2.4. Animals and Experimental Design

Sixteen 3-year-old male beagle dogs (Marshall, USA) were fed with commercial pellets (2025C) (Harlan, IN, USA) and tap water ad libitum before the experiment began. All teeth were scaled and polished using a piezo-type ultrasonic scaler (BonArt, LA, USA) under general anesthesia induced by a combination of medetomidine (0.01 mg/kg; Orion Pharma, Finland), tramadol (2 mg/kg; Samsung Pharm, Korea), zolazepam and tiletamine (2.5 mg/kg; Virbac, France) administered via intramuscular injection, and the teeth were brushed once daily for the following 2 weeks. Two weeks after scaling, all animals except one animal in the sham control group had ligation with silk-wire twisted ligatures (SWL) according to the described method by Kim et al. [12] on the left upper second premolar (PM2), third premolar (PM3), and fourth premolar (PM4) as well as the left lower PM3, PM4, and first molar (M1). The dogs were fed with soft moistened food to induce periodontitis for 8 weeks. After 8-week induction, clinical periodontal parameters were assessed and the dogs with SWL removed were assigned to five groups (Placebo, YH23537 75, YH23537 300, YH23537 900, YH14642 1,000 mg; three animals per group) based on CAL values. Placebo tablets, YH23537 tablets (37.5, 150, 450 mg), or YH14642 tablets (500 mg) were administrated orally twice a day.

The clinical periodontal parameters were recorded to evaluate the periodontal status before ligation, before treatment, and at 1, 2, 3, 4 weeks after treatment. These parameters included plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BoP).

The PI was rated as follows: PI 0: no plaque, PI 1: a film of plaque adhering to the free gingival margin and adjacent area of the tooth (not more than 1 mm), PI 2: moderate accumulation of soft deposits within the gingival pocket, or on the tooth and gingival margin (less than one half of crown), PI 3: abundance of soft matter within the gingival pocket and/or on the tooth and gingival margin (more than one half of the crown). Gingival index (GI) was rated as follows: (a) GI 0: absence of gingival inflammation, (b) GI 1: mild gingival inflammation—slight change in color of gingival margin and little change in texture, (c) GI 2: Moderate gingival inflammation—moderate glazing, redness, edema, hypertrophy, and/or bleeding on pressure, (d) GI 3: severe gingival inflammation, marked redness and hypertrophy, spontaneous bleeding and ulceration. Probing depth (PD) was defined as the distance between the gingival margin and the bottom of the probable pocket; clinical attachment level (CAL): the distance between the cement–enamel junction and the bottom of the probable pocket. CAL and PD were measured at three sites per tooth. No ligature casts were dislodged during the experiment. All measurements were taken by an experienced clinician (E.R. Lee, Seoul National University, Korea) using a Williams periodontal probe (Osung MND, Korea). Gingival recession was calculated using PD and CAL. Bleeding on probing (BoP) was assessed as probing (a) which was followed by no bleeding, referred to BoP 0 and (b) with bleeding, referred to as BoP 1.

The experiments were conducted in accordance with the rules for Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health following approval of the Institutional Animal Care and Use Committee of Yuhan Corporation (Authorization number, 14172). The animal housing was controlled at 23 ± 2°C with 50 ± 10% relative humidity under a 12/12 hr light/dark cycle throughout the experimental period.