This study examines circadian variations in growth factor release from platelet-rich fibrin (PRF) and explores correlations with cortisol levels, with the goal of optimizing the timing of PRF-based regenerative procedures.
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Research Article
Evaluation of Circadian Variation in Growth Factors of Platelet-Rich Fibrin: A Pilot Study
In This Article
Summary
Abstract
Platelet-rich fibrin (PRF) has extensive clinical applications in dentistry, particularly in the regeneration of soft and hard tissues, extraction socket healing, treatment of periodontal defects, sinus augmentation, and mucogingival procedures. PRF is a platelet-derived, growth factor-rich biomaterial prepared via a single-step centrifugation protocol. This study aimed to evaluate whether PRF-derived growth factors exhibit circadian variation between morning and evening hours. Twenty healthy volunteers participated, each providing six venous blood samples, three at 08:00 and three at 20:00. From each time point, one tube was used for complete blood count, one for serum cortisol analysis, and one for PRF preparation (centrifuged at 708 x gfor 12 min). Growth factors (IGF-1, PDGF, FGFR, VEGF, TGFβ-1) in PRF were measured at 24 h and 72 h using ELISA. Their levels were compared between time points and correlated with serum cortisol levels. FGFR levels were significantly higher at 72 h than at 24 h in evening samples. However, no significant differences were found for IGF-1, PDGF, VEGF, or TGFβ-1 between morning and evening. Cortisol levels were negatively correlated with PDGF at 72 h in morning samples and with IGF-1 at 72 h in evening samples; however, no other significant correlations were observed. These findings suggest that the timing of blood collection does not significantly influence PRF growth factor content, indicating flexibility in clinical protocols regarding sample timing.
Introduction
Platelet-rich fibrin (PRF) is a second-generation platelet concentrate commonly used in regenerative dentistry due to its autologous origin, ease of preparation, and biologically active content1. Formed by centrifugation of whole blood without anticoagulants, PRF provides a fibrin matrix rich in platelets and leukocytes that secrete growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta 1 (TGF-β1), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), and fibroblast growth factor receptor (FGFr). These biomolecules play central roles in cellular proliferation, angiogenesis, extracellular matrix remodeling, and tissue regeneration2. Compared with platelet-rich plasma (PRP), which requires anticoagulants and exogenous activation, PRF is produced via a single-step, anticoagulant-free process that yields a physiologic fibrin scaffold with entrapped leukocytes and platelets. This architecture supports a slower, more sustained release of growth factors and cytokines, potentially enhancing wound stability and early tissue repair while reducing handling complexity and cost1,2. Furthermore, the absence of anticoagulants and activators minimizes manipulation-related variability and may better preserve native platelet behavior, offering practical advantages for chairside use and standardization across operators2.
Despite its widespread clinical use, the temporal dynamics of PRF's bioactive components remain poorly understood. Circadian rhythm, an intrinsic 24-h biological cycle, influences various physiological systems, including immune function, hormone secretion, and hematological parameters3,4. Cortisol, the primary circadian hormone, peaks in the early morning and gradually declines throughout the day5. Importantly, studies have shown that platelet activity, aggregation capacity, and count may also be modulated by circadian mechanisms, with implications for coagulation and wound healing6.
Since PRF is derived directly from peripheral blood, the circadian timing of blood collection could potentially affect platelet activation status and the subsequent release of growth factors during fibrin polymerization. Although the diurnal variations in platelet counts and growth factors in platelet-rich plasma were previously analyzed by Aoto et al.7, there are no studies systematically examining whether PRF prepared at different times of the day exhibits measurable differences in its growth factor composition. Therefore, this pilot study aims to investigate the circadian variation of PRF-derived growth factors by comparing samples collected in the morning and evening from healthy individuals. In addition, we evaluated serum cortisol levels at each time point and explored their correlations with PRF growth factor release. Understanding such temporal variability may help optimize clinical protocols and contribute to the standardization of PRF-based regenerative treatments. The null hypothesis of this study was that circadian timing of blood collection does not significantly influence the concentration of growth factors released from platelet-rich fibrin (PRF); however, based on the known circadian regulation of platelet activity and hormone secretion, it was initially hypothesized that the time of blood collection (morning vs. evening) may alter PRF growth factor content. From a clinical perspective, clarifying whether PRF growth factor content is sensitive to circadian variation is important for the standardization of regenerative protocols. If growth factor release remains largely stable across the day, clinicians can be reassured that the timing of venipuncture may not substantially affect treatment outcomes, thus allowing flexibility in scheduling procedures. Conversely, if subtle differences emerge in select factors, such as those involved in angiogenesis or tissue repair, the timing of blood collection could be optimized in cases where maximal regenerative potential is desired, for example, in compromised healing conditions.
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Protocol
This study was conducted at the Department of Periodontology, Faculty of Dentistry, Çukurova University, Adana, Turkey, with the participation of 20 healthy volunteers. Prior to inclusion, all participants were informed about the aim, methodology, and procedures of the study, and written informed consent was obtained. Ethical approval was granted by the Çukurova University Faculty of Medicine Clinical Research Ethics Committee (Approval No: 125/55, Date: 16.09.2022). All procedures, including essential biosafety guidance for handling human blood and waste-disposal instructions for biohazardous materials and chemical reagents, were performed in accordance with NIH-CDC guidelines8. The reagents and the equipment used are listed in the Table of Materials.
1. Participant selection
Participants included in the study were healthy individuals between 18 and 25 years of age, without any known systemic disease, and who demonstrated adequate cooperation during the procedures. Individuals were excluded if they had any systemic condition, were pregnant or breastfeeding, or were using any medication or hormonal agents that could affect hematological or hormonal parameters.
2. Blood collection and group allocation
A total of six 10 mL venous blood samples were collected from each participant, three in the morning (08:00) and three in the evening (20:00). Sterile PRF tubes of additive-free glass (10 mL) were used in the study. In the morning session, Tube 1 was used for complete blood count (CBC), Tube 2 for serum cortisol analysis, and Tube 3 for PRF preparation followed by growth factor analysis. In the evening session, Tube 4 was used for CBC, Tube 5 for serum cortisol analysis, and Tube 6 for PRF preparation and growth factor analysis. Based on the time of blood collection, samples were allocated into two groups: Group 1 consisted of pure PRF prepared from morning blood (08:00), and Group 2 consisted of pure PRF prepared from evening blood (20:00).
3. PRF preparation
Blood samples were centrifuged at 708 x g for 12 min at room temperature using a centrifuge equipped with a 40° fixed-angle rotor and 10 mL glass tubes. The device used had a rotor radius adjustable between 88 mm and 110 mm; in this study, a radius of 88 mm was selected to standardize the protocol. No anticoagulants were used during the procedure. Following centrifugation, the resulting PRF were macroscopically evaluated to ensure proper formation, which is characterized by a smooth and homogeneous surface, an elastic and cylindrical shape, and a compact, firm consistency.
4. Growth factor ELISA analysis
PRF clots were carefully separated from the red blood cell layer using sterile scissors. Each clot was weighed, and RPMI-1640 culture medium was added in a 1:1 ratio based on weight (g = mL) in order to standardize the incubation environment across all samples and ensure consistent diffusion of soluble growth factors from the PRF matrix. This ratio allows for proportional extraction based on clot size, minimizing variability in growth factor concentrations due to unequal clot masses. Samples were incubated in an orbital shaker at 100 rpm and 3 °C for 24 h and 72 h to promote uniform diffusion of growth factors from the PRF matrix into the culture medium. Following incubation, the supernatants were collected and stored at −8 °C until analysis. Growth factor concentrations were quantified using commercial ELISA kits, including FGFR, VEGF, IGF-1, PDGF, and TGF-β1, following the manufacturer's preparation protocol9,10.
5. ELISA steps
Standards and samples (100 µL each) were added to the wells and incubated at 3 °C for 90 min. Wells were then washed three times with wash buffer. Next, 100 µL of biotin-labeled antibody was added to each well and incubated at 3 °C for 60 min, followed by three additional washes. Then, 100 µL of horseradish peroxidase (HRP) solution was added and incubated at 3 °C for 30 min. After another three washes, 100 µL of substrate reagent was added and incubated for 15 min. Finally, 50 µL of stop solution was added to each well, and absorbance was measured at 450 nm.
6. Statistical analysis
Categorical variables were expressed as frequencies and percentages, while numerical data were presented as mean ± standard deviation or median (minimum-maximum), as appropriate. Normality of numerical variables was assessed using the Shapiro-Wilk test. Variables that did not meet the assumption of normality, specifically the growth factor concentrations and cortisol levels, were analyzed using non-parametric tests, including the Wilcoxon signed-rank test for paired comparisons and Spearman's rank correlation coefficient for associations. All statistical analyses were performed using IBM SPSS Statistics software. A p-value <0.05 was considered statistically significant. All comparisons were conducted within-subject to control for inter-individual variability.
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Results
All 20 volunteer participants (10 female and 10 male, aged between 18 and 25) completed the study without any sample loss. Complete blood count results confirmed that platelet and hematological parameters were within normal reference ranges in all samples, validating the reliability of the PRF preparations.
Comparison of blood parameters between morning and evening
Cortisol levels were significantly higher in morning samples compared to evening (p < 0.001), reflecting t...
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Discussion
Based on the known circadian regulation of platelet activity and hormone secretion, it was initially hypothesized that the time of blood collection (morning vs. evening) may alter PRF growth factor content. This study aimed to test whether any such temporal variation exists, and if so, whether it correlates with serum cortisol levels and hematological parameters. The study was designed as a pilot investigation to explore circadian variation in PRF-derived growth factor release. Therefore, a formal sample size ca...
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Disclosures
The authors have nothing to disclose.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| CENTRIFUGE MACHINE | DUO | 913023410238 | |
| ELISA KITS | Fine Test, Wuhan FineBiotech Co., Ltd. | IGF-1 (Catalog no:EH0165) | |
| ELISA KITS | Fine Test, Wuhan FineBiotech Co., Ltd. | PDGF (Catalog no:EH3531) | |
| ELISA KITS | Fine Test, Wuhan FineBiotech Co., Ltd. | FGFR (Catalog no:EH0715) | |
| ELISA KITS | Fine Test, Wuhan FineBiotech Co., Ltd. | VEGF (Catalog no:EH0327) | |
| ELISA KITS | Fine Test, Wuhan FineBiotech Co., Ltd. | TGF-β1(Catalog no:EH0287) | |
| PRF Tubes |  | 6370 |
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