So much anticipation from the side of need patients has been caused by recent advances in tissue engineering technology. However, it seems that effective results can be hardly achieved unless we establish the proper principles for this technology. In this review paper, therefore, we discuss these problems and indicators from the viewpoint of the biological principles of hard tissue regeneration, which are as follows: 1) principles of hard tissue reconstruction, 2) bio-functional nano-composites for regeneration of hard tissue, 3) industrialization of growth factors for hard tissue reconstruction, 4) history and problems of medical ceramic materials, and 5) dentin matrices as a new autograft material for osseous regeneration.

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We examined the changes of osteoclasts and bone structure in vivo and in vitro under the condition of low activity of bone resorption induced by acetazolamide (AZ). AZ is a specific inhibitor of carbonic anhydrase II (CA II), which is the enzyme indispensable for substantial proton generation by osteoclasts. In vivo study, we investigated histologically and histomorphometrically the changes of trabecular bone and osteoclasts at the cartilage-bone junction of tibiae in rats injected subcutaneously with AZ for 21 days. The area of trabecular bone of AZ treated group increased compared with control group. Moreover, AZ treatment decreased about 20% the number of osteoclasts at the cartilage-bone junction. In vitro study, we examined the effects of AZ on osteoclasts using culture system of rat bone marrow. The number of osteoclasts on the bone slice was suppressed by AZ of a concentration from 10^-5 to 10^-4 M in a doze-dependent manner. The number of osteoclasts cultured with 10^-4 M AZ for 24 hours and with or without parathyroid hormone (PTH) on the bone slice and on the glass showed a significant decrease compared with the control group. Some osteoclasts treated with AZ showed morphological changes of apoptosis-like cell death, including shrinking of cytoplasm and fragmentation of nuclei. These results show that high-doze of AZ decreases bone resorption not only by inhibiting CA II but also by decreasing the number of osteoclasts via induction of cell death. This suggests that CA II plays an important role not only in proton formation but also in osteoclast survival.

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To determine the mechanisms and patterns of cleft palate induced by varied retinoic acid (RA) in different developmental stages of secondary palate, we detected the cell proliferation and apoptosis of palatal shelves by BrdU and TUNEL labeling on gestation day (GD) 10 and GD12 which RA was exposed, respectively. In GD10 RA treated group, the percentage of BrdU positive cells of embryonic palatal mesenchyme were lower than control group, while TUNEL expression was higher than control group. No significant difference was detected in GD12 RA treated group and in control group either in embryonic palatal mesenchyme or medial edge epithelium by BrdU and TUNEL labeling. Our data indicated that the mechanisms of RA induced cleft palate in the early developmental stage were the inhibition of proliferation of palatal mesenchymal cells and medial edge epithelium cells, as well as excessive induction of apoptosis.

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In this research, we aimed to clarify change of old odontoblasts as well as mineralization level of formative disturbances line upon administration of antineoplastics. Male Wistar rats weighing 100 g were given injection of 2 mg/kg of Vinblastine (VBL). 1, 3, 5, 7, and 14 days after the administration, perfusion fixation was performed and upper incisors were removed. Then specimens with and without demineralization were embedded in epoxy resin and were observed by light microscopy, contact microradiography and transmission electron microscopy. At 1 day after the administration, necrosis of many old odontoblasts, abnormality of direction of collagen secretion and plenty of collagen fibers deposition were seen. Detection of this alteration at even 7 days after the administration suggested that disorder of secretion function associated with microtubule abnormality continues for a long period. The collagen secreted after VBL administration did not contribute increase of thickness and density of the predentin collagen formed before the drug injection and was deposited extremely dense along distal end of the cells. Thus the original predentin remained with low collagen density and a wide interfibrillar space. Regarding with mineralization process of formative disturbances line, density of collagen fibers was extremely low with thick needle shaped crystals in hypermineralization layer, and the density was oppositely very high with small crystals in hypomineralization layer. Although irregular dentin formed at medial side of formative disturbances line increases with time, mineralization degree is very low. The reason for this phenomenon was considered not only due to embedment of vessels and cellular particles but also observation of an unmineralized region around thick dentinal tubule, thus resulting in a decrease of general mineralization level.

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Bone formed around implant was characterized and grouped into three categories by Nakada et al.^1,2) and Suwa et al.³⁾ In order to clarify these bone tissues, we have utilized radiography with a newly developed wavelength tunable and highly parallel Parametric X-ray (PXR) in the Laboratory for Electron Beam Research and Application (LEBRA) of Nihon University. Methods: Ti-alloy implants modified with coating or blasting were used in this study. These materials were implanted in the tibia of rabbits. After 1, 2 and 4 weeks of implantation, non-demineralized polished thin sections were cut. PXR was generated by a LINAC in the LEBRA of Nihon University. X-ray wavelength was tuned from 7 KeV, 0.177 nm to 16 KeV, 0.0775 nm; irradiation: 900 sec; sample-detector distance: 7.5 mm and recording film: Fuji Film Imaging Plate. The obtained images were processed by NIH image analyzer. Results: PXR radiography showed the clear difference between the newly formed bone and the compact bone. Estimated percentage for the newly formed and pre-existing bone were 8.27 % and 20.9 %, respectively. Conclusion: LEBRA-PXR could be a good tool for the quantitative analysis of bone formation.

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Micro FT-Raman spectroscopy was applied to examine the contents in a glass ampoule non-destructively. Water, linger solution, ethanol and nail color remover liquid were examined. Glass was sufficiently thin enough to obtain Raman spectrum from the contents in the ampoule. All the examined materials showed the fine Raman profiles. The results indicated that micro-FT-Raman has a potential to examine and check the contents and impurities in the ampoule non-destructively.

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