Naoto Osuga

Observations of pulpotomy in rats using in vivo Micro-CT —The changes after treatment of formocresol and calcium hydroxide pulpotomies or CO2 laser irradiation—

Abstract In general, pulpotomy is evaluated by pathological examination of sacrificed animals at each observing stage. Not only lots of small animals have to devote itself to experimentation, but also the result of research is lacking about the serial observation of an individual experimental animal. The aim of this study was to compare the relative effectiveness of the calcium hydroxide, formocresol and CO 2 laser using the R_mCT ® . Twenty male Wistar rats, 8 weeks of age, were used and imaged in present study. They were serially observed using the R_mCT ® on days 0, 3, 10, 30, 50 after pulpotomy, followed pathohistological examination. The R_mCT ® images revealed that the pulp capping agents were partly absorbed and a hard tissue-like was observed immediately beneath it, especially in the CO 2 laser group. Pathohistological examination showed a hard tissue-like appearance and inflammatory cell infiltration. The R_mCT ® images agreed with pathohistological finding on the days 50 after pulpotomy. Our research suggested that the R_mCT ® has made it possible to scan serial imaging of small animals such as experimental rats under anesthesia. Since its higher resolution enables clear observation of the entire hand of the rat, it is possible not only to compare the effectiveness of each pulpotomy method, but also to serially observe a single experimental animal during the process post-pulpotomy healing, growth, and aging. This study answered well for the animal protective law that to use the least of animal to got the best result.

Micro-CT observation of rat dental pulp healing after pulpotomy in in vivo study

Abstract We report the newly developed Micro-CT, which allows us to observe the individual animal over a long experimental period and to compare changes in pulp tissue in relation to growth and aging without considering individual differences. Further, we used pathological examination to prove similar the result observing from Micro-CT. We have examined wound healing of teeth after pulpotomy in rats, and could clearly observe histopathological changes in the affected teeth and the absorption of temporary filling material and pulp capping agents. In cases with breakage of the dental crown, the CT images agreed with the pathological observations, and it was possible to estimate the time of breakage. In vivo Micro-CT is possible to apply in continuous recording of small experimental animals, such as rats, under anesthesia and the result is sufficiently high. High-quality image was obtained in of the entire head region of the rat. It was suggested that this method can be used for long-term continuous observation of changes in the teeth conditions after pulpotomy in experimental animals. We report the newly developed Micro-CT, which allows us to observe the individual animal over a long experimental period and to compare changes in pulp tissue in relation to growth and aging without considering individual differences.

Pathological Analysis of Cell Differentiation in Cholesterol Granulomas Experimentally Induced in Mice.

In this study, cholesterin was implanted in the subcutaneous tissue in mice to induce the formation of cholesterol granuloma. Histological examination was carried out to determine the type and source of cells. The tissue surrounding the embedded cholesterin was examined histologically within the period of 6 months. Cell differentiation in cholesterol granulomas was investigated using ddY mice and GFP bone marrow transplanted mice. Cholesterin was embedded in mice subcutaneously and histopathological examination was carried out in a period of 6 months. Results showed that at 2 weeks, cholesterin was replaced partly by granulation tissues. The majority of cells in the granulation tissues were macrophages and foreign body giant cells and the center consists of small amount of fibroblasts, collagen fibers and capillaries. At 3 months, more granulation tissue was observed compared to 2 weeks. Similar cells were observed, however, there were more fibroblasts, collagen bundles and capillaries present compared to 2 weeks. At 6 months, the cholesterin was mostly substituted by fibrous tissues consisting mainly of fibroblasts and collagen fibers with some macrophages and foreign body giant cells. Specifically, the outer part of the tissue consists of fibroblasts, collagen bundles and capillaries and the inner portion is filled with collagen bundles. Immunohistochemistry revealed that macrophages and foreign body giant cells were positive to GFP and CD68 although the fibroblasts and capillaries in the outer portion of cholesterol granulomas were GFP negative. Some spindle shape fibroblasts were also GFP positive. Immunofluorescent double staining revealed that cells lining the blood vessels were both positive to GFP and CD31 indicating that those were endothelial cells and were actually derived from the transplanted bone marrow cells. The results suggest that macrophages, foreign body giant cells as well as fibroblasts and capillary endothelial cells are bone marrow derived mesenchymal cells.

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.

Application of an air-abrasive cutting apparatus in the pediatric dental field: Cutting using chitin-chitosan grains

Abstract Using an air-abrasive cutting apparatus, cavity preparation of dentin and artificially softened dentin was performed using alumina or chitinchitosan grains as cutting media. As a result of scanning electron microscopic observation, the following findings were obtained: 1.In cutting specimens that showed high values for Knoop hardness, alumina grains exhibited excellent cutting effects. 2.Regarding specimens after decalcification for each duration, the amount of cutting was significantly larger with alumina grains than with chitin-chitosan grains. 3.Regarding specimens cut using chitin-chitosan grains, the deepest cavity preparation was obtained in the specimens decalcified for 12hours. 4.Healthy dentin was effectively cut by the ejection of alumina grains, whereas artificially softened dentin was effectively cut by the selective use of chitin-chitosan grains. 5.Adhesion of a large amount of chitin-chitosan grains in the wet dentin specimens was observed. 6.The amount of chitin-chitosan grain adhesion was the largest in the wet dentin specimens decalcified 12 or 24 hours.