Iron deposition and ferritin heavy chain (Fth) localization in rodent teeth

Abstract

Background: An iron rich layer on the labial surface is characteristic of the enamel of rodent incisors. In order to address a role for iron content in continuously growing incisors during odontogenesis, we studied iron deposition patterns in enamel and dentine using Perls' blue staining and ferritin heavy chain (Fth) immunolocalization. Fth expression is regulated by iron level; therefore its localization can be used as a sensitive indicator for iron deposition.

Results: Sagittal sections of 4-week old rat incisors showed a gradual increase in iron level in the enamel organ from secretory to maturation stages. In addition, iron was detected in ameloblasts of erupting third molars of 4-week old rats, suggesting iron plays a role in both incisor and molar development. In odontoblasts, the presence of iron was demonstrated, and this is consistent with iron's role in collagen synthesis. Using postnatal 3-, 6-, 9-day old mice, the spatial and temporal expression of Fth in tooth development again indicated the presence of iron in mature ameloblasts and odontoblasts.

Conclusions: While these data do not explain what functional role iron has in tooth formation, it does highlight a significant molecular activity associated with the formation of the rodent dentition.

Figure 1

Perls’ blue staining of rat maxillary incisor. Sagittal section of 4-week old rat maxillary incisor was stained with Perls’ Prussian blue for detection of iron and counterstained with nuclear fast red. Panel A: Whole incisor observed under stereoscope with 12.5 x magnification. Panels B1 - B3: Boxed regions (1-3) in panel A are shown with 100x magnification. Panels C1, C2a - 2c, and C3a - 3d: Progression from secretory stage amelogenesis (C1) to late maturation stage amelogenesis (panel C3d) with 630x magnification. Enamel organ cells (ameloblasts, stratum intermedium, stellate reticulum and papillary layer cells) are labeled in panels C1 and C3d. The arrow in panel C3d shows a blood vessel surrounded by papillary layer cells. The following abbreviations are used: am, ameloblasts; si, stratum intermedium; sr, stellate reticulum; pl, papillary layer. Scale bars: A, 1 mm; B1-B3 and C1-C3d, 10 μm.

Figure 2

Immunohistochemical staining of Fth in maxillary incisor. Sagittal section of 4-week old rat maxillary incisor was stained with anti-Fth antibody and counterstained with hematoxylin. Panel A: Whole incisor observed under stereoscope with 12.5x magnification. Panels B1-B3: Boxed regions (1-3) shown in panel A are shown with 100x magnification. Panels C1, C2a - 2c and C3a - 3d: Progression from secretory stage amelogenesis (panel C1) through late maturation stage amelogenesis (panel C3d) with 630x magnification. Late stage ameloblasts and the overlying papillary layer cells are the most highly staining cells in this progression. Enamel organ cells (ameloblasts, stratum intermedium, stellate reticulum and papillary layer cells) are labeled in panels C1 and C3d. The arrow in panel C3d shows a blood vessel surrounded by papillary layer cells. The following abbreviations are used: am, ameloblasts; si, stratum intermedium; sr, stellate reticulum; pl, papillary layer. Scale bars: A, 1 mm; B1-B3 and C1-C3d,10 μm.

Figure 3

Iron detection in the rat maxillary third molar. Sagittal section of 4-week old rat maxillary molars were stained with DAB enhanced Perls’ blue for iron and counterstained with fast red. Panel A: Section of three molars observed under stereoscope with 16x magnification. Panel B: The third molar shown with 40x magnification. Panels C1 - C5: Regions 1 - 5 as identified in panel B are shown with 100x magnification. Panels D1 - D5: Ameloblasts and papillary layer cells, corresponding again to regions 1 - 5 as identified in panel B, are shown with 630x magnification. Ameloblasts (am) and papillary layer cells (pl) are labeled in panel D1. The arrows in D2 and D5 show iron deposits in papillary layer cells. Scale bars: A and B, 1 mm; C1-C5 and D1-D5, 10 μm.

Figure 4

IHC staining of Fth in rat maxillary third molar. Sagittal section of 4-week old rat maxillary molars stained with anti-Fth antibody and counterstained with hematoxylin. Panel A: Section of first, second and third molars observed under stereoscope with 16x magnification. Panel B: The third molar shown with 40x magnification. Panels C1 - C5: Regions identified as 1 - 5 in panel B are shown with 100x magnification. Panels D1 - D5: Ameloblasts and papillary layer cells, corresponding again to regions 1 - 5 indicated in panel B, are shown with 630x magnification. Ameloblasts (am) and papillary layer cells (pl) are labeled in panel D1. Scale bars: A and B, 1 mm; C1-C5 and D1-D5, 10 μm.

Figure 5

IHC staining of Fth in odontoblasts of rat maxillary incisor. Sagittal section of 4-week old rat maxillary incisor was stained with anti-Fth antibody and counterstained with hematoxylin. Panel A: Whole incisor observed under stereoscope with 12.5x magnification. Panels B1 - B6: Boxed regions (1 - 6) identified in panel A, spanning from outer enamel epithelia to odontoblasts, are shown with 100x magnification. Panels C1 - C6: Odontoblasts corresponding to the indicated regions 1 - 6 in panel A are shown with 630x magnification. Papillary layer cells (pl), ameloblasts (am), enamel (en), dentin (de), and odontoblasts (od) are labeled in panels B3 and C3. Scale bars: A, 1 mm; B1-B6 and C1-C6, 10 μm.

Figure 6

IHC staining of Fth in odontoblasts of rat maxillary third molar. Sagittal section of a 4-week old rat maxillary third molar stained with anti-Fth antibody and counterstained with hematoxylin. Panel A: the third molar observed under stereoscope with magnification 40x. Panels B1 - B6: Odontoblasts corresponding to indicated regions 1 - 6 identified in panel A are shown with 630x magnification. Ameloblasts (am), enamel (en), dentin (de), and odontoblasts (od) are labeled in panels A and B1. Scale bars: A, 1 mm; B1-B6, 10 μm.

Figure 7

IHC staining of Fth in mouse mandibular incisors. Sagittal section of postnatal (PN) 3, 6, and 9-day old mouse mandibular incisors were stained with anti-Fth antibody and counterstained with hematoxylin. Panel A: PN3 incisor observed with 100x magnification. Three pictures were overlapped to show the whole incisor. A1, box 1 in A shown with 630x magnification. Panel B: PN6 mandibular incisor observed with 100x magnification. Six pictures were overlapped to show the whole incisor. B1-B4, boxes 1-4 in B shown with 630x magnification. Panel C: PN9 mandibular incisor observed with 100x magnification. Seven pictures were overlapped to show the whole incisor. C1-C3, boxes 1-3 in C shown with 630x magnification. Papillary layer cells (pl), ameloblasts (am), enamel (en), dentin (de), and odontoblasts (od) are labeled. Scale bars: 50 μm.

Figure 8

IHC staining of Fth in mouse mandibular first molars. Sagittal section of postnatal (PN) 3, 6, and 9-day old mouse mandibular first molars were stained with anti-Fth antibody and counterstained with hematoxylin. Panel A: PN3 mandibular first molar observed with 100x magnification. Three pictures were overlapped to show the whole molar. A1, box 1 in A shown with 630x magnification. Panel B: PN-6 mandibular first molar observed with 100x magnification. Three pictures were overlapped to show the whole molar. B1, box 1 in B shown with 630x magnification. Panel C: PN-9 mandibular first molar observed with 100x magnification. Four pictures were overlapped to show the whole molar. C1 and C2, boxes 1 and 2 in C shown with 100x magnification. Ameloblasts (am), enamel (en), dentin (de), and odontoblasts (od) are labeled. Scale bars: 50 μm.