Interferon-γ enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects

JPOR-318; No. of Pages 10 journal of prosthodontic research xxx (2016) xxx–xxx

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Original article

Interferon-g enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects Peiqi Li DDSa, Yoshitomo Honda DDS, PhDb,*, Yoshiyuki Arima DDS, PhDa, Kenichirou Yasui DDS, PhDa, Kaoru Inami DDS, PhDa, Aki Nishiura DDS, PhDa, Yoshiya Hashimoto DDS, PhDc,**, Naoyuki Matsumoto DDS, PhDa a

Department of Orthodontics, Osaka Dental University, 8-1, Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan Institute of Dental Research, Osaka Dental University, 8-1, Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan c Department of Biomaterials, Osaka Dental University, 8-1, Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan b

article info

abstract

Article history:

Purpose: Interferon (IFN)-g is a major cytokine produced by immune cells that plays diverse

Received 2 October 2015

roles in modulating both the immune system and bone metabolism, but its role in autoge-

Received in revised form

nous bone grafting remains unknown. Here, we present that local IFN-g administration

29 December 2015

improved the efficacy of autogenous bone graft treatment in an experimental rat model.

Accepted 16 January 2016

Methods: An autogenous bone graft model was prepared with critically sized rat calvariae

Available online xxx

defects. Four weeks (w) after bone graft implantation, rats were treated locally with IFN-g or were not treated. The effect of IFN-g on bone formation was evaluated for up to 8 w with

Keywords:

micro-computed tomography, quantitative histomorphometry, and Von Kossa staining.

Autogenous bone graft

Osteoclastogenesis was assessed by tartrate-resistant acid phosphatase staining. Immuno-

Osteoclast

histochemistry staining or quantitative polymerase chain reactions were used to estimate

Interferon-g

the expression of osteoclast differentiation factor and inflammatory cytokines including

Bone formation

tumor necrosis factor (TNF)-a, a well-known stimulant of osteoclastogenesis and an inhibi-

Inflammation

tor of osteoblast activity, in defects. Results: Newly formed bone gradually replaced the autogenous bone grafts within 4 w, although severe bone resorption with osteoclastogenesis and TNF-a expression occurred after 6 w in the absence of IFN-g administration. IFN-g administration markedly attenuated bone loss, osteoclastogenesis, and TNF-a expression, while it enhanced bone formation at 8 w. Conclusion: Local IFN-g administration promoted bone formation in autogenous bone grafts possibly via regulating osteoclastogenesis and TNF-a expression. The data provide insights into the potential roles of IFN-g in autogenous bone grafting. # 2016 Published by Elsevier Ltd on behalf of Japan Prosthodontic Society.

* Corresponding author. Tel.: +81 72 864 3130. ** Corresponding author. Tel.: +81 72 864 3260. E-mail addresses: [email protected] (Y. Honda), [email protected] (Y. Hashimoto). http://dx.doi.org/10.1016/j.jpor.2016.01.002 1883-1958/# 2016 Published by Elsevier Ltd on behalf of Japan Prosthodontic Society.

Please cite this article in press as: Li P, et al. Interferon-g enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects. J Prosthodont Res (2016), http://dx.doi.org/10.1016/j.jpor.2016.01.002

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1.

Introduction

Autogenous bone grafting remains the gold standard for the treatment of bone loss due to trauma, tumor, surgery, and periodontal diseases [1–4]. Approximately 1 million dental bone grafting procedures were performed in the United States in 2006, and its number is thought to grow by more than 15% annually [5]. The bone graft is also applied in alveolar ridge reconstruction for implant therapy [6]. Nevertheless, it is widely accepted that the grafted bone often induces bone loss at a later stage in the recovery process [7,8]. The mechanism of bone loss following bone grafting is incompletely understood. However, it is thought that attenuated osteoclastic activity during bone turnover may be responsible for maintaining the bone formation of bone grafts [8–10]. Indeed, results from previous studies have shown that bisphosphonates, which are potent bone antiresorptive agents, can enhance bone graft efficacy during osseous wound healing by strongly inhibiting osteoclastogenesis [9,10]. However, severe side effects such as bisphosphonate-related osteonecrosis of the jaws and soft-tissue inflammation have been occasionally reported [11,12], which hinder applications of bisphosphonates for bone grafting in dental surgery. Therefore, alternative strategies for increasing the efficacy of autogenous bone grafts can improve for the success of bone regeneration in dentistry. Severe osteoclastogenesis is often associated with inflammatory responses [13,14]. Over-expression of inflammatory cytokines is known to cause activation of osteoclast and osteolysis, which are the main reasons for implantation failure with artificial bone grafts [15,16]. In particular, inflammatory cytokines such as tumor necrosis factor (TNF)-a and interleukin (IL)-1b collaborate with receptor activator of nuclear factor-kappa B ligand (RANKL) and dramatically enhance osteoclast precursor maturation and migration [17–19]. TNF-a has been reported to play a pivotal role in acute and chronic inflammation [20]. Thus far, only limited data are available regarding the role of inflammatory cytokines and osteolysis on postoperative recovery with autogenous bone grafts. Given these considerations, the overexpression of inflammatory cytokines in tissues surrounding bone defects is likely to facilitate the bone loss. The attenuation of these cytokines might be key for maintaining the efficacy of autogenous bone grafts. Interferon (IFN)-g is known to modulate multiple functions during immune responses [21]. IFN-g has been recently reported as an osteoclastogenesis-blocking cytokine that modulates inflammatory responses in vivo and in vitro at non-infectious sites [22–24]. Recent findings have demonstrated that IFN-g plays an important role in bone formation [25], suggesting its potential for enhancing bone grafting efficacy. However, few studies have explored the role of IFN-g in bone grafting, in contrast to the numerous studies on the role of IFN-g in inflammatory bone resorption in systemic diseases such as osteoporosis. The present study was designed to investigate whether local administration of IFN-g modulates inflammatory cytokine production and bone-turnover ability, thereby rescuing autogenous bone graft efficacy. To address this issue, we first

established a postoperative bone-loss model of autogenous bone graft treatment in critically sized defects of rat calvarias. Four weeks after the bone graft procedure, IFN-g was administrated at the graft sites. Morphometric changes in bone were assessed by micro-computed tomography (m-CT) and Von Kossa staining, and inflammatory cytokine production was evaluated by immunohistochemistry and quantitative real-time (q)PCR. To verify the mechanism whereby IFN-g regulates bone resorption, we measured the effect of IFN-g on osteoclastogenesis, using rat primary preosteoclast in vitro. Zoledronate (Zol) was used as a positive control for the inhibition of bone resorption.

2.

Materials and methods

2.1. Experimental autogenous bone graft model and drug injection Eight-week (w)-old male Sprague-Dawley rats (250–270 g; SHIMIZU Laboratory Supplies Co., Kyoto, Japan) were used for transplantation studies. The experimental strictly followed the protocol approved by the Animal Care and Use Committee of Osaka Dental University (Admission Number: 13-02016; March 29, 2013). A critically sized bone defect (diameter: 9 mm, depth: 1.0 mm) was created at the center of each rat skull using a Bond Trephine 8.0 drill (Implatex Co. Ltd., Tokyo, Japan). Calvarial bone was extracted from the defects and immediately fragmented into 0.1–1 mm3 pieces using a Bone Mill Mini Barrel (YDM Corporation, Tokyo, Japan). To prepare each autogenous bone graft, the shattered calvarial bone was added back to the bone-defect area without any intrusion or added solution (Appendix Fig. 1A). Each bone defect area was covered with an absorbable collagen GTR Membrane (KOKEN TISSUE GUIDE; Koken, Tokyo, Japan) after implantation. The operations were performed under strictly aseptic conditions. Grafted bone underwent bone remodeling and facilitated the generation of newly formed bone by 4 w, when subtle changes in TNF-a expression occurred (Figs. 1B and 4A; Appendix Fig. A1). Next, 4 w after the bone graft procedures were performed, 3 groups were prepared as follows: (i) a nodrug treatment control group, (ii) a Zol-injection group, and (iii) an IFN-g-injection group. Four rats were evaluated at each time point for each group (N = 4). After 4 w, 9 mg of IFN-g (Bioss, MA, USA) was locally injected (3 times/w) at the operation site (Fig. 1A). As for Zol, rats were treated with 100 mg of Zol (Cayman Chemical Company, Ann Arbor, MI, USA) injection once a week. IFN-g and Zol were dissolved in Otsuka normal saline (Otsuka, Tokyo, Japan) and used for injections. Although administration of saline alone could have been used for the control group, a no-drug-treatment control was used to represent conventional clinical procedures. The doses of IFN-g and Zol applied were based on previous dose described in the literature [22,26]. The timing of drug administration was determined by referring to the TRAP-staining results obtained with the control group in a pilot study, which showed strong osteoclast formation at 6 w post-treatment. To evaluate the osteogenesis dynamics after drug injection, the following 3 fluorescence labels were injected into the bone defects at the indicated time points following bone grafting: calcein

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Fig. 1 – Effect of in vivo IFN-g 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)-g 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 2 mm for the long and short bars, respectively. (C) Quantitative analysis of post-operative bone volumes/tissue volumes (BV/TV) and BMDs. Data show mean W SD (N = 4). *P < 0.05, **P < 0.01 (analysis of variance [ANOVA] with the Tukey-Kramer method).

(5 mg/kg; Wako Pure Chemical Industries Co., Osaka, Japan; 4 w), tetracycline (25 mg/kg; Wako Pure Chemical Industries Co.; 6 w), and Alizarin Red S (25 mg/kg; Wako Pure Chemical Industries Co.; 3 days before the animals were sacrificed).

2.2.

(TRAP) and alkaline phosphatase (ALP) staining with the TRAP/ALP Kit (Wako Pure Chemical Industries Co) was used for osteoclast identification and monitoring the activity of osteoblasts. After staining, sections were observed with a BZ9000 digital microscope (Keyence Co., Osaka, Japan).

m-CT experiments and data analysis 2.4.

Treated calvaria were scanned with an SMX-130CT micro-CT scanner (65 kV, 90 mA; Shimadzu, Kyoto, Japan) immediately after the rats were euthanized. Calvarial bones were measured in 3 dimensions, and their structural indices were calculated using a morphometric program (TRI/3D-BON; Ratoc System Engineering, Tokyo, Japan). The bone volume (BV) and total volume (TV) in the prepared defects were evaluated to calculate the BV/TV ratio (%). In addition to BV, we also quantified the bone mineral density (BMD) in terms of calcified bone tissue using cylindrical phantoms containing a hydroxyapatite (HA content: 200–800 mg/cm3).

2.3.

Histological observations

Samples were fixed in 4% paraformaldehyde for 16 h. Fourmicrometer-thick, non-decalcified frozen sections were obtained using the Kawamoto method [27]. Von Kossa staining, using the Von Kossa Method for Calcium Kit (Polysciences Inc., Warrington, England), was performed for the histological bone tissue observations. Tartrate-resistant acid phosphatase

Cell culture and staining

The Osteoclast Culture Kit V-4 (#OSC11, Cosmo Bio, Tokyo, Japan) was used for evaluating the effects of IFN-g on osteoclastogenesis. Primary preosteoclasts seeded at 1.9  105 cells/cm2 were exposed to culture medium containing 15 ng/mL RANKL, 100 ng/mL TNF-a, and increasing amounts of IFN-g (0–100 ng/mL; Bioss) for 5 days. The cells were fixed and stained with the TRAP Staining Kit (PMCAK04F-COS, Cosmo Bio). As for RANKL-immunostaining experiments, the UMR106 osteoblast cell line (CRL-1661; ATCC, Manassas, USA) was cultured for 3 days in alpha-Minimum Essential Medium containing 10% fetal bovine serum (FBS; Sigma, St. Louis, USA), antibiotics, 100 ng/mL TNF-a, and varying concentrations of IFN-g (0, 10, or 100 ng/mL). At day 3, cells were permeabilized with 1% Triton X-100 in PBS and blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS). Immunostaining was performed against anti-RANKL antibodies (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by Alexa 568-conjugated secondary antibodies (Biotium, CA, USA). DAPI

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(SouthernBiotech, Birmingham, AL, USA) was used for nuclear staining.

2.5.

Bone histomorphometry

Osteogenesis dynamics were measured using fluorescently labeled sections, which were observed under an LSM700 laserscanning microscope (Zeiss, Jena, Germany). Each dye was excited using lasers of different wavelengths, namely, 488 nm (calcein, green), 405 nm (tetracycline, blue), and 555 nm (Alizarin Red S, red). Mineral apposition rate (MAR, mm/day) was calculated as follows: the distance between colors was divided by prescribed days (14 days for 4–6 w and 11 days for 6–8 w).

2.6.

Statistical analysis

Statistical analysis was performed with Statcel3 software (OMS, Tokyo, Japan). For all experiments, values are reported as the mean  standard deviation (SD). For comparisons between 3 groups, differences were evaluated by one-way analysis of variance (ANOVA) with the Tukey–Kramer method.

3.

3.2. Effects of IFN-g administration on bone resorption following bone graft procedures

qPCR analysis and immunohistochemistry

Total RNA from bone defects was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany). qPCR was performed with a Universal ProbeLibrary set and a FastStart Universal Probe Master (Roche Diagnostics, Mannheim, Germany). The sequences of the primers and probes used are shown in Appendix Table A1. Expression of 18S mRNA was evaluated using a Gene Expression Assay (#4310893E, ThermoFisher Scientific Inc., Waltham, MA, USA). A comparative Ct method was used to calculate mRNA expression levels. Immunostaining was used to detect TNF-a production in bone defect sections. Endogenous peroxidase was blocked using a blocking solution (BLOXALL, Vector Labs, Burlingame, CA, USA) for 10 min. The sections were blocked in 5% goat serum (Vector Labs) for 30 min. The sections were then labeled for 30 min with an anti-TNF-a polyclonal antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:300 in PBS. After careful washing for 5 min, the sections were incubated for 30 min with an appropriate secondary antibody (Vector Labs). Subsequently, sections were washed for 5 min and processed using the VECTASTAIN ABC Kit (Vector Labs), and peroxidase activity was visualized by purple staining with an anti-TNF-a antibody and the VECTOR VIP Kit (Vector Labs).

2.7.

suggesting that the bone graft animal model used in this study replicated postoperative osteolysis (control group of Fig. 1B and C). Coincident with the osteolysis observed in the m-CT images, we also observed strong TRAP staining (6 and 8 w) and Rankl mRNA expression in the defects of the control group, indicating that osteolysis was due to activated osteoclasts and osteoclastogenesis (control group of Fig. 2A and C). Simultaneous augmentation of TNF-a staining and Tnf-a mRNA expression at the bone defects after 6 w supports the possibility that inflammatory responses were involved in postoperative osteolysis of the bone grafts (control group of Fig. 2B and C).

Results

3.1. Postoperative bone resorption following bone-graft procedures Fig. 1B and C shows the m-CT images and structural parameters representing morphometric changes of bone grafts at bone defect sites. As reported for bone resorption in clinical observations [28], grafted autogenous bones (without drug administration) showed a decrease in their radiopacities and BV/TV ratios in the 8 w control group,

m-CT images and related quantitative data for the Zol and IFNg groups in Fig. 1 show morphological changes occurring with autogenous bone grafts after the injection of each drug, beginning at 4 w post-surgery. The BVs and BMDs were significantly higher than that of the control group even at 8 w, suggesting that Zol and IFN-g administration markedly inhibited osteolysis (Fig. 1B and C). To elucidate the mechanism whereby IFN-g- and Zol administration suppressed osteolysis, we further evaluated inflammatory cytokine production in tissues surrounding the bone grafts by performing immunostaining and qPCR. TNF-a staining was robust at 8 w in the control and Zol groups (Fig. 2B), while administration of IFN-g markedly attenuated TNF-a staining. The mRNA expression of Rankl and Tnf-a was significantly lower in tissues treated with IFN-g compared with that observed in the control and Zol groups. No significant differences were found for IL-1b and Opg expression between the control and drug-treated groups. These in vivo results suggest that IFN-g and Zol administration both inhibited osteoclastogenesis in tissues surrounding the bone grafts, while the associated mechanisms were clearly different, with IFN-g likely modulating inflammatory cytokine production starting at 4 w.

3.3. IFN-g blocks TNF-a- and RANKL-induced osteoclastogenesis in vitro To evaluate its effect on osteoclastogenesis, we administered IFN-g to primary preosteoclasts and the osteoblastic cell line UMR106 in the presence of TNF-a and RANKL in vitro. TRAPstaining images and related quantitative data showed that IFN-g significantly inhibited osteoclast formation in a dosedependent manner (Fig. 3A and B). The expression of RANKL, a key regulator of osteoclastogenesis [29], was attenuated in UMR106 cells by IFN-g administration in a dose-dependent manner (Fig. 3C). These results supported that IFN-g may inhibit bone loss in vivo (at least in part) by hindering TNF-a/ RANKL-induced osteoclastogenesis in preosteoclasts and osteoblasts.

3.4.

Effects of IFN-g administration on bone formation

Careful observation of the m-CT images from the Zol and IFN-g group revealed that a large degree of coarse radiopacity occupied in the defects treated with Zol at 6 and 8 w

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Fig. 2 – Effect of IFN-g 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)-a immunostaining (purple). (A, B) Bars = 120 mm. (C) mRNA expression of genes closely related to osteoclast differentiation and bone resorption (Rankl, Tnf-a, IL-1b, and Opg) in bone defects. Data show the mean W SD (N = 4). #Control vs. IFN-g, *Zol vs. IFN-g; *,#P < 0.05, **,##P < 0.01 (ANOVA with the Tukey–Kramer method).

post-surgery, while the radiopacity were integrated in the IFNg-injection groups (Zol and IFN-g groups of Fig. 1B). To evaluate these differences, we next studied bone-metabolism by histochemical analysis (Fig 4). Fig. 4A shows Von Kossastaining results; the dark brown regions show mineralized tissues associated with bone (Fig. 4A). Significant bone degradation was seen at 8 w in the control group. In the Zol group, grafted autogenous bone (arrow) remained in the defect and integrated slowly. The bone fragments (asterisk) were still visible after 8 w in the Zol group. In contrast, bone fragments were not observed in the IFN-g group, and newly formed bone occupied in the bone defect even up to 8 w. We next measured the fluorescent labeling of mineral deposits and ALP expression to assess the osteogenesis dynamics in each group (Fig. 4B–D). Three fluorescence colors representing mineral deposition over time indicated the occurrence of significantly higher mineral deposition in the IFN-g-defects, compared to that of the control group. Weak ALP expression was observed in the bone defects at 6 and 8 w post-treatment in the control group, while robust ALP expression was observed with the IFN-g and Zol groups, indicating that osteoblasts facilitated bone defect repair (Fig. 4D). These results suggest that IFN-g inhibited bone resorption, while enhancing osteogenesis.

4.

Discussion

Results from long-term clinical studies have indicated that bone implants degrade gradually over time, possibly due to

inflammatory reactions in tissues surrounding bone grafts [8]. In the present study, we demonstrated that IFN-g administration significantly reduced excess osteoclastogenesis and TNF-a expression, while it elevated bone formation, resulting in increased postoperative bone graft efficacy. Implanted bone resorption appears to be highly related to RANKL and TNF-a overexpression in critically sized defects of rat calvaria. Takayanagi et al. reported that IFN-g potently inhibits osteoclastogenesis by interfering with the RANK-RANKL signaling pathway [24]. In agreement, our in vitro results showed that IFN-g administration decreased TRAP staining and Rankl expression induced by bone grafts in vivo. In in vitro experiments, IFN-g potently inhibited TRAP staining in cells cultured in the presence of TNF-a and RANKL (Fig. 3A and B). In addition, IFN-g caused a marked decrease in RANKL expression by UMR106 osteoblasts in vitro. These results suggested that the attenuation of bone resorption induced by IFN-g administration may have been partially due to a direct effect of IFN-g in both preosteoclasts and osteoblasts. TNF-a is well-known regulator of chronic inflammation [20]. As mentioned above, TNF-a potently stimulates osteoclastogenesis [30]. Thus, it is possible that high TNF-a expression in bone defects facilitated osteoclastogenesis from 4 w post-surgery. Indeed, the complete inhibition of severe osteoclastogenesis (as determined by TRAP staining) and bone resorption coincided with reduced TNF-a expression following IFN-g administration (Figs. 1 and 2). In the present study, we did not determine the exact mechanism

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Fig. 3 – Inhibitory effect of IFN-g on osteoclastogenesis in vitro. (A) TRAP staining in TNF-a/RANKL-induced osteoclasts. Primary rat bone marrow macrophages were cultured for 5 days in the presence of RANKL (15 ng/mL) and TNF-a (100 ng/ mL) with varying concentrations of IFN-g (0–100 ng/mL). Bar = 50 mm. (B) Quantitative data of TRAP-positive cells per well. Data show the mean W SD (N = 4). *P < 0.05, **P < 0.01 (ANOVA with the Tukey–Kramer method). (C) Effect of IFN-g on RANKL expression in the UMR106 osteoblast cell line. UMR106 cells were cultured for 3 days in the presence of 100 ng/mL TNF-a and varying concentrations of IFN-g (0–100 ng/mL). Nuclear staining is shown in blue and RANKL staining is shown in red. Bars = 20 mm.

whereby IFN-g attenuates TNF-a expression in bone defects at 4 w after bone grafting. However, Iwakura and Ishigame reported that IFN-g indirectly decreased TNF-a secretion by interfering with immune cells (such as Th17 cells) during chronic inflammation [31]. Results from recent studies have indicated that stimulated osteoclast can secrete TNF-a [32]. As shown in the present study, IFN-g reduced osteoclastogenesis (Figs. 2 and 3). Thus, IFN-g may suppress TNF-a production by interfering with various immune cells and osteoclasts; this suppression hindered the deterioration of osteoclastogenesis induced by the spontaneous feedback system between the osteoclastogenesis and TNF-a expression. Anti-TNF-a antibody treatment attenuates bone loss in arthritis [33], suggesting its potential for inhibiting

TNF-a-dependent, postoperative bone loss with autogenous bone grafts. However, this antibody is known to lack the capability of inducing bone formation [33]. In the present study, IFN-g administration promoted bone formation in vivo, which induced an almost complete repair of critically sized defects after 6 w, in contrast to findings with the control group (Figs. 1 and 4). This finding may have been due to the dual effects of IFN-g on osteoblast activity and its attenuation of TNF-a expression. Both exogenous and endogenous IFN-g enhance osteoblastogenesis in human mesenchymal stem cells in vitro [34]. Previous studies have reported that TNF-a potently inhibits osteoblast differentiation [19] and mediates bone formation [2]. In the present study, IFN-g administration completely decreased TNF-a expression in vivo compared with the control (Fig. 2B). The

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Fig. 4 – Synergistic enhancement of osteogenic capacity following IFN-g 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 4 w after the bone graft; tetracycline at 6 w; 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 4–6 w and 11 days for 6–8 w). Data show the mean W SD (N = 4). #Control vs. IFN-g, #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), 1.8 mm and 120 mm; mineral apposition and ALP staining, 100 mm and 120 mm.

enhanced bone formation induced by IFN-g administration appears to reflect a suitable environment enabling osteoblasts to form new bone. To date, various studies have reported effects of bisphosphonate [35–37] and IFN-g [24] in osteoclasts. For example, bisphosphonate induces apoptosis in osteoclasts [38] after adsorption to the bone matrix [36], whereas IFN-g treatment attenuates osteoclastogenesis via the RANKL-signaling pathway [24]. In the present study, RANKL and TNF-a expression levels at 6 and 8 w post-surgery differed in bone defects treated with Zol or IFN-g. TNF-a production was low in the IFN-g-injection group at 8 w postsurgery, compared to that observed in the Zol-injection group. These results suggest that Zol and IFN-g administration function through different mechanisms during bone resorption at autogenous bone graft sites. In light of the complications associated with Zol administration in

dentistry, IFN-g administration might serve as an alternative option to bisphosphonate administration. However, the relative efficacies of IFN-g and Zol administration remain unclear because of the limited conditions utilized in the present study. The rapid advance of stem cell engineering [39] has stimulated interest in the role of immune systems in stem cell-based bone regeneration therapy [40]. The findings presented here demonstrate that IFN-g administration can enhance bone graft efficacy, possibly by modulating the secretion of inflammatory cytokines related to bone resorption. However, certain limitations were associated with the present study. Unfortunately, we could not determine the cause of bone resorption at 4 w after post-operation. Understanding that trigger would provide key information for enhancing bone graft efficacy. Furthermore, basic information regarding the optimal concentration and timing

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of IFN-g administration would be useful for clinical applications.

Conflicts of interest

5.

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Conclusions

We generated an experimental bone loss model using autogenous bone grafts in rat calvariae. Without drug treatment, severe osteolysis with Tnf-a and Rankl overexpression occurred in bone defects 6 w after bone grafting. Postoperative IFN-g injection potently inhibited bone loss by modulating osteoclastogenesis, resulting in the enhancement of bone mass. In addition, IFN-g inhibited expression of the inflammatory cytokine TNF-a, a potent inhibitor of osteoblast activity. These results suggest that TNF-a is likely to play a critical role in postoperative bone loss following autogenous bone graft treatment. Hence, administering a modulator of osteoimmune function such as IFN-g may enhance the efficacy of autogenous bone grafts—the current gold standard for bone regeneration— in dentistry.

Acknowledgements This study was supported in part by a JSPS KAKENHI Grant number 25861904 and Osaka Dental University Research Founds (Grant no. 15-08) in Japan.

Appendix Fig. A1. Table A1.

Fig. A1 – Histological images of bone defects at 1 w 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)-a staining. Arrows: grafted autogenous bones. Bars: low magnification images, 1.8 mm; high magnification images, 120 mm.

Table A1 – Sequences of primer and probe used for qPCR analysis. mRNA Rankl Opg Tnf-a IL-1b

Sequence Forward Reverse Forward Reverse Forward Reverse Forward Reverse

Probe No. 0

5 -AGACACAGAAGCACTACCTGACTC-3 50 -GGCCCCACAATGTGTTGTA-30 50 -TGAGGTTTCCAGAGGACCAC-30 50 -GGAAAGGTTTCCTGGGTTGT-30 50 -GCCCAGACCCTCACACTC-30 50 -CCACTCCAGCTGCTCCTCT-30 50 -TGTGATGAAAGACGGCACAC-30 50 -CTTCTTCTTTGGGTATTGTTTGG-30

0

Accession No.

2

NM_057149.1

76

NM_012870.2

119 78

X66539.1 NM_031512.2

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Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpor. 2016.01.002.

references [1] Elsalanty ME, Genecov DG. Bone grafts in craniofacial surgery. Craniomaxillofac Trauma Reconstr 2009;2:125–34. [2] Jimi E, Hirata S, Osawa K, Terashita M, Kitamura C, Fukushima H. The current and future therapies of bone regeneration to repair bone defects. Int J Dent 2012;2012:148261. [3] Movahed R, Pinto LP, Morales-Ryan C, Allen WR, Wolford LM. Application of cranial bone grafts for reconstruction of maxillofacial deformities. Proc (Bayl Univ Med Cent) 2013;26:252–5. [4] Kim YK, Lee J, Yun JY, Yun PY, Um IW. Comparison of autogenous tooth bone graft and synthetic bone graft materials used for bone resorption around implants after crestal approach sinus lifting: a retrospective study. J Periodontal Implant Sci 2014;44:216–21. [5] Lupovici J. Regeneration of the anterior mandible: a clinical case presentation. JIRD 2009;1:31–4. [6] Masaki C, Nakamoto T, Mukaibo T, Kondo Y, Hosokawa R. Strategies for alveolar ridge reconstruction and preservation for implant therapy. J Prosthodont Res 2015;59:220–8. [7] Widmark G, Andersson B, Ivanoff CJ. Mandibular bone graft in the anterior maxilla for single-tooth implants: presentation of a surgical method. Int J Oral Maxillofac Surg 1997;26:106–9. [8] Toker H, Ozdemir H, Ozer H, Eren K. Alendronate enhances osseous healing in a rat calvarial defect model. Arch Oral Biol 2012;57:1545–50. [9] Myoung H, Park JY, Choung PH. Effects of a bisphosphonate on the expression of bone specific genes after autogenous free bone grafting in rats. J Periodontal Res 2001;36:244–51. [10] Belfrage O, Isaksson H, Tagil M. Local treatment of a bone graft by soaking in zoledronic acid inhibits bone resorption and bone formation. A bone chamber study in rats. BMC Musculoskelet Disord 2012;13:240. [11] Reid IR, Cornish J. Epidemiology and pathogenesis of osteonecrosis of the jaw. Nat Rev Rheumatol 2012;8:90–6. [12] Kharazmi M, Persson U, Warfvinge G. Pharmacovigilance of oral bisphosphonates: adverse effects manifesting in the soft tissue of the oral cavity. J Oral Maxillofac Surg 2012;70:2793–7. [13] Ollivere B, Wimhurst JA, Clark M, Donell IST. Current concepts in osteolysis. J Bone Joint Surg-BV 2012;94-B:10–5. [14] Childs LM, Paschalis EP, Xing L, Dougall WC, Anderson D, Boskey AL, et al. In vivo RANK signaling blockade using the receptor activator of NF-kB:Fc effectively prevents and ameliorates wear debris-induced osteolysis via osteoclast depletion without inhibiting osteogenesis. J Bone Miner Res 2002;17:192–9. [15] Van der Meulen J, Koerten HK. Inflammatory response and degradation of three types of calcium phosphate ceramic in a non-osseous environment. J Biomed Mater Res 1994;28:1455–63. [16] Lange T, Schilling AF, Peters F, Haag F, Morlock MM, Rueger JM, et al. Proinflammatory and osteoclastogenic effects of beta-tricalciumphosphate and hydroxyapatite particles on human mononuclear cells in vitro. Biomaterials 2009;30:5312–8.

[17] Wei S, Kitaura H, Zhou P, Patrick Ross F, Teitelbaum SL. IL-1 mediates TNF-induced osteoclastogenesis. J Clin Investig 2005;115:282–90. [18] Gallo J, Rasˇka M, Mra´zek F, Pet’ek M. Bone remodeling, particle disease and individual susceptibility to periprosthetic osteolysis. Physiol Res 2008;57:339–49. [19] Kitaura H, Kimura K, Ishida M, Kohara H, Yoshimatsu M, Takano-Yamamoto T. Immunological reaction in TNFalpha-mediated osteoclast formation and bone resorption in vitro and in vivo. Clin Dev Immunol 2013;2013:181849. [20] Popa C, Netea MG, van Riel PL, van der Meer JW, Stalenhoef AF. The role of TNF-alpha in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res 2007;48:751–62. [21] Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferongamma: an overview of signals, mechanisms and functions. J Leukoc Biol 2004;75:163–89. [22] Kohara H, Kitaura H, Fujimura Y, Yoshimatsu M, Morita Y, Eguchi T, et al. IFN-g directly inhibits TNF-a-induced osteoclastogenesis in vitro and in vivo and induces apoptosis mediated by Fas/Fas ligand interactions. Immunol Lett 2011;137:53–61. [23] Ji JD, Park-Min KH, Shen Z, Fajardo RJ, Goldring SR, McHugh KP, et al. Inhibition of RANK expression and osteoclastogenesis by TLRs and IFN-g in human osteoclast precursors. J Immunol 2009;183:7223–33. [24] Takayanagi H, Ogasawara K, Hida S, Chiba T, Murata S, Sato K, et al. T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. Nature 2000;408:600–5. [25] Duque G, Huang DC, Dion N, MacOritto M, Rivas D, Li W, et al. Interferon-g plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice. J Bone Miner Res 2011;26:1472–83. [26] Holen I, Walker M, Nutter F, Fowles A, Evans CA, Eaton CL, et al. Oestrogen receptor positive breast cancer metastasis to bone: inhibition by targeting the bone microenvironment in vivo. Clin Exp Metastasis 2015. [27] Kawamoto T, Kawamoto K. Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using kawamot’s film method (2012). In: Hilton MJ, editor. Skeletal development and repair. Humana Press; 2014. p. 149–64. [28] Peng W, Kim IK, Cho HY, Pae SP, Jung BS, Cho HW, et al. Assessment of the autogenous bone graft for sinus elevation. J Korean Assoc Oral Maxillofac Surg 2013;39: 274–82. [29] Xing L, Schwarz EM, Boyce BF. Osteoclast precursors, RANKL/ RANK, and immunology. Immunol Rev 2005;208:19–29. [30] Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–42. [31] Iwakura Y, Ishigame H. The IL-23/IL-17 axis in inflammation. J Clin Investig 2006;116:1218–22. [32] Li H, Hong S, Qian J, Zheng Y, Yang J, Yi Q. Cross talk between the bone and immune systems: osteoclasts function as antigen-presenting cells and activate CD4+ and CD8+ T cells. Blood 2010;116:210–7. [33] Saidenberg-Kermanac’h N, Corrado A, Lemeiter D, deVernejoul MC, Boissier MC, Cohen-Solal ME. TNF-alpha antibodies and osteoprotegerin decrease systemic bone loss associated with inflammation through distinct mechanisms in collagen-induced arthritis. Bone 2004;35:1200–7. [34] Duque G, Huang DC, Macoritto M, Rivas D, Yang XF, SteMarie LG, et al. Autocrine regulation of interferon gamma in mesenchymal stem cells plays a role in early osteoblastogenesis. Stem Cell 2009;27:550–8.

Please cite this article in press as: Li P, et al. Interferon-g enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects. J Prosthodont Res (2016), http://dx.doi.org/10.1016/j.jpor.2016.01.002

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[35] Kellinsalmi M, Monkkonen H, Monkkonen J, Leskela HV, Parikka V, Hamalainen M, et al. In vitro comparison of clodronate, pamidronate and zoledronic acid effects on rat osteoclasts and human stem cell-derived osteoblasts. Basic Clin Pharmacol Toxicol 2005;97:382–91. [36] Xu XL, Gou WL, Wang AY, Wang Y, Guo QY, Lu Q, et al. Basic research and clinical applications of bisphosphonates in bone disease: what have we learned over the last 40 years? J Transl Med 2013;11:303. [37] Ralte S, Bhattacharyya A. Effect of bisphosphonate on osteoclast of bone. FMAR 2014;2:56–62.

[38] Ito M, Amizuka N, Nakajima T, Ozawa H. Ultrastructural and cytochemical studies on cell death of osteoclasts induced by bisphosphonate treatment. Bone 1999;25:447–52. [39] Egusa H, Sonoyama W, Nishimura M, Atsuta I, Akiyama K. Stem cells in dentistry – Part I: stem cell sources. J Prosthodont Res 2012;56:151–65. [40] Kaku M, Akiba Y, Akiyama K, Akita D, Nishimura M. Cellbased bone regeneration for alveolar ridge augmentation – cell source, endogenous cell recruitment and immunomodulatory function. J Prosthodont Res 2015;59:96–112.

Please cite this article in press as: Li P, et al. Interferon-g enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects. J Prosthodont Res (2016), http://dx.doi.org/10.1016/j.jpor.2016.01.002