In-vitro bioactivity, biocompatibility and dissolution studies of diopside ...

Materials Science and Engineering C 68 (2016) 89–100

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In-vitro bioactivity, biocompatibility and dissolution studies of diopside prepared from biowaste by using sol–gel combustion method Rajan Choudhary a, Jana Vecstaudza b, G. Krishnamurithy c, Hanumantha Rao Balaji Raghavendran c, Malliga Raman Murali c, Tunku Kamarul c, Sasikumar Swamiappan a,⁎, Janis Locs b a

Department of Chemistry, School of Advanced Sciences, VIT University, Vellore -632014, Tamil Nadu, India Rudolfs Cimdins Riga Biomaterials Innovations and Development Centre, Institute of General Chemical Engineering, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Riga, Latvia c Tissue Engineering Group (TEG), Department of Orthopaedic Surgery (NOCERAL), Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia b

a r t i c l e

i n f o

Article history: Received 13 January 2016 Received in revised form 21 April 2016 Accepted 27 April 2016 Available online 4 May 2016 Keywords: Diopside Topic: Eggshell Fuels Apatite Topic: SBF circulation Bone marrow cells.

a b s t r a c t Diopside was synthesized from biowaste (Eggshell) by sol–gel combustion method at low calcination temperature and the influence of two different fuels (urea, L-alanine) on the phase formation temperature, physical and biological properties of the resultant diopside was studied. The synthesized materials were characterized by heating microscopy, FTIR, XRD, BET, SEM and EDAX techniques. BET analysis reveals particles were of submicron size with porosity in the nanometer range. Bone-like apatite deposition ability of diopside scaffolds was examined under static and circulation mode of SBF (Simulated Body Fluid). It was noticed that diopside has the capability to deposit HAP (hydroxyapatite) within the early stages of immersion. ICP-OES analysis indicates release of Ca, Mg, Si ions and removal of P ions from the SBF, but in different quantities from diopside scaffolds. Cytocompatability studies on human bone marrow stromal cells (hBMSCs) revealed good cellular attachment on the surface of diopside scaffolds and formation of extracellular matrix (ECM). This study suggests that the usage of eggshell biowaste as calcium source provides an effective substitute for synthetic starting materials to fabricate bioproducts for biomedical applications. © 2016 Elsevier B.V. All rights reserved.

1. Introduction By assisting body's response to self-repair, biomaterials were also found to assist in regenerating the tissues when stem cells are transplanted [1]. Scientists are developing novel and improved biomaterials to replace diseased or damaged tissues, cells and even entire injured body parts for regenerative medicine. It is a great challenge to extend the functionalities of biomaterials. For the past few decades, new and cost effective technologies have been adopted for the production of calcium based biomaterials from biowaste (e.g. eggshells) [2]. It has been found that a dry eggshell contains approximately 94–96% calcium carbonate. Globally, tons of hen eggs are produced to satisfy the increasing demands of food and nutrition manufacturing industries [3]. After usage of eggs on a large scale, the shells are considered as waste. This eggshell waste is either left as such in the open environment to decay or employed for land fillings. This unintentional motive leads to the soil as well as environmental pollution. In order to avoid such man-made damages to the natural environment, high emphasis is given in the conversion of these wastes into useful products. Reuse of such biowastes has proved to be an efficient method to prevent ⁎ Corresponding author. E-mail address: [email protected] (S. Swamiappan).

http://dx.doi.org/10.1016/j.msec.2016.04.110 0928-4931/© 2016 Elsevier B.V. All rights reserved.

pollution and landfills [4]. The concept of recycling biowastes has been employed for the preparation of different calcium containing bioceramics such as wollastonite, akermanite, larnite, hydroxyapatite, tri-calcium phosphate and their biological behavior has been studied [5–9]. In early 1970's, L.L. Hench introduced a new class of surface reactive silicates that can form chemical interfacial bonding between neighboring host tissues and implant [10]. The deposition of hydroxycarbonate apatite layer (HCA) on the implanted surface of the bioactive silicate was observed which induces the bone healing and stimulates bone regeneration [11]. Compatibility of bioactive silicates with tissues, physiological body environment, and their excellent mechanical strength makes them potential candidates for several applications in the field of hard tissue engineering [12,13]. Literature survey reveals that wollastonite, larnite, bregidite, forsterite, akermanite, diopside, merwinite etc. have been studied for dental restoration, bone substitutes, drug delivery system and tissue engineering applications. Due to the advanced biofunctionalities such as improved mechanical properties, slow degradation, superior HAP deposition and excellent in vivo biocompatibility, diopside is preferred over other silicate and phosphate bioceramics [14, 15]. Studies indicate that the reason for low degradation of diopside in SBF is due to the presence of magnesium ion in the crystal lattice. The

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bond energy of Mg–O is higher as compared to Ca–O bond, this makes crystal system more stable and prevents the rapid degradation of diopside [16]. Previous reports show that diopside can be used for load bearing applications as it has remarkable mechanical strength with a fracture toughness of 3.5 MPa m1/2 and bending strength 300 MPa, which is double to that of the bone [17]. Whereas, the commercially available calcium phosphate bioceramic implants are found to possess poor mechanical properties and hence cannot be used for stress and load-bearing applications [18]. Different methods like co-precipitation process, sintering, sol–gel method, solid-solution are reported for the preparation of diopside. The diopside prepared through these methods is found to be a suitable material for hard tissue engineering applications [14,17,19–22]. Raw materials used in these methods were Ca(NO3)2·4H2O, Mg(NO3)2·6H2O, doloma powders (CaO·MgO), TEOS (Tetraethyl orthosilicate), MgCl2·6H2O, ethanol, CaCO3, MgO, SiO2. In 2016, Ghomi et al. synthesized diopside at 800 °C by sol–gel method by using calcium nitrate as the calcium source. It was found that the low intensity diopside peaks got enhanced after sintering the scaffold at 1200 °C [23]. However, these methods involve higher thermal treatment which affects the particle size, morphology, porosity and nature of the product [24]. In the current report, we have attempted to synthesize pure diopside by sol–gel combustion method by using two different fuels. Eggshell biowaste was used as a substitute for the calcium source. Sol–gel combustion method is cost effective, energy efficient, low temperature process and produces highly homogeneous product [25]. Evolution of large amount of gases (NH3, H2O, and CO2) during combustion process help to disperse heat, and avoids oxides from getting sintered [26]. These advantages make sol–gel combustion method as preferable one over conventional synthesis routes. The calcination temperature required for different fuels was optimized and influence of fuels on particle size, porosity, surface area and morphology was investigated. The single phasic diopside obtained after calcination was subjected for cellular studies and HAP deposition ability in SBF under static and refreshing conditions. 2. Experimental procedure 2.1. Materials and methods Raw eggshells, Magnesium nitrate LR (99.0% SDFCL), L-alanine AR (99.5% SDFCL), Urea, pure (99% HIMEDIA), Tetraethyl orthosilicate (TEOS) (98%, Acros Organics), Concentrated Nitric Acid LR (69–72%, SDFCL), Ethylene Diamine Tetra Acetic Acid LR (98.0%, SDFCL), Eriochrome Black-T AR (99%, SDFCL), Ammonia Solution Extrapure AR (25%, SRL), Ammonium Chloride GR (98.8%, MERCK), 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), Concentrated Hydrochloric Acid LR (35–38%, SDFCL), Calcium Chloride AR (98%, Qualigen Fine Chemicals), Sodium Sulphate Anhydrous AR (99.5%, SDFCL) and Tris(hydroxymethyl)aminomethane AR (99.8%, SDFCL) were used in the present study. 2.2. Extraction of calcium from raw eggshell and estimation of calcium ions by EDTA titration Eggshells were collected from VIT hostel mess and fresh uncrushed eggshells were separated from compressed, torn out or broken shells. These selected eggshells were washed manually under running distilled water to remove dirt particles deposited on the surface of eggshells. In order to eliminate microbial contamination and unnecessary protein coatings, the washed eggshells were boiled at 100–110 °C for about 2 h with continuous stirring using glass rod. After boiling, the eggshells were transferred to filter paper to remove water droplets from the surface and later subjected into hot air oven drying at 150 °C until the shells

are completely dried. The dried raw eggshells were finally crushed and grinded to fine powders by using mortar and pestle. The eggshell powder was then characterized by XRD and FTIR spectroscopy to support the fact that calcium carbonate is the main constituent present in eggshells as reported earlier [6,7]. Eggshell solution approximately equivalent to 1 M calcium nitrate solution was prepared by the addition of 15 mL conc. nitric acid to 10 g of eggshell powder and the resultant solution was filtered and made up to 1 L by adding distilled water. The calcium ion concentration was estimated by EDTA titration by following the procedure described previously [5]. The concentration of calcium ion (Ca2+) present in the eggshell solution was found to be 0.98 M. 2.3. Synthesis of diopside Diopside was prepared by sol–gel combustion method using urea as a fuel is termed as EDU while for L-alanine as EDL. Eggshell solution, magnesium nitrate, TEOS, urea, L-alanine and conc. Nitric acid were used as starting materials. Stock solutions of magnesium nitrate (1 M), urea (2 M) and L-alanine (2 M) were prepared separately by using double distilled water in 100 mL standard volumetric flask. Equimolar concentration of eggshell solution and magnesium nitrate solution was mixed thoroughly in a beaker. L-alanine solution was pipetted out from stock solution and transferred into the beaker with uniform stirring to obtain homogeneous mixture of starting materials. A clear solution was obtained by adding 2 M TEOS to the beaker. Similar experimental procedure was carried out in another beaker except that urea was used as a fuel in place of L-alanine. The solutions were stirred vigorously until the disappearance of transparent layer. The pH of reaction mixture in both beakers was adjusted to 1 by dropwise addition of conc. nitric acid to carry out the rapid hydrolysis of TEOS [27]. Beaker containing urea as the fuel took 2 h to form viscous gel at room temperature by using magnetic stirrer whereas the beaker containing L-alanine as a fuel took 17 h of stirring and half an hour heating at 40 °C on magnetic stirrer to form a transparent gel. Thus, the gel formation time varies with fuel even though the composition of reaction mixture is same. This may be due to the formation of different polymeric network by different fuels. The gel containing beakers were kept undisturbed for 2–3 days to strengthen the gel and then dried at 150 °C in hot air oven for 8 h to obtain blocks of solid masses. The dried gel masses were decomposed separately at 400 °C for 1 h in preheated muffle furnace. Decomposition occurs in presence of oxygen to attain complete combustion of fuel by the oxidants. The resultant precursor contains residual carbon and nitrate groups as impurities. In order to eliminate these impurities the precursor was crushed to fine powder using agate mortar and pestle and calcined at 800 °C (EDU) and 900 °C (EDL) for 6 h in alumina crucibles to achieve the pure phase of diopside. 2.4. Material characterization Heating Microscope with automatic image analysis (Heating microscope EM-201, Hesse instruments) was used to observe and characterize the sintering process in situ from room temperature till 1400 °C. Prior to heating Microscopy analysis diopside powder samples were shaped by pressing into mold to obtain samples with correct shape for analysis. Pure phase identification of synthesized diopside was studied by X-Ray Diffractometer (Bruker, D8 advance, Germany), using CuKα, Ni filtered radiation. Functional groups of synthesized diopside were examined by FTIR (IR Affinity-1, Shimadzu FTIR spectrophotometer) using KBr method. For FTIR analysis calcined diopside samples were grinded into fine powder separately using agate mortar and pestle. The fine diopside powder was mixed with KBr powders and again grinded. Finally, the diopside and KBr mixture was manually pelletized using pellet press. Scanning electron microscopy (SEM-CARL ZEISS) was used for morphological characterization and Energy dispersive X-ray spectroscopy (EDX- OXFORD Inc.) for elemental analysis of diopside. Brunauer–Emmett–Teller (BET) method with nitrogen gas adsorption


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for specific surface area, pore size and volume analysis was used. The equipment was gas sorption system Quadra orb SI and Quadra Win (Quanta chrome Instruments, United States of America). Before BET measurements samples were degassed at 100 °C for 24 to remove physically absorbed moisture. ICP-OES (PerkinElmer, ICP-OES Optima 5300 DV) was used to determine the ionic concentration of fresh SBF and SBF collected after bioactivity studies.

humidified atmosphere of 95% air and 5% carbon dioxide. hMSCs has the tendency to attach and proliferate in the cell culture flask. For subsequent passaging, the cells in passage 0 (P0) were washed with 1× PBS and then trypsinized (TrypLE, Gibco, Invitrogen, USA) for 3 min in a CO2 incubator at 37 °C until complete cell detachment observed. Cells were sub-cultured upto passage 2 (P2) and the used for further experiments.

2.5. In vitro bioactivity assay

2.7. Cell seeding

Calcined samples of EDU and EDL were grinded separately in mortar and pestle to achieve fine powders for the preparation of pellets. The resultant powders were pelletized into compact circular shaped disks with 13 mm diameter and 3 mm thickness by using hydraulic pellet press under a pressure of about 20 MPa and compressed for at least 2–3 min. The pellets were carefully removed from pellet press machine and dried at 150 °C to eliminate atmospheric moisture from their surface. These dried pellets were ready for bioactivity studies by immersing it in SBF solution to examine the formation of HAP layer on the surface of the pellet. The composition and concentration of SBF similar to that of human blood plasma was prepared in single batch by dissolving the reagents of analytical grade in the sequence as proposed by Kokubo and Takadama with constant stirring in double distilled water [28]. The pH of SBF was maintained at 7.40 by using 1 M HCl. The diopside pellets were soaked in SBF solution and kept in an incubator maintained at 37 °C and the SBF solution was continuously refreshed after every 24 h. Apatite formation ability of the pellet surface was tested by recording powder XRD on 7th, 14th, 21st and 28th day after immersing the diopside pellets in SBF solution without shaking. The major reason behind SBF replacement was to facilitate HAP nucleation on the scaffold surface by continuous supply of phosphate and calcium ions from the SBF. Every 7 days once the samples were taken out from SBF, washed with double distilled water and dried in desiccator at room temperature before the surface being characterized by the powder X-ray diffractometer. The SBF removed after every 7th day was stored in refrigerator and ionic concentration of calcium, magnesium, phosphorus and silicon was determined by ICP-OES. A separate set of bioactivity studies was also performed as per above mentioned procedure without refreshing the SBF for 28 days. The immersed surface was characterized by XRD, FTIR and EDX to assess HAP deposition and changes occurred on the surface before and after the immersion in SBF. The ionic concentration of calcium, magnesium, phosphorus and silicon in SBF after 28 days of immersion was characterized by ICP-OES.

Multipotent stromal cells were enzymatically detached using 3 mL of TrypLE after reaching 80% of confluence at passage 2 (P2). A cell suspension was prepared and seeded onto the EDU and EDL in dropwise manner, at the density of 1 × 106 cells/mL. 2.8. Cell attachment analysis 2.8.1. Scanning electron microscopy analysis Scanning electron microscopy analysis was carried out to observe surface topography of hBMSCs seeded on EDU and EDL. The specimens at day 3 were fixed overnight in 4% glutaraldehyde in 0.1 M cacodylate buffer and post-fixed for 1 h in 1% aqueous osmium tetroxide. These specimens were processed with three consecutive washing steps in distilled water before being dehydrated through a graded ethanol series (50, 70, 80, 90, 95 and 100%). The specimens were subsequently dried at a critical point using critical point drier (Bal Tec, CPD030). The specimens were mounted on aluminium stub and sputter coated with gold before being examined using a digital scanning electron microscope (JSM 6400; JEOL, Tokyo, Japan). 2.8.2. Confocal microscopy analysis Confocal microscopy analysis was carried out to determine the cell density on the surface. The hBMSCs seeded on EDU and EDL samples (day 3 and day 12) were stained with Hoechst 33342 nucleic acid stain (Life Technologies, Invitrogen, USA). Samples without cells were used as control. This molecular probe binds to A–T regions of DNA and emits blue fluorescence at 460 nm. Scaffolds were stained according to the protocol provided in the manufacturer's instruction. After 20 min of incubation, the scaffolds were washed with 1 × PBS and observed using upright laser confocal microscope (LSM 5 PASCAL, Zeiss, Germany). Randomly selected spots (n = 5) from three confocal images of each samples at day 3 and 12 for EDU and EDL were used and number of blue stained nuclei was calculated using image-J software. Number of blue staining is proportional to the number of cells. 3. Result and discussion

2.6. hMSC isolation and culture Ethical approval for human bone marrow collection was obtained from the medical ethics committee of University of Malaya Medical Centre (MECID.NO: 201412-859). Human bone marrow aspirate were obtained from subjects (50–80 years old) undergoing total knee replacement. Written informed consent was obtained prior to sample collection. Human bone marrow stromal cells (hMSCs) isolation method was employed as reported earlier [29]. Briefly, bone marrow mono-nucleated cells were separated using standard Ficoll-Pague gradient centrifugation (density 1.073 g/mL) according to manufacturer's instruction. Following that, density gradient centrifugation at 2200 rpm for 25 min was performed. The middle layer which is in rich mono-nuclear cell (MNCs) were obtained and washed three times with 1 × PBS (Gibco, Invitrogen, USA). The MNCs were then suspended in culture medium DMEM-LG (Gibco, Invitrogen, USA) containing 100 U mL−1 of penicillin and 100 μg mL−1 of streptomycin supplemented with FBS (Gibco, Invitrogen, USA). Cell number was determined using the Trypan blue exclusion method. About 2 × 108 cells were seeded onto T-75 culture flask and incubated at 37 °C in a

Sol–gel combustion method involves stoichiometric addition of raw materials in aqueous state and stirring for homogeneous mixing of all contents to obtain a dense gel. Four different steps such as hydrolysis, polycondensation, polymerization and evaporation take place simultaneously in a sequence leading to the formation of polymeric gelly network. The rate of hydrolysis and polycondensation reaction will occur more rapidly under acidic conditions [27]. TEOS in presence of acidic medium will get hydrolyzed to silanol (Si–O–H) and ethanol (C2H6O). A polymeric network is formed by the polycondensation reaction of silanol, ethanol and fuel. The fuel plays dual role as complexing agent during gel formation and reducing agent in combustion process. The nitrate ions present in the reaction mixture, acts as an oxidizing agent. The gel is converted into solid powder mass by means of evaporation of the solvent molecules in hot air oven. The completely dried powder was decomposed in muffle furnace. During combustion an exothermic redox chemical reaction takes place between fuel (reductant) and nitrates (oxidant) which helped to achieve phase formation of diopside with the liberation of carbon dioxide, water and ammonia [30]. Porosity and thermochemistry of the

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product was affected by the amount of gases released during the decomposition of fuel. Finally the decomposed precursor was grinded well and calcined thermally in absence of air to eliminate the residual carbon and nitrate impurities. Calcination temperature was optimized by phase evolution study for the preparation of pure diopside.

3.1. Characterization of pure diopside 3.1.1. Heating microscope Heating microscope with an in situ observation during the whole thermal treatment process is irreplaceable tool in ceramic material development. In present study heating microscopy was employed to evaluate processes during thermal treatment of diopside precursors obtained through combustion of different fuels. Fig. 1a represents the heating microscope curves (sample crossection area changes [%] versus temperature [°C]) for EDU and EDL. It protrudes the thermal behavior of diopside affected by the fuel. Fig. 1b shows sample crossection area and changes in shape due to thermal events. Utilization of different fuel changed the thermochemistry and the chemical composition of diopside precursors. The density of EDU was found to be higher than EDL. After heat treatment of precursor pellet, it was found to become porous and expanded when L-alanine was used as a fuel. Both of the structures could be suitable for biomedical applications because bone has dense (cortical bone) and porous (spongy bone) parts. Two structurally different materials with different properties could be obtained by changing the fuel used in the synthesis. Overall thermal behavior of diopside precursors was similar till 700– 710 °C (Fig. 1a — # 3 and 4′). Both EDU and EDL lost moisture at initial points (# 1, 2, 2 and 3′) and then slowly compact due to loss of organic residues till 700–710 °C. From this temperature, the difference in

Fig. 1. Heating microscope curves and images of eggshell derived diopside powders with urea (EDU) and L-alanine (EDL) used as fuel in sol–gel combustion synthesis.

thermal behavior was observed. Precursor derived from urea compressed into half of its initial size from room temperature till 710 °C. The change in cross section area (34%) was observed due to an intense compaction process from 710 °C to 820 °C (Fig. 1b No 4′and 5′). This temperature interval corresponds to sintering process. The sample lost its shape and started flowing from the bottom corners around 1300 °C while at 1400 °C the sample was totally deformed and vanished its initial shape (Fig. 1b). The cross section area of EDL precursor compacted by 25% of its initial state from room temperature to 770 °C (Fig. 1a — # 1–4). Expansion of the EDL was happened in two steps (Fig. 1a): 1) from 770 to 910 °C (# 4, 5) and 2) from 810 to 855 °C (point No 5 to point No 6). First step expanded the sample for 10% and second for 55% from compacted state at point No 4. Stepwise expansion might be due to production of different volatile compounds (e.g. carbon dioxide, nitrous oxides etc.) releasing at different temperatures. This could also be attributed to different amount of gases released. Stepwise expansion was followed by negligible crossection area changes till the expanded structure collapsed between 1260 and 1400 °C (points No 8 and No 9 in Fig. 1a). It can be stated that different fuels showed different thermal behavior for the same material at identical heat treatments. Differences might be due to different amounts of residual organic matter, particle size and shape. All of this depends on the choice of fuel. Overall maximum heat treatment temperature should not exceed 1200– 1250 °C, sintered product could be obtained starting from 750 °C [31]. Prolonged exposure at maximum temperature might lead to melt or collapse of the material. 3.1.2. FT-IR analysis The presence of functional groups associated with diopside was determined by FTIR spectroscopy. Phase evolution study of EDU and EDL at different temperatures were demonstrated in Fig. 2A and B respectively. The FTIR spectra of EDU precursor (Fig. 2a) showed an intense nitrate band along with bending vibrational modes of O–Ca–O, O–Mg–O and O–Si–O and symmetric stretching of Si–O and Si–O–Si functional groups. The presence of all characteristic functional groups was noticed in FTIR spectra proved that phase formation of EDU was achieved after combustion (Fig. 2a). When diopside precursor was calcined at 800 °C, appearance of characteristic functional group and complete removal of nitrate (Fig. 2b) was observed. Non-bridging bending vibrational modes of O–Ca–O was found at 414 cm− 1 and peaks in the range of 455 cm− 1 to 503 cm− 1 showed non-bridging bending modes of O– Mg–O bond. Sharp dual peaks at 632 cm−1 and 675 cm−1 was O–Si–O bending mode while at 854 cm−1 to 960 cm−1 was assigned to Si–O symmetric stretching. An intense peak at 1062 cm−1 corresponds to the symmetric stretching of Si–O–Si functional group. Fig. 2B represents infrared absorption spectra of EDL. EDL Precursor obtained at 400 °C showed broad bands of O–Ca–O, O–Mg–O, Si–O–Si and absorbed water molecules. The absence of O–Si–O and Si–O bands indicated the formation of diopside phase was not achieved during combustion (Fig. 2c). As the temperature increased to 900 °C (Fig. 2d), partitioning of broad bands into characteristic peaks was noticed representing presence of all the characteristic functional groups. The non-bridging bending vibrational modes of O–Ca–O at 422 cm−1 and O–Mg–O non-bridging bending modes in the range of 455 cm−1 to 501 cm−1 was observed. The band at 634 cm−1 and 675 cm−1 represents bending O–Si–O mode. Dual peaks at 860 cm− 1 and 960 cm− 1 showed symmetric stretching modes of Si–O bond. Sharp intense peak at 1062 cm−1 symbolized to Si–O–Si symmetric stretching mode. These observations suggested that diopside prepared from eggshell biowaste using different fuels have similar pattern of functional groups as reported in earlier findings [32]. 3.1.3. X-Ray diffraction (XRD) Fig. 3 represents phase evolution study of diopside by powder-XRD. XRD patterns (Fig. 3A(a)) of EDU sample revealed the formation of


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Fig. 2. FTIR spectra of the (a, c) precursor and (b, d) diopside sample prepared by using (A) Urea as a fuel (B) L-alanine as a fuel.

amorphous diopside phase at 400 °C. EDU precursor calcined at 800 °C for 6 h showed formation of single phasic diopside (Fig. 3A(b)). Thus, the calcination temperature for the crystalline diopside prepared by eggshell by using urea as a fuel was optimized at 800 °C which is found to be very low when compared with earlier reports [5,9]. Contrastingly XRD pattern of EDL precursor (Fig. 3B(c)) didn't show any diopside phase, when the precursor was calcined at 800 °C for 6 h it showed diopside as a major phase and akermanite as the secondary phase (Fig. 3B(d)). Akermanite is an analogue of diopside with chemical formula Ca2MgSi2O8 and appearance of akermanite as a secondary phase is usually found during phase transformation of diopside. Further, when the calcination temperature was increased to 900 °C, complete removal of akermanite peak was observed with an increase in intensity and crystallinity of diopside peaks as shown in Fig. 3B(e). The thermochemistry involved during the combustion reaction of the fuel may be the major cause for the phase formation of diopside. Thus the thermochemistry of combustion reaction of urea is found to be optimum for the diopside formation than L-alanine. XRD patterns (Fig. 3C and D) of the EDU and EDL matched perfectly with the standard JCPDS data card 96-900-1308. The crystal system was found to be monoclinic and the lattice parameters for EDU sample are a = 9.75102 Å, b = 8.90445 Å, c = 5.24748 Å whereas for EDL sample the calculated values are a = 9.75348 Å, b = 8.92121 Å, c = 5.24944 Å. The effect of change in fuel in the lattice parameter values of the diopside is evident from the results. The crystallite size for both the systems was calculated by using Debye-Scherer equation from full width at half maximum values obtained from corresponding XRD patterns [33]. The intensed diffraction peak (2θ = 29.842) selected to determine crystallite size of diopside for the system was found to be exactly same and the calculated average crystallite size value was in the range of 35– 40 nm. Earlier reports showed that pure phase of diopside was obtained by calcining the samples in the range of 1100 °C–1300 °C temperature. Monticellite and akermanite phases were the secondary phases generally observed during the preparation of diopside [14,17,19,20,31,34]. Recently, diopside was synthesized at 800 °C for 2 h and it was found that the phase formation of diopside was just initiated and the phase appears to have amorphous nature with low intensity peaks [23]. Present results indicated that diopside precursor calcined at 800 °C and 900 °C for 6 h possess highly crystalline and sharp intense peaks corresponding to formation of single phasic diopside. Thus, calcination temperature of bioactive silicates can be brought down by changing the fuels and increasing the calcination time.

3.1.4. BET analysis Brunauer–Emmett–Teller (BET) N2 adsorption method was used for specific surface area (SSA), pore size and volume determination of diopside materials. These characteristics are of crucial importance in biomaterial surface science and in further processing of powders into larger objects. Prior to analysis samples were degassed at 100 °C for 24 h to remove physically adsorbed moisture. Values of SSA were determined from BET isotherms. Calculation of particle size dBET was done according to Eq. (1), where ρ — calculated density of diopside, 3.278 g/cm3 [35], SSA — specific surface area and dBET — particle size. dBET ¼ 6=ðρ SSAÞ

ð1Þ

Table 1 showed change in fuel (urea or L-alanine) has significant effect on the characteristics of the calcined diopside powder. EDL sample possess higher SSA than EDU powders. In fact SSA is relatively small, therefore particle size calculated by Eq. (1) is in sub micrometer range — 0.626 μm for urea and 0.526 μm for L-alanine. EDU has bigger pore volume and average pore diameter, while EDL has smaller pore volume and smaller average pore diameter. It has been reported that SSA for diopside prepared by solid-state reaction route at 1300 °C was 1.31 m2/g and average particle size was 4.75 μm [18]. The impact of calcination temperature on SSA of diopside powders obtained by solid-state reaction method was also studied [36]. They reported 9.9, 3.7, 2.8 and 3.3 m2/g as SSA values for diopside powders calcined at 1100, 1150, 1200 and 1250 °C. SSA values of diopside powders prepared by sol– gel combustion route are approximately the same. Fig. 4a and b showed the adsorption and desorption isotherms (type IV) [37] of EDU and EDL. Isotherms have hysteresis loop which is an indicator of mesoporosity (2–50 nm after IUPAC pore classification) [38]. Pore size distribution (Fig. 4c and d) of EDU sample was found to be in the range of 5 nm to 30 nm while EDL sample has desultory one in the range from 1 nm to 30 nm. From the results it was observed that EDU sample is having more uniform pore characteristics than EDL sample. 3.1.5. Surface and elemental analysis The surface morphology of EDL powder shows porous rock like morphology having tiny pores covering entire surface (Fig. 5A and B). The SEM image of EDU powder after calcination indicates irregular morphology with few smaller particles (Fig. 5C, D). The overall surface porosity of EDU and EDL was found to be in micron size. Particles appear as grains with micro and macro structure scattered irregularly over the surface. The formation of pores on the surface of EDU and EDL

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Fig. 3. Phase evolution XRD patterns of (A) EDU and (B) EDL at different calcination temperatures and indexed XRD patterns (C) EDU calcined at 800 °C; (D) EDL calcined at 900 °C.

samples may be due to the elimination of volatile matters during combustion and calcination process. Porous surface of bioceramics plays a major role in bioactivity as it enhances the deposition of HAP [39]. The EDX spectra of calcined EDL (Fig. 5E) and EDU (Fig. 5F) powders shows the presence of all essential elements such as Calcium, Magnesium, Silicon and Oxygen in stoichiometric ratio. This analysis further

Table 1 Powder characteristics determined by BET method for EDU and EDL samples. Sample.

Specific surface area, m2/g.

Pore volume, cm3/g.

Average pore diameter, nm.

Particle size dBET, μm.

EDU. EDL.

2.925 3.478

0.0186 0.0091

25.4 10.4

0.626 0.526

strengthens the data that pure phase of diopside was achieved at 800 °C and 900 °C respectively using two different fuels.

3.2. In-vitro bioactivity studies Bioactive silicates are commercially available for clinical use in various compositions as Bioglass® (45S5), Ceravital and NovaMin. The high success rate of bioactive silicates in clinical application is due to their unique surface reactivity [11]. Bioactive silicates form a natural bonelike apatite (HAP) layer more rapidly at their interface with natural bone in the aqueous physiological environment. A sequence of chemical and biological interactions at material-tissue interface takes place resulting in the development of bonding with bone followed by ingrowth of bone tissues. Surface characteristics such as morphology,


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Fig. 4. BET isotherms with adsorption and desorption processes shown for diopside materials obtained with different fuels: (a) urea; (b) L-alanine and cumulative and differential pore size distributions for EDU (c) and EDL (d) powders.

porosity, particle size, and roughness are the major factors responsible for the biological behavior of bioceramics [1]. Initially, Ca2 + and Mg2 + ions from the surface of diopside get exchanged with hydrogen ions (H+ or H3O+) present in SBF. It occurs within a couple of minutes after immersion in SBF. The loss of calcium and magnesium from the structure causes breakdown of silica network to form silanol with negative surface charge. This silanol

later repolymerizes into a silica-rich layer on the surface. The surface charge plays a key role in the formation of HAP layer on the immersed surface. The Ca2 + and PO34 − ions are attracted towards silicon rich layer resulting into a CaO–P2O5 film. Thus, the accumulation of these ions results into calcium phosphate layer formation on the immersed surface. The calcium phosphate layer later nucleates and crystallizes into amorphous calcium deficient carbonated apatite

Fig. 5. SEM/EDX micrographs EDL (A, B, E) and EDU (C, D, F) after calcination.

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layer. This carbonated HAP layer mimics the naturally occurring inorganic bone mineral hydroxyapatite and permits neighboring tissues to bind directly to the implant surface. Further, a chemical bonding occurs between the implant and natural tissues. As the immersion period increases, the thickness of bonding also increases to a certain micrometer level which is necessary for an implant to provide mechanical support to the tissues [11,40,41]. 3.3. Analysis of apatite layer formation in refreshed SBF 3.3.1. XRD analysis after bioactivity studies Comparative HAP deposition on the surface of EDU and EDL was analyzed by powder-XRD. XRD patterns (Fig. 6A) of the EDU samples after immersion in SBF for a different period of time with refreshment of SBF solution after every 24 h shows HAP peaks as secondary phase after 7 days whereas diopside remains as a primary phase. When the surface was analyzed after 14 days, the HAP peaks emerged into major intense peaks. The surface was partially covered by amorphous HAP phase but few low-intensity diopside peaks were also noticed (Fig. 6c). But after 21 days, diopside phase was entirely covered by highly amorphous apatite layer with complete disappearance of diopside peaks (Fig. 6d). Thus, diopside prepared using urea as fuel reveal remarkable HAP deposition within 21 days. The precipitation of small peaks corresponding to apatite on the surface of EDL was observed within 7 days (Fig. 6e). The intensity of these peaks got slightly increased after 14 days but diopside phase still dominated the immersed surface (Fig. 6f). After 21 days, the apatite peaks got enhanced and few more HAP peaks emerged as minor phase (Fig. 6g). The intensity of diopside peaks was reduced with the passage of immersion time (after 28 days) and amorphous HAP phase covers up the immersed surface. This behavior proved slow apatite formation ability of EDL in SBF (Fig. 6h). The diffraction peaks of HAP observed in XRD patterns of EDU and EDL matched exactly with standard HAP pattern (JCPDS data card 901–1092). Since, previous findings conclude apatite deposition ability of pure diopside after 3rd, 7th and 14th days [14,22,31,34]. Hence, we have attempted to study in vitro HAP deposition on the surface of pure diopside for an extended period of immersion in the SBF (four weeks). Report on apatite deposition behavior of diopside scaffold after 28 days showed slower bioactivity than EDU [42]. These findings proposed that EDU and EDL have the capability to deposit apatite within the

early stages of immersion. Excellent bone-like apatite formation was observed for EDU samples whereas slower HAP formation was observed in the case of EDL. This difference is due to the variation in their surface morphology, particle size, roughness, and porosity induced by different fuels. In addition, an earlier report also discloses that increase in calcination temperature, pore diameter gets reduced and packing of surface particles became more compact [24]. Consequently, these factors were found to affect the precipitation of Ca–P phase on the surface of diopside bioceramics. This analysis also suggested that immersion time and circulation of SBF play a key role to study HAP formation behavior of bioceramics. 3.3.2. FTIR analysis of diopside after immersion in SBF The surface of diopside samples after soaking in SBF was characterized by FTIR spectroscopy to analyze absorption bands of different functional groups to confirm apatite formation. The FTIR spectra (Fig. 7) of EDU and EDL recorded after immersion in SBF showed that O–Ca–O, O–Mg–O, and Si–O–Si groups were completely replaced by phosphate groups. This observation proved hydrolysis of metal ions during early stages of immersion in SBF. The FTIR spectra (curve a in Fig. 7) of EDU after immersion in SBF showed bending vibration modes of phosphate at 478 cm− 1 while stretching vibration bands of phosphate in the range of 970 cm−1and 1082 cm−1. Band at 1402 cm−1 corresponds to carbonate functional group [8]. A similar FTIR spectrum was observed for EDL (curve b of Fig. 7). The sharp peak at 499 cm−1 corresponds to bending vibration of the phosphate group. Dual peaks in the range of 962 cm−1 and 1060 cm−1 represent stretching vibrations of the phosphate group. The above-discussed spectra of EDU and EDL were found similar to previous reports [14,43–45]. 3.3.3. Surface and elemental analysis of diopside surface after bioactivity studies The morphology of deposited apatite on diopside sample (EDU and EDL) surfaces after immersion in SBF was studied by scanning electron microscopy (SEM). The surface of EDU was observed to form nonuniform globules after 21 days of immersion in SBF (Fig. 8a). The magnified image of EDU surface (Fig. 8b) revealed the existence of clustered particles deposited on globules represents precipitated HAP. The immersed surface of EDL revealed an appearance of bubble like morphology (Fig. 8c). The tiny particles scattered over the surface correspond to bone-like apatite (Fig. 8d). The appearance of cracks on the

Fig. 6. XRD patterns of EDU (A) and EDL (B) after immersion in SBF.


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The immersed surface of EDU indicated the absence of silicon peak and existence of calcium, phosphorus, oxygen as major peaks and magnesium as minor peak (Fig. 8e). The EDX pattern (Fig. 8e) showed that apatite layer deposition on the immersed surface which replaces silicon and reduces the concentration of magnesium. Thus, EDX analysis revealed that apatite layer covered the entire surface of EDU within 21 days immersion in SBF. In the case of EDL, the immersed surface showed the existence of Ca, Mg, Si, P and O as major constituents (Fig. 8f). This observation protrudes that the surface contains dual phases (diopside as well as HAP). The low intense phosphorus peak signifies that apatite layer formation has started on the immersed surface and is still in progress. Therefore, EDL possesses slow apatite deposition ability in comparison with EDU even after 28 days. 3.4. In vitro bioactivity analysis of diopside without refreshing the SBF medium

Fig. 7. FTIR spectra of EDU (a) and EDL (b) after immersion in SBF.

surface of EDU and EDL is due to drying process in hot air oven. Earlier reports on surface appearances of diopside after immersion in SBF indicate lath-like and leaf-like morphologies [14,31] while in the current study, different morphological structures from those reported in the literature were observed. The elemental composition of EDU and EDL surface immersed in SBF showed the presence of phosphorus peak in EDX spectra (Fig. 8e and f).

When the calcined EDU and EDL pellets were immersed in SBF at static condition for 28 days a distinct behavior of diopside samples was observed. XRD pattern of EDU surface after the 28th day (Fig. 9A) showed the existence of diopside phase with few characteristic HAP peaks. The intensity of diopside peaks got decreased due to the precipitation of HAP on the immersed surface. In case of EDL no HAP peaks were detected (Fig. 9B) and the surface still possesses intense diopside peaks. This activity confirmed inactive nature of EDL in terms of HAP formation in static SBF. Thus, EDU showed better HAP deposition ability than EDL. These observations were found in agreement with the fact that circulation of SBF provides the plentiful supply of ions necessary for HAP layer formation [46,47]. The elemental composition of EDU and EDL surface after immersion in static SBF was studied by EDX spectra. The presence of phosphate peak in EDX spectrum (Fig. 9c) clearly differentiates the deposition of apatite on the immersed surface of EDU. The surface of EDU sample showed the occurrence of intense P peak along with the existence of Ca, Mg, Si and O elements after 28 days. But in case of EDL intense Ca, Mg, O and Si peaks was detected without any P peak (Fig. 9d). Therefore, current activity concluded that circulation of SBF plays a vital role in predicting the apatite formation ability of a bioceramic. 3.5. Elemental ionic concentration in SBF after bioactivity studies It has been reported that the changes in ionic concentration in SBF is due to 1) leaching of ions from the immersed sample into the SBF,

Fig. 8. SEM/EDX micrographs of EDU (a, b, e) after 21 days and EDL (c, d, f) after 28 days immersion in SBF.

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Fig. 9. Bioactivity XRD patterns of EDU (A); EDL (B) and Elemental analysis of EDU (c) and EDL (d) without SBF circulation after 28 days.

2) This enriched ionic medium initiate formation of HAP layer on the immersed surface. As the result of this, the concentration of calcium and phosphorus gets reduced in the SBF [48]. The concentration of Ca, Mg, P and Si ions in fresh SBF was found to be 76.30 mg/L, 41.77 mg/L, 35.20 mg/L and − 1.678 mg/L respectively. The silicon concentration in negative value reveals its absence in fresh SBF. Fig. 10a shows Ca, Mg, P and Si ions release kinetics in refreshed SBF for EDU and EDL samples after three weeks of immersion time. The ionic release profile indicates release of Ca, Mg and Si ions, and removal of P

ions from the SBF. The Ca ion concentration increased rapidly during the initial stage of immersion and decreased after the 7th day. The leaching of Ca ions in SBF was rapid from EDU as compared to EDL. Similarly, the concentration of Mg and Si ions was increased in the early stage, later decreased slightly. This behavior suggests partial leaching of Mg and Si ions from EDU and EDL. Phosphorus is an essential ion required for apatite formation which is supplied from SBF. It was found that P content kept on decreasing throughout the immersion period. The consumption of P ions from SBF was observed to be higher for

Fig. 10. The change in ionic concentration of Ca, Mg, P and Si ions in SBF refreshed after every 24 h (a); SBF without circulation (b) after bioactivity studies.


EDU than EDL. This study also states that during initial immersion, the leaching of Ca and Si ions in the high concentration from EDU enriched the SBF. These ions are required to induce bone-like apatite formation on the surface. As a result, phosphorus from SBF was rapidly consumed by EDU and initiated hydroxyapatite layer deposition within 7 days. But in case of EDL, poor leaching and consumption of ions resulted into slow bioactivity. The EDU and EDL samples immersed in static SBF up to four weeks indicate similar ionic release profile (Fig. 10b). Static SBF condition reveals that increase in immersion time causes an increase in ionic concentration of Ca, Mg and Si ions while a decrease in P ion. Although there was plentiful Ca and Si ions in SBF but the reduced P content might not be sufficient to form Ca–P phase on the immersed surface of EDU and EDL. Thus, the major reason behind poor bioactivity of EDU and EDL in static SBF is due to insufficient supply of essential ions required for HAP deposition. The ion release behavior from EDU and EDL samples reveal enhanced bioactivity in refreshed SBF than static SBF. The changes in ionic concentration obtained in present work show similarity with earlier reported articles and satisfy the reactions involved in SBF for the HAP deposition [31,34]. 3.6. Cell attachment studies of diopside In previous studies, it has been highlighted greatly that the nanoscale properties such as surface topography and roughness of the samples disseminate cell morphology and that SEM images showed the structure and orientation of the cells that have contacted the samples surface [49]. In the present study, the SEM micrographs of bare samples revealed that the EDU has rough topography when compared with EDL (Fig. 11a and d). In addition, it has been found that both samples have successfully supported hBMSCs attachment. Furthermore, the cells attached to the EDU showed spherical and flatten morphology, meanwhile only flat cells have been found on the EDL (Fig. 11b and e). At 800× magnification, the architecture around the cells mimic extracellular matrix (ECM) and form a continuous layer of fusiform over the surface of the samples (Fig. 11c and f). Confocal images were used to confirm the presence of the live hBMSCs onto the EDU and EDL. Fluorescence microscopy images (day

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3 and 12) of the cells cultured on EDU (Fig. 11g and i) and EDL samples (Fig. 11h and j). Hoechst staining method revealed the adherence of the cells on the surface of samples. It clearly demonstrated that the topography and the composition of the samples provided an optimal physiological environment for cell attachment and, therefore, was biocompatible. In previous studies, the number of nuclei was calculated at different time points to describe the proliferative properties of hBMSCs when contact with the materials. In the present study, it was found that there was no significant difference in term of the number of nuclei observed between day 3 and 12 in EDU and EDL, respectively (Fig. 11g and h). It could be the reason of the material properties itself that may have stimulated the cells to differentiate and, therefore the proliferative potential of the cells would have been ceased temporarily. 4. Conclusion The main objective of the work was achieved by synthesizing pure diopside at low temperature by sol–gel combustion method by using two different fuels and their in vitro bioactivity and biocompatibility was analyzed. Utilization of eggshell biowaste as calcium source provides an effective substitute for synthetic starting materials. This motive also suggests that biowaste sources can be utilized to prepare useful bioceramics for biomedical applications. The variation in thermochemistry of the fuels was found to be the reason for the change in calcination temperature. Thermal analysis by heating microscopy indicates that different fuels possess different thermal behavior for the same material on identical heat treatments. Thus, rapid combustion rate of fuels and sol– gel combustion route helped in bringing down the calcination temperature of diopside samples by almost 300–500 °C. In vitro bioactivity analysis shows that diopside can induce HAP formation on its surface within few days of immersion and with the increase in immersion time the surface can be completely covered by hydroxyapatite layer. The difference in the surface characteristics such as morphology, porosity, particle size, surface area and calcination temperature induced by different fuels was the major cause of difference in bioactivity. Moreover, the behavior of diopside samples under static SBF condition expresses poor HAP layer deposition while excellent bioactivity was observed under refreshment of SBF after every 24 h. This indicates

Fig. 11. SEM micrographs of EDU and EDL. Bare samples (A and D-×100), Samples seeded with hMSCs (B and E-×100) and (C and F-×800). : extracellular matrix (ECM) and fluorescence microscopy of cells attached to the samples on day 3 and 12. EDU and EDL were washed with warm PBS twice and stained with Hoechst blue dye prior for fluorescence microscopy viewing.

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that circulation of SBF plays a key role in determining bioactive nature of bioceramics. Cellular studies show cell attachment, formation of an extracellular matrix on the surface and increased cell proliferation. Thus, current report suggests that diopside can be prepared at low temperature with an enhanced biological activity that can find various applications in hard tissue engineering for dental restorations, dental fillings, and bone substituents. Acknowledgement Authors present their sincere thanks to VIT management for providing necessary help to accomplish current research. This research was financially supported by Vellore Institute of Technology Research Grants for Engineering, Management and Science (VITRGEMS) (VITRGEMSCHEMISTRY 2013). Authors thank DST-FIST for the XRD, SEM facility and SAIF/IIT Madras for ICP-OES facility. This research was supported by University of Malaya's, HIR-MoE Grant (Ref. No. — UM.C/625/1/ HIR/MOHE/CHAN/03, Acc. No. — A000003-50001). References [1] W. Cao, L.L. Hench, Bioactive materials, Ceram. Int. 22 (1996) 493–507. [2] C. Balazsi, F. Weber, Z. Kover, E. Horvath, C. Nemeth, Preparation of calcium– phosphate bioceramics from natural resources, J. Eur. Ceram. Soc. 27 (2007) 1601–1606. [3] N. Verma, V. Kumar, M.C. Bansal, Utilization of egg shell waste in cellulose production by Neurospora crassa under wheat bran-based solid state fermentation, Pol. J. Environ. Stud. 21 (2012) 491–497. [4] J.P.H. Van Wyk, Biotechnology and the utilization of biowaste as a resource for bioproduct development, Trends Biotechnol. 19 (2001) 172–177. [5] U. Anjaneyulu, S. Sasikumar, Bioactive nanocrystalline wollastonite synthesized by sol–gel combustion method by using eggshell waste as calcium source, Bull. Mater. Sci. 37 (2014) 207–212. [6] R. Choudhary, S. Koppala, S. Sasikumar, Bioactivity studies of calcium magnesium silicate prepared from eggshell waste by sol–gel combustion synthesis, J. Asian Ceram. Soc. 3 (2015) 173–177. [7] R. Choudhary, S. Koppala, A. Srivastava, S. Sasikumar, In-vitro bioactivity of nanocrystalline and bulk larnite/chitosan composites: comparative study, J. Sol-Gel Sci. Technol. 74 (2015) 631–640. [8] S. Sasikumar, R. Vijayaraghavan, Low temperature synthesis of nanocrystalline hydroxyapatite from egg shells by combustion method, Trends Biomater. Artif. Organs 19 (2006) 70–73. [9] W.-F. Ho, H.-C. Hsu, S.-K. Hsu, C.-W. Hung, S.-C. Wu, Calcium phosphate bioceramics synthesized from eggshell powders through a solid state reaction, Ceram. Int. 39 (2013) 6467–6473. [10] L.L. Hench, An Introduction to Bioceramics, second ed. Imperial College Press, London, 2013. [11] D.C. Greenspan, NovaMin and tooth sensitivity—an overview, J. Clin. Densitom. 21 (2010) 61–65. [12] W. Paul, C.P. Sharma, Ceramic drug delivery: a perspective, J. Biomater. Appl. 17 (2003) 253. [13] M. Catauro, F. Bollino, F. Papale, S. Piccolella, S. Pacifico, Sol–gel synthesis and characterization of SiO2/PCL hybrid materials containing quercetin as new materials for antioxidant implants, Mater. Sci. Eng. C 58 (2016) 945–952. [14] C. Wu, J. Chang, Degradation, bioactivity, and cytocompatibility of diopside, akermanite, and bredigite ceramics, J. Biomed. Mater. Res. 83B (2007) 153–160. [15] T. Nonami, Developmental study of diopside for use as implant material, Mater. Res. Soc. Proc. 252 (1992) 87–92. [16] V. Vallet-Regi, A.J. Salinas, J. Roman, M. Gil, Effect of magnesium content on the in vitro bioactivity of CaO–MgO–SiO2–P2O5 sol–gel glasses, J. Mater. Chem. 9 (1999) 515–518. [17] T. Nonami, S. Tsutsumi, Study of diopside ceramics for biomaterials, J. Mater. Sci. Mater. Med. 10 (1999) 475–479. [18] M.A. Sainz, P. Pena, S. Serena, A. Caballero, Influence of design on bioactivity of novel CaSiO3–CaMg(SiO3)2 bioceramics: in vitro simulated body fluid test and thermodynamic simulation, Acta Biomater. 6 (2010) 2797–2807. [19] L. Ghorbaniana, R. Emadi, M. Razavi, H. Shin, A. Teimouri, Synthesis and characterization of novel nanodiopside bioceramic powder, JNS 2 (2012) 357–361. [20] S. Yamamoto, T. Nonami, H. Hase, N. Kawamura, Fundamental study on apatite precipitate ability of CaO–MgO–SiO2 compounders employed pseudo body solution of application for biomaterials, J. Aust. Ceram. Soc. 48 (2012) 180–184.

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