Preparation of nanocrystalline forsterite by

Materials Science and Engineering C 77 (2017) 811–822

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Preparation of nanocrystalline forsterite by combustion of different fuels and their comparative in-vitro bioactivity, dissolution behaviour and antibacterial studies Rajan Choudhary a, Prasanth Manohar b, Jana Vecstaudza c, Maria Josefa Yáñez-Gascón d, Horacio Pérez Sánchez d, Ramesh Nachimuthu b, Janis Locs c, Sasikumar Swamiappan a,⁎ a

Department of Chemistry, School of Advanced Sciences, VIT University, Vellore 632014, Tamil Nadu, India School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India c Rudolfs Cimdins Riga Biomaterials Innovations and Development Centre of RTU, Institute of General Chemical Engineering, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Riga, Latvia d Universidad Católica San Antonio de Murcia (UCAM), 30107 Guadalupe, Spain b

a r t i c l e

i n f o

Article history: Received 16 December 2016 Received in revised form 27 March 2017 Accepted 31 March 2017 Available online 3 April 2017 Keywords: Silicates Oxidant/fuel ratio Heating microscopy BET SBF circulation Antibacterial

a b s t r a c t This study presents different fuels (Glycine and Urea) that can be used to synthesize nanocrystalline forsterite by the sol-gel combustion method. The weight change of precursor during thermal treatment was studied by thermo-gravimetric analysis (TGA). Pure forsterite was characterized by heating microscopy, Fourier transform infrared spectroscopy, X-ray Diffraction, Brunauer-Emmett-Teller, Scanning Electron Microscopy, and Energy dispersive X-ray spectroscopy. The HAP (hydroxyapatite) deposition ability, degradation and dissolution behaviour of forsterite was examined in simulated body fluid (SBF). The combusted forsterite precursor showed distinct thermal behaviour for each fuel when analyzed by heating microscopy. BET analysis showed that the particle size of forsterite synthesized using glycine was 28 nm, specific surface area 65.11 m2/g and average pore diameter 16.4 nm while using urea 1.951 μm, 0.939 m2/g, and 30.5 nm are the respective parameters. The dissolution of forsterite pointed to the consumption of Ca and P ions from SBF, the negligible release of Si ion into the SBF and these ionic interactions with SBF can be altered as per the material properties. The forsterite showed good antibacterial activity against S. aureus but lower activity against E. coli. The bactericidal activity of forsterite indicated that it can be used to inhibit biofilm formation in dental, bone implants and bacterial infection during surgical operations. © 2017 Published by Elsevier B.V.

1. Introduction It has been estimated that about 60% of artificial bone substitutes are bioceramics, and much attention has focused on the use of different bioceramics for the bioactive fixation of artificial implants [1]. Calcium sulphates and calcium phosphates have a long clinical history for repairing and reconstructing several bone defects. However, some major drawbacks associated with these bioceramics are its high degradation rate and insufficient mechanical stability [2,3]. As a result, their applications are restricted to bone, dental fillers and coating over metallic implants. A new class of surface reactive bioceramics in the form of bioactive silicates has emerged during the last 10 years. Bioactive silicates have the unique ability to form direct chemical bonding with living bones and tissues, show lower degradability, possess osteogenic activity and ⁎ Corresponding author. E-mail address: [email protected] (S. Swamiappan).

http://dx.doi.org/10.1016/j.msec.2017.03.308 0928-4931/© 2017 Published by Elsevier B.V.

promote osteogenesis and angiogenesis [4,5]. These superior characteristics over calcium phosphates and sulphates make bioactive silicates first choice biomaterials for the development of artificial orthopaedic, bone, teeth and dental implants, and have led to a growing interest among researchers to predict and study their bio-functionalities. Bioactive silicates include a series of bioceramics, such as wollastonite, larnite, diopside, akermanite, merwinite, bredigite etc., which have been widely studied to predict their bioactive nature [6–9]. However, more scientific research is required to investigate the use of bioactive silicates as load/stress bearing implants. In this respect, Mg and Si-containing bioceramics have attracted the attention of many ceramists [10– 11]. Silicon and magnesium occur as trace minerals in the human body and their deficiency could lead to fatal disorders [12,13]. It is believed that silicon is required in the initial stages of the calcifying process, for the differentiation of osteoblast cells, growth, development and mineralization of young bone [14]. Previous findings suggest that magnesium contributes to skeletal mass gain and the metabolism of minerals [15, 16]. Forsterite (with the chemical formula Mg2SiO4) is a dimagnesium

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silicate belonging to the olivine group. Preliminary studies have reported on its bioactive nature and suggest that its mechanical strength is superior to that of HAP and bioglasses [10,11,17–19]. These advanced functionalities make forsterite a potential bioactive silicate for the repair and reconstruction of hard tissue injuries. Literature also reports that in some cases elution of magnesium from bioactive glasses in higher concentration can retard the precipitation of Hap [20]. It has been observed that at some stage during implant preparation or surgical implantation bacterial infections can take place. Microorganisms adhere to the biomaterial surfaces by depositing proteins and extracellular matrixes [21]. This represents the initial stages of bacterial infection and leads to the formation of a biofilm on the surfaces. After such biofilm formation bacteria become more resistant to antibiotics and cannot be entirely eliminated by antimicrobial agents [22]. Currently available antibiotics have lost power against these bacterial infections due to the development of resistant bacterial strains. Moreover, highly expensive and less effective antimicrobial agents cannot be utilized for long term applications as they are easily washed away by body fluids and unable to withstand temperature and the pH conditions of the body [23,24]. Hence, the best way to avoid such bacterial contaminations is by imparting antibacterial properties to the bioceramics. Earlier studies reported that bioactive glass possess reasonable antibacterial activity while synthesized HAP didn't exhibit any antibacterial activity [23, 25–27]. Hence, HAP doped with silver, copper or zinc and composites of HAP with chitosan and silver do have bactericidal properties [23,28,29]. A recent report claimed that the increase in the concentration of nanoforsterite in 58S bioactive glass/forsterite composites showing increased antibacterial activity [30]. Several synthesis routes developed to prepare pure forsterite include the microwave method [31], sol-gel method [32,33], combustion process [34], citrate-nitrate [35], and the alkoxy method [36]. The formation of enstatite (MgSiO3) and periclase (MgO) as binary phases has been found to be difficult to avoid. Thus, conventional methods use a high temperature of about 1200–1600 °C to obtain pure forsterite, although such thermal treatment causes variations in particle size, morphology, surface roughness and biological activity. In the last fifteen years numerous methods have been developed to eliminate these secondary phases and reduce the calcination temperature of forsterite [37–41]. In recent years, utilization of sol-gel combustion method has gained the attention of scientists. This methodology combines non-alkoxide sol-gel process with solution combustion synthesis. The main feature of this route is that the precursor undergoes dual thermal treatment via combustion process and calcination. During combustion the phase formation takes place which is further refined by calcination at different temperatures. In this preparatory route the parameters associated with fuel (ignition temperature, nature of fuel (exothermicity), oxidant fuel ratio) play an important role [42]. According to the principle of propellant chemistry, the organic fuel and oxidant (metal nitrate) must be in unity for effective and stoichiometric combustion. It has been reported that maximum amount of heat is released when fuel and oxidizer are in stoichiometric ratio. The heat generated during combustion provides additional temperature to undergo self-propagating reaction [43]. Consequently, product with desired properties such as chemical homogeneity, purity, crystallinity and high surface area is obtained. Literature survey reveals that the utilization of few fuels has been reported for the formation of forsterite [35,44]. But the role of different fuels on the formation of forsterite and its bioactivity is not compared so far. Hence, in the present work, dual thermal treatment route (solgel combustion) is attempted to study the possibilities to achieve nanocrystalline forsterite at low temperature with other fuels like glycine and urea. The effect of fuels on thermochemistry, surface properties and reactivity was also evaluated. The bone-like apatite layer deposition on the surface of forsterite was investigated under static and refreshed simulated body fluid. To the best of our knowledge, the antibacterial activity of pure forsterite has not been reported. For this reason we study

its antibacterial potential against the gram positive and gram negative bacteria responsible for causing biofilm formation in dental and bone implants, which drastically increases the risk of implant rejection. 2. Experimental procedure 2.1. Materials and methods The following chemicals were used in the present study: Magnesium nitrate LR (99.0% SDFCL), Glycine AR (99.5% SDFCL), Urea, pure (99% HIMEDIA), Tetraethyl Ortho Silicate (98%, Acros Organics), Conc. Nitric Acid LR (69–72%, SDFCL), Sodium Chloride AR (99.9%, SDFCL), Sodium Bicarbonate AR (99%, Nice Chemicals), Potassium Chloride AR (99.5%, SDFCL), Di-potassium Hydrogen Orthophosphate AR (99.0%, SDFCL), Magnesium Chloride AR (99.0%, SDFCL), Conc. Hydrochloric Acid LR (35–38%, SDFCL), Calcium Chloride AR (98%, Qualigen Fine Chemicals), Sodium Sulphate Anhydrous AR (99.5%, SDFCL), Tris(hydroxymethyl)aminomethane AR (99.8%, SDFCL) and MullerHinton agar (Hi-media Laboratories pvt. Ltd., Mumbai). The particle size dBET of forsterite was calculated by using equation given below. Where ρ = forsterite density, 3.275 g/cm3 [45], SSA = specific surface area, dBET = particle size, assuming particles were spherical and nonporous. dBET ¼ 6=ðρ SSAÞ

ð1Þ

2.2. Synthesis of forsterite Forsterite nanoparticles were synthesized by sol-gel combustion using glycine as a fuel is labelled as FG while for urea as FU. Magnesium nitrate (oxidant), glycine (fuel), urea (fuel), tetraethyl ortho silicate (TEOS) and nitric acid (catalyst) were used as starting materials for the synthesis of forsterite. The stoichiometric fuel to oxidant molar ratio was determined by dividing oxidizing valence of metal nitrate by reducing valence of respective fuels. Initially, glycine (2.22 M), urea (3.33 M), and magnesium nitrate (2 M) stock solutions were prepared separately in 100 ml standard volumetric flask by dissolving in deionized water. Magnesium nitrate and glycine were mixed in a beaker at room temperature and later 2 M aqueous tetraethyl ortho silicate (TEOS) was added carefully. A transparent layer of TEOS was observed to form over the surface of solution, which was stirred vigorously to obtain a homogeneous mixture. Similar procedure was followed in separate beaker and urea was used instead of glycine as a fuel. It has been reported that hydrolysis and polycondensation rate can be influenced by using an acid or base as a catalyst. At lower pH the silica particles bears little ionic charge and aggregate into long chains leading to polymeric gel network. This effect can be produced at pH 1.7 [46]. Hence, concentrated HNO3 was added dropwise to adjust the pH of the solution to 1.7 ± 0.2. It was found that both reaction mixtures took different time interval to form viscous gel under constant stirring at room temperature. Later, the beakers were heated at 50 °C on magnetic stirrer to strengthen the gel and kept undisturbed for aging. Aging causes stiffness, strengthening and shrinkage of the gel network when kept undisturbed at room temperature, these variations play a major role during drying, calcination or sintering [47]. After 3–4 days of aging, a compact mass of gel was obtained with small cracks on the surface. The gels were then dried into powders in a hot air oven at 70 °C. Finally, the dried powders of FG and FU were combusted at 400 ± 3 °C in a preheated muffle furnace for 30 min. The combustion process occurs in the presence of atmospheric air as an exothermic reaction between nitrates (oxidizing agent) and fuel (reducing agent). During this exothermic reaction, the internal temperature of the mixture increases, leading to the complete decomposition of fuel by the oxidant and formation of a forsterite phase accompanied by secondary phases in the form of impurities. These secondary phases


can be removed by the calcination process. Therefore, the decomposed precursors were manually ground into fine powders and calcined separately in alumina crucibles at different temperatures to optimize their thermal conditions and phase evolution. 2.3. In vitro bioactivity studies The bone-like apatite formation ability of materials is usually tested in SBF solution [48]. The forsterite powders were ground individually for 10–15 min to obtain fine powders, which were subsequently pelletized into 13 mm diameter and 3 mm thickness circular disks using a hydraulic pellet press. These scaffolds were dried in hot air oven at 150 °C for 2 h to eliminate atmospheric moisture and later immersed in 50 ml SBF solution in two separate conical beakers, which were incubated at 37 °C for 28 days without shaking to study HAP deposition on the surface of the immersed forsterite scaffolds. The SBF was refreshed every 24 h to accelerate HAP nucleation by maintaining a plentiful supply of calcium and phosphorus ions for exchange at the interface of the SBF and scaffold surface. In a separate bioactivity analyses, forsterite scaffolds were prepared as mentioned above and immersed in static SBF for 28 days to compare how dynamic and static SBF influences HAP deposition on the immersed surface. In the case of bioactivity studies with dynamic SBF, the scaffolds were removed after every 7th days while in the case of static SBF the scaffolds were taken out directly after the 28 days. The forsterite scaffolds were washed with deionized water, dried in a desiccator at room temperature and characterized by XRD, FTIR, SEM and EDX techniques to analyse HAP layer deposition on their surface. The SBF removed after every 7th day was stored in the refrigerator and ionic concentration of calcium, magnesium, phosphorus and silicon was determined in SBF by ICP-OES. 2.4. Antibacterial activity of forsterite A simple and cost effective agar diffusion method was used to assess the antibacterial activity of pure forsterite. In this test, 13 mm diameter circular disks of forsterite were prepared using a conventional hydraulic pellet press. The amount of forsterite in each disk varied from 100 mg to 500 mg to evaluate the effect of weight (i.e. concentration) on the zone of inhibition (mm) obtained in the antibacterial study carried against biofilm-forming pathogens: Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli). Susceptibility tests were performed with different concentrations of forsterite (100, 200, 300, 400, 500 mg) on Muller-Hinton agar. The inoculums of S. aureus and E. coli isolated from clinical samples were adjusted to the turbidity of 0.5 McFarland turbidity standards (1 × 108 CFU/ml) and swabbed on to Muller-Hinton agar plates following Clinical Laboratory Standard Institute (CLSI, 2009) guidelines [49]. The prepared disks were placed in the centre of the plate and incubated overnight for 48 h to evaluate the zone of inhibition. 2.5. Characterization Thermogravimetric analysis (SDT Q600 V20.9 Build 20, Universal V4.5A TA Instruments) was used to study thermal stability of dried forsterite precursor at a heating rate of 20 °C/min, N2 purge (100 ml/min) from room temperature to 1200 °C. The balance sensitivity of instrument is 0.1 μg, calorimetric accuracy/precision is ±2% and alumina sample pan. Phase evolution of forsterite was studied by Xray Diffractometer (Bruker, D8 advance, Germany), using Cu Kα, Ni filtered radiation. The maximum angular accuracy allowed for 2θ deviation is ±0.010 with XRD. The functional group and chemical nature of the combusted and calcined forsterite samples was examined by FTIR (IR Affinity-1, Shimadzu FTIR spectrophotometer). The FTIR spectrum was recorded from 4000 to 400 cm−1 region with 4 cm−1 resolution.

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A Heating Microscope with automatic image analysis (EM-201, Hesse instruments) was used to observe and characterize the sintering process in situ at temperatures ranging from room temperature to 1400 °C. Scanning electron microscopy (FESEM-Merlin Compact VP,CARL ZEISS, Germany) was used for morphological characterization and Energy dispersive X-ray spectroscopy (Bruker, Quantax 200, Software: Esprit 1.9) for elemental analysis of forsterite. The surface area, particle size and pore volume was analyzed by Brunauer-Emmett-Teller (BET) (gas sorption system Quadrasorb SI and Quadra Win). ICP-OES (PerkinElmer, ICP-OES Optima 5300 DV) was used to determine the ionic concentration of fresh and SBF collected after the bioactivity studies. The instrument was calibrated with standard solution for each ion analysis. The wavelength used was as follows: Ca - 317.933 nm, Mg - 285.213 nm, P - 213.617 nm and Si - 251.611 nm. 3. Results and discussion The Sol-gel combustion method was used to synthesize nanocrystalline forsterite powders using two different fuels (glycine and urea). The mechanism involved in the preparation of pure bioceramics with a highly crystalline nature, chemical homogeneity and uniform particle size is very well explained elsewhere [50]. In this study, two different fuels were used as complexing agents in the sol-gel method and as reductants in the combustion process. The choice of suitable complexing agents for phase formation plays a crucial role in the sol-gel combustion method. A good fuel should not release toxic gasses or produce violent reactions [51]. Certain fuels such as hydrazine are very rarely used due to their carcinogenic activity [52]. The heat generated and gasses released during combustion also differ from one fuel to another, affecting the properties of the final product [53]. A review of the relevant literature showed that the most accepted fuels are citric acid (C6H8O7), glycine (C2H5NO2) and urea (CH4N2O), all of which contain amine and carboxylate groups that make them as appropriate for water soluble synthesis routes [54]. 3.1. Thermal analysis In the present study thermogravimetric analysis (TGA) was used to study the overall weight loss behaviour of dried forsterite precursors. Fig. 1 shows the TGA curves (weight loss in percentage versus thermal treatment temperature in °C) of the forsterite precursors. The weight loss pattern followed by FG sample was about 11% till 150 °C assigned evaporation of water, whereas 35% of weight loss was observed between 200 and 350 °C corresponds to combustion reaction associated with redox reaction between oxidants (nitrate) and reductants (glycine). Further, 25% weight loss was observed till 530 °C due to elimination of organic residues. The thermogravimetric analysis (TGA) for FU was observed in three steps. Initially till 175 °C, 20% weight loss was observed due to dehydration of the dried precursor. The distinct peak at 303 °C was found due to explosive decomposition reaction between nitrate and urea. This process leads to nearly 40% of weight loss up to 330 °C. Later 20% of material got removed because of carbonaceous impurities between 375 and 500 °C. In both cases considerable weight loss was not observed after 550 °C. Similar weight loss patterns were observed when forsterite was prepared by using citric acid and sucrose as chelating agents [35,44]. 3.2. Characterization of forsterite after combustion 3.2.1. FTIR and XRD analysis The FTIR spectra of forsterite precursor after combustion at 400 °C is shown in Fig. 2. The forsterite precursors derived from stoichiometric oxidant/fuel ratio shows presence of functional groups at similar wavenumbers (Fig. 2a, b). The Mg\\O bending (420 cm−1), Si\\O bending (670 cm− 1), Si\\O stretching (870–970 cm− 1) bands were observed. The band associated with NO3 − vibrational modes around

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Fig. 1. Thermal analysis (TGA) of FG (a) and FU (b) precursor.

1450 cm−1 might be due to combustion of organic groups. These observations were found similar to reported literature [36,44,55]. XRD (Fig. 2c, d) pattern after combustion shows the formation of amorphous forsterite precursor. After the combustion of FG (Fig. 2c) periclase (MgO) peaks was detected as a primary phase while in the case of FU (Fig. 2d) a distinct XRD pattern was noticed with well-defined intensity peaks corresponding to forsterite. Apart from current finding, no reports have been found claiming the appearance of forsterite phase at 400 °C. Such disparities observed between forsterite samples prepared using different fuels might be due to difference in exothermicity of fuel used. This finding further strengthen that urea possess higher exothermicity than glycine leading to phase formation of forsterite after combustion [42,53]. The FTIR and XRD pattern of the sample after combustion at 400 °C proves that the oxidants and fuel taken in stoichiometric ratio undergoes complete decomposition of fuel. This causes to liberate heat energy which leads to occurrence of self-propagating reaction between fuel and oxidant to form desired product. The higher exothermicity of urea during combustion resulted into phase formation of forsterite.

Above results were found similar to previous finding stating that the urea possesses higher exothermicity than glycine [42]. 3.2.2. Heating microscope analysis Heating microscopy is a valuable tool for a powdered material to analyse its behaviour during the sintering process since it allows geometric changes in the samples to be observed in situ at every set time point of the experiment. It is possible to determine the start and end point of compaction, sintering, expansion and melting processes. In the present study a heating microscope was used to characterize the overall thermal behaviour of combusted forsterite powders obtained by sol-gel combustion with two different fuels. Fig. 3 shows the heating microscope curves (cross-section area of the sample in percentage versus thermal treatment temperature in °C) of the FG (glycine) and FU (urea) powders. The cross-section area changes at the beginning and the end of the experiment can be appreciated as well. It can be seen that the thermal behaviour of the forsterite powders obtained using the different fuels is not the same. However, the general similarities are: 1) neither of the powders melted; 2) final compaction due to sintering starts

Fig. 2. FTIR spectra (a, b) and XRD pattern (c, d) of forsterite after combustion.


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Fig. 3. Heating microscopy curves and images of combusted forsterite powders prepared using glycine (FG) and urea (FU) as fuel.

around 950–1100 °C (point 2 and 5 in Fig. 3). FU compacted more than FG, perhaps due to differences in grain size. FU clearly lost water up to 150 °C (point 2 in Fig. 3) and compacted rapidly from 450 to 550 °C and slowly from 550 to 925 °C. Unlike FG, FU showed a clearly distinguishable end point of sintering (point 6) around 1150 °C. For each sample the overall thermal processes during heat treatment can be summarized as follows: for FG – from room temperature till 1100 °C gradual expansion of the sample occurs, after that sample compacts due to sintering; for FU – from room temperature till the end of complete sintering (point 6). Two distinguishable and relatively rapid sample shape changes occur – a) loss of water, b) final compaction due to sintering. From here it can be stated that choice of fuel is the crucial step in sol-gel combustion process. 3.3. Characterization of forsterite after calcination 3.3.1. FT-IR analysis of forsterite The transformation of the precursor into pure forsterite after calcination was studied by FTIR spectroscopy (Fig. 4). After calcination (Fig. 4a, b), the nitrate functional group was completely eliminated from the spectra and IR spectrum with all essential functional groups of forsterite was observed. The band in the range of 464 cm− 1 (Fig. 4a) and 470 cm− 1 (Fig. 4b) was assigned to modes of octahedral MgO6. The sharp peaks appearing in the range of 500–615 cm−1 show SiO4 bending vibration while stretching modes of SiO4 bond were seen from 837 to 1000 cm−1 (Fig. 4a, b). The forsterite prepared using different fuels at different temperatures reveal the presence of absorption bands at similar wavenumbers. The band positions mentioned in both IR spectra for the preparation of forsterite were found to be similar to those described in earlier reported results [44,55]. 3.3.2. X-ray diffraction (XRD) The phase evolution of forsterite precursor after calcination at different temperatures is shown in Fig. 5. When the combusted precursor of FG was calcined at 700 °C (Fig. 5a), dual phases corresponding to forsterite and periclase were observed. There was decrease in intensity of periclase peak when compared with the pattern obtained after combustion. When the calcination temperature was increased to 800 °C (Fig. 5b), the surface comprises forsterite (99%) as major phase accompanied by a less intense periclase (1%) minor peak. Therefore, the calcination temperature at 900 °C led to the formation of single phasic crystalline forsterite with the complete disappearance of periclase phase (Fig. 5c). The presence of characteristic intense forsterite peaks at 900 °C

Fig. 4. FTIR spectra of the FG (a), and FU (b) after calcination.

confirms the temperature for pure phase formation of forsterite using glycine as a fuel. Further calcination at 1000 °C (Fig. 5d), revealed XRD pattern similar to that was achieved after 900 °C. This study proposes that forsterite attained stable phase after calcination at 900 °C above which negligible variation in the pattern was noticed. The FU precursor after calcination at 800 °C (Fig. 5e), indicates the initiation of forsterite phase formation. The surface was covered by dual phases of forsterite and periclase. When the calcination temperature of forsterite was increased from 900 °C–1000 °C (Fig. 5f, g), the phase was refined into crystalline and dominant forsterite peaks but no significant effect was noticed on the periclase. Previous studies mentioned that during the synthesis of forsterite impurities such as periclase and enstatite commonly occur, which are very difficult to avoid since high temperature treatment is required to obtain pure forsterite [31– 33,56]. Literature reports several reasons such as slow diffusion rate of reaction system for magnesium silicates from oxides, particle size, for the appearance of these impurities [33,46]. The permanent disappearance of the periclase phase from forsterite pattern was observed after calcination at 1100 °C (Fig. 5h). The variation in optimum calcination temperature for urea and glycine as fuel is due to the difference in their exothermicity [53]. Urea promotes high flame temperature during combustion, this causes increase in crystallinity, particle size and agglomerated particles [42]. The high surface area and nanoparticle size of FG promoted faster diffusion rate and resulted into phase purity at lower temperature (discussed in 3.3.4 BET analysis section). The XRD pattern of FG at 900 °C (Fig. 5A) and FU at 1100 °C (Fig. 5B) matched exactly with the standard JCPDS pattern (96-900-0320) and also indexed to the same. The crystal system of forsterite was found to be orthorhombic. The lattice parameters calculated from the powder XRD were similar for both the fuels used to prepare forsterite: a = 4.75237 Ǻ, b = 10.20296 Ǻ, c = 5.98133 Ǻ. The average crystallite size of the peak of major intensity (2θ = 36.5) was calculated using Debye Scherer's equation [57]. The influence of calcination temperature on crystallite size was also determined by comparing their average

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Fig. 5. Phase evolution XRD patterns of forsterite after calcination at different temperatures and indexed XRD patterns of FG calcined at 900 °C (A) and FU calcined at 1100 °C (B).

crystallite size values of FG and FU samples as given in Table 1. The average crystallite size of FG and FU was found to be in the range of 40– 46 nm. It is clear from these results that pure phase formation of forsterite can be achieved at a low temperature, while an increase in

Table 1 Influence of calcination temperature on crystallite sizes of FG and FU samples. S. no.

Sample

Calcination temperature (°C)

Crystallite size (nm)

1 2 3 4 5

FG FG FU FU FU

800 °C 900 °C 900 °C 1000 °C 1100 °C

27–32 nm 40–46 nm 20–25 nm 28–32 nm 40–46 nm

calcination temperature increases the crystallinity of the forsterite with the elimination of undesirable phases. 3.3.3. BET analysis Specific surface area (SSA) is an important property when surface reactions or further processing of materials have to be discussed. Since many reactions occur on implant surfaces a thorough description of the specific surface area (SSA), pore volume, pore size and particle size of forsterite powders is presented in Table 2. The BrunauerEmmett-Teller (BET) adsorption method with nitrogen gas as adsorbate was used in this study. Prior to analysis, samples were degassed at 100 °C for 24 h, while SSA was determined from the BET isotherms following the BET equation [58]. Isotherms were of type IV as shown in Fig. 6a and b.

R. Choudhary et al. / Materials Science and Engineering C 77 (2017) 811–822 Table 2 Powder characteristics of forsterite powders after BET analysis. Sample

Calcination temperature

Specific surface area, m2/g

Pore volume, cm3/g

Average pore diameter, nm

Particle size dBET

FG FU

900 °C 1100 °C

65.111 0.939

0.2711 0.0072

16.4 30.5

28 nm 1.951 μm

From the results (Table 2) it can be seen that use of different fuels has a great impact on the SSA of forsterite powders. Glycine produces the highest SSA (65 m2/g), while urea produces forsterite with rather small SSA of 0.9 m2/g. These differences could have different effects on the bioactivity of the obtained forsterite powders. Earlier reports showed that the SSA values for forsterite are ranged from 14.95 m2/g to 44.139 m2/g [35,59]. A recently report showed that about 159 m2/g and 83 m2/g of specific surface area of forsterite was observed when 6 and 3 g of surfactant (cetyltrimethylammonium bromide) was used [41]. In the current study, forsterite produced with glycine as fuel produced a better result than those described in the literature even without adding any additional agents. A high specific surface area is usually associated with small particle sizes. The particle size dBET of FG was 70 times higher than that of FU (Table 2), so the choice of fuel determines whether forsterite microparticles or nanoparticles will be produced. The dBET was calculated according to Eq. (1). The pore diameter was smaller and pore volume bigger for FG than for FU (Table 2). This supports the SSA data because a large amount of small pores will contribute to a higher SSA of the material. Porosity curves calculated by the DHT (Density Functional Theory) method are shown in Fig. 6c and d. Curves demonstrate distinct differences in pore size distributions of both analyzed forsterite samples. Forsterite combusted with glycine (FG) had pores measuring 8–20 nm, while forsterite combusted with urea had pores with a size of 15–30 nm. Based on the pore volume data, FU particles can be considered less porous than FG particles. Both of the samples had broad multimodal pore size distributions in micro- and mesoporous regions. Regarding the material characteristics according to the BET analysis, the potential biomaterial would be FG, given its valuable features such as particle size in the nanorange, high specific surface area and micro-meso porosity [60]. 3.3.4. Surface morphology and elemental analysis of forsterite The surface of the calcined forsterite powders was analyzed by SEM to study the appearance of particles and their morphologies (Fig. 7). The

Fig. 6. BET isotherms with adsorption and desorption processes for FG (a), FU (b) and Cumulative and differential curves of pore width and pore volume of FG (c) and FU (d) powders.

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surface structure of FG calcined at 900 °C shows irregular particles deposited on the surface (Fig. 7a). The SEM image of FG appears to have crater like-morphology with tiny pores under higher magnification (Fig. 7b). The FU sample fired at 1100 °C (Fig. 7d and e) shows the presence of tiny aggregated particles forming globules blancs morphology. The elemental composition of FG and FU after calcination was evaluated by EDX as illustrated in Fig. 7c and f. The EDX spectrum shows the presence of characteristic major intense peaks of magnesium, silicon and oxygen elements and further supports the molecular formula of forsterite as Mg2SiO4. No extra peaks were observed in the spectra, meaning that no impurities existed in the prepared forsterite powders. These results further support that pure forsterite can be prepared by sol-gel combustion using different fuels. 3.4. In vitro bioactivity studies Bone-like apatite formation on the surface is necessary for an implant to bond with natural bone [61–64]. The series of chemical and biological reactions that occurs on the surface of bioactive glasses in the body environment results in the formation of a hydroxyapatite layer (HAP), which mimics the apatite present on the surface of bone present in the body [65,66]. It has been suggested that the formation of a `Si\\OH functional group on the surface of bioactive silicates in a physiological environment aids nucleation and the growth mechanism of the bone-like apatite layer. The presence of a negative charge on the `Si\\OH group reacts initially with the positively charged calcium ions present in the SBF and forms amorphous calcium silicate on the interface. These interfacial reactions continue until the surface is completely covered by a layer of Ca ions. Later, the negatively charged carbonate or phosphate ions are attracted towards the interface and the apatite layer grows at the expense of the ions in the SBF that is saturated with respect to apatite [67]. Thus, silicon seems to be an important aspect in predicting the bioactive behaviour of silicate bioceramics. The detailed in vitro apatite deposition mechanism has been explained elsewhere [68,69]. 3.5. Analysis of apatite layer formation in SBF under dynamic conditions 3.5.1. FT-IR analysis Fig. 8 represents the major changes that occur in the FT-IR spectra of FG and FU after immersion in SBF. The absorption band of MgO was found to disappear, perhaps due to the dissolution of metallic oxides during the early stages of immersion. The band in the range 472 cm−1–505 cm−1 correspond to bending vibration of the characteristic phosphate group (Fig. 8a, b). The broad band at 1018 cm−1 (Fig. 8a) and 1022 cm−1 (Fig. 8b) represents the stretching vibration of the phosphate group. The formation of phosphate group on the surface caused shifting in Si\\O bands. These findings were found similar to earlier reports claiming apatite deposition on forsterite surface [19,33,70,71]. 3.5.2. XRD analysis A comparative apatite deposition analysis between FG and FU was carried in the physiological environment. The XRD patterns of FG scaffolds before and after immersion in SBF are illustrated in Fig. 9(A, A1). Till 14 days, no HAp peak was observed on the immersed surface (Fig. 9a). After three weeks, hydroxyapatite appeared as a minor phase on the surface with a slightly amorphous nature and the intensity of the forsterite peaks began to decrease (Fig. 9b). These observations suggest that the nucleation of Hap was initiated between 14th and 21st day. When the immersed surface was studied after 28 days, amorphous HAp peaks were observed (Fig. 9c). Hence, the immersion time for FG was further extended to one more week (35 days) to predict any further deposition on the surface. It was found that the surface became highly amorphous in nature and a clear HAP peak was noticed (Fig. 9A1). This peak was found to match with standard hydroxyapatite pattern (JCPDS: 09-0432). In the case of the FU scaffold, no apatite deposition

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Fig. 7. SEM/EDX images of FG (a, b, c) and FU (d, e, f) after calcination.

was found on the surface even after 28 days of immersion (Fig. 9d–f) and the intensity of the forsterite peaks was still unaffected. This reflected the poor degree of hydroxyapatite deposition in FU. Hence, FU requires another 15 days of incubation for bone-like apatite formation on the surface. FG possesses an excellent capability for HAp deposition. This observation suggests that high surface area and nanoparticles favour good apatite deposition than micron range [72,73]. Moreover, the high temperature reduces pore diameter and induces dense packing of the surface particles [74]. These factors cause major variations in the respective microstructure and surface architecture, which play a key role in determining the bioactivity response of bioceramics [75]. Present report shows enhanced apatite formation as compared with previous articles [17,33]. Thus, glycine derived nanoforsterite showed good ability to form apatite layer concluding it as a potential material for several biomedical applications such as a bone substitute to repair diseased, damaged or worn out hard tissues.

Fig. 8. FTIR spectra of FG (a) and FU (b) after immersion in SBF.

3.5.3. SEM/EDX analysis after bioactivity The SEM and EDX images of the forsterite scaffolds after immersion in SBF are shown in Fig. 10. After four weeks the immersed surface of FG was entirely changed and got covered by a bubble-like morphology (Fig. 10a, b). Pearl-like particles spread over the whole surface where deposits of hydroxyapatite. SEM images of the FU surface are presented in Fig. 10c and d. The surface appears to have fatty brain-like morphology. The composition of immersed surface was analyzed by EDX was found to contain calcium and phosphorus along with magnesium, silicon and oxygen (Fig. 10e, f). This indicates FG deposits bone-like apatite partially on the immersed surface while in case of FU has delayed Ca\\P precipitation. 3.5.4. In vitro bioactivity of forsterite in static SBF Fig. 11 represents XRD bioactivity patterns of FG and FU immersed in SBF for 28 days without refreshing. The analysis was carried out to examine the influence of SBF replacement on the deposition of bone-like apatite. After 28 days the immersed surface was found to be similar to that of forsterite before immersion, and no HAP deposition was noticed. The analysis suggests that SBF replacement and immersion time play a vital role in the formation of a HAP layer on the surface of bioceramics [69,76]. 3.5.5. Dissolution behaviour of forsterite after immersion in SBF The ionic concentration of Ca, Mg, Si and P in SBF before and after the biomineralization assay under static and dynamic conditions was determined by ICP-OES. Fig. 12a shows the variation in ionic concentration of the four ions in SBF refreshed every 24 h. The ionic concentration of Mg, Ca, and P in fresh SBF was found to be 41.77 mg/L, 76.30 mg/L and 35.20 mg/L respectively. The zero value (0 mg/L) of the silicon concentration reflects its absence in SBF. During the first few days of immersion, Mg and Si ions leached from both forsterite scaffolds (FG and FU) in SBF. As a result, the concentration of Mg and Si ions increased rapidly accompanied by a gradual decrease in the Ca concentration while no change in the concentration of P ion in SBF was observed until day 7. SBF was enriched with the essential ions responsible for apatite deposition (Ca, Si ions) but the consumption of P ions by FG and FU scaffolds had not yet begun. These observations point to a slow HAP formation ability of the FG and FU samples during the early stage of immersion. After the 7th day, the concentration of Ca, Mg, Si and P ions continuously decreased in SBF. This slight decrease in concentration of Mg and Si ion suggests partial leaching from FG and FU. The sharp reduction in the content of Ca and P ion from SBF throughout the immersion time was due to the consumption of these ions that are needed for bone-like apatite deposition. The Ca and P ion was more rapidly consumed by the FG sample than by FU. Thus, the dissolution behaviour of FG states superior


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Fig. 9. XRD patterns of FG (A and A1) and FU (B) after immersion in SBF.

HAP deposition than FU maintained under dynamic condition. The poor consumption of Ca, P ions from SBF and negligible leaching of Si ions into the SBF might be the main reasons for the poor bioactivity of FU. The overall mechanism of forsterite bioactivity in SBF as discussed in the scientific literature was found to be similar to our results [17,77]. The forsterite samples (FG and FU) prepared using different fuels and immersed in static SBF for 28 days showed a similar ionic release profile as shown in Fig. 12b. SBF under static conditions led to an increase in the ionic concentration of Mg and Si while the Ca and P contents decreased as the immersion period lengthened. The major difference noticed was the higher dissolution of Mg ion than Ca ion in SBF after four weeks. Although SBF contains sufficient calcium and silicon ions, the deficiency of an adequate supply of phosphorus might have suppressed the ability of forsterite samples to form Ca\\P phase on the immersed surface. The reason for this negligible bioactivity of FG and FU in uncirculated SBF would be the absence of the ions required for HAP deposition. This dissolution behaviour of forsterite samples suggests better bioactivity in SBF under dynamic conditions rather than in static SBF. 3.6. Antibacterial activity of forsterite The antibacterial activity of forsterite prepared using both fuels was studied against the biofilm-forming bacteria, S. aureus and E. coli. The

size of the zone of inhibition around the disk was considered to reflect the amount of FG/FU diffused out of the disks to prevent the growth of bacteria. In the case of FG, the inhibition zone for S. aureus increased from 20 mm to 25 mm (considering the disk size of 13 mm) as the concentration of FG increased (Fig. 13a, b), but when the concentration was increased to 300 mg the zone of inhibition decreased to 17 mm (Fig. 13c), perhaps due to combinational differences. In the case of FG acting against E. coli, the inhibition was not as strong as that observed for S. aureus, although the clear zones (14 mm–16 mm) increased with the concentrations of FG (Fig. 13d, e). Forsterite urea (FU) produced a clear inhibition zone (14 mm– 17 mm) against S. aureus at 300 mg–500 mg concentrations (Fig. 13f, g, h). In the case of E. coli, no clear zone of inhibition was observed around the disk but it was found the FU had potential to inhibit bacterial growth (Fig. 13i). Our results indicate that forsterite had a strong impact on antibacterial activity and, interestingly, the size of the inhibition zones increased as the incubation time increased, confirming the prolonged activity of forsterite. It was observed that S. aureus was inhibited more than E. coli by both FG and FU. The bactericidal activity of FG was greater than that of FU due to variation in their respective surface properties, such as surface area and nanoregime particle size [78]. This disparity might be the major reason for the difference in the diffusion rate of MgO from the forsterite samples. Thus, forsterite can be used for surface coating on implants or as a bactericidal bioceramic to

Fig. 10. SEM/EDX images of FG (a, b, e); FU (c, d, f) after immersion in SBF.

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Fig. 11. XRD pattern of FG (A) and FU (B) without circulation of SBF till 28 days.

prevent bacterial infections (biofilm formation) against the above resistant organisms in biomedical implants. 4. Conclusion Forsterite nanopowders were chemically synthesized by the sol-gel combustion method. The different combustion rates and exothermicity of the fuels used resulted in phase formation of pure forsterite at different temperatures. The calcination temperature of forsterite samples was reduced by almost 300 °C by the utilizing current synthesis scheme. Different characterization techniques revealed formation of crystalline forsterite. The thermal (decomposition and sintering) behaviour of the forsterite prepared using different fuels was found dissimilar to each other. The surface properties of the forsterite were found to be strongly influenced by the difference in their calcination temperatures. High exothermicity of urea promoted crystallization of forsterite during combustion while glycine assisted in forming nanoforsterite having high surface

area and porous nature. An increase in immersion time and SBF circulation led to the characteristic HAP peaks growing in size to cover a larger portion of the forsterite immersed surface. The bioactivity study showed that forsterite nanopowders (FG) possess a better apatite formation ability than micron sized powders (FU). The dissolution behaviour of forsterite prepared using glycine pointed to the rapid consumption of Ca and P ions from SBF, while in the case of urea-derived forsterite there was poor utilization of Ca, P ions from SBF. The forsterite samples revealed a clear zone of inhibition against S. aureus but a smaller zone against E. Coli. These results suggest forsterite had a strong effect on the growth of these resistant bacterial species. Acknowledgement The authors present their sincere thanks to VIT management for providing the necessary help to carry out this research, which was financially supported by Vellore Institute of Technology Research Grants for

Fig. 12. The change in ionic concentration of Ca, Mg, P and Si ions in SBF refreshed every 24 h (a); SBF without circulation (b) after bioactivity studies.


Fig. 13. Antibacterial activities of forsterite sample at different concentrations; FG against S. aureus: (a) 100 mg, (b) 200 mg, (c) 300 mg, and FG against E. coli: (d) 200 mg, (e) 300 mg; andle FU against S. aureus: (f) 300 mg, (g) 400 mg, (h) 500 mg and FU against E. coli: (i) 300 mg.

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