Functionalized titanium oxide surfaces with phosphated carboxymethyl cellulose: characterization and bonelike cell behavior

The performance of dental or orthopedic implants is closely dependent on surface properties in terms of topography and chemistry. A phosphated carboxymethylcellulose containing one phosphate group for each disaccharide unit was synthesized and used to functionalize titanium oxide surfaces with the aim to improve osseointegration with the host tissue. The modified surfaces were chemically characterized by means of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The investigation of the surface topography was performed by atomic force microscopy measurements before and after the polysaccharide coating. In vitro biological tests using osteoblastlike cells demonstrated that functionalized TiO(2) surfaces modulated cell response, in terms of adhesion, proliferation,and morphology. Phosphated carboxymethylcellulose promoted better cell adhesion and significantly enhanced their proliferation. The morphology of cells was polygonal and more spread on this type of modified surface.These findings suggest that the presence of a phosphate polysaccharide coating promotes osteoblast growth on the surface potentially improving biomaterial osseointegration.     

Human osteoblast cell spreading and vinculin expression upon biomaterial surfaces

Any biomaterial implanted within the human body is influenced by the interactions that take place between its surface and the surrounding biological milieu. These interactions are known to influence the tissue interface dynamic, and thus act to emphasize the need to study cell-surface interactions as part of any biomaterial design process. The work described here investigates the relationship between human osteoblast attachment, spreading and focal contact formation on selected surfaces using immunostaining and digital image processing for vinculin, a key focal adhesion component. Our observations show that a relationship exists between levels of cell attachment, the degree of vinculin-associated plaque formation and biocompatibility. It also suggests that cell adhesion is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation.     

Competitive protein adsorption on micro patterned polymeric biomaterials, and viscoelastic properties of tailor made extracellular matrices

Cell adhesion on biomaterial surfaces and the vitality of anchorage dependent cells is affected by several parameters of an adsorbate layer which is intentionally or spontaneously formed. Surface pre-treatments and several conditioning steps prior and during to the cell/biomaterial contact affect the composition, orientation, quantity and viscoelasticity of the interfacing layer between cells and biomaterial. This work was performed to elucidate the response of cells on two modified biomaterial surfaces based on protein or carbohydrate adsorbates: (a) Masked UV irradiations opened a simple route to obtain chemically patterned substrates controlling serum protein adsorption and cell adhesion. It is possible to achieve structures of subcellular size and to produce immobilized gradients. In order to examine the protein matrix deposited on these substrates we applied a quartz microbalance technique (QCM-D) capable to extract viscoelastic data in addition to the mass uptake during plasma protein deposition. It was found that the quantity and viscosity of surface bound albumin is lowered when the surface is modified (patterned) by UV exposure. Hence, the UV modification promotes the competitive adsorption of cell adhesion proteins from the media or upon secretion by the cells and yields to the observed cell patterns. (b) Another tissue engineering technique, using immobilized, modified and/or cross linked hyaluronic acid (HA), an important extra cellular matrix component in vivo, is also examined by QCM-D. Our data demonstrate that HA can be modified by an activation with a carbodiimide, followed by the application of an alpha,omega-bisamino polyethyleneglycol. The QCM-D data can be interpreted as a stiffening of the HA layer combined with the release of hydration water. Further, the hydration state and the viscoelastic behaviour of surface bound ultrathin HA hydrogels was examined. Quantification of viscoelastic parameters of thin films of ECM by QCM-D is valuable for the interpretation of durotaxis, describing effects of mechanical substrate parameters on the adhesion and motility of cells.     

Cytoskeletal Mechanisms of the Formation of Heterocellular Contacts

We studied the interaction of two main cell types, epitheliocytes and fibroblasts, in a mixed culture. Heterotypic cells had a different cytoskeleton organization and expressed different cell adhesion molecules, cadherins. In spite of this, when the cells contacted in the mixed cultures, a heterophilic contact was formed and the actin cytoskeleton of an epitheliocyte at the site of contact was reorganized: the marginal actin bundle was decomposed and actin structures were formed in its place, that were typical for the fibroblast lamella. No changes were observed in the actin organization of the fibroblast. In architecture, the heterophilic adhesion contacts resembled the contacts between fibroblasts. Both heterophilic and homophilic contacts were transient, rather than constant structures. The formation of heterophilic contacts in mixed cultures can serve as a model of formation of a tissue system consisting of epithelium and mesenchyme.     

Cytoskeletal mechanisms of heterocellular contact formation

We studied the interaction of two main cell types, epitheliocytes and fibroblasts, in a mixed culture. Heterotypic cells had a different cytoskeleton organization and expressed different cell adhesion molecules, cadherins. In spite of this, when the cells contacted in the mixed cultures, a heterophilic contact was formed and the actin cytoskeleton of an epitheliocyte at the site of contact was reorganized: the marginal actin bundle was decomposed and actin structures were formed in its place, that were typical for the fibroblast lamella. No changes were observed in the actin organization of the fibroblast. In architecture, the heterophilic adhesion contacts resembled the contacts between fibroblasts. Both heterophilic and homophilic contacts were transient, rather than constant structures. The formation of heterophilic contacts in mixed cultures can serve as a model of formation of a tissue system consisting of epithelium and mesenchyme.     

Fibroblast response is enhanced by poly(L-lactic acid) nanotopography edge density and proximity

The development of scaffolds for use in tissue engineering applications requires careful choice of macroscale properties, such as mechanical characteristics, porosity and biodegradation. The micro- and nano-scale properties of the scaffold surface are also an important design criterion as these influence cell adhesion, proliferation, and differentiation. The cellular response is known to be affected by surface topography but the mechanisms governing this remain unclear. Homogenous poly(L-lactic acid) was textured with surface nanotopographies by two-stage replication molding of heterogeneous demixed polymer films. Initial cell adhesion was improved on nanotextured surfaces compared with smooth controls, but subsequent cell density was significantly reduced on the roughest surfaces. Improvements in cell response were found to correlate with focal contact and actin microfilament development. Cell response was found to trend both with the surface density of topography edges and with inter-topography spacing, indicating possible roles for edges stimulating cell adhesion/proliferation or for spacing to modulate the ability of integrin-ligand bonds to cluster and form focal adhesions. This study furthers understanding of the geometric properties of surface nanotopographies that affect cellular response. It is hoped that identification of the mechanisms governing cell-topography interactions will allow rule-based design of biomaterial surface to engineer specific cellular responses.     

Improved initial osteoblast functions on amino-functionalized titanium surfaces

Adhesion and spreading of cells on biomaterials are integrin-mediated processes. But recent findings indicate a key role of the cell membrane associated matrix substance hyaluronan (HA) in interface interactions. Because HA is a negatively charged molecule we assume that a biomaterial surface with an opposed charge could boost the first contact of the cell to the surface. Polished cp titanium (R(a)=0.19 microm) was coated with an amino-group containing plasma polymer (Ti PPA). For this purpose, a microwave excited, pulsed, low-pressure plasma was used. Additionally, collagen was immobilized on Ti PPA with polyethylene glycol diacid (PEG-DA), catalyzed by carbodiimide (CDI). The physico-chemical surface analytical techniques like XPS, FT-IR, water contact angle and zeta-potential verified the retention of the allylamine precursor structure. Human osteoblasts were cultured in serum-free Dulbecco's modified Eagle medium (DMEM). Adhesion and cell cycle phases were calculated by flow cytometry. Spreading and actin cytoskeleton were visualized by confocal microscopy. Gene expression of osteogenic markers was detected by real-time RT-PCR. Ti PPA is significantly advantageous concerning initial adhesion and spreading during the first hours of the cell contact to the surface. The proliferation of osteoblasts is positively influenced. Gene expression of the differentiation marker bone sialoprotein was upregulated after 24h. Our results demonstrate that functionalization of titanium with positively charged amino-groups is sufficiently enough to significantly improve initial steps of the cellular contact to the material surface.     

Influence of polylysine on adhesion of fibroblasts to glass substrates visualized by total internal reflection microscopy

The cell adhesion topography of mouse fibroblasts growing on glass substrates has been investigated. In order to compare cell adhesion on covered and uncovered glass, substrates were partly exposed to a solution with 0.1 mg/ml polylysine (300 kDa) for 15 min before incubation with cell suspension. After cultivation for 1, 3, 6, and 24 h their adhesion was visualised by total internal reflection microscopy. In the presence of polylysine, cells incubated for 1 h were strongly attracted to the substrate, leading to a typical cell adhesion topography characterised by round concavities under the ventral cell membrane with an approximate diameter of 1 μm. The cavity-surrounding rims were tightly bound to the glass surface. During further cell cultivation, the topography changed into a well-organised adhesion pattern with focal contact areas on the periphery of the cells. In contrast to the polylysine-mediated adhesion, cells growing on untreated surfaces did not exhibit the cavity-like topography at any stage of cultivation, but a more point spread adhesion with a dense clustering of contact-forming areas.     

Probing cytoskeleton organisation of neuroblastoma cells with single-cell force spectroscopy

Single-cell force spectroscopy is an emerging technique in the field of biomedicine because it has proved to be a unique tool to obtain mechanical and functional information on living cells, with force resolution up to single molecular bonds. This technique was applied to the study of the cytoskeleton organisation of neuroblastoma cells, a life-threatening cancer typically developing during childhood, and the results were interpreted on the basis of reference experiments on human embryonic kidney cell line. An intimate connection emerges among cellular state, cytoskeleton organisation and experimental outcome that can be potentially exploited towards a new method for cancer stadiation of neuroblastoma cells.     

Cellular responses to substrate topography: role of myosin II and focal adhesion kinase

Although two-dimensional cultures have been used extensively in cell biological research, most cells in vivo exist in a three-dimensional environment with complex topographical features, which may account for at least part of the striking differences between cells grown in vivo and in vitro. To investigate how substrate topography affects cell shape and movement, we plated fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars. Compared to cells on flat surfaces, 3T3 cells on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability. These responses may be attributed to stabilization of cell adhesion on pillars coupled to myosin II-dependent contractions toward pillars. Moreover, using FAK-/- fibroblasts we showed that focal adhesion kinase, or FAK, is essential for the responses to substrate topography. We propose that increased surface contact provided by topographic features guides cell migration by regulating the strength of local adhesions and contractions, through a FAK- and myosin II-dependent mechanism.     

Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy

Adhesion of adherent cells on structured surfaces is influenced by the surface pattern given. Here, we designed a structured gold relief surface based on cell adhesion patterns we had previously observed. We analysed the geometric parameters and the overall distribution of focal adhesion kinase in focal adhesions on unstructured glass surfaces using optical microscopy. The basic structural elements obtained from this analysis were arranged in regular clusters that resembled the shape of a polarised migratory cell. In time-lapse studies we observed that the cells adhere preferentially to the gold pads and adopt the shape of the clusters. Staining of the actin cytoskeleton revealed that the actin filaments are aligned to the gold pads of the elementary structure.     

Integrin beta 1 cytoplasmic domain dominant negative effects revealed by lysophosphatidic acid treatment.

Integrin receptors localize to focal contact sites and interact with the cytoskeleton via the beta 1 cytoplasmic domain. To study the role of this domain in adhesion, we have expressed in NIH 3T3 cells a cDNA consisting of the interleukin 2 receptor alpha subunit extracellular and transmembrane domains, connected to the integrin beta 1 cytoplasmic domain (IL2R-beta 1). Since the extracellular domain of the chimeric protein has no role in adhesion, this protein could uncouple adhesion from intracellular events. As expected, in a cell line expressing IL2R-beta 1, this chimera was directed to focal contact sites. Unexpectedly, the cells exhibited normal adhesion to fibronectin (FN). However, when a rapid reorganization of the cytoskeleton was induced using lysophosphatidic acid (LPA), IL2R-beta 1 cells detached from FN in contrast to wild-type cells. The detachment in response to LPA could be prevented with cytochalasin D, an inhibitor of actin polymerization. These results imply that a beta 1 cytoplasmic domain, which is uncoupled from adhesion, can compete with the cytoplasmic domain of native integrin beta 1 for cytoskeletal proteins. As a consequence, the IL2R-beta 1 protein acts as a dominant negative effector of adhesion by disrupting the integrin-cytoskeleton connection.     

Biomimetic implant coatings

Biomaterials and tissue engineering technologies are becoming increasingly important in biomedical practice, particularly as the population ages. Cellular responses depend on topographical properties of the biomaterial at the nanometer scale. Structures on biomaterial surfaces are used as powerful tools to influence or even control interactions between implants and the biological system [; ]. The influence of nanometer sized surface structures on osteoblastlike cell interactions was tested with niobium oxide coatings on polished titanium slices (cp-Ti grade 2). The aim of the study was to investigate the influence of nanoscopic surface structures on osteoblast interactions in order to support collagen I production and cell adhesion. The coatings were done by means of the sol-gel process. The surface structure was adjusted by annealing of the metaloxide ceramic coatings due to temperature depended crystal growth. The applied annealing temperatures were 450, 550 and 700 degrees C for 1 h, corresponding to Ra-numbers of 7, 15 and 40 nm. The surfaces were characterized by means of AFM, DTA/TG, diffractometry and white light interferometry. The cell reactions were investigated concerning adhesion kinetics, migration, spreading, cell adhesion, and collagen I synthesis. The smooth surface (Ra=7 nm) resulted in the fastest cell anchorage and cell migration. The closest cell adhesion was reached with the surface structure of Ra=15 nm. The roughest surface (Ra=40 nm) impedes the cell migration as well as a proper spreading of the cells. The best results concerning cell adhesion and spreading was reached with an intermediate surface roughness of Ra=15 nm of the niobium oxide coating on cp-titanium slices.     

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to he a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.     

Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes

Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma. Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E. coli, A. tumefaciens) to recipient (bacterial or plant) cells. Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes. Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells. Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer. Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins.     

Fluorescence-mediated visualization of actin and myosin filaments in the contractile membrane-cytoskeleton complex of Dictyostelium discoideum

An intact complex that consisted of the cell membrane and cytoskeleton was prepared from Dictyostelium amoebae by an improved version of the method previously used by CLARKE et al. (1975). Proc. Natl. Acad. Sci. USA., 72: 1758-1762. After cells had attached tightly to a polylysine-coated coverslip in the presence of a divalent cation, the upper portions of the cells were removed with a jet of microfilament-stabilizing solution squirted from a syringe. The cell membranes left on the coverslip were immediately stained with tetramethylrhodamine-conjugated phalloidin for staining of actin filaments, and with antibody against myosin from Dictyostelium and a fluorescein-conjugated second antibody for staining of myosin. Networks of actin filaments and numerous rod-like structures of myosin (myosin filaments) aligned along them were observed on the exposed cytoplasmic surfaces of the cell membranes. These networks were similar to those observed in the cortex of fixed whole cells. Addition of ATP to these intact complexes of cell membrane and cytoskeleton caused the aggregation of both actin and myosin into several dot-like structures of actin on the cell membrane. Similar dot-like structures were also seen in the cortex of fixed whole cells, and their changes in distribution correlated with the motile activity of the cells. Transmission electron microscopy showed that these dot-like structures were composed of an electron-dense structure at the center, from which numerous actin filaments radiated outwards. These observations suggest that these novel dot-like structures are organizing centers for cortical actin filaments and may possibly be related to the adhesion of cells to the substratum.     

Nuclei deformation in HaCaT keratinocytes cultivated on aligned fibrous substrates

Substrate topography influences cell shape, direction and rate of migration, nucleus shape, and gene expression levels. This influence is commonly studied using substrates with predefined surface structure and chemical composition. In the current work, we studied the state of HaCaT keratinocyte nuclei and actin cytoskeleton on poly(ε-caprolactone) scaffolds obtained by electrospinning. Two types of fibrous scaffolds were prepared and characterized. In the random scaffolds, the fibers were arranged in a nonsystematic fashion; however, most of the fibers had the same direction in the aligned scaffolds. When cultured on the aligned scaffolds, HaCaT cells exhibited oriented actin filaments and had more elongated nuclei.     

Ultrastructure and surface topography of aggregates of human limb mesenchymal cells (HLM15) in vitro.

Tissue-like aggregates of human embryo fibroblasts can be created in vitro by limited aspiration of cells released from substrate during subcultivation. Aggregates increase in size, exhibit intercellular junctions, display a surface topography characteristic of cellular movement, elaborate an extracellular matrix and possess features of cellular death and phagocytosis. These cells, when introduced to a new culture environment, do not migrate away from one another as is common when a primary culture is started from tissue fragments. Instead, cells exhibit continued contact with each other, and develop complex junctional structures during that association. Cellular aggregates generated in this manner may provide a useful system for providing further information on cellular adhesion, intercellular communication, morphogenetic cell movements and the mechanisms of cell death.     

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

Force measurements in E-cadherin-mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42

We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell-cell adhesion. The force required to separate E-cadherin-expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin-expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin-based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.