## 4 Materials and Methods  
This study was approved by the ethics committee by the authors' institute, and informed consent was obtained.  
**PRP preparation**  
Thirty milliliters of whole blood were drawn from each patient and then mixed with 1.25 mL anticoagulant solution (citrate phosphate dextrose adenine-1; CPDA-1) in a plain tube. The PRP was prepared by two double spin centrifugation processes using a bench-top centrifuge machine (Model: 5430R; Eppendorf, Germany). For the first double spin PRP preparation, 900 g for 5 min and then 1500 g for 15 min centrifugations were performed. The lower 3 mL of the plasma was termed PRP A2 (Figure 3) [39]. For the second double spin PRP preparation, centrifugation of 1500 rpm for 5 min and then 20 min at 6300 rpm were performed. Finally, half of the superficial plasma layer was removed, and the platelet pellet was suspended in the remaining half of the plasma volume termed PRP B2 (Figure 3) [38]. The platelet numbers of the PRPs were counted by an automatic hematology analyzer (Model: Sysmex XT-1800i; Kobe, Japan) after preparation.  
Figure 3: Two types of PRP preparations. PRP A2, 900 g for 5 min and then 1500 g for 15 min; PRP B2, 1500 rpm for 5 min and then 6300 rpm for 20 min.  
**Evaluation of the cytokine composition in the 3-chamber co-culture device and the conventional 24-well culture plate at different time points**  
To study the platelet-derived cytokines in 3-chamber co-culture device and conventional culture well (24-well plate; Figure 4), 40 \(\upmu\)L PRP and 40 \(\upmu\)L culture medium were added to one sub-chambers of the co-culture device (Figure 5a). Then, 100 \(\upmu\)L of culture medium were added to each of the other two empty sub-chambers (Figure 5b). After gelling of the PRP overnight, 700 \(\upmu\)L of culture medium were applied to the whole culture well to cause the culture medium to cross the central barrier (Figure 5c). The platelet-derived cytokines released in the medium would diffuse to all 3 sub-chambers, as shown in Figure 5d. The same procedure was performed in a 24-well plate as a control. After gelling of the PRP overnight, 900 \(\upmu\)L of culture medium were applied to the 24-well culture well. The supernatant was collected at 0 h (immediately after culture medium exchange between each co-culture chamber), 24 h, 48 h, 72 h, and 96 h. TGF-81 and PDGF-AB were analyzed by using a commercial immunoassay kit (Human TGF-81 and Human PDGF-AB Quantikine(r) ELISA kit; R&D Systems, Minneapolis, MN, USA). The analytical protocol followed the manufacturer's instructions as previously described [40]. In addition, a serial dilution of the provided standard TGF-81 and PDGF-AB solution was utilized to set up the calibration curve. Thus, the actual concentration of the TGF-81 and PDGF-AB could be calculated based on the calibration curve. All samples were assayed in triplicate. The whole procedure was shown in Figure 6.  
**Statistical Analysis**  
Statistical analyses were performed with the SPSS software program (v 23.0; IBM Corp). The measured data are presented as the arithmetic mean and the standard deviation (SD). To compare platelet concentration between whole blood, PRP A2, and PRP B2, analysis of variance (ANOVA) followed by Bonferroni post hoc test was used. The concentrations of the cytokines in each incubation time were analyzed by Kruskal-Wallis test (nonparametric analysis of variance). A Bonferroni post hoc test was conducted to compare multiple values from each of the preparation conditions. Linear correlations between the platelet and cytokines content were analyzed in terms of the Pearson correlation coefficient. Correlations of cytokines between 3-chamber co-culture device and conventional 24-well plate at five time points were analyzed by the Pearson correlation coefficient. A \(p\)-value of <0.05 was considered significant.  
Figure 4: (**a**) 3-chamber co-culture device. (**b**) Conventional 24-well culture plate.