CytocompatibiliQ of two coating

materials,amorphousaluminaand
siliconcarbide,using human
differentiatedcell cultures
Abdessamad Naji and Marie-FranCoiseHarmand
INSERM U. 306. Universith de Bordeaux I/, 146, rue LBo-Saignat, 33076 Bordeaux CBdex, France
(Received 30 November 1989; revised 26 January 1990; accepted 10 July 1990)

The cytocompatibility of two coating materials, amorphous alumina and silicon carbide deposited by radio-
frequency sputtering, was studied using alveolar bone osteoblasts and gingival fibroblasts from human
healthy tissues. Cytocompatibility was assessed at the level of both the basic (attachment, proliferation
and cell protein content) and the specific features (intracellular alkaline phosphatase activity and the
cytoskeleton) of the cells in direct contact with the coating. Titanium was used as the reference material.
The results showed that both silicon carbide and amorphous alumina are cytocompatible for human
fibroblasts and osteoblasts, whereas titanium appears the least cytocompatible of all the three substrates.
Moreover, the amorphous alumina coating seems slightly bioactive. It seems that these coatings,
particularly amorphous alumina, could be used to protect alloys against corrosion, and consequently
combine the good mechanical properties of the alloys with the good biocompatibility of the coatings. These
coatings seem to perform more suitably than titanium if the strength of the bond between the coating and
the underlying alloys is strong enough to give a stable composite material.

Keywords: Alumina, biocompatibiiity. osteoblasts, fibroblasts

Cobalt-chromium or nickel-chromium-based alloys have
good mechanical properties, but have a tendency to corrode.
To protect a metal or an alloy against corrosion, it can be
modified by additives which increase its resistance against
corrosion, but this procedure modifies its mechanical
properties. One can modify its surface state, for example, by
polishing, but this procedure does not increase the resistance
against corrosion in vivo for very long. A third procedure is to
interpose, between the metal and the biological corrosive
medium, an impermeable and biocompatible barrier, which
could be also bioactive.
Therefore, we studied two coatings, amorphous alumina
and silicon carbide, and evaluated their cytocompatibility,
taking titanium as reference material. Titanium is now
considered to have the best performance regarding resistance
to corrosion. Moreover, it possesses good physico-chemical
and mechanical properties, which permit the titanium and its
alloys to be used in preferance to the cobalt-chromium or
nickel-chromium alloys and this has broadened the field of
application. For some authors, titanium is relatively inert and
considered toxicologically benign’. However, very
Correspondence to Dr M.F. Harmand.
few biological studies have been undertaken on titanium
concerning its biocompatibility either in vivo or in vitro.
In this study, we used two human differentiated cell
types’ belonging to the implantation site, when considering
dental implants: alveolar bone osteoblasts and gingival
fibroblasts.
The results show that silicon carbide coating and
titanium exhibit an equivalent level of cytocompatibility, but
no bioactivity; the amorphous alumina coating has better
performance because it is both cytocompatible and slightly
bioactive.
MATERIAL AND METHODS
Samples
Borosilicated glass discs, 15.5 mm in diameter and 1.5 mm
thick, were coated with amorphous alumina or silicon
carbide using radio-frequency (RF) sputtering (Laboratoire
de Physique des Materiaux, CNRS, Meudon, France). The
reference material consisted of discs (1 5.5 mm in diameter,
1 mm thick) of commercially pure titanium (T40 grade 1,

Q 199 1 Butterworth-Heinemann Ltd. 0142-9612/91/070690-05

690 Biomaterials 199 1, Vol 12 September

 Cvtocompatibility of amorphous alumina and silicon carbIde: R Nal, and M.-F. Harmand

IS0 standard), mechanically polished using successive three times in PBS. Cover slips were mounted on the slides
diamond pastes (30 to 1 pm). The polystyrene of the culture with glycerol at 50% (v/v) in PBS. The cells were examined
wells (Nunclon@ Delta) was the negative control. All the with an epifluorescence light microscope (Leitz) and photo-
materials studied were sterilized by y-rays (2.5 Mrad). graphed with 400 ASA film (Kodak - Ektachrome DX).
Samples are characterized by Rutherford Back Scattering
(RBS) (2S3B. Bordeaux, France). The analysis was performed
RESULTS
in the centre of the sample to verify the composition of the
deposit and measure its thickness.
The RBS spectra of the Alz03 sample confirmed the presence
of A1203 on the glass substrate. The main thickness of the
Cell culture layer was 780 nm.
Healthy alveolar bone and gingival tissue wereobtained from The RBS spectra of the SIC sample confirmed the
volunteers undergoing endodontic surgery. The cell culture presence of a Sic deposit on the glass substrate; the main
was established in 25 cm* tissue culture flasks (Corning), thickness of the layer was 720 nm. Moreover, in both cases
using the explant method3. Both osteoblasts and fibroblasts the RBS spectra indicated Pb traces in the glass substrate.
were cultivated at 37°C in Iscove’s modified Dulbecco’s The SEM study of the surface state of the three
medium (IMDM, Gibco) supplemented with 10% (v/v) fetal materials showed (Figure I) that amorphous alumina
calf serum (FCS) (Boehringer Mannheim). The medium was surface state was smooth and homogeneous, but contained
renewed every 3 d. When the cells formed a confluent
monolayer between the explants, they were carefully
detached and harvested using 0.2% (w/v) trypsin (Boehringer
Mannheim) in Hank’s balanced salt solution Ca++ and Mg+’
free. Cell cultures were used at the third passage.

Experimental procedure
The samples were placed in 15.5 mm multiwell plates
(Nunc), covering the bottom of each well. The assay
procedure was identical for both osteoblasts and fibroblasts;
cells were seeded at low density on to the material
(5 X 1 O3 cells/cm’), and the wells were filled up to 500,~l
by IMDM supplemented with 10% (v/v) FCS and kept at
37°C in an air atmosphere containing 5% CO2 and 95%
humidity. The medium was changed every 3 d. Four wells
per series were killed at 3 and 6 h for cell attachment assay
and at 1, 3, 6, 9, 15, 2 1 and 27 d for cell proliferation
assay.
The cells were detached with 0.1% (w/v) trypsin in
Hank’s balanced salt solution Ca++ and Mg++ free, rinsed
three times with complete medium, and counted in triplicate
using an haemocytometer. The data were expressed as the
mean + SEM.
The cell suspensions obtained after cell counting are
suspended in 400~1 of distilled water. All samples under-
went three times a freezing-thawing-shaking cycle. We
used 200~1 of the resulting suspension for cell protein
determination, according to the method of Lowry et a1.4 and
100,~l of osteoblast suspension for the evaluation of
intracellular alkaline phosphatase activity according to
Luben et al.5.

Indirect immunofluorescence staining of cytoskeleton

Cells cultured in contact with the samples were washed
twice in IMDM, fixed in 80% acetone (v/v) in phosphate
buffered saline (PBS) for 10 min at room temperature, air-
dried, and hydrated in PBS, pH 7.2, for 10 min. The excess
of buffer was removed. The microtubules were labelled with
mouse monoclonal antitubulin a antibody or antiactin
antibody (Amersham) at 37°C for 30 min. at a dilution of
1 : 750, in a humid atmosphere. The control experiment was
carried out using a specific monoclonal antibody against
RNA polymerase of odospora Comata’. The cells were
washed three times in PBS and treated in the same
conditions with the secondary antibody consisting of
fluorescein conjugated sheep antimouse IgG antibody F/gore 1 Surface state of (a) amorphous alumma, (6) skon carbide and
(Amersham) at a dilution of 1 : 25. The cells were washed (c) titanium (bars = 10 ,um).

Bomatenals 199 7. Vol 12 September 691

Cytocompatibility of amorphous alumina and silicon carbide: A Naji and M.-E Harmand

some granulations, the main diameter of which was 0.22 to final cell density was higher than that which is on the
0.63pm (Figure la), probably constituted by cn/stallized negative control (+ 12%, P < 0.05). Osteoblast growth was
grains of alumina. The silicon carbide surface state was better than fibroblast growth on the alumina; the first phase
perfectly smooth and homogeneous (Figure 76). whilst of growth was similar on both the alumina and the negative
titanium surface state was slightly irregular (Figure Ic). control, while enhanced on to alumina during the second
No significant difference was observed concerning phase of growth (+ 10%. P < 0.05). This slight significant
both fibroblast (Figure 2a) and osteoblast (Figure 2b) enhancement of both the osteoblast and the fibroblast growth
attachment on to three of the four substrates studied: on amorphous alumina has to be taken into consideration.
amorphous alumina, titanium and negative control. Osteoblast On titanium, fibroblast (Figure 3a) and osteoblast
attachment on to silicon carbide was quite normal, whereas (Figure 36) growth behaved the same, quite comparable
fibroblast attachment was inhibited -30% (P < 0.01) at 6 h. during the first phase of growth until day 10 and day 15 for
Fibroblast proliferation (Figure 3a) was slightly inhibited fibroblasts and osteoblasts, respectively. Thereafter, it was
on silicon carbide, from the outset of the incubation period, significantly inhibited, since the final cell density is 16%
which could be correlated with the above result, since cell (P < 0.05) and 25% (P < 0.01) less than on the negative
density diminished by 36% (P < 0.01) at day 9 compared to control for fibroblasts and osteoblasts, respectively. It is
the negative control. However, at day 2 1, cell density was interesting to note that osteoblast growth behaviour was
comparable to that measured on the negative control. On the identical on both titanium and silicon carbide.
contrary, during the first experimental phase of growth, Whatever the substrate and the cell model used,
osteoblast proliferation (Figure 36) was comparable on fibroblasts (Figure 4a) or osteoblasts (Figure 4b), the total
silicon carbide and negative control, whilst during the cell protein content was not significantly modified, which
second phase of growth, it was significantly inhibited; at day seems to demonstrate a steady metabolic state.
27, cell density was 27% (P < 0.01) less than on the Concerning osteoblast phenotype expression, intra-
negative control. cellular alkaline phosphatase activity was not modified in
On the amorphous alumina, fibroblast (Figure 3~3) contact with amorphous alumina, or silicon carbide when
proliferation was comparable to that on silicon carbide, compared to the negative control. On the contrary, this
slightly inhibited until day 6 (-37%, P < 0.01) but thereafter, osteoblast specific enzyme activity was significantly decreased
the fibroblast proliferation rate increased and at day 21, the on titanium, -30% (P < 0.01) less, at 8 X 1 O4 cells/cm*
(day 21). than on the negative control (Figure 5).
The study of osteoblast cytoskeleton, both the micro-
tubular (Figure 6a-d) and microfibrilar (notshown) systems,
a b shows that the cytoskeleton was very well expressed on all
of the substrates studied. However, it was particularly well
developed on both the titanium (Figure 6~) and the negative
control (Figure 6d) where the cells appeared more widely
spread. Comparable results were obtained using human
gingival fibroblasts (not shown).

DISCUSSION

The comparative study of two coating materials, amorphous

alumina and silicon carbide deposited by RF sputtering on to
borosilicated glass discs, was performed with regards to the
3 6 3 6 reference material widely used in implantology, titanium.
Time (hours) Two types of differentiated cells from human origin were
Figure 2 Cell attachment of (a) fibroblasts, (b) osteoblasts. (a) Negative
used: alveolar bone osteoblasts and gingival fibroblasts.
control, (0) amorphous alumina, f LI) silicon carbide. (0) titanium. This study proves to be interesting on several levels.

co
0 5 10 15 20 5 10 15 20 25 5 10 5 10 15

Time(days) (Ceils/cm2)x104

Figure 3 Cell proliferation kinetics of (a) fibroblasts. (b) osteoblasts. Figure 4 Cellprotein content of (a) fibroblasts, (bJ oSteoblasts. (0) Negative
(0) Negative control, (0) amorphous alumina, (A) silicon carbide, (0) titanium. control, (17) amorphous alumina, (A) silicon carbide, (0) titanium.

692 Biomaterials 199 1, Vol 72 September

 Cytocompatibility of amorphous alumina and s~hcon carbide: R Naj/ and M.-F. Harmand

function, as assessed by intracellular alkaline phosphatase

activity and cytoskeleton appearance, is identical on both the
substrates. We can conclude that amorphous alumina has a
high level of cytocompatibility, which could be associated, at
least for osteoblasts, with a certain level of bioactivity. Cell
proliferation enhancement associated with unchanged cell
phenotype expression, as assessed per cell unit, means an
increase in the total cell culture function, which by definition
is bioactivation.
Secondly, silicon carbide looks cytocompatible both
on basal and specific cytocompatrbrlrty levels. However,
fibroblast and osteoblast attachment is not highly satisfactory,
and during the second phase of osteoblast growth, osteoblast
proliferation is very significantly reduced by 30%.
Thirdly, the most striking result was obtained with
titanium which appears the least cytocompatible of the four
substrates studied. This cytoincompatibility is expressed on
the basal level, since the second phase of growth exhibits a
reduced proliferation rate for both fibroblasts and osteoblasts,
and on a specific level, since intracellular alkaline phosphatase
I 1 I L 1 activity is decreased by 30%. These results show that
(3 2 4 6 8 10 titanium is not as biocompatible as generally claimed, since it
reduces the basal functions of cells, attachment and
proliferation, and diminishes the specific function of the
osteoblast, i.e. the synthesis of alkaline phosphatase, which
Figure 5 Osteoblast intracellularphosphatase activity. (e) Negative control,
signifies a reduction in the degree of cellular differentiation.
(0) amorphous alumina, (A) silicon carbide, (0) titanium.
An explanation could be that the surface of the titanium
sample is not so smooth as the one of the SIC and AI,O,
Firstly, it showed that the amorphous alumina is as samples. However, experiments performed with specular
cy-tocompatible as is the negative control, whatever the cell polished titanium gave identical results’.
model used on the level of basal cytocompatibility and on the A second explanation could be the release of toxic
level of specific cytocompatibility. On the level of basal corrosion products from titanium. Titanium in principle is
cytocompatibility, cell attachment and cell protein content resistant to the corrosion due particularly to its high reactivity
are similar on both the alumina and the negative control, but with oxygen, which creates a layer of titanium oxides*
cell proliferation appears slightly but significantly enhanced (Ti,O,, TiO,, TiO). TiO, is the most stable oxide for the
on amorphous alumina (+ lo%, P < 0.05). On the level of passivatron phenomenon’. However, this passivation does
specific cytocompatibility, i.e. cell phenotype expression, cell not prevent but only reduces the corrosion. This phenomenon

Figure 6 Microtubule system immunofluorescence of osteoblasts at day 8: (a) on amorphous alumina. lb/ on skon
carbide, (c) on titanium, (d) on negative control (magnification X 750).

B/omatenals 199 1, Vol 12 September 693

Cytocompatibility of amorphous alumina and silicon carbide: A Naji and M.-F. Harmand
can explain the late effects of titanium on cells, which appear
after day 15. A rate of titanium corrosion was measured in
contact with an electrolyte consituted of 0.17 M NaCI,
2.7 X 1 Oe3 M ethylene-diaminotetra-acetic acid (EDTA) in
Hank’s balanced salt solution. The release of Ti is about
1.2 X 1 0m3 pg/mm’/d ‘O,” . Other studies have already
shown that normal human fibroblasts present a good
tolerance for titanium. The morphological cellular aspect is
not modified. However, the cells undergo an abnormal
release of L.D.H. (lactate dehydrogenase) in the medium’*.
Those materials, silicon carbide and particularly amor-
phous alumina which is associated with a bioactive effect,
display good resistance against biodegradation. These
coatings permit the combination of the mechanical qualities
of alloys which are not very biocompatible, with the properties
of those ceramics which are not mechanically resistant, but
biocompatible. Studies of corrosion performed by potentio-
dynamic technique of a chromium-cobalt alloy which was
covered by these coatings showed a disappearance of
corrosion current and a high resistance to the biodegra-
dation13,14 . However, the coatings must efficiently adhere or
bind to the alloys. It seems that chromium could induce a
certain chemical continuity by developing oxides which are
partially soluble in the ceramic.
The implantation of these materials is endo-osseous or
juxta-osseous, so they will interact, in particular with bone.
Our results show that the osteoblasts are more sensitive
than gingival fibroblasts, i.e. their proliferation is less
modified than the osteoblast proliferation when in contact
with silicon carbide. This in vitro study is rather short, when
compared to the in vivo implantation time. However, the
in vitro sensitivity of the osteoblasts shows some cyto-
incompatibilitywith titanium.This result leads us to question
the real biocompatibility of titanium in viva for a long
time.
Finally, if the corrosion phenomenons of the alloys
could be eliminated by protective coatings which would be
biocompatible and/or bioactive, new materials could be
elaborated, which have a better performance than materials
based on titanium. In this study, amorphous alumina, which
is both biocompatible and bioactive, gives an excellent
example.
694 Biomaterials 199 1. Vol 12 September
ACKNOWLEDGEMENTS
We would like to thank Dr Berdeu (2S3B, Bordeaux, France)
for performing the RBS analysis of the samples.
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