Comparative Assessment of the Cytotoxicity of Various Substrates in Organ Culture and Cell Culture: A Scanning Electron Microscopy Study

A comparative study of the behavior of chick embryo endothelial cells grown on various substrates was performed in order to establish the reliability and the limitation of both cell and organ culture methods. Following substrates were analyzed to compare these two different culture techniques: bovine serum albumin, pigskin gelatin and albumin + gelatin cross-linked by glutaraldehyde or carbodiimide, fibrin glue and negative control (Thermanox*). Parameters of cell growth and adhesion were calculated and compared with electron microscopic observations of cell morphology and of the extracellular matrix. Both culture methods provided complementary results and led to a similar classification of the biomaterials. However, the cell culture method exhibited a higher sensitivity to the surface properties of biomaterials which limited further experimentation. This was well illustrated by glutaraldehyde cross-linked protein membranes which did not support the growth of dispersed cells but enabled the formation of a cellular tissue in organ culture, thus allowing a cytocompatibility assessment. Endothelial cell morphology and extracellular matrix elaborated on biomaterials were compared to chicken blood vessels. Both methods showed that cells grown on fibrin glue and on gelatin or albumin + gelatin cross-linked by carbodiimide had a microscopic morphology similar to that of vessel wall. Organ and dispersed cell cultures provide complementary information relative to the cell behavior towards vascular prosthesis materials

Reliability of Human Fresh and Frozen Gingiva Explant Culture in Assessing Dental Materials Cytocompatibility

An explant culture of human gingival epithelium has been set up in order to provide a valuable test for evaluating the cytocompatibility of dental material s. In an attempt to supply a bank of gingiva ex plants, frozen and freshly excised specimens were cultured in parallel. Optical and scanning electron microscopy showed an early release of cuboidal cells forming a dense layer around the explants. Afterwards, cultures evolved differently . Spread cells grew and migrated more rap idly in fresh than in frozen explant cultures but their adhesion to substratum increased earlier in frozen ones. Epithelial phenotype of cells had been immunologically characterized by using a battery of monoclonal antibouies to cytokeratins (CKs). We found a time increasing expression of CKs 5, 6, 13 , 14115 , 16 and 17, whereas amounts of CKs 1, 2, 10 and 11 , specific for terminal differentiation , remained constant. The freezing procedure decreased the yield of CKs but did not modify the electrophoretic pattern . These results suggested that the differentiation of epithelial cells might proceed as in vivo. As an application, the cytocompatibility of precious (Au, Pd, Ag) and non-precious (Ni-Cr) alloys was assessed, the reference metals being Ti, which was chosen for its cytocompatibility and Cu, which was chosen for its cytotoxicity. Alloys differed by their ability to modulate cell proliferation and migration . Pd and Au exhibited a high migration potential, whereas Au-Pd and Ti allowed efficient cell proliferation but restricted migration . Reduced migration and proliferation attested the low cytocompatibility of Ag. The toxicity of Cu and Ni-Cr prevented cell migration. These result s showed the availability of this method for selecting biomaterials

Generación de una Línea Transgénica de Pez Cebra para Estudiar la Remodelación de Uniones Adherentes

El remodelado dinámico de los contactos adhesivos intercelulares o uniones adherentes (UAs) mediado por la proteína cadherina epitelial E (cadh-E) es clave para coordinar los movimientos migratorios celulares durante la embriogénesis animal. Debido a la simplicidad estructural y a la accesibilidad experimental, el embrión de pez cebra (Danio rerio) se ha establecido como un modelo para estudiar migración celular y para entender la dinámica del tráfico vesicular y distribución de cadh-E en UAs. En el presente proyecto se propuso generar una línea transgénica estable de pez cebra (cadh-E-KikGR) para la expresión de cadh-E fusionada a la proteína fotoconvertible KikGR para visualizar in vivo la remodelación de UAs. En una primera etapa se analizó la expresión endógena nativa de cadh-E y se comparó con la expresión transgénica de cadh-E fusionada a la proteína reportera fluorescente GFP (Green Fluorescent Protein) como una primera estrategia de monitoreo. Se observó que cadh-E no se localiza en membranas como la cadh-E endógena por lo que la fusión no sería funcional una vez expresada en el embrión. El clonado de KikGR, de menor tamaño, en reemplazo de GFP en el vector final de expresión permitirá la obtención del embrión transgénico de interés

Phase control and measurement in digital microscopy

The ongoing merger of the digital and optical components of the modern microscope is creating opportunities for new measurement techniques, along with new challenges for optical modelling. This thesis investigates several such opportunities and challenges which are particularly relevant to biomedical imaging. Fourier optics is used throughout the thesis as the underlying conceptual model, with a particular emphasis on three--dimensional Fourier optics. A new challenge for optical modelling provided by digital microscopy is the relaxation of traditional symmetry constraints on optical design. An extension of optical transfer function theory to deal with arbitrary lens pupil functions is presented in this thesis. This is used to chart the 3D vectorial structure of the spatial frequency spectrum of the intensity in the focal region of a high aperture lens when illuminated by linearly polarised beam. Wavefront coding has been used successfully in paraxial imaging systems to extend the depth of field. This is achieved by controlling the pupil phase with a cubic phase mask, and thereby balancing optical behaviour with digital processing. In this thesis I present a high aperture vectorial model for focusing with a cubic phase mask, and compare it with results calculated using the paraxial approximation. The effect of a refractive index change is also explored. High aperture measurements of the point spread function are reported, along with experimental confirmation of high aperture extended depth of field imaging of a biological specimen. Differential interference contrast is a popular method for imaging phase changes in otherwise transparent biological specimens. In this thesis I report on a new isotropic algorithm for retrieving the phase from differential interference contrast images of the phase gradient, using phase shifting, two directions of shear, and non--iterative Fourier phase integration incorporating a modified spiral phase transform. This method does not assume that the specimen has a constant amplitude. A simulation is presented which demonstrates good agreement between the retrieved phase and the phase of the simulated object, with excellent immunity to imaging noise