Hervé Lesot

Immunofluorescent localization of collagens, fibronectin, and laminin during terminal differentiation of odontoblasts.

Abstract The localization of collagens types I, II, III, IV, laminin, and fibronectin was analyzed in mouse embryonic molars by indirect immunofluorescence. Using affinity-purified antibodies, all these antigens except collagen type II were detected in tooth germs and particularly at the epithelio-mesenchymal junction. Collagens type I, type IV, and laminin were localized at the junction before, during, and after odontoblasts terminal differentiation. The staining patterns corresponding to type III collagen and fibronectin were modified during the polarization of odontoblasts. Collagen type III present at the epithelio-mesenchymal junction could no longer be detected in this region when odontoblasts were polarized. Fibronectin, surrounding preodontoblasts, was confined to the epithelio-mesenchymal junction when odontoblasts were fully polarized. Previous studies had shown that the presence of a basement membrane and associated material was a prerequisite for the polarization of odontoblasts. Therefore, the redistribution of collagen type III and fibronectin was discussed in terms of fibronectin-collagen interactions and transmembranous control of the cytoskeleton activity in the differentiating odontoblasts.

Odontoblast stimulation in ferrets by dentine matrix components

The possible effects of isolated dentine matrix components on odontoblast secretory activity were investigated in vivo by implantation of lyophilized fractions of these components into cavities prepared in ferret canine teeth. After implantations as short as 14 days there was significant deposition of reactionary dentine by the odontoblasts beneath the cavity and this response increased in a non-linear manner with time of implantation. In contrast, control cavities lacking the dentine matrix components showed no evidence of reactionary dentine deposition. Examination of teeth at early periods of implantation (2 and 5 days) indicated that odontoblast death had not occurred as a result of the operative procedures and that the response was one of stimulation of existing odontoblasts rather than that of induction of a new generation of odontoblast-like cells. The mechanisms of odontoblast stimulation by the dentine matrix components remain to be elucidated, but could be mediated by growth factors within the dentine matrix preparations.

Epigenetic Signals during Odontoblast Differentiation

Odontoblast terminal differentiation occurs according to a tooth-specific pattern and implies both temporo-spatially regulated epigenetic signaling and the expression of specific competence. Differentiation of odontoblasts (withdrawal from the cell cycle, cytological polarization, and secretion of predentin/dentin) is controlled by the inner dental epithelium, and the basement membrane (BM) plays a major role both as a substrate and as a reservoir of paracrine molecules. Cytological differentiation implies changes in the organization of the cytoskeleton and is controlled by cytoskeleton-plasma membrane-extracellular matrix interactions. Fibronectin is re-distributed during odontoblast polarization and interacts with cell-surface molecules. A nonintegrin 165-kDa fibronectin-binding protein, transiently expressed by odontoblasts, is involved in microfilament reorganization. Growth factors (TGFβ1,2,3/BMP2,4, and 6), expressed in tooth germs, signal differentiation. Systemically derived molecules (IGF1) may also intervene. IGF1 stimulates cytological but not functional differentiation of odontoblasts: The two events can thus be separated. Immobilized TGFβ1 (combined with heparin) induced odontoblast differentiation. Only immobilized TGFβ1 and 3 or a combination of FGF1 and TGFβ1 stimulated the differentiation of functional odontoblasts over extended areas and allowed for maintenance of gradients of differentiation. Presentation of active molecules in vitro appeared to be of major importance; the BM should fulfill this role in vivo by immobilizing and spatially presenting TGF(3s. Attempts are being made to investigate the mechanisms which spatially control the initiation of odontoblast differentiation and those which regulate its propagation. Analysis of molar development suggested that odontoblast differentiation and crown morphogenesis are interdependent, although the possibility of co-regulation requires further investigation.

Development of the vestigial tooth primordia as part of mouse odontogenesis.

The mouse functional dentition comprises one incisor separated from three molars by a toothless diastema in each dental quadrant. Between the incisor and molars, the embryonic tooth pattern also includes vestigial dental primordia, which undergo regression involving apoptosis in their epithelium. Apoptosis appears to play an important role in achieving the specific tooth pattern in the mouse. We documented similarities in the folding mechanism allowing the formation of the dental lamina in mice as well as in reptiles. While further budding on this dental lamina gives rise to many individual simple tooth primordia in crocodiles and lizards, budding morphogenesis of several simple tooth primordia appears to be integrated in the mouse, giving rise to enamel organs of a complex nature. The differentiation of a mammalian tooth germ during both ontogeny and phylogeny might thus include the concrescence (connation) of more primordia, putatively corresponding to simple teeth in mammalian ancestors.

Immunolocalization of BMP-2/-4, FGF-4, and WNT10b in the Developing Mouse First Lower Molar

Intercellular signaling controls all steps of odontogenesis. The purpose of this work was to immunolocalize in the developing mouse molar four molecules that play major roles during odontogenesis: BMP-2, −4, FGF-4, and WNT10b. BMP-2 and BMP-4 were detected in the epithelium and mesenchyme at the bud stage. Staining for BMP-2 markedly increased at the cap stage. The relative amount of BMP-4 strongly increased from E14 to E15. At E15, BMP-4 was detected in the internal part of the enamel knot where apoptosis was intense. In contrast to TGFβ1, BMP-2 and −4 did not show accumulation at the epithelial-mesenchymal junction where the odontoblast started differentiation. When odontoblasts became functional, BMP-2 and BMP-4 were detected at the apical and basal poles of preameloblasts. BMP-2, which induces ameloblast differentiation in vitro, may also be involved physiologically. The decrease in FGF-4 from E14 to E15 supports a possible role for the growth factor in the control of mesenchymal cell proliferation. The relative amount of FGF-4 was maximal at E17. The subsequent decrease at E19 showed correlation with the withdrawal of odontoblasts and ameloblasts from the cell cycle. WNT10b might also stimulate cell proliferation. At E14-15, WNT10b was present in the mesenchyme and epithelium except for the enamel knot, where the mitotic activity was very low. At E19 there was a decreasing gradient of staining from the cervical loop where cells divide to the tip of the cusp in the inner dental epithelium where cells become postmitotic. The target cells for FGF-4 and WNT10b appeared different.

Dental Epithelial Histomorphogenesis in vitro

Recent developments in tooth-tissue engineering require that we understand the regulatory processes to be preserved to achieve histomorphogenesis and cell differentiation, especially for enamel tissue engineering. Using mouse first lower molars, our objectives were: (1) to determine whether the cap-stage dental mesenchyme can control dental epithelial histogenesis, (2) to test the role of the primary enamel knot (PEK) in specifying the potentialities of the dental mesenchyme, and (3) to evaluate the importance of positional information in epithelial cells. After tissue dissociation, the dental epithelium was further dissociated into individual cells, re-associated with dental mesenchyme, and cultured. Epithelial cells showed a high plasticity: Despite a complete loss of positional information, they rapidly underwent typical dental epithelial histogenesis. This was stimulated by the mesenchyme. Experiments performed at E13 demonstrated that the initial potentialities of the mesenchyme are not specified by the PEK. Positional information of dental epithelial cells does not require the memorization of their history.

Vascularization of Engineered Teeth

The implantation of cultured dental cell-cell re-associations allows for the reproduction of fully formed teeth, crown morphogenesis, epithelial histogenesis, mineralized dentin and enamel deposition, and root-periodontium development. Since vascularization is critical for organogenesis and tissue engineering, this work aimed to study: (a) blood vessel formation during tooth development, (b) the fate of blood vessels in cultured teeth and re-associations, and (c) vascularization after in vivo implantation. Ex vivo, blood vessels developed in the dental mesenchyme from the cap to bell stages and in the enamel organ, shortly before ameloblast differentiation. In cultured teeth and re-associations, blood-vessel-like structures remained in the peridental mesenchyme, but never developed into dental tissues. After implantation, both teeth and re-associations became revascularized, although later in the case of the re-associations. In implanted re-associations, newly formed blood vessels originated from the host, allowing for their survival, and affording conditions organ growth, mineralization, and enamel secretion.

Epithelial histogenesis during tooth development

This paper reviews the current understanding of the progressive changes mediating dental epithelial histogenesis as a basis for future collaborative studies. Tooth development involves morphogenesis, epithelial histogenesis and cell differentiation. The consecutive morphological stages of lamina, bud, cap and bell are also characterized by changes in epithelial histogenesis. Differential cell proliferation rates, apoptosis, and alterations in adhesion and shape lead to the positioning of groups of cells with different functions. During tooth histo-morphogenesis changes occur in basement membrane composition, expression of signalling molecules and the localization of cell surface components. Cell positional identity may be related to cell history. Another important parameter is cell plasticity. Independently of signalling molecules, which play a major role in inducing or modulating specific steps, cell-cell and cell-matrix interactions regulate the plasticity/rigidity of particular domains of the enamel organ. This involves specifying in space the differential growth and influences the progressive tooth morphogenesis by shaping the epithelial-mesenchymal junction. Deposition of a mineralized matrix determines the final shape of the crown. All data reviewed in this paper were investigated in the mouse.

Patterning by heritage in mouse molar row development

It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mouse molar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions, and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the “primary enamel knot” (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK. We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transient signaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which may explain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago.

Morphogenetic fields within the human dentition: A new, clinically relevant synthesis of an old concept☆

This paper reviews the concept of morphogenetic fields within the dentition that was first proposed by Butler (Butler PM. Studies of the mammalian dentition. Differentiation of the post-canine dentition. Proc Zool Soc Lond B 1939;109:1–36), then adapted for the human dentition by Dahlberg (Dahlberg AA. The changing dentition of man. J Am Dent Assoc 1945;32:676–90; Dahlberg AA. The dentition of the American Indian. In: Laughlin WS, editor. The Physical Anthropology of the American Indian. New York: Viking Fund Inc.; 1951. p. 138–76). The clone theory of dental development, proposed by Osborn (Osborn JW. Morphogenetic gradients: fields versus clones. In: Butler PM, Joysey KA, editors Development, function and evolution of teeth. London: Academic Press, 1978. p. 171–201), is then considered before these two important concepts are interpreted in the light of recent findings from molecular, cellular, genetic and theoretical and anthropological investigation. Sharpe (Sharpe PT. Homeobox genes and orofacial development. Connect Tissue Res 1995;32:17–25) put forward the concept of an odontogenic homeobox code to explain how different tooth classes are initiated in different parts of the oral cavity in response to molecular cues and the expression of specific groups of homeobox genes. Recently, Mitsiadis and Smith (Mitsiadis TA, Smith MM. How do genes make teeth to order through development? J Exp Zool (Mol Dev Evol) 2006; 306B:177–82.) proposed that the field, clone and homeobox code models could all be incorporated into a single model to explain dental patterning. We agree that these three models should be viewed as complementary rather than contradictory and propose that this unifying view can be extended into the clinical setting using findings on dental patterning in individuals with missing and extra teeth. The proposals are compatible with the unifying aetiological model developed by Brook (Brook AH. A unifying aetiological explanation for anomalies of tooth number and size. Archs Oral Biol 1984;29:373–78) based on human epidemiological and clinical findings. Indeed, this new synthesis can provide a sound foundation for clinical diagnosis, counselling and management of patients with various anomalies of dental development as well as suggesting hypotheses for future studies.