Fig. 5.1 • Centrifuge for preparation of PRF.
Fig. 5.2 • Pellets of PRF extracted from the tube and separated from the PPP.
Fig. 5.3 • Pellets of PRF on sterile gauze pads, ready for use.
Fig. 5.4 • A PRF membrane, obtained by means of compression.
PLATELET CYTOKINES
The ELISA method has been used to measure the levels of cytokines in acellular plasma. The values obtained were compared with the different PRP production techniques. A statistical and comparative analysis by Dohan et al. showed that with the PRF protocol, cytokines were absent in the acellular plasma and fibrin exudate, remaining trapped in the fibrin meshes. In particular:
- There were no significant differences between the levels of PDGF-BB, TGF-1 and IGF-1 measured in PPP and in PRF exudate;
- The levels of PDGF-BB and TGFβ-1 in PPP and in PRF were lower than those obtained using the PRP technique;
- The levels of IGF-1 in PPP and in PRF clot exudate were higher than those found in PRP.
Putting all these data together, it can be concluded that cytokines remain trapped in the fibrin matrix even after exudation. This would explain why their concentration in PPP and in PRF clot exudate is low, compared with their concentration in PRP.
In the PRF protocol, the negative charges on the interior surface of the tube and the absence of anticoagulants together induce platelet activation. In theory, cytokines produced through centrifugation, being small, soluble molecules, should collect at the top of the test tube; instead, they are not found to concentrate either in the PPP or in the exudate. This is explained by the fibrin mesh formation process which, as already described, is slow in PRF, allowing better incorporation of the cytokines and a more homogeneous three-dimensional organisation of the matrix; none of this occurs in the PRP protocol, which is instead characterised by rapid formation of the fibrin mesh. Albeit with different values, IGF-1, too, follows this pattern. But the high concentration of IGF-1 in PPP and in exudate, compared with its concentration in PRP, is due to the fact that it is a circulating cytokine and the studies were conducted on the platelet concentrate PRP, whose production, of course, involves discarding PPP, which is the part in which most circulating IGF-1 is found.
An Austrian study in 2006 (Leitner et al.) compared the different commercially available PRF preparation systems: Vivostat PRF Preparation Kit®, PCCS Platelet Concentrate Collection System®, Harvest® SmartPReP 2 APC 60 Process and Fibrinet® Autologous Fibrine & Platelet System. The preparations provided by these systems contain large numbers of platelets in small plasma volumes. PDGF-AB levels were measured throughout the five days after preparation. The Vivostat PRF Preparation Kit, PCCS Platelet Concentrate Collection System and Harvest SmartPReP 2 APC 60 Process showed comparable levels of platelet production and PDGF-AB release over the 120 hours. Lower levels were recorded with the Fibrinet system, but only because this system requires a lower volume. Indeed, growth factor release ability was the same for all four systems.
IMMUNE CYTOKINES
Dohan et al., again using the ELISA method, also evaluated immune regulation and the inflammation retrocontrol abilities of PRF. In detail, they compared the concentrations of the inflammatory cytokines TNF-α, IL-1β, IL-6, IL-4 and VEGF present in PPP supernatant and in PRF clot exudate with those present in samples of completely activated blood (i.e., left to coagulate in a test tube) and in samples of non-activated blood (obtained by taking the sample with anticoagulant). They obtained the following results:
- As with the previous experiment, there emerged no major differences between the concentrations of cytokines present in PPP and in clot exudate;
- The levels of IL-1β, IL-6, TNF-α and IL-4 in the PRF clot exudate were far higher than those found in the completely activated and non-activated blood. Only the concentration of VEGF in the PRF clot exudate was found to be significantly lower than the value recorded in the completely activated blood.
This study showed increases in all the inflammatory cytokines present in the supernatant (PPP) and in the PRF clot exudate, contrary to what was observed in the non-activated and activated blood. Given that the origin of these cytokines can only be leucocytic, it can be concluded that during PRF preparation (i.e., during centrifugation), leucocytes, too, undergo increased degranulation. Like platelet cytokines, leucocytic cytokines become trapped in the fibrin matrix during polymersiation and are then released gradually. On the other hand, the level of VEGF in the completely activated blood exudate was significantly higher than the levels recorded in the PRF clot exudate, the PPP and the non-activated blood. This higher concentration is due to the fact that VEGF is probably produced as a result of platelet activation, while leucocytic secretion of VEGF is negligible. Moreover, the low levels of VEGF found in the PPP and in the PRF exudate confirm that it becomes trapped in the fibrin meshes.
The PRF clot, being extremely rich in pro- and anti-inflammatory cytokines, plays an important role in immune defences. These cytokines have chemotactic properties and, through neovascularisation, are easily able to access the injured site. Because of this immune regulatory and inflammatory retrocontrol capacity (IL-4), PRF can be used in surgery to reduce post-operative infections.
CELL BEHAVIOUR
Doglioli, in a 2005 French study, observed cell behaviour in the presence of PRF. Four cell lines were analysed:
- Gingival fibroblasts;
- The activity of alkaline phosphatase (ALP) in human osteoblasts (degree of bone mineralisation);
- Human keratinocytes;
- Human pre-adipocytes;
- Human mesenchymal cells.
Treated and non-treated gingiva were compared histologically in the same patient. After 3.5 months, the gingiva treated with PRF showed a marked accumulation of type I collagen and better-quality elastic fibres. von Kossa staining showed the quantity of bone nodule formation by osteoclasts: 11.2/cm2 in the control group and 63/cm2 in the treated group (+480%).
The results obtained showed that the presence of the PRF membrane stimulates proliferation of all the cell lines. The stimulation lasts for around five weeks, after which the cells return to their usual replication rate.
The PRF exudate is equally active: the growth factors continued to stimulate cell proliferation for several weeks. Furthermore, the stimulation of bone mineralisation is significant; in fact, after seven days it was already possible to note mineralised nodules.
EFFECTS ON HEALING TISSUE
Fibrin stimulates the process of angiogenesis, or new blood vessel formation. This is a phenomenon that requires an extracellular matrix to allow the migration, division and phenotypical alteration of endothelial cells. The fibrin matrix fulfils all these requirements.
It does so thanks to the three-dimensional structure of the fibrin gel and the simultaneous action of cytokines trapped in the meshes.
Furthermore, the gel contains many pro-angiogenic factors: FGFb, VEGF, PDGF. In addition, expression of integrin αvβ3 by endothelial cells favours cell binding to fibrin, fibronectin and vitronectin.
It is known that fibrin is a natural source of immune system support.
Fibrin and fibrinogen degradation products (FDP) stimulate the migration of neutrophils and increase the expression of the CD11 and CD18 receptors on the membrane, promoting adhesion of neutrophils to the endothelium. In addition, fibrinogen degradation products modulate neutrophil phagocytosis and the enzymatic degradation process. Neutrophils are known to be the first cells to reach a wound, followed by macrophages. It has been shown that macrophage colonisation of an injury site is controlled by fibronectin, by chemical and physical properties of the fibrin, and by chemotactic agents trapped in the fibrin meshes.
The fibrin matrix guides the repair of damaged tissue, acting on the metabolism of the epithelial cells and fibroblasts.
Around the edges of a wound, epithelial cells lose their apical-basal polarity and spread laterally. Subsequently, they migrate towards the matrix formed from fibrinogen, fibronectin and vitronectin.
Fibrin, fibronectin, PDGF and TGF-β are essential in regulating the expression of integrin, and in fibroblast proliferation and migration within a wound.
Thanks to the expression of two plasminogen activators, fibroblasts develop an important proteolytic activity that allows them to move within the fibrin clot.
It has been demonstrated that fibroblast migration within fibrin gel is excellent when there are plenty of crossed bonds between the α chains. This is one of the significant differences between PRF and PRP. Following fibrin degradation, fibroblasts begin synthesising collagen.
During haemostasis and healing processes, circulating stem cells are trapped in the fibrin clot thanks to initial neovascularisation. These trapped cells promote vascular and tissue repair.
Thus, PRF works like a net to catch stem cells, especially when the process of angiogenesis is well advanced. In this way, undifferentiated stem cells, originating from the bone marrow, contribute to the regeneration of all the cell types in bone and also in other tissues. They do this by differentiating into different cell types.
PRF AND BONE TISSUE
The fibrin matrix associated with morphogenetic bone proteins (MBP) has angiotrophic, haemostatic and osteoconductive properties. Trapped in the fibrin structure, these proteins are released gradually, just as all the cytokines caught in natural fibrin and the cytokines in PRF usually are; transplanted into muscle, they have the ability to induce the formation of bone.
Choukroun et al. conducted an experiment to document the bone-regenerating capacity of PRF. Following the enucleation of a cyst, the resulting cavity fills with blood, allowing the formation of a clot. The fibrin clot matrix attracts and traps circulating stem cells. Physiologically, the wound takes between 6 and 12 months to heal. However, when the cyst cavity is filled with PRF, the healing process is speeded up. Filling cyst cavities with PRF led to the formation of dense, cortical bone in two and a half months. It was concluded that PRF, being better organised, is better able to guide stem cells and, thus, the healing process.
