Cytoskeleton and surface structures of cells directly attached to the tooth in the rat junctional epithelium

OBJECTIVE: It is still an open question whether cells directly attached to the tooth (DAT) cells are migratory or non-migratory cells. The purpose of this study was to examine cytoskeletal and surface structures of DAT cells that might be involved in migration. METHODS: We investigated the distribution of stress fibers composed of actin filaments in DAT cells using phallacidin fluorescent dye methods in a confocal laser scanning microscope. To observe the three-dimensional structure of the DAT cell surface, the osmium maceration scanning electron microscope (SEM) method, which removes various soluble materials between DAT cells and the enamel, was employed. RESULTS: Stress fibers were found in the most apically located DAT cells, and were arranged in parallel to the presumable cervical-line, whereas some of the fibers ran parallel to the tooth axis in the more coronally located DAT cells. The parallel arrangement to the tooth axis of the fibers may be involved with migration for turnover, and the parallel accumulation to the presumable cervical-line may be concerned with the cervical contraction of DAT cells. Osmium maceration SEM images at high magnification revealed the existence of microvilli-like structures on the enamel surfaces (facing to the tooth surface) of DAT cells after removal of the soluble matrices. The thicknesses of the microvilli-like structures on the enamel surfaces and cell processes of intercellular bridges were significantly different. CONCLUSION: DAT cells possess stress fibers arranged in parallel to the tooth axis and to the presumable cervical-line in the cytoplasm, and microvilli-like structures on their enamel surfaces. These results suggest that these structures contribute to DAT cell migration.     

The localization of epithelial root sheath cells during cementum formation in rat molars

The purpose of this study was to investigate the distribution of epithelial cells and the fate of the basement membrane along the root surface of rat molars during cementogenesis, and to test the hypothesis that the Hertwig's epithelial root sheath (HERS) cells remain on the root surface if mineralization is inhibited. To demonstrate the HERS cells and basement membrane, immunohistochemistry with antibodies against keratin and laminin were used. The dentin matrix mineralization was inhibited by a single injection of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP). A modified Gomori staining method was used to monitor the inhibition of mineral formation in dentin and cementum. Paraffin sections were stained with haematoxylin-eosin. and freeze-dried sections were used for Gomori and immunohistochemical stainings. We found that the formation of acellular cementum was suppressed above the dentin with inhibited mineralization. Instead, a hyperplastic matrix, different from acellular cementum, covered the dentin. This hyperplastic cementum had keratin- and laminin-positive cells incorporated; such cells were never incorporated in normal acellular cementum. The later formation of cellular cementum correlated, in controls, with the disappearance of HERS cells from the root surface. Treatment with HEBP resulted in a persistent presence of epithelial cells, interpreted as an inhibition of their disappearance. In conclusion, there is evidence that the cells of HERS are involved in the development of both acellular and cellular cementum. The developmental processes of these tissues appear in some way to be influenced by or associated with the initial mineralization of the dentin.     

Topical application of Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces cell cycle arrest in the rat gingival epithelium in vivo

Ohara M, Miyauchi M, Tsuruda K, Takata T, Sugai M. Topical application of Aggregatibacter actinomycetemcomitans cytolethal distending toxininduces cell cycle arrest in the rat gingival epithelium in vivo. J Periodont Res 2011; 46: 389–395. © 2011 John Wiley & Sons A/S Background: Aggregatibacter actinomycetemcomitans is one of the etiological pathogens implicated in the onset of periodontal disease. This pathogen produces cytolethal distending toxin (CDT) that acts as a genotoxin to induce cell cycle arrest and cellular distension in cultured cell lines. Therefore, CDT is a possible virulence factor; however, the in vivo activity of CDT on periodontal tissue has not been explored. Here, CDT was topically applied into the rat molar gingival sulcus; and the periodontal tissue was histologically and immunohistochemically examined. Materials and Methods: Recombinant purified A. actinomycetemcomitans CDT was applied to gingival sulcus of male Wistar rats and tissue samples were immunohistochemmically examined. Results: One day after application, infiltration of neutrophils and dilation of blood vessels in the gingival connective tissue were found. At day three, desquamation and detachment of cells in the junctional epithelium was observed. This abrasion of junctional epithelium was not observed in rats treated with mutated CDT, in which a His274Ala mutation is present in the CdtB subunit. This indicates the tissue abrasion may be caused by the genotoxicity of CdtB. Expression of the proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, was significantly suppressed using CDT treatment in the junctional epithelium and gingival epithelium. Conclusion: Using the rat model, these data suggest CDT intoxication induces cell cycle arrest and damage in periodontal epithelial cells in vivo.     

Bundle formation of principal fibers in rat molars

The bundling of principal fibers was investigated in tangential sections through the tooth-related portion in developing rat molars by light and electron microscopy. When root dentin calcification began, cross sections of principal fibers emerged as fibril aggregates in the narrow intercellular spaces in a densely packed population of periodontal ligament cells. Subsequently, these cells changed shape and location to widen the intercellular spaces. The fibril aggregates became thicker in these spaces. With root development, the collagen fibrils formed loosely aggregated bundles and the periodontal ligament cells extended cell processes between the bundles. The cell processes usually contained microfilaments suggestive of actin filaments, and as the cell processes extended and came in close apposition, they formed delimited compartments. These compartments appeared to be a sheath-like structure, and the loose fibril bundles developed into tight fibril bundles in the compartments. Finally the principal fibers consisted of many tight fibril bundles, which were partially or entirely surrounded by cell processes and cell bodies. The findings suggest that the sheath-like, cellular compartments cause the tight bundling of the principal fibers.     

Evidence for the existence of intraepithelial nerve endings in the junctional epithelium of rat molars: an immunohistochemical study using protein gene product 9.5 (PGP 9.5) antibody

Innervation of the junctional epithelium was investigated in rat molars by means of immunohistochemistry for protein gene product 9.5 (PGP 9.5) at light and electron microscopic levels. In comparison with our previous study on same tissues using neurofilament protein (NFP)-antibody, the PGP 9.5-immuno-staining further disclosed numerous nerve fibers in the gingiva of rat molars and revealed the existence of a well-developed plexus of PGP 9.5-positive nerve fibers. The interproximal portion also contained numerous nerve fibers.
Observation of horizontal sections revealed a denser innervation toward the inner junctional gingival epithelium than the outer marginal epithelium. The nerve fibers, beaded in appearance and extending from the nerve bundles in the lamina propria, penetrated into the junctional epithelial layer and were distributed throughout the junctional epithelium, with some nerves being located near the epithelial surface. Non-neuronal cells showing PGP 9.5-immunoreactivity were absent in the junctional epithelium. In immunoelectron microscopy, the axoplasm of nerves in the gingiva was filled with electron-dense reaction products of PGP 9.5, except for the cell organellae. The nerve fibers were devoid of Schwann cell investment and terminated among the epithelial cells in the junctional epithelium, frequently beneath the epithelial surface. The intraepithelial nerve endings contained various kinds of vesicles including large-cored ones, supporting the presence of peptidergic innervation shown by previous studies. These findings confirmed the usefulness of PGP 9.5-immunohisto-chemistry for the identification of delicated nerve fibers in dental tissue, and suggested the dense network of nerve fibers that may serve as sensory receptor and other functions in the junctional epithelium.     

Positional and ultrastructural changes in peripheral pulp capillaries correlate with the active phase of dentin deposition and mineralization in rat molars

Objectives

The mechanisms regulating positional and ultrastructural changes in peripheral pulp capillaries during dentinogenesis have yet to be fully elucidated. This study aimed to clarify the relationship between the spatiotemporal localization and ultrastructure of peripheral capillaries and the dentin deposition and mineralization rate.

Effects of mechanical stimulation on cell cycle duration in rat gingival fibroblast progenitor cells

The aim of this investigation was to estimate cell cycle duration in rat gingival fibroblast progenitor cells in steady-state control and during sustained mechanical stimulation. Elastics (0.15 mm thick) were inserted between maxillary M1 and M2 of 8 wk-old male rats which were labelled with H3-TdR and killed in groups of 6-7 animals together with equal-sized groups of labelled control animals at intervals between 1-168 h. Autoradiographs of consecutive mesio-distal sections were used to determine grain counts for H3-TdR-labelled cells in the connective tissue of the gingival papilla between M2 and M3. Median cell cycle times (MCC) were estimated from plots of mean and median grain counts against time. Under steady-state conditions, MCC for heavily-labelled and lightly-labelled paravascular cell populations and for labelled extravascular cells were 144, 76 and 50 h, respectively. Mechanical stimulation caused a significantly faster rate of reduction of total grain counts relative to controls in all three cell populations and a decline of estimated MCC to 115, 50 and 21 h in heavily labelled and lightly labelled paravascular cells and labelled extravascular cells, respectively. These findings indicate that mechanical stimulation induces faster progression of gingival fibroblast progenitor cells through the cell cycle.     

An immunohistochemical study of the attachment mechanisms in different kinds of adhesive interfaces in teeth and alveolar bone of the rat

BACKGROUND AND OBJECTIVE: This study was designed to examine the histological and immunohistochemical nature of different kinds of adhesive interfaces in the rat molar region under identical experimental conditions and to discuss the structural and functional similarities between these adhesive interfaces. MATERIAL AND METHODS: Four kinds of adhesive interfaces - an initial attachment layer for principal fibers on the developing alveolar bone surface, a reattachment layer for principal fibers on resorbed alveolar bone surface, cement lines on the alveolar bone surface unrelated to the principal fibers, and the cemento-dentinal junction - were examined in 25-d-old male Wistar rats. Routine histological staining, immunohistochemical staining for bone sialoprotein and osteopontin, and digestion tests with trypsin were conducted. RESULTS: The adhesive interfaces showed very similar histological and immunohistochemical features: they were intensely hematoxylin-stainable, deficient in collagen fibrils, and rich in bone sialoprotein and osteopontin. After trypsin treatment the four adhesive interfaces had lost immunoreactivity to bone sialoprotein and osteopontin, and the two adjacent tissue parts held together finally separated at the adhesive interfaces. CONCLUSION: The above findings suggest that (i) the different types of adhesive interfaces in the rat molar region have a common structure in that they are filled with highly accumulated bone sialoprotein and osteopontin and deficient in collagen fibrils; (ii) accumulated bone sialoprotein and osteopontin are closely associated with the adhesion at the interfaces; and (iii) the adhesive interfaces have a similar developmental process.     

A fluid-phase endocytotic capacity and intracellular degradation of a foreign protein (horseradish peroxidase) by lysosomal cysteine proteinases in the rat junctional epithelium

We investigated the co-localization of lysosomal cathepsins B, H and L, and horseradish peroxidase (HRP) in junctional epithelial (JE) cells both as a fluid-phase endocytotic marker to demonstrate the fluid-phase endocytotic capacity of JE cells, and to understand the morphological relationships of the endocytosed foreign substances to lysosomal cathepsins in these cells. The diaminobenzidine (DAB) histochemical and cytochemical methods and immunohistochemical avidin-biotin-peroxidase complex and immunocytochemical post-embedding colloidal gold methods were used. Under light microscopy, DAB reaction products based on HRP were found in JE but were rare or absent in the oral sulcular epithelium and oral epithelium. Immunolabeling for cathepsins B and H was found in the granular structures of the cells, but no cathepsin L was identified. With electron microscopy, DAB reaction products, which indicated both HRP and the azurophil granules of neutrophils, were endocytosed into JE cells. Using a post-embedding technique, gold particles indicating HRP were present on the plasma membrane of JE cells, at the periphery of electronlucent vacuoles, and in the electrondense granules. Gold particles indicating cathepsin B or H were found in the electrondense granules. With different sizes of colloidal golds, the co-localization of cathepsin B or H with HRP was indicated only in the electrondense portion of the larger vacuoles consisting of electronlucent and -dense parts. This study provided the first morphological data which indicate that JE has a fluid phase endocytotic capacity, and which suggest that the lysosomal cathepsins B and H are involved in the intracellular degradation of foreign substances invading through the gingivl sulcus in JE cells.     

肌动蛋白在大鼠磨牙及其发育中的分布研究

目的：探讨肌动蛋白在大鼠磨牙及其发育中的时空分布及意义。方法：建立大鼠牙胚发育模型。用免疫组化方法检测肌动蛋白在成年和发育期磨牙中的表达。结果：肌动蛋白在钟状期外釉上皮细胞，分泌前期成釉细胞和成牙本质细胞及中间层细胞之间，硬组织形成期成釉细胞Tomes'突和成牙本质细胞胞浆呈阳性表达。结论：肌动蛋白在大鼠磨牙发育过程中的分布具有时空特异性。与细胞的分化程度和功能状态密切相关。     

实验性正畸牙齿移动中RANKL在牙周组织中表达的研究

目的 观察实验性正畸牙齿移动过程中，大鼠牙周组织中转录因子NFKB受体活化剂配体（receptor activator of NF-kB ligand／RANKL）的表达及定位，进一步探讨RANKL的来源、作用机制以及与正畸牙齿移动中骨改建的关系。方法 采用SP免疫组织化学方法检测RANKL在正畸大鼠牙周组织中的表达，并采用计算机图像分析的方法对各组RANKL的表达强度进行半定量分析。结果 实验性正畸牙齿移动过程中：①RANKL主要表达于牙槽骨表达的成骨细胞、破骨细胞和牙周膜成纤维细胞胞浆和胞膜中。②RANKL在牙周组织内的表达和分布在时间上是不均衡的，随时间的增加呈先增高后回降的趋势，峰值在第5天。③RANKL在牙周组织内的表达和分布在部位上是不均衡的，压力侧的表达量高于张力侧，牙槽骨表面的表达量高于近牙骨质侧的牙周膜。结论 RANKL的表达与正畸牙槽骨改建过程中破骨细胞的生命过程密切相关，RANKL通过旁分泌、自分泌机制作用于破骨细胞前体、成熟破骨细胞膜表达的受体RANKL，促进破骨细胞前体的分化、成熟，激活成熟破骨细胞的功能，加速骨改建。     

Regeneration of nerve fibres in the peri-implant epithelium incident to implantation in the rat maxilla as demonstrated by immunocytochemistry for protein gene product 9.5 (PGP9.5) and calcitonin gene-related peptide (CGRP)

The response of nerve fibres in the peri-implant epithelium to titanium implantation was investigated with an experimental model using rat maxilla and immunohistochemical techniques. The latter employed antibodies to protein gene product 9.5 (PGP9.5), and to calcitonin gene-related peptide (CGRP). In control rats without an implantation, a dense innervation of PGP9.5- and CGRP-positive nerve fibres was recognized throughout the junctional epithelium, as has been previously reported. A titanium-implantation induced a remarkable inflammatory reaction, as well as the destruction of covering epithelial cells. By 3-5 days post-implantation, inflammatory reaction showed a tendency to disappear, and the peri-implant epithelium showed proliferation and down-growth along the implant. At this stage, no nerve fibres were found around the peri-implant epithelium. At 10 days, a few nerve fibres reached the basal cell layers of the peri-implant epithelium, and entered it 15 days after implantation when the peri-implant epithelial cells showed morphological features roughly resembling those of normal junctional epithelial cells. At the complete osseointegration stage (days 20-30), the PGP9.5- and CGRP-positive nerve fibres, thin and beaded in appearance, were found distributed in the peri-implant epithelium. After 20 days, the numerical density of the intraepithelial nerves in the peri-implant epithelium appeared the same as, or less than, that in the normal junctional epithelium. These findings indicate that the peri-implant epithelium shows the same innervation as that in normal junctional epithelium, and that the intraepithelial nerve fibres in the peri-implant epithelium might have diverse functions, which have been suggested in the literature.     

Immunotoxic effects of smokeless tobacco on the accessory cell function of rat oral epithelium

Smokeless tobacco (ST) is known to adversely effect the oral mucosa, but knowledge about the influence on immune defence is limited. Few studies have investigated the effect of ST on the local immune response. In the present study, we have assessed the effect of a crude Swedish moist snuff (SS) extract, alkaloids, and nitrosamines on T-cell mitogenic response to Con A using epithelial cells, including Langerhans cells, of the rat oral mucosa as accessory cells. SS extract at a concentration of 4% reduced the T-cell proliferation by 50% (IC₅₀= 4%). Pretreatment of either oral epithelial cells or T-cells with SS extract also gave a significant inhibition of T-cell proliferation. This effect was not obtained following preincubation with SS components as alkaloids and different tobacco-specific nitrosamines (TSNA). None of the tested compounds were found to possess any mitogenic properties. This in vitro study showed that SS extract can evoke an immunosuppressive effect on mitogen-driven T-cell proliferation using cells from oral epithelium as accessory cells. This effect was more pronounced when SS extract was employed compared to when the single SS components were used alone.     

Immunocytochemical examination of the presence of amelogenin during the root development of rat molars

The presence of enamel proteins, especially amelogenins, during root development has been a subject of controversy for a long time. Whereas some studies have reported the presence of enamel proteins on the root surface, others were not able to detect them at these places. Since microwave (MW) processing has been shown to improve the antigen retention in mineralised tissues, we have applied MW techniques to ultrastructurally analyse the presence of amelogenin during root formation in rat molars. Upper molar tooth germs from 12 and 13-day-old Wistar rats were fixed in 0.1% glutaraldehyde + 4% formaldehyde under MW irradiation. They were then decalcified in 4.13% EDTA, dehydrated in graded concentrations of ethanol and embedded in LR White Resin. Ultrathin sections were processed for post-embedding colloidal gold immunolabelling using a chicken egg yolk antibody against a 24 kDa rat amelogenin. Then, the grids were incubated with a rabbit anti-chicken IgG secondary antibody and with a protein A-gold complex. The immunoreactivity for 24-kDa amelogenin was detected in the cytoplasm of the epithelial diaphragm cells--the most apical portion of the Hertwig's epithelial root sheath (HERS), and in less amounts on the adjacent dental papilla extracellular matrix. Amelogenin was no longer observed at advanced stages of root dentinogenesis or later, during cementogenesis. The restricted presence of amelogenin at the early stages of root formation suggests that this protein may play a role in the differentiation of ectomesenchymal cells into root odontoblasts.     

体外培养的SD大鼠三种牙周细胞表达骨钙素和碱性磷酸酶的比较研究

目的：旨在研究SD大鼠牙周的类成牙骨质细胞、成骨细胞和牙周韧带成纤维细胞表达骨钙素和碱性磷酸酶的情况,探讨区分此三种细胞的方法。方法：体外分别培养12周龄SD大鼠第一磨牙牙周的上述三种细胞,利用计算机图像分析仪和免疫酶联仪对细胞分别进行骨钙素免疫组化染色和碱性磷酸酶活力定量分析。结果：骨钙素在类成牙骨质细胞和成骨细胞中呈强阳性表达,在牙周韧带成纤维细胞中几乎不表达。成骨细胞碱性磷酸酶活性最强,在成纤维细胞中适中,在类成牙骨质细胞中几乎无活性。结论：体外培养的SD大鼠三种牙周细胞表达骨钙素和碱性磷酸酶有各自特点,可以依此对二三种细胞加以区分。     

Changes in root canal microbiota during the development of rat periapical lesions

Periapical lesions are reproducibly induced in rats by pulp exposure and infection from the oral cavity. Lesions expand rapidly between day 7 and day 15-20 (active phase), with slowed expansion thereafter. In the present study we characterized the root canal microbiota present during the active phase of lesion development in this system. The mandibular first molars of Sprague-Dawley rats were exposed on day 0. The teeth were extracted after 7 days ( n = 10 animals) and 15 days ( n = 10), and the microbiota present in roots was isolated and characterized. The number of colonies isolated per tooth was similar on day 7 (1.53 ± 0.64 colony-forming units × 10³) and day 15 (1.49 ± 0.37 colony-forming units × 10³). No colonies were isolated from unexposed control teeth. Anaerobic bacteria increased significantly between day 7 (24.3 ± 5.7%) and day 15 (47.3 ± 7.5%), and the proportion of gram-negative organisms increased from day 7 (24.3 ± 6.1) to day 15 (46.9 ± 6.8). The predominant bacteria included, on day 7: Streptococcus and Bacteroides species; on day 15: species of Streptococcus, Bacieroides, Prevotclla, Neisseria and Peptostreptococcus. Streptococcus oralis, Streptococcus mitis, Streptococcus rattus, Bacteroides pneumosinles and Bacteroides ureolyticus were frequently isolated at both points. Although approximately the same mean number of different species (∼3.5) was isolated per tooth on both day 7 and 15, the overall diversity of the isolates increased on day 15. These results demonstrate that the root canal microbiota becomes increasingly gram-negative and anaerobic with the emergence of Peptostreptococcus, Bacteroides, Prevotella and Neisseria during the period of rapid lesion expansion in this model.     

Histochemical study of the influence of transplanted teeth with periodontal ligament on the binding of peanut agglutinin in rat dorsal skin

Our previous study showed that the expression of carbohydrate residues in junctional epithelium (JE) after resection is closely related to attachment and stratification along the dental root surface. However, the influence of the periodontal ligament on carbohydrate expression has still not been clarified. In this study we examined the relationship between the presence of periodontal ligament and the expression of carbohydrate residues on epithelium regenerating along root surfaces. We transplanted extracted rat molars with or without periodontal ligament tissue, repeatedly frozen and thawed teeth with ligament and demineralized teeth without ligament into the dorsal skin of rats. After 2, 3, 5, 7, 10, 14 or 21 d, dorsal cutaneous tissues containing transplanted teeth were resected, fixed, decalcified and embedded in paraffin. Serial sections were stained histochemically with the HRP-conjugated lectin. peanut agglutinin (PNA) to observe the expression of carbohydrate residues in regenerating epithelium. Histochemical observation revealed that lectin binding reactions were changed from the characteristics of skin to those of JE when the regenerating epithelium was attached and stratified along the tooth with unfrozen or frozen tissue. These results suggested that the structural formation and expression of PNA in regenerating epithelium around the root surface were influenced by not only the tooth but also by the periodontal ligament.     

Proliferative activities of epithelial and connective tissue cells in the rat periodontal regeneration using argyrophilic nucleolar organizer regions staining

BACKGROUND AND OBJECTIVE: It is still an open question why long junctional epithelium can proliferate and occupies the root surface following periodontal surgery or experimentally produced periodontitis, and why the epithelium repopulated once on the root surface is replaced by the connective tissue. The aim of this study is to investigate the proliferative activity of the newly formed regenerative connective tissue and long junctional epithelium during wound healing by staining argyrophilic proteins of the nucleolar organizer regions (AgNORs). METHODS: Regenerative connective tissue and long junctional epithelium were experimentally created by insertion of a rubber piece between maxillary molars of rats for 1 week. After removal of the rubber, AgNORs parameters including nuclear area (NA), AgNORs area (AA), AgNORs percentage nuclear area (APNA), AgNORs number (AN) and nuclear number (NN) in regenerative connective tissue and long junctional epithelium were measured and analyzed statistically. RESULTS: APNA in long junctional epithelium after 1 and 4 weeks was over two times greater than that in the regenerative connective tissue. AA in long junctional epithelium was significantly higher than in regenerative connective tissue at 1 and at 4 weeks post-treatment. AN was higher in the central portion than at the root surface except at 20 weeks. APNA and AA decreased remarkably in long junctional epithelium at 12 weeks post-treatment (approximately half at 4 weeks), whereas in regenerative connective tissue, they did not change distinctly. CONCLUSIONS: These results imply that long junctional epithelium cannot supply sufficient epithelial cells because of their significantly low rates of proliferation, consequently long junctional epithelium becomes shorter after 12 weeks, whereas the proliferative activity of regenerative connective tissue maintains the same level of proliferation, and ultimately long junctional epithelium is replaced by regenerative connective tissue.     

Preparation of midsagittal semithin Epon sections of the entire secretory ameloblast population in the maxillary rat incisor

Abstract – To study cytological changes in the migrating cell layer of the ameloblasts in the rat incisor with semithin Epon sections the ideal section plane would consequently be along the midsagittal plane of the incisor. However, the small dimensions and the complex three-dimensional structure of the apical part of the odontogenic organ covered by alveolar bone make it difficult to orientate and position sections in the midsagittal plane. Serial sectioning of the apical half of the maxillary incisor in the horizontal and sagittal planes clarified the relation of the midsagittal plane of the odontogenic organ to the alveolar bone, and the changes of the sectional profile of the odontogenic organ as a function of its position on the transverse axis of the incisor. From this information a method was designed whereby the two axes of the midagittal plane were marked directly on the block surface before sectioning, and the position of sections in the midsagittal zone of the ameloblasts standardized.