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Horizontal Versus Fixed-Angle Centrifugation of PRF: Optimization of C-PRF

Contributors

Richard J. Miron

Masako Fujioka-Kobayashi

Tomoyuki Kawase

Yufeng Zhang

Chapter Highlights

 Advantages of horizontal centrifugation versus fixed-angle centrifugation

 Systematic evaluation of horizontal centrifugation using 24 different protocols

 Optimization of H-PRF in both liquid and solid formulations

 Histologic evaluation of L-PRF versus H-PRF

 Optimization of C-PRF using horizontal centrifugation

Video 3-1

The first two chapters of this textbook discussed the history of platelet concentrates and their commercial and biologic evolution. The aim of this chapter is to discuss centrifugation parameters that may best optimize the production of PRF. A great deal of work has been conducted at both the preclinical and clinical level to maximize the regenerative potential of PRF, and this chapter provides a more detailed overview of these methods and takes a closer look at the cell types found in PRF. It also discusses the work conducted by our laboratories to improve PRF using horizontal centrifugation by evaluating a range of 24 different protocols in a systematic and standardized way. Thereafter, comparative histologic evaluation compares PRF membranes produced following fixed-angle versus horizontal centrifugation. Lastly, data from the previous chapter is expanded upon to explain the concept of C-PRF.

The previous chapter provided a brief overview of the advantages of horizontal centrifugation of PRF. Previously, publications by our group found that horizontal centrifugation was superior at accumulating platelets and leukocytes when compared to standard fixed-angle centrifugation utilized to produce PRF.¹ Both solid-based and liquid-based PRF matrices were obtained with up to a fourfold increase in platelet/leukocyte numbers and/or concentrations.¹

This chapter discusses in greater detail the ability of horizontal-PRF (H-PRF) to better separate cell layers in blood based on their density. As previously shown (see Table 2-1), platelets are the lightest of the blood cells, with white blood cells (WBCs) and red blood cells (RBCs) similar in size and density (Fig 3-1). Therefore, the optimization of centrifugation parameters becomes extremely relevant should the clinician desire to maximize the harvest of cell types following centrifugation.

Fig 3-1 SEM showing the relative size of RBCs, platelets, and WBCs (leukocytes). Note the much larger size of RBCs and WBCs when compared to platelets as well as the similarity in size between WBCs and RBCs.

When cells are separated in a blood collection tube based on their density, it is important to understand that their ability to separate is based on differences in g-force produced at the RCF-min versus RCF-max of the tube (as reviewed in greater detail in chapter 4). Note that the greater the angulation of the tube, the greater the difference in g-force differential that exists between the RCF-min of the tube versus the RCF-max (Fig 3-2).

Fig 3-2 Graphic demonstrating the difference between the RCF-min and RCF-max on both fixed-angle and horizontal centrifugation systems. Note that because the tubes are completely horizontal on a horizontal centrifugation system, the gradient difference between the RCF-min and RCF-max is much greater, resulting in much better layer separation.

One avenue that is extremely relevant to the production of PRF has been the effect of the larger radius from the rotor found when centrifugation is carried out on a horizontal versus fixed-angle centrifuge. To illustrate this point further, an overlapping image of a centrifugation tube is provided in Fig 3-3. If a certain g-force can be expected at the RCF-clot, a much smaller RCF-max will be produced on a fixed-angle centrifuge (Fig 3-3a) when compared to a horizontal centrifuge (Fig 3-3b). While certain commercial entities such as the L-PRF system will report the g-force values specifically at the RCF-clot, note how in Fig 3-3b, the same RCF-clot value on both fixed-angle and horizontal systems displays a much greater RCF-max value if the tube is horizontal. This allows for higher g-force productions at the RCF-max on a horizontal centrifuge, which means faster and better ability for the heavier cells (ie, RBCs) to be pulled down to the bottom of the tube more effectively.

Fig 3-3 (a and b) Demonstration that even at the exact same RCF-clot value on either fixed-angle or horizontal centrifugation, the final RCF-max value on a fixed-angle centrifugation system is much smaller than that observed on a horizontal system due to the greater distance from the centrifuge rotor. (c) The introduction of 13-mL tubes has led to claims that they are better able to concentrate cells. While this is true compared to 10-mL tubes, if centrifugation remains carried out on a fixed-angle device, the difference between the RCF-min and RCF-max remains inferior to that on a horizontal centrifuge, even if a 10-mL tube is used.

More recently, an introduction of longer tubes (13-mL tubes versus 10-mL) has been proposed as a way to further concentrate cells (Fig 3-3c). While this concept is true, note that should these tubes be utilized on a fixed-angle centri-fugation device, the difference between the RCF-min and RCF-max will still equate to less than that obtained with a 10-mL tube in a horizontal centrifuge. These longer tubes still pose the main issue related to cells accumulating on the back distal surface of PRF tubes without proper cell layer separation, as reviewed in the previous chapter.

Larger Radius, Higher RCF, Shorter Spin Time

While not peer-reviewed in the same way as our published studies, the Internet is rife with support for horizontal centrifugation. Drucker Diagnostics, for example, clearly demonstrate how tubes centrifuged in a horizontal manner experience a much larger radius, resulting in a higher RCF and more efficient pull-down forces (https://druckerdiagnostics.com/horizontal-vs-fixed-angle/). Furthermore, they have shown that the time required for complete centrifugation on a horizontal rotor is only two-thirds of the time required on a fixed-angle centrifuge (Fig 3-4). This is particularly important because PRF is subject to clotting over time, so saving a few critical minutes by switching to horizontal centrifuge can translate to better layer separation and greater accumulation of cells following the protocol.²

Fig 3-4 Graphic from Drucker Diagnostics demonstrating that centrifugation carried out on a horizontal centrifuge requires only two-thirds the time required on a fixed-angle centrifuge. Therefore, a 12-minute protocol on a fixed-angle rotor would take only 8 minutes with a horizontal system. (Adapted from https://druckerdiagnostics.com/horizontal-vs-fixed-angle/.)

The time required for complete centrifugation on a horizontal rotor is only two-thirds of the time required on a fixed-angle centrifuge.

Accumulation of Cells in PRF Tubes

One thing commonly found following centrifugation is the angle produced after completion of the spin cycle (Fig 3-5).² In a study by Takahashi et al, the distribution of cells and growth factors (GFs) in PRF following centri-fugation using two different centrifugation devices was investigated.³ Blood samples were obtained in tubes and immediately centrifuged to prepare PRF using two protocols. Both matrices were compressed, embedded in paraffin, and subjected to immunohistochemical examination.³

Fig 3-5 (a) Following centrifugation on fixed-angle centrifuges, blood layers do not separate evenly, and as a result, an angled blood separation is observed. In contrast, horizontal centrifugation produces an even separation. (Reprinted with permission from Miron et al.¹) (b) Layer separation following either L-PRF or H-PRF protocols. L-PRF clots are prepared with a sloped shape, and multiple red dots are often observed on the distal surface of PRF tubes; H-PRF, on the other hand, is prepared with a horizontal layer separation between the upper plasma and lower RBC layers.

Following histologic assessment, it was observed that leukocytes and plasma proteins were localized on the back walls of PRF tubes (referred to as distal surface), including the interface corresponding to the buffy coat (Figs 3-6 and 3-7).³ These cells were being accumulated on the back distal surfaces only when utilizing fixed-angle centrifugation due to the gravitational pull (see Fig 3-8; see also Video 3-2).

Fig 3-6 Experimental setup describing the orientation of PRF membranes during histologic assessment. The proximal surface describes the inner tube wall (generally receiving the smallest g-force), whereas the distal surface is the outer tube wall, where cells generally accumulate during centrifugation at high g-force. (a) Regions in compressed A‐PRF or concentrated GF (CGF) matrix. This image is the proximal surface. (b) Centrifugal force and distal and proximal surfaces of A‐PRF or CGF matrix. (Reprinted with permission from Takahashi et al.³)

Fig 3-7 Distribution of PDGF‐BB in A‐PRF and CGF matrices. (a and d) Region 1. (b and e) Region 2. (c and f) Region 3. Note that the majority of cells and GFs accumulated on the back distal surfaces of PRF tubes. (Reprinted with permission from Takahashi et al.³)

Fig 3-8 Illustrations comparing fixed-angle and horizontal centrifuges. With horizontal centrifugation, increased separation of blood layers based on density is achieved due to the increased difference in RCF-min and RCF-max. Following centrifugation on fixed-angle centrifuges, blood layers do not separate evenly, and as a result, an angled blood separation is observed. In contrast, horizontal centrifugation produces even separation. Owing to the large RCF values (~200g–700g), the cells are pushed toward the outside and downward. On a fixed-angle centrifuge, cells are pushed toward the back of centrifugation tubes and then downward/upward based on cell density. These g-forces produce additional shear stress on cells as they separate based on density along the back walls of centrifugation tubes. In contrast, horizontal centrifugation allows for the free movement of cells to separate into their appropriate layers based on density, allowing for better cell separation and less trauma/shear stress on cells. (Adapted from Miron et al.¹)

Video 3-2

More intriguing was the fact that the type of tube (plain glass versus silica-coated plastic) had a significant impact on the final distribution of cells found within PRF clots when the clots produced at different centrifugation speeds were investigated histologically.⁴ The specific role of tubes for the production of PRF has been such a hot topic as of late that an entire book chapter is dedicated solely to this topic (see chapter 5).

Cells were being accumulated on the back distal surfaces only when utilizing fixed-angle centrifugation.

Evaluation of PRF via Horizontal Centrifugation

As the field continues to progress, it has become clear that horizontal centrifugation offers numerous advantages. A simple evaluation of three protocols previously tested has demonstrated the very obvious improved blood cell layer separation observed with H-PRF when compared to L-PRF or A-PRF (Fig 3-8; see chapter 2).

In a study titled “Histological comparison of platelet rich fibrin clots prepared by fixed-angle versus horizontal centrifugation,” Fujioka-Kobayashi et al compared L-PRF with H-PRF, observing the morphology of cells and their localization on the surface of PRF clots by SEM and histologically by transaxial frozen sections by means of a film method.⁵ It was consistently observed that L-PRF clots demonstrated a sloped separation between the upper plasma and the bottom RBC layers according to the angle of the rotor. Interestingly, red dots were often observed on the distal walls of the tubes in the upper layers, consisting of aggregations of RBCs, leukocytes, and platelets by SEM and histology⁵ (Fig 3-9). Clots produced on the horizontal centrifuge showed much smoother cell layer distribution and separation along the tube surfaces when compared to L-PRF.

Fig 3-9 Characterization of A-PRF, L-PRF, and H-PRF. (a and b) The morphology and size of A-PRF, L-PRF, and H-PRF after centrifugation using the manufacturer’s recommended centrifugation protocols and tubes. (c) SEM of A-PRF, L-PRF, and H-PRF clots. Scale bar = 20 μm. (Reprinted with permission from Zhang et al.⁶)

Microscopic and histologic observation of L-PRF and H-PRF

PRF clots were further investigated by SEM and histologic assessment for cell distribution and surface configurations (Figs 3-10 to 3-13). Three distinct patterns were observed on the distal walls of L-PRF clots, including RBC clusters on the smooth or wavy fibrin clot surfaces as well as clusters of leukocytes, platelets, and crushed RBCs (see Fig 3-10). Histologic observation further confirmed these pattern types (see Fig 3-11). The border between the plasma and RBC layers included more dense fibrin networks covered with many blood cells (see Fig 3-10e). Many leukocytes were found at this layer (see Fig 3-11d).

Fig 3-10 SEM images of the distal surface of PRF clots prepared utilizing the L-PRF protocol. (a) The described areas observed by SEM. The L-PRF clot surfaces showed typically three types, as shown in b to d. (b) Clusters of RBCs were observed overlaying a smooth clot surface. (c) A wavy surface was observed, including RBCs. (d) The rough surface included leukocytes, platelets, and crushed RBCs. (e) The dense fibrin networks were observed, including RBCs at the border between the yellow plasma and RBC layers. (f) Many RBCs were observed within a fibrin network in the RBC layer. (Reprinted with permission from Fujioka-Kobayashi et al.⁵)

Fig 3-11 Histologic observation of the frozen section of L-PRF sectioned transaxially. (a) The panoramic view of the sections from the whole PRF clot including the RBC layer stained with hematoxylin. The L-PRF clots and RBC layer were separated by a fixed-angle centrifuge. The distal wall showed two typical patterns, shown in b and c. (b) Clusters of RBCs with a few leukocytes were located on fibrin networks on the distal surface. (c) The aggregated cluster consisting of platelets, leukocytes, and RBCs occasionally observed. (d) Many leukocytes were located at the border between the PRF clot and the RBC layer. (e) The aggregated clusters of cells containing leukocytes were occasionally observed within the RBC layer within the red buffy coat zone. (Reprinted with permission from Fujioka-Kobayashi et al.⁵)

Fig 3-12 SEM images of the distal surface of PRF clots prepared utilizing the H-PRF protocol. (a) The described areas observed by SEM. The H-PRF clot surfaces showed typically two types, as shown in b and c. (b) Few leukocytes and RBCs were observed on the smooth surfaces. (c) The rough surfaces included leukocytes, platelets, and RBCs. (d and e) The fibrin networks twisted around the leukocytes with platelets at the border between the yellow plasma and RBC layers. (f) Many RBCs were observed within a fibrin network in the RBC layer. (Reprinted with permission from Fujioka-Kobayashi et al.⁵)

Fig 3-13 Histologic observation of the frozen section of H-PRF sectioned transaxially. (a) The panoramic view of the sections from the whole PRF clot including the RBC layer stained with hematoxylin. The H-PRF clots and RBC layers were separated evenly and horizontally with no obvious accumulation of cells on the distal surface. The clots showed two typical patterns shown in b and c. (b) The fibrin networks were observed in the clots with many platelets and few leukocytes/RBCs. (c) Aggregated clusters consisting of leukocytes and a few RBCs were occasionally observed. (d) Many leukocytes were located at the border between the clots and RBC layer. (e) Aggregated clusters of cells containing leukocytes were occasionally observed in the RBC layer within the red buffy coat zone. (Reprinted with permission from Fujioka-Kobayashi et al.⁵)

Within H-PRF clots, two typical patterns were observed on the surface, including fewer blood cells on the smooth clot surfaces, with more found located on the rough surfaces (see Fig 3-12). Abundant platelets were found within the clots, with a few clusters located on the actual clot surface (see Fig 3-13). The border between the plasma and RBC layers included a dense fibrin network with many leukocytes.

Interestingly, aggregated clusters of platelets with leukocytes were found in both L-PRF and H-PRF within the RBC layer, approximately 5 mm below the precise separation typically referred to as the buffy coat zone. A representative summary figure is provided in Fig 3-14.

Fig 3-14 Graphic demonstrating cell distribution within PRF when centrifugation was carried out either by fixed-angle or horizontal centrifugation. Note that the majority of cells following the L-PRF protocol are found along the back distal surface of PRF tubes as well as primarily contained within the buffy coat layer. A more even distribution of cells was observed when horizontal centrifugation was utilized. (Reprinted with permission from Fujioka-Kobayashi et al.⁵)

Optimization of PRF Protocols

To this day, PRF has not been most efficiently optimized. Additionally, in the early 2000s, a variety of publications on the topic of PRF were utilized at different RCF/rpm parameters. As highlighted in chapter 4, many were actually using the same rpm values for devices with different rotor sizes (which completely changes the g-force) without understanding its pronounced impact on cell layer separation.

It is important to note that larger-radius centrifuges produce much greater g-force even at identical rpms.

One of the most common limitations to PRF is the fact that various protocols have never been investigated in studies. In 2014, Ghanaati et al discovered a way to further optimize the production of PRF using three different protocols and by gradually reducing RCF. By doing so, he discovered that more cells could be obtained in the upper PRF layers; this method has since been named the low-speed centrifugation concept (LSCC).⁷

In 2019, drastically better results were obtained utilizing horizontal centrifugation. While 20% to 30% better results were obtained with the LSCC, the ability to simply shift from fixed-angle to horizontal centrifugation led to as much as a fourfold increase in cells. Our research team then investigated 24 different protocols (instead of the original 3) to better optimize PRF.

Evaluation of 24 protocols for the production of PRF

While in previous studies few protocols were compared (generally at most three),⁷ the desire and emphasis of our research group was to better investigate for the first time the effect of numerous centrifugation parameters on the final production of PRF. As such, 24 different protocols were evaluated (Fig 3-15).⁸ All protocols were compared utilizing a recent method to quantify cells in PRF in 1-mL sequential layers pipetted from the upper layer downward until all 10 mL were harvested (see chapter 2 for methodology). In total, 960 complete blood counts (CBCs) were investigated. Both solid- and liquid-based PRF protocols were investigated following 24 protocols involving six RCF values (100g, 200g, 400g, 700g, 1000g, and 1200g) at four centrifugation times (3, 5, 8, and 12 minutes).

Fig 3-15 Clinical image demonstrating the plasma layer separation for the 24 protocols investigated in this study. Note that while some protocols reveal roughly identical plasma layer separation, the underlying cellular content in the various protocols may be drastically different. (Reprinted with permission from Miron et al.⁸)

Figure 3-16 demonstrates the overall final volume of plasma of each of the protocols, along with the total yield and concentrations of platelets and leukocytes above baseline values. Note that of each of the protocols, 200g for 5 minutes led to the highest concentration of platelets/leukocytes. The best yield of leukocytes was achieved after centrifugation for 8 minutes utilizing the 700g, 1000g, and 1200g protocols. Of those, the highest concentration was achieved at 700g for 8 minutes (owing to the reduced total plasma volume). Another noteworthy trend that was apparent was that as centrifugation time was increased, a general increase in percent yield was observed; however, generally speaking, an overall decrease in concentration was also observed (see Fig 3-16). Furthermore, it was apparent that certain centrifugation protocols that were too reduced in RCF (such as 100g) typically did not lead to adequate yield of cells. This relates with our group’s previous work on i-PRF demonstrating that these low centrifugation speeds and time (~800 rpm for 3–4 minutes) led to substandard concentrations of platelets and leukocytes (see chapter 2). Protocols that were too fast or lengthy (1000g or more) led to a reduction in yield and/or concentrations (as more cells then got pushed into the bottom layers or the volume of total plasma led to a reduction in concentration).

Fig 3-16 (a to e) Evaluation of 24 protocols utilized for the production of PRF. Data includes final volume (mL), total leukocyte and platelet yields (% of the total from 10 mL), as well as concentration of leukocytes and platelets above baseline values (% increase). (Reprinted with permission from Miron et al.⁸)

To simplify Fig 3-16, certain time points were removed from the graph to facilitate its understanding for the reader. Figure 3-17 demonstrates only two protocols (700g and 1000g) over time. Note that in Fig 3-17a, a general increase of centrifugation time is associated with an increase in platelet yield. Note, however, in Fig 3-17b that an increase in centrifugation time actually decreases the concentration of platelets (because the plasma volume is increased, so even if the total yield of platelets remains the same or even slightly higher, the actual concentration decreases).

Fig 3-17 Evaluation of protocols utilizing both 700g and 1000g RCF for the production of PRF. Both total yield of platelets (a) as well as concentration of platelets (b) are depicted. (Reprinted with permission from Miron et al.⁸)

In Fig 3-18, the 100g protocol is included as well. Notice here how the yield is extremely low in platelets (Fig 3-18a) as well as in concentration (Fig 3-18b). This is a result of the speed cycle simply being so slow that it is unable to accumulate or concentrate platelets in the upper layer. As alluded to in chapter 2, it is possible to centrifuge too slowly to the point where platelets and leukocytes do not actually accumulate effectively in the upper layers. This is a common misconception that many clinicians maintain due to inaccurate information provided by various manufacturers.

Fig 3-18 Previous graphs demonstrating 700g and 1000g protocols but with the addition of 100g results as well. It is very clear that the 100g protocol is not able to accumulate high yields of platelets (a), and the concentration remains low (b).

Centrifugation carried out too slowly is a common misconception that many clinicians maintain due to inaccurate information provided by various manufacturers.

In Fig 3-19, observe the 200g protocol. Notice how at the higher g-force, the cells are actually able to accumulate more efficiently (Fig 3-19a). More importantly, observe the concentration of platelets in Fig 3-19b following a 200g protocol for 5 minutes. Here the platelets are actually most concentrated after 5 minutes, and thereafter, even though their yield continues to rise (see Fig 3-19a), the concentration actually begins to decrease because of the increase in liquid-PRF volume. Therefore, a 200g to 300g centrifugation cycle is most effectively able to concentrate platelets and leukocytes (300g is the one chosen after further optimization).

Fig 3-19 Evaluation of protocols utilizing both 700g and 1000g RCF for the production of PRF but now with the addition of the 200g protocols. Both total yield of platelets as well as concentration of platelets are depicted. Note that when a 200g protocol is utilized, while the yield is still low (a), the concentration peaks at 5 minutes at a level higher than any other group (b). (Reprinted with permission from Miron et al.⁸)

Discussion

In general, platelets were evenly distributed throughout a variety of protocols within the upper three to six plasma-rich layers (see Fig 3-16), but it was obvious that WBCs required more pristine fine-tuning to reach adequate harmony within the upper plasma layers. Protocols within the 400g to 700g (5- to 8-minute) range were better able to accumulate and distribute platelets/leukocytes more evenly throughout the upper layers.

Currently, one standard in the field of PRF is the novel use of injectable i-PRF.⁹ Previously, our research group found that only slight increases in platelets and leukocytes were noted, with failure to adequately accumulate cells in the upper plasma layer owing to extremely low RCF values (60g) and centrifugation times (3–4 minutes) on fixed-angle centrifugation devices.¹ In a study titled “Injectable platelet-rich fibrin: Cell content, morphological, and protein characterization,”¹⁰ Varela et al observed only a slight increase in platelets (less than 33%) and leukocytes following i-PRF protocols, with decreases in VEGF reported when compared to that in whole blood. Altogether, these studies confirm that previously utilized i-PRF protocols (~60g for 3–4 minutes on a fixed-angle centrifuge) are inadequately effective at separating blood cell layers due to their considerable reduction in centrifugation speed and time. Furthermore, protocols at 100g or lower are inefficient at accumulating platelets and leukocytes in the upper plasma layer, highlighting the limits of the LSCC.

It was also observed within this study that the use of PRF produced using a protocol of 200g for 5 minutes resulted in the highest concentration of platelets and leukocytes. Within these studies, up to a fourfold increase in platelet/leukocyte concentration and/or yield was observed when compared to the results of previously utilized i-PRF protocols produced on a fixed-angle centrifuge.¹ Further unpublished data has found that a 300g protocol for 5 minutes resulted in the highest concentration of liquid-PRF, as discussed further in chapter 5. Nevertheless, based on the C-PRF protocols established in chapter 2, it remained obvious that better concentrations could be achieved by further modifying centrifugation parameters.

Establishing C-PRF on a Horizontal Centrifuge

In a recent study titled “Improved growth factor delivery and cellular activity using concentrated platelet-rich fibrin (C-PRF) when compared to traditional injectable (i-PRF) protocols,” Fujioka-Kobayashi et al aimed to investigate and optimize PRF in its most concentrated formulation.¹¹ As reviewed in chapter 2, the ability to centrifuge at higher centrifugation speeds and times leads to an accumulation directly at the buffy coat layer.¹² There was an approximately tenfold increase in baseline concentrations specifically in this 0.3- to 0.5-mL buffy coat layer directly above the RBC layer produced using higher centrifugation protocols.¹² The PRF obtained from this harvesting technique was given the working name concentrated-PRF (C-PRF). Figure 3-20 demonstrates a clinical photograph of standard liquid-PRF protocols versus those of C-PRF. It was hypothesized that based on the extensive increase in the yield of platelets and leukocytes, C-PRF would exhibit higher GF release as well as superior cellular activity. Therefore, the aim of this study was twofold. First, a new centrifugation protocol was developed on a horizontal centrifugation system with the aim of accumulating the greatest concentrations of platelets and leukocytes within the buffy coat. The second aim was to compare the total GF release of PRF obtained through this newly developed C-PRF protocol to that of PRF obtained through the clinically utilized liquid i-PRF protocol over a 10-day period and to investigate the regenerative properties of human gingival fibroblasts in vitro.

Fig 3-20 Visual representation of layer separation following either the i-PRF (300g for 5 minutes) or C-PRF protocol (3000g for 8 minutes). Plasma was collected from the buffy coat region within the 1-mL layer above the RBC layer. (Reprinted with permission from Fujioka-Kobayashi et al.¹¹)

Optimization of C-PRF protocols

Prior to initiating the C-PRF experiments, protocols of 3000g for 5 minutes, 8 minutes, and 12 minutes were compared to optimize the accumulation of cells within the buffy coat layer. It was found that the 5-minute protocol was unable to concentrate all cells within the buffy coat layer, with the platelets remaining in the upper 4 mL. Following the 8-minute protocol, the sequential pipetting method results revealed that the majority of platelets and leukocytes were located within the buffy coat layer 6 region (Fig 3-21). No further advantage was observed following the 12-minute protocol (data not shown). For comparison purposes, a standard i-PRF protocol resulted in a slight concentration of platelets and leukocytes in the upper 1-mL layer from which i-PRF was harvested (see Fig 3-21). Note that many platelets/leukocytes, however, remained in the RBC layers. To harvest C-PRF, a 1-mL layer within this buffy coat layer was collected and processed for further analysis (Fig 3-22).

Fig 3-21 The concentrations of different cell types found in each 1-mL layer of the 10-mL tube obtained through the i-PRF protocol (300g for 5 minutes) and the C-PRF protocol (3000g for 8 minutes). Note that in the PRF obtained through the i-PRF protocol, the majority of platelets and leukocytes were located in the 1-mL buffy coat layer. In the PRF obtained through the C-PRF protocol, although higher concentrations of platelets and leukocytes were found in the upper 1-mL layer, the majority of the platelets and leukocytes were actually located in the RBC layers. (Reprinted with permission from Fujioka-Kobayashi et al.¹¹)

Fig 3-22 Method to collect and concentrate C-PRF. Following centrifugation at higher speeds (2000g for 8 minutes), the majority of cells are located directly at the buffy coat layer. Instead of attempting to remove this layer with a long needle into the deep layers, it is highly advised to first remove the upper 4 mL of platelet-poor plasma (PPP), followed by collection of the C-PRF buffy coat layer.

Tips

 In clinical practice, it is best to harvest C-PRF by first removing the upper 3 to 4 mL of platelet-poor plasma (discard it). The remaining C-PRF layer can then be taken much more easily. It is much more difficult to attempt to retrieve this buffy coat zone with 5 mL over top of it; it is harder to concentrate it, and too much volume is often collected.

 Many centrifuges may not reach the 3000g speed. A 2000g protocol for 8 minutes will also achieve a C-PRF layer.

Comparative GF release between i-PRF and C-PRF

In the first set of experiments, the release of GFs including PDGF-AA, PDGF-AB, PDGF-BB, TGF-β1, VEGF, EGF, and IGF-1 from i-PRF and C-PRF was investigated by ELISA (Fig 3-23). The release of all GFs over the entire 10-day (240-hour) period was significant for both protocols, with the C-PRF protocol resulting in up to two- to threefold higher quantities. This demonstrated clearly that this newly developed protocol had much greater regenerative potential when compared to previously utilized i-PRF protocols.

Fig 3-23 Protein quantification of PDGF-AA (a), PDGF-AB (b), PDGF-BB (c), TGF-β1 (d), VEGF (e), EGF (f), and IGF-1 (g) at each time point over a 10-day (240-hour) period for PRF obtained through the i-PRF protocol and the C-PRF protocol, as determined by ELISA. Data represents the mean ± SE; an asterisk (*) indicates a value significantly higher than the other group (P < .05). (Reprinted with permission from Fujioka-Kobayashi et al.¹¹)

Biocompatibility and cellular activity of i-PRF and C-PRF

It was first observed that while i-PRF induced a twofold increase in cell migration when compared to that observed in the control, a significantly higher fourfold increase was observed when cells were cultured with C-PRF (Fig 3-24a). Furthermore, C-PRF also induced significantly higher cell proliferation at 3 and 5 days postseeding when compared to i-PRF (Fig 3-24b). Both i-PRF and C-PRF were able to significantly upregulate TGF-β 3 days postseeding (Fig 3-24c), and a significant 250% and 400% increase in the PDGF-AA was observed for i-PRF and C-PRF, respectively (Fig 3-24d). The analysis of COL1 immunostaining also revealed significantly higher COL1A staining for C-PRF when compared to i-PRF and control tissue culture plastic groups (Figs 3-24e and 3-24f).

Fig 3-24 (a and b) Cell migration and proliferation at 1, 3, and 5 days in human gingival fibroblast (HGF-1) cells. (c and d) RT-PCR analysis of mRNA levels of TGF-β and PDGF in human gingival fibroblasts treated with i-PRF and C-PRF at 3 days. (e and f) Quantitative and representative staining of collagen1 at 14 days. Data represents mean ± SE; an asterisk (*) indicates a value significantly higher than the control group (P < .05); a double asterisk (**) indicates a value significantly higher than all other groups (P < .05); a number sign (#) indicates a value significantly lower than all groups (P < .05). (Reprinted with permission from Fujioka-Kobayashi et al.¹¹)

Conclusion

In summary, the results from the 24-protocol investigation demonstrated clearly that certain protocols were better at increasing the amount of platelet/leukocyte yield (400g to 700g for 8 minutes), whereas others were more effective at concentrating PRF (200g to 300g for 5 minutes). Therefore, new protocols were designed accordingly for solid-PRF and liquid-PRF. While histologically it was observed that horizontal centrifugation led to better cell layer separation of blood cells as well as more even distribution of cells throughout the PRF clot, it was also histologically observed that the majority of cells accumulated on the back distal surfaces of PRF tubes when fixed-angle centrifugation was carried out. Lastly, a new C-PRF formulation was evaluated demonstrating up to a threefold increase in GF release during a 10-day period and further elicited fourfold increases in gingival fibroblast migration, PDGF gene expression, and collagen1 synthesis when compared to standard i-PRF protocols.

References

1.Miron RJ, Chai J, Zheng S, Feng M, Sculean A, Zhang Y. A novel method for evaluating and quantifying cell types in platelet rich fibrin and an introduction to horizontal centrifugation. J Biomed Mater Res A 2019;107:2257–2271.

2.Miron RJ, Dham A, Dham U, Zhang Y, Pikos MA, Sculean A. The effect of age, gender, and time between blood draw and start of centrifugation on the size outcomes of platelet-rich fibrin (PRF) membranes. Clin Oral Investig 2019;23:2179–2185.

3.Takahashi A, Tsujino T, Yamaguchi S, et al. Distribution of platelets, transforming growth factor-beta1, platelet-derived growth factor-BB, vascular endothelial growth factor and matrix metalloprotease-9 in advanced platelet-rich fibrin and concentrated growth factor matrices. J Investig Clin Dent 2019;10:e12458.

4.Tsujino T, Masuki H, Nakamura M, et al. Striking differences in platelet distribution between advanced-platelet-rich fibrin and concentrated growth factors: Effects of silica-containing plastic tubes. J Funct Biomater 2019;10:43.

5.Fujioka-Kobayashi M, Kono M, Katagiri H, et al. Histological comparison of platelet rich fibrin clots prepared by fixed-angle versus horizontal centrifugation [epub ahead of print 18 April 2020]. Platelets doi: 10.1080/09537104.2020.1754382.

6.Zhang J, Yin C, Zhao Q, et al. Anti-inflammation effects of injectable platelet-rich fibrin via macrophages and dendritic cells. J Biomed Mater Res A 2020;108:61–68.

7.Ghanaati S, Booms P, Orlowska A, et al. Advanced platelet-rich fibrin: A new concept for cell-based tissue engineering by means of inflammatory cells. J Oral Implantol 2014;40:679–689.

8.Miron RJ, Chai J, Fujioka-Kobayashi M, Sculean A, Zhang Y. Evaluation of 24 protocols for the production of platelet-rich fibrin. BMC Oral Health (in press).

9.Miron RJ, Fujioka-Kobayashi M, Hernandez M, et al. Injectable platelet rich fibrin (i-PRF): Opportunities in regenerative dentistry? Clin Oral Investig 2017;21:2619–2627.

10.Varela HA, Souza JCM, Nascimento RM, et al. Injectable platelet rich fibrin: Cell content, morphological, and protein characterization. Clin Oral Investig 2019;23:1309–1318.

11.Fujioka-Kobayashi M, Katagiri H, Kono M, et al. Improved growth factor delivery and cellular activity using concentrated platelet-rich fibrin (C-PRF) when compared with traditional injectable (i-PRF) protocols [epub ahead of print 7 May 2020]. Clin Oral Investig doi: 10.1007/s00784-020-03303-7.

12.Miron RJ, Chai J, Zhang P, et al. A novel method for harvesting concentrated platelet-rich fibrin (C-PRF) with a 10-fold increase in platelet and leukocyte yields. Clin Oral Investig 2020;24:2819–2828.