Characterisation of human gingival neural crest-derived stem cells in monolayer and neurosphere cultures.

Neural crest (NC)-derived stem cells (NCSC) have an exceptionally wide differentiation potential, but their use in regenerative therapy has been hampered by their scarcity in adult tissues and complex isolation protocols. Human oral mucosal gingiva may provide an attractive source of these cells as it contains NC-derived cells, the tissue is easily accessible and wound healing is fast and scarless with very little morbidity. To this end, we first investigated whether NC-derived cells are retained in adult gingiva by examining 8-months-old NC-reporter Wnt1-Cre/R26RYFP mice. We then hypothesised that gingival cell NC-like phenotype can be further enhanced by floating neurosphere cultures generated from standard human gingival fibroblast (GF) and pooled CFU-F (GSC) cultures. Findings showed that NC-derived cells are retained in the gingival connective tissue of aged mice. Human GFs and GSCs expressed NC-related genes nestin, Snai1, Twist1, Pax3, Sox9 and FoxD3, and generated neurospheres. This was mediated via calcium- and connexin 43-dependent cell communication, which is similar to neurospheres formed by neural progenitors. Cells in the spheres showed significantly increased expression of NC-related genes, and down regulation of fibroblast-related type I collagen. Structurally, the neurospheres were polarised with nestin positive cells located on the outer layers underlined with an extracellular matrix rich in molecules typical to embryonic NC. Sphere-derived cells expressed significantly elevated levels of neural markers, and differentiated into Tau, neurofilament-M and GFAP-positive cells suggesting neural differentiation potential. Thus, human GF and GSC cultures may provide an efficient source of NC-derived cells via enrichment by floating sphere cultures.

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules.

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1-2 microm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 microm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50-70 nm.

Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions.

Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40 nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1-5 nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.

Immunogold labelling of fibroblast focal adhesion sites visualised in fixed material using scanning electron microscopy, and living, using internal reflection microscopy.

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell-substrate interface was removed with detergent. The antigen blocking procedure, size of label (5 nm) and duration of silver-enhancement (6 min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided 'sectional' views through the cell. This enabled visualisation of vinculin exclusively within the cell-substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.