Objectives: Several studies have recently demonstrated that only marginal improvements in platelet and leukocyte concentrations are achieved following standard injectable platelet-rich fibrin (i-PRF) protocols. Due to these previous findings, a novel harvesting technique was recently developed to collect higher concentrations of platelets/leukocytes specifically from the buffy coat layer (C-PRF) following faster centrifugation protocols. The aim of this study was to investigate the regenerative properties and effects on growth factor release and cellular activity of PRF collected through this novel harvesting technique compared to standard i-PRF protocols.

Materials and methods: The upper 1-ml layer collected through standard i-PRF protocols at low centrifugation speeds was compared with 1 mL of C-PRF collected from the buffy coat layer following high centrifugation protocols (3000×g for 8 min on a horizontal centrifuge) to specifically concentrate cells within the platelet/leukocyte-rich buffy coat layer. Thereafter, the expression of seven different growth factors, including PDGF-AA, PDGF-AB, PDGF-BB, TGF-β1, VEGF, IGF-1, and EGF, was characterized for up to 10 days. Then, gingival fibroblast biocompatibility was investigated at 24 h (live/dead assay); migration was investigated at 24 h; proliferation was investigated at 1, 3, and 5 days; and the expression of PDGF and TGF-β was investigated at 3 days. Collagen 1 immunostaining was also quantified at 14 days.

Results: At all investigated time periods, a significant increase in growth factor release was observed in C-PRF. In particular, the release of PDGF-AA, TGF-β1, and EGF exhibited the highest increases when compared with that in i-PRF. While both i-PRF and C-PRF exhibited high biocompatibility and induced significantly higher fibroblast migration and proliferation when compared with that of the control tissue culture plastic group, C-PRF showed the greatest potential for cell migration and proliferation. Furthermore, C-PRF induced significantly higher mRNA levels of TGF-β and PDGF levels at 3 days and greater collagen 1 staining when compared with induced by i-PRF.

Conclusions: In the present study, it was found that C-PRF collected specifically from the buffy coat layer following higher centrifugation protocols exhibited an up to a threefold increase in growth factor release when compared with that exhibited by standard i-PRF. This significantly promoted higher gingival fibroblast migration, proliferation, gene expression, and collagen I synthesis.

Clinical relevance: The findings of the present study demonstrate that a more potent formulation of liquid platelet concentrate than that obtained from the upper plasma layer following a short and slow centrifugation protocol (i-PRF protocol) can be obtained for clinical use by specifically harvesting cells in the platelet- and leukocyte-rich buffy coat layer following an 8-min 3000×g centrifugation protocol (C-PRF protocol).