Ameloblastic carcinoma of the mandible: a case report.

BACKGROUND: Ameloblastic carcinoma is a malignant form of ameloblastoma and a very rare odontogenic tumor. We report a case of ameloblastic carcinoma that occurred after removal of a right-sided mandibular dental implant. CASE PRESENTATION: A 72-year-old female patient visited her family dentist with a complaint of pain around a lower right implant placed 37 years previously. Although the dental implant was removed with the diagnosis of peri-implantitis, the patient experienced dullness of sensation in the lower lip and was followed up by her dentist, but after no improvement. She was referred to a highly specialized institution where she was diagnosed with osteomyelitis and treated the patient with medication; however, there was no improvement. In addition, granulation was observed in the same area leading to a suspicion of malignancy, and the patient was referred to our oral cancer center. The diagnosis of squamous cell carcinoma was made after a biopsy at our hospital. Under general anesthesia, the patient underwent mandibulectomy, right-sided neck dissection, free flap reconstruction with an anterolateral thigh flap, immediate reconstruction with a metal plate, and tracheostomy. Histological analysis of the resected specimen on hematoxylin and eosin staining showed structures reminiscent of enamel pulp and squamous epithelium in the center of the tumor. The tumor cells were highly atypical, with nuclear staining, hypertrophy, irregular nuclear size, and irregular nuclear shape, all of which were suggestive of cancer. Immunohistochemical analysis showed that Ki-67 was expressed in more than 80% of the targeted area, and the final diagnosis was primary ameloblastic carcinoma. CONCLUSION: After reconstructive flap transplantation, occlusion was re-established using a maxillofacial prosthesis. The patient remained disease-free at the 1-year 3-month follow-up.

Pentapartite fractionation of particles in oral fluids by differential centrifugation.

Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers. For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Canonical exosome markers, including CD63, CD9, CD133, and HSP70, were found in all fractions, whereas CD81 and AQP5 were enriched in the 160K fraction, with non-negligible amounts in the 2K fraction. The 2K fraction also contained its characteristic markers that included short derivatives of EGFR and E-cadherin, as well as an autophagosome marker, LC3, and large multi-layered vesicles were observed by electronic microscopy. Most of the DNA and RNA was recovered from the 0.3K and 2K fractions, with some in the 160K fraction. These results can provide guideline information for development of purpose-designed OF-based diagnostic systems.

Expression and localization of aqua-glyceroporins AQP3 and AQP9 in rat oral epithelia.

Aquaporins (AQPs) are a family of small integral membrane proteins made up of 6 hydrophobic, a-helical, membrane-spanning domains surrounding a highly selective aqueous pore. AQP3, AQP7, and AQP9, termed aqua-glyceroporins, are known to be involved in the transport of water, glycerol, and other small molecules. In this study, we investigated the expression and localization of aqua-glyceroporins in rat oral stratified squamous epithelia of the palate, the buccal mucosa, the inferior aspect of the tongue, and the oral floor by using RT-PCR, immunofluorescence, and immunogold electron microscopy. AQP3 and AQP9 mRNAs were expressed in whole oral epithelium. Immunostaining for AQP3 was recognized in each type of epithelium. The results suggest that AQP3 synthesis begins predominantly in the cytoplasm of the basal cells. During the process of epithelial cell differentiation, AQP3 protein appears to accumulate and be transported to the plasma membrane, from where it is incorporated into the cornified or surface layers. The intracellular localization of AQP3 appears to correlate with the differentiation of keratinocytes, suggesting that it acts as an enhancer of the physiological permeability barrier together with membrane coating granules. The distribution pattern of AQP9 was limited to the marginal areas of the basal and suprabasal layers, which was different from that of AQP3. This difference in distribution between AQP3 and AQP9 suggests that AQP9 in rat oral epithelia acts as a channel by facilitating glycerol uptake from the blood through the endothelial cells of the capillary vessels to the oral stratified squamous epithelium. AQP3 and AQP9 facilitate both transcellular osmotic water flow and glycerol transport as pore-like passive transporters in the keratinocytes of oral epithelia, and may play a key role in not only hydration and the permeability barrier, but also cell proliferation, differentiation, migration, development, and wound healing by generating ATP.

Immunoelectron microscopic observation of connexin43 in rat odontoblasts.

Gap junctions play an important role in differentiation of odontoblasts. Gap junction protein, connexin 43 is expressed in odontoblast. However, the detailed localization in odontoblasts has yet to be fully investigated. We investigated the localization of connexin43 in rat odontoblasts immuno-electron microscopically. The rats were transcardially fixed with 1% paraformaldehyde in 0.1M phosphate buffer, and mandibles were decalcified with 10% ethylenediamine tetraacetic acid. Pre-embedding method was carried out for immuno-electron microscopic analysis. Microscopically, gap junctions were localized between bodies of odontoblasts, and between bodies and processes of odontoblasts. The gap junctions were labeled with gold particles that indicated connexin43. These results suggest that gap junctions between odontoblasts are definitely composed of connexin43 in rats, and our methods used in this study is useful to investigate localization of connexin43 immuno-electron microscopically.

Erupted complex odontoma delayed eruption of permanent molar.

Odontomas, benign tumors that develop in the jaw, rarely erupt into the oral cavity. We report an erupted odontoma which delayed eruption of the first molar. The patient was a 10-year-old Japanese girl who came to our hospital due to delayed eruption of the right maxillary first molar. All the deciduous teeth had been shed. The second premolar on the right side had erupted, but not the first molar. Slight inflammation of the alveolar mucosa around the first molar had exposed a tooth-like, hard tissue. Panoramic radiography revealed a radiopaque mass indicating a lesion approximately 1 cm in diameter. The border of the image was clear, and part of the mass was situated close to the occlusal surface of the first molar. The root of the maxillary right first molar was only half-developed. A clinical diagnosis of odontoma was made. The odontoma was subsequently extracted, allowing the crown of the first molar to erupt almost 5 months later. The dental germ of the permanent tooth had been displaced by the odontoma. However, after the odontoma had been extracted, the permanent tooth was still able to erupt spontaneously, as eruptive force still remained. When the eruption of a tooth is significantly delayed, we believe that it is necessary to examine the area radiographically. If there is any radiographic evidence of a physical obstruction that might delay eruption, that obstruction should be removed before any problems can arise. Regular dental checkups at schools might improve our ability to detect evidence of delayed eruption earlier.

The actin-specific reagent jasplakinolide induces apoptosis in primary rat parotid acinar cells.

Jasplakinolide is a reagent that stabilizes and polymerizes actin filaments and is a commonly used tool in cell biology. In primary rat parotid acinar cells, jasplakinolide partially inhibited the release of amylase induced by ß-adrenergic receptor activation, as previously reported. However, in confocal microscopic observation with fluorescence conjugated anti-actin antibody, the jasplakinolide-treated cells not only showed decreased fluorescence intensity and aggregation of cortical F-actin but also revealed events characteristic of apoptosis such as cell shrinkage, membrane blebbing and apoptotic body formation. Such characteristic events of apoptosis were confirmed by transmission electron microscopy. The occurrence of apoptosis in jasplakinolide-treated cells was further confirmed by biochemical analysis: a DNA ladder was detected by electrophoresis, and DNA fragmentation was revealed using ELISA with an antibody to single-stranded DNA. Moreover, the degradation of fodrin was detected in jasplakinolide-treated cells by Western blotting, and the K(+) release induced by the fluid secretagogue carbachol was impaired. Taken together, these results demonstrate that jasplakinolide induces apoptosis and suppresses the secretory functions of rat parotid acinar cells.

Effect of diode laser on proliferation and differentiation of PC12 cells.

This study investigated the effects of diode (GaAlAs) laser irradiation at an effective energy density of 5 or 20 J/cm(2) on cell growth factor-induced differentiation and proliferation in pheochromocytoma cells (PC12 cells), and whether those effects were related to activation of the p38 pathway. Laser irradiation at 20 J/cm(2) significantly decreased the number of PC12 cells, while no difference was seen between the 5 J/cm(2) group and the control group (p<0.05). Western blotting revealed marked expression of neurofilament and ß-tubulin, indicating greater neurite differentiation in the irradiation groups than in the control group at 48 hr. Irradiation also enhanced expression of phospho-p38. The decrease in number of cells after laser irradiation was accelerated by p38 inhibitor, while neurite differentiation was up-regulated by laser irradiation, even when the p38 pathway was blocked. This suggests that laser irradiation up-regulated neurite differentiation in PC12 cells involving p38 and another pathway.

Cementum- and periodontal ligament-like tissue formation by dental follicle cell sheets co-cultured with Hertwig's epithelial root sheath cells.

Dental follicle cells (DFCs) are believed contain the precursor cells of the periodontium and can form cell sheets by secreting extracellular matrix (ECM) proteins. Cell sheet engineering has been recently developed and applied successfully in the field of tissue regeneration. However, research on the in vitro characteristics of DFC sheets is lacking and an assessment of whether DFC sheets can produce periodontal tissues in vivo has not been reported. To test the characteristics and applicability of DFC sheets in this field, we established a co-culture system of rat DFCs and Hertwig's epithelial root sheath (HERS) cells in vitro, and included the following controls: a co-culture of DFCs and alveolar mucosa epithelial cells, DFCs with no cells in the upper chamber, and DFCs cultured without an upper chamber. After 3 weeks of co-culturing the cells, the DFC sheets were transplanted into adult male rats' omenta. One week after co-culturing DFCs with HERS cells, mRNA levels of collagen type I (COL-1), alkaline phosphatase (ALP), runt related transcription factor 2 (Runx 2) and bone sialoprotein (BSP) were increased significantly. In addition, after 3 weeks of co-culturing the cells, the numbers of ALP-, osteocalcin (OCN)-, BSP- and osteoprotegerin (OPG)-positive DFCs increased. The DFCs also produced more calcified nodules and exhibited an increased number of subcellular organelles, which are important for protein synthesis and secretion. Moreover, gap junctions were found between the experimental DFCs within the sheet. Five weeks of in vivo growth of DFC sheets pre-exposed to HERS cells led to the formation of cementum-like tissues, which were positive for OCN, BSP and OPG, as well as the formation of periodontal ligament-like tissues, which were positive for COL-1. In contrast, control cells only produced fibrous tissues. These results indicate that the DFC sheets induced by HERS cells are able to produce periodontal tissues through epithelial-mesenchymal interactions. Therefore, DFC sheets may be useful in the field of periodontium regeneration.

Epithelial cell rests of Malassez modulate cell proliferation, differentiation and apoptosis via gap junctional communication under mechanical stretching in vitro.

Epithelial cell rests of Malassez (ERM) are involved in the maintenance and homeostasis of the periodontal ligament. The objective of this study was to investigate the effect of mechanical stretching on cell growth, cell death and differentiation in the ERM. Cultured porcine ERM were stretched for 24 hr in cycles of 18% elongation for 1 sec followed by 1 sec relaxation. The numbers of cells and TUNEL-positive cells were then counted. The expression of mRNAs encoding gap junction protein α1 (Gja1), ameloblastin, bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 4 (BMP4) and noggin were evaluated using quantitative real-time PCR. The number of cells in the stretching group was approximately 1.3-fold higher than that in the non-stretching controls at 24 hr (p<0.01). Apoptotic cells ranged from 1.9-2.5% in the stretching group at 24 hr, but were only 0.6% in the control group (p<0.01). The expression of Gja1, ameloblastin and noggin mRNAs in the stretching group was decreased at 24 hr compared with in the non-stretching group (p<0.01), whereas the expression of BMP2 and BMP4 mRNAs in the stretching group was significantly higher than that in the control group (p<0.01). Incorporation of 18 α-glycyrrhetinic acid (18GA, a gap junction inhibitor) promoted proliferation and apoptosis and confirmed both the increase of BMP2 and BMP4 and the decline of Gja1, ameloblastin and noggin in ERM. Thus, the ERM modulate cell proliferation and apoptosis, and inhibit differentiation by reducing expression of Gja1 under mechanical stretching.

Osteosarcoma of maxilla with unusual image findings in child.

Osteosarcoma of the head and neck is relatively rare and accounts for less than 10 percent of all osteosarcomas in general. We report a case of osteosarcoma in which imaging and histopathology of the hard palate of an 11-year-old boy yielded atypical findings. An approximately 8×15mm lesion found in the center of the palate was hard and healthy in color. Subsequent biopsy resulted in a diagnosis of nonepithelial malignant tumor. No abnormalities were observed in the maxillary bone or tooth on panoramic or occlusal radiographs. Computed tomography images revealed a mass lesion approximately 7×9×9mm in size on the hard palate extending into the maxilla. The cortex of the maxilla adjacent to the lesion was unclear in parts. The internal structures were slightly inhomogeneous and its density was lower than that of muscle. On magnetic resonance images, the lesion was represented by low signal intensity on T1-weighted (T1W) images and high signal intensity on T2-weighted images with fat-suppression. The margin of the lesion was a little unclear and the internal structures were slightly inhomogeneous. The lesion was enhanced homogeneously on post-contrast T1W images with fat-suppression. The histopathological diagnosis was fibrogenesis-type osteosarcoma. No findings specific to osteosarcoma such as localized enlargement of the periodontal ligament space alongside the root, cortical destruction, periosteal ossification or osteogenesis were found in this case.

Clinico-pathologic conference: case 4. Langerhans cell histiocytosis (LCH).

A 13-month-old Japanese boy presented with painless swelling in a left mandible and cheek. Intraoral examination revealed swelling in the left mandible and hemorrhage of oral mucosa due to biting. CT images revealed a wide osteolytic lesion of the left mandible with floating teeth. Biopsy was carried out and histopathological diagnosis was discussed.

Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells.

During tooth root formation, dental follicle cells (DFCs) differentiate into osteoblasts/cementoblasts when they are in contact with pre-existing dentin. Since some factors of dentin matrix were also produced by dental papilla cells (DPCs) and could induce DFCs differentiation, we hypothesized that DPCs can directly promote DFCs differentiation and that differentiation could occur in a co-culture model. To test this hypothesis, we investigated the characteristics of DFCs that are influenced by DPCs in an in vitro co-culture and in vivo heterotopic transplant model. One week into the co-culture, there were significant increases in the mRNA level of bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), bone sialoprotein (BSP) and osteocalcin (OCN), and a decrease of the receptor activator of nuclear factor κB ligand (RANKL). Additionally, the number of BMP2-, OPG-, BSP- and OCN-positive DFCs increased whereas RANKL-positive DFCs decreased. Three weeks after co-culture, DFCs produced calcified nodules, accompanied with increased sub-cellular organelles for protein synthesis and secretion. In the heterotopic transplant model, the adult male rats were used as hosts, DFCs were transplanted into the omentum. In vivo 5-week growth of DFCs in the presence of DPCs led to the formation of bone-like tissues, positive for BSP, OCN and BMP2. In contrast, DFCs alone led to fibrous-like tissues. These results indicated that in the absence of pre-existing dentin, DPCs can stimulate osteogenesis and inhibit osteoclastogenesis in DFCs and suggested a novel strategy to promote DFCs differentiation.

Effect of stretching stress on gene transcription related to early-phase differentiation in rat periodontal ligament cells.

Mechanical stress such as occlusal and orthodontic loading has been suggested to induce a homeostatic and regenerative response in periodontal ligament (PDL), but the underlying mechanism remains to be clarified. The purpose of this study was to investigate expression of mRNAs encoding proteins involved in osteogenesis and homeostasis by PDL cells following application of tensile stress and characterize the relationship between such expression and the regenerative and homeostatic functions of the PDL. PDL cells were obtained from rats and stretched by 9% or 18% at a frequency of 6 cycles/min for 12 hr to 5 days in a FX-4000T™ culture system. After stretching, expression of mRNAs encoding collagen type I (Col-I), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4), heat shock protein 70 (HSP70) and basic fibroblast growth factor (bFGF) was investigated. The highest levels of Col-I, ALP and BMP-2 mRNA expression occurred at 12 hr, while those of BMP-4 and HSP70 occurred at 1 day and 5 days, respectively. Expression levels of Col-I, ALP, BMP-2, BMP-4 and HSP70 increased magnitude-dependently with stretching force in the stretching groups. In contrast, expression of bFGF mRNA showed statistically significant reduction in both stretching groups, with the largest reduction seen in the 9% stretching group (p<0.01). These results suggest that stretching of PDL cells provokes significant increases in expression of factors promoting osteogenic differentiation and HSP70, which protects PDL cells undergoing mechanical stress and contributes to maintenance of PDL homeostasis. However, expression of bFGF was restrained. Reduced expression of bFGF mRNA suggested that there was an optimum magnitude of stretching force for increasing expression.

A case of congenital midline fistula of the upper lip.

Congenital fistulas of the lip are commonly found in the lower lip and accompany cleft lip. They are seen as a symptom of Van der Woude syndrome, which is predominantly hereditary. In contrast, congenital fistulas of the upper lip are rare. A number of hypotheses have been proposed to explain the pathogenesis of fistulas of the upper lip, including fusion failure of facial prominences and absence of mesoblasts, suggesting a relationship between this condition and the development of cleft lip. The pathogenesis of this disorder has been attracting attention. We report the case of a 5-year-old girl with congenital fistula of the upper lip.

Sodium-calcium exchangers in rat ameloblasts.

Although the central role of ameloblasts in synthesis and resorption of enamel matrix proteins during amelogenesis is well documented, the Ca(2+)-transport/extrusion mechanism remains to be fully elucidated. To clarify Ca(2+)-transport in rat ameloblasts, we investigated expression and localization of Na(+)-Ca(2+) exchanger (NCX) isoforms and the functional characteristics of their ion transporting/pharmacological properties. RT-PCR and immunohistochemical analyses revealed expression of NCX1 and NCX3 in ameloblasts, localized in the apical membrane. In patch-clamp recordings, Ca(2+) efflux by Na(+)-Ca(2+) exchange showed dependence on external Na(+). Ca(2+) influx by Na(+)-Ca(2+) exchange, measured by fura-2 fluorescence, showed dependence on extracellular Ca(2+) concentration, and it was blocked by NCX inhibitors KB-R7943, SEA0400, and SN-6. These results showed significant expression of NCX1 and NCX3 in ameloblasts, indicating their involvement in the directional Ca(2+) extrusion pathway from cells to the enamel mineralizing front.

Congo red-binding protein in rough-phenotype Aggregatibacter actinomycetemcomitans is amyloid-like fiber.

Aggregatibacter actinomycetemcomitans is a pathogen associated with chronic and aggressive periodontitis and extra-oral infections. Fresh isolates of A. actinomycetemcomitans are fimbriated, forming small, rough-phenotype colonies on agar plates and also form biofilms. Recently, it has been reported that amyloid fibers are abundant in natural biofilms, and Escherichia coli and Salmonella spp. produce amyloid fibers that contribute to biofilm formation. This has yet to be reported, however, in A. actinomycetemcomitans. Amyloid binds the Congo red (CR) dye. In this study, therefore, we investigated amyloid formation in A. actinomycetemcomitans using a detection of CR-binding colonies on CR agar plates and CR-binding assay. All rough-phenotype strains formed dark red colonies and smooth-phenotype strains formed white or opaque red colonies on CR agar plates. Compared with smooth-phenotype strains, rough-phenotype strains showed higher CR-binding activity. CR-binding of rough-phenotype strain AKR was not affected by protease digestion or heating, whereas smooth-phenotype strain 29523 showed a marked reduction in CR-binding after both types of treatment. AKR showed amyloid-positive staining with CR to produce yellow green birefringence under polarized light, whereas 29523 showed amyloid-negative staining. These findings indicate that the CR-binding component of rough-phenotype A. actinomycetemcomitans is an amyloid-like fiber.

Morpho-physiological approach to the paracellular route for salivary secretion by isolated perfused submandibular gland.

A part of the salivary components are shifted from the blood via a trans- and/or paracellular route. The isolated, arterially perfused, salivary glands (submandibular and parotid glands) were used to assess paracellular transport functionally and morphologically. In the present study, the hydrostatic pressure of the perfusion was changed and the fluid secretion and paracellular transport of fluorescent dye in the isolated perfused submandibular gland were examined. The present findings lead to the conclusion that part of the paracellular transport could be driven by hydrostatic pressure, and that fluid movement drags the solutes.

Electrophysiological properties of AQP6 in mouse parotid acinar cells.

Salivary gland acinar cells secrete large amounts of water and electrolytes, where aquaporins (AQPs) are thought to be involved in the secretion. In the present study, we investigated expression/localization of AQP6, and the anion transporting properties of AQP6 in mouse parotid acinar cells. RT-PCR, western blotting and immunohistochemical analyses revealed expression of AQP6 in acinar cells, localized in apical membrane. Voltage ramp from -100 mV to +100 mV at a holding potential of -60 mV elicited outwardly-rectifying currents, in the presence of extracellular Cl(-) channel blockers and intracellular solution with 150 mM Cs(+). These outward currents were increased when extracellular Cl(-) was replaced by Br(-), NO(3)(-), I(-), or SCN(-), accompanying a negative shift of reversal potentials. The outward current was enhanced by extracellular Hg(2+). These results were consistent with the biophysical properties of transfected AQP6 oocytes or HEK cells, which indicate that the AQP6 channel is functionally expressed in parotid acinar cells, and suggest that AQP6 contributes to secretion of anions in parotid acinar cells.

Morphological evidence of paracellular transport in perfused rat submandibular glands.

The morphological change of the paracellular route for fluid secretion is still a long-standing question. The purpose of this study was to visualize alterations in the cytoskeleton structure of tight junctions caused by carbachol (CCh) and isoproterenol (IPR) treatment of perfused rat submandibular glands (SMGs), using freeze-fracture (FF) replicas of rapidly frozen tissues. Isolated SMGs from male Wistar rats were perfused and stimulated with 1 microM CCh and IPR. Specimens were immediately rapidly frozen with liquid helium by metal contact. After cutting and deep etching, FF replicas were obtained by rotary shadowing and were examined by transmission electron microscopy. After CCh/IPR stimulation, the strand particles of TJs rearranged with free ends and terminal loops. In the vertical fracture surface, cytoskeletal filaments beneath the plasma membrane were arranged in a thicker layer than those of the gland without stimulation. Contraction of the submembranous actin cytoskeleton during exocytosis elicited by CCh/IPR may cause rearrangement of TJ strands due to direct interactions between the TJ membrane particles and actin filaments via the tiny bridging structures. The rearrangement and movement of TJ membrane particles involves reconstruction of the subluminal membranous actin filament network through the intermediary of interstitial molecules and may modulate increased paracellular permeability after CCh/IPR stimulation.