Understanding Platelet-Rich Fibrin. Richard J. Miron
the highest concentration of liquid-PRF, as discussed further in chapter 5. Nevertheless, based on the C-PRF protocols established in chapter 2, it remained obvious that better concentrations could be achieved by further modifying centrifugation parameters.
Establishing C-PRF on a Horizontal Centrifuge
In a recent study titled “Improved growth factor delivery and cellular activity using concentrated platelet-rich fibrin (C-PRF) when compared to traditional injectable (i-PRF) protocols,” Fujioka-Kobayashi et al aimed to investigate and optimize PRF in its most concentrated formulation.11 As reviewed in chapter 2, the ability to centrifuge at higher centrifugation speeds and times leads to an accumulation directly at the buffy coat layer.12 There was an approximately tenfold increase in baseline concentrations specifically in this 0.3- to 0.5-mL buffy coat layer directly above the RBC layer produced using higher centrifugation protocols.12 The PRF obtained from this harvesting technique was given the working name concentrated-PRF (C-PRF). Figure 3-20 demonstrates a clinical photograph of standard liquid-PRF protocols versus those of C-PRF. It was hypothesized that based on the extensive increase in the yield of platelets and leukocytes, C-PRF would exhibit higher GF release as well as superior cellular activity. Therefore, the aim of this study was twofold. First, a new centrifugation protocol was developed on a horizontal centrifugation system with the aim of accumulating the greatest concentrations of platelets and leukocytes within the buffy coat. The second aim was to compare the total GF release of PRF obtained through this newly developed C-PRF protocol to that of PRF obtained through the clinically utilized liquid i-PRF protocol over a 10-day period and to investigate the regenerative properties of human gingival fibroblasts in vitro.
Fig 3-20 Visual representation of layer separation following either the i-PRF (300g for 5 minutes) or C-PRF protocol (3000g for 8 minutes). Plasma was collected from the buffy coat region within the 1-mL layer above the RBC layer. (Reprinted with permission from Fujioka-Kobayashi et al.11)
Optimization of C-PRF protocols
Prior to initiating the C-PRF experiments, protocols of 3000g for 5 minutes, 8 minutes, and 12 minutes were compared to optimize the accumulation of cells within the buffy coat layer. It was found that the 5-minute protocol was unable to concentrate all cells within the buffy coat layer, with the platelets remaining in the upper 4 mL. Following the 8-minute protocol, the sequential pipetting method results revealed that the majority of platelets and leukocytes were located within the buffy coat layer 6 region (Fig 3-21). No further advantage was observed following the 12-minute protocol (data not shown). For comparison purposes, a standard i-PRF protocol resulted in a slight concentration of platelets and leukocytes in the upper 1-mL layer from which i-PRF was harvested (see Fig 3-21). Note that many platelets/leukocytes, however, remained in the RBC layers. To harvest C-PRF, a 1-mL layer within this buffy coat layer was collected and processed for further analysis (Fig 3-22).
Fig 3-21 The concentrations of different cell types found in each 1-mL layer of the 10-mL tube obtained through the i-PRF protocol (300g for 5 minutes) and the C-PRF protocol (3000g for 8 minutes). Note that in the PRF obtained through the i-PRF protocol, the majority of platelets and leukocytes were located in the 1-mL buffy coat layer. In the PRF obtained through the C-PRF protocol, although higher concentrations of platelets and leukocytes were found in the upper 1-mL layer, the majority of the platelets and leukocytes were actually located in the RBC layers. (Reprinted with permission from Fujioka-Kobayashi et al.11)
Fig 3-22 Method to collect and concentrate C-PRF. Following centrifugation at higher speeds (2000g for 8 minutes), the majority of cells are located directly at the buffy coat layer. Instead of attempting to remove this layer with a long needle into the deep layers, it is highly advised to first remove the upper 4 mL of platelet-poor plasma (PPP), followed by collection of the C-PRF buffy coat layer.
Tips
In clinical practice, it is best to harvest C-PRF by first removing the upper 3 to 4 mL of platelet-poor plasma (discard it). The remaining C-PRF layer can then be taken much more easily. It is much more difficult to attempt to retrieve this buffy coat zone with 5 mL over top of it; it is harder to concentrate it, and too much volume is often collected.
Many centrifuges may not reach the 3000g speed. A 2000g protocol for 8 minutes will also achieve a C-PRF layer.
Comparative GF release between i-PRF and C-PRF
In the first set of experiments, the release of GFs including PDGF-AA, PDGF-AB, PDGF-BB, TGF-β1, VEGF, EGF, and IGF-1 from i-PRF and C-PRF was investigated by ELISA (Fig 3-23). The release of all GFs over the entire 10-day (240-hour) period was significant for both protocols, with the C-PRF protocol resulting in up to two- to threefold higher quantities. This demonstrated clearly that this newly developed protocol had much greater regenerative potential when compared to previously utilized i-PRF protocols.
Fig 3-23 Protein quantification of PDGF-AA (a), PDGF-AB (b), PDGF-BB (c), TGF-β1 (d), VEGF (e), EGF (f), and IGF-1 (g) at each time point over a 10-day (240-hour) period for PRF obtained through the i-PRF protocol and the C-PRF protocol, as determined by ELISA. Data represents the mean ± SE; an asterisk (*) indicates a value significantly higher than the other group (P < .05). (Reprinted with permission from Fujioka-Kobayashi et al.11)
Biocompatibility and cellular activity of i-PRF and C-PRF
It was first observed that while i-PRF induced a twofold increase in cell migration when compared to that observed in the control, a significantly higher fourfold increase was observed when cells were cultured with C-PRF (Fig 3-24a). Furthermore, C-PRF also induced significantly higher cell proliferation at 3 and 5 days postseeding when compared to i-PRF (Fig 3-24b). Both i-PRF and C-PRF were able to significantly upregulate TGF-β 3 days postseeding (Fig 3-24c), and a significant 250% and 400% increase in the PDGF-AA was observed for i-PRF and C-PRF, respectively (Fig 3-24d). The analysis of COL1 immunostaining also revealed significantly higher COL1A staining for C-PRF when compared to i-PRF and control tissue culture plastic groups (Figs 3-24e and 3-24f).
Fig 3-24 (a and b) Cell migration and proliferation