RESUMEN
The localization of osteopontin (OP) was examined in Meckel's cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix.
Asunto(s)
Cartílago/metabolismo , Condrocitos/metabolismo , Sialoglicoproteínas/biosíntesis , Animales , Cartílago/citología , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Inmunohistoquímica , Hibridación in Situ , Ratones , Osteopontina , Ratas , Ratas WistarRESUMEN
All dental root implants come in contact with the oral epithelium, and many complex factors are found to arise in this region. In order to perform a successful dental root implantation, it is necessary to clarify the interaction of the dental root implant material with the host defense mechanisms involved in the specific and nonspecific immune responses to many antigens in oral bacteria and their components. Recently, focusing on developing the dental root implant, the Nikon Corporation improved the surface characteristics of pure titanium even further by developing a hydroxyapatite (HA) layer formed on an anodic titanium oxide film containing Ca and P via hydrothermal treatment (SA treatment). However, since little is known about the effect of SA-treated pure titanium (HA/Ti) on the defense mechanisms of the oral membrane epithelium, we investigated (1) the in vitro proliferation of murine splenic B lymphocytes on the surface of HA/Ti in the presence of three lipopolysaccharide (LPS) concentrations and (2) interleukin-1alpha (IL-1alpha) production by the reaction of human peripheral blood mononuclear cells (PBM cells) on the surface of HA/Ti under the same concentrations. After culture, murine splenic lymphocytes were measured by uptake of 3H-thymidine, and cytokine release (IL-1alpha) from PBM cells was measured by ELISA. Results showed that HA/Ti had hardly any effect on the LPS-induced proliferation of B lymphocytes and IL-1alpha production. In vitro investigations of the effects of HA/Ti on the LPS-induced proliferation of murine splenic B lymphocytes and IL-1alpha from PBM cells might be a useful way of elucidating the defense mechanism between implants and the oral epithelium.