Enamel organic matrix: potential structural role in enamel and relationship to residual basement membrane constituents at the dentin enamel junction

Abstract

Although mature enamel is predominantly composed of mineral, a previously uncharacterized organic matrix layer remains in the post-eruptive tissue that begins at the dentin enamel junction and extends 200-300 μm towards the outer tooth surface. Identification of the composition of this layer has been hampered by its insolubility; however, we have developed a single step method to isolate the organic enamel matrix relatively intact. After dissociative dissolution of the matrix with SDS and urea, initial characterization by Western blotting and gel zymography indicates the presence of type IV and type VII basement membrane collagens and active matrix metalloproteinase-20. When combined with data from transgenic knockout mice and from human mutations, these data suggest that the enamel organic matrix (EOM) and dentin enamel junction may have a structural and functional relationship with basement membranes, e.g. skin. To clarify this relationship, we hypothesize a "foundation" model which proposes that components of the EOM form a support structure that stabilizes the crystalline enamel layer, and bonds it to the underlying dentin along the dentin enamel junction. Since we have also co-localized an active matrix metalloproteinase to this layer, our hypothesis suggests that, under pathologic conditions, MMP-mediated degradation of the EOM could destabilize the enamel-dentin interface.

Keywords: Basement membrane; MMP-20; dentin enamel junction; mature human enamel; type IV collagen; type VII collagen.

Figure 1

The enamel organic matrix within the inner enamel layer of mature teeth. An SEM image of the cuspal region of a maxillary third molar demonstrates that the enamel organic matrix is congruent with the dentin at the dentin enamel junction, and extends 200–300 µm towards the outer tooth surface. Notably, the organic matrix is thicker in the cuspal/occlusal regions. Scale bar: 500 µm. Key: E, enamel; EOM, enamel organic matrix; D, dentin.

Figure 2

(A) Enamel organic matrix resides in the inner region of enamel following complete demineralization. Before and after images of a whole maxillary third molar crown demineralized for 1 week as described in METHODS section. Following complete demineralization of enamel, the residual particulate organic matrix is seen along the dentin, predominantly in the cuspal regions (white arrowheads). The amount of residual organic matrix can be appreciated as it suspends from the dentinal surface. (B) Immunoblotting of enamel matrix fraction and an extract of whole tooth crown demonstrate the presence of type IV and VII collagen and MMP-20. Protein extracts exclusively from enamel and from a whole tooth crown were electrophoresed as described in METHODS section using 7.5% gels (type IV and type VII collagen) and a 4–20% linear gradient gel (MMP-20), respectively. For type IV collagen, the non-reduced sample displayed higher molecular weights with mobilities of 300 and 250 kDa, which shifted to a 300 kDa monomer and a 70 kDa fragment after reduction with DTT. Similarly, for type VII collagen, the non-reduced sample presented as a 280 and 250 kDa dimer, which shifted to a 75 kDa fragment after reduction. For MMP-20, 5–6 bands were detected at 55, 43–41, 24, 22 and 15–11 kDa. These bands correspond in size to the pro-form (55 kDa), activated forms (43 and 41 kDa), and fragments (24, 22, and 15–11 kDa) of MMP-20. Molecular weight estimates are based on standards co-electrophoresed on the same gel. Twenty and thirty micrograms of enamel matrix and crown extract protein were applied to these lanes, respectively. (C) Casein gel zymography demonstrates catalytically active MMP-20 bands are present in a human tooth crown extract. Thirty micrograms of protein were applied.

Figure 3

Hypothetical DEJ interface “foundation” model. This simplistic model includes the enamel and dentin matrix components that may contribute to the structural integrity of the dentin enamel interface. We hypothesize that degradation of the collagenous complex of the enamel organic matrix due to the proteolytic activity of MMP-20 and/or other proteases under pathologic conditions could be linked to DEJ instability and lead to enamel separation from the dentin.