Improved adhesion and growth of human osteoblast-like MG 63 cells onbiomaterials modified with carbon nanoparticles
L. Bacakova a,⁎, L. Grausova a, J. Vacik b, A. Fraczek c, S. Blazewicz c,A. Kromka d, M. Vanecek d, V. Svorcik e
a Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, CZ 142 20 Prague 4-Krc, Czech Republicb Nuclear Physics Institute, Academy of Sciences of the Czech Republic, CZ 250 68 Rez near Prague, Czech Republic
c AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Biomaterials, Al. Mickiewicza 30, 30-059, Cracow, Polandd Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnicka 10, CZ 162 53 Prague 6, Czech Republic
e Department of Solid State Engineering, Institute of Chemical Technology, Technicka 5, CZ 166 28 Prague 6, Czech Republic
Available online 18 July 2007
Abstract
Three types of materials modified with carbon particles were prepared: (1) carbon fibre-reinforced carbon composites (CFRC), materialspromising for hard tissue surgery, coated with a fullerene C60 layer, (2) terpolymer of polytetrafluoroethylene, polyvinyldifluoride andpolypropylene mixed with 4 wt.% of single- or multi-walled carbon nanotubes and (3) nanostructured or hierarchically micro- and nanostructureddiamond layers deposited on silicon substrates. The materials were seeded with human osteoblast-like MG 63 cells (density from 8500 cells/cm2
Carbon nanoparticles, such as fullerenes, nanotubes andnanodiamonds, are considered as promising building blocks forthe construction of novel nanomaterials for emerging industrialtechnologies, such as molecular electronics, advanced optics orstorage of hydrogen as a potential source of energy [1]. Inaddition, they are considered as promising materials forbiomedical applications, such as photodynamic therapy against
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tumors and infectious agents, quenching oxygen radicals,biosensor technology, simulation of cellular components, suchas membrane pores or ion channels, as well as controlled drugor gene delivery, particularly targeting the mineralized bonetissue [2–6]. Despite these exciting perspectives, relativelylittle is known about the influence of carbon nanoparticlespresent on the biomaterial surface on the adhesion and growthof cells.
In this paper, we present the influence of three types ofcarbon nanoparticle-modified materials on the adhesion andgrowth of cells. In the first set of experiments, a layer offullerenes C60 was deposited on carbon fibre-reinforced carboncomposites, i.e. materials which are promising for hard tissuesurgery [7]. The second set of experiments was carried out on aterpolymer of polytetrafluoroethylene, polyvinyldifluoride andpolypropylene mixed with single- or multi-walled carbonnanotubes, and finally, diamond layers (nanostructured orhierarchically micro- and nanostructured) for potential bioma-terial coating were prepared on a silicone substrate. Thesecarbon nanoparticle-containing materials were seeded withhuman osteoblast-like MG 63 cells, and the adhesion andsubsequent growth of these cells was investigated. Weobserved that all tested carbon nanoparticle-containing materi-als gave good support to adhesion and growth of bone-derivedcells, and they can be considered as promising for constructionof bone implants and bone tissue engineering.
2. Material and methods
2.1. Coating of carbon fibre-reinforced carbon composites(CFRC) with a fullerene layer
Two-dimensionally reinforced CFRC were prepared at theInstitute of Rock Structure and Mechanics, Acad. Sci. CR,Prague [7]. Commercially available woven fabric (made ofcarbon fibres Toray T 800) was arranged in layers, infiltratedwith a carbon matrix precursor (phenolic resin UMAFORMLE, Synpo Ltd., Pardubice, CR), pressed, cured, carbonised at1000 °C, and finally graphitised at 2200 °C. In order todecrease the material surface micro-scale roughness, which hasbeen shown to be non-appropriate for spreading and subse-quent growth of bone-derived cells [7], the CFRC were groundusing metallographic paper of 4000 grade. Thin fullerene (C60)layers, deposited on the CFRC, were prepared by evaporationof C60 in the Leybold Univex-300 vacuum system (roomtemperature of the substrates, deposition rate ≼1 Å/s,temperature of C60 evaporation in the Knudsen cells 400 °C,time of deposition about 15 min, thickness of the fullerenelayers b100 nm). The Raman analysis confirmed that thefullerene films were prepared with high quality and with nofragmentation or graphitization of C60 (Fig. 1A). One third ofthe CFRC samples (3×3 cm) was protected against fullerenecoating by a mask in order to achieve regionally selectivegrowth of the fullerene layer. The other completely uncoatedCFRC samples as well as tissue culture polystyrene (TCPS)dishes (Gama, Trhove Sviny, CR) were used as controlmaterials.
2.2. Preparation of carbon nanotube–polymer composites
Five grams of a terpolymer of polytetrafluoroethylene,polyvinyldifluoride and polypropylene (PTFE/PVDF/PP, den-sity of 1600 g/dm3, Aldrich Chemical Co., U.S.A.) weredissolved in 50 ml of acetone. Single-wall carbon nanohorns(SWNH) or high crystalline electric arc multi-wall nanotubes(MWNT-A; both from NanoCraft Inc., Renton, U.S.A.) weremixed with acetone in a sonicator for 5 min, and then with theterpolymer solution for 15 min to a concentration of 4 wt.%. Themixtures were then poured on to Petri dishes and left toevaporate the solvent. The surface wettability of the compositeswas studied by water drop contact angle measurements (DSA 10Kruss). The roughness was determined by surface profilometry(Hommel Tester T500, Hommelwerke Co., Germany).
2.3. Diamond layers for potential biomaterial coating
Nanocrystalline diamond (NCD) films were grown on (100)oriented silicon substrates (12 mm in diameter) by a microwaveplasma enhanced CVD method in the ellipsoidal cavity reactor[8]. The silicon substrates were either mechanically lapped tothe root mean square (rms) roughness up to 300 nm or polishedto atomic flatness (rms roughness about 1 nm). Prior to thedeposition process, the substrates were mechanically seeded inan ultrasonic bath using 5–10 nm diamond nanoparticles for40 min. The nanoparticles were purchased under the name“NanoAmando®” from NanoCarbon Research Institute Co.,Ltd. (Faculty of Textile Science and Technology, ShinshuUniversity, Tokita, Ueda, Nagano, Japan; http://nano-carbon.com). Physical and chemical properties of these water-dispersedultrananocrystalline diamond particles were described in detailsearlier [9]. In order to minimize nanoparticle clustering, thesurface of these particles was passivated by oxygen containinggroups. As observed by dynamic light scattering measurements,our liquid suspension contained only a very small amount ofnanoparticle clusters of 70 nm in diameter. The nucleationprocedure was then followed by the growth step, provided at aconstant methane concentration (1% CH4 in H2) and at a totalgas pressure of 30 mbar. The substrate temperature was 860 °Cand was measured by the two-color pyrometer working atwavelengths of 1.35 and 1.55 mm. The silicon substrates wereovercoated with NCD film on both sides, i.e. on the top andbottom side, respectively. Thus, hermetic sealing of the Sisubstrate minimized any unwanted bio-chemical reaction, whileopened Si area could result in pollution and disturbance of thesubsequent cell experiments. Finally, the deposited NCD filmswere treated in oxygen plasma to enhance the hydrophiliccharacter of the diamond surface. The water drop contact anglewas approximately 20°. The film thickness was 330 nm on theatomically polished side, as calculated from optical measure-ments [10]. The Raman spectra, measured using a 514.5 nmexcitation wavelength laser, displayed one dominant peakcentered at wavenumber 1333 cm-1 (optical phonon indiamond), which confirmed the diamond character of thedeposited films [11]. In addition, X-ray photoelectron spectros-copy (XPS) determined that the ratio of carbon in sp3
Fig. 1. A. Raman analysis of a thin C60 layer deposited on a carbon-fibre reinforced composite (CFRC) substrate. The quality of the fullerene layer is confirmed by ahigh peak Ag(2) at wavenumber 1468 cm−1, low peaks Hg(7) and Hg(8) and absence of D (disorder, ∼1350 cm−1) and G (graphitic, ∼1600 cm−1) bands, which aresigns of fragmentation and graphitization of C60, respectively. B–D: Immunofluorescence staining of β-actin in osteoblast-like MG 63 cells on day 2 after seeding onCFRC (B), CFRC coated with a fullerene layer (C) and tissue culture polystyrene dish (D). Microscope Olympus IX 50, digital camera DP 70, obj. 40. Bar=100 μm.
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hybridization, characteristic for diamond, to the sp2 C was morethan 95%. As confirmed by atomic force microscopy (AFM),two types of samples were prepared: (a) nanostructured surfaceswith rms of 8.2 nm, and (b) surfaces with hierarchicallyorganized micro- and nanostructure (i.e., surfaces with rmsroughness up to 301 nm were further patterned with nano-scalefeatures of rms roughness as low as 7.2 nm).
2.4. Cell culture on carbon nanoparticle-containing materials
For the cell culture, the fullerene-coated CFRCs wererepeatedly rinsed in phosphate-buffered saline and inserted onthe bottom of polystyrene Petri dishes (diameter 5 cm; Gama,Trhove Sviny, CR). Sterilization was avoided in order to preventpossible damage to the fullerenes due to heat, irradiation orchemicals, and the samples were used for short-term cell cultureonly (i.e., 1–2 days). The carbon nanotube–polymer compo-sites were sterilized by the H2O2-plasma method (Sterrad 120,ASP, Johnson & Johnson, U.S.A.), and the diamond layers byhot air at 160 °C for 2 h. Both of the last-mentioned groups ofsamples were inserted into 24-well polystyrene multidishes(TPP, Switzerland; well diameter of 1.5 cm). All materials were
then seeded with human osteoblast-like MG 63 cells (EuropeanCollection of Cell Cultures, Salisbury, UK) in densities rangingfrom 8500 to 25,000 cells/cm2 and incubated for 1 to 8 days inDulbecco-modified Eagle Minimum Essential Medium (Sigma,U.S.A., Cat. No. D5648) supplemented with 10% fetal bovineserum (Sebak GmbH, Aidenbach, Germany) and gentamicin(40 mg/mL, LEK, Ljubljana, Slovenia) at 37 °C in a humidifiedair atmosphere containing 5% of CO2. The morphology wasevaluated in cells fixed with cold 70% ethanol (−20 °C, 5 min)and stained with propidium iodide (PI, 5 mg/ml, 5 min). Thisdye for nucleic acids stained the nuclei preferentially but thecytoplasmic part of the cells was also stained, though faintly.The PI-stained specimens were also used for counting cells inthe earlier culture intervals (day 1 to 3). In the later intervals(days 4 to 7), when the cells were confluent and formedmultilayered regions, the cells were detached with a trypsin-EDTA solution (Sigma, U.S.A, Cat. No. T4174) in phosphatebuffered saline (PBS) for 10 min at 37 °C, resuspended in thecell culture medium (see above) and analyzed in a ViCell XRanalyzer (Beckman Coulter, U.S.A). The cell numbers wereexpressed as cell population densities per cm2 and used forconstruction of growth curves. The cell population doubling
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time (DT) was calculated as DT=(t− to)log 2/log Nt–log Nto,where to and t represent earlier and later time intervals afterseeding, respectively, and Nto and Nt the number of cells atthese intervals. In addition, molecules participating in theprocess of cell adhesion and spreading (i.e., integrin-associatedproteins vinculin and talin as well as beta-actin, an importantcomponent of the cytoplasmatic cytoskeleton), differentiation(calcium-binding extracellular matrix glycoprotein osteocalcin)and immune activation (intercellular cell adhesion molecule-1,ICAM-1, a ligand for β2 integrin receptors on immunocompe-tent cells) were visualized by immunofluorescence staining andtheir concentration per mg of protein was evaluated semiquan-titatively by the enzyme-linked immunosorbent assay (ELISA)described in detail in [12–14].
2.5. Statistics
The results were expressed as means±S.E.M. obtained from4–12 samples for each experimental group. Statistical analyseswere performed using SigmaStat (Jandel Corporation). Multiplecomparison procedures were performed by the OneWayAnalysisof Variance (ANOVA), Student–Newman–Keuls method. The pvalues equal to or less than 0.05 were considered significant.
Fig. 2. Population density (A) and adhesion area (B) of osteoblast-like MG 63 cellsreinforced carbon composites (CFRC) and CFRC coated with a fullerene laypolytetrafluoroethylene, polyvinyldifluoride and polypropylene (Ter), terpolymer mcrystalline electric arc multi-wall nanotubes (MWNT-A). D: Growth curves of MGhierarchically organized micro-and nanostructure (Micro-Nano). Mean±S.E.M. frosignificance: TCPS, CFRC, Ter: p≤0.05 compared to the values on tissue culture p
3. Results and discussion
3.1. MG 63 cells on CFRC with a fullerene layer
Grinding with metallographic paper of 4000 grade loweredthe surface roughness of CFRC about twice. As measured by aprofilometer (Rank Taylor Hobson Ltd., England), the depar-tures of the roughness profile from the mean line (i.e., Ra
parameter) decreased from 6.5±1.8 μm to 3.5±0.6 μm, and themean spacing of the adjacent local peaks (parameter S)lengthened from 38±11 μm to 96±49 μm. The fullerenecoating did not significantly change this surface microrough-ness but created a nanostructured pattern on the pre-existingmicroarchitecture of the CFRC surfaces. On day 2 after seeding,the cell population density (Fig. 2A) on these surfaces waslower (19,222±664 cells/cm2) than that on the control uncoatedmaterial and TCPS (44,286±4155 cells/cm2 and 67,276±7287 cells/cm2, respectively), which could be due to the rela-tively high hydrophobicity of the non-functionalized fullerenes[15]. It is known that hydrophobic materials promote pre-ferential adsorption of cell non-adhesive proteins from theserum of the culture media, such as albumin. In addition, the celladhesion-mediating extracellular matrix (ECM) proteins may be
on day 2 after seeding on tissue culture polystyrene dish (TCPS), carbon fibre-er (CFRC+full). C: Growth curves of MG 63 cells on a terpolymer ofixed with 4 wt.% of single-wall carbon nanohorns (SWNH) or 4 wt.% of high63 cells on TCPS, a nanostructured diamond layer (Nano) and a layer with
m 4–12 measurements, ANOVA, Student–Newman–Keuls method. Statisticalolystyrene, pure CFRC and pure terpolymer.
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adsorbed in a relatively rigid state, thus their specific amino acidsequences were less accessible for cell adhesion receptors [12].On the other hand, the water drop contact angle, measured bya static method in a material-water droplet system using areflection goniometer [12], was not able to reveal hydropho-bicity of the fullerene layer. The contact angle was non-measurable due to a complete absorption of the water drop intothe fullerene layer, which suggested a certain non-compactnessor porosity of this layer. The contact angle of the non-coatedCFRC was 99.5±1.0o.
The release of fullerenes into the culture media and theircytotoxic action seemed to be less probable in our experiments.The cell attachment and spreading on the uncoated regions of thefullerene-modified CFRC, and also on the bottom of thepolystyrene dishes containing fullerene-coated samples, weresimilar as in the control polystyrene dishes without fullerenesamples (data not shown). At the same time, the fullerene layerwas resistant to mild wear, represented by swabbing with cotton,rinsing with liquids (water, phosphate-buffered saline, culturemedia) and exposure to cells and proteolytic enzymes used forcell harvesting. After these procedures and/or one-year-storageat room temperature in dark place, the Raman spectra (Fig. 1A)did not change significantly. In addition, the fullerene-coated
Fig. 3. Immunofluorescence staining of β-actin in osteoblast-like MG 63 cells on dayand polypropylene (A), terpolymer mixed with 4% of single-wall carbon nanohornspolystyrene dish (D). Microscope Olympus IX 50, digital camera DP 70, obj. 20. B
CFRC surfaces were stronger and less prone to release carbonparticles, which is an important limitation of the potentialbiomedical use of CFRC [7]. Moreover, the spreading area ofcells on the fullerene-coated samples amounted to 3,182±670 mm2, while on both control surfaces it was only 1,888±400and 1,300±102 mm2 (Fig. 2B). This could be explained by thelow cell population density on the fullerene layer, whichprovided the cells with more space for them to spread. On theother hand, the nanostructure of the fullerene layer mightenhance the adsorption of vitronectin, i.e. an extracellular matrixprotein mediating preferential adhesion of osteoblasts over othercell types [16,17]. TheMG 63 cells on the fullerene layer formedfine dot-like vinculin-containing focal adhesion plaques and afine network of actin microfilaments (Fig. 1B–D), which is asign of cell vitality and effective binding between cell adhesionreceptors and extracellular matrix molecules adsorbed on thematerial surface [12–14,16,17].
3.2. MG 63 cells on carbon nanotube–polymer composites
Similarly as on the fullerene layer, the cells on PTFE/PVDF/PP mixed with single-wall carbon nanohorns (SWNH) or multi-wall nanotubes (MWNT-A) were well spread, polygonal, and
3 after seeding on a terpolymer of polytetrafluoroethylene, polyvinyldifluoride(B) or high crystalline electric arc multi-wall nanotubes (C) and tissue culturear=100 μm.
Fig. 4. Concentration of vinculin (A), talin (B), osteocalcin (C) and ICAM-1 (D) in osteoblast-like MG 63 cells on day 8 after seeding on the pure terpolymer ofpolytetrafluoroethylene, polyvinyldifluoride and polypropylene (Ter), terpolymer mixed with 4 wt.% of single-wall carbon nanohorns (SWNH) or 4 wt.% of highcrystalline electric arc multi-wall carbon nanotubes (MWNT-A) and tissue culture polystyrene (TCPS). Measured by ELISA per mg of protein; absorbance values ofcells from the modified terpolymers were expressed in % of values obtained from control cells grown on the unmodified terpolymer. Mean±S.E.M. from 4–12measurements, ANOVA, Student–Newman–Keuls method. Ter, TCPS: p≤0.05 compared to the values on the pure terpolymer and tissue culture polystyrene.
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contained distinct beta-actin filament bundles, whereas mostcells on the pure terpolymer were less spread or even round andclustered into aggregates (Fig. 3). The enzyme-linked immuno-sorbent assay (ELISA) revealed that the cells on the materialwith SWNH contained a higher concentration of vinculin andtalin, i.e. components of focal adhesion plaques (by 56±21%and 35±6%, respectively) in comparison with the values in cellson the pure terpolymer (Fig. 4A, B). The concentration ofosteocalcin, a marker of osteogenic cell differentiation, wassimilar in cells on both the SWNH-modified and the pureterpolymer, and also TCPS (Fig. 4C). However, in cells onsamples with MWNT-A, the concentration of osteocalcin wassignificantly lower (by 14±2% and 10±1%) than the concen-tration in cells on the pure terpolymers and TCPS (Fig. 4C). Thiscould be explained by a higher proliferation activity of thesecells, which might delay the process of cell differentiation [13].While on day 1 after seeding the initial cell population densitywas similar on the terpolymers with or without MWNT-A(40,340±5765 cells/cm2 and 35,489±5833 cells/cm2, respec-tively), on day 7, the cells on theMWNT-A-modified terpolymerreached a density 4.5 times higher (228,029±10,050 cells/cm2)than the density on the unmodified samples (50,300±5400 cells/cm2, Fig. 2C). The concentration of ICAM-1, an adhesion
molecule of the immunoglobulin superfamily and a marker ofcell immune activation, was not increased in cells on bothnanotube-modified terpolymers (Fig. 4D).
The improved adhesion and growth of MG 63 cells on thenanotube-modified terpolymer could be attributed to changes inits surface roughness (Ra=1.03±0.52 mm and Ra=1.08±0.30 mm in SWNH-and MWNT-A-modified terpolymer,respectively, compared to Ra=0.20±0.04 mm in the pureterpolymer) rather than to its surface wettability, which remainedunchanged and relatively low (contact angle 105.2±2.12° and101.0±1.35° in SWNH-and MWNT-A-modified material incomparison with 100.0±3.9° in the pure terpolymer).
3.3. MG 63 cells on diamond layers
On nanostructured diamond layers, the number of initiallyadhered cells (4269±462 cells/cm2) on day 1 after seeding wassimilar to that found on the control TCPS (3807±342 cells/cm2),whereas on the layers with a combined micro- and nanoarchi-tecture, this number was significantly lower (2648±187 cells/cm2). In addition, the cells on the latter samples were distributednon-homogeneously (Fig. 5). However, from day 1 to 3 afterseeding, the cells on the hierarchically micro- and nanostructured
Fig. 5. Propidium iodide staining of osteoblast-like MG 63 cells on day 1 (A–C) and 7 (D–F) after seeding on a nanostructured diamond layer (A, D), a layer withhierarchically organized micro-and nanostructure (B, E) and a tissue culture polystyrene dish (C, F). Microscope Olympus IX 50, digital camera DP 70, obj. 10.Bar=200 μm.
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layers, which are considered to resemble the architecture ofnatural tissues [18], showed the quickest proliferation. Theirdoubling timewas 23.4 h, whereas on the nanostructured layers, itwas 56.7 h, and on TCPS, the cells still remained in the lag phaseand have not yet started their proliferation (Fig. 2D). As a result,on day 3 after seeding, the cells on the diamond layers with acombined micro-and nanostructure reached the highest cell pop-ulation density (10,951±1349 cells/cm2 compared to 7678±721 cells/cm2 and 4047±621 cells/cm2 on nanostructureddiamond and TCPS, respectively). On day 7, the cell numbersboth on the diamond layers and on TCPS leveled out, reaching
from 216,624±10,236 cells/cm2 to 255,102±18,808 cells/cm2
(Figs. 2D and 5).
4. Conclusion and further perspectives
The tested carbon nanoparticle-containing materials sup-ported adhesion and growth of bone-derived cells. The carbonnanoparticle layers used in this study, such as fullerene layersand especially hard diamond coatings, could be used for surfacemodification of bone implants (e.g., bone-anchoring parts ofjoint prostheses or bone replacements) in order to improve their
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integration with the surrounding bone tissue. Polymers mixedwith carbon nanoparticles could serve for the construction ofthree-dimensional porous scaffolds for bone tissue engineering.These particles could create the nanostructure of the pore wallsand thus enhance the ingrowth of bone tissue and its miner-alization [18,19]. The adhesion of osteoblasts may be furtherenhanced by the derivatization of carbon nanoparticles withchemical functional groups or oligopeptidic ligands for celladhesion receptors [15,19,20]. However, more and deeperstudies on the potential cytotoxicity, immunogenicity, mutage-nicity and carcinogenicity of carbon nanoparticles [15] areneeded for these purposes and are in progress.
Acknowledgements
This study was supported by the Grant Agency of the CzechRepublic (Grants No. 204/06/0225 and 202/05/2233). We alsowish to express our thanks to Mrs. Ivana Zajanova (Inst.Physiol., Acad. Sci. CR) for her excellent technical assistanceand Mr. Robin Healey (Czech Technical University, Prague) forthe language revision of the manuscript.
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