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Dental Epithelial Histomorphogenesis in vitro
B. Hu1,2,
A. Nadiri1,
S. Bopp-Küchler1,
F. Perrin-Schmitt3 and
H. Lesot1,*
1 UMR INSERM 595 and
3 UMR 7104 CNRS-ULP INSERM U596, Faculté de Médecine, Université Louis Pasteur, 11 rue Humann, 67085 Strasbourg Cedex, France;
2 Molecular Laboratory for Gene Therapy, Faculty of Stomatology, Capital University of Medical Sciences, Beijing, P.R. China;
Correspondence: * corresponding author, Herve.Lesot{at}odonto-ulp.u-strasbg.fr
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ABSTRACT
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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.
Key Words: epithelial-mesenchymal interaction • epithelial histogenesis • positional information • odontogenesis • tissue engineering
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INTRODUCTION
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Recent approaches to tooth engineering (Dualibi et al., 2004; Ohazama et al., 2004b) have addressed several questions about the critical regulatory processes to be preserved. This is especially pertinent to enamel tissue engineering, a very problematic area. From the bud to the bell stage during development, the histomorphogenesis of the enamel organ ends with the formation of the inner dental epithelium (IDE), the outer dental epithelium (ODE), and the primary enamel knot (PEK), as well as the later segregation of the PEK cells in contact with the basement membrane (Coin et al., 1999). This involves differential mitotic activities, apoptosis, cell adhesion, and cell segregation. Since these activities are regulated in time and space, positional information is particularly important. Positional information is specified by gradients of morphogens, and by cell-cell and cell-matrix interactions (Capdevila and Belmonte, 1999; Franceschi, 1999; Osterfield et al., 2003). Dental epithelial histomorphogenesis is accompanied by changes: (1) in the composition of the basement membrane (BM) (Yoshiba et al., 1998; Nagai et al., 2001), partially regulated by matrix metalloproteinases and their inhibitors (Yoshiba et al., 2003); (2) in the location of signal molecules in the epithelium and mesenchyme (Nadiri et al., 2004); and (3) in the expression of cell-surface molecules, including integrins (Salmivirta et al., 1996; Lesot et al., 2002), receptors for signaling molecules (Fried et al., 2002; Ohazama et al., 2004a), and cadherins (Obara and Lesot, 2004).
Cultured re-associations between isolated dental mesenchyme and the enamel organ have showed that the cap-stage mesenchyme controls crown morphogenesis (Kollar and Baird, 1970; Schmitt et al., 1999). The goals of this study were: (1) to determine whether the dental mesenchyme at ED14 can control dental epithelial histogenesis; (2) to test the role of the PEK in specifying the potentialities of the dental mesenchyme; and (3) to analyze whether positional information of dental epithelial cells is memorized. For this purpose, we dissociated first lower molars from mouse embryos, by trypsin treatment, to separate the mesenchyme from the epithelium, and further dissociated the epithelium to obtain isolated cells. These cells were re-associated with a dental mesenchyme and cultured in vitro for up to 14 days. Tooth germs were taken at the cap stage, embryonic day (ED) 14, when the PEK is present and functional, and at the bud stage (ED13), when the PEK is just forming.
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MATERIALS & METHODS
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Tissue Preparation
ICR female mice were mated overnight, and the detection of the vaginal plug was considered as Embryonic Day 0 (ED0). The embryos were harvested at ED13 and ED14, and the first lower molars were isolated under a stereomicroscope (Leica MZ9, Leica Microsystèmes SA, Rueil-Malmaison, France). The dental epithelium and mesenchyme were dissociated from ED13 (n = 640) and ED14 (n = 1920) first lower molars by the use of 1% trypsin in Hanks’ buffer at 4°C (Schmitt et al., 1999). After tissue separation, the dental epithelia were further dissociated into single cells (see Appendix 1) and passed through a 70-µm nylon filter. The quality of the tissue and cell dissociation was checked by histology, scanning electron microscopy, and an inverted microscope (see Appendix 1). After centrifugation at 9000 g for 2 min, the pellet of epithelial cells was cut into fragments, re-associated with dental mesenchyme, and co-cultured. One mesenchyme was recombined with, respectively, 13 ± 1 x 103 epithelial cells at ED14 and 9.1 ± 0.9 x 103 cells at ED13. Cells were counted in a Malassez hemocytometer after being stained with trypan blue. These cell counts were analyzed with a statistical program, Sigmastat (SPSS Inc., Chicago, IL, USA). The experimental protocol was designed in compliance with the recommendation of the European Economic Community (86/609/CEE) for the care and use of laboratory animals.
Cultures
The re-associations were cultured from 12 hrs up to 14 days on a semi-solid medium, which consisted of DMEM/F-12 (Gibco, Invitrogen SARL, Cergy Pontoise, France) containing 20% fetal bovine serum (Cambrex Bioscience Verviers SPRL, Verviers, Belgium), and supplemented with ascorbic acid (0.18 mg/mL, Merck), L-glutamine (2 mM, Gibco, Invitrogen SARL, Cergy Pontoise, France), penicillin/streptomycin (50 units/mL, Gibco, Invitrogen SARL, Cergy Pontoise, France), and agar (0.36%, Sigma-Aldrich Chimie SARL, Lyon, Frnace). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. The medium was changed every two days. In this work, 382 re-associations were performed at ED14 and 83 at ED13.
Histology
All samples were fixed in Bouin-Hollande solution and embedded in paraffin, and serial sections (5 µm) were stained by the Mallory procedure.
BrdU Incorporation
We investigated cell proliferation by mapping the distribution of S-phase cells after incorporation of 5-bromo-2-deoxy-uridine (BrdU, cell proliferation kit; Amersham Life Science, Amersham Biosciences Europe GmbH, Orsay, France) (Coin et al., 1999). The samples were fixed in Bouin-Hollande solution, embedded in paraffin, and sectioned. After the immunodetection of BrdU, the serial sections were counterstained with eosin.
Immunostaining for Apoptosis
Apoptosis was detected on frozen sections (7 µm) with the use of a rabbit polyclonal anti-ssDNA antibody (1:250, DakoCytomation SAS, Trappes, France). The primary antibody was visualized by means of a detection kit from Vector Laboratories (Burlingame, CA, USA). The staining with a biotinylated anti-rabbit secondary antibody was visualized with avidin coupled to horseradish peroxidase. The color reaction was achieved with DAB substrate. The sections were counterstained with nuclear fast red or 25% hematoxylin.
In situ Hybridization
We used the Shh cDNA (a 642-bp mouse cDNA, a gift from Prof. Andrew McMahon, Harvard University, Boston, MA, USA) as a template to make digoxygenin-labeled RNA probe (Echelard et al., 1993). In situ hybridization was performed on 7-µm frozen sections.
3D Reconstructions
The contours of the epithelium were represented from serial histological sections with the use of a Leica DMRB (Leica Microsystèmes SA, Rueil-Malmaison, France) microscope equipped with a drawing chamber. The drawings were digitalized with the use of a Hamamatsu C2400 camera (Hamamatsu Photonics France SARL, Massy, France) connected to a digital imaging system, as has previously been described (Lesot et al., 1996).
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RESULTS
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Cultured ED14 Re-associations
After 12 hrs, the epithelial-mesenchymal junction was restored (Figs. 1A , 1F) in 8/8 re-associations. All epithelial cells showed a similar size and round shape. Necrosis and apoptosis were still frequent (Fig. 1F).
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Figure 1. In vitro development of re-associations between dental mesenchyme and dissociated epithelial cells from first lower molars at ED14. The re-associations were cultured for 12 hrs (A,F), 24 hrs (B,G), 2 days (C,H), 3 days (D,I,P,Q,R), 6 days (E,J), and 14 days (N,O,S,T). To visualize cell proliferation, we cultured ED14 first lower molars for 18 hrs in the presence of BrdU (K), and cultured re-associations for 54 hrs in vitro and 18 hrs in the presence of BrdU (P). Apoptosis was visualized after cells were stained for ssDNA on ED14 molars (L, arrow), and re-association was cultured for 3 days (Q, arrow). In situ hybridization for Shh was performed on first lower molars at ED14 (M), and re-associations were cultured for 3 days (R). After 14 days in culture, odontoblasts were functional and ameloblasts were polarized (N,O,S). Six cusps were observed, as can be seen on the histological horizontal section (O) or after 3D reconstruction of the mesenchyme (T). AM, ameloblast; BM, basement membrane; D, dentin; DE, dental epithelium; DM, dental mesenchyme; DP, dental papilla; IDE, inner dental epithelium; OD, odontoblast; ODE, outer dental epithelium; PEK, primary enamel knot; SEK, secondary enamel knot; SI, stratum intermedium; SR, stellate reticulum. Bar = 40 µm.
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Most of the necrosis and apoptosis disappeared after 24 hrs (32/32 samples) (see Appendix 2). The epithelium organized as a bud with 2 cell populations (Figs. 1B , 1G). The internal cells remained small and round, while cells in contact with the BM became larger and started to elongate (Fig. 1G).
After 2 days’ culture, the epithelium reached the early cap stage (Fig. 1C). The cells of the ODE organized as a single layer. They were much shorter than the prospective IDE cells. A group of cells condensed in the central part of the IDE, with the same organization as cells of the PEK in vivo: small and non-dividing cells with much apoptosis (Fig. 1H).
After 3 days, the re-associations reached a very transient cap stage (Fig. 1D). Epithelial histogenesis had progressed, and the stellate reticulum was better seen between the IDE or the PEK and the ODE (Fig. 1I). The PEK was still visible in 31 of 47 samples. After BrdU incorporation, most of the cells of the PEK in the re-association were BrdU-negative (Fig. 1P), as in the tooth germ at E14 (Fig. 1K). In situ hybridization for Shh (Fig. 1R) showed that the Shh-positive and BrdU-negative PEK cells had already started to segregate (Coin et al., 1999). In re-associations cultured for 3 days, apoptosis was detected in the internal cells of what remained from the PEK (Fig. 1Q), as was also observed in tooth germs at ED14 (Fig. 1L).
After 6 days, cusp formation was more prominent, and the stratum intermedium (SI) was visible (Figs. 1E, 1J) in all 44 samples. The mesenchymal cells in contact with the BM were still pre-odontoblasts (Fig. 1J). The cells from the odontoblast layer started to polarize only after 8 days (22/22 samples, not shown), and after 10 days they became secretory odontoblasts (22/22 samples, not shown). Gradients of predentin/dentin secretion were initiated at the tip of the cusps and progressed toward the cervical loop. The cytodifferentiation of odontoblasts and ameloblasts (Figs. 1N, 1O, 1S) was illustrated after 14 days in culture (9/9 samples). At this stage, the shape of the cultured re-association was examined with 3D reconstruction, illustrating the formation of 6 cusps (Fig. 1T), very close to the 7 cusps observed in the first lower molar.
Cultured ED13 Re-asssociations
After 12 hours, the epithelial-mesenchymal junction was restored in 8 of 8 samples. However, all epithelial cells showed the same size (Fig. 2A). After 24 hrs (10/10 samples), the epithelial cells organized as a bud (Fig. 2B) and began to differentiate into two groups: round cells in the center and elongated cells in contact with the BM (Fig. 2B). The condensation of the dental mesenchyme increased from 12 hrs to 24 hrs (compare Fig. 2A with Fig. 2B). After 2 days, 10 of 12 samples had reached the cap stage (Fig. 2C). A PEK-like structure was observed in 9 of 12 samples, and epithelial histogenesis progressed with the appearance of the stellate reticulum (SR). After 3 days’ culture, the existence of PEK was also confirmed by BrdU incorporation and in situ hybridization for Shh (not shown). After 4 days in culture (13/13 samples), the cervical loop developed, and the volume of SR increased (Fig. 2D). After 6 days, the cervical loop was still elongated, and cusps were apparent in all (11 samples) re-associations (Fig. 2E). At this stage, all epithelial compartments (IDE, ODE, SR, and SI) were distinct. Odontoblasts became functional after 10 days (9/9 samples, not shown), and ameloblasts differentiated after 12 days (8/8 samples, not shown). After 14 days, polarized ameloblasts were functional, and the crown was well-formed (Fig. 2F).
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Figure 2. In vitro development of re-associations between dental mesenchyme and dissociated epithelial cells from first lower molars at ED13. The re-associations were cultured for 12 hrs (A), 24 hrs (B), 2 days (C), 4 days (D), 6 days (E), and 14 days (F). DE, dental epithelium; DM, dental mesenchyme; DP, dental papilla. Bar = 40 µm.
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DISCUSSION
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The strict regulation of crown morphogenesis involves reciprocal epithelial-mesenchymal interactions. Tissue re-association experiments have shown that tooth crown morphogenesis is controlled by the cap-stage mesenchyme (Kollar and Baird, 1970; Schmitt et al., 1999). The purpose of this work was to determine whether the mesenchyme can also control dental epithelial histogenesis after all epithelial cell organization has been lost by cell dissociation (Fig. 3).
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Figure 3. Scheme for the dissociation-re-association experiments. (1) About 60 tooth germs at the cap stage were dissociated by trypsin treatment, so that the dental mesenchyme could be separated from the dental epithelia. (2) The epithelia were further dissociated into single cells. (3) After filtration and centrifugation, the pellet containing mixed epithelial cells from 4 sources (SR, IDE, ODE, PEK) was fragmented and re-associated with dental mesenchyme. (4) These re-associations were cultured for 3 days to achieve the cap stage and illustrate the restoration of epithelial histogenesis.
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Notable cell death (see Appendix 2) and the restoration of the epithelial-mesenchymal junction preceded the first sign of epithelial histogenesis in the re-association. Trypsin dissociation altered the cell surfaces, especially the membrane receptors and adhesive molecules, two systems involved in signaling (Obara and Lesot, 2004; Tucker et al., 2004). All the pre-existing positional information for the different epithelial cell groups present at the cap stage was lost, but clearly began to be re-established after 24 hrs, when the internal/external cell compartments began to be restored. Cell-cell and cell-matrix interactions are involved in this specification (Edwards and Streuli, 1995).
After 2 days in culture, a new step in epithelial histogenesis was achieved with the transient appearance of a condensed group of cells in the IDE, similar to the PEK. In vivo, the PEK consists of non-dividing cells, which express signaling molecules suggested to be involved in the control of crown morphogenesis (for review, see Thesleff and Sharpe, 1997). In the re-associations, the group of condensed non-dividing cells, as checked after BrdU incorporation, expressed Shh and Fgf-4 (not shown). Cusp formation, initiated after 4 days, showed that, even after epithelial cell dissociation, the dental mesenchyme can control the restoration of a functional PEK as well as the geometry of the segregation of the non-cycling PEK cells in contact with the BM (Coin et al., 1999). The cap-stage mesenchyme thus can instruct dental epithelial histogenesis.
To check whether the PEK is involved in specifying the potentialities of the dental mesenchyme, we performed experiments using tooth germs at ED13. The epithelium was still at the bud stage, and the precursor of the PEK was just starting to appear. Epithelial histogenesis and general morphogenesis progressed similarly in re-associations made with material from ED13 or ED14. The mesenchyme at ED13 induced the formation of a new PEK, and cusp formation was followed by odontoblast and ameloblast differentiation. The initial potentialities of the mesenchyme are thus not specified by the PEK.
During odontogenesis, epithelial histomorphogenesis results from differential cell proliferation, apoptosis, and cell migration. The coordination of these cellular activities probably requires positional information, specified by cell-matrix and cell-cell interactions, as well as by gradients of morphogens (Capdevila and Belmonte, 1999; Franceschi, 1999; Tabata and Takei, 2004). When the epithelial cells were dissociated from cap-stage enamel organs (ED14), the IDE, ODE, SR, and PEK were already distinct (Fig. 3). The BM also showed a different composition when in contact with the IDE or ODE (Yoshiba et al., 1998; Nagai et al., 2001). The trypsin dissociation led to the hydrolysis of the BM and also affected the extracellular matrix in the mesenchyme and cell surfaces in both the epithelium and mesenchyme (Lesot et al., 1981; Osman and Ruch, 1981). After trypsin dissociation, single epithelial cells from 60 teeth were mixed, filtrated, pelleted, and re-associated with dental mesenchyme. Despite the mixture of different cell populations and the complete loss of positional information (Fig. 3), a newly formed continuous BM was deposited, after 12 hrs, at the junction between the mesenchyme and epithelial cells. As a consequence of their new position, epithelial cells were reprogrammed to adapt to their immediate environment (i.e., in contact or not with a mesenchyme), which conditioned their shape.
After 4 days in culture, the IDE and ODE differentiated in the re-associations, as occurs in vivo. This is also controlled by the dental mesenchyme (Olive and Ruch, 1982). Since the dental and peridental mesenchymes in vivo show differences in immunostaining for BMP-2 and BMP-4 (Nadiri et al., 2004), functional differences may be expected in the 2 areas. Furthermore, regional changes in the BM composition during the bud to cap transition, depending on whether it is in contact with the IDE or ODE, might result from the combined effects of MMPs and TIMPs (Yoshiba et al., 2003). These local changes in the BM might be involved in differential signaling through integrins (Tsuruta et al., 2003). Positional identity was suggested to be based on short-range information, and also to be interpreted by cells in terms of their developmental history (Wolpert, 2003). The fate of the re-associations suggests that epithelial histogenesis of the enamel organ requires no long-term memory (Hu et al., 2005).
In conclusion, these experiments confirmed the role of the cap-stage mesenchyme in the control of tooth morphogenesis. They further demonstrated that the mesenchyme can induce disorganized epithelial cells to restore a complete histogenesis of the enamel organ. Initially, the potentialities of the mesenchyme are not specified by the PEK. In this system, the positional information does not require memorization of cell history, as seen when epithelial cells from E14 were used. This epithelial cell plasticity (i.e., their ability to undergo conversion between different epithelial cell types) is a prerequisite for enamel tissue engineering.
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ACKNOWLEDGMENTS
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The authors thank A. Ackerman for his assistance in histology. In addition to financial support from the INSERM, this work was also financed by the Institut Français pour la Recherche Odontologique (2003), the National Natural Science Foundation of China (Grants No. 30125042 and 30170254), a Beijing Health Science Key Grant (1999–04), and a Frontier Research Project H15 on Tooth Morphogenesis (2003–2007) from the Japanese ESSCT Ministry.
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.
Received for publication November 30, 2004.
Revision received March 1, 2005.
Accepted for publication March 6, 2005.
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Journal of Dental Research, Vol. 84, No. 6,
521-525 (2005)
DOI: 10.1177/154405910508400607
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ABSTRACT
INTRODUCTION