Richard J. Miron - Understanding Platelet-Rich Fibrin

Figure 2-25depicts a 700g force performed on a horizontal centrifuge for 8 minutes (the selection of this protocol is presented in chapter 3). Interestingly, it was observed that more leukocytes, platelets, lymphocytes, and monocytes were much more evenly distributed throughout the PRF layers when compared to fixed-angle centrifugation. Unlike either the L-PRF or A-PRF protocols, a general increase in leukocyte numbers was observed (127% original values), and a 2.4-fold increase in platelet concentration was observed. This represents over a fourfold increase in leukocytes when compared to A-PRF protocols and a twofold increase when compared to L-PRF. Furthermore, this method concentrated 99.7% of all platelets and 53% of all leukocytes within the plasma layer. 50

Fig 2-25The concentration of cell types in each 1-mL layer utilizing the solid-PRF horizontal centrifugation protocol (700g for 8 minutes). Notice that most of the platelets as well as WBCs are now more evenly distributed throughout the upper plasma layer. (Adapted from Miron et al. 50)

Within that study, horizontal centrifugation was proposed as a means to better separate cell layers based on density ( Video 2-3). Two advantages were noted utilizing horizontal centrifugation. First, a completely horizontal tube produced from a swing-out bucket allows for the greatest differential between the minimum and maximum radius found within a centrifugation tube (see Fig 2-24a). This allows for a greater ability to separate cell layers based on disparities between the RCF-min and RCF-max produced within a tube. Second, a fixed-angle centrifuge results in more trauma to cells. Because centrifugation typically pushes cells outward and downward, cell layer separation is always observed in an angulated fashion toward the back distal surface of PRF tubes using fixed-angle centrifugtion (see Fig 2-24b). Furthermore, during the centrifugation process, cells are pushed toward the outer wall and then typically migrate either up or down the centrifugation tube based on density. Larger cells (such as RBCs) entrap smaller cells such as platelets during the centrifugation process and drag them downward along the back centrifugation wall into the RBC layer as a result of this cell accumulation against the back wall (see Fig 2-24c). In contrast, PRF produced via horizontal centrifugation and separation allows the direct flow-through of cells (see Fig 2-24c).

Video 2-3

Therefore, horizontal centrifugation allows cells to migrate freely throughout the blood layers. This allows for better separation of cell types (along with the greater differentiation in RCF values between RCF-min and RCF-max), resulting in higher final concentrations of desired cells (platelets and leukocytes) within the appropriate final blood layers. Furthermore, cells are less likely to suffer potential damage along the back wall of centrifugation tubes produced using high g-forces (~200–700g) following fixed-angle centrifugation. We therefore introduced this concept as “gentler centrifugation,” whereby cells are more freely able to separate between layers without the necessary friction produced on the back wall of fixed-angle centrifuges such as those produced on the fixed-angle IntraSpin and Process for PRF devices. 50This concept has been expanded with a series of research investigation launched to better optimize PRF. This is presented in greater detail in chapter 3of this textbook.

During the centrifugation process on fixed-angle centrifuges, cells are pushed toward the outer distal wall and then typically migrate either up or down the centrifugation tube based on density. Larger cells (such as RBCs) entrap smaller cells such as platelets during the centrifugation process and drag them downward along the back centrifugation wall. These platelets and leukocytes don’t make it to the upper PRF membrane.

Biologic Activity of PRF on Immune Cells

In an extensive systematic review by Strauss et al investigating the biologic properties of PRF, 1,746 studies were identified, of which 53 were included. 55Because PRF is capable of improving angiogenesis in vivo, it was reported that PRF enhanced the proliferation, migration, adhesion, and osteogenic differentiation of a variety of different cell types along with cell signaling activation. Furthermore, it was concluded that PRF reduced inflammation, suppressed osteoclastogenesis, and increased the expression of various GFs in mesenchymal cells. 55

Several interesting and very recent studies have investigated the effects of PRF and its involvement with macrophage polarization from proinflammatory M1 toward proresolving M2 phenotypes. 56Macrophages are extremely important cells during the healing process and can be involved with either secretion of proinflammatory markers (M1) or proresolution markers (M2). These studies are extremely relevant to PRF.

In a study by Nasirzade et al, murine primary macrophages and a human macrophage cell line were exposed to saliva and lipopolysaccharides (LPS) with and without PRF lysates. 56The expression of the proinflammatory M1 marker genes IL-1β and IL-6 were greatly decreased, and PRF-conditioned medium enhanced the expression of tissue-resolution markers. It was therefore concluded that PRF holds an anti-inflammatory activity and shifts the macrophage polarization from an M1 toward an M2 phenotype. 56In a second study on this topic, it was also reported that i-PRF reduced proinflammatory M1 phenotype of macrophages along with activated dendritic cells around muscle defects injected with bacterial suspension ( Figs 2-26and 2-27). 41

Fig 2-26The expressions of CD11b in tissues from control (a and c) and i-PRF groups (b and d) . Scale bar in a and b = 20 μm; scale bar in c and d = 10 μm. (e) Quantification of the immunohistochemical staining was calculated by the percentage of CD11b-positive cells in all cells in the area of the same size. **** P < .0001. Error bars indicate SD. (Reprinted with permission from Zhang et al. 41)

Fig 2-27Effects of i-PRF on the maturation of dendritic cells. (a and b) Sections of immunofluorescence with antibodies against CD11c and CD86. Scale bar = 20 μm. (c) Relative expressions of maturation-related and inflammatory-related genes in DC2.4. (d and e) Immunoblotting and relative intensity of NF-κB signaling pathway and maturation-related markers of DC2.4 stimulated by the whole blood (WB) or i-PRF in the presence of LPS. * P < .05; ** P < .005; *** P < .0005; **** P < .0001; ns, no significant difference. Error bars indicate SD. (Reprinted with permission from Zhang et al. 41)

While the study of PRF on various cell types such as immune cells has only begun, it appears that it acts to reduce the proinflammatory response and further decrease common oral cavity inflammatory responses to LPS. Thus, this work may in part explain the observed clinical decreases in postoperative pain reported in the clinical chapters discussed later in this textbook.