Immunologic investigation
MINST
The relationship of clinical periodontal parameters and MMP-3 and TIMP-1 levels was analysed to assess the efficacy of PACNs in periodontal therapy. Concentrations of immunologic markers, MMP-3, and TIMP-1 in saliva were assessed at baseline and at 8 weeks following therapy.For MMP-3/TIMP-1 detection, unstimulated saliva samples were collected using a Salivette (SARSTEDT AG and Co) saliva sample collection kit. Unstimulated saliva samples were collected at baseline and at 8 weeks following therapy (MINST or MINST + PACNs).Immunologic investigation was performed by the same examiner (RB). Samples were centrifuged at 3500 rpm (2 min) and aliquoted, and a protease inhibitor cocktail (1 mg/mL) was added to each sample. All samples were stored at −80 °C and evaluated using enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions. MMP-3 and TIMP-1 concentration in saliva samples were examined using commercial ELISA kits (Elabscience) and a Multiskan Microplate Photometer (Thermo Fisher Scientific) at a 450-nm wavelength. Results were calculated using the standard curves formed by each assay for saliva and are given in pg/mL.