Inactivation of MMP-2 and -9 on Dentin by Crosslinkers

Method: Powdered dentin prepared from extracted human teeth was demineralized with 10% H₃PO₄ for 10 min and incubated for 1 or 5 min with 1 or 5 v/w% glutaraldehyde (GA), 1 or 5 wt% grape seed extract (GS), 0.1 or 0.5 wt% riboflavin/UVA (R) or riboflavin-5-monophospate/UVA (RP), 10% sumac extract (S), 20µM or 200µM curcumin (CR). MMP-2 and MMP-9 activities were detected by gelatin zymography. Untreated mineralized and demineralized powder served as control. For in situ zymography mid-coronal dentin slabs (0.5 mm) from freshly extracted intact third molars were etched for 10 sec with 10% H₃PO₄ and subsequently treated with the same crosslinkers for 1 or 5 min. In situ zymography was performed using fluorescein-conjugated gelatin and the gelatinolytic activity was observed with a multi-photon confocal microscope (Zeiss, LSM 510). 

Result: Zymography revealed reduced MMP-9 in dentin both after 1 and 5 min crosslinker treatment especially with GS1, GS5, R1, R5, RP5 and S groups compared to controls. Almost complete inhibition of MMP-2 activity was detected for all pretreated groups. The MMP-2 inhibition was ranked in the following order: GS5>GS1>RP5>S>R1>R5>RP1>CR200>GA1>GA5>CR20. Similarly, in situ zymography confirmed the results showing much lower fluorescence intensity at pretreatment groups compared to controls after 48 hrs incubation. 

Conclusion: The study clearly indicated that the activity of MMP-2 and MMP-9 in acid-etched dentin is reduced by crosslinkers.