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Cell turnover in the odontogenic organ of the rat incisor as visualized by graphic reconstructions following a single injection of 3H-thymidine.

Turnover of cells within the odontogenic organ was studied in three dimensions by preparing serial sections of incisors from young male rats killed at various times following a single intraperitoneal injection of 1 muCi/g body weight of 3H-thymidine. Radioautographs showed that at 1 hour after injection labeled cells were present in all cell layers throughout the entire depth of the odontogenic organ. They were encountered frequently within the inner dental epithelium and stratum intermedium but appeared less abundant within the stellate reticulum and outer dental epithelium. With time, the frequency of labeled cells in each layer declined progressively, and more rapidly at the anterior and labial side of the odontogenic organ than toward its posterior and lingual side. Hence labeled cells were observed over the longest time interval in regions where cell layers were in closest proximity to the opening of the apical foramen, that is, near the apical and cervical loops. By 32 days after injection, numerous labeled cells could still be identified within the outer dental epithelium and stellate reticulum near the apical loop (bulbous part of the odontogenic organ) and the outer dental epithelium near the cervical loops ("U"-shaped part of the odontogenic organ). These findings support the hypothesis that cells originate within the bulbous part of the odontogenic organ and migrate anteriorly through the "U"-shaped and root sheath parts of the odontogenic organ during renewal of the incisor. It appears that individual stem cell compartments may be maintained for surface (outer/inner dental epithelium) and intermediate layers (stellate reticulum/stratum intermedium) in the odontogenic epithelium.

Animals↗

A fine-structural survey of the pulpal innervation in the rat mandibular incisor.

The innervation of the rat incisor pulp has been studied using transmission electron microscopy and light microscopy. Transverse sections of mandibular incisor pulp (380-460 gm rats) from numerous positions in the long axis of the tooth were examined systematically in the electron microscopy. Quantitative data on total axon populations were obtained. The nerve fibers were found to pass through the lingual half of the pulp from the apical end to within 2 mm of the incisal tip. Although the nerve fibers were seen to lie amongst the connective tissue cells between the blood vessels, the electron microscopic observations showed that the blood vessels are not innervated. Throughout their pulpal course the nerve fibers showed no trace of perineurial investment. Virtually all the axons were unmyelinated. Total numbers of axons were small (233-328) and peak diameters of 0.3-0.4 microM confirmed the observed immature appearance of the nerve supply. Obvious nerve endings were seldom observed and the axons showed no structural association with odontoblasts. The evidence indicates that, although most axons terminate near the incisal end of the tooth, no specific structure is supplied. The qualitative features of the axons do not suggest autonomic function; however, they are consistent with a sensory role.

Animals↗

Induction of a specific (LM) protein in the submandibular gland of the rat by repeated amputation of the lower incisor teeth.

Chronic administration of the beta-adrenergic agonist isoproterenol (IPR) leads to marked hyperplastic/hypertrophic enlargements of the parotid and submandibular glands in rats and mice with concomitant changes in the composition of both the glands and the saliva. Conspicuous among the alterations of the submandibular saliva is the appearance of a 13,000 Mr protein, termed LM (large mobile) protein. Repeated amputation of the lower incisor teeth also causes enlargements of the major salivary glands in rats. In this study, we have compared the enlargements of submandibular glands of rats produced by IPR administration or teeth amputation with respect to the relative levels of the LM protein in gland extracts and saliva. Administration of IPR-HCl (40 mg/kg) twice daily for 5 days or amputation of the lower incisor teeth 3 times a week for 3 weeks resulted in a 2.2-fold increase in the weight of the submandibular gland. Amputation for one week led to a 1.4-fold increase in gland weight. Double immunodiffusion in agar antibodies against the purified LM protein gave a single precipitin line with gland extracts and saliva of IPR-treated and teeth-amputated rats, indicating immunological identity of the reacting antigens. No precipitin lines were seen with gland extracts or saliva of untreated rats. Immunoblots of pooled saliva obtained from IPR-treated or teeth-amputated rats revealed a single protein band of the same electrophoretic mobility in SDS-polyacrylamide gels when stained using anti-LM antibodies. The relative concentrations of LM protein in gland extracts and saliva were measured by a solid-phase enzyme-linked immunoabsorption assay using antibodies against the purified LM protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Wear striations on the incisors of ceropithecid monkeys as an index of diet and habitat preference.

Wear striations on the incisiors of Old World monkeys were examined in order to determine associations between the distributions of striations, diet and habitat preference. Significant differences exist between the Cercopithecinae and the Colobinae in respect to the orientation of incisor wear striations. In the Colobinae striations are oriented in a predominantly mesiodistal direction. In the Cercopithecinae straitions usually have a labiolingual orientation. Comparisons of terrestrial and arboreal genera indicate that significant differences exist between the two groups in respect to the density of striations on the occlusal wear facets of maxillary central incisiors. Arboreal and terrestrial monkeys also differ in the frequency of individual incisor wear facets completely devoid of wear striations.

Animals↗

Variation in the convexity of the human maxillary incisor labial surface.

Labial surface convexity of the maxillary central incisors ( ILC ) is classified with a new five grade ranked scale. More than 2,000 individuals representing 20 worldwide populations were studied. Principle findings are 1) sexual dimorphism is not significant, 2) antimere asymmetry is moderate, 3) labial convexity is negatively associated (r = -0.48) with labial surface double- shovelling , and 4) significant differences occur between several populations. Convexity is most marked in African and Asiatic Indian populations, particularly Bushmen . Europeans have intermediate degrees of convexity, and American Indians the least; Eskimos have the highest amount of convexity among Native Americans. Pacific Islanders are intermediate; Melanesians show the strongest expression of incisor labial convexity in the Pacific.

Anthropometry↗

The eruption pattern of the permanent incisors and first permanent molars in Australopithecus (Paranthropus) robustus.

This study aims to reassess the claim that the eruption sequence of the permanent incisor and first permanent molar teeth of Australopithecus (Paranthropus) robustus is identical with that in modern Homo sapiens. Eight fossil hominid mandibles of equivalent dental developmental age were chosen for comparative study. Emphasis has been placed upon the comparative timing of events within the growth period rather than eruption sequence alone. The results of this study indicate that Homo sapiens and Australopithecus (Paranthropus) robustus share the same pattern of permanent molar and incisor eruption and that this is significantly different from the pattern of eruption shared by the great apes, Australopithecus africanus and Australopithecus afarensis.

Haplorhini↗

Osteoclast induction in periodontal tissue during experimental movement of incisors in osteoprotegerin-deficient mice.

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor (TNF) receptor superfamily that negatively regulates osteoclastogenesis. The receptor activator of the NFKB ligand (RANKL) is one of the key regulatory molecules in osteoclast formation and binds to OPG. In this study, it was suggested that OPG and RANKL are involved in alveolar bone remodeling during orthodontic tooth movement. We examined RANKL localization and osteoclast induction in periodontal tissues during experimental movement of incisors in OPG-deficient mice. To produce orthodontic force, an elastic band was inserted between the upper right and left incisors for 2 or 5 days, and the dissected maxillae were examined for cytochemical and immunocytochemical localization of tartrate-resistant acid phosphatase (TRAP), vacuolar-type H(+)-ATPase, and RANKL. Compared to wild-type OPG (+/+) littermates, TRAP-positive multinucleated cells were markedly induced in the periodontal ligament (PDL) on the compressed side and in the adjacent alveolar bone of OPG-deficient mice. These multinucleated cells exhibited intense vacuolar-type H(+)-ATPase along the ruffled border membranes. Because of accelerated osteoclastic resorption in OPG-deficient mice, alveolar bone was severely destroyed and partially perforated at 2 and 5 days after force application. In both wild-type and OPG-deficient mice, RANKL expression became stronger at 2 and 5 days after force application than before force application. There was no apparent difference in intensity of RANKL expression between OPG (+/+) littermates and OPG-deficient mice. In both wild-type and OPG-deficient mice, expression of RANKL protein was detected in osteoblasts, fibroblasts, and osteoclasts mostly located in resorption lacunae. These results suggest that during orthodontic tooth movement, RANKL and OPG in the periodontal tissues are important determinants regulating balanced alveolar bone resorption.

Acid Phosphatase↗

Vascular turnover of a continuously growing organ: the rat incisor.

The vascular tissue supplying the pulp of the continuously growing and developing rat incisor was quantitatively analyzed in six female rats, weighing approximately 200 gm each. One hour after a single administration of 3H-TdR, 1 microCi/gm body weight, the rats were sacrificed and the lower left jaws excised and processed histologically. Every 20th section was evaluated for venular and arterial count, endothelial cell count, and percentage of labeled endothelial cells. It was found that the proliferative capacity of the vascular tissue of the rat incisor is confined to its most apical 3.0 mm-a zone defined as the vascular progenitor compartment. Daily production of endothelial vein cells and of veins within the progenitor compartment were calculated to be 7,392 cells/day and two veins/day, respectively. Generation time of these cells was found to be 3.56 days. Endothelial cell production within the progenitor compartment provides for the formation of new vessels and for the enlargement and elongation of existing ones. The vessels originating within the progenitor compartment move in an incisal and centripetal direction, while simultaneously undergoing a continuous process of aging, until their ultimate disintegration.

Animals↗

The effect of osmium postfixation and uranyl and lead staining on the ultrastructure of young enamel in the rat incisor.

Enamel crystallites are electron opaque without osmium or heavy metal staining and give a crystalline electron diffraction pattern. Since the opacity and diffraction pattern are abolished from ultrathin sections of young enamel by floating on distilled water (Bishop and Warshawsky, 1982), the possibility that aqueous staining may also remove crystallites was tested. In addition, the effect of osmium postfixation on crystallite structure was examined. Rat incisors fixed by perfusion with a mixture of aldehydes were either nonosmicated or osmicated prior to dehydration. Incisor segments in the region of inner enamel secretion were embedded in the same Epon block to ensure reliable comparison. Osmicated enamel was more intensely stained with toluidine blue and more electron opaque than nonosmicated enamel. No other structural differences were seen. However, crystallites in osmicated enamel were more resistant to grid demineralization and electron beam damage. Routine staining was done by floating sections on solutions of uranyl acetate and lead citrate; sections were also floated on similar solutions from which the heavy metals were omitted. These solutions removed the electron opaque crystallites from the youngest enamel. Stained sections showed electron opaque crystallite-like structures similar to unstained enamel. When sections that were extracted by the solutions from which the metals were omitted were restained, they appeared identical to routinely stained enamel. It was concluded that staining of young enamel removes the crystallites and reveals only the organic matrix.

Aging↗

In vivo experimentation on rat incisor enamel organs through a surgical window.

Experimental agents administered systemically are costly and often toxic to animals. An in vivo technique has been developed whereby a surgical window in the alveolar bone allows selected areas of the rat incisor enamel organ and underlying enamel to be exposed to various drugs, radiolabeled molecules, and molecular weight markers. Sherman rats weighing 100 gm were anesthetized and the inferior surface of each hemimandible was surgically exposed. A slow-speed dental hand drill was used to drill a small hole through the alveolar bone overlying the secretion or maturation zones of the enamel organ. The wound was closed and during recovery the mechanical trauma to the underlying tissue moved away from the hole due to the continuous eruption of the tooth. Two to 5 days later the hole was reexposed and microinjections of 3H-proline, 125I-salmon calcitonin, vinblastine sulphate, and normal saline (as control) were administered through the hole with a micromanipulator and a microliter syringe. Radioautographic detection of 3H-proline incorporation in secretory ameloblasts and enamel at 10 minutes, 30 minutes, 1 hour, 4 hours, 1 day, and 2 days after microinjection was identical to that obtained previously by systemic injection. Two hours after microinjection of vinblastine sulphate the cellular response was again identical to that following systemic injection; 125I-salmon calcitonin (M.W. approximately 3,600D) was used as a molecular weight marker and was seen to diffuse into the enamel of the maturation zone at 10 minutes after microinjection. This study has demonstrated the feasibility of this new technique for experimentation on rat incisor enamel organs.

Alveolar Process↗

The initiation of osteodentin formation in the rat incisor after adriamycin administration.

In order to study the initiation of osteodentin formation in rat incisors, animals were injected intravenously with adriamycin (5 mg/kg body weight), and killed from 2 to 7 days after injection by perfusion with a 2.5% buffered glutaraldehyde solution. Control animals, injected with only physiological saline, were treated in the same manner. Three days after adriamycin injection aggregations of mesenchymal cells were observed along the mesial and lateral walls of the pulp chamber. Between 3 days and 7 days osteodentin production was observed at the sites where the mesenchymal aggregations were previously observed. Electron microscopic observations revealed that the cells involved in the aggregates were larger and contained more profiles of rough endoplasmic reticulum and secretion granules than the unaffected pulp cells. The osteodentin matrix first appeared as a scant deposition of collagen fibers between the cells. As more collagen fibers were deposited the matrix became much denser. Some cells that initially formed the mesenchymal aggregates were completely enclosed by the increased deposition of the matrix. It therefore appears that osteodentin formation, as observed in the rat incisor pulp after adriamycin administration, is the result of an abnormal differentiation of pulp mesenchymal cells.

Animals↗

Root-analogue versus crown-analogue dentin: a radioautographic and ultrastructural investigation of the mouse incisor.

The present paper reports on differences between the root- and crown-analogue dentin portions of the continuously growing mouse incisor. Conventional light microscopy and radioautography were used to study dentin formation and the uptake of [3H]-proline and [3H]-serine. It was found that, although the dentin apposition rate along the crown-analogue part (covered by enamel) equalled or slightly exceeded that along the root-analogue part (covered by cementum), the processing of predentin into dentin was considerably faster in the root aspect. Comparison of the two dentin portions at the ultrastructural level revealed that differences occurred in the morphology of the secretory granules of the odontoblast layer. Two types of granules were observed: those that were and those that were not loaded with electron-dense particles of 30 nm diameter. While the former type was most frequent along the crown-analogue aspect of the incisor, the latter type was particularly found along its root-analogue aspect. This difference may reflect differences between the two dentin portions in the composition of the noncollagenous matrix.

Animals↗

Ultrastructural location of complex carbohydrates in developing rat incisor enamel.

The ultrastructural distribution of complex carbohydrates in an early formation stage of rat incisor enamel was investigated by staining with the periodic acid-thiocarbohydrazide-silver proteinate reaction (PA-TCH-SP) for vicinal glycol-containing glycoconjugates, the phosphotungstic acid-chromic acid mixture (PTA) for glycoproteins, and the cationic dyes alcian blue or bismuth nitrate for sulfated glycoconjugates. In order to remove selectively sulfated complex carbohydrates, half of the serial sections obtained were digested with a bovine testicular hyaluronidase prior to staining. Far fewer electron-dense deposits were observed with the PA-TCH-SP method on hyaluronidase-treated sections, especially those subsequently treated for 48 hours with TCH. On the other hand, the minimal staining obtained with PTA was much more intense on sections treated with hyaluronidase where linear fiberlike structures were observed. With cationic dyes, staining of dotlike alignment structures and ground substance was obtained but was completely abolished by hyaluronidase treatment. Cuprolinic blue in a critical electrolyte concentration, ruthenium hexamine trichloride used with aldehyde during fixation, as well as rapid-freezing followed by freeze-substitution validate that this dotlike distribution is not an artefact of processing. The staining results demonstrated that the glycoproteins and sulfated complex carbohydrates in developing rat incisor enamel each display a specific distribution pattern. The glycoproteins were present as fiberlike structures and the sulfated carbohydrates appeared as dotlike formations located close to the surface of the fiberlike structures, and/or in the spaces between them.

Animals↗

Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors.

In the continuously erupting rat incisor the ameloblasts progress through distinct stages associated with the secretion and maturation of enamel. We have examined the possibility that the so-called "postsecretory" ameloblasts of the maturation stage of amelogenesis remain biosynthetically active and are engaged in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel organ cells during the early maturation stage of amelogenesis in rat incisors by using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late secretion through the first complete modulation from ruffle-ended to smooth-ended ameloblasts were examined. Specific immunolabelling was found within the rough endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout these regions. The heaviest intracellular immunolabelling was found within secretory granules and lysosomes (multivesicular type). Quantitative analyses showed that the Golgi saccules and the multivesicular lysosomes of modulating ameloblasts were generally less immunoreactive compared to similar organelles in ameloblasts secreting the inner enamel layer. Radioautographic studies confirmed that ameloblasts of the maturation stage incorporated 3H-leucine and 3H-methionine and secreted labelled proteins into the enamel layer. Grain counts indicated that ameloblasts from the first ruffle-ended band incorporated about two-fold less 3H-methionine and secreted about tenfold less labelled proteins into the enamel compared to ameloblasts secreting the inner enamel layer. The results of this study confirm that ameloblasts do not terminate biosynthesis and secretion of enamel proteins once the final layer has been deposited on the surface of the developing enamel. They continue to form and release new proteins during the maturation stage which intermix with older proteins laid down initially during the secretory stage of amelogenesis. Secretory activity for enamel proteins has been detected in ameloblasts up to at least the second ruffle-ended phase of maturation, at which point the enamel matrix is partially soluble in EDTA.

Ameloblasts↗

Cyclic localization of actin and its relationship to junctional complexes in maturation ameloblasts of the rat incisor.

The patterns of fluorescence associated with maturation ameloblasts of mandibular incisors labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) for the detection of F-actin were investigated in normal and fluoride-treated rats. In normal rats, bands of smooth-ended ameloblasts (SA) exhibited intense fluorescence at their proximal ends only. Bands of ruffle-ended ameloblasts (RA) exhibited strong fluorescence at their distal ends as well as at their proximal ends. Regional differences in degree of intensity within the bands and between bands were displayed. In the apical part of the RA bands the proximal fluorescence was intense; it then decreased in an incisal direction; and it finally was absent close to the adjacent SA band. The incisal extension of strong proximal fluorescence in RA bands was short in early maturation and long in late maturation. The fluorescence pattern at both ends of the ameloblasts was cyclically repeated throughout the region of ameloblast modulation corresponding to the numbers of SA bands. In rats receiving 113 ppm fluoride in their drinking water for 2 months the number of fluorescence and ameloblast modulation cycles was reduced equally indicating that the cyclic F-actin localization is a phenomenon related to ameloblast modulation. Electron microscopy revealed that areas of strong fluorescence contained filament bundles, presumably actin filaments, in relation to continuous junctions occluding the interameloblast spaces. Areas of weak or no fluorescence were related to discontinuous macular junctions. The results suggest that the changes in F-actin distribution correlate well with junctional complex development, and therefore, possible functions related to the intermeloblast spaces within the RA bands may be redistributed as the ameloblasts are carried incisally by the erupting incisor.

Actins↗

Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse-chase labeling with L-[35S]- and L-[methyl-3H]-methionine.

The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl-3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.

Animals↗

Fate of odontoblasts and blood capillaries in the incisal region of the rat incisor pulp.

Transmission electron microscopy of thin sections of the rat incisor pulp revealed that in the middle region of the incisor there were fenestrated capillaries in the "predentinal plexus" and that this region contained the tallest odontoblasts. The odontoblasts gradually became shortened in the incisal part of this region; the fenestrated capillaries in the predentinal plexus changed to continuous type capillaries. Almost all the odontoblasts had degenerated near the incisal end of the tooth. The predentinal plexus disappeared in this region, but the "subodontoblastic capillary plexus" persisted. In a specific region just beneath the worn incisal end, numerous macrophages and polymorphonuclear neutrophils appeared and scavenged the degenerating cells, possibly including the odontoblasts.

Animals↗

Correlated alkaline phosphatase histochemistry and quantitative backscattered electron imaging in the study of rat incisor ameloblasts and enamel mineralization.

The different functional conditions of pre-ameloblasts, secretory ameloblasts, and maturation ameloblasts in 9 day rat incisors were recognized using high resolution light microscopic alkaline phosphatase histochemistry: Digital backscattered electron imaging was performed using the block surfaces from which thin sections were taken for histochemical study. It was possible to correlate exact locations in histochemical sections with positions in the block face at all stages of enamel mineralization from early secretion through late maturation. The first steep increase in the rate of mineralization of completed enamel matrix occurs after the first transition from smooth ended ameloblasts to ruffle ended ameloblasts. In the 9 day rat incisors used for this purpose, there are only two smooth to ruffle cyclical transitions, and the width of successional smooth ended bands of ameloblasts in the maturation cycling process is always narrow. Nevertheless, there seems to be a good correlation between mineralization increase and the acquisition of the high alkaline phosphatase activity in the deeply enfolded distal cytoplasm of the ruffle-ended maturation stage ameloblasts.

Alkaline Phosphatase↗