Platelet Rich Fibrin In Regenerative Dentistry ... REPACK
Course Description: The use of platelet concentrates has had a long history of use in various fields of medicine as an autologous source of growth factors fabricated utilizing centrifugation of blood under various conditions. While platelet-rich plasma (PRP) was proposed as a first-generation platelet concentrate over 3 decades ago, over the past 10 years, platelet rich fibrin (PRF) has seen a steady increase in utilization for a variety of medical procedures due to its lack of anti-coagulation factors favoring fibrin clot formation and faster wound healing. More recently, further research has demonstrated that shorter and slower centrifugation spin cycles ('the low-speed concept' now termed advanced-PRF, A-PRF+) additionally favors wound healing by incorporating higher populations of white blood cells and progenitor cells within the PRF fibrin matrix leading to higher growth factor release within the local microenvironment. Parallel to these findings, the development of a liquid injectable PRF (i-PRF) provides a new formulation of liquid PRF without using anti-coagulation factors that may specifically be combined with currently available bone biomaterials favoring particle stability, angiogenesis and tissue integration. This course aims to highlight the recent advancements made with respect to the newest formulations of platelet concentrates and systematically presents when, where and why specific platelet concentrates may be utilized to further speed wound healing and tissue regeneration for various clinical indications faced in routine daily dental practice. Objectives: ??? Provide the biological background and scientific rationale for why platelet concentrates speed wound healing ??? Introduce the low-speed centrifugation concept and the theory behind these advanced PRF formulations??? .
Platelet rich fibrin in regenerative dentistry ...
The aim of this study was to evaluate 24 protocols for the production of platelet rich fibrin (PRF) produced via horizontal centrifugation to better understand cell separation following protocols at various times and speeds.
Platelet concentrates, including platelet-rich plasma (PRP) and platelet-rich fibrin (PRF), have been widely utilized in many fields of medicine as a scaffold capable of facilitating tissue regeneration . PRP was first introduced over 20 years ago as a specific regenerative modality aimed at concentrating platelets from whole blood [2,3,4,5,6]. PRP contains an array of naturally derived autologous growth factors, including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF). This set of growth factors is responsible for facilitating new blood vessel formation (angiogenesis) as well as inducing the cell migration and proliferation of various cell types [2,3,4,5,6]. While PRP has been widely utilized in many fields of medicine for over 2 decades, one of the major reported limitations includes its use of anticoagulants, known suppressors of clotting and thereby wound healing [2, 7, 8]. Nevertheless, the simplicity and low cost of harvesting peripheral blood and concentrating blood-derived growth factors and cells using a centrifuge has long been considered an effective and easy-to-obtain source of natural growth factors for tissue regeneration [9, 10].
The factors that affect fibrin clot formation and structure include genetic factors, acquired factors (such as abnormal concentration of thrombin and factor XIII in plasma, blood flow, platelet activation, oxidative stress, hyperglycemia, hyperhomocysteinemia, medications, and cigarette smoking), and other parameters (such as microgravity, pH, temperature, reducing agents, and concentration of chloride and calcium ions) . All donors were confirmed to be without any of the above conditions. The CBC of the donors were also investigated prior to beginning the experiments to confirm standard cell count ranges.
PRF has gained tremendous momentum in recent years as a natural concentration of autologous growth factors capable of stimulating tissue regeneration. Despite its widespread use, to date, very little scientific data exist from studies investigating centrifugation protocols. In 2014, lower centrifugation speeds were proposed as a means to better accumulate growth factors and cells within the upper platelet-rich layers by modifying L-PRF protocols from 700g down to 200g . An approximately 20% increase in platelet concentration could be observed following these lower speed centrifugation protocols . While these previous methods based on histological observations allow for a relative estimate of the cells found in the various blood cell layers, the new methodology recently proposed by our group  allows for the precise quantification and concentration of cells in 1 mL incremental layers following centrifugation. This protocol provides researchers with a better ability to understand the events occurring following centrifugation at various protocols.
To address the focused question: in patients with freshly extracted teeth, what is the efficacy of platelet-rich fibrin (PRF) in the prevention of pain and the regeneration of soft tissue and bone compared to the respective control without PRF treatment?
Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.
Figure 1. Time-course of degradation images of a platelet-rich plasma (PRP)-FM. The PRP fraction after centrifugation with GBs; CaCl2 was not added (A). The PRP fraction after centrifugation with both GBs and added CaCl2. The PRP-FM has formed (B). The PRP-FM after digestion with tissue-plasminogen activator (t-PA) for 20 min (C), 3 h (D), and 24 h (E). Abbreviation: GBs, glass beads. Arrows indicate the remaining PRP-FM. Surface microstructures of PRP-FM (F) and platelet-rich fibrin (PRF) (G). Similar observations were obtained from other three independent blood samples.
Figure 4. Digestion time-dependent changes in white blood cell (WBC) counts. Total WBC counts released from a platelet-rich plasma-FM as a raw number (A) and a percentage (B) (N = 7).
Figure 5. Scanning electron microscopy images of the platelets and other substances present in the digestion mixture at the indicated time points. Platelet-rich plasma (PRP) before clotting (A). PRP-FM digested with tissue-plasminogen activator for 3 h (B), 6 h (C), and 24 h (D).
Figure 8. Histograms of particle size, which is expressed as electronic volume (EV), in all populations (A), and CD41+ platelets in the gated populations (B). (C) The percentage of CD41+ platelets in the gated populations. Platelets derived from a platelet-rich plasma (PRP)-FM and whole-blood-derived platelet-rich fibrin (PRF) clots; large and small red thrombi are compared with platelets contained in the PRP fraction before clotting (N = 6 or 12).
Figure 9. Differences in the pathways of clot formation. (A) In vitro platelet-rich fibrin (PRF) cloy formation. (B) In vivo primary hemostasis of the extrinsic pathway. (C) In vivo secondary hemostasis of the extrinsic pathway.
The purpose of this study was to evaluate the effects of the growth factor within platelet-rich fibrin (PRF) in proliferation and differentiation of osteoblast and to observe the effectiveness of PRF.