|Year : 2023 | Volume
| Issue : 1 | Page : 23-27
Effect of two different antifibrinolytic agents on platelet-rich fibrin: An in vitro comparative study
Vishnu Ponnusamy, H Esther Nalini, R Renuka Devi, Nivetha Raja
Department of Periodontology, K.S.R. Institute of Dental Science and Research, Tiruchengode, Tamil Nadu, India
|Date of Submission||21-Nov-2022|
|Date of Decision||26-Jan-2023|
|Date of Acceptance||03-Feb-2023|
|Date of Web Publication||18-Mar-2023|
Dr. H Esther Nalini
Department of Periodontology, K.S.R. Institute of Dental Science and Research, Tiruchengode - 637 215, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Platelet-rich fibrin (PRF) is a potent autologous regenerative material used in periodontal regeneration. PRF collapses easily into the periodontal defect due to its faster resorption rate and lesser rigidity. The degradation time of PRF has a direct impact on the clinical outcome. Aim: This study aimed to compare and analyze the effect of two different antifibrinolytic agents on the degradability of PRF membranes. Materials and Methods: Twenty-one PRF membranes were randomly divided into three groups: Group 1 – Conventional PRF obtained by adding normal saline (Control), Group 2 – PRF obtained by adding 200 mg tranexamic acid (TXA), and Group 3 – PRF obtained by adding 250 mg epsilon aminocaproic acid (EACA). After storing at room temperature in normal saline, PRF membranes were retrieved at 1st, 2nd, and 3rd weeks and the percentage of remaining weight was calculated. ANOVA is used for the statistical comparison of the data between groups. Results: In comparison to the baseline, the mean PRF weight was decreased in all groups in the 1st, 2nd, and 3rd weeks. In the 1st, 2nd, and 3rd weeks, the mean percentage of the remaining weight of PRF membrane in Group 1, Group 2, and Group 3 were 69.53 ± 12.96, 36.23 ± 9.28, and 17.43 ± 4.75 respectively. The statistical significance with the percentage of the remaining weight of the PRF was obtained in 3rd week with a P = 0.029. Conclusion: TXA efficiently delayed the degradability of the PRF membrane compared to EACA under experimental conditions.
Keywords: Antifibrinolytics, degradation, platelet-rich fibrin, preparation, regeneration
|How to cite this article:|
Ponnusamy V, Nalini H E, Devi R R, Raja N. Effect of two different antifibrinolytic agents on platelet-rich fibrin: An in vitro comparative study. SRM J Res Dent Sci 2023;14:23-7
|How to cite this URL:|
Ponnusamy V, Nalini H E, Devi R R, Raja N. Effect of two different antifibrinolytic agents on platelet-rich fibrin: An in vitro comparative study. SRM J Res Dent Sci [serial online] 2023 [cited 2023 Mar 24];14:23-7. Available from: https://www.srmjrds.in/text.asp?2023/14/1/23/372002
| Introduction|| |
Periodontal disease is an infectious disease affecting the supporting structures of the teeth resulting in progressive attachment and bone loss. The treatment for periodontal disease aims to stop the disease progression as well as to restore the damaged periodontal apparatus. The regeneration of the periodontal apparatus is a complex process that requires coordinated development of bone, cementum, and periodontal ligament. The orchestration of biological processes such as cell migration, adhesion, and proliferation occurs during periodontal regeneration. Conventional periodontal surgery results in repair, not regeneration. Numerous regenerative techniques using hard and soft-tissue grafts are available to promote the regeneration of lost periodontal apparatus.
The class of naturally occurring proteins known as growth factors exhibits a variety of dominant local properties. They play a key role in the biological process such as cell migration, adhesion, and proliferation. Numerous cytokines and growth factors are present in the platelets which are essential for periodontal wound healing.,
Platelet-rich fibrin (PRF) is a second generation of platelet concentrates introduced by Choukroun in 2000. It consists of platelets, leucocytes, stem cells, and cytokines embedded in a fibrin matrix with a tetra molecular structure. This PRF is a naturally occurring fibrin matrix that primarily concentrates platelets and growth factors. PRF membrane is indicated in alveolar bone reconstruction, implant surgery, sinus augmentation, socket preservation, and root coverage procedures. The protocol for the preparation of PRF was first described by Choukroun et al. (2700 rpm for 12 min). PRF membranes not only act as scaffolds but also release potent growth factors such as transforming growth factor β1, vascular endothelial growth factor, insulin-like growth factor, and platelet-derived growth factor-AB. This platelet gel has superior mechanical and handling properties and it can be utilized either alone or in combination with bone grafts.,
PRF has a higher degradation rate and lesser rigidity compared to other barrier membranes. Due to its faster resorption rate and lesser rigidity, the PRF membrane collapsed easily into the periodontal defect thereby causing failure in space maintenance property in guided tissue regeneration (GTR). Fibrinolysis is the physiological process that eliminates excess fibrin and improves better fibrin clot formation and wound healing. Antifibrinolytic agents are the drugs that reversibly block the plasminogen and plasmin lysine binding site thereby retards fibrinolysis., The most commonly used antifibrinolytic agents in dentistry are tranexamic acid (TXA) and Epsilon aminocaproic acids (EACA), both are synthetic lysine amino acid derivatives., These agents are used as hemostatic agents and mouthwashes to control postoperative bleeding in patients on anticoagulant therapy and coagulation disorders.
In our study, we modified the PRF membrane by adding antifibrinolytic agents such as TXA and EACA with blood samples before centrifugation. Thus, the aim of this study was to compare and analyze the effect of two different antifibrinolytic agents on the degradability of PRF membranes.
| Materials and Methods|| |
This in vitro study was carried out in the Department of Periodontology, K. S. R Institute of Dental Science and Research, Tiruchengode, Tamilnadu after getting approval from the KSRIDSR Institutional Ethical Committee (Ref. No: 320/KSRIDSR/IEC/2022). All procedures performed in the study were conducted in accordance with the ethical standards given in 1964 Declaration of Helsinki, as revised in 2013. All the study participants gave the written informed consent for their participation in the study.
The sample size was estimated using G*Power software (version 220.127.116.11; Heinrich-Heine-University Dusseldorf, Dusseldorf, Germany). For the power of study at 80%, the total sample size required for the study is 21 which were divided into 7 samples in each group.
A volume of 52.5 ml of venous blood was collected from 7 different systemically healthy student volunteers. Each collected blood sample was then divided into 3 glass tubes resulting in 21 tubes of blood samples. Group 1: 2.5 ml of Normal saline (n = 7), Group 2: 2.5 ml of 200 mg TXA (n = 7), and Group 3: 2.5 ml of 250 mg EACA (n = 7) was added to the test tube containing collected blood [Figure 1]. The PRF was prepared based on Choukroun's protocol (2700 rpm for 15 min). After centrifugation, test tubes contain the following layers: upper straw-colored fluid, middle layer of PRF, and lower layer of red blood cells (RBCs). The middle layer of PRF is separated from the lower layer of RBC [Figure 2].
|Figure 1: Protocol for PRF preparation in three groups. PRF: Platelet-rich fibrin|
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|Figure 2: PRF membrane obtained in three groups. PRF: Platelet-rich fibrin|
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PRF membranes were retrieved and pressed between the sterile gauze pieces. They were thoroughly dried with blotting paper to remove excess saline thereby avoiding errors during weight measurement. The baseline weight was measured using a micro-weighing machine (REMI 1MLH). PRF membranes were stored in Eppendorf tubes containing normal saline for 3 weeks at room temperature (7 in each group). At the end of 1st, 2nd, and 3rd weeks, PRF membranes were retrieved from Eppendorf tubes, dried with blotting paper, and weighted gain. The percentage of the remaining weight of PRF was calculated in each Eppendorf tube at 1st, 2nd, and 3rd weeks. The percentage of the remaining weight was calculated by using the formula ([Weight of PRF at 1/2/3 week ÷ Weight of PRF at baseline] ×100). The mean weight of PRF and the percentage of the remaining weight were also calculated in each group.
Statistical analysis was carried out with Statistical Package for Social Sciences software [SPSS IBM Corp. Released (2012),Version 21.0, Armonk, NY]. A one-way ANOVA test was used to compare the mean weight and percentage of remaining weight between groups at different time intervals. The statistical significance was set at P < 0.05.
| Results|| |
The mean weight of PRF in Group 1 (Normal saline), Group 2 (200 mg TXA), and Group 3 (250 mg EACA) at baseline, 1st, 2nd, and 3rd weeks were 1.78 ± 0.81, 1.31 ± 0.77, 0.69 ± 0.44, and 0.33 ± 0.21, respectively [Table 1]. In comparison to the baseline, the mean PRF weight was decreased in all groups in the 1st, 2nd, and 3rd weeks.
|Table 1: Mean platelet-rich fibrin weight in each group at different intervals|
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The mean percentage of the remaining weight of PRF in Group 1 (Normal saline), Group 2 (200 mg TXA), and Group 3 (250 mg EACA) in the 1st, 2nd, and 3rd weeks were 69.53 ± 12.96, 36.23 ± 9.28, and 17.43 ± 4.75, respectively [Table 2]. The percentage of the remaining weight of PRF at the 1st, 2nd, and 3rd week was maximum in Group 2 (200 mg TXA) followed by Group 3 (250 mg EACA) and least in Group 1 (NS-Control) [Figure 3]. When the percentage of the remaining weight of PRF in 3rd week was compared statistically between the three groups using ANOVA, statistical significance was obtained with a P = 0.029 [Table 3].
|Figure 3: Difference in percentage of remaining weight of PRF between groups. PRF: Platelet-rich fibrin|
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|Table 2: Percentage of the remaining weight of platelet-rich fibrin in each group at different intervals|
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|Table 3: Difference in percentage of remaining weight of platelet-rich fibrin across groups using ANOVA|
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With the above results, it was found that TXA and EACA were significantly better than the control group in delaying the PRF membrane degradation.
| Discussion|| |
PRF is the widely used autologous biomaterial for periodontal regeneration. The fibrin matrix of the PRF serves as a scaffold for the colonization of mesenchymal stem cells. Numerous data are available in the literature about the clinical and radiographic effectiveness of PRF in treating the intrabody defects of periodontitis patients. PRF is used as a membrane with a coronally advanced flap in the treatment of gingival recession and to increase the width of attached gingiva around teeth as well as implants. PRF degrades rapidly and hence several methods such as heat compression, and antifibrinolytic agents are proposed to prolong the lifespan of PRF.
Yamashita et al. histologically reported that PRF disappeared completely in 28 days of the rat model. Sam et al. evaluated the degradation rate of the collagen membrane and the PRF membrane. PRF membrane maintains its physical property for up to 7 days and degraded about 36% of initial weight at the end of 1st week whereas fish and bovine collagen membranes degraded about 8% and 3% respectively. Panda et al. reported that the combination of PRF with a barrier membrane is more effective to treat intrabony defects in periodontitis patients than the use of a barrier membrane alone.
Senghore and Harris reported that in patients undergoing third molar extraction, an intravenous preoperative dose of TXA is effective in reducing excessive postoperative bleeding. Forbes et al. found that in hemophilia and Christmas disease patients, 1 g TXA for 5 days decreased blood loss and the need for blood transfusions during dental extraction. Sitek et al. compared the stability of PRP-fibrin gel with and without TXA. The gel with TXA remained stable for 28 days whereas without TXA degrades completely. Radha and Varghese reported that TXA 200 mg, 150 mg, and 50 mg showed equal inhibitory effects in inhibiting the degradation of PRF membrane. Dechtham and Aschaitrakool reported that TXA mixed technique increased the longevity of the PRF membrane and it had no negative effect on the weight and appearance of the PRF membrane. EACA 250 mg is the second most commonly used antifibrinolytic agent after TXA in dentistry. Hence, in our study 250 mg EACA was used and compared against 200 mg TXA. Both PRF membranes prepared with and without antifibrinolytic agents were stored in normal saline at room temperature. The normal saline was used as medium because it is isotonic as saliva and storage at room temperature to mimic the temperature of the oral cavity.
In this present study, we proposed that adding two different antifibrinolytic agents may delay the degradation of PRF thereby prolonging the barrier membrane and space maintenance property of PRF. This is the first study to compare the effect of TXA and EACA mixed techniques in inhibiting the degradation of the PRF membrane. We found that TXA-mixed PRF membranes degraded by about 44% and EACA-mixed PRF membranes by about 54% at the end of 3rd week. The degradation rate of TXA and EACA mixed PRF membranes was longer than conventional PRF membranes. This is primarily due to the mechanism of action of antifibrinolytics. Antifibrinolytics block the interaction of plasminogen and plasmin with the lysine residues of the PRF fibrin matrix thereby retards fibrinolysis. However, following the preparation, the concentration of TXA and EACA in the PRF membrane was unknown.
Dohan Ehrenfest et al. reported that PRF contains 97% platelets and more than 50% of WBCs. The weight of PRF decreased in both control and test groups due to the release of platelets and WBCs embedded in the fibrin matrix during fibrinolysis. In our study, the PRF membrane weight gradually reduced in relation to the rate of fibrinolysis.
Limitations of our study include (1) small sample size, (2) PRF degradation may be influenced by local and systemic factors such as heat, pH, and various enzymes, and (3) Under in-vivo conditions, the characteristics and impact of antifibrinolytics on PRF may change, (4) We did not evaluate the impact of antifibrinolytic agents on PRF's ability to release growth factors. Antifibrinolytic agents are contraindicated in pregnancy and systemic diseases such as disseminated intravascular coagulation, severe renal impairment, cerebral hemorrhage, porphyria, and massive hematuria. TXA interacts with oral contraceptives such as conjugated estrogen, ethinyl estradiol, estradiol valerate, and estetrol and exacerbates the thrombotic risk thereby concomitant usage is avoided. It is not recommended to combine TXA and EACA because TXA may intensify the thrombogenic effect of EACA in clinical situations.
| Conclusion|| |
Under experimental conditions, both TXA and EACA prolonged the longevity of PRF membranes. TXA is more effective in inhibiting PRF membrane degradation compared to EACA. In antifibrinolytic agents mixed PRF technique, no negative effects in the appearance and weight of PRF were observed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]