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Competing Roles of Substrate Composition, Microstructure, and Sustained Strontium Release in Directing Osteogenic Differentiation of hMSCs Sunil Kumar Boda,†,‡ Greeshma Thrivikraman,†,‡,§ Bharati Panigrahy,⊥ D. D. Sarma,*,⊥ and Bikramjit Basu*,‡,∥ ‡

Laboratory for Biomaterials, Materials Research Centre, §Centre for Nano Science and Engineering, ⊥Solid State and Structural Chemistry Unit, and ∥Centre for Biosystems Science and Engineering, Indian Institute of Science, Bengaluru 560 012, India S Supporting Information *

ABSTRACT: Strontium releasing bioactive ceramics constitute an important class of biomaterials for osteoporosis treatment. In the present study, we evaluated the synthesis, phase assemblage, and magnetic properties of strontium hexaferrite, SrFe12O19, (SrFe) nanoparticles. On the biocompatibility front, the size- and dosedependent cytotoxicity of SrFe against human mesenchymal stem cells (hMSCs) were investigated. After establishing their non-toxic nature, we used the strontium hexaferrite nanoparticles (SrFeNPs) in varying amount (x = 0, 10, and 20 wt %) to consolidate bioactive composites with hydroxyapatite (HA) by multi-stage spark plasma sintering (SPS). Rietveld refinement of these spark plasma sintered composites revealed a near complete decomposition of SrFe12O19 to magnetite (Fe3O4) along with a marked increase in the unit cell volume of HA, commensurate with strontium-doped HA. The cytocompatibility of SrHA-Fe composites with hMSCs was assessed using qualitative and quantitative morphological analysis along with phenotypic and genotypic expression for stem cell differentiation. A marked decrease in the stemness of hMSCs, indicated by reduced vimentin expression and acquisition of osteogenic phenotype, evinced by alkaline phosphatase (ALP) and collagen deposition was recorded on SrHA-Fe composites in osteoinductive culture. A significant upregulation of osteogenic marker genes (Runx2, ALP and OPN) was detected in case of the SrHA-Fe composites, whereas OCN and Col IA expression were similarly high for baseline HA. However, matrix mineralization was elevated on SrHA-Fe composites in commensurate with the release of Sr2+ and Fe2+. Summarizing, the current work is the first report of strontium hexaferrite as a non-toxic nanobiomaterial. Also, SrHA-based iron oxide composites can potentially better facilitate bone formation, when compared to pristine HA. KEYWORDS: strontium hexaferrite, hydroxyapatite, Rietveld refinement, human mesenchymal stem cells, osteogenesis, nanobiomaterial

1. INTRODUCTION Strontium ranelate is used clinically to treat osteoporotic patients in order to reduce the risk of bone fractures. However, the oral administration of this drug has been implicated to cause side effects such as cardiovascular complications, allergic skin reactions, and kidney failures.1 To overcome these systemic effects, researchers are developing a host of strontium-releasing orthopedic biomaterials, such as strontium-doped bioglasses and calcium phosphate-based ceramics. The chemical similarity of Sr2+ to Ca2+ in terms of ionic size and polarity facilitates easy ion exchange of Sr2+ with Ca2+ in the bone tissue.2 The biological significance of strontium ion (Sr2+) stems from its role in bone homeostasis, wherein it inhibits osteoclast activity and encourages osteoblast growth and differentiation.3 The cellular mechanisms of action of strontium on bone remodeling are based on the fact that Sr2+ induces apoptosis in osteoclast cells preventing bone © XXXX American Chemical Society

resorption, while it is actively incorporated into the apatite of the neobone formed.4 At the molecular level, strontium has been identified to play a key role in bone formation by activating calcium sensing receptors (CaSR) in osteoblasts, which trigger osteoprotegerin (OPG) production. OPG is a protein secreted by osteoblasts, which competitively binds to receptor activator of nuclear kappa beta ligand (RANKL) expressed on osteoblast cell surfaces during osteoclastogenesis. The OPG:RANKL ratio regulates the formation of healthy bones.5 Special Issue: Focus on India Received: July 15, 2016 Accepted: September 2, 2016

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DOI: 10.1021/acsami.6b08694 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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solution was prepared by stirring equal volumes of Sr(NO3)2 and Fe(NO3)3.9H2O solutions at 45 °C on a hot plate. The molar ratio of Sr2+/Fe3+ in the above solution mixture corresponded to 1:8. Incidentally, earlier studies suggest a Sr2+/Fe3+ molar ratio of 1:8 and annealing temperature ≥700 °C to be optimal for the crystallization of phase pure SrFe12O19.17,18 The PVA solution was then mixed with the nitrate solution and the resulting sol was dehydrated at 70 °C. Subsequently, the dried precursors underwent a self-ignition reaction to form a very fine brown foamy amorphous powder mixture of SrFeO3−x and α-Fe2O3.19 A higher amount of strontium is added to ensure the formation of amorphous precursors and inhibition of the crystallization of α-Fe2O3. The obtained powder was crushed into fine particles before thermal annealing. The assynthesized particles were annealed at temperatures of 400, 700, and 900 °C for 2 h in a muffle chamber furnace. The annealed powders have been respectively referred to as SrFeNPs_400, SrFeNPs_700, and SrFeNPs_900 throughout the paper. 2.1.2. Synthesis and Consolidation of Hydroxyapatite (HA) and Strontium Hexaferrite (SrFe) Nanocomposites. Hydroxyapatite was synthesized by the suspension precipitation route, as described in our earlier works.20 Briefly, calcium oxide (CaO) was dispersed in deionized water at a concentration of 18.6 g/L followed by stirring on a magnetic hot plate until the temperature of the suspension reached 80 °C. Phosphoric acid, H3PO4 (0.17 M) was added dropwise to the CaO dispersion along with continuous stirring. Simultaneously, the pH of the solution mixture was maintained at ∼10, with the help of ammonium hydroxide (NH4OH). After 4 h, the precipitated reaction product was filtered and calcined at 800 °C. For preparing composites of HA-SrFe, the calcined HA and SrFeNPs_900 powders were mixed in proportions of 10 and 20 wt % of strontium hexaferrite and ballmilled for 16 h in a planetary ball mill (Fritsch Pulveristee, Germany) using a ball to powder ratio of 4:1. The homogeneous powder mixtures were packed in 15 mm graphite dyes and consolidated by multi-stage spark plasma sintering to obtain dense pellets of HA-SrFe composites. The densification was carried out using SPS (Dr. Sinter, Model 515S, SPS syntax Inc., Japan) under vacuum. The sintering conditions used during SPS have been listed in the Table S1. The bulk sample density for the spark plasma-sintered pellets was determined by the Archimedes method using deionized water as the fluid. 2.2. Materials Characterization. 2.2.1. Phase Assemblage. Phase characterizations of the thermally annealed powders of strontium hexaferrite, HA-SrFe ball-milled powders and spark plasma sintered composites were carried out using X-ray diffractometer (X’Pert Pro PaNalytical, Netherlands) operated at 40 kV and 30 mA equipped with a CuKα (1.5418 Å) source. The scans were recorded at a scan rate of 1.5°/min with a step size of 0.025° and 2θ varying between 15−80° or 10−90°. Phase identifications of the samples were accomplished with the help of Inorganic Crystal Structure Database (ICSD). For determining the phase composition and lattice parameters of the major and minor phase components/impurities, Rietveld refinement of the XRD data was performed with the help of computer software ‘General Structure Analysis System - GSAS’.21 Further, Fourier transform infrared spectroscopy (FTIR) was employed for the chemical characterization of the annealed SrFeNPs by scanning the diffuse reflectance spectra over a range of 400−4000 cm−1 using a Bruker Alpha FTIR spectrophotometer. 2.2.2. Microstructure and Crystallite Size Distribution. Size distributions of the magnetic nanoparticles annealed at 400, 700, and 900 °C were determined by transmission electron microscopy (TEM). The annealed powders were drop cast on a Formvar coated copper grid and visualized under a TEM, operated at 300 kV in the bright-field mode. The size distribution of the particles was estimated by measuring the particle sizes from the bright field mode captured electron micrographs in TEM. In case of the spark plasma sintered composites, the microstructure of the specimens was visualized on the fractured surfaces using scanning electron microscopy. The elemental distribution was mapped over the area of the specimens with the help of an EDAX attachment to the SEM (Inspect F50, FEI, USA). 2.2.3. Magnetic Characterization. The room-temperature magnetic properties of the annealed strontium hexaferrite samples

Among the strontium-releasing biomaterials, the most commonly studied osteoinductive implants are the strontiumdoped bioactive glasses. It was shown that strontium incorporation into the well-known 45S 5s bioactive glass upregulated the isoprenoid pathway leading to enhancements in the level of membrane cholesterol and other lipid content in human mesenchymal stem cells (hMSCs).6 It is reported that the dissolution of bioactive glasses to release calcium and silicon ions promotes osteogenesis and infiltration of the bone tissue into the implant, as the scaffold degrades. However, the mechanical properties of bioactive glass scaffolds are also lowered in simulated body fluid (SBF) in vitro and in vivo, in a time-dependent manner.7 This has prompted the fabrication of bioactive glass and glass-ceramics for load bearing applications. While developing such materials, their bioactivity or bonebonding ability was not compromised. The criteria for designing such osseointegrating mechanically tough implants have been deliberated in a dedicated review.8 Recently, research from our own group has demonstrated the short-term and long-term osseointegration of strontium containing glass ceramic, designated as LG26Sr in comparison to commercially available Biograft (HABG manufactured by IGFL, Bioceramics, India) in rabbit animal model.9,10 Another class of strontium eluting biomaterials is Sr2+ doped calcium phosphates, such as hydroxyapatite (HA) and βtricalcium phosphate (β-TCP), which have been investigated more from a crystallographic perspective. First-principle calculations combined with Rietveld refinement of synchrotron data revealed a greater distortion of the HA (P 63/m) lattice, when calcium substitution by strontium occurs at the Ca(2) site as compared to Ca(1) site.11 This differential lattice expansion of strontium substituted HA arises as a consequence of the larger Ca(1) sites and smaller Ca(2) sites.12 In our previous work, we discerned a similar lattice expansion of HA as a result of the high temperature sintering induced diffusion of iron from magnetite (Fe3O4) into HA lattice, which occurred at levels similar to that of tooth enamel and bone.13 Such bone mimicking magneto-responsive composites elicited osteogenic differentiation of human mesenchymal stem cells aided by magnetic field stimulation.14 Another recent study also established the strength reliability and degradation of strontium-substituted magnesium phosphate scaffolds as bioresorbable bone substitutes.15 In the above perspective, we investigate the potential of pristine strontium hexaferrite (SrFe12O19), a permanent magnetic material as a nanobiomaterial and its size dependent cytotoxicity. Further, the phase stability, microstructure and release profiles of essential ions (Ca, Sr, P, and Fe) from HAxSrFe12O19 (x = 0, 10, and 20 wt %) composites and their osteogenic effect on human mesenchymal stem cells (hMSCs) are analyzed.

2. EXPERIMENTAL SECTION 2.1. Materials and Methods. 2.1.1. Synthesis of Strontium Hexaferrite (SrFe12O19) Nanoparticles. SrFe12O19 NPs were synthesized by the sol−gel auto combustion method. During the synthesis, poly(vinyl alcohol) (PVA) was used as the protective/stabilizing agent. The precursors for the synthesis are strontium nitrate (Sr(NO3)2, Sigma-Aldrich), iron(III) nitrate nonahydrate (Fe(NO3)3.9H2O, Thomas Baker), and poly(vinyl alcohol) (PVA, Mol wt. 125,000, Sigma). A modified procedure without the use of sodium hydroxide (NaOH) as a coprecipitant was followed for the synthesis of strontium hexaferrite.16 Briefly, an aqueous solution of PVA was first prepared by dissolving 1 mg of PVA in 100 mL of water. The nitrate aqueous B


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ACS Applied Materials & Interfaces (SrFeNPs_400, SrFeNPs_700 and SrFeNPs_900) were recorded using a LakeShore vibrating sample magnetometer (VSM). The saturation magnetization, remnant magnetization and coercive field parameters for the ball-milled HA-SrFe powders and SPSed pellets were also recorded. For the measurement, the field increment per data point was fixed at 0.0025 Tesla (T), while sweeping the field from −1 to 1 T. 2.3. In Vitro Cell Culture Experiments. Human mesenchymal stem cells (hMSCs) were procured from the Institute for Regenerative Medicine, Texas A&M HSC COM, USA. Also, ethical clearance certification was obtained from the Institutional Committee for Stem Cell Research and Therapy (ICSC-RT), IISc, Bangalore, India. All the cell culture products used in the study were purchased from Invitrogen. For the study, the cryopreserved cell stock was revived and cultured in complete growth medium containing α-MEM (alpha modified eagle’s medium), 20% FBS (fetal bovine serum), 1% antibiotic antimycotic and 2 mM L-glutamine. The cells were maintained in a CO2 incubator (Sanyo MCO-18AC, USA) at 37 °C, 5% CO2, and 95% humidity. Upon reaching 70−80% confluency, the cells were harvested using 0.05% trypsin-EDTA and used for experiments. 2.3.1. Cytotoxicity Evaluation of Strontium Hexaferrite Nanoparticles. For the cytotoxicity assessment of SrFeNPs_400, SrFeNPs_700 and SrFeNPs_900, hMSCs were harvested at 70− 80% confluency and seeded in a 96 well plate at a density of 5,000 cells per well. The cells were allowed to adhere to the bottom of the wells for 24 h, followed by addition of the magnetic nanoparticles at concentrations of 10, 50, 100, 500, and 1000 μg/mL. After exposure of the cells to nanoparticles for 24 h, the cell viability was recorded by MTT assay, following the regular protocols. In brief, 200 μL of cell culture media containing 15% MTT (3(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; Sigma) was added per well of the 96 well plate and incubated for 4 h. The purple formazan crystals formed by the enzymatic activity of the live cells was solubilized in DMSO and the absorbance was read at 595 nm using a microplate reader (BioRad). In parallel, live/dead imaging of the cells exposed to different doses of the strontium hexaferrite nanoparticles was carried out, using a combination of fluoroscein diacetate (FDA: 25 μg/mL) and propidium iodide (PI: 10 μg/mL) for live and dead cells, respectively. The cell monolayers cultured on glass coverslips were treated with predetermined doses of the magnetic nanoparticles as mentioned earlier. After exposure for 24 h, the cells were stained for 15 min using the above combination of live and dead stains and imaged under a fluorescence microscope (Nikon). 2.3.2. hMSC Viability, Adhesion, and Osteogenic Differentiation on SPSed HA-SrFe Composites. For further analysis of stem cell viability, adhesion and osteogenic differentiation, dense cylindrical pellets (15 mm diameter and 2 mm thickness) consolidated by SPS were used. The SPSed composites were polished to similar roughness and steam sterilized in an autoclave at 121 °C and 15 psi for 20 min. Approximately 5,000 cells were seeded on each sample with the tissue culture glass coverslip used as control. For assessing the viability and adhesion of hMSCs on the SPSed HA-based composites, the culture experiments were performed in complete α-MEM media without the addition of osteogenic supplements. By using previously described protocols, the viability was evaluated by MTT assay after 3 days and 5 days of culture while live/dead imaging of cells was performed after 5 days of culture. The adhesion of hMSCs on the various substrates was qualitatively assessed by the number of focal adhesions formed at the substrate−cell interface, as observed by immunofluorescence microscopy. This is indicated by the expression of vinculin, a cytoskeletal protein that regulates cell-matrix interactions.22 For osteogenic differentiation of hMSCs, the cells were initially cultured in α-MEM for 72 h, followed by the addition of osteogenic supplemented media. The StemPro Osteogenesis Differentiation Kit procured from Thermo Fisher Scientific was used for cell culture through the differentiation assays. As per the manufacturer’s protocol, complete differentiation medium containing 10% (v/v) of the osteogenic supplements in StemPro Osteocyte/Chondrocyte Differentiation Basal medium was prepared freshly prior to each experiment.

At predetermined time points (7 and 14 days), the expression of alkaline phosphatase and collagen was detected by BCIP-NBT and Sirius red staining, respectively followed by imaging under an optical microscope. Also, the phenotypic expression levels of vimentin, a mesenchymal stem cell marker was determined by immunofluorescence microscopy. After 21 days of osteogenic induction culture, calcium mineralization was analyzed by Alizarin Red S (ARS) staining and scanning electron microscopy. The protocols for the each of the above differentiation studies are briefly outlined in the following subsections. 2.3.3. Immunofluorescence Microscopy (vinculin and vimentin). After 5 days of hMSC culture on the SPSed HA-based composites, vinculin expression was analyzed by immunofluorescence microscopy. The cells were fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature. For immunostaining, the cultured hMSCs were permeabilized with 0.1% Triton-X in phosphate buffer saline (PBS) for 5 min and incubated with blocking buffer (1% FBS, 1% BSA, 0.1% Triton-X and 0.01% NaN3) for 30 min to prevent non-specific binding of antibodies. The primary labeling for vinculin (Rabbit monoclonal antibody, Invitrogen) was performed at 1:200 dilution in blocking buffer for 1 h at room temperature. Subsequently, the samples were washed thrice in PBS and labeled with Alexa Fluor 488 Goat AntiRabbit secondary antibody at 1:500 dilution in blocking buffer for 45 min. This was followed by staining of the nuclei with DAPI (Invitrogen) and actin cytoskeleton by Alexa Fluor 546 C5 maleimide conjugate (Invitrogen). For visualization of vinculin in single cells, immunofluorescence microscopy was carried out at 100× magnification under an oil immersion lens (Nikon). In the case of vimentin, the cells cultured for 14 days in osteogenic induction media were labeled with Rabbit monoclonal vimentin (abcam), following a similar staining protocol as mentioned above. 2.3.4. Alkaline Phosphatase (ALP) Activity by BCIP-NBT Staining. After 7 days of hMSC culture on the HA-based composites in osteogenic induction media, ALP activity was assessed with the help of BCIP-NBT (5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride) kit procured from ThermoFisher Scientific. Briefly, the cell monolayers on each sample were washed with PBS and fixed with 4% PFA for 1 min. A short fixation period was chosen as longer fixation times can inactivate ALP. The staining buffer was prepared freshly by adding a BCIP-NBT solution mixture in 1:2 volume ratio to a wash buffer (100 mM Tris, 150 mM NaCl and 1 mM MgCl2) as per the manufacturer’s instructions. The samples were immersed in the staining buffer and incubated in dark for 20 min. Subsequently, the samples were rinsed in PBS and imaged under a bright field optical microscope (Nikon Eclipse LV100, Japan). 2.3.5. Deposited Collagen Detection by Sirius Red Staining. The deposited collagen in the cell monolayers after 14 days of culture in osteogenic induction media was detected by Sirius red staining. The cells adhered on all the samples were washed with PBS and fixed with 4% PFA for 30 min. Post-fixation, each of the samples were stained with 1 mL of Sirius red (Biocolor) for 1 h at room temperature. Subsequently, the samples were thoroughly washed in acid-salt wash buffer to remove the unbound dye and visualized under a bright-field optical microscope (Nikon Eclipse LV100, Japan). 2.3.6. Gene Expression Analysis Using Semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). After 14 days of culture in the presence of osteogenic supplements, total RNAs were extracted from the cells using RNA isolation kit (Ambion, Thermofisher Scientific). The purity and quantity of total extracted RNA samples were then examined using spectrophotometric OD260 and OD280 measurements. Subsequently, 2 μg of the total RNA was first reverse transcribed into cDNA using a RevertAid first-strand cDNA synthesis kit (Fermentas corporation, MD, USA) following the manufacturer’s instructions. The PCR reaction was carried out as follows. After an initial 5 min denaturation step at 94 °C, the samples were subjected to 35 cycles of denaturation for 30 s at 94 °C; annealing for 30 s at appropriate annealing temperature for the primer set under study (55−60 °C), and extension for 1 min. A final 10 min extension step at 72 °C was performed. The amplified products were resolved by electrophoresis in 1.5% agarose gel and were quantified by C


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ACS Applied Materials & Interfaces scanning with a gel documentation system after subsequent staining with ethidium bromide dye. The intensity for each gene was quantified by Quantity One software (Bio-Rad, CA, USA). The sequence of primers, annealing temperature, and product size are shown in Table S2. 2.3.7. Matrix Mineralization by Alizarin Red S (ARS staining) and SEM. The extent of mineralization/calcium deposition in the secreted extracellular matrix (ECM) on each sample was determined by Alizarin Red S staining after 21 days of osteogenic induction culture. After fixation of the cells in 4% PFA, the samples were stained with 2% Alizarin red (sigma) solution (pH 4.1−4.3) for 45 min at room temperature. The samples were carefully washed with deionized water to remove the excess dye and imaged under an optical microscope (Nikon Eclipse LV100, Japan). For the observation of calcium phosphate nodules under SEM, the samples were fixed in 3% (v/v) glutaraldehyde (LobaChemie) in PBS for 20 min. The fixed cells were dehydrated in an increasing gradient series of ethanol (30, 50, 70, 95 and 100%), sputter coated with gold, and imaged in a scanning electron microscope in the secondary electron mode. 2.3.8. In Vitro Ion Dissolution Studies. The elemental concentrations of calcium, phosphorus, strontium and iron released from the SPSed composites into cell culture media were evaluated by inductively coupled plasma mass spectrometry (ICP-MS). At different time points (0, 4, 7, and 10 days), the cell culture media from hMSC culture were retrieved for ICP-MS analysis. The dissolution medium was replenished every 3−4 days for further culture and ion dissolution study in vitro. The retrieved cell culture media were diluted with 3% Nitric acid to solubilize the ions, if precipitated. A standard plot of concentration versus counts for each of the elemental isotopes was generated by recording the standards (100, 500, and 1000 ppb) using a quadrupole inductively coupled plasma mass spectrometry (Thermo Fisher Scientific X Series II) . From the standard plots, the unknown concentrations of the ions released from the cylindrical SPSed pellets were determined. 2.4. Statistical Analysis. The statistical analysis for all the data presented in this study was carried out using IBM SPSS Statistics 20 software. All experiments were carried out in triplicate. The analysis of variance (one way ANOVA) was adopted to determine the statistical significance between the cell response on HA-SrFe composites and the control. For data analysis, Tukey and Games-Howell tests were employed to determine the statistical significance at p < 0.05, where p denotes the probability that there is no significant difference between the means.

error as well as parameters for refinement of crystallite size, peak shifts and particle strain-related effects. The calculated XRD pattern was generated by considering the following structural symmetries of the constituent phases − HA (P63/m), SrFe12O19 (P63/mmc), Fe3O4 (Fd3̅m), α-Fe2O3 (R3̅c), SrCO3 (Pmcn) and CaCO3 (R3̅c). The fit between the experiment and calculated patterns was evaluated by Rietveld agreement factors such as RBragg/R(F2) (%), χ2 and goodness of fit (GOF). Compositions (wt %) of the constituent phases were determined for each sample from the refined scale factors of the experimental data, as analyzed below. 3.1.1. Annealed SrFeNPs. Strontium hexaferrite (SrFe12O19) is a synthetic magnetic material possessing M-type hexaferrite structure similar to that of magnetoplumbite, a naturally occurring magnetic material.26 It crystallizes in the hexagonal structure with a space group symmetry P63/mmc, with 64 ions per unit cell (2 formula units of SrFe12O19) located at 11 different symmetry sites. The 24 Fe3+ ions are arranged in 5 different symmetry sites −3 octahedral, 1 tetrahedral and 1 bipyramidal and the 5 different sublattices about each type of Fe atom, 3 parallel and 2 antiparallel contribute toward the strong ferrimagnetism of strontium hexaferrite.27 Figure 1 shows the XRD patterns of the thermally annealed SrFeNPs after Rietveld refinement. The high intensity reflections of the constituent phases for each sample have been indicated in the refined patterns color-coded with the particular phase. It can be clearly seen from Figure 1A that the phase formation of strontium hexaferrite (SrFe12O19) did not occur by annealing as synthesized PVA capped SrFeNPs at 400 °C (SrFeNPs_400) for 2 h. Instead, the predominant phase detected was magnetite (Fe3O4) along with SrCO3 formed by the probable reaction of SrO with the amorphous carbon/CO2 generated from PVA. In case of SrFeNPs annealed at 700 and 900 °C (SrFeNPs_700 and SrFeNPs_900), a near complete phase formation of SrFe12O19 (93−95 wt %) was observed along with hematite αFe2O3 (4−7 wt %) as an impurity. Table 1 summarizes the phase compositions and lattice parameters of the thermally annealed SrFeNPs along with Rietveld refinement patterns showing the degree of agreement between the observed and calculated diffraction patterns. The annealed SrFeNPs were further characterized by FTIR for their chemical composition. Figure S1 shows ATR-FTIR spectra of SrFeNPs annealed at 400, 700, and 900 °C. Figure S1A shows distinct and sharp absorption peaks at 1445 and 866 cm−1 corresponding to the asymmetric stretch and bending carbonate vibrations of SrCO3 respectively.28 Also a broad OH stretch due to hydroxyl group from PVA may be noted. Further, strong absorptions at 560 cm−1 by Fe3O4 and a weak Fe−O vibration at 472 cm−1 by α-Fe2O3 mask the characteristic 590 and 550 cm−1 peak signals from SrFe12O19.29 However, absorption at 445 cm−1 confirms the presence of SrFe12O19 in the SrFeNPs_400 sample. In the case of SrFeNPs_700, very weak absorptions for SrCO3 and OH stretch from PVA along with strong absorptions for SrFe12O19 at 590, 550, and 445 cm−1 can be observed (Figure S1B).18 The annealing of the powders at 900 °C led to complete removal of the carbonate and PVA with pronounced absorptions for SrFe12O19, as shown in Figure S1C. In both SrFeNPs_900 and SrFeNPs_700, the Fe−O vibration at 472 cm−1 due to α-Fe2O3 has been concealed by strong absorptions of SrFe12O19 in the same region. 3.1.2. Ball-Milled HA-SrFe Powders. Rietveld refinement of the XRD data for the ball-milled HA-SrFe powders indicated a

3. RESULTS The HA-based magnetic composites were consolidated by spark plasma sintering under the sintering conditions depicted in Table S1. The heating cycle used for pure hydroxyapatite (HA) was identical to the one optimized in our earlier studies to achieve maximum densification.23 In case of the HA-based composites of SrFe12O19, the holding times were shorter and the peak holding temperature was set to 1000 °C. This was in view of earlier reports on the decomposition of SrFe12O19 to Fe3O4 when SPSed at 950 °C for 2 min.24 Nevertheless, all the samples were densified to 95−98% of theoretical density (% ρth). Further, a critical analysis of the phase compositions of the SrFeNPs and HA-based (ball-milled and SPSed) composites was carried out considering the importance of composition, size and dose dependent cellular toxicity of nanomaterials.25 3.1. Phase Analysis by Rietveld Refinement. Rietveld refinement of the X-ray diffraction data was carried out with the help of General Structure Analysis System - GSAS software. The least-squares refinement was performed taking into account the structure and symmetry of the constituent phases, the scale factor for adjustment of the peak intensities, parameters for the background, peak profile and instrumental D


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ACS Applied Materials & Interfaces Figure 1. continued

marked (color-coded) in the refined patterns and phases have been indicated below the designated sample labels (SrHA10Fe_SPS and SrHA20Fe_SPS).

ternary phase mixture consisting predominantly of hydroxyapatite (HA) along with SrFe12O19 and α-Fe2O3 as the minor constituents. For the 10 wt % SrFe12O19 composition (HA10SrFe_B), ball-milling for 16 h did not significantly affect the phase composition in terms of the projected/ calculated wt % of SrFe12O19 from SrFeNPs_900. In the case of HA20SrFe_B, (Table S3) suggests a minor conversion of SrFe12O19 to α-Fe2O3 during ball-milling. This indicates the greater phase stability of SrFe12O19, when dispersed in small amounts in hydroxyapatite (HA). Also, our observations of the stability of SrFe12O19 in ball-milled powders are in line with an earlier study, which reported considerable amorphization of SrFe12O19 upon prolonged ball-milling for 42 h, without any phase transformation.24 Further, HA gave a much better fit for the hexagonal P63/m compared to the lower symmetry monoclinic P21/b phase. The Rietveld agreement parameters [RBragg/R(F2) (%), χ2 and goodness of fit (GOF)] were smaller for HA fit with the hexagonal phase than the monoclinic structure (data not shown). Figure S2 shows the Rietveld refined XRD patterns of HA-SrFe ball-milled powders along with the constituent phases, indicated in the figure legend. Overall, the phase compositions determined by Rietveld refinement are in close agreement with the weight percentages used for preparing the composite ball-milled powders. 3.1.3. Spark Plasma-Sintered Composites. Rietveld refinement of the SPSed HA-based composites strikingly depicts the decomposition of SrFe12O19 into magnetite (Fe3O4), indicating low thermal stability under the SPS conditions. Figure 1B shows the Rietveld refined XRD patterns of the SPSed HAbased composites. In our previous study, we observed a greater phase retention of the magnetic ferrite (Fe3O4) embedded in hydroxyapatite matrix, when hot-pressed at 950 °C in inert atmosphere.13 Similarly, we expected HA to impart thermal stability to strontium hexaferrite. However, a near complete thermal degradation of SrFe12O19 was observed in both the SPSed composites. This is in line with earlier reports, wherein densification of SrFe12O19 by SPS at 950 °C was accompanied by one-third of the phase decomposing to magnetite (Fe3O4), even for a small sintering time of 2 min.24 In our study, the total heating cycle duration for consolidation of the HA-SrFe composites by SPS was 18 min. Hence, it may also be reasoned that the long sintering duration of the cycle promoted the near complete decomposition of SrFe12O19. Interestingly, no XRD peak corresponding to strontium oxide (SrO) was detected in both the composites. Further, the XRD pattern of SPSed HA-10 wt %SrFe12O19 gave a much better fit for a two phase mixture comprising of HA and Fe3O4. On the other hand, SPSed HA-20 wt %SrFe12O19 was characterized by a four phase mixture, primarily consisting of HA and Fe3O4 along with SrFe12O19 (∼0.4 wt %) and calcite/CaCO3 (∼1.3 wt %) as the minor phases. It may be emphasized that the Rietveld fitting for HA-20 wt %SrFe12O19 greatly improved only upon the inclusion of these minor phases. Therefore, a meager retention of SrFe12O19 as well as minor decomposition of HA into CaO can be implicated. The CaO further reacted with carbon from the graphite die to form calcite (CaCO3) during SPS. Another characteristic feature noted in the SPSed HA-based composites

Figure 1. (A) Rietveld refined XRD patterns of SrFeNPs annealed at different temperatures. The high intensity reflections for the constituent phases have been marked (color-coded) in the refined patterns and phases have been indicated below the designated sample labels (SrFeNPs_400, SrFeNPs_700, and SrFeNPs_900). (B) Rietveld refined XRD patterns of SPSed HA-SrFe12O19 composites. The high intensity reflections for the constituent phases have been E


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Table 1. Phase Compositions and Lattice Parameters of Thermally Annealed SrFeNPs Determined from Rietveld Refinement of XRD Data phase abundance (wt %) and lattice parameters (Å) of the ferromagnetic component

phase abundance and lattice parameters of impurity

RBragg/ R(F2)(%)

χ2

goodness of fit (GOF)

SrFeNPs_400

Fe3O4 ; Fd3m ̅ (80.02 ± 0.15%) a = b = c = 8.3453(2) α = β = γ = 90° V = 581.203 ± 0.032

7.44

1.48

1.2

SrFeNPs_700

SrFe12O19; P63/mmc (95.45 ± 0.04%) a = b = 5.8834(7) c = 23.0547(1) α = β = 90°; γ = 120° V = 691.112 ± 0.017 SrFe12O19; P63/mmc (93.26 ± 0.01%) a = b = 5.8803(4) c = 23.0388(6) α = β = 90°; γ = 120° V = 689.909 ± 0.009

SrCO3; Pmcn (19.98 ± 0.15%) a = 5.0895(9) b = 8.6127(8) c = 6.0363(9) α = β = γ = 90° V = 264.609 ± 0.269 α-Fe2O3; R3c̅ (4.54 ± 0.04%) a = b = 5.0346(9) c = 13.7415(6) α = β = 90°; γ = 120° V = 301.656 ± 0.043 α-Fe2O3; R3c̅ (6.74 ± 0.01%) a = b = 5.0134(7) c = 13.6654(6) α = β = 90°; γ = 120° V = 297.453 ± 0.068

10.62

1.31

1.1

7.85

1.214

1.1

samplea

SrFeNPs_900

a

SrFeNPs _400 = SrFe12O19 nanoparticles annealed at 400 °C

Table 2. Phase Compositions and Lattice Parameters of SPSed HA-SrFe12O19 Composites Determined from Rietveld Refinement of XRD Data sample SrHA10Fe_SPS

SrHA20Fe_SPS

phase abundance (wt %) and lattice parameters (Å) of HA

phase abundance and lattice parameters of ferrites

RBragg/R(F2) (%)

χ2

goodness of fit (GOF)

SrHA; P63/m (90.60 ± 0.08%) a = b = 9.4322(5) c = 6.8947(4) α = β = 90°; γ = 120° V = 531.223 ± 0.055 SrHA; P63/m (81.92 ± 0.59%) a = b = 9.4338(9) c = 6.8987 (5) α = β = 90°; γ = 120° V = 531.719 ± 0.032

Fe3O4 ; Fd3̅m (8.40 ± 0.51%) a = b = c = 8.4165(6) α = β = γ = 90° V = 596.205 ± 0.225

18.06

4.92

2.2

SrFe12O19; P63/mmc (0.38 ± 0.13%) a = b = 5.8903 (1) c = 23.1883 (9) α = β = 90°; γ = 120° V = 696.749 ± 2.457 Fe3O4; Fd3̅m (16.40 ± 0.18%) a = b = c = 8.4302 (4) α = β = γ = 90° V = 599.129 ± 0.086 CaCO3; R3c̅ (1.32 ± 0.15%) a = b = 4.9819(4) c = 17.0163(9) α = β = 90°; γ = 90° V = 365.757 ± 0.181

28.01

6.30

2.5

experimentally by Rietveld refinement of XRD data.11 However, the refinement of atomic occupancies was not possible because of the presence of multiple phases in the composites. Table 2 summarizes the phase compositions of SPSed HA-based composites along with the lattice parameters of the constituent phases. On the basis of the predominant phase fractions of Fe3O4 and SrHA arising from the observed increase in unit-cell volume of HA by Sr-doping, the SPSed

was a significant increase in the lattice parameters/unit-cell volume of HA. In comparison to the ball-milled powders with unit-cell volumes of 527.886 Å3 and 528.373 Å 3 for HA10SrFe_B and HA20SrFe_B, respectively, the hydroxyapatite (HA; P63/m) in SPSed HA-10 wt %SrFe12O19 and HA-20 wt %SrFe12O19 underwent lattice expansion to 531.223 and 531.719 Å3, respectively. These lattice volumes match for 1 atom % doping of Sr2+ at the Ca sites in HA, as predicted previously by density functional theory (DFT) calculations and F


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Figure 2. (A) Bright-field TEM images of thermally annealed SrFeNPs at 400, 700, and 900 °C showing the morphology and particle size distribution. (B) SEM microstructures of SPSed HA, SrHA10Fe, and SrHA20Fe composites revealing coarsened HA grains, nanocrystallites, and micrometer-sized grains, respectively.

the crystallite size distributions can significantly influence the magnetic behavior and cytotoxicity of the nanoparticles. Figure 2B presents the microstructures of the spark plasma sintered HA, SrHA10Fe and SrHA20Fe composites, respectively. The fractured surfaces of the specimens imaged under scanning electron microscope divulged a marked difference in the grain sizes. The SPSed HA exhibits a coarse grained microstructure with grain size range of 10−20 μm. The grain coarsening in HA is attributed to higher sintering temperature (1100 °C) and longer holding times (5 min each) in between the steps during the multi-stage SPS (see Table S1). Similar grain growth of HA with sintering temperature during SPS was reported by Gu et al.31 On the other hand, the SrHA10Fe_SPS and SrHA20Fe_SPS present submicrometer (200−300 nm) and micrometer-sized (1−3 μm) grains, respectively, because of the relatively lower sintering temperature (1000 °C) and shorter holding times (2 min each). Further, the microstructure of SrHA20Fe revealed the formation of pits at the grain

composites are henceforth referred to as SrHA10Fe_SPS and SrHA20Fe_SPS throughout the paper. 3.2. Particle Size Distribution of SrFeNPs and Microstructure of SPSed SrHA-Fe Composites. The particle size and morphology of the annealed SrFeNPs were determined using transmission electron microscopy (TEM). Figure 2A shows representative bright-field TEM images of thermally annealed SrFeNPs at 400, 700, and 900 °C, respectively. As analyzed from XRD, SrFeNPs_400 contains cuboidal nanocrystallites of magnetite (Fe3O4) with a size range between 20− 30 nm. With an increase in the annealing temperature to 700 °C, the SrFe12O19 phase formation occurred along with agglomeration of the crystallites with the appearance of distorted hexagonal platelet - like structures, as observed in a previous study.30 The agglomerated SrFeNPs_700 and SrFeNPs_900 possessed crystallite size ranges from 50−60 nm and 70−100 nm, respectively. It may be emphasized that G


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Figure 3. Room-temperature magnetic behavior of (A) annealed SrFeNPs at 400, 700, and 900 °C; (B) ball-milled HA-SrFe powders, (C) spark plasma-sintered/SPSed HA, and (D) SPSed SrHA-Fe composites.

boundaries as well as small precipitates at the intersection of grain boundaries. Elemental mapping of the composites with the help of EDAX showed a uniform distribution of the constituent elements (Ca, P, O, Fe, and Sr) with few Fe-rich clusters in some microstructural regions (Figure S3). This indicates the possible codiffusion of Fe and Sr into the apatite matrix under the high-temperature and pressure conditions during SPS. As both Fe and Sr are implicated in anticatabolism of bone,32 the SPSed SrHA-Fe composites can be described as potential antiresorptive bone-mimicking biocomposites. 3.3. Magnetic Behavior of SrFeNPs and HA-Based Iron Oxide Composites. Strontium hexaferrite is a well-known hard permanent magnetic material used for recording media. On the other hand, magnetite (Fe3O4) is a soft magnetic material with a bulk saturation magnetization (Ms) of 96.5 emu/g.33 In the present study, the SrFeNPs, annealed at 400 °C (SrFeNPs_400) contains ∼80 wt % Fe3O4 as analyzed from XRD and a particle size of 20−30 nm. This can be correlated with an observed experimental Ms of ∼45 emu/g and Mr/Ms ratio of ∼0.2. In case of SrFeNPs_700 and SrFeNPs_900, the areas under the hysteresis loops are larger with high coercive fields (Hc) of ∼5kG, remnant magnetization (Mr) between ∼23−27 emu/g and saturation magnetization (Ms) ranging from ∼35−50 emu/g. Considering the presence of 5−7 wt % of antiferromagnetic hematite (α-Fe2O3) in SrFeNPs_700 and SrFeNPs_900 as well as nanosized crystallites, the Ms and Hc values recorded in our study are close to those reported for SrFe12O19 nanoribbons (Ms = 68 emu/g and Hc = 7 kG).34

Further, the hysteresis loops are more square-like in shape, resulting in the large magnetic hardness reflected by the Mr/Ms ratios of ∼0.6. The wavy nature of the SrFeNPs_700 and SrFeNPs_900 (Figure 3A) arises from the combinatorial response of the hard (SrFe12O19) and soft (α-Fe2O3) magnetic materials present in the polycrystalline powders. With regard to the ball-milled HA-SrFe powders, the recorded Ms and Mr (emu/g) are commensurate with the phase compositions detected in XRD. Ball-milling of the powder mixtures for 16 h caused a small increase in the decomposition of SrFe12O19 to α-hematite in HA20SrFe_B and correspondingly, a proportionate drop in Ms was recorded. In HA10SrFe_B, ball-milling of the powders did not induce any further decomposition of SrFe12O19, leading to equitable Mr and Ms. However, the intrinsic magnetic hardness (Mr/Ms) for the ball-milled powders ranged from 0.5−0.6 similar to SrFeNPs, while the coercive fields Hc were lowered to ∼2 kG, because of the deagglomeration of the SrFeNPs in the HA matrix during ball-milling. Figure 3B shows distinct ferromagnetic hysteresis loops recorded using a VSM at room temperature. The wavy nature of the hysteresis loop in SrFeNPs_900 disappeared upon ball-milling with hydroxyapatite powders. Figure 3C depicts a typical diamagnetic behavioral response of hydroxyapatite (HA) under an applied field swept from −1 to 1T. Figure 3D presents ferromagnetic hysteresis loops of spark plasma-sintered (SPSed) SrHA10Fe and SrHA20Fe composites recorded at room temperature using VSM. In line with the phase composition and nanocrystalline H


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3.4.2. hMSC Viability/Proliferation on SPSed SrHA-Fe Composites. In a similar manner as above, the viability of hMSCs on spark plasma sintered (SPSed) SrHA-Fe composites was evaluated after 3 and 5 days of culture. Figure 5A is a schematic representation of hMSC viability assessment on similar SPSed composites. The MTT assay results in Figure 5B indicate no significant difference in the viability of hMSCs on all the samples after 3 days of culture. However, on day 5, the cell viability/proliferation were significantly lower on SrHA10Fe and SrHA20Fe as compared to control and HA. Nevertheless, live/dead imaging of the cells after 5 days of culture did not reveal any sign of necrosis/apoptosis as indicated by the near absence of PI stained cells in all the composites (Figure 5C). The combination of MTT and live/ dead imaging results implicate the proliferative arrest of hMSCs on SrHA-Fe composites, marking the onset of stem cell differentiation. Further signatures of osteogenic differentiation were identified from the cell morphology and focal adhesion patterns on the SPSed SrHA-Fe substrates, as elaborated in the subsequent sections. 3.4.3. Altered adhesion and Spreading of hMSCs on SPSed SrHA-Fe Composites. The cell functionality was assessed quantitatively using cell morphological analysis and qualitatively using biochemical assays. Considering the fact that cell adhesion precedes cell spreading and subsequent changes in cell functionality, we now analyze the cell adhesion first. The adhesion of hMSCs to SPSed SrHA-Fe composites was assessed in terms of the number of focal adhesions formed at the cell−substrate interface as well as the degree of spreading of hMSCs on the substrates after 3 days of culture. Vinculin is a key focal adhesion marker protein that regulates the binding of cell surface receptors called integrins with the matrix/ substrate.22 Figure 6A shows focal adhesion complexes formed at the interface of hMSCs on spark plasma sintered (SPSed) SrHA-Fe composites and these complexes are detected by the green fluorescent signal from Alexa Fluor 488 conjugated to the secondary antibody. Figure S4A shows fluorescence images testifying increased degree of cell spreading on SrHA-Fe composites and Figure S4B present supplementary images highlighting distinct and larger number of focal adhesion complexes on similar composites. The degree of cell spreading/ morphology represented as cell area (μm2) in Figure 6B, was calculated using ImageJ software, as also reported in our previous work.35 The number of focal adhesions per cell was calculated with the help of ImageJ, following an image processing protocol optimized for quantification of focal adhesions.36 The small size of the focal adhesion complexes shown in Figure 6A indicates active cell migration on the substrates, as reported previously.37 Figure 6C presents the statistical analysis of the number of focal adhesions per cell formed on the SPSed SrHA-Fe composites. The hMSCs adhered on SrHA10Fe and SrHA20Fe exhibit larger number of focal adhesions per cell and extent of spreading as indicated by cell area in comparison to the control and HA. Although not statistically significant, the nanotopography of SrHA10Fe, due to large fraction of grain boundaries in ultrafine grained microstructure, resulted in lesser number of focal adhesions per cell as compared to the microtopography exhibited by SrHA20Fe. This is consistent with earlier findings wherein nanotopography decreased the expression of integrin subunits in hMSCs cultured on nanosized gratings of polystyrene.38 Further, the elastic stiffness varying over orders of magnitude (stiff/rigid (MPa) to compliant (kPa) surfaces) can induce

nature of SrHA10Fe_SPS, the complete decomposition of SrFe12O19 to Fe3O4 under the SPS conditions explains the near superparamagnetic hysteresis behavior (Hc = 132 G, Mr/Ms = 0.09). A similar decomposition of SrFe12O19 to Fe3O4 may be noted in SrHA20Fe_SPS along with microcrystallites in the microstructure. These characteristics contribute to the relatively broader hysteresis loops (Hc = 256 G, Mr/Ms = 0.2) observed in SrHA20Fe_SPS. Table 3 summarizes the room temperature Table 3. Room-Temperature Magnetic Properties of SrFeNPs and HA-Based Iron Oxide Composites Recorded Using Vibrating Sample Magnetometer (VSM) sample SrFeNPs_400 SrFeNPs_700 SrFeNPs_900 HA10SrFe_B HA20SrFe_B SrHA10Fe_SPS SrHA20Fe_SPS

remnant magnetization Mr (emu/g) 8.1 29.9 23.2 2.7 3.9 0.4 2.1

± ± ± ± ± ± ±

0.1 3.0 0.15 0.03 0.02 0.01 0.01

saturation magnetization Ms (emu/g)

Mr/ Ms

coercive field Hc (G)

± ± ± ± ± ± ±

0.18 0.62 0.66 0.59 0.54 0.09 0.19

146.7 5797 4898 2104 2070 132.5 256.8

44.9 47.8 34.8 4.5 7.2 4.2 10.5

0.1 4.0 0.1 0.01 0.02 0.01 0.04

± ± ± ± ± ± ±

2.0 23 99 4 30 6 7.8

magnetic properties of SrFeNPs and HA-based iron oxide composites. Overall, it is reconfirmed that phase composition and crystallite size have significant influence on the magnetic property of polycrystalline composite materials. 3.4. Stem Cell Functionality on SrFeNP and SrHA-Fe Composites in Vitro. 3.4.1. Size-, Composition-, and DoseDependent Cytotoxicity of SrFeNPs. The cytotoxicity assessment of SrFeNPs in hMSCs was carried out using the standard MTT assay. The adhered hMSCs were treated with concentrations ranging from 0−1,000 μg/mL. Further, the thermally annealed SrFeNPs exhibited different size distributions as in 20−30 nm for SrFeNPs_400, 50−60 nm for SrFeNPs_700 and 70−100 nm for SrFeNPs_900. Figure 4A is a schematic representation of the treatment of hMSCs with SrFeNPs. The MTT assay results, as shown in Figure 4B indicate negligible cytotoxicity of the SrFeNPs toward hMSCs after 24 h of exposure. A significant drop in cell viability was recorded only in case of hMSCs treated SrFeNPs_700 and SrFeNPs_900 at doses of 50 and 100 μg/mL, respectively. This mild decline in viability arises from both composition and size induced toxicity. Interestingly, at higher concentrations, a significantly higher cell proliferation was recorded by MTT assay. We further confirmed the viability of cells treated with the SrFeNPs at doses varying from 0−500 μg/mL by live/dead imaging. Using a combination of Fluoroscein diacetate (FDA) and propidium iodide (PI) for live and dead cells respectively, a small number of dead cells (PI stained) were detected in case of SrFeNPs_400 and SrFeNPs_700 (Figure 4C). Also, the aggregates of SrFeNPs sticking onto the cell surfaces can be observed in case of the larger SrFeNPs_700 and SrFeNPs_900 at higher concentrations. Summarizing, the nanoparticles of strontium hexaferrite do not cause significant cytotoxicity in vitro. Nevertheless, the SrFeNPs should be evaluated in vivo for biodistribution and clearance from body tissues as well as systemic toxic effects. This is important as we envisage potential applications for SrFeNPs in drug delivery, T2 proton relaxation-based contrast agents for magnetic resonance imaging (MRI) and hyperthermia-based anticancer treatment. I


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Figure 4. Cytotoxicity assessment of ultrafine bioceramic particulates: (A) Schematic depiction of cytotoxicity assessment of SrFeNPs on hMSCs; (B) MTT assay results indicating negligible toxicity of SrFeNPs in hMSCs after 24 h of exposure. Three samples were used for experiments and data shown is the mean ± sd of two independent experiments. * indicates statistically significant difference (p < 0.05) of the SrFeNP-treated samples w.r.t control. (C) Live−dead imaging of SrFeNPs treated hMSCs at different concentrations for 24 h (green = FDA = live; red = PI = dead; scale bar = 100 μm).

differentiation of progenitor cells.40 Thus, the activation of focal adhesion kinase (FAK) signaling pathway is commonly implicated in the induction of osteogenesis of hMSCs.41 This is reflected in the analysis of stem cell functionality as described in subsequent sections. 3.4.4. Decline in Stemness of hMSCs with a Concomitant Acquisition of Osteogenic Phenotype. Any study to analyze stem cell behavior on a biomaterial substrate should probe into two aspects, (a) stemness retention in culture and (b) changes in cell morphology and associated gene expression changes. To

differential patterns of focal adhesions and cytoskeletal reorganization in cells.39 However, in our study, the SPSed SrHA-Fe composites have Young’s moduli of the order of GPa, which may be indistinguishable by cellular traction forces. Hence, we attribute the increased cell spreading and focal adhesion patterns to the differential microstructural features of the SPSed SrHA-Fe composites. Also, tyrosine phosphorylation by focal adhesion kinase (FAK) located at the focal adhesion sites has a strong link to integrin transferred mechanotransduction and topographic signals necessary for osteogenic J


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Figure 5. Cytocompatibility assessment by quantitative cell viability and qualitative live dead analysis: (A) Schematic illustration of cell viability evaluation on SPSed SrHA-Fe composites. (B) MTT assay showing mild reduction in cell proliferation on SrHA10Fe and SrHA20Fe composites. Three samples were used for each experiment and data shown is the mean ± sd of two independent experiments. * and # indicate statistically significant difference (p < 0.05) of SPSed SrHA-Fe composites w.r.t control and HA, respectively. (C) Live−dead imaging of hMSCs on SrHA-Fe composites did not indicate significant cell death after 5 days of culture (green = FDA = live; red = PI = dead; scale bar = 100 μm).

dephosphorylation of BCIP and (ii) oxidative dimeration of dephoshorylated BCIP by NBT. Figure 7B corresponds to the phenotypic expression of vimentin in hMSCs, assessed by immunofluorescence microscopy. After 14 days of culture, a clear reduction in green fluorescent (Alexa Fluor 488) labeled vimentin intermediate filaments can be observed in cells cultured on SrHA20Fe and it is nearly absent in case of SrHA10Fe. Thus, a marked reduction in the stemness of hMSCs indicated by the low vimentin expression suggests that the hMSCs exhibit accelerated differentiation on the SPSed SrHA-Fe composites. Figure 7C depicts the level of deposited/secreted collagen by hMSCs cultured on similar substrates for 14 days. Post-staining with Sirius red, the bright field optical microscope images suggest that hMSCs cultured on SrHA10Fe secrete a greater amount of collagen as extracellular matrix (ECM). This observation is consistent with earlier reports on the induction of osteogenesis by nanotopographical cues combined with osteogenic growth factors.44 Summarizing, the decrease in vimentin expression is commensurate with an increase in osteogenic marker proteins (ALP and Collagen), aided by substrate composition, topographic cues, and osteogenic induction factors. 3.4.5. Enhanced expression of Multiple Genes Regulating Different Osteogenic Stages on SrHA-Fe Matrices. The osteogenic differentiation of hMSCs toward mature osteoblasts is driven by a set of specific genes. The culture of hMSCs on SrHA-Fe substrates resulted in selective differentiation towards osteogenic lineage, which was confirmed by semiquantitative PCR analysis of multiple osteogenic markers, namely, Runx2, Col IA, ALP, Osteocalcin (OCN) and Osteopontin (OPN).

analyze the former, we assayed the phenotypic expression of vimentin in hMSCs. A decline in the expression of vimentin, a marker specific for mesenchymal stem cells (MSCs) along with the enhancement in the expression of osteogenic proteins was observed, when hMSCs were cultured on SrHA-Fe composites. Vimentin is a type III intermediate filament (IF) protein, which is a marker for epithelial to mesenchymal transition that occurs during embryogenesis and cancer metastasis.42 Also, vimentin has been reported to inhibit activating transcription factor 4 (ATF-4) and down-regulate the expression of osteocalcin and mineralization in immature osteoblasts.43 Hence, we evaluated the expression of vimentin after 14 days of hMSC culture in osteoinduction media. At the same time point (14 days), osteocalcin (OCN) and osteopontin (OPN) mRNA levels have been determined by semiquantitative polymerase chain reaction (PCR). For all the differentiation assays, the complete growth media (α-MEM supplemented with 10% FBS) was replaced by osteogenic induction media (10% osteogenic supplements) after allowing 3 days for cell attachment and multiplication. The phenotypic expression of ALP, collagen and vimentin were qualitatively evaluated by optical/fluorescence microscopy. A quantitative assessment of the early markers (ALP and Col) along with late osteogenic markers (OCN and OPN) is assayed by PCR. These results are described in the next section. Figure 7A shows the alkaline phosphatase (ALP) levels detected by BCIP-NBT staining in hMSCs cultured on SPSed SrHA-Fe composites for 7 days. A distinct dark purple precipitate can be observed in the cells adhered on SrHA10Fe and SrHA20Fe, because of the enzymatic activity of ALP. The reaction in BCIP-NBT staining procedure occurs in two steps: (i) K


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Figure 6. Cell attachment to biomaterial substrate is mediated by focal adhesion complexes: (A) Focal adhesion complexes on SPSed SrHA-Fe composites visualized by immunostaining for vinculin (focal adhesion marker protein) after 3 days of culture. Vinculin was labeled with Alexa Fluor 488 (green), nuclei with DAPI (blue) and actin cytoskeleton by Alexa Fluor 546 (red). Scale bar = 10 μm. (B) Cell area/ μm2 and (C) number of focal adhesions per cell were quantified from a minimum of 25 cells per sample. Three replicates were used per experiment and data shown corresponds to the mean ± sd of two independent experiments. * and # indicate statistically significant difference (p < 0.05) of SPSed SrHA-Fe composites w.r.t control and HA, respectively.

Fe substrates did not have a significant influence over Col IA gene expression, an early stage marker that provides the organic matrix for mineralization. ALP, being one of the effector proteins responsible for the mineralization of the ECM, was upregulated significantly in SrHA-Fe composites, whereas it was undetectable in control and HA samples, as shown in Figure 8. Interestingly, in this case, a decrease in mRNA expression was noticed from SrHA10Fe to SrHA20Fe. The most striking effect of strontium incorporation in HA matrix is the marked stimulation of OPN gene expression, which is known to regulate the formation and remodeling of mineralized tissue. Especially, OPN was overexpressed at a very high level,

Figure 8A shows representative PCR bands for the multiple osteogenic genes analyzed after 14 days of hMSC culture. Figure 8B is the densitometric quantification of the PCR bands corresponding to the mRNA expression of osteogenic genes (Runx2, ALP, Col IA, OCN, and OPN). The expression of Runx2, an early stage differentiation marker was detected to be significantly elevated in cells cultured on SrHA-Fe composites, even after 14 days. Notably, the Runx2 expression in case of SrHA20Fe was almost triple that in the SrHA10Fe samples (0.82 versus 0.26, respectively), validating the potential anabolic effect of released Sr2+ ions by triggering the master switch of osteogenic differentiation. On the other hand, SrHAL


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Figure 7. Qualitative analysis of bone marker proteins as signatures of osteogenic differentiation - (A) Alkaline phosphatase (ALP) activity levels detected by BCIP-NBT staining after 7 days of hMSC culture. (B) Immunofluorescence labeling of vimentin, a mesenchymal stem cell marker with Alexa Fluor 488 (green), actin with Alexa Fluor 546 (red) and nuclei with DAPI (blue) indicates a decrease in vimentin expression in hMSCs cultured on SrHA-Fe composites for 14 days. (C) Deposited collagen detected by sirius red staining after 14 days of culture. All the above results correspond to hMSC culture in osteogenic supplemented media for different time periods and scale bar = 100 μm.

shown). The EDAX analyses (shown below the corresponding SEM images) confirm the CaP-rich nodules with traces of sodium and magnesium from the fixation buffer (PBS). The Ca/P ratios calculated from the EDAX data ranged between 1.1−1.2 instead of Ca/P of 1.67 for hydroxyapatite. Apart from adsorption of excess phosphate from the fixation/wash buffer, the low Ca/P ratios could also result from the incorporation of Sr and Fe released from the composites into the mineralized calcium nodules. This is in line with earlier literature evidence, which suggests a dose--dependent incorporation of Sr into the mineralized calcium phosphate nodules during the osteoblastic maturation of MC3T3-E1 osteogenic cells treated with strontium ranelate.45 Further, EDAX is a surface analytical technique for composition analysis. Therefore, it is probable that the composition of the mineralized nodules in the interior is different from that at the surface. 3.4.7. Dissolution of Osteoinductive Ions from the SPSed SrHA-Fe Composites. The dissolution of essential inorganic ions from the SPSed SrHA-Fe composites was determined by ICP-MS. Particularly, we have analyzed the release of strontium, calcium, iron, and phosphorus into the cell culture media as these ions play a pivotal role in inducing osteogenesis in hMSCs. Figure 10 shows the release profiles of these ions in terms of the concentrations measured at different time points in culture. The calcium release profile in Figure 10A indicates a greater assimilation of Ca2+ from the cell culture media by the osteogenically differentiating hMSCs cultured on SrHA10Fe and SrHA20Fe for matrix mineralization. However, the release pattern for phosphorus does not indicate a significant difference in the phosphate levels among the samples (Figure 10B). The inorganic phosphorus (Pi) involved in the mineralization of calcium phosphate might be masked by the presence of organic phosphates (β-glycerol phosphate) in the cell culture media. Figure 10C presents a significantly higher release of Sr2+ for SrHA10Fe and SrHA20Fe compositions. An initial burst release is followed by a sustained release of strontium for over 10 days

preferentially in cells cultured on SrHA20Fe, thereby confirming the accelerated osteogenesis in hMSCs within 14 days of culture. Finally, a significant upregulation of mineralization specific gene, OCN was recorded in all rigid HA platforms in comparison to control. Furthermore, no difference in the OCN expression was observed between SrHAFe composites and monolithic HA, suggesting the well-known superior osteoinductive capabilities of bioactive HA in directing MSCs to produce more calcified structure for bone tissue formation. Therefore, based on the gene expression analysis in the present study, it can be deduced that strontium promotes the differentiation of hMSCs toward osteogenic lineage via the upregulation of bone-specific genes, including Runx2, ALP, and OPN, but interestingly without significant effects on Col IA and OCN expression. 3.4.6. Terminal Osteogenesis of hMSCs Marked by Matrix Mineralization. The terminal stage of osteogenesis is indicated by mineralization of calcium phosphate on the extracellular matrix (ECM) secreted by the hMSCs committed toward osteoblastic lineage. The calcium deposits were visualized by Alizarin Red S (ARS) staining after 21 days of culture on SrHAFe composites. Figure 9A presents bright-field optical images, which reveal the extent of calcium deposition on the SPSed SrHA-Fe composites. Clearly, the calcium mineral deposition is significantly higher on SrHA10Fe and SrHA20Fe composites in comparison to the control and HA. This was further confirmed by quantification of the extracellular calcium bound to alizarin red dye, detected by elution of the ARS extracts and recording the absorbance at 450 nm, as shown in Figure 9B. We also performed scanning electron microscopy and energy dispersive X-ray spectroscopy (EDAX) analysis of the bonelineage committed hMSCs after 21 days of osteoinduction culture. Figure 9C shows distinct CaP nodules formed on the surfaces of the osteogenically differentiated hMSCs, cultured on SrHA10Fe and SrHA20Fe. Such prominent mineralization of CaP was not observed in case of the control and HA (data not M


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Figure 8. Semiquantitative RT-PCR analysis of osteoblast differentiation gene markers. (A) Gel panel showing bands corresponding to PCR end products obtained from control, HA, SrHA10Fe, and SrHA20Fe groups; (B) Densitometric quantification was performed using ImageJ software. The mRNA expression levels of Runx2, ALP, Col IA, OCN, and OPN were normalized to the expression level of GAPDH. Three samples were used for each experiment and data shown are the mean ± sd of two independent experiments. * and # indicate statistically significant difference (p < 0.05) of SPSed SrHA-Fe composites w.r.t control and HA, respectively.

and osteoinduction factors in driving the osteogenic differentiation of hMSCs on SrHA-Fe composites.

of culture. It may be noted that the concentrations indicated in the plots correspond to ion concentrations measured in replenished media at different time points and not the cumulative ion release. The release profiles of iron (Figure 10D) indicate a significant increase in Fe levels for SrHA10Fe and to a smaller extent in SrHA20Fe. The iron release from SrHA20Fe is seemingly hampered by the presence of multiple minor phases (CaCO3, SrFe12O19, and Fe3O4) as against the single secondary phase of Fe3O4 in SrHA10Fe. Although high iron concentrations can induce cytotoxicity mediated by oxidative stress, low levels of iron released from calcium phosphate cements have been shown to enhance osteogenesis and angiogenesis in mouse bone marrow stromal cells (mBMSCs) and human umbilical vein endothelial cells (HUVECs), respectively.46 Summarizing, a sustained release of Sr2+ and Fe2+, with a simultaneous depletion of Ca2+ and PO43− due to mineralization has been noted. Therefore, the spatiotemporal dynamics of bioinorganic ions implicated in bone homeostasis complement the surface topographic effects

4. DISCUSSION 4.1. SrFeNPs and SrHA-Fe Composites As Potent Orthopedic Biomaterials. Magnetic nanoparticles (MNPs) offer a wide scope for application in biomedical research because of their versatile synthesis methods, ease of size control leading to desired magnetic properties, and surface functionalization for targeted drug delivery and anticancer treatment by hyperthermia. The transition metals (Co, Ni, Fe) and metal ferrites (CoFe2O4, NiFe2O4) possess high saturation magnetization, but the leaching of Co2+ and Ni2+ present toxicity issues, limiting their application in the biomedical arena.47 In most cases, their toxicity has been circumvented by developing magnetic core−silica/CaP shell structures of the type MFe2O4@SiO2 (M = Co, Ni), which may be tedious for bulk synthesis and characterization.48 Therefore, magnetite (Fe3O4) and maghemite (γ-Fe2O3) are the most widely used MNPs for therapeutic treatments. N


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Figure 9. Biomineralization, an indicator of terminal stage osteogenesis of hMSCs: (A) Bright-field optical micrographs showing ARS stained calcium deposits formed on SPSed SrHA-Fe composites after 21 days of hMSC culture in osteoinduction media. Scale bar = 100 μm. (B) Quantification of extracellular calcium deposits by elution of calcium-ARS complex extracts and recording the absorbance at 450 nm. Three samples were used for each experiment and data shown are the mean ± sd of two independent experiments. * and # indicate statistically significant difference (p < 0.05) of SPSed SrHA-Fe composites w.r.t control and HA, respectively. (C) SEM images showing the formation of calcium phosphate (CaP) nodules on SPSed SrHA10Fe and SrHA20Fe composites; scale bar = 100 μm. The EDAX spectra recorded from the CaP nodules (indicated by arrows) on the cell surfaces are shown below the corresponding images.

study, the incorporation of strontium did not elicit any effect on the replication of MSCs, indicating a rapid onset of osteogenic differentiation within a week of culture. These results are consistent with the previous observations of Li et al.,56 demonstrating a delayed inhibition in MSC proliferation at day 5, upon treatment with strontium ranelate, an osteoporotic treatment drug. Therefore, the reduced cell growth observed in this study is subsequently marked by the increased genotypic/ phenotypic expression of osteoblastic markers and matrix mineralization, elucidating the interdependence of proliferation arrest and subsequent induction of differentiation. Another important factor that governs osseointegration is the intimate adhesion between the substrate and the cell/tissue in the vicinity. Especially, since stem cell fate regulation is largely influenced by the material features, an integrated information on a variety of factors such as cell area, number of FA points, spreading area and phenotypic/genotypic expression of differentiation markers can give a deep insight toward understanding and even predicting cell behaviors more precisely.57 As can be seen from the quantitative data depicted in Figure 6, the number of FA points on SrHA-Fe substrates was higher compared to HA and control, suggesting its effectiveness in accelerating the osteogenesis of MSCs compared to pure HA. It may be noted that apart from strontium release, we also invoke the role of microstructures in terms of surface topography to explain the bias of nanograined SrHA10Fe in eliciting the osteogenesis of hMSCs over their micron-sized grains in SrHA20Fe and even larger grains in SPSed HA. These findings

In this light, our observations of negligible toxicity of SrFeNPs over a size range from 30−100 nm and dosage up to 1000 μg/mL promise potential biomedical applications of these nanoparticles. Further, we intend to exploit the beneficial effects of strontium release from SrFeNPs and its composites for the development of osteoinductive biomaterials, which promote bone formation and reduce bone resorption. Furthermore, the incorporation of Sr2+ is proven to be effective in enhancing the bioactivity of biomaterials and, in particular, in osteoporosis treatment.49 Altogether, the objective of this study is two-fold. The first is to demonstrate the efficacy of Sr and Fe incorporated hydroxyapatite composites to trigger osteogenesis and suppress osteoclastogenesis for osteoporotic bone regeneration. This is achieved by the direct release of Sr and Fe from the composites. The therapeutic benefits of Fe are implicated in the increased risk of osteoporosis due to iron deficiency as well as in calcium homeostasis in healthy bone and teeth.50 The second objective is to illustrate the indirect effect of strontium incorporation manifested as enhanced degradation of HA.51 The consequent release of Ca2+ and PO43− results in an ion rich microenvironment conducive for osteogenic commitment of hMSCs. The dose-dependent increase in cell proliferation on Srdoped biomaterials has been confirmed by previous in vitro studies.49,52−54 Nuclear factor of activated T cell (NFATc)mediated Wnt signaling has been shown to be involved in the strontium-induced replication of osteoblasts, in a study reported by Fromigue et al.55 Conversely in the present O


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Figure 10. Competitive essential ion (Sr2+and Fe2+) release from SrHA-Fe composites implicated in hMSC osteogenesis. Elemental concentrations of (A) calcium, (B) phosphorus, (C) strontium, and (D) iron released into the cell culture media as determined by ICP-MS. All the experiments were carried out in triplicates and data shown corresponds to mean ± sd of three measurements from three individual experiments.

to extracellular stimuli,62 MAPK pathway partly mediates the response of MSCs to matrix signals by controlling Runx2 phosphorylation. Particularly in our study, the SrHA-Fe induced Runx2 activation, as shown in Figure 8, might be possibly mediated through ERK1/2 MAPK pathway. It can be noted that the initiation of Runx2 expression is the critical step in the commitment and differentiation of MSCs toward osteoblastic lineage and Sr in turn exerts an enhancing effect on ERK1/2 and p38 activation in order to promote the sustained transcriptional activity of Runx2.62 Besides, as Runx2 directly regulates osteogenic genes such as osteopontin (OPN) and osteocalcin (OCN), it is apparent that Sr can indirectly stimulate differentiation markers both at the gene and protein level, thus facilitating osteoblastic differentiation.63 Likewise, the expression of Col IA is one of the earlier events during osteogenesis, which is usually followed by sequential expression of ALP and OCN.64 The mRNA expression of Col IA was significantly higher than control for all the HA-based bioceramics. Similarly, consistent with an inverse relationship between vimentin and osteocalcin expression, the OCN levels were marked higher on HA and SrHA-Fe composites as compared to control. ALP, another important bone marker, was detected only on SrHA-Fe matrices. When compared to HA, the SrHA-Fe composites significantly promoted ALP production (Figure 7A) and robust collagen synthesis (Figure 7C) in

are in line with earlier literature reports that describe similar effect of fine and coarse grained dense (96−99%ρth) HA bioceramics on cell proliferation and cell-material interactions.58 On the contrary, both macroporosity and difference in grain sizes in resorbable β-TCP bioceramics did not elicit a significant difference in bone formation in vivo, while microporosity only influenced the rate of TCP degradation.59 Thus, differences in scaffold architecture and degradation can elicit differential biological responses, in vitro and in vivo. 4.2. Correlation of Gene Expression and Signaling Cascades Involved in Strontium-Induced Osteogenesis. Multiple molecular pathways have been implicated in strontium mediated osteogenesis, namely MAPK/ERK1/2, Wnt/NFATc signaling, FGF/FGFR signaling pathway and so on.60 Although MAPK activation has been implicated in many cellular signaling events, it is considered as one of the potential signal transduction pathway that regulates osteogenic differentiation of MSCs.61 Normally, hMSCs take 14−28 days to differentiate into osteoblasts, when cultured in the presence of osteogenic supplements. However, in our case, hMSCs cultured on SrHAFe matrices differentiated more rapidly than on monolithic HA or control samples, with enhanced mineralized matrix production at 21 days, along with early onset of osteogenic gene expression. Being the major signal transduction mechanism that regulates transcriptional activity in response P


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Figure 11. Schematic representation of the microstructure and strontium release triggered osteogenic differentiation of human mesenchymal stem cells on SrHA-Fe composites.

hMSCs; this agrees with the previous findings by Yang et al.65 Notably, a remarkable upregulation of OPN gene (∼10-fold) was recorded as an immediate early response in MSCs cultured on SrHA20Fe surfaces. High levels of osteopontin (OPN) are normally correlated with an increased concentration of free inorganic phosphate (Pi) in the culture medium.66 The growing evidence from literature implicates the participation of local inorganic phosphate signaling by NaPi transport via Pit1 in the induction of OPN.67 However, in our study, the higher inorganic phosphate (Pi) release due to a greater degradability of Sr-doped HA was probably masked by the presence of high amounts of organic phosphates in the culture media. It may be reiterated that one of the distinct outcomes of inorganic ion incorporation is the increased solubility of hydroxyapatite/ calcium phosphates. Subsequently, OPN has stimulated bone mineralization by mediating cell-matrix and matrix−matrix mineral adhesion (Figure 9). These findings are in accordance with the results of Schäck et al., wherein increased P i concentration induced upregulation of OPN and downregulation of ALP gene in osteogenic induction culture.68 Besides the assessment of osteogenic marker level based on mRNA and protein expression, the determination of mineralization in vitro is also crucial in evaluating terminal osteogenic differentiation of hMSCs. Since Sr2+ is largely known to exert dose-dependent increase in mineralization, it is not surprising to observe robust mineralization of extracellular matrix on SrHA-Fe platforms, as detected by ARS staining (Figure 9A). Summarizing, although nanotopography presented by nanofibrous scaffolds or nanosized surface features close to 100 nm are well-known to aid in osteogenesis,69 the surface topographic effects arising from the nanocrystallites in SrHA10Fe are surpassed by bioinorganic ionic cues as in greater strontium release from SrHA20Fe promoted higher matrix mineralization. In a nutshell, Figure 11 schematically depicts the microstructure and strontium release-dependent osteogenic differentiation of hMSCs cultured on SrHA-Fe composites.

NPs during spark plasma sintering of HA-SrFe12O19 powders led to the formation of Sr-doped HA and Fe3O4 predominantly. The magnetic properties of the annealed SrFeNPs and SrHAFe composites are commensurate with the phase compositions and crystallite/grain sizes determined using Rietveld refinement and electron microscopy, respectively. The spark plasmasintered SrHA-Fe composites with different grain sizes evoked cell spreading and large number of focal adhesion complexes along with enhanced osteogenic marker (Runx2, ALP, Col IA, OCN, and OPN) expression in hMSCs governed by the sustained release of strontium and/or other essential bioinorganic ions (Ca, P, and Fe). Matrix mineralization was similarly elevated on both SrHA10Fe and SrHA20Fe, suggesting the competing effects of presenting surface nano features in SrHA10Fe against the greater strontium release from SrHA20Fe. In summary, our study demonstrates the osteogenic potential of novel strontium incorporated SrHA-Fe bioceramics for orthopedic applications. Through our approach, a combinatorial release system of bioinorganic ions (Sr, Fe, Ca and P) essential for bioactivity has been achieved. Further, the slow release profiles of two therapeutically relevant inorganic ions (Sr and Fe) can synergistically regulate osteogenic differentiation in vitro and bone metabolism in vivo, as against the burst release of strontium in Sr-doped bioglasses and Sr-doped HA. In the future, we intend to apply such magnetoactive composites as culture platforms for an external magnetic field-assisted manipulation of stem cell response.14,70

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b08694. FTIR analysis of annealed SrFeNPs in Figure S1; Rietveld refinement of HA-SrFe ball-milled powders in Figure S2; elemental mapping of SPSed SrHA20Fe in Figure S3; additional fluorescence images showing cell spreading and focal adhesions on SPSed HA-based composites in Figure S4; heating cycle used for multistage spark plasma sintering of HA and its composites with SrFe12O19 in Table S1; primer sequences and PCR reaction conditions used for evaluating the expression of osteogenic marker genes in Table S2; phase compositions and lattice parameters deduced from the rietveld

5. CONCLUSIONS The competing effects of substrate composition, topography and sustained release of bioinorganic ions in eliciting the osteogenic differentiation of hMSCs are demonstrated in the present study. For the first time, we have shown that strontium hexaferrite (SrFe12O19), a hard magnetic material is relatively non-toxic in a size range from 30−100 nm and dosage up to 1,000 μg/mL. Further, the in situ decomposition of SrFe12O19 Q


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refinement of HA-SrFe ball-milled powders in Table S3 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +91-80-2293 2945. Fax: +91-80-2360 1310. *E-mail: [email protected]. Phone: +91-80-2293 3256. Fax: +91-80-2360 7316. Author Contributions †

S.K.B. and G.T. contributed equally

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the Department of Science and Technology (DST) and major funding support from Department of Biotechnology (DBT), Government of India via the ‘Translational Centre on Biomaterials for Orthopaedic and Dental Applications’ and through the project entitled ‘Stem cell fate processes on substrates with gradient in conductivity and stiffness’. A special note of thanks is due to Ariharan S. from the department of Material Science and Engineering, IIT Kanpur, for SPS of the samples. The authors also thank Dr. Ramananda Chakrabarti and his student Rahul Das Gupta from the Centre for Earth Sciences (CEAS), IISc, for helping in the ICP-MS measurements. Further, we thank Dr. Chandan Srivastava and his student, Sai Phaneendra Bachu from Materials Engineering, IISc, for his support with the magnetic data recording using VSM and Amsarajan from IPC, IISc, for FTIR recording. One of the authors, S.K.B., acknowledges the Council for Scientific and Industrial Research (CSIR) for providing scholarship (09/ 079 (2501)/2011-EMR-I dt. 16-11-2011) during the period of study.

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