RESUMEN
Bioengineered organs raised in vitro are candidate substitutes for natural organs in biological, pharmacological and clinical applications. We have studied cell kinetics in a human skin equivalent (HSE) using a combined immunohistochemical and flow cytometric approach. Morphological analysis has shown that, relative to unstimulated natural skin, cell proliferation mainly occurs in the basal layer of the epidermal equivalent. Immunohistochemical and flow cytometric measurements of the growth fraction suggested a cell turnover comparable to that of natural skin. Immunohistochemical labelling indices matched well with flow cytometric data. These observations are consistent with morphological and histochemical data demonstrating normal cell differentiation and tissue architecture in HSE and suggest that such HSE may be a usefull substitute for human skin.
Asunto(s)
Materiales Biocompatibles , Dermis/citología , Células Epidérmicas , Modelos Biológicos , Técnicas de Cultivo de Célula/métodos , Muerte Celular , Células Cultivadas , Dermis/química , Epidermis/química , Fibroblastos/química , Fibroblastos/citología , Citometría de Flujo/métodos , Humanos , Técnicas para Inmunoenzimas , Queratinocitos/química , Queratinocitos/citología , Antígeno Ki-67/análisisAsunto(s)
Implantación Dental Endoósea , Implantes Dentales , Encía/anatomía & histología , Sistema Linfático/anatomía & histología , Membrana Basal/anatomía & histología , Capilares/anatomía & histología , Colágeno , Tejido Conectivo/anatomía & histología , Pilares Dentales , Endotelio/anatomía & histología , Epitelio/anatomía & histología , Encía/irrigación sanguínea , Humanos , Microscopía ElectrónicaRESUMEN
Investigation was performed on healthy and inflamed human gingivae. In the healthy mucosa lymphatic vessels generally appeared as flattened channels with a reduced lumen. Only in very inflamed tissue were some more evident vessels with a distended wall detectable. Ultrastructurally, most of the vessels had the characteristics of capillaries and they were delimited by a thin and irregular endothelial wall with large intercellular spaces. These observations indicate that in the gingival tissues, which are continuously exposed to inflammatory agents and need a really efficient draining system, the pathway of interstitial exudation and cell migration may include both the lymphatic vessel system and the intercellular spaces of the permeable junctional epithelium.
Asunto(s)
Encía/citología , Gingivitis/patología , Sistema Linfático/citología , Sistema Linfático/patología , Humanos , Membrana Mucosa/citología , Membrana Mucosa/patologíaAsunto(s)
Endotelinas/química , Endotelio Linfático/citología , Endotelio Linfático/enzimología , Óxido Nítrico Sintasa/química , Secuencia de Aminoácidos , Animales , Bovinos , Células Cultivadas , Endotelinas/inmunología , Endotelio Linfático/inmunología , Inmunohistoquímica , Microscopía , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/inmunologíaRESUMEN
The development of antibodies to cell cycle-related antigens provides the basis for immunochemical studies on cell kinetics. Bromo-deoxyuridine (BrdU) incorporated by S-phase traversing cells is an exogenous marker of replicating cells, whereas proliferating cell nuclear antigen (PCNA) is an endogenous marker of replicating cells. We have applied monoclonal antibodies to BrdU and PCNA to study cell kinetics in tooth germ by immunohistochemistry and flow cytometry. BrdU-antibody reacted only with S phase-traversing cells in pulse-labelling experiments, whereas PCNA-antibody reacted with G1, S and G2-M phases traversing cells. Although the number of PCNA-positive cells largely exceeded the number of BrdU-labelled cells, the pattern distribution of immunoreactive cells was similar using BrdU- and PCNA-antibodies as revealed by immunohistochemistry. The use of PCNA-antibody allowed the detection of proliferating cells also in human tooth germ. It is suggested that combined identification of BrdU and PCNA on one side and growth factors, oncoproteins or differentiation markers on the other side may constitute a useful approach to understand the mechanisms of cell differentiation in tooth germ.
Asunto(s)
Bromodesoxiuridina/análisis , Antígeno Nuclear de Célula en Proliferación/análisis , Germen Dentario/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Antígenos , Ciclo Celular , Diferenciación Celular , División Celular , Citometría de Flujo , Fase G1 , Fase G2 , Sustancias de Crecimiento/análisis , Humanos , Inmunohistoquímica , Mitosis , Proteínas Oncogénicas/análisis , Ratas , Ratas Wistar , Fase S , Germen Dentario/citologíaRESUMEN
PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3-24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehyde-containing solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.
Asunto(s)
Anticuerpos Monoclonales , Núcleo Celular/inmunología , Proteínas Nucleares/análisis , Adulto , Aldehídos , Animales , Animales Recién Nacidos , Etanol , Femenino , Humanos , Inmunohistoquímica , Embarazo , Antígeno Nuclear de Célula en Proliferación , Ratas , Ratas Wistar , Fijación del TejidoRESUMEN
Nasal blood flow is finely regulated by local release of neurotransmitters, neuropeptides and other bioactive molecules acting via paracrine mechanisms. We have investigated the occurrence and distribution in human nasal mucosa of endothelin, a potent vasoconstrictor peptide, by immunocytochemistry and the effect of systemic administration of endothelin-1 on vascular perfusion of rabbit nasal mucosa by laser Doppler flowmetry. Endothelin-like immunoreactivity was demonstrated within vascular endothelial cells in both developing and mature human mucosa. Nasal epithelial cells and some connective tissue cells, presumed to be macrophages, also displayed specific immunostaining. In rabbits injected with endothelin-1, a potent and prolonged nasal vasoconstriction was observed. It is suggested that endothelin released locally may participate in the regulation of nasal blood flow via paracrine mechanisms. Since endothelin has growth-promoting actions on several cell types, it is also tentatively proposed that this regulatory peptide may play a role during development of the nose.