Carbon 44 (2006) 1106–1111
www.elsevier.com/locate/carbon
In vitro studies of carbon nanotubes biocompatibility
J. Chłopek
a
b
a,*
, B. Czajkowska b, B. Szaraniec a, E. Frackowiak c,
K. Szostak c,d, F. Béguin d
AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Biomaterials,
al. Mickiewicza 30, 30-059 Krakow, Poland
Collegium Medicum of the Jagiellonian University, Department of Immunology, ul. Czysta 18, 31-121 Krakow, Poland
c
Poznań University of Technology, ul. Piotrowo 3, 60-965 Poznań, Poland
d
CRMD, CNRS-University, 1B rue de la Férollerie, 45071 Orléans Cedex 02, France
Received 17 November 2005; accepted 22 November 2005
Available online 6 January 2006
Abstract
Cellular tests have been applied to study the biocompatibility of high purity multiwalled carbon nanotubes (MWNTs). The viability
of fibroblasts, osteoblasts and osteocalcin concentrations in osteoblasts cultures in the presence of nanotubes has been examined, as well
as the degree of cells stimulation, based on the amount of released collagen type I, IL-6 and oxygen free radicals. The high level of viability of the examined cells in contact with the nanotubes, the slight increase of collagen formation, the lack of pro-inflammatory IL-6
cytokine as well as the induction of free radicals, confirm a good biocompatibility of nanotubes, which is similar to that of polysulfone
currently used in medicine. The collagen synthesis induced on nanotubes by both fibroblasts and osteoblasts may be significant for future
medical applications of nanotubes, in particular as substrates for the regeneration of tissues.
2005 Elsevier Ltd. All rights reserved.
Keywords: Carbon nanotubes; Biocompatibility
1. Introduction
Carbon nanotubes, due to their specific structure/texture and properties, may play a significant role in the development of carbon materials for medicine, the main body of
which includes pyrocarbons, glassy carbon, carbon fibers,
carbon–carbon composites, and diamond-like layers [1–
4]. The medical applications of these materials are determined by the following properties: biocompatibility in
contact with blood, bone, cartilage and soft tissues;
biofunctionality understood as the ability of taking over
certain functions of tissues by a mutual adjustment of
implants and tissues properties [5–7]. Fields of current
applications of carbon biomaterials are given in Table 1.
*
Corresponding author. Fax: +48 12 633 15 93.
E-mail address: chlopek@uci.agh.edu.pl (J. Chłopek).
0008-6223/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbon.2005.11.022
The electronic structure and the surface morphology of
carbon nanotubes, which probably determine their biocompatibility, are typical of graphite-like structures [11].
They can be distinguished by a tubular construction in
the nanometer range and by high strength and Young
modulus [12]. The combination of these properties may
open new fields of application, including those in medicine.
Like other fibrous materials, nanotubes can be used as substrates for the regeneration of tissue. Due to their high electrical conductivity, the latter process can be additionally
assisted by electrostimulation during the cell cultures and
the tissue formation [13].
Two forms of carbon nanotubes can exist: single-wall
(SWNTs) and multiwalled (MWNTs) [14,15]. They can
be open-ended after specific chemical treatments [16], or
may have closed tips. In the latter case they may be used
as biosensors [17]. The accessible canals of open-ended
nanotubes may facilitate the migration of metabolites or
J. Chłopek et al. / Carbon 44 (2006) 1106–1111
1107
Table 1
Examples of applications of carbon biomaterials [8–10]
Type of material
Function
Type of implant
Area of medicine
Carbon–carbon composites
Braided carbon fibers
Bone fixation
Tissue knitting, reconstruction of joint
ligaments and tendons
Filling bone and cartilage losses
Coating of metal implants—corrosion
protection
Blood flow regulation
Screws, plates, nails, stems of endoprosthesis
Surgical sutures, ligament and tendon’s prosthesis
Bone surgery
Orthopedics
Disks and rings
Joint endoprosthesis, screws
Bone surgery
Bone surgery
Heart valves
Cardiology
Unwoven carbon fabric
Coatings of diamond-like
carbon (DLC)
Glassy carbon
growth agents, and may also be used as a drug carrier [18].
Very good mechanical properties, the possibility of surface
machining as well as the ability to form functional groups
constitute good fundaments for the use of nanotubes in the
fabrication of composite materials.
The possibility of forming composites with polymer
matrices, both biostable (polysulfone, PEEK), and bioresorbable (PLA, PGLA, co-polymers) is of particular
importance in the case of medical applications [19–21].
This opens opportunities for the manufacture of multifunctional implants useful in many areas of medicine. However,
with resorbable polymers, the relation between the resorption time and the time of tissue healing is of significant
importance. A too high resorption rate may lead to a
release of nanotubes from the composite materials into
the living body.
In the case of the ceramic matrix composites, an
improvement of the fracture toughness can be expected,
which may be particularly important in the case of manufacturing reinforced nanostructure ceramics. The nanocomposite system of nanotubes reinforced hydroxyapatite
[22] may be included in this category.
The use of the advantages of nanotubes in medical
applications, particularly their tubular morphology and
their excellent electrical and mechanical properties relies
heavily on their biocompatibility. Although both the nature of carbon and positive experiences to date with various
forms of carbon would suggest also a good biocompatibility of nanotubes, basic cellular tests must be performed in
order to allow projects and application works to be opened
in medicine. In the present work, the viability of fibroblasts, osteoblasts and osteocalcin concentrations in osteoblasts cultures in the presence of high purity multiwalled
carbon nanotubes (MWNTs) has been examined, as well
as the degree of cells stimulation, based on the amount of
released collagen type I, IL-6 and oxygen free radicals.
MWNTs, free of any disordered carbon, that makes the
purification very easy by simple dissolution of the catalyst
precursor in HCl. The elemental analysis on the purified
material gives: C = 96 wt%, H = 0.85 wt%, Mg = 200 ppm
and Co = 2 wt%. The low magnesium and cobalt content
shows that the treatment in hydrochloric acid is very
efficient.
The structure and texture of the purified carbon nanotubes was controlled by scanning electron microscopy
(SEM, Hitachi S 4200) and transmission electron microscopy (TEM, Philips CM 20). For the TEM observations,
the samples were ground in ethanol and dropped over a
copper holey grid covered by amorphous carbon. The
SEM picture presented in Fig. 1 shows a very dense and
entangled network of nanotubes, where graphitic particles,
disordered carbon and nanocapsules are completely absent.
The TEM observation (Fig. 2) demonstrates that this
material consists exclusively of multiwalled nanotubes.
The high-resolution images show that the central canal is
quite well defined (from 2 to 5 nm in diameter) and the
walls consist of an average number of 10–15 continuous
layers oriented in parallel to the tube axis. Most of the carbon nanotubes have closed tips and for only few of them
cobalt particles are encapsulated near the tip or inside the
canal. The histogram of the outer diameters (Fig. 3) shows
a good calibration of the carbon nanotubes, with outer
diameters mostly in the range from 10 to 15 nm. The histogram of the inner diameters (diameters of the central
canal), ranges mainly in a narrow range from 2 to 5 nm.
2. Experimental
2.1. Preparation and characterization of the nanotubes
In our experiments, we used high purity multiwalled carbon nanotubes (MWNTs) prepared by catalytic decomposition of acetylene on a CoO/MgO solid solution catalyst,
according to the process described in Refs. [23,24]. This
process allows a large scale and selective production of
Fig. 1. SEM micrograph of the purified nanotubes produced by the
catalytic decomposition of acetylene at 600 C over the CoO/MgO solid
solution.
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J. Chłopek et al. / Carbon 44 (2006) 1106–1111
with nanotubes. The level of secreted collagen type I,
IL-6 and osteocalcine was also defined. The degree of
macrophages activation to secrete free radicals under the
influence of nanotubes was determined indirectly. The
following cellular lines, from American Type Culture
Collection (ATCC), and the media recommended by
ATCC were used:
Fig. 2. TEM image of the purified nanotubes produced by the catalytic
decomposition of acetylene at 600 C over the CoO/MgO solid solution.
Fig. 3. Histogram of the outer diameters of the purified carbon nanotubes
produced by the decomposition of acetylene at 600 C over the CoO/MgO
solid solution.
Even if MWNTs with outer diameters up to 30 nm were
observed during the TEM observations, it can be concluded that the inner diameter always remains in the same
range, being probably more strictly controlled by the catalyst particle size.
The above described nanotubes together with polysulfone PSU (C27H26O6S)n from Aldrich (molecular mass
M = 26,000, glassy transition Tg = 190 C, density
d = 1.24 g/cm3) have been used for the samples preparation. The choice of PSU resulted from its good biocompatibility (this polymer is currently used in medicine) and the
necessity to manufacture a composite material, in which
the nanotubes can modify the mechanical and biological
properties. Polysulfone was dispersed in methylene chloride (CH2Cl2), poured onto a Petri platter and then covered
with nanotubes. After solvent evaporation, a thin polymer
film containing the nanotubes with a thickness of about
1.8 lm was obtained. A pure polysulfone film was used
as a reference sample.
2.2. Cellular tests
The cellular viability was determined after 24 h, 48 h
and 7 day cultures in order to get an initial evaluation of
the biocompatibility of pure polysulfone and polysulfone
• Human osteoblastic line hFOB 1.19 ATCC CRL-11372
in 1:1 mixture of Ham’s F12 medium and Dulbecco’s
modified Eagle’s medium with 2.5 · 10 3 mol l 1 L-glutamine and 0.3 mg/ml G418 with 10% of foetal bovine
serum.
• Human fibroblastic line HS-5 ATCC CRL-11882 in
RPMI with 15% bovine serum.
The cell cultures were run in 12-well plates at the bottom
of which the samples of polysulfone and polysulfone with
nanotubes were placed, and 2 cm3 of the cells suspension
in the culture medium were added. The cultures were run
in an incubator under 5% CO2/95% air atmosphere at
37 C (fibroblasts) or 34 C (osteoblasts) over 7 days. After
24 h, 48 h and 7 days, the viability of the cells was
examined by Cell Titer 96 Aqueous One Solution Cell
Proliferation Assay (Promega) [25]. The main reagent in
this method contains a tetrazolium compound (MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolim, inert salt) and an electron
coupling reagent (phenazin ethosulfate; PES). The MTS
tetrazolium compound is bioreduced by the cells into a coloured formazan product which is soluble in the tissue culture medium. The quantity of formazan formed, measured
by the absorbance at 490 nm, is directly proportional to the
number of living cells in the culture. The results have been
presented in percentage, adopting 100% of transmittance
for reference cells, i.e. cultures without materials.
The level of secreted collagen I (the protein constituting
the building material of connective tissue as well as bones,
skin and vessel walls) was determined together with the
level of IL-6 cytokine (contributing in inflammation reactions) in the supernatant liquids over 7 day cultures of osteoblasts and fibroblasts. The ELISA (Enzyme-Linked
Immunoabsorbent Assay) test was used with bioproducts
and endogen reagents. In the case of 7-day cultures of osteoblasts, the level of osteocalcin, a protein specific of these
cells contributing mainly in bone rearrangement, was determined in the supernatant liquids. The ELISA test was used,
with the DSL10-7600 Active Human Osteocalcin Enzyme
Linked Immunosorbant Assay Kit, produced by Diagnostic System Laboratories.
The chemiluminescence of mouse peritoneum macrophages in RPMI with 15% foetal bovine serum was
examined using a luminometer Lucy 1 (Anthos, Salzburg,
Austria), in order to check if the tested nanotubes activate the macrophages to produce free radicals, which could
prove their pathogenic character. 50 lg of nanotubes, 50 lg
of carbon particles (carbonized phenol–formaldehyde
J. Chłopek et al. / Carbon 44 (2006) 1106–1111
1109
resin) and opsonized with the mouse serum—zymosan—
were added to the cells (at a concentration of 5 · 105),
and photon emission was measured over a period of
60 min.
3. Results and discussion
The examination of fibroblasts and osteoblasts viability
on polysulfone films covered with nanotubes indicates a
small decrease of the viability of all the examined cells, as
compared to the viability obtained on pure polysulfone
films (Fig. 4). This decrease may be related to the nature
of the substance itself, as well as to its surface state. In
the case of other carbon materials, the effect of their surface
roughness on the cells viability was found to be of importance [26]. The same effect can be expected for the case of
Fig. 4. Viability of fibroblasts and osteoblasts in contact with PSU and
PSU + nanotubes: ((a) fibroblasts and (b) osteoblasts). The results
represent the average ± SD of duplicates from 8 different experiments.
Fig. 6. Production of collagen on PSU and PSU + nanotubes. The results
represent the average ± SD of duplicates from 8 different experiments.
polysulfone containing nanotubes. The profilographic tests
presented in Fig. 5 confirm a higher degree of roughness for
the samples covered with nanotubes. A similar good cellular behaviour of carbon nanofibers has already been
observed [27,28].
The amount of collagen type I formed is slightly higher
on PSU surfaces covered with nanotubes than on pure
PSU, as particularly shown in Fig. 6 for fibroblasts and
osteoblasts. These results are very encouraging for the
application of nanotubes in tissue engineering. A more
intensive collagen synthesis may lead to the regeneration
of soft and bone tissues, where nanotubes may constitute
an excellent substrate for their growth.
The level of pro-inflammatory cytokine IL-6 has been
also determined in the supernatants over the fibroblast cultures of the examined materials. The IL-6 can be characterized by multidirectional influences and is considered as one
of the major factors regulating the defensive mechanisms.
Its main role is to participate in the immune response,
the blood formation and the inflammatory reactions, since
it is the main stimulator for the generation of the acute
phase proteins by liver. The IL-6 is generated mainly by
macrophages, monocytes, and also by fibroblasts, endothelium cells and lymphocytes. An increased concentration of
IL-6 in macrophages and fibroblasts cultures would point
out a same level of stimulation of these cells as in the
50
pg/ml
40
30
20
10
0
Fb
Fig. 5. Profilograms of PSU (a) and PSU + nanotubes (b). (Ra—Arithmetic mean roughness value.)
PSU
PSU+nanotubes
Fig. 7. IL-6 release from fibroblasts cultured on PSU and PSU + nanotubes. The results represent the average ± SD of duplicates from 4
different experiments.
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J. Chłopek et al. / Carbon 44 (2006) 1106–1111
and osteoblasts may be significant for future medical applications of nanotubes, in particular as substrates for the tissue regeneration.
The detrimental effects of a possible release of nanoparticles into the human body should be investigated in the
future.
References
Fig. 8. Osteocalcin concentrations in osteoblasts culture. The results
represent the average ± SD of duplicates from 4 different experiments.
Fig. 9. Chemiluminescence of macrophages (Mf) stimulated with zymosan, carbon particles and nanotubes.
inflammatory reaction [29]. In fact, Fig. 7 shows that there
is not any induction of pro-inflammatory IL-6 by PSU and
PSU with nanotubes.
The same effect was observed for osteocalcin released
from osteoblasts (Fig. 8). The presence of nanotubes does
not affect the osteocalcin release.
The amount of free radicals and the degree of cells stimulation has been measured by the chemiluminescence
method. The aim of this test was to find out whether the
macrophages might undergo activation and release free
radicals in contact only with carbon nanotubes, or with
carbon particles of size similar to nanotubes. As shown
in Fig. 9, neither the nanotubes, nor the carbon particles
of similar dimension which were obtained by carbonization
of phenol–formaldehyde resin, can activate the macrophages to release free radicals, which under specific circumstances could be toxic for both the surrounding cells and
tissues.
4. Conclusion
The cellular tests performed in this study confirm a good
biocompatibility of nanotubes, which is similar to that of
polysulfone currently used in medicine. The high level of
viability of the examined cells in contact with the nanotubes, the unchanged level of osteocalcin released from
osteoblasts, the lack of pro-inflammatory IL-6 cytokine
as well as free radicals induction, point out a good cellular
biocompatibility of nanotubes. The slight increase of collagen formation induced on nanotubes by both fibroblasts
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