Biomimetic biphasic curdlan-based scaffold for osteochondral tissue engineering applications - Characterization and preliminary evaluation of mesenchymal stem cell response in vitro.

Osteochondral defects remain a huge problem in medicine today. Biomimetic bi- or multi-phasic scaffolds constitute a very promising alternative to osteochondral autografts and allografts. In this study, a new curdlan-based scaffold was designed for osteochondral tissue engineering applications. To achieve biomimetic properties, it was enriched with a protein component - whey protein isolate as well as a ceramic ingredient - hydroxyapatite granules. The scaffold was fabricated via a simple and cost-efficient method, which represents a significant advantage. Importantly, this technique allowed generation of a scaffold with two distinct, but integrated phases. Scanning electron microcopy and optical profilometry observations demonstrated that phases of biomaterial possessed different structural properties. The top layer of the biomaterial (mimicking the cartilage) was smoother than the bottom one (mimicking the subchondral bone), which is beneficial from a biological point of view because unlike bone, cartilage is a smooth tissue. Moreover, mechanical testing showed that the top layer of the biomaterial had mechanical properties close to those of natural cartilage. Although the mechanical properties of the bottom layer of scaffold were lower than those of the subchondral bone, it was still higher than in many analogous systems. Most importantly, cell culture experiments indicated that the biomaterial possessed high cytocompatibility towards adipose tissue-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells in vitro. Both phases of the scaffold enhanced cell adhesion, proliferation, and chondrogenic differentiation of stem cells (revealing its chondroinductive properties in vitro) as well as osteogenic differentiation of these cells (revealing its osteoinductive properties in vitro). Given all features of the novel curdlan-based scaffold, it is worth noting that it may be considered as promising candidate for osteochondral tissue engineering applications.

Preparation of highly wettable coatings on Ti-6Al-4V ELI alloy for traumatological implants using micro-arc oxidation in an alkaline electrolyte.

Pulsed micro-arc oxidation (MAO) in a strongly alkaline electrolyte (pH > 13), consisting of Na2SiO3⋅9H2O and NaOH, was used to form a thin porous oxide coating consisting of two layers differing in chemical and phase composition. The unique procedure, combining MAO and removal of the outer layer by blasting, enables to prepare a coating suitable for application in temporary traumatological implants. A bilayer formed in an alkaline electrolyte environment during the application of MAO enables the formation of a wear-resistant layer with silicon incorporated in the oxide phase. Following the removal of the outer rutile-containing porous layer, the required coating properties for traumatological applications were determined. The prepared surfaces were characterized by scanning electron microscopy, X-ray diffraction patterns, X-ray photoelectron spectroscopy, atomic force microscopy and contact angle measurements. Cytocompatibility was evaluated using human osteoblast-like Saos-2 cells. The newly-developed surface modifications of Ti-6Al-4V ELI alloy performed satisfactorily in all cellular tests in comparison with MAO-untreated alloy and standard tissue culture plastic. High cell viability was supported, but the modifications allowed only relatively slow cell proliferation, and showed only moderate osseointegration potential without significant support for matrix mineralization. Materials with these properties are promising for utilization in temporary traumatological implants.

Enhancement of Biomimetic Enzymatic Mineralization of Gellan Gum Polysaccharide Hydrogels by Plant-Derived Gallotannins.

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.

COMPARISON OF THE RADIATION SENSITIVITY OF THE BREAST CANCER CELL LINE MCF7 AND HUMAN ADIPOSE-DERIVED STEM CELLS.

A comparison between breast cancer cell line MCF7 and human adipose-derived stem cells (ADSC) after irradiation by the same doses of megavoltage X-rays was performed. The cell growth, the induction of apoptosis and the expression of selected genes were analyzed. Irradiated MCF7 related to its control sample grows slower than ADSC and it undergoes apoptosis in much higher levels than ADSC. This was confirmed by real-time polymerase chain reaction as well, where the expression of apoptotic genes was found to be considerably higher for MCF7 than for ADSC. From the results of this project, it could be stated that MCF7 is more radiosensitive than ADSC.

Laser-synthesized nanocrystalline, ferroelectric, bioactive BaTiO3/Pt/FS for bone implants.

The goal of our study is to design BaTiO3 ferroelectric layers that will cover metal implants and provide improved osseointegration. We synthesized ferroelectric BaTiO3 layers on Pt/fused silica substrates, and we studied their physical and bio-properties. BaTiO3 and Pt layers were prepared using KrF excimer laser ablation at substrate temperature Ts in the range from 200°C to 750°C in vacuum or under oxygen pressure of 10 Pa, 15 Pa, and 20 Pa. The BaTiO3/Pt and Pt layers adhered well to the substrates. BaTiO3 films of crystallite size 60-140 nm were fabricated. Ferroelectric loops were measured and ferroelectricity was also confirmed using Raman scattering measurements. Results of atomic force microscopy topology and the X-ray diffraction structure of the BaTiO3/Pt/fused silica multilayers are presented. The adhesion, viability, growth, and osteogenic differentiation of human osteoblast-like Saos-2 cells were also studied. On days 1, 3, and 7 after seeding, the lowest cell numbers were found on non-ferroelectric BaTiO3, while the values on ferroelectric BaTiO3, on non-annealed and annealed Pt interlayers, and on the control tissue culture polystyrene dishes and microscopic glass slides were similar, and were usually significantly higher than on non-ferroelectric BaTiO3. A similar trend was observed for the intensity of the fluorescence of alkaline phosphatase, a medium-term marker of osteogenic differentiation, and of osteocalcin, a late marker of osteogenic differentiation. At the same time, the cell viability, tested on day 1 after seeding, was very high on all tested samples, reaching 93-99%. Ferroelectric BaTiO3 films deposited on metallic bone implants through a Pt interlayer can therefore markedly improve the osseointegration of these implants in comparison with non-ferroelectric BaTiO3 films.

Applications of zeolites in biotechnology and medicine - a review.

Zeolites are microporous tectosilicates of natural or synthetic origin, which have been extensively used in various technological applications, e.g. as catalysts and as molecular sieves, for separating and sorting various molecules, for water and air purification, including removal of radioactive contaminants, for harvesting waste heat and solar heat energy, for adsorption refrigeration, as detergents, etc. These applications of zeolites were typically related with their porous character, their high adsorption capacity, and their ion exchange properties. This review is focused on potential or already practically implemented applications of zeolites in biotechnology and medicine. Zeolites are promising for environment protection, detoxication of animal and human organisms, improvement of the nutrition status and immunity of farm animals, separation of various biomolecules and cells, construction of biosensors and detection of biomarkers of various diseases, controlled drug and gene delivery, radical scavenging, and particularly tissue engineering and biomaterial coating. As components of scaffolds for bone tissue engineering, zeolites can deliver oxygen to cells, can stimulate osteogenic cell differentiation, and can inhibit bone resorption. Zeolites can also act as oxygen reservoirs, and can improve cell performance in vascular and skin tissue engineering and wound healing. When deposited on metallic materials for bone implantation, zeolite films showed anticorrosion effects, and improved the osseointegration of these implants. In our studies, silicalite-1 films deposited on silicon or stainless steel substrates improved the adhesion, growth, viability and osteogenic differentiation of human osteoblast-like Saos-2 cells. Zeolites have been clinically used as components of haemostatics, e.g. in the Advanced Clotting Sponge, as gastroprotective drugs, e.g. Absorbatox® 2.4D, or as antioxidative agents (Klinobind®). Some zeolites are highly cytotoxic and carcinogenic, e.g. erionite. However, in other zeolites, the antiproliferative and pro-apoptotic effects can be used for tumor therapy.

Application of whey protein isolate in bone regeneration: Effects on growth and osteogenic differentiation of bone-forming cells.

Recently, milk-derived proteins have attracted attention for applications in the biomedical field such as tissue regeneration. Whey protein isolate (WPI), especially its main component ß-lactoglobulin, can modulate immunity and acts as an antioxidant, antitumor, antiviral, and antibacterial agent. There are very few reports of the application of WPI in tissue engineering, especially in bone tissue engineering. In this study, we tested the influence of different concentrations of WPI on behavior of human osteoblast-like Saos-2 cells, human adipose tissue-derived stem cells (ASC), and human neonatal dermal fibroblasts (FIB). The positive effect on growth was apparent for Saos-2 cells and FIB but not for ASC. However, the expression of markers characteristic for early osteogenic cell differentiation [type-I collagen (COL1) and alkaline phosphatase (ALP)] as well as ALP activity, increased dose-dependently in ASC. Importantly, Saos-2 cells were able to deposit calcium in the presence of WPI, even in a proliferation medium without other supplements that support osteogenic cell differentiation. The results indicate that, depending on the cell type, WPI can act as an enhancer of cell proliferation and osteogenic differentiation. Therefore, enrichment of biomaterials for bone regeneration with WPI seems a promising approach, especially due to the low cost of WPI.

Cytocompatibility assessment of Ti-Zr-Pd-Si-(Nb) alloys with low Young's modulus, increased hardness, and enhanced osteoblast differentiation for biomedical applications.

Ti-based alloys have increased importance for biomedical applications due to their excellent properties. In particular, the two recently developed TiZrPdSi(Nb) alloys, with a predominant ß-Ti phase microstructure, have good mechanical properties, such as a relatively low Young's modulus and high hardness. In the present work, the cytocompatibility of these alloys was assessed using human osteoblast-like Saos-2 cells. Cells grown on the alloys showed larger spreading areas (more than twice) and higher vinculin content (nearly 40% increment) when compared with cells grown on glass control surfaces, indicating a better cell adhesion. Moreover, cell proliferation was 18% higher for cells growing on both alloys than for cells growing on glass and polystyrene control surfaces. Osteogenic differentiation was evaluated by quantifying the expression of four osteogenic genes (osteonectin, osteocalcin, osteopontin, and bone sialoprotein), the presence of three osteogenic proteins (alkaline phosphatase, collagen I, and osteocalcin) and the activity of alkaline phosphatase at different time-points. The results demonstrated that TiZrPdSi and TiZrPdSiNb alloys enhance osteoblast differentiation, and that cells grown on TiZrPdSiNb alloy present higher levels of some late osteogenic markers during the first week in culture. These results suggest that the TiZrPdSi(Nb) alloys can be considered as excellent candidates for orthopaedical uses. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 834-842, 2018.

Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities.

Polyphenols are known for their antimicrobial activity, whilst both polyphenols and the globular protein ß-lactoglobulin (bLG) are suggested to have antioxidant properties and promote cell proliferation. These are potentially useful properties for a tissue-engineered construct, though it is unknown if they are retained when both compounds are used in combination. In this study, a range of different microbes and an osteoblast-like cell line (human fetal osteoblast, hFOB) were used to assess the combined effect of: (1) green tea extract (GTE), rich in the polyphenol epigallocatechin gallate (EGCG), and (2) whey protein isolate (WPI), rich in bLG. It was shown that approximately 20-48% of the EGCG in GTE reacted with WPI. GTE inhibited the growth of Gram-positive bacteria, an effect which was potentiated by the addition of WPI. GTE alone also significantly inhibited the growth of hFOB cells after 1, 4, and 7 days of culture. Alternatively, WPI significantly promoted hFOB cell growth in the absence of GTE and attenuated the effect of GTE at low concentrations (64 µg/mL) after 4 and 7 days. Low concentrations of WPI (50 µg/mL) also promoted the expression of the early osteogenic marker alkaline phosphatase (ALP) by hFOB cells, whereas GTE inhibited ALP activity. Therefore, the antioxidant effects of GTE can be boosted by WPI, but GTE is not suitable to be used as part of a tissue-engineered construct due to its cytotoxic effects which negate any positive effect WPI has on cell proliferation.

Growth of a TiNb adhesion interlayer for bioactive coatings.

Surface bioactivity has been under intensive study with reference to its use in medical implants. Our study is focused on coatings prepared from an electroactive material which can support bone cell adhesion. Until now, hydroxyapatite films have usually been utilized as a chemically-active surface agent. However, electrically-active films could set a new direction in hard tissue replacement. As a base for these films, it is necessary to prepare an intermediate film, which can serve as a suitable barrier against the possible diffusion of some allergens and toxic elements from the substrate. The intermediate film also improves the adaptation of the mechanical properties of the basic material to an electroactive film. The aim of our work was to select an implantable and biocompatible material for this intermediate film that is suitable for coating several widely-used materials, to check the possibility of preparing an electroactive film for use on a material of this type, and to characterize the structure and several mechanical properties of this intermediate film. TiNb was selected as the material for the intermediate film, because of its excellent chemical and mechanical properties. TiNb coatings were deposited by magnetron sputtering on various substrates, namely Ti, Ti6Al4V, stainless steel, and bulk TiNb (as standard), and important properties of the layers, e.g. surface morphology and surface roughness, crystalline structure, etc., were characterized by several methods (SEM, EBSD, X-ray diffraction, nanoindentation and roughness measurement). It was found that the structure and the mechanical properties of the TiNb layer depended significantly on the type of substrate. TiNb was then used as a substrate for depositing a ferroelectrically active material, e.g., BaTiO3, and the adhesion, viability and proliferation of human osteoblast-like Saos-2 cells on this system were studied. We found that the electroactive BaTiO3 film was not only non-cytotoxic (i.e. it did not affect the cell viability). It also enhanced the growth of Saos-2 cells in comparison with pure TiNb and with standard tissue culture polystyrene wells, and also in comparison with BaTiO3 films deposited on Ti, i.e. a material clinically used for implantation into the bone.

Interaction of human osteoblast-like Saos-2 cells with stainless steel coated by silicalite-1 films.

This paper investigates the interaction of human osteoblast-like Saos-2 cells with stainless steel covered by a film of densely inter-grown silicalite-1 crystals with defined outer and inner surfaces. The chemical composition of this film, labeled as SF(RT), was tuned by heat treatment at 300°C and 500°C (labeled as SF(300) and SF(500), respectively) and characterized by X-ray photoelectron spectroscopy (XPS), water drop contact angle (WCA) measurements and scanning electron microscopy (SEM). The number, the spreading area and the activity of alkaline phosphatase of human osteoblast-like Saos-2 cells in cultures on the silicalite-1 film were affected by the chemical composition of its outer surface and by its micro-porous structure. The number and the spreading area of the adhered osteoblast-like cells on day 1 was highest on the surface of SF(RT) relative to their adhesion and spreading on a glass cover slip due to the SF(RT) topology. However, SF(300) markedly supported cell growth during days 3 and 7 after seeding.

Evaluation of the potential of chitosan/ß-1,3-glucan/hydroxyapatite material as a scaffold for living bone graft production in vitro by comparison of ADSC and BMDSC behaviour on its surface.

The opinion regarding the origin of adult stem cells that should be used for living bone construct generation is strongly divided in the scientific community. In this study, the potential of chitosan/ß-1,3-glucan/hydroxyapatite (chit/glu/HA) material as a scaffold for bone regeneration applications was evaluated by behaviour comparison of adult stem cells derived from both origins-adipose derived mesenchymal stem cell (ADSC) tissue and bone marrow derived mesenchymal stem cells (BMDSCs). In the case of ADSC isolation, low and high negative pressures were applied during a liposuction procedure in order to determine if negative pressure settings may have an impact on subsequent cell behaviour in vitro. The obtained results demonstrated that the chit/glu/HA material is a promising candidate to be used for living bone graft production in vitro as both ADSCs and BMDSCs revealed a satisfactory proliferation and differentiation ability on its surface. Nevertheless, BMDSCs would be a better choice of adult stem cells since they were better spread, more strongly attached and showed a more superior proliferation and differentiation ability than ADSCs when cultured on the chit/glu/HA scaffold. However, if BMDSCs cannot be isolated, ADSCs may be used for bone construct production but lipoaspirate should be collected under low negative pressure (-200 mm Hg), as high negative pressure (-700 mmHg) applied during liposuction surgery may retard subsequent ADSC proliferation and type I collagen production.

Adhesion and differentiation of Saos-2 osteoblast-like cells on chromium-doped diamond-like carbon coatings.

Diamond-like carbon (DLC) thin films are promising for use in coating orthopaedic, dental and cardiovascular implants. The problem of DLC layers lies in their weak layer adhesion to metal implants. Chromium is used as a dopant for improving the adhesion of DLC films. Cr-DLC layers were prepared by a hybrid technology, using a combination of pulsed laser deposition (PLD) from a graphite target and magnetron sputtering. Depending on the deposition conditions, the concentration of Cr in the DLC layers moved from zero to 10.0 at.%. The effect of DLC layers with 0.0, 0.9, 1.8, 7.3, 7.7 and 10.0 at.% Cr content on the adhesion and osteogenic differentiation of human osteoblast-like Saos-2 cells was assessed in vitro. The DLC samples that contained 7.7 and 10.0 at.% of Cr supported cell spreading on day 1 after seeding. On day three after seeding, the most apparent vinculin-containing focal adhesion plaques were also found on samples with higher concentrations of chromium. On the other hand, the expression of type I collagen and alkaline phosphatase at the mRNA and protein level was the highest on Cr-DLC samples with a lower concentration of Cr (0-1.8 at.%). We can conclude that higher concentrations of chromium supported cell adhesion; however DLC and DLC doped with a lower concentration of chromium supported osteogenic cell differentiation.

Cell interaction with modified nanotubes formed on titanium alloy Ti-6Al-4V.

Nanotubes with diameters ranging from 40 to 60nm were prepared by electrochemical oxidation of the Ti-6Al-4V alloy in electrolyte containing ammonium sulphate and ammonium fluoride. The nanotubes were further modified with calcium and phosphate ions or were heat treated. Polished Ti-6Al-4V alloy served as a reference sample. The spreading of human osteoblast-like cells was similar on all nanotube samples but lower than on polished samples. The number of initially adhered cells was higher on non-modified nanotubes, but the final cell number was the highest on Ca-enriched nanotubes and the lowest on heat-treated nanotubes. However, these differences were relatively small and less pronounced than the differences in the concentration of specific molecular markers of cell adhesion and differentiation, estimated by their intensity of immunofluorescence staining. The concentration of vinculin, i.e. a protein of focal adhesion plaques, was the lowest on nanotubes modified with calcium. Collagen I, an early marker of osteogenic cell differentiation, was also the lowest on samples modified with calcium and was highest on polished samples. Alkaline phosphatase, a middle marker of osteogenic differentiation, was observed in lowest concentration on nanotubes modified with phosphorus and the highest on heat-treated samples. Osteocalcin concentrations, a late marker of osteogenic cell differentiation, were similar on all tested samples, although they tended to be the highest on heat-treated samples. Thus, osteogenic differentiation can be modulated by various additional treatments of nanotube coatings on Ti-6Al-4V implants.

Bonding and bio-properties of hybrid laser/magnetron Cr-enriched DLC layers.

Chromium-enriched diamond-like carbon (DLC) layers were prepared by a hybrid technology using a combination of pulsed laser deposition (PLD) and magnetron sputtering. XRD revealed no chromium peaks, indicating that the layers are mostly amorphous. Carbon (sp(2) and sp(3) bonds) and chromium bonds were determined by XPS from C 1s, O 1s, and Cr 2p photoelectron peaks. Depending on the deposition conditions, the concentration of Cr in DLC layers moved from zero to 10 at.% for as-received sample surfaces, and to about 31 at.% after mild sputter-cleaning by argon ion cluster beam. It should be noted that the most stable Cr(3+) bonding state is in Cr2O3 and Cr(OH)3, and that there is the toxic Cr(6+) state in CrO3. The surface content of hexavalent chromium in the Cr 2p3/2 spectra is rather low, but discernible. The population density of Saos-2 cells was the highest in samples containing higher concentrations of chromium 7.7 and 10 at.%. This means that higher concentrations of chromium supported the cell adhesion and proliferation. In addition, as revealed by a LIVE/DEAD viability/cytotoxicity kit, the cells on all Cr-containing samples maintained high viability (96 to 99%) on days 1 and 3 after seeding. However, this seemingly positive cell behavior could be associated with the risk of dedifferentiation and oncogenic transformation of cells.

Collagen-lactoferrin fibrillar coatings enhance osteoblast proliferation and differentiation.

Lactoferrin is a milk-derived glycoprotein with anabolic effects on the bone tissue. In this study, artificial extracellular matrices (aECM) consisting of collagen type I fibrils formed in the presence of lactoferrin at two different concentrations (0.5 and 1 mg mL(-1) ) were prepared on the surface of poly(lactic-co-glycolic acid) (PLGA) foils. The aim of the study was to investigate the effects of aECM on the adhesion, growth and osteogenic differentiation of human osteoblast-like Saos-2 cells. On days 1 and 3 after seeding, higher numbers of cells were found on samples with collagen and collagen-lactoferrin coatings (particularly on those formed at the higher concentration of lacroferrin) than on control microscopic glass coverslips. Cells on coatings formed in the presence of lactoferrin had more numerous and better developed vinculin-containing focal adhesion plaques. On day 7, cells on coatings with and without lactoferrin produced significantly higher levels of osteocalcin than cells on control polystyrene cell culture dishes, the highest average values being found on samples with the lower concentration of lactoferrin. Expression of collagen I and alkaline phosphatase was on a similar level in cells on all tested samples and control polystyrene. Thus, lactoferrin promotes adhesion, growth and osteogenic differentiation of Saos-2 cells and is promising as a bone implant coating component.

Interaction of human osteoblast-like Saos-2 and MG-63 cells with thermally oxidized surfaces of a titanium-niobium alloy.

An investigation was made of the adhesion, growth and differentiation of osteoblast-like MG-63 and Saos-2 cells on titanium (Ti) and niobium (Nb) supports and on TiNb alloy with surfaces oxidized at 165°C under hydrothermal conditions and at 600°C in a stream of air. The oxidation mode and the chemical composition of the samples tuned the morphology, topography and distribution of the charge on their surfaces, which enabled us to evaluate the importance of these material characteristics in the interaction of the cells with the sample surface. Numbers of adhered MG-63 and Saos-2 cells correlated with the number of positively-charged (related with the Nb2O5 phase) and negatively-charged sites (related with the TiO2 phase) on the alloy surface. Proliferation of these cells is correlated with the presence of positively-charged (i.e. basic) sites of the Nb2O5 alloy phase, while cell differentiation is correlated with negatively-charged (acidic) sites of the TiO2 alloy phase. The number of charged sites and adhered cells was substantially higher on the alloy sample oxidized at 600°C than on the hydrothermally treated sample at 165°C. The expression values of osteoblast differentiation markers (collagen type I and osteocalcin) were higher for cells grown on the Ti samples than for those grown on the TiNb samples. This was more particularly apparent in the samples treated at 165°C. No considerable immune activation of murine macrophage-like RAW 264.7 cells on the tested samples was found. The secretion of TNF-α by these cells into the cell culture media was much lower than for either cells grown in the presence of bacterial lipopolysaccharide, or untreated control samples. Thus, oxidized Ti and TiNb are both promising materials for bone implantation; TiNb for applications where bone cell proliferation is desirable, and Ti for induction of osteogenic cell differentiation.

Innovative surface modification of Ti-6Al-4V alloy with a positive effect on osteoblast proliferation and fatigue performance.

A novel approach of surface treatment of orthopaedic implants combining electric discharge machining (EDM), chemical milling (etching) and shot peening is presented in this study. Each of the three techniques have been used or proposed to be used as a favourable surface treatment of biomedical titanium alloys. But to our knowledge, the three techniques have not yet been used in combination. Surface morphology and chemistry were studied by scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Fatigue life of the material was determined and finally several in-vitro biocompatibility tests have been performed. EDM and subsequent chemical milling leads to a significant improvement of osteoblast proliferation and viability thanks to favourable surface morphology and increased oxygen content on the surface. Subsequent shot-peening significantly improves the fatigue endurance of the material. Material after proposed combined surface treatment possesses favourable mechanical properties and enhanced osteoblast proliferation. EDM treatment and EDM with shot peening also supported early osteogenic cell differentiation, manifested by a higher expression of collagen type I. The combined surface treatment is therefore promising for a range of applications in orthopaedics.

Improved adhesion and differentiation of endothelial cells on surface-attached fibrin structures containing extracellular matrix proteins.

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.

On the role of Nb-related sites of an oxidized ß-TiNb alloy surface in its interaction with osteoblast-like MG-63 cells.

ß-Stabilized titanium (Ti) alloys containing non-toxic elements, particularly niobium (Nb), are promising materials for the construction of bone implants. Their biocompatibility can be further increased by oxidation of their surface. Therefore, in this study, the adhesion, growth and viability of human osteoblast-like MG 63 cells in cultures on oxidized surfaces of a ß-TiNb alloy were investigated and compared with the cell behavior on thermally oxidized Ti, i.e. a metal commonly used for constructing bone implants. Four experimental groups of samples were prepared: Ti or TiNb samples annealed to 600 °C for 60 min in a stream of dry air, and Ti and TiNb samples treated in Piranha solution prior to annealing. We found that on all TiNb-based samples, the cell population densities on days 1, 3 and 7 after seeding were higher than on the corresponding Ti-based samples. As revealed by XPS and Raman spectroscopy, and also by isoelectric point measurements, these results can be attributed to the presence of T-Nb2O5 oxide phase in the surface of the alloy sample, which decreased its negative zeta (ζ)-potential in comparison with zeta (ζ)-potential of the Ti sample at physiological pH. This effect was tentatively explained by the presence of positively charged defects acting as Lewis sites of the surface Nb2O5 phase. Piranha treatment slightly decreases the biocompatibility of the samples, which for the alloy samples may be explained by a decrease in the number of defective sites with this treatment. Thus, the presence of Nb and thermal oxidation of ß-stabilized Ti alloys play a significant role in the increased biocompatibility of TiNb alloys.