Preparation of CGF
This research was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangzhou, People’s Republic of China). CGF was extracted from the healthy Sprague Dawley (SD) rats by venipuncture of the abdominal aortic vein. In brief, the blood was collected in sterile Vacuette tubes (Beijing Ruidexy Medical Technology Limited, China) without anticoagulant solutions. CGF was prepared according to Sacco’s protocol [27]. These tubes were then immediately centrifuged (Medifuge CGF, MF200,Silfradent Srl, Italy) using a program with the following characteristics: This centrifuge device used a program with the characteristics: 2,700 rpm 2 min, 2,400 rpm 4 min, 2,700 rpm 4 min, and 3,000 rpm 3 min [28]. Immediately after CGF preparation, a CGF extract was prepared. Briefly, CGF was transferred into a syringe and was gently compressed in the syringe so a liquid fraction (CGF extract) can be fabricated, the extracts were filtered by a 0.22 µm sterile syringe filter (Solarbio, China). The final CGF concentration (5%, 10% and 20%) in the experimental groups was calculated on the basis of the volume of CGF that was added to the total volume of culture medium containing 10% fetal bovine serum (FBS). The control group only received 10% FBS.
Enzyme-linked Immunosorbent Assay (ELISA)
Growth factors extracted from the CGF were prepared according to previously reported protocols with slight modifications [29]. In brief, the CGF clot was soaked in 1 mL DMEM (Cyagen Biosciences Inc. Guangzhou, China) without FBS in a 15 mL flacon tube and incubated at 37 °C. The medium was collected continuously for 14 days. Following collection, 1 mL fresh DMEM was added to the tube. All the collected extracts were stored at -80 °C. Finally, the Elisa kit (Elabscience Biotechnology Co., Ltd) was used to quantify the representative growth factor of BMP-2 released from the CGF extract according to the manufacturer’s instructions. All the assays were performed in triplicate.
The structure of CGF tested by Scanning electron microscope (SEM)
To verify the structure of CGF, CGF was lyophilized and examined by SEM (fei-q25, American FEI company). In brief, CGF was pre-frozen at − 60 °C for 24 h and then dehydrated for 24 h using a freeze dryer (SCIENTZ-10N, Ningbo, China). The samples were then observed, and representative images were captured.
Isolation, Culture And Identification Of Rat BMSCs
BMSCs were harvested from 2-week-old healthy SD female rats. After euthanasia, bone marrow was flushed out from tibia and femur with SD Rat Bone Marrow Mesenchymal Stem Cell Basal Medium supplemented with 10% (v/v) Fetal Bovine Serum, 1% (v/v) Glutamine and 1% (v/v) Penicillin-Streptomycin (Cyagen Biosciences Inc. Guangzhou, China) in standard sterile condition. Cells were incubated under the condition of 5% CO2 at 37 °C in a cell incubator. Cells were passaged every 3 days using 0.25% (w/v) trypsin-EDTA solution. The third passage cells were used to perform the flow cytometry identification according to the method as reported [30]. After identification, the third passage cells were used in our experiments.
Cell Proliferation Assay
To detect the capacity of CGF promoting cell proliferation, CCK-8 assay was performed. In brief, BMSCs were seeded in 96-well plates at a density of 2000 cells/well in the culture medium. After 24 h of culture, DMEM medium was replaced with different concentrations (5%, 10%, and 20%) of CGF extracts or FBS for 1, 3, and 5 days in the same conditioning culture (37 °C, 5% CO2). The cell proliferation rates were determined with CCK-8 (Solarbio, China) using a microplate reader (Bio-Rad, Hercules, USA) at a wavelength of 450 nm. Eight repetitive wells were used for each group, the maximum and minimum values were excluded, and the remaining data were used for statistical analysis.
Migration Assay
To detect the capacity of CGF on the migration of BMSCs, the transwell migration assay was performed. The transwell migration assay was performed according to the method reported [31]. In brief, BMSCs were suspended at a density of 1 × 104/well and loaded into the top chamber of a 24-well, 8 µm pore-size transwell plate (Corning, NY, USA). Then, complete medium from different groups with CGF or medium was added to the lower chamber. After 24 h, un-migrated cells that remained in the upper chambers were removed by wiping the top of the insert membranes with cotton swabs, while the migrated cells that passed through the membrane pores were stained with 0.5% crystal violet for several minutes and counted under an optical microscope (BX53, OLYMPUS CORPORATION, Japan).
Alizarin Red Staining And Alkaline Phosphatase Activity Assay
To test the osteogenic differentiation and mineralization ability of CGF on BMSCs, Alkaline phosphatase activity assay and Alizarin Red Staining were performed after osteogenic differentiation for 14 days. According to the CCK-8 and migration results, 10% CGF extract was selected to perform the osteogenic differentiation experiment. Briefly, BMSCs were seeded in 12-well plates at a density of 2 × 104 cells/cm2. After incubation for 12 h in complete medium, the medium was changed to medium containing with 0%, 5%, 10%, or 20% CGF extract, the cells were divided into 4 groups: as follows: i) The control group (Con), cultured with the complete medium without osteogenic induction medium; ii) 10 CGF% group, cultured with the medium supplemented with 10% CGF extract without osteogenic induction medium; iii) Osteogenic induction group (OI), cultured in the presence of osteogenic induction medium; and iv) OI + 10% CGF group, cultured with the medium supplemented with 10% CGF extract in the presence of osteogenic induction medium. After cultured for 14 days, Alizarin Red staining and ALP activity were performed. For Alizarin Red staining, after gently rinsing with ddH2O, the cells were stained in a solution of 2% Alizarin Red at pH 4.1 for 20 min and then washed with ddH2O. The samples were air-dried, and images were captured under a light microscope (BX53, OLYMPUS CORPORATION, Japan). Additionally, the bound alizarin red was dissolved in 200 ll 100 mM hexadecylpyridinium chloride (Cyagen Biosciences Inc. Guangzhou, China) and the absorbance (OD value) of the supernatant was measured at 578 nm. After staining, plates were digitally photographed and the acquired images were analyzed.
For the ALP activity assay, the cell supernatant in each group was collected after cultured for 14 days. ALP activity of BMSCs was measured using Lab Assay ALP (Cyagen Biosciences Inc. Guangzhou, China), according to the manufacturer’s protocol. ALP activity was normalized to total protein determined by BCA protein assay kit (Beyotime). The optical density (OD) of the ALP protein was measured at 520 nm according to the manufacturer’s instruction. ALP activity was calculated using the ALP level normalized to the total protein.
Quantitative Real-time Pcr (RT-qPCR)
The mRNA expressions of Runx2, Ocn, Smad1, and Smad5 were detected by RT-qPCR. Briefly, BMSCs were cultured with cell culture media without CGF extract, or with 10% CGF extract, OI, and OI + 10% CGF extract. After 14 days of culture, the cells were collected to perform TR-qPCR and Western Blot. For the RT-qPCR, total RNA was isolated from cells by use of the RNeasy mini Kit (Qiagen, Valencia, CA, USA), in accordance with the manufacturer’s instructions. cDNA was synthesized by use of the SuperScript III First-Strand synthesis system (Invitrogen). This cDNA was then analyzed by real-time PCR analysis by use of the 7900HT fast real-time PCR system (Applied Biosystems, Tokyo, Japan). The SYBR green assay with SYBR Premix EX taq (Tli RnaseH plus; TaKaRa Bio, Otsu, Japan) was used for amplification of all target transcripts. Expression values were normalized to β-actin. The sequences of the specific primers for the target genes were listed as follows (Sangon Biotech, China): Runx2 (forward: 5’-GACCAGTCTTACCCCTCCTA-3’ and reverse: GGCAGTGTCATCATCTGAAA-3’); Ocn (forward: 5’-ATGGCGTATTAGAGGCAGCA-3’ and reverse: 5’-GGTTTCGTGAAGAGCGCCA-3’); Smad1 (forward: 5’-CAGCGTGTTGGTGGATGGT − 3’ and reverse: 5’-CCGTGGTGGGATGAAAGC − 3’); Smad5 (forward: 5’-AACACCAGGCGGCACATCGG − 3’ and reverse: 5’-GACGGTGGTGGGGTGGAAGC-3’); β-actin (forward: 5’-GGAGATTACTGCCCTGGCTCCTA − 3’ and reverse: 5’-GACTCATCGTACTCCTGCTTGCTG − 3’).
Western Blot
The protein expressions of BMP-2, p-Smad1/5/8, and Smad1/5/8 were detected by western blot. Briefly, the cell samples were lysed in RIPA lysisbuffer (Beyotime, China). Protein concentrations were measured using a BCA protein kit (Beyotime, China). Twenty microliters of protein were loaded per lane on a 10% SDS-PAGE gel for electrophoresis and then transferred to 0.22 mm PVDF membranes (Bio-Rad, USA).
Statistical analysis
All analyses were performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). All experiments were performed at least in three independent repeats. All data are shown as the mean ± SD, a two-sided unpaired Student’s t-test or analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons were used for statistical analysis of differences between groups. A statistical difference between experimental groups was regarded as significant when the p value was < 0.05.