WAKAO Kohichi Nihon University, School of Dentistry, Department of Anatomy.Instructor, 歯学部, 助手 (60175057)
KUWATA Fumiyuki Nihon University, School of Dentistry, Department of Biochemistry.Assistant Prof, 歯学部, 講師 (60120440)
SHIMOKAWA Hitoyata Tokyo Medical and Dental University, Faculty of Dentistry, Department of Biochem, 歯学部, 助教授 (80014257)
|Budget Amount *help
¥6,500,000 (Direct Cost : ¥6,500,000)
Fiscal Year 1995 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1994 : ¥5,400,000 (Direct Cost : ¥5,400,000)
To clarify the effects of TGF-beta on tooth development, mouse molars were organ cultured in medium containing TGF-beta1 and then change in the morphology and gene expressions of TGF-beta and extracellular matrices were studied.
MATERIALS AND METHODS : To study the gene expression of TGF-beta in vivo, DDY mouce embryos from E13 to E18 and 0 to 7 days afeter birth were exmined. For in vitro experints, the first molar tooth germs of DDY mice were removed from embryos at E18 days of gestation. Utilizing a floating method, the culture medium consisted of alpha-MEM supplemented with 10% FBS and antibiotics under humidified conditions with 5% CO_2/50%O_2/45% air at 37ﾟC.TGF-beta1 (5ng/ml) was added to the same medium and cultured. As controls for the effects of TGF-beta, IL-1beta (1,000 pg/ml) and EGF (50 ng/ml) , were each added to respective cultures containing the same medium. Each material was examined by light and electron microscopy. Gene expressions of TGF-beta and extracellular matrix
was studies by in situ hybridization as described by Nomura (1985). The cDNA,bovine amelogenin 860 bp, alpha2R2 encoding at the C-terminal non-helical region (1011-1171) of rat alpha2 (I) collagen 900 bp (Genovese et al.1984) , Eco RI fragment of bovine osteonectin cDNA 324-bp and mouse TGF-beta1 cDNA of 1,600 bp were subcloned. Single-stranded ^<35>S-labeled sense and antisense RNA probes were generated, respectively.
RESULTS : In the normal culture conditions, dentin was formed 4 days after incubation and enamel was formed after 7 days. When a incubated in TGF-beta1, a thick layr of dentin was formed after 2 days, and enamel was secreted after 4 days of organ culture and 7 days after incubation, transitional stage-ameloblasts were found along the enamel surface. When incubated with IL-1beta, there was no effect on hard tissue formations but dental follicles had proliferated more than those in tooth germs cultured under normal conditions. When the incubated with EGF,cells of the enamel organ proliferated, but differentiation of ameloblasts was not obvious. In the culture incubated with TGF-beta, gene expression of TGF-beta was stronger than that in cultures incubated under normal conditions.
This result shows that TGF-beta may affect the differentiation of odontoblasts and ameloblasts, as well as the synthesis of extracellular matrix. Less