Word of caution: negative impact of mouthwashes on folded Platelet-Rich Fibrin (F- PRF) membrane viability

doi:10.21203/rs.3.rs-990808/v1

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Word of caution: negative impact of mouthwashes on folded Platelet-Rich Fibrin (F- PRF) membrane viability

https://doi.org/10.21203/rs.3.rs-990808/v1

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Backround: The number of clinical application 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 postsurgical management is the application of mouthwashes especially chlorhexidine-digluconate, which is recommended in order to prevent postoperative infections. In some cases there is possibility that there is contact between the mouthwash and PRF membrane. The impact of mouthwashes on cell viability of folded F-PRF was tested.

Methods: 3 mouthwash brands were tested: Chorsodyl, Listerine 6 in 1 and Elmex Sensitive Plus using MTT viability assay after 30 seconds treatment and 72 hours treatment (twice daily for 30 seconds). The membrane samples were incubated in cell culture conditions.

Results: 30 seconds of mouthwash treatment diminished the fresh F-PRF viability significantly 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 can not launch optimal bone morphogenesis. The MTT assay is cheap, reliable and simple method to assess the platelet and cellular viability and potential regenerative capacity of F-PRF membrane, or any platelet-rich product. The isolation of the PRF membrane from oral liquids and/or application of less aggressive mouthwashes is recommended for at least 5-7 days after PRF surgery.

Dentistry

Head & Neck Surgery

mouthwash

F-PRF

viability

MTT assay

regenerative dentistry

During the last two decades the number of reports concerning the clinical application of different platelet-rich membranes (PRF) in oral surgery and implant dentistry has increased. There are many articles on the treatment of oroantral fistulas, preparation of sticky bone for augmentation and postextraction socket healing as well [1–6]. The integral part of the postoperative management is the bleaching using chlorhexidine (CHX) – digluconate twice or three times daily for 30 seconds to kill or decrease the number of the pathogenic bacteria in order to prevent postoperative infections [2, 4, 6]. Sometimes in spite of the doctors’ recommendations the patients use mouthwashes more frequently at home due to postoperative malodor or bad flavor. Mouthwash can modify a healthy oral microbiome however frequent mouthwash use can destroy it [7, 8]. CHX has a deleterious effect on gingival fibroblast viability, collagen synthesis and wound healing [27].

Undoubtedly there is a short or long-term direct contact between liquids in the oral cavity (saliva, drinks, mouthwashes etc.) and the implanted PRF membranes through the holes of sutures depending on the speed of epithelization and closure of wounds. If the PRF membrane cannot be separated perfectly from oral cavity there is a chance for contact. Even some surgical techniques leave the wound opened and margins were not closed primarily after PRF membrane implantation [2, 5].

The main effect of platelet-rich products is based on the theory of regenerative properties of the autologous cells like leukocytes (LCs), platelets (PLs) and stem cells. They release cytokines and growth factors for 2-3 weeks in vitro after play crucial role in the bone and soft tissue regeneration [9].

PRF is a special mixture of individual cells entrapped in a freshly nascent fibrin clot which later serves as autoscaffold. Fibrin network is permeable for liquids so these cells are very vulnerable and the harmful agents of mouthwashes can injure or destroy the cell viability. The decrease of viable cells diminishes the cytokine and growth factor release and the chance for an optimal local tissue morphogenesis. In spite of many successful clinical cases that were reported concerning the clinical application of PRF membranes with postoperative mouthwash bleaching, some failed cases forced us to investigate the possible reasons. One of the possible reasons is the effect of postoperative mouthwashes.

The chosen MTT viability assay first described by Mosmann is based on the conversion of yellow tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to the purple colored formazan pigment using mitochondrial enzymes in viable cells [10]. The assay has been widely used in studies on chemosensitivity, cell stimulation in immunology, cytotoxicity, fungal, worm and cellular studies [11–18] .

Few studies on solid tissues were published, but have not examined any PRF membrane [15, 17, 19].

Since activity of mitochondrial enzymes involved in the very vulnerable respiratory chain is required for cellular survival, assessment of this enzymatic activity can be used as a surrogate marker of cell viability. Mitochondrial enzyme activity will decline and ultimately cease in lethally damaged cells. As autolysis advances, the activity of mitochondrial enzymes in the respiratory chain (e.g., succinic dehydrogenase and cytochrome oxidase) approaches zero in 24 to 36 hours [20–22].

Viability assay after 30 seconds treatment

Twelve tubes of blood were collected from a volunteer donor into 9 ml Vacuette tube without anticoagulants (Gerner BioOne, Germany). Folded F-PRF membrane was created from 50 ml plasma as reported earlier [23]. It contained many PLs and LCs as well.

The membrane was cut into 20 uniform pieces and four groups were created (consisting of 5 pieces each). Group #1 (G1) served as negative control without any treatment. In G2 the pieces were submerged into 0.2% chlorhexidine - digluconate (Chorsodyl®, GSK,UK) for exactly 30 seconds at room temperature. Same process was performed in G3 with Listerine® 6 in 1 (Johnson&Johnson Consumer Inc.,USA) and in G4 with Elmex® Sensitive Plus (Colgate-Palmolive, Poland). None of them contained alcohol. After treatment the F-PRF pieces were washed in PBS for 10 seconds to remove the mouthwash.

Subsequently a viability assay was performed to assess the cellular viability of the membrane. A fresh MTT (Sigma, USA) solution was made, the powder was dissolved in PBS and stored at 4°C. F-PRF pieces were incubated separately in 500 µl 0.1% MTT for 1 hour at 37°C in 24 well plates. The reaction was halted by transferring the specimens into distilled water for 1 minute.

The formazan pigment produced by mitochondrial enzymes is not water soluble, but after extraction using a suitable organic solvent, its concentration can be measured by spectrophotometer. The maximal absorbance was found @563 nm in spectrophotometer (Jenway 6300, UK). After weighing, the samples were incubated in 24 well plates overnight at 37°C in 1 ml ethylene glycol monomethyl ether (methyl cellosolve, Sigma) in order to extract the formazan. The resulting color density is proportional to the mitochondrial enzyme activity, reflective of cell viability, regardless of cell cycle state. The absolute viability index was expressed as optical absorbance @563 nm/mg F-PRF/ml Cellosolve but in Figure 1 it was transformed to relative %, the fresh 30 second control group (G1) average was set at 100%.

Viability assay after 3 days

20 other F-PRF membrane pieces from 12 tubes of blood were also assessed after 72 hours of incubation in cell culture conditions in a CO₂ incubator (Heracell 150, Germany). The samples were incubated in 1 ml Dulbecco’s Minimal Essential Medium (DMEM) with 0.1% trypsin (Sigma, USA) and 0.4 mg/ml EDTA at 37°C in 5% CO₂ in a 24 cell well plate. This condition mimics the physiological conditions including feeding the cells by tissue culture medium (Dulbecco’s Minimal Essential Medium-DMEM) and the unfavorable effect of proteolytic enzymes (trypsin). The DMEM with trypsin was replaced every day with a fresh one.

Each day 15 samples (3 groups, G2-4) were submerged in mouthwashes in the same way as previously described, twice daily every 12 hours (7 times all). After treatment the F-PRF pieces were washed in PBS for 10 seconds to remove the mouthwash.

Between the mouthwash baths the F-PRF pieces were kept in the incubator. The control group (G1, 5 pieces) was not treated with mouthwash but it was managed in the same way. The viability assay was performed after 3 days of incubation.

The cell viability indexes can be seen in Figure 1. There was a remarkable decrease even after a short 30 second mouthwash treatment, especially G2 (chlorhexidin) as it showed a 22% decrease. In the case of Listerine (G3) and Elmex (G4) the viability drop was more moderate but still 15-19%. There was significant difference between G1 and the treated groups (p<0.01).

After 3 days ~50% viability lost was detected in the Listerine and chlorhexidine group. Elmex treatment showed a better result (56% viability). The viability results presented significant difference (p<0.001) between the control group (G1) and the treated groups (G2-4) after 30 seconds and 72 hours as well. There was no significant difference among the 3 treated groups (G2-4).

The viability of cellular elements of any PRF membrane is crucial for satisfactory tissue and bone regeneration. PRF is a kind of transient between a cell mixture and a solid tissue. The most numerous cells are PLs, and LCs but red blood cells (RBCs) and some stem cells are also found. In the absence of mitochondria, RBCs do not play important role in this assay. The F-PRF “viability” is the result of mixed viability indexes of different cells. PLs are the most metabolically active “cells” without nuclei. PLs have 5-8 mitochondria so they represent a remarkable viability index in F-PRF apart from LCs [24]. PLs and lymphocytes have the highest oxygen consumption rate in oxidative phosphorylation while monocytes have a moderate one and the neutrophils mostly use glycolysis [25]. The MTT assay is based on the activity of oxidative phosphorylation enzymes predominantly, so it represents the number of active PLs and lymphocytes mostly and less the neutrophils.

One of the main advantages of the F-PRF membrane is the homogenous cell distribution [23]. It was confirmed also by the viability test because the standard deviation (SD) was not too high within the different experimental groups. It means that each sample contained a similar number of cells in comparison to the L-PRF membrane where the cellular distribution is zonal and inhomogeneous [26].

MTT assay is a cheap, simple and reliable method and does not require laborious process. It provided proper quantitative results in F-PRF viability testing. The assay is suitable for any platelet-rich product viability research to test the impact of any agent on cell viability and can show the predictable potential of local tissue morphogenesis.

The viability of the cells of F-PRF decreased after a 30 second mouthwash treatment and the fall was dramatic after 3 days of treatment in spite of cell culture conditions. Chlorhexidine especially had negative impact on cell viability but Listerine and Elmex showed similar result without significant difference. The disintegration of the F-PRF membrane in the presence of proteolytic enzyme is quite slow during first week but accelerates during 2nd week as reported earlier [23]. The samples could not withstand the double rigor of mouthwash and enzyme treatment. After 72 hours the G1 control group lost only 9% of its viability so the difference (9% vs ~50%) is the result of the mouthwash treatment.

There is some stress between the conventional postoperative surgical aim (kill pathogenic bacteria) and the principle of regenerative dentistry (preserve the regenerative cell viability). We have to find the balance between the two aspects so dental surgeons should have a proper approach and thinking also on cell level in PRF surgery. The chance for better cell survival means better tissue regeneration. In spite of successful reports using PRF membranes in maxilla without primarily wound closure and postoperative chlorhexidine bleaching the potential harmful effect of mouthwashes does exist [2, 6].

The low part of oral cavity has a longer potential contact so in mandible regeneration using PRF membrane alone or in sticky bone there is probably a higher risk. The ideal bleaching solution prevents the postoperative infection but does not have an unfavorable impact on cells and healthy microbiome.

Conclusion: Intact platelets, leukocytes and stem cells of PRF play a crucial role in the local bone augmentation releasing cytokines and growth factors. The routinely administered postoperative mouthwashes especially CHX decreases the cell viability of PRF. 30 seconds of mouthwash treatment diminished the fresh F-PRF viability significantly by 15-21%. After 72 hours (2x30 sec/day) of treatment the viability loss was ~50%. The isolation of PRF membrane from oral liquids and the application of less aggressive mouthwashes are recommended for at least 5-7 days after PRF surgery.

CHX: chlorhexidine

DMEM-Dulbecco’s Minimal Essential Medium

F-PRF: folded platelet-rich fibrin

LCs: leukocytes

MTT assay: methyl tetrazolium assay

PLs: platelets

RBCs: red blood cells

Statement on ethics approval and consent

Blood samples were collected with the informed consent. Experiments were in accordance with the ethical standards of the Regional Research and Ethic Commitee of Győr.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

Not applicable

Authors' contributions

LC designed the experiment and adopted the viability assay and interpreted the data.

ÁB was involved in the phlebotomy and PRF membrane preparation.

ZTB and RG were involved in study design, search of relevant references and set the clinical consequences of the study.

DC performed the viability assay.

TK and JK were a major contributor in writing the manuscript and in searching the optimal laboratory technical requirements.

All authors read and approved the final manuscript.

Acknowledgements

We would like to express our thanks to John Kowalchuk for his support in editing the manuscript.

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No competing interests reported.

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Word of caution: negative impact of mouthwashes on folded Platelet-Rich Fibrin (F- PRF) membrane viability

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Abstract

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Introduction

Materials And Methods

Viability assay after 30 seconds treatment

Viability assay after 3 days

Results

Discussion

Abbreviations

Declarations

References

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