Migration and Differentiation of GFP–transplanted BoneMarrow–derived Cells into Experimentally InducedPeriodontal Polyp in Mice

松本歯科大学大学院歯学独立研究科博士（歯学）学位申請論文要旨 ; 大学院歯学独立研究科　硬組織疾患病態解析学講座（主指導教員：川上　敏行　教授

Pathological Examination of Experimentally Induced Periodontal Polyp in Mice

The mechanism in the formation of periodontal polyp has been established in several histological studies but details on cell differentiation and/or proliferation have not been elucidated. In the present study, we established a convenient and possible experimental system using ddY mice. Briefly, pentobarbital sodium (Somnopentyl) was injected into the abdominal cavity of the mouse followed by access cavity preparation on maxillary first molar using low speed ½ round bur (Merufa Inc), exposing the pulp and then allowed to perforate the floor of the pulp chamber. Observation was done over time until 6 months using micro CT (m_CT) image photography. Results with transmission image using m_CT showed theexpansion in the width of the periodontal ligament in the furcation area. The lesion was excised as one mass and examined histopathologically. The granulation tissue was covered with stratified squamous epithelium. The present experimental technique has been confirmed to be effective in analyzing the formation of periodontal polyp induced by mechanical perforation

Reactions to Bioabsorbable Suture Thread Embedded in Rat Subcutaneous Tissue

We examined the subcutaneous tissue reactions in rats to bioabsorbable suture thread using histopathological methods. Using Wister rats, Vicryl®, a bioabsorbable suture thread, was embedded into the subcutaneous tissue and histopathological examination was carried out after 4 weeks. Cholesterin crystals were used for the control. Furthermore, immunohistochemistry for CD68 was done. Histopathological examination showed proliferation of granulation tissues inboth experimental and control groups. The majority of cells in the granulation tissues were macrophages and giant cells. Fibroblasts were also observed in the proliferating granulation tissues surrounding the embedded bioabsorbable suturethread. Immunohistochemistry revealed that macrophages and giant cells were positive to CD68. The results suggest that the embedded bioabsorbable suture thread is not only fabricated to undergo absorption but also for phagocytosis bymacrophages and foreign body giant cells

Overview of Cytological Dynamics of Periodontal Ligament Inflammatory Lesions

Cyto-pathological features of the periodontal ligament tissue inflammatory lesions have somehow been carried out but detailed cellular dynamics remain unclear. Therefore, in this review, we overviewed mainly our recent experimental model studies. That is performed using using ordinary ddY mice and BMP bone marrow transplanted mouse model. Regaring the experimental apical inflammatory periodontitis, at four weeks, micro-CT confirmed the presence of a radiolucent image at the apex of the tooth, which was then removed for histological examination. The results showed granulation tissue with fibrosis gradually formed at the periphery of an abscess. Next, if perforation were large, granulation tissue would grow to form periodontal polyp. 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. 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

Histochemical Characteristics of Tertiary Dentin Due to Calcium Hydroxide Paste in Rats

Calcium hydroxide is mainly used for dental pulp capping and it is thought that it induces hard tissue formation far better than other materials. Experimentally verifying this fact, Nishikawa et al revealed that bone-like dentin corresponding to tertiary dentin is rapidly formed when calcium hydroxide is applied directly to the pulp. Utilizing the same experimental system, histochemical study of the newly formed hard tissue (reparative dentin) was carried out and the results thereof were reported. Thick and irregular reparative dentin was formed in the pulp cavity and partial narrowing of the root canal was observed in m-CT. Histopathologically, the irregular reparative dentin increased its thickness obliterating the root canal having a diff erent Azan staining of aniline blue compared to primary dentin. Numerous cellular inclusion bodies were also trapped inside the thick dentin. Furthermore, with Schmorl’s thionine picric acid staining,thick reparative dentin was noted around the pulp cavity and dentin. The dentin was densely stained with picric acid with diff erent staining ability from the surrounding dentin. In addition, it was clearly confi rmed that many cells were trapped in reparative dentin. The results of the experiment suggest that the characteristics of the newly formed reparative dentin is comparable to tertiary dentin

マウスにおける実験的根尖部炎症病変の確立

Abstract: Although studies on the formation of apical periodontitis have somehow been carried out but detailed cellular dynamics remain unclear. We recently established an experiment that could easily be performed using ddY mice. First, under general anesthesia using isoflurane inhalation, the coronal portion of the maxillary first molar was penetrated using a round bur and drill with water irrigation causing pulp exposure until the root apex. Micro computed tomography (R_mCT) was taken over time during observation. Four weeks later, R_mCT confirmed the presence of a radiolucent image at the apex of the tooth, which was then removed for histological examination. The results showed that granulation tissue with fibrosis had gradually formed at the periphery of the abscess. The present method confirmed the effectiveness of the experimental mode to exmine the formation of chronic inflammatory lesions at the root apex.根尖部炎症病変のマウスモデルを作製した。マウスをイソフラン吸入により麻酔し、上顎第1大臼歯の歯冠部をラウンドバーにより貫通させた後、水の灌注により穴をあけて根尖まで歯髄を露出させた。処置の4週間後、根尖部にX線透過性部位が存在することが、マイクロコンピュータートモグラフィーにより確認された。この部位について組織学的検査を行った結果、線維増生を示す肉芽組織が膿瘍の周辺に徐々に形成されることが明らかになった。以上より、本モデルは根尖部の慢性炎症病変の形成を調べる上で有用であることが示された

焼結ルチル二酸化チタン上の骨芽細胞様細胞の増殖およびアルカリホスファターゼ活性

The purpose of this study is creation of biomaterials from titanium dioxide (TiO2). This TiO2 has known for photocatalysis and osteogenesis. For the purpose of applying this function to orthodontic brackets and coating materials for implant, the relationship between surface of sintered and cell proliferation were examined. In addition, crystal structure and the surface property of sintering TiO2 were investigated. TiO2 were sintered at 1300°C for use as samples. We examined surface roughness, x-ray diffraction and scanning electron microscopy to make observations of the surface properties and texture. Moreover, mouse osteoblast-like cell line, MC3T3-E1 was cultured on sintered TiO2 in order to evaluate the cell proliferation and ALP. For the samples sintered at 1300°C, the crystalline phase of rutile-type TiO2 was confirmed.5000-fold magnified SEMimages of the surface of the unsintered samples, needle-like TiO2 crystals were pressure welded and showed mutual overlap, with pores occurring among the crystals. Sintering at 1300°C produced numerous small pores. Rutile TiO2 as a starting material was sintered at 1300°C and subjected to a cell culture experiment in which MC3T3-E1 cells were cultured on the sample, followed by viable cell counting and cell morphology observationon days 7, 14, 21, and 28 of culture. In the test of cell proliferation, sintered at 1300°C samples was found to remarkable cell proliferation even after time had passed. ALP activity of cells on 1300°C TiO2 sample, the values were 110% and 126% on days 14 and 28 of culture, respectively. These changes were calculated using polystyrene dish as the reference condition. Thus, TiO2 sintered at 1300°C showed good compatibility and increase in the ALP activity in MC3T3-E1 cells

Trends and Practical Issues in Collaboration between Communities and Schools: Focusing on School Management Councils and Community Schools

This paper aimed to summarize trends in educational policies, pedagogical research, and educational practices concerning communities and schools, and to clarify the practical matters of school-based community development, especially practical development and issues concerning collaboration between communities and schools, with a focus on school management councils and community schools. To this end, we examined trends in educational policies in terms of collaboration between communities and schools. Next, we explored trends in pedagogical research and educational practices from three perspectives (educational methods, educational administration, and social education), and their regional development and issues. Then, from the perspective of collaboration between different types of schools and between schools and local communities, we investigated discuss two local practices (Waki-Cho in Yamaguchi Prefecture and Niseko-Cho in Hokkaido) that promote advanced practices through the establishment of school management councils. Finally, based on the field research, we examined the significance of these practices and future topics

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

マウスにおける実験的に発生させた歯根膜ポリープへのGFP移植骨髄由来細胞の移動と分化

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.2016博士（歯学）松本歯科大