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UBC Theses and Dissertations

Effect of manipulation of leucocyte and platelet rich fibrin on quality and quantity of the membrane Wintermute, Robin Marya


Background: Leucocyte and platelet rich fibrin (L-PRF) is a membrane containing fibrin matrix with platelets and leucocytes made via blood centrifugation. Molecules released from L-PRF have been shown to regulate wound healing-related gene expression in human gingival fibroblasts. It is not well-understood how the length of time elapsed between blood collection and centrifugation affects the final L-PRF. We hypothesized that greater elapsed time would be associated with smaller L-PRF membranes, containing fewer leucocytes and platelets, and altered expression of gingival fibroblast wound healing-related genes. Methods: Blood samples, collected from 18 healthy individuals, were centrifuged immediately (T-0) or after 1-, 2-, 4-, and 6-minute storage. Each L-PRF was measured. L-PRF from T-0 and T-6 from 11 of the individuals 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 to measure the expression of select genes. Additional pairs of membranes from T-0 and T-6 were used for visualization of leucocyte nuclei for counting. Platelets were additionally identified by immunostaining. Results: Immediate centrifugation (T-0) resulted in the largest membrane for each participant, on average 26% larger than T-6 membranes, with individual variation in membrane mass. The mean mass for the T-0 membranes was 1.805 g (1.252 g to 2.322 g) compared to 1.283 g (0.570 g to 1.858 g) for the T-6 membranes (p < 0.001). While individual L-PRF releaseates showed 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 < 0.03). The quantities of leucocytes and platelets were higher in T-0 samples compared to the T-6 samples (p < 0.01). Conclusions: Longer elapsed time between blood collection and centrifugation resulted in smaller L-PRF membranes. Storing blood before centrifugation likely lowers the L-PRF’s biological effect. Immediate centrifugation appears to result in optimal quantitative and qualitative properties of L-PRF for clinical use.

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