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The Localization of Matrix Metalloproteinase-20 (MMP-20, Enamelysin) in Mature Human Teeth

¹ Institute of Dentistry, University of Oulu, PO Box 5281, FIN-90014 University of Oulu, Finland;
² Oulu University Hospital, Oulu, Finland;
³ Oral Pathology Unit and Biomedicum Laboratory Diagnostics (HUCH), University of Helsinki, Helsinki, Finland; and
⁴ Faculty of Dentistry, University of Toronto, Toronto, ON, Canada;

View larger version (47K): [in this window] [in a new window]   Figure 1. MMP-20 is detected in sound dentin, dentinal fluid, and odontoblasts and pulp tissue of sound and carious teeth. Western blottings were performed with monoclonal MMP-20 antibody (1 µg/mL). Molecular-weight standards (kDa) are marked on the left. (A) MMP-20 in different dentin protein fractions of sound tooth. Each dentin sample contained 60 µg of protein according to direct spectrophotometric analysis. In the first guanidine extraction (lane 1), MMP-20 immunoblotting was negative. In subsequent EDTA extractions (lane 2; first EDTA extraction; lane 3, the last EDTA extraction) and the second guanidine extraction (lane 4), a double-band of 46 and 43 kDa (arrows) corresponding to the active form of human MMP-20 (Llano et al., 1997) with higher- and lower-molecular-weight forms of MMP-20 was detected. As a positive control for MMP-20 detection, we used conditioned odontoblast culture medium (lane 5) as described previously (Väänänen et al., 2001), in which a 46-kDa active form of the MMP-20 band was observed. (B) In dentinal fluid (lane 1) concentrated from 26 teeth, a band of 46 kDa (arrow) as well as a 57-kDa band (arrow) corresponding to the proform of human MMP-20 (Llano et al., 1997) were detected. Also, complexed and truncated forms of MMP-20 were detected. In the conditioned odontoblast cell culture medium (lane 2), a 46-kDa band was observed, as in Fig. 1A. (C) MMP-20 in odontoblasts and pulp tissue. Tissues of two teeth were combined to yield odontoblast (lanes 1 and 3) and pulp tissue samples (lanes 2 and 4). The results of the MMP-20 Western blotting were similar in tissues obtained from carious (lanes 1 and 2) and sound (lanes 3 and 4) teeth. In odontoblasts, in addition to a double-band of 46 and 43 kDa (arrows), complexed forms as well as several truncated forms of MMP-20 were observed. In odontoblasts, a distinct band of approximately 110 kDa was observed, which was not usually seen in the pulp tissue samples. Otherwise, the bands observed in pulp tissue were similar to the bands of odontoblasts.

View larger version (164K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining of mature human teeth demonstrating MMP-20 in odontoblasts and dilated dentin tubuli of caries lesions. Key: Ob = odontoblasts, P = pulp, Cd = crown dentin, Rd = root dentin, Pd = predentin. (A) Staining of a sound tooth. Positive staining in the odontoblast cell layer was detected in 3/4 of sound and 4/4 of carious teeth, with increasing intensity in the apical direction. The sound tooth not demonstrating positive staining in the odontoblast cell layer required extensive demineralization, which may have affected its immunoreactivity. The rectangles show the area of higher magnification in (B) and (C). (B) Higher magnification of the coronal dentin-pulp border showing positive odontoblasts and weaker staining in the predentin area. Positive staining in the predentin area was observed in 3/4 of sound and 2/4 of carious teeth. (C) Higher magnification from the root dentin area showing higher density of stained odontoblasts and stronger staining in the predentin area as compared with that observed in the coronal part of the pulp chamber (B). (D) Higher magnification from the area in (C). Positive staining is observed in the cell bodies of the odontoblasts, mostly in their pulpal side. (E) In all the carious teeth, some dilated dentinal tubuli were positively stained (arrows). (F) In the pulp of all sound and carious teeth capillaries, endothelial cells showed positive staining. Also, few pulpal fibroblasts were MMP-20-positive in 3/4 of sound and 3/4 of carious teeth. (G) Negative control, with non-immune serum instead of MMP-20 antibody, showing no staining.

Journal of Dental Research, Vol. 81, No. 9, 603-607 (2002)
DOI: 10.1177/154405910208100905


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