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Adhesion, growth and differentiation of human osteoblast-like cells on thermally oxidized Ti and TiNb substrates

Bacakova L., Vandrovcova M., Jirka I., Novotna K., Stary V.

Engineering of Biomaterials

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2013

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Vol. 16, no. 122-123 spec. iss.

75--76

EN

Metallic materials are essential for construction of load-bearing bone implants, such as replacements of hip, knee and other joints. For these applications, modern materials used in advanced tissue engineering, e.g. resorbable porous or fibrous polymeric and ceramic scaffolds are mechanically insufficient, even if these materials enable the ingrowth of bone cells and bone tissue formation. Therefore, searching for new metallic materials and their surface modifications improving their biocompatibility and osseointegration is still desirable. As first metallic materials for bone implantation, AISI 316L stainless steel and Co-Cr alloys were used. In the 1950’s, the Ti-6Al-4V alloy was developed. These materials are still frequently used for construction of implants because of their relatively low price [1]. However, these materials are biomechanically incompatible with the bone tissue, because their Young’s modulus is markedly higher (110-220 GPa) than that of the bone (10-40 GPa). Implants with high stiffness take over a considerable part of load from the bone. This phenomenon, referred as “stress-shielding effect”, can then cause the bone resorption and loosening of the implant [1]. Also chemical compositions of the mentioned metallic materials limit their biocompatibility, because they contain harmful elements as V, Al, Co and Cr, which can act as cytotoxic, catabolic, immunogenic or even carcinogenic agents [2,3], and can also cause serious neurological problems [4]. Due to these adverse reactions, new types of Ti-alloys have been developed, namely low-rigidity β-type Ti alloys, containing non-toxic and non-allergenic elements (Nb, Ta, Zr etc.) and having good mechanical properties and workability [4,5]. The goal of this study was to evaluate the adhesion, growth and differentiation of osteoblast-like MG-63 and Saos-2 cells on titanium-niobium alloys after their surface modification by thermal oxidation at two different temperatures (165°C and 600°C). Pure titanium (treated at 165°C and 600°C) and polystyrene culture dishes (PS) were used as control materials. Possible immune activation of the cells was tested by the levels of TNF-alpha secreted to the cell culture media by murine macrophage-like RAW 264.7 cells cultured on the tested materials. On samples treated at 165°C, the number of initially adhered MG-63 and Saos-2 cells was on an average higher on TiNb than on Ti or PS. On day 3 after seeding, the trend of the cell numbers remained similar, with the highest cell density found on TiNb. Similar results were obtained on samples treated at 600°C, where the difference in cell number between TiNb and Ti samples became more apparent. This cell behavior could be attributed to a less negative zeta potential on TiNb samples. In samples treated at 165°C, the zeta potential of TiNb surfaces was on the average less negative than on Ti surfaces, but this difference was not significant. However, in samples treated at 600°C, this difference became much more pronounced, which was probably due to the formation of T-Nb2O5 phase on the surface of the TiNb samples. This phase was of a crystalline structure, while at 165°C, the structure of Nb2O5 was amorphous. In addition, both Ti and TiNb samples treated at 600°C contained rutile, while the samples treated at 165°C contained anatase in their surface layer. It has been shown that rutile films deposited on PEEK enhanced the adhesion and growth of osteoblasts more than anatase films [6]. This phenomenon was explained by an increase in the material surface wettability, and particularly to the presence of –OH- groups on the rutile films. The expression of collagen I and osteocalcin, i.e. an early and late marker of osteogenic cell differentiation, respectively, was higher on Ti than on TiNb samples, and this difference was more apparent in samples treated at 165°C. At the same time, no considerable immune activation of the cells on all tested samples was found. The production of TNF-α by RAW 264.7 cells was very low in comparison with cells grown in the presence of bacterial lipopolysaccharide, and also significantly lower than on untreated samples. These results indicate that TiNb substrates increased the proliferation of human bone cells, while pure Ti rather supported the cell differentiation. The effect on cell proliferation was more apparent in samples treated at the higher temperature (600°C), while the effect on cell differentiation was more pronounced at the lower temperature (165°C). None of the tested samples induce significant cell proinflammatory activation. Thus, all tested samples are suitable as carriers for bone cells; only an appropriate application (i.e., requiring either proliferation or quick differentiation of osteogenic cells) should be selected.

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Human osteoblast-like MG 63 cells in cultures on carbon fibre-reinforced carbon composites coated with zirconium nitride

Paul J., Bacakova L., Douderova M., Stary V., Vyskocil J., Lisa V., Ślósarczyk A., Zima A.

Engineering of Biomaterials

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2007

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Vol. 10, no. 67-68

5-8

EN

Zirconium nitride is considered as a promising material for strengthening for surface of various materials, especially those designed for hard tissue surgery. In this study, five groups of materials were prepared: non-modified carbon fibre-reinforced carbon composites (CFRC), CFRC ground with metallographic paper, non-ground CFRC with a layer of ZrN deposited by magnetron sputtering, ground CFRC with a ZrN layer deposited by the arc technique, and ground CFRC with a ZrN layer deposited by a magnetron. We found that all samples gave good support for the adhesion and growth of human-osteoblast-like MG 63 cells, though the cell numbers on these materials were often lower than on standard cell culture polystyrene dishes and microscopic glass coverslips. Nevertheless, ZrN films can be considered as suitable materials for surface modification of bone implants in order to improve their mechanical properties and their integration with the surrounding tissue.

3

The study of the surface properties of C/C composites

Tolde Z., Stary V., Douderova M.

Inżynieria Biomateriałów

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2006

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R. 9, nr 58-60

16-17

4

Adhesion, growth and differentiation markers in human osteoblast-like cells cultured on surface-modified metallic materials designer for bone implants

Bacakova L., Kabatova J., Lisa V., Stary V., Fencl J.

Inżynieria Biomateriałów

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2006

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R. 9, nr 58-60

1-3

EN

A series of metallic materials with different surface treatments were prepared: pure machined titanium (T), titanium polished by diamond paste (TL), machined Ti6Al4V alloy (TS), Ti6Al4V alloy polished by diamond paste (TSL), Ti5Al2.5Fe alloy treated by electro-erosion (A) and Ti5Al2.5Fe plasma-sprayed with Ti (PL). The materials were seeded with human osteoblast-like cells MG 63. One day after seeding, the highest cell numbers were obtained on the samples of medium surface roughness (T and TS; Ra 0.63-0.30 um and 0.89-0.57 um, respectively). From day 1 to 4, the cell proliferation was the quickest on the samples with the lowest surface roughness (TL and TSL; Ra 0.17-0.13 for both materials). The cells on TL also contained the highest concentration of integrin adhesion molecules with alpha V chain, i.e. receptors for vitronectin and fibronectin. One day 8 after seeding, the cell on all metallic samples as well as tissue culture polystyrene reached similar population densities. The cells on electro-eroded Ti5Al2.5Fe (samples A; Ra 15.27-0.74 um) contained the highest concentration of osteocalcin and osteopontin, i.e. markers of osteoblastic differentiation. Thus, the latter newly developed material could be considered as promising for construction of bone implants well anchored in the surrounding bone tissue.

5

3D evaluation of the surface roughness using stereo images made in SEM - influence on osteoblast cell growth

Douderova M., Stary V., Tolde Z., Bacakova L.

Inżynieria Biomateriałów

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2006

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R. 9, nr 58-60

13-14

6

Influence of surface properties of carbon based materials on cell spreading

Douderova M., Bacakova L., Stary V.

Inżynieria Biomateriałów

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2004

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R. 7, nr 37

17-17

EN

Carbon materials are generally well tolerated by animal cells. The possibility of applying carbon fiber reinforced carbon (CFRC) composite materials is given by their excellent biocompatibility and porosity, coupled with a modulus which can be tailored to be similar to that of bone. This makes them an attractive material for bone plates and implants in orthopaedic and dental surgery. It is known that the volume properties of a material usually have little or no influence on the surrounding living tissue cells. In general, biocompatibility is controlled mainly by the interface between biomaterial and living tissue cells. The literature and our study indicate that the interaction at the interface is specifically controlled by the surface morphology (i.e. especially by surface roughness), and by the chemical state of the surface - by hydrophobia (wettability), free chemical bonds and present chemical groups, etc. Never-theless, biocompatibility can be improved by a suitable change of these parameters. There are several possible methods for influencing the roughness and chemical state of the surface. One way to change the surface properties is by preparing a suitable coating. The properties of the surface are controlled by process technology, and the grinding and polishing of the substrate can be used for roughness control. Till now we studied the influence of the surface on the cell adhesion and on the rate of the cell growth. There, we have studied the influence of a surface coating of CFRC using a several types of layers on the base of carbon. In our present contribution we continue in this work using the surfaces of CFRC in native and polished states, both covered by layers of amorphous carbon, or titanium with carbon or pyrolitic graphite. The vascular smooth muscle cells were grown on these surfaces. The purpose of this paper is to find the influence of the surface on the important parameter of tissue cell growth - the spreading of cells. The main topic of this work is therefore the measurement and statistical evaluation of the cell area on the various types of surfaces. It will be shown, that the cell spreading is strongly influenced by various surface roughness and also its chemical state.

7

Adhesion, differentiation and immune activation of human osteogenic cells in cultures in carbon-fibre reinforced carbon composites

Bacakova L., Stary V., Glogar P., Lisa V.

Inżynieria Biomateriałów

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2003

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R. 6, nr 30-33

8-9

8

Osteoblast-like MG63 cells in cultures on carbon fibre-reinforced carbon composites

Bacakova L., Stary V., Hornik J., Glogar P., Lisa V., Kofronova O.

Inżynieria Biomateriałów

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2001

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R. 4, nr 17-19

11-12

9

Measurement of surface properties of pyrolytic graphite for biomedical applications

Stary V., Bacakova L., Glogar P., Hornik J., Jirka I., Svorcik V.

Inżynieria Biomateriałów

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2001

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R. 4, nr 17-19

10-11

10

Engineering properties of pyrolytic graphite surfaces

Hornik J., Chmelik V., Nevoralova M., Stary V.

Inżynieria Biomateriałów

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2001

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R. 4, nr 17-19

13-14

11

Improved biocompatibility of carbon-fibre-reinforced carbon composites in vitro after their polishing and coating with a carbon-titanium layer

Bacakova L., Stary V., Kofrooova O.

Inżynieria Biomateriałów

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1998

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R. 1, nr 4

22--24

EN

The surface of unidirectionally reinforced carbon-carbon composites was modified either by polishing or coating with a carbon-titanium layer. In culture conditions; the composites were seeded with vascular smooth muscle cells derived from the rat aorta. On both types of modified samples, the number of initially adhered cells, degree of their spreading and their subsequent growth were significantly higher than on untreated samples, and in the case of carbon-titanium-covered composites, also higher than on standard plastic culture dishes Sterilin. These results obtained in vitro suggest possible good biointegration of the polished and carbon-titanium-covered carbon-carbon composites with the surrounding tissue in situ after their use in transplantation medicine for the construction of artificial implants.

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Adhesion and growth of cells in culture on carbon-carbon composites with different surface properties

Bacakova L., Stary V., Glogar P.

Inżynieria Biomateriałów

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1998

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R. 1, nr 2

3--5

EN

The biocompatibility of unidirectionallv reinforced carbon-carbon composites (carbon fibre T300, phenolformaldehyde resin based matrix) with different surface roughness and chemical composition was tested in cell culture conditions. The surface of the composites was polished, covered with amorphous or pyrolytic carbon and seeded with rat aortic smooth muscle cells. Coating with amorphous carbon significantly lowered the number of initially adbered cells. In these samples, the surface roughness had no significant effect on the number of initially adbering cells nor their subsequent proliferation, In contrast, coating with pyrolitic carbon improved significantly both cell adhesion and growth, especially on the polished surfaces. In addition, the layer of pyrolitic carbon was more resistant to mechanical damage than the film of amorphous carbon. It is concluded that polished composites covered by pyrolytic carbon could be suitable for the future application in medicine and biotechnology.