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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 Correspondence: * corresponding author, mark.tummers{at}helsinki.fi
Mouse molars are normally not capable of continuous growth. We hypothesized that the mouse molar has intrinsic potential to maintain the epithelial stem cell niche and assessed this potential by growth in vitro. Although the tooth germs flattened considerably, they developed a mineralized crown and a root. However, histologically, the root surface was composed of 3 structurally different regions affecting the fate of the dental epithelium. The anterior and posterior aspects maintained the morphological and molecular characteristics of the cervical loop of a continuously growing incisor, with a continuous layer of ameloblasts. The epithelium making contact with the supporting filter resembled Hertwig’ss epithelial root sheath. The top of the cultured molar exposed to air lacked epithelium altogether. We conclude that the fate of the epithelium is regulated by external cues influenced by culture conditions, and that the molar has the intrinsic capacity to grow continuously.
Key Words: in vitro • root development • molar • tooth
During tooth development, the crown area develops first, and only then does root formation begin. The formation of the root is a complex process, and the developmental mechanisms are not well-understood (Thomas, 1995; Nanci, 2003). One of the key features of root initiation is a structural change in the cervical loop. During crown formation, the epithelium of the cervical loop consists of a basal layer of epithelium that loops around a core of loosely aggregated stellate reticulum cells in the center (Fig. 1  ). The epithelium of the basal layer is known on the outside as outer enamel epithelium and on the inside as inner enamel epithelium. During root formation, the stellate reticulum is lost, and only the basal layer remains in the form of a double layer of epithelium. This structure is known as Hertwig’s epithelial root sheath (HERS), and generally no new ameloblasts will form from the epithelium after this structure is formed.
The HERS fragments during advancing root development and forms a fenestrated network of cells known as the epithelial cell rests of Malassez (ERM) (Nanci, 2003). This enables the surrounding mesenchymal follicular cells to penetrate the epithelial layer and reach the root surface, consisting of dentin. These follicular cells subsequently differentiate into cementoblasts and will form the cementum that covers the root surface. The entire structure between alveolar bone and tooth is known as the periodontal ligament, and consists of several cell types and matrix molecules (Diekwisch, 2001). In sharp contrast to molars that grow roots, the original structure of the cervical loop is maintained in continuously growing molars (Tummers and Thesleff, 2003) and continuously growing incisors (Harada et al., 1999). There is limited understanding of the mechanism of root elongation and the regulation of the cell fate in root epithelium. The root epithelium apparently has a limited growth capacity (Thomas, 1995). Disruption of Patched, the hedgehog receptor, led to shorter roots and the disturbance of eruption (Nakatomi et al., 2006). The roots of the transgenic mouse where the transcription factor NHI-C/CTF was knocked out failed to develop (Steele-Perkins et al., 2003). Data on cell-fate decisions of the root epithelium come mostly from the continuously growing rodent incisor, which is divided into 2 halves: the lingual side, representing a root analogue; and the labial side, representing a crown analogue. In the incisor, the ameloblast-inducing activity of BMP4, expressed in the adjacent dental mesenchyme, is inhibited by Follistatin expression in the dental epithelium on the lingual root analogue side (Wang et al., 2004). This causes the lingual side of the incisor to be enamel-free, while the labial side has enamel. Interestingly, Follistatin expression has been shown to be regulated by expression of Activin in the dental follicle (Wang et al., 2004). Hence, the interplay among 3 tissue layers—dental follicle, dental mesenchyme, and the intermediate epithelium—regulates the differentiation of the epithelium. Since molars are not normally capable of continuous growth, we cultured molars in vitro to test the intrinsic potential of the mouse molar to maintain the epithelial stem cell niche. Previously, a serum-free system was developed to study cementogenesis, where cap-stage teeth were grown for up to 31 days, and roots developed normally (Slavkin et al., 1989). We excised lower molars at the bell stage, when crown formation was near completion and root formation had not yet commenced, placed them in a Trowell-type in vitro culture system, and studied their development in vitro for 3 to 6 wks, looking at macroscopic development, histology, and expression of molecular markers, to track the fate of the cervical loop in this system.
The tissues were obtained from NMRI mice (Mus musculus), bred in the Transgenic Animals Unit (Helsinki, Finland). Animals were treated according to the standards established by the University of Helsinki. First molars (often with the second molar bud attached) were collected from mice at developmental stages E16 (n > 200), E17 (n > 50), and 2 days post-natal (n = 12). The tooth germs were dissected from the surrounding tissues under a stereomicroscope by means of disposable needles (Sahlberg et al., 2002). The surrounding tissues were removed except for some oral epithelium at the cusp side, which was removed after a week in culture. Each tooth germ was placed on a separate filter (polycarbonate Nuclepore® filters, pore size 0.1 m, Costar Nuclepore, Cambridge, MA, USA) on the lingual or buccal aspect. In some cultures, the filter was first covered with Matrigel (BD Biosciences, San Jose, CA, USA), which facilitated the growth of the tooth germs slightly, but was not essential in any way. The filters were placed on a metal grid under which culture medium was present (DMEM, Glutamax 10%, Fetal Calf Serum 10%, Penicillin Streptomycin, all from Invitrogen, Auckland, NZ, and 100 µg/mL ascorbic acid from Sigma-Aldrich, St. Louis, MO, USA). The molars were placed carefully on the filters. The medium was changed every 3 to 4 days. Tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 1 hr at room temperature, and decalcified in 2% paraformaldehyde and 12.5% EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 1 wk. After dehydration, they were embedded in paraffin and cut in 7-µm sections. Pictures of the cultures were taken before the medium was changed (Nikon SMZ-U stereomicroscope combined with the Olympus Dp11 digital camera). The pictures were processed with Adobe Photoshop.
Immunohistochemistry
Radioactive in situ Hybridization
Macroscopic Development Molar tooth germs from the bell stage of development (E16 and E17) were grown in vitro for 3–6 wks (Fig. 2 ; APPENDIX and video). Tooth germs collected from mice 2 days post-natally (n = 12) failed to show any root growth in culture (data not shown). All of the E16 (n > 200) and E17 (n > 50) stage tooth germs grew a structure from the mineralized crown that became apparent around 10 to 14 days after the start of the culture, resembling a root in the sense that it appeared distinct from the crown area under the stereomicroscope. This root-like structure will hereafter be referred to as the ‘in vitro root’ The mineralization of the crown was seen as a distinct darkening in the crown area as early as 1 wk after the start of the culture. Two distinct ‘root’ tips were initiated (Figs. 2, 3C). After 6 wks of in vitro culture, the growth of the root-like structure slowed, and the 2 separate root tips often merged. The tooth germ flattened considerably during culture, which is typical for culture of all tissues (Fig. 3A).
Microscopic Development Histological analysis of sections showed that there were 3 distinct areas in the in vitro root, and none of these represented the typical arrangement of cell layers covering the mouse molar root, i.e., HERS and its fragmentation to ERM (Fig. 1B  ). First, the epithelium making contact with the supporting filter consisted of differentiated elongated ameloblasts in the crown region, with a clear demarcation from a continuous layer of cuboidal epithelium of the root (Fig. 3A). Here, odontoblast differentiation was initiated normally, as seen by the elongated odontoblasts bordering the root epithelium. Immunostaining with an anti-keratin antibody confirmed the identity of epithelial cells (Fig. 3B). The fragmentation of the root epithelium, resulting in the formation of epithelial cell rests of Malassez, was absent, although sometimes a break in the epithelial layer was observed between the ameloblasts and root epithelium. This could be a histological artifact. Second, the epithelium was largely missing on the top surface of the root, which was exposed to air during culture (Figs. 3A, 3B). Without the epithelium, no odontoblast differentiation took place here. Third, sections in the plane of the filter showed epithelium lining the root surface on the anterior and posterior root surfaces. Interestingly, here, typical root epithelium was lacking; instead, the cervical loop structure was maintained with stellate reticulum cells in the core, surrounded by inner and outer enamel epithelium (Figs. 3C-3E). Away from the cervical loop, the epithelium differentiated into elongated ameloblasts typical of the crown region, resulting in a continuous ameloblast layer from the crown to the tip of the root.
Proliferation and Molecular Regulation
Dentin Sialophosphoprotein (Dspp) is normally expressed in odontoblasts and preameloblasts, and similar expression was seen in the three-week cultured tooth germ. There was high expression in the odontoblast layer and in a small subsection of the ameloblast cell lineage (Fig. 4J ). High Bone sialoprotein 1 (Bsp1) expression was found in the entire odontoblast layer (Fig. 4K). Bsp1 in the in vivo root is also expressed in the cementoblasts, but comparable expression was not seen in vitro, indicating an absence of cementoblasts. The epithelial markers were missing at the top of the cultured specimens, due to the absence of epithelium, and the epithelium in contact with the filter lacked the ameloblast differentiation markers Jagged1 and Dspp (data not shown).
Our results indicated that root extension in the in vitro root was accompanied by modulation of the epithelial cells’ fate. There were 3 histologically different areas in the in vitro root. The topside had no epithelium whatsoever, the bottom resembled root epithelium of the HERS (although without fragmentation into ERM), and the anterior and posterior aspects had maintained the cervical loop, with its typical structure of a layer of basal epithelium that loops around a compartment of more loosely aggregated stellate reticulum cells in the center. Molecular markers, such as the Notch and FGF signaling pathway genes and the epithelial proliferation, showed patterns identical to those in the cervical loop of the continuously growing molar of the vole (Tummers and Thesleff, 2003) and the mouse incisor (Harada et al., 1999). This indicated that the morphological and molecular phenotypes of the mouse molar epithelium on the anterior and posterior aspects resembled those of continuously growing teeth, and that the epithelium did not acquire the root fate (see also additional discussion in the APPENDIX). This was possibly caused by the mechanical compaction of epithelial and mesenchymal tissue maintaining regulatory signaling, or regulatory signaling was affected differently due to local conditions. We have previously shown that, in the continuously growing molar of the sibling vole, 3 small root domains are present, nested in the continuously growing crown domain, represented by cervical loops and a continuous ameloblast layer (Tummers and Thesleff, 2003). Similarly, in the continuously growing incisor, the lingual and labial halves of the tooth represent a root and crown analogue, respectively. This typical subdivision of continuously growing teeth in different functional domains in vivo also occurred in the mouse molar in vitro. The epithelial free zone at the top of the explant has no comparable analogy in mammalian teeth, and likely is a tissue culture artifact. The ultimate cause of this epithelial modulation is unknown, but it may be due to the regression of peripheral tissues, which is typical in tissues grown in the Trowell-type set-up at the gas-medium interface. The absence of the dental follicle may have contributed to the absence of epithelium. Although the histology of the epithelium of the in vitro root that formed in contact with the filter resembled HERS, it did not fragment into the epithelial cell rests of Malassez typical of the normal root surface (Nanci, 2003). The unfragmented HERS resembled the continuous root sheath of fishes, amphibians, and non-crocodilian reptiles (Luan et al., 2006). The formation of a periodontal ligament was not observed. Cementoblasts were absent, possibly because the continuous HERS prevented the contact between the dental follicle cells and the root surface, and/or because there was no or very little dental follicle. Odontoblast and ameloblast formation was present in the in vitro root, with the exception of the top surface of the explants, where odontoblasts were absent in the region lacking the dental epithelium and its inductive effect on the mesenchymal pre-odontoblasts. Our findings indicate that it is possible to turn a low-crowned or brachydont molar into a continuously growing or hypselodont molar under the right in vitro culture conditions, and that mouse molars have the intrinsic capacity to grow continuously, although in vivo this capacity is repressed. We conclude that local regulatory cues are responsible for the determination of continuous growth and root development, and that these normal cues are disrupted in the in vitro tooth, due to physiological properties of the culture environment, or removal of external signaling compartments.
This work was supported by the Academy of Finland and the Sigrid Juselius Foundation. We thank Merja Mäkinen and Riikka Santalahti for their technical assistance.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication March 20, 2007. Revision received July 10, 2007. Accepted for publication July 11, 2007.
Journal of Dental Research, Vol. 86, No. 11,
1063-1067 (2007)
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