Sr2+-Substituted CaCO3 Nanorods: Impact on the Structure and

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Sr2+-substituted CaCO3 nanorods: impact on the structure and bioactivity Camila B. Tovani, Tamires M Oliveira, Alexandre Gloter, and Ana P. Ramos Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00017 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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Crystal Growth & Design

Figure 1. SEM images of rod carbonate particles with differ-ent sizes (horizontal lines), 50 nm (a-c), 100 nm (d-f), 200 nm (g-i), and 400 nm (j-l), and different Sr2+ molar fractions (X) (vertical lines), X = 0.10 (a-j), X = 0.50 (b-k), and X= 1.00 (c-l). 166x173mm (96 x 96 DPI)

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Figure 2. Dark field TEM images and EDS mapping of the 200-nm-diameter particles with different Sr2+ molar fractions X = 0 (a-c), X = 0.10 (d-f), X = 0.25 (g-i), X = 0.50 (j-l), X = 0.75 (m-o), and X = 1.00 (pr). The purple and green dots corresponded to Ca2+ and to Sr2+, respectively. 668x447mm (96 x 96 DPI)


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Figure 3: XRD patterns for the Sr2+-substituted CaCO3 particles with 200-nm diameter. The Sr2+ molar ratios (X) ranged from 0.00 to 1.00. The captions S, V, and C indicate the peaks assigned to the polymorphs strontianite (JPDCS 01-071-4899), vaterite (JPDCS 01-074-1867), and calcite (JPDCS 00-005-0586), respectively. 509x436mm (96 x 96 DPI)



Figure 4. Rotational average of the selected area diffraction pattern for nanorods assemblies with Sr2+ molar fractions X = 0.25 (a), X = 0.50 (b), X = 0.75 (c), and X = 1.00 (d) and the corresponding diffraction rings indexed for a Pmcn arago-nite-strontianite structure. 183x173mm (96 x 96 DPI)


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Figure 5. TEM bright field image of nanorods with Sr2+ molar fractions X = 0.25 (a), X = 0.50 (b), X = 0.75 (c), and X = 1.00 (d); TEM dark field image of the same nanorods with Sr2+ molar ratios X = 0.25 (e), X = 0.50 (f), X = 0.75 (g), and X = 1 (h); selected area diffraction patterns of single nanorods wit Sr2+ molar fractions X = 0.25 (i), X = 0.50 (j), and X = 0.75 (k); high-resolution images of samples X = 0.25 (l) and X =1.00 (m). The blue arrows indicate the crystalline domains in the HR-TEM image. 223x440mm (96 x 96 DPI)



Figure 6. (a) Raman spectra of the Sr2+-substituted CaCO3 particles with Sr2+ molar fractions ranging from 0.00 to 1.00, and (b) Position of the CO32- bands as a function of X. 472x732mm (96 x 96 DPI)


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Figure 7. SEM images of rod carbonate particles with 50 nm (a-c), 100 nm (d-f), 200 nm (g-i) and 400 nm (j-l) with X = 0.10 (a-j), X = 0.50 (b-k) and X= 1.00 (c-l) after 5 days SBF exposure. 399x413mm (96 x 96 DPI)



Figure 8. FTIR spectra of the rod carbonate particles with 200-nm diameter and Sr2+ molar fractions X = 0.10 (a), X = 0.50 (b), and X= 1.00 (c) before (black line) and after (red line) five days of exposure to SBF. 187x453mm (96 x 96 DPI)


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Sr2+-substituted CaCO3 nanorods: impact on the structure and bioactivity Camila B. Tovani†, Tamires M. Oliveira†, Alexandre Gloter‡, Ana P. Ramos†* †

Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil. E-mail: [email protected] . ‡

Laboratoire de Physique des Solides, Université Paris Sud, CNRS UMR 8502, F-91405 Orsay, France

ABSTRACT: Strontium is a natural trace element found in biominerals such as aragonitic coral skeletons and bone apatite. Sr2+substitution in biomaterials has been found to regulate the cellular metabolism thus enhancing bone healing. Even though Ca2+ substitution for Sr2+ has been described in many phosphate minerals, the impact of such substitution on bioactivity and structure in pure carbonate phases has not been explored. Therefore, here we used a biomimetic approach to synthesize carbonate particles with controlled size in which Ca2+ was progressively substituted for Sr2+. Through structural investigation by X-ray diffraction, Raman spectroscopy, electron microscopy techniques including high resolution transmission electron microscopy and electron diffraction we studied the precipitation mechanism of Sr-substituted CaCO3 nanorods showing that increasing Sr2+/(Ca2+ + Sr2+) molar fractions lead to stabilization of strontianite, a mineral from aragonite group, increasing the carbonate crystalline lattice and particle crystallinity. The in vitro bioactivity evaluation attested that the particles bioactivity is maintained even at high Sr2+ concentrations. These outcomes are fundamental for proper evaluation of the role Sr 2+ plays in carbonate based biomaterials properties and biomineralization and constitute a starting point to explore (Ca-Sr)CO3 particles as the next generation of bioactive materials for bone replacement.

14,15

INTRODUCTION The major challenge in the field of bone regeneration is to obtain a biomaterial with suitable bioactivity, biocompatibility and osteoconduction. The bioactivity, the ability of a biomaterial to induce apatite precipitation in biological .1 medium, is essential to biomaterial performance in vivo The apatite layer formed at the biomaterial/tissue interface 2 guides the first steps of the osteoconduction process. In this sense, among the countless materials that have been applied 3-6 to achieve this goal, biominerals have proven to be a prom7,6 ising choice. Biominerals are hybrid materials with excellent mechanical properties. Their organic component consists mainly of proteins and polysaccharides, whereas the most common inorganic phases are calcium carbonate (CaCO3), hydroxyapatite (Ca10(PO4)6(OH)2, designated HAp), and silica (SiO2). Carbonates and phosphates have been investigated as bone replacement materials—they chemically resemble the bone tissue inorganic phase (which has a hierarchical organization comprising a non-stoichiometric calci9 um-deficient apatite ), which accounts for their outstanding biocompatibility. Although the use of HAp to build implants is well established, CaCO3 has been increasingly studied as 10 2bone replacement material, not to mention that the CO3 ion is the main substitute in bone apatite (B-type substitu11 tion). This substitution underlies the many properties of biological apatite such as the high reactivity and enhanced collagen deposition and resorption of young bones as compared to stoichiometric HAp or HAp bearing A-type substi12,13 tution (OH group). This fact has motivated numerous studies regarding the conversion of CaCO3-based materials

to calcium phosphate minerals. Recently, we have employed CaCO3 tube-like particles as precursors to attain biomimetic HAp precipitation upon exposure to simulated 16 2+ 2+ body fluid (SBF). Ca and Sr have similar charge and size, 2+ 2+ so Ca can be replaced with Sr in the biological apatite 2+ lattice. Consequently, Sr , which is a trace element in the human body, preferentially accumulates in the skeleton. 2+ 2+ Moreover, Sr can substitute Ca in the carbonate crystal structure, and the former element occurs in aragonitic coral 17 skeletons and carbonate rocks. The mineral SrCO3 has been synthesized for application in the fields of biosensors and 18-20 optical materials. This mineral has also been used as a model to examine aragonite mineralization—SrCO3 is an isostructure of this CaCO3 polymorph, which is present in 21-22 natural systems like nacre, pearls, and corals. 2+

The fact that Sr is found mostly in young bone tissues has prompted several investigations into its biological role in bone tissue formation and into its application in osseocon23 24 25 ductive systems. In vitro and in vivo studies have shown 2+ that Sr administration has dual action: it increases osteoblast activity and diminishes osteoclast activity, to improve 26,27 bone mineral density and mechanical properties. Never2+ theless, issues concerning Sr oral administration must be 2+ considered. Sr incorporation in the bone tissue is less than 2+ one for every 10 Ca ions, which makes it difficult to en2+ hance bone properties by Sr oral administration. In this 2+ sense, Sr incorporation in the material that will be in con2+ tact with the tissue offers the advantage of direct Sr deliv28 ery at the specific bone site defect, at the desired dosage. In this regard, special attention has been devoted to substitut2+ 2+ 29-30 ing Ca for Sr in biomaterials to boost bone healing.


Crystal Growth & Design 2+

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SrCO3 particles have been used to produce Sr -rich phos32-34 2+ phate cements. The presence of Sr in the phosphate lattice leads to physical-chemical changes that directly impact the cement features, including mechanical resistance, 35 cellular response, and bioactivity. However, the innumerous methodologies to synthesize strontium rich compounds can generate products with similar composition but different physical chemical properties and consequently biological behavior. The lack regarding material characterization has 2+ caused contradictory reports about Sr participation in 36 biomaterial performance. To draw general conclusions 2+ about the role Sr plays in biomaterial performance, it is crucial to obtain materials with controlled size, crystallinity, and composition. In this context, the synthesis of a series of 2+ Sr -substituted carbonate particles with controlled features 2+ can be an important starting point to evaluate the role Sr has in biomaterial properties and to clarify some issues such as its impact on bioactivity and crystalline structure of car2+ 2+ bonates. Even though Ca substitution for Sr has been 9,37-42 described in many phosphate structures, such substitution has not been explored in pure carbonate phases. Therefore, here we used a biomimetic approach to synthesize 2+ 2+ particles in which Ca was progressively substituted for Sr 43 in the carbonate structure. The approach involved precipitating mineral particles in confined media, to mimic bio44 mineralization. Apart from synthesizing (Ca-Sr)CO3 particles for application in the field of bone regeneration, we also 2+ conducted a systematic study about how the Sr content affected the carbonate structural properties in order to ex2+ tract useful information concerning the biological role Sr plays in biomineralization.

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(SAED) patterns. High-resolution imaging (HR-TEM) and energy dispersive spectroscopy (EDS) mapping of the elements Ca and Sr were obtained on a single particle. The chemical groups were identified by Fourier-transform infrared spectroscopy (FTIR) coupled with an attenuated total reflectance (ATR) accessory (Shimadzu-IRPrestige-21), with -1 resolution of 2 cm . The X-ray diffraction patterns were acquired with a Bruker-AXS D5005 diffractometer with Cu Kα radiation. The peaks were indexed on the basis of the databank of the Joint Committee on Powder Diffraction. The coherent domain length was estimated in the direction of the planes (111) and (021) in the 2 = 25-27º region, according to the Scherrer equation. This length was also estimated by HRTEM. Interplanar distances (d values) allowed us to calculate the crystal lattice parameters and Mindat database was used to obtain the lattice parameters to the mineral strontianite. The Raman spectra were recorded on a MicroRaman LabRAM HR (HORIBA Jobin–Yvon, New Jersey, USA) combined with an Olympus microscope. A He/Ne laser operating at 0 = 632.81 nm was employed as the excitation source. The Digital Micrograph (Gatan) program was used to process the images.

EXPERIMENTAL (Ca-Sr)CO3 rod particle preparation. The particles were synthe43 sized by method described by Loste et al modified by To16 vani at al. Briefly, polycarbonate membranes with pore sizes measuring 50, 100, 200, and 400 nm were used as templates for rod particle growth. Poly(acrylic acid) (PAA) (0.1 wt%, -1 Sigma MW 1800 g.mol ) was dissolved in aqueous solutions containing CaCl2 (Merck P.A.), SrCl2(Synth P.A.), or a mix2+ 2+ 2+ ture of these salts containing Sr /(Ca + Sr ) molar frac2+ 2+ tions (X) ranging from 0 to 1. The total [Ca ] + [Sr ] concen-1 tration was 0.10 mol.L . The formation (Ca-Sr)CO3 particles occurred by a vapor diffusion mechanism in which (NH4)2CO3 decomposition at room temperature produces 2+ 2+ CO2(g) which reacts with Ca and Sr into the membrane pores. Bioactivity evaluation. Bioactivity assays are a classic strategy 45,46 to predict biological performance. Immersion in SBF at 37 ºC for five days helped us to evaluate particle bioactivity by its ability of inducing apatite formation in this simulated 47 biological medium . The procedure used to prepare SBF is 48 described elsewhere.

Figure 1. SEM images of rod carbonate particles with different sizes (horizontal lines), 50 nm (a-c), 100 nm (d-f), 200 nm 2+ (g-i), and 400 nm (j-l), and different Sr molar fractions (X) (vertical lines), X = 0.10 (a-j), X = 0.50 (b-k), and X= 1.00 (c-l).

Particle characterization. The morphology of the gold-coated particles was investigated by scanning electron microscopy (SEM) on a Zeiss-EVO 50 microscope under 20-kV accelerating voltage. A transmission electron microscope (TEM) FEI TECNAI G² F20 HRTEM operating at 200 kV was used to obtain the selected area TEM images and electron diffraction


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Table 1. Sr molar fraction (X) in the starting solutions and in the particles

RESULTS AND DISCUSSION Particles formation and chemical analysis. The (Ca-Sr)CO3 2+ 2+ particles emerged after the Ca and Sr ions were entrapped in the membrane pores used as templates. The positively 2+ 2+ charged Ca and Sr ions bound electrostatically to the negatively charged PAA chains. The polymer carrying the ions penetrated into the membrane pores, to form an organic matrix for subsequent mineralization. (NH4)2CO3 decomposition at room temperature produced CO2(g), which reacted 2+ 2+ with Ca and Sr ions and water entrapped in the membrane pores to give (Ca-Sr)CO3:

X in the solution

X in the product

0.00

0.00

0.10

0.09

0.25

0.20

0.50

0.50

0.75

0.80

1.00

1.00

(NH4)2CO3(s) → 2NH3(g) + CO2(g) +H2O(l) 2+

+

Ca (aq)+CO2(g) + H2O(l) → CaCO3(s) + 2H (aq)

Structural analysis. Figure 3 illustrates the powder XRD 2+

Figure 1 contains the SEM images of the particles, which presented well-defined rod morphology with diameters successfully tuned by the size of the template membrane pores. 2+ Table 1 summarizes the Sr molar fraction (X) in the final nanoparticles as evaluated by EDS coupled to a TEM micro2+ scope. The Sr molar fraction in the synthesized particles 2+ 2+ agreed with the Ca and Sr proportions in the starting solution and attested to incorporation of these ions in the 2+ 2+ rod particles. Figure 2 depicts the Ca and Sr distribution over the particle surface. Distribution was homogeneous, and there was no phase segregation.

patterns of the particles containing Sr at molar ratios (X) ranging from 0 to 1. The diffraction patterns evidenced that 2+ samples with Sr molar fractions X = 0.00 and X = 0.10 were a mixture of the calcite (space group R-3c) and vaterite (space group P63/mmc) CaCO3 polymorphs. The peaks related to vaterite were broader than the peaks relative to calcite, which indicated smaller particle size and poorer crystallinity.

2+

Figure 3. XRD patterns for the Sr -substituted CaCO3 parti2+ cles with 200-nm diameter. The Sr molar ratios (X) ranged from 0.00 to 1.00. The captions S, V, and C indicate the peaks assigned to the polymorphs strontianite (JPDCS 01-0714899), vaterite (JPDCS 01-074-1867), and calcite (JPDCS 00005-0586), respectively.

Figure 2. Dark field TEM images and EDS mapping of the 2+ 200-nm-diameter particles with different Sr molar fractions X = 0 (a-c), X = 0.10 (d-f), X = 0.25 (g-i), X = 0.50 (j-l), X = 0.75 (m-o), and X = 1.00 (p-r). The purple and green dots 2+ 2+ corresponded to Ca and to Sr , respectively.

This was attributed to the mechanism through which the particles originated. In nature, carbonate particles arise from 49 a sequence of precipitation and re-precipitation steps. The first step involves formation of a transient unstable amorphous precursor that is subsequently dissolved and reprecipitated in the vaterite phase, which will be later converted to calcite, the most thermodynamically stable CaCO3 polymorph. In this sense, the broader peaks verified for vaterite were due to its partial conversion to calcite. On another hand, besides the mineralization of CaCO3 be accompa50 nied by the competition between its different polymorphs as observed before, for the samples with X ranging from 0.25


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to X = 1.00 the Bragg reflections in the XRD pattern can be indexed with only one crystalline phase with strontianite structure (space group Pmcn) which is isomorphic with the 51 aragonite structure . The resulting polymorphs to the different X are summarized in the table 2. Table 2. Main polymorphs identified in the particles by XRD 2+ according to the Sr molar fractions (X). X

Polymorph

0.00

Calcite, Vaterite

0.10

Calcite, Vaterite

0.25

Strontianite

0.50

Strontianite

0.75

Strontianite

1.00

Strontianite

attested to the reduced degree of crystallinity in the latter 2+ samples. The samples with Sr molar fractions X = 0.25 and X = 0.50 exhibited very broad peaks, whilst the samples with 2+ Sr molar ratios X = 0.75 and X = 1.00 had better resolved peaks, which showed that crystallinity improved with higher 2+ Sr content in the samples with aragonite structure. According to the Scherrer equation, the coherent domains had typical size of ca. 10 nm for X = 0.25-0.50 to ca. 17 nm for X = 1.00. The diffraction peak shift to lower 2 values in the case of the samples with aragonitic structure was a consequence of larger d-spacing and lattice parameters due to incorpora2+ 2+ tion of the larger Sr ions. Increasing Sr content expanded the lattice parameters of the aragonite-strontianite structure by ca. 3 % on the basis of the (111) and (021) peak shifts, becoming similar to the values described in literature to strontianite (see Table 3). Table3. Interplanar distance (d-spacing), crystallite size (d), lattice parameter (a), and unity cell volume (V) obtained from the XRD patterns of the particles with 200-nm diamater 2+ and Sr molar ratios X ranging from 0.25 to 1.00 with strontianite structure

Figure 4. Rotational average of the selected area diffraction 2+ pattern for nanorods assemblies with Sr molar fractions X = 0.25 (a), X = 0.50 (b), X = 0.75 (c), and X = 1.00 (d) and the corresponding diffraction rings indexed for a Pmcn aragonite-strontianite structure. 2+

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In the aragonite structure, Ca ions coordinate to nine oxygen atoms, whereas these ions coordinate to six oxygen at.52 oms in the calcite and from 6 up to 8 in vaterite structures 2+ The presence of Sr ions, which have larger ionic radius as 2+ compared to Ca , favors coordination to nine oxygen atoms. For this reason, the aragonite structure is the most stable for 53 SrCO3. Similarly, other carbonates such as BaCO3 and PbCO3 precipitate with aragonite structure and have also been employed as models to study aragonite mineralization due to the difficult synthesis of pure aragonite in the labora54 2+ tory in the absence of additives. The samples with Sr molar fractions X = 0.00, X = 0.1, and X = 1.00 displayed better defined and sharper peaks as compared to the samples 2+ 2+ containing strong admixture of Ca and Sr ions, which

3

X

d spacing (nm)

d (nm)

a (nm)

V (nm )

0.25

0.345

10

0.499

0.240

0.50

0.347

11

0.502

0.245

0.75

0.350

10

0.506

0.251

1.00

0.355

17

0.513

0.261

Strontianite

-

-

0.511

0.260

The SAED (Figure 4) obtained for the nanorods with differ2+ ent Sr molar ratios X confirmed the XRD measurements. Lattice expansion and higher crystallinity occurred with 2+ increasing Sr molar fraction. For instance, the planes (002), (012), which were not visible at X = 0.25, became clear for the 2+ samples with Sr molar ratios X = 0.75-1.00, in agreement with the XRD patterns. Figure 5(a-d) and Figure 5(e-h) show the bright and the dark field TEM images recorded for the 200-nm-diameter rod particles. All the particles had nanorod structure with lateral width of ca. 200 nm and several micrometers in length. For X = 0.25, the dark field images (Figure 5e) revealed a rather homogeneous contrast, and the diffraction pattern obtained for a single rod (Figure 5i) showed a very broad ring with some preferred orientations for the nanodomains. The HR-TEM image of this sample (Figure 5l) confirmed that the nanorod consisted of crystalline domains measuring around 10 nm. Figures 5b and 5f contain the TEM images of the sample with 2+ Sr molar ratio X = 0.50. The corresponding diffraction pattern, exhibited in Figure 5j, clearly evidenced a preferential orientation and was indexed as a primary zone axis [01-1] with strong squared diffraction patterns from the (111) family planes. Many other spots assigned to fiber-type diffraction with rod oriented along the [100] direction emerged. The dark field image (Figure 5f) showed some well-diffracted nanocrystals with typical size still in the range of tens of nanometers. In accordance with the XRD measurements, the 2+ dark field images of the samples with Sr molar fraction X = 0.75 (Figure 5g) and X = 1.00 (Figure 5h) were typical of rods with higher crystallinity. The dark field contrast extended


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over the whole nanorod, with some heterogeneous intensity changes arising due to local texture misorientation. The 2+ corresponding diffraction patterns for the sample with Sr molar ratio X = 0.75 (Figure 5k) referred to a single orientation, which attested to long-range ordering. The HR-TEM 2+ images of the sample with Sr molar ratio X =1.00 (Figure 5m) exhibited crystalline domains that extended over several tens of nanometer, with surface lattice plane of (022) corresponding to a rod oriented along the [100] direction. This Figure also revealed the presence of disorders such as lattice distortions, grain boundaries, and some dislocations. Such defect explained why the XRD peaks of the 2+ samples with Sr molar ratios X = 0.75-1.0 were still rather broad despite their improved crystallinity.

2+

Figure 6. (a) Raman spectra of the Sr -substituted CaCO3 2+ particles with Sr molar fractions ranging from 0.00 to 1.00, 2and (b) Position of the CO3 bands as a function of X.

2+

Figure 5. TEM bright field image of nanorods with Sr molar fractions X = 0.25 (a), X = 0.50 (b), X = 0.75 (c), and X = 1.00 2+ (d); TEM dark field image of the same nanorods with Sr molar ratios X = 0.25 (e), X = 0.50 (f), X = 0.75 (g), and X = 1 (h); selected area diffraction patterns of single nanorods wit 2+ Sr molar fractions X = 0.25 (i), X = 0.50 (j), and X = 0.75 (k); high-resolution images of samples X = 0.25 (l) and X =1.00 (m). The blue arrows indicate the crystalline domains in the HR-TEM image.

Figure 6a displays the Raman spectra of the particles with 2+ Sr molar fractions ranging from 0.00 to 1.00. Two character-1 istic bands appeared at 275 and 150 cm in the spectrum of 2+ the sample with Sr molar fraction X = 0.00; these bands 2+ were related to the presence of calcite. Samples with Sr molar fraction X = 0.25-1.00 exhibited bands at 150 and 175 -1 cm , which confirmed formation of the polymorph arago55 nite, in accordance with the XRD data. Because the band assigned to vaterite overlapped with the band assigned to -1 calcite at 281 cm , we were not able to identify the presence of this polymorph in the Raman spectrum of the sample with 2+ Sr molar fraction X = 0.00. Because the frequency observed in the Raman spectra depends on lattice vibrations, Figure 6a demonstrated a general shift to lower wavenumbers with 2+ increasing Sr content in the samples. The main Raman 2-1 band around 1100 cm , assigned to CO3 vibration, shifted 2+ linearly to lower wavenumbers with increasing Sr molar ratio, as depicted in Figure 6b. The results we obtained for carbonate particles resemble the literature data reported for 2+ phosphates: Sr incorporation boosted phosphate crystallin31,32 2+ ity. According to the literature, Sr disturbed the apatitic 2+ structures when the Sr molar fraction was as high as X = 2+ 0.10. Higher Sr concentrations improved crystallinity as 2+ 2+ 31,56 Ca was replaced with Sr in the lattice structure. In this study, we noted improved particle crystallinity for increasing 2+ Sr molar ratios. Whereas the apatite structure did not


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2+

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2+

change upon Ca substitution with Sr , Sr favored formation of the polymorph aragonite in the case of carbonates.

Bioactivity evaluation. Figure 7 depicts the SEM images of the particles after exposure to SBF. The images revealed that 2+ all the particles, irrespective of size or Sr molar fraction, induced precipitation of a new phase consisting of spherical agglomerates of needle-like particles typical of hydroxyapatite.

Figure 7. SEM images of rod carbonate particles with 50 nm (a-c), 100 nm (d-f), 200 nm (g-i) and 400 nm (j-l) with X = 0.10 (a-j), X = 0.50 (b-k) and X= 1.00 (c-l) after 5 days SBF exposure.

Figure 8 shows the FITR spectra of the 200-nm diameter 2+ particles with Sr molar ratio X = 0.10 (a), X = 0.50 (b), and X= 1.00 (c) before (black line) and after (red line) exposure to SBF. All the spectra displayed bands ascribed to the presence 2-1 of the CO3 group at 1400 cm (asymmetric stretching) and -1 860 cm (out-of-plane bending vibration), confirming CaCO3 −1 formation. A narrow intense band at 1100 cm , assigned to ν3 3stretching of the PO4 group after exposure to SBF, confirmed that the needle-like particles corresponded to the apatite phase.

Figure 8. FTIR spectra of the rod carbonate particles with 2+ 200-nm diameter and Sr molar fractions X = 0.10 (a), X = 0.50 (b), and X= 1.00 (c) before (black line) and after (red line) five days of exposure to SBF.

Furthermore, for the particles synthesized in the absence and 2+ 3in the presence of Sr , the PO4 band was centered at 1040 -1 and 1020 cm , respectively, which agreed report of Wang et 56 2+ al and attested that Sr was released from the particles and subsequently incorporated in the phosphate lattice during its precipitation in SBF medium. This finding suggested that the


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mechanism through which apatite originated involved an interfacial dissolution-re-precipitation. Carbonate dissolu2+ 2+ tion at the surface raised the local Ca and Sr ion concen32+ tration and favored PO4 and Ca migration from the SBF leading to apatite precipitation. The apatite crystallization in 57 SBF is a complex phenomenon but its formation is thermo58 dynamically favored in such conditions. This is because SBF is strongly supersaturated with respect to HAp and not to 59 with respect to carbonates and other phosphates phases.

(5) Tran, P. A.; Fox, K.; Tran, N. Novel hierarchical tantalum oxide-PDMS hybrid coating for medical implants: One pot synthesis, characterization and modulation of fibroblast proliferation. J. Colloid Interface Sci. 2017, 485, 106-115. (6) Su, D.; Jiang, L.; Chen, X.; Dong, J.; Shao, Z. Enhancing the gelation and bioactivity of injectable silk fibroin hydrogel with laponite nanoplatelets. ACS Appl. Mater. Interfaces 2016, 8, 9619-9628. (7) Brunski, J. B.; Puleo, D. A.; Nanci, A. Biomaterials and biomechanics of oral and maxillofacial implants: current status and future developments. Int. J. Oral Maxillofac. Implants 2000, 15, 15-46.

CONCLUSIONS 2+

We have described a series of Sr -substituted rod-like CaCO3 particles in order to better understand the role played 2+ by Sr on the carbonate mineralization. The use of confined medium allowed the synthesis of cylindrical and monodisperse particles with tuned diameters. A detailed structural 2+ 2+ characterization confirmed that Sr and Ca were homogenously incorporated into the particles. Overall, crystallinity 2+ and lattice parameters augmented with rising Sr molar fraction in the carbonate particles. Bioactivity evaluation in vitro confirmed the potential application of these particles as 2+ a bioactive platform for Sr delivery. To sum up, high control over the physical-chemical features of the synthesized particles could be useful in a further detailed in vitro and in 2+ vivo investigation into the role Sr play in biomineralization.

AUTHOR INFORMATION

(8) Nandi, S. K.; Kundu, B.; Mukherjee, J.; Mahato, A.; Datta, S.; Balla, V. K. Converted marine coral hydroxyapatite implants with growth factors: in vivo bone regeneration. Mater. Sci. Eng., C 2015. 49, 816-823. (9) Habraken, W.; Habibovic, P.; Epple, M.; Bohner, M. Calcium phosphates in biomedical applications: materials for the future? Mater. Today 2016, 19, 69-87.

(10) Cruz, M. A. E.; Ruiz, G. C.; Faria, A. N.; Zancanela, D. C.; Pereira, L. S.; Ciancaglini, P.; Ramos, A. P. Calcium carbonate hybrid coating promotes the formation of biomimetic hydroxyapatite on titanium surfaces. Appl. Surf. Sci. 2016, 370, 459-468. (11) Dorozhkin, S. V. Bioceramics of calcium orthophosphates. Biomaterials 2010, 31, 1465-1485.

Corresponding Author *Ana P. Ramos, e-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank the São Paulo Research Foundation (FAPESP—grant 2014/24249-0 and 2017/08892-9) and the National Council of Technological and Scientific Development (CNPq- grant 44283412014-4) for financial support. Camila B. Tovani thanks FAPESP for the PhD scholarship.

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For table of contents use only

Sr2+-substituted CaCO3 nanorods: impact on the structure and bioactivity Camila B. Tovani†, Tamires M. Oliveira†, Alexandre Gloter‡, Ana P. Ramos†*

Synopsis: We have described a series of Sr2+substituted rod-like CaCO3 nanoparticles prepared by a biomimetic approach, in order to better understand the role played by Sr2+ on the carbonate mineralization. The physical and chemical properties associated to the bioactivity constitute a starting point to explore (Ca-Sr)CO3 particles as the next generation of bioactive materials for bone replacement.


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Sr2+-Substituted CaCO3 Nanorods: Impact on the Structure and

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