## 4 Discussion
Remarkably, the data indicate that the presence of L-PRF may also be linked to increased local levels of some pro-inflammatory mediators such as IFN-gamma and IL-1 beta, which have already been reported as produced and released by L-PRF preparations [9]. While these cytokines mediate inflammatory changes of the gingival tissues, including angiogenesis and edema [31] that could affect the soft tissue healing improvement, the present data suggest that the supraphysiological concentrations released by L-PRF on dental sockets of the third molars are not sufficient to increase postoperative discomfort. On the other hand, the concentrations of most cytokines decreased to levels similar to the control sides within seven days, with the persistent difference found only to VEGF and the anti-inflammatorycytokines IL-10 and the antagonist of the receptor of IL-1 (IL-1 RA), which might contribute to an immunomodulatory role for L-PRF, as previously proposed by [PERSON] et al. [8]. Another interesting finding was the identification of a strong positive correlation between the platelet count of the samples used to produce L-PRF and the local content of VEGF and, to a lower extent, TGF-\(\beta\)1. The relatively small sample from the present study may have impaired the identification of possible correlations for other analytes present at smaller concentrations. Still, this finding is in agreement with the idea that individual traits may impact biological factors related to the materials effectivity; in this case, the local release of growth factors, reinforcing the need for point-of-care quality assurance of individual L-PRF preparations, including platelet counts of whole blood and prepared membranes [32; 33]. Concerning the analysis of pain and healing of soft tissue, the present findings are in agreement with previous reports on good outcomes related to the use of L-PRF on extracted dental sockets [5; 10; 34]. L-PRF implanted in the third molar socket improved postoperative discomfort (\(p<0.05\)) and significantly enhanced soft tissue healing on the seventh and fourteenth day, a timeframe where several other studies also report good clinical results of L-PRF [5; 10; 34]. Furthermore, the present study provides direct evidence of the connection between released growth factors and clinical improvement by different outcome measures, represented mainly by the strong correlation of PDGF-bb concentration at the L-PRF site with the reduction of pain on the third day after surgery, and the moderate correlation with the cicatrization index at the 7 th and the 14 th days after surgery. It is essential to notice that PDGF is considered one of the essential mediators released by platelet aggregates. Its proven efficacy in the regeneration of both soft and hard tissue has even led to its approval by the FDA for clinical uses, such as periodontal therapy [31]. Together with the weak-to-moderate negative correlation of FGFb with pain scores on the third day, these findings should encourage further studies on the release of this growth factor by L-PRF, including larger samples and other biomolecular, histological, and histomorphometric analyses at different times of tissue repair. One of the main limitations of the present study is related to the fact that the sample size was calculated based on the primary clinical outcomes. While the resulting small sample indeed has proven sufficient for the detection of differences between L-PRF and blood clot, as well as a correlation between PDGF and clinical improvement at the L-PRF site, it might have been insufficient for the identification of direct relationships between the clinical and biochemical outcomes through the multiple regression analysis. We were also unable to identify L-PRF treatment's effects through the time-weighted endpoint of pain assessment SPID, employed mainly in clinical trials of analgesics. In this case, rather than a small sample, the very nature of L-PRF mechanisms, which are possibly anti-inflammatory rather than analgesic, may explain the results.