APF_nHAp (G5), APF_MWCNTO-GO (G6), and APF_nHAp/MWCNTO-GO (G7) formed a cluster separate from negative control (G1), nHAp

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(G3), and MWCNTO-GO (G4), wherein fluoride was absent. Groups containing pure MWCNTOGO, without (G4) or with fluoride pretreatment (G6) also clustered. Fluoride treatment groups

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showed higher protection from erosion after brushing, compared to non-fluorides. Thus, Raman spectroscopy combined with multivariate analysis may be useful for rapid identification of effective treatment strategies.

3.3. SEM analysis

Figures 7-10 shows SEM micrographs of root bovine dentin after application of materials’ and after erosion-abrasion and remineralization cycles. SEM micrographs of artificial saliva group (negative control), before cycling, illustrate dentin surface with the absence of smear layer and tubule exposure by acid etching (arrows) (Fig. 7A). After cycling, the tubules are partially occluded in the negative control (Fig 9A). SEM micrograph of dentin after nHAp treatment revealed partial dentin tubule exposure and some deposits in tubule entrance (Fig. 7B). SEM micrographs confirm the Raman results that nHAp treatment resulted in lower phosphate peak area than in negative control (Fig. 2A). In micrographs, there is a formed layer with low exposure of dentin surface and some level of protection (Fig. 7B). 13

Journal Pre-proof This formed layer of CaF2 crystals on the surface promotes an additional barrier and occluding the dentin tubules [49]. Kunam, Manimaran, Sampath and Sekar [50] observed a partial occlusion of the dentin tubules after treatment with NaF 2%. It was reported that NaF reacts with the calcium of the dentin resulting in the formation of calcium fluoride crystals, but with reduced size, and are not effective in occlusion of the tubules. In contrast, the same group after erosion-abrasion showed partially or totally exposed (arrows) dentin tubules (Fig. 9B). The SEM micrographs showed that before cycles, the MWCNTO-GOs formed agglomerates on dentin (arrows) (Fig. 7C) and after erosion, these agglomerates are not visible, and the dentin tubules are partially occluded (Fig. 9C). The SEM analysis corroborates with previous results where

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the MWCNTO-GO formed agglomerates on dentin because they have an affinity with dentin

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collagen [30]. After erosion they are not visible, however, it seems that helped in the deposition of crystals on tubule entrance (Fig. 9C).

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The micrographs of the APF group (positive control) (Fig. 8A) showed partially exposed dentin tubules after APF treatment and totally occluded tubules after erosion-abrasion-

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remineralization cycles (Fig. 10A). The SEM micrographs of dentin after erosion-abrasion in APF

10A).

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group suggested precipitation of mineral crystallites homogeneously distributed on the dentin (Fig.

The dentin of APF_nHAp group exhibited opened dentin tubules (Fig. 8B) and after erosion-

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abrasion, they are totally occluded by a thick granular layer (Fig. 10B). On the dentin surface of APF_MWCNTO-GO group, the dentin tubules are exposed (arrows) or occluded by some

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MWCNTO-GO agglomerates (circle) (Fig. 8C). After cycles, most of dentin tubules were totally occluded by a thick granular layer (Fig. 10C). The micrograph of APF_nHAp/MWCNTO-GO group reveals the absence of dentin tubule exposure and a thick layer covering the dentin surface (Fig. 8D). After erosion-abrasion cycles, the dentin was partially covered by a thin granular layer and some sites with tubules exposure (arrows) (Fig. 10D). The dentin surface coverage with the isolated materials, nHAp and MWCNTO-GOs (without fluoride association), showed to be sparse coverage and with reduced obliteration of the dentinal tubules (Figs 7B, C). However, these same materials when associated with a previous application of fluoride produced a greater obliteration of the dentin tubules forming a uniform layer on the surface (Figs 8B, C). This positive association of nHAp with fluoride was also observed by Kunam, Manimaran, Sampath and Sekar [50] where it observed a complete obliteration of the dentin tubules with the association of nHAp combined with 2% NaF. A thick layer with a large amount of precipitate in the form of agglomerates was produced. In this case, the effect of synergism provided by the use of fluoride associated with treatment with nHAp may be reported. 14

Journal Pre-proof The present study is relevant because it presents data on dentin coating with biomaterials, as well as their stability in consecutive cycles of demineralization-remineralization and abrasion. The reason for conducting a study on dentin erosion was due to the occurrence of frequent exposure of dentinal tubules in the non-carious cervical lesions. This lesion often occurs where the intratubular fluid moves through stimuli in exposed dentin, which generates pain sensitivity. In addition, the multi-walled carbon nanotube (MWCNTO-GO) is an emerging material with excellent properties, such as chemical inertia, robust mechanical properties, and especially, collagen and dentin affinity [24]. Akasaka, Nakata, Uo and Watari [24] showed that MWCNT adhered easily to the dentin and cementum surfaces due to the elevated organic content in comparison to the enamel and this was

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why in our study dentin was selected and not enamel. The MWCNTO-GO based biomaterials are

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viable to be applied only to the dentin structure as previously reported by our group [30]. This is because it does not effectively bind to enamel. There are significant differences in the organic matrix

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portion between enamel and dentin and the enamel is composed mainly of minerals. In this research,

applied on dentin with an exposed tubule.

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one of the focuses was the evaluation of the role of MWCNTO-GO in the anti-erosive effect when

Raman microscopy proved to be useful in obtaining chemical and structural information of

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dentin after biomaterials application and after erosion/abrasion processes. Combined with multivariate analysis, it provided possibility of rapidly evaluating treatment efficacy.

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The association between fluoride and nHAp (APF_nHAp) allowed a better affinity of nHAp with the inorganic portion of dentin and that the association between fluoride and MWCNTO-GO

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(APF_MWCNTO-GO) resulted in the better affinity of the carbon nanotubes with the portion inorganic and organic dentin, through union with type I collagen. Raman spectroscopy indicated that a chemical bond between the carbon nanotubes and the dentin collagen occurs in the MWCNTO-GO and APF_MWCNTO-GO groups. This interaction indicates the formation of a protective layer for the surface of the dentin and for the collagen giving possible protection against erosion. A protective effect of the APF_nHAp treatment was observed due to the maintenance of inorganic content relative to the phosphate component. The adhesion of the carbon nanotubes to the dentin was observed due to the presence of the peaks referring to these components in the spectra after the processes of erosive cycling and abrasion. The SEM micrographs showed the formation of a protective layer after application of the biomaterials and that it was partially or totally removed after the erosive cycling and abrasion processes.

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4. Conclusion

The APF_nHAP/MWCNT-GO composite has shown to be a promising material since it has binding characteristics both to the inorganic and organic portion of the dentin, reduced solubility and mainly to the coloration in gray shade near the white, which would not interfere with the aesthetic in a possible clinical application. MMR parameter analysis confirmed the binding capability of

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MWCNTO-GO-based materials to dentin.

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References

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Journal Pre-proof Figures and tables captions Table 1 Flow chart describing the stages of analysis, number of samples, and analytical techniques used for the experimental and control groups Fig. 1 Average Raman spectrum of root dentin surfaces after application of materials’ (A-C). Mean and standard deviation (n = 10) of integrated area (a.u. - arbitrary units) of the Raman peaks after application of materials’ (D-F). The spectrum showed the differences in the intensity of the peaks attributed to phosphate (960 cm-1) (A), carbonate (1070 cm1

) (B) and amide I (1666 cm-1) (C), among the experimental groups. Statistical comparisons considering phosphate (PO43-

) (D), carbonate (CO3-2) (E) and amide I (F) peaks. Dunnett's Multiple Comparison Test: between negative control (NC) and treatments; between positive control (PC) and treatments. Tukey's Multiple Comparison Test: among treatment

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groups. Legend of groups: C - Control; APF – Acidulated Phosphate Fluoride; nHAp - nanohydroxyapatite; MWCNTOGO - Multi-walled carbon nanotubes oxide and graphene oxide; APF_nHAp - APF + nanohydroxyapatite;

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APF_MWCNTO-GO - APF + MWCNTO-GO; APF_nHAp/MWCNTO-GO - APF + nHAp/MWCNTO-GO composite

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Fig. 2 Average Raman spectrum of root dentin surfaces after erosion-abrasion and remineralization cycles (A-C). Mean and standard deviation (n = 10) of integrated area (a.u. - arbitrary units) of the Raman peaks after erosion-abrasion and

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remineralization cycles (D-F). The spectrum showed the differences in the intensity of the peaks attributed to phosphate (960 cm-1) (A), carbonate (1070 cm-1) (B) and amide I (1666 cm-1) (C), among the experimental groups. Statistical

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comparisons considering phosphate (PO43-) (D), carbonate (CO3-2) (E) and amide I (F) peaks. Dunnett's Multiple Comparison Test: between negative control (NC) and treatments; between positive control (PC) and treatments. Tukey's Multiple Comparison Test: among treatment groups. 1- Statistical significant difference from nHAp; 2- Statistical

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significant difference from MWCNTO-GO; 3- Statistical significant difference from APF_nHAp; 4- Statistical significant difference from APF_nHAp/MWCNTO-GO. Legend of groups: C - Control; APF – Acidulated Phosphate Fluoride; nHAp - nanohydroxyapatite; MWCNTO-GO - Multi-walled carbon nanotubes oxide and graphene oxide;

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APF_nHAp - APF + nanohydroxyapatite; APF_MWCNTO-GO - APF + MWCNTO-GO; APF_nHAp/MWCNTO-GO APF + nHAp/MWCNTO-GO composite

Fig. 3 Images of the dentin surface obtained by the camera attached to the sample holder Raman spectrometer after MWCNT-GO (A) and APF_MWCNT-GO (C) treatments. Raman spectra of the dentin surface after MWCNT-GO treatments (B, D) showed: the D’ band (black arrow: peak at 1333 cm-1), the G’ band (blue arrow: peak at 1586 cm-1) both related to MWCNT-GO and the peak at 960 cm-1 related to PO43- of dentin (red arrow) Fig. 4 Mean and standard deviation (n = 10) of carbonate/phosphate ratio obtained by Raman spectroscopy measurements, after application of materials’ (A) and after erosion-abrasion cycles (B). Statistical comparisons, Dunnett's Multiple Comparison Test: between negative control (NC) and treatments; between positive control (PC) and treatments. Tukey's Multiple Comparison Test: among treatment groups. 1- Statistical significant difference from APF_nHAp. Legend of groups: C - Control; APF – Acidulated Phosphate Fluoride; nHAp - nanohydroxyapatite; MWCNTO-GO - Multi-walled carbon nanotubes oxide and graphene oxide; APF_nHAp - APF + nanohydroxyapatite; APF_MWCNTO-GO - APF + MWCNTO-GO; APF_nHAp/MWCNTO-GO - APF + nHAp/MWCNTO-GO composite.

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Journal Pre-proof Fig. 5 Mineral-to-matrix ratio (MMR) of dentin obtained by Raman spectroscopy measurements, after application of materials’ (A, C) and after erosion-abrasion cycles (B, D) of phosphate peak at 960 cm-1 (A, B) and carbonate peak at 1070 cm-1 (C, D). Statistical comparisons, Dunnett's Multiple Comparison Test: between negative control (NC) and treatments; between positive control (PC) and treatments. Tukey's Multiple Comparison Test: among treatment groups. 1- Statistical significant difference from nHAp. Legend of groups: C - Control; APF – Acidulated Phosphate Fluoride; nHAp - nanohydroxyapatite; MWCNTO-GO - Multi-walled carbon nanotubes oxide and graphene oxide; APF_nHAp APF + nanohydroxyapatite; APF_MWCNTO-GO - APF + MWCNTO-GO; APF_nHAp/MWCNTO-GO - APF + nHAp/MWCNTO-GO composite

Fig. 6 Scatter plot of PC1 and PC4 PC2 each group before (A) and PC 4 and PC5 after erosion-abrasion cycles (B): G1: C – artificial saliva (negative control); G2: APF – Acidulated Phosphate Fluoride; G3: nHAp - nanohydroxyapatite; G4:

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MWCNTO-GO - Multi-walled carbon nanotubes oxide and graphene oxide; G5: APF_nHAp - APF + nanohydroxyapatite; G6: APF_MWCNTO-GO - APF + MWCNTO-GO; G7: APF_nHAp/MWCNTO-GO - APF +

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nHAp/MWCNTO-GO composite

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Fig. 7 Representative SEM micrographs of dentin surface treated with different materials, considering non-fluoridated groups. Before erosion-abrasion and remineralization cycles, the dentin surface of negative control group is free of smear

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layer and some tubules are opened (arrows) (A). The dentin tubules are partially or totally occluded by a granular layer of nHAp on the surface of nHAp group (arrows) (C). On the dentin surface of MWCNTO-GO group, the dentin tubules are

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images show detail of the region

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partially occluded by some MWCNTO-GO agglomerates (arrows) (D) (1000). High power magnification (10,000)

Fig. 8 Representative SEM micrographs of dentin surface treated with different materials, considering fluoridated groups. Before erosion-abrasion and remineralization cycles, in the dentin surface of positive control group (APF), the

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dentin tubules are occluded or partially opened (arrows) (A). The dentin tubules are partially or totally occluded by a granular layer of nHAp on the surface of APF_nHAp group (arrows) (B). On the dentin surface of APF_MWCNTO-GO group, the dentin tubules are exposed (arrows) or occluded by some MWCNTO-GO agglomerates (circle) (C). APF_nHAP/MWCNT-GO treatment resulted in tubule occlusion (D) (1000). High power magnification (10,000) images show detail of the region

Fig. 9 Representative SEM micrographs of dentin surface treated with different materials, considering non-fluoridated groups. After erosion-abrasion and remineralization cycles, the dentin surface of negative control group is free of smear layer and some tubules are opened (arrows) (A). The dentin tubules are partially or totally exposed (arrows) on the surface of nHAp group (arrows) (B). On the dentin surface of MWCNTO-GO group, the dentin tubules are partially occluded (arrows) (C) (1000). High power magnification (10,000) images show detail of the region

Fig. 10 Representative SEM micrographs of dentin surface treated with different materials, considering fluoridated groups. After erosion-abrasion and remineralization cycles, in the dentin surface of positive control group (APF), the dentin tubules are totally occluded (A). The dentin tubules are totally occluded on the surface of APF_nHAp group (B). On the dentin surface of APF_MWCNTO-GO group, almost all tubules are occluded (C). APF_nHAP/MWCNT-GO

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Table 1. Flow chart describing the stages of analysis, number of samples, and analytical techniques used for the experimental and control groups SAMPLE GROUPS After application After of materials’ erosion/abrasion 1st Raman (n = 10) 2nd Raman (n = 9) Negative control: ↓ ↓ C - Artificial saliva (n = 10) 1st SEM (n = 1) 2nd SEM (n = 1) Non1st Raman (n = 10) 2nd Raman (n = 9) fluoridated nHAp (n = 10) ↓ ↓ (n = 30) 1st SEM (n = 1) 2nd SEM (n = 1) 1st Raman (n = 10) 2nd Raman (n = 9) MWCNTO-GO (n = 10) ↓ ↓ µ-EDXRF st nd 1 SEM (n = 1) 2 SEM (n = 1) 1st Raman (n = 10) 2nd Raman (n = 9) Dentin Positive control: ↓ ↓ Samples APF (n= 10) 1st SEM (n = 1) 2nd SEM (n = 1) selection (n = 70) 1st Raman (n = 10) 2nd Raman (n = 9) APF_nHAp (n = 10) ↓ ↓ 1st SEM (n = 1) 2nd SEM (n = 1) Fluoridated (n = 40) 1st Raman (n = 10) 2nd Raman (n = 9) APF_MWCNTO-GO (n = 10) ↓ ↓ 1st SEM (n = 1) 2nd SEM (n = 1) 1st Raman (n = 10) 2nd Raman (n = 9) APF_nHAp/MWCNTO-GO ↓ ↓ composite (n = 10) 1st SEM (n = 1) 2nd SEM (n = 1)

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Analytical tools: µ-EDXRF (sample selection), Raman spectroscopy (1 st and 2nd measurements) and SEM (1st and 2nd measurements). Legend: nHAp – nanohydroxyapatite; MWCNTO-GO - Muli-walled carbon nanotube oxide and graphene oxide composite; APF - Acidulated Phosphate Fluoride gel.

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Dentin was modified by carbon nanotubes and nanohydroxyapatite composites Raman spectroscopy and multivariate analysis allows a prompt non-contact diagnosis. SEM revealed the interaction between biomaterials and dentin. Fluoride allowed a better affinity of composites with dentin. Biomaterials formed a layer which was partially or totally removed after erosion.

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