2.5. Matrix Metalloproteinases (MMPs) Inhibition Test

A generic MMP Assay Kit (SensoLyte^® Generic MMP colorimetric assay kit-catalogue No. 72095, AnaSpec Inc., Lot# 131-029, Fremont, CA, USA) was used to examine the MMP inhibition effects of DA-MBN and DA-MBN-Zn in dentin tubules. The teeth were sectioned parallel to the occlusal surface using a low-speed diamond saw with irrigation to expose the mid-coronal dentin surface. The exposed surface was polished with 320, 600-grit SiC for 60 s to remove the smear layer. The teeth were stored in DW at 37 °C for 24 h and sectioned vertically with respect to the exposed surface into a total of 50 beams (1.0 × 1.0 × 4.0 mm). The dentin beams were treated with 37% phosphoric acid for 15 s and washed with DW for 15 s. The dentin beams were randomly divided into 10 groups (5 beams per group; Table S1; DW, DW + inhibitor, Ac (50%), Ac + DA, Ac + DA-0.1%MBN, Ac + DA-0.5%MBN, Ac + DA-1.0%MBN, Ac + DA-0.1%MBN-Zn, Ac + DA-0.5%MBN-Zn, Ac + DA-1.0%MBN-Zn). The dentins were immersed in 400 µL of DW (control), 400 µL of acetone (50%, acetone (Ac)/deionized water (DW) 1:1 vol%), 400 µL of DW/MMP inhibitor, or 400 µL of adhesive-acetone mix (1:1 vol%) and continuously stirred for 5 min. Next, the beams were immediately transferred to a 96-well plate that contains 250 μL of a generic MMPs substrate and treated at 37 °C for 2 h. After removing the beams from the plate, the total MMP activity was spectrophotometrically determined by measuring the absorbance of each well at 412 nm in a plate reader (Synergy HT microplate reader, BioTek Instruments, Winooski, VT, USA) [36].