Preparation and Culture of ASCs and EPCs
Animal procedures were conducted in accordance with the protocol approved by IACUC of China Medical University. Sprague-Dawley (SD) rats, 4-week- old, were euthanized by CO2. The subcutaneous white adipose tissue of the inguinal region was cut off, while the blood vessels and other tissues were removed. The fresh tissue was washed three times with PBS containing 1% penicillin/streptomycin and minced by sterile surgical scissors. The minced tissue was digested with 0.1% collagenase typeⅠ(Gibco)at 37℃ for 1h and then centrifuged at 1000 rpm for 5 min. The upper layer of fat was removed, the remaining cell pellet was resuspended and filtered through a 70 μm filter. After another centrifugation for 5min, the cell pellet was resuspended in the culture medium consisting of low-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone), 10% fetal bovine serum (FBS) (Gibco), and 1% penicillin/streptomycin. The suspension was transferred to a flask and cultured at 37℃ with 5% CO2 in a humidity atmosphere[23]. The culture medium was replaced 24h after transferring into flasks[24]. The medium was replenished every 2-3 days, and the cells were passaged after 80% confluence. The ASCs at passage 3 were used for the following experiment.
After 4-week-old SD rats were sacrificed with CO2, their femurs were taken, and the bone marrow was evenly beaten. Single-cell suspension was obtained by centrifugation with Histopaque-1083 (Sigma), and mononuclear cell layer was separated by mononuclear cell separation solution. CD34+ cells were separated by immunomagnetic bead method. Cells were resuspended in EGM medium( Lonza, Cologne, Germany) and seeded into culture flasks and cultured at 37℃ with 5% CO2 in a humidity atmosphere. The culture medium was replaced 24h to remove nonadherent cells.
The medium was replenished every 2-3 days, and the cells were passaged after 80% confluence. The EPCs at passage 3 were used for the following experiment.
Flow Cytometry Analysis and Immunofluorescence Staining
1*106 ASCs and EPCs at passage 3 were harvested, washed with 10% FBS/PBS and centrifuged at 1000 rpm, 5 min to gather cell pellets. For flow cytometry analysis, ASCs were stained with FITC-conjugated rat anti-CD70, Cy5.5-conjugated rat anti-CD90, PE-conjugated rat anti-CD45 and Alexa Fluor 647-conjugated rat anti-CD34 antibodies at a concentration of 2 mg/ml at 4℃[25]. EPCs were stained with PE-conjugated rat anti-CD31, Alexa Fluor 647-conjugated rat anti-CD34, FITC-conjugated rat anti-CD45 and FITC-conjugated rat anti-CD133, at a concentration of 2 mg/ml at 4℃. Mouse IgG was served as negative controls. Processed specimens were washed with 2 ml of 10% FBS/PBS for 30 min. After resuspension in 500μl PBS, the cell pellets were tested by flow cytometry with 10,000 events recorded for each condition. The results were analyzed by FACS Express software. For immunofluorescence staining, EPCs at passage 3 were co-stained with DPBS-E containing 10 mg/ ml DiI-labeled acLDL (Biomedical Technologies) for 60 min at 37℃, then observed under fluorescence microscopy.
Co-culture of ASCs and EPCs in vitro
To determine the optimal ratio of EPCs and ASCs in bone regeneration, six groups were divided for experimental observation, namely ASCs alone, EPCs alone, and EPCs: ASCs at ratios of 1: 1, 1: 2, 1: 5, and 1:10. Cells were seeded in 12-well plates at the density of 1*105 cells per well and induced with EGM/CM media (EGM media: complete media ratio of 1:1) or EGM/OS media (EGM media: OS media ratio of 1:1) for 7 days, which was prepared for ALP activity assay as previously reported.
Alkaline phosphatase activity assay
ALP activity was detected by using an ALP assay kit (Sigma) following the manufacturer’s instructions. In brief, cells were mixed with an alkaline buffer solution (1.5 M, pH10.3) containing 10 mM p-nitrophenyl phosphate as a substrate and NaOH solution (3 M) was used as stop solution. The optical density was measured at 405 nm with a microplate reader. ALP activity was normalized by the DNA content and expressed as nmol of p-nitrophenol produced per minute per mg of total DNA. The implanted samples were smashed in liquid nitrogen, and lysed in 1 ml harvest buffer for 1 hour, then homogenized carefully to further lyse cells. After a centrifugation at 2000 rpm for 10 min, 10 ml supernatant were harvested for ALP activity assay.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated using Trizol reagent (Invitrogen) according to manufacturer's instructions.Reverse transcription of total RNA was performed by RT-PCR (Invitrogen) using the reverse transcription first chain synthesis system.Real-time PCR reaction was performed with synthetic cDNA. Specific primers were used for PCR amplification to analyze the expression of osteoblastic marker genes including OCN, Col1a1, BMP2, vascular endothelial growth factor (VEGF), Cadherin5 (cdh5), and von Willebrand factor (vWF). According to the manufacturer's instructions, real-time PCR was performed using SYBR GREEN PCR Master Mix on ABI PRISM 7500 sequence detection system.PCR conditions were 94°C 1 min, 95°C 30 s, and 58°C 40 s, with a total of 35 cycles. All reactions were repeated three times and normalized to GAPDH. Comparative ct was used to calculate the relative difference of PCR results.
Table 1 The primer sequences used for qRT-PCR
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Forward sequence
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Reverse sequence
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OCN
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5'-tctttctcctttgccggc-3'
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5'-caccgtcctcaaattctccc-3'
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Col1a1
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5'-gcaacagtcgcttcacctaca-3'
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5'-caatgtccaagggagccacat-3'
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BMP2
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5'-tccgctccacaaacgagaaa-3'
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5'-aaaggcatgatagcccggag-3'
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VEGF
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5'-ccgaaaccatgaactttctgc-3'
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5'-gacttctgctctccttctgtc-3'
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cdh5
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5'-ggcaatcaa ctgtgctctcc-3'
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5'-cttcgtgga ggagctgatct-3'
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vWF
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5'-ccggaagcgaccctcaga-3'
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5'-cgg tcaattttgccaaagatct-3'
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Von Kossa staining
After ASCs/EPCs induced with osteogenic medium for 3 weeks, the formation of mineralized nodules in vitro was detected by Von kossa staining. Briefly described below, 1% silver nitrate solution was added to the culture medium for 45 min under ultraviolet light, rinsed with distilled water, and treated with 3% sodium thiosulfate for 5 minutes. After another rinse, specimens were re-stained and washed with ethanol, then finally performed microscopic observation, image acquisition and analysis.
Matrigel tubule formation assay
24 hours before the experiment, the Matrigel (BD Corporation, USA) was moved from -20°C to 4°C refrigerator to fully melt. 50 μL of Matrigel was added to each well in a 96-well plate and placed in a 37°C, 5% CO2 incubator for 2 h to coagulate. Cells of different groups were seeded to the 96-well plate containing matrigel (5 duplicate wells per group), placed in the incubator, and observed under a microscope every 3 h. When there was obvious blood vessel formation, photographs were taken immediately. Image-Pro Plus 6.0 software was used to analyze the number and relative length of each component tube.
Fabrication of HA/Col scaffold
According to a previous method of thermally induced phase separation[26], the scaffold preparation protocol is briefly described as follows. 1 g of hydroxyapatite (HA)(Sangon Biotech, Shanghai, China) powder was dissolved into 10 ml of suspension with deionized water. After stirring at room temperature (RT) for 5h, the Ha powder was fully dispersed with ultrasonic. Then, 40ml (5 mg/mL) collagen(Kele Biological Technology Co. Ltd, Chengdu, China)solution was mixed with the Ha solution and stirred at RT for 2h to form a Ha-collagen mixture (HA:Col=8:2). Subsequently, the mixture was transferred to a circular mold with a diameter of 8 mm and a thickness of 3 mm, pre-frozen at a temperature of -40°C for 24 hours, and then freeze-dried at a temperature of -55°C using a constant temperature freeze dryer.
Scanning Electron Microscope (SEM)
Field Emission SEM S-4800 (Hitachi, Japan) was employed to observe the microstructure of the HA/Col scaffold, as well as the morphology and behavior of the cells grown in the scaffold. The co-cultured ASCs/EPCs were inoculated on HA/Col scaffolds. After 3 days of culture, the cells were fixed with 2.5% glutaraldehyde fixation, and the samples were dehydrated with ethanol in grades. After dehydration, gold spraying was performed for 15-20 seconds. All samples were analyzed at 1.0 kv
Rat critical-sized cranial bone defect model
The in vivo experimental protocol was approved by IACUC of China Medical University. Thirty-two male SD rats at eight-week old underwent surgery under general anesthesia, induced by 5% isoflurane/O2 gas inspiration and maintained by 1–2% isoflurane/O2 by a facial mask. The scalps were shaved cleanly, disinfected with iodophors, and infiltrated with 0.1–0.5 ml of a local anesthetic agent of 2% lidocaine with epinephrine (0.01 mg/ml). The skin and periosteum were incised along the midline to expose the cranial bone surface[27]. A trephine bur with diameter of 8mm was used to create a standardized, round, segmental defect around the sagittal suture, maintaining the underlying dura mater intact. A single implant of 1*106 cells mixed with the scaffold was inserted into each defect. The periosteum and skin were sutured in layers with non-absorbable 4-0 prolene sutures. For 2 days after surgery, the rats were treated with carprofen for analgesia and penicillin for prevention of infection. Animals were divided into 4 groups randomly: group 1, blank group; group 2, hydroxyapatite/collagen scaffold only (HA/Col); group 3, HA/Col +ASCs, and; group 4, HA/Col +ASCs+EPCs. At the end of the eighth week after surgery, animals were euthanized with CO2 and the repaired calvaria bones were harvested for the following analyses.
Analysis of bone regeneration
Micro-CT (Latheta LCT200) was used to scan the harvested samples for 3D imaging analysis. Meanwhile, Bone Mineral Density (BMD, g/cm²) were performed with LUNAR PIXImus bone densitometer and analyzed by
LUNAR PIXImus software according to the manual book of the equipment. A total of 6 samples were analyzed in each group. On the computerized scan, 5 regions of interest (ROI) of each slide were selected to measure the BMD of the defect area, and the average of these values was taken as the final result.
For histological analysis, six samples per group were decalcified and cut into 5mm sections, half of which were used for hematoxylin-eosin (H&E) staining and the other half for immunohistochemistry analysis. Digital images of each slide were acquired using a digital camera mounted to a microscope. Newly formed bone areas in the total defect area were calculated manually at 10×magnification by using NIH ImageJ software.
Analysis of Blood Vessel Ingrowth
VEGF was detected by immunohistochemistry in paraffin embeded and decalcitonized bone sections. As previously mentioned, VEGF is largely present in the subendothelial matrix.Half of the samples in each group were analyzed independently.The primary antibody of goat VEGF(Santa Cruz Biotechnology, Santa Cruz, CA) was diluted at a ratio of 1:300, and the hrp-conjugated rabbit anti-goat antibody(Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) was diluted at a ratio of 1:500 to 1% BSA.The peroxidase activity was observed with diaminobenzidine.Both the negative control group and the positive control group were included under the same conditions.Negative control staining was performed on the same bone plates without primary antibodies.The kidney sections as positive group were stained with the same primary and secondary antibodies.AxioImager software was used for image acquisition.Using the NIH Image J software, the number of blood vessels in the entire implant area of each sample displayed by VEGF staining was calculated manually in a 10x magnification manner.
Statistical analysis
The test results were expressed as mean ± standard deviation, and statistical analysis was performed by SPSS 22.0 software (IBM SPSS Statistics, IBM, Armonk, NY). T-test was used for the comparison of the data of two groups and one-way ANOVA was for the data of multiple samples. P <0.05 was considered as statistically significant.