In this month’s edition, I wanted to discuss the evolution of platelet rich fibrin (PRF) therapy. PRF has been used clinically in order to stimulate tissue regeneration in a variety of medical fields. Platelet rich plasma (PRP) was first introduced over 20 years ago following studies that showed the role of platelets during tissue regeneration. While PRP was widely used and successful, it required the use of anti-coagulants. After the development of PRP, the aim was to remove the use of anti-coagulants. This was termed leukocyte and PRF (L-PRF). Since anti-coagulants were not used, it was important to begin the centrifugation process shortly after blood collection to separate the layers before clotting began. Following centrifugation, a fibrin clot is obtained in the upper platelet rich layer. Other cells found in this layer include platelets and leukocytes in the fibrin matrix. (Fig 1)
Figure 1: Cell types found in a PRF matrix
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 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 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).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. 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. (Fig 2)
Figure 2: PRF Matrix being removed from tube for use
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 basic lab experiments 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. (Fig 3)
Fig 3: Comparison of fixed-angle versus horizontal centrifuge cell accumulation
By utilizing a horizontal swing-out bucket centrifugation system (Bio-PRF), it becomes possible to fully 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. (Fig 4)
Fig 4: Bio-PRF horizontal centrifuge
Typical i-PRF protocols have been shown to favor a 1.5-3 fold increase in platelets and leukocytes. Novel centrifugation tubes and protocols developed by Miron and colleagues 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. (Fig 5)
Figure 5: Instrumentation required for BIO PRF fabrication
Horizontal centrifugation is the next evolution in the use of PRF therapy in clinical dentistry. Horizontal centrifugation produces both solid and liquid formulations with both higher concentrations and number of platelets and leukocytes when compared to fixed-angle centrifuges. Further research is in development in this interesting field and I will surely keep you posted online on the Canadian Implant Dentistry Network (CIDN) Facebook forum and website.