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

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.

Keywords: L-PRP; centrifugation; cytokine; fibrin; growth factor; leukocyte; mesenchymal stem cells; nanofiber; platelet.

Figure 1

Schematic illustration of the experimental design. (A) Human whole blood was collected and centrifuged at 100 and 800× g for 10 min to form L-PRP₁₀₀ and L-PRP₈₀₀. These L-PRPs were submitted to a second spin at 400 xg for 10 min to form L-PRP_100-400 and L-PRP_800-400 after the removal of 70% of the supernatant; (B) h-AdMSCs were resuspended in each L-PRP, followed by the formation of a fibrin nanofiber network after activation with thrombin/Ca²⁺. Activated platelets and leukocytes secrete GFs and cytokines that in an appropriate balance stimulate cell proliferation.

Figure 2

Concentration of (A) platelets, (B) leukocytes, (C) lymphocytes, and (D) granulocytes, obtained by measuring each L-PRP in a hematologic analyzer. The calculated ratio of (E) platelet/leukocyte and (F) lymphocyte/granulocyte. Note: * p < 0.05.

Figure 3

(A) Schematic illustration representing the concentration of the blood components in each L-PRP. (B) The calculated concentration factors relative to the whole blood. Note: * p < 0.05.

Figure 4

Characterization of the fibrin nanofibers. (A) SEM images showing the morphology of the networks. (B) Frequency of fiber diameter measured using ImageJ software, highlighting their mean diameter; (i) fibrin₁₀₀, (ii) fibrin_100-400, (iii) fibrin₈₀₀, and (iv) fibrin_800-400.

Figure 5

Kinetics of the GFs (A) TGF-β1, (B) PDGF-BB, and the inflammatory cytokines (C) IL-8 and (D) TNF-α released from the nanofibers over 72 h.

Figure 6

Assessment of h-AdMSC behavior seeded in the four types of fibrin nanofibers. (A) Cell viability on the seventh day of incubation in (i) fibrin₁₀₀, (ii) fibrin_100-400, (iii) fibrin₈₀₀, and (iv) fibrin_800-400. (B) Number of living and dead h-AdMSCs in the nanofibers incubated for seven days and calculated from three different images using ImageJ software. Note: ** Significantly different from fibrin₈₀₀ and fibrin_800-400 (p < 0.05). (C) Proliferation kinetics for h-AdMSCs seeded in the fibrin networks of the L-PRP preparations. Note: *p < 0.05.