Ameloblast differentiation in the human developing tooth: effects of extracellular matrices

Abstract

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin alpha2beta1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.

Fig. 1

H&E staining of a human developing bell stage incisor. (A) Sagittal section of dental primordium in the stage of hard tissue deposition (250 mm CRL, 26^th week) was stained with H&E. Dentin is stained pink and enamel is stained purple. The box shows the location of Fig. 1B, 1C and 1D. Scale bar: 1000μm. (B) Magnification of box B in Fig. 1A. Prior to dentin formation, the preameloblasts obtain an elongated form, which was maintained during initial dentin deposition. Prior to enamel matrix secretion, these cells reduced their length. Basement membrane (BM) is labeled with a green dash line. Scale bar: 200 μm. (C) The inner enamel epithelial cells obtain a cuboidal outline and differentiate to preameloblasts (PA) with a columnar shape. The preameloblasts are separated from the dental mesenchyme by a BM, indicated with green dash line. Scale bar: 100 μm. (D) Preameloblasts elongate and differentiate into presecretary ameloblasts (PSA). At this stage the basement membrane is lost and the cells are in direct contact with the dentin matrix. Scale bar: 100μm. (E) Magnified section of box E from Fig. 1B. The transition of ameloblasts from tall columnar presecretary ameloblasts (PSA) to short columnar secretory ameloblasts (SA), which produce enamel matrix (E) as indicated by arrows. Dentin (D) and odontoblasts (OD) are also indicated. Scale bar: 100 μm.

Fig. 2

Diagrams of human and mouse ameloblasts differentiation. Human presecretory ameloblasts polarize and dramatically elongate as basement membrane (labeled as BM in red) disappears and the dentin matrix directly contacts the presecretory ameloblast cells. As compared to ameloblast differentiation in human, mouse ameloblasts gradually elongate and the basement membrane stays in place until enamel matrix synthesis is initiated, with the result that secretory stage rodent ameloblasts are longer than human secretory stage ameloblasts.

Fig. 3

Changes in ECM at the transition from presecretory to secretory ameloblasts in the human primary tooth incisor. (A) Trichrome staining showed that dentin was stained blue and the developing enamel matrix staining dark red. The presecretary ameloblasts easily pulled away from the dentin matrix after the loss of the basement membrane, resulting in sectioning artifacts. am: ameloblasts; od: odontoblasts; si: stratum intermedium. (B) In a serial section, type IV collagen (red fluorescent immunostaining) was localized between the epithelium and mesenchyme as indicated by arrows, and then disappeared at the presecretory stage of ameloblast differentiation. (C) Laminin 5α, another major component of the basement membrane was immunolocalized with a pattern similar to that of type IV collagen. (D) In situ hybridization with a type I collagen probe showed a rapid up-regulation in the secretory stage of odontoblasts (arrows). (E) In situ hybridization with amelogenin probe showed abundant expression in secretary ameloblasts (double arrow), accompanied with a brief upregulation of amelogenin in the odontoblasts (single arrow). (F) Ameloblastin was detected in the secretary ameloblasts. (G) Amelogenin immunoreactivity was detected in the enamel matrix and secretory ameloblasts. Weak signals were also detectable in the odontoblasts. Positive signals are indicated by arrowheads. Scale bar: 50 μm.

Fig. 4

The effects of basement membrane matrix and signaling from dental pulp cells on the cultured ameloblast lineage cells grown in 3 dimensional (3-D) Matrigel. The conditions used for co-culture, 3D culture and monolayer culture were illustrated in Fig.4A. a: ameloblast lineage cells (in blue); m: Matrigel (in purple); p: pulp cells (in red). (B) H&E staining of the cross sections of 3-D culture showed ameloblast lineage cells grown in Matrigel co-cultured with pulp cells (a+m+p) formed larger acini than the acini formed by ameloblast lineage cells in Matrigel (a+m) without signaling from pulp cells. The larger acini formed by ameloblast lineage cells co-cultured with both Matrigel and pulp (a+m+p) displayed more amelogenin immunoreactivity than ameloblast lineage cells grown in Matrigel alone (a+m) (Fig.4C). qPCR results showed that Matrigel and signaling from pulp cells highly upregulated the expression of amelogenin in ameloblast lineage cells (P = 0.0045) (Fig.4D). (E) The expression level of integrin α2 was significantly up-regulated by Matrigel (a+m) and co-cultured pulp cells (a+m+p) as compared with the expression level of ameloblast lineage cells in monolayer culture (a) (P = 0.0039). The presence of Matrigel significantly increased the expression of integrin beta 7 (P = 0.0046). (F) Either pulp cells co-cultured with ameloblast lineage cells (a+p) or co-cultured with ameloblast lineage cells (a+m+p) in Matrigel up-regulated expression of type I collagen as compared to the expression level in the monolayer cultured pulp cells (p) (P = 0.01216). One-way ANOVA analysis, n=3.

Fig. 5

The effects of type I collagen on cultured ameloblast lineage cells. (A) Type I collagen promoted a more elongated cell morphology (arrows) as observed through a phase contrast image. (B) Ameloblast lineage cells bound to type I collagen via α2β1. Type I collagen significantly promoted adhesion of ameloblast lineage cells (p = 0.0001), however, the binding of ameloblast lineage cells to type I collagen was almost completely blocked by integrin α2β1 antibody (P = 0.0002), and partially blocked by integrin β1 antibody (P = 0.0015). Mouse IgG, used as a control, did not have an effect on type I collagen binding (P = 0.3952). (C) Cell type-specific binding activity of type I collagen. Ameloblast lineage cells, dental pulp cells and HEK 293 cells were plated on dishes coated with type I collagen. Type I collagen dramatically increased adhesion of ameloblast lineage cells (P = 0.0003) and HEK 293 cells (P = 0.0321), however, slightly decreased adhesion of dental pulp cells to cell culture plates (P = 0.006). (D) Type I collagen significantly inhibited the expression of PCNA (P = 0.0014). However, no significant differences were observed in the expression of amelogenin with or without the presence of type I collagen (P = 0.2143). Student t-test, n=3.