MSC Isolation and Identification
MSC was isolated from femurs of 3 weeks old male SD rats, as previously described . Before performing any experiment, cells were passaged for 3 to 6 times. The surface marker expressions of MSC, including CD90, CD44, CD34, CD45, and CD11b/c, were analyzed by flow cytometry assay, as described in the previous study [23, 24].
A total of 40 male SD rats, 8-12 weeks old, weighing 350-500 g, were obtained from Laboratory Animal Center of Dalian Medical University, China. Rats were housed in an environment with a temperature of 22 ± 1 ºC, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 hr.
After one week of adaptation, 40 SD rats were accurately weighed and randomly divided into two groups (n=20/group): Short-Wave treatment group (SW) and control group (Con). All rats underwent surgery in order to establish the femoral shaft fracture and intramedullary fixation model.
For in vivo bioluminescent assays to the MSC homing, 20 nude mice of femoral shaft fracture were injected MSC labeled by the GFP (MG), and then randomly divided into two groups, including a Short-Wave treatment group (MG+SW) and control group (MG) (n=10/group).
Stabilized Fracture Model
Stabilized right femur fractures were established by intramedullary fixation in 8 - 12-week-old male SD rats. A 0.25-mm titanium alloy pin was inserted inside the medullar canal of the femur. A three-point bending device was then used to produce closed fractures of the femoral shaft with a standardized force . In 20 male nude mice, transverse osteotomy with a 1 mm bone fracture was created in the middle of the right femur in the same way. Bupremorphine was subcutaneously administered (0.5 mg/kg) for pain control.
All the 20 nude mice received injections with 5×106/50μl GFP-labeled MSC (C57BL/6 mouse strain) (CYAGEN Company, China) three days after the femoral fracture. Cells were injected by tail vein using a microinjector.
Three days after the operation, Short-Wave therapy was applied in a Short-Wave treatment group. Briefly, animals were fixed in an apparatus so that they could not turn around. The treatment regimen was applied to the right thigh. The shortwave generator (Curapuls 970, Netherlands) operated at a frequency of 27.12MHz. A micro-heat continuous-wave Short-Wave exposure for 10 min was applied once a day. A similar procedure was carried out in the control group; however, the device was turned off.
For the in vitro study, the isolated rat osteoblasts in treatment group underwent Short-Wave irradiation. The protocol of Short-Waves therapy irradiation on cells was based on a previous study. The two non-contact applicators were of perpendicular contraposition and 10 centimeters away from each other. The cell culture flask was placed in the middle of the applicators. A micro-heat continuous-wave Short-Wave exposure for 90 min was applied twice on day 1 in the open air at 37℃. The control group received a sham Short-Wave treatment by turning off the Short-Wave generator. On day 2, the culture medium from each group was collected for HIF-1 and SDF-1protein level analysis, and the medium was used in MSC cell wound healing assay and trans-well chamber test.
The fracture healing was assessed by plain anteroposterior radiographs at day 7, 14, 21 and 28. The same X-ray machine and settings were used for all radiographs every seven days after fracture. Bone defects were analyzed using Xsys software.
Micro-CT scanning was performed to assess callus. Quantification for the volumes of the bony calluses as determined as previously described [27, 28]. The region of interest (ROI) was set within 800 μm (50 slices) around the defect edge. We applied a fixed threshold of less than 330 for new calcified cartilage or unmineralized cartilage. Three D microstructural image data were reconstructed by Inveon Research Workplace software. After 3D reconstruction, bone volume fraction (BV/TV) were automatically determined to confirm fracture healing.
In Vivo Fluorescence Assays
Ten nude mice per group received anesthesia and were examined by the IVIS imaging system at day 7 after the surgery. The identical parameter settings were used for all samples: f number: 1, field of view: 22, binning factor: 18, luminescent exposure (seconds): 10. The IVIS imaging examination and rates of photons were calculated and performed according to methods reported in a previous study .
Histological Analysis and Immunofluorescence Imaging
Twenty nude mice (MG: n=10, MG+SW: n=10) were sacrificed for histological analysis and immunofluorescence imaging at day 28 after operation. The femoral bone of nude mice and rats were sectioned, preserved, decalcification, and embedded in paraffin along the longitudinal axis. For morphological analysis, 5 μm slices were sectioned, deparaffinized, and stained using hematoxylin and eosin. The immunofluorescence staining was performed as previously described . Tissue slides of nude mice were stained with antibodies (A0516, Beyotime, China) against GFP to track exogenously delivered MSC labeled by GFP. A four-channel confocal laser scanning microscope was used to analyze all the samples. GFP-positive cells were automatically counted in five fields on × 100 magnification by ImageJ software. Sixteen rats were sacrificed for histological analysis at day 14 (n=4/group) and day 28 (n=4/group) after operation. It is on the same time end points for rats and mice.
Enzyme-Linked Immunosorbent Assay (ELISA)
The plasma of SD rats received by heart puncture at day 3, 7 and 14 after operation (n=4/group/time point). The concentration SDF-1 in plasma of SD rats was analyzed using citrulline ELISA kit (CSB-E13414r, Cusabio Biotech, China). In culture media of osteoblasts (SD rat strain), the concentration of HIF-1 (SEA798Ra, USCN, China) and SDF-1 content was also detected using the ELISA kit.
Quantitative Reverse Transcription-Polymerase Chain Reaction (q RT-PCR)
Callus of sacrificed SD rats was collected and snap-frozen in liquid nitrogen at day 3, 7 and 14 after operation (n=4/group/time point). RNA isolation and subsequent cDNA synthesis (Bio-Rad, 170-8891) were performed as previously described . A total of 50 ng of cDNA was amplified with custom-designed q RT-PCR primers (Table 1) (Thermo Fisher Scientific). A melt curve was generated to analyze the purity of amplification products. The expression levels of mRNA were normalized to the average of β-actin. Relative expression of mRNA was evaluated by using the comparative CT method (ΔΔCt) .
Osteoblasts Culture and Identification
Osteoblasts were obtained from calvaria of one-day-old neonatal SD rats using the method of collagenase-pancreatic enzyme digestion as detailed in reference . After two passages, alkaline phosphatase staining was utilized to identify the osteoblast cells (Figure2c).
Small Interference RNA Transfection
We inhibited HIF-1α expression by siRNA in osteoblasts (SD rat strain). Synthetic siRNA oligonucleotide specific for HIF-1α (NM_024359) (59 to 39: UUUAUCAAGAUGGGAGCUCTT) and nontargeting siRNA were obtained from Sangon (Shanghai, China).
Osteoblasts Culture Medium
Osteoblasts (SD rat strain) were seeded in 6-well plates for 24 h to 80–90% confluence. Three kinds of interventions were provided: 1) Short-Wave continuous irradiation for 180 min; 2) 200 μmol/L CoCl2-stimulated hypoxia condition in fracture; 3) siRNA inhibition of HIF-1α. Eight kinds of osteoblasts culture mediums were obtained from single or combined interventions for the follow-up experiment (Figure 1).
Wound Healing Assay
MSC (SD rat strain) was cultured on six-well plates to confluency and monolayers and wounded with a sterile 200 μL pipette tip. The cultures were washed with PBS to remove detached cells and stimulated with 8 kinds of osteoblasts culture medium fluid 1.5 ml in each well. Photographs were collected at 0, 24, and 48 hours.
Trans-well Chamber Test
The tests were performed in Boyden Chambers (Corning, 3422, Lowell, MA). Eight kinds of osteoblasts (SD rat strain) culture medium was seeded in the bottom chamber. The top chambers filled with MSC (SD rat strain) starved overnight were inserted. Twenty-four hours later, inserts were removed and washed. The cells that migrated to the bottom side were accumulated.
Osteoblasts and MSC cells (both are SD rat strain) lysates were prepared, and western blots were performed as previously described . The 30 μg of protein was loaded in each lane for reducing electrophoresis. Primary antibodies were used for β-actin (Sigma; A2228) diluted 1:10000, HIF-1α (Gene Tex; GTX127309) diluted 1:2000, SDF-1 (CST; 3740) diluted 1:1000, FAK (Sangon Botech; D160324) diluted 1:3000, phosphor-FAK (Sangon Botech; D160324) diluted 1:2000, F-actin (Abcam; Ab205) diluted 1:2000, β-catenin (wanleibio; WL0962a) diluted 1:500 and CXCR4 (Abcam; ab124824) diluted 1:1000.
Statistical analysis was performed by SPSS 22.0 for Windows (SPSS, Chicago, USA). The results are shown as the mean value ± standard deviation. The differences between groups were analyzed by t-test or analysis of variance (ANOVA). Two-tailed P values were computed, and P<0.05 was considered to be statistically significant.