Mmp 8

KP‐457 and GM‐6001 were tested for their ability to inhibit MMP‐ and ADAM‐catalyzed cleavage of substrates in a fluorescence‐based assay. Human ADAM17, ADAM10, and MMP17 were from R&D Systems (Minneapolis, MN, http://www.rndsystems.com), and human MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, and MMP14 were from EMD Millipore. MMP1, MMP2, MMP8, MMP9, MMP13, and MMP17 were activated using p‐aminophenylmercuric acetate (Sigma‐Aldrich) before testing. Fluorogenic substrates for measuring activity of ADAM10 and ADAM17 [Nma‐LAQAVRSSK(Dnp)r‐NH₂, based on the cleavage site of TNF‐α]; MMP1, MMP9, MMP13, and MMP14 [Dnp‐P‐Cha‐GC(Me)HAK(N‐Me‐Abz)‐NH₂]; MMP3 [MOCAc‐RPKPVE‐Nva‐WRK(Dnp)‐NH₂]; and MMP2, MMP8, and MMP17 [MOCAc‐PLGL‐A₂pr(Dnp)‐AR‐NH₂] were all provided by Peptide Institute (Osaka, Japan, https://www.peptide.co.jp/en) and used as substrates. In addition, inhibitory activities were measured using LC/MS/MS with a GPIbα‐based substrate peptide (KKTIPELDQPPKLRGVLQGHLESSRNDPFLHPDF), a C terminal‐based standard peptide (VLQGHLESSRNDPFLHPDF), and an internal standard peptide (VTTGKGQDHSPFWGF). These peptides were synthetized by Scrum (Tokyo, Japan, http://www.scrum‐net.co.jp/english/top_en.htm).

Histological sections (3 μm thick) were deparaffinized and rehydrated. Afterwards, sections were heated in a citrate buffer solution for antigen retrieval and incubated with methanol and a 6% hydrogen peroxide solution to quench the endogenous peroxidase activity. The sections were incubated with 1% BSA (bovine serum albumin; Sigma-Aldrich) for 45 min to block nonspecific reactions. The sections were incubated with the following primary antibodies at the indicated dilutions overnight at 4°C: 1:50 SOX-5 (H-90 Santa Cruz Biotechnology), 1:200 MMP-13 (SAB4501900 Sigma-Aldrich), 1:200 MMP-8 (SAB4501895 Sigma-Aldrich), 1:100 IHH (AV45230 Sigma-Aldrich), and 1:250 Col2 (M2139 Santa Cruz Biotechnology). The Advanced HRP system (Dako, Carpinteria, Calif., USA) was used to detect antibodies and specimens were counterstained with Mayer’s hematoxylin, dehydrated and mounted for observation and quantification [18 (link)].
Quantification was performed using WCIF ImageJ software. Three sections from each sample were selected, and the surface, middle, and deep sections were obtained, calibrated and color deconvolution was performed. In Vectors, H DAB was chosen and the brown image was selected. After many tests, the Threshold interval was selected (0 to 180) and the colored Area Fraction was obtained and compared to the total area of each studied section.

Frozen saliva samples were thawed and centrifuged at 14000 rpm for 15 minutes and the supernatants were collected. The levels of MMP-8, MMP-9, OPG (Sigma-Aldrich, St. Louis, MO, USA), and RANKL (MyBioSource, CA, USA, and PeproTech EC, London, UK) were determined using enzyme linked immunosorbent assays (ELISA) according to the manufacturers' instructions. Optical densities were determined using a microplate reader (FLUOstar OPTIMA, BMG Labtech, Germany). The final concentrations are presented in pg/mL.