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 International Journal of Dentistry and Oral Health
Sci Forschen
Open HUB for Scientific Researc h ISSN 2378-7090 | Open Access

RESEARCH ARTICLE Volume 8 - Issue 1

Word of Caution: Negative Impact of Mouthwashes on Folded Platelet-Rich

Fibrin (F-PRF) Membrane Viability
Lajos Csönge1,*, Ágnes Bozsik1, Zoltán Tóth Bagi2, Róbert Gyuris3, Dóra Kinga Csönge4, and János Kónya5
1
West Hungarian Regional Tissue Bank, Petz A. University Teaching Hospital, Győr, Hungary
2
Private praxis, Budapest, Hungary
3
Private praxis, Eger, Hungary
4
Pepperdine University, Malibu, CA, USA
5
Dent-Art-Technik Ltd, Győr and Széchenyi University, Győr, Hungary

*Corresponding author: Lajos Csönge, MD, West Hungarian Regional Tissue Bank, Petz A. University Teaching Hospital, Győr, H-9024, Hungary,
E-mail: [email protected]

Received: 29 Nov, 2021 | Accepted: 09 Dec, 2021 | Published: 16 Dec, 2021

Citation: Csönge L, Bozsik A, Bagi ZT, Gyuris R, Csönge DK, et al. (2021) Word of Caution: Negative Impact of Mouthwashes on Folded Platelet-Rich
Fibrin (F-PRF) Membrane Viability. Int J Dent Oral Health 8(1): dx.doi.org/10.16966/2378-7090.385
Copyright: © 2021 Csönge L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract
Background: The number of clinical applications of different Platelet-Rich Fibrin (PRF) membranes has increased in regenerative medicine including
regenerative dentistry. Intact platelets, leukocytes and stem cells of PRF play a crucial role in the local bone augmentation releasing cytokines and
growth factors. An integral part of the post-surgical management is the application of mouthwashes, especially chlorhexidine digluconate, which
is recommended in order to prevent postoperative infections. In some cases, it is possible that there is contact between the mouthwash and PRF
membrane. Therefore, the impact of mouthwashes on cell viability of folded F-PRF was tested.
Method: 3 mouthwash brands were tested: Chorsodyl, Listerine 6 in 1, and Elmex Sensitive Plus using MTT viability assay after 30-second treatment
and 72-hour treatment (twice daily for 30 seconds). The membrane samples were incubated in cell culture conditions.
Results: A 30-second mouthwash treatment significantly diminished the fresh F-PRF viability by 15-21% depending on the agent. After 72 hours of
treatment, the viability loss was ~50%.
Conclusion: The decreased number of platelets and other blood cells cannot launch optimal bone morphogenesis. The MTT assay is a cheap,
reliable, and simple method to assess the platelet and cellular viability and potential regenerative capacity of F-PRF membrane, or any type of
platelet-rich product. The isolation of the PRF membrane from oral liquids and/or application of less aggressive mouthwashes are recommended for
at least 5-7 days after PRF surgery.

Keywords: Mouthwash; F-PRF; Viability; MTT assay; Regenerative dentistry

Abbreviations: CHX: Chlorhexidine; DMEM: Dulbecco’s Minimal Essential Medium; F-PRF: Folded Platelet-Rich Fibrin; Lcs: Leukocytes; MTT
Assay: Methyl Tetrazolium Assay; PBS: Phosphate Buffered Saline; Pls: Platelets; Rbcs : Red Blood Cells

Introduction mouthwash use can destroy it [7,8]. CHX has a deleterious effect on
gingival fibroblast viability, collagen synthesis, and wound healing [9].
Over the last two decades, the number of reports concerning the
clinical application of different platelet-rich membranes (PRF) in Undoubtedly, there is either a short or long-term direct contact
oral surgery and implant dentistry has increased. There are many between liquids in the oral cavity (saliva, drinks, mouthwashes, etc.)
articles on the treatment of oroantral fistulas, preparation of sticky and the implanted PRF membranes through the holes of sutures
bone for augmentation, and post-extraction socket healing as well [1- depending on the speed of epithelialization and closure of wounds. If
6]. The integral part of postoperative management is rinsing using the PRF membrane cannot be separated perfectly from the oral cavity,
Chlorhexidine-Digluconate (CHX) twice or three times daily for 30 there is a chance for contact. Moreover, even some surgical techniques
seconds to kill or decrease the number of pathogenic bacteria, in order left the wound open and margins are not closed primarily after PRF
to prevent postoperative infections [2,4,6]. Sometimes, in spite of membrane implantation [2,5].
doctors’ recommendations, patients use mouthwash more frequently
at home due to postoperative malodor or bad taste in their mouths. The main effect of platelet-rich products is based on the theory of
Mouthwash can modify a healthy oral microbiome, however, frequent regenerative properties of the autologous cells like leukocytes (LCs),

Int J Dent Oral Health | IJDOH 1

 Sci Forschen
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platelets (PLs), and stem cells. They release cytokines and growth to the mitochondrial enzyme activity and, reflective of cell viability,
factors for 2-3 weeks in vitro, playing a crucial role in bone and soft regardless of the cell cycle state.
tissue regeneration [10].
The main aim of the study is to investigate the impact of
PRF is a special mixture of individual cells entrapped in a freshly mouthwashes on F-PRF viability and adapt MTT viability assay in
nascent fibrin clot, which later serves as auto scaffold. Fibrin network platelet-rich product studies.
is permeable for liquids, so these cells are very vulnerable and harmful
agents of mouthwashes can injure or destroy cell viability. The decrease
Materials and Methods
of viable cells diminishes cytokine and growth factor release, and thus Viability assay after 30-second of treatment
limits the chance for an optimal local tissue morphogenesis. In spite
Twelve tubes of blood were collected from one volunteer donor
of many successful reported clinical cases concerning the clinical
into a 9 ml Vacuette tubes without anticoagulants (Gerner BioOne,
application of PRF membranes with postoperative mouthwash
Germany). A folded F-PRF membrane was created from 50 ml plasma
rinsing, it is the few failed cases that compelled us to investigate the as reported earlier [24]. It contained many PLs and LCs as well.
possible reasons as to why. One of those possible reasons is the effect
of postoperative mouthwashes. The membrane was cut into 20 uniform pieces and four groups
were created (consisting of 5 pieces each). Group #1 (G1) served as
The chosen MTT viability assay first described by Mosmann is based a negative control without any treatment. In group 2 (G2) the pieces
on the conversion of yellow tetrazolium salt, 3-(4,5-dimethylthiazol- were submerged into 0.2% chlorhexidine - digluconate (Chorsodyl®,
2-yl)-2,5-diphenyltetrazolium bromide (MTT) to the purple-colored GSK, UK) for exactly 30 seconds at room temperature. In group 3
formazan pigment using mitochondrial enzymes in viable cells [11]. (G3), pieces were submerged into Listerine® 6 in 1 (Johnson & Johnson
The assay has been widely used in studies on chemosensitivity, cell Consumer Inc., USA) and in group 4 (G4) the pieces were submerged
stimulation in immunology, cytotoxicity, fungal, worm, and cellular into Elmex® Sensitive Plus (Colgate-Palmolive, Poland) for 30 seconds
studies [12-19]. at room temperature. None of the mouthwashes contained alcohol.
There have only been a few studies published on solid tissues and After treatment the F-PRF pieces were washed in PBS for 10 seconds
none have examined PRF membranes [16,18,20]. to remove the mouthwash.
Since activity of mitochondrial enzymes involved in the very Subsequently a viability assay was performed to assess the cellular
vulnerable respiratory chain is required for cellular survival, viability of the membrane. A fresh MTT (Sigma, USA) solution was
assessment of this enzymatic activity can be used as a surrogate made, while the powder was dissolved in PBS and stored at 4°C. F-PRF
marker of cell viability. Mitochondrial enzyme activity will decline pieces were incubated separately in 500 µl 0.1% MTT for 1 hour at
and ultimately cease in lethally damaged cells. As autolysis advances, 37°C in 24 well plates. The reaction was halted by transferring the
the activity of mitochondrial enzymes in the respiratory chain specimens into distilled water for 1 minute.
(e.g., succinic dehydrogenase and cytochrome oxidase) approaches The absolute viability index was expressed as optical absorbance
zero in 24 to 36 hours [21-23]. The formazan pigment produced @563 nm/mg F-PRF/ml Cellosolve but in figure 1 it was transformed
by mitochondrial enzymes is not water soluble, but after extraction to relative percent, the fresh 30-second control group (G1) average was
using a suitable organic solvent, its concentration can be measured set at 100%. Unpaired two-sample t-tests were performed to compare
by a spectrophotometer. The maximal absorbance found was @563 viability index differences of the four groups.
nm via spectrophotometer (Jenway 6300, UK). After weighing, the
samples were incubated in 24 well plates overnight at 37°C in 1 ml Viability assay after 3 days
ethylene glycol monomethyl ether (methyl cellosolve, Sigma) in order 20 other F-PRF membrane pieces from 12 tubes of blood were also
to extract the formazan. The resulting color density is proportional assessed after 72 hours of incubation in cell culture conditions in a

Figure 1: Viability results after mouthwash treatment of F-PRF membranes.

Citation: Csönge L, Bozsik A, Bagi ZT, Gyuris R, Csönge DK, et al. (2021) Word of Caution: Negative Impact of Mouthwashes on Folded Platelet-
Rich Fibrin (F-PRF) Membrane Viability. Int J Dent Oral Health 8(1): dx.doi.org/10.16966/2378-7090.385
2
 Sci Forschen
Open HUB for Scientific Researc h
International Journal of Dentistry and Oral Health
Open Access Journal

CO2 incubator (Heracell 150, Germany). The samples were incubated The viability of the cells of F-PRF decreased after a 30-second
in 1 ml Dulbecco’s Minimal Essential Medium (DMEM) with 0.1% mouthwash treatment and the fall was dramatic after 3 days of
trypsin (Sigma, USA) and 0.4 mg/ml EDTA at 37°C in 5% CO2 in treatment in spite of cell culture conditions. Chlorhexidine in
a 24 cell well plate. This condition mimics physiological conditions particular had a negative impact on cell viability but Listerine and
which includes feeding the cells by tissue culture medium (Dulbecco’s Elmex showed similar results without significant difference. The
Minimal Essential Medium-DMEM) and the unfavorable effect of disintegration of the F-PRF membrane in the presence of a proteolytic
proteolytic enzymes (trypsin). The DMEM with trypsin was replaced enzyme was quite slow during the first week but accelerated during the
every day with a fresh one. 2nd week as reported earlier [24]. The samples could not withstand the
double rigor of mouthwash and enzyme treatment. After 72 hours the
Each day 15 samples (3 groups, G2-4) were submerged in
G1 control group lost only 9% of its viability so the difference (9% vs
mouthwashes in the same way as previously described, twice daily
~50%) was the result of the mouthwash treatment.
every 12 hours (7 times in all). After treatment the F-PRF pieces were
washed in PBS for 10 seconds to remove the mouthwash. There is some stress between the conventional postoperative
surgical aim (kill pathogenic bacteria) and the principle of
Between mouthwash baths the F-PRF pieces were kept in the regenerative dentistry (preserve regenerative cell viability). We have
incubator. The control group (G1, 5 pieces) was not treated with to find the balance between these two aspects so dental surgeons can
mouthwash but it followed the same procedure. The viability assay was have a proper approach and thinking on cell levels in PRF surgery.
performed after 3 days of incubation. The chance for better cell survival means better tissue regeneration. In
spite of successful reports using PRF membranes in maxilla without
Results
primary wound closure and postoperative chlorhexidine rinsing, the
The cell viability indexes can be seen in figure 1. There was potential harmful effect of mouthwashes does exist [2,6].
a remarkable decrease even after a short 30 second mouthwash
The lower part of the oral cavity potentially has a longer contact, so
treatment, especially G2 (CHX), as it showed a 22% decrease. In the
in mandible regeneration using PRF membrane alone or in sticky bone
case of Listerine (G3) and Elmex (G4) the viability drop was more
there is probably a higher risk. The ideal rinsing solution prevents
moderate but still reached 15-19%. There was significant difference
postoperative infection but does not have an unfavorable impact on
between G1 and the treated groups (p<0.01).
cells and a healthy microbiome.
After 3 days ~50% viability loss was detected in the Listerine and
chlorhexidine groups. The Elmex treatment showed a better result Conclusion
(56% viability). The viability results presented a significant difference Intact platelets, leukocytes, and stem cells of PRF play a crucial role
(p<0.001) between the control group (G1) and the treated groups (G2- in the local bone augmentation releasing cytokines and growth factors.
4) after 30 seconds and after 72 hours as well. There was no significant The routinely administered postoperative mouthwashes especially
difference (p<0.001) among the 3 treated groups (G2-4). CHX decreases the cell viability of PRF. A 30-second mouthwash
treatment diminished the fresh F-PRF viability significantly by 15-
Discussion
21%. After 72 hours (2 × 30 sec/day) of treatment, the viability loss
The viability of cellular elements of any PRF membrane is crucial was ~50%. The isolation of the PRF membrane from oral liquids and
for satisfactory tissue and bone regeneration. PRF is a kind of transient the application of less aggressive mouthwashes are recommended for
between a cell mixture and a solid tissue. The most numerous cells at least 5-7 days after PRF surgery.
are PLs and LCs but red blood cells (RBCs) and some stem cells can
The in vitro F-PRF viability test results do not show the same results
also be found. In the absence of mitochondria, RBCs do not play an
presumably as in clinical conditions. The tissue culture conditions
important role in this assay. The F-PRF “viability” is the result of mixed
combined with trypsin mimic the effect of oral saliva- but further
viability indexes of different cells. PLs are the most metabolically active investigations are necessary to identify the ideal conditions.
“cells” without nuclei. PLs have 5-8 mitochondria, so they represent
a remarkable viability index in F-PRF apart from LCs [25]. PLs and Declarations
lymphocytes have the highest oxygen consumption rate in oxidative
Statement on ethics approval and consent
phosphorylation while monocytes have a moderate rate and the
neutrophils mostly use glycolysis [26]. The MTT assay is based on the Blood samples were collected with informed consent. Experiments
activity of oxidative phosphorylation enzymes predominantly, so it were in accordance with the ethical standards of the Regional Research
mostly represents the number of active PLs and lymphocytes, and less and Ethic Committee of Győr. Approval date: 18th January 2021. ID
so the neutrophils. number: 2/2021
One of the main advantages of the F-PRF membrane is the Consent for publication
homogenous cell distribution [24]. This was confirmed by the viability
Not applicable
test because the standard deviation (SD) was not too high within the
different experimental groups which means that each sample contained Availability of data and materials
a similar number of cells in comparison to the L-PRF membrane where
The datasets used and/or analysed during the current study are
the cellular distribution is zonal and inhomogeneous [27].
available from the corresponding author on reasonable request.
MTT assay is a cheap, simple, and reliable method and does not
Competing interests
require laborious processing. It provided proper quantitative results
in F-PRF viability testing. The assay is suitable for any platelet-rich The authors declare that they have no competing interests.
product viability investigation to test the impact of any agent on
Funding
cell viability, and can show the predictable potential of local tissue
morphogenesis. Not applicable

Citation: Csönge L, Bozsik A, Bagi ZT, Gyuris R, Csönge DK, et al. (2021) Word of Caution: Negative Impact of Mouthwashes on Folded Platelet-
Rich Fibrin (F-PRF) Membrane Viability. Int J Dent Oral Health 8(1): dx.doi.org/10.16966/2378-7090.385
3
 Sci Forschen
Open HUB for Scientific Researc h
International Journal of Dentistry and Oral Health
Open Access Journal

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LC designed the experiment and adopted the viability assay and
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cell viability: application to the quantitation of cytotoxic and growth
ÁB was involved in the phlebotomy and PRF membrane preparation. inhibitory lymphokines. J Immunol Methods 70: 257-268.
ZTB and RG were involved in study design, search of relevant 13. Klebe RJ, Harris JV (1984) A technically simple “non-lethal” vital
references, and set the clinical consequences of the study. staining procedure for viral plaque and cell transformation assays.
DC performed the viability assay. Brief report. Arch Virol 81: 359-362.

JK was a major contributor in writing the manuscript and in 14. Levitz SM, Diamond RD (1985) A rapid colorimetric assay of fungal
viability with the tetrazolium salt MTT. J Infect Dis 152: 938-945.
searching the optimal laboratory technical requirements.
15. Pagé M, Bejaoui N,Cinq-Mars B, Lemieux P (1988) Optimatization
All authors read and approved the final manuscript.
of the tetrazolium-based colorimetric assay for the measurement
Acknowledgements of cell number and cytotoxicity. Int J Immunopharmac 10: 785-793.
We would like to express our thanks to John Kowalchuk for his 16. Comley JC, Townson S, Rees MJ, Dobinson A (1989) The further
support in editing the manuscript. application of MTT-formazan colorimetry to studies on filarial worm
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Citation: Csönge L, Bozsik A, Bagi ZT, Gyuris R, Csönge DK, et al. (2021) Word of Caution: Negative Impact of Mouthwashes on Folded Platelet-
Rich Fibrin (F-PRF) Membrane Viability. Int J Dent Oral Health 8(1): dx.doi.org/10.16966/2378-7090.385
4

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