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J. Mater. Sci. Technol., 2013, 29(7), 628e632

Forsterite Nanopowder: Structural Characterization and Biocompatibility

Evaluation
M.A. Naghiu1), M. Gorea1)*, E. Mutch2), F. Kristaly3), M. Tomoaia-Cotisel1)
1) Department of Chemical Engineering, Babes-Bolyai University, Cluj Napoca, Romania
2) Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK
3) Department of Mineralogy and Geology, University of Miskolc, Hungary
[Manuscript received October 22, 2012, in revised form February 13, 2013, Available online 8 April 2013]

Forsterite, a new biocompatible material was synthesized from Mg(NO3)2$6H2O and TEOS by using the solegel
method. The material was then heated at 800, 900 and 1000
C. The forsterite was noticed as the main
crystalline phase in the material fired at 900 and 1000
C, while periclase (MgO) was present in all the
samples. The tests confirm that in the first two samples forsterite is present as crystallites <60 nm, while in
the sample synthesized at 1000
C it forms aggregates of micrometre-sized grains. In vitro test was
performed by immersing the forsterite powder in the simulating body fluids (SBF) and hydroxyapatite
formation on the surface was investigated. We could evidence the formation of hydroxyapatite on the
forsterite surface after 7 days of immersion. The MTT test confirmed that forsterite powders dissolution
promote osteoblast proliferation of the human-type osteoblasts with no significant cytotoxicity effects.

KEY WORDS: Forsterite; Nano particles; Biocompatibility; Grain size; Fourier-transform infrared (FTIR) spectroscopy

1. Introduction Ceramics with Si and Mg contents have been tested for

In the last decades, a wide range of biomaterials have been
produced and tested as replacements for the bone tissue. Espe-
cially bioceramic materials were investigated, because of their
low production costs, simple technology of fabrication and
increased compatibility with hard tissues[1e5].
The evolution of research on bioceramics produced promising
alternative materials that could be used for replacing or sus-
taining parts of the human skeleton[6,7]. Thus, it is important to
investigate new, higher-quality bioceramic materials with an
increasingly better biocompatibility. Previous research has
demonstrated that silicon is an essential element for the human
bone. Magnesium is also of special importance, being closely
related to the mineralization of calcied tissues[8], indirectly
inuencing the mineral metabolism[9].
In the last years, a special focus was placed on the study of
forsterite bioactive ceramics as important materials for medical
applications[10].
* Corresponding author. Dr.; Tel.: 40 264 593877; E-mail address:
mgorea@chem.ubbcluj.ro (M. Gorea).
1005-0302/$ e see front matter Copyright 2013, The editorial ofce of
Journal of Materials Science & Technology. Published by Elsevier
Limited. All rights reserved.
http://dx.doi.org/10.1016/j.jmst.2013.04.007
obtaining bone implants materials[10e14]. Forsterite (Mg2SiO4) is
a phase in the MgOeSiO2 system that shows high biocompati-
bility and good mechanical properties; thus it may be tested for
orthopaedic, maxillary-facial surgery and dentistry applications,
for dental and bone prostheses[11,12].
In spite of the high demand on the market, the production of
pure nanocrystalline forsterite with controlled grain size is still a
challenge for the industry. The main cause is the slow speed of
the reaction of obtaining Mg silicate from oxides; this is related
to the systems relative low diffusion rate that controls the for-
mation of enstatite and/or of magnesium oxide as end products.
As a result, a relative high ring temperature, between 1200 and
1600
C is needed leading to the formation of coarser
powders[15,16].
Great interest in special methods for synthesis of powders is
related not only to a high level of mixing of initial components,
but also to the possibility for ceramics to inherit morphology and
other characteristics of initial materials[17]. Many alternatives for
forsterite synthesis by different techniques have been reported,
for example heating MgO and SiO2 powders (up to 1525
C)[18]
mechanical activation method[19e25], co-precipitation[26],
molten-salt approach[27], polymer precursor method[28] and sole
gel techniques[2,29,30].
In the last 20 years, the solegel method was used with a
continuously increasing interest. The basic advantages of using
this method are represented by lower processing temperatures,
 Author's personal copy

M.A. Naghiu et al.: J. Mater. Sci. Technol., 2013, 29(7), 628e632 629

and higher homogeneity and purity of the nal products. The
hydrolysis of alkoxides metals or the precipitation methods are
frequently used for obtaining homogeneous gels that are then
processed for obtaining ne ceramics[16].
To conclude, the goal of the present paper was to synthesize
forsterite by using the solegel method starting from hydrated
magnesium nitrate and tetraethyl orthosilicate (named also tet-
raethoxysilane, TEOS); the forsterite nanopowder containing
small amounts of MgO was subsequently characterized for its
structure and grain size, as well as for its in vitro biocompati-
bility (SBF and MTT tests).
2. Materials and Methods
2.1. Preparation of forsterite nanopowder
In order to synthesize the forsterite powder, it was used hex-
ahydrated magnesium nitrate (Mg (NO3)2$6H2O, 99.5% purity,
Merck) and tetraethyl orthosilicate (TEOSeC8H20O4Si, Merck)
as raw materials, and polyvinyl alcohol (PVA), sucrose and nitric
acid as binder and pH regulators. The molar Mg:Si ratio was 2:1.
Magnesium nitrate (0.5685 mol) was dissolved into magnesium
nitrate into 200 ml distilled water, and then TEOS was added in the
needed amount. Sucrose (sucrose:TEOS ratio of 4:1) solution was
prepared into 400 ml distilled water and then it was added as drops
into the magnesium nitrate and TEOS mixture. This solution was
homogenized for 2 h by using a magnetic stirring device.
After that, polyvinyl alcohol (PVA:TEOS 0.8:1) was pre-
viously dissolved into 120 ml distilled water. Nitric acid was
further used to bring the solution to pH 1; the solution was
then stirred again for 2 more hours. The mixture was heated up
to 80
C and then stirred for 2 h. The nal solution thus obtained
was preserved for 24 h at room temperature, in order to allow the
gel formation. Subsequently, the gel was heated at 100
C in air
to obtain its complete dehydration; the product was represented
by a thick black gel. In the last stage, the dry gel was red at 800,
900 and 1000
C, with 2 h plateau at the maximum temperature.
The heating rate of gel was 5
C/min for each gel sample.
2.2. Characterization of the forsterite nanopowders
The phase composition of the synthesized powders was
investigated by using a Bruker D8 Advance diffractometer
(CuKa radiation, 40 kV and 40 mA, parallel-beam geometry
obtained by Gbel-mirror). For phase evaluation we have used
DiffracPlus EVA software, with components identication by
Search/Match on ICDD PDF2-2005 database.
The crystallite size of the forsterite powders was determinated
by using the Scherrer equation:
The in vitro bioactivity of the obtained powder was evaluated
by soaking prepared forsterite nanopowder in the SBF solution
for 7 and 28 days at a solid/liquid ratio of 1.5 mg/ml without
refreshing the soaking medium. The SBF solution has been
prepared according to the procedure described by Kokubo and
Takadama[31]. The experiment was carried out in a water bath, at
37
C. At the end of the experiment, the obtained powder was
ltered and gently washed with distilled water in order to remove
the SBF solution, then dried at 100
C. The potential formation
of hydroxyapatite on the surface of the forsterite powder was
investigated by the means of IR spectroscopy using KBr pellets
on a FTIR JASCO 6100 unit in the 4000e400 cm1 spectral
range, with a resolution of 4 cm1.
The SEM back-scattered electron images (BSE) were obtained
on a Hitachi TM-1000 low-vacuum benchtop microscope with
15 kV accelerating voltage on uncoated samples (working dis-
tance 14e16 mm), while the energy-dispersive analysis (EDS)
was carried out using a Si-drift detector with an ultra-thin Be
window.
2.3. Cytotoxicity test
The cytotoxicity test was performed by adding forsterite
powder extracts in contact with osteoblasts cells. For this we
have used U20S-type human osteoblasts cells in a Dulbeccos
Modied Eagles Medium (DMEM, Sigma 500 ml) supple-
mented with 10% fetal bovine serum (FBS) and 1% Penicilline
Streptomycin. The forsterite powder has been sterilized before
being tested. The dissolution extract of forsterite was prepared by
mixing forsterite powder with Dulbeccos Phosphate Buffered
Saline (PBS, Sigma) at an average volume of 200 mg/ml. After
24 h incubation at 37
C, the mixture was centrifuged, with the
supernatant being collected. The cell suspension was adjusted for
a density of 3  103 cells/ml. Then, 1 ml of cell suspension was
added into each sample placed in a 24-well plate that was sub-
sequently incubated with 5% CO2 supply at 37
C for 24 h.
1 ml of the diluted extract of forsterite powder with concen-
trations of 25, 12.5, and 6.25 mg/ml has been added to each
sample, each concentration being used for three different
samples.
After the cells were incubated for 1, 2, and 5 days the mixture
was removed; to each sample we added 500 ml DMEM and 50 ml
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide
(MTT) solution. The well plates were then incubated for 2 h. This
was followed by the addition of 0.8 ml of 2-propanol in order to

Kl
b
tcosq

where b is the width of peak at half of its height, l is the

wavelength (0.154 nm), q is the Bragg angle, K is a constant
(0.9) and t is the apparent crystallite size. We have selected
three distinctive peaks showing the highest intensities that are
clearly separated in the X-ray diffraction (XRD) pattern.
The size and shape of the grains were evaluated by scanning
electron microscopy, on a SEM JEOL 5600 LV unit and by using
a Shimadzu Sald-7101. Particle size distribution was investigated Fig. 1 X-ray diffraction patterns for the forsterite synthesized at various
by using Counter Coulter equipment, also. temperatures.
 Author's personal copy
630 M.A. Naghiu et al.: J. Mater. Sci. Technol., 2013, 29(7), 628e632
Fig. 2 SEM images of the forsterite powder red at: (a) 800
C, (b) 900
C, (c) 1000
C.
prevent an interaction between the cells and the MTT. The optical
density was measured by using the SkanIt Software 2.5.1.
2.4. Statistical analysis
For each experiment, the data were collected with N 3 and
expressed as means  standard deviation (SD). Statistical anal-
ysis was done using the Tukey test. The differences were
considered statistically signicant at p <0.05.
3. Results and Discussion
3.1. Characterization of the forsterite powder and phase
composition
Fig. 1 illustrates the XRD patterns on the forsterite powder
obtained by using Mg (NO3)2$6H2O and TEOS and red at 800,
900 and 1000
C. The patterns show that the sample red at
800
C has a lower crystallinity degree, the pattern being
dominated by the peaks of periclase, MgO. The increase of the
synthesis temperature is accompanied by the formation of well-
crystallized forsterite. Periclase is present also in the sample
obtained at 1000
C; nevertheless, enstatite peaks were not
identied (probably the intensities being below the equipments
limit of detection).
The calculated grain size is 1e19 nm at 800
C, 30e40 nm at
900
C and 45e60 nm at 1000
C. SEM images on the forsterite
powder are presented in Fig. 2. The sample red at 800
C
(Fig. 2(a)) does not reveal the presence of forsterite crystals.
However, at 900
C (Fig. 2(b)) small crystals are visible. The
Fig. 3 Grain size distribution for the forsterite nanopowders synthe-
sized at various temperatures.
forsterite powder red at 1000
C (Fig. 2(c)) is well-crystallized;
the individual crystals show relatively spherical morphologies
but also crystalline aggregates are present.
3.2. Grain size distribution
Fig. 3 illustrates the grain size distribution (cumulative and
differential curves) in the forsterite powders obtained at various
temperatures (800, 900 and 1000
C). The grains red at 800,
and 900
C show a single, very similar size interval: 0.010e
0.028 mm (83%) and 100% of the total particles <0.043 mm in
the rst case, and 0.010e0.034 mm (82%) and maximum
0.064 mm grain size for the second one.
The crystal sizes calculated based on the XRD patterns are
very close to the measured ones. The size distribution in the third
sample, red at 1000
C evidences three distinctive grain size
intervals: 71e17 mm (16%), 17e0.5 mm (68%) and 0.500e
0.119 mm (16%). This grain size increase can be assigned to
strong particle aggregation phenomena due to large specic
surfaces and to supercial energy of nanoparticles at high
temperatures.
3.3. Cytotoxicity assay
We have selected the sample synthesized at 900
C to be
tested for biocompatibility due to the presence of nanosize
grains.
Fig. 4 illustrates the results of the MTT assay of forsterite
powders dissolution (at concentrations of the extract of 6.25,
Fig. 4 Results of the MTT test performed on forsterite powders in
different concentrations, after different intervals of cell culture
using U20S-type osteoblast cells. Data are presented as mean
values; they were considered as statistically signicant at
p <0.05 difference in the same day when compared using the
Tukey test.
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M.A. Naghiu et al.: J. Mater. Sci. Technol., 2013, 29(7), 628e632
Fig. 5 FTIR spectrum of the forsterite powder red at 900
C placed in
a SBF solution for various periods of time.
Fig. 6 XRD patterns of the forsterite nanopowder before and after 7
days, and after 28 days of soaking in SBF (F forsterite;
B brucite; A hydroxyapatite).
Fig. 7 SEM micrographs and EDS spectrum of the forsterite nanopowders after immersion in SBF solution for: (a) 7 days, (b) 28 days.
631
12.5 and 25%) after different times (1, 2 and 5 days) of cell
culturing as compared to the control sample, C.
We can notice that the cell proliferation rate in the forsterite
solution for all the studied concentrations increases proportion-
ally to the culturing time. As compared to the control sample, the
cell proliferation is diminished at lower concentration (6.25%),
especially after one day. After 2 days, the cell proliferation effect
is insignicant at low concentration, 6.25%, it decreases at
12.5% concentration and then increases again at 25%. After 5
days, the effect is similar to the one observed in the control
sample at 6.25, and 12.5%; a slight decrease was noticed in the
25% concentration solution.
The constant increase of the proliferation rate at all concen-
trations represents a conrmation that the forsterite powder
samples promoted the proliferation of osteoblasts cells in the
absence of cytotoxicity effects. Also, it is worthy to notice that
MgO present in small amounts does not play a role in dimin-
ishing the powders biocompatibility. Based on our results, we
can state that the forsterite synthesized as described above shows
a good compatibility with the osteoblast cells.
3.4. In vitro evaluation of bioactivity
As in the previous case, in order to evaluate the powders
bioactivity we have used the sample synthesized at 900
C given
the nanometre-size of its particles and the main crystallized
component in this sample is forsterite. The evaluation was based
on the comparison of Fourier-transform infrared (FTIR) spectra
after various time intervals.
Fig. 5 presents the FTIR spectra of the studied forsterite
nanopowders before and after they were placed in the SBF so-
lution. Prior to the immersion, the typical absorption bands are
located at 420 cm1 representing the MgO6 octahedron, at 513
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632 M.A. Naghiu et al.: J. Mater. Sci. Technol., 2013, 29(7), 628e632
and 620 cm1 for the SiO4 bending and at 890 and 1010 cm1
for the SiO4 stretching.
In correlation with the duration of soaking in SBF, new bands
can be observed, assigned to OeH, CeO and PeO bonds. The
band corresponding to the CO3 2 (CeO stretching) located at
1428 and at 1486 cm1 is present even after 7 days of
experiment.
The absorption bands at 1629 and 3437 cm1, corresponding
to the OH bond in the newly formed hydroxyapatite (HAP) are
clearly visible already after 7 days of soaking.
After 28 days, the previously mentioned OH bands increase in
intensity; more than that, they are accompanied now by the sharp
band at 3690 cm1. At the same time, the band at 476 cm1
corresponding to the PO4 group occurs. The PeO stretching
vibration of the PO4 group is clearly visible at 1074 cm1, in
spite of its partial overlap with the SieO vibration band corre-
sponding to the SiO4 tetrahedron.
3.5. Characterization of the formed hydroxyapatite
The XRD patterns of the forsterite nanopowder before and
after soaking in SBF for 7 and 28 days are illustrated in Fig. 6.
Only the typical peaks of forsterite and brucite are visible after 7
days. Brucite forms as a result of the hydration of MgO from the
starting powder. In the sample immersed for 28 days in SBF
solution the typical peaks for hydroxyapatite are clearly visible.
Thus, we have demonstrated that hydroxyapatite can form on the
surface of the forsterite nanopowder by soaking into SBF
solution.
The surface morphology in BSE images and the EDS spec-
trum of the forsterite nanopowder soaked into the SBF solution
for 7, and 28 days are presented in Fig. 7. The EDS spectrum
after 7 days indicates the presence of calcium and phosphorous,
as well as the area where only the forsterite exists (Cl adsorbed
from SBF solution). With increasing the soaking time for 28
days the clusters of HAP aggregates increased in size, so that the
samples surface became more compact. The corresponding EDS
spectrum shows that these grains are relatively enriched in Ca
and P.
4. Conclusions
The forsterite nanopowder was obtained by using the solegel
method, with Mg nitrate and TEOS as raw materials. The syn-
thesis was performed at temperatures of 800, 900 and 1000
C.
The grain size distribution has shown that the powder is
nanometre-in size in the case of the lower temperatures, in most
cases below 40 nm. In the case of the sample synthesized at
1000
C, the sizes increased due to particle agglomeration at
higher temperatures.
The in vitro bioactivity test has proven that the forsterite
nanopowder is highly reactive and biocompatible; thus, it can be
used as bioactive material in bone reconstruction. The FTIR,
XRD and SEMEDS analyses conrmed the formation of hy-
droxyapatite on the nanoforsterite samples starting with the 7
days of immersion into the SBF solution.
The MTT test has shown that the osteoblasts spreading rate
increases with the cultivation time, in the absence of any cyto-
toxicity signs. These results show that the forsterite nanopowder
obtained by using the method applied in this study represents a
biocompatible material that allows that formation of HAP during
its immersion into SBF solution.
Acknowledgements
This work was nancially supported by research project 171/
2012. One of the authors (M. A. Naghiu) thanks the nancial
support of the Sectoral Operational Programme for Human Re-
sources Development 2007e2013, co-nanced by the European
Social Fund, under the project No. POSDRU/107/1.5/S/76841,
76841. F. Kristaly thanks for support of the European Union and
the European Social Fund under the grant agreement No.
TMOP-4.2.1.B-10/2/KONV-2010-0001.
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