PRF.24 The aim was to develop a second-generation platelet concentrate focused on anticoagulant removal. Because anticoagulants were removed, a much quicker working time was needed, and centrifugation had to begin shortly after blood draw (otherwise, the blood would naturally clot). Furthermore, high g-force centrifugation protocols were initially utilized in an attempt to separate blood layers prior to clotting. The final spin cycle (initial studies ranged from 2500–3000 rpm for 10–12 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 its ability to release GFs over an extended period of time while the fibrin clot was being degraded.25 Over the years, PRF has been termed L-PRF (for leukocyte platelet-rich fibrin) due to the discoveries that several leukocytes remained incorporated in PRF and that white blood cells play a central and key role in the tissue healing process. The most commonly utilized protocol today is a spin cycle at 3000 rpm for 10 minutes or 2700 rpm for 12 minutes (RCF-max = ~700g, RCF-clot = ~400g).
Several other advantages also existed during clinical use because it avoided the need for dual-spin protocols requiring pipetting or various specialized tube compartments, which made the overall procedure much more user-friendly, cheaper, and faster when compared to PRP. Original protocols were purposefully designed to spin at high centrifugation speeds with the main aim of phase separation to occur as quickly as possible in order to separate the red corpuscle base layer from the upper plasma layer prior to clotting. Following centrifugation, a platelet-rich fibrin mesh was formed, giving it the working name PRF26–28 (Fig 1-4). PRF has since been highly researched, with over 1,000 publications dedicated to this topic alone.
Fig 1-4 Layers produced after centrifugation of whole blood. A PRF clot forms in the upper portion of tubes after centrifugation.
Additionally, research teams from around the world have demonstrated the impact of leukocytes on tissue healing.29–34 While it was once thought that the additional benefit of leukocyte incorporation into PRF was its main properties in improved host defense to foreign pathogens,29–34 it has since been shown in well-conducted basic research studies that leukocytes are pivotal to tissue regeneration and favor faster wound healing also.11,35–37 In dentistry, where the oral cavity is filled with bacteria and microbes, the inclusion of leukocytes was initially thought to play a pivotal role in wound healing by participating in the phagocytosis of debris, microbes, and necrotic tissues, as well as directing the future regeneration of these tissues through the release of several cytokines and GFs and orchestrating cell-to-cell communication between many cell types.
Tissue engineering with PRF
Tissue engineering has been an emerging discipline over the past decade, with major breakthroughs routinely being made every year. At its simplest foundation, tissue engineering requires three parameters: (1) a scaffold responsible to support tissue ingrowth, (2) cells that may act to promote tissue regeneration, and (3) GFs that stimulate the overall wound healing events. Unlike the majority of biomaterials currently available on the market, PRF actually contains each of these three properties (Fig 1-5). For comparative purposes, routine bone allografts contain a scaffold (mineralized cortical/cancellous bone) and GFs embedded in its bone matrix (such as bone morphogenetic protein 2 [BMP-2]) but have no cells. Recombinant human GFs typically have a GF (for instance, rhBMP-2) and a carrier (collagen sponge) but also lack cells. Certain stem technologies typically contain cells and also a delivery system (for instance a nanocarrier delivery system) but lack GFs. The ability to actually contain each of the three tissue engineering properties within a single biomaterial is quite rare and, more importantly, usually extremely expensive (think recombinant GFs and/or stem cell technology).
Fig 1-5 Three main components of PRF all derived naturally from the human body. These include (1) cell types (platelets, leukocytes, and red blood cells); (2) a provisional ECM 3D scaffold fabricated from autologous fibrin (including fibronectin and vitronectin); and (3) a wide array of over 100 bioactive molecules, including most notably PDGF, TGF-β, VEGF, IGF, and EGF.
PRF, on the other hand, is a particularly simple and inexpensive way to utilize the three principles of tissue engineering by utilizing a 3D scaffold (fibrin) that incorporates both regenerative host cells (platelets and leukocytes) and various GFs. These include PDGF, TGF-β, and VEGF, each of which is crucial during the regeneration process. Furthermore, the concentrated leukocytes (as opposed to simply platelets) in PRF have been well implicated as key regulators of tissue healing and formation.26–28,31,38
Snapshot of PRF
PRF is considered a second-generation platelet concentrate with a longer GF release profile.
Centrifugation protocols are shorter and do not need any chemical additives such as anticoagulants.
PRF falls more in line with tissue engineering principles in that it is not only an accumulation of cells and GFs but also a scaffold (fibrin matrix).
PRF incorporates leukocytes, which are key cells in pathogen defense and biomaterial integration.
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 discussed later in this textbook, major discoveries were made several years later from basic research laboratories. Following extensive clinical use and research with the original L-PRF protocol, it was discovered in 2014 by Dr Shahram Ghanaati that centrifugation carried out at relatively high centrifugation speeds (~700g) led to the great majority of leukocytes being located either at the buffy coat zone (between the red blood cell layer and the upper plasma layer) or more commonly at the bottom of centrifugation tubes (Fig 1-6).39 It was expressed that the longer the centrifugation time is carried out, the more likely it is that cells get pushed further down the centrifugation tube. Similarly, the faster the spin centrifugation speed (higher g-force), the greater the proportion of cells found in the lower levels of centrifugation tubes.
Fig 1-6 Histologic observation of leukocytes following centrifugation. Resulting white blood cells have been shown to be contained basically in the layers between the plasma PRF layer and the red blood cell clot. This finding demonstrated quite clearly that the g-force was excessive, necessitating the development of newer protocols aimed to improve the retention of leukocytes within the PRF matrix. (Reprinted with permission from Ghanaati et al.39)
Pioneering research within his laboratory led to the development of an advanced PRF (A-PRF) whereby lower centri-fugation speeds (~200g) led to a higher accumulation of platelets and leukocytes more evenly distributed throughout the upper PRF layers. These newer protocols more favorably led to a higher release and concentration of GFs over a 10-day period when compared to PRP or L-PRF.19 In 2015 to 2017, our research team further demonstrated that optimization of PRF could be achieved by reducing not only centrifugation speed but also the time involved. The A-PRF protocol was therefore modified from 14 minutes at 200g as originally described in 2014 down to an 8-minute protocol.19
Following an array of basic research studies on this topic, 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, scientifically liquid fibrinogen but often referred to as liquid-PRF