NBD peptides have been described previously (16,18). A cell-permeable, wild type NBD peptide (DRQIKIWFQNRRMKWKK-TALDWS-WLQTE) was provided by GL Biochem (Shanghai) Ltd. (Shanghai, China) as lyophilized powder. Immediately prior to use, 20 mM stock solutions of the NBD peptide were prepared in dimethyl sulfoxide (DMSO). Stocks were diluted in culture medium to the final concentration required.

RAW264.7 cells [American Type Culture Collection (ATCC) no. TIB-71; Cell Bank of Chinese Academy of Sciences, Shanghai, China] were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 µg/ml streptomycin at 37°C in an atmosphere containing 5% CO₂. Fresh media were added every 2 days. To study osteoclast formation, RAW264.7 cells were cultured in complete α-minimum Eagle's medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) in the presence of NBD peptide at concentrations of 0 or 20 µM (18), and simultaneously stimulated with soluble recombinant mouse RANKL (100 ng/ml; R&D Systems, Inc., Minneapolis, MN, USA) (19). The media were replaced every 2 days.

RAW264.7 cells grown as aforementioned were cultured in the presence of soluble RANKL (100 ng/ml). After 4 days of culture, cells were fixed with 4% formaldehyde at room temperature for 10 min and histostained for tartrate-resistant acid phosphatase (TRAP) using a TRAP staining kit (cat. no. 387A; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), according to the manufacturer's protocol and as previously described (20,21). Cells were also immunostained with fluorescein isothiocyanate-labeled phalloidin (F-actin, 5 µg/ml; cat. no. P5282-1MG; Sigma-Aldrich; Merck KGaA) for 60 min, and with 4′,6-diamidino-2-phenylindole (1 µg/ml; Sigma-Aldrich; Merck KGaA) for 5 min at room temperature. Cells were analyzed using a Nikon microscope (Nikon Corporation, Tokyo, Japan) under ×10 magnification, and images were captured and analyzed with NIS-Elements F2.20 (Nikon Eclipse 80i; Nikon Corporation). TRAP⁺ multinucleated cells, containing >3 nuclei, were counted as osteoclasts. The average number of nuclei in osteoclasts was detected from the immunostaining images.

Bovine femoral cortical bone (22), purchased fresh from a butcher's shop, was cut into 0.1 cm slices, which were cleaned by ultrasonication in sterile distilled water at 40 kHz for 30 min at room temperature, autoclaved at 120°C and 0.4 MPa, and immersed in DMEM. The medium was changed three times every 30 min, and the slices were stored in DMEM at 4°C. Prior to each experiment, bone slices were preheated at 37°C for 1 h in 48-well plates containing 1 ml α-MEM. Subsequently, RAW264.7 cells stimulated with RANKL were added, with or without NBD peptide. After 4 days, the cells on the slices were fixed with 4% formaldehyde at room temperature for 10 min and observed under scanning electron microscopy (SEM; SU8010FE-SEM; Hitachi, Ltd., Tokyo, Japan). Subsequently, cells were removed by ultrasonication at 40 kHz for 30 min at room temperature and resorption lacunae were observed under the electron microscope. The total area of bone lacunae on the slices was quantified using NIS Elements-AR software (Nikon Corporation).

New Zealand white rabbits were provided by the Animal Experiment Center of Zhejiang University (Hangzhou, China). All procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University. Briefly, male rabbits (n=6/group; age, 4 months; weight, 2.5–3.0 kg) were selected for the osteomyelitis model (23). Rabbits were housed in stainless steel cages under controlled conditions (temperature, 23±2°C; relative humidity, 55±10%; ventilation, >10 times/h; 12-h light/dark cycle). All animals had free access to food and water throughout the acclimation and experimentation periods, and were maintained according to the Animal Experiment Center of Zhejiang University. Animals were anesthetized with Sumianxin II (10 mg/kg, 0.2 ml/kg; Dunhua Shengda Animal Medicine Co., Ltd., Dunhua, China; also known as xylazine hydrochloride), and a bone defect (8×8×3 mm) was created at the lateral inferior border of the mandible beside the middle joint. Subsequently, 100 µl 5% sodium morrhuate containing 5.0×10⁷ cfu/ml S. aureus (ATCC no. 25923; Department of Microbiology of Zhejiang University) was injected into the bone defect, which was then sealed with bone wax. After 6 weeks, rabbits were separated into the following three groups: i) Untreated controls, ii) treated with debridement surgery, and iii) treated with debridement surgery plus 500 µg/kg NBD peptide, which was injected into the defect area. During debridement, dead, damaged and infected tissue was removed from the bone defect. A total of 4 and 8 weeks following surgery, rabbits were sacrificed and specimens were harvested.

A total of 4 and 8 weeks following surgery, rabbits were euthanized and bone mineral density (BMD) was examined in the surgical area by dual energy X-ray absorptiometry (24). In addition, bone morphology was analyzed by radiographic analysis using CBCT (25) (NewTom 3G QR-DVT9000; NewTom, Verona, Italy).

In vitro experiments were conducted three times to obtain the mean and standard error from independent experiments. In vivo experiments were conducted on six animals/group. Statistical analysis was performed using SPSS 17.0 software package (SPSS, Inc., Chicago, IL, USA) by one-way analysis of variance with Tukey's post hoc test for comparisons between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

RAW264.7 cells were treated with DMSO or the NBD peptide and were stimulated with RANKL in vitro, in order to induce osteoclastogenesis (Fig. 1A). Addition of the NBD peptide resulted in a 71±5.6% reduction in the number of osteoclasts formed (P<0.05; Fig. 1B). In addition, there was a 55±1.5% reduction in the average number of nuclei per osteoclast (P<0.05; Fig. 1C). Osteoclast morphology in bone slices was analyzed by electron microscopy. Consistent with the TRAP staining results, the number and size of osteoclasts were reduced in the NBD group compared with in the DMSO group (Fig. 1A).

Osteoclast function in vitro was measured according to lacunae area on bone slices using scanning electron microscopy (Fig. 2A). There was a 95±3.7% reduction in total lacunae area in the NBD group compared with in the DMSO group (P<0.05; Fig. 2B).

A bone defect was created at the lateral inferior border of the mandible beside the middle joint of laboratory rabbits, and a 100-µl suspension of S. aureus (5.0×10⁸ cfu/ml) was injected into the bone defect. After 6 weeks, redness and swelling in the surgical field of rabbits were observed, thus confirming that osteomyelitis had been established. Then animals were divided into three experimental groups: Untreated, treated with debridement, and treated with debridement plus NBD peptide. After another 4 and 8 weeks, BMD and bone morphology of the surgical area were analyzed by dual energy X-ray absorptiometry and CBCT. As shown in Fig. 3, the size of the bone defect was gradually decreased in a 3-dimensional manner at 4 and 8 weeks in the debridement plus NBD peptide group compared with in the debridement group, as determined by CBCT.

Dual energy X-ray absorptiometry revealed that there were no significant differences in BMD between the three different time-points in the control group (P>0.05; Fig. 4A). Conversely, BMD was increased by 51±6.1 and 85±2.2% at 4 and 8 weeks, respectively, in the debridement group when compared with the baseline value at week 0 (P<0.05; Fig. 4A). Notably, additional 18±4.5 and 42±5.1% increases in BMD were observed in the debridement plus NBD peptide group when compared with the debridement group at 4 and 8 weeks, respectively (P<0.05; Fig. 4A).

The bone defects were also analyzed in three different dimensions by CBCT. Similar to the BMD results, the control group exhibited no significant differences in the 3-dimensional areas of the bone defect at the three time-points analyzed (P>0.05 Fig. 4B-D). Conversely, the bone defect area was decreased by 23.3±2.1, 21.6±2.5 and 28±2.6% in the mesiodistal, buccolingual and vertical directions, respectively, at 4 weeks; and by 36.7±3.1, 32.6±2.5 and 39.2±2.9%, respectively, at 8 weeks in the debridement group (P<0.05; Fig. 4B-D). Notably, injection with the NBD peptide resulted in increased inhibition of bone resorption, with an additional 10±1.1, 20±3.1 and 11.4±2.2% reduction in bone defect areas at 4 weeks; and 15±3.6, 34.6±3.1 and 18.3±4.5% reductions at 8 weeks, when compared with debridement alone (P<0.05) (Fig. 4B-D).