Enhancing Platelet-Rich Fibrin: Influence of Hyaluronic Acid and Collagen on Growth Factor Release and Biomechanical Properties in Vitro

Summary

This study evaluates how hyaluronic acid and collagen affect the release of growth factors and the mechanical properties of platelet-rich fibrin.

Abstract

Platelet-Rich Fibrin (PRF) is an autologous matrix rich in platelets, leukocytes, and growth factors that support tissue regeneration. Enhancing its structural and biological properties through biomaterial supplementation may improve clinical outcomes. This study evaluated the effects of adding hyaluronic acid (HA) and collagen to PRF on growth factor release and mechanical strength. In the current study, venous blood samples were collected from 20 healthy volunteers (seven tubes per participant). The tubes were used for complete blood count analysis, obtaining pure PRF and supplemented PRF with HA (HA-PRF) and collagen (COL-PRF), mechanical testing, and scanning electron microscopy (SEM) analysis of pure PRF, HA-PRF, and COL-PRF, respectively. All samples were centrifuged at 708 x g for 12 min. Growth factor levels were measured at 24 h and 72 h via ELISA. PRF membranes were also subjected to tensile and elongation testing using a testometric device. At 72 h, platelet-derived growth factor (PDGF) levels were significantly higher in HA-PRF and COL-PRF than in controls. Other growth factors showed no significant differences. Mechanical testing revealed greater elongation and tensile strength in the COL-PRF group compared to both HA-PRF and control. These findings suggest that HA and collagen enhance PRF's mechanical performance. In particular, collagen improves structural durability, potentially extending PRF's resorption time. This supports its application as a membrane substitute in bone augmentation procedures.

Introduction

Extensive research has been conducted on natural and synthetic biomaterials to enhance soft and hard tissue wound healing. Autologous biomaterials naturally present in the body significantly contribute to repair, regeneration, and healing by providing essential biochemical signals¹. Synthetic alloplastic materials have also demonstrated promising outcomes and extensive use in regenerative dentistry. However, these materials possess significant disadvantages, such as avascularity and the potential to induce foreign body reactions². Increasing the concentration of autologous biomaterials at the wound site has emerged as a common strategy to accelerate the healing process and overcome these limitations³.

Platelets and fibrin are crucial autologous biomaterials supporting wound healing and tissue regeneration⁴. Recent advancements in platelet concentrates, such as fibrin glue, platelet-rich plasma (PRP), platelet-rich fibrin (PRF), advanced platelet-rich fibrin (A-PRF), titanium-prepared platelet-rich fibrin (T-PRF), injectable platelet-rich fibrin (I-PRF), and concentrated growth factor (CGF), represent significant progress in regenerative dentistry^5,6,7. These developments aim to provide more effective and reliable therapeutic options for tissue healing and regeneration^8,9.

PRF is obtained through centrifugation of blood, yielding a completely natural biomaterial that provides the three critical components required for tissue engineering: cells, growth factors, and scaffolding¹⁰. PRF comprises platelets and leukocytes. Adjustments in centrifugation speed and duration can increase macrophage and leukocyte counts, which are essential for host defense and wound healing¹¹. These cells release numerous growth factors, including transforming growth factor-beta 1 (TGF-β1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor-I (IGF-I), promoting cell migration, proliferation, and differentiation^12,13.

Since PRF preparation does not involve anticoagulants, it naturally creates a three-dimensional fibrin scaffold meeting the primary criteria for tissue engineering¹⁴. Over time, fibrin not only retains various cell types but also allows the gradual release of growth factors. This controlled release profile significantly enhances angiogenesis, cellular behavior, and ultimately tissue regeneration^7,13.

PRF has been combined with various materials such as tannic acid,bioactive glass,metals,polymers, composites, and 1% HA Gel to enhance its biological and mechanical properties^15,16,17. The selection of materials to be combined with PRF should be guided by the specific requirements of the intended tissue regeneration, including not only mechanical properties but also critical factors such as biocompatibility, porosity, and overall biological performance¹⁶. While numerous studies have demonstrated the positive impact of autologous growth factors present in PRF on wound healing, the effects of the incorporation of hyaluronic acid (HA) and collagen into PRF on growth factors remain unexplored. Therefore, the present study aims to evaluate the influence of hyaluronic acid and collagen on the growth factor content and mechanical properties of PRF. To ensure consistency and reproducibility, HA and collagen volumes were standardized at 0.5 mL per 10 mL blood sample, based on preliminary testing. This volume was selected as optimal for stable clot formation across HA and collagen conditions. Based on previous research, the centrifuge protocol was 708 x g for 12 min¹⁸.

Protocol

This research was conducted at the Department of Periodontology, Faculty of Dentistry, Cukurova University, with the participation of 20 healthy volunteers (10 male and 10 female). Written informed consent was obtained from all individuals after providing detailed information about the purpose and methods of the study. Ethical approval was obtained from the Çukurova University Medical Faculty Ethics Committee (approval number: 89/130; date: February 4, 2023). The reagents and the equipment used are listed in the Table of Materials.

A preliminary test using blood samples from two volunteers was conducted to determine the optimal dose of HA and collagen for PRF preparation. In this test, volumes of 1.0 mL, 0.5 mL, and 0.25 mL for both biomaterials were added to blood tubes prior to centrifugation. The resulting PRF clots were macroscopically evaluated for clot formation to obtain a smooth and homogeneous surface, an elastic and cylindrical shape, and a compact and firm consistency. 1.0 mL volume showed no clot formation after centrifugation, while the addition of both 0.25 mL and 0.5 mL resulted in macroscopically adequate PRF formation. Based on these observations, 0.5 mL was selected as the maximum volume for both HA and collagen, as it consistently yielded stable and homogeneous PRF clots. These preliminary samples were not included in the main experimental groups or subjected to statistical analysis.

In this study, a commercially available cross-linked hyaluronic acid (HA) gel was used. According to the manufacturer, this product has a concentration of 24 mg/mL and a molecular weight of approximately 3 million Daltons. The collagen used was a type I collagen derived from equine tendon. Both products were supplied in sterile syringes and were used without any dilution or modification. A volume of 0.5 mL of HA or collagen was aseptically added directly into 10 mL of freshly collected venous blood prior to centrifugation. The flow-chart of the protocol is described in Figure 1.

1. Participant selection

The inclusion criteria were: (1) Participants who were systemically healthy and cooperative; (2) Individuals with adequate cooperation.

The exclusion criteria were: (1) Individuals with systemic diseases; (2) Pregnant or breastfeeding individuals; (3) Individuals using medications or with conditions that cause bleeding disorders; (4) Smokers.

2. Blood collection and group allocation

Seven 10 mL tubes of venous blood were collected from each participant.

The tubes were used for the following purposes: Tube 1: A complete blood count was performed (CBC); Tube 2: Pure PRF was prepared for ELISA; Tube 3: 0.5 mL of hyaluronic acid was added before centrifugation (HA-PRF); Tube 4: 0.5 mL of collagen was added before centrifugation (COL-PRF); Tube 5: Pure PRF was prepared for mechanical testing and SEM; Tube 6: HA-PRF was prepared for mechanical testing and SEM; Tube 7: COL-PRF was prepared for mechanical testing and SEM.

The study groups were defined as follows: Group 1: Pure PRF (control); Group 2: PRF with 0.5 mL hyaluronic acid (HA-PRF); Group 3: PRF with 0.5 mL collagen (COL-PRF).

3. PRF preparation

The blood samples were centrifuged at a relative centrifugal force of 708 x g for 12 min at room temperature using a centrifuge with a 40° fixed-angle rotor and 10 mL glass tubes. It was ensured that the rotor radius was between 88 mm and 110 mm. No anticoagulants or other dilution factors were used. The macroscopic clot assessment was performed immediately after centrifugation. A successful clot was defined as one presenting a smooth and homogeneous surface, an elastic and cylindrical shape, and a compact and firm consistency. Clots exhibiting granular accumulation or phase separation were classified as inadequate.

4. Growth factor ELISA analysis

PRF clots were separated from the red blood cell layer using sterile forceps.Each clot was weighed, and RPMI-1640 culture medium was added in a 1:1 ratio based on weight (g = mL). The samples were incubated at 37 °C in an orbital shaker for 24 h and 72 h. The supernatants were collected and stored at −80 °C until analysis. IGF-1, PDGF, FGF, VEGF, and TGF-β1 levels were quantified using commercial ELISA kits according to the manufacturer's instructions.

4.1 ELISA procedure

100 µL of standards and samples were added to the wells and incubated at 37 °C for 90 min. The wells were washed three times with wash buffer. 100 µL of biotin-labeled antibody was added and incubated at 37 °C for 60 min. The wells were washed three times. 100 µL of HRP was added and incubated at 37 °C for 30 min. The wells were washed three times. 100 µL of substrate reagent was added and incubated for 15 min. 50 µL of stop solution was added, and the absorbance was measured at 450 nm.

5. Scanning Electron Microscopy (SEM)

The samples were freeze-dried at <0.1 mPa for 8 h. The samples were coated with 15 nm gold-palladium. The samples were imaged using a field emission scanning electron microscope at 10 kV and 24,000x magnification.

6. Mechanical testing

The PRF membranes were fixed without preload and tested at a speed of 1 mm/min until rupture. The maximum load (F) and cross-sectional area (A) were recorded. The tensile strength was calculated using the formula S = F/A. The elastic modulus and toughness were evaluated using the stress-strain curve.

7. Statistical analysis

Numerical data were expressed as mean ± standard deviation and median (min-max). The normality of the data was tested using the Shapiro-Wilk test. Intra-group comparisons were performed using the paired t-test or the Wilcoxon signed-rank test. Inter-group comparisons were conducted using repeated-measures ANOVA or the Friedman test. Bonferroni-adjusted post hoc tests were performed when necessary. Time-dependent changes were analyzed using nested ANOVA. All analyses were conducted using statistical software. A p-value of <0.05 was considered statistically significant.

Results

All 20 volunteer participants completed the study without any sample loss. Platelet and hematological parameters were within the normal reference range in all complete blood count results.

Scanning Electron Microscopy (SEM)

SEM images at 6,000× and 24,000× magnification revealed distinct fibrin morphologies among the groups. The control PRF exhibited a...

Discussion

This study investigated the effects of incorporating HA and collagen into platelet-rich fibrin on its biological and mechanical properties. The study proposed that enriching PRF with these biomaterials prior to centrifugation may enhance its regenerative potential by modulating growth factor release and improving structural integrity. The findings support this hypothesis, particularly for collagen, which consistently improved mechanical performance and altered fibrin network architecture.

The ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CENTRIFUGE MACHINE	DUO	913023410238
ELISA KITS	REED BIOTECH LTD
INJECTABLE COLLAGEN	CSD Sri	1840
INJECTABLE HYALURONIC ACID	Forest Hills Lab Korea Co.Ltd
MECHANICAL TEST	The Testometric Company Limited
PRF Tubes	Hema&Lab.Saglik Ürünleri Imalat Paz.San.ve Tic.Ltd.St Ankara/TÜRKIYE	6370
SEM	FEI