In the present study, all the experiments were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China. CGF was prepared according to the following protocol as previously described (14): briefly, 5 ml venous blood was drawn from the inferior vena cava of Wistar rats (weighing 250 g). Blood was collected into a sterile tube without any anticoagulants and immediately centrifuged in a Medifuge centrifuge (Silfradent S.R.L., Sofia, Italy). Following centrifugation, the blood had separated into 3 layers due to the different densities of the blood components. CGF was the middle layer; it was mechanically separated using scissors and gently compressed into a thin membrane. Each membrane was soaked in 5 ml fresh Dulbecco's modified Eagle's medium (DMEM; Gibco, New York, NY, USA) without fetal bovine serum (FBS) in a 15 ml centrifuge tube (Corning Glassworks, Corning, NY, USA). The medium, named CGF extract, was collected 7 days later and centrifuged at 400 × g for 5 min to pellet the platelets and red blood cells. All the extractions were stored at −80°C for future use.

The CGF membrane was fixed in 2.5% glutaraldehyde solution and 1% osmium tetroxide (both from Sigma-Aldrich, St. Louis, MO, USA). It was dehydrated serially in 50, 70, 80, 90 and 100% ethanol. After drying, the sample was coated with gold, and images were captured under a scanning electron microscope (SEM; S-3400N; Hitachi High Technologies America, Schaumburg, IL, USA). Fibrin fiber diameter and percentage porosity were measured on the SEM images using ImageJ software (version 1.49; Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Briefly, the diameters of 6 randomly selected fibers were measured using the tool. Percentage porosity was determined by the particle analysis function in the image analysis software.

We hypothesized that the growth factors, which are trapped within the fibrin meshes of CGF, are released in a controlled manner over a long-term period. Thus, we examined the state of the growth factors released from the CGF extract over time. Briefly, the CGF membrane was soaked in 5 ml DMEM without FBS in a 15 ml flacon tube and incubated at 37°C. The medium was collected on days 1, 3, 5, 7, 9, 11 and 13 and following collection, 5 ml fresh DMEM was added to each tube. All the collected extracts were stored at −80°C. TGF-β1 levels were determined using a commercially available ELISA kit (Elabscience, Wuhan, China) according to the manufacturer's instructions. All the assays were performed in triplicate.

The RSC96 SCs (obtained from Chinese Academy of Sciences, Shanghai, China) were seeded into 6-well plates at a density of 5×10⁵ cells/well. After reaching confluence, a scratch was made on the cell monolayer using a P10 pipette tip according to the protocol previously described by Liang et al (20). Cellular debris was then washed off using phosphate-buffered saline (PBS). DMEM or CGF were then added to the culture plates. Images of the cells were captured using an Olympus DP-50 digital microscope camera (Olympus Optical Co., Ltd., Tokyo, Japan) immediately after wounding and again 24 h later. Reference points were made on the outer bottom of the plates to mark the location of the scratch in order to obtain the same field. The distance between the edges of the scratch was measured using cellSens Entry software (Olympus Life Science, Hamburg, Germany). The migration rate was calculated using the following equation: migration rate = (D₀ − D₂₄)/D₀ ×100, as previously described (21). D₀ is the distance at the time of making the scratch wound, and D₂₄ is the corresponding distance 24 h later.

The protein expression of integrin β1 and phosphorylated (p-)FAK was examined by western blot analysis. The RSC96 SCs were treated with DMEM or CGF extract supplemented with 10% FBS (Gibco, Melbourne, Australia). At 1, 2 and 3 days, the cells were washed twice using PBS, and then lysed in ice-cold lysis buffer [radioimmunoprecipitation assay buffer with 1% phenylmethylsulfonyl fluoride (Biyuntian, Beijing, China)] on ice for 1 h. The cell lysates were collected and centrifuged at approximately 400 × g. The protein concentrations were determined using the bicinchoninic acid (BCA) assay. Equal amounts of protein (40 µg) were loaded onto a 10% SDS-polyacrymide gel and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After blocking in Tris-buffered saline with 0.05% Tween-20 (TBST) containing 5% skimmed powdered milk for 90 min, the membranes were washed thrice with TBST and then incubated with primary antibodies against either integrin β1 (polyclonal antibody, rabbit anti-rat, 1:500, Cat. no. bs-0486R; Bioss, Wuhan, China) or p-FAK (polyclonal antibody, rabbit anti-rat, 1:1,000, cat no. AP0437; ABclonal, Cambridge, MA, USA) overnight at 4°C. The samples were eluted 4 times with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit, 1:1,000, cat. no. AS014; ABclonal). An ECL chemiluminescence assay was used for the chemiluminescence-based immunodetection of HRP. The intensities of the bands (representative of protein levels) were determined using ImageJ software (version 1.49; Wayne Rasband, National Institutes of Health). β-actin was used to normalize target proteins.

During RNA intereference (RNAi), siRNAs may serve as guides for the enzymatic cleavage of complementary RNAs, thereby inhibiting sequence-specific gene expression. RNAi is an effective tool for the analysis of gene function (22). In the this study, a plasmid vector synthesizing siRNA was constructed in order to suppress the gene expression of integrin β1 in the SCs. Briefly, siRNA were synthesized by Sangon Biotech (Shanghai, China). The siRNAs with the targeting sequences GAAGGGTTGCCAACCAAGT, GACATGGATGCTTACTGCA and GGTGGCTTTGATGCAATCA were labeled as siRNA-1, siRNA-2 and siRNA-3, respectively. All the siRNAs were annealed and ligated into the pRNA-H1.1 plasmid between the HindIII and the BamHI sites. The normal plasmid without siRNA was used as a control, labeled as siRNA-N. A volume of 20 µl of the recombinant plasmids was transformed into 100 µl E. coli strain JM109, and the cells were then plated onto Luria-Bertani (LB) plates containing 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 100 mg of ampicillin, and 15 g of agar per liter and incubated at 37°C. The colony was picked up and the plasmids were extracted. All the plasmids were verified by DNA sequence analysis.

The RSC96 cells were cultured in dishes to obtain 70–90% confluence without antibiotics. The cells were then transfected with siRNA-1, siRNA-2, siRNA-3 or siRNA-N, using transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The medium was replaced with DMEM supplemented with 10% FBS 4 h after transfection. The protein expression of integrin β1 after gene silencing was determined using western blot analysis as described above.

The mRNA expression of integrin β1 following the application of siRNA was analyzed by RT-qPCR. Total RNA was extracted using TRIzol reagent (Invitrogen) and was then reverse transcribed using the RevertAid kit (Takara Bio, Otsu, Japan) according to the manufacturer's instructions. SYBR-Green qPCR Super Mixture (Takara Bio) was used to assess gene expression on an Exicycler 96 Real-time PCR system (Bioneer, Daejeon, Korea). The samples were normalized to β-actin. The following primer sequences were used: rat integrin β1 forward, 5′-TTGCCAACCAAGTGACATA-3′ and reverse, 3′-GGTAGTCTTCAGCCCTCTT-5′; and rat β-actin forward, 5′-GGAGATTACTGCCCTGGCTCCTAGC-3′ and reverse, 3′-GGCCGGACTCATCGTACTCCTGCTT-5′. The following amplification conditions were used: 95°C for 10 min followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. All the samples were run in triplicate in each experiment. Changes in gene expression were calculated using the 2^−ΔΔCt method.

The migration of the RSC96 SCs following integrin β1 gene silencing was examined using 6.5 mm Transwell chambers with 8 µm pores (Corning Costar, Corning, NY, USA). CGF or DMEM supplemented with 20% FBS was added to the lower wells to act as a chemoattractant. The cells were trypsinized, washed in PBS and then added to the upper Transwell chambers (1×10⁴ cells/well) and allowed to migrate for 24 h. The non-migrated cells were removed from the upper chambers using a cotton swab. The migrated cells were fixed with paraformaldehyde, stained with 0.5% crystal violet (Amresco, Solon, OH, USA), and then counted in 5 fields/well under a microscope (×200 magnification).

Data are expressed as the means ± standard deviation and were analyzed using one-way analysis of variance (ANOVA) with the Tukey HSD comparison test or using the independent-samples t-test. A P-value <0.05 was considered to indicate a statistically significant difference.

The biological properties of CGF were evaluated as a first step. CGF was generated by centrifuging whole blood. There were 3 layers following centrifugation (Fig. 1A): the upper layer is a clear fluid which is the blood serum; the middle layer is CGF which is composed of a large and dense polymerized fibrin block with aggregated platelets and growth factors; the bottom layer is composed of red blood cell debris. CGF was mechanically separated using scissors and then compressed into a thin membrane. Surface parameters such as topography and hardness markedly affect cell behavior and the release of growth factors (10,12,23). SEM analysis (Fig. 1B) revealed that CGF had a fiber-like appearance. The diameter of the fibers was 0.36±0.14 µm and the percentage porosity was 40.44±2.97%. The fibrin fiber network in CGF was sparse, with many platelet aggregates or red blood cells trapped in the spaces between the fibrin fibers.

TGF-β1 is one of the growth factors secreted by platelets (24) and has neuroprotective effects (25,26). Thus, the secretion of TGF-β1 was measured by ELISA in the present study. In a sustained-release test, it was found that CGF slowly released TGF-β1 for up to 13 days (Fig. 2). The level of TGF-β1 increased from 75 pg/ml on day 1 to 130 pg/ml on day 7. Subsequently, the level slowly decreased to 60 pg/ml on day 13.

To examine the effects of CGF on the migration of RSC96 SCs, a scratch wound-healing assay was performed (Fig. 3). The scratch assay is a straightforward and economical method used to observe cell migration in vitro (20). It imitates, to a certain extent, the migration of cells in vivo. CGF significantly increased the migration rate of the RSC96 SCs in comparison with DMEM. The migration rate of the cells in the CGF group was 4.25-fold greater than that of the cells in the DMEM group (P<0.05).

Integrin β1 is essential for the migration of SCs (6,19). FAK is a cytoplasmic tyrosine kinase that plays critical roles in integrin-mediated signal transductions, and it is phosphorylated following activation (27). Fig. 4 shows the protein expression of integrin β1 and p-FAK. There was a statistically significant (P<0.05) increase in the integrin β1 and p-FAK levels following treatment with CGF compared with DMEM. The integrin β1 levels increased 1.62-fold on day 1, 1.54-fold on day 2, and 1.14-fold on day 3. The p-FAK levels were increased 1.46-fold on day 1, 1.80-fold on day 2 and 1.40-fold on day 3. These results suggest that integrin β1 is involved in the CGF-induced migration of SCs, and that the FAK signaling pathway is activated.

In order to reveal whether integrin β1 plays an essential role in the CGF-induced migration of SCs, integrin β1 was silenced by different siRNAs. As shown in Figure 5A, no significant change in the integrin β1 mRNA level was observed in the siRNA-N group compared with that the normal (untransfected) cells. Transfection with siRNA-1 decreased the integrin β1 mRNA level by approximately 50%, transfection with siRNA-2 decreased the integrin β1 mRNA level by 70–80% and transfection with siRNA-3 reduced the integrin β1 mRNA level by 30–40%. Fig. 5B and C show the protein expression of integrin β1 following transfection. No significant change was observed in the siRNA-N group. Among the targeted siRNA groups, transfection with siRNA-2 significantly reduced the protein expression of integrin β1 by almost 70%. Thus, siRNA-2 was selected for use in the subsequent experiments.

Transwell migration assays were performed to detect changes in cell migration after integrin β1 was downregulated by siRNA-2. Fig. 6 shows that Lipofectamine 2000, which was used as a transfection reagent, did not affect the migration of the SCs. Following the application of siRNA-2, the cell migration rates were decreased significantly by 67% in the DMEM-treated group, and by 50% in the CGF-treated group. Moreover, the migration rate of the cells in the CGF treatment group was significantly higher than that of the cells in the DMEM treatment group. This demonstrates that CGF promotes the migration of SCs even after integrin β1 silencing, indicating that integrin β1 plays only a partial role in the CGF-induced migration of SCs, and that other pathways are also involved in this process.