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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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Potential activation of the immune system on metallic materials for bone implants

Stranavova L., Bacakova M., Novotna K., Bacakova L., Fencl J.

Engineering of Biomaterials

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2012

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Vol. 15, no. 116-117 spec. iss.

130--131

EN

Titanium and stainless steel are metallic materials that have been in use for a long time in orthopedics, traumatology and stomatology. These metals are strong, corrosion-resistant and biocompatible. However, metallic materials have some disadvantages in comparison with the natural bone, particularly their relatively high specific weight and toughness. For example, the Young's modulus of AISI316L stainless steel, Co-Cr alloys and Ti-6Al-4V alloy, i.e. materials frequently used for implantation into bone, ranges between 110-220 GPa, while the Young's modulus of bone tissue is 10-40 GPa [1]. In addition, these metals can release cytotoxic, allergenic and immunogenic ions, which can affect their biocompatibility [2, 3]. Implantation is a special type of transplantation process, in which the implant is inserted into the body, usually in order to replace an irreversibly damaged tissue. However, the immune system recognizes the implant as a foreign substance and attacks it with its effector mechanisms. Just as it can reject other types of transplants, the immune system can reject an artificial implant. To prevent rejection of an implant, it is important to study the potential activation of the immune system. This study has investigated the biocompatibility of samples made of pure titanium (according to quality standard ISO 5832-2) and corrosion-resistant steel (quality standards ISO 5832-1 and AISI 316L), obtained from Beznoska Ltd. (Kladno, Czech Republic), and the potential activation of the immune system by these materials. In addition to Fe, the steel samples contained C (max. 0.025 wt.%), Si (0.6 wt.%), Mn (1.7 wt.%), P (max. 0.025wt.%), S (max. 0.003 wt.%), Cr (17.5 wt.%), Ni (13.5 wt.%), Mo (2.8 wt.%), and Cu (max. 0.1 wt. %). The materials were used in the form of square samples (9x9 mm or 30x30 mm, thick¬ness 1 mm). Both the Ti samples and the steel samples were ground with SiO2. The surface of the steel samples was then treated by polishing with Al2O3 paste (grain size up to 1 um), while the surface of the Ti samples, i.e. a material not suitable for polishing, was finished by brushing using another type of Al2O3 paste with slightly larger grains. Thus, the surface of the steel samples was finally smoother and glossy, while the Ti surface was rougher and matte. For the in vitro biocompatibility tests, human osteoblast-like MG 63 cells (European Collection of Cell Cultures, Salisbury, UK) were used. The smaller samples (9x9 mm) were inserted into polystyrene 24-well cell culture plates (TPP, Trasadingen, Switzerland; well diameter 1.5 cm). Each well contained 25 000 cells (approx. 14 150 cells/cm2) and 1.5 ml of Dulbecco's Modified Eagle Minimum Essential Medium (DMEM; Sigma, USA, Cat. No. 10270-106) supplemented with 10% foetal bovine serum (FBS; Gibco, Cat. No. 10270-106) and gentamicin (40 /jg/ml, LEK, Slovenia). These samples were used for evaluating the size of the cell spreading area (day 1), and for evaluating cell shape and cell viability (days 1, 4 and 7 after seeding). The size of the cell spreading area was measured using Atlas Software (Tescan Ltd., Brno, Czech Republic). The viability of the cells was determined by the LIVE/ DEAD viability/cytotoxicity kit for mammalian cells (Invitrogen, Molecular Probes, USA). The larger samples (30x30 mm) were inserted into GAMA polystyrene dishes (diameter 5 cm; GAMA Group Joint-Stock Company, Ceske Budejovice, Czech Republic) and seeded with 300 000 cells/dish (approx. 15 300 cells/cm2) suspended in 9 ml of the above mentioned culture medium. These samples were used for evaluating the cell number on days 1, 4 and 7 after seeding, using a Beckman Vi-CELL XR Cell Analyser automatic cell counter. For the in vitro analysis of markers of osteogenic differentiation and cell immune activation, human osteoblast-like MG 63 cells (European Collection of Cell Cultures, Salisbury, UK) were used. The samples (9x9 mm) were inserted into polystyrene 24-well cell culture plates (TPP, Trasadingen, Switzerland; well diameter 1.5 cm). Each well contained 25 000 cells (approx. 14 150 cells/cm2) and 1.5 ml of Dulbecco's Modified Eagle Minimum Essential Medium (DMEM; Sigma, USA, Cat. No. 10270-106) supplemented with 10% foetal bovine serum (FBS; Gibco, Cat. No. 10270-106) and gentamicin (40 jg/ml, LEK, Slovenia). The cells were cultured for 1, 4, or 7 days at 37°C in a humidified atmosphere of 5% of CO2 in the air. On day 4 after seeding, the medium was changed; one half of the samples contained standard medium DMEM with 10% foetal bovine serum and gentamicin (40 jg/ml) mentioned above, and the second half contained osteogenic medium, i.e. the standard medium further supplemented with ß-glycerophosphate, L-glutamin, ascorbic acid, dihydroxyvitamin D3, dexamethason, 10% foetal bovine serum and gentamicin (40 jg/ml). Using an Enzyme-Linked ImmunoSorbent Assay (ELISA), we measured the concentration of the Inter¬cellular Adhesion Molecule-1 (ICAM-1, a marker of cell immune activation) and osteocalcin (a marker of osteogenic cell differentiation). These measurements were performed in homogenates of cells on days 4 and 7 after seeding, and the concentration of both markers was measured per cell or per mg of protein. On day 7, the amount of osteocalcin was measured and compared in cells cultured in the standard and osteogenic media. We also measured TNF-а and IL- 1ß, i.e. other markers of cell immune activation. These cytokines are important mediators of the inflammatory response, and they are involved in a variety of cellular activities, including cell proliferation and differentiation. We measured the secretion of these markers into the cell culture medium in murine macrophage-like RAW264.7 cells (American Type Culture Collection, Manassas, VA). The samples (9x9 mm) were inserted into polystyrene 24-well cell culture plates (TPP, Tra- sadingen, Switzerland; well diameter 1.5 cm). Each well contained 30,000 (approx. 16 980 cells/cm2) cells and 1.5 ml of the culture medium. RAW 264.7 cells were cultured in the RPMI-1640 medium (Sigma; 10% fetal bovine serum, 40 jg/mL gentamicin). After 7 days of cultivation, the cell culture medium was collected and used for measuring the concentration of TNF-а and IL-1ß by a sandwich ELISA using commercially available kits. A mouse TNF-а kit and an IL- 1ß Quantikine ELISA kit were used for the RAW 264.7 cells. Both kits were purchased from R and D Systems (Minneapolis, MN) and used according to the manufacturer's protocol. The results indicated that the number of initially adhering MG 63 cells on day 1 after seeding was significantly lower on the titanium (5320±390 cells/cm2) and on the stainless steel (4110±370 cells/cm2) than on the control polystyrene culture dishes (7740±350 cells/cm2). However, on day 4 after seeding, the cell population density on both metallic materials became significantly higher than on the control polystyrene dishes (75200±2890 cells/cm2 on Ti and 90 870±2350 cells/cm2 on steel vs. 56440±1180 cells/cm2 on polystyrene). This suggests faster cell proliferation on both metallic materials than on polystyrene. At the same time, the cell number on the stainless steel samples was significantly higher than on the Ti samples. On day 7, the differences in the number of adhered cells on the two metals and on the control polystyrene substrate was on an average similar (from 328780±680 cells/cm2 to 362200±760 cells/cm2). The cell viability on all tested materials was almost 100% in all culture intervals. The morphology of the cells adhered on the studied materials was similar to the morphology of the cells on the control polystyrene dishes, i.e. the cells were mostly flat and polygonal, and the size of their cell spreading areas was similar on all tested materials. The cells were distributed homogeneously on the entire material surface, and on day 4 they started to form confluent cell layers. On day 4, we measured the amount of ICAM-1 by the ELISA test. This immunoglobulin molecule is typically expressed on cells of the immune system, but it is also expressed on other cell types, including MG 63, during their immune activation, e.g. by an artificial growth support. In this case, ICAM-1 molecules on cells are bound byß2-integrin receptors on inflammatory cells (for a review, see [4]). Surprisingly, titanium seemed to be more immunogenic than stainless steel, which was indicated by a higher concentration of ICAM-1 per cell and mg of protein in cells on day 4 after seeding. However, on day 7, there was no difference between the concentrations of ICAM-1 per cell and mg of protein in cells on titanium and on stainless steel. The second molecule that we measured was osteocalcin, a calcium-binding extracellular matrix glycoprotein, an important marker of the bone formation process. The concentration of osteocalcin on day 4 in the standard culture medium was higher in MG 63 cells on the titanium and stainless steel than on the control polystyrene samples. This could be explained by the fact that the metals are harder than polystyrene. It is known that harder substrates promote osteogenic cell differentiation, while softer substrates direct the cell differentiation towards neural or muscle phenotype [5]. In addition, the osteogenic differentiation was further supported by the osteogenic medium, as indicated by a higher concentration of osteocalcin in cells grown in this medium compared to cells in the standard medium on day 7 after seeding. On day 7 after seeding murine macrophage-like RAW264.7 cells on the tested materials, the concentration of TNF-а in the culture medium ranged on an average from 57.10 to 79.39 pg per 2000000 cells. The concentration of TNF-а in the medium from Ti and Fe was significantly higher than in the medium from the control polystyrene dishes. The highest value (79.39 pg/2000000 cells) was found in the medium taken from RAW264.7 cells on Ti. The second molecule that we tested was IL-1ß. No significant differences in the concentration of IL-1ß were detected in the culture medium obtained from RAW264.7 cells on all tested materials. In other words, neither type of metallic material, i.e. Ti and Fe, evoked significantly higher production of IL-1ß by RAW 264.7 cells than standard polystyrene cell culture dishes. It can be concluded that the tests of biocompatibility and immune activation confirmed that titanium and stainless are promising for construction of bone implants and for good integration with the surrounding bone tissue.

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Application of cellulose-based biomaterials in vascular tissue engineering - a review and our experience

Bacakova L., Novotna K., Parizek M., Havelka P., Sopuch T.

Engineering of Biomaterials

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2012

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Vol. 15, no. 116-117 spec. iss.

128--130

EN

Artificial vascular replacements used in current clinical practice are fabricated from polyethylene terephthalate (PET, e.g. Dacron) orpolyterafluoroethylene (PTFE, e.g. Teflon). Older materials used earlier for constructing vascular prostheses are polyamide (Nylon), polyvinyl alcohol (Ivalon) and polyacrylonitrile (Orlon). New promising materials include polyurethane and a wide range of biodegradable synthetic or nature-derived polymers, which are usually designed as temporary scaffolds for vascular cells forming a new regenerated blood vessel wall (for a review, see [1]). One of the nature-derived polymers is cellulose and its derivatives and composites with other materials. Cellulose is the most abundant biopolymer on Earth. It is a polysaccharide consisting of a linear chain of several hundred to over ten thousand ß(1\to 4) linked D-glucose units [2,3]. Cellulose is the structural component of the primary cell wall of green plants, many forms of algae and the oomycetes. In plant cells, cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes [4]. Some species of bacteria secrete cellulose to form biofilms. For industrial use, cellulose is mainly obtained from wood pulp and cotton. For tissue engineering applications, bacterial cellulose has been predominantly used, mainly that synthesized by Acetobacterxylinum. Bacterial cellulose is identical to plant cellulose in chemical structure, but it can be produced without contaminant molecules, such as lignin and hemicelluloses, and does not require intensive purification processes. In addition, it is remarkable for its mechanical strength, its ability to be engineered structurally and chemically at nano-, micro-, and macroscales, its biocompatibility and chemical and morphologic controllability [5]. Bacterial cellulose has been used for experimental engineering of bone tissue [6], cartilage [7], skin [8], heart valve [9], and also for urinary reconstruction and diversion [10]. One of the first attempts at vascular tissue engineering was made with cellulose fibers, which were used for constructing three-dimensional vascularized tissue in vitro. These fibers were immobilized with fibronectin in order to improve cell adhesion, and were seeded with bovine coronary artery smooth muscle cells. These cells proliferated on the scaffolds and, after they formed multilayers on the fibers, the fibers were removed by enzymatic digestion using cellulase. The remaining smooth muscle cell aggregates maintained lumens after this procedure, and thus mimicked newly-formed blood vessels [11]. Similarly, three-dimensional nanofibrous scaffolds with micropores made of bacterial cellulose allowed attachment and proliferation of human saphenous vein smooth muscle cells on the surface and also in the inside of the scaffolds [12]. In addition, the mechanical properties of nanofibrous bacterial cellulose scaffolds, evaluated by the shape of the stress-strain response, were reminiscent of the properties of the carotid artery, most probably due to the similarity in architecture of the nanofibril network [13]. The adhesion and growth of vascular endothelial cells was also supported by cellulose-based scaffolds, namely by nanofibrous bacterial cellulose or cellulose acetate scaffolds, especially if these scaffolds were functionalized with RGD-containing oligopeptides, i.e. ligands for integrin adhesion receptors on cells [14, 15], or if they were combined with chitosan [16]. The angiogenic response to bacterial cellulose was also observed under in vivo conditions, i.e. after implantation of these scaffolds in the form of dorsal skinfold chambers into Syrian golden hamsters [17]. Cellulose has also been used for creating tubular structures designed for replacing small-caliber vessels. Hollow-shaped segments of bacterial cellulose were created with a length of 10 mm, an inner diameter of 3.0-3.7 mm and a wall thickness of 0.6 -1.0 mm. These grafts were used to replace the carotid arteries of eight pigs. After a follow-up period of 3 months, seven grafts (87.5%) remained patent, whereas one graft was found to be occluded. All patent grafts developed a single inner layer of endothelium with a basement membrane and a thin layer of collagen, followed by a concentric medial layer containing smooth muscle cells and cellulose, and an outer layer of fibrous cells [18]. Similarly, bacterial cellulose grafts 4 cm in length and 4 mm in internal diameter were implanted bilaterally in the carotid arteries of eight sheep. Although 50% of the grafts occluded within 2 weeks, all patent grafts developed a confluent inner layer of endothelial- like cells [19]. In addition, the mechanical properties of tubular structures created from bacterial cellulose seemed to be advantageous for vascular tissue engineering. For example, these structures exhibited a compliance response similar to that of human saphenous vein [20]. In our experiments, we have concentrated on cellulose-based materials modified with oxidation and/or functionalization with biomolecules. We have prepared fibrous scaffolds made of non-oxidized viscose, dialdehyde cellulose and 6-carboxycellulose with 2.1 wt.% or 6.6 wt.% of -COOH groups. In addition, all these material types were functionalized with arginine, i.e. an amino acid with a basic side chain, or with chitosan, in order to balance (compensate) the relatively acid character of oxidized cellulose molecules. Two groups of samples with and without functionalization were then seeded with vascular smooth muscle cells (VSMC) derived from the rat thoracic aorta by an explantation method [21]. We found that the oxidized cellulose with 2.1 wt.% of-COOH groups was the most appropriate of all the tested materials for colonization with VSMC. The cells on this material achieved an elongated shape, while they were spherical in shape on the other materials. In addition, the numbers of cells obtained in one week after seeding and the concentration of alpha-actin and SM1 and SM2 myosins, measured per mg of protein, were significantly higher on oxidized cellulose with 2.1 wt.% of -COOH groups. Functionalization with arginine and chitosan improved the cell adhesion, but usually only slightly. The most apparent increase in cell number after functionalization was observed on oxidized cellulose with 2.1 wt.% of -COOH groups functionalized with chitosan, and on viscose functionalized with chitosan or arginin. However, the cells on all samples proliferated slowly and with a non-significant increase in cell population densities from day 1 to 7 after seeding. This suggests that cellulose-based materials can be used in applications where high proliferation activity of vascular smooth muscle cells is not desirable. They can therefore be used on vascular prostheses, where excessive VSMC proliferation can lead to the restenosis of the graft. Alternatively, cell proliferation might be enhanced by some other more efficient modification. This would require further research.

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Vascular smooth muscle cells in cultures on biofunctionalized cellulose-based scaffolds

Novotna K., Bacakova L., Lisa V., Havelka P., Sopuch T., Klepetar J.

Engineering of Biomaterials

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2009

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Vol. 12, no. 89-91

21--24

EN

Viscose, dialdehyde cellulose and oxidized 6-car-boxycellulose with 2.1 or 6.6wt.% of –COOH groups were prepared. The materials were subsequently functionalized with arginine or chitosan. Both unmodified and biofunctionalized materials were seeded with vascular smooth muscle cells. The morphology of the adhered cells indicated that oxidized 6-carbo-xycellulose with 2.1% content of –COOH groups was the most appropriate of all tested materials for potential use in tissue engineering. The shape of the cells on this material was elongated, which demonstrates adequate adhesion and viability of the cells, while the morphology of the cells on other tested materials was spherical. Moreover, the stability of 6-carboxycellulo-se with 2.1wt.% of –COOH groups in the cell culture environment was optimal, with a tendency to degrade slowly with time. The highest stability was found on the viscose samples, whereas there was very low stability on oxidized 6-carboxycellulose with 6.6 wt. % of –COOH groups, and also on dialdehyde cellulose. Functionalization with arginine or chitosan increased the number of adhered cells on the materials, but not markedly. We did not obtain a significant elevation of the cell population densities with time on the tested samples. These results suggest the possibility of using a cellulose-based material in such tissue engineering applications, where high proliferation activity of cells is not convenient, e.g. reconstruction of the smooth mu-scle cell layer in bioartificial vascular replacements.

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Micro- and nanopatterned surfaces for guided adhesion, growth and phenotypic maturation of cells

Bacakova L., Filova E., Grausova L., Vandrovcova M., Parizek M., Novotna K., Svorcik V., Vacik J., Rypacek F., Kromka A., Heitz J., Shard A.

Engineering of Biomaterials

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2009

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Vol. 12, no. 89-91

18--21

EN

Micropatterned surfaces were created by UV light-irradiation of polytetrafluoroethylene through a metallic mask, by successive plasma polymerization of acrylic acid and 1,7-octadiene, or by creation of prominences and grooves by deposition of fullerenes C60 through a metallic mask. All these surface types were capable of inducing regionally-selective adhesion, proliferation and phenotypic maturation of vascular endothelial cells, vascular smooth muscle cells or human bone-derived MG 63 cells. Nanopatterned surfaces created by tethering GRGDSG oligopeptides through polyethylene oxide chains on a polymeric surface promoted spreading, formation of focal adhesion plaques and DNA synthesis in vascular smooth muscle cells. Surfaces nanopatterned with nanocrystalline diamond gave good support for the adhesion, growth and metabolic activity of osteoblast-like MG 63 cells.