Accepted Manuscript Preparation and structural characterization of bioactive bredigite (Ca7MgSi4O16) nanopowder Maryam Rahmati, Mohammadhossein Fathi, Mehdi Ahmadian PII: S0925-8388(17)33568-5 DOI: 10.1016/j.jallcom.2017.10.132 Reference: JALCOM 43529 To appear in: Journal of Alloys and Compounds Received Date: 20 February 2017 Revised Date: 17 September 2017 Accepted Date: 17 October 2017 Please cite this article as: M. Rahmati, M. Fathi, M. Ahmadian, Preparation and structural characterization of bioactive bredigite (Ca7MgSi4O16) nanopowder, Journal of Alloys and Compounds (2017), doi: 10.1016/j.jallcom.2017.10.132. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Preparation and structural characterization of bioactive bredigite (Ca7MgSi4O16) nanopowder Maryam Rahmati* , Mohammadhossein Fathi, Mehdi Ahmadian RI PT Biomaterials Research Group, Department of Materials Engineering, Isfahan University of Technology, Isfahan, 8415683111, Iran. SC Abstract Nanostructured bioceramics are expected to have better bioactivity than coarser M AN U crystals. This work aims to prepare, characterize and evaluate the bioactivity of bredigite nanopowder, synthesized by modified sol–gel method. In vitro bioactivity assessment was performed by soaking the samples in the simulated body fluid (SBF) and immersing it for apatite formation on the surface. X-ray diffraction (XRD), and TE D field emission scanning electron microscopy (FESEM) techniques were utilized to characterize the nanopowders. The morphological and compositional changes of the samples, after soaking in SBF, were studied and analyzed by scanning electron EP microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive spectroscopy (EDS), and inductively-coupled plasma optical emission spectroscopy AC C (ICP-OES). Results showed that the particle size of pure bredigite was around 38– 48 nm. During immersion in SBF, the dissolution rate of the bredigite nanopowder was higher than that of the bredigite micro size powder, and apatite was formed on it after 3 days of soaking. Keywords: Sol–gel processes; Grain size; Apatite; Nano powder; Bioactivity; Bredigite. * Corresponding author. Tel.: +983113912750; fax: +983113912752. E-mail address: email@example.com (M. Rahmati) 1 ACCEPTED MANUSCRIPT 1. Introduction Calcium phosphate ceramic such as hydroxyapatite is good candidate for bone tissue regeneration in biomedical industries due to their biocompatibility, low density, RI PT chemical stability and their compositional similarity to human bone. Weak mechanical properties such as low fracture toughness the value of about 1 MPa m1/2 as compared with 2–12 MPa m1/2 for human bone and higher Young's modulus than cortical bone SC limit the clinical applications of HA Therefore, pure hydroxyapatite cannot be used as heavy loaded implants[1-3]. M AN U New studies have demonstrated that glass and ceramics containing MgO–CaO–SiO2 components and glass-ceramic have excellent bioactivity for bone tissue engineering application[4-7]. Mg is one of the important element in human body besides Ca and Si. It is closely associated with mineralization of calcined tissues, and indirectly TE D influences mineral metabolism. Due to the enhanced mechanical properties of MgO– CaO–SiO2 based bioceramics compared to calcium phosphates, there has been an increasing interest in these ceramics. Bredigite with chemical formula of EP Ca7MgSi4O16 is one of the bioactive ceramics[5,11-12]. Various techniques have been applied to synthesize this bioceramics including sol–gel method[5,11], combustion AC C method and mechanical activation[13-14]. However, the particle sizes of the as prepared bredigite were mostly in the range of 1-10 µm. Bioceramics at nano-level have shown interesting functional properties due to larger surface area to volume ratio and ultrafine structure similar to biological apatite, which would have a great impact on implant-cell interaction in body environment. Additionally, nanosized bioceramics have shown better bioactivity compared to coarser crystals[8,15-16]. 2 ACCEPTED MANUSCRIPT The present study aims to synthesize, characterize bredigite nanopowder by a modified sol–gel method, and evaluate the in vitro behavior of prepared bredigite nanopowder in simulated body fluid (SBF). Modified sol–gel method is a combination RI PT of two methods. This method is essentially a sol-gel method for synthesis of pure powder, and then a short time ball-milling process was also used for preparing smaller SC nanometer powders 2. Materials and methods M AN U 2.1. Synthesis of bredigite nanopowder Bredigite nanopowder was prepared using a modified sol–gel method. In this process, tetraethyl orthosilicate (TEOS) ((C2H5O)4Si, Merck) was mixed with double distilled water and 2M HNO3 (mole ratio= TEOS:H2O:HNO3=1 : 4 : 0.08) and was kept TE D to hydrolyze for 30 min under stirring. Then, magnesium nitrate hexa-hydrate (Mg(NO3)2.6H2O, Merck) and calcium nitrate tetra-hydrate (Ca(NO3)2·4H2O, Merck) were added in 150 ml of ethanol as a solvent, while the mole ratio of TEOS: EP Mg(NO3)2.6H2O: Ca(NO3)2. 4H2O was about 4:1:7 . As-prepared solutions were mixed AC C and stirred for 5 h at room temperature. The solution was maintained at 60°C for 24 h, and then dried in an oven at 120 ◦C for another 48 h. The dried gel was calcinated at 1150 ◦C for 3 h at heating rate of 5 °C/min. Finally, the calcinated powder was milled for 5h using a high-energy planetary ball mill (Fretch Pulverisette 5) with zirconia vial and balls at ambient temperature. Powder to ball weight ratio of 1:10, and a rotational speed of 300 rpm were selected. 2.2. Characterization of nanopowders 3 ACCEPTED MANUSCRIPT Thermal behavior of bredigite powder dried at 120◦C was monitored using a thermogravimetric and differential thermal analyzer(TG–DTA). The dried powder was heated from room temperature up to 1100◦C in air with a heating rate of 10◦C/min. RI PT In order to identify the phase structure and composition of powder, XRD pattern of the obtained powder was recorded over the 2θ range of 20–60◦ (step size of 0.05 degree per 1 second) by using a Philips diffractometer(40 kV, 30 mA) with monochromated Cu SC Kα radiation(λ = 0.15406 nm). The pattern was then analyzed by PANalytical X’Pert High Score software. The obtained experimental patterns were compared with standards M AN U compiled by the Joint Committee on Powder Diffraction and Standards(JCDPS). The crystallite size of prepared powder was also determined using Williamson-Hall equation (Eq. (1)). β cos θ = 0.89 λ D + 2 A ε 2 sin θ TE D Where β is the full width at half maximum(FWHM) of diffraction peaks, λ is the wavelength of the used X-ray(λ = 0.15406 nm), θ is the diffraction angle, d is the average crystallite size, ε is internal lattice strain, and A is a coefficient depending on EP the strain distribution. AC C . FESEM (Hitachi, S4160) technique was utilized to characterize the morphology of the synthesized bredigite powder. 2.3. In vitro bioactivity evaluation The pellet-shape samples with a diameter of 12 mm were prepared by pressing the nanopowders under a force of 80 kN. Bioactivity evaluation of the obtained nanopowder was investigated by soaking the samples in the simulated body fluid (SBF) at pH 7.5 ± .01 for 3, 7, 14 and 28 days. The solid: liquid ratio was 3:2 mg/ml without refreshing the soaking medium. The SBF was prepared according to the procedure 4 (1) ACCEPTED MANUSCRIPT described by Bohner and Lemaitre. The soaking experiment was carried out in a shaking bath maintained at 37°C. At various soaking time, the samples were removed from SBF and dried at 100°C for 48 h. The apatite formation on the surface of the RI PT samples was examined by FTIR(Bomem, MB 100, the spectrum was recorded in the range of 4000–600 cm-1 with 2 cm-1 resolution), SEM(Philips XL30) and EDS. The concentrations of Ca, P and Mg ions of SBF solutions after soaking the samples were SC determined by ICP-OES(Perkin Elmer, Optima 7300DV). The concentrations of Ca, P and Mg ions was recorded in the range of 318.049-317.836 nm, 213.555-213.697nm M AN U and 285.126-285.309nm respectively. Additionally, the changes in pH of soaking solutions were also measured at pre-determined time intervals for 0–28 days using an electrolyte-type pH meter. 3.1. Thermal analysis TE D 3. Results In order to determine the mechanism of ceramic formation during heat treatment EP process, thermo-gravimetric analysis was performed. Figure 1 shows the TG–DTA curves of the bredigite gel after drying at 120◦C for 48 h. The weight loss occurs within AC C three main stages leading to the total weight loss of 16%. The first stage takes place at below 380°C which might be due to the loss of physically adsorbed water molecule into the bredigite gel and consequences the weight loss of 5%[7,13-14]. The second stage occurs below 600°C owing to the loss of organic compounds, i.e. alkoxy groups, decomposition of precursor and burning of carbon and elimination of residual nitrates introduced by metal nitrate during the preparation of the sol[6-7,20]. The weight loss in this region is around 9%. The third stage observed below ~ 850°C, is attributed to the 5 ACCEPTED MANUSCRIPT crystallization of bredigite powder[6-7,21]. DTA curve of the obtained powder (Figure 1) exhibits one small endothermic peak at 310°C, two sharp endothermic peaks at 570°C and 600°C, and one exothermic peak at 800°C. The first endothermic peak corresponds RI PT to the dehydration of the precipitated complex, and the latter two endothermic peaks are associated with decomposition of compounds; meanwhile, exothermic peak is assumed SC to be the result of crystallization of bredigite powder. 3.2. Phase structure analysis M AN U Figure 2 shows the XRD patterns of bredigite powder before and after ball-milling process. The XRD patterns of the sample correspond to bredigite(Ca7MgSi4O16) (PDF #036-0399) and show a strong peak around 32◦ indicating the formation of a single phase. according to Williamson-Hall equation, the crystallite size of bredigite TE D powder, before ball-milling, is about 280-430 nm which decrease to 36 nm after milling. The morphological shape and particle size of bredigite powder is shown in figure 3. Results shows the spherical shapes particles with uniform sizes are synthesized by the EP performed process. The average particle size of the bredigite powders measured from FESEM was around 38–48 nm. Wu and Chang synthesized a bredigite powder by AC C sol-gel method and calcination for 3 h at 1150 °C with particle size in the range of 1-10 µm. In another study, Huang and Chang also synthesized nanocrystalline bredigite powder by a simple combustion method, and the pure bredigite powder was obtained with particle size in the range of 234-463 nm after calcination at 650 °C for 4 h. Mirhadi et al. synthesized bredigite powder with particle size of about 1 µm via 20 h mechanical activation and annealing at 1200°C for 1 h. In this paper, bredigite nanopowders were prepared by modified sol-gel method, calcined at 1150 °C for 3 h, 6 ACCEPTED MANUSCRIPT and ball milled for 5 h with particle size in the range of 38-48 nm. The nanometer-sized grains and the high-volume fraction of grain boundaries and dislocation in nanostructured bioceramics may enhance osteoblast adhesion, bone-building cells RI PT proliferation, osteointegration, and the deposition of calcium phosphate on the surface of these bioceramics[22-23]. SC 3.3. In vitro bioactivity evaluation The FTIR spectra of the bredigite nanopowders before and after soaking in the SBF M AN U are shown in figure 4. Before immersing the sample in SBF, the bands related to the characteristic peaks of bredigite could be observed at 1100–1000, 960, and 873 cm−1 (SiO4 stretching) and at 616 cm−1(SiO4 bending) confirming the formation of bredigite powder. After soaking the samples in the SBF for 28 days, the intensity of silicate absorption bands decreased and the new peaks are appeared. These peaks consist of v3 TE D vibration peak of PO4-3 groups located in the range of 1030- 1090 cm-1 and, the bands of CO32- groups observed in the range of 1415-1490 cm-1 and at 872 cm-1. Additionally, EP the bands assigned to hydroxyl(OH-) groups appear at 3571 cm-1, 632 cm-1 and 1619 cm-1. According to the obtained results, it is obvious that the bone-like apatite layer AC C is formed on the surface of bredigite. The presence of carbon in the precipitated apatite structure plays an important role in forming the chemical bonding to the living bone. This carbonate-containing hydroxyapatite layer can be reproduced in vitro in the SBF. 3.4. Morphology and distribution of apatite Figure 5 shows SEM micrographs of bredigite nanopowder, before and after soaking in SBF for different periods of time. The grains of bredigite exhibited spherical 7 ACCEPTED MANUSCRIPT morphology before soaking the samples(figure 5a). After soaking for 3 days, numerous uniform wormlike crystallites with size of smaller than 100 nm which is a typical morphology of apatites was evident(figure 5b). The number and size of these sediments RI PT were enhanced by increasing the soaking time upon 7 days(figure 5c). After soaking for 14 days, the lath-like apatite crystallites became more compact and the surface is continually being covered by apatite(figure 5d). Further soaking upon 28 days results in SC the formation of new and tiny sediments of the apatite(figure 5e). Studies on other bioceramics such as tri-calcium silicate ceramics showed the same morphology of the M AN U apatite[25-26]. According to the results, it is obvious that bredigite nanopowder can develop bone-like apatite on the surface after soaking for 3 days in the SBF, showing enhanced in vitro apatite forming ability as compared to bredigite powders prepared by common sol–gel method. The bioceramics of micron size, on the contrary, present a low TE D surface area and have a strong crystal-to-crystal bond. Bredigite at nano-level would have highly functional properties due to its grain size, large surface area to volume ratio and ultrafine structure similar to biological apatite, which would have a great impact on EP implant-cell interaction in body environment. The EDS spectra of the bredigite nanopowder, after soaking in the SBF for 28 days, are shown in figure 6. The EDS AC C spectrum indicates that the tiny ball-like particles are composed of Ca and P, but Mg and Si in the EDS spectrum originate from the bredigite. 3.5. Changes of pH and ions concentration in SBF solution Figure 7 shows the changes of the concentrations of Ca, P, Si and Mg in the SBF containing bredigite pellets after different soaking periods of time. The Mg and Ca concentrations in the SBF were increased, while the P concentration was decreased. The 8 ACCEPTED MANUSCRIPT decreasing of P ions concentration continued due to the consumption of these ions during the formation of apatite on the surface of the pellets. The Ca ions concentration of bioactive ceramics containing CaO in the SBF normally increases rapidly probably RI PT due to the degradation of bioceramic and Ca2+ dissolution. The released Ca ions may increase the supersaturating degree of the SBF, and facilitate the apatite nucleation on the pellet surface. In fact, the Ca ions concentration is controlled by both the release of SC Ca ions from the bioceramics and the formation of the bone-like apatite. While the sample is bredigite bioceramic, the degradation rate overcomes the deposition rate, M AN U resulting in a continuous increase in a concentrations of Ca ion. The Mg ions concentration in SBF from the beginning up to 3 days decreased partially but from 3 days up to 28 days increased again slightly. The increase of Mg ions concentration in SBF is due to its dissolution and the exchange with the H+ ions. Hafezi-Ardakani et al. TE D  have stated that the Mg may be able to enter the forming hydroxyapatite nuclei and thus inhibits their evolution to tiny apatite crystals, because this element cannot be accommodated in the hydroxyapatite structure. The Mg2+ ions substitution into the EP apatite lattice causes noticeable changes in its physical and chemical properties. Replacing the magnesium ions with calcium ions leads to formation of the amorphous AC C calcium phosphate(Ca, Mg)9(PO4)6. In fact, inorganic ions such as magnesium and sodium, suppress the crystallization of apatite and at higher concentrations favor the formation of amorphous calcium phosphate. Consequently, the decrease of Mg2+ ions in the SBF might be due to replacing with Ca2+ ions in the hydroxyapatite structure. The researchers reported that the glasses containing magnesium oxide had a lower rate of apatite growth than the other glasses but they had the potential to serve as the biodegradable composite implants that can repair load-bearing defects in osseous 9 ACCEPTED MANUSCRIPT tissue. The small change of Si ions concentration in SBF is due to its dissolution of bredigite. The dissolution of Si ions plays an important role as nuclies for apatite formation. Figure 8 shows the changes of pH in SBF after different soaking periods RI PT of time. The pH of the SBF increased after the first 1 days of soaking; and by increasing the soaking time to 28 days, it remained almost unchanged. Increasing the pH in the early stage of soaking is due to the exchange of Ca2+ with the H+ ions and increase in the OH- ion concentration. Kokubo et al. have shown that apatite formation was SC also much sensitive to the concentration of OH- ions in the surrounding solution, and M AN U that an increase in the pH of the solution would favor the deposition of apatite on the surface. Bredigite powder, synthesized by sol-gel method, does not contain nanoparticles. On the other hand, although it was reported that the powder prepared via mechanical TE D activation is nanostructured, the particle size was measured to be about 1 µm and the mentioned process requires a long time. The mechanism of apatite formation on surface bioactive glass, glass-ceramics and EP ceramics containing CaO-SiO2 and MgO-CaO-SiO2 components, after soaking in the SBF, might be similar to each other. According to figure 9, hydroxyl groups on the AC C surface of bioceramics in contact with the SBF are known to act as nuclies for apatite formation. The ionic exchange between Ca2+ ions of the bredigite and H+ ions within the SBF increases the ions activity to product the apatite in the SBF solution and led to formation of silanol(Si-OH-) in the surface layer: Si-O-Ca-O-Si + 2H+ =2 Si-OH- + Ca2+ (2) The hydrated silica could provide specific favorable sites for the apatite nucleation on the surface of bioceramics. Finally, a negative charge is produced on the surface by 10 ACCEPTED MANUSCRIPT forming the functional group (Si-O-). The following reaction takes place on the surface of the bioceramics[35-36]. Si-OH- +OH- = Si-O- + H2O (3) RI PT There is also the release of Mg2+ ions. Previous studies of researchers showed that Mg in bioglass could cause a decrease of the solubility of the glass. The higher Mg-O bond energy makes it difficult to release from crystal lattice when compared with the Ca-O SC bond. In addition, Mg atoms in crystal lattice inhibit the Ca atoms releasing, which also decrease the solubility of the glass. The Mg content indirectly affects the apatite M AN U formation ability and the dissolution of the bioactive ceramics in CaO-SiO2-MgO system. The Ca ions in the SBF are initially attracted to the interface between the powder and solution, and are combined with the negative phosphate ions in the fluid. This results in the formation of an amorphous calcium phosphate, which later TE D crystallizes into bone-like apatite[36-37, 11]. The approach utilized in this study, i.e. combination of sol-gel and mechanical activation methods, led to the production of nanoparticles with size of about 38-48 nm. EP Besides, this approach provides several advantages such as economic benefits, saving time and the desired purity of the final product. The nanoparticles of bredigite AC C bioceramic are expected to have higher bioactivity and greater capability to form bonelike apatite layer as compared to micron-sized bredigite. Furthermore, the profile of Ca, Mg and P concentration and pH in SBF is similar to that of CaO-SiO2-MgO based bioactive ceramics such as diopside. The diopside and bredigite are similar in terms of their composition. Obtained bredigite nanopowder can be a good candidate for biomedical applications such as preparing polymer-bioceramic composite foam and scaffolds, nanoparticles and 11 ACCEPTED MANUSCRIPT nanofibers for drug delivery, surface treatment of biodegradable Mg alloy, and electrophoretic deposition nanostructured coating for human body implants[34,38-40]. RI PT 4. Conclusions Single-phase bredigite nanopowder was synthesized by a modified sol–gel method. Particle size of the prepared bredigite nanopowder was in the range of 38– 48 nm. The SC in vitro bioactivity test showed that bredigite nanopowder possessed bone-like apatite layer formation ability earlier compared to the micro size bredigite. After 3 days M AN U soaking, the numerous uniform wormlike of apatite crystallites were formed on the surface with the size of smaller than 100 nm. Further soaking upon 28 days resulted in the formation of new and tiny sediments of the apatite. The mechanism of apatite formation on the surface of bredigite in the SBF seems to be related to the dissolution of Ca(II) ions from the bioceramics in the early stage of immersion and exchange with H+ TE D ions leading to the formation of silanol (Si-OH-) in the surface layer. The Ca ions are attracted to the interface, and are combined with the negative phosphate ions in the EP fluid. It is suggested that bredigite nanopowder was able to present the improved behavior of bioactivity, and could be a potential candidate for biomedical applications AC C such as bone repair tissue engineering. Acknowledgement This work is supported by Isfahan University of Technology. 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Ramakrishna, Electrospun PHBV nanofibers containing HA and bredigite nanoparticles: Fabrication, characterization and evaluation of mechanical properties SC and bioactivity, Comp. Sci. and Tech. 121 (2015) 115.  M. Eilbagia, R. Emadia, K. Raeissia, M. Kharazihaa, A. Valiani, Mechanical and M AN U cytotoxicity evaluation of nanostructured hydroxyapatite-bredigite scaffolds for bone AC C EP TE D regeneration, Mater. Sci. Eng. C.68 (2016) 603. 17 ACCEPTED MANUSCRIPT Figure captions Figure 1. TG–DTA curves of gel bredigite nanopowders dried at 120 ◦C for 48 h. Figure 2. XRD patterns of prepared bredigite nanopowders via modified sol-gel bredigite by sol-gel method. Figure 3. FESEM micrographs of bredigite nanopowders. RI PT method, calcined at 1150°C for 3 h, and finally ball milled for 5 h, and prepared SC Figure 4. FTIR spectra of bredigite nanopowder soaked in SBF solutions for various period of times. M AN U Figure 5. SEM micrograph of the bredigite powders after soaking in the SBF for various period of times: (a) 0 day, (b) 3 days, (c) 7 days, (d) 14 days and (e) 28 days. Figure 6. EDS spectrum of the bredigite powder after soaking in the SBF for 28 days. Figure 7. Changes of ions concentrations in the SBF solution after soaking the bredigite TE D for various periods: (a) Ca, (b) P, (c) Mg, and (d) Si. Figure 8. Changes of pH in the SBF solution after soaking the bredigite for various periods. EP Figure 9. Schematic representation of the mechanism of apatite formation on the AC C surface of bredigite in the SBF. 18 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT The dear reviewer, Journal of Alloys and Compounds Elsevier nanopowder Highlights: 1. The particle size of pure bredigite was around 38– 48 nm. RI PT Preparation and structural characterization of bioactive bredigite (Ca7MgSi4O16) SC 2. The dissolution rate of the bredigite nanopowder was higher than that of the bredigite micro M AN U size powder AC C EP TE D 3. The bone-like apatite was formed on the surface bredigite after 3 days of soaking.