Abstract
Background
This in vitro study evaluates the effects of adding ascorbic acid (vitamin C) and cholecalciferol (vitamin D3) to platelet-rich fibrin (PRF) matrices on growth factor release, cytokine levels, and mechanical properties.
Methods
Twenty healthy volunteers (10 males and 10 females) participated in the study. Seven tubes of venous blood were collected from each participant. Samples were divided into three groups: control (PRF), vitamin C-augmented PRF (VitC-PRF), and vitamin D-augmented PRF (VitD-PRF). All PRF samples were prepared using a standard centrifugation protocol (2700 rpm, 12 min). Growth factors (IGF-1, PDGF, FGF-2, VEGF, TGF-β1) and inflammatory cytokines (IL-1β, TNF-α) were analysed by ELISA at 24 and 72 h. Mechanical properties were evaluated by scanning electron microscopy (SEM), tensile strength, and elongation tests.
Results
The VitC-PRF group demonstrated significantly higher tensile strength and elongation compared to both the control and VitD-PRF groups (p < 0.05). FGF-2 and PDGF levels were highest in the VitD-PRF group, while the control group exhibited the highest levels of IGF-1 and TGF-β1. IL-1β levels were significantly lower in the VitC-PRF group compared to the other groups, with no significant differences in TNF-α levels between groups.
Conclusions
The addition of ascorbic acid and cholecalciferol to PRF enhanced its mechanical properties and exhibited favorable biological effects on growth factors and inflammatory processes. These findings suggest that vitamin-enriched PRF could be a promising approach for optimizing periodontal regeneration.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12903-025-07620-9.
Keywords: Platelet-rich fibrin, Ascorbic acid, Cholecalciferol, Growth factors, Inflammatory cytokines, Periodontal regeneration.
Background
Regenerative medicine and dentistry have advanced rapidly with the aim of optimizing biological processes for the repair and regeneration of damaged tissues. Among various autologous platelet concentrates, platelet-rich fibrin (PRF) has gained wide acceptance due to its ability to release growth factors and cytokines that promote cellular migration, proliferation, differentiation, and angiogenesis, thereby supporting fundamental mechanisms of tissue regeneration [1, 2]. As a result, PRF has become a valuable biomaterial in periodontal and dental surgical procedures, primarily to enhance wound healing [3–5].
The regenerative potential of PRF can be further enhanced through its combination with different biomaterials and pharmacological agents [6–9]. PRF also serves as a local drug delivery system, increasing the concentration of therapeutics at target sites while minimizing systemic side effects [10, 11]. Recent strategies to augment PRF have included the incorporation of antibiotics to boost antimicrobial efficacy and various vitamins to improve regenerative capacity [6, 7, 12].
Among these, vitamin C (ascorbic acid), known for its potent antioxidant properties and support for collagen synthesis [13], and vitamin D (cholecalciferol), recognized for its regulatory effects on bone metabolism and anti-inflammatory activity [14], are of particular interest in the context of periodontal tissue repair. Vitamin C functions as a cofactor for prolyl and lysyl hydroxylases, promoting collagen maturation and extracellular matrix stability, while its antioxidant action regulates redox-sensitive transcription factors such as NF-κB, thereby reducing the expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) and supporting fibroblast activity and angiogenesis [15].
Vitamin D, acting through the vitamin D receptor (VDR), stimulates osteoblastic differentiation, enhances matrix mineralization, and suppresses proinflammatory mediators, contributing to balanced bone remodeling and improved tissue healing [16]. Through these mechanisms, both vitamins act synergistically to enhance the regenerative processes of periodontal tissues [17]. Evidence suggests that the addition of these vitamins prior to centrifugation may positively modulate growth factors, cytokines, and mechanical properties within PRF, thereby potentially improving regenerative outcomes in periodontal therapy [12, 18]. Considering the proven benefits of vitamins C and D in dental applications and periodontal tissue repair processes, it may be suggested that the addition of these vitamins prior to centrifugation may positively influence the growth factors, cytokines, and mechanical properties of PRF, thereby enhancing its regenerative capacity and contributing to more favorable outcomes in periodontal treatment. Therefore, the aim of this in vitro study was to evaluate the effects of ascorbic acid and cholecalciferol supplementation on the levels of growth factors, cytokines, and mechanical properties of PRF.
Methods
Study design and ethical approval
This in vitro study was conducted at the Department of Periodontology, Faculty of Dentistry, Çukurova University. Ethical approval was obtained from the Ethics Committee of Çukurova University Faculty of Medicine (Date: 04.02.2023, Approval number: 130/90). All participants provided written informed consent prior to enrollment.
Participant selection
A total of 20 healthy volunteers (10 males and 10 females) were included in the study. Inclusion criteria were the absence of systemic diseases and good cooperation. Exclusion criteria were the presence of systemic disease, known allergy to vitamin C or D, pregnancy or lactation, use of medications causing bleeding disorders, and a history of vitamin C or D deficiency treatment in the past six months.
PRF preparation protocol
PRF membranes were prepared by centrifuging blood samples for 12 min at 2700 rpm using a fixed-angle centrifuge (Duo, Nice, France; RCF average 708 g, 88–110 mm radius, 40° rotor angle). For the vitamin groups, ascorbic acid (water-based, pH 6.0–7.0) (Redox-C® 500 mg/5 mL ampule, Bayer Türk Kimya San. Ltd Şti.) or cholecalciferol (oil-based, neutral pH) (Devit-3® 300,000 I.U./1 mL, DEVA Holding A.Ş.) was added to blood samples prior to centrifugation. Vitamin C and vitamin D were freshly drawn from sealed ampoules under sterile conditions and immediately added to the blood samples to minimize oxidation or photodegradation at room temperature (23 ± 1 °C). Immediately after addition of vitamin solutions to the blood samples, tubes were gently inverted twice to ensure uniform dispersion without mechanical agitation or hemolysis. Centrifugation commenced within 30 s after mixing. The resulting PRF clots were separated and weighed.
Pilot study for vitamin dose selection
A pilot experiment was conducted with three volunteers to determine the optimal vitamin doses. Blood samples were supplemented with 0.25, 0.5, and 1 mL of vitamin solutions before centrifugation. The selected doses were within physiological human range without toxicity and these doses were chosen to mimic maximal biologically relevant local concentrations without interfering with PRF formation. The final PRF samples were macroscopically assessed for clot formation and consistency. Inadequate PRF formation was detected with 0.5 or 1 mL of vitamin D, while satisfactory PRF structure was seen with all tested doses of vitamin C. Based on these observations, 1 mL of vitamin C and 0.25 mL of vitamin D were chosen for the main study.
Blood collection and group allocation
Seven tubes (~ 10 mL each) of venous blood were collected from each participant:
Tube 1: Complete blood count and baseline vitamin C and D levels
Tubes 2–3: PRF preparation for growth factor/cytokine assessment and for mechanical/SEM analyses (control group)
Tubes 4–5: PRF preparation with the addition of 1 mL ascorbic acid (Vitamin C group: VitC-PRF) for growth factor/cytokine assessment and mechanical/SEM analyses
Tubes 6–7: PRF preparation with the addition of 0.25 mL cholecalciferol (Vitamin D group: VitD-PRF) for growth factor/cytokine assessment and mechanical/SEM analyses
All blood samples were collected in additive-free, plain glass tubes (no silica or silicone coating) to avoid cytotoxic or pro-coagulant interference. Baseline complete blood count was analyzed using an automated hematology analyzer. Serum vitamin C and D levels were determined by high-performance liquid chromatography and chemiluminescence immunoassay respectively.
Thus, three study groups were established (Fig. 1):
Fig. 1.
Representative PRF samples of control, VitD-PRF and VitC-PRF
Group 1: Standard PRF (Control)
Group 2: PRF with ascorbic acid (VitC-PRF)
Group 3: PRF with cholecalciferol (VitD-PRF)
Growth factor and cytokine analysis
PRF samples were weighed and incubated in HyClone™ RPMI-1640 medium. At 24 and 72 h, samples were collected and stored for analysis. Growth factors (IGF-1, PDGF, FGF-2, VEGF, TGF-β1) and cytokines (IL-1β, TNF-α) were quantified by ELISA (Reed Biotech®) following the manufacturer’s instructions. Measurements were read at 450 nm and reported in pg/mL or ng/mL.
Scanning electron microscopy (SEM) analysis
For microstructural analysis, PRF samples were freeze-dried at < 0.1 mPa for 8 h and sputter-coated with gold-palladium. SEM images were obtained at 6000X and 24,000X magnifications using a Quanta 650 Field Emission SEM (10 kV).
Mechanical testing
Tensile strength and elongation were evaluated using a Testometric M500-25kN universal testing machine. PRF samples were clamped without initial tension and subjected to uniaxial extension at 1 mm/min until failure (Fig. 2). The maximum force (N) and elongation at break (mm) were recorded, and tensile strength was calculated (S = F/A). Elastic modulus and toughness were determined from the stress-strain curve.
Fig. 2.
Tensile strength and elongation analysis of PRF samples
Statistical analysis
Numerical data were summarized as mean ± standard deviation and median (minimum–maximum). The Shapiro-Wilk test assessed normality. Paired t-tests or Wilcoxon signed-rank tests were used for within-group comparisons, as appropriate. Friedman and Bonferroni-adjusted Wilcoxon signed-rank tests were used for group comparisons. Nested ANOVA evaluated time and group effects. Statistical significance was set at p < 0.05. Analyses were performed using IBM SPSS Statistics Version 20.0.
Results
A total of 20 healthy volunteers (10 males and 10 females) were included. All hematological parameters and baseline vitamin levels were within normal reference ranges for all participants. PRF formation was visually confirmed in all tubes immediately after centrifugation, and no delay or inhibition of fibrin polymerization was observed following vitamin supplementation.
SEM analysis
Scanning electron microscopy revealed distinct differences in surface morphology among the groups (Fig. 3). The control PRF displayed a granular structure and uniform surface integrity, with micro-porosities visible at high magnification. While VitC-PRF showed a denser and more compact structure with clustered particles and irregular amorphous spaces; VitD-PRF exhibited pronounced granularity, particle aggregation, and dendritic, crystal-like formations at higher magnification.
Fig. 3.
Representative scanning electron microscopy (SEM) images of PRF samples in each group (A) Control group: granular surface and regular micro-porosity; (B) VitC-PRF: dense and compact structure with clustered particles; (C) VitD-PRF: granular surface and dendritic, crystal-like features. Images shown at 6,000× and 24,000× magnification
Mechanical properties
Tensile strength and elongation at break demonstrated statistically significant differences among the groups (p < 0.05) (Table 1). The VitC-PRF group showed significantly higher tensile strength (3.8 ± 1.1 N/mm²) compared to the control group (2.9 ± 0.5 N/mm², p = 0.0115), while VitD-PRF (3.3 ± 0.5 N/mm²) did not differ significantly from either (Fig. 4).
Table 1.
Summary of mechanical properties of PRF Membranes. Tensile strength and elongation values summarized as mean ± SD and median (min–max) for all study groups
| Group | (p) | ||||||
|---|---|---|---|---|---|---|---|
| PRF(Control) | VitC-PRF | VitD-PRF | p | PRF vs. VitC-PRF | PRF vs. | VitD-PRF vs. | |
| (n=20) | (n=20) | (n=20) | VitD-PRF | VitC-PRF | |||
| Tensile | 2,9±0,5 | 3,8±1,1 | 3,3±0,5 | ||||
| Strength | 2,8 (2,5-3,7) | 3,4 (2,6-5) | 3,1 (2,7-3,8) | 0,001 | 0,0115 | 0,1105 | 0,6199 |
| (N/mm2) | |||||||
| Elongation | 13,9±4,7 | 24,9±4,6 | 17,4±6,9 | 0,001 | 0,001 | 0,0252 | 0,001 |
| (mm) | 13,9 (8,2–21,2) | 26,2 (17,9–30,2) | 16,2 (10,2–28) | ||||
| Mean ± SD |
Fig. 4.
Comparison of elongation (mean ± SD) between PRF groups
For elongation, the VitC-PRF group (24.9 ± 4.6 mm) exceeded both control (13.9 ± 4.7 mm, p = 0.001) and VitD-PRF (17.4 ± 6.9 mm, p = 0.0252) (Fig. 5).
Fig. 5.
Comparison of tensile strength (mean ± SD) values between PRF groups
Growth factor analysis
The results of the growth factor analysis are summarized in Table 2.
Table 2.
Growth factor levels and statistical comparisons at 24 h and 72 h. Summary of growth factor measurements and statistical significance across time points and groups
| Group | Group (Time) t | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Time | PRF(Control) | VitC-PRF | VitD-PRF | p | PRF vs. VitC- | PRF vs. VitD- | VitD-PRF vs. VitC- | p | |
| (n=20) | (n=20) | (n=20) | PRF | PRF | PRF | ||||
| FGF-2 | 24 Hour | 7,4±3,1 | 3,4±2,5 | 7,7±1,3 | 0,001 | 0,002 | >0,999 | 0,005 | 0,067 |
| 8,6 (0,9–10,6) | 2,1 (1,3–8,3) | 7,5 (5,6–10,0) | |||||||
| 72 Hour | 8,3±3,5 | 5,3±2,2 | 8,2±2,3 | <0,001 | <0,001 | >0,999 | 0,013 | ||
| 9,7 (1,0–12,3) | 5,8 (1,8-8,5) | 9,0 (0,9–10,8) | |||||||
| p | <0,001 | 0,017 | 0,225 | ||||||
| IGF-1 | 24 Hour | 14,0±3,0 | 9,8±2,7 | 12,2±2,8 | <0,001 | <0,001 | 0,246 | 0,022 | 0,593 |
| 14,4 (8,0–18,1) | 10,1 (1,1–13) | 11,6 (6,8–19,1) | |||||||
| 72 Hour | 14,9±3,3 | 10,5±1,6 | 12,6±3,6 | <0,001 | <0,001 | 0,053 | 0,173 | ||
| 15,5 (8,7–19) | 10,2 (8,8–14,9) | 11,5 (9,1–20,6) | |||||||
| p | <0,001t | 0,360 | 0,809 | ||||||
| PDGF | 24 Hour | 1,9±1,8 | 1,0±0,3 | 2,7±2,7 | 0,003 | 0,053 | >0,999 | 0,003 | <0,001 |
| 1,5 (0,7-7,3) | 0,9 (0,5-1,6) | 1,5 (0,6–12) | |||||||
| 72 Hour | 1,7±1,6 | 1,1±0,4 | 6,6±5,6 | 0,003 | >0,999 | 0,053 | 0,003 | ||
| 1,2 (0,6-6,9) | 1,0 (0,5-2,0) | 4,8 (0,6–16,0) | |||||||
| p | <0,001 | 0,191 | 0,002 | ||||||
| VEGF | 24 Hour | 403,0±134,5 | 314,3±64,7 | 363,8±92,6 | 0,043 | 0,053 | >0,999 | 0,173 | <0,001 |
| 368,8 (262,7–809,4) | 302,0 (215,9–437,8) | 326,4 (267,6–598,4) | |||||||
| 72 Hour | 410,0±137,0 | 317,6±63,9 | 148,5±205,0 | <0,001 | >0,999 | <0,001 | 0,003 | ||
| 373,6 (271,7–813) | 307,1 (211,3–460,8) | 70,1 (23,4–735) | |||||||
| p | 0,002 | 0,861 | 0,002 | ||||||
TGF- 1 |
24 Hour | 879,5±494,7 | 549,5±329,8 | 443,1±223,0 | 0,002 | 0,246 | 0,002 | 0,246 | 0,161 |
| 641,8 (303,9–1686,4) | 376,7 (147,1–1213,3) | 423,2 (162,3–770,7) | |||||||
| 72 Hour | 660,6+371,1 | 601,4±264,6 | 363,8±200,9 | 0,116 | >0,050 | >0,050 | >0,050 | ||
| 479,5 (230,2–1303,8) | 678,1 (209,5–1023,2) | 243,1 (118,9–719,4) | |||||||
| p | <0,001 | 0,627 | 0,502 | ||||||
| Mean ± SD | |||||||||
| tNested ANOVA |
FGF-2
Increased significantly from 24 to 72 hours in the control and VitC-PRF groups.
VitD-PRF group showed the highest FGF-2 values at 24 hours (Fig. 6).
Fig. 6.
FGF-2 Levels at 24 and 72 h
IGF-1
Control group exhibited a significant increase from 24 to 72 hours; no significant changes in VitC-PRF or VitD-PRF groups (Fig. 7).
Fig. 7.
IGF-1 Levels at 24 and 72 h
PDGF
Control group showed a decrease over time, while VitD-PRF showed a significant increase from 24 to 72 hours. PDGF was consistently higher in VitD-PRF compared to VitC-PRF (Fig. 8).
Fig. 8.
PDGF Levels at 24 and 72 h
VEGF
Control group showed an increase, while VitD-PRF showed a decrease from 24 to 72 hours. Both control and VitC-PRF had higher VEGF at 72 hours compared to VitD-PRF (Fig. 9).
Fig. 9.
VEGF Levels at 24 and 72 h
TGF-β1
Control group showed a decrease over time. No significant temporal changes in VitC-PRF or VitD-PRF (Fig. 10).
Fig. 10.
TGF-β1 Levels at 24 and 72 h
Inflammatory cytokine analysis
The results of the inflammatory cytokine analysis are summarized in Table 3.
Table 3.
Inflammatory cytokine levels (IL-1β, TNF-α) in Control, VitC-PRF, and VitD-PRF groups at 24 and 72 hours
| Group | Group (Time)t | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Time | PRF(Control) | VitC-PRF | VitD-PRF | p | PRF vs. | PRF vs. | VitD-PRF vs. | p | |
| (n=20) | (n=20) | (n=20) | VitC-PRF | VitD-PRF | VitC-PRF | ||||
TNF- |
24 Hour | 14,3±2,6 | 14,4±3,4 | 15,5±2,6 | 0,428 | >0,050 | >0,050 | >0,050 | 0,166 |
| 13,6 (11,4–22,9) | 13,9 (9,2–21,3) | 15,2 (12,1–20) | |||||||
| 72 Hour | 15,1±3,1 | 16,1±3,7 | 17,1±4,8 | 0,486 | >0,050 | >0,050 | >0,050 | ||
| 14,2 (12,2–26,2) | 16,4 (9,7–22,2) | 16,5 (9,9–27,7) | |||||||
| P | <0,001 | 0,228 | 0,232 | ||||||
IL-1 |
24 Hour | 278,5±206,3 | 28,7±6,8 | 314±210,8 | <0,001 | <0,001 | >0,999 | <0,001 | 0,114 |
| 236,4 (26–591) | 29,4 (17,8–49,1) | 389,9 (37,4–582,4) | |||||||
| 72 Hour | 311,8235,6 | 60,4±57,1 | 435,9±146,2 | 0,342 | <0,001 | ||||
| 257,3 (30,9–680,6) | 35,1 (23,1–252,3) | 484,5 (141–623,5) | <0,001 | <0,001 | |||||
| p | <0,001 | 0,008 | 0,008 | ||||||
| Mean ± SD | |||||||||
| tNested ANOVA |
IL-1β
Significantly lower in the VitC-PRF group compared to control and VitD-PRF at both 24 and 72 hours (p < 0.05) (Fig. 11).
Fig. 11.
IL-1β cytokine levels in each group at 24 and 72 h
TNF-α
No statistically significant differences between groups (Fig. 12).
Fig. 12.
TNF-α cytokine levels in each group at 24 and 72 h
Discussion
The present study evaluated the effects of ascorbic acid (vitamin C) and cholecalciferol (vitamin D3) supplementation on the release of growth factors, inflammatory cytokines, and mechanical properties of platelet-rich fibrin (PRF). The findings indicate that the addition of these vitamins can modulate the biological activity and biomechanical characteristics of PRF, with potential implications for optimizing periodontal regeneration.
Our data demonstrate that vitamin C-enriched PRF exhibited superior tensile strength and elongation capacity compared to both control and vitamin D-enriched PRF. These findings are consistent with previous reports suggesting that vitamin C plays a critical role in collagen cross-linking and fibrin stabilization, thereby enhancing the structural resilience of biomaterials [13, 19, 20]. The improved mechanical properties observed in the Vit C-PRF group may contribute to extended matrix resorption time and greater tissue integration, as suggested by studies examining various PRF modifications and their impact on clinical outcomes.
Scanning electron microscopy further revealed that vitamin supplementation leads to pronounced changes in the surface morphology of PRF. Vitamin C yielded a denser and more compact microstructure, while vitamin D resulted in irregular, crystal-like features. The dendritic, crystalline structures observed in the VitD-PRF samples may reflect localized calcium-phosphate precipitation or vitamin D-mediated mineralization, consistent with receptor-regulated osteogenic signaling. Additionally, the presence of butylated hydroxyanisole as an excipient in the vitamin D formulation may have contributed to the formation of crystalline deposits, since phenolic antioxidants can undergo surface crystallization or interact with calcium ions under specific physicochemical conditions. These microstructural alterations may underlie the observed differences in mechanical properties and could influence cellular adhesion and proliferation in vivo.
Our results indicate that vitamin D-enriched PRF exhibited elevated levels of FGF-2 and PDGF, in line with the known capacity of vitamin D to stimulate gene transcription of growth factors involved in mitogenic and regenerative pathways [21–23]. The decrease in VEGF levels observed in the VitD-PRF group from 24 to 72 h may reflect the modulatory role of vitamin D in angiogenic signaling. Vitamin D, through VDR-mediated regulation, can downregulate VEGF expression [24] as part of its anti-inflammatory and tissue-stabilizing actions, particularly during the resolution phase of wound healing. This transient suppression might therefore indicate a shift from an early proangiogenic response toward controlled matrix maturation. In contrast, the control group showed higher levels of IGF-1 and TGF-β1. The significant increase in FGF-2 from 24 to 72 h in the Vit C-PRF group supports the hypothesis that vitamin C fosters an environment conducive to angiogenesis [25] and matrix synthesis [15], corroborated by reports of increased collagen production and osteoblastic differentiation in the presence of ascorbic acid.
A notable finding was the marked reduction in IL-1β in the Vit C-PRF group, consistent with vitamin C’s well-documented anti-inflammatory and antioxidant effects [26]. These results suggest that vitamin C enrichment could attenuate the local inflammatory response, potentially favoring wound healing and tissue regeneration. Conversely, vitamin D did not significantly reduce pro-inflammatory cytokines in this study, though previous work suggests its immunomodulatory impact may depend on context and concentration.
The ability to tailor PRF’s biological and mechanical properties via vitamin enrichment holds promise for advancing regenerative protocols in periodontology and oral surgery. Enhanced tensile strength and anti-inflammatory activity could translate into improved handling characteristics and clinical outcomes, such as increased membrane stability, better wound closure, and reduced risk of postoperative complications. Furthermore, the capacity for controlled release of key growth factors supports the development of customized biomaterials targeting specific phases of healing.
Although PRF has been extensively studied, variations in preparation protocols continue to hinder consistent interpretation of results. The RCF, rotor size and angulation, RPM, centrifugation time, and tube composition have been proven to be critical parameters [27, 28]. Miron et al. [28, 29] emphasized reporting RCF, as it markedly influences cell content and growth factor release. Tube composition also plays a role [30–32]; silica- or silicone-coated tubes can be cytotoxic, inducing apoptosis [30].
The angled rotor centrifuge used in this study directs cells to the tube walls, possibly complicating separation due to density differences. In contrast, novel techniques using horizontal rotors may yield a more uniform cell distribution and higher concentration with minimal damage [33]. Protocol refinements include concentrated PRF at higher RCF to target the buffy coat, hydrophobic tube coatings to delay clotting, and cooling systems to extend working time [32].
Despite these promising findings, several limitations must be acknowledged. The study was performed in vitro, and extrapolation to in vivo or clinical settings should be made cautiously. Although the sample size was adequate for preliminary assessment, its limited nature may reduce statistical power and affect the reliability and generalizability of the results. Expanding the sample size in future studies would allow stronger and more meaningful conclusions to be drawn. In addition, the final concentration of the vitamins after adding into the blood samples and the dose normalized to blood/PRF volume were not analyzed which may weaken the biological comparability and reproducibility of the results. Although pH and osmolarity were not directly measured, both vitamin preparations were sterile injectable formulations with physiological pH ranges, minimizing any potential alteration in coagulation environment. Furthermore, only two incubation time periods (24 h, 72 h) were selected for the current study. Advanced studies with extended intervals could reveal late-phase release dynamics of PRF. Long-term outcomes such as resorption kinetics and clinical effectiveness require validation in animal and human studies. Similarly, the cytokine panel was restricted to IL-1β and TNF-α as representative proinflammatory markers. Future analyses incorporating IL-6, IL-10, MCP-1, and RANKL/OPG pathways should be planned to comprehensively characterize the immunomodulatory potential of vitamin-enriched PRF. Only single doses and forms of vitamins were investigated; exploring different concentrations, vitamin combinations, and additional biomodifiers could yield further insights.
Future research should focus on evaluating vitamin-enriched PRF in animal models and randomized clinical trials, incorporating different patient populations, defect types, and follow-up durations. Investigating the impact of patient-specific factors (e.g., age, sex, systemic health) and various biomaterial combinations will be essential to optimize protocols for personalized regenerative therapies.
Conclusion
This study demonstrates that the addition of ascorbic acid and cholecalciferol to platelet-rich fibrin can enhance its mechanical durability and modulate the release of growth factors and inflammatory cytokines. Vitamin C-enriched PRF showed superior tensile strength, elongation, and anti-inflammatory properties, while vitamin D-enriched PRF promoted increased levels of key regenerative growth factors. These findings suggest that vitamin supplementation of PRF represents a promising and innovative strategy for improving the efficacy of regenerative protocols in periodontal therapy. Further preclinical and clinical studies are warranted to validate these results and to optimize dosing regimens for translational applications.
Supplementary Information
Acknowledgements
Not applicable.
Abbreviations
- Vitamin C
Ascorbic acid
- Vitamin D3
Cholecalciferol
- PRF
Platelet-rich fibrin
- VitC-PRF
Vitamin C-augmented PRF
- VitD-PRF
Vitamin D-augmented PRF
- VDR
Vitamin D receptor
- SEM
Scanning Electron Microscopy
- IGF-1
Insulin-like Growth Factor
- PDGF
Platelet-derived growth factor
- FGF-2
Fibroblast growth factor 2
- VEGF
Vascular endothelial growth factor
- TGF-β1
Transforming growth factor
- IL-1β
Interleukin-1-beta
- TNF-α
Tumor necrosis factor alpha
- nm
Nanometer
- pg/mL
Picograms per milliliter
- ng/mL
Nanograms per millilitre
- RCF
Relative centrifugal force
- Rpm
Revolutions per minute
Authors’ contributions
MO and MCH contributed to study conception and design. BA and BET performed clinical procedures, BY performed ELISA analysis, OUT performed the data analysis and prepared the manuscript. All authors critically revised the manuscript. All authors read and approved the final manuscript.
Funding
This study was supported by Cukurova University Research Fund, Project number TDH 2024–16601.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethical approval and consent to participate
This in vitro study was conducted at the Department of Periodontology, Faculty of Dentistry, Cukurova University. Ethical approval was obtained from the Ethics Committee of Cukurova University Faculty of Medicine (Date: 04.02.2023, Approval number: 130/90). All participants provided written informed consent prior to enrollment. The study protocol and informed consent were in full accordance with the ethical principles of the Declaration of Helsinki ( https://www.wma.net/policies-post/wma-declaration-of-helsinki/)
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;101(3):e37-44. [DOI] [PubMed] [Google Scholar]
- 2.Miron RJ, Zucchelli G, Pikos MA, Salama M, Lee S, Guillemette V, Fujioka-Kobayashi M, Bishara M, Zhang Y, Wang H-L. Use of platelet-rich fibrin in regenerative dentistry: a systematic review. Clin Oral Invest. 2017;21(6):1913–27. [DOI] [PubMed] [Google Scholar]
- 3.Farshidfar N, Ahmad P, Estrin N, Zhang Y, Miron RJ. Use of Platelet-Rich fibrin (PRF) on palatal wound healing: A narrative review with clinical recommendations. J Periodontal Res. 2025. 10.1111/jre.13413. Epub ahead of print. PMID: 40492943 [DOI] [PubMed]
- 4.Miron RJ, Fujioka-Kobayashi M, Bishara M, Zhang Y, Hernandez M, Choukroun J. Platelet-rich fibrin and soft tissue wound healing: a systematic review. Tissue Eng Part B Rev. 2017;23(1):83–99. [DOI] [PubMed] [Google Scholar]
- 5.Quirynen M, Sculean A, Blanco J, Wang HL, Donos N. Introduction and overview on autogenous platelet concentrates. Periodontol 2000. 2025;97(1):7–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Fang X, Li Y, Wang Y, Cai R, Ao Q. Platelet-derived biomaterials for targeted drug delivery and tissue repair. J Mater Chem B. 2025;13(5):1573–85. 10.1039/D4TB02477J. [DOI] [PubMed]
- 7.Ozcan M, Kabaklı SC, Alkaya B, Isler SC, Turer OU, Oksuz H, Haytac MC. The impact of local and systemic penicillin on antimicrobial properties and growth factor release in platelet-rich fibrin: in vitro study. Clin Oral Invest. 2023;28(1):61. [DOI] [PubMed] [Google Scholar]
- 8.Miron RJ, Gruber R, Farshidfar N, Sculean A, Zhang Y. Ten years of injectable platelet-rich fibrin. Periodontol 2000. 2024;94(1):92–113. [DOI] [PubMed] [Google Scholar]
- 9.Niemczyk W, Kępa M, Żurek J, Aboud A, Skaba D, Wiench R. Application of Platelet-Rich fibrin and concentrated growth factors as carriers for antifungal Drugs—In vitro study. J Clin Med. 2025;14(14):5111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Niemczyk W, Żurek J, Niemczyk S, Kępa M, Zięba N, Misiołek M, Wiench R. Antibiotic-Loaded Platelet-Rich fibrin (AL-PRF) as a new carrier for antimicrobials: A systematic review of in vitro studies. Int J Mol Sci. 2025;26(5):2140. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Thamaraiselvan M, Jayakumar ND. Efficacy of injectable platelet-rich fibrin (i-PRF) as a novel vehicle for local drug delivery in non-surgical periodontal pocket therapy: a randomized controlled clinical trial. J Adv Periodontol Implant Dent. 2024;16(2):94. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Kulkarni M, Sowmya N, Gayathri G, Triveni M. Effects of ascorbic acid augmented albumin platelet-rich fibrin on the wound healing activity of human gingival fibroblasts: an in vitro trial. J Korean Assoc Oral Maxillofac Surg. 2024;50(4):206–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Alberts A, Moldoveanu E-T, Niculescu A-G, Grumezescu AM. Vitamin C: a comprehensive review of its role in health, disease prevention, and therapeutic potential. Molecules. 2025;30(3):748. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Christakos S, Dhawan P, Verstuyf A, Verlinden L, Carmeliet G. Vitamin D: metabolism, molecular mechanism of action, and pleiotropic effects. Physiol Rev. 2016;96(1):365–408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.D’Aniello C, Cermola F, Patriarca EJ, Minchiotti G. Vitamin C in stem cell biology: impact on extracellular matrix homeostasis and epigenetics. Stem Cells Int. 2017;2017(1):8936156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Posa F, Di Benedetto A, Colaianni G, Cavalcanti-Adam EA, Brunetti G, Porro C, Trotta T, Grano M, Mori G. Vitamin D effects on osteoblastic differentiation of mesenchymal stem cells from dental tissues. Stem Cells Int. 2016;2016(1):9150819. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Fawzy El-Sayed KM, Cosgarea R, Sculean A, Doerfer C. Can vitamins improve periodontal wound healing/regeneration? Periodontol 2000. 2024;94(1):539–602. [DOI] [PubMed] [Google Scholar]
- 18.Sherif MA, Anter E, Graetz C, El-Sayed KF. Injectable platelet-rich fibrin with vitamin C as an adjunct to non-surgical periodontal therapy in the treatment of stage-II periodontitis: a randomized controlled clinical trial. BMC Oral Health. 2025;25(1):772. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Murererehe J, Uwitonze AM, Nikuze P, Patel J, Razzaque MS. Beneficial effects of vitamin C in maintaining optimal oral health. Front Nutr. 2022;8:805809. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Van der Velden U. Vitamin C and its role in periodontal diseases–the past and the present: a narrative review. Oral Health Prev Dent. 2020;18(2):a44306. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Gurlek A, Pittelkow MR, Kumar R. Modulation of growth factor/cytokine synthesis and signaling by 1α, 25-dihydroxyvitamin D3: implications in cell growth and differentiation. Endocr Rev. 2002;23(6):763–86. [DOI] [PubMed] [Google Scholar]
- 22.Mostafa NZ, Fitzsimmons R, Major PW, Adesida A, Jomha N, Jiang H, Uludağ H. Osteogenic differentiation of human mesenchymal stem cells cultured with dexamethasone, vitamin D3, basic fibroblast growth factor, and bone morphogenetic protein-2. Connect Tissue Res. 2012;53(2):117–31. [DOI] [PubMed] [Google Scholar]
- 23.Jamali N, Song YS, Sorenson CM, Sheibani N. 1, 25 (OH) 2D3 regulates the proangiogenic activity of pericyte through VDR-mediated modulation of VEGF production and signaling of VEGF and PDGF receptors. FASEB Bioadv. 2019;1(7):415–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Irani M, Seifer DB, Grazi RV, Irani S, Rosenwaks Z, Tal R. Vitamin D decreases serum VEGF correlating with clinical improvement in vitamin D-deficient women with PCOS: a randomized placebo-controlled trial. Nutrients. 2017;9(4):334. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.May JM, Harrison FE. Role of vitamin C in the function of the vascular endothelium. Antioxid Redox Signal. 2013;19(17):2068–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Gęgotek A, Skrzydlewska E. Antioxidative and anti-inflammatory activity of ascorbic acid. Antioxidants (Basel). 2022;11(10):1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Miron RJ, Chai J, Fujioka-Kobayashi M, Sculean A, Zhang Y. Evaluation of 24 protocols for the production of platelet-rich fibrin. BMC Oral Health. 2020;20(1):310. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Miron RJ, Pinto NR, Quirynen M, Ghanaati S. Standardization of relative centrifugal forces in studies related to platelet-rich fibrin. In., vol. 90: Wiley Online Library; 2019: 817–820. [DOI] [PubMed]
- 29.Miron RJ, Fujioka-Kobayashi M, Sculean A, Zhang Y. Optimization of platelet‐rich fibrin. Periodontol 2000. 2024;94(1):79–91. [DOI] [PubMed] [Google Scholar]
- 30.Miron RJ, Kawase T, Dham A, Zhang Y, Fujioka-Kobayashi M, Sculean A. A technical note on contamination from PRF tubes containing silica and silicone. BMC Oral Health. 2021;21(1):135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Tunalı M, Özdemir H, Küçükodacı Z, Akman S, Yaprak E, Toker H, Fıratlı E. A novel platelet concentrate: titanium-prepared platelet‐rich fibrin. Biomed Res Int. 2014;2014(1):209548. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Miron RJ, Horrocks NA, Zhang Y, Horrocks G, Pikos MA, Sculean A. Extending the working properties of liquid platelet-rich fibrin using chemically modified PET tubes and the Bio-Cool device. Clin Oral Investig. 2022;26(3):2873–8. [DOI] [PubMed] [Google Scholar]
- 33.Farshidfar N, Apaza Alccayhuaman KA, Estrin NE, Ahmad P, Sculean A, Zhang Y, Miron RJ. Advantages of horizontal centrifugation of platelet-rich fibrin in regenerative medicine and dentistry. Periodontology 2000. 2025;1–52. [DOI] [PubMed]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.