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Preparation and characterization of a novel nano-structured merwinite scaffold prepared by freeze casting method

| Proceedings Book | INST | Sharif University of Technology
Proceedings of the 5thInternational Conference on Nanostructures (ICNS5)
6-9 March 2014, Kish Island, Iran
Preparation and characterization of a novel nano-structrued merwinite scaffold prepared by
freeze casting method
Nader Nezafatia, Masoud Hafezia*, Ali Zamaniana, Mana Yasaei b, Mohammadreza Badr Mohammadia
a Biomaterials group, Nanotechnology and advanced materials Department, Materials & Energy Research Center,
b Pardis Pajoohesh Fanavaran Yazd, BT center, Yazd Science and Technology Park, Yazd, Iran
Abstract: The aim of this study is to investigate mechanical properties and in vitro bioactivity of a nano-structured
merwinite scaffold fabricated by water-based freeze-casting method. The scaffold sintered at 1350° C to obtain a uniform
microstructure. Evaluation of apatie formation on the surface of the scaffold was conducted by soaking in simulated body
fluid (SBF). The cell morphology of the scaffold was also assessed. The morphological results showed that the lamellar
and unidirectional aligned channels were produced by freeze casting. It observed that spherical particles were formed on
the surface of the scaffold after 5 days of soaking. The compressive strength was reached to 1.92±0.05 MPa. The result of
cell attachment indicated that the porous merwinite scaffold was non-toxic to osteoblast cells and the cells spread well.
Keywords: Bioceramic; Scaffold; Nano-structured Merwinite; Freeze casting; Bioactivity
Recently, merwinite has been introduced as a biomaterial
consisting of a calcium magnesium silicate composition.
Very few studies have done on physicochemical and
mechanical properties of merwinite. However, no study
has been performed on fabrication of merwinite scaffold
[1, 2]. Several techniques such as gas bubbling [3], salt
leaching [4], and addition of porogens have been used to
realize porous scaffolds [5]. Recently, Freeze casting was
used to develop scaffolds for biomedical application [6, 7,
8]. The scaffolds, as a bone substitute, should have some
properties such as good cell ingrowth and proliferation,
adequate pore dimension, bioactivity and suitable
mechanical properties [9].
The present work focuses on the preparation of porous
nano-structured merwinite by freeze casting method. The
mechanical properties, ability of hydroxyapatite formation
and cell attachment of merwinite scaffold were studied.
Materials and method
In our previous study, nanocrystaline merwinite powders
were synthesized using the solgel method. The physical
and in vitro and in vivo evaluations of synthesized
nanostructured merwinite have been described previously
[1,10]. In this study, particle size of synthesized merwinite
powder was calculated by Laser particle size analyzer
(LPSA) method. The powder specifications which used
for preparation of suspension are shown in Table 1. In the
first step, merwinite concentration of 15vol% was
prepared. To prepare stable slurry, a small amount (5 wt%
of merwinite content) of a commercially available
dispersant (Dolapix CE 64) was used. Furthermore, to
provide an initial strength of the scaffold, a 5% wt of
polyvinylalkohol (PVA) was added to the suspension.
Freezing of the slurries was done by pouring into a PTFE
mold, placed on a Cu cold finger whose temperature is
controlled using liquid nitrogen and a ring heater with
cooling rate of 4°C/min. Frozen samples were freeze dried
at a low temperature and a low pressure for 24 h.
Sintering of the green bodies was done in an air furnace at
heating rate of 1C/min at 300 C and kept for 1h. This
heating process continued by treatment at 600 C at the
same rate. After that, the temperature elevated up to 1350
C at 2C/min and remained for 3h. The scaffolds were
then cooled at room temperature in the furnace. Samples
of 10 *20 mm2 were used for compression tests.
Compression tests were carried out on a testing machine
(Instron 5565, Santam Testing Machine) with a cross head
speed of 1 mm/min. The identification of phases after
sintering was carried out by X-ray diffraction (Philips
PW3710 diffractometer). Transmission electron
microscopy (TEM: GM200 PEG Philips, Netherland,
working at 200 kV) was used for characterizing the
morphology of nano-structured merwinite. For this
purpose, the particles were deposited onto Cu grids, which
support a carbon film by deposition from a dilute
suspension in ethanol. In order to evaluate bioactivity, the
scaffold was immersed in simulated body fluid (SBF) for
5 days at 36.5 ± 1.5 ◦C. The microstructure, apatite
formation and osteoblastic cells adhesion of the samples
were analyzed by scanning electron microscopy (SEM)
[SEM-Stereoscan S360-Cambridge 1990].
Table 1. The specification of the synthesized powder
Particle size (µm)
Powder density (gcm-3)
Results and Discussion
The freeze casting method was used to produce porous
merwinite scaffold. The compressive strength of the
porous merwinite scaffold was tested and the result has
been shown in Fig. 1.It is noted that the compressive
BIO 018
Proceedings Book | INST | Sharif University of Technology |
Proceedings of the 5thInternational Conference on Nanostructures (ICNS5)
6-9 March 2014, Kish Island, Iran
strength and and Young's modulus were 1.92±0.05 MPa
and 48.2±0.3 MPa, respectively. The resultant liquid
phase during sintering process at 1350 °C caused a
suitable binding between particles and reduction in pores.
Fig. 1. The load displacement curves of the scaffolds prepared
at 4 C/min and sintered at 1350 C
According to the XRD pattern (Fig. 2) of sintered
samples, the only phase was related to merwinite (JCPDS
25-0161). No processing residue or secondary phases
were found in the materials.
Fig. 2. XRD pattern of merwinite scaffolds sintered at 1350 C.
Figure 3 shows TEM image of milled nanoapatite powder.
It determined that each particle owned an irregular shape
with a grain size of about 150-300 nm which has been
composed of some smaller particles aggregated to others.
Fig. 3. TEM image of milled nanoapatite powder
Fig. 4(a) shows the morphology of the sintered porous
merwinite scaffold. The nano-structured merwinite
scaffolds possessed unidirectional aligned channels with
lamellar structure. Macroscopic aligned pores of the
merwinite scaffolds were formed almost uniformly over
the entire sample. The porous structure of the merwinite
scaffolds was a replica of the ice structure when
merwinite slurries were frozen. These pores were
generated during sublimation of the ice and sintering. A
dendritic structure, which has been presented on the
internal walls of the lamellae, is shown in Fig. 4(a). These
features reach the adjacent pore walls and, therefore,
connecting struts are produced. The aligned channels and
the dendritic structure on the internal surface can act as a
guiding pattern for cell growth, which will improve the
osteoconduction characteristics [11]. Figure 4(b) shows
that there is a layer of spherical particles formed on the
merwinite scaffold surface thoroughly after immersion in
SBF for 5 days. High-magnification of SEM images
(Figure 4(c)) revealed that each spherical granule
consisted of a large number of tiny and needle-like
A typical osteoblast attachment to a merwinite scaffold
surface after 3 days of culturing is shown in Figure 5.
Cells exhibited a considerable filopodias spread on the
surface of merwinite scaffold after 3 days of culturing. It
was obvious that osteoblast cells adhered on the
merwinite scaffold. Figure 5 also shows that cells
proliferated to form a monolayer. One potential
explanation for cell attachment of merwinite scaffold is
that the released ions can promote cell adhesion by
mediating cellular integrin interactions associated with
signal transduction pathways [12]. Further studies are
needed to confirm this hypothesis. Our results
demonstrated that this scaffold was amicable for the
attachment and proliferation of osteoblast cells.
Fig.4. (a) SEM images of surface of merwinite scaffold after 5
days of soaking in SBF solution at different magnifications of:
b) 1000 and c) 5000.
| Proceedings Book | INST | Sharif University of Technology
Proceedings of the 5thInternational Conference on Nanostructures (ICNS5)
6-9 March 2014, Kish Island, Iran
Fig 5.SEM image of merwinite scaffold after 3 days of
In this research, a nano-structured merwinite scaffold,
with cooling rate of 4C/min and sintering temperature of
1350 C, was fabricated and its mechanical and in vitro
bioactivity properties were evaluated. They showed a
proper biomineralization behavior in SBF solution after 5
days of soaking and also an acceptable cell attachment.
However, further in vivo studies are required to explore
the applicability of this scaffold.
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... The class of calcium silicates also includes the ceramic components of the ternary system CaO-SiO 2 -MgO [11][12][13], such as diopside (CaMgSi 2 O 6 ), akermanite (Ca 2 MgSi 2 O 7 ) and merwinite (Ca 3 MgSi 2 O 8 ). Their multifunctional properties recommend them as candidates for the development of materials suitable for the treatment of bone tissue injuries, as well as its regeneration [12][13][14][15][16][17]; this is due to Ca and Mg ions [1,18] that promote the process of mineralization through apatite deposition [3,19] and enhance cell proliferation and differentiation [1,20,21]. Some researchers prepared larnite and rankinite through the sol-gel combustion method [22], but also monticellite and diopside from eggshell waste via the combustion route [23], with good results in terms of mechanical strength, bioactivity, antibacterial activity, as well as cell adhesion, proliferation and differentiation. ...
Full-text available
In this work, calcium magnesium silicate ceramics were processed through the sol–gel method in order to study the crystalline and morphological properties of the resulting materials in correlation with the compositional and thermal parameters. Tetraethyl orthosilicate and calcium/magnesium nitrates were employed as sources of cations, in ratios specific to diopside, akermanite and merwinite; they were further subjected to gelation, calcination (600 °C) and thermal treatments at different temperatures (800, 1000 and 1300 °C). The properties of the intermediate and final materials were investigated by thermal analysis, scanning electron microscopy, energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction and Rietveld refinement. Such ceramics represent suitable candidates for tissue engineering applications that require porosity and bioactivity.
Full-text available
The need to improve synthetic materials for human bone replacement is significant. Nanostructured bioceramics are expected to have better bioactivity than microcrystals. Merwinite (Ca3MgSi2O8) ceramic is a new bioceramic with good biocompatibility. The aim of this study was the preparation, characterization and evaluation of the bioactivity of nanostructured merwinite. Nanostructured merwinite was synthesized by a sol-gel process. Evaluation of the bioactivity was preformed by immersing the nanostructured merwinite in a simulated body fluid (SBF) and the formation of apatite on the surface of the immersed nanostructured merwinite was investigated. The results showed that hydroxyapatite (HAp) was formed after soaking for 7 days. Osteoblast viability and proliferation was measured by a cell proliferation kit I (MTT). Our study indicated that nanostructured merwinite possessed an apatite-formation ability, show a good bioactivity and might be used for preparation of new biomaterials.
We fabricated a novel type of porous HA scaffold with a dense shell/porous core structure by freezing a hydroxyapatite (HA)/camphene slurry in-situ. During freezing, the camphene dendrites from the mold wall grew 2-dimensionally by pushing the HA particles into the remaining slurry, which resulted in the formation of a camphene layer/concentrated HA particles layer as the surrounding skin of the sample. After removing the frozen camphene and sintering the HA walls at 1250 °C for 3 h, a dense shell integrated with a porous core was formed in-situ. We prepared two types of porous HA scaffold, a porous HA cylinder with a dense shell and a 3-D HA scaffold, consisting of periodic HA networks with a dense shell/porous core structure. These novel scaffolds would be expected to have improved mechanical integrity due to the use of a dense shell, as well as efficient bone ingrowth inside pores formed in a porous core.
Ice-templated structure formation of water-based suspensions was investigated from very slow (<1 μm s -1) to very fast (>100 μm s -1) solidification velocities by analysing the microstructural development in the frozen state and the green state. Maps for the microstructural development were established, showing transitions from planar to lamellar and lamellar to isotropic microstructure with dependence on particle size, solids content and solidification velocity. © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Polymer sponge replication method was used in this study to prepare the macroporous hydroxyapatite scaffolds with interconnected oval shaped pores of 100–300μm with pore wall thickness of ∼50μm. The compression strength of 60wt.% HA loaded scaffold was 1.3MPa. The biological response of the scaffold was investigated using human osteoblast like SaOS2 cells. The results showed that SaOS2 cells were able to adhere, proliferate and migrate into pores of scaffold. Furthermore, the cell viability was found to increase on porous scaffold compared to dense HA. The expression of alkaline phosphate, a differentiation marker for SaOS2 cells was enhanced as compared to nonporous HA disc with respect to number of days of culture. The enhanced cellular functionality and the ability to support osteoblast differentiation for porous scaffolds in comparison to dense HA has been explained in terms of higher protein absorption on porous scaffold.
Freeze-casting, the templating of porous structures by the solidification of a solvent, have seen a great deal of efforts during the last few years. Of particular interest are the unique structure and properties exhibited by porous freeze-casted ceramics, which opened new opportunities in the field of cellular ceramics. The objective of this review is to provide a first understanding of the process as of today, with particular attention being paid on the underlying principles of the structure formation mechanisms and the influence of processing parameters on the structure. This analysis highlights the current limits of both the understanding and the control of the process. A few perspectives are given, with regards of the current achievements, interests and identified issues.
In this research, the synthesis of nanocrystalline merwinite (2SiO2–3CaO–MgO) bioactive ceramic was carried out by the sol–gel method. After crushing, obtained sol–gel derived bioceramic powder pressed uniaxially to produce cylindrical-like pellets, followed by sintering at 1300 °C. Via immersion in simulated body fluid (SBF) for various time intervals, the formation of apatite was characterized. Scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), and Fourier transform infrared spectroscopy (FT-IR) studies were conducted both before and after immersion in SBF. The crystallization temperature of the merwinite was determined by thermal analysis. Attained results confirmed formation of apatite layer within the first day of soaking. Accordingly it can be concluded that merwinite is bioactive and might be used for preparation of implantable biomaterials.
Porous, hollow ceramic components were produced by freeze casting technique. For this purpose aqueous slurries with high solid contents were prepared which were stable against freezing down to at least −5 °C. Ice cores were made by coating steel components with freezing water which were subsequently dip-coated with the ceramic suspensions. After freeze drying which removes both, the ice core and the frozen suspension liquid, and sintering, ceramic components with a high amount of open porosity including steel parts could be achieved. As an example hydroxyapatite was used for showing the opportunities of the freeze casting technology among others for applications in the field of bone replacement. The influence of the solid content of the hydroxyapatite slurries on the ice crystal growth has been investigated by means of compact hydroxyapatite bodies which were prepared by freeze casting using ice moulds with cylindrical cavities.
The impact of bone diseases and trauma in developed and developing countries has increased significantly in the last decades. Bioactive glasses, especially silica-based materials, are called to play a fundamental role in this field due to their osteoconductive, osteoproductive and osteoinductive properties. In the last years, sol-gel processes and supramolecular chemistry of surfactants have been incorporated to the bioceramics field, allowing the porosity of bioglasses to be controlled at the nanometric scale. This advance has promoted a new generation of sol-gel bioactive glasses with applications such as drug delivery systems, as well as regenerative grafts with improved bioactive behaviour. Besides, the combination of silica-based glasses with organic components led to new organic-inorganic hybrid materials with improved mechanical properties. Finally, an effort has been made to organize at the macroscopic level the sol-gel glass preparation. This effort has resulted in new three-dimensional macroporous scaffolds, suitable to be used in tissue engineering techniques or as porous pieces to be implanted in situ. This review collects the most important advances in the field of silica glasses occurring in the last decade, which are called to play a lead role in the future of bone regenerative therapies.
Highly open porous biodegradable poly(L-lactic acid) ¿PLLA scaffolds for tissue regeneration were fabricated by using ammonium bicarbonate as an efficient gas foaming agent as well as a particulate porogen salt. A binary mixture of PLLA-solvent gel containing dispersed ammonium bicarbonate salt particles, which became a paste state, was cast in a mold and subsequently immersed in a hot water solution to permit the evolution of ammonia and carbon dioxide within the solidifying polymer matrix. This resulted in the expansion of pores within the polymer matrix to a great extent, leading to well interconnected macroporous scaffolds having mean pore diameters of around 300-400 microm, ideal for high-density cell seeding. Rat hepatocytes seeded into the scaffolds exhibited about 95% seeding efficiency and up to 40% viability at 1 day after the seeding. The novelty of this new method is that the PLLA paste containing ammonium bicarbonate salt particles can be easily handled and molded into any shape, allowing for fabricating a wide range of temporal tissue scaffolds requiring a specific shape and geometry.