Histological comparison of Platelet rich fibrin clots prepared by fixed-angle versus horizontal centrifugation

Fujioka‐Kobayashi, Masako; Kono, Michihide; Katagiri, Hiroki; Benoit, Sandrine; Zhang, Yufeng; Sculean, Anton; Miron, Richard J.

doi:10.1080/09537104.2020.1754382

Cited by 52 publications

(51 citation statements)

References 28 publications

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“…In 2018, we developed a novel method that directly determines platelet counts via digestion using tissue plasminogen activator and demonstrated its validity and accuracy in comparison with those of the subtraction method [ 3 ]. To date, micrographic examination methods using scanning electron microscopy (SEM) and immunohistochemical techniques have been the only practical options for evaluating platelet distribution [ 4 , 5 , 6 , 7 ]. However, the data obtained using these methods are often too fragmented to adequately evaluate platelet distribution in three-dimensional (3D) structures by reconstruction.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Near-Infrared Imaging of Platelets in Platelet-Rich Fibrin (PRF) Matrices: Comparative Analysis of Bio-PRF, Leukocyte-Rich PRF, Advanced-PRF and Concentrated Growth Factors

Aizawa

Tsujino

Watanabe

et al. 2020

IJMS

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Platelet-rich fibrin (PRF) is a fibrin matrix enriched with platelets. The PRF matrix is thought to form a steep gradient of platelet density around the region corresponding to the buffy coat in anticoagulated blood samples. However, this phenomenon has not yet been proven. To visualize platelet distribution in PRF in a non-invasive manner, we utilized near-infrared (NIR) imaging technology. In this study, four types of PRF matrices, bio-PRF, advanced-PRF (A-PRF), leukocyte-rich PRF (L-PRF), and concentrated growth factors (CGF) were compared. Blood samples collected from healthy, non-smoking volunteers were immediately centrifuged using four different protocols in glass tubes. The fixed PRF matrices were sagittally divided into two equal parts, and subjected to modified immunohistochemical examination. After probing with NIR dye-conjugated secondary antibody, the CD41+ platelets were visualized using an NIR imager. In L-PRF and CGF, platelets were distributed mainly on and below the distal surface, while in bio-PRF and A-PRF, platelet distribution was widespread and homogenous. Among three regions of the PRF matrices (upper, middle, and lower), no significant differences were observed. These findings suggest that platelets aggregate on polymerizing fibrin fibers and float up as a PRF matrix into the plasma fraction, amending the current “gradient” theory of platelet distribution.

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Section: Introductionmentioning

confidence: 99%

Quantitative Near-Infrared Imaging of Platelets in Platelet-Rich Fibrin (PRF) Matrices: Comparative Analysis of Bio-PRF, Leukocyte-Rich PRF, Advanced-PRF and Concentrated Growth Factors

Aizawa

Tsujino

Watanabe

et al. 2020

IJMS

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“…Even though the preparation of solid PRF is simple, as it basically requires the spontaneous coagulation of centrifuged blood, the protocols have been refined towards gaining an ideal ratio of the PRF clot and its content of platelets, leucocytes, and consequently also growth factors. However, apart from swing-out rotors causing a superior separation of the phases than fixed angle rotors [ 4 ], it is the clotting time, the g-force and the centrifugation time that define the properties of the PRF, also reflected by the various acronyms, including leukocyte-rich L-PRF and advanced A-PRF [ 5 ], most of which are, however, linked to a certain brand of blood collection tubes and centrifuges [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

“…Considering that in L-PRF, platelets and leucocytes are restricted to the buffy coat, A-PRF (200 g) protocols showed a rather equal distribution of platelets in the PRF [ 10 , 11 ]. More recently, PRF protocols prepared via horizontal centrifugation using plain glass tubes without clot activators to prepare H-PRF have been shown to lead to more favorable accumulation of platelets and leukocytes in the upper plasma layers [ 4 , 12 ]. Nevertheless, L-PRF and H-PRF membranes undergo spontaneous fibrinolysis within a 3 week period upon subcutaneous implantation in immunocompromised rodents [ 13 ], and thus are not ideal for clinical application requiring long-term volume stability serving as dermal fillers [ 14 , 15 ] or as a replacement for soft tissue grafts in dentistry [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

“…They used plastic tubes with a hydrophobic surface to delay coagulating following the L-PRF protocol, thereby generating a large yellow plasma fraction [ 11 , 20 ]. This yellow clot consists of an almost cell-free liquid platelet-poor plasma (PPP) and the small cell-rich buffy coat [ 4 ]. To block fibrinolysis and simultaneously to provoke albumin coagulation, the PPP is subjected to heated at 75 °C for 10 min.…”

Section: Introductionmentioning

confidence: 99%

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Liquid Platelet-Rich Fibrin and Heat-Coagulated Albumin Gel: Bioassays for TGF-β Activity

et al. 2020

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Liquid platelet-rich fibrin (PRF) can be prepared by high centrifugation forces separating the blood into a platelet-poor plasma (PPP) layer and a cell-rich buffy coat layer, termed concentrated PRF (C-PRF). Heating the liquid PPP was recently introduced to prepare an albumin gel (Alb-gel) that is later mixed back with the concentrated liquid C-PRF to generate Alb-PRF. PRF is a rich source of TGF-β activity; however, the overall TGF-β activity in the PPP and the impact of heating the upper plasma layer remains unknown. Here, we investigated for the first time the in vitro TGF-β activity of all fractions of Alb-PRF. We report that exposure of oral fibroblasts with lysates of PPP and the buffy coat layer, but not with heated PPP, provoked a robust increase in the TGF-β target genes interleukin 11 and NADPH oxidase 4 by RT-PCR, and for IL11 by immunoassay. Consistent with the activation of TGF-β signaling, expression changes were blocked in the presence of the TGF-β receptor type I kinase inhibitor SB431542. Immunofluorescence and Western blot further confirmed that lysates of PPP and the buffy coat layer, but not heated PPP, induced the nuclear translocation of Smad2/3 and increased phosphorylation of Smad3. The immunoassay further revealed that PPP and particularly BC are rich in active TGF-β compared to heated PPP. These results strengthen the evidence that not only the cell-rich C-PRF but also PPP comprise a TGF-β activity that is, however, heat sensitive. It thus seems relevant to mix the heated PPP with the buffy coat C-PRF layer to regain TGF-β activity, as proposed during the preparation of Alb-PRF.

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“…Centrifugation protocols were originally refined with the goal of an equal distribution and accumulation of platelets within the platelet-poor plasma and the buffy coat layer moving from the original L-PRF protocol (700 g , 12 min) towards a slower A-PRF protocol (210 g , 8–12 min) in fixed-angle centrifuges [ 9 ]. Recently, horizontal centrifugation was introduced, allowing a faster and sharper separation of the cells according to their density in H-PRF [ 13 ]. The impact of gravitation force ( g -force) and time on the gradient of platelets and leucocytes in the tubes was measured in anticoagulated blood [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Relative Centrifugal Force (RCF; G-Force) Affects the Distribution of TGF-β in PRF Membranes Produced Using Horizontal Centrifugation

Kargarpour

Nasirzade

Panahipour

et al. 2020

IJMS

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Solid platelet-rich fibrin (PRF) is produced with centrifugation tubes designed to accelerate clotting. Thus, activated platelets may accumulate within the fibrin-rich extracellular matrix even before centrifugation is initiated. It can thus be assumed that platelets and their growth factors such as transforming growth factor-β (TGF-β) are trapped within PRF independent of their relative centrifugal force (RCF), the gravitation or g-force. To test this assumption, we prepared PRF membranes with tubes where clotting is activated by a silicone-coated interior. Tubes underwent 210 g, 650 g and 1500 g for 12 min in a horizontal centrifuge. The respective PRF membranes, either in total or separated into a platelet-poor plasma and buffy coat fraction, were subjected to repeated freeze-thawing to prepare lysates. Gingival fibroblasts were exposed to the PRF lysates to provoke the expression of TGF-β target genes. We show here that the expression of interleukin 11 (IL11) and NADPH oxidase 4 (NOX4), and Smad2/3 signaling were similarly activated by all lysates when normalized to the size of the PRF membranes. Notably, platelet-poor plasma had significantly less TGF-β activity than the buffy coat fraction at both high-speed protocols. In contrast to our original assumption, the TGF-β activity in PRF lysates produced using horizontal centrifugation follows a gradient with increasing concentration from the platelet-poor plasma towards the buffy coat layer.

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Histological comparison of Platelet rich fibrin clots prepared by fixed-angle versus horizontal centrifugation

Cited by 52 publications

References 28 publications

Quantitative Near-Infrared Imaging of Platelets in Platelet-Rich Fibrin (PRF) Matrices: Comparative Analysis of Bio-PRF, Leukocyte-Rich PRF, Advanced-PRF and Concentrated Growth Factors

Quantitative Near-Infrared Imaging of Platelets in Platelet-Rich Fibrin (PRF) Matrices: Comparative Analysis of Bio-PRF, Leukocyte-Rich PRF, Advanced-PRF and Concentrated Growth Factors

Liquid Platelet-Rich Fibrin and Heat-Coagulated Albumin Gel: Bioassays for TGF-β Activity

Relative Centrifugal Force (RCF; G-Force) Affects the Distribution of TGF-β in PRF Membranes Produced Using Horizontal Centrifugation

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