solidi

status

pss

physica

phys. stat. sol. (a) 205, No. 9, 2146 2153 (2008) / DOI 10.1002/pssa.200879729

www.pss-a.com

applications and materials science

Improved adhesion,
growth and maturation of human bone-derived cells
on nanocrystalline diamond films
Milan Kopecek**, 1, Lucie Bacakova*, 1, Jiri Vacik***, 2, Frantisek Fendrych****, 3, Vladimir Vorlicek3,
Irena Kratochvilova3, Vera Lisa1, Emilie Van Hove4, Christine Mer4, Philippe Bergonzo4,
and Milos Nesladek*****, 3, 4
1
2
3
4

Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic
Nuclear Physics Institute, Academy of Sciences of the Czech Republic, 25068 Husinec Rez, Czech Republic
Institute of Physics, Academy of Sciences of the Czech Republic, Na Slovance 2, 18221 Prague 8, Czech Republic
CEA-LIST, Centre dEtudes Saclay, Bat. 451, p. 84, 91191 Gif Sur Yvette, France

Received 16 April 2008, revised 28 July 2008, accepted 29 July 2008

Published online 25 August 2008
PACS 78.67.Bf, 81.05.Uw, 87.17.Ee, 87.17.Rt, 87.85.jj, 87.85.Lf
*****

Corresponding author: e-mail lucy@biomed.cas.cz, Phone: +420 2 9644 3743, Fax: +420 2 9644 2488
e-mail kopecek@biomed.cas.cz, Phone: +420 739 589 951, Fax: +420 2 9644 2488
*****
e-mail vacik@ujf.cas.cz, Phone: +420 266 173 129, Fax : +420 220 941 130
*****
e-mail fendrych@fzu.cz, Phone: +420 266 053 111, Fax: +420 286 890 527
*****
e-mail Milos.NESLADEK@cea.fr, Phone: +331 69088704, Fax: +331 69087679
*****

Nanocrystalline diamond (NCD) films were deposited on microscopic glass slides using the MW PECVD method (20
Torr, 710 C, 0.8 1% CH4). After the growth period, the
films were oxidized and subsequently hydrogenated, and
some of them were doped with boron (NCD-B; 3,000
30,000 ppm B : C; leading to ~ 101 cm for the highest
doped films). The neutron depth profiling showed that in the
near surface region (< 800 nm) the boron (10B + 11B) content
in the highest doped sample was about (1.9 0.3) 1021 B
cm3 (i.e., 1.1 0.2 at% of B). The films were seeded
with human osteoblast-like MG 63 cells (~ 17,000 cells/cm2).
On day 3 after seeding, the cell number on NCD
(56,280 1,090 cells/cm2) was significantly higher than that
on NCD-B (by 27 3%), glass slides (by 22 3%) and poly-

styrene wells (by 36 3%). On day 7, the cell numbers on

both NCD and NCD-B films (351,170 16,530 cells/cm2 and
310,020 10,410 cells/cm2, respectively) became significantly higher than the values on glass slides and polystyrene dishes
(218,800 12,340 cells/cm2 and 223,400 9,290 cells/cm2,
respectively). Immunofluorescence staining showed that the
cells on both NCD films assembled fine streak- or dot-like
focal adhesion plaques containing alphav integrins or talin,
and a mesh-like beta-actin cytoskeleton. The cells on NCD-B
were brightly stained for osteocalcin, an important marker of
osteogenic differentiation. Thus, both tested nanocrystalline
diamond films gave good support for the adhesion, growth
and maturation of bone-derived cells.

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1 Introduction Nanocrystalline diamond (NCD) has

attracted considerable attention as a promising material for
advanced biomedical applications. In the form of a powder,
diamond with grain sizes of ~5 nm, so called ultradispersed
diamond (UDD), can be prepared by detonation synthesis
from a mixture of trinitrotoluene and hexogene [1], and
could serve as a carrier for controlled drug and gene delivery, for advanced imaging technologies or as antioxidant
and anti-inflammatory agents [24]. Thin NCD films can

be deposited using microwave plasma-enhanced chemical

vapor deposition (MW PECVD) from methane hydrogene
mixtures. So called ultrananocrystalline diamond (UNCD)
can be prepared from methane in Ar rich plasmas or
fullerene C60 precursors [1, 5]. NCD films exhibit a wide
range of unique physicochemical properties, such as mechanical hardness, chemical and thermal resistance, excellent optical transparency and controllable electrical properties. Biocompatible NCD films could markedly improve
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Original
Paper
phys. stat. sol. (a) 205, No. 9 (2008)

the mechanical and other physical properties of medical

implants, particularly those designed for hard tissue surgery. When deposited on metallic or polymeric implants,
these films would prevent metal ion migration and polymer
leaching. Nanocrystalline diamond has already been used
for coating the head and cup of artificial joint replacements,
e.g., prostheses of temporomandibular joints [6]. In addition, NCD films could be applied in the bone-anchoring
stems of articular prostheses or other permanent bone implants in order to improve their integration with the surrounding bone tissue. In earlier studies in vitro, performed
by us or other authors, NCD provided excellent substrates
for the adhesion, growth and metabolic activity of osteoblasts, as well as non-osteogenic cell types, such as human cervical carcinoma HeLa cells, rat pheochromocytoma PC12 cells or bovine pulmonary artery endothelial
CPAE cells [2, 711]. Type IIa diamond, microcontactprinted with laminin, also supported adhesion of mouse
cortical neurons and neurite outgrowth [12]. In studies in
vivo, diamond layers, deposited by an MW CVD method
on TiAl6V4 probes implanted into a rabbit femur, showed
very high bonding strength to the metal base and also to
the surrounding bone tissue, without any problems from
corrosion [13]. In addition, NCDs are promising building
blocks for constructing biomarkers, microchips, nanorobots or biosensors [2, 11, 1416].
An interesting issue is that the sensing applications of
NCD can be markedly improved by doping NCD with boron [1719]. In addition, the presence of charged states of
the ionized B acceptors on the diamond film surface may
also influence cell colonization. Therefore, in the present
study, we investigated adhesion, growth and maturation of
human osteoblast-like MG 63 cells in cultures on NCD
layers deposited on microscopic glass slides and doped
with boron. The cells were counted in three time intervals
and the cell population doubling times were calculated.
The size of the cell spreading area was measured, and the
cells were immunocytochemically stained against selected
molecular markers of cell adhesion, spreading and maturation. These markers involved integrin adhesion receptors
with alphav chain, an integrin-associated protein talin,
cytoskeletal protein beta-actin and a non-collagenous calcium-binding extracellular matrix (ECM) glycoprotein osteocalcin, an important marker of osteogenic cell differentiation participating in bone tissue mineralization.
2 Material and methods
2.1 Preparation and characterization of nanocrystalline diamond (NCD) films The NCD films of
the thickness 250 30 nm were deposited using a microwave plasma-enhanced chemical vapour deposition (MW
PECVD) technique on 1 mm thick microscopic glass slides
(Corning 1737) cut into square samples of 1 1 cm, utilizing a lab-made microwave plasma-enhanced chemical vapour deposition (PE-CVD) reactor at CEA in Paris, France,
(enabling application of the high microwave powers, typically 2 kW, at relatively low pressures, i.e., 20 mbar). The
www.pss-a.com

2147

substrate temperature was 710 C, and an atmosphere of

0.81% CH4 in H2 at a total gas pressure of 20 Torr was
used. After the growth period, the films were exposed to
oxygen plasma and subsequently hydrogenated. Some of
the NCD films were doped with boron (NCD-B). The boron doping used trimethylboron (TMB) mixures with H2 in
a concentration of 3,00030,000 ppm B: C leading to
~ 101 cm for the highest doped films [18]. As discussed later the doping ratio of 3,000 ppm led to a B concentration of about a 12 1020 cm3, i.e., this incorporation is about a factor 10 lower than in our previous works
[18], probably due to the TMB-H2 mixture quality.
The physico-chemical properties of the NCD and
NCD-B layers were characterized by Raman spectroscopy.
Micro-Raman spectra were measured at room temperature
( = 514.5 nm, Ramascope, Model 1000, Renishaw, GB).
The polarization of the scattered light was not analyzed;
the frequencies of the Raman features observed were taken
directly from the spectra without any fitting. Seven to
twelve sites were examined on each sample.
The content and depth distribution of boron in the subsurface layer was analyzed using neutron depth profiling
(NDP) utilizing the 10B(nth, alpha)7Li exothermal nuclear
reaction induced by thermal neutrons [20].
The topography of the films was investigated by
atomic force microscopy (AFM). The AFM measurements
were performed with NTEGRA Prima NT MDT system
(Ireland) under ambient conditions using the Tapping
Mode. This AFM mode consists of oscillating the cantilever at its resonance frequency (160 or 270 kHz) and lightly
tapping the tip on the surface during scanning. The laser
deflection method is used to detect the RMS (Root-MeanSquare) amplitude of cantilever oscillation. Analysis of AR
(Average Roughness) and AH (Average Height) was also
performed on 8 sites of 5 m 5 m for each sample.
The surface wettability of the films was determined
from the contact angle measured by a static method in a
lab-made material-water droplet system using a reflection
goniometer.
2.2 Cells and culture conditions The samples
were sterilized with 70% ethanol for 1 hour, inserted into
24-well polystyrene multidishes (TPP, Switzerland;
diameter 15 mm) and seeded with human osteoblast-like
MG 63 cells (European Collection of Cell Cultures, Salisbury, UK). Each dish contained 30,000 cells (i.e.,
16,985 cells/cm2) and 1.5 ml of Dulbeccos modified
Eagles Minimum Essential Medium (DMEM; Sigma,
USA, Cat. No D5648) supplemented with 10% foetal bovine serum (FBS; Sebak GmbH, Aidenbach, Germany) and
gentamicin (40 g/ml, LEK, Ljubljana, Slovenia). The
cells were cultured for 1, 3 and 7 days at 37 C in a humidified air atmosphere containing 5% of CO2.
2.3 Evaluation of cell number and spreading On
days 1 and 3 after seeding, the cells were rinsed with
phosphate-buffered saline (PBS), fixed in 70% cold etha 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

M. Kopecek et al.: Improved adhesion, growth and maturation of human bone-derived cells

nol (20 C, 10 minutes) and stained with a combination

of fluorescent cell membrane dye Texas Red C2-maleimide
(Molecular Probes, Invitrogen, Cat. No T6008; 20 ng/ml in
PBS) and nuclear dye Hoechst #33342 (Sigma, USA;
5 g/ml in PBS). The cells were then counted on microphotographs taken under an Olympus IX 50 microscope
equipped with a DP 70 digital camera. On day 1 after seeding, the size of the cell spreading area (i.e., cell area projected on the material) was also measured using the software Atlas (Tescan S.R.O., Brno, Czech Republic). For
each experimental group, in total 32 microphotographs
taken from two samples were used.
On day 7 after seeding, when the cells were confluent
and formed multilayered regions (which disabled counting
on microphotographs), the cells were released from the
cultivation substrates with trypsin-EDTA solution (Sigma,
USA, Cat. No T4174) in PBS and counted in a Brker
haemocytometer. As control substrates, unmodified glass
slides and polystyrene wells were used.
2.4 Growth curves and cell population doubling time The cell numbers obtained on days 1, 3 and 7
after seeding were used for the construction of growth
curves and calculation of cell population doubling time
(DT) using the following equation:
DT = log 2 (t t0)/(log Nt log Nt0)

(1)

where t0 and t represent earlier and later time intervals after

seeding, respectively, and Nt0 and Nt the number of cells at
these intervals.
2.5 Immunofluorescence staining of vintegrins, talin, b-actin and osteocalcin The cells
were rinsed twice in PBS, fixed with cold methanol
(20 C, 5 minutes), pre-treated with 1% bovine serum albumin in PBS containing 0.05% Triton X-100 (Sigma, St.
Louis, MO, USA) for 20 minutes at room temperature, and
incubated with the following primary antibodies: rabbit
anti-integrin v subunit polyclonal antibody (Chemicon International Inc., Temecula, CA, USA; Cat. No. AB1930),
mouse monoclonal anti-human talin (Chemicon International Inc., Cat No. MAB3264), mouse monoclonal antibeta-actin (clone AC-15, Sigma, St. Louis, MO, USA, Cat.
No. A-5441) or rabbit polyclonal anti-human osteocalcin
(Chemicon International Inc., Cat. No. AB1857). The antibodies were diluted in PBS to concentrations of 1:100 to
1:400 and applied overnight at 4 C. After rinsing with
PBS, the secondary antibodies, represented by goat antimouse F(ab)2 fragment of IgG (for samples stained with
the monoclonal antibodies; dilution 1:1000) or goat antirabbit F(ab)2 fragment of IgG (for samples stained with
the polyclonal antibodies; dilution 1:5000) were added for
1 hour at room temperature. Both secondary antibodies
were conjugated with Alexa Fluor 488 and purchased
from Molecular Probes, Eugene, OR, USA (Cat. No.
A11017 and A11070, respectively). After incubation with
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

the secondary antibodies, the cells were rinsed twice in

PBS, mounted under microscopic glass coverslips in a
Gel/Mount permanent fluorescence-preserving aqueous
mounting medium (Biomeda Corporation, Foster City, CA,
USA) and evaluated under an epi-fluorescence microscope
(IX-50, Olympus, Japan) equipped with a digital camera
(DP-70, Olympus, Japan).
2.6 Statistics The quantitative data on the physicochemical properties of the material was presented as mean
S.D. (Standard Deviation) from 7 to 12 measurements.
The quantitative data obtained in the cells was presented as
mean S.E.M. (Standard Error of the Mean) from 9 to 32
measurements. The statistical analyses were performed using SigmaStat (Jandel Corporation). Multiple comparison
procedures were made by the One Way Analysis of Variance (ANOVA), StudentNewmanKeuls method. The p
values equal to or less than 0.05 were considered significant.
3 Results and discussion Representative Raman
spectra of both NCD and NCD-B samples, recorded under
the same conditions, are compared in Fig. 1. The diamond
peak was seen at 1335 and 1330 cm1 in the NCD and
NCD-B sample, respectively, i.e., there was a small upshift (down-shift) with respect to the bulk diamond value
(1332.5 cm1 [21]). The first shift indicates a compressive
strain in the undoped film [21], the second one the incorporation of the boron atoms into the NCD network [22, 23]. It
allows us to estimate the boron doping in NCD-B as [B]
12 1020 cm3 for our lower doped samples (3000 ppm)
based on our calibration and literature data. Indeed, such
estimate is rather crude; nevertheless it is consistent with
the absence of the feature at 1220 cm1 in our spectra,
which is typical of higher doping levels [22, 23] and the
compressive strain induced by the incorporation of the boron
into the diamond lattice.
G
D

Intensity [a.u.]

2148

solidi

status

physica

pss

"NCD"
DIA

B-DOPED

"NCD"
UNDOPED

500

1000

1500

2000

-1

Raman shift [cm ]

Figure 1 Typical Raman spectra of the undoped and B-doped

(3,000 ppm) NCD films. To enable the comparison, the intensity
for the undoped curve was reduced appropriately.
www.pss-a.com

Original
Paper
phys. stat. sol. (a) 205, No. 9 (2008)

2149

In addition, there are the G and (weak) D bands characteristic for the non-diamond (graphite-like) phase and the
features at about 1140 and 1480 cm1 (labelled NCD),
originating in the organic species (transpolyacetylene) at
grain boundaries. Though these features do not belong to
the vibrations of the nanocrystalline network itself, they
always appear in the NCD materials and act as some
NCD fingerprints (cf. [23, 24] and references therein).
The intensity of these features is considerably enhanced by
the boron doping (cf. [23]). In the NCD-B sample, there is
also a weak and broad band near 500 cm1. Such a band is
observed in heavily doped epitaxial and polycrystalline
diamond films [25].
The Raman features are superimposed on the photoluminescence (PL) background. This PL is stable in the
undoped samples. In the B-doped sample its intensity decreases with the irradiation time, leaving the Raman features unaffected. Nitrogen related defects are usually responsible for most of the PL in diamond films. Nitrogen
and especially hydrogen play a similar role in nondiamond
carbon films. Especially, in polymer-like a-C:H films
(PLC), the PL may obscure the observation of Raman
features under the visible excitation. The observed decrease of the PL intensity in the NCD-B film may be due
to the suppression of the PLC component by the illumination [26].
For highly doped samples (30,000 ppm), the neutron
depth profiling showed that within the near surface region
(<800 nm) the boron (10B + 11B) content was nearly constant, see Fig. 2, with the value of (1.9 0.3) 1021 B cm3,
i.e., 1.1 0.2 at% of B (the error is associated with a multistep procedure of the B depth distribution measurement
based on the comparative study with the 10B reference
samples). This value is consistent with the boron evaluation content obtained by the Raman analysis (see above) on
the sample with 10 lower doping levels (3,000 ppm),
pointing to a rather linear incorporation ratio with the Bcontent in the gas phase [18, 19, 23, 27].

For the biological tests, only the low TMB-doped films

(i.e., 3,000 ppm) were used in this work. Increasing the
doping level above 3,000 ppm leads to different surface
morphologies and grain coarsening which we tried to avoid
for comparison with undoped film, i.e., we wanted to keep
a similar surface morphology as much as possible.
The AFM analysis revealed that both of the NCD and
NCD-B (3,000 ppm, TMB) surfaces were composed of
nano-sized grains. The RMS, AR and AH parameters of
NCD films were 18.23 nm, 13.16 nm and 56 8 nm, respectively, whereas in NCD-B these values amounted only
to 10.00 nm, 8.12 nm and 31 5 nm, respectively. On the
other hand, the shape of the prominences on NCD-B was
more conical and sharper than on the NCD surfaces, where
the peaks of the irregularities were mostly oblong and
rounded (Fig. 3). This suggests a coarsening of the film
morphology upon introduction of TMB into the gas phase.
On day 1 after seeding, the cells on all tested substrates
adhered in similar numbers (15,700 1,220 cells/cm2 to
20,100 1,430 cells/cm2), and were well spread by a similar
area (1,620 130 m2 to 2,510 700 m2). The cells were
polygonal in shape and assembled dot- and streak-like focal adhesion plaques, i.e., cell membrane microdomains,
through which the cells adhere to the adhesion substrate.
These plaques contained integrin adhesion receptors

10

surface

Concentration [ B / cm ]

22

10

10

DEPTH PROFILE OF B IN NCD-B

21

10

20

10

19

10

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Depth [mm]
Figure 2 Depth distribution of 10B isotope (with natural abundance of 18.8%) in the B-doped nanocrystalline diamond
(30,000 ppm), measured using the NDP method.
www.pss-a.com

Figure 3 (online colour at: www.pss-a.com) AFM 3D reconstruction of the surface of NCD (A) and NCD-B (3,000 ppm, B)
film. NTEGRA Prima NT MDT system (Ireland).
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

solidi

2150

M. Kopecek et al.: Improved adhesion, growth and maturation of human bone-derived cells

with alphav chain (Fig. 4AC), i.e., receptors for ECM

proteins vitronectin and fibronectin, which are present in
the serum of the culture medium, and are spontaneously
adsorbed on the material surfaces, if its physical and
chemical properties are appropriate for this adsorption [28].
The focal adhesion plaques also contained talin, and integrin-associated protein, and were apparent mainly at cell
periphery (Fig. 4DF). The focal adhesion plaques in cells
on both NCD layers were usually smaller and thinner than
those in cells grown on glass. This feature might be due to
the nanostructure of the adhesion substrate, which resembled, at least to a certain degree, the architecture of the
natural ECM [29, 30]. It has been reported that the cells
adhering to physiological ECM molecules, especially those
arranged in 3D matrices, are attached through fine focal
adhesion sites, whereas large and well apparent focal adhesion plaques are rather an artifact of the classical cell culture system on smooth planar surfaces [31]. The cells on
all substrates also developed beta-actin-containing filaments arranged in parallel bundles or in a mesh-like cytoskeleton (Fig. 4GI). The beta-actin fibres are known to
be connected to focal adhesion sites and to participate in
the delivery of extracellular signals inside the cells [28].
From days 1 to 3 after seeding, the cells on NCD films
proliferated with the shortest cell population doubling time
(Tab. 1). As a result, on day 3 after seeding, the cells on

____

material/DT (hours) days 1 to 3

*

PS
glass
NCD
NCD-B

29.8 2.3
25.1 1.2
21.3 1.1PS
27.4 2.3

NCD reached a significantly higher population density

(56,280 1,090 cells/cm2) in comparison with the values
on NCD-B (40,850 1,920 cells/cm2), glass slides
(44,100 1,810 cells/cm2) and polystyrene wells (36,220
1,760 cells/cm2) (Fig. 5A).

A. Day 3

NCD_B
Glass
PS

70
60
50

NCD

40
30
20
10
0

____

PS

____
J

____

Glass

____

____
L

NCD
NCD_B
Glass
PS

400

Cells / cm2 (x 1000)

36.6 0.7
41.5 1.0PS
36.2 0.5glass
33.0 0.3PS, glass, NCD

The data are given as mean S.E.M. (Standard Error of Mean) from 9
measurements. ANOVA, Student-Newman-Keuls method. Statistical significance: PS, Glass, NCD: p 0.05 in comparison with the sample of the same
label.

B. Day 7
____

days 3 to 7

____

____
E

Table 1 Cell population doubling time (DT) between days 1 to 3

or between days 3 to 7 after seeding of human osteoblast-like MG
63 cells on polystyrene cell culture dishes (PS), microscopic glass
slides (Glass), nanocrystalline diamond films (NCD) and borondoped NCD films (NCD-B).

Cells / cm2 (x 1000)

status

physica

pss

350

NCD_B
NCD
Glass
PS

300
250
200
150
100
50
0

____

____

____

Figure 4 (online colour at: www.pss-a.com) Immunofluorescence of alphav integrins (A C), talin (D F), beta-actin (G I)
and osteocalcin (J L) in human osteoblast-like MG 63 cells on
days 1 (G I), 3 (A F) or 7 (J L) after seeding on microscopic
glass slides (A, D, G, J) and slides coated with non-doped (B, E,
H, K) or boron-doped (C, F, I, L) nanocrystalline diamond films.
Olympus IX 50 microscope, DP 70 digital camera, obj. = 100,
bar = 20 m.
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

PS

Glass

NCD

NCD_B

Figure 5 Number of human osteoblast-like MG 63 cells on days

3 (A) and 7 (B) on polystyrene cell culture dishes (PS), microscopic glass slides (Glass), nanocrystalline diamond films (NCD)
and boron-doped NCD films (NCD-B). Mean S.E.M. (Standard
Error of Mean) from 32 (A) and 9 (B) measurements. ANOVA,
Student Newman Keuls method. Statistical significance: PS,
Glass, NCD, NCD-B: p 0.05 in comparison with the sample of
the same label.
www.pss-a.com

Original
Paper
phys. stat. sol. (a) 205, No. 9 (2008)

Between days 3 to 7 after seeding, the shortest cell

population doubling time was found in cells growing on
boron-doped NCD films (Table 1). On day 7, the cell
numbers on both NCD (351,170 16,530 cells/cm2)
and NCD-B (310,020 10,410 cells/cm2) films became
significantly higher than the values on glass slides
(218,800 12,340 cells/cm2) and polystyrene dishes
(223,640 9,290 cells/cm2) (Fig. 5B).
Although the cells growing on both NCD and NCD-B
films reached a high state of confluence with the formation
of multilayered regions, they did not show spontaneous detachment of the surfaces, observed in MG 63 cells on oxygen-terminated NCD films in our earlier studies [8, 9, 32].
This tendency to cell detachment could be explained by a
relatively high hydrophilia of O-terminated surfaces (contact angle about 2035). Highly hydrophilic surfaces are
known to bind the adsorbed ECM molecules with relatively weak forces [28, 33], which could lead to the detachment of these molecules especially at later culture intervals, when they bind a large number of cells. It is generally known that the cell adhesion and growth is maximal
on moderately hydrophilic surfaces [28, 34]. This was the
reason why we concentrated on less hydrophilic
H-terminated NCD films in the present study, because we
expected a more stable and long-lasting cell adhesion on
these surfaces.
Thus, the adhesion and growth activity of MG 63 cells
on both NCD layers was similar or even higher in comparison with the values obtained on standard cell culture
substrates, represented by polystyrene dishes and glass.
This favorable cell behavior was probably due to the surface nanostructure of both NCD layers. The material
nanostructure has been reported to be highly supportive for
colonization with cells. Nanostructured surfaces are considered to improve the adsorption of the cell adhesionmediating ECM molecules mentioned above, e.g., fibronectin, vitronectin or collagen, from the serum of the
culture medium or body fluids. These molecules can also
be synthesized by cells and deposited on the material surface. On nanostructured surfaces, these proteins are
adsorbed in an appropriate amount and probably also in a
favorable geometrical conformation, which enables good
accessibility of specific amino acid sequences in these proteins by cell adhesion receptors, e.g., integrins [29, 30]. In
addition, surfaces with nano-sized irregularities, including
NCD, have a higher surface area in comparison with flat or
microstructured surfaces, which renders them intrinsically
more reactive toward moisture and oxygen [35]. Moreover,
nanostructured surfaces are believed to promote preferential adhesion of osteoblasts over other cell types. This
event has been explained by the preferential adsorption of
vitronectin on nanostructured surfaces, due to its relatively
small molecule in comparison with other more complicated
ECM proteins, and its preferential recognition by osteoblasts [29, 30]. This advantageous cell behavior would
lower the risk of fibrous encapsulation of the bone implant.
www.pss-a.com

2151

As shown by reflection goniometry, both NCD and

NCD-B layers used in this study were relatively hydrophobic, having a water drop contact angle of 8590. Despite this, the MG 63 cells on these surfaces adhered in
similar initial numbers, grew with similar cell population
doubling time and on day 3 after seeding, they reached
similar cell population densities in comparison with the
values obtained on highly hydrophilic oxygen-terminated
NCD layers with contact angles of 2035 [79, 32]. It is
known that on hydrophilic surfaces, the cell adhesionmediating ECM molecules are adsorbed in a more flexible
form, which allows their reorganization by cells and thus
the access of cell adhesion receptors to specific binding
sites on these molecules. Conversely, the contact with hydrophobic surfaces usually leads to increased rigidity and
denaturation of these molecules and their lower availability
for cell adhesion [28, 34]. Our results suggest that the surface nanostructure can compensate, at least partly, the hydrophobicity of a material, e.g. by keeping the adsorbed
cell adhesion-mediating ECM molecules in a more appropriate spatial conformation, which mimics the physiological shape of these molecules, and thus improve their binding to cell adhesion receptors. In accordance with this, the
creation of surface nanoroughness on a terpolymer of
polytetrafluoroethylene, polyvinyldifluoride and polypropylene by mixing this material with carbon nanotubes
markedly improved its colonization with MG 63 cells, although this material remained similarly hydrophobic (contact angle from 99.3 6.6 to 104.5 2.2) as the unmodified terpolymer (contact angle 100.0 3.9) [7]. Also on
hydrophobic (contact angle about 100) but nanostructured
fullerene C60 layers, the adhesion and growth of MG 63
cells was similar as on standard cell culture substrates, i.e.,
polystyrene dishes and microscopic glass coverslips [7].
Moreover, as mentioned above, the cells on nanostructured
hydrophobic surfaces were less prone to spontaneous detachment at later culture intervals (day 5 and 7) [8, 9, 32].
On the other hand, in experiments in vivo, the number
of connective tissue cells at O-terminated NCD layers, deposited on titanium substrates and implanted into the abdominal subdermal layer of Wistar rats, was significantly
higher and the scar tissue formation was less tight than in
the region adjacent to the H-terminated NCD films, although the surface morphology of both types of NCD samples was similar [36].
Interestingly, the cells growing on boron-doped NCD
reached significantly lower population densities on days 3
and 7 than on non-doped NCD films (Fig. 5). Similar cell
behavior was observed in the presence of other boroncontaining materials. Incubation of MG 63 cells with
2-aminoethoxydiphenylborate (2-APB) resulted in a blockage of store-operated Ca2+ channels, followed by lengthening of the S and G2/M phases of the cell cycle [37].
On the other hand, supplementation of rats with boron
in drinking water improved the mechanical properties of
the bone tissue [38]. In accordance with this finding, the
MG 63 cells, grown on boron-doped NCD surfaces, were
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2152

solidi

status

physica

pss

M. Kopecek et al.: Improved adhesion, growth and maturation of human bone-derived cells

more brightly stained for osteocalcin, an important extracellular matrix glycoprotein involved in bone tissue
mineralization (Fig. 4JL).
However, for the above mentioned effects on Ca2+
channels or on mechanical properties of the bone, boron or
boron-containing molecules were diluted in a water environment, freely accessible for cell membrane and capable
of entering the cells. In the case of boron-doped NCD the
situation is different. Boron atoms in NCD-B are either located in the lattice (mainly) or around the grain boundaries
[18, 23, 27]. Their release from NCD is very limited because of their strong chemical bonds. Even at elevated
temperatures, the mobility of the boron atoms is very low.
At 1000 C (in a vacuum of 686 Torr and under electric
field of 200 V), the diffusion coefficient of boron in the
diamond was found to be only 3.50 1014 cm2/s [39].
The different behaviour of cells on NCD and NCD-B
films might be, however, related to their distinct electrochemical reactivity and difference in the surface potential.
This difference is due to changes in the occupation of the
surface states caused by thermal excitation of holes from
the B-acceptor level (the sample with the concentration of
2 1020 cm3 are nearly metallic, i.e., close to the Motts
metallic transition [40]). The changes in the occupation of
the surface states may affect the bonding of various biomolecules [19] (including cell adhesion-mediating
ECM molecules like fibronectin and vitronectin) to the
H-terminated surfaces. They can aggravate the formation
of a thin layer (of bio-molecules) on the diamond surface
which is needed for the adhesion and growth of cells. This
remains, however, an open issue that deserves further investigation.
Another possible explanation of a lower growth activity of MG 63 cells on NCD-B surfaces is the shape of the
irregularities, which can act as less appropriate for the cellmaterial interaction. Although the irregularities on NCD-B
films were smaller than those on NCD, their thin conical
shape (with sharp peaks) could provide mechanical stress
to the cell membrane and complicate the attachment and
spreading of cells, including those newly formed after mitosis [41].
Nevertheless, it can be concluded that both non-doped
and boron-doped nanocrystalline diamond films gave good
support for the adhesion and growth and maturation of osteogenic cells, and can be used in tissue engineering applications and construction of bone implants. This is a great
advantage over other carbon nanoparticles, such as fullerenes and nanotubes, which can behave as cytotoxic and
genotoxic agents, especially in a free form dispersed in cell
culture media [42, 43]. For example, the nanocrystalline
diamond layers could be used for the surface modification
of bone implants (e.g., bone-anchoring parts of joint prostheses or bone replacements) in order to improve their integration with the surrounding bone tissue.
Acknowledgements This study was supported by the
Grant Agency of the Czech Republic (contracts No. 204/06/0225
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

and 101/060226) and Acad. Sci. CR (KAN 400480701 and KAN

200100801). We also thank Mrs. Ivana Zajanova for her help in
immunocytochemical staining. Mrs. Sherryl Ann Vacik, Pangrac
& Associates, Port Aransas, Texas, USA is gratefully acknowledged for her language revision of the manuscript.

References
[1] K. Iakoubovskii, G. J. Adriaenssens, K. Meykens, M. Nesladek, Y. A. Vul, and V. Y. Osipov, Diam. Relat. Mater. 8,
1476 (1999).
[2] A. M. Schrand, H. Huang, C. Carlson, J. J. Schlager,
E. Osawa, S. M. Hussain, and L. Dai, J. Phys. Chem. 111, 2
(2007).
[3] K. Bakowicz and S. Mitura, J. Wide Bandgap Mater. 9, 261
(2002).
[4] C. C. Fu, H. Y. Lee, K. Chen, T. S. Lim, H. Y. Wu, P. K.
Lin, P. K. Wei, P. H. Tsao, H. C. Chang, and W. Fann, Proc.
Natl. Acad. Sci. USA 104, 727 (2007).
[5] L. C. Qint, D. Zhoutt, A. R. Krauss, and D. M. Gruen,
Nanostruct. Mater. 10, 649 (1998).
[6] M. J. Papo, S. A. Catledge, Y. K. Vohra, and C. J. Machado,
Mater. Sci. Mater. Med. 15, 773 (2004).
[7] L. Bacakova, L. Grausova, J. Vacik, A. Fraczek, S. Blazewicz, A. Kromka, M. Vanecek, and V. Svorcik, Diam.
Relat. Mater. 16, 2133 (2007).
[8] L. Grausova, L. Bacakova, A. Kromka, S. Potocky, M. Vanecek, M. Nesladek, and V. Lisa, J. Nanosci. Nanotechnol.,
in press (2008).
[9] L. Grausova, A. Kromka, L. Bacakova, S. Potocky, M. Vanecek, and V. Lisa, Diam. Relat. Mater., in press (2008).
[10] M. Kalbacova, M. Kalbac, L. Dunsch, A. Kromka, M. Vanecek, B. Rezek, U. Hempel, and S. Kmoch, phys. stat. sol.
(b) 244, 4356 (2007).
[11] P. Bajaj, D. Akin, A. Gupta, D. Sherman, B. Shi, O. Auciello, and R. Bashir, Biomed. Microdevices 9, 787 (2007).
[12] C. G. Specht, O. A. Williams, R. B. Jackman, and
R. Schoepfer, Biomaterials 25, 4073 (2004).
[13] S. Rupprecht, A. Bloch, S. Rosiwal, F. W. Neukam, and
J. Wiltfang, Int. J. Oral Maxillofac. Implants 17, 778 (2002).
[14] A. Hartl, E. Schmich, J. A. Garrido, J. Hernando, S. C. Catharino, S. Walter, P. Feulner, A. Kromka, D. Steinmller,
and M. Stutzmann, Nature Mater. 3, 736 (2004).
[15] P. Christiaens, V. Vermeeren, S. Wenmackers, M. Daenen,
K. Haenen, M. Nesladek, M. vandeVen, M. Ameloot, L.
Michiels, and P. Wagner, Biosens. Bioelectron. 22, 170
(2006).
[16] X. Xiao, J. Wang, C. Liu, J. A. Carlisle, B. Mech,
R. Greenberg, D. Guven, R. Freda, M. S. Humayun,
J. Weiland, and O. Auciello, J. Biomed. Mater. Res. B Appl.
Biomater. 77, 273 (2006).
[17] W. Zhao, J. J. Xu, Q. Q. Qiu, and H. Y. Chen, Biosens. Bioelectron. 22, 649 (2006).
[18] M. Nesladek, J. J. Mares, D. Tromson, C. Mer, P. Bergonzo,
P. Hubik, and J. Kristofik, Sci. Technol. Adv. Mater. 7, S41
(2006).
[19] C. E. Nebel, D. Shin, B. Rezek, N. Tokuda, H. Uetsuka, and
H. Watanabe, J. R. Soc. Interface 4, 439 (2007).
[20] J. Vacik, J. Cervena, V. Hnatowicz, V. Havranek, J. Hofmann, S. Posta, D. Fink, and R. Klett, Nucl. Instrum. Methods Phys. Res. B 142, 397 (1998).
www.pss-a.com

Original
Paper
phys. stat. sol. (a) 205, No. 9 (2008)

[21] A. M. Zaitsev, Optical Properties of Diamond (Springer

2001).
[22] R. J. Zhang, S. T. Lee, and Y. W. Lam, Diam. Relat. Mater.
5, 1288 (1996).
[23] P. W. May, W. J. Ludlow, M. Hannaway, P. J. Heard, J. A.
Smith, and K. N. Rosser, Diam. Relat. Mater. 17, 105
(2008).
[24] W. Kulisch, C. Popov, S. Boycheva, M. Jelinek, P. N. Gibbon, and V. Vorlicek, Surf. Coat. Technol. 200, 4731 (2006).
[25] M. Bernard, C. Baron, and A. Deneuville, Diam. Relat.
Mater. 13, 896 (2004).
[26] S. Xu, M. Hundhausen, J. H. Ristein, B. Yan, and L. Ley,
J. Non-Cryst. Solids 164 166, 1127 (1993).
[27] P. W. May, W. J. Ludlow, M. Hannaway, P. J. Heard, J. A.
Smith, and K. N. Rosser, Chem. Phys. Lett. 446, 103 (2007).
[28] L. Bacakova, E. Filova, F. Rypacek, V. Svorcik, and
V. Stary, Physiol. Res. 53, S35 (2004).
[29] T. J. Webster, C. Ergun, R. H. Doremus, R. W. Siegel, and
R. Bizios, J. Biomed. Mater. Res. 51, 475 (2000).
[30] R. L. Price, M. C. Waid, K. M. Haberstroh, and T. J. Webster, Biomaterials 24, 1877 (2003).
[31] A. J. Engler, M. A. Griffin, S. Sen, C. G. Bonnemann, H. L.
Sweeney, and D. E. Discher, J. Cell. Biol. 166, 877 (2004).
[32] L. Grausova, L. Bacakova, A. Kromka, M. Vanecek, and
V. Lisa, Diam. Relat. Mater, submitted.
[33] L. Bacakova, E. Filova, D. Kubies, L. Machova, V. Proks,
V. Malinova, V. Lisa, and F. Rypacek, J. Mater. Sci. Mater.
Med. 18, 1317 (2007).

www.pss-a.com

2153

[34] L. Bacakova, K. Walachova, V. Svorcik, and V. Hnatowicz,

J. Biomater. Sci., Polym. Ed. 12, 817 (2001).
[35] K. F. Chong, K. P. Loh, S. R. Vedula, C. T. Lim,
H. Sternschulte, D. Steinmller, F. S. Sheu, and Y. L.
Zhong, Langmuir 23, 5615 (2007).
[36] F. R. Kloss, L. A. Francis, H. Sternschulte, F. Klauser,
R. Gassner, M. Rasse, E. Bertel, T. Lechleitner, and
D. Steinmller-Nethl, Diam. Relat. Mater., in press (2008),
DOI:10.1016/j.diamond.2007.12.061.
[37] D. Labelle, C. Jumarie, and R. Moreau, Cell Prolif. 40, 866
(2007).
[38] M. R. Naghii, G. Torkaman, and M. Mofid, Biofactors 28,
195 (2006).
[39] A. Suarez, M. A. Prelas, T. K. Ghosh, R. V. Tompson, S. K.
Loyalka, W. H. Miller, and D. S. Viswanath, J. Wide Bandgap Mater. 10, 15 (2002).
[40] S. Alehashem, F. Chambers, J. W. Strojek, G. M. Swain,
and R. Ramesham, Anal. Chem. 67, 2812 (1995).
[41] L. Bacakova, V. Stary, O. Kofronova, and V. Lisa, J. Biomed. Mater. Res. 54, 567 (2001).
[42] A. Dhawan, J. S. Taurozzi, A. K. Pandey, W. Shan, S. M.
Miller, S. A. Hashsham, and V. V. Tarabara, Environ. Sci.
Technol. 40, 7394 (2006).
[43] M. Bottini, S. Bruckner, K. Nika, N. Bottini, S. Bellucci,
A. Magrini, A. Bergamaschi, and T. Mustelin, T. Toxicol.
Lett. 160, 121 (2006).

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim