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Cell seeding, Adsorption, Collagen scaffold, Fibroblasts, Tissue engineering, Optical density

The process of cell seeding oil biocompatible scaffolds has a major impact oil the morphological evolution of an engineered tissue because it involves all the key factors of tissue formation: cells, matrix, and their mutual interactions. In order to characterize the efficiency of cell seeding techniques, mainly static parameters are used such as cell density, cell distribution, and cell viability. Here, we present an experimental model that incorporates an optical density meter providing real-time information oil the cell seeding velocity, a relevant dynamic parameter of cell-matrix interaction. Our setup may be adapted to fit various Cell seeding protocols. A modified fluorimetric cuvette is used as bioreactor culture flask. The optical density of the magnetically stirred cell suspension is recorded by a digital optoelectronic device. We performed calibration experiments in order to prove that, in our experimental conditions, optical density depends linearly on the number of cells in the unit volume Of Suspension. Control studies showed that, during the time course of a typical experiment (up to 10 h), the cells (murine 3T3 fibroblasts) neither aggregated nor adhered significantly to the walls of the cuvette. Hence, our Setup yields the number of cells attached to the scaffold as a function of time. In order to analyze the experimental seeding curves, we built a kinetic model based on Langmuir’s adsorption theory, which was extended to include a preliminary step of integrin function recovery. We illustrate the proposed approach by two sets of experiments that involved trypsin-EDTA or only EDTA treatment (no trypsin) used to detach the cells from the culture flasks. The data indicate that in both cases cell-matrix adhesion has a sequential, two-step dynamics, but kinetic parameters and attachment site availability depend on the experimental protocol.

Structural-Changes, Serine Chemotaxis, Photocycle, Receptor, Protein, Diffraction, Resolution, Membrane, Insights, Domain

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota(1-3). This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis(4). The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically 5 to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 Angstrom resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.

Biological cells; Effects of electromagnetic radiations; Membrane

The effects of low-level microwaves (2.45 GHz) on the membrane of human erythrocytes were studied measuring the hemoglobin loss and osmotic resistance of erythrocytes exposed to different power densities (0.025-10.0 mW/cm(2)) at different irradiation times. A significant increase of the hemoglobin loss by exposed erythrocytes as well as a strong dependence of the rate of the increase of hemoglobin loss on the initial level of spontaneous hemolysis were observed, It was found that at low power densities (0.84 and 1.36 mW/cm(2)), the hemolysis degree increases quasi-linearily with the exposure time, while at higher density (5 mW/cm(2)), this tendency is reversed after first 10 h of irradiation, It appears like long-term irradiation exerts a protective effect against spontaneous hemolysis caused by blood ageing. The osmotic fragility test performed on samples exposed to 5 mW/cm2 at different irradiation times showed that the osmotic resistance of exposed erythrocytes increases in time, reaching a maximum at the end of irradiation (60 h), while the osmotic resistance of the controls is constant.

Computerized videomicroscopy; Photoreceptor rods orientation; Ouabaine; Dark current

It was shown earlier [E. Kovacs, T. Savopol, A. Dinu, The polar behavior of frog photoreceptors, Biochim. Biophys. Acta, 1273 (1995) 217-222] that intact photoreceptor rod outer segments (ROS) are permanent dipoles, their polarity being, very probably, related to the dark current which flows along the cell in dark-adapted, living specimens [R.D. Penn, W.A. Hagins, S. Yoshikami, Dark current and photocurrent in retinal rods, Biophys. J. 10 (1970) 380-412; J.L. Schnapf, Dependence of the single photon response on longitudinal position of absorbtion in toad rod outer segment, J. Physiol. 343 (1983) 147-158] The light induced change in the dark current (the ‘photocurrent’) is the electrophysiological response of the photoreceptor cell to the light stimulus, being large enough to account for the visual system’s ability to detect single photons in the rod outer segments [R.D. Penn, W.A. Hagins, Kinetics of the photocurrent of retinal rods, Biophys. J. 12 (1972) 1073-1094]. The dark current is maintained by cell metabolism and is present only in freshly isolated, dark-adapted, living cells. If the cell polarity is related to the charge accumulation due to the dark current, it must also depend on cell metabolism. In order to check if the observed cell polarity is dependent indeed on cell energy, the sensitivity of photoreceptor orientation ability to Na+/K+-ATP-ase inhibitor, ouabaine was tested. It was found that photoreceptor rods loose their electrical polarity in the presence of ouabaine. That means that cell polarity is dependent on functioning of the membrane ion pumps, which provide a continuous flow of the dark current along the cell. Computerized videomicroscopy catching of photoreceptor cells orientation patterns in static electric fields thus seems to be a possible test for structural and functional integrity of photoreceptor cells to be used before performing elaborated microelectrophysiological measurements. (C) 1998 Elsevier Science S.A. All rights reserved.

Rod outer segment; Photoreceptor cell; Electric field; Orientation

It was observed that the outer segments of the frog visual rods orient along the direction of an externally applied static electric field. The orientation ability of the rod outer segments seems to be fuelled by the cell energy. The dipolar moment per rod was determined using a model which considers rod outer segments as rigid dipoles interacting with the electric field in a viscous medium. The mean dipolar charge of ROS was determined as being (2.10 +/- 0.17) . 10(-14) C.