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Zymographic Analysis and Characterization of MMP-2 and -9 Forms in Human Sound Dentin

A. Mazzoni1, F. Mannello2, F.R. Tay3, G.A.M. Tonti2, S. Papa4, G. Mazzotti1, R. Di Lenarda5, D.H. Pashley3, and L. Breschi*,5

1 Department of SAU & FAL, University of Bologna, Italy;
2 Institute of Histology and Laboratory Analysis, and
4 Institute of Morphological Sciences, University ‘Carlo Bo’ of Urbino, Italy;
3 Department of Oral Biology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA; and
5 Unit of Dental Sciences and Biomaterials, Department of Biomedicine, University of Trieste, Via Stuparich, 1, I-34129 Trieste, Italy


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  		Figure. Gelatin zymograms of MMPs from dentin extracts after sonication and ammonium sulphate treatments. MMP-2 and -9 forms present in whole blood and their relative molecular masses expressed in kDa are reported in Std lanes. (A) MMP forms detected in representative dentin extracts, after demineralizing/extraction with 0.87 M citric and 0.26 M acetic acids (lanes 1 and 2, respectively), after 0.5 M EGTA and 0.5M EDTA (lanes 3 and 4, respectively). (B) MMP forms detected in citric-acid-demineralized dentin extracts after activation with 2 mM APMA for 30, 60, and 90 min (lanes 1, 2, and 3, respectively). (C) Inhibition of MMPs in EDTA (lanes 1 and 4), acetic acid (lanes 2 and 4), and citric acid (lanes 3a and 3b) after pre-incubation with 2 mM 1,10-phenanthroline (lanes 1, 2, and 3a) and 0.5 M EDTA (lanes 3b, 4, and 5). (D) Western blot analysis of MMPs present in whole blood and citric-acid-demineralized dentin extracts (lanes Std and Dent), with the use of monoclonal anti-MMP-2 (72 kDa) and MMP-9 antibodies (92 kDa).

 




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