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Versican is a large chondroitin sulfate proteoglycan enriched in the extracellular matrix, and it has at least four different isoforms, termed V0, V1, V2, and V3. Although several studies have demonstrated that versican is stably expressed in various developing organs, the expression of versican isoforms during tooth development has not been elucidated yet. Therefore, the present study was to investigate the expression of versican isoforms in the developing mouse molars. The mandibular first molars from embryonic day (E) 11.5 to postnatal day (PN) 21 were used to investigate the expression of versican isoforms by immunohistochemistry, and the gene expressions of versican ( Vcan) isoforms from E13.5 to PN7 were analyzed by quantitative real-time PCR. The results exhibited different expressing patterns of versican isoforms—the stellate reticulum (SR) and the dental mesenchymal cells adjacent to Hertwig’s Epithelial Root Sheath (HERS) only expressed V1 and the mature odontoblasts mainly expressed V2, while the dental papilla and the ameloblasts might both express V0/V1/V2. These results suggested that different versican isoforms may act different roles in the tooth development, and we speculated that V0/V1 might be intimately involved in the cell proliferation while V2 was associated in the cytodifferentiation.

Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities, including hypodontia and supernumerary teeth, aberrant crown and root morphologies, and enamel defects, although the underlying mechanistic link is poorly understood. As most CLP genes are expressed throughout the oral epithelium, the authors hypothesized that the expression of CLP genes may persist in the dental epithelium and thus, in addition to their earlier role in labiopalatine development, may play an important functional role in subsequent tooth patterning and amelogenesis. To address this, the authors generated a unique conditional knockout model involving the major CLP gene, Irf6, that overcomes the previously reported perinatal lethality to enable assessment of any posteruption dental phenotypes. A dental epithelium–specific Irf6 conditional knockout ( Irf6-cKO) mouse was generated via a Pitx2-Cre driver line. Dental development was analyzed by microcomputed tomography, scanning electron microscopy, histology, immunohistochemistry, and quantitative polymerase chain reaction. Irf6-cKO mice displayed variable hypodontia, occasional supernumerary incisors and molars, as well as crown and root patterning anomalies, including peg-shaped first molars and taurodontic and C-shaped mandibular second molars. Enamel density was reduced in preeruption Irf6-cKO mice, and some shearing of enamel rods was noted in posteruption incisors. There was also rapid attrition of Irf6-cKO molars following eruption. Histologically, Irf6-cKO ameloblasts exhibited disturbances in adhesion and polarity, and delayed enamel formation was confirmed immunohistochemically. Altered structure of Hertwig’s epithelial root sheath was also observed. These data support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. This data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual.

The tooth root is an important part of the tooth that works together with the surrounding periodontium to maintain the tooth in the alveolar socket. The root develops after crown morphogenesis. While the molecular and cellular mechanisms of early tooth development and crown morphogenesis have been extensively studied, little is known about the molecular mechanisms controlling tooth root formation. Here, we show that β-catenin is strongly expressed in odontoblast-lineage cells and is required for root formation. Tissue-specific inactivation of β-catenin in developing odontoblasts produced molars lacking roots and aberrantly thin incisors. At the beginning of root formation in the mutant molars, the cervical loop epithelium extended apically to form Hertwig’s epithelial root sheath (HERS), but root odontoblast differentiation was disrupted and followed by the loss of some HERS inner layer cells. However, the outer layer of the HERS extended without the root, and the mutant molars finally erupted. The periodontal tissues extensively invaded the dental pulp. These results indicate that there is a cell-autonomous requirement for Wnt/β-catenin signaling in the dental mesenchyme for root formation.

Cementogenesis starts with the differentiation of cementoblasts. Mature cementoblasts secrete cementum matrix. Cementum components are similar to bone; moreover, cementoblasts possess many characteristics similar to those of osteoblasts. Runx2 and osterix, the transcriptional factors for osteoblast differentiation, participate in tooth formation. However, the characteristics of Runx2 and osterix during the differentiation process of cementoblasts remain unclear. In this study, we examined the immunolocalization patterns of Runx2, osterix, and osteopontin during rat molar tooth formation. Periodontal ligament cells and osteoblasts located on the alveolar bone surface showed immunoreactivity for Runx2. Colocalization of Runx2 and osterix was detected in cementoblasts, which penetrated the ruptured Hertwig's epithelial root sheath and attached to root dentin. Moreover, osteopontin was observed in Runx2-positive cementoblasts facing the root surface. However, the cells adjacent to cementoblasts showed only Runx2 reactivity. Neither Runx2 nor osterix was seen in cementocytes. These results suggest that both Runx2 and osterix are important for differentiation into cementoblasts. Additionally, osterix may be indispensable for transcription of osteopontin expression. (J Histochem Cytochem 57:397–403, 2009)

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.

Hertwig’s epithelial root sheath (HERS) cells are a unique population of epithelial cells in the periodontal ligament compartment. To date, their functional role has not been fully elucidated. Our hypothesis was that HERS cells may be involved in regulating differentiation of periodontal ligament stem cells (PDLSCs) and forming cementum in vivo. In this study, we found that HERS cells may be capable of promoting PDLSC differentiation and undergoing epithelial-mesenchymal transition in vitro. Immunohistochemical staining, Western blot analysis, a transwell co-culture system, and in vivo transplantation were used to characterize the interplay between HERS cells and PDLSCs, as well as the epithelial-mesenchymal transition (EMT) of HERS cells. TGFβ1 was capable of inducing the epithelial-mesenchymal transition of HERS cells through activating the PI3K/AKT pathway. Furthermore, HERS cells were able to form cementum-like tissue when transplanted into immunocompromised mice. Abbreviations: bone marrow mesenchymal stem cell, BMMSC; bone sialoprotein, BSP; hydroxyapatite/tricalcium phosphate, HA/TCP; Hertwig’s epithelial root sheath, HERS; osteocalcin, OCN; periodontal ligament, PDL; periodontal ligament stem cell, PDLSC; phosphatidylinositol 3-kinase, PI3K.

During cementogenesis, dental follicular cells penetrate the ruptured Hertwig's epithelial root sheath (HERS) and differentiate into cementoblasts. Mechanisms involved in basement membrane degradation during this process have not been clarified. Perlecan, a heparan sulfate (HS) proteoglycan, is a component of all basement membranes. Degradation of HS of perlecan by heparanase cleavage affects a variety of biological processes. We elucidated immunolocalization of perlecan and heparanase in developing murine molars to clarify their roles in cementoblast differentiation. At the initial stage of root formation, perlecan immunoreactivity was detected on the basement membrane of HERS. Weak heparanase immunoreactivity was detected in HERS cells. HERS showed intense staining for heparanase as root formation progressed. In contrast, labeling for perlecan disappeared from the basement membrane facing the dental follicle, and weak immunoreactivity for perlecan was detected on the inner side of the basement membrane of HERS. These findings suggest that perlecan removal is an important step for root and periodontal tissue formation. Heparanase secreted by the cells of HERS may contribute to root formation by degrading perlecan in the dental basement membrane.

Surgical endodontic therapy (apical surgery) is a treatment alternative aimed at removing periapical inflammatory tissue followed by apical resection and retro-filling of the root canal. These procedures are performed through a trans-osseous approach. Terminology pertinent to this article include: apical (periapical) curettage--a surgical procedure to remove diseased tissue from the alveolar bone in the apical region of a pulpless tooth; apical cyst--a cyst in bone at the apex of a pulpless tooth. It is believed that such cysts arise after the death of the pulp from noxious physical, chemical, or bacterial stimulation of epithelial rests of Malassez; apicoectomy (apical resection) amputation of the apical portion of the root and removal of soft tissue in the bone; epithelial rests of Malassez--cords, strands, or clusters of ectodermal cells in the periodontal ligament (or sometimes alveolar bone) derived from remnants of Hertwigs epithelial root sheath. These cells frequently begin proliferating when inflammation occurs in the periodontal ligament and are believed to be responsible for the genesis of the epithelial lining of apical cysts.

Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig’s epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed.

There is increasing evidence that the cells of the epithelial root sheath synthesize enamel matrix proteins and that these proteins play a fundamental role in cementogenesis and periodontal tissue formation. Emdogain, enamel matrix derivative (EMD), is a porcine enamel matrix derived product used to enhance regeneration of the peridontium after inflammatory destruction. Today, little is known about EMD's potential regenerative properties on cell function. The purpose of this study was to investigate the effects of EMD on mouse fibroblasts (L 929 cells) and rat marrow stromal osteoblasts. For effects on cell proliferation, the L 929 cell lines were plated in 24-well culture plates at an initial density of 10,000 cell/mL and allowed to attach. Following a 24-h incubation within Dulbecco's modified eagle medium (DMEM) enriched with 10% fetal bovine serum, DMEM supplemented with 0 (Control), 50 μg/mL and 100 μg/mL of EMD was added and cultures maintained for 96 h. Cell proliferation was measured at 24, 48, 72 and 96 h as the total cell number per well and cell morphology was investigated. Osteoblasts were digested from mouse tibia marrow and were plated in similar manner as with L 929 cells, while the observation periods were 2, 6, 8 and 10 days in this group. Although both cell types were able to maintain their original cell morphology throughout the tests, in both cell groups the number of cells in the EMD groups at each observation period were not significantly different than that in the control group (ANOVA, p > 0.05). Moreover, EMD failed to show any impact on cell growth with higher concentration (ANOVA, p > 0.05). These results suggest that although EMD had no cytotoxic effect on mouse fibroblasts and stromal marrow osteoblasts, the same material failed to enhance the growth of both cell types.

After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also “typical” mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins.

The epithelial root sheath in the cat is a multilayered structure that consists of confluent outer and inner epithelial strata enclosing a central layer of cells, which are morphologically like those of the stratum intermedium. A continuous PAS-positive membrane separates the epithelial structure from the developing pulp. On the dental sac surface, the basement membrane is discontinuous, and even absent in some regions. This situation remains until the root and cementum are completely formed.