Notch Signaling Affects Oral Neoplasm Cell Differentiation and Acquisition of Tumor-Specific Characteristics.

Histopathological findings of oral neoplasm cell differentiation and metaplasia suggest that tumor cells induce their own dedifferentiation and re-differentiation and may lead to the formation of tumor-specific histological features. Notch signaling is involved in the maintenance of tissue stem cell nature and regulation of differentiation and is responsible for the cytological regulation of cell fate, morphogenesis, and/or development. In our previous study, immunohistochemistry was used to examine Notch expression using cases of odontogenic tumors and pleomorphic adenoma as oral neoplasms. According to our results, Notch signaling was specifically associated with tumor cell differentiation and metaplastic cells of developmental tissues. Notch signaling was involved in the differentiation of the ductal epithelial cells of salivary gland tumors and ameloblast-like cells of odontogenic tumors. However, Notch signaling was also involved in squamous metaplasia, irrespective of the type of developmental tissue. In odontogenic tumors, Notch signaling was involved in epithelial-mesenchymal interactions and may be related to tumor development and tumorigenesis. This signaling may also be associated with the malignant transformation of ameloblastomas. Overall, Notch signaling appears to play a major role in the formation of the characteristic cellular composition and histological features of oral neoplasms, and this involvement has been reviewed here.

Migration and Differentiation of GFP-transplanted Bone Marrow-derived Cells into Experimentally Induced Periodontal Polyp in Mice.

Perforation of floor of the dental pulp is often encountered during root canal treatment in routine clinical practice of dental caries. If perforation were large, granulation tissue would grow to form periodontal polyp. Granulation tissue consists of proliferating cells however their origin is not clear. It was shown that the cells in granulation tissue are mainly from migration of undifferentiated mesenchymal cells of the bone marrow. Hence, this study utilized GFP bone marrow transplantation mouse model. The floor of the pulp chamber in maxillary first molar was perforated using ½ dental round bur. Morphological assessment was carried out by micro CT and microscopy and GFP cell mechanism was further assessed by immunohistochemistry using double fluorescent staining with GFP-S100A4; GFP-Runx2 and GFP-CD31. Results of micro CT revealed alveolar bone resorption and widening of periodontal ligament. Histopathological examination showed proliferation of fibroblasts with some round cells and blood vessels in the granulation tissue. At 2 weeks, the outermost layer of the granulation tissue was lined by squamous cells with distinct intercellular bridges. At 4 weeks, the granulation tissue became larger than the perforation and the outermost layer was lined by relatively typical stratified squamous epithelium. Double immunofluorescent staining of GFP and Runx2 revealed that both proteins were expressed in spindle-shaped cells. Double immunofluorescent staining of GFP and CD31 revealed that both proteins were expressed in vascular endothelial cells in morphologically distinct vessels. The results suggest that fibroblasts, periodontal ligament fibroblasts and blood vessels in granulation tissue were derived from transplanted-bone marrow cells. Thus, essential growth of granulation tissue in periodontal polyp was caused by the migration of undifferentiated mesenchymal cells derived from bone marrow, which differentiated into fibroblasts and later on differentiated into other cells in response to injury.

Functional Role of HSP47 in the Periodontal Ligament Subjected to Occlusal Overload in Mice.

We carried out an experiment to induce traumatic occlusion in mice periodontal tissue and analyzed the expression of HSP47. Continuous traumatic occlusion resulted to damage and remodeling of periodontal ligament as well as increase in osteoclasts and bone resorption. Four days after traumatic occlusion, osteoclasts did not increase but Howship's lacunae became enlarged. That is, the persistent occlusal overload can destroy collagen fibers in the periodontal ligament. This was evident by the increased in HSP47 expression with the occlusal overload. HSP47 is maintained in fibroblasts for repair of damaged collagen fibers. On the other hand, osteoclasts continue to increase although the load was released. The osteoclasts that appeared on the alveolar bone surface were likely due to sustained activity. The increase in osteoclasts was estimated to occur after load application at day 4. HSP47 continued to increase until day 6 in experiment 2 but then reduced at day 10. Therefore, HSP47 appears after a period of certain activities to repair damaged collagen fibers, and the activity was returned to a state of equilibrium at day 30 with significantly diminished expression. Thus, the results suggest that HSP47 is actively involved in homeostasis of periodontal tissue subjected to occlusal overload.

Histological Evaluation of Periodontal Ligament in Response to Orthodontic Mechanical Stress in Mice.

The purpose of the study was to determine the cell dynamics in periodontal ligament in response to mechanical stress during orthodontic movement. Following Waldo's method, a square sheet of rubber dam was inserted in between the first and second maxillary molars in 10 ddY mice leaving the stress load for 3 hours. After 3 days and at 1 week, cell count on pressure and tension sides of the periodontal ligament was determined. Furthermore, the type of cell present after mechanical stress was identified using GFP bone marrow transplantation mouse model. Immunohistochemistry was carried out at 0 min (immediately after mechanical stress), 24 hours, 1 week, 2 weeks and 6 months. Temporal changes in the expression of GFP-positive bone marrow derived cells were examined. Moreover, double immunofluorescent staining was performed to determine the type of cell in the periodontal ligament. Cell count on the tension side tremendously increased 3 days after mechanical stress. At 1 week, spindle and round cell count increased compared to the control group. These changes were observed on both tension and pressure sides. Cell count on pressure side at 3 days (22.11+/-13.98) and at 1 week (33.23+/-11.39) was higher compared to the control group (15.26+/-8.29). On the tension side, there was a significantly increased at 3 days (35.46+/-11.85), but decreased at 1 week (29.23+/-13.89) although it is still higher compared to the control group (AD+/-SD: 10.37+/-8.69). Using GFP bone marrow transplantation mouse model, GFP positive cell count increased gradually over time in 6 months. GFP positive cells were also positive to CD31, CD68 and Runx2 suggesting that fibroblasts differentiated into osteoclasts and tissue macrophages. In conclusion, mechanical stress during orthodontic movement promoted the increase in the number of cells in the periodontal ligament on both tension and pressure sides. The increase in the number of cells in the periodontal ligament is believed to be due to the migration and cell division of undifferentiated mesenchymal cells.

Cytological Kinetics of Periodontal Ligament in an Experimental Occlusal Trauma Model.

Using a model of experimental occlusal trauma in mice, we investigated cytological kinetics of periodontal ligament by means of histopathological, immunohistochemical, and photographical analysis methods. Periodontal ligament cells at furcation areas of molar teeth in the experimental group on day 4 showed a proliferation tendency of periodontal ligament cells. The cells with a round-shaped nucleus deeply stained the hematoxylin and increased within the day 4 specimens. Ki67 positive nuclei showed a prominent increase in the group on days 4 and 7. Green Fluorescent Protein (GFP) positivity also revealed cell movement but was slightly slow compared to Ki67. It indicated that restoration of mechanism seemed conspicuous by osteoclasts and macrophages from bone-marrow-derived cells for the periodontal ligament at the furcation area. It was suggested that the remodeling of periodontal ligament with cell acceleration was evoked from the experiment for the group on day 4 and after day 7. Periodontal ligament at the furcation area of the molar teeth in this experimental model recovered using the cells in situ and the bone-marrow-derived cells.

Role of extracytoplasmic function sigma factors in biofilm formation of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis has been implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis biofilm formation in the subgingival crevice plays an important role in the ability of the bacteria to tolerate stress signals outside the cytoplasmic membrane. Some bacteria use a distinct subfamily of sigma factors to regulate their extracytoplasmic functions (the ECF subfamily). The objective of this study was to determine if P. gingivalis ECF sigma factors affect P. gingivalis biofilm formation. METHODS: To elucidate the role of ECF sigma factors in P. gingivalis, chromosomal mutants carrying a disruption of each ECF sigma factor-encoding gene were constructed. Bacterial growth curves were measured by determining the turbidity of bacterial cultures. The quantity of biofilm growing on plates was evaluated by crystal violet staining. RESULTS: Comparison of the growth curves of wild-type P. gingivalis strain 33277 and the ECF mutants indicated that the growth rate of the mutants was slightly lower than that of the wild-type strain. The PGN_0274- and PGN_1740-defective mutants had increased biofilm formation compared with the wild-type (p < 0.001); however, the other ECF sigma factor mutants or the complemented strains did not enhance biofilm formation. CONCLUSION: These results suggest that PGN_0274 and PGN_1740 play a key role in biofilm formation by P. gingivalis.

Promotion of transplanted bone marrow-derived cell migration into the periodontal tissues due to orthodontic mechanical stress.

BACKGROUND: Bone marrow-derived cells (BMCs) have abilities of cell migration and differentiation into tissues/organs in the body and related with the differentiation of teeth or periodontal tissue including fibroblasts. Then, we examined the effect of orthodontic mechanical stress to the transplanted BMC migration into periodontal tissues using BMC transplantation model. MATERIAL AND METHOD: BMC from green fluorescence protein (GFP) transgenic mice were transplanted into 8-week-old female C57BL/6 immunocompromised recipient mice, which had undergone 10 Gy of lethal whole-body-irradiation. Five mice as experimental group were received orthodontic mechanical stress using separator between first molar (M1) and second molar (M2) 1 time per week for 5 weeks and 5 mice as control group were not received mechanical stress. The maxilla with M1 and M2 was removed and was immunohistochemically analyzed using a Dako Envision + Kit-K4006 and a primary anti-GFP-polyclonal rabbit antibody. Immunohistochemically stained was defined as positive area and the pixel number of positive area in the periodontal tissue was compared with the previously calculated total pixel number of the periodontal tissue. RESULTS: The immunohistochemistry revealed that GFP positive cells were detected in the periodontal tissues, both in the experimental and control specimens. The ratio of pixel number in the examination group showed 5.77 ± 3.24 % (mean ± SD); and that in the control group, 0.71 ± 0.45 % (mean ± SD). The examination group was significantly greater than that of control group (Mann-Whitney U test: p<0.001). CONCLUSION: These results suggest that orthodontic mechanical stress accelerates transplanted BMC migration into periodontal tissues.

Heat shock protein27 expression and cell differentiation in ameloblastomas.

The expression of HSP27 and some CKs were examined the 40 cases of typical solid/multicystic ameloblastoma using immunohistochemical techniques. In order to examine the relevance of HSP in cell differentiation, we focused on the cytoskeletal expression of CK. CK19 is a marker of typical odontogenic epithelium widely observed in follicular and plexiform types of ameloblastomas. Since staining with CK14 is one of the measures of the differentiation potential of squamous cells and is extensively expressed in both follicular and plexiform types, it implies that squamous differentiation of each type can occur. CK8 was strongly detected in tumor nests in plexiform type but weakly detected in follicular type. It was considered that the expression of HSP27 in plexiform type correlated with the expression of CK8 suggesting that HSP27 might have regulated the expression of CK8.

Differential expression of canonical and non-canonical Wnt ligands in ameloblastoma.

BACKGROUND: Canonical and non-canonical Wnt signaling pathways modulate diverse cellular processes during embryogenesis and post-natally. Their deregulations have been implicated in cancer development and progression. Wnt signaling is essential for odontogenesis. The ameloblastoma is an odontogenic epithelial neoplasm of enamel organ origin. Altered expressions of Wnts-1, -2, -5a, and -10a are detected in this tumor. The activity of other Wnt members remains unclarified. MATERIALS AND METHODS: Canonical (Wnts-1, -2, -3, -8a, -8b, -10a, and -10b), non-canonical (Wnts-4, -5a, -5b, -6, 7a, -7b, and -11), and indeterminate groups (Wnts-2b and -9b) were examined immunohistochemically in 72 cases of ameloblastoma (19 unicystic [UA], 35 solid/multicystic [SMA], eight desmoplastic [DA], and 10 recurrent [RA]). RESULTS: Canonical Wnt proteins (except Wnt-10b) were heterogeneously expressed in ameloblastoma. Their distribution patterns were distinctive with some overlap. Protein localization was mainly membranous and/or cytoplasmic. Overexpression of Wnt-1 in most subsets (UA = 19/19; SMA = 35/35; DA = 5/8; RA = 7/10) (P < 0.05), Wnt-3 in granular cell variant (n = 3/3), and Wnt-8b in DA (n = 8/8) was key observations. Wnts-8a and -10a demonstrated enhanced expression in tumoral buddings and acanthomatous areas. Non-canonical and indeterminate Wnts were absent except for limited Wnt-7b immunoreactivity in UA (n = 1/19) and SMA (n = 1/35). Stromal components expressed variable Wnt positivity. CONCLUSION: Differential expression of Wnt ligands in different ameloblastoma subtypes suggests that the canonical and non-canonical Wnt pathways are selectively activated or repressed depending on the tumor cell differentiation status. Canonical Wnt pathway is most likely the main transduction pathway while Wnt-1 might be the key signaling molecule involved in ameloblastoma tumorigenesis.

Heat Shock Protein Overexpression-Mediated Periodontal Ligament Regeneration: A Fundamental Approach to Generate a Potential Biomaterial.

The periodontal ligament (PDL) is a cell-rich fibrous connective tissue supporting the tooth roots. The tissue helps to maintain homeostasis and exhibits regenerative and repairing ability, which is mediated by the heat shock protein (HSP). Here, we experimentally created PDL tissue with notable ability to regenerate hard tissue and evaluated it as a potential biomaterial. We immunohistochemically examined the mechanical load-induced HSP overexpression in mouse PDL. Following mechanical load application and release, HSP70 localization in the PDL was altered immediately, suggesting that the HSP70 function may differ with the timing of its expression in PDL. HSP70 expressed in the cytoplasm and nucleus of fibroblasts in PDL on the tension side not only participated in periodontium repair, but also functioned as a molecular chaperone during protein expression involved in osteogenesis to restructure injured tissue. This study highlights the potential of artificially created highly functional PDL tissues as biomaterials.

Comparative examination of subcutaneous tissue reaction to high molecular materials in medical use.

Polypropylene (PP), Polyethylene (PE) and polytetrafluoroethylene (FE) are high molecular materials in medical use. They are also used as the negative control materials for ISO 10993-6 international standard biological evaluation of medical devices. We examined tissue reactions to these materials embedded subcutaneously in the dorsal area of male ddY mice. One week and 12 weeks after embedding, the tissue surrounding the embedding site was removed and then histopathological examination was performed. Our results demonstrate that the basic histopathological reaction is the formation of fibrous capsule consisting of granulation tissue around the embedded materials. Based on our results, we believe that the high molecular materials such as, PP, PE and FE, can be considered for medical use as a biomaterials within the body.

Immunohistochemical expression of heat shock protein27 in the mouse dental pulp after immediate teeth separation.

AIM: After immediate teeth separation, expression of HSP27 in the mouse dental pulp was examined. Immunohistochemistry was performed to examine the incidence of HSP27 expression. MATERIALS AND METHODS: A total of 36 8-week-old ddY mice were used as experimental subjects and a wedge was inserted in between maxillary right molars. The wedge was removed 30 min or 3 h after insertion. Animals were immediately sacrificed after the removal of wedge or until 1 week later and serial sections from paraffin-embedded tissues were prepared. Immunohistochemistry was carried out to examine the expression of HSP27. The untreated side served as the control. RESULTS: In the control group, the endothelial cells and some pulp fibroblasts weakly expressed HSP27 suggesting that the expression is due to mechanical stress brought about by physiological masticatory force and pressure from the tongue. In both 30 min and 3 h experimental groups, HSP27 expression was highest at 24 h after wedge removal and the expression remained the same or started to decrease thereafter. The expression decreased at the same level as that of the control group 1 week after wedge removal. CONCLUSION: HSP27 may serve as an indicator of stimulus strong enough to show its expression.

Notch signaling and ghost cell fate in the calcifying cystic odontogenic tumor.

Notch signaling is an evolutionarily conserved mechanism that enables adjacent cells to adopt different fates. Ghost cells (GCs) are anucleate cells with homogeneous pale eosinophilic cytoplasm and very pale to clear central areas (previous nucleus sites). Although GCs are present in a variety of odontogenic lesions notably the calcifying cystic odontogenic tumor (CCOT), their nature and process of formation remains elusive. The aim of this study was to investigate the role of Notch signaling in the cell fate specification of GCs in CCOT. Immunohistochemical staining for four Notch receptors (Notch1, Notch2, Notch3 and Notch4) and three ligands (Jagged1, Jagged2 and Delta1) was performed on archival tissues of five CCOT cases. Level of positivity was quantified as negative (0), mild (+), moderate (2+) and strong (3+). Results revealed that GCs demonstrated overexpression for Notch1 and Jagged1 suggesting that Notch1-Jagged1 signaling might serve as the main transduction mechanism in cell fate decision for GCs in CCOT. Protein localizations were largely membranous and/or cytoplasmic. Mineralized GCs also stained positive implicating that the calcification process might be associated with upregulation of these molecules. The other Notch receptors and ligands were weak to absent in GCs and tumoral epithelium. Stromal endothelium and fibroblasts were stained variably positive.

Immunohistochemical expression of hard tissue related factors in the mouse dental pulp after immediate teeth separation.

We examined change of Runx2 and ALP expression in mouse tooth pulp which exposed to teeth separation experiment by immunohistochemistry as a model for conservative dentistry treatment. 8-week-old 36 male ddY mice were used and wedge was inserted between upper 1st and 2nd molars. The wedge was removed 30 minutes as well as 3 hours after the insertion and the samples were prepared extending up to 1 week of time period for regular histopathological and immunohistochemical examinations for ALP and Runx2 expression. The opposite sides without wedge insertion were taken as controls. In the control group pulp, weak expressions of Runx2 and ALP in the vessel endothelial cells as well as the pulp cells were revealed, suggesting the appearance of these genes upon mechanical stress induced by mastication and tongue pressure etc. On the other hand in the experiment group, Runx2 expression increased both in 30-minute and 3-hour teeth separation group. The expression became maximum at 24 hours. Then it gradually decreased and became similar level with the control group at 1-week after the wedge insertion. Similarly ALP expression increased after the wedge insertion and was maximum at 24 hours and then gradually decreased to the levels similar with the control group. These results suggest that when immunohistochemical expression of Runx2 as well as ALP was used as an index, no severe damage occur upon clinical application of wedge insertion.

Strawberry gingivitis as the first presenting sign of Wegener's granulomatosis: report of a case.

Wegener's granulomatosis is a rare multi-system disease characterized by the classic triad of necrotizing granulomas affecting the upper and lower respiratory tracts, disseminated vasculitis and glomerulonephritis. Oral lesions as a presenting feature are only encountered in 2% of these cases. Hyperplastic gingival lesions or strawberry gingivitis, is a characteristic sign of Wegener's granulomatosis. The latter consists of reddish-purple exophytic gingival swellings with petechial haemorrhages thus resembling strawberries. Recognition of this feature is of utmost importance for timely diagnosis and definitive management of this potentially fatal disease. A case of strawberry gingivitis as the first presenting sign of Wegener's granulomatosis affecting a 50-year-old Malay male is reported here. The differential diagnosis of red lesions that may present in the gingiva is discussed.

Differential expression of Notch receptors and their ligands in desmoplastic ameloblastoma.

BACKGROUND: In mammals, the Notch gene family encodes four receptors (Notch1-4), and all of them are important for cell fate decisions. Notch signaling pathway plays an essential role in tooth development. The ameloblastoma, a benign odontogenic epithelial neoplasm, histologically recapitulates the enamel organ at bell stage. Notch has been detected in the plexiform and follicular ameloblastoma. Its activity in the desmoplastic ameloblastoma is unknown. METHOD: Notch1-4 and their ligands (Jagged1, Jagged2 and Delta1) were examined immunohistochemically in 10 cases of desmoplastic ameloblastoma. RESULTS: Ameloblastoma tumor epithelium demonstrated positive expression for Notch1 (n = 5/10), Notch3 (n = 8/10), Notch4 (n = 10/10), Jagged1 (n = 6/10) and Delta1 (n = 5/10), but no reactivity for Notch2 (n = 10/10) and Jagged2 (10/10). Expression patterns were distinct with some overlap. Positive activity was detected largely in the cell membrane and cytoplasm of peripheral and central neoplastic epithelial cells, and sometimes in the nucleus. Staining score was highest for Notch4. Stromal components namely endothelial cells and fibroblasts showed overexpression for Notch4 but were mildly or non-reactive for the other Notch members and their ligands. CONCLUSIONS: These findings suggest that Notch receptors and their ligands may play differing roles during the development of the desmoplastic ameloblastoma with Notch4 probably playing a greater role in the acquisition of tissue-specific cellular characteristics in the desmoplastic ameloblastoma.

Cell culture in vivo by means of diffusion chamber system.

In a diffusion chamber (DC) system, cells are cultured in vivo - hence making it possible to minimize infection and foreign material contamination. In view of this merit, we devised a technique to combine a DC system and a scaffold to the end of incubating sufficient host cells for grafting. In the present study, PLGA sponge and rat bone marrow cells were encapsulated inside a DC and then placed inside the abdominal cavities of rats. DCs were removed at two or four weeks after grafting. At four weeks after grafting, fibrous and calcified tissue matching the shape of the PLGA sponge was formed. These results suggested that the PLGA sponge was an effective scaffolding material in inducing three-dimensional tissue formation and that combination with a DC system resulted in a cell mass matching the scaffold shape. In addition, the cells were cultured in vivo - which meant that DC culturing did not require special incubation facilities or technologies after grafting.

Gene expression of Jagged2 in mandibular condylar cartilage development.

Expression pattern of Jagged2 gene in mandibular condylar cartilage was examined by means of in situ hybridization (ISH) technique. At E14, Jagged2 mRNA signals appeared in cytoplasm of proliferating chondrocytes. From E15 to E19, Jagged2 mRNA was detected throughout almost all cytoplasm in all layers. However, the distribution pattern was not uniform. These results suggest that Jagged2 plays an essential role for mandibular condylar cartilage morphogenesis and development.

Immunohistochemical characteristics of developing mandibular angle in fetal mice.

Mouse mandibular angle development started as a coagulation of mesemchymal cells on the 15(th) fetal day. On the 16(th )fetal day, cells of the central portion of the cell coagulation showed metachromasia to toluidine blue, and type 2 collagen positive chondrocytes were immunohistochemically detected. After the 17(th) fetal day, cartilaginous osteogenesis occurred with invasion of capillaries. At the same stage, membranous (perichondral) ossification occurred in the periphery of the chondrocyte mass. These proliferating chondrocytes showed positive reactions to type 2 collagen, type 1 collagen and osteopontin. These results suggest that the characteristics of mandibular angular cartilage are slightly different from those of normal physiological articular cartilage.

Expression of Notch1 and Math1 in mandibular condyle cartilage in neonatal mice.

On the basis of the cellular morphological changes in the cartilaginous area, the mandibular condylar cartilage is histopathologically composed of four different cell layers--fibrous, proliferative, maturative, and hypertrophic. Reaction for Notch1 was present in the hypertrophic cells only. However, Math1 was locally distributed in the hypertrophic layer and partially in the proliferative layer. The expression patterns of Notch1 and Math1 were slightly different. These results suggest that the morphogenesis regulation factors of Notch1 and Math1 may play some role in mandibular condylar cartilage. Positive reactions to osteopontin, as a control, were detected in the cytoplasm of all layers, although they varied from published data.