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

Zahra Kargarpour,Richard J Miron,Jila Nasirzade,Layla Panahipour,Reinhard Gruber

doi:10.3390/ijms21207629

Zahra Kargarpour, Richard J Miron + Show 3 more

Open Access

https://doi.org/10.3390/ijms21207629

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Abstract

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.

Highlights

Platelet-rich fibrin is produced by the centrifugation of venous blood that undergoes coagulation [1]
We report here that the total transforming growth factor-β (TGF-β) activity is similar among the Platelet-rich fibrin (PRF) membranes produced using different protocols; despite the proposed binding of TGF-β to the fibrin-rich matrix, there is a clear gradient of TGF-β activity within the PRF membrane towards the buffy coat produced at higher centrifugation speeds
To evaluate the effect of different g-forces on activation of TGF-β signaling, oral fibroblast cells were exposed to 30% of the PRF lysates prepared at different g-forces but normalized to the length of the PRF membrane (Table 1)

Summary

Introduction

Platelet-rich fibrin is produced by the centrifugation of venous blood that undergoes coagulation [1]. The recommended time between the blood drawing and start of centrifugation is 60–90 s; otherwise the size of the PRF membrane is negatively affected [18] It is the size of the PRF membrane that matters but attention has been paid to the distribution of platelets within the PRF membranes [8], as platelets are rapidly activated during the coagulation cascade and thereby entrapped in the developing fibrin-rich extracellular matrix. Fibronectin [21] and vitronectin [22], both being part of the blood clot [23], bind TGF-β In support of these molecular principles, immunostainings of PRF revealed that TGF-β1 is present throughout the matrix, with an accumulation on the distal surface from PRF clots produced using fixed-angle centrifuges [24], refining studies on the release of TGF-β into the supernatant of PRF [9,10]. We report here that the total TGF-β activity is similar among the PRF membranes produced using different protocols; despite the proposed binding of TGF-β to the fibrin-rich matrix, there is a clear gradient of TGF-β activity within the PRF membrane towards the buffy coat produced at higher centrifugation speeds

Results

Cell Culture

Preparation of PRF Lysates

RT-qPCR Analysis and Immunoassay