Involvement of SASH1 in the Maintenance of Stable Cell-Cell Adhesion.

SASH1 is an adapter and signaling protein that contains SH3 and SAM domains responsible for protein-protein interactions. SASH1 downregulation has been observed in many tumors. We examined localization of SASH1 in cultures of normal IAR-20 epithelial cells and HT-29 colorectal cancer cells using immunofluorescence staining and confocal microscopy. IAR-20 normal epithelial cells and HT-29 cells with epithelial phenotype formed stable linear adherens junctions (AJs) associated with circumferential actin bundles. In both IAR-20 and HT-29 cells, SASH1 was co-localized with zones of circumferential actin bundles and linear AJs. SASH1 was not detected in lamellipodia. IAR-20 and HT-29 cells treated with Epidermal Growth Factor underwent epithelial-mesenchymal transition (EMT). We observed significant differences in the course of EMT between IAR-20 and HT-29 cultures. IAR-20 cells underwent partial EMT acquiring migratory phenotype but retaining E-cadherin in unstable radial AJs. SASH1 was present in these contacts. Disappearance of AJs was observed in HT-29 cell undergoing a complete EMT, which also resulted in disruption of stable cell-cell adhesion. SASH1 was lost from the zones of cell-cell interaction. SASH1 depletion by means of RNA interference in IAR-20 cells led to destruction of stable linear AJs and acquisition of mesenchymal phenotype by some of the cells. These data indicate involvement of SASH1 in the maintenance of stable cell-cell adhesion.

Role of Epithelial-Mesenchymal Transition in Tumor Progression.

Epithelial-mesenchymal transition (EMT) is a fundamental process of morphogenesis whereby epithelial cells acquire the mesenchymal phenotype. Multiple data suggest a critical role of EMT in tumor progression. In carcinomas, EMT can be initiated and promoted by many oncogenic signaling pathways, hypoxia, and signals of tumor microenvironment resulting in epithelial cells losing their cell polarity and cell-cell adhesion and gaining the migratory and invasive properties. Downregulation of expression of the cell adhesion protein E-cadherin is considered a poor prognostic factor in cancer. Many tumors are characterized by incomplete EMT, where tumor cells acquire mesenchymal characteristics but retain their epithelial markers, in particular, E-cadherin. In cells with the hybrid epithelial-mesenchymal phenotype, E-cadherin is accumulated in adherens junctions which are less stable than adherens junctions in normal epithelial cells. E-cadherin-based adherens junctions are essential for efficient collective migration and invasion of carcinoma cells, and their survival in metastasis. The plasticity of the hybrid epithelial-mesenchymal phenotype improves adaptive capabilities of cancer cells. By undergoing EMT, carcinoma cells become resistant to chemotherapy and acquire the ability to suppress immune response. Emergence of cancer stem cells after EMT activation has been observed in many types of carcinoma.

Synergistic and long-lasting antibacterial effect of antibiotic-loaded TiCaPCON-Ag films against pathogenic bacteria and fungi.

Implant-related bacterial infections remain a serious problem that is not solved yet. Herein we combined several antibacterial agents to achieve synergistic effects and broader protection of widely used metallic implants. Titanium samples with microcontainers for drug, produced by selective laser sintering, were coated with Ag-doped biocompatible and bioactive TiCaPCON film and loaded with an antibiotic (gentamicin or a mixture of gentamicin and amphotericin B). Bactericide release tests demonstrated that the release rate of one bactericide agent (Ag+ ions or gentamicin) depended on the presence of the other antibacterial component. The antibacterial activity of the biocide-doped samples was evaluated against clinically isolated Escherichia coli O78 (E. coli), Staphylococcus aureus (S. aureus) bacteria, and Neurospora crassa wt-987 (N. crassa) spores. It was found that samples loaded with low gentamicin concentration (0.2 and 0.02 mg/cm2), i.e. 10 and 100 times less than the standard gentamicin concentration (2 mg/cm2), demonstrated a superb antibacterial activity against E. coli bacteria. We showed that a crosslinking reaction between gentamicin and TiCaPCON film proceeded either through the formation of amide bonds or via the electrostatic interaction between amine groups of gentamicin and COOH groups of TiCaPCON and led to the formation of relatively stable drug/film conjugates that prevented a rapid dissolution of gentamicin and ensured its long-term (for 72 h) antibacterial protection. Leaching of silver ions provided an effective antibacterial protection after the depletion of the drug reservoirs. The obtained results clearly show a synergistic antibacterial action of Ag+ ions and gentamicin against S. aureus bacteria. In addition, in the presence of Ag+ ions, the antifungal activity of samples loaded with a mixture of gentamicin and amphotericin B against N. crassa fungus was observed to increase. Thus, it is demonstrated that silver can be successfully coupled with different types of antibiotics to provide innovative hybrid metal-ceramic bioconstructions that are able to deliver precise doses of bactericide agents within a certain period of time and are equally effective against Gram-negative E. coli bacteria, Gram-positive S. aureus, and N. crassa fungus.

Antibacterial Performance of TiCaPCON Films Incorporated with Ag, Pt, and Zn: Bactericidal Ions Versus Surface Microgalvanic Interactions.

It is very important to prevent bacterial colonization at the early postoperative stages. There are four major strategies and their corresponding types of antibacterial surfaces specifically designed to fight infection: bactericide release, anti-adhesion, pH-sensitive, and contact-killing. Herein, we aimed at determining the antibacterial efficiency of different types of bactericidal ions and revealing the possible contribution of surface microgalvanic effects arising from a potential difference on heterogeneous surfaces. We considered five types of TiCaPCON films, with Ag, Zn, Pt, Ag + Zn, and Pt + Zn nanoparticles (NPs) on their surface. The Ag-modified film demonstrated a pronounced antibacterial effect at a very low Ag ion concentration of 0.11 ppb in physiological solution that was achieved already after 3 h of immersion in Escherichia coli ( E. coli) bacterial culture. The Zn-containing sample also showed a noticeable antibacterial effect against E. coli and Staphylococcus aureus ( S. aureus) strains, wherein the concentration of Zn ions was 2 orders of magnitude higher (15 ppb) compared with the Ag ions. The presence of Ag NPs accelerated the leaching of Zn ion out of the TiCaPCON-Ag-Zn film, but no synergistic effect of the simultaneous presence of the two bactericidal components was observed. After the incubation of the samples with Ag, Zn, and Ag + Zn NPs in E. coli and S. aureus suspensions for 24 and 8 h, respectively, all bacterial cells were completely inactivated. The Pt-containing film showed a very low Pt ion release, and therefore the contribution of this type of ions to the total bactericidal effect could be neglected. The results of the electrochemical studies and Kelvin probe force microscopy indicated that microgalvanic couples were formed between the Pt NPs and the TiCaPCON film, but no noticeable antibacterial effect against either E. coli or S. aureus strains was observed. All ion-modified samples provided good osteoblastic cell attachment, spreading, and proliferation and therefore were concluded to be nontoxic for cells. In addition, the TiCaPCON films with Ag, Pt, and Zn NPs on their surface demonstrated good osteoconductive characteristics.

Multifunctional Ti-(Ca,Zr)-(C,N,O,P) films for load-bearing implants.

Films of Ti-Ca-P-C-O-(N), Ti-Ca-C-O-(N) and Ti-Zr-C-O-(N) were deposited by DC magnetron sputtering or ion implantation-assisted magnetron sputtering of composite targets TiC0.5 + 10%Ca10(PO4)6(OH)2, TiC0.5 + 20%(CaO + TiO2) and TiC0.5 + 10%ZrO2 in an Ar atmosphere or reactively in a gaseous mixture of Ar + 14%N2. The microstructure, elemental and phase composition of films were studied by means of X-ray diffraction, transmission electron microscopy, scanning force microscopy, X-ray photoelectron spectroscopy and energy-dispersive X-ray spectroscopy. The films were characterized in terms of their hardness, Young's modulus, elastic recovery, adhesion strength, and friction and wear both in air and under physiological solution. Particular attention was paid to the analysis of deformation and fracture for various film/substrate systems during scratch testing. The biocompatibility of the films was evaluated by both in vitro and in vivo experiments. In vitro studies involved the investigation of adhesion, spreading, and proliferation of MC3T3-E1 osteoblasts and IAR-2 epithelial cells, morphometric analysis, actin cytoskeleton, focal contacts staining, alkaline phosphatase activity and von Kossa staining of osteoblastic culture. Cell culture experiments demonstrated an increase of osteoblastic proliferation on Ca- and P-incorporated films. In vivo studies were fulfilled by subcutaneous implantation of Teflon plates coated with the tested films in mice and analysis of the population of adherent cells on their surfaces. The results obtained show that multicomponent nanostructured Ti-(Ca, Zr)-(C, N, O, P) films possess a combination of high hardness, wear resistance and adhesion strength, reduced Young's modulus, low friction coefficient and high biocompatibility.

Characteristics and in vitro response of thin hydroxyapatite-titania films produced by plasma electrolytic oxidation of Ti alloys in electrolytes with particle additions.

The enhancement of the biological properties of Ti by surface doping with hydroxyapatite (HA) is of great significance, especially for orthodontic applications. This study addressed the effects of HA particle size in the electrolyte suspension on the characteristics and biological properties of thin titania-based coatings produced on Ti-6Al-4V alloy by plasma electrolytic oxidation (PEO). Detailed morphological investigation of the coatings formed by a single-stage PEO process with two-step control of the electrical parameters was performed using the Minkowski functionals approach. The surface chemistry was studied by glow discharge optical emission spectroscopy and Fourier transform infrared spectroscopy, whereas mechanical properties were evaluated using scratch tests. The biological assessment included in vitro evaluation of the coating bioactivity in simulated body fluid (SBF) as well as studies of spreading, proliferation and osteoblastic differentiation of MC3T3-E1 cells. The results demonstrated that both HA micro- and nanoparticles were successfully incorporated in the coatings but had different effects on their surface morphology and elemental distributions. The micro-particles formed an irregular surface morphology featuring interpenetrated networks of fine pores and coating material, whereas the nanoparticles penetrated deeper into the coating matrix which retained major morphological features of the porous TiO2 coating. All coatings suffered cohesive failure in scratch tests, but no adhesive failure was observed; moreover doping with HA increased the coating scratch resistance. In vitro tests in SBF revealed enhanced bioactivity of both HA-doped PEO coatings; furthermore, the cell proliferation/morphometric tests showed their good biocompatibility. Fluorescence microscopy revealed a well-organised actin cytoskeleton and focal adhesions in MC3T3-E1 cells cultivated on these substrates. The cell alkaline phosphatase activity in the presence of ascorbic acid and ß-glycerophosphate was significantly increased, especially in HA nanoparticle-doped coatings.

Design, characterization and testing of Ti-based multicomponent coatings for load-bearing medical applications.

A comparative investigation of multicomponent thin films based on the systems Ti-Ca-C-O-(N), Ti-Zr-C-O-(N), Ti-Si-Zr-O-(N) and Ti-Nb-C-(N) is presented. TiC(0.5) + 10%CaO, TiC0.5 + 20%CaO, TiC0.5 + 10%ZrO2, TiC0.5 + 20%ZrO2, Ti5Si3 + 10%ZrO2, TiC0.5 + 10%Nb2C and TiC0.5 + 30%Nb2C composite targets were manufactured by means of self-propagating high-temperature synthesis, followed by DC magnetron sputtering in an atmosphere of argon or in a gaseous mixture of argon and nitrogen. The films were characterized in terms of their structure, chemical composition, surface topography, hardness, elastic modulus, elastic recovery, surface charge, friction coefficient, and wear rate. The biocompatibility of the films was evaluated by both in vitro and in vivo experiments. In vitro studies involved the investigation of the proliferation of Rat-1 fibroblasts and IAR-2 epithelial cells on the tested films, morphometric analysis and actin cytoskeleton staining of the cells cultivated on the films. In vivo studies were fulfilled by subcutaneous implantation of Teflon plates coated with the tested films in mice and analysis of the population of cells on the surfaces. The films deposited under optimal conditions showed high hardness in the range of 30-37 GPa, significant reduced Young's modulus, low friction coefficient down to 0.1-0.2 and low wear rate in comparison with conventional magnetron-sputtered TiC and TiN films. The surface of all films was negatively charged with an outstanding shift between the Ar and Ar + N2 Zeta potential curves that reaches 5 mV at the highest pH values. We did not detect statistically significant differences in the attachment, spreading and cell shape of cultured IAR-2 and Rat-1 cells on the Ti-Ca-C-O-(N), Ti-Zr-C-O-(N) (TiC0.5 + 10%ZrO2 target), Ti-Si-Zr-O-(N) films and the uncoated substrata. The adhesion and proliferation of cultured cells in vitro was perfect at all investigated films. Assessment of the population of cells covering on the Teflon plates coated with the Ti-Ca-C-O-(N) and Ti-Zr-C-O-(N) films after 16 weeks of subcutaneous implantation revealed the high biocompatibility level of tested films and absence of inflammatory reactions in mice. Contrary, the epitheliocytes and fibroblasts cultivated on the Ti-Zr-C-O-(N) (TiC0.5 + 20%ZrO2 target) and Ti-Nb-C-(N) films had disturbing actin cytoskeleton.

Toward bioactive yet antibacterial surfaces.

The fabrication of antibacterial yet biocompatible and bioactive surfaces is a challenge that biological and biomedical community has faced for many years, while no "dream material" has been developed so far. The primary goal of this study was to establish an optimal range of Ag concentration and its state of agglomeration in bioactive nanocomposite TiCaPCON films which would provide a strong bactericidal effect without compromising the material biocompatibility and bioactivity. To obtain samples with different Ag content and redistribution, two different methods were employed: (i) TiCaPCON films deposition by magnetron sputtering of composite TiС0.5-Ca3(РО4)2 target followed by Ag(+) ion implantation and (ii) Ag-doped TiCaPCON films obtained by co-sputtering of composite TiС0.5-Ca3(РО4)2 and Ag targets. In order to reveal the antibacterial role of Ag nanoparticles and Ag(+) ions, both separate and in synergy, part of the samples from the first and second groups was subjected to additional ion etching to remove an Ag rich surface layer heavily populated with Ag nanoparticles. All resultant films were characterized with respect to surface morphology, chemical composition, surface roughness, wettability, and Ag(+) ion release. The antibacterial and antifungal effects of the Ag-doped TiCaPCON films were evaluated against clinically isolated Escherichia coli O78 (E. coli) and Neurospora crassa wt-987 spores. The influence of the surface chemistry on spreading, proliferation, and early stages of MC3T3-E1 osteoblastic cell differentiation was also studied. Our data demonstrated that under optimal conditions in terms of Ag content and agglomeration, the Ag-doped TiCaPCON films are highly efficient against E. coli bacteria and, at the same time, provide good adhesion, spreading, proliferation and differentiation of osteoblastic cells which reflect high level of biocompatibility and bioactivity of the films. The influence of Ag(+) ions and nanoparticles on the MC3T3-E1 osteoblastic cells and E. coli bacteria is also discussed.

Changes in regulation of cell-cell adhesion during tumor transformation.

Cadherin-mediated cell-cell adhesion defines the integrity of most tissues. Cell-cell adherens junctions are dynamic structures whose functional state is regulated by interactions of cadherin with beta-catenin, p120, and actin cytoskeleton structures. Small GTPases of the Rho family and GTPase Rap1 play the central role in the formation and maintenance of cell-cell adhesion. Aberrant activation of signaling pathways, transcriptional repression of the E-cadherin gene, ectopic expression of N-cadherin, and disturbances in regulation of adhesive and transcriptional functions of beta-catenin stimulate tumor progression.

Role of the microtubular system in morphological organization of normal and oncogene-transfected epithelial cells.

To understand better the role of the microtubular system in the development and maintenance of morphological organization of nonpolarized and polarized cells of the same origin we examined the effects of two microtubule-specific drugs, colcemid and taxol, on discoid cultured epithelial rat cells of the IAR-2 line and on polarized cells obtained from this line by transfection of mutated N-ras oncogene; morphometric, immunomorphologic, and videomicroscopic methods were used. Depolymerization of microtubules by colcemid did not cause major changes in the discoid shape of IAR cells but altered organization of actin cortex; in particular, it led to disappearance of circumferential bundle of actin microfilaments. Taxol reorganized the normal network of microtubules radiating from the perinuclear centers into numerous arrays of short microtubules not associated with any centers. Taxol-treated cells had wider circumferential bundles of microfilaments than control cells and morphometric analysis showed that their contours were closer to geometric circle than those of control or of colcemid-treated cells. These data show that function of the microtubular system is essential for maintenance of the characteristic morphological organization of discoid cells; we propose to name this function "contra-polarization." Contra-polarization is not prevented and is even promoted by taxol; this result suggests that a decentralized system of microtubules is sufficient for this function. In contrast, maintenance of polarized morphology of IAR-2 cells transfected by the N-ras oncogene is inhibited not only by colcemid but also by taxol and thus requires the presence of a normal centralized microtubular system.

Myosin-dependent contractile activity of the actin cytoskeleton modulates the spatial organization of cell-cell contacts in cultured epitheliocytes.

The spatial organization of cell-cell adherens junctions is distinct in cultured cells from two different tissue types, specifically, epitheliocytes and fibroblasts. In epitheliocytes, contacts are localized tangentially, along contacting cell edges and in association with circumferential actin bundles. Contacts between fibroblasts are radially oriented; that is, they are perpendicular to the overlapping edges of the cells and are associated with straight bundles of actin filaments. In the present study, we establish that the spatial organization of cell-cell contacts in the epithelial cell line IAR-2 can be converted from the typical tangential pattern to the radial pattern observed in fibroblasts. This transition can be induced by treatment with two agents, phorbol 12-myristate 13-acetate and nocodazole, which have different modes of action. Inhibition of myosin contractility reverses tangential-to-radial conversion of cell-cell contacts. These data suggest that formation of radially aligned contacts depends on modulation of contractility within the actin cytoskeleton through the myosin motor protein. The results open the possibility that modulation of the spatial organization of cell-cell contacts may play important roles in regulating organization and physiological functions of epithelial tissues.

Cell-cell contact changes the dynamics of lamellar activity in nontransformed epitheliocytes but not in their ras-transformed descendants.

We investigated the structural and functional alterations of active lamellae during initial cell-cell collision and establishment of cell-cell contacts in wounded cultures of nontransformed rat epitheliocytes (IAR-2 line) and their ras-transformed descendants (C4 line). Typically, the leading edges of nontransformed cells formed multiple transient contacts followed by establishment of small, stable contacts that would undergo lateral expansion. Formation and expansion of the contact area was accompanied by accumulation of the cell-cell adhesion molecules E-cadherin, beta-catenin, and plakoglobin. During lateral expansion, the circumferential bundles of actin filaments, characteristic of IAR-2 cells, disassembled at the site of stable contact forming a concave arc-like actin bundle between adjacent cells at the expanding edge. Pseudopodial activity was completely inhibited in the contact zone and partially inhibited at the free lamellar edges adjacent to the zone of contact. Con A-coated beads on the plasma membrane at the zone of contact stopped undergoing centripetal transport but now moved along the cell-cell boundary. On the other hand, ras-transformed cells developed overlapping lamellae and exhibited no detectable change in activity of lamellae, localization of adhesion molecules, and organization of the actin cytoskeleton. We propose that contact-induced reorganization of cell surface adhesion molecules and the underlying cortical cytoskeleton leads to development of lateral traction that may be an essential element in inducing expansion of the contact and in inhibiting local pseudopodial activity.

Dynamics of active lamellae in cultured epithelial cells: effects of expression of exogenous N-ras oncogene.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

Dynamics of contacts between lamellae of fibroblasts: essential role of the actin cytoskeleton.

We investigated actin cytoskeletal and adhesion molecule dynamics during collisions of leading lamellae of nontransformed and oncogene-transformed fibroblasts. By using real-time video microscopy, it was found that during lamellar collision there was considerable overlapping of leading lamellae followed by subsequent retraction. Overlapping of nontransformed fibroblasts was accompanied by formation of beta-catenin-positive contact structures organized into strands oriented parallel to the long axis of the cell that were associated with bundles of actin filaments. Maintenance of such cell-cell contact structures critically depended on the contractility of actin cytoskeleton, as inhibition of contractility with serum-free medium or 2,3-butanedione 2-monoxime (BDM) resulted in loss of strand formation. Strand formation was recovered when cells in serum-free medium were incubated with the microtubule inhibitor nocodazole, which is known to increase contractility. Oncogene-transformed fibroblasts reacted to collisions with responses similar to nontransformed fibroblasts but did not develop well-organized cell-cell contacts. A model is presented to describe how differences in the organization of the actin cytoskeleton could account for the structurally distinct responses to cell-cell contact by polarized fibroblastic cells versus nonpolarized epithelial cells.