Cell culture

The biological effects of AMD3100 or BMP-2 on mesenchymal cells were evaluated with pluripotent mesenchymal progenitor C3H10T1/2 cells and myoblastic C2C12 cells which were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). Mouse bone marrow stromal cells (BMSCs) were harvested according to the previous report [38] and the passage 2–3 were used in experiments. C3H10T1/2, C2C12 cells, and BMSC were cultured at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Lonza Group Ltd., Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA), 100 U/mL penicillin, 100 mg/mL streptomycin sulfate and 2 mM glutamine. Recombinant BMP-2 proteins were purchased from PeproTech Inc. (New Jersey, USA) and Daewoong Pharmaceutical company (Seoul, Korea), respectively. L-ascorbic acid, β-glycerophosphate and AMD3100 were all purchased from Sigma-Aldrich (#A5602, St. Louis, MO, USA).

Alkaline phosphatase (ALP) activity assay

To evaluate the effect of AMD3100 and BMP-2 on Alkaline phosphatase (ALP) activity, C3H10T1/2 cells were plated in 12-well plates (Corning® Costar®; Sigma-Aldrich) at a concentration of 5 × 105 cells/well (1.3 × 105 cells/cm2) and cultured in osteogenic media (20% FBS with 1% antibiotics in DMEM, 10 mM β-glycerophosphate and 50 µg/ml l-ascorbic acid 2-phosphate) for 9 days. One day after seeding, the media were replaced every 2 days with the addition of AMD3100 (100 µM) and/or BMP-2 (0.5 µg/mL). For example, A-B-B-B indicated that the cells were cultured in media containing AMD3100 for the first 2 days and then replaced with BMP-2-containing media on the 3rd, 5th, and 7th days, followed by final cell harvesting on the 9th day after cell seeding. AB-AB-AB-AB group represents concomitant application of both AMD3100 and BMP2 when the media was replaced. The cells were then harvested and lysed using ProteoJET lysis buffer (Fermentas, St. Leon-Rot, Germany). The cell extracts were incubated with p-nitrophenyl phosphate substrate (Sigma-Aldrich) in a 0.05 M glycine buffer containing 0.05 mM MgCl2 (pH 10.5) at 37 °C for 30 min. ALP activity was evaluated by measuring absorbance at 405 nm using a microplate reader (Infinite® M200, Tecan U.S., Inc. Mannedorf, Switzerland) and normalized to the total protein content of cell lysates.

ALP staining assay

ALP staining was performed using an ALP detection kit (Sigma-Aldrich), according to the manufacturer’s instructions. Twelve-well plates with osteogenic media comprising 100 uM AMD3100 and/or 0.5 µg/mL BMP-2, both of which were replaced every 2 days for a total of 8 days (similar to the procedure for the ALP activity assay), were seeded with C3H10T1/2 cells at a concentration of 5 × 104 cells/well (5.3 × 104 cells/cm2) in triplicate. Subsequently, each well containing the cells was washed with phosphate-buffered saline (PBS). The fixation solution was applied for 45 s. After reaction with ALP substrate, the solution was removed and the cells were washed three times using PBS. The ALP-positive cells were identified by red staining.

Calcium accumulation assay

C3H10T1/2 cells were seeded on 12 well plates (5 × 104 cells/well) and were incubated in 5% FBS DMEM osteogenic media with 100 µM AMD3100 and/or 0.5 µg/ml BMP-2 for 8 days. Calcium levels were determined colorimetrically using a calcium assay kit (Calcium Colorimetric Assay Kit, Abcam, and Cambridge, MA, USA) by following the manufactures’ protocol. The optical density was measured at 595 nm using a microplate reader (Infinite® M200, Tecan U.S., Inc. Mannedorf, Switzerland) and normalized to total protein content.

Cell migration assay

The effects of AMD3100 and/or BMP-2 treatment on cell migration were evaluated with a transwell migration assay as previously described [38]. C2C12 cells and BMSC (3 × 105 cells/well) were suspended in 100 µL serum-free DMEM and loaded into upper wells (Corning® Transwell™; Sigma-Aldrich). After the serum-free media containing 1 µM AMD3100 or 0.1 µg/mL BMP-2 were added in the upper chamber for 24 h with serum free media, AMD3100 and/or BMP-2 were added the lower chamber containing 2% FBS-containing media to for a further 24 h. For migration assay, cells were washed with 600 µL PBS and fixed with 600 µL ethanol for 10 min. After washing again with 600 µL PBS, the cells remaining on the inner surface of the upper chambers were removed with cotton swabs. For example, to demonstrate the effect of the sequential treatment of AMD3100 and subsequent BMP-2, the medium in the upper chamber was replaced with 1 µM of AMD3100 in 300 mL of serum-free DMEM and incubated for 1 h, following which the medium in the lower chamber was replaced with 0.1 ug/mL BMP-2 (AMD3100 → BMP-2, marked as A → B in the figure).

The chambers were stained with 600 µL 0.2% crystal violet (Junsei Chemical Co. Ltd, Tokyo, Japan) for 30 min at room temperature, and the cells in four random visual fields were counted (100 × magnification).

Colony-forming unit (CFU) assay after AMD3100 injection

To analyze the in vivo effect of AMD3100 on hematopoietic progenitor cell mobilization, AMD3100 or PBS was administered by intraperitoneal (IP) injection at a concentration of 2 mg/kg, which was determined based on a previous study that reported the effect of optimal mobilization of HSPCs 1 h after AMD3100 injection [39]. For the CFU assay, peripheral blood (PB) was obtained 1 h after the injection using the cardiac puncture method. Single-cell suspensions of PB were obtained after ammonium chloride lysis to exclude the red blood cells. The centrifuged cells were counted using with 3% acetic acid with methylene blue and were plated into 35-mm dishes (1 × 105 cells / dish). The cells were incubated in MethoCult media (GF M3434, StemCell Technologies, Vancouver, Canada), and the hematopoietic colonies were counted and scored after incubation for 14 days. The number of hematopoietic CFU cells were analyzed.

Mouse critical-size calvarial defect model: effect of AMD3100 and BMP-2

Seven-week-old female C57BL/6 mice (Hyochang Science, Daegu, Korea) were used as the critical-size calvarial defect models to evaluate the efficacy of combined treatment of AMD3100 and BMP-2 treatment for orthotopic bone formation. All the animal experiments were approved by the Institutional Animal Care and Use committee Kyungpook National University (authorization number; KNU 2010–30). PBS or AMD3100 (2 mg/kg; Sigma-Aldrich) was administered by IP injection before surgical implantation. One hour later, all the animals (n = 32) were anesthetized by an IP injection of ketamine (100 mg/kg) and xylazine (5 mg/kg). Subsequently, the scalp was opened at the midline exposing the underlying calvaria. The calvarial defect was made on the parietal bone of mice using a 6 mm-diameter dental trephine bur under PBS irrigation. The dura mater was preserved without damage. After removing the 6 mm-diameter calvarial bone segment, a 6 mm collagen scaffold (Teruplug, Terudermis Olympus Terumo Biomaterials Co., Japan) was prepared. The PBS or BMP-2 (5 µg in 50uL of PBS) loaded scaffold was implanted in the defect area. The overlying soft-tissue wound was sutured immediately with 4–0 silk.

To evaluate the effect of continuous injection of AMD3100 and sequential treatment of AMD3100 and BMP-2 in bone regeneration, the two experimental settings were used in mouse calvarial defect model. Initially, BMP-2- or PBS-loaded collagen scaffolds were implanted into the calvarial defects. Overall experimental settings were shown in Table 1.

Table 1 Experimental settings for AMD3100 and/or BMP-2 administration

The first animal experiment was intended to evaluate the effect of continuous injection of AMD3100 on BMP-2-induced bone formation, for which PBS or AMD3100 (2 mg/kg) was administered by IP injection on postoperative days 3 and 6. The animals were divided into the following four groups (n = 4 / group); group C–C-C, the PBS-loaded collagen scaffold was inserted in the calvarial defect and PBS was administrated by IP injection on postoperative days 3 and 6 after the surgery; group A-A-A, the PBS-loaded scaffold was implanted, and AMD3100 was administered by IP injections 1 h before the surgery with additional injections on days 3 and 6; group B-C–C, the BMP-2-loaded scaffold was inserted, and PBS was administered by IP injections on days 3 and 6; group AB-A-A, the BMP-2-loaded scaffold was implanted, and AMD3100 was administered by IP injections 1 h before the surgery with additional injections on postoperative days 3 and 6.

The second animal experiment was performed to evaluate the effect of sequential treatment, for which BMP-2 was locally administered in the calvarial defect after AMD3100 IP injection.

The animal was divided into four groups (n = 4 / group); group C–C-C, the PBS-loaded scaffold was implanted in the calvarial defect, and PBS was administered by IP injections on postoperative days 3 and 6; group A-C–C, the PBS-loaded scaffold was implanted in the calvarial defect, AMD3100 was administered by IP injections 1 h before the surgery, and PBS was administered by IP injections on days 3 and 6; group C-B-B, the PBS-loaded scaffold was implanted in the calvarial defect, and BMP-2 was injected into the scaffold on days 3 and 6; group A-B-B, AMD3100 was administered by IP injections 1 h before implantation of the PBS-loaded scaffold in the calvarial defect, and BMP-2 was injected locally into the scaffold on postoperative days 3 and 6.

After 28 days, the mice were euthanized by CO2 inhalation and the calvarial specimens were harvested for histology and micro-computed tomography (micro-CT) analyses.

Micro-CT analysis of bone formation

New bone formation at the calvarial bone defect site were evaluated with micro-CT. The mouse calvarial specimens containing the PBS or BMP-2-loaded scaffold were excised, trimmed and fixed in 4% paraformaldehyde for 24 h at room temperature. They were then transferred into PBS and stored at 4 °C until the time for micro-CT analysis. The specimens were scanned using micro-CT (Skyscan 1072–32, Bruker Corporation, Kontich, Belgium) under an aluminum filter measuring 0.5 mm using an X-ray voltage of 50 kV, anode current of 200 mA, isotropic voxel size of 10 μm, and exposure time of 1.2 s with a pixel size of 2.5 μm. Serial axial images were reformatted to generate three-dimensional image reconstructions using TomoNT software (Skyscan software). The regenerated bone volume (mm3), trabecular number (1/mm), trabecular separation (mm) and trabecular thickness (mm), were measured in each group.

Histological evaluation of bone formation

After taking the micro-CT measurements, the specimens were fixed in cold 4% paraformaldehyde solution for 10 days and then decalcified in 18% ethylenediaminetetraacetic acid (EDTA). The samples were then dehydrated and embedded in paraffin. The sections (10 μm in thickness) were subjected to hematoxylin and eosin (H & E) and trichrome staining. Areas of newly formed bone (measured in mm2) were analyzed histomorphometrically using i-solution software (Image & Microscope Technology, Korea).

Statistical analysis

All the experiments were repeated two to three times independently, with four samples included in each treatment group. All data were expressed as mean ± standard deviation (SD). The differences between different test conditions were compared by the Tukey–Kramer post-hoc test, with the level of significance was set at p < 0.05. All the experiments were repeated at least in triplicate.