The ultrastructural architecture of the tissue/hard-tissue replacement interface.

This investigation examined the tissue response and interfacial bonding between bone and hard-tissue replacement (HTR) using scanning (SEM) and transmission (TEM) electron microscopy. Twenty adult male Sprague-Dawley rats were anesthetized and a hole (1.0 mm deep by 2.0 mm wide) was drilled in the calvarium. Subsequently, HTR was implanted and the wound closed. The implants and surrounding tissues were removed at 7, 14, 28, and 56 days and prepared for examination by SEM or TEM. Scanning electron microscopic analysis revealed a typical inflammatory response that subsided by day 14. At that time, a fine layer of collagen fibrils (fibrous envelope) was observed covering the polymeric surface. Energy dispersive x-ray analysis (EDXA) showed no sign of mineralization. Ultrastructural analysis demonstrated that the fibrous envelope was bilaminar; it consisted of a relatively undifferentiated cellular layer adjacent to the polymer and an outer fibrous region. Scanning electron microscopic analysis of 28-day implants showed that osteoblasts had migrated onto the outer surface of the fibrous envelope and that calcification had been initiated as judged by EDXA. Electron microscopic examination confirmed previous observations of an undifferentiated cellular layer along the interfacial boundary, but also showed both macrophages and foreign-body giant cells. At 56 days, bone was observed to contact and cover the fibrous envelope surrounding the polymeric bead; however, EDXA showed that the fibrous envelope remained noncalcified. Transmission electron microscopic analysis revealed that the inner cellular layer was beginning to mature, as indicated by the presence of numerous cellular organelles. This maturation was accompanied by an increased incidence of macrophages as well as foreign-body giant cells. Within the time constraints of the experimental design, it is apparent that a bilaminar layer of cells and fibers remains between the HTR and the bone. Additional studies will be necessary, over extended time periods, to determine whether the bilaminar layer remains a constant feature between the HTR and the surrounding bone or whether this region is gradually supplanted by the ingrowing bone.

Ultrastructural architecture of pulmonary small-granule cell clusters in adult Syrian golden hamster.

Throughout the epithelial lining of the respiratory system is a class of cells with characteristics similar to Amine Precursor Uptake and Decarboxylation (APUD) polypeptide hormone-producing cells. In the intrapulmonary airways, these small-granule cells (SGCs) occur either singly or in organized clusters. Although no specific peptide has yet been identified, subclasses have been postulated based on granule geometry or light microscopic staining. The present study characterizes the architectonic and cellular organization of clustered SGCs in the adult Syrian golden hamster. Two morphologically distinct cells can be defined in such clusters, "light" and "dark." Thid distinction was based primarily on differences in the electron density of the cytoplasmic matrix rather than on the remarkable variations in cellular organelles or dense-core secretory vesicles. Both cell types were normal as judged by uniform spherical nuclei, chromatin organization, and distribution of cellular organelles. The "dark" cells, however, presented the profile of a cell actively involved in synthesis with a markedly dilated perinuclear cisterna and endoplasmic reticulum. Additionally, the "dark" cells contained membrane-delimited structures containing concentric membranous whorls, clear vacuoles, and lipofuscin granules. Occasionally, cells were observed to contain features of both cell types, suggesting that they may represent a continuum of common cell lineage. Accordingly, in the absence of additional morphologic or biochemical data, the "light" and "dark" cells most probably correspond to different stages of functional activity or age-related changes of a single type of cell. Unmyelinated nerve endings were occasionally interposed between cells, but synaptic specializations were not observed. Beneath the clusters, nerve fibers were also present, but they were never observed to penetrate the basal lamina or contact any of the SGCs. Of equal occurrence were elements of the vascular system and smooth muscle, suggesting that some SGCs in the adult hamster may function in a paracrine or endocrine manner. Such knowledge is essential to any study attempting to delineate the functional role or roles of these enigmatic organoids.

Three-dimensional reconstruction of a small-granule paracrine cell cluster in an adult hamster bronchus.

Amine precursor uptake and decarboxylation (APUD) small-granule cells were stained by periodic acid-Schiff (PAS)-lead hematoxylin in 0.5-micron etched Epon sections of adult hamster lung fixed for transmission electron microscopy. The leading edge of a small-granule cell cluster was identified in a segmental bronchus as a single PAS-positive cell. From 256 serial thin sections through its entirety, a three-dimensional wooden reconstruction of the cluster and morphometric estimates of the apical and basal surfaces, cell volume, and intracytoplasmic distribution of mitochondria and small granules was made. Of moderate size, the body consisted of 16 small-granule cells, 11 forming its ovoid core with five outlying cells diverging at the margin; these were pyramidal, possessing wide bases and thin apical processes. At the bronchial surface, processes from the 11-cell core emerged together, whereas the divergent cells emerged in groups of two and three. Ten Clara-like cells and one ciliated cell encircled the core. Altogether they formed a pseudostratified epithelium in contrast to the surrounding simple columnar epithelium. Deeper in the cluster, numerous cytoplasmic extensions interdigitated with those from adjacent cells, and toward the base the Clara-like and APUD cells were increasingly interposed. In marked contrast to the apical cytoplasm, the infranuclear cytoplasm of the latter was densely packed with ca. 1,000 A electron-dense granules; and the basal, presumptively secretory face of each cell was five to six times greater than the area exposed to the bronchial lumen. Judged by granule size and ultrastructure, only one APUD cell type was recognized in the reconstructed cluster. Beneath it many fibrocytic processes were separated from the APUD cells by only the thickness of the basal lamina. Two fascicles of smooth muscle approached the cluster within 0.4-0.8 micron. Unmyelinated nerve fibers came as close but contacted only the muscle. Capillaries, in contrast, came no closer than 15 micron from the base of the body. Evidently, 1) fibrocytes and smooth muscle are more likely targets for secretions from such a paracrine body than cells reached through the blood-stream, and 2) not all small-granule cell clusters are innervated.

Morphologic and cytochemical characteristics of amine-containing globule leukocytes in rat tracheal epithelium.

Amine-containing cells in the tracheal epithelium are typically of the small-granule type (diameter approximately 100 nm). However, in the rat, another amine-containing cell type has been identified that possesses the amine-handling features of the APUD-series of cells (amine precursor uptake and decarboxylation) but not the ultrastructural characteristics. It has been postulated that these cells may be related to cutaneous melanocytes. In this study, fluorescent cells were present in the laryngeal and tracheal epithelial lining of adult Sprague-Dawley rats following freeze-drying and exposure to formaldehyde vapor (FIF or formaldehyde-induced fluorescence). Microspectrofluorimetry revealed an emission maximum at 493 nm. The excitation maximum could not be calculated but appeared to be around or below 350 nm (to record spectra below requires the use of quartz optics). Yellow fluorescence also emanated from serotonin-containing mast cells (excitation and emission maxima: 401/515 nm). Tracheal segments processed according to the aqueous formaldehyde ( AFIF ) technique, for the demonstration of 5- hydroxytryptophan (5-HTP) or serotonin (5-HT), failed to identify fluorescent cells in the epithelial lining even though connective-tissue mast cells were evident. Subsequent treatment of AFIF -fixed sections with formaldehyde and HCl vapors ( AFIF -HCl) resulted in the formation of a fluorogenic compound within numerous cells in the tracheal lining (455/537 nm). This spectral shift and increase in intensity of fluorescence following acidification are characteristic for standards and/or cells that contain tryptamine, tryptophan, or peptides with NH2-terminal tryptophan and are markedly different from microspectrofluorimetric data reported for the phenylethylamines or serotonin. It is therefore postulated that these cells contain a closely related beta-(3-indolyl) ethylamine-like compound, serotonin excluded. The morphology of the fluorescent cells was similar when prepared according to the FIF or AFIF -HCl techniques. Conjunctive staining, the examination of a single section first by fluorescence microscopy and subsequently by other histochemical and cytochemical methods, demonstrated that the fluorescent granules were also methylene blue, alcian blue, periodic-acid Schiff, and ferric- fericyanide positive. Subsequent correlative electron microscopic examination of Epon-embedded AFIF -HCl-treated tracheal sections demonstrated that these amine-containing cells were globule leukocytes.

Comparative biology of small granule cells and neuroepithelial bodies in the respiratory system. Short review.

Small granule epithelial cells and clusters of these cells termed neuroepithelial bodies are widely distributed in the respiratory systems of vertebrates, according to observations made in 24 species. The cells are thinly scattered along extrapulmonary and intrapulmonary airways from the larynx to the entrances of the alveoli. As a rule they escape notice unless demonstrated by special light microscopic methods including silver impregnation, PAS-lead hematoxylin staining, or immunocytochemical localization of a marker enzyme, neuron-specific enolase, all of which reveal small granule cells as a class. They are also recognizable in electron micrographs by their characteristic 1,000 to 3,000 A dense-core granules. These can differ in appearance from cell to cell within the same or different species, and small granule cells also can differ in their anatomical relationships or in their content of pharmacological agents like serotonin and peptide hormones bombesin, calcitonin, or leu-enkephalin. Current evidence thus clearly points out the heterogeneity of the class, although it is inadequate to characterize the presumptive endocrine or paracrine function of any of its members specifically. This brief review gives an idea of the range of animals surveyed for small granule cells as well as similarities and differences among cells from different species. It concludes with comments on practical methods for revealing these cells and guidance to sources of detailed information about them.

The effects of carbon monoxide and hypoxic hypoxia on amine-containing cells in the tracheal epithelium of young rabbits.

Endogenously fluorescent, singly occurring amine-containing cells in tracheal epithelium were examined in 3-, 10-, and 28-day-old rabbits. These cells are pyramidal in shape with the apex projected toward the tracheal lumen. The cytoplasm exhibits a yellow fluorescence which is predominantly supranuclear. Occasional, infranuclear, fluorescent cytoplasmic processes project from the cells. The numbers of fluorescent cells per unit length of trachea increase with age. Acute exposure of 10-day-old rabbits to 13% O2 decreases the number of detectable fluorescent cells in the tracheal compared to controls exposed to room air. Similarly, exposure to 750 ppm carbon monoxide decreases the number of fluorescent epithelial cells appearing in tracheas of 10- and 28-day-old rabbits. These results suggest that the amine-containing epithelial cells of the trachea respond to tissue hypoxia and that decreased airway pO2 is not necessary to elicit a response.