Philias R. Garant

The demonstration of complex gap junctions between the cells of the enamel organ with lanthanum nitrate.

During enamel maturation the cells of the enamel organ are characterized by the presence of numerous mitochondria, coated vesicles, and microvilli suggesting that their function might be the transport of substances from the vascular compartment into the underlying enamel. Another prominent feature of the papillary cells is the presence of extensive intercellular contacts usually described as tight junctions. These areas of apparent membrane fusion occur between large exopthytic cytoplasmic processes and the indented surfaces of adjacent cells. Mitochondria are frequently observed to be in close relation to these junctions. Utilizing the method of prestaining with uranyl acetate and the lanthanum nitrate tracer technique it is now established that these junctions are of the gap variety. Thus the papillary layer contains intercellular junctions which are morphologically equivalent to those junctions believed to be responsible for intercellular ionic coupling. The demonstration of gap junctions provides evidence that a direct cytoplasmic route for the movement of ions or small metabolites does exist within the enamel organ.

STRUCTURE AND ORGANIZATION OF ODONTOBLASTS

Differentiation of odontoblasts involves cell‐to‐cell recognition, contact stabilization involving the formation of attachment specializations, cytoplasmic polarization, development of the protein synthetic and secretory apparatus, and the active transport of mineral ions. The secretory odontoblast is characterized by an extensive rough‐surfaced endoplasmic reticulum, a highly developed Golgi complex, and the presence of specific secretion granules. Type I collagen, a major constituent of dentin matrix, appears to be secreted by the odontoblast into predentin at the proximal portion of the odontoblast process, the major cytoplasmic process extending from the odontoblast cell body into the dentin. The odontoblast process contains a rich network of microtubules and microfilaments. The proximal portion of the process is also a site of fluid‐phase endocytosis. Adjacent odontoblasts are held together by numerous macula adherens junctions and a well‐developed distal junctional complex adjacent to the predentin. Junctional strands of the occludens type have been observed to be a component of this junctional complex. Tracer studies employing horse‐radish peroxidase indicate that this junctional complex does not form a tight barrier to the diffusion of tissue fluid from the interodontoblast spaces into the predentin. Many well‐developed gap junctions are formed between adjacent odontoblasts and between odontoblasts and the fibroblasts that make up the subodontoblastic layer. Ca‐ATPase activity is demonstrated in the Golgi complex and mitochondrial cristae and along the distal plasma membranes of odontoblasts. ALPase activity is also intense along the entire odontoblast cell surface. The osmium tetroxide‐pyroantimonate technique for calcium localization demonstrates prominent reaction precipitates in mitochondria of odontoblasts. Energy‐dispersive x‐ray microanalysis of anhydrously fixed and processed odontoblasts detected Ca and P peaks throughout the cytoplasm. A sulfur peak is noted in the distal cytoplasm of odontoblasts and in matrix vesicles. Together, these results demonstrate the complexity and variety of cell functions involved in dentinogenesis.

In-vitro cultivation of human oral keratinocytes

Abstract The technique of Rheinwald and Green (1975b) for the serial cultivation of foreskin keratinocytes was modified to facilitate the cultivation of gingival and buccal keratinocytes. We used 7–10-day epithelial outgrowths from excised tissue fragments as a source of keratinocytes, whereas Rheinwald and Green used enzymically-dissociated keratinocytes from freshly obtained foreskin. Mitomycin-C treatment as opposed to gamma irradiation was used to suppress the growth of fibroblast feeder layers. Single oral keratinocytes proliferated to form colonies resembling a stratified squamous epithelium. Light and electron microscopy revealed structures usually associated with epithelia, i.e. desmosomes, tonofilaments, tonofibrils, thickened cell membranes and a glycocalyx on the free surface of the epithelium. Oral keratinocytes underwent 20–40 doublings before senescence.

Ultracytochemical demonstration of ATP-dependent calcium pump in ameloblasts of rat incisor enamel organ

SummaryThe enamel organ of the growing rat incisor was fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of Ca- and Mg-activated membrane ATPase by a one-step lead technique at alkaline pH. To inhibit nonspecific alkaline phosphatase, 5 mM levamisole was added to the incubation media. Intense Ca- and Mg-ATPase activity was demonstrated in the cell surfaces of the secretory ameloblasts, except at the proximal and distal junctional complexes and the gap junctions in the lateral and basal cell surfaces. Deep plasma membrane invaginations at the proximal and distal parts of Tomes processes facing interrod- and rod-enamel growth regions exhibited the strongest enzymatic reaction. Mg-ATPase activity was also shown to be present in the plasma membranes of secretory ameloblasts but it was less intense than Ca-ATPase. Except for a slight reaction in the Golgi membranes, all other cell organelles of the secretory ameloblasts and the adjacent enamel matrix were free of enzymatic reaction. However, when the tissues were incubated in media lacking levamisole, a prominent enzymatic reaction was observed in the newly secreted enamel matrix of the rod and interrod growth regions as well as on the plasma membranes of the cells. In maturation ameloblasts of both ruffleended and smooth-ended types, a weak reaction for Ca- and Mg-ATPase was restricted to basal cell surfaces facing the papillary cell layer. In tissues incubated in media lacking levamisole, a variable deposition of reaction products was observed in the Golgi membranes, mitochondrial membranes, tubular elements of smooth endoplasmic reticulum in the ruffled border zone, and along the plasma membranes of the ruffled border. Throughout the secretory and maturation stages, a moderate and/or weak enzymatic reaction for both Ca- and Mg-ATPase was seen in the plasma membranes of the cells of the stratum intermedium and the papillary layer when incubated in media with levamisole. Omission of substrate ATP and/or the enzyme activator CaCl2 from the incubation media for Ca-ATPase produced a negative reaction in the tissues examined. When the calmodulin blocker trifluoperazine was administered to the rats intravenously, Ca-ATPase activity was almost completely abolished from the plasma membranes of secretory ameloblasts, but not of other cell types.

Fate of annular gap junctions in the papillary cells of the enamel organ in the rat incisor

SummaryTo investigate the mechanisms whereby annular gap junctions in the papillary cells of the enamel organ are degraded intracellularly, continuously growing rat incisors were examined by electron microscopy of routine thin sections as well as for the cytochemical localization of inorganic trimetaphosphatase activity. Routine thin-section analysis revealed small flat or undulated gap junctions, hemi-annular gap junctions between an invaginated cell process and a cell body, and fully internalized cytoplasmic annular gap junctions. Both hemi-annular and annular gap junctions usually contain various organelles and/or inclusions, such as mitochondria, endoplasmic reticulum, ribosomes, vesicles, and lysosomes in the cytoplasm confined by the junctional membranes. Annular gap junctions are sometimes fused with vesicular or tubulovesicular structures. Cytochemistry of inorganic trimetaphosphatase activity revealed an intense enzymatic reaction within a system of tubular structures and round or oval dense bodies. Both structures are believed to correspond to primary lysosomes. A part of the Golgi apparatus also shows a weak reaction. Although hemi-annular gap junctions never show enzymatic reaction, annular gap junctions sometimes contain reaction products throughout their interior cytoplasm and inclusions. Fusion of annular gap-junctional membranes with reaction-positive tubular structures is also observed. In one instance, revealed in serial sections, an annular gap junction was encircled entirely by a reaction-positive structure. These results suggest that cytoplasmic annular gap junctions are formed by endocytosis of hemi-annular gap junctional membranes from the cell surface and then degraded intracellularly by lysosomal enzymes.

Expression and role of epidermal growth factor receptors during differentiation of cementoblasts, osteoblasts, and periodontal ligament fibroblasts in the rat

Periodontium is a collective term describing tooth supporting tissues that develop and function effectively as a unit (Hoffman, 1960; Schroeder, 1986). These include the alveolar bone, cementum, periodontal ligament (PDL), and gingiva. Cementum is a specialized mineralized tissue covering the entire root surface and forms the interface between root dentin and the PDL and has a unique function of attachment of PDL collagen fibers to the root surface. Traditionally, cementum has been classified as cellular and acellular cementum depending on the presence and absence of cementocytes in cementum but further grouped into intrinsic or extrinsic fiber cementum depending on the presence of the collagen fibers formed by cementoblast or by fibroblasts (i.e., Sharpey’s fibers), respectively (Jones, 1981; Schroeder, 1986). Formation of acellular extrinsic fiber cementum (AEFC) has been investigated during root formation in rodents (Lester, 1969; Selvig, 1964; Stern, 19641, dogs, and man (Bosshardt and Schroeder, 1991a,b, 1992; Owens, 1974,1978; Schroeder, 1992; Sequeira et al., 1992). These studies indicate that cementogenesis in rats differs from that in the dog and human. A discussion of the structure and development of all types of cementum in the many different animals studied to date is beyond the scope of this article. The focus of this paper is on the formation of the initial layer of AEFC by cells originating in the dental follicle proper during root formation and on the role of epidermal growth factor (EGF) and its receptor (EGF-R) in maintaining the fibroblast phenotype in the PDL. The PDL, a dense connective tissue located between the alveolar bone and cementum, is composed of cellular as well as extracellular matrix components. The PDL contains numerous actively secreting fibroblasts and a small number of undifferentiated paravascular fibroblast precursors. Epithelial cells of the rests of Malassez, derived from the breakup of the epithelial root sheath, endothelial cells, and macrophages are also present. In addition, a small number of cementoblastic cells are located adjacent to the root surface, while osteoblastic cells such as preosteoblasts and osteoblasts are present along the alveolar bone surface. The collagen fibers, consisting of type I and I11 collagen, are the major structural components of the ligament. They are organized into principal fibers that sus-

Calmodulin blocker inhibits Ca++-ATPase activity in secretory ameloblast of rat incisor

SummaryThe effects of the calmodulin blocker, trifluoperazine (TEP), on membrane-bound Ca++ -ATPase, Na+ -K+ -ATPase (EC 3.6.1.3.) and the ultrastructure of the enamel organ were investigated in the lower incisors of normal and TFP-injected rats. The rats, of about 100 g body weight, were given either 0.2 ml physiological saline or 100 μg TFP dissolved in 0.2 ml physiological saline through a jugular vein and fixed by transcardiac perfusion with a formaldehyde-glutaraldehyde mixture at 1 and 2 h after TFP administration. Non-decalcified sections of the enamel organ less than 50 μm in thickness, prepared from dissected lower incisors, were processed for the ultracytochemical demonstration of Ca++-ATPase and Na+-K+ -ATPase by the one-step lead method at alkaline pH. In control saline-injected animals the most intense enzymatic reaction of Ca++-ATPase was demonstrated along the plasma membranes of the entire cell surfaces of secretory ameloblasts. Moderate enzymatic reaction was also observed in the plasma membranes of the cells of stratum intermedium and papillary layer. Reaction precipitates of Na+-K+-ATPase activity were localized clearly along the plasma membranes of only the cells of stratum intermedium and papillary layer. The most drastic effect of TFP was a marked disappearance of enzymatic reaction of Ca++-ATPase from the plasma membranes of secretory ameloblasts, except for a weak persistent reaction in the basolateral cell surfaces of the infranuclear region facing the stratum intermedium. The cells of stratum intermedium and papillary layer, however, continued to react for Ca++-ATPase even after TFP treatment. Similarly, Na+-K+-ATPase activity in these cells was not inhibited by TFP administration. Ultrastructural examination of secretory ameloblasts revealed that administration of TFP caused no considerable cytological changes and did not act as a cytotoxic agent. These results suggest that secretory ameloblasts may have an active Ca++ transport system, which is modulated by an endogenous calmodulin.

A freeze-fracture study of ruffle-ended post-secretory ameloblasts.

The post-secretory portion of the rat incisor enamel organ was prepared for routine transmission electron microscopy and freeze-fracture replication in order to define further the structural surface features of the ruffle-ended ameloblasts. Surface views of the distal plasma membrane of the ruffle-ended ameloblasts revealed a well-developed zonula occludens junction with from six to ten rows of tight junctional strands. Gap junctions were also observed just proximal to the tight junctional strands. The membranes of the ruffled border contained a rich supply of intramembrane particles (IMP). The IMPs were approximately 7 to 8 nm in diameter and preferentially located on the P-face profiles of the membrane. The density of IMPs on the membranes of the ruffled border was higher than that on the lateral borders of the cell. It is suggested that the IMPs of the ruffled border may represent enzymatic proteins in the basal cell membrane of absorptive ameloblasts. In addition, the large, highly-developed zonula occludens appeared structurally capable of sealing the intercellular spaces between the ruffle-ended ameloblasts.

A study of post-secretory maturation ameloblasts in the cat by transmission and freeze-fracture electron-microscopy

Permanent canine and molar tooth germs of kittens were processed for thin-sectioning and freeze-fracture replication. Maturation ameloblasts were divided into ruffle-ended (RA), smooth-ended (SA) and intermediate (IA). RA had an extensive distal ruffled border consisting of deep membrane invaginations, forming complicated extracellular channels. Adjacent RA were connected by extensive distal junctional complexes (zonulae occludentes). The SA had flattened distal cell surfaces and few coated and smooth vesicles in the distal cytoplasm. Adjacent SA were connected by proximal zonulae occludentes, but had only maculae occludentes at their distal ends so permitting broad lateral extracellular spaces to communicate directly with the enamel surface. IA near the RA layer had a ruffled border consisting of deep and narrow membrane invaginations and pinosomes filled with granular material. IA next to the SA layer had no ruffled border but had pinosomes that seemed to originate directly from the distal cell surfaces. IA were polarized and connected by proximal and distal junctional complexes consisting of either zonulae or fasciae occludentes and associated gap junctions.

Calmodulin in rat incisor secretory ameloblasts as revealed by protein A-gold immunocytochemistry

SummaryThin sections of aldehyde-fixed, undecalcified, embedded rat incisor enamel organ were incubated with sheep antiserum to bovine testes calmodulin to reveal the sites of antigen-antibody reaction at the ultrastructural level in secretory ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabelling was localized intensely on free polyribosomes and those attached to rough-surfaced endoplasmic reticulum but only rarely observed in the cisternal space. The nuclei, mitochondria, cytosol, and plasma membranes were also immunoreactive. The Golgi membranes and related vesicles, secretion granules, and lysosomes were unlabelled. The proximal and distal cell web junctional complex systems were not immunoreactive. These findings suggest that calmodulin location reflects its synthetic site and multifunctional roles in the immunolabelled cytoplasmic components of secretory ameloblasts.