Contribution of mesenchymal proliferation in tooth root morphogenesis

Abstract

In mouse tooth development, the roots of the first lower molar develop after crown formation to form 2 cylindrical roots by post-natal day 5. This study compared the morphogenesis and cellular events of the mesial-root-forming (MRF) and bifurcation-forming (BF) regions, located in the mesial and center of the first lower molar, to better define the developmental mechanisms involved in multi-rooted tooth formation. We found that the mesenchyme in the MRF showed relatively higher proliferation than the bifurcation region. This suggested that spatially regulated mesenchymal proliferation is required for creating cylindrical root structure. The mechanism may involve the mesenchyme forming a physical barrier to epithelial invagination of Hertwig's epithelial root sheath. To test these ideas, we cultured roots in the presence of pharmacological inhibitors of microtubule and actin polymerization, nocodazole and cytochalasin-D. Cytochalasin D also inhibits proliferation in epithelium and mesenchyme. Both drugs resulted in altered morphological changes in the tooth root structures. In particular, the nocodazole- and cytochalasin-D-treated specimens showed a loss of root diameter and formation of a single-root, respectively. Immunolocalization and three-dimensional reconstruction results confirmed these mesenchymal cellular events, with higher proliferation in MRF in multi-rooted tooth formation.

Keywords: cell proliferation; histochemistry; molar; odontogenesis; tooth development; tooth root.

Figure 1.

Morphogenesis of mouse lower molar tooth root. (A-F) The tooth root and BF regions were designated and examined by HE staining at PN3, PN5, and PN8. The boxed area in (B) demarcates the condensed mesenchymal cells. (A′-F′) Pan-CK-positively stained epithelial cells, including HERS, were visualized at higher magnifications (A′′-F′′). (G) The distances between the end parts of the buccolingual pan-CK-positive epithelial cells were measured (n = 10). MRF, mesial-root-forming region; BF, bifurcation-forming region; mc, mesenchymal condensation. Scale bars: 100 μm (A, A′, B, B′, C, C′, D, D′, E, E′, F, F′), 50 μm (A′′, B′′, C′′, D′′, E′′, F′′).

Figure 2.

Localization patterns of Ki67, a cell proliferation marker, during tooth root development. (A-H) Ki67-positive cells were detected in the dental papillae and HERS in a specific spatiotemporal manner. (A, E) Mesenchymal cells with Ki67-positive staining, located between the HERS, are observed mainly in the MRF region at PN3 and PN5. (I) Ratio of Ki67-positive cell numbers to the whole-cell numbers were examined in the 100-μm² areas (n = 10). MRF, mesial-root-forming region; BF, bifurcation-forming region. Scale bars, 50 μm (A-H).

Figure 3.

Treatments with the pharmacological inhibitors during in vitro cultivations at PN3 for 4 days. (A-F) Ki67 and (G-L) PCNA immunostaining after 4 days’ cultivation at PN3. Arrows indicate the bulbous ends of HERS in the CD-treated specimens. All scale bars are 50 μm.

Figure 4.

Morphogenesis of in vitro cultivated lower molar tooth. (A, B) DMSO control specimens show morphogenesis similar to that of normal tooth root development. (D, E) The nocodazole-treated specimens show a narrow distance between both sides of the HERS tissues in the MRF (single asterisk) but a normal gap in the BF (double asterisk) regions. The arrowheads indicate HERS discontinuities. (G, H) After cytochalasin-D treatment, the histology and localization patterns of pan-CK-positive epithelial cells showed much wider gaps between the end margins of both HERS tissues with the aggregated structures in MRF and BF regions. (C, F, I) Three-dimensional reconstruction after the immunostaining against pan-CKs. (J) The distances between the HERS were measured. (K) Summary and schematic diagram of tooth root and bifurcation morphogenesis. (C, F, I) The epithelial tissues in the tooth root, which show positive staining of the pan-CKs, were reconstructed. Red indicates the dental papillae facing the epithelium. All scale bars are 100 μm.