(64) Effect of Manipulation of Leucocyte and Platelet Rich Fibrin on Membrane Quality

Introduction: Leucocyte and platelet rich fibrin (L-PRF) is a membrane containing fibrin matrix with platelets and leucocytes made via centrifugation of blood, used for wound healing applications in clinical practice. Many of the positive clinical effects of L-PRF have been proposed to be mediated by growth factors and cytokines released from the membranes. For instance, molecules released from L-PRF have been shown to regulate wound healing-related gene expression in human gingival fibroblasts. However, there is a paucity of data regarding standardization of L-PRF generation and how variations in storage time may affect its biological properties. We hypothesized that greater elapsed time between blood collection and centrifugation would be associated with smaller L-PRF membranes and altered expression of wound healing-related genes in gingival fibroblasts.

Methods: Blood samples were collected from 18 healthy individuals. The samples were centrifuged immediately (T-0) or after 1-, 2-, 4-, and 6-minute storage at room temperature. L-PRF was isolated from each sample and mass measured by weighing with precision balance. Samples from T-0 and T-6 were each incubated 48 hours in cell culture medium at 37°C to create L-PRF “releaseate”. Human gingival fibroblasts were incubated 48 hours with releaseate, followed by RNA isolation and real-time polymerase chain reaction (RT-PCR) to measure the expression of select genes. Additional pairs of membranes from T-0 and T-6 were fixed, sectioned, and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei of leucocytes for counting. Platelets were identified by immunostaining using anti-CD42a antibody.

Results: Immediate centrifugation (T-0) resulted in the largest membrane for each participant, on average 26% larger than T-6 membranes, with a large individual variation in membrane mass. The mean mass for the T-0 membranes was 1.805 g (range 1.252 g to 2.322 g) compared to 1.283 g (range 0.570 g to 1.858 g) for the T-6 membranes (p < 0.001). While L-PRF releaseates from different individuals showed some variability in gingival fibroblast responses, there was a significant difference between the mean relative mRNA expression of T-0 samples compared to T-6 samples for fibroblast growth factor 2, vascular endothelial growth factor alpha (p < 0.001), and matrix metalloproteinase 3 (p < 0.05). The quantities of leucocytes and platelets were significantly higher in T-0 samples compared to the T-6 samples (p < 0.01).

Conclusions: Longer elapsed time between blood collection and centrifugation generally resulted in smaller L-PRF membranes, with significant individual variation. Storing blood samples for 6 minutes before centrifugation alters the biological effect of the L-PRF membranes. Thus, immediate centrifugation appears to result in optimal quantitative and qualitative properties of L-PRF for clinical use.

Lead Author’s Contributions to the Research Project: The Lead Author was responsible for the L-PRF preparation, data collection regarding L-PRF mass, preparation of L-PRF releasate, performing the data analysis, and the composition of the abstract.