Understanding Platelet-Rich Fibrin. Richard J. Miron
properties of PRF have been a topic of much interest in recent years following the clinical observation that PRF seems to reduce postoperative swelling and pain. In a study titled “Effects of liquid platelet-rich fibrin on the regenerative potential of hPDLCs cultured under inflammatory conditions,” our group investigated the effects of PRF on human periodontal ligament cells (hPDLCs) under inflammatory conditions. As a model, hPDLCs were investigated using a migration and proliferation assay. To investigate hPDLC differentiation, alkaline phosphatase (ALP) assay, Alizarin Red Staining, and gene expression levels of Runx2, Col1a1, and OCN were conducted. Furthermore, cells were cultured with LPS to induce an inflammatory condition to investigate the ability of PRF to impact inflammatory resolution. All assays were compared to PRP (lower in WBCs).
Osteogenic differentiation demonstrated that liquid-PRF significantly induced greater ALP activity and more mineralized nodules when compared to PRP and controls (Fig 2-28). According to the experimental timeline, cells were pretreated with or without LPS to induce an inflammatory condition for 7 days, and then liquid-PRF was added to the culture media for an additional incubation period of 7 days (Fig 2-29a). Immunofluorescence images demonstrated that LPS induced more p65 expression (a marker for inflammation; Fig 2-29b), while the addition of liquid-PRF decreased its expression level. Furthermore, other inflammation markers including IL-1β and TNF-α were also significantly downregulated, as confirmed by real-time polymerase chain reaction (RT-PCR; Fig 2-29c). In summary, it was concluded that liquid-PRF displayed an anti-inflammatory response when hPDLCs were cultured with LPS.
Fig 2-28 Differentiation of hPDLCs. (a and b) Effects of PRP and liquid-PRF on ALP activity were detected by ALP staining and ALP activity test, respectively. (c) Alizarin Red S staining showed the mineralized nodules in each group after induction for 14 days. (d) The semiquantification of mineralization level. (e) Relative gene expression levels of Col1a1, OCN, and Runx2 after being treated with PRP or liquid-PRF for 14 days. Error bars correspond to the mean ± SD; significant differences are indicated: *P < .05; **P < .01.
Fig 2-29 Liquid-PRF can decrease the inflammation induced by LPS. (a) The timeline of experimental inflammatory condition stimulation. (b) Immunofluorescence staining of p65 in hPDLCs after being cultured with or without LPS and/or liquid-PRF. (c) The relative gene expression levels of inflammatory markers including IL-1β, TNF-α, and p65. Error bars correspond to the mean ± SD; significant differences are indicated: *P < .05; **P < .01; ns, not statistically significant vs control group.
In a final experiment, it was observed that liquid-PRF promoted the osteogenic differentiation of hPDLCs even when cultured in an inflammatory environment. Briefly, cells pretreated and cultured with LPS resulted in an intense reduction in mineralization nodule formation (Figs 2-30a and 2-30b). As the previous experiment demonstrated the ability for liquid-PRF to decrease the inflammatory response, it was further found that liquid-PRF could actually reverse a decrease in mineralization observed by LPS and resulted in a significant upregulation of expression markers Runx2, Col1a1, and OCN (Fig 2-30c). These findings indicate that the anti-inflammatory effect and regenerative potential of liquid-PRF can counterbalance the negative inflammatory effect induced by LPS. Later chapters address these findings more specific to the periodontal field, because PRF has been shown to improve the regeneration of intrabony and furcation defects not only by improving GF release but also by counterbalancing the inflammatory response induced by LPS (see chapter 10).
Fig 2-30 Liquid-PRF can promote the osteogenic potential of hPDLCs in an inflammatory environment induced by LPS. (a) Alizarin Red S staining indicated the odontoblastic differentiation of hPDLCs in the presence of LPS and/or liquid-PRF. (b) Mineralization level. (c) Gene expression levels of Runx2, Col1a1, and OCN in inflammatory condition. Error bars correspond to the mean ± SD; significant differences are indicated: *P < .05; **P < 0.01; ns, not statistically significant vs control group.
Furthermore, it has also been shown that PRF exerts potent antibacterial properties. In another study by our group, PRF was separated into solid and leachate components, and the PRF clots were divided into five equal layers to explore the specific antibacterial aspects of PRF. Both antimicrobial tests and flow cytometric analysis revealed that PRF produced using horizontal centrifugation demonstrated a significantly better antibacterial effect than L-PRF and was strongly correlative with immune cell numbers and types (Fig 2-31). In addition, our results demonstrated that the antimicrobial ability of PRF clots were less efficient than the wet PRF containing leachate, which suggest a promising application guidance to retain the liquid components of PRF for better anti-infection properties during clinical use. This study is presented in greater detail in chapter 3.
Fig 2-31 Antibacterial properties of L-PRF and H-PRF. (a) Photographs of PRF obtained after centrifugation by both protocols. Note the horizontal layer centrifugation in H-PRF versus the angled layering in L-PRF. (b) Weight and size measurements of PRF matrices. (c and d) Photographs and quantitative analysis of bacterial colony of Staphylococcus aureus and Escherichia coli incubated with L-PRF or H-PRF clots for 4 hours. (e and f) Photographs and quantification of the inhibition zone of L-PRF and H-PRF membranes incubated with S aureus or E coli after 24 hours. *P < .05; **P < .01; ***P < .001; ns, not statistically significant.
The Effect of Age, Sex, and Time on the Size Outcomes of PRF Membranes
Two topics that were heavily questioned for many years were (1) How long does the clinician have from the start of blood draw? and (2) Why do colleagues observe so much variability in clot size even from blood draws coming from the same patient? In 2019, we addressed this topic in a publication where the final PRF size outcomes were compared following centrifugation that took place after 0, 30, 60, 90, and 120 seconds in both male and female patients of different age categories. Each participant donated six vials of blood, and centrifugation was begun precisely after 0, 30, 60, 90, and 120 seconds.57
As depicted in Fig 2-32, by 90 seconds already a drop in membrane size of 13% was observed, and by 120 seconds this dropped even further. The entire goal of centrifugation is to separate layers based on density, so when blood remains sitting in a centrifugation tube for 120 seconds, it is certain that some of the fibrinogen and thrombin are beginning to convert into fibrin. Thereafter, when centri-fugation begins, it becomes harder and harder to separate layers (and most importantly cell types as a result). Therefore, centrifugation should be carried out between 60 and 90 seconds after blood draw. It generally takes 15 seconds to fill the tube