## 3 Discussion
To maximize the advantages of PRP treatment, the optimal PRP formulation (leukocyte and platelet count, cytokines), platelet activation methods (thrombin, type 1 collagen, calcium gluconate), type of carrier vehicle (fibrin matrix, scaffold), and dosing regimen (single versus multiple injections, intraoperative versus delayed injection) [34]. Unfortunately, the best preparation protocols of PRPs, the precise cytokines combination, and the best timing to treat a specific disease at its different injury level are still unknown. Cytokines within the \(\alpha\)-granules of platelets are believed to be responsible for tissue healing. Knowledge of the kinematics of cytokine release is fundamental to PRP application, especially the optimal timing and interval of injection of PRP in different kinds and stages of the disease. [PERSON] et al. compared the cellular composition and cytokine-release kinetics of five different PRPs at 1 h, 24 h, 72 h, and 7 days after PRP preparation [13]. They found the fibroblast growth factor (FGF) and transforming growth factor (TGF) release quickly and decreased over time. On the other hand, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were constantly released and sustained over 7 days. Therefore, the cytokine content was not necessarily proportional to the platelet count of the PRPs. In our study, we compared concentrations of TGF-\(\beta\)1 and PDGF-AB and at five time points after PRP preparation. The variations in the concentrations of different cytokines at different time points suggested that each type of cytokine release has individual dynamics. However, there are studies that reported a correlation between the platelet counts and the cytokine concentrations in PRP [35; 36]. In our study, however, there was only a weak ([r] = \(-\)0.084) correlation of TGF-\(\beta\)1 and platelet concentration at 0 h. The correlation of PDGF-AB and platelet concentration was even negative ([r] = \(-\)0.4) at time zero. This corresponded to the results of [PERSON] et al. [13] that the cytokine content was not necessarily proportional to the cellular composition of the PRPs. The objective of using a 3-chamber co-culture device in PRP studies is because it works as a screening platform. The design could avoid the gelling effect of PRP [15; 17] and provide more variables, e.g., diseased tissue or cells at different stages, when co-cultured with different PRPs. The relatively smaller culture area makes it possible to culture cells from scant specimens because sometimes the diseased tissue is very few. This helps clinicians to screen a tailored treatment to the extent of the disease process. For example, to find out the best PRP preparation for mild versus severely degenerative rotator cuff tear, only a few specimens are needed to be harvested during surgery. More types of PRPs could be co-cultured with cells from different tissues to see their potential to facilitate cell proliferation than in conventional culture wells with a larger diameter. This study confirmed the high correlation of cytokines in the 3-chamber co-culture device and conventional 24-well plate. This implies the 3-chamber co-culture device might replace conventional ones in further PRP studies. The other findings of this study are the concentrations of TGF-\(\beta\)1 and PDGF-AB at specific time points. TGF-\(\beta\)1 is a key growth factor to stimulate collagen synthesis, cell proliferation, recruitment, and reduced scar formation in healing tendons [4]. PDGF-AB is an anabolic cytokine and had a role in inhibiting inflammation and pain as well as enhancing the biosynthesis of cartilage and bone matrix [37]. The best time point to start PRP treatment for a specific disease, however, is yet to be elucidated. In the current study, TGF-\(\beta\)1 and PDGF-AB showed consistent concentrations through 96 h after the preparation. These data may help clinicians to design individualized treatment protocols at different time points when PRP injection is considered. There are still limitations regarding this study.