Effect of Smoking on Growth Factors in Platelet-Rich Fibrin: Ex-Vivo Pilot Study

Summary

This protocol evaluates the effect of smoking on platelet-rich fibrin (PRF) by comparing growth factor release and blood parameters between smokers and non-smokers using a standardized and reproducible method.

Abstract

Platelet rich fibrin (PRF) and growth factors are routinely used to accelerate hard and soft tissue regeneration in periodontal surgical procedures, dental implant applications and oral surgery for the last 30 years in the field of dentistry. The aim of this study was to evaluate the effect of smoking on autologous growth factors within PRF. The study population consisted of 20 male participants (10 smokers/10 non-smokers) and 20 female participants (10 smokers/10 non-smokers). Each participant provided two tubes of venous blood (approximately 7 ml each). The first tube was used for a complete blood count (CBC). The second tube was centrifuged at 708 x g for 12 min, and the resulting PRF was analyzed using enzyme-linked immunosorbent assay (ELISA) to determine the levels of growth factors (Platelet-derived growth factor (PDGF), Transforming growth factor-beta (TGF-β), Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF) and Insulin-like growth factor (IGF)). The results showed that smoking resulted in a decrease in mean platelet volume (MPV), platelet distribution width (PDW) and platelet larger cell ratio (P-LCR) values (p<0.05) in the whole population. In females, smoking led to a decrease in red blood cell (RBC), MPV, PDW and P-LCR levels, while in male subjects, it resulted in an increase in hematocrit (HCT) values (p<0.05). It was also found that smoking led to an increase in FGF and TGFβ-1 within PRF (p<0.05). Although current results showed a slight increase in certain growth factors in smokers, the clinical relevance of this finding should be investigated in further studies.

Introduction

The utilization of autologous platelet concentrates in various fields of dentistry began in the 1980s and 1990s. Since then, platelet concentrates and growth factors have been routinely employed in periodontal surgical procedures, dental implant applications, and oral surgery to accelerate the regeneration of hard and soft tissues¹. Platelets containing growth factors play crucial roles in cell migration, proliferation, differentiation, and angiogenesis, all of which are integral to tissue regeneration. Autologous platelet concentrates are produced by centrifuging venous blood at different speeds, with or without the use of thrombin and anticoagulants². The growth factors present in platelet-rich fibrin (PRF) include platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF), among others^3,4. These growth factors are known to influence various cellular processes essential for tissue healing and regeneration⁵. Factors affecting the levels and efficacy of these growth factors in PRF include the patient's age and sex, PRF preparation method, platelet count in the collected blood, platelet distribution width (PDW), and mean platelet volume (MPV)as well as patient’s general health status, the presence of systemic conditions, and external influences such as smoking⁶.

Smoking poses a significant health hazard, particularly impacting coagulation physiology and directly affecting platelet function⁷. Cigarette smoke contains numerous toxic compounds, including nicotine, carbon monoxide, and reactive oxygen species, which are known to affect platelet behavior and vascular biology^8,9 resulting in altered degranulation and release of growth factors^8,10. Therefore, smoking may potentially influence the biological composition and functionality of platelet-rich fibrin (PRF) through multiple interconnected mechanisms such as disrupting cellular homeostasis and modulating cytokine production^9,11 changing fibrin clot formation and architecture, including alterations in fibrin fiber thickness, density, and cross-linking^12,13, modulating inflammatory responses and endothelial dysfunction which can further impact leukocyte activity and the regenerative microenvironment^9,14. All of these factors may potentially affect the quality and quantities of bioactive molecules incorporated into the PRF matrix and influence the mechanical stability of PRF and the kinetics of growth factor release^9,11.

The impact of smoking on the biological properties of platelet concentrates, including platelet-rich fibrin (PRF) has been investigated in recent studies. Comparative studies evaluating smokers and non-smokers have reported differences in growth factor release, fibrin architecture, and cellular content of PRF^10,11. In particular, smoking has been associated with alterations in key growth factors such as transforming growth factor beta-1 (TGF-β1), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), as well as with structural changes in the fibrin network^15,16. While Lara et al. (2023) reported that leukocyte- and platelet-rich fibrin (L-PRF) obtained from smokers and non-smokers exhibited similar fibrin architecture, cellular distribution, and uniaxial tensile properties, suggesting that smoking may not significantly affect the biomechanical behavior of PRF in vitro¹⁷. Srirangarajan et al. reported that smoking may influence the membrane properties of platelet-rich fibrin (PRF) and advanced platelet-rich fibrin (A-PRF), including their structural and morphological characteristics¹⁸.

Although previous studies have investigated the effects of smoking on platelet concentrates and PRF, the available evidence remains limited and inconsistent, and the underlying biological and molecular mechanisms have not been fully clarified¹⁷. These discrepancies may be attributed to variations in study design, differences in age and sex distribution across study populations, inconsistencies in PRF preparation protocols, and patient-related factors such as smoking intensity and duration^11,19. In addition, most existing studies have focused primarily on general platelet behavior, fibrin architecture, membrane morphology, or broad biological characteristics of PRF, whereas quantitative data on specific PRF-derived growth factors remain scarce, inconsistent, and frequently restricted to single-time-point analyses. As a result, it is still unclear whether smoking alters the quantitative early release profile of PRF-derived growth factors under standardized conditions.

Furthermore, the time-dependent release pattern of these growth factors in smokers versus non-smokers, as well as the potential dose-related association with smoking intensity, has not been clearly established. Therefore, standardized and reproducible studies are needed to evaluate both the quantity and temporal release kinetics of PRF-derived growth factors in relation to smoking status^20,21. Against this background, the rationale, aim, and hypothesis of the present study were formulated as follows. The positive effects of autologous growth factors in PRF on wound healing have been demonstrated in numerous studies, especially during surgical procedures in dentistry, although contradictory results also exist, probably related to individual differences in PRF characteristics. The aim of this study was to investigate the effect of smoking on the biological activity of platelet-rich fibrin by quantitatively evaluating growth factor levels at different time points and assessing their association with smoking intensity. The null hypothesis of the current study was that smoking does not alter the release kinetics and levels of growth factors in platelet-rich fibrin. It also states that smoking intensity is not associated with measurable differences in these growth factor levels between smokers and non-smokers.

Protocol

This study was conducted at the Department of Periodontology, Faculty of Dentistry, Cukurova University, with the participation of 20 male and 20 female volunteers. All participants provided written informed consent after being informed about the purpose, methodology, and procedures of the study. Ethical approval was obtained from the Ethics Committee of the Faculty of Medicine, Cukurova University (Approval No: 125/54, 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 guidelines²². The reagents and the equipment used are listed in the Table of Materials.

1. Patient Selection

Participants included in the study were healthy individuals between 18 and 30 years of age, without any known systemic diseases, and who demonstrated adequate cooperation during the procedures. Individuals were excluded if they had any systemic disease, were pregnant or breastfeeding, had any bleeding disorders, or were using systemic medications that could affect hemostatic parameters. An equal number of male and female participants were included in the study to ensure balanced sex distribution between the groups. Smoking status was determined based on self-reported information obtained from the participants. Individuals who reported current daily cigarette smoking were classified as smokers, whereas individuals who reported no current smoking history were classified as non-smokers. Former smokers and occasional smokers were excluded to maintain clear group allocation. In addition, the number of cigarettes smoked per day was recorded for each smoker, and correlations between smoking intensity and the study outcome parameters were analyzed. No biochemical validation (e.g., cotinine measurement) was performed.

The sample size was determined based on feasibility considerations and the number of eligible subjects available during the study period. The present study was conceived as a preliminary exploratory clinical investigation.

2. Blood collection and group allocation

A total of four study groups were established: non-smoking female participants (n = 10), non-smoking male participants (n = 10), smoking female participants (n = 10), and smoking male participants (n = 10 Blood samples were collected from 20 healthy men (10 smokers and 10 non-smokers) and 20 healthy women (10 smokers and 10 non-smokers). Two blood collection tubes (approximately 7 mL each) were obtained from each participant. The first tube (sterile glass tube with ethylenediaminetetraacetic acid as an anticoagulant) was used for complete blood count analysis, including white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), platelet (PLT), mean platelet volume (MPV), platelet distribution width (PDW), platelet large cell ratio (P-LCR), and platelet crit (PCT) values. The second tube (sterile glass tube with no anticoagulant) was centrifuged for PRF preparation, and the resulting PRF was analyzed for growth factor levels using ELISA. The weight of the obtained PRF was measured and recorded.

3. PRF preparation

All PRF samples were prepared under strictly standardized conditions to reduce inter-sample variability. Venous blood was drawn from each participant using the same procedure and centrifuged immediately under identical settings. Centrifugation was performed at 708 × g for 12 min at room temperature using a centrifuge equipped with a 40° fixed-angle rotor and 10 mL glass tubes. Although the rotor radius of the device could be adjusted between 88 mm and 110 mm, a radius of 88 mm was consistently used in the present study to standardize the protocol. No anticoagulants were added at any stage of the procedure. Macroscopic evaluation of PRF formation was carried out immediately after centrifugation. Proper PRF formation was defined as the presence of a clearly identifiable fibrin clot located between the red blood cell layer and the plasma layer. In addition, the clot was required to exhibit a consistent gel-like structure with sufficient mechanical integrity to allow separation with sterile forceps. Only samples demonstrating a well-formed and clearly separated PRF clot were included in the study. Samples showing incomplete clot formation or poor layer separation were excluded from analysis, and when necessary, additional blood samples were collected from the participants during the same session.

4. Growth factor ELISA analysis

PRF samples were processed for growth factor analysis under strictly standardized experimental conditions to ensure comparability among participants. Following preparation, each PRF clot obtained from the 40 volunteers was separated from the red blood cell layer using sterile scissors and weighed on a precision balance. Cell culture medium was then added to each clot in proportion to its weight. After medium addition, the samples were incubated for 72 h on an orbital shaker operating at a slow speed. At the predefined evaluation time points of 24 h and 72 h, the media surrounding the PRF clots, representing PRF-derived exudates/supernatants, were collected and transferred into separate microcentrifuge tubes for subsequent ELISA analysis. The collected samples were stored at -80 °C until use. To minimize protein degradation and technical variation, all specimens were handled according to the same protocol, analyzed within the same experimental period, and thawed only once prior to measurement. Samples were kept on ice during relevant processing steps whenever applicable.

Quantitative analysis of IGF-1, PDGF, FGF, VEGF, and TGF-β1 levels was performed using commercially available ELISA kits in accordance with the manufacturer’s instructions. All measurements were carried out in duplicate, and the mean values were used for statistical analysis. Standard curves were prepared for each assay, and only readings within the linear detection range were included in the final evaluation. The same reagents, equipment, and incubation conditions were used throughout the study, and careful pipetting procedures were applied to reduce technical variability.

5. ELISA steps

For each assay, 100 µL of standards and sample aliquots were dispensed into the designated wells and incubated at 37 °C for 90 min. The wells were then washed three times with wash buffer. Subsequently, 100 µL of biotin-labeled antibody solution was added to each well, followed by incubation at 37 °C for 60 min and a further three washing steps. Next, 100 µL of horseradish peroxidase (HRP) solution was applied and incubated at 37 °C for 30 min. After three additional washes, 100 µL of substrate solution was added and allowed to react for 15 min. Finally, 50 µL of stop solution was added to each well, and absorbance was measured at 450 nm using a microplate reader.

6. Statistical analysis

The sample size per group was 20; therefore, the assumption of normality could not be ensured with sufficient confidence. Distributional properties of the variables were evaluated using graphical methods, including histograms and Q-Q plots. Considering the limited sample size and the uncertainty regarding normality assumptions, primary analyses were conducted using non-parametric tests, which are more appropriate for small samples and data that may not follow a normal distribution. The study groups were initially stratified by sex to ensure balanced distribution; however, statistical analyses were performed based on smoking status. Sex-based comparisons were not included in the primary analysis, as the study was not designed or powered to detect differences between male and female groups.

The Mann-Whitney U test was used for comparisons between two independent groups, while the Wilcoxon signed-rank test was applied for paired comparisons. Correlations between continuous variables were assessed using Spearman’s rank correlation coefficient^23,24. To further evaluate the longitudinal effect of time and the interaction between time and smoking status, repeated-measures ANOVA was also performed as a secondary analysis. Despite the relatively small group size, repeated-measures ANOVA is generally considered robust to moderate deviations from normality when group sizes are similar. Therefore, repeated-measures ANOVA results were reported to support the consistency of the findings and to assess time and interaction effects. Statistical analyses were performed using a statistical software package. A significance level of 0.05 was used for all tests.

DATA AVAILABILITY:
All raw data generated and analyzed during this study have been deposited in the open-access repository Zenodo and are publicly available. The dataset can be accessed via the following DOI:10.528/zenodo.19204955.

Results

A total of 20 males (10 smokers and 10 non-smokers, aged between 18 and 25) and 20 females (10 smokers and 10 non-smokers, aged between 18 and 25) healthy participants were included in the study.

Evaluation of the Relationship Between Smoking and Blood Parameters
The comparison of blood parameters based on smoking status is presented in Table 1 and the correlation between the number of cigarettes smoked and blood parameters is shown in Table 2

Discussion

In this study, we aimed to evaluate the impact of smoking on autologous growth factors within Platelet-Rich Fibrin (PRF), a widely used material in dental surgeries to enhance wound healing. The findings indicated that smoking slightly affects blood parameters. In addition, the evaluation of the effects of smoking on the growth factors levels at different time points revealed that smoking influences the release profile of growth factors with these effects being correlated with the number of cigarettes smoked. Each of the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge (40° fixed-angle rotor)	N/A (Duo Centrifuge (Hettich))		708 x g, 12 min
ELISA Kit - FGF	Fine Test (Wuhan Fine Biotech)	EH0541
ELISA Kit - IGF-1	Fine Test (Wuhan Fine Biotech)	ER0030
ELISA Kit - PDGF	Fine Test (Wuhan Fine Biotech)	ER1240
ELISA Kit - TGFβ-1	Fine Test (Wuhan Fine Biotech)	EH0287
ELISA Kit - VEGF	Fine Test (Wuhan Fine Biotech)	EH0327
Eppendorf Tubes	N/A
Glass Blood Collection Tubes (10 mL)	N/A		No anticoagulant
HRP Solution	Included in ELISA kit
Micropipettes & Tips	N/A
Microplate Reader	N/A		450 nm
Orbital Shaker	N/A		Slow program, 72 h
Precision Scale	N/A
RPMI-1640 Cell Culture Medium	HyClon
Statistical analyses	IBM SPSS Statistics Version 20.0 (Armonk, NY: IBM Corp.)
Sterile Scissors	N/A
Stop Solution	Included in ELISA kit
Substrate Solution	Included in ELISA kit
Wash Buffer	Included in ELISA kit