Methodological Variations Affect the Release of VEGF in Vitro and Fibrinolysis’ Time from Platelet Concentrates

Abstract

Blood Concentrates (BCs) are autologous non-transfusional therapeutical preparations with biological properties applied in tissue regeneration. These BCs differ in the preparation method, in fibrin network architecture, growth factors release as well as in platelet/cell content. Methodological changes result in distinct matrices that can compromise their clinical effectiveness. The present study evaluated the influence of different g-forces and types of tubes in the release of vascular endothelial growth factor (VEGF) from platelet-rich fibrin (PRF) as a function of time. The PRF-like samples were obtained with three g-forces (200, 400, and 800 x g) for 10 minutes in pure glass tubes or in polystyrene-clot activator tubes. Scanning and Transmission electron microscopy was used to morphometric analyzes of PRF’s specimens and flow cytometry was used to quantify VEGF slow release until 7 days. Our results showed that platelets were intact and adhered to the fibrin network, emitting pseudopods and in degranulation. The fibrin network was rough and twisted with exosomic granulations impregnated on its surface. An increase in the concentration of VEGF in the PRF supernatant was observed until 7 days for all g forces (200, 400 or 800 x g), with the highest concentrations observed with 200 x g, in both tubes, glass or plastic. Morphological analyzes showed a reduction in the diameter of the PRF fibers after 7 days. Our results showed that g-force interferes with the shape of the fibrin network in the PRF, as well as affect the release of VEGF stored into platelets. This finding may be useful in applying PRF to skin lesions, in which the rapid release of growth factors can favor the tissue repair process. Our observations point to a greater clarification on the methodological variations related to obtaining PRF matrices, as they can generate products with different characteristics and degrees of effectiveness in specific applications.

The G-Force Conundrum in PRF Generation- Management of a Problem Hidden in Plain Sight

“…[3][4][5][6][7] A myriad of centrifuges with different radii are designed and used in practice, which results in inappropriate architecture and cell content of L-PRF. 1,[3][4][5][6][7] RCF is an important parameter in the production of L-PRF and must be calculated for each centrifuge, especially if this parameter is not pre-set on the machine. 3 Most often than not, running these centrifuges at 2700 RPM results in a g-force in an excess of 700g.…”

“…3 Most often than not, running these centrifuges at 2700 RPM results in a g-force in an excess of 700g. [1][2][3][4][5][6][7] At the same time, there is no provision for adjusting or changing the g-force through analog or digital means. 1,2 If the center of the centrifuge to tube end distance in millimeters is known, by applying the abovementioned formula (Figure 1), the RPM can be altered to generate a force of 400g resulting in a L-PRF of better quality.…”

“…[1][2][3] This reduction in the g-force is extremely beneficial; Amable et al, 4 showed that changes in RCF significantly influence the platelet yield in platelet rich plasma when centrifuge time and temperature are kept constant. 4 In a recent study, 5 it was observed that the organization of the fibrin matrix and the release kinetics of growth factors are influenced by factors such as centrifugation time, g-force, type of rotor, model of the centrifuge as well as the type of tubes used for blood collection. 5 When the g-force and RPM relationship is not appropriate, it results in the preparation of a clot of much smaller size, weaker biological significance and lower fibrin polymerization even when a stable centrifuge was used.…”

“…4 In a recent study, 5 it was observed that the organization of the fibrin matrix and the release kinetics of growth factors are influenced by factors such as centrifugation time, g-force, type of rotor, model of the centrifuge as well as the type of tubes used for blood collection. 5 When the g-force and RPM relationship is not appropriate, it results in the preparation of a clot of much smaller size, weaker biological significance and lower fibrin polymerization even when a stable centrifuge was used. [1][2][3][4][5][6] This has a negative effect on the release of growth factors as well.…”

“…5 When the g-force and RPM relationship is not appropriate, it results in the preparation of a clot of much smaller size, weaker biological significance and lower fibrin polymerization even when a stable centrifuge was used. [1][2][3][4][5][6] This has a negative effect on the release of growth factors as well. Lowering the RPM controls and reduces the g-forces and results in an increase in cell number, platelets and growth factors such as VEGF and TGF.…”