Resumen de Optimized Platelet-Rich Fibrin With the Low-Speed Concept: Growth Factor Release, Biocompatibility, and Cellular Response
Masako Fujioka Kobayashi, Richard J. Miron, Maria Hernandez, Umadevi Kandalam, Yufeng Zhang, Joseph Choukroun
Background: Over the past decade, use of leukocyte platelet-rich fibrin (L-PRF) has gained tremendous momentum in regenerative dentistry as a low-cost fibrin matrix used for tissue regeneration. This study characterizes how centrifugation speed (G-force) along with centrifugation time influence growth factor release from fibrin clots, as well as the cellular activity of gingival fibroblasts exposed to each PRF matrix.
Methods: Standard L-PRF served as a control (2,700 revolutions per minute [rpm]-12 minutes). Two test groups using low-speed (1,300 rpm-14 minutes, termed advanced PRF [A-PRF]) and low-speed + time (1,300 rpm-8 minutes; A-PRF+) were investigated. Each PRF matrix was tested for growth factor release up to 10 days (eight donor samples) as well as biocompatibility and cellular activity.
Results: The low-speed concept (A-PRF, A-PRF+) demonstrated a significant increase in growth factor release of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1, epidermal growth factor, and insulin-like growth factor, with A-PRF+ being highest of all groups. Although all PRF formulations were extremely biocompatible due to their autogenous sources, both A-PRF and A-PRF+ demonstrated significantly higher levels of human fibroblast migration and proliferation compared with L-PRF. Furthermore, gingival fibroblasts cultured with A-PRF+ demonstrated significantly higher messenger RNA (mRNA) levels of PDGF, TGF-β, and collagen1 at either 3 or 7 days.
Conclusions: The findings from the present study demonstrate modifications to centrifugation speed and time with the low-speed concept favor an increase in growth factor release from PRF clots. This, in turn, may directly influence tissue regeneration by increasing fibroblast migration, proliferation, and collagen mRNA levels. Future animal and clinical studies are now necessary.
Over 15 years ago, platelet-rich fibrin (PRF) was introduced as an autogenous source of blood growth factors that could serve as a tool for tissue regeneration in modern medicine.1 The concepts were derived from the fact that a first-generation platelet concentrate, platelet-rich plasma (PRP), was being heavily used in various fields of medicine despite bearing the negative aspect of containing anticoagulants, thereby preventing the full coagulation cascade important for tissue wound healing.2-4 PRF (since renamed leukocyte PRF [L-PRF] due to its higher leukocyte content) does not contain anticoagulants and further provides a three-dimensional fibrin matrix that may be used as a scaffold for a variety of procedures including serving the function of a barrier membrane in guided bone regeneration and guided tissue regeneration procedures.5-7 Since its introduction in 2001,1 PRF has been extensively used in dentistry for a variety of procedures, and its effectiveness has been demonstrated for extraction socket management,8 gingival recessions,9-11 intrabony defect regeneration,12,13 and sinus elevation procedures.7 Major advantages include having completely immune-compatible growth factors collected at relatively no costs without anticoagulants.14-17 While initial and early experiments revealed PRP contained high concentrations of autologous growth factors (up to 6 to 8 times higher than normal blood concentrations), including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β1,18 PRF has since been shown to release even higher total growth factors over a more extended period of time.19 One primary proposed reason for a slower release of growth factors over time is the ability of the fibrin matrix to hold proteins within its fibrin network as well as cells capable of further release of growth factors into their surrounding microenvironment.6,20-23 Leukocytes have been shown to be highly important immune cells capable of directing and recruiting various cell types during the wound healing process.24-26 Since high centrifugation forces are known to shift cell populations to the bottom of collection tubes (whereas PRF is collected from the top one-third layer), it was recently hypothesized that by reducing centrifugation speed (G-force), an increase in leukocyte numbers may be achieved within the PRF matrix.27 It was since shown that with decreased centrifugation G-force (now termed advanced PRF [A-PRF]), an increase in total leukocyte numbers within PRF matrix scaffolds was observed.27 Furthermore, and in agreement with this hypothesis, it was shown that the release of several growth factors, including PDGF, TGF-β1, VEGF, epidermal growth factor (EGF), and insulin-like growth factor (IGF), were significantly higher in A-PRF compared with L-PRF and PRP.19 Since centrifugation force has been shown to have a direct impact on growth factor release from within PRF scaffolds,19 the aim of the present study is to further investigate whether centrifugation time would similarly further improve growth factor release from within PRF scaffolds. In principle, less centrifugation time would reduce cell pull-down by centrifugation forces, which would theoretically increase the total number of cells left contained within the top layer (PRF matrix). Furthermore, since at present it remains completely unknown what influence these changes to centrifugation protocols will have on tissue regeneration, effects of each PRF matrix, including L-PRF, A-PRF, and A-PRF+, were investigated for the first time on human gingival fibroblast (HGF) cell biocompatibility and cell activity. Cells were therefore cultured with growth factors from each PRF matrix (L-PRF, A-PRF, and A-PRF+) and investigated for cell migration, proliferation, growth factor release, and collagen synthesis in vitro.
