Enrichment of Platelet-Rich Fibrin Using Cobalamin and Alpha-Tocopherol to Modulate Growth Factor Release

Summary

This protocol describes an approach for biologically fine-tuning platelet-rich fibrin through enrichment with cobalamin and alpha-tocopherol prior to centrifugation, enabling controlled modulation of growth factor and cytokine release under in vitro conditions.

Abstract

Platelet-rich fibrin (PRF) is an autologous biomaterial widely used in regenerative dentistry due to its biocompatibility and sustained release of growth factors. Recent research has shifted toward biologically modulating PRF to enhance its regenerative performance without altering its fundamental preparation principles. This study presents a protocol for biologically fine-tuning PRF through enrichment with cobalamin and alpha-tocopherol prior to centrifugation and evaluates their effects on growth factor and proinflammatory cytokine release under in vitro conditions. Peripheral blood samples obtained from healthy volunteers were processed using a fixed centrifugation protocol. Cobalamin or alpha-tocopherol was added to whole blood before centrifugation to obtain bioactively modified PRF matrices. The release profiles of platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were quantified at 24 h and 72 h using an enzyme-linked immunosorbent assay. Cobalamin-enriched PRF was associated with significant intergroup differences in TGF-β1 levels and reduced release of proinflammatory cytokines, indicating a shift toward a more favorable regenerative microenvironment. In contrast, increases observed in FGF-2 and IGF-1 were time-dependent and detected across all groups, without demonstrating a cobalamin-specific effect. Alpha-tocopherol-enriched PRF exhibited a selective increase in VEGF release, suggesting a potential proangiogenic influence. These findings demonstrate that targeted vitamin enrichment enables controlled modulation of PRF bioactivity while preserving its autologous and anticoagulant-free nature. This protocol provides a practical framework for biologically fine-tuning PRF and supports its use as a tunable regenerative platform for future translational and clinical applications.

Introduction

Platelet-rich fibrin (PRF) is a second-generation autologous platelet concentrate that has been widely adopted in periodontology, implant dentistry, and oral surgery because of its biocompatibility, ease of preparation, and capacity for sustained release of growth factors^1,2. PRF is obtained by centrifugation of whole blood without the use of anticoagulants or exogenous activators, resulting in a three-dimensional fibrin matrix that entraps platelets and leukocytes. These cellular components serve as endogenous reservoirs of growth factors, including platelet-derived growth factor (PDGF), transforming growth f....

Protocol

This experimental study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and received approval from the Clinical Research Ethics Committee of Çukurova University (Approval No: 139/25; Date: 8^th December, 2023). Prior to participation, all individuals were informed about the study procedures, and written informed consent was obtained from each participant. All procedures involving human blood samples were performed in compliance with institutional biosafety regulations. Appropriate personal protective equipment (PPE), including gloves, masks, and laboratory coats, was used throughout the experimental process. Bi....

Results

Successful preparation of control, cobalamin-enriched, and alpha-tocopherol-enriched platelet-rich fibrin (PRF) was achieved in all participants. Macroscopic examination confirmed the formation of well-structured PRF clots with preserved fibrin integrity and sufficient mechanical stability, allowing standardized handling, incubation, and subsequent biochemical analyses (Table 1).

Discussion

The present study demonstrates that platelet-rich fibrin (PRF) can be biologically fine-tuned through targeted enrichment with cobalamin and alpha-tocopherol prior to centrifugation, resulting in distinct and reproducible modulation of growth factor and cytokine release profiles. These findings reinforce the evolving concept that PRF is not merely a passive autologous scaffold but a biologically responsive matrix whose regenerative behavior can be adjusted through carefully selected bioactive interventions without alteri.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alpha-Tocopherol (Vitamin E)	AKSU FARMA, Istanbul, Turkey	A11HA03	Ampule EVIGEN 2 mL
Centrifuge (40° fixed-angle rotor)	Process for PRF, Nice, France	913023410238	Duo Centrifuge
Cobalamin (Vitamin B12)	DEVA HOLDING, Istanbul, Turkey	B03BA01	Ampule DODEX 1000 mcg/mL
Eppendorf Tubes	Axygen	PCR-02-C	200 piece
Glass Blood Collection Tubes	Hema & Tube (Istanbul, Turkey)	HNM8080	(10 mL) No anticoagulant
HRP Solution	Included in ELISA kit
Human ELISA Kit - FGF	Wuhan Fine Biotech	EH0541	Fine Test
Human ELISA Kit - IGF-1	Wuhan Fine Biotech	ER0030	Fine Test
Human ELISA Kit - PDGF	Wuhan Fine Biotech	ER1240	Fine Test
Human ELISA Kit - TGFβ-1	Wuhan Fine Biotech	EH0287	Fine Test
Human ELISA Kit - VEGF	Wuhan Fine Biotech	EH0327	Fine Test
Human IL-1β ELISA Kit	Wuhan Fine Biotech	EH0185	Fine Test
Human TNF-α ELISA Kit	Wuhan Fine Biotech	EH0302	Fine Test
Micropipettes & Tips	Eppendorf	3123000063	1 Set
Microplate Reader	Biochrome	EZ Read 400	450 nm
Orbital Shaker	Stuart	SSM3	Mini Gyro-Rocke
Precision Scale	Sartorius	H51-D	1 piece
RPMI-1640 Cell Culture Medium	HyClone, Cytiva, USA	SH30027.01	500 mL
Sterile Scissors	ASA Dental (Istanbul, Turkey)	0305-1	1 piece
Stop Solution	Included in ELISA kit
Substrate Solution	Included in ELISA kit
Wash Buffer	Included in ELISA kit