Platelet concentrates have been utilized in medicine for over two decades, owing to their ability to rapidly secrete autologous growth factors and ultimately speed wound healing. They have gained tremendous momentum as a regenerative agent derived from autologous sources capable of stimulating tissue regeneration in a number of medical fields (Miron 2021; Anfossi et al. 1989; Fijnheer et al. 1990). Many years ago, it was proposed that by concentrating platelets utilizing a centrifugation device, growth factors derived from blood could be collected from a platelet-rich plasma layer and later utilized in surgical sites to promote local wound healing (Anfossi et al. 1989; Fijnheer et al. 1990). Today, it has been well established that platelet concentrates act as a potent mitogen capable of (Fig. 1):

1. Speeding the revascularization of tissues (angiogenesis) through vascular endothelial growth factor (VEGF) release (Choukroun & Miron 2017; Kobayashi et al. 2016)
2. Acting as a potent recruitment agent of various cells including stem cells through platelet-derived growth factor (PDGF) release (Choukroun & Miron 2017; Kobayashi et al. 2016)
3. Inducing the prompt multiplication of various cell types found in the human body (proliferation) (Choukroun & Miron 2017; Kobayashi et al. 2016); Fujioka et al. 2017)

In dentistry, platelet concentrates were introduced over 20 years ago by Marx and colleagues with the aim of concentrating blood proteins as a natural source of growth factors that could stimulate vascularization (angiogenesis) and new tissue growth based on the fact that blood supply is pivotal for regeneration of all tissues (Upputuri et al. 2015). Wound healing has been described as a 4-step process which includes 1) hemostasis, 2) inflammation, 3) proliferation, and 4) maturation (Gosain & DiPietro 2024; Eming, Brachvogel et al. 2007; Eming, Kaufmann et al. 2007).

It is interesting to point out that the use of platelet concentrates has slowly and gradually gained in popularity with a dramatic increase being observed in the past 5-10 years.

Platelet-rich plasma (PRP), as its name implies, was designed to accumulate platelets in supra-physiological doses within the plasma layer following centrifugation. The main aim of PRP was to isolate and further concentrate the highest quantity of platelets and their associated growth factors for regenerative purposes and thereafter re-implanting this supra-concentrated clot at sites of local injury (Miron 2021).

Initial protocols typically ranged in duration from 30 minutes to 1 hour based on the centrifugation/collection systems and protocols utilized. Since lengthy protocols were utilized, anti-coagulants were added to blood collection tubes. These anticoagulants were typically various concentrations of bovine thrombin and calcium chloride.

Despite its growing success and continued use, several reported limitations existed with respect to reaching its full healing potential. Mainly, the use of anti-coagulants was shown to limit wound healing (Miron 2021). Simply put, when injury occurs leading to an open wound, a blood clot is one of the first and most crucial steps that occurs in order for healing to take place. Shortly thereafter, cells and growth factors get trapped within this newly formed extracellular matrix and the wound healing process/cascade begins. By limiting the body’s ability to form a clot-stable tissue, wound healing is delayed. Several studies have now demonstrated the superior outcomes of PRF when compared to PRP simply by removing anticoagulants from their formulations (Miron 2021).

Another drawback of PRP was the fact that it remained liquid by nature due to its use of anti-coagulants. Therefore when compared to PRF, PRP demonstrates a quick initial burst of growth factors being released whereas PRF demonstrates a slower and more gradual release of growth factors over an extended period of time that has since been shown to significantly improve cell growth and tissue regeneration (Lucarelli et al. 2010; Saluja et al. 2011).

Owing to the main drawback that anticoagulants utilized in PRP prevented clotting, platelet-rich fibrin (PRF) was developed with the main aim of simply removing anti-coagulants (Choukroun et al. 2001). By doing so, a much quicker working time was needed and the practitioner absolutely required centrifugation to begin shortly after blood collection (otherwise the blood would naturally clot within a tube). The main advantage of this fibrin matrix is the ability for it to release growth factors over an extended period of time while the fibrin clot is being degraded (as opposed to PRP which remained in liquid form having a much faster growth factor release profile) (Dohan Ehrenfest et al. 2010). Over the years, PRF has also been termed L-PRF (for leukocyte and platelet-rich fibrin) owing to the discovery that several leukocytes remained incorporated in PRF.

Recently, a series of basic laboratory experiments revealed better means to optimize the production of PRF using horizontal centrifugation. Simply, horizontal centrifuges are routinely utilized in high-end research labs as well as in medical hospitals owing to their greater ability to separate layers based on density. Unlike a fixed-angle centrifugation system in which the tubes are actually inserted on a ~45 degree angle, horizontal centrifugation (often referred to as swing-out bucket centrifugation) offers the ability for tubes to swing out to 90 degrees once they are in rotation (Fig. 2).

This technology leads to a 4-times-greater cell content when compared to fixed-angle centrifugation (Fig. 3) (Miron et al. 2019). The major disadvantages of fixed-angle centrifugation is that during the spin cycles, cells are typically driven along the back wall of centrifugation tubes at high g-forces with relative difficulty to separate properly according to their cell density. This also exposes cells to higher compressive forces against the back wall and cells must then separate by travelling either up or down the inclined centrifugation slope based on their respective cell density differences. Since red blood cells are larger and heavier than platelets and leukocytes, they travel downwards, whereas lighter platelets travel towards the top of the tube where the PRF is collected. This makes It relatively difficult for the smaller cell types such as platelets and particularly leukocytes to reach the upper layers, especially granted that RBCs outnumber in particular WBCs typically by ~1000 fold. In summary, by utilizing a fixed-angle centrifuge, it is not possible to reach optimal accumulation of platelets or leukocytes as a result of their fixed-angle system design.

In general, 3 protocols are necessary for basic PRF therapy.

The first is the standard solid-PRF protocol to produce PRF membranes in which a high yield of platelets and leukocytes are harvested with an even distribution of cells within the upper 4-5 mL PRF layers. This is best achieved using a horizontal centrifugation system (700 RCF for 8 minutes). The second protocol is a liquid-PRF formulation capable of concentrating platelets and leukocytes within the upper 1mL layer (previously known as injectable-PRF or i-PRF). By utilizing a horizontal centrifugation system, higher concentrations are ensured (smaller volume though a higher concentration of cells). This protocol is achieved utilizing a 300 RCF for 5 minutes. The final and third protocol is that of concentrated-PRF (C-PRF) where cells are purposefully accumulated specifically towards the buffy coat layer using faster spin protocols. This is best achieved using a 2000 RCF for 8 minutes protocol and a resulting 0.3-0.5mL cell-rich zone may be collected exactly within the buffy coat (Fig. 4).

In dentistry, the majority of procedures are done utilizing the 700 RCF for 8 minutes protocol. Specifically in guided bone regeneration (GBR) procedures, the so-called ‘sticky bone’ protocol utilizes this spin rate when both liquid-PRF (blue or white) and solid-PRF (red) tubes are drawn simultaneously and centrifuged at the same time (Fig. 5). Always remember to draw liquid-PRF tubes first.