The role of surface wettability on hepatocyte adhesive interactions and function

In this paper the effect of surface wettability on hepatocyte morphology and function was studied, using clean and octadecylsylane (ODS)-coated glass as a model for hydrophilic and hydrophobic surfaces, respectively. C3A cells--a hepatoblastoma cell line, and freshly obtained porcine hepatocytes were cultured for a short-time period of up to 4 days on the above substrata. Hepatocyte adhesive interactions were characterized monitoring the initial cell attachment, the overall cell morphology, the formation of focal adhesions, and actin filaments. Since hepatocytes showed a clear tendency for homotypic adhesion on ODS, specific E-cadherin staining was used to visualize the intercellular contacts by immunofluorescence microscopy. Additionally, functional assays were carried out to monitor proliferation, metabolic activity, and albumin synthesis of C3A cells. It could be shown that both C3A cells and normal porcine hepatocytes spread better on hydrophilic glass; spreading being accompanied by the development of pronounced actin stress fibers and focal adhesion contacts. In contrast, on hydrophobic substrata predominant cell-cell interactions took place which led to intense E-cadherin staining in the intercellular contacts of porcine hepatocytes but not in C3A cells. On the other hand, metabolic activity and growth of C3A cells were reduced on hydrophobic ODS, but albumin synthesis was similar on both surfaces. It was concluded that the wettability of materials has a strong influence on the attachment and morphology of hepatocytes while the influence of surface properties on the functional activity of hepatocytes still remains to be elucidated.     

Membranes for biohybrid liver support systems--investigations on hepatocyte attachment,morphology and growth

The biological properties of four different membranes were studied regarding their possible application in biohybrid liver support systems. Two of them, one made of polyetherimide (PEI), and a second based on polyacrylonitrile-N-vinylpyrollidone co-polymer (P(AN-NVP)), were recently developed in our lab and studied for the first time. Together with pure polyacrylonitrile (PAN) membranes, the three preparations were characterised as ultra-filtration membranes. Their ability to support cell attachment, morphology, proliferation and function of human hepatoblastoma C3A cells was studied. The role of surface morphology for the interaction with hepatocytes was highlighted using a commercial, moderately wettable polyvinylidendifluoride (PVDF) membrane with micro-filtration properties. Comparative investigations showed strongest interaction of C3A cells with PAN membranes, as the focal adhesion contacts were more expressed and cell growth was also high. However, the functional activity in terms of albumin synthesis was reduced. Very similar results were obtained with the most hydrophobic PEI membrane. In contrast, the most hydrophilic membrane P(AN-NVP) was found to provoke stronger homotypic adhesion (E-cadherin expression) of C3A cells and less substratum attachment (focal adhesions), but enhanced albumin secretion. However, proliferation of C3A cells was lowered. Micro-porous PVDF membrane showed very good initial attachment, but the resulting cell material and cell-cell interaction were relatively poor developed. Among four membranes tested, PEI seems to be the most attractive membrane for biohybrid liver devices, as it provides good surface properties for hepatocytes interaction, but in addition it is highly thermostable, which would permit steam sterilisation. No simple relationship, however, between the wettability of the membranes and their ability to support hepatocyte adhesion and function was found in this study.     

Integrin expression and osteopontin regulation in human fetal osteoblastic cells mediated by substratum surface characteristics

Integrin-mediated adhesion of anchorage-dependent cells to scaffolds is a critical component of tissue engineering. We investigated integrin expression by the human fetal osteoblastic cell line, hFOB 1.19 (hFOB), as a function of substratum surface wettability. The influence of surface wettability on bone cell phenotype was also examined. Plasma-treated quartz (PTQ) and glass (PTG) (hydrophilic, contact angles of 0 degrees), octadecyltrichlorosilane-treated quartz (STQ) and glass (STG) (hydrophobic, contact angles above about 100 degrees), and tissue culture polystyrene were used for cell culture. hFOB cells cultured on hydrophilic substrata displayed well-developed actin stress fibers relative to cells on hydrophobic substrata. Western blot analysis revealed that hFOB cells cultured on hydrophobic substrata (STQ or STG) express lower levels of alphav and beta3 integrin subunits than do cells on hydrophilic substrata (PTQ or PTG). This effect was more pronounced in cells on STQ than on STG. These variations in integrin expression were lessened by extended culture time. Double- labeled integrin/actin immunofluorescence confirmed Western blot results, that is, cells cultured on PTQ displayed distinct, large plaques of alphav and beta3 subunits and integrin alphavbeta3, as well as their colocalization with actin stress fiber ends, whereas cells on STQ did not display integrin plaques after 24 h and displayed only minimal plaque formation after 3 days. Vinculin, a focal adhesion protein that mediates binding between the integrin and actin cytoskeleton, appeared in Western blots to mimic the variations of alphav and beta3 expression with respect to surface wettability. Interestingly, real-time RT-PCR analysis showed that hFOB cultured on hydrophobic substrata, which have downregulated alphav and beta3 integrin subunits, displayed greater steady state mRNA levels of osteopontin, an extracellular matrix (ECM) protein containing the Arg-Gly-Asp (RGD) integrin recognition sequence, than did cells cultured on hydrophilic substrata. Our results imply that substratum surface wettability regulates integrin-mediated bone cell adhesion and further influences the expression of bone cell-ECM complexes.     

Altered vitronectin receptor (alphav integrin) function in fibroblasts adhering on hydrophobic glass.

Function of integrins is crucial for adhesion, movement, proliferation, and survival of cells. In a recent study we found impaired fibronectin receptor function on hydrophobic substrata (G. Altankov et al. J Biomater Sci Polym Edn 1997;8:712-740). Here, we have studied the distribution and function of the vitronectin receptor (alphav integrin) in fibroblasts adhering on hydrophilic glass and hydrophobic octadecyl glass (ODS). The morphology of fibroblasts and the organization of actin cytoskeleton were studied and found to be altered on ODS, where the cells did not spread and possessed condensed actin. Pretreatment of the surfaces with serum or pure vitronectin improved cell morphology on both substrata, resulting in the development of longitudinal actin stress fibers. It was found with biotinylated vitronectin that comparable quantities of vitronectin were adsorbed from single vitronectin solutions or serum on glass and on hydrophobic ODS. The organization of the vitronectin receptors on the ventral cell surface was investigated in permeabilized cells showing normal focal adhesions in fibroblasts plated on glass but none of these structures on ODS. The distribution of alphav integrin on the dorsal cell surface was studied on nonpermeabilized living cells after antibody tagging. While fibroblasts adhering on plain or serum-treated glass developed a linear organization of alphav integrin, cells on plain and serum-treated ODS were not able to reorganize the vitronectin receptor. Studies on signal transduction with antiphosphotyrosine antibodies revealed co-localization of alphav integrin and phosphotyrosine in focal adhesions on glass and serum-treated glass. However, signaling was almost absent on plain ODS and weak on serum-treated ODS. It was concluded that alterations in vitronectin receptor function on the ventral cell surface caused by the hydrophobic material surface inhibit signal transfer and subsequent intracellular events that are important for the organization and function of integrins.     

Amount and surface structure of albumin adsorbed to solid substrata with different wettabilities in a parallel plate flow cell

In this article we studied the adsorption of serum albumin to substrata with a broad range of wettabilities from solutions with protein concentrations between 0.03 and 3.00 mg.mL-1 in a parallel-plate flow cell. Wall shear rates were varied between 20 and 2000 s-1. The amount of albumin adsorbed in a stationary state was always highest on PTFE, the most hydrophobic material employed and decreased with increasing wettability of the substrata. Increasing stationary amounts of adsorbed albumin were observed with increasing wall shear rates at the lowest protein concentration. Inverse observations were made at the highest protein concentration. Transmission electron micrographs of replicas from the albumin-coated substrata showed that proteins were mostly adsorbed in islandlike structures on the hydrophobic substrata. The tendency to form islandlike structures was shear rate- and concentration-dependent and disappeared gradually going to more hydrophilic substrata. On glass, the most hydrophilic material employed, a homogeneous, well distributed, fine knotted, reticulated structure was found. In conclusion, this study demonstrates that both the amount of adsorbed albumin as well as the surface structure of the adsorbed proteins are regulated by the substratum wettability. This observation may well account for the fact that substratum properties can be transferred by an adsorbed protein film to the interface with adhering cells or microorganisms.     

Morphological and functional studies of rat hepatocytes on a hydrophobic or hydrophilic polydimethylsiloxane surface

This study describes the morphological and functional behavior of rat hepatocytes on a polydimethylsiloxane (PDMS)-coated surface. Hepatocytes were cultured on hydrophobic or hydrophilic PDMS-coated surfaces in serum-free and serum-containing media. In the serum-free medium, almost all hepatocytes adhered onto the surface irrespective of the wettability, with a cell adhesion ratio of >90% at 24h. In the serum-containing medium, although they strongly adhered onto the hydrophilic surface (cell adhesion ratio >85%), the ratio on the hydrophobic surface was <15%. Furthermore, hepatocytes in the serum-free medium gradually formed spheroids irrespective of the surface characteristics; however, on the hydrophilic surface in the serum-containing medium, they maintained a monolayer configuration for up to 10 days, and their numbers gradually decreased over time. Expression levels of the functional activities (albumin secretion and ammonia removal) and the cell-cell adhesion molecules (cadherin and connexin-32) were higher in the hepatocytes that formed spheroids compared to those which assumed a monolayer configuration, and these levels were maintained for at least 10 days. These results suggest that the wettability of PDMS and the composition of the culture medium together control the cell adhesion, morphology and expression of functional genes in hepatocytes.     

Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis

While microporous biopolymer matrices are being widely tested as cell culture substrates in hepatic tissue engineering, the microstructural basis for their control of cell differentiation is not well understood. In this paper, we studied the role of void size of collagen foams in directing the induction of liver-specific differentiated morphology and secretory activities of cultured rat hepatocytes. Hepatocytes cultured on collagen foams with subcellular sized pore diameters of 10 microm assumed a compact, cuboidal cell morphology, rapidly achieving monolayer coverage, and secreted albumin at the rate of 40 +/- 8 pg/cell/d. Increasing the pore size to 18 microm elicited a distinctly spread cellular phenotype, with discontinuous surface coverage, and albumin secretion rates declined precipitiously to 3.6 +/- 0.8 pg/cell/d. However, when collagen foams with an even higher average void size of 82 microm were used, hepatocytes exhibited high degree of spreading within an extensive three-dimensional cellular network, and exhibited high albumin secretory activity (26 +/- 0.6 pg/cell/d). The effect of void geometry on cellular ultrastructral polarity was further analyzed for the three void size configurations employed. The distribution of the cell-cell adhesion protein, E-cadherin, was primarily restricted to cell-cell contacts on the 10 microm foams, but was found to be depolarized to all membrane regions in cells cultured on the 18 and 82 microm foams. Vinculin-enriched focal adhesions were found to be peripherally clustered on cells cultured on 10 microm foams, but were found to redistribute to the entire ventral surface of cells cultured on the 18 and 82 microm foams. Overall, we demonstrate the significance of designing pore sizes of highly adhesive substrates like collagen toward selective cell morphogenesis in 2-D or 3-D. Subcellular and supercellular ranges of pore size promote hepatocellular differentiation by limiting 2-D cell spreading or effecting 3-D intercellular contacts, while intermediate range of pore sizes repress differentiation by promoting 2-D cell spreading.     

Membranes for biohybrid liver support: the behaviour of C3A hepatoblastoma cells is dependent on the composition of acrylonitrile copolymers

Co-polymers based on acrylonitrile, N-vinylpyrrolidone, aminoethylmethacrylate and sodium methallylsulfonate were used to prepare flat membranes by phase inversion. The surface properties of membranes were characterised by water contact angle measurements, atomic force microscopy and X-ray photoelectron spectroscopy (XPS). Membrane permeability was estimated by porosity measurements with water as test liquid. Human C3A hepatoblastoma cells were plated on these materials. Cell-material interaction was characterised by overall cell morphology, formation of focal adhesion contacts and intercellular junctions. Furthermore, cell proliferation was measured and compared with the functional activity of cells as indicated by 7-ethoxycoumarin-O-deethylation. More hydrophilic materials reduced spreading of cells, formation of focal adhesion and subsequent proliferation while homotypic cell adhesion was facilitated in correlation with stronger expressions of intercellular junctions and improved functional activity. In contrast, membranes with stronger adhesivity enhanced cell proliferation but reduced the functional activity of cells. It was concluded that the co-polymerisation of acrylonitrile with hydrophilic co-monomers, such as N-vinylpyrrolidone, could be used to tailor membrane materials for the application in biohybrid liver support systems.     

Morphological evidence for a different fibronectin receptor organization and function during fibroblast adhesion on hydrophilic and hydrophobic glass substrata

A polyclonal antibody against the β1 subunit of the fibronectin (FN) receptor was used to mimic the early events of integrin receptor functioning to study the initial cellular processes during the organization of FN matrix on biomaterials. Hydrophilic glass and hydrophobic octadecylsilane (ODS) surfaces have been applied as models for different biocompatible materials. By immunofluorescence we could demonstrate that FN receptors organize on the dorsal cell surface of adhering fibroblasts in a specific linear pattern along with actin filaments, but only if the cells were attached to hydrophilic glass. In contrast, FN receptors were not reorganized on hydrophobic octadecylsilane (ODS). In parallel experiments, FN matrix formation after 72 h of incubation on the same substrata has been analyzed microscopically, and quantified by cell ELISA, in order to be further correlated with the integrin receptor functioning in contact with the biomaterials. It was found that FN structuring and the amount of FN matrix have been significantly diminished on ODS that was related to the observed changes in integrin receptor functioning. To learn more about the mechanism of this phenomenon, desorption of 125-FN from these substrata was studied and found to be significantly decreased on hydrophobic ODS. As a consequence, FN receptor (function) might be arrested on the ventral cell surface, thus the important role of β 1 integrins in the positional organization of the FN matrix may be disturbed. In light of these facts, antibody-induced clustering of FN receptor can be considered as a useful model for studying the early steps of FN matrix formation on biomaterials.     

Adhesion of mouse fibroblasts on hexamethyldisiloxane surfaces with wide range of wettability

Surface wettability is an important physicochemical property of biomaterials, and it would be more helpful for understanding this property if a wide range of wettability are employed. This study focused on the effect of surface wettability on fibroblast adhesion over a wide range of wettability using a single material without changing surface topography. Plasma polymerization with hexamethyldisiloxane followed by oxygen (O2)-plasma treatment was employed to modify the surfaces. The water contact angle of sample surfaces varied from 106 degrees (hydrophobicity) to almost 0 degrees (super-hydrophilicity). O2 functional groups were introduced on polymer surfaces during O2-plasma treatment. The cell attachment study confirmed that the more hydrophilic the surface, the more fibroblasts adhered in the initial stage that includes super-hydrophilic surfaces. Cells spread much more widely on the hydrophilic surfaces than on the hydrophobic surfaces. There was no significant difference in fibroblast proliferation, but cell spreading was much greater on the hydrophilic surfaces. The fibronectin adsorbed much more on a hydrophilic surface while albumin dominated on a hydrophobic surface in a competing mode. These findings suggest the importance of the surface wettability of biomaterials on initial cell attachment and spreading. The degree of wettability should be taken into account when a new biomaterial is to be employed.     

Cytomimetic engineering of hepatocyte morphogenesis and function by substrate-based presentation of acellular E-cadherin

Although cadherin-mediated intercellular contacts can be integral to the maintenance of functionally competent hepatocytes in vitro, the ability to engineer hepatocellular differentiated function via acellular E-cadherin has yet to be thoroughly explored. To investigate the potential of substrate-presented, acellular E-cadherin to modulate hepatocellular self-assembly and functional fate, rat hepatocytes were cultured at sparse densities on surfaces designed to display recombinant E-cadherin/Fc chimeras. On these substrates, hepatocytes were observed to recognize microdisplayed E-cadherin/Fc and responded by modulating the spatial distribution of the intracellular cadherin-complexing protein beta-catenin. Substrate-presented E-cadherin/Fc was also found to markedly alter patterns of hepatocyte morphogenesis, as cellular spreading and two-dimensional reorganization were significantly inhibited under these conditions, leading to multicellular aggregates that were considerably more three-dimensional in nature. Increasing cadherin exposure was also associated with elevated levels of albumin and urea secretion, two markers of hepatocyte differentiation, over control cultures. This suggested that cell-substrate cadherin engagement established more functionally competent hepatocellular phenotypes, coinciding with the notion that E-cadherin is a differentiation-inducing ligand for these cells. The morphogenetic and function-promoting effects of substrate-bound E-cadherin/Fc were further enhanced under conditions in which protein A was utilized as an anchoring molecule to present cadherin molecules, suggesting that ligand mobility may play an important role in the effective establishment of cell-to-substrate cadherin interactions. Interestingly, the percent increase in function detected for conditions of high cadherin exposure versus control cultures was found to be substantially higher at extremely low cell densities. This observation indicated that hepatocytes respond to substrate-presented E-cadherin even in the absence of native intercellular interactions and associated juxtacrine signaling. The incorporation of acellular E-cadherin on biomaterial substrates may thus potentially present a means to prevent hepatocellular dedifferentiation by maintaining liver-specific function in otherwise severely functionally repressive culture conditions.     

Endothelial cell motility, integrin receptor clustering, and microfilament organization are inhibited by agents that increase intracellular cAMP

Adhesion of human umbilical vein endothelial cells (ECs) to various extracellular matrix proteins is mostly mediated by receptors of the integrin family. The interaction of ECs with extracellular matrix proteins is accompanied by cell spreading, cytoskeletal organization, and clustering of the specific integrin receptors in complex supramolecular structures known as adhesion plaques or focal contacts. Little is known on the functional role of focal contacts in EC adhesion and motility and on the possibility to modulate their organization. In this article we report that an increase in intracellular cAMP levels severely impaired focal contact formation. This process did not affect cell attachment, but increased cell adhesion and strongly inhibited cell motility. ECs were treated with the cAMP-increasing agents forskolin and 2-chloro-adenosine or with the cAMP analogue 8-bromo-cAMP. When treated cells were seeded on purified vitronectin, fibrinogen, or fibronectin little modification in the number of attached cell was observed. In contrast ECs showed impaired organization of microfilaments and poorly developed clusters of beta 3- and beta 1-integrin receptors. On a vitronectin substrate, vinculin followed the distribution of beta 3-receptors. It was typically enriched at the focal contacts in control cells but was fragmented in small dots at the cell periphery in treated cells, as were bundles of actin stress fibers. Similarly, when forskolin was added to ECs spread on vitronectin or on fibrinogen, there was a progressive but reversible disruption of actin microfilaments and diffusion of beta 3 receptors. This was accompanied by a tighter adhesion of the cells to substrata. Migration of ECs in response to different matrix proteins was severely inhibited by cAMP-increasing agents. These data indicate that EC adhesion can occur very efficiently in the absence of fully developed beta 3- or beta 1-integrin receptor-containing focal contacts but suggest that the capacity to normally assemble focal contacts and cytoskeletal proteins is required for full cell spreading and migration.     

Cultivated human arterial smooth muscle displays heterogeneous pattern of growth and phenotypic variation

Human arterial smooth muscle grows in primary, as well as passaged, cultures with a heterogeneous pattern of monolayered regions alternating with multilayered with formation of tissue-like mounds or nodules. Cells in the monolayered region are large and well spread, whereas the multilayers are composed of smaller cells with poor spreading on tissue culture plastic. The two cell types were separated from passaged cultures of human arterial media. They were investigated with regard to capacity for attachment to tissue culture plastic, native collagen, and substrata coated with plasma fibronectin, the presence and distribution of cellular fibronectin and actin, as evaluated with specific staining, and the presence of cell-substratum contacts with interference reflection microscopy. The cells from monolayered regions were high adhesive and had abundant filamentous actin, often organized like stress fibers, numerous punctate and streak-like focal contacts with the substratum, and abundant fibronectin of which some was organized as streaks. In contrast, cells from multilayered regions were low adhesive, had very little filamentous actin, and had few or no stress fibers, fibronectin content was low and variable, and focal contacts with the substratum were poorly developed. The results demonstrate phenotypic heterogeneity in arterial smooth muscle cultures with expression toward a cell type with monolayered growth pattern or toward cells with a tendency for multilayered growth.     

Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer.

We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method. These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C. At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells. Stress fibers, peripheral bands, and focal contacts were established in similar ways. After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating. After mild agitation cells floated free from the dish surface without trypsin treatment. Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces. An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment. An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment. These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton. While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive. The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment.     

The formation of a spheroid of primary hepatocytes and the expression of liver-specific functions depend on the characteristics of polyurethane foam

Rapid formation of spherical multicellular aggregates (spheroids) of hepatocytes and expression of liver-specific functions are important in developing a hybrid artificial liver. The relation between these points and the surface characteristics of the culture substratum are investigated in this article. Polyurethane foam (PUF) was used as a culture substratum for hepatocytes. Rat, dog, and porcine primary hepatocytes spontaneously formed a spheroid within 1.5 days in the pores of a hydrophilic PUF with a contact angle of 53.4±2.7°. Rat and dog primary hepatocytes formed a spheroid within 3 and 7 days of culture, respectively, in hydrophobic PUF with a contact angle of about 100°. The rates of albumin secretion and ammonia metabolism at 5 days of culture of porcine hepatocytes were 31 μg of albumin/10⁶ nuclei/day and 0.018 μmol of NH₃/10⁶ nuclei/h, respectively, about 2 times higher than the values with hydrophobic PUF. It is very important to control the hydrophilicity of the PUF surface to develop an effective artificial liver module.     

Arrangement of type IV collagen and laminin on substrates with controlled density of -OH groups

Collagen IV (Col IV) and laminin (Lam) are the main structural components of the basement membrane where they form two overlapping polymeric networks. We studied the adsorption pattern of these proteins on five model surfaces with tailored density of -OH groups obtained by copolymerization of different ratios ethyl acrylate (EA) and hydroxyl EA (HEA): X(OH)=0, X(OH)=0.3, X(OH)=0.5, X(OH)=0.7, and X(OH)=1 (where X refers the ratio of HEA). Atomic force microscopy revealed substratum-specific adsorption patterns of Col IV and Lam, ranging from single molecules deposition on more hydrophilic substrata to the formation of complex networks on hydrophobic ones. Human umbilical endothelial cells were used to study the biological performance of adsorbed proteins, following the overall cell morphology, the quantities for cell adhesion and spreading, and the development of focal adhesion complexes and actin cytoskeleton. Surprisingly, two optima in the cellular interaction were observed-one on the most hydrophilic X(OH)=1 and other on the relatively hydrophobic X(OH)=0.3 substrate-valid for both Col IV and Lam. When the proteins were adsorbed consecutively, a hydrophobic shift to X(OH)=0 substratum was obtained. Collectively, these data suggest that varying with the density of -OH groups one can tailor the conformation and the functional activity of adsorbed basement membrane proteins.     

Endothelial cell formation of focal adhesions on hydrophilic plasma polymers.

Endothelial cell (EC) formation and distribution of both actin stress fibers and focal contacts on hydrophilic plasma polymers derived from gamma-butyrolactone (GBL) and n-vinylpyrrolidone (NVP) were examined to determine their ability to support endothelial cell growth in comparison to fibronectin. One hour after seeding, cells adhered and spread moderately on fibronectin with the development of defined actin stress fibers and focal adhesions compared to NVP and GBL, on which the cells were spread with poorly developed stress fibers and a perinuclear localization of vinculin. At 3 h, cells continue to spread more on fibronectin and NVP than GBL, and the cells on fibronectin had well-defined stress fibers terminating with sharp spikes of vinculin, typical of focal adhesions. At this time point, paxillin, a signaling component of focal adhesion complex, was predominantly localized at the focal contacts for well-spread EC on fibronectin and NVP, whereas it was almost entirely concentrated in the perinuclear region of less-spread cells on GBL. However, by 24h, cells were much more spread on all three surfaces with defined stress fibers and focal contacts although EC expression of vinculin and paxillin was moderate on GBL compared to fibronectin and NVP. These results suggest that EC can form cytoskeletal structures necessary for cell survival on plasma polymers, especially on more hydrophilic NVP, which could be exploited as interface material for seeding endothelial cells.     

Development of the cytodetachment technique to quantify mechanical adhesiveness of the single cell

Adhesion of cells to biomaterials or to components of the extracellular matrix is fundamental in many tissue engineering and biotechnological processes, as well as in normal development and tissue maintenance. Many cells on adhesive molecules will spread and form an organized actin cytoskeleton and complex transmembrane signaling regions called focal adhesions. Focal adhesions appear to function as both signaling and stabilizing components of normal adherent cell activity. To better understand adhesion formations between cells and their underlying substrata, we have designed, developed, and utilized a novel 'cytodetachment' methodology to quantify the force required to displace attached cells. We allowed bovine articular chondrocytes to attach and spread on a substratum of either fibronectin, bovine serum albumin, or standard microscope glass. The cytodetacher was then employed to displace the cells from the substratum. Our results demonstrate that a significantly greater force is required to detach cells from fibronectin versus the two other substrata, suggesting that a cell's actin cytoskeleton and perhaps focal adhesions contribute significantly to its mechanical adhesiveness. The cytodetacher allows us to directly measure the force required for cell detachment from a substratum and to indirectly determine the ability of different substrata to support cell adhesion.     

Polymeric membranes for hybrid liver support devices: The effect of membrane surface wettability on hepatocyte viability and functions

Extracorporeal therapies based on membrane hybrid liver support devices using primary hepatocytes are an interesting approach to the treatment of acute hepatic failure. In such devices, semipermeable polymeric membranes are effectively used as immunoselective barriers between a patient's blood and the xenocytes in order to prevent the immune rejection of the graft. The membranes may act also as the substratum for cell adhesion, thus favouring the viability and functions of anchorage-dependent cells such as the hepatocytes. Membrane cytocompatibility is expected to depend on the surface properties of the polymer, such as its morphology and its physico-chemical properties. In this paper, we report our investigation on the effect of the surface wettability of membranes on hepatocyte viability and functions. Polypropylene microporous membranes were modified to increase their surface wettability and were used as substrata for rat hepatocyte adhesion culture. Isolated hepatocytes were also cultured on collagen as a reference substratum. Hepatocyte viability generally improved as the cells were cultured on more wettable membranes. In agreement with the viability data, the increasing wettability of the membrane surface also improved some metabolic functions.     

The behaviour of human ameloblastoma tissue in cell culture

Human ameloblastoma tissue was investigated using cell culture techniques, transmission electron microscopy and fluorescent microscopy. Cultured cellular morphology was dependent on the type of substratum, with polygonal cells predominant on collagen substrata in contrast to an elongated cellular morphology on glass substrata. The presence of tonofilaments and desmosomes confirmed the epithelial origin of these cells. The distribution of Con A surface receptors and cytoplasmic actin in the same cell was studied using a double fluorochrome technique. Incubation with fluorescein isothiocyanate-labelled Con A at 37°C for increasing time periods resulted in the Con A receptors showing progressive changes in staining patterns from clusters, to caps to perinuclear globules. Sequential changes in cytoplasmic actin, labelled by a specific anti-actin auto-antibody traced with rhodamine-labelled goat anti-human globulin, corresponded to the Con A staining patterns.