Analysis in vitro of the cytotoxicity of potential implant materials.
I: Zirconia-titania sintered ceramics
Juliana Marchi,1 Valter Ussui,2 Carina S. Delfino,3 Ana H. A. Bressiani,2 Márcia M. Marques3
1
Centro de Ciências Naturais e Humanas—CCNH, Universidade Federal do ABC, UFABC, Santo André, SP, Brazil
Centro de Ciência e Tecnologia de Materiais—CCTM, Instituto de Pesquisas Energéticas e Nucleares—IPEN,
São Paulo, SP, Brazil
3
Departamento de Dentı́stica, Faculdade de Odontologia Universidade de São Paulo, São Paulo, SP, Brazil
2
Received 19 October 2009; revised 5 February 2010; accepted 23 February 2010
Published online 1 June 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31652
Abstract: Zirconia (ZrO2) is a bioinert, strong, and tough ceramic, while titania (TiO2) is bioactive but has poor mechanical properties. It is expected that ZrO2-TiO2 mixed ceramics
incorporate the individual properties of both ceramics, so that
this material would exhibit better biological properties. Thus,
the objective of this study was to compare the biocompatibility properties of ZrO2-TiO2 mixed ceramics. Sintered ceramics
pellets, obtained from powders of TiO2, ZrO2, and three different ZrO2-TiO2 mixed oxides were used. Roughnesses, X-ray
diffraction, microstructure through SEM, hardness, and DRIFT
characterizations were performed. For biocompatibility analysis cultured FMM1 fibroblasts were plated on the top of disks
and counted in SEM micrographs 1 and 2 days later. Data
were compared by ANOVA complemented by Tukey’s test. All
samples presented high densities and similar microstructure.
The H2O content in the mixed ceramics was more evident
than in pure ceramics. The number of fibroblasts attached to
the disks increased significantly independently of the experimental group. The cell growth on the top of the ZrO2-TiO2
samples was similar and significantly higher than those of
TiO2 and ZrO2 samples. Our in vitro experiments showed that
the ZrO2-TiO2 sintered ceramics are biocompatible allowing
faster cell growth than pure oxides ceramics. The improvement of hardness is proportional to the ZrO2 content. Thus,
the ZrO2-TiO2 sintered ceramics could be considered as potenC 2010 Wiley Periodicals, Inc. J Biomed Mater
tial implant material. V
INTRODUCTION
when properly stabilized with additives, are known as the
strongest and toughest single phase oxide ceramic.17–19
ZrO2 bioceramics, because of their excellent mechanical
properties (especially high strength and fracture toughness) and white color are considered as alternative to
Al2O3, however this ceramic has an inert character, presenting poor bioactivity.20,21 This ceramic has been used
for orthopedic and dental applications, such as artificial
knee, bone screws, and dental post crown.22 Moreover,
this ceramic is promising to be used in dental implants.
In fact, in vivo studies using ZrO2 as dental implant material exhibited biological and mechanical appropriate
properties.23–25
As implant material, ZrO2 ceramic, despite of the high
mechanical properties, have often clinically failed due to
lack of direct bonding with bone, which limited the osteointegration process through adhesion of anchorage dependent
cells.26 To circumvent this problem, many attempts to
improve the osteointegration process, and, consequently,
faster bone regeneration, have been proposed, such as coating27,28 or addition29 of bioactive ceramics, such as calcium
phosphate or bioactive glass; ablative surface modification;30 superficial irradiation with CO2 laser, which has
Pure titanium and titanium alloys are commonly used as
implant materials for dental and orthopaedic prostheses,
due to good mechanical properties and biocompatibility.1–5
The biocompatibility and corrosion resistance are generally
attributed to the titanium passivation oxide layer, which is
basically amorphous in crystal structure and morphologically homogeneous.6
Titanium oxide ceramic or titania (TiO2) is widely used
as pigment, catalyst, and more recently, as biomaterial.7–9
Indeed this ceramic can induce in vitro bone-like apatite formation.10,11 and in vivo stimulate osteoconductivity.12 This
bioactivity is based on its ability to bond directly and reliably
to living bone in a shorter period after implantation.13 Moreover, TiO2 ceramic are noncytotoxic when in contact with rat
hepatocytes and rat cardiomyocytes.14 Nevertheless, the
applications of such ceramic are limited in biomedical field
by their poor mechanical properties allowing its use only as
bioactive layers on the surface of implants. Many attempts to
improve these properties have been proposed, such as preparation of ceramics from nanostructured powders.15,16
Other ceramic oxides suitable for implant materials
are alumina (Al2O3) and zirconia (ZrO2). ZrO2 ceramics,
Res Part B: Appl Biomater 94B: 305–311, 2010.
Key Words: in vitro experiments, cell culture, inert ceramic,
zirconia-titania
Correspondence to: M. M. Marques; e-mail: [email protected]
Contract grant sponsors: CNPq; FAPESP
C 2010 WILEY PERIODICALS, INC.
V
305
promoted the fibroblast31 and osteoblast32 cell adhesion by
increasing the wettability properties of the ceramic surface.
An ideal implant material would have the mechanical
characteristics of ZrO2 along with the biological properties
of the TiO2 ceramics. Thus, a ceramic formed by mixing ZrO2
with TiO2 would have the most appropriate characteristics
for implant application. Fortunately, these two ceramics can
be mixed because both oxides have solid solubility to a large
extent. Studies on ZrO2-TiO2 sintered ceramic applications as
catalysts, dielectric materials, and photosensitive cells have
been published.33,34 However, studies aiming future biological
applications of the ZrO2-TiO2 sintered ceramics are not available in the literature.
In vitro studies enable the observation of cell culture
behavior of different materials, allowing the evaluation of
their biocompatibility and estimation of the biological
response to dental materials. The early assessment of the
histocompatibility of dental materials considered of fundamental importance for the clinical success of dental restorations. It is well known that cell adhesion, when in contact
with a material surface, is considered essential for bone-biomaterial interaction. This process has a great effect on cell
morphology and their ability to proliferation and differentiation. The attachment of anchorage-dependent cells, such as
fibroblasts and osteoblasts, to a biomaterial surfaces is a
complex process involving cell attachment and spreading,
local adhesion formation, and extracellular matrix formation
and reorganization.35 The critical parameter of osteoconduction is the initial number of well attached cells to the bone
substitute, which lead to significant differences in terms of
cell growth.36
In this work, sintered ceramics of ZrO2, TiO2, and three
different ZrO2-TiO2 mixed ceramics were prepared and the
biocompatibility properties of these ceramics were characterized aiming future biological implant applications.
MATERIALS AND METHODS
Samples preparation
To test the physical, mechanical, and biological properties of
the sintered ceramics (TiO2, ZrO2, and three different ZrO2TiO2 mixed oxides), 35 disks (2.5-mm height and 5-mm
diameter) resultant of powder pressing followed by sintering were used.
The TiO2 and ZrO2 powders were obtained following
the procedures described by Ussui et al. in 2003.37 All
ceramics were prepared by a coprecipitation technique.
Briefly, calculated amounts of zirconium oxychloride, titanium chloride, yttrium chloride, and titanium chloride solutions were mixed and added dropwise to an aqueous solution of ammonium hydroxide. The precipitates obtained
were filtered and washed successively with water, followed by ethyl and n-butyl alcohol and then dried and calcinated at 800 C. Prepared powders were uniaxially
pressed (100 MPa) and the obtained disks were sintered
at 1400 C for 4 h. Ceramic surfaces were roughly polished
with 15-lm diamond suspension.
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MARCHI ET AL.
Experimental groups
According to the proportions of the TiO2 and ZrO2 powders
of the sintered ceramic disks five different experimental
groups (n ¼ 7 per group) were obtained, as follows:
•
•
•
•
•
TiO2 : 100% TiO2
Zr40-Ti60: 40 wt % ZrO2 and 60 wt % TiO2
Zr50-Ti50: 50 wt % ZrO2 and 50 wt % TiO2
Zr60-Ti40: 60 wt % ZrO2 and 40 wt % TiO2
ZrO2: 100% ZrO2
Physical characterization
The sintered ceramic samples were characterized by density,
roughness, X-ray diffraction, microstructure, and hardness,
as described below.
Densities of sintered ceramics were measured through
immersion method.38 The density was calculated by comparison with the theoretical densities values that were
assumed to be 5.084 and 5.85 g cm3 for zirconium titanate
(ZrTiO4)39 and baddeleyte (ZrO2),40 respectively. The final
densities were expressed as percentage of the theoretical
densities considered as 100%.
Ceramic surface roughness was measured with a Mitutoyo Surftest 211 portable rugosimeter. Ra was estimated
using three different values parallel oriented. The crystal
structures were registered in a Rigaku DMAX 2000 diffractometer. The microstructure was observed through scanning
electron microscopy (SEM). Vickers hardness of the disks
was evaluated in a Buehler VMT-7 indentator, using 50N load
applied perpendicularly to the polished surface samples.
Biological characterization
The biocompatibility of the sintered ceramic samples was analyzed by cell proliferation assay using cell culture technique.
Cell culture
The cells were cultured as previously described.41,42 Briefly,
the FMM1 fibroblasts, a human gingival cell line, were used.
These cells were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum
(FBS, Cultilab, Campinas, SP, Brazil) and 1% antimycotic-antibiotic solution (10,000 U of penicillin, 10 mg of streptomycin, and 25 lg of amphotericin B per mL in 0.9% sodium
chloride; Sigma Chemical Company, St Louis, MO). The cells
were kept in an incubator at 37 C and a humidified 5% CO2
atmosphere. Cultures were supplied with fresh medium every other day. Cells between the 10th and the 14th passages
were used in all experimental procedures.
Cell proliferation assay
For testing the biocompatibility of the ceramics cultured
fibroblasts were seeded on the top of sterilized disks (six
from each experimental group) and the proliferation of
those cells were followed using scanning electron microscopy images of the disks. The ceramic disks were placed in
the bottom of Petri dishes (24-wells plates, Corning Costar,
Cambridge, MA) and then they were covered by DMEM.
Then, 105 cells were seeded on the top of each sample and
IN VITRO CYTOTOXICITY OF ZIRCONIA-TITANIA SINTERED CERAMICS
ORIGINAL RESEARCH REPORT
TABLE I. Final Density and Surface Roughness
of Ceramic Samples
Samples
TiO2
ZrO2 40-TiO2 60
ZrO2 50-TiO2 50
ZrO2 60-TiO2 40
ZrO2
Final Density
(% Theoretical)
94.7
94.0
94.8
93.8
95.2
6
6
6
6
6
0.3
0.5
0.4
0.3
0.4
Roughness
(lm)
0.53
0.61
0.53
0.64
0.40
6
6
6
6
6
0.14
0.07
0.03
0.07
0.03
the 24-wells plates were placed at 37 C in a CO2 incubator.
One and 2 days after seeding, three samples of each experimental group were fixed in 2.5% glutaraldehyde in a 0.1M
phosphate buffer solution (pH ¼ 7.4) overnight at 4 C for
further scanning electron microscopy (SEM) analysis.
FIGURE 1. X-ray diffraction patterns of ZrO2-TiO2 samples.
Scanning electron microscopy (SEM)
For SEM, 30 samples with cells (six for each experimental
group) were prepared for counting the attached cells. Additionally, five samples with no cells (one for each experimental group) were prepared for superficial microstructural observation. All samples were post fixed in 1% osmium
tetroxide in the 0.1M phosphate buffer solution (pH ¼ 7.4).
Samples were then dehydrated in ethanol and submitted to
chemical drying in hexa methyl disilazane (HMDS, Electron
Microscopy Sciences, Fort Washington, PA). Specimens were
then sputter-coated with gold (Sputtering SCD 020, Bal-Tec,
Liechtenstein). Scanning electron microscopy analysis was
carried out using a Philips equipment (XL 30, Nederland).
Cell counting
For cell counting, scanning electron micrographs of three
defined areas of each specimen were taken at the same
work distance (10 mm) and same magnification (500).
The cells were counted using the Image Pro Plus computational image software.43 The counting provided data for
obtaining cell growth curves.
RESULTS
Physical characterization
Final density and surface roughness measurements results
are shown in Table I. All samples presented high densities,
with values higher than 93% theoretical, showing no significant differences amongst the studied compositions. There
were no significant differences in roughness amongst the
groups. A significant negative correlation was observed showing that higher roughness was obtained for lower density
samples (Pearson linear correlation coefficient r ¼ 0.95).
X-ray diffraction pattern of samples are shown in Figure 1.
Reflections are compared to ICDD files. ZrO2, TiO2, and
ZrO2-TiO2 samples symmetry were identified as tetragonal
ZrO2, rutile, and zirconium titanate (ZrTiO4), respectively.
Moreover, in ZrO2 rich sample (ZrO2 60-TiO2 40), strong
reflections of monoclinic ZrO2 (Baddeleyte) were seen, and
in TiO2 rich sample (ZrO2 40-TiO2 60) reflections of TiO2
rich compound Zr5Ti7O24 were identified.
The mechanical property results, obtained through Vickers indentation technique, of all samples, except those of the
TiO2 group, are shown in Figure 2. It can be seen that ZrO2
sample showed the highest mean value of Vickers hardness
Statistical analysis
Correlation between roughness and densities values, as well
as between composition (as ZrO2 amount) and Vickers hardness were compared through Pearson linear correlation
coefficient.44
The number of cells counted in the scanning electron
micrographs, obtained in triplicate, is presented as mean 6
standard error of the mean. These data were compared by
analysis of variance (ANOVA) complemented by the Tukey’s
test. The level of significance was 5% (p 0.05).
Complementary analysis
Disks covered by DMEM solution had their superficial functional groups analyzed by diffuse reflectance infrared Fourier
transformed (DRIFT) technique (Thermo Nicolet, Nexus 400).
FIGURE 2. Vickers hardness (GPa) of ZrO2-TiO2 samples.
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FIGURE 3. Scanning electron micrographs of FMM1 cells grown on ZrO2-TiO2 substrates before and after different exposure time: (a) TiO2 surface before cell exposure; (b) Zr50-Ti50 surface before cell exposure; (c) Zr50-Ti50 surface before cell exposure; (d) Zr60-Ti40 surface before cell
exposure; (e) ZrO2 surface before cell exposure; (f) TiO2 surface after 1-day exposure; (g) Zr40-Ti60 surface after 1-day exposure; (h) Zr50-Ti50
surface after 1-day exposure; (i) Zr60-Ti40 surface after 1-day exposure; (j) ZrO2 surface after 1-day exposure; (k) TiO2 surface after 2-day exposures; (l) Zr40-Ti60 surface after 2-day exposures; (m) Zr50-Ti50 surface after 2-day exposures; (n) Zr60-Ti40 surface after 2-day exposures;
(o) ZrO2 surface after 2-day exposures.
(13.5 GPa). ZrO2-TiO2 mixed ceramic samples have intermediate values, depending on the composition (from 5.3 up to
8.1). The Pearson linear correlation coefficient r between
composition and Vickers hardness was estimated as r ¼
0.9835, showing a strong positive correlation. Higher Vickers
hardness values were observed in the samples with higher
ZrO2 additions.
Figure 3 illustrates the superficial microstructure of
each ceramic tested [Figure 3(a–e)]. The scanning electron
micrographs showed a similar overall microstructure of all
ceramics. The surfaces were mostly irregular and rough.
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MARCHI ET AL.
The ZrO2 samples [Figure 3(e)] presented grooves with different deepness and oriented in two directions.
Biocompatibility analysis
Figure 3 also exhibits representative scanning electron
micrographs of attached fibroblasts cells on ceramic substrates after 1 [Figure 3(f–j)] and 2 days [Figure 3(k–o)] after seeding. The FMM1 cells adhered and proliferated on
the top of samples of all groups. The overall morphology of
the fibroblasts was similar in all groups showing stellate or
fusiform aspects. On the top of the disks it was possible to
IN VITRO CYTOTOXICITY OF ZIRCONIA-TITANIA SINTERED CERAMICS
ORIGINAL RESEARCH REPORT
FIGURE 4. FMM1 cell growth on ZrO2-TiO2 substrates according to
different cell exposure time.
observe cells arranged in monolayer in a more sparse fashion in 1-day samples [Figure 3(f–j)] and more crowed in the
1-day samples [Figure 3(k–o)]. Two days after seeding only
samples of ZrO2-TiO2 mixed ceramics [Figure 3(l–n)]
showed confluent cell monolayers.
Figure 4 shows a graphic representation of the number
of FMM1 cells adhered to the ceramic samples surfaces in
function of the experimental time (1 and 2 days). The number of cells presented a significant increase, independently
of the experimental group (p 0.05). In both times the
number of cells on the top of ZrO2-TiO2 mixed ceramic samples were similar and significantly higher than those of TiO2
and ZrO2 samples (p 0.05).
Complementary analysis
The DRIFT spectrum of studied samples (Figure 5) shows
several functional groups. The main difference between the
spectra of the samples are the large band region, attributed
to the OAH stretching (3500–3750 cm1), that is more pronounced in the mixed ZrO2-TiO2 ceramics than that
observed in ZrO2 and TiO2 ceramics. These are attributed to
water adsorbed on samples surface of such materials.
with different compositions, to find the most appropriate
material to be indicated for biological implants. This study
has analyzed density, crystalline structure, Vickers hardness,
and biocompatibility of three different ZrO2-TiO2 sintered
ceramics and of individual ZrO2 and TiO2 ceramics. ZrO2TiO2 sintered ceramics presented significantly higher Vickers hardness than TiO2 ceramic and allowed faster adhesion
and growth of fibroblasts than both ZrO2 and TiO2 ceramics.
For measuring the biocompatibility of all ceramics tested
a human gingival fibroblast cell line was used. The attachment and growth of such cells on the top of ceramics was
observed in scanning electron micrographs. The cells
attached to the different ceramics were counted and the
results were compared. The cells attached and grew faster
on the top of the three ZrO2-TiO2 mixture ceramic samples
in comparison to the cells grown on the top of the ZrO2
samples. Somehow this finding was expected, due to the
presence of TiO2 in the mixtures. It is known thatTiO2
presents bioactivity based on its ability to bond directly and
reliably to living bone in a shorter period after implantation.13 Moreover, TiO2 ceramic are non cytotoxic when in
contact with other cells, such as rat hepatocytes and rat cardiomyocytes.14 Surprisingly, the cell attachment and growth
to the ZrO2-TiO2 mixed ceramics were also faster than those
observed in the TiO2 ceramic samples. It means that the affinity of the cells to the TiO2 ceramic has been improved by
mixing this oxide with ZrO2. Thus, besides the chemical
composition, other features, such as physical properties, of
the mixed ceramics should be responsible for this biocompatibility improvement.
In general, the attachment and spreading of cell are
affected by chemical compositions and physical properties
of sintered materials, such as porosity, roughness, and morphology. All sintered ceramics tested, although different in
chemical composition, presented similar physical properties
DISCUSSION
The development of biomaterials to replace hard biological
tissues is a challenge due to the complexity of preparation
of materials exhibiting simultaneously appropriate biocompatibility and mechanical properties. Individually, commonly
used bioceramics, such as alumina (Al2O3), zirconia (ZrO2)
and titania (TiO2), are still deficient in features ideal for
replacing hard biological tissues. However, a material that
combines the mechanical properties of ZrO2 with the biological characteristics of TiO2 in a mixture of ZrO2-TiO2 sintered ceramic, could exhibit the characteristics of a promising hard tissue substitution material. Fortunately, these two
ceramics can be mixed because both oxides have solid solubility to a large extent. Thus, it is of importance to test the
physical and biological characteristics of this mixed ceramic
FIGURE 5. DRIFT spectra of ZrO2-TiO2 samples surface after DMEM
immersion (1): OAH stretching; (2): CAH stretching (CH3); (3): CAH
stretching (CH2); (4): CAO stretching; (5): MeACO (Me ¼ Ti, Zr); (6):
OAH bending; (7): CAO (CO2
3 ); (8): TiAO bond vibration ; (9): ZrAO
bond vibration bond vibration.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | AUG 2010 VOL 94B, ISSUE 2
309
(e.g., roughness and porosity). Thus, in relation to the physical properties it was expected to observe similar behavior
in the cell attachment and growth. However, it was clear the
improved cell growth and attachment to the ZrO2-TiO2
mixed ceramics instead of individual ZrO2 and TiO2
ceramics. It means that more than physical properties the
chemical composition of such ceramics were the main factor
affecting the cell behavior.
At this point we have decided to further analyze the superficial characteristics of the tested ceramics. The functional
groups present at the surface of the ceramics were revealed
by the DRIFTS analysis. In fact, this study showed the presence of several functional groups; however the most striking
difference between pure ZrO2 or TiO2 and the mixed
ceramics was in the H2O content adsorbed on surface. The
mixed ceramics presented more evident H2O groups than the
pure ceramics. It is known that cells are more prompt to
adhere to wet surfaces, thus this difference could explain the
improved cell adherence and growth on mixed surfaces
ceramics than in the pure ceramics. Moreover, although it
was expected to have more adherence and growth on the
TiO2 that has known bioactivity, the water content on the
surface of the mixed ceramics could increase this bioactivity.
Moreover, the surface water content could also have influenced the biological behavior of ZrO2 and TiO2 that although
having differences in bioactivity lead to similar cell adherence
and growth. In fact, the DRIFT analysis showed similarity in
the water content on the surface of the pure ceramics.
This study showed that by mixing the ZrO2 and TiO2
powers it was possible to obtainmixedceramics that kept
the hardness of ZrO2 and improved the bioactivity of the
TiO2. The improved cell attachment and growth on the top
of the mixed ceramics could be due to the superficial water
content that was more pronounced in these ceramics than
in the pure ceramics. Our results showed that the increased
concentration of ZrO2 in the mixed ceramics increase their
hardness; however the biocompatibility was not influenced
by the concentration of the components in the mixture.
Thus, the preparation of the mixed ceramics must follow
the final clinical indication for this implant material. The
ZrO2-TiO2 mixed ceramics have potential bone graft application, but also can be used as dental implant materials. In
fact, the ZrO2-TiO2 mixed ceramics exhibit hardness allowing
their use to load bearing biomedical applications. If high
mechanical properties are required, samples containing
more than 50 wt % ZrO2 would be preferable.
Our in vitro experiments showed that the ZrO2-TiO2 sintered ceramics are biocompatible allowing faster cell growth
than pure oxides ceramics. The improvement of Vickers
hardness is proportional to the ZrO2 content. Thus, based
on physical and biocompatibility properties, the ZrO2-TiO2
sintered ceramics could be considered as potential implant
material. However, the ZrO2-TiO2 sintered ceramics must be
further analyzed to validate them as implant alternative
materials. The next step would be to test the tissue reaction
to these materials in vivo searching for osteoinductive activity when used as bone grafting implants along with osteointegration when used as dental implant material.
310
MARCHI ET AL.
Our in vitro experiments showed that the ZrO2-TiO2 sintered ceramics are biocompatible allowing faster cell growth
than pure oxides ceramics. The improvement of hardness is
proportional to the ZrO2 content. Thus, based on biocompatibility properties, the ZrO2-TiO2 sintered ceramics could be
considered as potential implant material.
ACKNOWLEDGMENTS
The authors thank Bruno B. Silva, Marilene M. Targino, and
Jenecir A. de Almeida for their help in experimental procedures. DRIFT analyses were done with the help of Antonio Carlos da Silva.
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