MMP activity in the hybrid layer detected with in situ zymography

A. Mazzoni, F. D. Nascimento, M. Carrilho, I. Tersariol, V. Papa, L. Tjäderhane, R. Di Lenarda, F. R. Tay, D. H. Pashley, L. Breschi

Research output: Contribution to journalArticlepeer-review

157 Scopus citations


Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study investigated the HL gelatinolytic activity by in situ zymography and functional enzyme activity assay. The hypotheses were that HLs created by an etch-and-rinse adhesive exhibit active gelatinolytic activity, and MMP-2 and -9 activities in dentin increase during adhesive procedures. Etched-dentin specimens were bonded with Adper Scotchbond 1XT and restored with composite. Adhesive/dentin interface slices were placed on microscope slides, covered with fluorescein-conjugated gelatin, and observed with a multi-photon confocal microscope after 24 hrs. Human dentin powder aliquots were prepared and assigned to the following treatments: A, untreated; B, etched with 10% phosphoric acid; or C, etched with 10% phosphoric acid and mixed with Scotchbond 1XT. The MMP-2 and -9 activities of extracts of dentin powder were measured with functional enzyme assays. Intense and continuous enzyme activity was detected at the bottom of the HL, while that activity was more irregular in the upper HL. Both acid-etching and subsequent adhesive application significantly increased MMP-2 and -9 activities (p < 0.05). The results demonstrate, for the first time, intrinsic MMP activity in the HL, and intense activation of matrix-bound MMP activity with both etching and adhesive application.

Original languageEnglish (US)
Pages (from-to)467-472
Number of pages6
JournalJournal of Dental Research
Issue number5
StatePublished - May 2012


  • MMP-2
  • MMP-9
  • biochemical assays
  • degradation
  • dentin bonding agent
  • human dentin

ASJC Scopus subject areas

  • Dentistry(all)


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