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ORIGINAL ARTICLE
Year : 2020  |  Volume : 10  |  Issue : 1  |  Page : 21-27

In vitro cytotoxicity of some hemostatic agents used in apicoectomy to human periodontal ligament and bone cells


1 Division of Endodontics, Faculty of Dentistry, Thammasat University, Pathumthani, Thailand
2 Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand

Date of Submission12-Jan-2019
Date of Decision24-Jan-2019
Date of Acceptance15-Feb-2019
Date of Web Publication27-Dec-2019

Correspondence Address:
Dr. Phumisak Louwakul
Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, 110 Suthep Road, Muang District, Chiang Mai 50200
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sej.sej_8_19

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  Abstract 

Aims: The aim of this study was to test the cytotoxicity of some hemostatic agents used in periapical surgery to primary human periodontal and bone cells.
Materials and Methods: Primary human periodontal ligament and bone cells were divided into five experimental and two control groups. In each of the experimental groups, the cells were cultured in complete media containing various concentrations of different hemostatic agents: epinephrine, aluminum chloride, aluminum sulfate, ferric sulfate, or tranexamic acid. The cytotoxicity was evaluated at 1 min, 5 min, and 24 h. Regular complete medium and sodium hypochlorite were used as positive and negative control groups, respectively. The number of viable cells was investigated using MTT assay. The data were analyzed statistically at the 95% confidence interval.
Statistical Analysis Used: The statistical analysis was done using Kruskal–Wallis and multiple comparisons tests.
Results: The cytotoxicity of all hemostatic agents was time and concentration dependent. Epinephrine and tranexamic acid showed mild or no toxicity to both human periodontal and bone cells at all-time points. Aluminum chloride, aluminum sulfate, and ferric sulfate were moderately to highly toxic to the cells. No significant difference was found between epinephrine and tranexamic acid (P < 0.05).
Conclusions: Epinephrine and tranexamic acid tended to be nontoxic or mildly toxic to periodontal ligament and bone cells. Both of them might be considered as the appropriate hemostatic agents for surgical endodontics.

Keywords: Apicoectomy, bone, hemostasis, periodontal ligament, toxicity


How to cite this article:
Phumpatrakom P, Ariyakriangkai W, Srisuwan T, Louwakul P. In vitro cytotoxicity of some hemostatic agents used in apicoectomy to human periodontal ligament and bone cells. Saudi Endod J 2020;10:21-7

How to cite this URL:
Phumpatrakom P, Ariyakriangkai W, Srisuwan T, Louwakul P. In vitro cytotoxicity of some hemostatic agents used in apicoectomy to human periodontal ligament and bone cells. Saudi Endod J [serial online] 2020 [cited 2020 Apr 4];10:21-7. Available from: http://www.saudiendodj.com/text.asp?2020/10/1/21/274195


  Introduction Top


Endodontic microsurgery is a minimally invasive procedure that results in faster healing and a better patient response than does the traditional technique.[1],[2],[3] The objective of the surgery is to eliminate diseased tissues and obtain an apical seal to prevent the ingress of residual irritants into the periradicular area.[4],[5] Inspection of root-end surface, root-end preparation, and retrofilling procedures needs adequate hemostasis, as well as dryness in the root-end cavity is extremely important.[6] Effective hemostasis enhances vision and root-end management. Besides all of the hemostatic agents, epinephrine has been widely used in the form of epinephrine-impregnated cotton pellets left in the bony crypt.[6] Since epinephrine provides excellent hemostasis, it has a potential risk to cause cardiovascular complications. The amount of epinephrine administered for hemostasis in endodontic microsurgery is approximately 0.21 − 2.38 mg, which is much higher than in other dental surgeries.[7] Therefore, clinicians should recognize the risk and safety of its use. In patients who have cardiovascular risk related to preexisting systemic disease, alternative hemostatic agents may be considered.

Various hemostatic agents have been used in the periapical surgery. These agents may be classified as mechanical, chemical, biological, and resorbable agents. The chemical agents, such as aluminum compounds and ferric sulfate, are commonly used hemostatic agents because they are clinically effective, easy to use, inexpensive, and commercial availability. The mechanism of action of both agents is protein precipitation. The literature shows the efficacy and superior short-term outcome of aluminum chloride over epinephrine.[8],[9] Ferric sulfate, widely used in pulpotomy of the primary teeth, is also effective in hemostasis and encourages a dry field for root-end filling.[10],[11] However, protein precipitates from these chemical agents, when not completely eliminated from the surgical site, and may cause a foreign body reaction that complicates healing.[9]

Tranexamic acid is a synthetic derivative of the amino acid lysine that exerts its antifibrinolytic effect through the reversible blockade of lysine binding sites on plasminogen molecules.[12] It has been used as a mouthwash to prevent bleeding in patients taking warfarin who require dental extractions or oral surgery.[13],[14] It has been found that 4.8% tranexamic acid has high potential to stop bleeding with no systemic involvement.[13]

Clinically, the hemostatic agent is applied directly to periodontal and bone tissues. Ideally, these agents should be nontoxic to the cells and tissues. Therefore, a cytotoxicity test is necessary to determine which agent has minimal toxicity and is appropriate in the endodontic surgical procedure. The purpose of this study was to test the cytotoxicity of some hemostatic agents used in periapical surgery to primary human periodontal and bone cells.


  Materials and Methods Top


Patient recruitment

This study was approved by the Human Experimentation Committee of the Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand (Ethics No. 34/2556). After oral and written informed consent, periodontal tissues were obtained from nonpathologically impacted third molars, with mature roots, that were extracted from healthy patients (aged 18–25 years) for routine purposes. Bone chips were obtained from healthy patients (aged 20–60 years) who required alveoloplasty, torus palatinus, or torus mandibularis removal for prosthodontic reasons. The teeth and bone chips were rinsed using sterile normal saline solution and stored on ice in separate containers with serum-free media. The containers were transported to the laboratory within 1 h and the cell cultures were immediately established by explant culture outgrowth.

Primary cell culture

Extracted teeth were carefully removed from their containers and placed onto separate 35-mm culture dishes (Nunc, Roskilde, Denmark) (one tooth per dish), under a laminar flow. The teeth were held by their crowns with the roots facing upward. Sections of 0.5-mm thickness of periodontal ligament tissue were removed from the apical and midroot areas using a No. 15 surgical blade. Primary human periodontal ligament cells (HPDLCs) were obtained from the extracted teeth. The peeled-off tissues were copiously washed in phosphate-buffered saline (PBS; Merck KGaA, Darmstadt, Germany), cut into small pieces sized 1 mm × 1 mm, transferred into new 35-mm culture dishes and cultured in Dulbecco's modified Eagle's medium containing L-glutamine (Gibco/Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco/Invitrogen) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere of 5% CO2 and 95% air.

To obtain primary human bone cells (HBCs), all bone chips were copiously washed in PBS solution, cut into smaller pieces, and digested in 0.25% trypsin-EDTA (Gibco/Invitrogen) to remove the rest of the adipose tissue and hematopoietic tissue. The bone chips were immediately transferred into 35-mm culture dishes and cultured in the regular complete medium, as previously described, at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

After the HPDLCs and HBCs reached confluence, they were subcultured at 1:3 ratio. Cells from the 3rd to 5th passage were used in the experiment. All experiments were performed at least in triplicate, using cells prepared from three different donors.

Preparation of hemostatic agents

The hemostatic agents used in the experimental groups 1–5 were epinephrine (Government Pharmaceutical Organization, Bangkok, Thailand), aluminum chloride (Ajax Finechem Pty Ltd, Taren Point, NSW, Australia), aluminum sulfate (Ajax Finechem Pty Ltd), ferric sulfate (Ajax Finechem Pty Ltd), and tranexamic acid (OLIC [Thailand] Limited, Ayutthaya, Thailand), respectively. Regular complete medium (Gibco/Invitrogen) and sodium hypochlorite (RCI Labscan, Bangkok, Thailand) were used as positive and negative control groups, respectively. Initial concentrations of epinephrine, ferric sulfate, and tranexamic acid, 0.1%, 31%, and 10%, respectively, were serially diluted with serum-free media into two-fold dilutions. Stock solutions of 30% aluminum chloride and aluminum sulfate were diluted to concentrations of 25%, 20%, 15%, 10%, and 5% sodium hypochlorite, used as negative control, was diluted to 5%, 2.5%, 1%, and 0.5%.

MTT cytotoxicity assay

Briefly, the HPDLCs and HBCs were separately seeded into 96-well plates at 5000 cells/well in regular complete media. The number of wells per sample and time of exposure was six. The cells were exposed to one of the test agents at specific time intervals of 1 min, 5 min, and 24 h. Cell morphology was monitored under an Olympus CK40 microscope (Olympus, Melville, NY, USA). Cell viability assay was measured using colorimetric qualification of 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich). Briefly, freshly mixed MTT solution (5 mg/mL MTT powder in PBS solution) was added to cells at 100 μL/well and incubated at 37°C and 5% CO2 for 3 h. After the designated time, each well was flushed with sterile PBS and added with 100 μL of dimethyl sulfoxide (Sigma-Aldrich). The mixed solution was measured using a spectronanometer with an absorbance at 550 nm (Sunrise; Tecan, Mannedorf, Switzerland). The optical density was measured and expressed as percentages of the positive control. Cytotoxicity was rated based on cell viability relative to the control, as described by Dahl et al.:[15] noncytotoxic >90% cell viability, slightly cytotoxic 60%–90% cell viability, moderately cytotoxic 30%–59% cell viability, and severely cytotoxic <30% cell viability.

Statistical analysis

The data were analyzed using the Kruskal–Wallis and multiple comparisons tests, using IBM SPSS statistics version 20 software (IBM Corporation, Armonk, NY, USA). The difference between experimental groups was considered to be statistically significant at P < 0.05.


  Results Top


The morphology of HPDLCs or HBCs under a light microscope was a mixed population containing both spindle-shaped cells and polygonal-like cells. Normal cell configurations were observed in the epinephrine and tranexamic acid groups, whereas protein precipitates were observed and obscured the cell morphology in the aluminum chloride, aluminum sulfate, and ferric sulfate groups [Figure 1].
Figure 1: Cell morphology after exposure to several hemostatic agents. (a) Epinephrine, (b) tranexamic acid, (c) aluminum chloride, (d) aluminum sulfate, (e) ferric sulfate, and (f) culture medium (positive control)

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Cytotoxicity of the hemostatic agents to HPDLCs and HBCs is shown in [Figure 2]. The pattern of response of both cell types was similar and tended to be dose dependent. However, the HBCs were more resistant to the hemostatic agents than the HPDLCs. At 1 and 5 min, both epinephrine and tranexamic acids were slightly toxic to HPDLCs but nontoxic to HBCs. At 24 h, they were highly toxic to the HPDLCs but moderately toxic to HBCs. Both aluminum chloride and aluminum sulfate had moderate toxicity to the HPDLCs at 1 and 5 min and highly toxic at 24 h. Both agents were moderately toxic to the HBCs at all-time points. Ferric sulfate was highly toxic to the HPDLCs but moderately toxic to the HBCs at all-time points.
Figure 2: Cytotoxicity of several hemostatic agents. Human periodontal cells (Left column) and human bone cells (Right column) were treated with the indicated doses of the hemostatic agents at 1 and 5 min and 24 h. The X-axis demonstrates the concentrations of the hemostatic agents. The Y-axis demonstrates the ratios of cell viability in the treated samples relative to those of the controls, set to 100. Error bars = SD

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Most of the clinical concentrations were significantly more toxic than the diluted agents [Figure 2]. One-minute exposure of epinephrine was slightly toxic to the HPDLCs. The epinephrine (5 min), aluminum sulfate, and tranexamic acid were moderately toxic but were significantly less toxic than the aluminum chloride and ferric sulfate (P > 0.05) as shown in [Figure 3]. For the HBCs, epinephrine and 1-min exposure of tranexamic acid had slight toxicity. Aluminum chloride was moderately toxic and significantly higher toxic than the others (P < 0.05).
Figure 3: Cytotoxicity of clinical dosages of the hemostatic agents at 1 and 5 min and 24 h. Human periodontal cells (Upper figure) and human bone cells (Lower figure) were treated with the clinical doses of the hemostatic agents at 1 and 5 min and 24 h. The X-axis demonstrates the concentrations of the hemostatic agents. The Y-axis demonstrates the ratios of cell viability in the treated samples relative to those of the controls, expressed in percentages. Error bars = SD

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  Discussion Top


MTT assay was used in this study to measure the cytotoxicity of various hemostatic agents to the cells. It is a colorimetric method that measures cell metabolic activity, based on the ability of mitochondrial succinate dehydrogenase to reduce the tetrazolium salt to its insoluble formazan crystal, which has a purple color. The assay is a quick, reproducible, and reliable method and thus may be considered as the gold standard of cell viability tests. There are some disadvantages to this method: the conversion to formazan depends on metabolic rate and on the number of mitochondria, resulting in many known interferences, the possibility of fluorescent interference from compounds being tested, and the numerous wash steps required.[16]

According to the ISO standard 10993-5, a cytotoxicity test should be carried out at the 24-h time point. At that time point, the clinical concentrations of all tested hemostatic agents were considered highly toxic to periodontal cells and moderately toxic to bone cells. Dilution of the agents decreased toxicity to those cells. These findings may be useful for further experiments. However, in clinical situations, application of the hemostatic agents in periapical surgery should be within 5 min of the occurrence of bleeding. Therefore, the critical time intervals selected in this study were only 1 and 5 min.

Considering the clinical concentrations of the hemostatic agents at 1 and 5 min, epinephrine was the least toxic agent to both HPDLCs and HBCs. Together with its excellent hemostasis, epinephrine should be considered as the first-choice hemostatic agent used in periapical surgery.[17],[18] Epinephrine has been used in humans for several purposes, but its use in cardiovascular patients involves some risk. Some evidence shows that it produces effective hemostasis with no evident change in blood pressure or pulse rate.[7],[11] Nevertheless, in patients with underlying cardiovascular diseases, the amount of epinephrine administered for hemostasis in endodontic microsurgery is approximately 0.21 − 2.38 mg, much higher than for other dental surgeries,[7] and may exert some effects on the systemic circulation.[19] Therefore, alternative hemostatic agents may be considered in patients who have cardiovascular risk related to preexisting systemic diseases.

Both aluminum compounds and ferric sulfate have also been successfully used as hemostatic agents in endodontic surgery.[8],[9],[11] At 1 and 5 min, aluminum chloride and aluminum sulfate were moderately to severely toxic to both types of cells, but ferric sulfate was highly toxic to HPDLCs. This result was consistent with that of a previous study,[15] in which ferric sulfate was the most toxic agent to gingival fibroblasts, followed by aluminum chloride and aluminum sulfate. Besides, the cytotoxicity of these hemostatic agents was concentration and time dependent.[20] Real-time, cell-analyzing research has confirmed that aluminum chloride-based hemostatic agents have a significant cytotoxic effect on gingival fibroblast cells at 48-h and 72-h time points.[21] Another study showed that 25% aluminum chloride was the most toxic agent to the gingival tissue of beagles when compared to 10% aluminum chloride and 20% aluminum sulfate.[22]

Direct contact of the aluminum compounds and ferric sulfate with the culture media caused flocculation that interfered with the observation of the cell morphology under light microscopy. However, we noticed that the cells were round and shrunken, shapes which were abnormal, and which confirmed the cytotoxicity of these agents. The mechanism causing the flocculation is due to the ability of these agents to induce protein coagulation and precipitation, which result in occlusion of small vessels, and hemostasis. However, these precipitates are highly toxic and may cause bone destruction and delayed wound healing if they were left in situ.[9]

Tranexamic acid, at the concentration of 4.8%, was mildly to moderately toxic to HPDLCs and HBCs. However, lower concentrations of tranexamic acid were nontoxic or mildly toxic to both types of cells. This finding is consistent with that of a previous study, which showed that 10% tranexamic acid was toxic to bovine explanted cartilage and murine chondrocytes, but 2.5% tranexamic acid did not affect cell viability.[23] Another study reported that tranexamic acid, at low concentrations, had no effect on cartilage explants in a minipig model.[24] These results suggest that tranexamic acid may be the best alternative hemostatic agent because of its lower cytotoxicity and its effective hemostasis.

This is an in vitro study that may be a useful preliminary screening method to identify the cytotoxicity of hemostatic agents; however, an in vitro study cannot provide data that are directly applicable to humans. It is difficult to establish an in vitro system with direct relevance to in vivo circumstances because isolated and cultivated primary cells usually differ strongly from the corresponding cell type in an organism. Therefore, the application of this study in vivo should be further investigated. In addition, further studies are also needed to investigate the hemostatic potential of different concentrations of tranexamic acid.


  Conclusions Top


Within the limitations of this study, it may be concluded that cytotoxicity of five different hemostatic agents was dose- and time-dependent. Epinephrine and tranexamic acid were none to mildly toxic to HPDLCs and HBCs and therefore should be considered as the most appropriate hemostatic agents for surgical endodontics.

Financial support and sponsorship

This study was financially supported by Intramural Endowment Fund, Faculty of Dentistry, Chiang Mai University.

Conflicts of interest

There are no conflicts of interest.

 
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