## 3 Results  
### Effects of PEF on Platelet Activation and Growth Factor Release  
PCs were subjected to nine different PEF treatments, varying the electric field (10-15 kV/cm) and pulse number (1, 7, and 15) with 1 Hz frequency. The pulse duration was the same in all conditions (2 \(\upmu\)s/pulse). Platelet activation was evaluated by flow cytometry, defining the activation by the percentage of CD62P+ platelets [21; 23]. The data showed a significant increase in CD62P+ platelets in comparison to the untreated group for all PEF conditions tested. In particular, platelet activation was enhanced with the increase in pulse number, whereas no differences were observed between the different electric fields applied (Figure 2a).  
The proteins released from platelets upon PEF stimulation were evaluated and compared to untreated PR. No differences in the total protein present in PR PEF were observed for the majority of PEF conditions applied when compared to untreated PR or PL F&T; moreover, one pulse of 15 kV/cm (PR PEF G) leads to a significant increase in the total protein in comparison to untreated PR (Figure 2b).  
Following this, the levels of GF described to be stored in platelets [24; 25] and relevant for MSC expansion, such as PDGF-BB, TGF-$1, VEGF, and IGF [26; 27; 28; 29; 30], were measured in the PR of all conditions tested. Our data demonstrate that PEF application leads to the release of GF to the PR, with higher release levels observed for TGF-$1, achieving similar levels to PL F&T (Figure 2c-f).  
Figure 2: Characterization of platelet activation and PR after PEF application to PCs. (**a**) Percentage of CD62P positive platelets after PEF treatment. (**b**) Total PR protein after PEF application. (**c**) PDGF-BB, (**d**) TGF-$1, (**e**) VEGF, and (**f**) IGF released to PR after application of PEF. Results plotted relative to the untreated condition. Mean \(\pm\) SD is shown. \(n\) = 3. ns = non-significant. \({}^{*}\)\(p\) < 0.05, \({}^{**}\)\(p\) < 0.01, \({}^{***}\)\(p\) < 0.001, \({}^{***}\)\(p\) < 0.0001.  
Our data demonstrate that the PEF conditions with 10 (PR PEF A, B, and C) and 12.5 kV/cm (PR PEF D, E, and F) pulses led to an increase in the PDGF concentration in the PR PEF when compared to the untreated group (Figure 2c).  
In addition, the levels of TGF-\(\beta\)1 were similar to those obtained in the PL F&T condition, which were significantly higher than the untreated PR. Moreover, comparing the PR PEF with untreated PR, we could observe that lower pulse intensities require a higher pulse number to achieve a significant increase in comparison to the untreated PR. Thus, a significant enhancement of TGF-\(\beta\)1 was observed for 7 pulses of 10 and 12.5 kV/cm (PR PEF B and E), and 15 pulses of 12.5 and 15 kV/cm (PR PEF F and I). The VEGF concentration in the PR PEF was similar to the untreated condition; however, PEF application led to a higher VEGF release if 1 pulse of 12.5 and 15 kV/cm (PR PEF D and G), and 15 pulses of 10 kV/cm (PR PEF C) were applied to cells. However, PEF application to platelets led to a significant reduction in IGF levels in the PR after the application of 15 pulses of 10 and 15 kV/cm.  
Of particular note, the levels of PDGF-BB or VEGF released from platelets after the application of PEF did not achieve PL F&T levels, which indicates that PEF application induces a controlled released of GF (Figure 2c,d).