Interferon-γ enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects  Peiqi Li, DDS, Yoshitomo Honda, DDS, PhD, Yoshiyuki Arima, DDS, PhD, Kenichirou Yasui, DDS, PhD, Kaoru Inami, DDS, PhD, Aki Nishiura, DDS, PhD, Yoshiya Hashimoto, DDS, PhD, Naoyuki Matsumoto, DDS, PhD  Journal of Prosthodontic Research  Volume 60, Issue 3, Pages 167-176 (July 2016) DOI: 10.1016/j.jpor.2016.01.002 Copyright © 2016 Japan Prosthodontic Society Terms and Conditions

Fig. 1 Effect of in vivo IFN-γ administration on postoperative bone loss in an experimental rat model of autogenous bone grafting with critically sized defects in rat calvariae. (A) Scheme of experiments. Four weeks after the bone graft, rats were injected with interferon (IFN)-γ and Zoledronate (Zol). A no-drug treatment control group was utilized as an intact autogenous bone graft model. Zol was used as a positive control. (B) Micro-computed tomography and bone mineral density (BMD) images of the bone defects. Scale bars=10 and 2mm for the long and short bars, respectively. (C) Quantitative analysis of post-operative bone volumes/tissue volumes (BV/TV) and BMDs. Data show mean±SD (N=4). *P<0.05, **P<0.01 (analysis of variance [ANOVA] with the Tukey-Kramer method). Journal of Prosthodontic Research 2016 60, 167-176DOI: (10.1016/j.jpor.2016.01.002) Copyright © 2016 Japan Prosthodontic Society Terms and Conditions

Fig. 2 Effect of IFN-γ administration on osteoclastogenesis and immune responses in defects treated with bone grafts. (A) Tartrate-resistant acid phosphatase (TRAP) staining: the presence of osteoclasts in tissue sections. (B) Tumor necrosis factor (TNF)-α immunostaining (purple). (A, B) Bars=120μm. (C) mRNA expression of genes closely related to osteoclast differentiation and bone resorption (Rankl, Tnf-α, IL-1β, and Opg) in bone defects. Data show the mean±SD (N=4). #Control vs. IFN-γ, *Zol vs. IFN-γ; *,#P<0.05, **,##P<0.01 (ANOVA with the Tukey–Kramer method). Journal of Prosthodontic Research 2016 60, 167-176DOI: (10.1016/j.jpor.2016.01.002) Copyright © 2016 Japan Prosthodontic Society Terms and Conditions

Fig. 3 Inhibitory effect of IFN-γ on osteoclastogenesis in vitro. (A) TRAP staining in TNF-α/RANKL-induced osteoclasts. Primary rat bone marrow macrophages were cultured for 5 days in the presence of RANKL (15ng/mL) and TNF-α (100ng/mL) with varying concentrations of IFN-γ (0–100ng/mL). Bar=50μm. (B) Quantitative data of TRAP-positive cells per well. Data show the mean±SD (N=4). *P<0.05, **P<0.01 (ANOVA with the Tukey–Kramer method). (C) Effect of IFN-γ on RANKL expression in the UMR106 osteoblast cell line. UMR106 cells were cultured for 3 days in the presence of 100ng/mL TNF-α and varying concentrations of IFN-γ (0–100ng/mL). Nuclear staining is shown in blue and RANKL staining is shown in red. Bars=20μm. Journal of Prosthodontic Research 2016 60, 167-176DOI: (10.1016/j.jpor.2016.01.002) Copyright © 2016 Japan Prosthodontic Society Terms and Conditions

Fig. 4 Synergistic enhancement of osteogenic capacity following IFN-γ administration and autogenous bone grafting. (A) Von Kossa staining. The upper and lower panels show low and high magnification images, respectively. The broken-line squares in the upper panel indicate the magnified area. The arrow points to grafted autogenous bone. Asterisk: representative bone fragment. (B) Fluorescence labeling of mineral apposition over time. Calcein (green), tetracycline (blue), and Alizarin Red S (red) staining is shown. Each drug was injected into the bone defects at the indicated date as follows: calcein at 4w after the bone graft; tetracycline at 6w; and Alizarin Red S at 3 days before the animals were sacrificed. The white square represents the area that was used for the calculated values shown in (C). (C) Analysis of the mineral apposition rate (MAR). Calculation of MAR: the distance between two colors per prescribe days (14 days for 4–6w and 11 days for 6–8w). Data show the mean±SD (N=4). #Control vs. IFN-γ, #P<0.05 (ANOVA with the Tukey–Kramer method). (D) Alkaline phosphatase (ALP) expression in bone defects. Bars: von Kossa staining (upper and lower panels), 1.8mm and 120μm; mineral apposition and ALP staining, 100μm and 120μm. Journal of Prosthodontic Research 2016 60, 167-176DOI: (10.1016/j.jpor.2016.01.002) Copyright © 2016 Japan Prosthodontic Society Terms and Conditions

Fig. A1 Histological images of bone defects at 1w after bone graft treatment. Upper and lower panels show low and high magnification images, respectively. The broken-line squares in the upper panel show the magnified area. (A) Von Kossa staining. (B) Tartrate-resistant acid phosphatase (TRAP) in osteoclasts. (C) Tumor necrosis factor (TNF)-α staining. Arrows: grafted autogenous bones. Bars: low magnification images, 1.8mm; high magnification images, 120μm. Journal of Prosthodontic Research 2016 60, 167-176DOI: (10.1016/j.jpor.2016.01.002) Copyright © 2016 Japan Prosthodontic Society Terms and Conditions