Synthesis and characterization of novel mesoporous strontium-modified bioactive glass nanospheres for bone tissue engineering applications

Journal Pre-proof Synthesis and characterization of novel mesoporous strontium-modified bioactive glass nanospheres for bone tissue engineering applications Amir Hossein Taghvaei, Forough Danaeifar, Christoph Gammer, Jürgen Eckert, Sadjad Khosravimelal, Mazaher Gholipourmalekabadi PII:

S1387-1811(19)30748-6

DOI:

https://doi.org/10.1016/j.micromeso.2019.109889

Reference:

MICMAT 109889

To appear in:

Microporous and Mesoporous Materials

Received Date: 16 August 2019 Revised Date:

8 November 2019

Accepted Date: 13 November 2019

Please cite this article as: A.H. Taghvaei, F. Danaeifar, C. Gammer, Jü. Eckert, S. Khosravimelal, M. Gholipourmalekabadi, Synthesis and characterization of novel mesoporous strontium-modified bioactive glass nanospheres for bone tissue engineering applications, Microporous and Mesoporous Materials (2019), doi: https://doi.org/10.1016/j.micromeso.2019.109889. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Synthesis and characterization of novel mesoporous strontium-modified bioactive glass nanospheres for bone tissue engineering applications

Amir Hossein Taghvaeia,1, Forough Danaeifara, Christoph Gammerb, Jürgen Eckertb,c, Sadjad Khosravimelald,e,f, Mazaher Gholipourmalekabadie,f

a

Department of Materials Science and Engineering, Shiraz University of Technology, Shiraz, Iran

b

Erich Schmid Institute of Materials Science, Austrian Academy of Sciences, Jahnstraße 12, 8700 Leoben, Austria

c

Department of Materials Science, Chair of Materials Physics, Montanuniversität Leoben, Jahnstraße 12, 8700 Leoben, Austria

d

Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran e

Cellular and Molecular Research Centre, Iran University of Medical Science, Tehran, Iran f

Department of Tissue Engineering & Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

Abstract Mesoporous bioactive glass nanoparticles (MBNs) have recently gained increasing attention as nanocarriers for co-delivery of therapeutic ions and molecules for treatment of hard tissue injuries. In the current study, novel Sr-substituted silica-based MBNs were synthesized through a template assisted sol-gel process. The results showed that the prepared nanoparticles are

1

Corresponding author. Tel: +989177038948, Email address: [email protected], [email protected]

1

amorphous with a spherical morphology and an average size between 86-115 nm, and their microstructure is not considerably changed by Sr incorporation. The produced nanoparticles have a disordered mesoporous structure and notably better dispersion, as well as higher textural properties including specific surface area (408-480 m2/g) and pore volume (0.54-0.62 cm3/g), compared to most Sr-modified mesoporous bioactive glasses synthesized to date. In-vitro mineralization experiments revealed that the synthesized nanospheres exhibit an excellent bioactivity within 3 days immersion in simulated body fluid (SBF) solution, irrespective of the Sr content. Moreover, the prepared nanospheres are ideal platforms for a sustained release of ibuprofen in SBF up to 7 days. In-vitro cell viability results proved that the ionic extracts of the Sr-loaded MBNs can markedly increase the proliferation of adipose tissue-derived stem cells (ADSCs), and Wharton's jelly-derived stem cells (WJSCs) compared to the performance of unloaded MBNs, at certain concentrations. Due to the spherical shape, excellent dispersion and promising physiochemical/biological properties, the newly synthesized Sr-loaded MBNs are encouraging filler materials for fabrication of nanocomposite scaffolds for treatment of bone defects.

Keywords: Mesoporous bioactive glass; Therapeutic ions; Bioactivity; Textural properties; Cell viability, Bone defects

1. Introduction Bioactive glasses (BGs) are very interesting materials for the regeneration of hard tissues, like bones, due to their bioactivity, osteoconductivity, osteoinductivity and their capability to enhance angiogenesis [1-4]. BGs can resorb in physiological fluids and their degradation products stimulate the osteogenesis through increasing cell proliferation and expression of osteogenic marker genes [1,2]. It is well-known that the bioactivity and the degradation rate of

2

BGs strongly depend on their composition, particles size and their textural properties, including specific surface area and pore volume. Mesoporous bioactive glasses (MBGs), as a new generation of bioceramics exhibit a higher degradation rate, in-vitro bioactivity and in-vivo bone formation in comparison with non-mesoporous BGs due to their notably higher surface area and pore volume [5-7]. Furthermore, compared to non-mesoporous BGs, MBGs can accommodate drugs, genes and growth factors inside their mesopores for a controlled release of such therapeutic molecules [8,9]. MBGs are usually synthesized by an acid-catalyzed template-assisted sol-gel method, like evaporation-induced self-assembly (EISS), in the form of micron-size particles or threedimensional (3D) porous scaffolds by combination of the EISS and replica methods [10-12]. In addition, 3D composite scaffolds exhibiting good osteogenic and mechanical properties can be prepared by introducing MBGs particles into a matrix of biopolymers [13-15]. It should be noted that the micron-size MBGs particles are frequently synthesized in an irregular and aggregated form and, therefore, their stability and uniform dispersion inside a polymeric matrix can be greatly suppressed. Furthermore, micron-size MBGs cannot be easily phagocytized by different cells and their insufficient enjectability can limit their use in some applications [16]. Recently, MBGs have been synthesized in the form of nanoparticles, in order to enhance their stability, enjectability and surface area for improving their drug-loading capacity and osteogenic potential [8,16]. Compared to micron-size MBGs, mesoporous bioactive glass nanoparticles (MBNs) are expected to have a more homogeneous dispersion inside the polymeric matrix, which in turn can level up the mechanical and biological properties of the prepared composite. Moreover, the small size of MBNs can facilitate their intracellular uptake by different cells, which can further increase their biological performance [8,17]. It should be noted that that MBNs are not only nanovehicles for delivery of therapeutic molecules, but they can be used as nanocarriers for a sustained release of therapeutic ions,

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stimulating the osteogenesis, angiogenesis and antibacterial properties. For instance, it was shown that Ag-doped MBNs can effectively internalize into dentinal tubules and inhibit the adherence and colonization of the E. faecalis bacteria, and they did not show any cytotoxicity on mesenchymal stem cells (MSCs) [18]. Furthermore, Lee et al. [19] demonstrated that MBNs can be used as nanocarriers for co-delivery of Ag+ ions and tetracycline or chlorhexidine, to regenerate infected dentin-pulp defects. Besides, Bari et al. [20] have recently produced MBNs with excellent textural properties, in-vitro bioactivity, and good antibacterial performance against different bacteria. Strontium is an important trace element in the body, which accumulates mostly in bones, with a fraction of about 0.035 % of the total calcium content [21]. Strontium plays a dual role in the bone remodeling process by increasing bone formation and decreasing bone resorption [22]. It has been shown that Sr2+ ions increase the cell proliferation and enhance the alkaline phosphate (ALP) activity and the expression of the osteoblastic-related genes [22-24]. On the other hand, Sr2+ ions can suppress osteoclastogenesis via upregulating the expression of osteoprotegerin (OPG) and preventing the expression of the receptor activator of nuclear factor kappa-B ligand (RANKL) in osteoblasts or MSCs [22]. Due to these promising biological roles, Sr2+ ions have been incorporated into MBGs in some works. Zhang et al. [25] showed that 3D MBG scaffolds modified with Sr exhibit good bioactivity, and a higher proliferation and osteogenic gene expression of osteoblastic-like cells (MC3T3-E1). Additionally, Wu et al. [26] indicated that while Sr addition notably deteriorates the textural properties of 3D MBG scaffolds, the bioactivity of the scaffolds is not impaired and osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) are greatly improved compared to MBG scaffolds alone. Although the potential of Sr-containing MBG scaffolds for bone regeneration and sustained delivery of therapeutic molecules has been studied in several works, the

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physiochemical properties and bioperformance of Sr-doped MBNs were rarely investigated. In the present work, novel Sr-doped MBG nanospheres with good dispersion, large surface area, excellent in-vitro bioactivity and promising biological properties were synthesized by a facile template-assisted sol-gel method. The influence of the different Sr contents on microstructure, physiochemical, textural and biological properties, as well as in-vitro mineralization, and drug release profile were systematically examined.

2. Experimental procedures 2.1. Sample preparation The Sr-modified MBNs with the compositions summarized in Table 1 were synthesized via a template-assisted sol-gel method, described in Ref. [16], with some modifications. The compositions were named as MBN0 (80SiO2-15CaO-5P2O5), MBN5 (80SiO2-10CaO-5SrO5P2O5)

and

MBN10

(80SiO2-10CaO-10SrO)

for

simplicity.

Initially,

1

g

poly(vinylpyrrolidone) (PVP) and 0.46 g NaOH were added to 120 ml double-distilled water, and then 1.4 g cetyltrimethylammonium bromide (CTAB) was added to this solution and stirred for 1 h. Subsequently, the precursors including calcium nitrate tetrahydrate (Ca(NO3)2.4H2O),

strontium

nitrate

(Sr(NO3)2),

triethylphosphate

(TEP)

and

tetraethylorthosilicate (TEOS) were added to the prepared solution and mixed for 24 h. The prepared suspension was hydrothermally treated at 353 K for a period of 48 h. The obtained mixture was centrifuged and the collected nanoparticles were washed three times with water and three times with ethanol, and then dried at 373 K for 10 hours. Finally, the powders were calcined at 823 K for 5 h using heating and cooling rates of 2 K/min.

2.2. Structural characterization

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To characterize the structure of the synthesized powders, wide-angle X-ray diffraction (XRD, PANalytical X’Pert PRO MPD) measurements were carried out in a 2θ range of 10-80 degrees, using Cu Kα radiation, with a step size of 0.02 degrees and a step time of 2 s. In addition, in order to study the mesoporous structure, small-angle XRD experiments (XRD, PANalytical X’Pert PRO MPD) with the same parameters as for the wide-angle ones were performed in the 2θ range of 1-8 degrees. The morphology, particle size and microstructure of the produced nanoparticles were studied by field-emission scanning electron microscopy (FE-SEM, TESCAN, MIRA3) coupled with energy dispersive spectroscopy (EDS) and high-resolution transmission electron microscopy (HRTEM, JEOL 2100F equipped with a CEOS imaging spherical aberration corrector). The size distribution of the prepared nanoparticles was determined using the microstructural image processing software (MIP 4.1 full; Nahamin Pardazan Asia). In addition, the local distribution of the elements in the prepared nanoparticles was determined by electron energy loss spectroscopy (EELS). For the TEM measurements, the nanoparticles were ultrasonically dispersed in ethanol and subsequently transferred to a carbon copper grid. The chemical bonds and surface functional groups were investigated by Fourier transform infrared spectroscopy (FTIR: IRAffinity-1S Shimadzu). The specific surface area, pore size distribution and pore volume of the synthesized nanoparticles were measured by the Brunauer-Emmet-Teller (BET) method. The mentioned parameters were determined by the Barrett-Joyner-Halenda (BJH) method, using the desorption branch of the isotherms.

2.3. Analysis of chemical composition The actual composition of the nanoparticles was determined according to the method suggested in Ref. [27] with some modifications. In brief, 50 mg of each sample were digested in 10 ml HF:HNO3:HCl (1:1:3) solution, and the resulting mixture was heated in a microwave at 423 K for 5 min and then at 503 K for another 5 min. The obtained solutions were cooled to room

6

temperature and subsequently diluted by distilled water to 100 ml. The composition of each solution was determined by inductively-coupled plasma atomic emission spectroscopy (ICPOES, Varian, 30-ES model). 2.4. Bioactivity investigations The in-vitro bioactivity of the synthesized nanoparticles was studied by soaking them in simulated body fluid (SBF), prepared according to the method suggested by Kokubo et al. [28]. For this purpose, the nanoparticles were soaked in the SBF at a ratio of 1.5 mg/1 ml and incubated at 37°C up to 7 days. The soaked samples were removed from the SBF after 3 and 7 days incubation and washed gently with distilled water and dried at 373 K for 10 h. Apatite formation was studied by FTIR and XRD measurements. In order to assess the bioactivity of the samples by SEM measurements, the nanospheres were compacted into the pellets before soaking in the SBF. The concentration of the Sr2+ ions released from pellets in the SBF after different immersion periods was evaluated by ICP measurements. The pH values of the SBF during the mineralization tests were measured by a pH meter.

2.5. Drug loading and delivery Ibuprofen (IBU) was employed as anti-inflammatory drug for studying the drug loading capability and release profile of the synthesized nanoparticles. For IBU loading, 200 mg of each powder were first added to 10 ml of hexane solution containing 33 mg/ml IBU and the resulting suspension was stirred for 24 h at room temperature in a sealed container. Afterwards, the particles were separated by centrifugation, washed with hexane and subsequently died at 340 K for 24 h. The IBU-loaded nanoparticles were characterized by FTIR and the content of the loaded IBU was measured by an UV-Vis spectrophotometer (Varian) at a wavelength of 264 nm, according to the method described elsewhere [29].

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In order to study the release profile of IBU, 45 mg of each drug-loaded sample were soaked in 30 ml SBF at 37 °C for several days. Afterwards, 2 ml of the obtained solution were taken for UV-Vis measurements after a certain incubation period and the same volume of fresh SBF (2 ml) was added to maintain the total volume of the solution at a constant value.

2.6. Cell viability The biocompatibility of the prepared nanospheres was evaluated by investigating the viability of different cells cultured in the nanospheres extracts. For preparation of the ionic extracts, 50 mg/ml of each sample (MBN0, MBN5 and MBN10) were soaked in Dulbecco`s Modified Eagle Medium (DMEM) at 37 °C for 24 h, according to ISO/EN 10993-5 [30]. The extracts were collected and freshly used for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For this purpose, 2 × 104 adipose tissue-derived stem cells (ADSCs), fibroblasts and Wharton's jelly-derived stem cells (WJSCs) were separately exposed to 25, 50 and 100 v/v% of the prepared extracts for 1, 3 and 7 days. The medium was changed after 3 days. At each time interval, the cells were treated with tetrazolium salt (MTT, Sigma-Aldrich, USA) for 4 h at 37 °C. Mitochondria of the living cells can reduce the tetrazolium dye MTT and form insoluble formazan crystals. The crystals can be dissolved in dimethyl sulfoxide (DMSO) and produce a purple color. The intensity of the color reflects the number of viable cells and their metabolic activity. After dissolving the formazan crystals in DMSO, the samples were shacked in dark and their optical density (OD) was measured using a plate reader (DANA, DA3200) at wave length of 570 nm. The viability of the cells in the control sample (the culture medium without extracts) was considered as 100% [31].

2.7. Statistical analysis

8

Data were analyzed by one-way ANOVA using SPSS statistical software (version 16.0; SPSS Inc, Chicago, IL). The distribution of data was defined by Kolmogorov‐Smirnov test. The results were expressed as mean ± SD. 3. Results and discussion 3.1. Compositional characterization The actual compositions of the synthesized MBNs are shown in Table 1. There are some differences between the designed (nominal) and the actual compositions. For instance, phosphorous is absent in the MBN0 and MBN5 samples, which indicates that this element is not incorporated into the MBN network upon synthesis. A phosphorous deficiency in the composition of the silica-based glasses synthesized under strong basic condition, like in case of the Stober process, has also been reported in previous studies [32]. This effect is caused by delayed hydrolysis of the TEP, in comparison with TEOS, which shows fast hydrolysis in basic conditions [32]. In other words, while the silica nanoparticles are spontaneously formed in basic solution, there is no available phosphorous species to enter the silica network, due to a lower hydrolysis rate of the TEP. Furthermore, TEP has a high water solubility, which can be simply removed during the centrifugation and subsequent washing process [32]. According to above discussion, one composition (MBN10) was produced without TEP addition during the synthesis for comparison. As shown in Table 1, all samples contain Na2O, originating from NaOH used during the synthesis. Moreover, there are some differences between the designed and the actual concentrations of Ca2+, and particularly Sr2+ ions. The fraction of incorporated ions can be affected by different parameters, like charge and ionic radii of the cations, the sequence of precursors addition, the pH and the temperature of the solution, and the type or content of the polymeric templates [29,32,33]. During the Stober process, the silica nanoparticles are initially formed and then grow via Ostwald-ripening [32]. The Ca2+ or Sr2+ cations are adsorbed on the surface of the negatively charged silica nanoparticles by

9

electrostatic attraction and formation of hydrated silicate or hydroxide species with [SiO4]4- and [OH]- groups, respectively [29,32]. Afterwards, the adsorbed ions can diffuse into the network of the silica nanoparticles during the calcination step [34]. The existence of several cations in the solution may decrease their adsorption on the surface of the silica nanoparticles due to electrostatic repulsion between the cations. In addition, larger ions (like Sr2+) may have a lower opportunity for surface adsorption due to a weaker electrostatic force with [SiO4]4- and [OH]groups. Furthermore, Sr(OH)2 has a larger water solubility than Ca(OH)2 [35], leading to a lower adsorption of Sr(OH)2 on the silica surface, and consequently, a smaller fraction of Sr in MBN5 and MBN10 compared to the designed values.

3.2. Particle size and morphology Fig. 1 displays FESEM images of the synthesized MBNs and the corresponding particle size distribution histograms. Irrespective of the composition, the particles have a spherical morphology with a good dispersion, and MBN0 and MBN5 display smoother surface compared to MBN10. The average particle size is 86 ±5 nm, 90 ±5 nm and 115 ±5 nm for MBN0, MBN5 and MBN10, respectively. It was shown that the type, content and time of addition of the metallic precursors during the Stober process can significantly affect the dispersion and size of the produced powders [32,36]. Previous researches indicated that the metallic precursors are usually added to the solution after the formation of the silica nanoparticles to prepare spherical and dispersed nanopowders [27,32,36,37]. However, a uniform size and excellent dispersion of the nanoparticles is achieved at the expense of a significant gap between the designed and actual composition [27,32,36,37]. The present work reveals that the prepared nanospheres, particularly MBN0 and MBN10, have still a good dispersion, although the silica precursor during the synthesis was introduced to the solution after addition of the metallic precursors. Moreover, according to Table 1, the differences

10

between the nominal and actual compositions are notably smaller compared to those reported for most BGs synthesized by the Stober process [27,32,36,37]. It is worth to note that the surfactants, specifically PVP, utilized during the synthesis of the nanospheres may contribute to their better dispersion by steric (polymeric) stabilization, besides the intrinsic electrostatic stabilization established in the basic solution [38]. Based on Fig. 1, MBN0 and MBN5 have an almost comparable average particle size, smaller than that of MBN10. The size discrepancy can originate from the difference between the ratio of Si and cations (Ca2+ and Sr2+) for MBN10 (80:20) compared to that of MBN0 and MBN5 (80:15). Additionally, it has been reported that Sr2+ with a lower field strength than Ca2+ can expand the glass network and thus increase the particle size [39]. The FESEM results reveal that MBN10 has a rougher particle surface (see the inset in Fig. 1(c)) than the other samples. The adsorption rate of smaller nanoparticles on the surface of larger ones and subsequent filling of the holes between small nanoparticles by molecular silica species can affect the surface roughness of the particles [27,40]. The content of metallic precursors can alter the equilibrium between the two mentioned phenomena, and consequently, change the surface roughness of the synthesized nanospheres.

3.3. Structural studies Fig. 2 depicts wide-angle XRD patterns of the synthesized MBNs. The absence of sharp Bragg peaks and the existence of broad diffuse diffraction maxima in the 2θ range of 20-35 degrees confirm their glassy nature. According to the patterns, no significant difference is observed for the shape, position and broadening of the diffuse maxima. This effect may signify that no remarkable discrepancies exist between the atomic structure of the MBNs. FTIR spectra of the MBNs are shown in Fig. 3. All samples exhibit the characteristic bands of silica-based glasses, located in the range of 400-1300 cm-1. The absorption band at

11

465 cm-1 is assigned to the rocking vibration mode of Si-O-Si, while the bands located around 802 cm-1 and 1070 cm-1 correspond to the symmetric and asymmetric Si-O-Si stretching modes, respectively [41,42]. The appearance of a small shoulder around 920 cm-1, particularly for MBN0 and MBN10, can be attributed to the stretching mode of Si-O groups, consisting of non-bridging oxygen (NBO) [43]. The intensity of this band, and consequently the number of NBOs, is determined by the concentration of the network modifier cations (Ca2+, Sr2+, Na+) in the silica network. According to Table 1, MNB5 has a higher silica content compared to the other samples, and a weaker intensity of the small shoulder at 920 cm-1 for this sample may indicate a lower content of NBO. The absorption band at 1630 cm-1 is assigned to the hydroxyl groups of water molecules adsorbed on the powders surface. Furthermore, the weak peaks (inset in Fig. 3) located around 1420 cm-1 and 1510 cm-1 for MBN0 are assigned to carbonate groups, coordinated with the Ca2+ cations on the surface of the nanoparticles [32]. Such absorption bands are very weak for MBN5 or MBN10 probably due to their lower Ca2+ content (see Table 1). According to Fig. 3, it is anticipated that the incorporation of Sr2+ ions does not notably affect the shape and position of the characteristic absorption bands and, hence, the atomic structure of the prepared glasses, as also confirmed by the XRD measurements (Fig. 2). The N2 adsorption-desorption isotherms of the nanoparticles and the corresponding pore size distribution, determined from the desorption branch, based on the BJH method are shown in Fig. 4. In addition, the BET surface area (SBET), the pore volume (VP) and the average pore size (DP) of the samples are listed in Table 2. As the figure shows, all samples exhibit IV type isotherms, with a well-defined step between P/P0 of 0.3 and 0.4, characteristic of MCM-41type mesoporous materials [44, 45]. The slope of the mentioned step is not notably changed in the samples, which demonstrates that the size distribution of the mesopores is not drastically changed. According to Fig. 4(a), by increase of the N2 pressure towards the saturation point (P/P0 ~ 1), a large quantity of gas is adsorbed through capillary condensation inside the large

12

pores that exist between the nanospheres. Based on Table 2, all samples have a large pore volume and a high surface area due to their fine particle size and mesoporous nature. It is worth noting that the synthesized nanospheres in the present work have a significantly better textural property compared to most Sr-containing MBGs that have been prepared recently [25,26,46]. Such a larger pore volume and surface are highly beneficial for loading therapeutic molecules, such as drugs, growth factors and genes [5,8]. According to Table 2, the pore volume and SBET of the MBN5 are slightly larger compared to those of MBN0, while these parameters are notably decreased for MBN10. Similar to the current results, suppressed textural properties were also observed in previous works for excessive replacement of Ca2+ with Sr2+ cations [26]. Indeed, formation of the MCM-41-type mesoporous structure is a consequence of the electrostatic interaction and subsequent self-assembly of the silicate species (I-) and the micelles containing the surfactant molecules (CTA+) [44]. The excessive addition of the Sr2+ cations, with a larger size compared to Ca2+ can disrupt the electrostatic interaction and interfere with the cooperative templating mechanism, which is essential for formation of the well-defined mesoporous structure. As a result, a more defective mesoporous framework, and thus a lower surface area and pore volume are expected for MBN10 compared to other samples. Fig. 4(b) depicts the pore size distribution of the synthesized nanospheres. MBN0 shows a narrow pore size distribution in the range of 2-4 nm with an average size of 2.8 ±0.1 nm. On the other hand, for MBN5 and MBN10, most of the mesopores have a size range between 2-3.5 nm and a smaller fraction of mesopores has a larger size, in the range of 3.5-5 nm. The average pore size for both MBN5 and MBN10 is 3.1 ±0.1 nm, which is a little larger than that of MBN0. Based on Fig. 4(b), a narrow pore size distribution for all samples is in line with a uniformity of the created mesopores inside the synthesized nanoparticles. In contrast, as the

13

figure shows, the larger pores existing between the nanospheres have a wide size distribution in the range of 25-130 nm. The microstructure and textural properties of the nanospheres were further studied by TEM analysis. Fig. 5 shows TEM images at different resolutions and the corresponding selected-area electron diffraction patterns (SAED) of the produced nanoparticles. The TEM images show no lattice fringes and the SAED pattern of each sample display only diffuse halo feature. Therefore, in agreement with the XRD data (Fig. 2), the TEM results confirm the glassy nature of the nanospheres. Additionally, high-resolution images (Figs. 5(b), (d) and (f)) reveal that the internal mesopores have a disordered (‘worm-like’) structure with a relatively uniform size and distribution. The Fourier-transform (FT) images (insets in the high-resolution images) reflect only diffuse patterns, confirming the disordered nature of the mesopores. The HRTEM images show that the mesopores are mainly around 3 ±0.1 nm for each sample, while few larger pores are observed (see Fig. 5(f)), perhaps due to the interconnection of smaller pores. The distribution of Ca and Sr inside the nanospheres was studied by EELS analysis (Fig. 6). It was confirmed that these elements are distributed homogeneously inside the nanoparticles. It has been suggested that such chemical homogeneity is a consequence of the interaction between the surfactant (CTAB) and inorganic species [29]. The mesoporous structure of the synthesized nanoparticles was further studied by lowangle XRD measurements, as shown in Fig. 7. In contrast to MCM-41-type mesoporous materials, which show sharp Bragg peaks around 2θ = 2 and 4 degrees, diffuse diffraction maxima with a comparable sharpness and broadening are observed for all samples in the 2θ range of 1-3 degrees. This result indicates a weak ordering of the mesoporous structure in the prepared nanoparticles, irrespective of their composition, as proved also by TEM. According to Fig. 7, the position of the diffuse diffraction maxima shifts to smaller angles with increasing Sr

14

content. This effect is possibly caused by a slight increase of the average pore size (see Table 2) or pore wall thickness, which is correlated with the larger radius of Sr2+ cations compared to Ca2+ ones.

3.4. Bioactivity assessment Fig. 8 presents FTIR spectra of the prepared nanospheres after immersion in SBF for different periods. Compared to the as-prepared samples (Fig. 3), new double bands are formed at 560 cm-1 and 600 cm-1 for all samples after soaking for 3 days (Fig. 8(a)). These bands are characteristics for the bending mode of the P-O bonds in crystalline hydroxyapatite (HA) [47]. Additionally, the band appearing at 960 cm-1 can be assigned to the Si-O bonds of silanol groups, formed during the ionic exchange of the glass with SBF. The bands at 875 cm-1, 1420 cm-1 and 1500 cm-1, particularly for MBN0, reflect the stretching vibration of the C-O bonds in the carbonate group [47]. This indicates that carbonated HA (CHA) is formed on the surface of MBN0. According to Fig. 8(b), the intensity of the HA bands levels up for each sample with increase of the soaking time up to 7 days, due to the enhanced content and crystallinity of the deposits. As the figure shows, the characteristic bands of HA are slightly sharper for MBN0 after 7 days immersion compared to other samples. This suggests that the Sr content can alter the crystallinity or thickness of the HA layer. The formation of bone-like HA and its morphology was further studied by SEM measurements. For this purpose, the synthesized nanospheres were compacted into pellets before immersion in SBF. Fig. 9 shows SEM images of the pellets after soaking in SBF for 3 and 7 days. Based on Fig. 9(b), the surface of the MBN0 disc is almost covered by HA crystals with a typical cauliflower-like morphology after 3 days immersion in SBF, which indicates an excellent bioactivity of this sample. In contrast, spherical deposits with a particle size between 50-100 nm are formed on the surface of the MBN5 and MBN10 discs after similar immersion

15

time (Figs. 9 (e) and (h)). These findings indicate that the presence of Sr2+ cations causes the morphological change of the bone-like HA from the well-known cauliflower-type to spherical shape. Similar results were found previously, indicating that the morphology of biomimetic HA is changed from plate-like for pure HA to sphere-like for Sr-substituted HA [48]. According to Fig. 9, the fraction of deposited HA increases with increase of the immersion period. The surface of all samples is nearly covered by HA crystals after 7 days of soaking in SBF. EDS analysis (not shown) revealed the presence of P on the surface of the pellets after the bioactivity experiments, and the Ca/P ratio was lower than 1.67 for all samples, which confirms the formation of bone-like calcium-deficient HA. Besides, for MBN5 and MBN10, the deposited HA undergoes a morphological variation with increasing soaking time from a spherical to a needle-like or cauliflower-like shape, respectively (Figs. 9(f) and (i)). By considering the same morphology of the HA on the MBN0 and MBN10 pellets after 7 days of immersion, slightly finer HA crystals are formed on the surface of the MBN10 pellets. It has been reported that the suppressed crystallinity of HA is a consequence of a partial substitution of Sr for Ca (both Ca-1 and Ca-2 sites) in the HA crystals [47]. The phase evolution of the synthesized nanospheres after immersion in SBF for 7 days was further studied by XRD analysis (Fig. 10). As can be seen, several diffraction peaks are visible at 2θ values of about 26, 32, 46.5 and 50 degrees for all samples, which are assigned to the (0 0 2), (2 1 1), (2 2 2) and (2 1 3) planes of HA [47]. Furthermore, the position of the main HA peak (located near 26 degrees) shifts somewhat to lower angles with increasing Sr content in the glass composition. The substitution of Sr

2+

ions with a larger size (118 pm) for Ca2+ ions

with smaller ionic radii (100 pm) in the HA lattice is responsible for the observed lattice expansion and a reduced crystallinity of the HA crystals, as mentioned above [47]. The presented results clarify that although Sr2+ cations marginally decrease the crystallinity of the HA deposits, the in-vitro bioactivity of the prepared nanospheres does not

16

significantly change. Indeed, formation of a mesoporous structure with a large pore volume and high surface area plays a crucial role for the excellent HA-forming ability of the synthesized nanoparticles. Even though excessive Sr addition is detrimental for the textural properties, the obtained surface area (> 400 m2/g) and pore volume (> 0.54 cm3/g) are still large enough for good chemical reactivity, and consequently, a promising in-vitro mineralization. According to the above results, a high bioactivity of the produced MBNs can be beneficial for their in-vivo bone bonding ability, as well as for enhanced osteoblast proliferation and differentiation [25]. The release of Sr2+ ions from the prepared pellets after soaking in SBF for different times was also studied. As s shown in Fig. 11, the concentration of Sr2+ ions gradually raises in the SBF with increase of the soaking period. As expected, the content of Sr2+ ions in the solution is higher for pellets of MBN10 compared to MBN5. It has been reported that Sr2+ ion concentrations within 0.1-1 mM (8.7-87.6 ppm) are highly favorable for stimulating bone formation, as well as for inhibiting osteoclastogenesis and bone resorption [49]. The present results indicate that the prepared samples are able to work as platforms for a sustained release of Sr2+ ions in a desired concentration range. It is worth to note that immersion of the nanospheres in the SBF does not significantly change its pH; moreover, the measured pH variations are almost similar for the three different samples. Additionally, the maximum pH value reached near 7.58, indicating that the surrounding environment of the synthesized nanospheres is safe for cell growth and differentiation.

3.5. In-vitro drug release Ibuprofen (IBU), as an anti-inflammatory drug, was loaded inside the mesopores and its release profile was studied in SBF, as shown in Fig. 12. The existence of the IBU molecules on the surface or inside the pores was confirmed by FTIR measurements (not shown for brevity). The

17

amounts of IBU loaded for MBN0, MNB5 and MBN10 are 8.5 ± 0.1, 8.4 ± 0. 1 and 8 ± 0.1 wt.%, respectively. As expected, the uptake of IBU is slightly reduced, due to a decrease of the surface area and pore volume with increase of the amount of Sr in the glasses. According to Fig. 12, the release profiles of IBU are similar for all samples, indicating a burst release up to 24 h, and a subsequent sustained release, with a decreasing rate up to 192 h. About 51% of the loaded IBU is released after 24 h and it gradually increases to about 70 % after 192 h. The initial burst release is related to IBU molecules, physically adsorbed on the external surface of the nanospheres or inside the mesopores. On the other hand, IBU molecules that are hydrogen-bonded with the hydroxyl groups inside the mesopores, diffuse out into the solution in a sustained way, when the SBF infiltrates into the pores and dissolves the IBU. In this case, the drug release takes place in a diffusion-controlled mechanism and the release profile can be well-described by the Higuchi model, according to the following equation [50,51]:

C = Kt 0.5 ,

(1)

where C is the drug concentration, K is a constant and t is the immersion time. The inset in Fig. 12 shows that the IBU concentration changes linearly with t0.5, which confirms that the release profile follows a diffusion-controlled mechanism for all samples between 24 h to 192 h. It should be noted that the diffusion of IBU into the bulk of SBF is also affected by degradation of the nanospheres and subsequent formation of HA crystals on their surface.

3.6. Cell viability The results obtained from the MTT assay for viability of different cells cultured with the ionic extracts of the synthesized MBNs at different concentrations are shown in Fig. 13. The cell

18

viability, particularly for ADSCs and WJSCs, exhibits a strong correlation with the concentration of the extracts. At day 3, the 100 v/v % extract shows a significantly lower viability for ADSCs and WJSCs, specifically for Sr-containing samples. This finding reveals that the concentration of Sr in the 100 v/v % extract probably exceeds the range favorable for cell growth, and consequently, results in cytotoxicity. On the other hand, the 25 and 50 v/v % extracts show good cytocompatibility for all samples after 3 and 7 days. Fig. 13 reveals that 25 v/v % extract of MBN10 has a larger viability for ADSCs after 3 days, compared to that of MBN0. A similar stimulatory effect on the WJSCs growth is detected after 7 days for MBN10 (25 v/v % extract), in comparison with MBN0.

Recent studies have reported that the

synergistic effect of Sr with other ions, particularly silicates, can drastically improve the proliferation and subsequent differentiation of MSCs [22,39]. It has been shown that Sr can activate the nuclear factor of activated T-cells (NFATc1) signaling pathway in MSCs, thereby increasing their proliferation [39]. According to Fig. 13, fibroblasts show less sensitivity to the concentrations of the extracts compared to MSCs, and Sr exhibits no substantial stimulating effect on the viability of fibroblasts.

4. Conclusions Silica-based MBNs with different fractions of Sr were synthesized by a facile templateassisted sol-gel method. Analysis of the chemical composition revealed that the incorporated contents of Sr are smaller than the designed values. The synthesized nanoparticles have a spherical morphology with a good dispersion, and their average particle size increases from 86 nm to 115 nm with increased Sr loading. BET analysis indicated that while the textural properties are suppressed with excessive Sr addition, the prepared nanospheres loaded with Sr still show a large exposed surface area (> 400 m2/g) and pore volume (> 0.54 cm3/g). HRTEM and low-angle XRD results confirmed that the prepared nanospheres possess a ‘worm-like’

19

mesoporous structure and the average size of the mesopores becomes slightly larger with increasing Sr content. Although the incorporation of Sr affects the crystallinity and morphology of the precipitated hydroxyapatite, the produced nanospheres exhibit promising invitro bioactivity. Furthermore, the prepared nanoparticles can be utilized as nanocarriers for sustained delivery of ibuprofen in the SBF. While the ionic extracts of the Sr-loaded nanospheres are not cytotoxic against fibroblasts, they show a stimulatory effect on the proliferation of ADSCs and WJSCs in a concentration dependent manner.

Acknowledgements A.H. Taghvaei is grateful to Shiraz University of Technology for supporting this research.

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Figure captions Fig. 1. FESEM images of MBN0 (a), MBN5 (b) and MBN10 (c) and corresponding particle size distribution histograms. Fig. 2. Wide-angle XRD patterns of the synthesized nanospheres. Fig. 3. FTIR spectra of the prepared nanospheres.

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Fig. 4. Nitrogen adsorption-desorption isotherms (a) and corresponding pore size distributions (b) of the prepared nanospheres. Fig. 5. TEM images with corresponding SAED patterns and HRTEM images with corresponding FT patterns for MBN0: (a) and (b), MBN5: (c) and (d), and MBN10: (e) and (f). Fig. 6. TEM image of MBN10 (a) and the corresponding mapping of Ca (b) and Sr (c) obtained by EELS analysis. Fig. 7. Low-angle XRD patterns of the produced nanospheres. Fig. 8. FTIR spectra of the nanospheres after soaking in SBF for 3 days (a) and 7 days (b). Fig. 9. SEM images of the discs prepared from MBN0 (a), MBN5 (d) and MBN10 (g) before immersion in SBF, MBN0 (b), MBN5 (e) and MBN10 (h) after soaking for 3 days and MBN0 (c), MBN5 (f) and MBN10 (i) after 7 days immersion in SBF, respectively. Fig. 10. XRD patterns of the nanospheres after 7 days immersion in SBF. Fig. 11. Concentration of Sr2+ ions released from compacted nanospheres after immersion in SBF for different periods. Fig. 12. Ibuprofen release profiles of the synthesized nanospheres in SBF and corresponding Higuchi plots shown in the inset. Fig. 13. MTT assay of various concentrations (25%, 50% and 100% v/v) of MBN0, MBN5 and MBN10 extracts (50mg/ml) for adipose tissue-derived stem cells, fibroblasts and Wharton's jelly-derived stem cells. * and ** indicate significant difference with control (cell culture medium without MBN extracts) and MBN0, respectively (p-value < 0.05). The viability of the cells in the control sample was considered as 100% (dashed line).

27

Table captions Table 1: Nominal and actual compositions of the synthesized nanospheres. Table 2: Textural properties of the prepared nanospheres.

28

Table 1 Nominal composition (at. %) 80 SiO2-15 CaO-5 P2O5 (MBN0) 80 SiO2-10 CaO-5 SrO-5 P2O5 (MBN5) 80 SiO2-10 CaO-10 SrO (MBN10)

Actual composition (at. %) 79.4 SiO2-16.7 CaO-3.9 Na2O 85.8 SiO2-10.7 CaO-0.8 Na2O-2.7 SrO 82.2 SiO2-12.6 CaO-0.2 Na2O-5 SrO

Table 2 Sample

Surface area (m2/g)

Pore volume (cm3/g)

Average pore size (nm)

MBN0

463

0.59

2.8

MBN5

480

0.62

3.1

MBN10

408

0.54

3.1

Fig. 10

Fig. 11

Fig. 12

Fig. 13

Fig. 1

Fig. 2

Fig. 3

(a)

(b)

Fig. 4

Fig. 5

Fig. 6

Fig. 7

(a)

(b) Fig. 8

(a)

(d)

(g)

(b)

(e)

(h)

(c)

(f)

(i)

Fig. 9

(c)

Highlights

•

Sr-doped mesoporous bioactive glass nanospheres with good dispersion were produced.

•

Worm-like mesopores with average size between 2.8-3.1 nm were formed.

•

The nanospheres exhibit excellent bioactivity, irrespective of Sr content.

•

The nanospheres are suitable platforms for sustain- release of ibuprofen.

•

The ionic extracts of the prepared nanospheres significantly improve cell viability.

Author Contribution Statement

1- Amir Hossein Taghvaei: Writing the manuscript, designing the compositions, methods, discussing the results and supervising the M.Sc. student (Forough Danaeifar) 2- Forough Danaeifar: nanoparticles synthesis and their characterization. 3- Christoph Gammer: HRTEM/EELS measurements. 4- Jürgen Eckert: HRTEM/EELS measurements. 5- Sadjad Khosravimelal: performing the biological experiments. 6- Mazaher Gholipourmalekabadi: Discussing the biological results.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: