MSC Isolation and Identification
MSC was isolated from femurs of 3 weeks old male SD rats as previously described . Cells had uniform long fusiform shape (Figure 1a). Before performing an 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, a 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. The control group received a sham treatment.
For in vivo bioluminescent assays to the MSC homing, 20 nude mice 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).
Stabilized Fracture Model
Stabilized 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 nude mice, femur fractures were produced in the same way. Bupremorphine was subcutaneously administered (0.5 mg/kg) for pain control.
Cells were injected by tail vein using a microinjector. All the 20 nude mice received 5×106/50μl MSC-GFP (CYAGEN Company, China) two days after the fracture was established.
Short Wave Treatment
Three days after the operation, short wave therapy was applied in a short wave treatment group and united treatment. 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; the current output was 30mA. A similar procedure was carried out on the control group with the device that was turned off.
For the in vitro study, a short wave treatment group received a 180 min-short wave irradiation, while the control group received a sham short wave treatment.
The fracture healing was assessed by plain anteroposterior radiographs. The same X-ray machine and settings were used for all radiographs every seven days. Bone defects were analyzed using Xsys software. Micro-CT scanning was performed to assess callus, and 3D microstructural image data were reconstructed by coneCT express software. The region of interest (ROI) was set within 800 μm around the defect edge and a thickness of 100 μm.
In Vivo Fluorescence Assays
Nude mice were examined by the IVIS imaging system at day 7 after the surgery. Ten mice in each group received anesthesia. The identical parameter settings for IVIS imaging were used for all samples: f number: 1, field of view: 22, binning factor: 18, luminescent exposure (seconds): 10. The methods of IVIS imaging examination and rate of photons calculation according to the description in the previous study .
Histological Analysis and Immunofluorescence Imaging
The femoral bone was 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 were stained with primary antibodies (CST; 2956S) against GFP to track exogenously delivered MSC labeled by GFP. A four-channel confocal laser scanning microscope LSM700 (Zeiss, Pleasanton, CA, USA) was used to analyze all the samples. GFP-positive cells were automatically counted in five fields on × 40 magnification by ImageJ software.
Enzyme-Linked Immunosorbent Assay (ELISA)
The SDF-1 in plasma was analyzed using citrulline ELISA kit (CSB-E13414r, Cusabio Biotech, China). The osteoblasts cultured supernatant of HIF-1 (SEA798Ra, USCN, China) and SDF-1 content was also detected using the ELISA kit.
Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Callus of sacrificed rats was collected and snap-frozen in liquid nitrogen. 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 qRT-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 (Figure1c).
Small Interference RNA Transfection
We inhibited HIF-1α expression by siRNA in osteoblasts. Synthetic siRNA oligonucleotide specific for HIF-1α (NM_024359) (59 to 39: UUUAUCAAGAUGGGAGCUCTT) and a nontargeting siRNA were obtained from Sangon (Shanghai, China).
Osteoblasts Cultured Supernatant
Osteoblasts 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) 0 μmol/L CoCl2-stimulated hypoxia condition in fracture; 3) siRNA inhibition of HIF-1α. Six kinds of osteoblasts cultured supernatants were obtained from single or combined interventions for the follow-up experiment.
Wound Healing Assay
MSC 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 osteoblasts cultured supernatant fluid 1.5 ml in different wells. Photographs were collected at 0, 24, and 48 hours.
Trans-well Chamber Test
The tests were performed in Boyden Chambers (Corning, 3422, Lowell, MA). Osteoblasts cultured supernatant was seeded in the bottom chamber. The top chambers filled with MSC 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 cell 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 1:50000, 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.
We performed Statistical analyses 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.