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The root apex of the tooth elongates until the completion of root development. Although the signaling molecules inducing root elongation have been studied, the characteristic of the cells having the ability to maintain the root elongation remains unclear. This study aimed to investigate the characteristics of the cells involved in the root elongation. Octamer-binding factor 3/4 (Oct3/4) is known as one of the key regulators in maintaining the pluripotency and self-renewal properties of embryonic stem cells. Bmi1, the polycomb-group transcriptional repressor, has emerged as a key regulator in several cellular processes including stem cell self-renewal and cancer cell proliferation. At the beginning of root formation, ameloblasts expressed Oct3/4 in the nucleus, except in the apex of the cervical loop, in which Bmi1and cyclinD were expressed. At PN6, the expression of Oct3/4 in the ameloblasts shifted from the nucleus to the cytoplasm, whereas ameloblastin-negative Hertwig's epithelial root sheath (HERS) cells expressed Bmi1 and cyclinD. By PN10, the cells in the apex of HERS began to express Oct3/4 in their nucleus, whereas Bmi1 and cyclinD began to decrease in their expressions. The odontoblasts consistently expressed Oct3/4 in their cytoplasm. Our results suggest that (1) Oct3/4 creates the border between the ameloblasts from the proliferative region of HERS, (2) Bmi1-positive cells would be one of the candidates resulting in root elongation and (3) the Oct3/4 expression in the cytoplasm of odontoblasts may be related to maintain the odontoblastic characteristics.

Although Hertwig's epithelial root sheath (HERS) performs an important function in the formation of the tooth root, the developmental mechanisms that control HERS growth and differentiation remain to be thoroughly elucidated. Bone morphogenetic protein 4 (BMP4), which is secreted by mesenchymal cells, acts on the dental epithelium as a regulator of cell differentiation during crown formation. In an effort to determine whether BMP4 specifically regulates the development of HERS in the dental epithelium, we assessed the localizations of BMP4, BMP receptor-IB (BMPR-IB), and BMPR-II during molar root formation in the mouse. HERS cells were shown to express BMPR-IB and BMPR-II. BMP4-positive cells were detected densely in the dental papillae around HERS, thereby suggesting that BMP4 participated in HERS formation. Beads soaked in BMP4, NOGGIN, or phosphate-buffered saline (PBS) were implanted into the pulp cavity under culture conditions, and the length of HERS was evaluated with regard to the proliferating cells. After 12 h, both groups exhibited a similar HERS developmental pattern, with the length and shape of HERS bearing a close resemblance to one another. However, after 48 h, the observed HERS elongation was significantly shorter in the BMP4-treated group. In addition, proliferative cell nuclear antigens were detectable only in the NOGGIN- and PBS-treated groups. These findings demonstrate that mesenchymally expressed BMP4 regulates HERS development by preventing elongation and maintaining cell proliferation. BMP4 may, therefore, prove useful as a root-formation regulatory agent in a variety of tissue-engineering applications.

To elucidate the mechanism of root formation in tooth development, we examined the role of insulin-like growth factor I (IGF-I) on early root formation in mandibular first molar teeth from 5-day-old mice. Immunohistochemistry revealed the specific localization of the IGF-I receptor in Hertwig's epithelial root sheath (HERS) in the tooth root. The effect of IGF-I on root development, especially on HERS, was subsequently examined in vitro. The control culture showed normal development of HERS and the periodontium, resembling that in vivo. However, the presence of 100 ng/ml IGF-I resulted in elongation of HERS and increased cell proliferation in its outer layer. These effects were negated by the addition of antibodies specific for IGF-I. Thus, we propose that IGF-I is involved in early root formation by regulating the mitotic activity in the outer layer of HERS.

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig's epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca(2+)-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca(2+)-binding proteins in the ERM has not so far been characterized. Among the three Ca(2+)-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca(2+) is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca(2+) is regulated only by calretinin. The expression of Ca(2+)-binding proteins is restricted in a developmental manner in the ERM.

The dental follicle (DF) surrounding the developing tooth germ is an ectomesenchymal tissue composed of various cell populations derived from the cranial neural crest. Human dental follicle cells (HDFC) are believed to contain precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Bone morphogenetic proteins (BMPs) produced by Hertwig’s epithelial root sheath or present in enamel matrix derivatives (EMD) seem to be involved in the control of DF cell differentiation, but their precise function remains largely unknown. We report the immunolocalization of STRO-1 (a marker of multipotential mesenchymal progenitor cells) and BMP receptors (BMPR) in DF in vivo. In culture, HDFC co-express STRO-1/BMPR and exhibit multilineage properties. Incubation with rhBMP-2 and rhBMP-7 or EMD for 24 h increases the expression of BMP-2 and BMP-7 by HDFC. Long-term stimulation of these cells by rhBMP-2 and/or rhBMP-7 or EMD significantly increases alkaline phosphatase activity (AP) and mineralization. Expression of cementum attachment protein (CAP) and cementum protein-23 (CP-23), two putative cementoblast markers, has been detected in EMD-stimulated whole DF and in cultured HDFC stimulated with EMD or BMP-2 and BMP-7. RhNoggin, a BMP antagonist, abolishes AP activity, mineralization, and CAP/CP-23 expression in HDFC cultures and the expression of BMP-2 and BMP-7 induced by EMD. Phosphorylation of Smad-1 and MAPK is stimulated by EMD or rhBMP-2. However, rhNoggin blocks only Smad-1 phosphorylation under these conditions. Thus, EMD may activate HDFC toward the cementoblastic phenotype, an effect mainly (but not exclusively) involving both exogenous and endogenous BMP-dependent pathways.

The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig's epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca(2+) concentration.