In vitro response of human osteoblasts to multi-step sol–gel derived bioactive glass nanoparticles for bone tissue engineering

Materials Science and Engineering C 36 (2014) 206–214

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

In vitro response of human osteoblasts to multi-step sol–gel derived bioactive glass nanoparticles for bone tissue engineering Jian Ping Fan a,⁎, Priya Kalia b, Lucy Di Silvio b, Jie Huang a a b

Department of Mechanical Engineering, University College London, London WC1E 7JE, UK Biomaterials, Tissue Engineering & Imaging, The Dental Institute, King's College London, Guy's Hospital, London SE1 9BT, UK

a r t i c l e

i n f o

Article history: Received 23 September 2013 Received in revised form 28 November 2013 Accepted 7 December 2013 Available online 15 December 2013 Keywords: Tissue engineering Nanoparticles Bioactive glass Sol–gel Hydroxyapatite Osteoblast

a b s t r a c t A multi-step sol–gel process was employed to synthesize bioactive glass (BG) nanoparticles. Transmission electron microscopy (TEM) revealed that the BG nanoparticles were spherical and ranged from 30 to 60 nm in diameter. In vitro reactivity of the BG nanoparticles was tested in phosphate buffer saline (PBS), Tris-buffer (TRIS), simulated body fluid (SBF), and Dulbecco's modified Eagle's medium (DMEM), in comparison with similar sized hydroxyapatite (HA) and silicon substituted HA (SiHA) nanoparticles. Bioactivity of the BG nanoparticles was confirmed through Fourier transform infrared spectroscopy (FTIR) analysis. It was found that bone-like apatite was formed after immersion in SBF at 7 days. Solutions containing BG nanoparticles were slightly more alkaline than HA and SiHA, suggesting that a more rapid apatite formation on BG was related to solutionmediated dissolution. Primary human osteoblast (HOB) cell model was used to evaluate biological responses to BG nanoparticles. Lactate dehydrogenase (LDH) cytotoxicity assay showed that HOB cells were not adversely affected by the BG nanoparticles throughout the 7 day test period. Interestingly, MTS assay results showed an enhancement in cell proliferation in the presence of BG when compared to HA and SiHA nanoparticles. Particularly, statistically significant (p b 0.05) alkaline phosphatase (ALP) activity of HOB cells was found on the culture containing BG nanoparticles, suggesting that the cell differentiation might be promoted by BG. Real-time quantitative PCR analysis (qPCR) further confirmed this finding, as a significantly higher level of RUNX2 gene expression was recorded on the cells cultured in the presence of BG nanoparticles when compared to those with HA and SiHA. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The current gold standard for bone regeneration treatment is autografting, whereby the patient's own bone is harvested and implanted into the trauma site of the patient [1]. However, with the limited availability of autologous bone, and a rise in demand for orthopedic implants, the need for new bone regenerative materials has become an element of global significance. One attractive method of addressing this issue has been the use of bone tissue engineered 3D scaffolds, which act as 3D platforms for bone morphogenesis [2–4]. Conventionally, a bioceramic is used as a filler material in a polymeric matrix to improve on structural properties and induce bioactivity to encourage effective biological interaction [5]. Other methods include the use of bioceramics as bioactive coatings on inert materials, particularly for implants required in load-bearing situations, where a thin bioactive layer is deposited onto the bioinert materials. Over the decades, there has been considerable research into bioceramics for biomedical applications, ranging from the bioinert ceramics such as zirconia and alumina to the bioactive ceramics such ⁎ Corresponding author. Tel.: +44 20 7679 3907; fax: +44 20 7388 0180. E-mail address: [email protected] (J.P. Fan). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.009

as bioactive glasses and calcium phosphate ceramics [6]. Bioinert materials are typically biologically inactive and incapable of forming a direct bond with the host tissue. On the other hand, bioactive materials are capable of promoting bone formation through chemical reactivity with its surroundings. The focus would therefore be on bioactive glasses or ceramics as they form bone-like apatite on the surface in vivo and in vitro, thus capable of bonding directly to tissue material [7–9]. Hydroxyapatite (HA), which has a calcium to phosphorus (Ca/P) ratio of 1.67, has been extensively used in biomedical applications due to its chemical similarities to the inorganic components of bone [10–13]. More recently, silicon substituted hydroxyapatite (SiHA) has become an attractive alternative due to its increased bioactivity and osteoconductivity [11,14,15]. Reports have suggested that, with the inclusion of trace amounts of silicon in HA, improvements to osteoblast attachment, proliferation and differentiation were observed [12]. In vivo studies carried out by Patel et al. [11] further showed that SiHA had enhanced bioactivity when compared to HA. Bioactive glasses have also been seen as a set of promising biomaterials for various biomedical applications. The Class A properties of bioactive glasses set it apart from HA, wherein the former has both osteoinductive and osteoconductive properties whilst the latter only being osteoconductive [6,16]. Studies have shown that the high levels

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of bioactivity of bioactive glasses were due to their ability to rapidly form a hydroxycarbonate apatite (HCA) layer [17]. Although important in the process of bone regeneration, it has been found that the dissolution products of Si and Ca ions from the degradation can up-regulate and activate genes which enhance osteoblastic proliferation and differentiation, leading to rapid bone regeneration [18–20]. First introduced in 1969, the classical melt-derived Bioglass® 45S5 has been widely studied and successfully used in numerous commercial products [4,16]. However, the melt-derived process, which involves the quenching of oxides at high temperature to obtain this glass, has its limitations such as requiring high processing temperature and a narrow Class A compositional range. To overcome this, bioactive glasses were synthesized by the sol–gel process [21]. The sol–gel process is a relatively low temperature method of obtaining bioactive glasses from the hydrolysis and polycondensation of metal hydroxides, alkoxides and inorganic salts. Further benefits such as better bone bonding ability, higher degradation rates and the ability to incorporate various cation inclusions into the sol–gel network have made this method of bioactive glass synthesis very attractive [22,23]. Studies have shown that fundamental changes in pH, precursor concentration and processing temperature have been able to change the silica networks and hence affect the final glass structure [24,25]. During the sol–gel reaction, an acidic reaction leads to the formation of a linear or random branched polymer, whilst under a basic reaction, clustered polymer branches are formed [25,26]. Stöber et al. [24] demonstrated the use of basified water as a morphological catalyst to successfully synthesize monodispersed silica particles from the acidic hydrolysis of tetraalkyl silicates. Expanding on the usage of a basified catalyst to obtain homogenous sized and shaped particles, Hong et al. [27,28] successfully synthesized bioactive glass nanoparticles in the range of 30–100 nm diameters through a sol–gel and co-precipitation method. Nanomaterials which are one dimension less than 100 nm are classified as nanoparticles [29]. As a comparison to living organisms, proteins are typically 5 nm in size whilst organelles fall in the 100–200 nm domain [30]. The potential benefits of nanoparticles are their inherent high surface to volume (S/V) ratios, allowing increased solubility, and hence increased bioactivity. Recent research on the mesoporosity of BG nanoparticles also point towards their potential as platforms for drug delivery and imaging [31]. Employing sol–gel synthesis of bioactive glass with an alkali morphological catalyst, this study firstly sets out to obtain BG nanoparticles of a homogenous shape and size, allowing for a narrow size distribution. Subsequently, the in vitro response and bioactivity of these biomaterials were tested with physiological fluids of phosphate buffer saline (PBS), Tris-buffer (TRIS), simulated body fluid (SBF) and Dulbecco's modified Eagle medium (DMEM). It was crucial to understand the cellular responses of primary human osteoblast (HOB) cells in culture with BG nanoparticles, such as cytotoxicity, cell proliferation and cell differentiation, which were investigated in this study for their potential application in coatings and scaffolds for bone tissue engineering. 2. Materials and methods 2.1. Material preparation BG nanoparticles of composition 58% SiO2\37% CaO\5% P2O5 (mol%) were synthesized by modifying the method described by Hong et al. [28]. Briefly, 21.38 g of tetraethyl orthosilicate (TEOS; Aldrich) was dissolved in 120 mL of ethanol with the pH adjusted to pH 1.9 using 0.1 M nitric acid. Separately, 15.46 g of calcium nitrate (Ca(NO3)2; Aldrich) was dissolved in 200 mL of deionized water and then mixed together with the TEOS solution. 1.164 g of ammonium dibasic phosphate ((NH4)2HPO4; Aldrich) was dissolved in 3 L of deionized water, the pH was adjusted to pH 11 using ammonium hydroxide (NH3 28% in H2O; Aldrich). Using a peristaltic pump, the solution containing TEOS and calcium nitrate was slowly dripped into the ammonium dibasic phosphate solution and stirred vigorously. During this process, the

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pH value of the solution was maintained at pH 11 using ammonium hydroxide. The precipitate obtained was aged for 48 h and dried in the oven at 90 °C. The precipitate was then crushed and sintered at a temperature of 680 °C to eliminate any residual substances of nitrates and silanol groups [32]. HA and SiHA nanoparticles were synthesized using the precipitation method in which 1.953 g of calcium hydroxide (Ca(OH)2; Aldrich) and 1.717 g of phosphoric acid (H3PO4 85% concentrate; Aldrich) was dissolved/diluted in 200 mL distilled water respectively. For SiHA containing 1.0 wt.% Si, 0.197 g TEOS was added to the phosphoric acid and mixed homogenously. Using a peristaltic pump, the acid was slowly dripped into the calcium hydroxide solution under stirring. Ammonium hydroxide was added during the reaction to maintain the pH above 10 [33]. 2.2. Material characterization 2.2.1. Evaluation of nanoparticles The morphology of the synthesized nanoparticles was examined using a transmission electron microscope (JEOL 1010 TEM) with an accelerating voltage of 80 kV. The nanoparticles were suspended in ethanol using a sonicator (Branson 250 Sonicator) before being collected on TEM copper grids for imaging. The surface morphology and chemical composition of the nanoparticles were analyzed by a scanning electron microscope (JEOL JSM-6301F field emission SEM) equipped with an energy-dispersive X-ray (EDX) spectroscope (INCA X-sight Oxford Instruments) detector. Further quantitative chemical composition analysis was determined by X-ray fluorescence (XRF) (London & Scandinavian Metallurgical Co.). Crystal structure of the nanoparticles was analyzed by X-ray diffraction (XRD) using an X-ray diffractometer (Bruker D4 Endeavor) with copper Kα radiation using 2θ values between 5° and 80° with a 0.05° step size and a count rate of 2 s/step. A Fourier transform infrared spectroscope (Perkin Elmer 2000 FTIR) was utilized to characterize the functional groups of the biomaterials produced. The nanoparticles were crushed with potassium bromide (KBr) and then compacted into thin disk for FTIR analysis. An average of 20 scans was recorded for each spectrum, which was normalized against pure KBr. 2.2.2. In vitro testing The nanoparticles were immersed in phosphate buffer saline (PBS), Tris-buffer (TRIS), simulated body fluid (SBF) and Dulbecco's modified Eagle medium (DMEM). PBS was prepared by dissolving one tablet (PBS; Aldrich) in 200 mL of deionized water to obtain a final pH of 7.4. TRIS buffer was made by dissolving tris(hydroxymethyl)aminomethane (Aldrich) with deionized water and adjusted to pH 7.4 using hydrochloric acid (1 M HCL; Aldrich). SBF, closely resembles the ion concentration of blood plasma, was prepared accordingly to the method reported previously [8], and adjusted to a final pH of 7.4. The nanoparticles with a final concentration of 0.1 mg/mL were incubated at 37 ºC for 1, 4, 7, 14, 21 and 28 days. At each time point, the pH of the supernatant was measured. The reacted nanoparticles in SBF were removed, rinsed in de-ionized water, and dried in an air circulation drying oven, and the changes in surface structure was analyzed by FTIR. 2.3. In vitro biological study 2.3.1. Cell culture HOB cells, obtained by a method previously described [34] were cultured in 25 cm2 sterile tissue culture flasks at 37 °C in a humidified air atmosphere of 5% CO2. The culture medium used was Dulbecco's modified Eagle medium (DMEM) media, supplemented with 10% fetal calf serum (FCS), L-ascorbic acid (150 g/mL), and L-glutamine, penicillin and streptomycin (100 units/mL). Once confluent, the HOB cells were collected by trypsinizing adherent cells and resuspended in DMEM and cell viability was assessed using the Trypan blue exclusion test.

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2.3.2. Nanoparticle loading and cell seeding The nanoparticles were sterilized by thorough rinsing in ethanol (H CCH OH 100% absolute; Analar Normapur). After drying, DMEM medium containing nanoparticles of 0.2 mg/mL was prepared and sonicated to break up any agglomerates. 0.5 mL of HOB cells in DMEM (8 × 104 cells/mL) was seeded onto 24-well culture plates containing 0.5 mL nanoparticles and DMEM solution of 0.2 mg/mL to make a final concentration of 0.1 mg/mL. The plates were then incubated at 37 °C in a humidified air atmosphere of 5% CO2 for a period of 4 and 7 days. 2.3.3. Cell morphology Changes in HOB cell morphology post-interaction with the nanoparticles were examined by a scanning electron microscope (JEOL JSM7401FF.E.SEM). After 7 days, glass slips containing HOB cells cultured with the nanoparticles were fixed in a glutaraldehyde solution (2.5% CH2(CH2CHO)2 in H2O; Aldrich) and stained with Osmium tetroxide (OsO4 99.8%; Aldrich). The osmium stained samples were then rinsed to remove residual OsO4 and dehydrated using a CO2 exchanger. Dried samples were coated with a thin layer of gold (Gatan High Resolution Ion Beam Coater) and observed under SEM. 2.3.4. Lactate dehydrogenase assay In vitro toxicology was assessed using the lactate dehydrogenase (LDH) assay to test the amount of cytoplasmic LDH released into the medium [35]. The LDH master mix which contained equal amounts of assay substrate, dye and cofactor was prepared according to the manufactures' guidelines (TOX7; Aldrich). 50 μL of the culture medium from each sample was transferred into a 96-well plate and to it 100 μL of the LDH master mix was added. The samples were then incubated at room temperature for 30 min before being read using a plate reader (Opsys MR Dynex) at an absorbance of 490 nm with a background absorbance of 690 nm. 2.3.5. Cell viability assay The viability of HOB cells after culturing with BG nanoparticles was determined using the CellTiter 96® AQueous non-radioactive cell proliferation assay (Promega Corporation). The tetrazolium compound (MTS) is biologically reduced by cells into a formazan product. 20 μL of 5% phenazine methosulfate (PMS; Promega Corporation) mixed in MTS solution was aliquoted into a 96-well plate containing 100 μL of the cells in DMEM and incubated for 4 h at 37 ºC in a humidified air atmosphere of 5% CO2. Subsequently, the absorbance was measured at a wavelength of 490 nm (Opsys MR Dynex Plate Reader).

freeze–thaw cycle 3 times to compromise the integrity of the cell walls. A series of standard concentrations (0, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 μg/mL) in saline sodium citrate buffer (SSC) at pH 7 were prepared. Cell lysate and the series standards were aliquoted into a 96-well plate, to which Hoechst 33258 was added in a 1: 1 ratio. The plate was read (Hidex Chameleon Platform Multilabel Detector) at a wavelength of 460 nm. 2.3.8. Alkaline phosphatase activity assay The ALP activity from the cell lysate was measured via the enzyme alkaline phosphatase which cleaves the phosphate group from p-nitrophenyl phosphate to p-nitrophenol [39]. Equal amounts of cell lysis and the substrate reagent which contained p-nitrophenyl phosphate, magnesium chloride hexahydrate, triton X-100 (12.5 v/v %) and 0.1 M glycine (pH 10.3) from each sample were transferred into a 96-well plate. A series of standard concentrations (0, 1.563, 3.125, 6.25, 12.5, 25, 50 and 100 μg/mL) were prepared from the dilution of p-nitrophenol with 0.1 M glycine (pH 10.3). The samples were then incubated at 37 ºC for 15 min before being read at an absorbance of 405 nm. 2.3.9. Real-time quantitative PCR analysis Runx2, the gene associated with osteoblastic differentiation, was determined by real-time qPCR analysis. After 4 and 7 days of culture of HOB cells in the presence of BG nanoparticles, the culture medium was removed and replaced with 1 mL Tri Reagent (AM8738; Ambion). RNA was then extracted from each Tri Reagent treated sample with chloroform and quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). Once the concentration of RNA in each sample was determined, 200 ng/20 μL (RNA/H2O) concentration was calculated and mixed thoroughly with 20 μL of reverse transcription (RT) master mix (Applied Biosystems). High-capacity complementary (cDNA) reverse transcription was carried out using a thermal cycler (Applied Biosystems Venti 96-Well Thermal Cycler) in which the extracted RNA is transcribed into cDNA. Once completed, 10 μL of the cDNA from each sample was mixed with 15 μL of TaqMan gene expression master mix (Life Technologies) and a gene master mix which contains the gene associated with osteoblastic differentiation, Runx2, and the house-keeping gene 18S. The Taqman gene expression master mix contained Taq polymerase which is used to extend the short primers. Primers are used to bind the short cDNA fragments when amplification is carried out to obtain more DNA. Real-time qPCR analysis for 18S and Runx2 was performed using a real-time PCR machine (Taqman 7900HT). 3. Results

2.3.6. alamarBlue® assay Cell proliferation was evaluated using alamarBlue® assay, which is based on the reduction of resazurin by mitochondrial metabolic activity of the cells. The blue colored resazurin is reduced to resorufin which is pink in color and highly fluorescent [36]. The proliferation of HOB cells in contact with BG nanoparticles was measured following the method described previously [37]. After 4 and 7 days of culture, media from the 24-well plate was replaced with 1 mL of 10% alamarBlue® solution in phenol free medium and incubated for 4 h at 37 ºC in a humidified air atmosphere of 5% CO2. At the end of the incubation period, 100 μl of the supernatant was transferred into a 96-well plate and read at wavelength 570 nm with a reference wavelength of 630 nm (Opsys MR Dynex Plate Reader). 2.3.7. DNA assay The DNA content of the cell lysate after incubation with the nanoparticles was determined fluorimetrically. The Hoechst 33258 (B2883; Aldrich) is DNA specific and binds to contiguous adenine–thymine base pairs thus emitting fluorescence at 460 nm when excited at 355 nm [38]. After 4 and 7 days of incubation, the DMEM was replaced with 1 mL distilled water. Cell lysate was obtained by repeating a

3.1. Evaluation of nanoparticles 3.1.1. Nanoparticle characterization TEM examination revealed the spherical shape of the BG nanoparticles synthesized, as shown in Fig. 1a. The diameter of the nanoparticles mainly ranged from 30 to 60 nm. HA and SiHA nanoparticles synthesized were observed to be rod-shaped with a mean length-to-width aspect ratio of 3.5. Particle sizes ranged from 40 to 130 nm in length. EDX analysis of the BG nanoparticles (Fig. 2) confirmed the ternary system of the glass, containing the elements Si, Ca and P. The experimental compositions of the nanoparticles used in this study are listed in Table 1. XRF analysis found the BG composition to be almost consistent with the nominal values, suggesting that this multi-step synthesis method proved an effective way of accurately producing homogenous nanoparticles of intended composition. Fig. 3 shows the XRD patterns of BG, HA and SiHA nanoparticles. For the BG nanoparticles, a broad band between the 2θ angles of 20° and 40° was observed with no diffraction maxima being detected, suggesting the amorphous nature of glass, as expected. Crystalline phases of HA and SiHA were verified by XRD diffractive peaks at 2θ = 25.8° (002),

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Fig. 1. TEM micrographs of the nanoparticles synthesized showing shape and size of BG (a), HA (b) and SiHA (c).

31.7° (211), 32.1° (112), and 32.9° (300) and at 2θ = 31.7° (211), 32.1° and (112), respectively. No other peaks were observed in comparison with the HA standard (JCPDS 9-432), thus confirming phase purity of the HA and SiHA nanoparticles. 3.1.2. pH of the immersion solutions A significant difference in the pH levels was measured when BG nanoparticles were immersed over a 28 day period in PBS as compared to HA and SiHA. Throughout the immersion period, the BG in PBS fluid remained at a stable pH of 9.5 whilst HA and SiHA samples maintained an average stable pH of 7.2 and 7.5 respectively (Fig. 4a). This large difference in pH was only observed in PBS. Overall, the pH of TRIS, SBF and DMEM across all nanoparticle types remained relatively neutral, with BG nanoparticles recording slightly higher levels of alkalinity when compared to solutions containing HA and SiHA nanoparticles. 3.1.3. In vitro bioactivity The FTIR spectra of the BG nanoparticles before and after 7 days of immersion in SBF are shown in Fig. 5, which showed a decrease in transmittance intensity and a broadening of peaks. Before immersion in SBF (0 days), a strong band in the region of 1080–1200 cm− 1 and of 890 cm− 1 was observed which corresponded to the asymmetric stretching modes of Si\O\Si, whilst the band at 470 cm− 1 corresponded to the bending mode of this group [40–42]. A Si\O\Ca bond was observed at 960 cm−1 [42–44], suggesting that after thermal treatment at 680 °C, the calcium was introduced into the glass network as a network modifier, forming non-bridging oxygen bonds [45]. After 7 days of immersion in SBF, a weak twin band was observed around the wave number 600 cm−1 and 570 cm−1 which corresponds to the υ4 antisymmetric bending mode of P\O bonds in the amorphous calcium phosphate [9,46]. Another set of peaks formed at 1460 cm−1

and 1420 cm−1 after immersion was attributed to the formation of C\O bonds. These C\O bonds are from the υ3 vibration mode of carbonate ions, which have undergone peak splitting [40,47]. The presence of P\O and C\O bonds suggests the formation of bone-like apatite and indicates the bioactivity of the nanoparticles [40,42,48]. 3.2. In vitro biocompatibility evaluation 3.2.1. Cell morphology The morphology of HOB cells following exposure to the nanoparticles was observed under light microscopy. As shown in Fig. 6a, the cells appeared flattened and have anchored onto the surface of the wells after 24 h. There was no difference in cell morphology observed when cultured in the presence of the various nanoparticles over both time points. The detailed interaction of HOB cells with the nanoparticles was further examined using SEM. As shown in Fig. 6c, after 7 days of culture, cells exhibited flattened morphologies with filopodia extensions on the surface similar to that of the tissue culture plastic (TP) control. 3.2.2. In vitro cell viability The cytoplasmic lactate dehydrogenase (LDH) released into the medium by cells was used to assess the cytotoxicity of the nanoparticles as this release would suggest that cell membranes have been compromised. Compared to cells cultured in the absence of nanoparticles, LDH releases from HOB cells cultured with the nanoparticles were higher, with SiHA recording the highest release after 4 days, which indicated some levels of cytotoxicity of the nanoparticle towards HOB cells. However, at 7 days of incubation, the LDH release decreased across all samples and recorded no significant difference between BG, HA, and SiHA, similar to that of the TP control. (See Fig. 7). 3.2.3. In vitro cell proliferation The metabolic activity of cells during 7 days of culture was measured using the alamarBlue® assay. Result in Fig. 8a showed that at day 4, cell proliferation levels on the HA and SiHA samples were comparable to the BG sample. However, at day 7, the measured absorbance showed significantly more cells proliferating on the HA sample when compared to BG (p b 0.05). The CellTiter 96® Aqueous MTS assay was also used to investigate cell attachment and proliferation. TP was used as the negative control and normalized at 100% cell viability. In Fig. 8b, all three biomaterials Table 1 XRF measurements of experimental nanoparticle compositions (mol%).

Fig. 2. EDX curves of BG nanoparticles showing the presence of Si, Ca, P and O peaks.

Sample

SiO2 (mol%)

CaO (mol%)

P2O5 (mol%)

BG HA SiHA

56.0 – 1.7

38.5 63.0 62.8

5.5 37.0 35.5

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Fig. 3. XRD patterns of BG, HA and SiHA nanoparticles with identified reflection at major peaks.

displayed very similar growth trends, and at day 4 showed high percentage of activity (N90% viable cells) when normalized against the negative control. Interestingly, at 7 days of culture, there was no significant change in the percentage of viable cells in BG samples whilst cell viability in HA and SiHA samples decreased by approximately 15% and 10% respectively. 3.2.4. Cell alkaline phosphatase (ALP) enzyme activity ALPase enzymatic activity of cells on test materials in culture recorded an overall increase in ALP activity of the cells as the incubation period increased from 4 to 7 days. In comparison to the negative control (TP), there was no significant difference in the level of ALP activity measured for both 4 and 7 days. The total DNA obtained from each sample

Fig. 4. The pH changes of buffered solutions PBS (a) and DMEM (b) after immersion of BG, HA and SiHA nanoparticles.

demonstrated that for both time points, cells cultured with BG nanoparticles produced less DNA than HA and SiHA. The ALP activity was normalized against DNA content per sample to determine the ALP production per cell (Fig. 9). At day 4, cells cultured with BG were at comparable levels to TP, but indicated significantly higher (p b0.05) ALP expression than HA and SiHA. After 7 days, ALP expression by cells cultured with BG remained significantly higher than SiHA, and also expressed significantly higher ALP levels than the TP group which decreased by almost half from the levels of the first time point. 3.2.5. Real-time qPCR analysis The house-keeping gene 18S was used as a marker for the polymerase chain reaction (PCR). In Fig. 10, significant differences in transcription

Fig. 5. FTIR spectra of BG nanoparticles before and after immersion in SBF for a period of 7 days.

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Fig. 6. Light microscopy images of HOB cells in culture with the presence of BG nanoparticles after 24 h (a) and 7 days (b); SEM micrograph of HOB cell morphology cultured in the presence of nanoparticles after 7 days (c).

levels of the osteogenic gene RUNX2 were observed when compared over 4 and 7 day periods. The expression of RUNX2 by HOB cells was comparable for all three biomaterials on day 4 in contrast to TP, which expressed significantly higher levels of the osteogenic gene (p b 0.05). As the culture progressed, it is interesting to observe that the levels of expressed RUNX2 by HOB cells cultured with BG became significantly higher than HA and SiHA. 4. Discussion BG, HA and SiHA nanoparticles are widely used in bone tissue engineering applications as nanofillers for scaffolds or implant coatings [12,13]. Understanding the reaction of these nanoparticles within a physiological environment and its interaction with living matter is extremely crucial as it can open new doors to the future development and application of non-invasive yet highly impactful treatments in the biomedical field. The physico-chemical properties such as particle morphology, concentration, bioactivity and stability are highly influential at the material–tissue interface [49,50]. Although the nanoparticles are chemically similar to its larger micro-sized counterparts, due to the large S/V ratio, these nanoparticles can result in significantly different biological responses. Salata [51] reported that the surface of natural bone contains submicron features and surface roughness of approximately 100 nm. Thus, the synthesized nanoparticles in this study, which were in the range of 30–60 nm in diameter, would be highly suitable in providing for this submicron surface texture when applied as a nanofiller or implant coating. This type of surface is believed to improve cell adhesion and proliferation [49–53]. The crystal structure of bioactive glasses or bioceramics would play a significant role in its degradation and thus affect the biological consequences since the stability of the nanoparticles and subsequent ionic release through degradation affect its bioactivity and biocompatibility. Generally, the higher the solubility of the bioactive glass or bioceramic,

Fig. 7. Cytotoxicity of the nanoparticles on HOB cells cultured in DMEM at 4 and 7 days measured through the LDH assay at 460 nm. Data represents the mean ± SD, n = 3.

the faster is the rate of apatite formation [10]. Since no crystalline phases were observed in the BG nanoparticles as oppose to HA and SiHA in Fig. 3, this would explain the rapid rise in pH levels of PBS observed as bioactive glasses have been known to exhibit faster dissolution rates than crystalline bioceramics [46]. Changes in pH levels of the physiological fluids upon immersion of the nanoparticles are indicative of ionic release. Partly, this increase in pH upon immersion forms the beginnings of the formation mechanism of apatite, which begins with the rapid exchange of Ca2+ with H+ and H3O+ ions giving rise to pH levels in the solution [23,54]. The higher levels of alkalinity from the solutions containing BG nanoparticles corroborate with this mechanism in which, during the early stage of apatite formation, the rapid exchange of ions lead to an increase in concentration of silionol (Si-OH) [20,54]. A significantly high alkali level of PBS containing BG nanoparticles was observed

Fig. 8. Cell proliferation of HOB cells cultured with nanoparticles monitored using alamarBlue® assay at 4 and 7 days (a), percentage cell viability of HOB cells cultured with nanoparticles as compared to negative control TP monitored using CellTiter 96® Aqueous MTS assay at 4 and 7 days (b). Data represents the mean ± SD, n = 6, *: BG to HA at day 7 (p b 0.05).

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Fig. 9. Osteogenic expression of HOB cells in ratio of ALP/DNA cultured for 4 days and 7 days. Data represents the mean ± SD, n = 6, *: BG to HA, SiHA at day 4 (p b 0.05), **: BG to SiHA, TP at day 7 (p b 0.05).

due to PBS having a lower buffering capacity when compared to the other solutions. This would also suggest that due to the different buffering capacities of various solutions, the mechanisms of reaction between the nanoparticles and the surrounding interface can be affected, inferring that buffering capacities of physiological solutions also play an important role in determining the mechanisms of reaction between the nanoparticles and the surrounding interface. A comparison of apatite formation rates on bioactive glasses immersed in SBF has shown a rapid formation within an hour, with the growth of apatite observed to increase with immersion time [21,40,43,47,55]. The bioactivity of the BG nanoparticles derived in this experiment showed a fast formation of HCA, as confirmed in Fig. 5. Its formation rate was in agreement with the findings of Hong et al. [28], which showed the formation of bone-like apatite on bioactive glass nanoparticles within 7 days of soaking in SBF. The formation of an apatite layer is important to the bone bonding capabilities of a biomaterial, as it provides a suitable surface for HOB cell attachment and proliferation, allowing it to interact with the host bone [23]. Knowing the cytotoxicity of the nanoparticles is the first step in determining the suitability of the BG nanoparticles derived from the multi-step sol–gel route as a nanofiller or implant coating. Although a decrease in cell viability was noticed after 7 days of culture, results indicated no significant differences in cytotoxicity levels between BG with that of HA and SiHA nanoparticles, suggesting the non-cytotoxic

Fig. 10. Real-time qPCR analysis of the relative expression for the osteogenic gene of RUNX2 in the HOB cells after 4 days and 7 days. Data represents the mean ± SD, n = 6, *: BG to TP at day 4 (p b 0.05), **: BG to HA, SiHA at day 7 (p b 0.05).

behavior of BG. This decrease in cell viability levels over the two time points could point to the possibility of HOB cells remodeling at early stages of culture, forming a new sub-population of cells specifically adapted to the new environment introduced [19]. HOB cells were able to proliferate when cultured with BG, HA and SiHA nanoparticles, as observed in Fig. 8, suggesting that all three biomaterials initiated positive cellular responses, following a normal growth pattern and metabolic activity. MTS assays further demonstrated enhanced cell proliferation on the BG sample, with a statistically higher number of viable cells than on HA and SiHA samples. Studies have shown that bioactive glasses, especially those obtained from the sol– gel route display remarkably high bioactivity [21,28,47] and therefore corroborate with the findings from this study. It suggests that cell proliferation was influenced by the rapid apatite formation, and thus lead to a favorable osteoblast response when cultured with BG nanoparticles. Cell proliferation on HA nanoparticles was also in agreement with studies which have confirmed its stimulatory effects on osteoblastic proliferation [58,59]. Comparison studies between nano- and microsize HA particles showed that cell proliferation favored nanosize HA, further reinforcing the positive HOB cell response with BG, since HA and SiHA used in this study were nanoscale [58]. The enhanced cell proliferation observed from the MTS assays could be attributed to the dissolution products of bioactive glasses. Biologically active ions of calcium and silicon are known to induce osteostimulation, giving rise to rapid bone formation through the stimulation of HOB cells to enter an active cycle of mitosis and subsequently differentiate into mineralized mature bone [19,20,56]. DNA concentration in the cell lysate is indicative of the level of osteoblastic proliferation, whilst ALP is a key osteoblastic marker of differentiation. Normalizing the ALP activity against DNA concentration, the 4 day time point showed cells cultured in the presence of BG expressed significantly higher levels of ALP activity per cell than HA and SiHA. Interestingly, as culture progressed, increases in ALP activity of HOB cells cultured with HA and SiHA nanoparticles would suggest that cells cultured with BG were inclined to enter cell differentiation at an earlier stage of culture [57]. This might explain the decrease in ALP activity of HOB cells cultured with BG because at later stages of culture, mineralization was already in progress. ALP activity of HOB cells is known to diminish over extended periods of culture due to the onset of mineralization, with the possibility of mineralization occurring as early as 6 days of culture [19,56]. On the other hand, the leaching of ions from the nanoparticles is known to enhance osteoblastic phenotypes which could explain the decrease in ALP activity of HOB cells in the negative control (TP). Effah Kaufmann et al. [18] and Hench [20] showed that biomaterials are dynamic materials constantly undergoing transformation reactions during culture, releasing biologically active ions for the upregulation and activation of osteogenic genes. Results from real-time qPCR supported the results from the ALP/DNA experiment, in which TP demonstrated high levels of RUNX2 expression at day 4, but decreased significantly at day 7. On the other hand, expression levels of RUNX2 of HOB cells cultured with the three types of biomaterials (BG, HA and SiHA) were relatively maintained throughout culture. From the results of ALP and RUNX2 expression, it is conclusive that all three biomaterials are capable of osteostimulation, however, the higher levels measured throughout culture with the multi-step sol–gel derived BG demonstrated that this biomaterial is superior in enhancing the rate of the osteogenic cell cycle, from proliferation to differentiation and subsequently mineralization. It is crucial to note, however, that further testing is required to confirm the claim that HOB cells cultured on BG have progressed to a terminal differentiation stage, leading to mineralization. Although studies have confirmed that the dissolution products of bioactive glasses are capable of positively stimulating osteoblast cell responses, leading to rapid bone regeneration, it is important to confirm that the nanoparticles synthesized by the multi-step route also support such functions before any application either as a nanofiller or coating can be considered.

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J.P. Fan et al. / Materials Science and Engineering C 36 (2014) 206–214 Jian Ping Fan holds an undergraduate degree in Mechanical Engineering and Business Finance from University College London (UCL) and is currently pursuing his PhD under the supervision of Jie Huang. His research involves the synthesis and characterization of novel bioactive glass nanoparticles and their application towards improving composite scaffold structural integrity. His work also includes collaborative efforts with Barts and the London School of Medicine and Dentistry on antimicrobial testing and with the Dental Institute Guy's Hospital London on the biocompatibility of novel biomaterials developed at UCL.

Lucy Di Silvio is Professor of Tissue Engineering and Research Director of the Biomaterials, Biomimetics & Biophotonics Division at the Dental Institute, King's College London (KCL), UK. Her research activity focuses on interdisciplinary research based on developing and delivering products and therapies that re-establish tissue and organ function impaired by disease, trauma or congenital abnormalities— interfacing basic science and translation to clinical application. She has over 24 years of experience in the field of biomaterials/tissue engineering and has published over 110 papers in peer-reviewed journals in this field, numerous book chapters and is Editor of “Cell materials Interactions”.

Priya Kalia is a Research Associate in the Biomaterials, Biomimetics and Biophotonics division of the Dental Institute, King's College London. She completed a BSc(Hons) in Molecular Genetics and Molecular Biology at the University of Toronto, Canada in 2002, and her PhD at University College London in 2007. Dr. Kalia has experience with testing biomaterials and nanomaterials for medical applications, particularly bone and cartilage tissue engineering, and the testing of new tissue engineering therapies in vitro, as well as small and large in vivo models. She has published in peer-reviewed journals in her field and given presentations on her work at the national and international level.

Jie Huang is a lecturer in Biomaterials in the Department of Mechanical Engineering at UCL. She obtained her BSc from East China University of Science and Technology in 1989, and PhD from Queen Mary, University of London, UK in 1997. Dr Huang is a Fellow of the Institute of Materials, Minerals and Mining and has extensive experience in the development and characterization of bioceramics and biocomposites for biomedical applications. She has published over 60 papers and contributed to four book chapters.