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 Table of Contents  
ORIGINAL ARTICLE
Year : 2011  |  Volume : 17  |  Issue : 4  |  Page : 149-154

Evaluation of the cytotoxicity of ventilation tube, nasal packing, and spongostan used in ear-nose-throat surgeries


1 Department of Ear, Nose and Throat, Education and Research Hospital, Sivas, Turkey
2 Department of Microbiology, University of Cumhuriyet, Sivas, Turkey

Date of Web Publication29-Mar-2012

Correspondence Address:
Emine Elif Altuntas
Department of Otorhinolaryngology, Cumhuriyet University School of Medicine, Sivas, TR-58140
Turkey
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Source of Support: The present study was supported by Cumhuriyet University Scientific Research Projects (CÜBAP) Project no T-408, Conflict of Interest: None


DOI: 10.4103/0971-7749.94492

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  Abstract 

Purpose: The aim of this study was to determine biocompatibility of spongostan, merocell, and ventilation tube using agar diffusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide tests in cell culture and to evaluate the results in terms of clinical practices. Materials and Methods: The experimental procedures involved in this study were approved by the Animal Ethics Committee of Cumhuriyet University (B.30.2.CUM.0.01.00-50/4), and the study was conducted following accepted guidelines for the care and use of laboratory animals for research. The present study was supported by Cumhuriyet University Scientific Research Projects (CÜBAP) Project no T-408. L929 mouse fibroblast cell culture was used in the present study. In this study, indirect toxic effects of the leakage products from the materials were tested with the agar overlay test, and also direct toxic effects of leakage materials occurring at different time and leakage products from the materials were tested with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Results: As a result of evaluating the 21 days' leakage material of the ventilation tube a moderate (+2), 24 and 48 h of leakage material of the merocell low degree and 24-hour leakage material of the spongostan limited cytotoxicity were found. Conclusion: Many biomaterials cytotoxicity study were found in the literature as well as in our study shown by MTT and agar diffusion tests they were not toxic.

Keywords: Agar overlay, Cytotoxicity, Merocell, MTT test, Spongostan, Ventilation tube


How to cite this article:
Altuntas EE, Sümer Z. Evaluation of the cytotoxicity of ventilation tube, nasal packing, and spongostan used in ear-nose-throat surgeries. Indian J Otol 2011;17:149-54

How to cite this URL:
Altuntas EE, Sümer Z. Evaluation of the cytotoxicity of ventilation tube, nasal packing, and spongostan used in ear-nose-throat surgeries. Indian J Otol [serial online] 2011 [cited 2019 Apr 23];17:149-54. Available from: http://www.indianjotol.org/text.asp?2011/17/4/149/94492


  Introduction Top


Today, widespread use of artificial biomaterials in close contact with living tissues brings together the concept of biocompatibility. Materials designed to be implemented into a living system and to perform functions of organs and tissues partially or wholly are called biomaterials, and these materials are required to be biologically compatible, non-toxic or non-carcinogenic, chemically inert and stable, have sufficient mechanical strength and adequate weight and density, produced in large numbers, have an easy fabrication process, and be cost effective. [1]

The capability of a biomaterial placed in a living tissue to stay inert without causing any change in the surrounding soft or hard tissue is called biocompatibility. [2],[3],[4] Biological reaction occurring against a biomaterial placed on or in body tissues is evaluated by in vitro biocompatibility tests and the most important step in evaluating biocompatibility is choosing the appropriate test method. [5],[6],[7],[8],[9]

Although we found some animal studies in literature evaluating biocompatibility of spongostan, merocell, and ventilation tube used in ear-nose-throat surgeries and investigating whether they have any negative effects on the success rate of surgery, we could not find any cytotoxicity studies carried out with cell culture and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT test) test. [10],[11],[12],[13]

The MTT test is widely used in the assessment of drugs and chemicals, but is less well accepted in the study of toxicity of biomaterials. If so, why did we want to apply the MTT test for the evaluation of cytotoxicity? Spongostan, merocell, and ventilation tube are in contact with body fluids and tissues for a long time. The structural elements of these materials whether the release or not is unknown in this process. For this reason, our aim was to determine biocompatibility of these three materials using agar diffusion and MTT tests in cell culture and to evaluate the results in terms of clinical practice.


  Materials and Methods Top


In the present study, time-dependent cytotoxic effects of non-absorbable ventilation tube (Shepard design TE-7011 lot no: #0594), nasal packing (Merocel, REF:400402X1, Lot no:68076200), and absorbable spongostan (absorbable gelatin sponge, lot no: GS010T 00415/1) used in ear-nose-throat surgeries were studied in vitro using agar-overlay and MTT tests measuring the activity of mitochondrial succinate dehydrogenases.

Preparation of study materials

Ventilation tube as a whole, spongostan in 1x1 cm 2 pieces, and nasal packing cut into 0.5-cm pieces were placed into sterilized tubes; 5 mL phenol-free Dulbeccos's Modified Eagle's Medium (DMEM) was added; and leakage materials were collected after storing the ventilation tube for 21 days, spongostan for 24 h and 7 and 14 days, and nasal packing for 24 and 48 h at +37 o C.

Cell culture

L929 mouse fibroblast cell culture was used in the present study.

Preparation of the media used

The media used for cell culture was prepared by adding 1 mL penicillin-streptomycin (PAA cat no: P11-010, lot no: P01009-1013) and 10 mL fetal bovine serum (FBS) (Biological industries lot no: 715442) into 89 mL DMEM (PAA: cat no: E 15-806, lot no: E 80609-2477).

Preparation of the passage

Passages were made to maintain the L929 mouse fibroblast cell lines to be used during the study and to keep these cells live. To create the passages, the media in the flask where cells adhere and grow was aspirated, cells were washed by FBS-free-DMEM, and rinsed using trypsin/EDTA solution (0.05% trypsin + 0.02% EDTA) (Multical cat: 325.042-E1, lot no: 325042081). Trypsin was aspirated and placed in an incubator at 37 o C for 5 min to allow the cells to detach from the surface of the flask. Cell suspension was obtained by adding DMEM. The cell suspension prepared was passaged by dividing into two flasks. The same process was repeated by following the cell proliferation in the cell culture plates and the cell culture line was maintained.

Agar overlay test

The aim of this test was to determine, using agar as the media, the indirect toxic effects of the leakage products from the materials tested. ISO 2009 10933-5 protocol was used in determining cytotoxicity by agar overlay method. [14],[15]

According to the said protocol, three ventilation tube samples and seven spongostan and merocell samples of 0.25 cm 2 were cut in a sterile environment to test their effects directly on the cells. To study the leakage materials, the samples prepared using the said materials were kept in neutral red-free 5 mL DMEM (ventilation tube kept for 21 days, spongostan for 24 h and 7 and 14 days, and nasal packing for 24 and 48 h), and leakage samples were collected. These samples were used in agar overlay test by loading 50 μ of these samples onto 6-mm Whatmann Paper No 4.

Preparation of 1% agar

1% agar was prepared with FBS-free 10 mL DMEM and 0.1 gr agar (Agarose, Prona Lot:71447PR), sterilized in 120 o C autoclave for 25 min, and 2 mL FBS was added after cooling to 37-38 o C. DMEM in cell  Petri dish More Detailses was aspirated and 0.5 mL agar was added to each. Cell petri dishes were incubated for 30 min for the agar to solidify.

Preparation of 0.01% neutral red solution

0.01% neutral red solution was prepared with FBS-free 15 mL DMEM and 0.5 mL neutral red (Neutral red, Biochrom KG, Germany, Lot no: 079 B).

Application of the test

Cells grown in flasks were transferred to cell culture petri dishes having a diameter of 3.5 cm with a target of 2.5 × 10 5 cell/mL and incubated for 24-48 h in a 5% CO 2 incubator. After the cells covered the base of the petri dish, the plating media was removed and cells were covered with 0.5 mL agar. Whatmann papers soaked with leakage material were placed in the middle of the petri dish after the agar solidified, and the petri dish was incubated for 24 h at 37 o C. At the end of 24 h, 1 mL neutral red solution was added to each petri dish and 30 min was allowed for the dye to diffuse into cells. Colour alterations in petri dishes were examined macroscopically. After the excess dye was aspirated, a microscopic evaluation was also done and photographed [Figure 1]. Zone index was scored. [16],[17]
Figure 1: (a) Fibroblast cells reproduce in the base of plate after application of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (×10). (b) After application of MTT assay Formosan crystals collapsed cells (×10). (c ) Unpainted cell reproduce in the base of petri dish for agar overlay test. (d) Neutral red dye taken living cells for agar overlay test (e) Living cells that not taken neutral red dye for agar overlay. (f) Cytotoxicity (-) view in the agar overlay test (g) Cytotoxicity (+1) view in the agar overlay test (toxic limit)

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According to this, Decolorization Zone Index Scores: 0=no decolorization zone, 1=decolorization zone only under the disc, 2=decolorization zone maximum 5 mm around the disc, 3=decolorization zone maximum 5-10 mm around the disc, 4=decolorization zone more than 10 mm around the disc, 5=full decolorization of the culture petri dish.

In order to determine whether the reaction occurring in cells is reversible or irreversible, 1 mL 0.01% neutral red solution was added to each petri dish and the cells were allowed to stain for 30 min. Excess dye was removed and macroscopic and microscopic controls were repeated.

MTT test

Toxic effects occurring at different times as a result of a direct contact of leakage materials were determined by this test.

In determining toxic effects by MTT, the solution in which the leakage products got collected was prepared by adding FBS, L-glutamine, and penicillin-streptomycin into phenol red-free DMEM. [14],[15],[18] The said solution was also used as cell culture media.

The leakage material collected was frozen to -20 o C until the cytotoxicity test. Cells grown in flasks were scraped off the surface and transferred to cell culture plates with a target of 2 × 10 5 cell/mL and 100 μL of samples were transferred to 96 cell culture plates and incubated for 24 h at 37 o C in 5% CO 2 incubator for them to cover the surface. Base of the wells were covered with cell, media was removed, and 100 μL of leakage materials were placed into each well followed by incubation for another 24 h at 37 o C in 5% CO 2 incubator. After the incubation, 100 μL of 5 mg/mL DMSO was added into each well and incubated for 4 h at 37 o C in 5% CO 2 incubator followed by adding 10 μL DMSO on each well. When formazan crystals were fully dissolved, microplates were shaken gently for 10 min at vertex until color changed from yellow to blue/purple. Optical density of formazan in microplates was assessed using a 450 nm wavelength ELISA (EL 312 Mikroplate, Bio-Tek, Biokinetics Reader) reader.

Ethical considerations

The present study was supported by Cumhuriyet University Scientific Research Projects (CÜBAP) Project no T-408. The experimental procedures involved in this study were approved by the Animal Ethics Committee of Cumhuriyet University (B.30.2.CUM.0.01.00-50/4), and the study was conducted following accepted guidelines for the care and use of laboratory animals for research.

Statistical analysis

SPSS 14.0 programs (SPSS Inc., Chicago, IL, USA) were used to record and evaluate the results obtained. Kruskal Wallis variance analysis was used for the data obtained in MTT test while one-way variance analysis (ANOVA) test and Wilcoxon test were used for repeated measures.

ANOVA was used for the results of the leakage materials from the study materials (21 days for ventilation tube, 24 and 28 hours for merocell, and 24 hours and 7 and 14 days for spongostan) while Mann Whitney U Test was used to detect the group causing the difference.


  Results Top


Results obtained in MTT test

Optical Density (OD) values (450 nm) of the cytotoxicity data of the leakage samples of the study materials determined by MTT test are shown in [Table 1].
Table 1: Optical density values (450 nm) of the cytotoxicity data of the leakage samples of the study materials determined by MTT test (450 nm)

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In the evaluation of the results of MTT test performed on 21 days' leakage material of the ventilation tube, the difference between the groups was found to be statistically significant (P<0.05). Results obtained from the positive control group were the ones causing the difference. As a result of evaluating the 21 days' leakage material of the ventilation tube by agar overlay method, a moderate (+2) cytotoxicity, which was parallel to the one in MTT test, was found. Evaluation of 24 and 48 hours' leakage materials of nasal packing revealed a statistically significant difference between positive and negative control groups (P<0.05). According to this, although a low cytotoxicity, which was parallel to that in MTT was observed, when 24 and 48 hours' leakage materials were evaluated by agar overlay method, 24 and 28 hours' leakage materials were not cytotoxic as the difference was not statistically significant when compared with non-intervened group.

In the evaluation of spongostan leakage materials by MTT test, the difference between the groups was found to be statistically significant (P<0.05). However, results obtained from the negative control groups were the cause of the said difference. No statistically significant difference was observed between 7 and 14 days' spongostan leakage samples and positive controls and non-intervened group (P>0.05). While the difference between the initial 24 hours leakage samples and positive controls was statistically significant, there was no difference between the said 24 hours leakage samples and non-intervened group. For this reason, while a limited cytotoxicity was observed in the initial 24 hours' spongostan leakage material, no cytotoxicity was found in 7 and 14 days' leakage materials.

Results obtained in the agar overlay test are summarized in [Table 2].
Table 2: Cytotoxicity evaluation of the substances with agar overlay test

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  Discussion Top


The number of biomaterials used in medicine has increased rapidly in recent years parallel to technological developments. Biomaterials are mostly used in vitro as a part of or complementary to a surgical procedure. When a biomaterial is placed into a human being's body for any reason, this causes at the histopathological level a scar formation, acute inflammation, chronic inflammation, granulation tissue, foreign body reaction, and fibrosis respectively, at the implantation area. As a result, the duration and/or severity of the inflammation occurring due to activation of various systems for scar healing at the cellular level is accepted as the biocompatibility. [19],[20],[21],[22]

While evaluating biomaterials, most of the times the first target is to evaluate their physical and mechanical properties while biological properties are not treated the same. As any material that is in contact with tissue or blood poses a potential risk for health by causing some reactions, the advantages and disadvantages of such materials should be taken into consideration before clinical application, and their biocompatibilities should be evaluated by cytotoxicity tests. [2],[8],[23],[24]

Although there are many studies in the literature on ventilation tube, spongostan, and merocell packing used in ear nose and throat surgeries, we could find only one cell level study where cytotoxic effects of spongostan were evaluated. In a study of Cenni et al., [25] biocompatibility of hemostatic gelatin sponge was evaluated by neutral red and amido black staining cytotoxicity tests. The undiluted extract of the test material was found to be cytotoxic, but hemostatic gelatin sponge was reported not to be cytotoxic as no cytotoxicity was observed when diluted extract was used. Similarly, in our study, although a limited cytotoxicity was found in the initial 24 hours' leakage material from spongostan, the said cytotoxicity was not observed in the 7 and 14 days' leakage materials.

Results of different viability tests used in biocompatibility studies are not in agreement with each other as different target points are evaluated in these tests. For example, neutral red test targets membrane integrity while MTT test targets mitochondrial activity. [23] Among the biocompatibility tests, MTT test is considered the most reliable one as it is a fast yielding and sensitive test, which allows evaluating very low-level toxicities. [9],[26] In their studies on cytotoxicity, Saw et al., [27] Coa et al., [23] and Sletten et al. [28] preferred using neutral red, which is a sensitive and numeric test. In our study, we too used MIT, neutral red, and agar overlay test methods that allow differentiating live cells from dead ones.

In their study where they evaluated cytotoxicity of three different hemostats using two different methods, Hexig et al. [29] reported that results of different tests showed inconsistency in terms of the degree of cytotoxicity of the materials. In the said study, seprafilm, which is a widely used absorbable hemostatic material, was and/or could be cytotoxic. The reason of such a conflicting comment about its cytotoxicity is that seprafilm was found to be cytotoxic in one of the two different methods while the other method did not reveal such an effect. For this reason, they reported that one or more numeric cytotoxicity tests having high reliability should be done to prevent falsenegative and positive test results before reaching definite conclusions about biocompatibility of a biomaterial. In our study, a limited cytotoxicity was found in the initial 24 hours' leakage materials of ventilation tube, merocell nasal packing, and spongostan when MTT test was used, but it should be kept in mind that results may be different when different tests are used.


  Conclusions Top


Results obtained in our study do not reveal any information about whether other biological responses such as allergy, inflammation, and mutagenicity develop in relation to these biomaterials.

Another important point to keep in mind is that these biomaterials cause fibrosis or cellular changes in varying degrees. It is doubtless that our study does not explicitly show whether these biomaterials have cytotoxic effects at the cell level as the structural elements of these materials were not evaluated individually, but only leakage materials collected at different times were evaluated. For this reason, we believe that using chemical surface analysis techniques, measuring secretion of elements, and performing physical surface characterization for each element constituting these biomaterials would be more appropriate to evaluate cellular cytotoxicity in the future studies.

The studies in the literature are generally the animal studies carried out by spongostan, merocell, or ventilation tube, and even though they reveal different results on the cytotoxicity of these materials they report that these materials are not toxic in general. The results we obtained in our study support this information.

As we could not find any studies in the literature assessing biocompatibility of the materials by using MTT test, we believe that our study could be the first to investigate biocompatibility of these materials by MTT test and provide a basis for future studies.


  Acknowledgment Top


The authors wish to warmly thank Professor Haldun Sümer for her critical appraisal of the manuscript, and statistical analysis.

 
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