# Improved adhesion, growth and maturation of human bone-derived cells on nanocrystalline diamond films

Miloslav Kopecek, Lucie Bacakova, Jiri Vacik, Frantisek Fendrych, Vladimir Vorlicek, Irena Kratochvilova, Vera Lisa, Emilie van Hove, Christine Mer, Philippe Bergonzo, et al.

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## To cite this version:

Miloslav Kopecek, Lucie Bacakova, Jiri Vacik, Frantisek Fendrych, Vladimir Vorlicek, et al. Improved adhesion, growth and maturation of human bone-derived cells on nanocrystalline diamond films. Physica Status Solidi A (applications and materials science), 2008, Special Issue: Hasselt Diamond Workshop 2008 - SBDD XIII, 205 (9), pp.2146-2153. 10.1002/pssa.200879729 . cea-05459333v3

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Submitted on 23 Feb 2026

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Improved adhesion, growth and maturation of human bone-derived cells on nanocrystalline diamond films

Milan Kopecek*,¹, Lucie Bacakova*¹, Jiri Vacik², Frantisek Fendrych⊥,³, Vladimir Vorlicek³, Irena Kratochvilova³, Vera Lisa¹, Emilie Van Hove⁴, Christine Mer⁴, Philippe Bergonzo⁴, and Milos Nesladek†, 3, 4

1 Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague 4, Czech Republic

2 Nuclear Physics Institute, Academy of Sciences of the Czech Republic, 25068 Husinec - Rez, Czech Republic

3 Institute of Physics, Academy of Sciences of the Czech Republic, Na Slovance 2, 18221 Prague 8, Czech Republic

4 CEA-LIST, Centre d'Etudes Saclay, Bat. 451, p. 84, 91191 Gif Sur Yvette, France

* 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

Abstract

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 p ~ 10⁻¹ Ω 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 cm⁻³ (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 polystyrene 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 αv 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.

**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 [2–4]. 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 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, 7–11]. Type IIa diamond, microcontact printed 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 TiAl<sub>6</sub>V<sub>4</sub> 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, 14–16].

An interesting issue is that the sensing applications of NCD can be markedly improved by doping NCD with boron [17–19]. 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 alpha<sub>v</sub> 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 substrate temperature was 710 °C, and an atmosphere of 0.8–1% CH<sub>4</sub> in H<sub>2</sub> 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) mixtures with H<sub>2</sub> in a concentration of 3,000–30,000 ppm B:C leading to ρ ~ 10<sup>-1</sup> Ω 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 1–2 × 10<sup>20</sup> cm<sup>-3</sup>, i.e., this incorporation is about a factor 10 lower than in our previous works [18], probably due to the TMB-H<sub>2</sub> 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 (λ = 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 <sup>10</sup>B(n<sub>th</sub>, alpha)<sup>7</sup> Li 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-Mean-Square) amplitude of cantilever oscillation. Analysis of AR (Average Roughness) and AH (Average Height) was also performed on 8 sites of 5 µm × 5 µm 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 Dulbecco's modified Eagle's Minimum Essential Medium (DMEM; Sigma, USA, Cat. No D5648) supplemented with 10% foetal bovine serum (FBS; Sebak GmbH, Aidenbach, Germany) and gentamicin (40 µg/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 ethanol (–20 °C, 10 minutes) and stained with a combination of fluorescent cell membrane dye Texas Red C₂-maleimide (Molecular Probes, Invitrogen, Cat. N° T6008; 20 ng/ml in PBS) and nuclear dye Hoechst #33342 (Sigma, USA; 5 µg/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. N° T4174) in PBS and counted in a Bürker 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:

$$ DT = \log 2 (t - t_0)/(\log N_t - \log N_{to}) \quad (1) $$

where $t_0$ and $t$ represent earlier and later time intervals after seeding, respectively, and $N_{to}$ and $N_t$ the number of cells at these intervals.

## 2.5 Immunofluorescence staining of αv-integrins, talin, β-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 αv subunit polyclonal antibody (Chemicon International Inc., Temecula, CA, USA; Cat. N° AB1930), mouse monoclonal anti-human talin (Chemicon International Inc., Cat N° MAB3264), mouse monoclonal antibeta-actin (clone AC-15, Sigma, St. Louis, MO, USA, Cat. N° A-5441) or rabbit polyclonal anti-human osteocalcin (Chemicon International Inc., Cat. N° 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. N° A11017 and A11070, respectively). After incubation with 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), Student–Newman–Keuls 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 cm⁻¹ 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 cm⁻¹ [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] ~1–2 × 10²⁰ cm⁻³ 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 cm⁻¹ 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.

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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.

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 cm⁻¹ (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 cm⁻¹. 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 ( ) the boron (¹⁰B + ¹¹B) content was nearly constant, see Fig. 2, with the value of (1.9 ± 0.3) × 10²¹ B cm⁻³, 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 B-content in the gas phase [18, 19, 23, 27].

<figure><img src="image_4.png" /></figure>

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.

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.

<figure><img src="image_5.png" /></figure>

Figure 3. AFM 3D reconstruction of the surface of NCD (A) and NCD-B (3,000 ppm, B) film. NTEGRA Prima NT MDT system (Ireland).
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 µm² to 2,510 ± 700 µm²). 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 with alpha<sub>v</sub> chain (Fig. 4A–C), 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. 4D–F).

<figure><img src="image_6.png" /><figcaption>Figure 4. Immunofluorescence of alpha<sub>v</sub> 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 µm</figcaption></figure>

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. 4G–I). 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 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).
<table><thead><tr><th>material/DT (hours)</th><th>days 1 to 3</th><th>days 3 to 7</th></tr></thead><tbody><tr><td>PS</td><td>29.8 ± 2.3*</td><td>36.6 ± 0.7</td></tr><tr><td>glass</td><td>25.1 ± 1.2</td><td>41.5 ± 1.0<sup>PS</sup></td></tr><tr><td>NCD</td><td>21.3 ± 1.1<sup>PS</sup></td><td>36.2 ± 0.5<sup>glass</sup></td></tr><tr><td>NCD-B</td><td>27.4 ± 2.3</td><td>33.0 ± 0.3<sup>PS, glass, NCD</sup></td></tr></tbody></table>

* 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.

Table 1 Cell population doubling time (DT) between days 1 to 3 or between days 3 to 7 after seeding of human osteoblast-like MG63 cells on polystyrene cell culture dishes (PS), microscopic glass slides (Glass), nanocrystalline diamond films (NCD) and boron-doped NCD films (NCD-B).

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).

<figure><img src="image_7.png" /></figure>

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.

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 hydrophilicity of O-terminated surfaces (contact angle about 20–35°). 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 adhesion mediating 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.

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 85-90°. 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 20-35° [7-9, 32]. It is known that on hydrophilic surfaces, the cell adhesion mediating 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, polyvinylidifluoride 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 boron-containing 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 more brightly stained for osteocalcin, an important extracellular matrix glycoprotein involved in bone tissue mineralization (Fig. 4J-L).
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 × 10⁻¹⁴ 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 cm⁻³ are nearly metallic, i.e., close to the Mott's 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 cell material 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 N° 204/06/0225 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.

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