Centrifugation Conditions in the L-PRP Preparation Affect Soluble Factors Release and Mesenchymal Stem Cell Proliferation in Fibrin Nanofibers.

Santana Santana,Melo Melo,Luzo Luzo,Lana Lana

doi:10.3390/molecules24152729

Abstract

Leukocyte and platelet-rich plasma (L-PRP) is an autologous product that when activated forms fibrin nanofibers, which are useful in regenerative medicine. As an important part of the preparation of L-PRP, the centrifugation parameters may affect the release of soluble factors that modulate the behavior of the cells in the nanofibers. In this study, we evaluated the influences of four different centrifugation conditions on the concentration of platelets and leukocytes in L-PRP and on the anabolic/catabolic balance of the nanofiber microenvironment. Human adipose-derived mesenchymal stem cells (h-AdMSCs) were seeded in the nanofibers, and their viability and growth were evaluated. L-PRPs prepared at 100× g and 100 + 400× g released higher levels of transforming growth factor (TGF)-β1 and platelet-derived growth factor (PDGF)-BB due to the increased platelet concentration, while inflammatory cytokines interleukin (IL)-8 and tumor necrosis factor (TNF)-α were more significantly released from L-PRPs prepared via two centrifugation steps (100 + 400× g and 800 + 400× g) due to the increased concentration of leukocytes. Our results showed that with the exception of nanofibers formed from L-PRP prepared at 800 + 400× g, all other microenvironments were favorable for h-AdMSC proliferation. Here, we present a reproducible protocol for the standardization of L-PRP and fibrin nanofibers useful in clinical practices with known platelet/leukocyte ratios and in vitro evaluations that may predict in vivo results.

Highlights

In the past few years, the benefits of autologous leukocyte- and platelet-rich plasma (L-PRP)have been evidenced in the treatment of many types of diseases [1,2,3,4,5,6]
Aside from growth factors (GFs) released from the platelets’ alpha granules, L-PRP contains inflammatory cytokines secreted from leukocytes that act in synergy to modulate the migration, proliferation, and differentiation of autologous cells through different pathways that lead to tissue regeneration [7,8,9,10,11]
L-PRP activation led to the formation of fibrin nanofibers rich in GFs and cytokines that are suitable for supporting cells, and whose response depend on the anabolic/catabolic balance of the microenvironment (Figure 1B)

Summary

Introduction

In the past few years, the benefits of autologous leukocyte- and platelet-rich plasma (L-PRP)have been evidenced in the treatment of many types of diseases [1,2,3,4,5,6]. Modern classifications systems consider the platelet and leukocyte levels, aside from other conditions, such as the number of centrifugation spins, activation, the presence of erythrocytes, and guided applications [15,16,17,18]. Whether conducted manually or by machine, L-PRP is prepared by centrifuging the patient’s whole blood, in which platelets, leukocytes, proteins, and other components are concentrated in a small fraction of plasma, with their levels adjusted by varying the centrifugation conditions [19,20]. As blood centrifugation is a separation process based on the size and density of the components as well as the packing behavior of the erythrocytes, different recoveries of platelets and leukocytes could be obtained in L-PRP from a first spin [19]. Choosing the centrifugation conditions for L-PRP preparation is crucial to achieving an optimized performance

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