Distribution of Matrixmetalloproteinase-2 in Human Coronal Dentin

Boushell, Lee W.

Distribution of Matrixmetalloproteinase-2 in Human Coronal Dentin Public Deposited

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March 20, 2019

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Boushell, Lee W.
- Affiliation: School of Dentistry, Department of Operative Dentistry

Abstract

It has been reported that matrixmetalloproteinase-2 (MMP-2) is present in dentin, but its distribution and significance in human dentin are not well understood. OBJECTIVE: To identify MMP-2 and determine its distribution in human coronal dentin. METHODS: Immunohistochemistry was used to investigate the distribution of MMP-2 in coronal dentin. Freshly extracted human premolars and 3rd molars (age range 12-30) were fixed with formaldehyde, demineralized with 10% EDTA (pH=7.4) and embedded in paraffin. Five [micrometer] serial sections were made and subjected to analysis using a monoclonal anti-MMP-2 antibody with an avidin-biotin-complex method. Immunoreactivity was visualized with 3,3'- diaminobenzidine substrate and observed under light microscopy. ImageJ software was used to calculate the relative amount/distribution of MMP-2. Based on immunohistochemical results, crowns of freshly extracted human 3rd molars (age range 15-32) were sectioned, pulp and predentin tissue was removed, dentin matrix was extracted with EDTA and guanidine- HCl, pH7.4, and subjected to Western blot analysis with monoclonal anti-MMP-2 antibody and zymography. RESULTS: Immunohistochemical analysis revealed immunoreactivity for MMP-2 throughout human coronal dentin. However, intense immunoreactivities were identified in a 90-200 [micrometer] zone adjacent to the pre-dentin as well as a 9-10 [micrometer] wide zone adjacent to the dentinoenamel junction (DEJ). Biochemical strategies detected MMP-2 as ~66 and ~72 kDa proteins (mature and proform of MMP-2, respectively). Furthermore, gelatinolytic activity was detected in the extracts. CONCLUSION: The results indicate that MMP-2 may be involved in extracellular matrix organization and dentin mineralization in predentin matrix. In addition, its concentration in the zone immediately adjacent to the DEJ and the retained enzymatic activity after demineralization suggest that MMP-2 may play a role in the spread of early dentin caries along DEJ. Supported by NIH grants DE10489, DE015876 and the UNC School of Dentistry.

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