Biocompatibility, Cytotoxicity and Bioactivity

of Amorphous Carbon Films

Sandra E. Rodil1 , René Olivares2 , Higinio Arzate2 , and Stephen Muhl1

1
Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de
México, Circuito Exterior s/n, CU, MEX-04510 México D. F.
2
Facultad de Odontologı́a, Universidad Nacional Autónoma de México, Circuito
Exterior s/n, CU, MEX-04510 México D. F.
ser38@zinalco.iimatercu.unam.mx

Abstract. In this work we investigate the possibility of using an amorphous car-

bon layer as a bioactive coating that promotes bone ingrowth for medical implants
in direct contact with bone. The initial step was to confirm the tissue compatibil-
ity of the amorphous carbon coatings by studying cellular adhesion, proliferation
and viability with human osteoblasts and fibroblasts, cultured on the carbon sur-
face. To investigate the bone-bonding capabilities of the sputtered a-C coatings,
“in vitro” mineralisation studies were performed. For the biomineralisation assays,
human osteoblasts were grown on a-C samples for periods up to 14 days, leading
to the formation of mineralised structures that were morphologically and biochem-
ically investigated by the scanning electron microscope and X-ray microanalysis,
respectively.

1 Introduction

There is an increasing interest in the development of novel coatings or sur-

face modification treatments to improve the bone-bonding ability of metal
implants [1]. An essential condition for an artificial material to bond directly
to living bone is the formation of bone-like apatite on its surface. Direct bone-
implant joint formation is important since if the ingrowing bone is separated
from the implant by an intervening soft tissue layer, excessive micromove-
ments in the interface may occur. These micromovements are known to pro-
duce negative reactions such as destruction of the surface oxide layer and wear
of the implant surface, and these can be accompanied by corrosion and wear
debris accumulation, causing the subsequent failure of the implant [2]. The
parameters that are important for long-term implant life include the biocom-
patibility of the chosen implant material, the surface chemistry and physics,
the macro- and microstructure, the surgical implant procedure, the time and
mode of implant loading and the implantation bed itself. Therefore, the bio-
compatibility of an implant is only one of parameters influencing the tissue
response to an implant material; other factors must be satisfied for a device
to be suitable for implantation. Particularly, for bone ingrowth many factors
have been shown to be important: (a) mechanical factors, such as micro-
motion and loading; (b) geometrical factors: the surface of ingrowth fixation
G. Messina, S. Santangelo (Eds.): Carbon, The Future Material for Advanced Technology Ap-
plications, Topics Appl. Phys. 100, 55–75 (2006)
© Springer-Verlag Berlin Heidelberg 2006
56 Sandra E. Rodil et al.

implants must have a geometry that accommodates the bone ingrowth, and
therefore noncemented porous coated implants have been proposed where
the pore size and the interface gap play major roles; and (c) materials fac-
tors: the composition of the surface layer is a very important determining
factor [3]. Porous coatings on titanium (Ti) and cobalt–chrome (Co–Cr) al-
loys are the most commonly used materials for odontological and orthopedic
implants. However, the failure rates today demonstrate that there is still
scope for improvement [4]. Other potential implant materials include mostly
ceramics, such as, hydroxyapatite [5], tricalcium phosphate [6] and carbon-
based ceramics (pyrolitic carbon, glassy carbon) [7]. All have been shown
to be conducive to bone ingrowth (osteoinductive), but they have not been
used for load-bearing implants due to strength concerns. Here is where the
surface modification comes into play; by modifying the surface chemistry of
an implant, it is possible to retain the mechanical and geometrical conditions
needed to assure the long-term stability of the implant, together with enhanc-
ing a particular biological response such as bone ingrowth [8]. Both pyrolitic
and glassy carbon have been shown to be osteoinductive, but they are not me-
chanically stable. Therefore carbon-based coatings having graphite-like prop-
erties deposited on mechanically stable substrates are interesting systems to
evaluate for their potential to enhance bone ingrowth. Amorphous carbon
(a-C) and diamond-like carbon (DLC) films are known as bioinert materials
with no toxic reactions with living organism [9, 10]. Furthermore, the combi-
nation of high hardness, low coefficient of friction, high wear and corrosion
resistance, with the bioinert character is a good reason for selecting carbon
films as a surface finish on biomedical implants. Previous studies have shown
that these films exhibited good compatibility with different cell types, includ-
ing macrophages, fibroblasts, human myeloblasts and human embryo kidney
cells [11, 12, 13, 14, 15, 16]. They have also shown excellent haemocompati-
bility, reduced platelet adhesion, activation and aggregation, which are good
indicators of reduced thrombus formation [17, 18, 19]. Another important fac-
tor in favor of the amorphous carbon films as a biocompatible surface is that
their properties (surface energy, electrical conductivity, tribological proper-
ties, etc.) can be tailored by the deposition conditions or doping/bonding
using additional elements to induce specific biological responses [20, 21]. To-
day, two main biomedical applications of carbon-based coatings can be seen:
those of DLC in blood-contacting devices (stents and heart valves); and the
use of DLC to reduce wear in load-bearing joints. Coronary artery stents
and heart valves are affected by platelet activation due to blood contact with
the biomaterial or the release of metallic ions. This activation is an impor-
tant trigger for thrombosis. To solve this situation, the use of coatings on
metallic stents and heart valves has been suggested. Of these coatings, car-
bon coating is the most frequently used. In vitro studies [22, 23] have shown
that DLC significantly reduces the release of metal ions and diminishes the
platelet activation, therefore lower rates of acute and subacute thrombosis
 Biocompatibility, Cytotoxicity and Bioactivity 57

are expected. Although, recent clinical trials showed no statistical differences

between metal bare and coated stents [24, 25, 26]. While, for coated heart
valves, clinical trials have demonstrated that the most bio-haemocompatible
material is pyrolitic carbon, either as a solid or a coating [27, 28]. On the
other hand, the use of diamond-like carbon for orthopedic implants has been
motivated by the low coefficient of friction, high wear resistance and hardness
of the films. These properties are needed to improve the lifetime of total hip
or knee replacements. The standard design of a hip replacement comprises a
metal or sometimes a ceramic head running against an ultra-high-weight poly-
ethylene acetabular cup. This can last for up to 15 years, which is not enough
for young, active patients, and moreover, early failures are usually reported.
The failure is mainly a consequence of generation of polyethylene wear debris
resulting from the relative movement of the femoral head and the acetabular
cup. Thus, coatings to prevent such wear have been suggested and DLC is one
of the potential candidates [29, 30]. Minor applications of carbon films in the
biomedical field include coating of polymer-based medical products, such as
catheters, drainage tubes or polymer contact lenses [31]. Not much research
has focused on studying the bone-related cell responses of amorphous carbon
films. The studies of the bone-forming activity have been performed on cell
populations extracted from bone tissue: osteoblasts. Du et al. [32] studied
the morphological behavior of osteoblast on DLC and amorphous carbon ni-
tride deposited on silicon substrates, showing that cells were able to attach,
spread and proliferate on the surfaces without apparent impairment of cell
physiology. However, the ability of carbon surfaces to promote bone growth
or biomineralization has not been studied. The formation and maintenance of
viable bone in close proximity to the surface of biomaterials are essential for
the stability and clinical success of noncemented orthopedic/dental implants.
The osteoblast is the cell type responsible for the deposition of bone within
the interfacial zone between the implant and host tissue. A better knowledge
of the osteoblasts’ function could enhance current understanding of the cellu-
lar/molecular events that occur at the tissue–implant interface, and with this
information one could produce biomaterials engineered to elicit specific re-
sponses, such as enhanced osteoblastic mineral deposition. The bone-forming
activity of osteoblasts entails an initial phase of attachment, proliferation, dif-
ferentiation and extracellular matrix synthesis, and a second phase of bone
matrix mineralization. Mineralization in osteoblasts, cultures in vitro can be
demonstrated in two main ways: detection of calcium and phosphate deposits
in the cell layer or detection of bone-matrix proteins that are known to regu-
late “ex novo” bone formation. For example, bone sialoprotein is specifically
expressed by osteoblasts depositing bone ex novo [33]. Osteoblasts cells are
anchorage-dependant, and their development depends strongly on the ini-
tial cellular attachment [34]. In this paper we investigate cellular adhesion
and subsequent cellular functions: proliferation and deposition of a miner-
alized matrix. The cellular adhesion and proliferation in conjunction with
58 Sandra E. Rodil et al.

viability tests constitute the best combination of cytotoxicity–biocompatibil-

ity tests, while the analysis of the production of mineralized matrix on the
implant materials represents the most appropriate bioactivity test for bone-
bonding applications. The bioactivity was monitored by direct observation
of the morphology of the deposited mineralized matrix, as well as the ex-
pression of some biochemical parameters of osteoblastic phenotypes: alkaline
phosphatase (ALP) activity and bone sialoprotein (BSP).

2 Experimental Details
Two sets of experiments were realized to evaluate the biocompatibility and
bioactivity of the a-C coatings. In the first set, we studied the cytotoxicity us-
ing 1 cm2 stainless steel (AISI316L) squares as substrates. From profilometer
measurements the surface roughness was between 0.05–0.1 µm with randomly
distributed scratches. The second set, used to evaluate the bioactivity, was of
stainless steel discs of 15 mm in diameter: the same size as that of the wells
in the culture plates. The surface of the discs was sand-blasted with SiO2
particles to obtain a uniform average roughness of 2 µm. In an earlier study
we confirmed that human osteoblast attachment on amorphous carbon films
was enhanced on substrates with an average surface roughness greater than
1 µm [35].

2.1 Film Deposition

Prior to deposition, the substrates were ultrasonically cleaned in acetone

and isopropanol for 30 min, respectively, and then air-dried. A thin layer of
titanium was deposited as an interface layer to increase the adhesion between
the amorphous carbon film and the stainless steel (SS) substrate. The Ti layer
was deposited using a pulsed magnetron sputtering system using argon as the
precursor gas and a high-purity, 99.99%, Ti target. The a-C were deposited
using a high purity hollow cathode graphite target, 7 cm2 , in a DC magnetron
sputtering system and an argon plasma. The Ti/SS substrates were initially
cleaned by an argon plasma for 10 min. The base pressure prior deposition
was less than 2 · 10−4 Pa and the carbon films were deposited at 4 Pa using
0.4 A and argon flow rate of 20 sccm for 5 min, giving a film thickness of
150 nm. For the characterization of the film properties, a-C films under the
same conditions were deposited on silicon and quartz substrates. All layers
were deposited at room temperature.

2.2 Film Characterization

The films deposited on silicon substrates were used to obtain the thickness us-
ing a DEKTAK profilometer. The optical absorption in the ultraviolet–visible
 Biocompatibility, Cytotoxicity and Bioactivity 59

range was measured for the samples deposited on quartz using an UV–VIS
UNICAM spectrometer in the range 1.5–3.5 eV. Spectroscopic ellipsometry
was also used to study the optical properties and thickness of the films.
The energy range was 1.5–5 eV, and the ellipsometric parameters were ob-
tained using a Jobyn Yvon photoelastic modulated ellipsometer. The optical
constants were obtained by fitting spectroscopic ellipsometry measurements
with a two-oscillator Tauc–Lorentz model [36]. Surface composition was in-
vestigated with X-ray photoelectron spectroscopy using a Thermo Scientific
Multilab and Al Kα-radiation.

2.3 Cell Preparation

Human alveolar bone-derived cells (HABDC) were obtained by a conventional

explant technique [37]. The cells were cultured in 75 cm2 flasks in a standard
culture medium composed of: Dulbecco’s Modified Eagle’s Medium (DMEM),
supplemented with 10% fetal bovine serum (FBS) and antibiotic solution
(Streptoycin 100 mg/ml and penicillin 100 U/ml, Sigma Chem Co.). The cells
were incubated in a 100% humidified environment at 37◦ C in an atmosphere
of 95% air and 5% CO2 .

2.4 Cytotoxicity Test

The cytotoxicity was evaluated on the a-C films, the SS substrate, the Ti coat-
ing and a plastic positive control. All samples were sterilized by autoclave.
The cytotoxicity study include three different tests: adhesion after 24 h, prolif-
eration and viability up to 7 d. The HABDCs were plated at an initial density
of 1 · 104 /well and left to adhere for 3 h. After this time, 600 µl of the culture
medium were added. For the adhesion tests the cells were kept for 24 h and for
the proliferation and viability assays the cells were left in the culture plates
for 1, 3 and 7 d. The experimental and control cultures were treated every
2 d with fresh media. To determine the number of attached cells, we followed
the same procedure in each case; that is, after incubation, the unattached
cells were removed with a phosphate buffered saline solution (PBS) and the
attached cells were fixed with 3.5% paraformaldehyde. The evaluation of the
number of attached cells was performed by staining the cellular membranes
and measuring the optical density at the corresponding color. To perform the
staining, the fixed cells were incubated with 0.1% toluidine blue dye for 3 h.
The dye was extracted with sodium dodecyl sulfate (SDS) and the optical ab-
sorption measured with a microplate enzyme-linked immune assay (ELISA)
reader at 600 nm. The number of cells was then determined by correlating the
absorbance of the experimental samples with a standard correlation curve.
For the proliferation test we followed the same procedure for each incubation
time. On the other hand, for the cell viability the conversion of the yellow
dye (3-[4,5-dimethylthiazolyl-2-y]-2,5-diphenyltetrazolium bromide, MTT) to
60 Sandra E. Rodil et al.

blue formazan was used as a marker of cell viability or cell enzymatic activ-
ity (mitochondrial). The cells were plated and incubated as described above.
After each term 10 µl of MTT were added and incubated for 3 h. Then,
the supernatant was removed and 600 µl of dimethyl sulfoxide (DMSO) were
added to each well. After 60 min of slow shaking the absorbance was read at
570 nm which gives a reading directly proportional to the number of viable
cells.

2.5 Bioactivity

Evaluation of the biomineralization consisted of the morphological examina-

tion of the mineralized extracellular matrix and the evaluation of a selection
of proteins involved in the bone-formation process.

2.5.1 Morphological Assay

The samples (a-C, Ti, SS and positive control) were cultured in the same
way as for the proliferation assay until they reached confluence. Then, we
added the mineralization medium: 50 µg/ml of ascorbic acid, 10 mM of
β-glycerophosphate and 100 nM of dexamethasone. These compounds are
known to accelerate the mineralization process. The media was changed every
2 d. Biomineralization was evaluated after 7, 14 and 21 d. Following culti-
vation the samples were prepared for observation in the scanning electron
microscope (SEM) in the following way: cell cultures were fixed with 4%
formaldehyde in 0.1 M phosphate buffer solution (pH 7.3), then dehydrated
in graded ethanol and finally sputter-coated with gold. The microscope was
a CAMBRIDGE-LEICA STEREOSCANN 440 SEM.

2.5.2 Protein Synthesis

Alkaline Phosphatase Activity

In this case, the cells were incubated in the culture medium, i.e., no min-
eralization medium was added, for 3, 7 and 14 d. The alkaline phosphatase
activity was determined in cell lysates, obtained by treatment of the cultures
with 0.1 Triton X100 in PBS. The lysates were analyzed by measuring the
release of p-nitrophenol from p-nitrophenyl phosphate in an alkaline buffer
solution, and the colorimetric determination of the product (p-nitrophenol).
Alkaline phosphatase catalyzes the cleavage of a phosphate group from a va-
riety of compounds, including p-nitrophenyl phosphate, which is colorless.
However, one product, p-nitrophenol, is yellow in basic solutions. The ap-
pearance and intensity of yellow color thus indicates the degree to which the
substrate has been acted upon by the enzyme. Normalizing the results to
the time and the total protein concentration gave us information about the
 Biocompatibility, Cytotoxicity and Bioactivity 61

specific activity of the ALP in each of the surfaces. The total protein con-
tent of the lysates was determined using a commercially available kit (Macro
BCA, Pierce Chemical Co.). The ALP activity is reported as nanomoles of
p-nitrophenol produced per minute normalized to the milligrams of total
protein content (nmol min−1 mg protein−1 ). The samples were produced in
triplicate and the measurements repeated at least three times, giving a total
of nine data values for each incubation period.

Western Blot Analysis of Bone Sialoprotein

Similarly, the HABDCs were plated on surfaces in triplicate at an initial den-

sity of 1 · 104 /well in a culture medium in 24-well culture plates; control cells
were cultured directly on tissue culture polystyrene. The cells were cultured
for 7 and 14 d. Western blots allowed us to determine the molecular weight of
the protein and to measure the relative amounts of the protein present in the
different samples. The proteins were separated by gel electrophoresis. The
proteins were transferred to a sheet of special blotting paper called nitrocel-
lulose. The proteins retained the same pattern of separation they had on the
gel. The blot was incubated with a generic protein (such as milk proteins)
to bind to any remaining sticky places on the nitrocellulose. An antibody
was then added to the solution which is able to bind to its specific protein.
The antibody has an enzyme (e.g., alkaline phosphatase or horseradish per-
oxidase) or dye attached to it that cannot be seen at this time. The location
of the antibody is revealed by incubating it with a colorless substrate that
the attached enzyme converts to a colored product that can be seen and
photographed. The bandwidth is proportional to the quantity of the protein,
so the analysis gives us a qualitative assessment of the protein content. The
relative level of the protein was assessed by measuring the integrated density
of all pixels in each band, excluding the local background and normalizing to
the area of the reference. The results are expressed as intensity per mm2 .

Bone Sialoprotein Inmunofluorescence

For this test, the cells were culture for 14 and 21 d in the culture medium.
After the incubation period, cells were fixed using 4% paraformaldehyde for
15 min. After washing with PBS they were processed for immunofluorescence:
cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min followed
by blocking with 1% ovalbumin in PBS for 30 min. A primary monoclonal
antibody to BSP was used, followed by corresponding Alexa Fluor 488 (green
fluorescence) conjugated goat secondary antibody. The samples were exam-
ined by epifluorescence under a conventional fluorescence microscope.
62 Sandra E. Rodil et al.

Fig. 1. (a) Absorption coefficient determined by three different techniques: analy-

sis of reflectance and transmittance spectra, photothermal deflexion spectroscopy
and ellipsometry spectroscopy. (b) Refractive index and extinction coefficient as a
function of energy

3 Results
3.1 Film Properties

Because of the diversity of properties that amorphous carbon films exhibit,

it is very important to perform as complete as possible a characterization of
the type of film used. The results of this evaluation for the present study are
described below. The average surface roughness remained nearly constant at
2 µm after coating the substrate. In a previous paper, we reported the energy
loss spectra of amorphous carbon films [38] deposited by magnetron sputter-
ing and demonstrated that the films are nearly 100% sp2 bonded, although
a quantitative sp2/sp3 fraction could not be obtained since the π peak was
 Biocompatibility, Cytotoxicity and Bioactivity 63

Fig. 2. Visible Raman spectra. Results of the decomposition using two Gaussian
peaks for G and D

too wide, indicating that more than one configuration was present and thus
impeding the area calculation [39]. It is well known that the optical and elec-
trical properties of amorphous carbon depend not only on the quantity of
sp2 bonds but also on their distribution or configuration. The optical mea-
surements showed that the films were highly absorbing for energies above
1 eV, having a Tauc optical gap of about 1 eV. The absorption coefficient
as a function of the energy, as determined by three independent techniques,
is shown in Fig. 1a. The E04 falls below the measured range, and therefore
we only report the√Tauc gap that results from an extrapolation to zero of
the line fitting the α × energy vs. energy spectra in the high energy range.
Typical data of the refractive index and extinction coefficient are shown in
Fig. 1b. The existence of the small gap is in agreement with a small degree of
clustering of the sp2 phase detected by the analysis of the Raman spectrum,
shown in Fig. 2 together with the results of the decomposition of the Raman
spectra into two Gaussians: D and G peaks. The XPS analysis demonstrated
that the surface composition consisted predominantly of carbon, and oxygen
as a surface contaminant (Fig. 3). We checked for the presence of metal atoms
from the substrate or the Ti coating but no signal was found, indicating that
the a-C film completely and uniformly covers the substrate.
According to this characterization we can conclude that the films were uni-
formly deposited on the rough stainless steel substrates, copying the surface
topography. The physical and structural properties, optical gap, refractive
index and bonding characteristics of the films suggest that the films used for
this investigation are graphite-like amorphous carbon films.
64 Sandra E. Rodil et al.

Fig. 3. XPS spectra of the sample, general view

3.2 Cytotoxicity

In vitro methods provide a necessary and useful adjunct to “in vivo” studies
for selecting materials as candidates for implants or biomaterials. This study
includes two stages; the first, cytotoxicity tests, is carried out to detect any
toxic effects of the potential materials. The second stage allows a more thor-
ough evaluation of the biocompatibility of the material in relation to its end
use, in our case the biomineralization tests. The cytotoxicity of amorphous
carbon films has been tested with different cell lines, and in general it is cat-
alogued as a biocompatible material. However, considering the differences in
film properties and the importance to test the material using cells specific
for the proposed application of the medical device, we decided to evaluate
the cytotoxicity using primary human osteoblasts on the sputtered a-C films.
Toxicity in vitro is a negative or deleterious effect that induces changes in the
cellular response, such as cell death, reduced cell adhesion and proliferation,
altered cellular morphology, and inhibition of biosynthetic functions. All of
these processes were evaluated. Cell death was not observed during the pe-
riod of evaluation up to 7 d. The cellular adhesion, on the other hand, which
is very important for this specific type of cells, was actually improved by the
films in comparison to either the stainless steel substrate, the Ti coating and
the positive control. This is observed in Fig. 4, in which we plot the number
of attached cells after 24 h on each surface. The a-C had the highest number
of attached cells compared to all other materials (ANOVA p < 0.05). Cell
proliferation was evaluated for periods up to 7 d. Figure 5 shows that the
number of cells increased as a function of the incubation time for all sur-
faces. The number of cells on the a-C films doubled at three d of incubation,
while the doubling time for the Ti films was about 7 d. On the stainless
steel the number of cells remained constant at about 1.2 · 104 even after 7 d.
 Biocompatibility, Cytotoxicity and Bioactivity 65

These results were further confirmed by the cell viability assay (MTT test),
which is very important since the combined proliferation/metabolic test is a
way of providing information about the cell growth and metabolic activity
of the cells. The results of the MTT assay (Fig. 6) are presented as the opti-
cal absorbance at 570 nm. We found high levels of MTT conversion (higher
absorbance, high metabolic activity), compare to the control, for Ti and a-
C films, and this increased with time. This increment reflects the increased
number of cells on the surfaces and therefore confirms that there are a greater
number of both dividing and metabolically active cells. In accordance to the
lower proliferation rate measured for the SS substrate, the enzymatic activity
of the osteoblasts cells on SS is lower. Finally, scanning electron microscopy
allowed the observation of the cell morphology. Osteoblasts with dorsal ruf-
fles close to each other connected by filopodia, attached and spread on the
substrate were observed during the different incubation times (not shown).
The results of the cytotoxicity tests suggested that the amorphous carbon
and titanium coatings have no negative affect on cell adhesion, viability and
proliferation. The cell number and absorbance values obtained for a-C and Ti
resulted significantly higher with respect to the positive control and the SS
substrate, indicating the absence of toxic effects. Morphologically, the cells
were in good condition and well attached to the surfaces.

3.3 Bioactivity
3.3.1 Morphological Analysis
Secondary electron image examination at 14 and 21 d of cultured osteoblasts
on the surfaces revealed a continuous cell multilayer and an extracellular
matrix that completely covered the surface. Figure 7 shows the images at
different magnifications for the a-C surfaces at 14 and 21 d. Figure 8 shows
corresponding images for SS and Ti coatings. The extracellular matrix was
composed of both noncollagenous and collagenous components, with small
(< 1µm) spherical structures or mineral deposits. These mineral deposits
were seen interspersed among the osteoblast cell layer or within the fibrillar
matrix. The images are similar to those reported in other works [40, 41, 42],
although the incubation periods used in our work are relatively shorter. By
comparing Figs. 7 and 8 we can observe that similar results were obtained
for the SS substrate, a-C and Ti coatings. Therefore, by simple morpholog-
ical analysis of the mineralization process it is impossible to determine any
substrate-dependent effect, emphasizing the importance of making quantita-
tive measurements, such as those obtained by the analysis of specific proteins.

3.3.2 Proteins
Chemical reactions in living organisms occur rapidly at moderate tempera-
tures and under mild conditions primarily because of the catalytic action of
66 Sandra E. Rodil et al.

Fig. 4. Number of attached cells after 24 h of incubation for the SS substrate, a-C
and Ti coatings and the plastic culture dish

a-C
24000
SS
Ti
Control
Number of Cells

20000

16000

12000

8000
0 1 2 3 4 5 6 7
Days
Fig. 5. Osteoblast proliferation for 1, 3 and 7 d of incubation. The initial plated
cells was 1 · 104 per surface

specialized proteins called enzymes. Each step in the chain of a biochemical

reaction is usually catalyzed by a specific enzyme. A large number of proteins
are involved in preparing the matrix for mineralization: phosphatases that
regulate extracellular phosphate concentrations, kinases that regulate matrix
protein phosphorylation and metalloproteinases that degrade the extracellu-
lar matrix. In order to mineralize, osteoblasts express these compounds into
the extracellular matrix, specifically alkaline phosphatase, bone sialoprotein,
osteocalcin and osteopontin. The rate of an enzyme-catalyzed reaction may
be measured by (a) the disappearance of substrate, which is the substance
acted upon by the enzyme and changed to the product, or (b) the appear-
 Biocompatibility, Cytotoxicity and Bioactivity 67
2.0
a-C
1.8 SS
Ti
Absorbance at 570 nm

1.6 Control

1.4

1.2

1.0

0.8

0.6
3 4 5 6 7
Days
Fig. 6. Cell viability (MTT test) expressed as the absorbance at 570 nm

ance of the product. The ALP activity was measured by the product quan-
tity as described above. The levels of this enzyme in the various surfaces
are shown in Fig. 9, as a function of the incubation time. ALP plays a vital
but yet undefined role in bone mineralization. It is thought to regulate phos-
phate transport. The enzyme is glycosylated and attached to the cytoplasmic
membrane on its external surface, where it interacts with extracellular ma-
trix proteins. The results showed that all the cultures presented a significant
induction of the ALP activity, which is in some way expected since we use os-
teoblasts, which produce high ALP concentrations. However, the activity was
significantly higher for the amorphous carbon coatings compared to the other
surfaces (ANOVA p < 0.05). An increase in the activity and expression of
ALP is a strong marker of both the osteoblast phenotype and mineralization.
The other bone-related protein investigated in this work was bone sialo-
protein (BSP), a phosphorylated glycoprotein. BSP is a major structural pro-
tein of the bone matrix that is specifically expressed by fully differentiated
osteoblasts. The expression of BSP is normally restricted to mineralized con-
nective tissue of bones and teeth, where it has been associated with mineral
crystal formation and is considered a potent nucleator of hydroxyapatite.
Western blot analysis was performed to determine the expression of bone
sialoprotein on the surfaces. Semiquantitative information about the level
of BSP expression was obtained by densitometric studies, and both images
are shown in Fig. 10. BSP was expressed on all surfaces after 7 and 14 d
of culture. For a-C, SS and Ti we observed a progressive increase in BSP
expression with time, while in the tissue plastic control, the level decreased.
Remarkably, the level of BSP expression was much higher on a-C than in any
other surface.
Because of the difficulty in analyzing the samples to obtain the compo-
sition of the mineralized matrix, particularly of the small spherical nodules,
68 Sandra E. Rodil et al.

Fig. 7. SEM images of the mineralized matrix in the amorphous carbon surfaces
after A, B 14 d. C and D 21 d. Bar: A 2µm, B 10µm, C 2µm, D 30µm
 Biocompatibility, Cytotoxicity and Bioactivity 69

Fig. 8. SEM images of the mineralized matrix in the SS substrate after A 14 d, B

21 d. SEM images of the mineralized matrix in the Ti coatings after C 14 d and D
21 d. Bar: A, 10 µm; B, 2 µm; C, 30 µm; D, 2 µm
70 Sandra E. Rodil et al.

Fig. 9. Alkaline phosphatase (ALP) activity

Fig. 10. Western blot analysis of bone sialoprotein (top) and densitometric analysis
of the bands, showing the intensity per µm2 (bottom)
 Biocompatibility, Cytotoxicity and Bioactivity 71

we determined the distribution of bone sialoprotein on the surfaces by im-

munofluorescence labeling. This technique allowed us to confirm that the
small nodules shown in the SEM images corresponded to mineral deposits
containing high concentrations of BSP. Figure 11 shows the different images
of the BSP distribution on the a-C coating, and similar images were obtained
for the other surfaces (not shown). An important proportion of the cells were
immunoreactive to BSP for all the time periods (14 and 21 d). BSP was
present in both the cell layer and the extracellular matrix. Active osteoblasts
contain BSP in a large juxtanuclear mass reminescent of the Golgi region, so
the underlying cellular layer was clearly seen. However, fluorescence intensity
was stronger in the spherical nodules that appeared at 14 d of incubation
and grew into big mineral deposits, forming large mineralized layers, as those
observed in Figs. 9d and 7a by SEM.

4 Discussion
The present study describes an in vitro model to study the response of human
osteoblasts to well-characterized a-C coatings in comparison with titanium
coatings and the stainless steel substrates. We performed both short- and
medium-term studies to determine the cytotoxicity and the biomineraliza-
tion of the different surfaces. We observed an enhanced cellular adhesion and
proliferation/viability on the a-C coatings, demonstrating the biocompatibil-
ity of the material. Moreover, we demonstrated the formation of a calcified
matrix with bone sialoprotein and alkaline phosphatase being expressed.
By comparison between the different surfaces, we showed that osteoblast
cells respond differently to biomaterial in both short- and medium-term cul-
tures. The SEM examination of the osteoblast cultures in the presence of
β-glycerophosphate, dexamethasone and ascorbate revealed the formation of
a stratified matrix mixed with cell layers and the mineralized nodules. The
ALP activity and Western blot were performed to determine the time de-
velopment and levels of two important proteins, ALP and BSP, associated
with the mineralization process. Our results showed different responses to the
biomaterial, and in both cases the amount of protein expressed on the a-C
surfaces was larger, suggesting that the film properties promote the differen-
tiation and mineralization of human osteoblast.
A good correlation between proliferation and mineralization results was
obtained. It is well established that proliferation stops when the mineraliza-
tion process is initiated [43] and that mineralization initiates once the cells
have formed a certain amount of extracellular matrix. This is clearly reflected
in our results. Figure 4 shows that cell proliferation attained a saturation level
at about 7 d, after which the ALP activity increased dramatically (Fig. 9).
This was important to establish the moment at which the mineralization
medium should be added to the culture, since once the culture medium is
72 Sandra E. Rodil et al.

Fig. 11. Distribution of the bone sialoprotein on the amorphous carbon surfaces
after A, B 14 d. C, D 14 d. A, C and B 10X; D 20X
 Biocompatibility, Cytotoxicity and Bioactivity 73

rich in phosphate ions, mineralization will occur, but without the appropri-
ate matrix, the mineralization obtained is dystrophic and does not necessarily
resemble the bone formation in vivo.
Human osteoblast cultures may be regarded as a potential in vitro model
to study biomaterial/bone tissue interactions. However, in order to deter-
mine the differences between biomaterial-induced responses, morphological
analysis of the cell layers grown on the surface is not sufficient. If possible,
structural characterization of the mineral deposits must be performed, but
standard immunofluorescence or staining techniques are more appropriate to
quantitatively compare the response of the osteogenic cells to the different
materials.
In conclusion, we have shown that graphite-like amorphous carbon is a
potential candidate for further research as a bioactive coating to induce bone
formation. Many issues must still be investigated, for example, film proper-
ties, and more important, film–substrate adhesion must be improve to the
point that no failure is possible in long-term applications. Mechanical tests
associated with the torque resistance that the coating should support during
the insertion of the implant into the bone must be performed. Similarly, other
biological tests, such as genotoxicity and in vitro bone growth, need to be
carried out.

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Index
a-C, 55 G band, 63
sp3 /sp2 bonding ratio, 62
haemocompatibility, 56, 57
alkaline phosphatase (ALP), 58–61, 66, hardness, 56, 57
67, 70, 71
mineralization, 57, 60, 64–67, 71
bioactivity, 55, 58, 60, 65, 73 MTT test, 65, 67
biocompatibility, 55–58, 64, 71
biomaterials, 57, 64, 73 optical properties, 59
biomedical implants, 55–57, 64, 73
bone sialoprotein (BSP), 57, 58, 61, 66, proliferation, 57, 59, 64, 65, 71
67, 70–72
Raman spectroscopy, 63
coatings, 56–58, 67, 71–73
refractive index, 63
bioactive coatings, 55
biomedical coatings, 57
scanning electron microscopy (SEM),
cytotoxicity, 58, 59, 64, 65, 71
60, 65, 68, 69, 71
D band, 63
diamond-like carbon (DLC), 56, 57 tribological properties, 56

electrical properties, 63 viability, 58, 59, 65, 71

friction coefficient, 56, 57 wear resistance, 56, 57