MMP activity in the hybrid layer detected with in situ zymography.

Dentinal proteases are believed to play an important role in the degradation of hybrid layers (HL). This study inves- tigated the HL gelatinolytic activity by in situ zymogra- phy 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 bot- tom 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 activi- ties (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 adhe- sive application.