Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols

Objectives: To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-alpha), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-beta(1)) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240 g/8 min or 416 g/10 min. Methods: Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1 h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6 h, 24 h and then every 48 h to 21 days. Cytokines and GFs were measured by ELISA at 1 h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. Results: There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p = 0.01) effect of time was noted. All cytokines were detected after 6 h of PRF clot culture until day 21. GF were detected at 1 h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p = 0.01) correlation was noticed between all the proteins evaluated. Conclusions: Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-beta(1) and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals.