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

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Proceedings of the 5thInternational Conference on Nanostructures (ICNS5)
6-9 March 2014, Kish Island, Iran
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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,
Karaj,Iran
b Pardis Pajoohesh Fanavaran Yazd, BT center, Yazd Science and Technology Park, Yazd, Iran
*mhafezi@merc.ac.ir
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
.
Introduction
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)
3.45
3.1698
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
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Proceedings Book | INST | Sharif University of Technology |
59
Proceedings of the 5thInternational Conference on Nanostructures (ICNS5)
6-9 March 2014, Kish Island, Iran
BIO
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
crystals.
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.
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Proceedings of the 5thInternational Conference on Nanostructures (ICNS5)
6-9 March 2014, Kish Island, Iran
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Fig 5.SEM image of merwinite scaffold after 3 days of
culturing.
Conclusion
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.
References
[1] Daniel Arcos, Maria Vallet-Regi. “Sol–gel silica-
based biomaterials and bone tissue regeneration”. Acta
Biomater. 6 (2010), pp. 28742888.
[2] M. Hafezi-Ardakani, F. Moztarzadeh, M. Rabiee, A.R.
Talebi. “Synthesis and characterization of
nanocrystallinemerwinite (Ca3Mg (SiO4)2) via solgel
method”. Ceram Inter. 37 (2011), pp 175–180
[3] Y. S. Nam, J. J. Yoon, T. G. Park, “A novel
fabrication method of macroporous biodegradable
polymer scaffolds using gas foaming salt as a porogen
additive”, J. Biomed. Mater. Res. (Appl. Biomater),
(2000), pp. 1-7.
[4] S.B. Lee, Y.H. Kim, “Study of gelatin coating
artificial skin: fabrication of gelatin scaffolds using a salt-
leaching method”. Biomater. 26 (2005), pp. 1961-1968
[5] A.Atala, R.P.Lanza, “Methods of tissue engineering”,
Academic Press. 34 (2002), pp. 21-41.
[6] S. Deville. “Freeze-Casting of porous ceramics: A
review of current achievements,” Advan Eng Mater. 3
(2008), pp.155-169.
[7] T. Waschkies, R. Oberacker, M. J. Hoffmann,
“Investigation of structure formation during freeze-casting
from very slow to very fast solidification velocities,” Acta
Mater. 59 (2011), pp.5135-5145.
[8] T. Moritz, H. Richter, “Ice-mould freeze casting of
porous ceramic components”. J Euro Ceram Soc. 27
(2007), pp. 4595-4601.
[9] G. Tripathi, B. Basu. “A porous hydroxyapatite
scaffold for bone tissue engineering: Physico-mechanical
and biological evaluations”. Ceram Inter. 38 (2012), pp.
341349.
[10] M. Hafezi-Ardakani, F. Moztarzadeh, M. Rabiee,
A.R. Talebi, M. Abasi-shahni, F. Fesahat, F. Sadeghian.
“Sol-gel synthesis and apatite-formation ability of
nanostructure merwinite (Ca3MgSi2O8) as a novel
bioceramic”. J Ceram Proces Res. 11 (2010), pp 765-768.
[11] B.H. Yoon, C.S. Park, H.E. Kim, Y.H. Koh, “In situ
fabrication of porous hydroxyapatite (HA) scaffolds with
dense shells by freezing HA/camphene slurry”, Mater.
Lett. 62 (2008), 17001703.
[12] H. Zreiqat, C. R.Howlett, A. Zannettino, P. Evans, G.
Schulze-Tanzil, C. Knabe and M. Shakibaei “Mechanisms
of magnesium-stimulated adhesion of osteoblastic cells to
commonly used orthopaedic implants” J. Biomed. (2002),
pp 17584.
... 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. ...
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