SELECTIVE OSTEOBLASTIC CELL MICRO-ARRAYS ON

DIAMOND FILMS

Bohuslav Rezek, Lenka Michalíková, Egor Ukraintsev, Alexander Kromka

Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnicka 10, Prague 6, Czech Republic

Marie Kalbacova

Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine, UK, Ke Karlovu 2, Prague 2, Czech Republic

Keywords: Cell adhesion, Proteins, Diamond, Biotechnology, Biosensors, Osteoblasts, Atomic force microscopy.

Abstract: Unique combination of chemical and biocompatible properties with semiconducting properties makes

diamond an attractive material for merging solid state and biological systems. Microscopic chemical

patterning of diamond films by hydrogen and oxygen surface atoms is used for self-assembly of human

osteoblastic cells into micro-arrays. The cell adhesion and assembly on the diamond is further controlled

and optimized by cell and protein (fetal bovine serum - FBS) concentration. The cells are characterized by

fluorescence microscopy of actin fibers and nuclei. The protein adsorption is studied by atomic force

microscopy (AFM). The cells are arranged into arrays on O-terminated patterns. The best cell selectivity is

achieved for the lowest cell concentrations of 2500 cells/cm

. Higher cell concentrations enable to colonize

unfavorable H-terminated regions due to mutual cell communication. Based on AFM, the proteins are

present on both H- and O-terminated surfaces, however, pronounced differences in the thickness, surface

roughness, morphology, and phase images indicate different conformation of the proteins and hence the cell

selectivity. There is no cell selectivity when no protein is supplemented in the medium. These results may

be applicable in tissue engineering, implants, bio-electronics, and biotechnology in general.

1 INTRODUCTION

Diamond is not only a gem but also a promising

technological material. Its properties include high

hardness, fracture toughness, low friction

coefficient, high Young modulus, increased wear

resistance and a variety of substrates onto which it

can be prepared (Potocky, 2007). Although diamond

is considered inert, its surface can be functionalized

by various atoms or molecules (Rezek, 2007a). This

gives rise to striking properties (Nebel, 2003).

For instance, electrical conductivity and electron

affinity of diamond are strongly influenced by the

O- or H-termination of the diamond surface

(Kawarada, 1996; Maier, 2001). The differences are

mainly caused by the surface dipole of C-H and C-O

bonds (Tachiki, 2003). O-terminated diamond is

highly resistive, whereas H-terminated surface

induces p-type surface conductivity even on an

undoped diamond (Maier, 2001). These features can

be applied for field-effect transistor (FET) devices

(Rezek, 2007b; Garrido, 2003).

Furthermore, O-terminated surfaces are

hydrophilic while H-terminated surfaces are

hydrophobic. H-terminated surfaces were thus found

less favorable for osteoblastic cell adhesion,

spreading and viability compared to O-terminated

surfaces (Kalbacova, 2007a). On the other hand, H-

terminated diamond surface is an ideal starting point

for covalent attachment of biomolecules (Yang,

2002). Chemical functionalization can also lead to

bio-passivation or bio-active properties (Bajaj,

2007).

Unique combination of the mechanical,

chemical, and biocompatible properties (Tang, 1995,

Kalbacova, 2007a) with semiconducting properties

makes diamond an attractive material for merging

solid state and biological systems (Yang, 2004;

Rezek, 2007b). Hence the hydrogen and oxygen

surface patterns are highly relevant for bio-

electronics as well as for tissue engineering.

347

Rezek B., Michalíková L., Ukraintsev E., Kromka A. and Kalbacova M. (2009).

SELECTIVE OSTEOBLASTIC CELL MICRO-ARRAYS ON DIAMOND FILMS.

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 347-354

DOI: 10.5220/0001434203470354

 SciTePress

Characterization of the interaction between cells and

surfaces is essential for cell-based biosensors,

engineered tissue therapies and the optimization of

implant biomaterials. Cells recognize their

environment and consequently start to change it by a

production of appropriate extracellular matrix

(ECM) proteins to form the basis for cell spreading,

increased adhesion and expression of differentiated

phenotypes (Schakenraad, 1989). This complex and

flexible process is dependent on culture conditions,

including the underlying substrate and the pre-

adsorbed protein layer.

Until now, the research on the cell-diamond

interfaces has been focused on overall homogeneous

surface terminations (Yang, 2004; Kalbacova,

2007a; Rezek, 2007c; Song, 2007). In this work we

show selective adhesion and arrangement of

osteoblast-like cells on NCD thin films that are

microscopically patterned with H- and O-terminated

regions (Michalikova, 2008). We control initial cell

density and serum concentration in medium

influencing cellular colonization of patterned

susbtrate. Furthermore, we employ atomic force

microscopy (AFM) to characterize the structural

properties of mediating proteins (fetal bovine serum,

a crucial component for the cell growth) adsorbed

onto the diamond micro-patterns. The data are used

to discuss the selectivity of the cell adsorption on the

patterns, i.e. to what degree the cell adhesion and its

selectivity is driven by serum adsorption and

conformation on H- and O-terminated surfaces or by

a direct effect of diamond surface dipoles on the

cells. We also provide perspectives for potential bio-

electronic applications.

2 MATERIALS AND METHODS

Diamond films are grown on (100) oriented silicon

substrates (13 mm in diameter, 500 µm thickness,

RMS roughness of < 0.6 nm) by microwave plasma

process using total gas pressure 50 mbar, substrate

temperature 800°C, 1% CH

in H

and total power

2.5 kW. This process results in a growth of

continuous, smooth and high quality nanocrystalline

diamond film (Potocky, 2007; Kromka, 2008). X-ray

photocurrent spectroscopy (XPS) detects that the

films are 95% pure diamond (Zemek, 2006). The

diamond film thickness is 300-400 nm. Average

crystal size is 50 nm, RMS roughness at 1x1 µm

area is 15-20 nm as measured by AFM using

standard silicon tips of nominal radius < 10 nm. The

silicon substrates are coated with NCD film on both

sides, silicon is thus hermetically encapsulated in the

diamond.

The diamond films were further chemically

cleaned in acids (97.5% H

+ 99% powder

KNO

) at 200°C for 30 minutes. The surface was

then hydrogenated at 800°C for 10 minutes. NCD

films were lithographically processed to generate

alternating H- and O-terminated patterns of 30 to

200 µm widths. A positive photoresist ma-P 1215

(micro resist technology GmbH, Germany) was

applied. NCD films with lithography mask were

treated in oxygen radio-frequency plasma (300W

power, 3 minutes process time) to oxidize the

surface and hence to generate the hydrophilic

patterns. Then the sample was rinsed in a stripper,

de-ionized water and dried. This process removed

possible surface contamination (Rezek, 2006a). The

H-/O-termination quality was proved by a scanning

electron microscope (SEM; JEOL Superprobe 733).

Electronic measurements detected a surface

conductivity of 10

-5

S/sq on the H-terminated

surfaces (Kozak, 2008). Surfaces with O-termination

were highly resistive. The NCD samples were

sterilized in 70% ethanol for 10 minutes prior to cell

plating. The device concept is schematically shown

in Figure 1.

Figure 1: Schematic picture of silicon substrate

hermetically coated with NCD layer with stripe-like

patterns having hydrogen or oxygen surface termination.

Cell adhesion on the O-terminated region is also

schematically indicated.

SAOS-2 cells (human osteoblast-like cell line)

(DSMZ GmbH), were grown in McCoy’s 5A

medium (BioConcept) supplemented with heat

inactivated fetal bovine serum (FBS; Biowest) of

various concentrations (0-15%), penicillin (20 U/ml)

and streptomycin (20 µg/ml). Note that the SAOS-2

is a standard and well-defined cell line. Thus the

results can be compared between various series of

experiments as well as with reports in the literature.

Cells were plated at the densities of 2,500 and

10,000 cells/cm

using a droplet technique: substrate

surface was covered by 100 µl droplet of cell

BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices

348

Figure 2: Nanocrystalline diamond film with 200μm wide H-/O-terminated patterns: (a) optical (bright field) image prior to

cell plating showing optically transparent and featureless surface, (b) scanning electron microscopy image prior to cell

plating where bright stripes correspond H-termination and dark stripes O-termination of the diamond surface due to their

opposite electron affinity, (c) fluorescent microscopy image of osteoblastic cells cultivated on the substrate. The alignment

cross is used for correlation of the surface termination micro-patterns with the cells.

Figure 3: Fluorescent microscopy images of osteoblastic cells (SAOS-2) cultivated in McCoy's medium supplemented with

15% FBS for 2 days on H-/O-terminated stripes of different widths (30μm, 60μm, 100μm and 200μm) on diamond films.

Initial cell concentration was 2,500 cells/cm

. The fluorescence shows actin stress fibers (green) and nuclei (blue). Scale bar

is 100μm.

suspension in the appropriately supplemented

medium, let to incubate for 2 h (adhesion time), and

then 1.4 ml of the medium was added. In case of 0%

FBS, the cells were plated and incubated for 2 h in

the medium without the serum. Then the 15% FBS-

supplemented medium was added to facilitate

further cell cultivation. After plating, the cells were

cultivated for 48 hours in 5% CO

at 37°C.

An advantage of the applied droplet technique is

a precise control of the applied number of cells on

the sample. A disadvantage is slightly non-

homogenous distribution of cells over the sample

with lower concentration on the edge and higher

concentration in the middle of the sample.

Therefore, the microscopic images were taken from

comparable areas on the samples.

Adhesion and morphology of SAOS-2 cells were

characterized by fluorescent staining of actin stress

fibers (phalloidin-Alexa 488 - 1:100, Molecular

Probes) and nuclei (DAPI - 1:1000, Sigma)

according to the protocol in Ref. (Kalbacova,

2007b). The staining was visualized using the E-400

epifluorescence microscope (Nikon); digital images

were acquired with a DS-5M-U1 Color Digital

Camera (Nikon).

As the adhesion and growth of osteoblastic cells

is mediated by proteins, the adsorption, adhesion,

and conformation of FBS itself on the H- and O-

terminated diamond was also investigated. Polished

IIa (100) mono-crystalline diamonds were used as

substrates to minimize the contribution from surface

morphology of NCD films. The mono-crystalline

diamond surface was H- or O-terminated using the

same procedures as for NCD films. A droplet of

15% FBS in the McCoy’s 5A medium was applied

on the diamond substrates for 10 min. Then the

whole samples were immersed in the fluid cell

containing the same FBS/McCoy’s medium and

characterized by AFM (Ntegra, NTMDT).

AFM measurements were performed in the

medium using doped silicon cantilevers

(BSMulti75Al) with the typical force constant of 3

N/m, resonance frequency 75 kHz in air (30 kHz in

the medium), and nominal tip radius <10 nm.

Surface morphologies were investigated in

oscillating-mode AFM (OM-AFM), where the tip-

surface interaction is controlled by adjusting the

AFM amplitude set-point ratio. Free oscillation

amplitude of 60 nm and the set-point ratio of 50%

were typically used. The parameters were optimized

not to influence the soft FBS layer yet to provide

optimal resolution and contrast. A nanoshaving

procedure (Rezek, 2006b; Rezek, 2007a) was

applied to evaluate the protein layer thickness. First,

SELECTIVE OSTEOBLASTIC CELL MICRO-ARRAYS ON DIAMOND FILMS

349

Figure 4: Fluorescent microscopy images of osteoblastic cells (SAOS-2) cultivated for 2 days on 100μm H-/O-terminated

stripes on diamond films: (a) low initial cell seeding concentration (2,500 cells/cm

), (b) high initial cell seeding

concentration (10,000 cells/cm

), and (c) cells bridging of unfavorable H-terminated regions. The fluorescence shows actin

stress fibers (green) and nuclei (blue). Scale bar is 100μm.

Figure 5: Fluorescent microscopy images of osteoblastic cells (SAOS-2) cultivated for 2 days on 200μm H-/O-terminated

stripes on diamond films in McCoy's medium supplemented with different fetal bovine serum (FBS) concentrations (0, 5,

10, and 15%). Scale bar is 100μm.

a region of 2x2 μm

was scanned in contact AFM

(C-AFM) and then re-measured across somewhat

larger area by OM-AFM. The force applied during

C-AFM was approx. 200 nN. The interaction forces

in OM-AFM are orders of magnitude lower. The

FBS layer thickness was then determined as the

difference between average height values across 1

of the FBS layer surface and 1 μm

of the

nanoshaved area where FBS was removed. Several

regions were probed on each sample to determine

the error bar from root-mean-square (RMS)

roughness values and statistical errors.

Autocorrelation function of the images was

calculated to determine typical lateral feature size

(Lx).

3 RESULTS

Correlation of oxygen- and hydrogen-terminated

micro-patterns on the diamond films with patterns of

cell adhesion on such structures is illustrated in

Figure 2. Figure 2(a) shows a bright field image of

the micro-structured sample in optical microscope

before cell seeding. The surface is featureless as the

patterns are optically invisible. Figure 2(b) presents

SEM image of the sample, where H- and O-

terminated patterns (width of 200 µm) are clearly

identified due to their different electronic properties.

The bright stripes correspond to the H-terminated

NCD surface, having negative electron affinity

(Maier, 2001). The dark stripes represent the O-

terminated NCD surface. Fluorescently stained

human osteoblasts adherent on 200 µm wide

patterned surface are presented in Figure 2(c). By

correlating a position of the alignment mark in SEM

and fluorescent microscopy pictures, it is evident

that the osteoblastic cells preferentially colonize the

O-terminated (hydrophilic) patterns.

Figure 3 shows that the cells adhere

preferentially onto O-terminated stripes

independently of the stripe width in the range of 30-

200µm. Two types of cell adhesion patterns are

detectable. Cells on the narrow stripes (30µm –

smaller than the cell size) are elongated and form

cell-by-cell arrays. On wider stripes (60, 100, and

200µm – bigger than the cell size) the cells spread

and fill the entire width of the stripe. At the micro-

pattern borders they form a sharp boundary.

Osteoblast adhesion onto the NCD surface is

affected by the initial cell seeding concentration.

Figure 4 illustrates higher selectivity for cell

adhesion on the O-terminated surface at lower initial

cell seeding density (2,500 cells/cm

). There is still

some free space for cell spreading and expansion

within the hydrophilic region. On the other hand,

cells plated at the higher density (10,000 cells/cm

)

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350

colonize not only hydrophilic areas but also

unfavorable hydrophobic regions (Figure 4(b)).

Figure 4(c) presents an abnormally long single cell

(left image side) as well as clusters of several cells

(right image side) that can bridge and colonize the

hydrophobic area.

Figure 5 demonstrates the influence of different

initial FBS concentrations (0, 5, 10, and 15%) in the

culture medium on the cell attachment onto the H-

/O-patterned surface. The range of serum

concentrations 5-15% does not significantly affect

the cell adhesion pattern. The cells follow the H-/O-

terminated micro-patterns in the same way as shown

in the previous figures. In a sharp contrast, cells

plated in FBS-free medium colonize the surface

independently of the micro-patterns. The cell

selectivity is obviously determined by the FBS

proteins.

Figure 6 shows OM-AFM topography image of

the FBS layer on diamond with stripe-like patterns

of hydrogen and oxygen surface terminations. The

diagonal lines in the background are due to polishing

of the diamond substrate. The roughness of diamond

substrate is about 0.6 nm. On this background one

can see clear stripes on the O-terminated surface.

There are also some small scattered islands of

similar thickness on the H-terminated stripes, most

likely due to certain degree of non-specific

adsorption. When the height of stripes is probed by

the nanoshaving method, we find that the layer

thickness of the layer adsorbed on O-terminated

diamond is 4 ± 2 nm. Even on H-terminated surface

(outside of the islands) there is a thin layer of 1.5 ± 2

nm. Hence the FBS layer is present on both types of

diamond surfaces, although in the different

thickness.

Figure 6: AFM topography image of a fetal bovine serum

(FBS) layer on the diamond with stripe-like patterns of H

and O surface terminations.

Figure 7: AFM force curves on H- and O-terminated

diamond with the adsorbed FBS layer.

Figure 8: AFM measurements in FBS/McCoy’s medium

on hydrogen- and oxygen-terminated diamond surfaces

with adsorbed FBS layers: topography and phase image on

(a-b) FBS/H-terminated diamond (c-d) FBS/O-terminated

diamond.

Figure 7 shows force curves obtained by force

spectroscopy on H- and O-terminated diamond. The

force curves exhibit 500 ± 100 pN interaction

between tip and surface on both H- and O-

terminated diamond. Similar forces and shapes were

found between cantilevers functionalized by bovine

serum albumin and glass surfaces after deposition of

proteins (Popov, 2007). Hence also the protein

molecules from FBS are present on both H- and O-

terminated diamond.

Figure 8 shows the detailed topography and

phase images on both types of surfaces. Values of

RMS roughness and lateral feature size (Lx) are also

given. The roughness of FBS layer on O-terminated

diamond (1.7 nm) is about two times higher

SELECTIVE OSTEOBLASTIC CELL MICRO-ARRAYS ON DIAMOND FILMS

351

compared to H-terminated diamond (0.6 nm). The

topographic features are different, with a kind of

ridge-like shapes around valleys on H-terminated

diamond and hillock-like shape on O-terminated

diamond. Correspondingly, the feature size is also

different, about 10 nm on H-terminated diamond and

20 nm on O-terminated diamond. A pronounced

difference is detected also in the AFM phase images.

AFM phase image of the adsorbed layer on H-

terminated diamond is dominated by dark dots

correlated with protrusions in morphology. On O-

terminated diamond, brighter spots having darker

boundaries are correlated with the hillocks.

4 DISCUSSION

In correlation with previous reports on homogeneous

surface termination of diamond (Kalbacova, 2007a,

Bacakova, 2007) we find that in case of H/O micro-

patterns the cells colonize preferentially hydrophilic

(O-terminated) stripes forming confluent arrays with

sharp edges separating O- and H- terminated

regions. The cells generally did not show any

decreased viability, however some of them

(preferentially on hydrophobic region) remain

rounded for an extended period of time exhibiting

poor cell-substratum-compatibility (Liu, 2007;

Michalikova, 2008). Evolution of cell morphology

on hydrophobic surfaces is slower, but otherwise not

remarkably different than that observed for human

osteoblasts (hFOB) (Liu, 2007) or SAOS-2 on more

hydrophilic surfaces – it is an example of the time-

cell-substratum-compatibility-superposition

principle.

Also noteworthy is bridging of unfavorable H-

terminated regions as illustrated in Figure 4(c). This

is obviously enabled by connection to the cells on

the O-terminated regions because solitaire cells on

the H-terminated regions exhibit bad adhesion and

reduced metabolic activity (Kalbacova, 2007a;

Kalbacova, 2008). To reach the optimal status on

unfitting surface, cells will communicate with each

other, exchanging growth factors and various stimuli

as well as produce extracellular matrix (ECM) and

thus modify the surface with proteins and

proteoglycans underneath to overcome the

inhospitable environment. It is known that proteins

adsorbed onto the substrate surface do not become

permanently immobilized. They will be

enzymatically degraded, denatured, they undergo

conformational and configuration changes and will

even be replaced by other proteins (Zeng, 1999).

However, when more cells are able to gently attach

to hydrophobic surface in a specific pattern (forming

a bridge between two hydrophilic stripes) then these

cells may form ECM faster due to support from their

proliferating neighbors, thus masking unsuitable

properties of the surface. This may be very useful

mechanism for bio-electronic applications as it

enables to overgrow electrically conductive H-

terminated surface when it is surrounded by O-

terminated regions at small enough dimensions.

As the cell adsorption is protein mediated, a

question arises whether the specific cell adsorption

is due to direct effect of diamond surface dipoles on

the cells or due to differences in protein adsorptions

on the micro-patterns. Figure 5 clearly demonstrates

that cells plated without protein (FBS-free medium)

do not sense any chemical micro-patterning, whereas

cells plated in FBS-supplemented medium clearly

follow the hydrophilic patterns. It proves that the

cell selectivity is driven by the FBS protein

adsorption. Since protein adsorption is much more

rapid than the transport of cells to the surface, it is

expected that the interaction of host cells with the

material is determined by the nature of this adsorbed

protein layer.

AFM study of the protein layers revealed that

FBS adsorbs on both types of diamond surfaces.

This is in agreement with previous reports that

albumin adsorbs on both hydrophilic and

hydrophobic surfaces (Browne, 2004). Here, the

adsorbed thickness differs by few nm. It should be

noted that FBS layer is a soft matter so there is some

uncertainty in determining its thickness by AFM

because even in OM-AFM the height may be

underestimated (Rezek, 2007a; Rezek, 2007b).

Another influence on the observed step in the height

across the nanoshaved region may be wear of the

substrate material. As the flat bulk diamond is very

hard compared to proteins and its wear is extremely

low, only the FBS layer was penetrated and removed

by the nanoshaving forces applied here.

The cell selectivity is thus not determined merely

by FBS layer presence. More subtle differences must

be considered for explaining the selective

adsorption, such as protein denaturation on

hydrophobic surfaces (Ukraintsev, 2007; Zeng,

1999). Detailed studies of surface morphology

revealed clear differences in surface roughness,

morphological features and phase images between

the protein layers on H- and O-terminated diamond.

By comparison with the literature (Browne, 2004) ,

where similar difference in topography on

polystyrene substrates were shown, the most

important factor for the cell growth on diamond

seems to be the wetting property of the surface

BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices

352

rather than any other specific property of the

diamond films.

One has to critically consider that the actual

composition of the adsorbed layers may be different

on H- and O-terminated diamond because various

proteins (albumin, fibronectin, vitronectin, etc.) from

FBS may influence the cell adhesion in different

ways. Further experiments are needed to elucidate

these details.

5 CONCLUSIONS

Chemical patterning of diamond films by hydrogen

and oxygen surface atoms enables self-assembly of

human osteoblastic cell micro-arrays. The cell

adhesion and assembly on diamond can be further

controlled and optimized by biochemical factors.

The cells strongly prefer O-terminated patterns. The

best selectivity is achieved for lower initial cell

concentrations (2,500 cells/cm

), regardless of

surface geometry and commonly used protein (FBS)

concentrations (5 to 15%). Widths of the patterns

affect the shape of adhered cells in the following

way: i) good cell spreading with a sharp boundary

was observed on broader stripes and ii) elongated

cell chains were observed on stripes which were

narrower than the cell size. Higher initial

concentration of cells enables colonization of less

favorable H-terminated surface regions, which are

electrically conductive and can be employed in

electronic devices. A non-preferential cell adhesion

is found when the initial cell adhesion occurs

without the serum presence. Hence the cell

selectivity is driven by the FBS properties on H- and

O-terminated surfaces. AFM detected presence of

the FBS layer on both types of surfaces. However,

the layer thickness and microscopic morphology are

rather different. This may be the reason for the cell

selectivity. Further experiments are needed to

elucidate details of the selectivity, such as particular

composition of the adsorbed layers and so on.

Nevertheless, the presented data may already

provide valuable information for application of

diamond films in tissue engineering, implants, bio-

electronics, and biotechnology in general. We

speculate that similar cell behavior will occur also

when using other cell lines.

ACKNOWLEDGEMENTS

This work was supported by the Academy of

Sciences of the Czech Republic contracts

KAN400100701, AV0Z10100521, and Czech

Ministry of Education, Youth and Sport projects LC-

510 and MSM0021620806. The authors would like

to express their thanks to Ing. Vlastimil Jurka, Zdena

Poláčková, Dr. Zdeněk Potměšil (all Inst. Phys.

ASCR) for a kind assistance with photolithography,

and surface treatments, Jana Sovová (Inst. Inh. Met.

Disorders, 1st Fac. Med., Charles Uni.) for technical

assistance and Mgr. Veronika Barešová (Inst. Inh.

Met. Disorders, 1st Fac. Med., Charles Uni.) for a

kind assistance with fluorescent microscopy.

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