Platelet rich fibrin (PRF) has been utilized clinically as a platelet concentrate capable of stimulating tissue regeneration. Interestingly, several protocols have been proposed with little data obtained regarding the final cell counts following centrifugation. The aim of the present study was to compare different commercially available centrifuges and their respective protocols utilizing a novel method to quantify cells. One millimeter blood layers following centrifugation were sequentially pipetted from the upper layer downward until all 10 mL were harvested in sequential samples. Thereafter, each sample was sent for CBC analysis to accurately quantify precisely cell numbers within each separate blood layer following centrifugation. The results from this study revealed that L-PRF protocols (2700 rpm × 12 min) produced a clot with the majority of platelets and leukocytes concentrated within the buffy coat with relatively no cells found within the first 4 mL of L-PRF. Slower centrifugation protocols produced using the A-PRF protocols (1300 rpm × 8 min) produced a more evenly distributed number of platelets throughout PRF. Injectable-PRF (i-PRF) protocols produced the highest concentration of leukocytes/platelets, however, the total number of leukocytes and platelets were significantly lower owing to the decreased total volume collected. Horizontal centrifugation produced a significant increase in both the number and concentration of platelets and leukocytes (up to 3.5× higher for either solid/liquid PRF). When compared to either fixed or angled centrifuge (InstraSpin, Process for PRF). In conclusion, the present study revealed a novel/accurate method to quantify cells following PRF protocols. Furthermore, PRF produced via horizontal centrifugation accumulated a higher number and concentration of platelets/leukocytes when compared to either fixed-angle centrifugation. K E Y W O R D S blood platelets, centrifugation, fibrin, platelet rich fibrin

Objectives Platelet-rich fibrin (PRF) has gained tremendous momentum in recent years as a natural autologous growth factor derived from blood capable of stimulating tissue regeneration. Owing to its widespread use, many companies have commercialized various centrifugation devices with various proposed protocols. The aim of the present study was to compare 3 different commercially available centrifuges at both high and low g-force protocols. Materials and methods PRF was produced on three commercially available centrifuges including the IntraSpin Device (IntraLock), the Duo Quattro (Process for PRF), and Salvin (Salvin Dental). Two separate protocols were tested on each machine including the original leukocyte and platelet-rich fibrin (L-PRF) protocol (~700 RCF max (~400 RCF clot) for 12 min) as well as the advanced platelet-rich fibrin (A-PRF+) protocol (~200 g RCF max (~130 g RCF clot) for 8 min). Each of the tested groups was compared for cell numbers, growth factor release, scanning electron microscopy (SEM) for morphological differences, and clot size (both weight and length/width).Results The present study found that PRF clots produced utilizing the low-speed centrifugation speeds (~200 g for 8 min) produce clots that (1) contained a higher concentration of evenly distributed platelets, (2) secreted higher concentrations of growth factors over a 10 day period, and (3) were smaller in size. This was irrespective of the centrifugation device utilized and consistently observed on all 3 devices. The greatest impact was found between the protocols utilized (up to a 200%). Interestingly, it was further revealed that the centrifugation tubes used had a much greater impact on the final size outcome of PRF clots when compared to centrifugation devices. It was found that, in general, the Process for PRF tubes produced significantly greater-sized clots when compared to other commercially available tubes. The Salvin Dental tubes also produced significantly greater PRF clots when compared to the IntraLock tubes on each of the tested centrifugation devices. Conclusions The present study demonstrated the reproducibility of a scientific concept (reduction in RCF produces PRF clots with more evenly distributed cells and growth factors) utilizing different devices. Furthermore, (and until now overlooked), it was revealed for the first time that the centrifugation tubes are central to the quality production of PRF. Future research investigating tube characteristics thus becomes critically important for the future optimization of PRF.

Platelet-rich fibrin (PRF) has been widely used owing to its ability to stimulate tissue regeneration. To date, few studies have described the antibacterial properties of PRF. Previously, PRF prepared by horizontal centrifugation (H-PRF) was shown to contain more immune cells than leukocyte- and platelet-rich fibrin (L-PRF). This study aimed to compare the antimicrobial effects of PRFs against Staphylococcus aureus and Escherichia coli in vitro and to determine whether the antibacterial effects correlated with the number of immune cells. Blood samples were obtained from eight healthy donors to prepare L-PRF and H-PRF. The sizes and weights of L-PRF and H-PRF were first evaluated, and their antibacterial effects against S. aureus and E. coli were then tested in vitro using the inhibition ring and plate-counting test methods. Flow-cytometric analysis of the cell components of L-PRF and H-PRF was also performed. No significant differences in size or weight were observed between the L-PRF and H-PRF groups. The H-PRF group contained more leukocytes than the L-PRF group. While both PRFs had notable antimicrobial activity against S. aureus and E. coli, H-PRF demonstrated a significantly better antibacterial effect than L-PRF. Furthermore, the antimicrobial ability of the PRF solid was less efficient than that of wet PRF. In conclusion, H-PRF exhibited better antibacterial activity than L-PRF, which might have been attributed to having more immune cells.

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Purpose Platelet-rich fibrin (PRF) has been proposed as promising biomaterials with the advantages of host accumulation of platelets and leukocytes with entrapment of growth factors and fibrin scaffold. However, limitations including fast resorption rate (~ 2 weeks) restricts its clinical application. Recent studies have demonstrated heating treatment can prolong PRF degradation. Current published articles used the method of 75 °C for 10 min to obtain longer degradation, while few studies investigated the most suitable temperature for heating horizontal PRF. Our present study was to discover and confirm the optimum temperature for heat treatment before obtaining H-PRF gels by investigating their structure, mechanical properties, and bioactivity of the H-PRF gels after heating treatment. Methods In the present study, 2-mL upper layer of horizontal PRF was collected and heated at 45 °C, 60 °C, 75 °C, and 90 °C to heat 2-mL upper layer of horizontal PRF for 10 min before mixing with the 2-mL lower layer horizontal PRF. The weight, solidification time and the degradation properties were subsequently recorded. Scanning electron microscopy (SEM) and rheologic tests were carried out to investigate the microstructure and rheologic properties of each H-PRF gel. The biological activity of each H-PRF gel was also evaluated using live/dead staining. Results H-PRF gel prepared at 75 °C for 10 min had the fast solidification period (over a tenfold increase than control) as well as the best resistance to degradation. The number of living cells in H-PRF gel is greater than 90%. SEM showed that H-PRF gel becomes denser as the heating temperature increases, and rheologic tests also revealed that the heat treatment improved the mechanical properties of H-PRF gels when compared to non-heated control group. Future clinical studies are needed to further support the clinical application of H-PRF gels in tissue regeneration procedures. Conclusions Our results demonstrated that the H-PRF gel obtained at 75 °C for 10 min could produce a uniform, moldable gel with a short time for solidification time, great rheologic behavior and, high percent of live cells in PRF gel. A promising use of the commonly utilized PRF gel was achieved facilitating tissue regeneration and preventing degradation.