Effect of Centrifugation Methods on the Regenerative Potential of Concentrated Platelet-Rich Fibrin: An In Vitro Study

Abstract

Objective

A new type of platelet concentrate used in regenerative periodontal therapy, called concentrated platelet-rich fibrin (C-PRF), contains more growth factors than previous generations of platelet concentrate. Compared to fixed-angle centrifugation, a recently discovered technique called horizontal centrifugation can produce C-PRF with a more evenly distributed quantity of platelets and improved biological properties. This study aims to determine the effect of horizontal and fixed-angle centrifugation methods on the release of transforming growth factor β1 (TGF-β1) and platelet-derived growth factor AB (PDGF-AB), as well as on the migration and proliferation of osteoblast cells.

Materials and Methods

This study used two test groups: a horizontal group and a fixed-angle group. The release of growth factors TGF-β1 and PDGF-AB was measured at five time points: the first, third, seventh, 10^th, and 14th days of incubation, using the enzyme-linked immunosorbent assay. To determine the effect of C-PRF type on osteoblast cell migration, a scratch wound healing assay was conducted 24 hours after incubation. Osteoblast cell proliferation was counted using a 450-nm microplate reader to determine the number of cells in the well plate on the first, third, and fifth days, with the Cell Counting Kit-8. The data were examined using two-way ANOVA (analysis of variance) and post hoc LSD (least significant difference).

Results

The findings indicated a statistically significant increase in the release of TGF-β1 and PDGF-AB in the horizontal group compared to the fixed-angle group over a 14-day period. The horizontal group's TGF-β1 release increased by a factor of 1.5 compared to the fixed-angle group, peaking on day 7. Following a 24-hour incubation period and microscopic examination, the horizontal group exhibited greater osteoblast migratory activity than the control group and the fixed-angle centrifugation group. Horizontal centrifugation C-PRF had a significantly greater effect on osteoblast proliferation than fixed-angle centrifugation. The horizontal group's proliferation rate surpassed that of the fixed-angle group by up to 1.5 times on the third day of observation.

Conclusion

Compared to fixed-angle centrifugation, the horizontal method of preparing C-PRF has a greater effect on the release of growth factors and the behavior of osteoblasts.

Keywords

horizontal centrifugation - fixed-angle centrifugation - concentrated platelet-rich fibrin - TGF-β1 - PDGF-AB - proliferation - migration

Introduction

In regenerative periodontal therapy, tissue engineering aims to generate new alveolar bone, cementum, and periodontal ligament attachment to surrounding tooth tissue. The primary elements of tissue engineering are bone grafts, biological materials, and membranes. Biological materials are expected to enhance both the initial wound healing phase and overall tissue regeneration by serving as a reservoir of cytokines, growth factors, and human protein products.[1] Platelet concentrate is a common type of biological material.[2]

Platelet-rich plasma (PRP) is a first-generation platelet concentrate that is widely used in regenerative procedures for periodontal treatment. Despite the success of PRP, concerns were raised over the autologous properties of the procedure involving an anticoagulant that may affect its regenerative potential.[3] Therefore, platelet-rich fibrin (PRF) was developed by removing the anticoagulating agent. Centrifugation protocols operating at higher rpm/RCF (relative centrifugal force) values produce solid fibrin clot matrices rich in leukocytes, known as leukocyte and PRF (L-PRF) membranes. However, further evidence showed that the majority of platelets and leukocytes were pushed out of the buffy coat layer due to the high centrifugal force and into the red blood cell layer at the bottom of the tube, thus excluding them from the PRF membrane. To overcome this limitation, pioneering research on the low-centrifugation concept was conducted. This demonstrated a greater concentration of platelets and leukocytes in the upper layer of PRF after reducing the duration and speed of centrifugation.[4]

This optimization increased the release of growth factors and enhanced the regenerative potential of the PRF. This protocol development also revealed that, with a 3 to 5 minute spin cycle at a low g-force, the plasma will remain in a liquid state for 15 to 20 minutes before coagulation occurs. This liquid PRF, known as injectable PRF (i-PRF), can be extracted and injected directly into tissues. It offers better handling properties and clinical applications than L-PRF.[5] Further findings revealed that a higher centrifugation protocol (2,000 RCF for 8 minutes) produced concentrated PRF (C-PRF), demonstrating a 10-fold increase in platelet and leukocyte concentration. This is thought to be due to the larger disparity between the speed of the centrifuge and the horizontal instruments, which are more effective at distributing platelets, leukocytes, and growth factors across the blood layer and at segregating cell types according to density.[6] A previous study demonstrated that C-PRF had the highest concentrations of platelets and leukocytes compared to L-PRF exudate and the combination of the two. An experiment on interdental papilla tissue regeneration in male Sprague-Dawley rats concluded that C-PRF stimulated vascularization more effectively than the other PRF groups.[7]

The fixed-angle centrifugation method has been used to produce platelet concentrates for many years. Recently, the horizontal centrifugation approach has been used to manufacture PRFs.[8] Because it applies more uniform pressure to the blood-filled tube throughout the screening procedure, this approach is being studied as a means of achieving a more even distribution of leukocytes and platelets on the PRF membrane. This method produces PRF with improved regenerative potential, known as horizontal PRF.[8] [9]

Previous studies have shown that PRF may affect the release activity of growth factors, as well as cell migration and proliferation, in the process of wound healing.[9] [10] [11] A review of these studies concluded that, in addition to a higher neutrophil count, A-PRF significantly increased the release of growth factors, such as transforming growth factor β1 (TGF-β), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), within 10 days.[12] The migration speed of human dental pulp stromal cells was observed to be at its highest after being cultured in 10% A-PRF for 24 hours compared to the DMEM (Dulbecco's Modified Eagle Medium) control group.[13] Wang et al found that the group receiving PRF showed an increase in osteoblast cell proliferation starting on the third day and peaking on the fifth day.[10] A similar study comparing C-PRF with the earlier generation of PRF, i-PRF, was carried out by Fujioka-Kobayashi et al.[11] This study found that the C-PRF group showed increased cell proliferation activity on the third and fifth days, with a gradual rise in growth factor release over the 10-day observation period.[11]

Materials and Methods

Fixed-Angle and Horizontal C-PRF Preparation

All research procedures involving human beings have passed the institutional ethical feasibility assessment with code 106/UN1/KEP/FKG-RSGM/EC/2024. Blood samples were collected from three volunteers who consented to having their blood used as study material. Donors were selected based on the following inclusion criteria: (1) aged between 18 and 60; (2) platelet count >150,000 mc/L; and (3) hemoglobin >12 g/dL.

Six PET tubes containing 10 mL of blood (BD Vacutainer, United States) were divided into two groups and centrifuged at 2,000 × g RCF for 8 minutes using both a fixed-angle centrifuge (EBA 280, Hettich, Germany) and a horizontal centrifuge (Hunan Biojoint, China; [Fig. 1]). Following preparation, the harvesting procedures adhered to the C-PRF optimization protocol from an earlier study by Miron et al.[6] A 1 mL layer of liquid, which separated the plasma and red blood cell corpuscles and was located on top of the buffy coat, was harvested from each tube individually using a syringe with an 18G needle (Terumo, Japan). Once identified, the C-PRF sample was placed in a six-well culture plate (Iwaki, Japan) containing 5 mL of cell culture medium (Gibco, Thermo Fisher Scientific, United States) and processed and observed as described below.

Fig. 1 The two types of centrifuge used in this study, (a) fixed-angle centrifuge and (b) horizontal centrifuge.

Growth Factor Release Quantification

Samples were incubated in a CO₂ incubator at 37 °C (Binder, Germany) to allow growth factors to be released into the cell culture medium. This was done in order to determine the concentrations of growth factors released by horizontal and fixed-angle C-PRF on days 1, 3, 7, 10, and 14. Five milliliters of culture medium were obtained at each observation period and stored at −80°C in a freezer (New Brunswick Innova U535, Eppendorf, Germany). Five milliliters of fresh culture medium were added to the well plate. This process was repeated until the final day of observation. Following the manufacturer's instructions, quantification was performed using TGF-β1 (detection range = 31.2–2,000 pg/mL, RK00055, ABclonal Technology, United States) and PDGF-AB (detection range = 78.13–5,000 pg/mL, ELK1089, ELK Biotechnology, United States). The absorbance value was determined at 450 nm using a microplate reader (iMark, Bio-Rad, United States).

Osteoblast Cell Culture

A stock of 100 μL of MG-63 osteoblast cells was placed in a centrifugation tube filled with 10 mL of phosphate-buffered saline, and the osteoblast cells were extracted. The tube was then centrifuged for 10 minutes at 24 °C and 2,000 rpm. The pellet and the supernatant are the two components resulting from the centrifugation process. The pellets were then homogenized by dissolving them once more in the culture medium. After transferring the solution to a petri dish, the dish is filled with the culture medium until it reaches a volume of 7 mL. The petri dishes are then incubated for 24 hours at 37°C with 5% CO₂. Osteoblasts can be extracted once the cells have proliferated to reach confluence.[14]

Osteoblast Cell Migration

The osteoblast cells were immersed in the control medium and horizontal and fixed-angle C-PRF in a six-well plate and then incubated for 24 hours. The scratch wound healing assay method was used to observe osteoblast cell migration under a microscope, with the percentage of area closure by osteoblast cells being measured ([Fig. 2]).[15]

Fig. 2 Visual documentation of C-PRF after centrifugation with an RCF at 2,000 g for 8 minutes in a fixed-angle centrifuge and a horizontal centrifuge. C-PRF, concentrated platelet-rich fibrin; RCF, relative centrifugal force.

Osteoblast Cell Proliferation

Culture media with a cell density of 2.5 × 10³ were placed in a 96-well plate and cultured at 37°C in an incubator with 5% CO₂. Observations were made after 1, 3, and 5 days of incubation. At each time point, 10 μL of Cell Counting Kit-8 solution (Abbkine, China) was added to each well. The plate was then incubated for an additional 2 hours at 37 °C in an incubator with 5% CO₂. Osteoblast cell counts were determined using a microplate reader set to 450 nm.[10]

Statistical Analysis

All experiments were performed in triplicate. The means and standard errors were calculated and the data obtained in this study were analyzed statistically using a two-way ANOVA (analysis of variance) and an LSD (least significant difference) post hoc test in IBM SPSS Statistics.

Results

Growth Factor Release

This study measured the concentration of released growth factors (TGF-β1 and PDGF-AB) using ELISA (enzyme-linked-immunosorbent assay) over a period of 14 days. According to the data shown in [Fig. 3], there was a statistically significant difference in the release of TGF-β1 and PDGF-AB between the horizontal C-PRF group and the fixed-angle C-PRF group. TGF-β1 release was similar in both groups on the first day of observation, then gradually increased on day 3, reaching a peak on day 7, with the horizontal group's concentration increasing 1.5 times more (4,408 pg/mL) than the fixed-angle group's (3035 pg/mL). After that, it gradually decreased on days 10 and 14.

Fig. 3 TGF-β1 concentration at each observation time point over 14 days in horizontal and fixed-angle C-PRF groups. C-PRF, concentrated platelet-rich fibrin.

The release of PDGF-AB in both groups followed a similar pattern to that of TGF-β1. Horizontal groups were statistically significant compared to fixed-angle groups at each observation time point, as shown in [Fig. 4].

Fig. 4 PDGF-AB concentration at each observation time points over 14 days in horizontal and fixed-angle C-PRF groups. C-PRF, concentrated platelet-rich fibrin; TGF-β1, transforming growth factor-β1.

Osteoblast Cell Migration

Following 24 hours of incubation and observation under a microscope, the horizontal C-PRF group exhibited the highest percentage of area coverage by osteoblasts (35.7%). [Fig. 5] shows that osteoblast migration activity was higher in the horizontal C-PRF group than in the control group (30.44%) and the fixed-angle C-PRF group (32.38%).

Fig. 5 Migration activity of osteoblast after 24 hours of incubation.

Osteoblast Cell Proliferation

Compared to the fixed-angle centrifugation group, the C-PRF horizontal centrifugation group indicates a higher osteoblast proliferation rate. As shown in [Fig. 6], on the third day of the observation period, the growth of the horizontal group (0.7193) was up to 1.5 times higher than that of the fixed-angle group (0.4597).

Fig. 6 Proliferation rate of osteoblast from the C-PRF horizontal group and fixed-angle group on each observation day. C-PRF, concentrated platelet-rich fibrin.

Discussion

The release pattern of the growth factors TGF-β1 and PDGF-AB exhibited by C-PRF in this study is consistent with previous findings, which showed cellular activity and growth factor release over a period of 10 days, after which it began to diminish.[16] Other studies reveal that, in both liquid and solid PRF, five types of growth factor (including TGF-β1) exhibit an increasing release pattern starting from the first hour after incubation, peaking on day 7, and then declining on day 10.[17] The production of TGF-β1 and PDGF-AB in both groups may be related to the physiological state of the dominant cell responsible for generating growth factors in C-PRF, specifically, platelets and leukocytes.

Growth factors, such as TGF-β1 and PDGF-AB, are produced by activated platelets and leukocytes. The number, distribution, and intensity of activated cells in PRF affect growth factor release. An increase in the number of platelets and leukocytes is proportional to growth factor release.[18] [19] The angulation of the tube during horizontal centrifugation prevents cells from being forced against the tube's distal wall, thereby limiting the possibility of them crashing into the tube and becoming damaged. Horizontal centrifugation decreases the likelihood of cell damage by separating the blood cells from the plasma in the middle of the tube rather than pushing them towards the wall of the tube. Horizontal centrifugation results in a more uniform distribution of cells in the upper layer than fixed-angle centrifugation, which concentrates cells at the distal wall.[9] Miron et al conducted a study analyzing cell blood counts to quantify cell numbers, and found that horizontal centrifugation significantly increased platelet and leukocyte numbers and concentrations compared to fixed-angle centrifugation.[6] Horizontal centrifugation resulted in a more uniform distribution of platelets and leukocytes across PRF matrices compared to fixed-angle centrifugation.[20]

The results of this study showed that the C-PRF from the horizontal centrifugation group had a higher concentration of both TGF-β1 and PDGF-AB than the fixed-angle centrifugation group at all observation times. These findings are consistent with those of previous studies. Investigations into the effect of rotor angle (fixed-angle or horizontal) on growth factor release have reported that horizontal centrifugation leads to a twofold increase in the release of PDGF, VEGF, and fibroblast growth factor 2 compared to fixed-angle centrifugation.[21] Another study by Gheno et al also exhibited a significant increase in PDGF and VEGF release from PRF produced by horizontal centrifugation, with a concentration twice that of PRF produced by fixed-angle centrifugation.[22] In contrast to these findings, Al-Maawi et al found no statistically significant difference between horizontal and fixed-angle centrifugation protocols in terms of the release of TGF-β1 and VEGF from liquid PRF matrices over 7 days of observation.[23]

C-PRF releases a higher concentration of growth factors, particularly PDGF-AB and TGF-β1, than previous generations of PRF.[6] PDGF-AB and TGF-β1 attract osteoblasts and enhance their migration to bone remodeling sites. This chemotaxis is followed by osteogenic proliferation and differentiation.[24] [25] Prior studies have examined the ability of the growth factors BMP-2, PDGF-AB, and TGF-β1 to induce migration in mesenchymal progenitor cells, obtaining data on the chemotactic index with no significant difference among BMP-2, PDGF, and TGF-β1. This study found that both PDGF and TGF-β increased primary osteoblast migration similarly to BMP-2.[26] The results of the observations showed a significant increase in osteoblast cell proliferation activity on days 1, 3, and 5. These findings align with those of a previous study which found that proliferation begins 24 hours after damage occurs, increasing until it reaches its maximum level on day 5.[27] The finding that osteoblast cell migration and proliferation peak after 5 days also correlates with the release of PDGF-AB and TGF-β1, which reach their maximum concentration after 3 to 7 days. Observation of osteoblast cell proliferation using fixed-angle and horizontal centrifugation on the first and third days revealed no significant difference. However, horizontal centrifugation showed a significant difference, indicating that C-PRF has a better capacity to increase proliferation in the horizontal group than in the fixed-angle group. This aligns with the 2020 findings of Fujioka-Kobayashi et al, who discovered that horizontal centrifugation substantially increases cell proliferation, migration, and differentiation activities, which are precursors to soft and hard tissue regeneration.[9] Additionally, a more recent study by Fujioka-Kobayashi et al showed that horizontal centrifugation significantly increased cell proliferation within 5 days compared to the control group.[16]

A systematic review of 13 studies comparing horizontal and fixed-angle centrifugation concluded that 84.6% of researchers favored horizontal centrifugation, 15.4% reported no difference, and none favored fixed-angle centrifugation.[28] This suggests that horizontal centrifugation may lead to enhanced clinical outcomes when C-PRF is applied in various regenerative procedures. The results of the present study also support this suggestion. However, it is important to note that the small sample size and controlled variability in this study may have influenced the statistical significance of the data. Another limitation of this study is that the observation period was considerably short, since long-term observation may have affected the results. Additionally, the number of studies currently available on the regenerative potential of C-PRF application in clinical trials is limited. Therefore, further development and subsequent research are important to ensure the clinical relevance of these promising findings.

Conclusion

Previous studies have shown evidence that the centrifugation method affects the regenerative potential of PRF, including growth factor release, cell migration, and proliferation. This in vitro study demonstrated that horizontal centrifugation is associated with higher TGF-β1 and PDGF-AB release, as well as enhanced osteoblast cell migration and proliferation, compared to fixed-angle centrifugation. Further in vivo and clinical studies with larger sample sizes, greater variability, longer observation periods, and more comprehensive experiments on regenerative potential are required to validate the significance of these findings.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgement

We extend our gratitude to the Faculty of Dentistry, Universitas Gadjah Mada for partially funding this work through the Program DAMAS 2024 No. 3905/UN1/KG/Set.KG1/LT/2024.

Informed Consent

For this study, informed written consent was provided to conduct the outlined experiments prior to the blood collection from volunteers.

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Publication History

Article published online:
07 November 2025