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Anodic titanium dioxide (TiO 2 ) nanostructures, i.e., obtained by electrochemical anodization, have excellent control over the nanoscale morphology and have been extensively investigated in biomedical applications owing to their sub-100 nm nanoscale topography range and beneficial effects on biocompatibility and cell interactions. Herein, we obtain TiO 2 nanopores (NPs) and nanotubes (NTs) with similar morphologies, namely, 15 nm diameter and 500 nm length, and investigate their characteristics and impact on stem cell adhesion. We show that the transition of TiO 2 NPs to NTs occurs via a pore/wall splitting mechanism and the removal of the fluoride-rich layer. Furthermore, in contrast to the case of NPs, we observe increased cell adhesion and proliferation on nanotubes. The enhanced mesenchymal stem cell adhesion/proliferation seems to be related to a 3-fold increase in activated integrin clustering, as confirmed by immunogold labeling with β1 integrin antibody on the nanostructured layers. Moreover, computations of the electric field and surface charge density show increased values at the inner and outer sharp edges of the top surfaces of the NTs, which in turn can influence cell adhesion by increasing the bridging interactions mediated by proteins and molecules in the environment. Collectively, our results indicate that the nanoscale surface architecture of the lateral spacing topography can greatly influence stem cell adhesion on substrates for biomedical applications.

Tunneling nanotubes (TNTs) are thin membranous tubes that interconnect cells, representing a novel route of cell-to-cell communication and spreading of pathogens. TNTs form between many cell types, yet their inception mechanisms remain elusive. We review in this study general concepts related to the formation and stability of membranous tubular structures with a focus on a deviatoric elasticity model of membrane nanodomains. We review experimental evidence that tubular structures initiate from local membrane bending facilitated by laterally distributed proteins or anisotropic membrane nanodomains. We further discuss the numerical results of several theoretical and simulation models of nanodomain segregation suggesting the mechanisms of TNT inception and stability. We discuss the coupling of nanodomain segregation with the action of protruding cytoskeletal forces, which are mostly provided in eukaryotic cells by the polymerization of f-actin, and review recent inception mechanisms of TNTs in relation to motor proteins., Competing Interests: The authors declare no conflict of interest.

In the present work we investigate the key factors involved in the interaction of small-sized charged proteins with TiO 2 nanostructures, i.e. albumin (negatively charged), histone (positively charged). We examine anodic nanotubes with specific morphology (simultaneous control over diameter and length, e.g. diameter - 15, 50 or 100nm, length - 250nm up to 10μm) and nanopores. The nanostructures surface area has a direct influence on the amount of bound protein, nonetheless the protein physical properties as electric charge and size (in relation to nanotopography and biomaterial's electric charge) are crucial too. The highest quantity of adsorbed protein is registered for histone, for 100nm diameter nanotubes (10μm length) while higher values are registered for 15nm diameter nanotubes when normalizing protein adsorption to nanostructures' surface unit area (evaluated from dye desorption measurements) - consistent with theoretical considerations. The proteins presence on the nanostructures is evaluated by XPS and ToF-SIMS; additionally, we qualitatively assess their presence along the nanostructures length by ToF-SIMS depth profiles, with decreasing concentration towards the bottom., Statement of Significance: Surface nanostructuring of titanium biomedical devices with TiO 2 nanotubes was shown to significantly influence the adhesion, proliferation and differentiation of mesenchymal stem cells (and other cells too). A high level of control over the nanoscale topography and over the surface area of such 1D nanostructures enables a direct influence on protein adhesion. Herein, we investigate and show how the nanostructure morphology (nanotube diameter and length) influences the interactions with small-sized charged proteins, using as model proteins bovine serum albumin (negatively charged) and histone (positively charged). We show that the protein charge strongly influences their adhesion to the TiO 2 nanostructures. Protein adhesion is quantified by ELISA measurements and determination of the nanostructures' total surface area. We use a quantitative surface charge model to describe charge interactions and obtain an increased magnitude of the surface charge density at the top edges of the nanotubes. In addition, we track the proteins presence on and inside the nanostructures. We believe that these aspects are crucial for applications where the incorporation of active molecules such as proteins, drugs, growth factors, etc., into nanotubes is desired., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Sterilization is the final surface treatment procedure of all implantable devices and is one of the key factors which have to be considered before implementation. Since different sterilization procedures for all implantable devices influence mechanical properties as well as biological response, the influence of different sterilization techniques on titanium nanotubes was studied. Commonly used sterilization techniques such as autoclaving, ultra-violet light sterilization, hydrogen peroxide plasma sterilization as well as the not so frequently used gaseous oxygen plasma sterilization were used. Three different nanotube diameters; 15 nm, 50 nm and 100 nm were employed to study the effects of various sterilization techniques. It was observed that autoclave sterilization resulted in destruction of nanotubular features on all three studied nanotube diameters, while UV-light and both kinds of plasma sterilization did not cause any significant morphological changes on the surfaces. Differences between the sterilization techniques employed influenced cytocompatibility, especially in the case of nanotubes with 100 nm diameter., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nanoscale topography in improving vascular response in vitro was established previously on various titanium surfaces. In the present study different surface nanotopographies that is different diameters of titanium dioxide (TiO2 ) nanotubes (NTs) were fabricated by electrochemical anodization and conditioned with highly reactive gaseous oxygen plasma. The morphology of different diameter NTs was studied by scanning electron microscopy and atomic force microscopy, while changes in chemical composition on the surface before and after plasma treatment were determined by X-ray photoelectron spectroscopy. Performance of human coronary artery endothelial cells (HCAEC) on those conditioned surfaces was studied in regard to cell proliferation, released IL-6 protein and immunofluorescence microscopy (IFM). We show that HCAEC function is dependent on the diameter of the TiO2 NTs, functioning far less optimally when bound to 100 nm TiO2 NTs as compared to Ti foil, 15 nm NTs or 50 nm NTs. There were improved, morphological cell shape changes, observed with IFM, between HCAEC growing on oxygen-rich plasma-treated versus nontreated 100 nm NTs. These endothelialized conditioned Ti nanosurfaces could elucidate optimization conditions necessary for vascular implants in coronary arteries., (© 2016 Wiley Periodicals, Inc.)

Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices.

The adhesion of cells to vertically aligned TiO2 nanotubes is reviewed. The attraction between a negatively charged nanotube surface and a negatively charged osteoblast is facilitated by charged protein-mediators like proteins with a quadrupolar internal charge distribution, fibronectin and vitronectin. It is shown that adhesion and spreading of osteoblasts on vertically aligned TiO2 nanotube surfaces depend on the diameter of the nanotubes. Apparently, a small diameter nanotube surface has on average more sharp convex edges per unit area than a large one, leading to stronger binding affinity on its surface.

Intercellular membrane nanotubes (ICNs) are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant) urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are discussed.

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Membrane nanotubes are a morphologically versatile group of membrane structures (some resembling filopodia), usually connecting two closely positioned cells. In this article, we set morphological criteria that distinguish the membrane nanotubes from filopodia, as there is no specific molecular marker known to date that unequivocally differentiates between filopodia and protruding nanotubes. Membrane nanotubes have been extensively studied from the morphological point of view and the transport that can be conducted through them, but little is known about the way they connect to the adjacent cell. Our results show that the nanotubes may connect to a neighboring cell by anchoring junctions. Among cell adhesion proteins, N-cadherin, β-catenin, nectin-2, afadin and the desmosomal protein desmoplakin-2 were immune-labeled. We found that N-cadherin and β-catenin are concentrated in nanotubes, while the concentrations of other junction-involved proteins are not increased in these structures. On the basis of data from transmission electron microscopy, we propose a model of the nanotube attachment where the connection of nanotubes is stabilized by several anchoring junctions, most likely adherens junctions that are formed when the nanotube is sliding along the target cell membrane. [ABSTRACT FROM AUTHOR]

Cells interact by exchanging material and information. Two methods of cell-to-cell communication are by means of microvesicles and by means of nanotubes. Both microvesicles and nanotubes derive from the cell membrane and are able to transport the contents of the inner solution. In this review, we describe two physical mechanisms involved in the formation of microvesicles and nanotubes: curvature-mediated lateral redistribution of membrane components with the formation of membrane nanodomains; and plasmamediated attractive forces between membranes. These mechanisms are clinically relevant since they can be affected by drugs. In particular, the underlying mechanism of heparin’s role as an anticoagulant and tumor suppressor is the suppression of microvesicluation due to plasma-mediated attractive interaction between membranes. [ABSTRACT FROM AUTHOR]

We used sub-micron metal rod decorated surfaces, 'nano-lawn' structures, as a substrate to study cell-to-cell and cell-to-surface interactions of primary murine astrocytes. These cells form thin membranous tubes with diameters of less than 100 nm and a length of several microns, which make contact to neighboring cells and the substrate during differentiation. While membrane protrusions grow on top of the nano-lawn pillars, nuclei sink to the bottom of the substrate. We observed gondola-like structures along those tubes, suggestive of their function as transport vehicles. Elements of the cytoskeleton such as actin fibers are commonly believed to be essential for triggering the onset and growth of tubular membrane protrusions. A rope-pulling mechanism along actin fibers has recently been proposed to account for the transport or exchange of cellular material between cells. We present evidence for a complementary mechanism that promotes growth and stabilization of the observed tubular protrusions of cell membranes. This mechanism does not require active involvement of actin fibers as the formation of membrane protrusions could not be prevented by suppressing polymerization of actin by latrunculin B. Also theoretically, actin fibers are not essential for the growing and stability of nanotubes since curvature-driven self-assembly of interacting anisotropic raft elements is sufficient for the spontaneous formation of thin nano-tubular membrane protrusions. [ABSTRACT FROM AUTHOR]

We indicate that membrane nanotubes may have an important role in directed transport of membrane vesicles between different membrane-enclosed compartments in cells. We present experimental evidence that small blebs of phospholipid nanotube may travel along the nanotube and act as vehicles for transporting the enclosed solution. We have also observed similar small membrane blebs of a long membrane tube in red blood cells. In both cases the small vesicles seem to be a distended integral part of the membrane tube and not independent vesicles entrapped within the tube. [Copyright &y& Elsevier]

Crystallization alters the characteristics of TiO2 nanosurfaces, which consequently influences their bio-performance. In various biomedical applications, the anatase or rutile crystal phase is preferred over amorphous TiO2. The most common crystallization technique is annealing in a conventional furnace. Methods such as hydrothermal or room temperature crystallization, as well as plasma electrolytic oxidation (PEO) and other plasma-induced crystallization techniques, present more feasible and rapid alternatives for crystal phase initiation or transition between anatase and rutile phases. With oxygen plasma treatment, it is possible to achieve an anatase or rutile crystal phase in a few seconds, depending on the plasma conditions. This review article aims to address different crystallization techniques on nanostructured TiO2 surfaces and the influence of crystal phase on biological response. The emphasis is given to electrochemically anodized nanotube arrays and their interaction with the biological environment. A short overview of the most commonly employed medical devices made of titanium and its alloys is presented and discussed. [ABSTRACT FROM AUTHOR]

Facile crystallization of titanium oxide (TiO2) nanotubes (NTs), synthesized by electrochemical anodization, with low pressure non-thermal oxygen plasma is reported. The influence of plasma processing conditions on TiO2 NTs crystal structure and morphology was examined by X-ray diffraction (XRD) and scanning electron microscopy (SEM). For the first time we report the transition of amorphous TiO2 NTs to anatase and rutile crystal structures upon treatment with highly reactive oxygen plasma. This crystallization process has a strong advantage over the conventional heat treatments as it enables rapid crystallization of the surface. Thus the crystalline structure of NTs is obtained in a few seconds of treatment and it does not disrupt the NTs' morphology. Such a crystallization approach is especially suitable for medical applications in which stable crystallized nanotubular morphology is desired. The last part of the study thus deals with in vitro biological response of whole blood to the TiO2 NTs. The results indicate that application of such surfaces for blood connecting devices is prospective, as practically no platelet adhesion or activation on crystallized TiO2 NTs surfaces was observed. [ABSTRACT FROM AUTHOR]

Nanostructured TiO 2 nanotubes (NTs) of diameters from 15 to 100 nm were fabricated by an electrochemical anodization process. Biofilm-positive strains of Bacillus cereus and Pseudomonas aeruginosa behaved similarly on all TiO 2 NTs as well as on native titanium (Ti) foil. The adhesion and growth of mesenchymal stem cells (MSc), embryonic stem cells (ESc), and pure cardiomyocytes derived from ESc exhibited significant differences. MSc as well as ESc were, in contrast to cardiomyocytes, able to adhere, and grow on TiO 2 NTs. A correlation between NTs diameter and cell behaviour was however observed in the case of MSc and ESc. MSc were not in a physiological state in the case of 100 nm TiO 2 NTs, while ESc were not able to grow on 15 nm TiO 2 NTs. It can be stated that these differences can be assigned to different diameters of the NTs but not to the chemistry of the surface. This is the first study describing the comprehensive behaviour of both eukaryotic and prokaryotic cells on TiO 2 NTs. On the basis of obtained results, it can be concluded that new generation of medical devices providing selective cell behaviour can be fabricated by optimizing the nanoscale morphology of TiO 2 . [ABSTRACT FROM AUTHOR]

Abstract: Intercellular membrane nanotubes (ICNs) serve as a very specific transport system between neighboring cells. The underlying mechanisms responsible for the transport of membrane components and vesicular dilations along the ICNs are not clearly understood. The present study investigated the spatial distribution of anisotropic membrane components of tubular shapes and isotropic membrane components of spherical shapes. Experimental results revealed the preferential distribution of CTB (cholera toxin B)–GM1 complexes mainly on the spherical cell membrane, and cholesterol–sphingomyelin at the membrane leading edge and ICNs. In agreement with previous studies, we here propose that the spatial distribution of CTB–GM1 complexes and cholesterol–sphingomyelin rafts were due to their isotropic and anisotropic shapes, respectively. To elucidate the relationship between a membrane component shape and its spatial distribution, a two-component computational model was constructed. The minimization of the membrane bending (free) energy revealed the enrichment of the anisotropic component along the ICN and the isotropic component in the parent cell membrane, which was due to the curvature mismatch between the ICN curvature and the spontaneous curvature of the isotropic component. The equations of motion, derived from the differentiation of the membrane free energy, revealed a curvature-dependent flux of the isotropic component and a curvature-dependent force exerted on a vesicular dilation along the ICN. Finally, the effects of possible changes in the orientational ordering of the anisotropic component attendant to the transport of the vesicular dilation were discussed with connection to the propagation of electrical and chemical signals. [Copyright &y& Elsevier]

Abstract: The physical properties of organic nanotubes attract increasing attention due to their potential benefit in technology, biology and medicine. We study the effect of ion size on the electrical properties of cylindrical nanotubes filled with electrolyte solution within a modified Poisson–Boltzmann (PB) approach. For comparison purposes, small hollow nanospheres filled with electrolyte solution are considered. The finite size of the particles in the inner electrolyte solution is described by the excluded volume effect within a lattice statistics approach. We found that an increased ion size reduces the number of counterions near the charged inner surface of the nanotube, leading to an enlarged electrostatic surface potential. The concentration of counterions close to the inner surface saturates for higher surface charge densities and larger ions. In the case of saturation, the closest counterion packing is achieved, all lattice sites near the surface are occupied and an actual counterion condensation is observed. By contrast, the counterion concentration at the axis of the nanotube steadily increases with increasing surface charge density. This growth is more pronounced for smaller nanotube radii and larger ions. At larger nanotube radii for small ion size counterion condensation may also be observed according to the Tsao criterion, i.e. the counterion concentration at the centre is independent of the number of counterions in the system. With decreasing radius the Tsao condensation effect is shifted towards physiologically unrealistic surface charge densities. [Copyright &y& Elsevier]