The evolution of Platelet Rich Fibrin
Platelet concentrates have been utilized in medicine for over 3 decades owing to their ability to rapidly secrete growth factors. They have gained tremendous momentum as a regenerative agent derived from autologous sources capable of stimulating tissue regeneration in a number of medical fields. Many years ago, it was proposed that by concentrating platelets utilizing a centrifugation device, autologous 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.
Today, it has been well established that platelet concentrates act as a potent mitogen capable of:
Speeding the revascularization of tissues (angiogenesis)
Acting as a potent recruitment agent of various cells including stem cells (chemotaxis)
Inducing the prompt multiplication of various cell types found in the human body (proliferation)
Various systematic reviews from multiple fields of medicine have now demonstrated their ability to support the regeneration of a number of cell types and tissues. Below we review the evolution of platelet concentrates.
Platelet Rich Plasma (1990s)
Work led by Dr. Robert Marx in the 1990s led to the popular working name ‘platelet rich plasma’ (PRP). The goal of PRP was to collect the largest and highest concentrations of platelets/growth factors to be later utilized for regenerative purposes. The PRP protocol required extensive centrifugation time (typically over 30 minutes). During this process, the use of anti-coagulants (namely bovine thrombin or calcium chloride) was absolutely necessary in order to prevent clotting owing to the lengthy centrifugation times. The final composition of PRP contained over 95% platelets, known cells responsible for the active secretion of growth factors involved in wound healing.
Two reported drawbacks of PRP have since been reported in the literature. First, centrifugation times were deemed long (>30 minutes) and not practical for a variety of surgical procedures that may routinely be performed in many clinical practices. Furthermore, despite improving wound healing, it has since been revealed that clotting in general, is a necessary component of normal physiological wound healing. This limitation prevented optimal wound healing and led to the development of platelet rich fibrin.
Leukocyte and Platelet Rich Fibrin: L-PRF (2000-2010)
Owing to the drawback that the anticoagulants utilized in PRP prevented clotting, pioneering work led by Dr. Joseph Choukroun and Dr. David Dohan led to the development of platelet rich fibrin (PRF). The aim of PRF was to develop a second-generation platelet concentrate with anti-coagulant removal. Since anti-coagulants were removed, a much quicker working time was needed and the practitioner absolutely required that centrifugation began shortly thereafter blood draw (otherwise blood would clot). Furthermore, high g-force centrifugation protocols were utilized in order to separate blood layers in attempt to separate blood layers prior to clotting. The final spin cycle (2700 RPM for 12 minutes = ~700g), resulted in a plasma layer composed of a fibrin clot with entrapment of platelets and leukocytes. The main advantage of this fibrin matrix was the ability for it to release growth factors over an extended period of time while the fibrin clot was being degraded (as opposed to PRP which is a liquid/gel).5 Over the years, PRF has been termed L-PRF (for leukocyte and platelet rich fibrin) owing to the discovery that leukocytes play a central and key role during tissue regeneration.
Advanced and injectable Platelet Rich Fibrin: A-PRF and i-PRF (2014-2018)
While much of the research performed in the late 2000s and early 2010s was dedicated to the clinical uses and indications of L-PRF, major discoveries were made several years following extensive clinical use of L-PRF. In 2014, an oral maxillofacial surgeon in Germany by the name of Dr. Shahram Ghanaati observed histologically that following centrifugation at high g-forces (~700g – utilized in L-PRF protocols), the majority of leukocytes and platelets were in fact located at the base of L-PRF clots or even worse, within the red corpuscle blood layer at the bottom of centrifugation tubes. Pioneering research within his laboratory led to the development of an advanced platelet rich fibrin (A-PRF) whereby lower centrifugation speeds (~200g) led to a PRF membrane with more evenly distributed platelets. These newer protocols more favorably released a higher concentration of growth factors over a 10 day period when compared to PRP or L-PRF. 7 In 2016/2017, Kobayashi and colleagues then demonstrated that further optimization of platelet rich fibrin could be further achieved by not only reducing centrifugation speed, but also time. The A-PRF protocol was therefore modified from 14 minutes at 200g, down to 8 minutes at 200g.
Following an array of basic research studies, it was observed that by further reducing the g-force and also the time, it was possible to obtain a plasma layer that had not yet converted into fibrin (ie a liquid PRF). In a study titled: “Injectable platelet rich fibrin (i-PRF): opportunities in regenerative dentistry?” Miron and colleagues then demonstrated that with even lower centrifugation speeds and times (~60g for 3 minutes), a liquid platelet rich fibrin (termed injectable-PRF or i-PRF) could be obtained following centrifugation. While these protocols typically produced minimal volumes (~1-1.5mL), it was shown that both platelets and leukocytes were even more highly concentrated when compared to L-PRF or A-PRF. This liquid-PRF layer could be utilized clinically for approximately 15-20 minutes during which time fibrinogen and thrombin had yet converted to a fibrin matrix (ie remained liquid). This has since been utilized for the injection into various joints/spaces similar to PRP, however with the reported advantages of a longer growth factor releasing time. Furthermore, the concept of ‘sticky’ bone was also developed. Importantly, a different type of tube (plastic) must be utilized to minimize clotting.
Horizontal centrifugation: Bio-PRF and C-PRF (2019-present)
Very recently, Miron and colleagues have demonstrated through a series of studies that horizontal centrifugation is capable of producing significantly greater concentrations of platelets and leukocytes when compared to the currently available fixed-angle centrifugation devices most commonly utilized to produce L-PRF or A-PRF. It was reported that one of 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. This exposes cells to high compressive forces against the back wall and must thereafter travel either up or down the inclined centrifugation tube based on cell density differences. Since red blood cells are larger and heavier than platelets and leukocytes, they typically travel downwards, whereas lighter platelets travel towards the top of the tube where PRF is collected. Noteworthy however, it was shown that on a fixed angle centrifuge, cells accumulate at the back walls of centrifugation tubes and the larger red blood cells entrap the smaller platelets/leukocytes below them and drag them into the red corpuscle layer. By utilizing a fixed-angle centrifuge, it is not possible to reach total accumulation of platelets or leukocytes as a result of this fixed-angle.
By utilizing a horizontal swing-out bucket centrifugation system (Bio-PRF), it becomes possible to better separate cells and blood layers based on their density without necessitating cells to accumulate/damage on the back walls of centrifugation tubes. By utilizing horizontal centrifugation (Bio-PRF), it therefore becomes possible to isolate a higher number and concentration of platelets, leukocytes and monocytes when compared to either the L-PRF or A-PRF protocols.
Furthermore, while typical i-PRF protocols have been shown to favor a 1.5-3 fold increase in platelets and leukocytes, novel centrifugation tubes and protocols have shown improvements in cell concentration/accumulation by utilizing the Bio-PRF system between 10-15 fold. While the i-PRF protocol was previously deemed a highly-concentrated liquid-PRF protocol, newer protocols utilizing the Bio-PRF system have consistently produced a concentrated-PRF (C-PRF) with over 10-15 times greater concentrations of platelets and leukocytes when compared to i-PRF. Today, C-PRF has been established as the most highly concentrated PRF protocol described in the literature.