Colloids and Surfaces B: Biointerfaces 68 (2009) 8387

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium

Zouhir E. Allouni a , Mihaela R. Cimpan a , Paul J. Hl b,c, , Tore Skodvin d , Nils R. Gjerdet a,b
a
Department of Clinical DentistryBiomaterials, University of Bergen, Bergen, Norway
b
Orthopedic Biomaterials, Haukeland University Hospital, Bergen, Norway
c
Department of Surgical Sciences, University of Bergen, Bergen, Norway
d
Department of Chemistry, University of Bergen, Bergen, Norway

a r t i c l e i n f o a b s t r a c t

Article history: The physicochemical characterization of nanoparticles in suspension is a prerequisite for the adequate
Received 27 May 2008 assessment of their potential biological effect. Little is known to date about the colloidal stability of TiO2
Received in revised form 8 September 2008 nanoparticles in cell culture medium. This study investigates the effect of particle concentration, ionic
Accepted 16 September 2008
strength, pH, and the presence of fetal bovine serum (FBS) and human serum albumin (HSA) on the
Available online 25 September 2008
colloidal stability of TiO2 nanoparticles in RPMI cell culture medium, by sedimentation measurements,
dynamic light scattering, and electrokinetic measurements (-potential). TEM revealed that the particles
Keywords:
were polydisperse, with diameters ranging from 15 to 350 nm. The agglomeration rate and sedimen-
Titanium dioxide
Nanoparticles
tation rate increased with particles concentration. The size of the agglomerates at 100 mg/L TiO2 was
Colloidal stability signicantly reduced, from 1620 160 to 348 13 and 378 15 nm, upon the addition of 10% (v/v) FBS
Sedimentation and 1% (w/w) HSA, respectively. The isoelectric point of TiO2 in water was 2.9 and the measured -potential
Physiological medium in RPMI was 16 2 mV at pH 7.4. A slight increase in the -potential of TiO2 in RPMI was observed upon
Albumin the addition of FBS and HSA. The addition of FBS and HSA prevented high agglomeration, leading to a
stable dispersion of TiO2 nanoparticles for at least 24 h, possibly due to steric stabilization of the particles.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Titanium and its alloys are among the most commonly used
implant materials [1]. The TiO2 passivation lm formed on the
surface does not have high resistance to abrasion and as a result
particulate wear debris of different sizes, from nano- to micron-
sized particles, are released in the surrounding soft and hard tissues
[25], and can also reach distant organs (liver, spleen, and lung)
[6]. Biodistribution, movement of nanomaterials through tissues,
phagocytosis, and endocytosis of nano-sized material are all likely
to have an impact on their potential toxicity [7]. In turn, these pro-
cesses are most likely to depend on nanoparticle size and surface
properties.
Concerns about the health and environmental risks of nanopar-
ticles are increasing. Numerous in vitro studies report that
nano-sized particles are more biologically active than equivalent
micron-sized particles of the same chemical composition, e.g. TiO2
[8,9]. The toxicity of nanoparticles has been connected with several
physicochemical characteristics such as the shape of the particles,
their specic surface area, the primary particles size, their agglom-
Corresponding author at: Orthopedic Biomaterials, Haukeland University
Hospital, rstadveien 21, NO-5009 Bergen, Norway. Tel.: +47 55586269.
E-mail address: paul.hol@kir.uib.no (P.J. Hl).
eration state, their surface potential (-potential), and their surface
chemistry [10]. Thus, it was argued that the exceptional physico-
chemical properties and the unique kinetics of nanomaterials in
biological solution should be considered prior to in vitro toxicity
studies [1012]. Protein adsorption onto the nanoparticles surface
in biological uids, and the type, amount and conformation of
the adsorbed proteins are also important factors to be taken into
consideration when testing biological responses to nanoparticles
[13,14].
The agglomeration and subsequent sedimentation of nanopar-
ticles raise concerns when considering size-, time-, and dose-
dependent toxicity [15]. Most of the recent studies have focused
on physicochemical property-dependent toxicity. However, less
emphasis has been placed on approaches to predict nanoparticles
disposition [16].
The colloidal stability of TiO2 nanoparticles has been studied
mainly in connection with their use as colloidal photocatalyst for
water detoxication [17,18]. Few studies have been done on the col-
loidal stability of TiO2 nanoparticles in cell culture medium [1921].
Such studies are needed in order to corroborate the observed
biological effect with the size of the particles, the size of their
agglomerates, and with the surface potential. The necessity of
understanding nanoparticles behavior in a biological environment
is now shared by nanomedicine, nanotoxicology, and nanobiology
[22].

0927-7765/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2008.09.014
84 Z.E. Allouni et al. / Colloids and Surfaces B: Biointerfaces 68 (2009) 8387
This paper presents the agglomeration and sedimentation
behavior of TiO2 nanoparticles in RPMI, a widely used cell cul-
ture medium. In addition, the shape of the particles, their N2 -BET
(BrunauerEmmettTeller) specic surface area, the primary par-
ticles size, and their surface potential (-potential) were also
measured. The inuence of fetal bovine serum (FBS), a commonly
used supplement for cell culture, and of human serum albumin
(HSA), the main component of blood plasma [23], on the nanopar-
ticles colloidal stability and surface potential were investigated.
2. Materials and methods
2.1. Materials and reagents
TiO2 nanopowder (99.9%, SigmaAldrich, Product Number
634662, St. Louis, USA), a mixture of anatase and rutile phases, was
used without further purication. RPMI 1640 (Roswell Park Memo-
rial Institute; Lonza Group Ltd., Cat. No. BE12-167F, Belgium) cell
culture medium, a high ionic strength solution, was used. RPMI con-
tains low amounts of amino acids, vitamins, and glucose. Slurries
of TiO2 in RPMI, 101000 mg/L, were used immediately after a 2-
min sonication. Suspensions of 100 mg/L TiO2 with 0.001, 0.01, 0.1,
and 1% (w/w) of HSA (9699%, SigmaAldrich, SL04521, St. Louis,
USA) in RPMI were prepared, sonicated for 30 s and placed for 3 h
in a shaking machine prior to analysis. In the same manner, sus-
pensions of TiO2 were prepared in RPMI to which 1, 5, and 10%
(v/v) of FBS (Lonza Group Ltd., Cat No. DE14-801E, Belgium) were
added. The pH isoelectric point of TiO2 nanoparticles was deter-
mined in water, which corresponds to the point of zero charge [18],
and in NaCl (99.99%, Merck, Darmstadt, Germany) concentration
of 0.12 M, which is close to the ionic strength of physiological solu-
tions. Deionized water was used for the preparation of suspensions.
The change of pH in the medium was obtained by adding the nec-
essary quantity of 0.25 M of HCl (Merck, Darmstadt, Germany) or
NaOH (Merck, Darmstadt, Germany).
2.2. Experimental methods
2.2.1. TEM image analysis
The samples were placed on a (Cu) mesh grid, lyophilized
overnight at room temperature, and analyzed with a Transmission
Electron Microscope (JEM-1230) at an electron emission of 60 kV.
2.2.2. Surface area
The specic surface area of the TiO2 nanopowder was deter-
mined by using the BET method according to the nitrogen
adsorption. The measurement was made with a volumetric adsorp-
tion apparatus (ASAP 2020, Micromeritics, USA) after degassing in
helium for 1 h at 150 C.
2.2.3. Sedimentation study
The sedimentation rate was determined by monitoring the opti-
cal absorbance (at 337 nm) as a function of time, during a time
interval of 6 and 24 h, by UVvis spectrophotometry (PerkinElmer
& Co. GMBH, Model 554, berlingen, Germany). All measurements
were made at 25 C in square cuvettes with 1 cm light path, the
center of the light beam striking the cuvette 1.5 cm above its bottom.
2.2.4. Agglomerate size measurement
Dynamic light scattering (DLS) [24], also known as photon
correlation spectroscopy, was used to determine the hydrody-
namic diameter and the polydispersity index (PI) [25] of the stable
agglomerates. Diluted samples (from 5 to 100 mg/L) were used to
avoid multiple scattering. The measurements were conducted with
the Zetasizer (Nano ZS, Malvern Instruments Ltd., UK). The mean
hydrodynamic diameter (Z-average mean) was calculated from the
autocorrelation function of the intensity of light scattered from the
particles. The software used was DTS Nano version 5.03, supplied
by the manufacturer (Malvern Instruments Ltd.).
2.2.5. -Potential measurement
The rate of particle movement under the inuence of an external
oscillating electrical eld with a voltage of 150 V (electrophoretic
mobility) was measured with the Zetasizer (Nano ZS, Malvern
Instruments Ltd., UK). The measured electrophoretic mobilities
were converted to -potentials by the instrument software (Dis-
persion Technology Software, version 5.03, Malvern Instruments
Ltd., UK) using Henrys equation:
2 f (a)
Ue =
3
where Ue is the electrophoretic mobility, is the dielectric con-
stant,  is the zeta potential,  is the viscosity of the dispersant,
and f(a) is the Henry function. The Smoluchowski approximation,
f(a) = 1.5, was used for high ionic strength media and the Hckel
approximation, f(a) = 1, was used for low dielectric medium.
3. Results and discussion
3.1. Particle properties
TEM (Fig. 1) revealed that the TiO2 nanoparticles were spher-
ical and polydisperse with a particle diameter ranging from 15
to 350 nm. The particles were slightly agglomerated in water
(Fig. 1a) and highly agglomerated in RPMI without FBS (Fig. 1b).
The measured N2 -BET specic surface area of TiO2 nanopowder
was 19.6 m2 /g and the calculated [26] average diameter DBET was
72 nm. The -potential of the nano-TiO2 particles (100 mg/L) mea-
sured in RPMI, was 16 2 mV at pH 7.4, showing that the surface
of TiO2 is negatively charged in cell culture medium.
3.2. Effect of concentration
Once in solution the particles did not retain their nano-size. The
TiO2 particles agglomerated rapidly in RPMI medium (Fig. 2). After
3 h the hydrodynamic diameter of the agglomerates increased from
793 to 2833 nm as the particles concentration increased from 5 to
50 mg/L (Fig. 2). The concentration signicantly affected the rate of
agglomeration.
Particle agglomeration led to sedimentation. The sedimentation
was faster as the concentration increased (Fig. 3). This indicates
that the particle concentration affected the rate and degree of the
agglomeration by enhancing the rate of direct particle-to-particle
interaction.
3.3. Effect of the ionic strength and pH
The isoelectric point (pHiep ) of TiO2 in water was 2.9 (Fig. 4). This
pHiep is remarkably lower than the one reported for the Degussa
P25 TiO2 (pHiep 6.26.9) [18,27,28]. However, the empirical pHiep
of TiO2 reported in the literature ranges from 2 to 8.9, showing a
certain degree of scatter [29]. This scatter is mainly due to the insuf-
cient surface purity of TiO2 particles [30]. In 0.12 M NaCl solution,
the pHiep of TiO2 was shifted to the pH value of 6.4 (Fig. 4), which
is close to the pH of physiological solutions. This shift was assumed
to be due to the specic adsorption of Na+ on TiO2 surface [31].
The hydrodynamic diameter and the settling rate of nanoparti-
cles reach their maximum around pHiep . Consequently, the ionic
strength diminished the colloidal stability of TiO2 in cell cul-
ture medium. Nevertheless, the -potential measured in RPMI
 Z.E. Allouni et al. / Colloids and Surfaces B: Biointerfaces 68 (2009) 8387 85

Fig. 1. TEM picture of TiO2 particles in (a) distilled and deionized water (bar = 500 nm) and (b) RPMI (bar = 500 nm).

Fig. 2. Time evolution of the hydrodynamic diameter of the stable agglomerates for
different concentrations of TiO2 particles in RPMI measured by the DLS technique.
(16 2 mV) was higher in its absolute value than the -potential
measured at the same pH in 0.12 M NaCl, which was close to zero
mV (Fig. 4). This is probably due to the complexity of the cell culture
medium with the presence of not only various ionic salts, but also
a mixture of low amounts of amino acids, vitamins, and glucose.
Fig. 4. -Potential as a function of pH for 30 mg/L TiO2 particles, suspended in water
and in 0.12 M NaCl.
3.4. Effect of proteins
FBS (5 or 10%, v/v) is generally added to cell culture medium for
optimal cell growth. Therefore, its effect on the colloidal behavior of
TiO2 is highly relevant for the evaluation of the cytotoxic potential
of TiO2 nanoparticles. The colloidal stability of TiO2 nanoparticles
increased as the concentration of FBS increased (Fig. 5). The par-
ticles were stable for up to 24 h in RPMI containing 10% (v/v) of
FBS. The results from the DLS measurements (Table 1) were consis-

Table 1
Mean hydrodynamic diameters, polydispersity index (PI) and -potentials of TiO2
(100 mg/L) for different concentrations (%, v/v) of FBS in RPMI.

Samples Mean hydrodynamic PIb S.D.a -Potentials

diameter (nm) S.D.a (mV) S.D.a

TiO2 + 0% FBS 1620 160 0.42 0.08 16 2

TiO2 + 0.5% FBS 561 13 0.32 0.04 12 1
TiO2 + 1% FBS 449 17 0.27 0.03 11 1
TiO2 + 2% FBS 440 8 0.29 0.04 11 1
TiO2 + 5% FBS 375 2 0.26 0.01 11 1
TiO2 + 10% FBS 348 13 0.25 0.01 9 1
a
All measurements are performed at least in triplicate.
b
Fig. 3. Sedimentation curves of TiO2 particles at various initial concentrations in PI is a dimensionless number extrapolated from the autocorrelation function
RPMI expressed by absorbance A, relative to its t = 0 value, A0 . which gives an indication about how broad the size distribution is.
86 Z.E. Allouni et al. / Colloids and Surfaces B: Biointerfaces 68 (2009) 8387

Table 2
Mean hydrodynamic diameters and polydispersity indexes (PI) of TiO2 (100 mg/L)
for different concentrations (%, w/w) of HSA in RPMI.

Samples Mean hydrodynamic diameter PIb S.D.a

(nm) S.D.a

0.1% HSA
TiO2 + 0% HSA 1620 160 0.42 0.08
TiO2 + 0.01% HSA 558 46 0.34 0.05
TiO2 + 0.1% HSA 481 14 0.32 0.01
TiO2 + 1% HSA 378 15 0.24 0.02
a
All measurements are performed at least in triplicate.
b
PI is a dimensionless number (between 0 and 1) extrapolated from the autocor-
relation function which gives an indication about how broad the size distribution
is.

Fig. 5. Sedimentation curves of TiO2 particles (100 mg/L) for different concentra-
tions (%, v/v) of FBS in RPMI.

tent with the sedimentation curves (Fig. 5). In the presence of 10%
(v/v) of FBS the mean hydrodynamic diameter of the agglomerates
was reduced to about 1/5 (Table 1) of the mean agglomerate size
measured in RPMI alone. The decrease of PI (Table 1) shows that the
size distribution becomes narrower in presence of FBS. Even though
the -potential of TiO2 nanoparticles increased from 16 2 mV in
RPMI alone to 9 1 mV in RPMI containing 10% (v/v) FBS (Table 1),
the suspension remained stable.
Protein adsorption to the surface of particles often plays a
decisive role in governing disposition. Albumin is the dominant
protein in FBS, and it is well established that albumin shows high
afnity toward TiO2 particles under different experimental con-
ditions [3235]. Albumin is recognized as a principal component
of blood and the most prevalent protein in plasma [14,23]. It was Fig. 7. -Potential as a function of HSA concentration for 50 mg/L TiO2 particles.
revealed that the TiO2 surface exhibits albumin-preferred adsorp-
tion property when used in mixture with human brinogen [33,36].
The increased stability could be due to albumin adsorption
TiO2 agglomerates sedimented rapidly in the absence of albumin,
onto the solid surface. A slight increase in the -potential of TiO2
whereas in presence of 1% (w/w) HSA, the sedimentation rate was
was observed upon the addition of HSA (Fig. 7). The measured
close to zero over a 20-h period (Fig. 6). In the same manner as
-potential of HSA in RPMI was 9 1 mV at pH 7.4. This shows
in the presence of FBS, the mean hydrodynamic diameter of TiO2
that, as TiO2 , albumin is also negatively charged in physiological
agglomerates was reduced to about 1/5 in the presence of 1% (w/w)
pH. Rezwan et al. revealed that serum proteins such as albumin
HSA (Table 2). This shows that TiO2 nanoparticles are stable in sus-
adsorb on oxide surfaces which are of the same charge [37], and
pension when they are in the presence of albumin, which is in
that the electrostatic interaction plays a very important role in such
accordance with a previous study by Vamanu et al. [21] on the sta-
adsorption. It was suggested that divalent cations (mainly calcium)
bility of TiO2 nanoparticles in Dulbeccos Modied Eagles Medium
binding is a major mechanism whereby serum proteins adsorb to
containing HSA.
TiO2 [3839]. Under physiologic conditions, the albumin molecule
binds Ca2+ ions to its electrostatic sites (such as carboxyl, RCOO ),
serving as bridges between the negatively charged protein and the
TiO2 surface (TiIV O ). Thus, the likely adsorbed serum protein led
to stable colloidal dispersion of TiO2 nanoparticles, probably by a
steric stabilization mechanism.
In order to evaluate in vitro the effects of primary particles and
small agglomerates on cells and their uptake, it is necessary to
maintain a stable suspension of particles throughout the length
of the exposure. Our results show that mixing TiO2 in RPMI with
FBS (rotation for 3 h) allowed the reduction of agglomeration which
enhanced the particles dispersion in cell culture medium.

4. Conclusion

Particle concentration, ionic strength, and the presence of pro-

teins are all factors that inuence the colloidal stability of TiO2
nanopowder in a cell culture medium, such as RPMI. We have seen
Fig. 6. Sedimentation curves of TiO2 particles (100 mg/L) for different concentra- that in RPMI without proteins TiO2 nanoparticles agglomerate and
tions (%, w/w) of HSA in RPMI. sediment rapidly at high concentrations. The presence of FBS and
 Z.E. Allouni et al. / Colloids and Surfaces B: Biointerfaces 68 (2009) 8387
HSA in RPMI notably enhanced the stability of TiO2 nanoparticles
in suspension for a period of 24 h, which improved the particle dis-
position in the cell culture medium. Further studies are needed
to investigate plasma proteins adsorption onto TiO2 nanoparticles.
This can give relevant information about the amount and type of
adsorbed proteins, as well as the inherent protein-particle interac-
tions.
Conict of interest
None.
Acknowledgments
The authors would like to thank the Meltzer Foundation for
nancial support. Special thanks to Prof. R. Anwander for the nitro-
gen adsorption measurement, to C.I. Vamanu for TEM analysis, and
to Mr. O.J. Lundberg for the technical assistance.
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