Temporal and spatial localization of the dentin matrix proteins during dentin biomineralization

Abstract

Formation of bone and dentin are classical examples of matrix-mediated mineralization. The mineral phase is essentially the same in these two tissues and primarily consists of a carbonated hydroxyapatite, but the difference lies in the crystal size and shape. There are three components that are necessary for proper mineralization, namely the proper synthesis and secretion of the non-collagenous proteins (NCPs), self-assembly of the collagenous matrix, and delivery of calcium and phosphate ions to the extracellular matrix. Three major NCPs present in the dentin matrix are dentin matrix protein 1 (DMP1), dentin phosphophorin (DPP), and dentin sialoprotein (DSP). In this study, we show the temporal and spatial localization of these NCPs and correlate their expression with the presence of collagenous matrix and calcified deposits in developing mouse incisors and molars. DMP1, an acidic protein, is present predominantly at the mineralization front and in the nucleus of undifferentiated preodontoblast cells. DPP, the major NCP, is present in large amounts at the mineralization front and might function to regulate the size of the growing hydroxyapatite crystals. For the first time, we report the localization of DPP in the nucleus of preodontoblast cells, suggesting a signaling function during the odontoblast differentiation process. DSP is localized predominantly in the dentinal tubules at the site of peritubular dentin, which is highly mineralized in nature. Thus, the precise localization of DMP1, DPP, and DSP in the dentin tissue suggests that a concerted effort between several NCPs is necessary for dentin formation.

Figure 1

Expression of dentin matrix protein 1 (DMP1), dentin phosphophorin (DPP), and dentin sialoprotein (DSP) in bone and teeth as determined by Western blotting. Total protein extracts were prepared as described in Materials and Methods from mouse molars and calvaria bone in postnatal 3-day-old mice. (A) Anti-DMP1 polyclonal antibody. (B) Anti-DPP polyclonal antibody. (C) Anti-DSP polyclonal antibody. (D) Coomassie blue staining of total proteins isolated from 3-day-old mouse molar and calvaria bone.

Figure 2

Localization of collagen, mineral deposits, DMP1, DSP, and DPP in 18-day-old mouse embryos. Developing heads were collected from E18.5 mouse embryo, fixed, embedded, and cut into 5-μm-thick sections. DMP1 was expressed only in the osteoblasts of alveolar bone (C′, arrows). DPP and DSP were not expressed at this stage of development. Presence of collagenous matrix and calcium deposits were determined by Masson's trichrome stain (A,A′) and von Kossa stain (B,B′), respectively. Both were seen in trace amounts only in the mineralized matrix of bone. (C,C′) Anti-DMP1 antibody. (D,D′) Anti-DPP antibody. (E,E′) Anti-DSP antibody. Bo, bone; P, dental pulp. Bars: A–E = 20 μm; A′–E′ = 10 μm.

Figure 3

Localization of collagen, mineral deposits, DMP1, DPP, and DSP in a postnatal 1-day-old mouse (P1). (A,A′) The incisor is at the late bell stage and trichrome Masson staining showed the deposition of the collagen matrix in the dentin and in the surrounding bone. (B,B′) Sparse deposits of calcified deposits were observed in dentin, whereas abundant calcified matrix was observed in the surrounding bone. (C,C′) DMP1, (D,D′) DPP, and (E,E′) DSP were localized to secretory odontoblasts near the cuspal region, which were actively synthesizing a mineralized matrix. Arrows indicate mineralized dentin and bone matrix. A′–E′ represent the boxed areas in A–E. Am, ameloblast; O, odontoblast; P, pulp. Bars: A–E = 20 μm; A′–E′ = 10 μm.

Figure 4

Localization of collagen, mineral deposits, DMP1, DPP, and DSP in a postnatal 3-day-old mouse incisor. (A,A′ and B,B′) Progressive increase in collagen deposition and calcified matrix formation, respectively. DMP1 was localized at the mineralization front, which was between predentin and dentin, and in the odontoblasts (C,C′, arrow). (C) The surrounding alveolar bone also showed DMP1 localization. However, DMP1 was transiently expressed in the ameloblasts (C′, arrowhead). DPP and DSP were both localized at the mineralization front (D′,E′, arrow). DSP was highly expressed in the cytoplasm of the odontoblasts (E′, arrowhead). Both DPP and DSP were expressed in the ameloblasts. Bars: A–E = 20 μm; A′–E′ = 10 μm.

Figure 5

Localization of collagen, mineral deposits, DMP1, DPP, and DSP in postnatal 3-day-old mouse molars. Deposition of collagen and mineralized matrix are seen in dentin (A,A′ and B,B′) and in the surrounding bone (A″,B″). Localization of DMP1, DPP, and DSP were predominantly expressed at the mineralization front as shown in C–E, respectively. Arrows in C′–E′ indicate the mineralization front. Localization of these proteins is clearly seen in the alveolar bone as indicated by arrows in C″–E″. Bars: A–E = 20 μm; A′–E′= 10 μm; A″–E″ = 2 μm.

Figure 6

Localization of collagen, mineral deposits, DMP1, DPP, and DSP in a postnatal 5 day mouse first molar. Deposition of collagen and mineralized matrix are observed (A,A′,B,B′). Localization of DMP1, DPP, and DSP were predominantly observed at the mineralization front (C′–E′, arrow). All three matrix proteins were also localized in the nucleus of pulp cells. The insert in E depicts the localization of DSP throughout the cytoplasm in the odontoblasts. A′–E′ are higher magnification from the corresponding boxed portion from A–E. P, pulp; O, odontoblasts; Am, ameloblasts. Bars: A–E = 20 μm; A′–E′ = 10 μm.

Figure 7

Localization of collagen, mineral deposits, DMP1, DSP, and DPP in a postnatal 7 day mouse first molar. Deposition of collagen and mineralized matrix seen in A, B, A′, and B′. A′–E′ are higher magnification from the corresponding boxed portion from A–E. Arrows indicate localization of proteins at the mineralization front. Localization of DMP1, DPP, and DSP observed at the mineralization front and at the cervical loop region. DSP is localized in the bone at this stage of development. Bars: A–E = 20 μm; A′–E′ = 10 μm.

Figure 8

Localization of collagen, DMP1, DSP, and DPP in a postnatal 20-day-old mouse incisor. Abundant collagenous matrix are localized in A,A′, and A″. DMP1 is localized at the mineralization front and also observed in the nucleus of differentiating odontoblasts (B,B′,B″). DPP is localized at the mineralization front and in the nucleus of the odontoblasts (C,C′,C″). DSP is observed in the dentinal tubules and around the plasma membrane of the odontoblasts (D,D′,D″). DSP is also localized in the early formed enamel matrix (E′) and in the dentin matrix (E″). Am, ameloblast; De, dentin; En, enamel; O, odontoblast; P, pulp. Bars: A–E = 20 μm; A′–E′ = 10 μm; A″–E″ = 2 μm.

Figure 9

IHC analysis of a 3-day-old mouse first molar with preimmune serum of DMPs. (A) DMP1. (B) DPP. (C) DSP. Am, ameloblast; O, odontoblast; P, pulp. Bar = 20 μm.