Marie-Danielle Nagel

In Vitro Assessment of Encapsulated C3A Hepatocytes Functions in a Fluidized Bed Bioreactor

In the present in vitro model, the authors intended to assess viability and functionality of hepatocytes encapsulated into alginate beads and submitted to a fluidized bed motion in a bioreactor. Human immortalized C3A line was chosen as cell model. Two controls consisting of (1) cells cultured on flasks and (2) cells encapsulated in alginate beads under static conditions were implemented. The cell functions studied were total protein, albumin, urea, and ammonia synthesis, as well as ammonia removal in the case of overdose. The comparison among the three cases studied showed that the three‐dimensional structure of alginate offered a suitable environment for cell functions. In addition, the fluidized bed bioreactor enhanced the mass transfer and thus increased the amount of species released out of the beads, as compared with the static case. Ammonia detoxification only appeared reduced by encapsulation. The concept of a fluidized bed bioartificial liver was thus validated by this in vitro model, which indicated that cell functions could be efficiently retained. In addition, as far as urea and protein synthesis and release were concerned, the use of the C3A cell line, in combination with encapsulation and fluidization technology, offered a real potentiality for the purpose of extracorporeal liver supply.

Antiproliferative and proapoptotic actions of okra pectin on B16F10 melanoma cells

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG‐I) on melanoma cell growth and survival in vitro. We added okra RG‐I containing an almost pure RG‐I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2‐hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin‐3 (Gal‐3) protein.

Characterization of biomaterials polar interactions in physiological conditions using liquid–liquid contact angle measurements: Relation to fibronectin adsorption

Wettability of biomaterials surfaces and protein-coated substrates is generally characterized with the sessile drop technique using polar and apolar liquids. This procedure is often performed in air, which does not reflect the physiological conditions. In this study, liquid/liquid contact angle measurements were carried out to be closer to cell culture conditions. This technique allowed us to evaluate the polar contribution to the work of adhesion between an aqueous medium and four selected biomaterials widely used in tissue culture applications: bacteriological grade polystyrene (PS), tissue culture polystyrene (tPS), poly(2-hydroxyethyl methacrylate) film (PolyHEMA), and hydroxypropylmethylcellulose-carboxymethylcellulose bi-layered Petri dish (CEL). The contributions of polar interactions were also estimated on the same biomaterials after fibronectin (Fn) adsorption. The quantity of Fn adsorbed on PS, tPS, PolyHEMA and CEL surfaces was evaluated by using the fluorescein-labeled protein. PolyHEMA and CEL were found to be hydrophilic, tPS was moderately hydrophilic and PS was highly hydrophobic. After Fn adsorption on PS and tPS, a significant increase of the surface polar interaction was observed. On PolyHEMA and CEL, no significant adsorption of Fn was detected and the polar interactions remained unchanged. Finally, an inverse correlation between the polarity of the surfaces and the quantity of adsorbed Fn was established.

Aging affects passive stiffness and spindle function of the rat soleus muscle

Aging affects many motor functions, notably the spinal stretch reflexes and muscle spindle sensitivity. Spindle activation also depends on the elastic properties of the structures linked to the proprioceptive receptors. We have calculated a spindle efficacy index, SEI, for old rats. This index relates the spindle sensitivity, deduced from electroneurograms recording (ENG), to the passive stiffness of the muscle. Spindle sensitivity and passive incremental stiffness were calculated during ramp and hold stretches imposed on pseudo-isolated soleus muscles of control rats (aged 4 months, n=12) and old rats (aged 24 months, n=16). SEI were calculated for the dynamic and static phases of ramp (1-80 mm/s) and for hold (0.5-2mm) stretches imposed at two reference lengths: length threshold for spindle afferents discharges, L(n) (neurogram length) and slack length, L(s). The passive incremental stiffness was calculated from the peak and steady values of passive tension, measured under the stretch conditions used for the ENG recordings, and taking into account the muscle cross-sectional area. The pseudo-isolated soleus muscles were also stretched to establish the stress-strain relationship and to calculate muscle stiffness constant. The contralateral muscle was used to count muscle spindles and spindle fibers (ATPase staining) and immunostained to identify MyHC isoforms. L(n) and L(s) lengths were not significantly different in the control group, while L(n) was significantly greater than L(s) in old muscles. Under dynamic conditions, the SEI of old muscles was the same as in controls at L(s), but it was significantly lower than in controls at L(n) due to increased passive incremental stiffness under the stretch conditions used to analyze the ENG. Under static conditions, the SEI of old muscles was significantly lower than control values at all the stretch amplitudes and threshold lengths tested, due to increased passive incremental stiffness and decreased spindle sensitivity at L(s). The muscle stiffness constant values were greater in old muscles than in controls, confirming the changes in elastic properties under passive conditions due to aging. Aging also altered the intrafusal fibers: it increased the mean number of intrafusal fibers and the contents in the slow, neonatal and developmental isoforms intrafusal of MyHC have been modified. These structural modifications do not seem great enough to counteract the loss of the spindle sensitivity or the spindle efficacy under passive conditions and after the nerve was severed. However, they may help to maintain the spindle afferent message under natural conditions and under fusimotor control.

Cyclic AMP--dependent aggregation of Swiss 3T3 cells on a cellulose substratum (Cuprophan) and decreased cell membrane Rho A.

Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling.

cAMP levels in cells attached to AN69 and Cuprophan: cAMP dependence of cell aggregation and the influence of serum

We have examined the link between the aggregation or spreading of cells adhering to substrata of differing biocompatibility and activation of the cyclic AMP (cAMP) pathway. We compared the rate at which the Mouse Swiss 3T3 fibroblasts attached to Cuprophan (CU), AN69 and a control plastic in the presence and absence of foetal calf serum (FCS). Serum had no effect on the kinetics of cell attachment to CU or AN69. Cells incubated in culture medium containing 10% FCS aggregated on CU, whereas they spread on AN69 and plastic. Aggregated cells contained significantly higher concentrations of cAMP than cells spreading, and aggregation was prevented by treatment with miconazole, an inhibitor of adenylyl cyclase. cAMP-dependent cell aggregation occurred on all three substrata in serum-free medium, suggesting that proteins adsorbed onto AN69 and plastic in the presence of serum helped protect the cells. Far less serum protein was adsorbed onto CU than onto AN69 or plastic, consistent with the similar increases in cAMP in cells attached to CU with or without serum.

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose‐coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N‐cadherins. The levels of N‐cadherin and β‐catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less β‐catenin protein. Immunoprecipitation and immunostaining showed that both N‐cadherins and β‐catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell–cell interactions and cell functions in 3D cultures.

Collagen type I together with fibronectin provide a better support for endothelialization

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.

Probing Fibronectin−Surface Interactions: A Multitechnique Approach

The development of adhesive as well as antiadhesive surfaces is essential in various biomaterial applications. In this study, we have used a multidisciplinary approach that combines biological and physicochemical methods to progress in our understanding of cell-surface interactions. Four model surfaces have been used to investigate fibronectin (Fn) adsorption and the subsequent morphology and adhesion of preosteoblasts. Such experimental conditions lead us to distinguish between anti- and proadhesive substrata. Our results indicate that Fn is not able to induce cell adhesion on antiadhesive materials. On adhesive substrata, Fn did not increase the number of adherent cells but favored their spreading. This work also examined Fn-surface interactions using ELISA immunoassays, fluorescent labeling of Fn, and force spectroscopy with Fn-modified tips. The results provided clear evidence of the advantages and limitations of each technique. All of the techniques confirmed the important adsorption of Fn on proadhesive surfaces for cells. By contrast, antiadhesive substrata for cells avoided Fn adsorption. Furthermore, ELISA experiments enabled us to verify the accessibility of cell binding sites to adsorbed Fn molecules.

Activation of the cyclic AMP pathway in cells adhering to biomaterials: regulation by vitronectin– and fibronectin–integrin binding

Our previous studies have shown that cells adhering to biomaterials in serum-free conditions increase their content of cyclic AMP (cAMP) and become aggregated. In cells on an acrylonitrile membrane (AN69), these biochemical and morphological changes are prevented by adding 10% foetal calf serum (FCS) to the medium; cells on the cellulose membrane Cuprophan (CU) remain unaffected. The present study examines the roles of vitronectin (VN)- and/or fibronectin (FN)-integrin binding in this inhibition. Competitively blocking VN- and FN-receptors with echistatin increased intracellular cAMP significantly and caused cells on AN69 to aggregate, but did not modify cAMP-dependent cell aggregation on CU. VN or FN adsorbed onto CU also inhibited cAMP production by attached cells and prevented their aggregation, whereas adsorbed BSA had no effect. Therefore, the binding of VN or FN to cell-surface integrins seems to limit the activation of the cAMP pathway initiated by the substratum itself.