Animal model with diabetes and periodontitis and simvastatin treatment
F344 rats (male, 6 weeks old) were purchased from Shizuoka Laboratory Center (Shizuoka, Japan). To minimize distress, all rats were housed three per a cage in specific pathogen-free conditions on a 12 h light/dark cycle at constant temperature (22 °C) and received standard chow and water ad libitum. After acclimation for 1 week, rats were randomly assigned to three groups: control group (C), diabetes with periodontitis group (DP), or diabetes with periodontitis treated with SIM group (DPS). After fasting for 18 h, DP and DPS groups were intravenously injected with STZ (50 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.1 M citrate buffer. The C group was injected with citrate buffer alone. Seven days after injection (day 0), blood glucose levels were measured from the tail vein using a glucometer (Accu-Check active system, Roche, Mannheim, Germany). Rats with fasting blood glucose levels over 300 mg/dl were considered diabetic. On day 0, all rats were intraperitoneally injected with a mixture of zoletil 50 (30 mg/kg; Virbac, Carros, France) and rompun (10 mg/kg; Bayer Korea, Ansan, Korea). Following injection, periodontitis was induced in the DP and DPS groups by ligatures of the mandibular first molars with dental floss to induce biofilm formation. SIM (30 mg/kg/day, Chong Kun Dang Pharm, Seoul, Korea) was given daily via oral gavage to the DPS group, and saline was given to the DP group. The C group was sacrificed on day 0 (n = 6), and DP and DPS groups were sacrificed on days 3 (DP, n = 6; DPS, n = 7), 10 (DP, n = 4; DPS, n = 4), and 20 (DP, n = 7; DPS, n = 9). The experimental protocol is presented in Fig. 1a. The maintenance of diabetes was confirmed by measuring body weight and the fasting glucose level during the experimental period. The animal protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University (Approval Number: 2015-0345).
Serum biochemistry
Serum was obtained from all rats under fasting conditions at sacrifice. Levels of serum triglyceride (TG), total cholesterol (CHO), and low-density lipoprotein (LDL) were measured using a Konelab prime 30i automated analyzer (Thermo scientific, Waltham, MA, USA).
Histological analysis of alveolar and tibial bones
Mandibles dissected from rats were fixed with 10% neutral-buffered formalin for 24 h and embedded in paraffin after decalcification with 10% ethylenediaminetetraacetic acid (EDTA) for 2 months. Sagittal sectioned mandibles were cut at 4 μm thickness. Sections, including clear dental pulp of the mesial and distal roots of the first molar, were selected and stained with hematoxylin and eosin (H&E). For determination of alveolar bone loss, the distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) was measured in the distal area, and the alveolar bone area was measured in the region of interest (ROI) of the furcation. Then, the alveolar bone area in the ROI was divided by the ROI. The ROI was 0.8 mm downward from the top of the furcation. For the evaluation of alveolar bone formation, the light pink unmineralized osteoid area between the alveolar bone surface and osteoblasts was measured in the ROI, which was 0.5 mm downward from the ABC in the furcation. For the determination of tibial bone loss, tibiae were fixed with 10% neutral-buffered formalin. After 24 h of fixation, left tibiae were decalcified with 10% EDTA for 1 month after which paraffin embedding was performed. The paraffin blocks were sectioned as previously described, and the sections containing the clear proximal epiphyseal growth plate were selected and H&E stained. The ROI for trabecular bone extended 1.0 mm from 1.0 mm below the growth plate toward the diaphysis (excluding the outer cortical shell). The area of remaining trabecular bone in the ROI was measured and then divided by the ROI. An Aperio AT2 Digital Whole Slide scanner (Leica Microsystems Inc., Buffalo Grove, IL, USA) and Aperio ImageScope software (version 12.3.2.2013, Aperio Technologies Inc., Vista, CA, USA) were used for the slide scanning and analysis, respectively.
Micro-computed tomography analysis of tibiae
Right tibiae were scanned using micro-computed tomography (m-CT, Skyscan1076, Kontich, Belgium). The scanning parameters were set at 70 kV and 139 μA with an exposure time of 1475 ms and a resolution of 18 μm. The trabecular bone structures extending 2 mm from 0.7 mm below the epiphyseal growth plate were chosen as the ROI [24], and three-dimensional images of the specimens were reconstructed. Bone volume fraction (BVF), BMD, bone surface density (BSD), and trabecular number (Tb. N) in the ROI were calculated using the NRecon software (Bruker SkyScan, Aartselaar, Belgium).
Tartrate-resistant acid phosphatase staining
To identify osteoclast formation in tibiae sections, tartrate-resistant acid phosphatase (TRAP) staining was performed using a TRAP stain kit (Wako chemicals, Osaka, Japan). Methyl green (Trevigen, Gaithersburg, MD, USA) was used for the counterstaining, and slides were scanned as described above. The number of TRAP-positive cells in the ROI were counted along the trabecular bone surface, divided by the bone length, and then divided by the percentage of bone area remaining in the ROI. The ROI was area extended 1.0 mm from 1.0 mm below the growth plate toward the diaphysis (excluding the outer cortical shell).
Immunohistochemistry staining
To assess sclerostin expression in tibiae, tibiae sections were deparaffinized in xylene and rehydrated through ethanol series. Sections were incubated with Trypsin Enzymatic Antigen Retrieval Solution (Abcam, Cambridge, UK) for 15 min at 37 °C followed by immersion in 3% H2O2 in methanol for 20 min to block endogenous peroxidase. After blocking in normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 20 min, the sections were incubated overnight at 4 °C with goat anti-sclerostin antibody (1:50 dilution, R&D Systems, Minneapolis, MN, USA). After incubation with anti-goat secondary antibody (Vector Laboratories) for 20 min, sections were developed with 3,3-diaminobenzidine tetrahydrochloride substrate chromogen system (DAKO, Botany, Australia) and counterstained with methyl green. Slides were scanned as described above. The number of sclerostin-positive osteocytes in the ROI was divided by the number of total osteocytes in the ROI. The ROI was the same as the ROI for osteoclast counting.
Statistical analysis
All statistical comparisons were made using a statistical analysis program (SPSS, Chicago, IL, USA). Significant differences were determined using one-way analysis of variance with Scheffé’s post hoc test. A value of P < 0.05 was considered statistically significant. Data are expressed as the mean ± standard error (SE).
