Study design and ethics statement
All procedures were carried out according to the guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. Male Sprague-Dawley (SD) rats were purchased from Sun Yat-sen University Animal Experimental Center (Guangzhou, China). Rats were maintained under standard laboratory conditions, with free access to food and water, and housed prior to experiments in an animal room under standard conditions (23 ± 2 °C; 60 ± 10% humidity; 12 h light/dark cycle).
Preparation and composition analysis of platelet-rich plasma
Ten 12-week-old SD rats were anesthetized by intraperitoneal injection of 2.5% pentobarbital sodium (40–45 mg/kg). The preparation of PRP was strictly aseptic. Ten milliliter whole blood was drawn from each rat via intracardiac puncture. A total of 9 ml of blood sample was drawn from each rat into 10-ml syringes containing 1 ml sodium citrate anticoagulant, of which 0.5 mL was used for blood count (quantitative analysis of platelet and leukocyte counts). The remaining 9.5 ml was transferred into a sterile test tube and centrifuged twice to obtain fresh PRP [19]. After the first centrifugation (400 g, 10 min), the blood was divided into three layers, with the plasma in the supernatant, a buffy coat layer in the middle, and red blood cells at the bottom. Then, the supernatant was pipetted into another sterile test tube for second centrifugation (800 g, 10 min). The 3/4 upper fraction of the plasma (platelet-poor plasma) was discarded, and the remaining liquid was platelet-rich plasma (about 1.5 mL). 10% CaCl2 (C5670, Sigma-Aldrich) plus 1000 U/ml thrombin from bovine plasma (T8021, Solarbio, Beijing, China) was used for activation of the alpha granules in platelets and was added into the apheresis platelets at a volume ratio of 1:10. The activated PRP-containing supernatant was collected and filtered through 0.22-μm aseptic membrane filter and then aliquoted and stored at − 80 °C to avoid repeated freezing and thawing. A 0.2 mL sample of PRP was reserved for the detection of the growth factors.
As mentioned above, the platelets and leukocyte concentrations in the samples of whole blood (0.5 mL) and PRP were measured with the Mindray BC-5000Vet analyzer. ELISA kits were utilized to quantify the concentrations of rat transforming growth factor-beta (TGF-β, LER822–1, Laizee, Shanghai, China) based on the manufacturer’s protocol.
BMSCs isolation and culture
To investigate the effects of PRP, the BMSCs were induced by Dex (D4902, Sigma-Aldrich). Different interventions were used to assess a series of functional assays, including the control group, the Dex group, and the Dex + PRP group. In cell proliferation assay, migration assay, and osteogenic differentiation assay, the concentration of Dex was 10 μM. The concentration of Dex was 100 μM in the apoptosis experiment.
Bone marrow stem cells (BMSCs) of 4-week-old SD rats were obtained from the femur and tibia according to the method described by Shen [6] and were cultured with complete medium:Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, MA, USA) containing 10% fetal bovine serum (FBS, Gibco), 1% penicillin-streptomycin (Gibco) in an incubator at 37 °C and 5% CO2. After 24 h, the medium was changed and the attached cells were washed by phosphate-buffered saline (PBS, Gibco). The culture medium was refreshed every 2 days until adherent cells reach 80–90% confluence, then the cells were passaged for three to five passages for all further experiment use.
Cell proliferation assay
Effect of PRP on Dex-induced BMSC proliferation
To determine the optimal PRP concentration that could promote the proliferation of BMSCs, we first carried out a Cell-counting kit 8 (CCK-8) cell proliferation assay. BMSCs were inoculated in 96-well plates with 3 replicate wells in each group of 3 × 103/well. After 24 h of complete adherence, 100 μL complete medium containing 0, 2.5, 5, 10, and 20% PRP was added. The proliferation of BMSC was measured with a CCK-8 assay kit (K1018–1, ApexBio, MA, USA) at 24, 48, and 72 h. The optimum PRP concentration obtained in this experiment was used in all subsequent experiments.
The CCK-8 kit was used to explore the proliferation rate of BMSCs treated with Dex. A total of 3000/well BMSCs were inoculated into 96-well plates. The cells were divided into three groups: (1) control group, (2) Dex group, and (3) Dex + PRP group. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay reader (SpectraMax Plus 384, Molecular Devices, Sunnyvale, CA, USA).
Flow cytometry
The cell cycle was measured by flow cytometry. The cells were inoculated in a 6-well plate with 1.5 × 105 cells/well. After the cells were completely attached to the wall of the wells, the serum-free medium was incubated overnight, and different interventions were conducted. 2 ml medium was added to each well. After 24 h of induction, the cells were digested by trypsin and fixed overnight with 75% iced ethanol. After being washed by PBS, propidium iodide (PI, C0080, Solarbio, Beijing, China) and RNase A(R8020, Solarbio) were added and then detected by flow cytometer (BD FACSCalibur; BD Biosciences). The results were presented as proliferation index PrI (percentage of cells at S and G2M phase), which was calculated as PrI = (S+ G2 /M) / (G 0 /G1 + S+ G2 /M).PrI indicated the proportion of cells in active proliferation [20].
BMSCs migration assay
BMSCs migration assay was performed by 24-well plates with 8 μm transwell (Corning) upper chambers. 1 × 104/well BMSCs were seeded into the upper chamber, and 600-uL of the complete medium was added into the lower chamber according to the experimental grouping. After incubating for twelve hours, non-migrative cells on the upper surface of the transwell membrane were wiped with a cotton swab. Then the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 20 min, stained with 0.5% crystal violet for 15 min and washed with PBS. It was then observed with a light microscope (Nikon NI-U). Five different visual fields were randomly selected and photographed, and the migratory cells were counted by Image J software (National Institutes of Health, Bethesda, MD, USA).
TUNEL assay
The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was used to estimate the effect of PRP on BMSCs cell apoptosis according to the manual [21]. After 48 h of culture with the serum-free medium in the presence or absence of PRP, the cells were fixed with 4% paraformaldehyde for 25 min at 37 °C, incubated with 0.3% Triton X-100 for 5 min, and washed with PBS twice between each step. The cells were incubated with TUNEL reagent (C1086, Beyotime, Shanghai, China) according to the manufacturer’s instructions, and the nuclei were counterstained with DAPI (C1002, Beyotime) for five minutes. The samples were observed and imaged under a fluorescence microscope (Olympus IX 71).
Alkaline Phosphatase (ALP) and Alizarin Red (ALR) staining
Bone marrow mesenchymal stem cells were seeded on a 24-well plate at a density of 1 × 105 cells/well. After 7 days of using osteogenic differentiation induction medium (RAXMX-90021, Cyagen, Guangzhou, China), ALP staining (Beyotime) was performed. 14 days later, alizarin red staining (Cyagen) was performed according to the manufacturer’s protocol. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for half an hour, and then stained. Before taking pictures, each well was washed 3 times with double distilled water.
Western blotting
The protein was extracted from the cells using radio immunoprecipitation assay (PC101, RIPA) lysis buffer (EpiZyme, Shanghai, China). The protein concentration was determined using Thermo Scientific™ Pierce™ BCA (23227). An equal amount of protein was separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with protein-free fast blocking buffer (PS108, EpiZyme), it was incubated with primary antibodies against Runx2 (Affinity, AF5186), Bcl-2 (Affinity, AF6139), Caspase-3 (Huabio, ET1608–64), and GAPDH (EpiZyme, LF205) at 4 °C overnight, and then incubated with the secondary antibody at 37 °C for 1 h. Thereafter, the proteins were visualized using Omni ECL reagent (SQ201, ECL; EpiZyme) under e-Blot (Touch Imager, Shanghai, China). The gray densitometric was analyzed using Image J (USA).
