Radiopacity and mechanical properties of dental adhesives with strontium hydroxyapatite nanofillers

journal of the mechanical behavior of biomedical materials 101 (2020) 103447

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Radiopacity and mechanical properties of dental adhesives with strontium hydroxyapatite nanofillers E.V. Carvalho a, b, D.M. de Paula c, D.M. Andrade Neto a, L.S. Costa d, D.F. Dias e, V.P. Feitosa c, f, P.B.A. Fechine a, * a

Group of Chemistry of Advanced Materials (GQMat) - Department of Analytical Chemistry and Physical-chemistry, Federal University of Cear� a - UFC, Campus do Pici, CP 12100, 60451-970, Fortaleza, Brazil Technology Center, Christus University Center, Fortaleza, 60160-230, Brazil c Research Division, Paulo Picanço School of Dentistry, Fortaleza, 60325-218, Brazil d Institute of Chemistry, University of Campinas – UNICAMP, Campus Universit� ario Zeferino Vaz - Bar~ ao Geraldo, CP 6154, CEP 13083-970, Campinas, SP, Brazil e Laboratiry X-Ray (LRX) - Department of Physic, Federal University of Cear� a - UFC, Campus do Pici, CP 6030, 60451-970, Fortaleza, Brazil f Post-Graduate Program in Dentistry - Department of Restorative Dentistry, Federal University of Cear� a, Fortaleza, Brazil b

A R T I C L E I N F O

A B S T R A C T

Keywords: Strontium hydroxyapatite Radiopacity Dental resin

Objectives: Dental resins filled with hydroxyapatite (HAp) nanoparticles have significantly revolutionized restorative dentistry offering advantages such as remineralization potential and increase of polymerization. However, these materials have limited radiopacity hindering the diagnosis of secondary caries. The present study investigated the development of a new radiopaque dental adhesive by incorporating novel pure strontium hy­ droxyapatite nanoparticles [Sr10(PO4)6(OH)2, SrHAp] synthesized by a simple hydrothermal method. Methods: Strontium phosphates were prepared using co-precipitation (SrHAp0h) and hydrothermal treatment for 2 and 5h (SrHAp2h and SrHAp5h). The crystallinity, crystallite size, textural and morphology features of the nanoparticles were characterized by XRD, FT-IR, micro-Raman and TEM. Strontium hydroxyapatite (SrHAp) or calcium hydroxyapatite (HAp) nanoparticles were then incorporated (10 wt%) into an adhesive resin of a commercial three-step etch-and-rinse adhesive to evaluate the radiopacity of disk-shaped specimens, degree of conversion (DC) assessed by FT-IR and mechanical properties by three-point bending test. The unfilled adhesive was used as negative control. Results: While SrHAp0h and SrHAp2h resulted a phase mixing, the pure and highly crystalline phase of strontium hydroxyapatite free of calcium was obtained with 5h hydrothermal treatment (SrHAp5h). The TEM images revealed nanorods morphology for SrHAp5h. SrHAps incorporated into adhesive showed higher radiopacity, no alteration on DC despite slightly reducing the mechanical properties. Significance: Although the mechanical properties are slightly impaired, incorporation of SrHAp nanoparticles offers potential method to improve radiopacity of restorative dental resins, easing the tracking of actual remi­ neralization effects and enabling diagnosis of recurrent caries.

1. Introduction Resin composites have become the primary material for restorative dentistry. However, several studies indicate that resin composite has less durability than amalgam (Orrego et al., 2017; Elkassas et al., 2017). Therefore, adhesive compositions and bonding procedures have been improved by addition of different inorganic nanofillers. In dental bio­ materials, hydroxyapatite (HAp) nanoparticles have shown to be an adequate filler for adhesive resins to improve their adhesion to dental

hard tissues and preserve mechanical properties after water aging (Andrade Neto et al., 2016). HAp crystallites represent the main constituent of the mineralized dental structures (Vassal et al., 2019). The apatite forms have beneficial dental applications due to its biocompatibility in addition to biological and chemical similarity to the dental structures that comprises hexag­ onal prisms of Ca10(PO4)6(OH)2. This similarity of structures facilitates integration with the mineral components of teeth and can be used to induce tooth remineralization (Elkassas et al., 2017; Mazumder et al.,

* Corresponding author. E-mail address: [email protected] (P.B.A. Fechine). https://doi.org/10.1016/j.jmbbm.2019.103447 Received 21 June 2019; Received in revised form 23 September 2019; Accepted 23 September 2019 Available online 23 September 2019 1751-6161/© 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Synthesis scheme for SrHAp nanoparticles.

2019; Melnikov and Gonçalves, 2015). In addition, control of crystallite size and morphology may result in increased biocompatibility (Andrade Neto et al., 2016). Nevertheless, the clinical diagnosis of teeth restored with resin composites fillings is still difficult due to the low radiopacity of fillers, what difficult to verify the occurrence of secondary caries by radiographs. Restoration is often replaced without necessity thanks to the difficulty of evaluating this material (Zanatta et al., 2019). Therefore, an alternative strategy to overcome the radiopacity drawback of HAp and restorative composites is to dope bioactive trace elements with higher atomic weight into HAp by ion substitution. There are many reports of calcium ion substitution in apatites by bivalent cations (Gopi et al., 2014; Huang et al., 2016; Aina et al., 2012; Krishnan et al., 2016). Among the bioactive metal ions, strontium ions (Sr2þ) has better performance in biomaterials for bone regeneration (Huang et al., 2016; Aina et al., 2012). Comparative studies have demonstrated the superiority of strontium ions incorporated in the structure of HAp in stimulating osseointegration and biomechanical properties with respect to the Zn and Mg ions (Tao et al., 2016). Strontium is, like calcium, a group II element and, from a chemical point of view, they behave similarly (Shahid et al., 2014; Habibovic and Barralet, 2011) in the human body. It is reported that Sr2þ can reduce bone resorption by inhibiting osteoclast activity and improve bone formation by stimu­ lating osteoblast activity (Tao et al., 2016; Masala et al., 2014). Sr-substituted apatite structure shows increased radiopacity due to its high atomic number and weight compared to Ca leading to an increased absorption of X-ray energy (Wang et al., 2007). Strontium is also known to favors cellular proliferation (Sarkar et al., 2019), exhibit weak anti­ bacterial activity (Liu et al., 2016; Guida et al., 2003; Brauer et al., 2012; Jayasree et al., 2017) and cariostatic properties in most animal caries and epidemiological studies reported (Jayasree et al., 2017; Curzon, 1985). The addition of Sr to glasses for dental restoratives is designed to enhance radiopacity. Shahid and collaborators report the effect on es­ thetics (translucency) and radiopacity of substitution of Ca2þ ion by Sr2þ on production of radiopaque glass ionomer cements. They concluded that the level of radiopacity is found to increase linearly with Sr, without adverse effects on visual properties of the cement (Shahid et al., 2014; Meininger et al., 2019). In particular, strontium hydroxyapatite (SrHAp) would serve as a viable alternative to overcome the disadvantages of HAp such as radi­ opacity, and track the sustained release of Sr2þ ions from SrHAp for proving the true remineralization, which is difficult with Ca-containing materials (Lei et al., 2017). In addition, there are reports that the pres­ ence of nanometric apatites significantly increases the bioactivity and

biocompatibility of the biomaterials (Sadat-Shojai et al., 2013), besides being able to improve the mechanical properties when associated with resins (Andrade Neto et al., 2016). Thus, the aim of this investigation was demonstrate a simple method to synthesize pure SrHAp, and study its potential as filler to dental ad­ hesives, in order to obtain a dental material improved in terms of radiopacity and physicochemical properties. The first hypothesis is that the addition of SrHAp nanoparticles will effectively improve the radio­ pacity, and the second one is that the presence of the nanoparticles in the adhesive does not alter significantly the mechanical properties. 2. Materials and methods 2.1. Synthesis of SrHAp nanoparticles The preparation of SrHAp was adapted from the method previously described by Andrade Neto and collaborators (Andrade Neto et al., 2016). A solution of H3PO4 0.3 mol.L 1 (Quimex) was added to Sr(NO3)2 0.5 mol.L 1 solution (Dinamica, 99.67%) (molar ratio Sr/P ¼ 1.67) under vigorous stirring. The pH of solution was adjusted to pH ¼ 9.0 by adding NH4OH (Dinamica, 30%). A white precipitate was formed, and the suspension was stirred for 2 h. Thereafter, the precipitate was washed with distilled water and vacuum filtered. A part of precipitate was reserved (specimen SrHAp0h). The remaining powder was dispersed in NH4Cl 0.1 mol.L 1 solution (Vetec, 99.5%) with 1:10 weight ratio between the precipitate and the solution. The sus­ pension was placed in a Teflon autoclave and covered with stainless steel to receive the hydrothermal treatment at 150 � C for 2 h and 5h (obtaining specimens SrHAp2h and SrHAp5h, respectively) (Andrade Neto et al., 2016). Finally, the material was filtered, washed with distilled water and dried. The synthesis reaction occurs according to the equation (1). A summary of the synthesis and the samples provided herein is shown in the Fig. 1. 10 SrðNO3 Þ2ðaq:Þ þ 6 H3 PO4ðaq:Þ þ 2 NH4 OHðaq:Þ → Sr10 ðPO4 Þ6 ðOHÞ2ðsÞ þ 18 HNO3ðaq:Þ þ 2NH4 NO3ðaq:Þ

(1)

2.2. Characterization techniques The samples were characterized with various techniques in order to obtain morphological crystalline structure information. X-ray-Diffrac­ tion (XRD) and transmission electron microscopy (TEM) were used to obtain information concerning the composition, size and morphology of the nanoparticles, as well as the influence of the time of hydrothermal 2

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treatment on these properties. Fourier Transformed Infrared Spectros­ copy (FTIR), Raman Spectroscopy were performed to further confirm our evidences obtained with XRD and TEM. Additionally, these char­ acterization techniques helped us to indicate a mechanism for the syn­ thesis of our SrHAp nanoparticles.

and dentin. The samples (n ¼ 5) and aluminum steps template (#1 mm) were placed over ultraspeed periapical film (Eastman Kodak Co., Rochester, USA) and then captured. The film was exposed for 0.20 s, with total filtration equivalent to 3.81 mm of Al at 70 kV, 7 mA, and 300-mm target film distance with a Time x 70C dental X-ray unit ~o Paulo, Brazil) and manually processed in fresh chemicals (Gnatus, Sa using temperature-time table. The radiograph was digitized using a scanner with a transparent adapter and 600 dpi resolution (CX-4200, Samsung Engineering Co., Ltd., Seoul, Korea). The image was saved as uncompressed TIFF files. The digital image was analyzed using a computer graphics program (ImageJ, National Institutes of Health, Bethesda, USA). Three reference points (40 x 40 pixels) of each sample were randomly chosen and the optical density of each one was measured according to the radiopacity of aluminum steps template. The average gray scale values from 0 (black) to 255 (white) for selected regions of the image were measured using the histogram tool. The histogram provides numeric data in pixels for the optical density in the selected area, based on the 256 values of gray scale available in the system, providing the mean and standard deviation. The three readings were obtained from each sample and the arithmetic mean calculated, corresponding to the density value of the item as previously descripted (de Moraes Porto et al., 2014).

2.2.1. X-ray diffraction The specimens (n¼5) were characterized by x-ray diffraction (XRD), in order to obtain the structural information of the strontium apatite due to the heating process. It was used a diffractometer model X-Pert PRO MPD-Panalytical with cobalt radiation (Co Kα 1, λ ¼ 1.78887 Å). The measurements were made at room temperature, with range between 20 and 60� with step of 0.01� /min. The identification of the crystalline phases was performed using the software X-pert HighScore Plus version 3.0.4 (Panalytical) with PDF 22004 data base (The International Center for Diffraction Data). The Rietveld refinement method was employed using the code GSAS through the graphical interface EXPGUI. We calculated the diffraction peaks profiles by the Thompson-Cox- Hastings Pseudo-Voigt function. The Full width at half maximum (FWHM) was used to calculate the crystallite size by the Scherrer equation. In the analysis, the refined parameters: scale factor, background, lattice pa­ rameters, shift, isotropic displacement parameter, anisotropic factor of deformation, atomic position and occupation. In this case, it used the polynomial function of Chebyschev with 12 terms for the background correction.

2.2.6. Degree of conversion (Feitosa et al., 2014a) Micro-Raman spectroscopy analysis (n ¼ 3) was used to assess the degree of conversion (DC) of adhesive 10 min after light-curing. The micro-Raman spectrophotometer (Xplora, Horiba JobinYvon, Paris, France) was firstly calibrated using a silicon standard sample supplied by the manufacturer. HeNe laser with 3.2 mW power and 532 nm wavelength was employed with 1.5 μm spatial resolution, 2.5 cm 1 spectral resolution associated with 10x magnification lens (Olympus, London, UK) to attain approximately 60� 70 μm field area. The degree of conversion was calculated based on a previous investigation (Feitosa et al., 2014a) by means of the Formula 2. � � Rcured DC ¼ 1 � 100 (2) Runcured

2.2.2. FT-IR spectroscopy For FTIR analysis, the obtained powders (n¼5) were mixed with potassium bromide (KBr) in 1:10 weight ratio. After the pressing pro­ cess, the spectra were recorded from 4000–450 cm 1 wave number with 32 scans and a resolution of at 4 cm 1 using the FT-IR equipment FT-IR 8300 (Shimadzu Inc., Tokyo, Japan). 2.2.3. Raman Spectroscopy The Raman spectroscopy was performed on an equipment model LabRAM HR of HORIBA Scientific. The spectral excitation (n¼3) was performed with a laser using the line of 632.8 nm, with adjustable filter D1 (effective potencies of 151 μW) and 3 accumulations of 10 s.

Where R is the ratio between the heights of 1639 cm 1 and 1609 cm 1 peaks of uncured and light-cured adhesive were attained after baseline correction. Three readings were undertaken on the top surface of each specimen (Feitosa et al., 2014a).

2.2.4. Transmission electron microscopy (TEM) TEM images were performed using JEOL JEM 2100 LaB6 TEM (ac­ celeration voltage 200 kV). The samples were dispersed in isopropyl alcohol and ultrasonicated for 15 min. One drop of the dispersion was placed onto ultrathin carbon/holey carbon, 400 mesh copper grid. After deposition, the samples were dried at room temperature overnight prior to the obtainment of the images. The TEM images were acquired with the sample on a single-tilt sample holder using Gatan MSC798 TV camera, Gatan Digital Micrograph. Size distribution curves were ob­ tained by manually measuring the width and length of approximately 100 rod-like nanoparticles. The dimensions of different samples were compared by using one-way ANOVA and Tukey’s test (p < 0.05).

2.2.7. Three-point bending flexural test (Feitosa et al., 2014b) SrHAp or HAp adhesives were poured into stick-shaped silicone molds of 1 mm thickness, 8 mm length, 1 mm width and covered by a transparent strip. The adhesives were irradiated for 20 s with an LED light-curing unit DB-685 (1100 mW/cm2; Dabi Atlante, Ribeirao Preto, Brazil). The specimens were subjected to a three-point flexural test for measuring modulus of elasticity (n ¼ 5). Specimens were tested on threepoint bending set-up in a universal testing machine (Instron 4484; Ins­ tron Inc., Canton, USA), with 500N load cell at 0.5 mm/min crosshead speed. Load-displacement curves were converted to stress-strain curves. Width and thickness of the specimens were measured and the Young’s modulus was calculated as previously described (Sato et al., 2017). Data were expressed in MPa.

2.2.5. Radiopacity The dentin adhesives (Scotchbond Multi Purpose, 3M, St. Paul, USA) incorporated with no fillers (negative control), with calcium containing HAp or with SrHAp (0h, 2h and 5h) were poured into disc-shaped sili­ cone molds of 6 mm diameter, 1 mm height and covered by a trans­ parent strip. The composition of the commercial adhesive is based on hydroxyethyl-methacrylate (HEMA), bisphenol-A-diglycidyl dimetha­ crylate (BisGMA), photoinitiator and coinitiator. Hydroxyapatite nano­ particles were added at 10 wt%. The calcium-containing HAp nanoparticles were produced as previously reported (Andrade Neto et al., 2016). The adhesives were light-cured for 20s with an LED light-curing unit DB-685 (1100 mW/cm2; Dabi Atlante). One extracted third molar was cut longitudinally in mesial-distal direction to obtain a 1 mm-thick slab, which was used to analyze the radiopacity of enamel

2.2.8. Statistical analysis The results of degree of conversion were statistically analyzed by Shapiro-Wilk normality test (p > 0.05) and after proving normal data, the data were analyzed by one-way ANOVA and Tukey’s test. The data from all experiments were normal and analyzed by parametric tests. The level of significance was 5%. All data were statistically analyzed with Sigma Stat version 3.5 software (Systat, San Jose, USA).

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ratio remains almost constant (~1.34–1.37). In addition, we evidenced a change in the relative intensity for the peak [112], which exhibited a higher intensity than would be expected. This behaviour is better seen for the sample HApSr-5h, which the peaks [211] and [112] are separated from each other and the peak [112] exhibited a higher intensity (Fig. 2B). This difference in the relative intensity can be attributed to preferential orientation of the SrHAp nanoparticles. This behaviour were already observed in strontium hydroxyapatite crystals reported in the literature (Melnikov and Gonçalves, 2015; Hori et al., 2017). Finally, we calculated the crystallite size values through Scherrer’s equation considering the FWHW of all diffraction peaks of SrHAp and SrHPO4 phases (Gonçalves et al., 2012). We evidenced a decrease in the crystallite size of Sr10(PO4)6(OH)2 phase as the hydrothermal treat­ ment time increase. 3.2. Vibrational spectroscopy Fig. 3A shows the FT-IR spectra of synthesized nanoparticles and there is no significant spectral differences between the strontium phos­ phate samples and the HAp nanoparticles (Aina et al., 2013). Table 2 presents FTIR and Raman vibrational modes obtained from the samples SrHAp0h, SrHAp2h and SrHAp5h, compared to data from literature (Silva et al., 2003; Nelson and Williamson, 1982). FTIR spectra present vibrational modes in ~460 cm 1 assigned to mode ν2 (PO4)3–. Bands localized around 560 and 590 cm 1 are associ­ ated with the modes of ν4 (PO4)3–. The band at ~946 cm 1 is typical of mode ν1 (PO4)3. Broad bands at ~1020 and ~1400 cm 1 (sharp shoul­ der) are assigned to mode ν3 (PO34 ) and to mode ν1 (CO23), respectively. The band at 1451 cm 1, which appears only in the spectra of SrHAp0h sample, is usually associated with mode ν3 (CO23 ). In addition, FTIR spectroscopy of the powder showed a clear hydroxyl vibration band, at both 630 cm– 1 and 3500 cm 1. This large band corresponds to physically adsorbed water. However, bands that correspond to modes ν1 (CO23 ) and H2O decrease in the other samples as the heat treatment time increases, which is an expected behaviour. Raman spectra offer information about the phosphate and the hy­ droxyl bonds. All the specimens show the typical bands of HAp (Table 2) (Aina et al., 2013). Raman spectra of synthesized specimens (Fig. 3B) exhibited the most intense band at around 950 cm 1 that is attributed to symmetric stretching of the P–O bond (ν1 PO34 ). The spectra of speci­ mens SrHAp0h, SrHAp2h and SrHAp5h present a profile similar to that of the HAp with a slight displacement for smaller wavelength, justified by replacing of Ca2þ ion by Sr2þ (O’Donnell et al., 2008).

Fig. 2. XRD patterns for the SrHAp specimens. (A) Full-range diffractogram and (B) selected range of 002, 211 and 112 peaks.

3. Results 3.1. XRD SrHAp0h and SrHAp2h specimens showed Sr10(PO4)6(OH)2 phase (Hexagonal system) (Fig. 2A), according with reported data (JPCDS n� 00-033-1348). Nevertheless, it observed peaks attributed to a secondary phase of strontium hydrogen phosphate, SrHPO4 (JPCDS n� 00-0120359). The structural parameters results, refined by the Rietveld method (Table 1), indicate that the hydrothermal treatment promotes a phase transformation in which SrHAp is produced from SrHPO4 phase. Consequently, the specimen with a longer hydrothermal treatment time (SrHAp5h) presented a single phase: Sr10(PO4)6(OH)2. As shown in Table 1, the goodness of fit (S) and weighted profile R-factor (Rwp) of the Rietveld refinement were calculated and their values were satisfactory. The lattice parameters to the phase SrHAp (Table 1) in all specimens are in according with previously reported publication (Zhang et al., 2014). The a parameter value of phase Sr10(PO4)6(OH)2 shows a small increase with increasing hydrothermal treatment time, while the a/c Table 1 Results of XRD and TEM. Specimen

XRD Results

TEM results

Rwp (%)

S

Identified phases

Fraction (%)

Lattice parameters (Å)

12.26

1.398

α - SrHPO4

27.16

a ¼ 7.2094 b ¼ 6.8083 c ¼ 7.2772

33.1

Sr10(PO4)6(OH)2

72.83

28.2

α - SrHPO4

14.26

a ¼ 9.7937 b ¼ 9.7937 c ¼ 7.2851 a ¼ 7.2069 b ¼ 6.8101 c ¼ 7.2760

Sr10(PO4)6(OH)2

85.73

23.5

Sr10(PO4)6(OH)2

100

a ¼ 9.8020 b ¼ 9.8020 c ¼ 7.2780 a ¼ 9.8595 b ¼ 9.8595 c ¼ 7.2173

Size of the crystallite

Width (nm)

Length (nm)

Average (nm) SrHAp0h

SrHAp2h

SrHAp5h

Letters

a,b

12.47

12.85

1.615

1.997

33.2

21.9

12.7 � 12 a

50.6 � 27 a

20.3 � 10 b

89.4 � 53 b

34.0 � 14 c

105.0 � 57 c

and c indicate that the average width and length for the SrHAp particles are significant different at the level of 0.05 at the Turkey test. 4

E.V. Carvalho et al.

SrHAp2h

– – 987.7 – 1047.4 1032.7 1023.1 949.6 – – 595.7 578.6 570.6 438.8 421 – – 986 – 1051.3 1032.7 1020.7 948.9 – – 594.3 580.4 571.9 439.5 420.1

TEM images and particle-size distribution curves are shown in the Fig. 4A–C. All specimens synthesized herein possess a significant portion of population of particles with well-defined rod-like morphology. However, for the specimen SrHAp0h (Fig. 4A) we could evidence a larger number of particles with non-defined morphology, in comparison with the other specimens. It was measured the width and length for the particles with welldefined rod like morphology (Table 1). The values of width and length for specimens SrHAp0h, SrHAp2h and SrHAp5h were 12.7 � 12.0, 50.6 � 27.0 nm; 20.3 � 10.0, 89.4 � 53.0 nm and 34.0 � 14, 1 � 57.0 nm, respectively. One-way ANOVA and Tukey’s test evidenced that the average values for width and length are significant different among the synthesized specimens. Moreover, in the micrographs with higher magnification, it was possible to visualize that the growth of the SrHAp nanoparticles were through orientated attachment mechanism (Penn and Banfield, 1998) (See Fig. 5A and B). For instance, the growths of the particles seen in the Fig. 5B were through the [100] direction. Although we evidenced just orientated attachment in our micrographs, we do not exclude that other coalescence mechanisms could occurred in our samples. 3.4. Radiopacity

– 1070 1048 – 1075 1048 1029 962 – – 608 592 584 448 432

The digitized radiograph is shown in the Fig. 6A. The enamel radi­ opacity (5.41 � 0.27 mm Al) was the highest. The radiopacity of the specimens SrHAp0h (0.74 � 0.4 mm Al), SrHAp2h (1.1 � 0.3 mm Al) and SrHAp5h (0.8 � 0.1 mm Al) were statistically higher compared to HAp-containing and control adhesives (p < 0.05) (Fig. 6B). 3.5. Degree of conversion and Young’s modulus

3429.7 – 1442.9 1389.9 1088.8 1072.4 1019.7 945.0 880.4 – 619.2 597.3 561.1 405.5 466.1 3440.7 1450.9 1433.3 1377.3 1088.8 1072.4 1019.8 946.0 870.7 – 622.9 589.2 560.2 405.9 458.0

DC test did not show statistical difference between the adhesives (p ¼ 0.904) (Fig. 6C). Young’s modulus of adhesives is showed in Fig. 6D. Control adhesive (1230 � 201 MPa) attained highest modulus with statistical difference compared to further adhesives. HAp (1107 � 211 MPa) was statistically higher than the strontium apatites in Young’s modulus. The values for the samples SrHAp0h, SrHAp2h and SrHAp5h were 145 � 36, 180 � 52 and 193 � 127 MPa. 4. Discussion The present study showed a simple and inexpensive method for synthesis of pure SrHAp nanoparticles with high crystallinity and defined morphology. Dental resins were doped with these nanoparticles to evaluate the radiopacity and mechanical properties of this new ma­ terial. The first hypothesis was accepted due to the resin doped with SrHAp exhibit a higher radiopacity compared to other adhesives. The second hypothesis was partially accepted because the DC was not changed as a function of the used nanoparticles. However, the Young’s modulus was lower to the incorporated adhesives. Regarding the confirmation of obtaining the strontium apatite nanoparticles, XRD analysis is conclusive. Once the diffraction patterns of the samples produced herein match satisfactorily with strontium apatite samples synthesized in the literature (JPCDS n� 00-033-1348) (Melnikov and Gonçalves, 2015; Gonçalves et al., 2012). Additionally, the increase in the cell parameters of the SrHAp nanoparticles in com­ parison to those ones of calcium apatite reported in a previous publi­ cation of our group also indicates the success in the preparation of SrHAp nanoparticles (Andrade Neto et al., 2016) (See Table 1). The increase in the cell parameters caused by the substitution of Ca for Sr is well-consolidated in the literature, and it is related to greater ionic radio of Sr (Geng et al., 2015, 2016). The characterization results (XRD and TEM) confirmed that the morphology of SrHAp5h nanoparticles, submitted to hydrothermal

H2O ν3 (CO23 ) ν3 (CO23 ) ν3 (CO23 ) ν3 (P–O)(so) ν3 (P–O)(so) ν3 (P–O)(so) ν1 (O–P–O)(ss) ν2 (CO23 ) OH free ν4 (O–P–O)(b) ν4 (O–P–O)(b) ν4 (O–P–O)(b) ν2 (P–O)(ss) ν2 (P–O)(ss)

1630 1469 1454 1415 1088vs 1065vs 1035vs 962s 874w 631s 602s 574s 565s 474w 462w

SrHAp2h

3430.9 – – – 1094.0 1066.8 1027.2 947.2 – – 619.2 589.8 561.0 415.5 459.2

HAp (Nelson and Williamson, 1982; O’Donnell et al., 2008)

SrHA0

– – – – 1047.5 1034 1029.5 950.2 – – 595.6 578.9 570.9 437.3 421.1

3.3. TEM

SrHA0 HAp (Nelson and Williamson, 1982; O’Donnell et al., 2008)

FT-IR Specimens

Table 2 Assignment of the bands obtained from the FTIR and Raman spectra of samples.

SrHAp5h

RAMAN

SrHAp5h

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Fig. 3. FTIR (A) and Raman (B) spectra for the SrHAp specimens.

treatment, was entirely rod-shaped, as predicted by XRD. The growth of HAp particles under hydrothermal conditions is a well-known result (Andrade Neto et al., 2016). Therefore, the same behaviour was ex­ pected for the SrHAp nanoparticles due to its structural similarity. XRD analysis showed that hydrothermal treatment induces preferential orientation in the [112] direction (See Fig. 2A and B). This behaviour is characteristic of nanorods, fact that has been confirmed through TEM (Figs. 4 and 5). Additionally, we found a secondary phase (SrHPO4) in the specimens SrHAp0h and SrHAp2h. This phase was converted to Sr10(PO4)6(OH)2, as the samples were submitted to longer hydrothermal treatments. We can speculate a mechanism in which this conversion happens by the analysis of the size of the particles. The dimensions of the particles were assessed by two methods, XRD and TEM analysis (See Table 1). In order to drive our conclusions concerning the mechanism we selected the TEM data, once this technique measures directly the size of the particles instead of the crystallite size (dimension measured by XRD). Furthermore, literature reports that XRD agrees well with TEM for particle smaller than 50–60 nm (Uvarov and Popov, 2013), which is not our case. Once the length of the nanorod increased with time of hydrothermal treatment, we suggest that the time of synthesis is essential to dissolve SrHPO4 phase under basic medium, releasing Sr2þ and PO34 ions (re­ action 3). These ions could have two paths: i) be attached to the already formed nanorods, through classical Lamer theory (You and Fang, 2016); ii) react with OH ions to form new SrHAp crystals (see reaction 4) that coalesced by shape-directed nanoparticle attachment followed by straightening and orientation yielding nanorods with larger lengths (Liao et al., 2012). We believe that the second path of the Sr2þ and PO34 ions for the synthesis of our SrHAp particles is consistent due to following assumptions: a) we can see greater number well-defined rod-like nanoparticles for the samples SrHAp2h and 5h, and b) the length of the measured nanords by TEM increase as a function of the time of hydrothermal treatment; c) furthermore, we observed, for the samples HApSr2h and 5h nanoparticles, the process of oriented attach­ ment coalescence process (See Fig. 5A and B); d) it was evidenced an increase in the relative intensity of the peak [112] for the sample HApSr5h, compared to the other samples (See Fig. 2A and B); e) Geng and co-authors reported that Sr2þ ions induce an accelerated growth of

the c-plane, producing particles greater aspect ratio (Geng et al., 2015); f) The reactions of dissolution and recrystallization were already re­ ported for the formation HAp crystals in the work of Stojanovi�c and collaborators, and we extrapolate for the synthesis proposed herein (Stojanovi�c et al., 2016). 3 SrHPO4ðsÞ þ OHðaq:Þ → Sr2þ ðaq:Þ þ PO4ðaq:Þ þ H2 OðlÞ

(3)

3 10Sr2þ ðaq:Þ þ 6PO4ðaq:Þ þ 2OHðaq:Þ →Sr10 ðPO4 Þ6 ðOHÞ2ðsÞ

(4)

In our previous publication (Andrade Neto et al., 2016), in which we used a similar hydrothermal methodology to prepare nanorods of HAp, we did not find any other calcium phosphate phase, however the SrHPO4 phase evidenced herein. The absence of additional phases in the hy­ drothermal synthesis of HAp is due to the greater solubility of CaHPO4 compared to SrHPO4, which causes the dissolution reaction to occur even before the hydrothermal treatment. SrHAp or Sr-doped apatite materials have shown potential for several applications, such as bioelectronics materials (AL-Wafi et al., 2017), drug delivery (Scudeller et al., 2017), dental implants (Hori et al., 2017; Park et al., 2010), and bone regeneration (Frasnelli et al., 2017; Landi et al., 2007; Ehret et al., 2017). However, to the best of our knowledge, there is not report on the potential application of SrHAp as radiopaque agent. Nevertheless, Sr-based materials have already been applied as radiopaque agent for mainly injectable calcium phosphate cements for orthopedic applications, and as filler for dental materials. According to the American Dental Association (ADA) recommenda­ tion, adequate radiopacity is one of requisites of dental materials (Ob­ stacles to the developm, 1989). Filler type and amount of radiopaque fillers influence the degree of radiopacity of resin-based materials (Kapila et al., 2015). The radiopacity of dental adhesives aids the clinical to distinguish a restorative material from the adjacent decalcified dentin and secondary caries avoiding replacing the restoration unnecessarily. Inadequate opacity of adhesive resins used under composite restorations makes diagnosis more challenging and confusing. Radiographic detec­ tion of secondary caries because the thick layer of adhesive system makes incipient caries invisible (Kurs¸un et al., 2012). Several methods have been introduced for assessing the radiopacity of dental materials, including digitalized and digital radiography, 6

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Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103447

Fig. 4. TEM micrographs (Left) (obtained at 50,000 magnification) and particle size-distribution curves of the samples (right) for the samples (A) SrHAp0h, (B) SrHAp2h and (C) SrHAp5h.

absorbance densitometry, and spectrophotometry (Poorsattar Bejeh Mir and Poorsattar Bejeh Mir, 2012). In this study, it was used the X-ray system as method of reporting the equivalent aluminum radiopacity of dental adhesives (See Fig. 6) (Gu et al., 2006). According to IS04049:2009 (International Organization for Standardization ISO, 2009), the aluminum step wedge may be produced from either a single block or stacking 1 mm-thick strips of aluminum. Each dental material should have radiopacity level equal to or greater than that of same thickness of aluminum step. Although of the SrHAp radiopacity has been lower than the enamel radiopacity, it was higher than that of control adhesive and adhesive containing calcium-based hydroxyapatite nanofillers. Indeed, the higher atomic mass of strontium in comparison to calcium may afford such augment in overall radiopacity of the adhesive. This result would help clinicians to visualize a line of restorative material separation with the subtractive dentin, decreasing the chances of misdiagnosing secondary

caries. Besides this, it makes possible to visualize the proximity of the restorations with dental pulp. Nevertheless, the crystal structure of fully substituted strontium hydroxyapatite is not as stable as the structure of calcium based hydroxyapatite (Sadat-Shojai et al., 2013; Zhang et al., 2014). Therefore, it is reasonable to expect some diminishing in me­ chanical properties. According to Leitune and collaborators’ study, the dental adhesive incorporated with HAp particles showed a radiopacity similar to the control and was lower than that of the dentin, thus not being favorable for this goal. However, it could increase the biocom­ patibility, conversion and bond strength (Leitune et al., 2013). The addition of some kind of fillers could prevent the hybrid layer degra­ dation and preserve its bonding efficacy overtime, inhibiting the collagen degradation (Toledano et al., 2012). The longevity of dental restorations is related to the quality of the polymer formed. The addition of filler decreases the relative amount of polymeric matrix, decreasing the stress of contraction and hydrolytic 7

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Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103447

Fig. 5. TEM micrographs evidencing growth coalescence process for the SrHAp nanoparticles prepared herein (obtained at 200,000 magnifications). Samples in the micrographs: (A) SrHAp2h and (B) SrHAp5h.

Fig. 6. (A) Image of X-ray exposed films with aluminum steps, tooth slab and different specimens: a – control; b – HApSr0h; c – HApSr2h; d – HApSr5h and e HAp. Results of (B) radiopacity in mmAl, (C) degree of conversion and (D) Young’s modulus of the modified dental adhesives. Different letters indicate significant dif­ ference (p < 0.05).

degradation. Inorganic fillers are less prone to degradation than poly­ meric matrix when they are adequately bonded to the polymer, which was not the case herein, once the nanofillers were not silanated (Sun et al., 2011). Non-polymerized monomers could be leached from the adhesive, leading to degradation of the hybrid layer (Ferracane, 2006). In the present study, the addition of SrHAp and HAp into dental adhesive did not influence the DC when compared to control. However, the presence of the particles reduced the Young’s modulus of the adhesives due to the formation of clusters that affected the polymer chains, leaving fragility points in the polymer. Further studies should be carried out to better understand the performance of the presence of nanoparticles incorporated in dental adhesives.

5. Conclusions The results of this study confirm that the hydrothermal method is a simple and efficient chemical synthesis route for the preparation of pure SrHAp nanoparticles. XRD patterns, Raman and FT-IR spectra indicated the crystalline phase of SrHAp5h, without secondary phases. Increased duration of hydrothermal treatment promoted the rearrangement of atoms in the crystalline lattice results in higher purity producing nanorod shape confirmed by TEM. Therefore, the dental adhesive incorporated with SrHAp may be able to improve the radiopacity of adhesives, despite slightly reducing the Young’s modulus. However, the degree of conversion is not affect by either calcium- or strontium8

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Journal of the Mechanical Behavior of Biomedical Materials 101 (2020) 103447

containing HAp nanofillers.

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