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Inhibition of Apoptosis in Early Tooth Development Alters Tooth Shape and Size

Division in Anatomy and Developmental Biology, Department of Oral Biology, Research Center for Orofacial Hard Tissue Regeneration, Oral Science Research Center, College of Dentistry, Brain Korea 21 Project for Medical Science, Yonsei Center of Biotechnology, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul, 120-752, Korea; and
² Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Japan

View larger version (124K): [in a new window]   Figure 1. Morphological observations of the in vitro cultured teeth of control (A–C) and 100 µM z-VAD-fmk-treated (D,E) groups from 0 to 4 days after dissection under the dissecting microscope. Z-VAD-fmk-treated tooth germs were cultured in a medium containing z-VAD-fmk for 2 days and then cultured in the control medium. (F–H) Di.I. microinjections were performed in the mesenchymal parts of the buccal and lingual lateral sides of a tooth germ, so that we could understand the patterning for tooth formation in the buccolingual axis of the tooth germ. (A) In vitro cultured tooth germ at E13.5, at the onset of culture. (B) In vitro cultured tooth germ showing specific shape. The tooth germ was observed in the distal part of a cultured 1st molar tooth germ. (C) The distal region of the control cultured tooth germ includes the 2nd molar. (D) The size of the tooth germ was not altered after a two-day culture. (E) After 4 days, the shape of the tooth germ in the experimental group was larger than that of the control, especially the length of the mesiodistal diameter, although there were no significant alterations in buccolingual length. (F) At E13.5, the lateral sides of the tooth germ were labeled with Di.I. injection. (G) Di.I.-labeled cells had migrated along the mesiodistal axis and into the tooth germ. (H) After 96 hrs, Di.I.-positive cells were examined along the mesiodistal axis, and some of these cells were recruited into the tooth germ area. An asterisk indicates that the 2nd molar was not formed in the z-VAD-fmk-treated tooth germ after 96 hrs. Mes, mesial; Dis, distal; B, buccal; L, lingual; M1, first molar; M2, second molar. Scale bar: 200 µm.

View larger version (70K): [in a new window]   Figure 2. H&E staining (A,B), PCNA immunohistochemistry (C,D), and TUNEL staining (E,F) were observed after the in vitro tooth germs were cultured for 7 days from E13.5. Control group (A,C,E). The z-VAD-fmk-treated group was cultured for 2 days in medium containing the z-VAD-fmk, then post-cultured in the control medium for 5 days (B,D,F). To examine the formation of the calcified tooth, we cultured the tooth germ for 2 days using a modified Trowell’s culture method, then transplanted it into the mouse kidney capsule for 3 wks (G,H). (I) Occlusal view of (G). (J) Occlusal view of (H). (A) Tooth germ is composed of dental papilla (DP), inner dental epithelium (IDE), and outer dental epithelium. (B) The histological structures are similar to those of the control. (C,D) Blue arrows indicate PCNA-positive cells. Both the control and the z-VAD-fmk-treated tooth germs show a similar expression pattern of PCNA. PCNA-positive cells were localized in the inner dental epithelium and dental papilla. (E) Apoptotic bodies appeared with a broad localization pattern in the cultured tooth in the inner dental epithelium and the dental papilla of the control tooth. Red arrows indicate the apoptotic bodies. (F) Few apoptotic bodies were detected after 100-µM z-VAD-fmk treatment. Red arrows indicate the apoptotic bodies. (G) Six cusps were recognized in the control-cultured tooth. The whole shape of the calcified tooth was similar to a rectangular structure. (I) Occlusal view of (G). (H) Tooth shape of the 100-µM z-VAD-fmk-treated tooth germ was altered. The mesiodistal diameter became longer, and the crown height was significantly decreased compared with that of the control. (J) Occlusal view of (H). (I,J) The calcified teeth, both the control and z-VAD-fmk-treated, showed 6 cusps [(B1, B2, B3, and L1, L2, L3), according to the nomenclature of Gaunt (1955, 1961)]. DP, dental papilla; IDE, inner dental epithelium. White arrowheads indicate the mesial direction. Scale bars: A-F, 100 µm; G-J, 200 µm.

View larger version (30K): [in a new window]   Figure 3. Mesiodistal diameter and crown height of calcified teeth after treatment with various concentrations of z-VAD-fmk. This value is plotted (on a log scale) against the concentration of z-VAD-fmk treatment. (A) The mesiodistal diameter is directly proportional to the concentration of z-VAD-fmk. (B) The crown height is inversely related to the concentration of z-VAD-fmk. (C) The mesiodistal diameter and crown height of calcified teeth were measured under the microscope. Diameter, mesiodistal diameter; height, crown height. (D) Numbers of PCNA- and TUNEL-positive cells. There were no significant differences between the control and z-VAD-fmk-treated groups in PCNA. However, apoptosis was significantly reduced in the z-VAD-fmk-treated group. *P < 0.01.

View larger version (20K): [in a new window]   Figure 4. Schematic diagram of the putative mechanism of macrodontia. Disruption of apoptosis would result in macrodontia.