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Modulation of Epithelial Cell Fate of the Root in vitro

¹ Developmental Biology Program, Institute of Biotechnology, Viikki Biocenter, PO Box 56, FIN-00014, Helsinki, Finland; and
² Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata-cho 2–5–1, Okayama 700–8525, Japan

View larger version (37K): [in this window] [in a new window]   Figure 1. The tooth is an epithelial-mesenchymal organ going through distinct stages during early development. First the crown is formed (A). Only then will root formation begin. During the cap stage, the cervical loop is formed, which will give rise to either the adult epithelial stem cell niche, typical of continuously growing teeth (Aa), or to Hertwig’s epithelial root sheath (HERS), typical of root formation (Ab). After the crown is completed, root development starts by the formation of the HERS (B). The root elongates, and the HERS gives rise to the epithelial cell rests of Malassez (ERM) (B). Components of the periodontal ligament can invade the space between the cells of the ERM and reach the dentin surface. In the in vitro root, there are 3 distinct areas with different properties (C). In the perpendicular section, the epithelium making contact with the filter does not differentiate into ameloblasts, but resembles HERS epithelium, creating a continuous root sheath without fragmentation. Typically, there is a clear border between ameloblasts from the crown and the root epithelium from the root area. At the top of the culture, where the tissue is exposed to air, the epithelium is missing altogether. No odontoblast formation occurs here. In a section in the plane of the filter, the cervical loop structure characteristic of continuously growing teeth is maintained on both lateral aspects. Ameloblast and odontoblast differentiation occurs here in continuous cell layers from crown to root tip. Oee, outer enamel epithelium; iee, inner enamel epithelium; sr, stellate reticulum; ERM, epithelial cell rests of Malassez; HERS, Hertwig’s epithelial root sheath; PDL, periodontal ligament; ant, anterior; pos, posterior.

View larger version (98K): [in this window] [in a new window]   Figure 2. Advancing development of the in vitro root culture of an E17 tooth germ grown for 41 days, shown from a top view. See also the video sequence of the in vitro growth in the APPENDIX. Asterisks, cusps; arrows, root tips; arrowheads, border between crown and root; M1, first molar; M2, second molar; oe, oral epithelium. Scale bars are 200 µm.

View larger version (99K): [in this window] [in a new window]   Figure 3. Histology of in vitro-cultured molar after 3 wks in culture. Hematoxylin & eosin (HE) staining of a section perpendicular to the filter plane, showing a clear boundary between crown and root epithelium (A). The localization of keratin, an epithelial marker, confirms the border between elongated ameloblasts of the crown and cuboidal cells of the root epithelium (B). This panel also shows the lack of epithelium on the top of the root. HE staining of a section parallel to the plane of the filter (C), showing detail of the cervical loop area (D). The anterior and posterior aspects of the protrusion show continuous differentiation of ameloblasts, unlike the perpendicular section (A,B), and the cervical loop has a core of stellate reticulum (D). Incorporation of BrdU showing the epithelial proliferation in the cervical loop area concentrated in the inner enamel epithelium (E). Scale bars are 200 µm in A-C, 100 µm in D-E. Re, root epithelium; mes, mesenchyme; am, ameloblasts; od, odontoblasts.

View larger version (140K): [in this window] [in a new window]   Figure 4. Analysis of gene expression patterns in the cervical loop. Localization of Fgf10 in the mesenchyme near the cervical loop (A), Fgf3 expression is similarly located (arrows) (B), Bmp4 is expressed in the mesenchyme opposing differentiating ameloblasts (C), Notch1 is expressed in the stellate reticulum and stratum intermedium (arrows pointing to cervical loop) (D), Notch2 is similarly expressed (E), Lunatic fringe is expressed in outer enamel epithelium (arrow) (F), Jagged1 is expressed in differentiating ameloblasts (arrow) (G), Delta1 is expressed locally in differentiating odontoblasts (arrows) (H), Hes1 is expressed in stellate reticulum and stratum intermedium similar to Notch1 (arrow pointing to cervical loop) (I), Dspp is expressed in odontoblasts (arrow) mostly in the root and a subset of the ameloblast population (arrowhead) (J), and Bsp1 is expressed in odontoblasts of crown and root (K) in sections of four-week-old in vitro cultures of E16 tooth germs. L shows a schematic representation of the tooth cultures with the subdivision in the crown and root area and the location of the cervical loop. All scale bars are 200.µ.m.

Journal of Dental Research, Vol. 86, No. 11, 1063-1067 (2007)
DOI: 10.1177/154405910708601108


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