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Vitamin D deficiency appears during pregnancy with a relatively high prevalence, especially when the last trimester occurs during winter(1, 2). The resulting maternofetal vitamin D deficiency has been associated with neonatal hypocalcemia(3, 4) and a delay in neonatal growth, and it may also induce disturbances in enamel formation(4). However, limited data have been obtained on the molecular basis of vitamin D action in the early stages of human development(5).

The intracellular action of 1,25(OH)2D3, the active form of vitamin D, is initiated by binding of the hormone to specific VDR in nuclei of target cells. The 1,25(OH)2D3-VDR complex binds as homodimers or heterodimers with other nuclear receptors(6). These complexes bind to target genes and regulate gene expression in a tissue and stage-specific manner(7). These vitamin D-dependent molecules mediate the biologic actions of the hormone. Among the various vitamin D-responsive genes, calbindin-D28k is expressed in teeth(8). Kim et al.(9) identified target cells for 1,25(OH)2D3 in developing rodent teeth by autoradiography. Immunoreactivity for VDR(8) is present in all progenitor cells and in differentiated ameloblasts and odontoblasts. At this latter stage of development, the expression of VDR, calbindin-D9k, and calbindin-D28k is jointly up-regulated by 1,25(OH)2D3(18). Therefore, ameloblasts and odontoblasts, as well as progenitor dental cells, have been described to be target cells for 1,25 (OH)2D3 in rodents(8).

Calbindin-D28k has been demonstrated in mature odontoblasts of human formed teeth(10), whereas VDR has been described only in central pulp cells(11). Immunocytochemical(12) and in situ hybridization studies(13) in developing human fetal teeth have demonstrated the presence of another vitamin D-dependent [for review, see Lowe et al.(14)] matrix protein: osteonectin. However, the expression of potential vitamin D-responsive proteins in human ameloblasts has not been investigated. In the present study, the temporospatial appearance of VDR and a vitamin D-dependent calcium-binding protein (calbindin-D28k) was investigated by immunocytochemistry during human odontogenesis.

METHODS

Immunolocalization in human teeth was performed as follows.

Tissue collection and preparation. Tooth germs (n = 50) from 15 human embryos and fetuses were obtained from legally approved medical abortions induced by prostaglandins, spontaneous abortions, and newborn infants. Informed consent was obtained according to the guidelines of the Declaration of Helsinki. The fetuses studied ranged from 8 to 26 wk of gestation. Embryonic stage and fetal age were determined by anthropometric measurements and/or histologic maturation. For VDR immunodetection in tooth germ, 10 mandibles were frozen immediately after delivery, plunged into liquid nitrogen, and stored frozen at -80°C. After embedding in Tissue-Teck O.C.T Coump (Miles Scientific, Elkhart, IN), serial 7-μm sections were cut sagittally with a cryostat at -20°C, transferred onto poly-L-lysine (Sigma Chemical Co., la Verpillère, France) coated glass slides, and stored at-20°C. For calbindin-D28k immunodetection in the forming tooth, five mandibles were fixed in 10% formalin for several days at 4°C, decalcified or not, dehydrated, and embedded in paraffin. Furthermore, the forming postnatal tooth germs (n = 10) were obtained with the parent's consent either from deceased children during autopsy for diagnosis and/or scientific purposes or from surgical hemimandibulectomy. Sections (5 μm) were cut, deparaffinized, and rehydrated. For calbindin-D28k in the formed tooth, 10 premolars extracted for orthodontic reasons were collected and immediately immersed in 4% buffered phosphonoformatic acid. Dental pulps were microdissected under a microscope, dehydrated, and embedded in paraffin.

VDR fluorescence immunolocalization. To saturate nonspecific binding sites, each section was treated with normal goat serum (Sigma Chemical Co.) for 30 min, diluted to 1:30 in PBS (0.1 mol/L, Eurobio) at room temperature. They were then incubated with monoclonal rat anti-VDR (Chemicon, Temecula, CA) diluted to 1:20, 1:50, and 1:100 in PBS. Sections were maintained overnight at 4°C in a humid atmosphere. For control staining, VDR antibody was replaced by rat IgG (Nordic, Tilburg, The Netherlands) with the same dilutions. After rinsing in PBS, sections were incubated with biotinylated anti-rat IgG antibodies (Sigma Chemical Co.) diluted to 1:50 for 1 h at 37°C. They were then rinsed in PBS (0.1 mol/L) and incubated with streptavidin-FITC complex (FITC, Sigma Chemical Co.) for 30 min (dilution 1:200) at 37°C. Sections were then rinsed in PBS containing 2% BSA and maintained in PBS with 2% BSA for 2 h in an agitator. Finally, sections were mounted in hydrophilic fluorescence medium (Biosys, Compiègne, France) and examined under epifluorescence with an Axioplan light microscope(Zeiss).

Calbindin-D28K immunolocalization. Antibodies were raised against rat kidney calbindin-D28k in rabbits(15). Their cross-reactivity with human calbindin-D28k has been classically established(16). All rinsings and incubations were performed in 0.05 mol/L Tris-HCl at pH 7.3 (Tris). Endogenous peroxidases were blocked by a 30-min treatment with 3% H2O2 in distilled water. Sections were then rinsed and incubated for 30 min with normal goat serum diluted to 1:10 with 1% BSA. They were then incubated for 1 h at 37°C with 1:1000 rabbit polyclonal anti-calbindin-D28k antibodies diluted in Tris-1% normal goat serum, and then rinsed. The sections were then incubated for 30 min with 1:100 peroxidase secondary antibodies (anti-rabbit IgG; Sigma Chemical Co.). The sections were rinsed and incubated with rabbit anti-peroxidase complex (Sigma Chemical Co.) at a dilution of 1:100 for 30 min at 37°C. The sections were finally rinsed with Tris. Immunoreactive sites were visualized with 3,3′-diaminobenzidine (Sigma Chemical Co.; 5 mg/10 mL) in 0.005 mol/L Tris-HCl, pH 7.6, with 0.03% H2O2 for 10 min. Sections were rinsed in water, stained with Harris hematoxylin, dehydrated, mounted in DePex(BDH Laboratory, Poole, UK), and examined with an Orthoplan light microscope(Zeiss).

RESULTS

VDR immunolabeling during early stages of tooth formation (Fig. 1). Bud, cap, and bell stages in human mandibles from 8 to 26 wk were examined (Fig. 1, A-F). Immunolabeling for VDR was detected in the dental epithelium throughout morphogenesis and histodifferentiation (Fig. 1, A, bud stage, and B, cap stage). Buccal epithelium (Fig. 1C) and mandibular bone (Fig. 1D) were used as positive controls for VDR immunoreactivity. Negative controls (Fig. 1F, cap stage) with nonspecific IgGs provided low background levels. Morphologic staining illustrates the detailed appearance of the cap stage epithelium, especially enamel knot (Fig. 1E), as VDR staining was very marked in this area (Fig. 1B).

Figure 1
figure 1

(A) VDR immunolabeling in bud stage tooth germ (second primary molar of a 10-wk-old human fetus; ×400, antibody 1:50). The staining appears present mainly in the epithelial cells.N, Nucleus; C, cytoplasm. (B) VDR immunolabeling in cap stage tooth germ (first primary molar of a 16-wk-old human fetus;×200, antibody 1:50). The staining is distributed mainly in the epithelium, but adjoining ectomesenchymal cells are also immunolabeled.IDE, Inner dental epithelium; ODE, outer dental epithelium; EK, enamel knot. (C) VDR immunolabeling in the mandibular bone of a 26-wk-old human fetus (×200, antibody 1:50). The first control tissue, forming bone, contains immunopositive osteoblastic cells at several stages of differentiation and maturation. OP, Osteoprogenitor cells; OB, osteoblasts; OC, osteocytes;MO, osteoid matrix. (D) VDR immunolabeling in buccal epithelium of a 26-wk-old human fetus (×400, antibody 1:50). The second control tissue, buccal epithelium, appears to show stained cells, with characteristic subcellular distribution of immunoreactive patches inside the nuclei (white arrows). N, Nucleus; E, epithelium; B, basal membrane; C, connective tissue.(E) Section of cap stage tooth germ (second primary molar of an 11-wk-old human fetus; ×200) which illustrates the morphology corresponding to that in B. DL, Dental lamina; EK, enamel knot; DM, dental ectomesenchyme; IDE, inner dental epithelium; ODE, outer dental epithelium. (F) Immunocytochemical control in cap stage tooth germ (second primary molar of an 11-wk-old human fetus; ×200). The background level obtained when the primary antibodies are omitted appears low. DL, Dental lamina;EO, enamel organ; DM, dental ectomesenchyme.

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VDR and Calbindin-D28k immunolabeling during differentiation of ameloblasts and odontoblasts (Fig. 2). In the bell stage tooth germ, the terminal differentiation of ameloblasts and odontoblasts may be followed from the cervical loop to the lateral sides (Fig. 2A), as far as the tip (Fig. 2B) of the cusp. VDR were detected in the dental epithelium (Fig. 2A), even before complete ameloblast differentiation. In the dental mesenchyme, immunoreactive VDR were also present in morphologically undifferentiated cells (Fig. 2A) and in fully polarized odontoblasts (Fig. 2B). VDR labeling was mainly nuclear with, however, some cytoplasmic immunoreactivity (Fig. 2, A, B,and E). VDR was also distributed in distinct patches inside the nucleus (Fig. 2, A and E). Calbindin-D28k was immunostained in bell stage preameloblasts in undecalcified forming tooth (Fig. 2C) and was present in the cytoplasm of polarized odontoblasts (Fig. 2D) in undecalcified formed teeth.

Figure 2
figure 2

VDR and calbindin-D28k immunolabeling in differentiating and differentiated ameloblasts and odontoblasts. (A) VDR immunolabeling in bell stage of a second primary molar in a 26-wk-old human fetus: lateral aspect of the cusp (×400, antibody 1:50). Nuclear labeling appears condensed in defined areas (small arrows).IDE, Inner dental epithelium containing preameloblasts;DM, dental ectomesenchyme. (B) VDR immunolabeling in bell stage of a second primary molar in a 26-wk-old human fetus: dental papilla located at the cusp tip (×400, antibody 1:50). Odontoblasts as well as other cells of the dental ectomesenchyme contain immunoreactivity for VDR.O, Odontoblasts; DP, adjacent dental papilla.(C) Calbindin-D28k immunolabeling in bell stage tooth germ (first primary molar of a 16-wk-old human fetus; ×400, antibody 1:1000). Calbindin-D28k is present during the differentiation of ameloblastic cells but not detected in the ectomesenchyme. In odontoblasts, the immunonegativity shown on decalcified samples must be interpreted carefully, as decalcifying agents may artefactually extract calbindin-D from the tissues(17). PA, Preameloblasts; DM, dental mesenchyme. (D) Calbindin-D28k immunolabeling in mature dental pulp(premolar of a 12-y-old child). Only odontoblasts are labeled in formed dental pulp. O, Odontoblasts; DP, dental pulp. (E) VDR immunolabeling in bell stage of a second primary molar in a 26-wk-old human fetus: enamel organ located at the cusp tip (×400, antibody 1:50). Similar to that seen in B, nuclear labeling inside ameloblasts appears condensed in defined areas (small arrows). A, Ameloblasts; E, enamel.

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Calbindin-D28k in differentiated ameloblasts (Fig. 3). The presence of calbindin-D28k was related to the differentiation of ameloblasts. No immunoreactivity was present in dental lamina (Fig. 3A), in bud and cap stages (not shown), at which ameloblasts and odontoblasts were still not differentiated. In developing bell-staged teeth, the cervical loop area (Fig. 3B) contained immunonegative cells. In contrast, ameloblasts appeared to contain calbindin-D28k at the secretion stage (Fig. 3C). The immunoreactivity was evenly distributed in all ameloblasts (Fig. 3C). Immunoreactive controls were negative (Fig. 3D). The immunoreactivity of ameloblasts at the maturation stage was alternatively positive and negative (Fig. 3E). All cells were negative in the reduced enamel organ (Fig. 3F). Odontoblasts were immunonegative in these decalcified samples.

Figure 3
figure 3

Distribution of calbindin-D28k immunostaining in tooth germs of a 1-mo-old infant. (A) Calbindin-D28k immunostaining in the dental lamina of a first premolar (×400, antibody 1:1000). No immunoreactive cells are observed. DL, Dental lamina. (B) Calbindin-D28k immunostaining in the cervical loop of a permanent incisor germ(×400, antibody 1:1000). Similar to dental lamina, all tissues are immunonegative. IDE, Inner dental epithelium; ODE, outer dental epithelium; DM, dental ectomesenchyme. (C) Calbindin-D28k immunostaining in the same tooth (×400, antibody 1:1000). Immunolabeling is restricted to ameloblasts. A, Secretion stage ameloblasts. (D) Control staining in a serial section vs C(×400). Low background is present throughout the section. A, Ameloblasts. (E) Calbindin-D28k immunostaining in the same tooth(×400, antibody 1:1000). A, Irregular immunolabeling in maturation stage ameloblasts. Solid arrowheads correspond to maximal staining, empty arrowheads correspond to minimal staining.(F) Calbindin-D28k immunostaining in the same tooth (×400, antibody 1:1000). Immunolabeling is negligible. A, Ameloblasts in reduced enamel organ.

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DISCUSSION

VDR distribution in human forming teeth. Tooth morphogenesis results from invagination of the epithelium inside the ectomesenchyme of the first branchial arch(17, 18). This process and the subsequent histodifferentiation and terminal cytodifferentiation of ameloblasts and odontoblasts are secondary to epithelio-mesenchymal interactions(19). When overtly differentiated, polarized cells elaborate mineralized tissues, i.e. enamel for ameloblasts and dentin for odontoblasts(20). These processes result from a coordinated action of growth factors on progenitor and differentiated cells(17–20). Although numerous in vivo and in vitro investigations have been devoted to rodents, the identification of target cells for growth factors and hormones has been less extensively studied in human teeth. The receptors for various growth factors such as nerve growth factor receptors, p75NGF, and trkA(21, 22), and epithelial growth factor receptor(23–26) have been identified during human dental development. VDR have been previously shown in human teeth, but only in fully formed teeth(11).

The primary objective of the present study was therefore to immunolocalize VDR from early developmental stages until the process of mineralization in human forming teeth. To validate the immunochemical methodology and in agreement with previous observations(27–29), the presence of VDR was investigated in epithelium and forming bone. VDR has been detected throughout the differentiation process of forming bone, in progenitor cells, as well as osteoblasts and osteocytes. Moreover, the subcellular pattern of VDR (diffuse in the cytoplasm and distributed in distinct patches in the nucleus) was obtained in all immunopositive cell types and closely corresponded to the previously described VDR distribution(11).

Using these immunofluorescent techniques, the present study showed that VDR may also be expressed in early stages of tooth morphogenesis, in contrast to previous studies in which VDR was studied only after the bell stage had already been reached(8, 9, 11, 30). Epithelium and mesenchyme were immunoreactive for VDR in bud, cap, and bell stages of tooth germs. Consequently, vitamin D may control these early stages of odontogenesis. Morphogenesis and the differentiation pattern of ameloblasts and odontoblasts are both affected in vitamin D-deficient rats(31). At the present time, numerous vitamin D-dependent molecules in other tissues [for review, see Berdal et al.(32)] have also been suggested to contribute to epithelio-mesenchymal interactions leading to tooth morphogenesis and cell differentiation(19, 20). These molecules may therefore be potentially involved in the control of development by vitamin D, as may be the case for matrix proteins [fibronectin(33) and collagen type I(34)], growth factors [(nerve growth factor(35) and transforming growth factor-β(36)] and their receptors [epidermal growth factor receptor(37)], and transcription factors [Msx-2(38)].

VDR and Calbindin-D28k in human ameloblasts and odontoblasts. Defects in dental mineralization observed in dietary vitamin D deficiency or vitamin D-resistant rickets are classically described(39, 40). They were essentially considered to be related to calcium and phosphorus imbalances-hypocalcemia leading to enamel hypoplasia and hypophosphatemia leading to interglobular dentin formation(41). Furthermore, VDR were detected in human teeth essentially in cells not involved in the elaboration of dental tissues(11). In contrast, the present study supports that VDR are present in ameloblasts and odontoblasts of human forming teeth, as shown in rats(8). Vitamin D could therefore play a role in the control of the formation of mineralized tissues by acting on these cells. After their terminal differentiation, ameloblasts and odontoblasts secrete an extracellular matrix which ultimately undergoes mineralization(42). This process results from a coordinated expression of matrix proteins and molecules involved in calcium handling (calcium pump, calbindin-D9k, and calbindin-D28k). Most of these molecules are commonly synthesized under the control of 1,25(OH)2D3 in the tissues involved in calcium homeostasis: bone, intestine, or kidney(14). A vitamin D-dependent expression of calbindin-D(8) has been specifically demonstrated in rat ameloblasts and odontoblasts(43). The second objective of this study was therefore to investigate the developmental pattern of expression of one of these proteins. Calbindin-D28k was immunolocalized with the antibodies used previously in rat teeth(44–46), because they are cross-reactive with human calbindin-D28k(16). The present data in human samples closely correspond to the immunolabeling observed in rodents(10, 32, 44–48), especially in ameloblasts during the presecretion, secretion, and maturation stages of amelogenesis. The observed variations support the notion that calcium transport and homeostasis are finely tuned throughout the two steps of enamel mineralization, as previously proposed(44).

In conclusion, the present study supports the notion that vitamin D may control human odontogenesis from morphogenesis up until complete mineralization. The control of vitamin D synthesis and/or supply may therefore be critical for tooth development during pregnancy and in young children. This factor should be considered additional to the general nutritional status of the mother and child, as its imbalance leads to dental dysplasia but also to a secondary increased susceptibility to dental decay(49).