Indian Journal of Dental ResearchIndian Journal of Dental ResearchIndian Journal of Dental Research
HOME | ABOUT US | EDITORIAL BOARD | AHEAD OF PRINT | CURRENT ISSUE | ARCHIVES | INSTRUCTIONS | SUBSCRIBE | ADVERTISE | CONTACT
Indian Journal of Dental Research   Login   |  Users online: 5041

Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size         

 


 
Table of Contents   
ORIGINAL RESEARCH  
Year : 2016  |  Volume : 27  |  Issue : 2  |  Page : 145-150
Effect of electron-beam irradiation on antimicrobial, antibiofilm activity, and cytotoxicity of mouth rinses


1 Nitte University Centre for Science Education and Research, Nitte University, Mangalore, Karnataka, India
2 NUCSReM, Nitte University, Mangalore, Karnataka, India
3 Department of Biosciences, Mangalore University, Mangalore, Karnataka, India
4 Department of Physics, Mangalore University, Mangalore, Karnataka, India
5 Department of Microbiology, K. S. Hegde Medical Academy, Nitte University, Mangalore, Karnataka, India

Click here for correspondence address and email

Date of Submission05-Mar-2015
Date of Decision29-Mar-2015
Date of Acceptance20-Apr-2016
Date of Web Publication30-May-2016
 

   Abstract 


Background: Oral health diseases are common in all regions of the world. Mouth rinses are widely used generally by population as a port of daily oral care regimen. In addition to antimicrobial activity, mouth rinses possess certain cytotoxic effects. Electron-beam (E-beam) radiation is a form of ionizing energy known to induce structural, physical, and chemical changes in irradiated products. In this study, the modulatory effects of E-beam in irradiated mouth rinses were evaluated for its biological activities.
Materials and Methods: The antimicrobial activities of nonirradiated and irradiated mouth rinses were evaluated for its antimicrobial and antibiofilm activities against oral pathogens, Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, and Candida albicans. The antimicrobial activity was evaluated by disc diffusion method and antibiofilm activity was evaluated by O'Toole method. The cytotoxicity was evaluated against human gingival fibroblast (HGF) cells by 3-(4, 5 Dimethythiazol-yl)-2,5-Diphenyl-tetrazolium bromide assay.
Results: Colgate Plax (CP) exhibited the antimicrobial activity against the tested pathogens, and a significant (P< 0.05) increase was observed against S. aureus at 750 Gy irradiation. Further, CP significantly (P< 0.05) suppressed S. mutans, S. aureus, and C. albicans biofilm. Listerine (LS) inhibited S. mutans and C. albicans biofilm. Whereas irradiated CP and LS significantly (P< 0.05) suppressed the biofilm formed by oral pathogens. The suppression of biofilm by irradiated mouth rinses was dose- and species-dependent. There was no significant (P > 0.05) difference in the cytotoxicity of irradiated and nonirradiated mouth rinses on HGF cells. However, an increased percentage viability of HGF cells was observed by mouth rinses irradiated at 750 Gy.xs
Conclusion: The E-beam irradiation enhanced the antibiofilm activity of mouth rinses without modifying the cytotoxicity.

Keywords: Antibiofilm activity, antimicrobial activity, cytotoxicity, electron-beam irradiation, mouth rinses

How to cite this article:
Geethashri A, Kumar B M, Palaksha K J, Sridhar K R, Sanjeev G, Shetty A V. Effect of electron-beam irradiation on antimicrobial, antibiofilm activity, and cytotoxicity of mouth rinses. Indian J Dent Res 2016;27:145-50

How to cite this URL:
Geethashri A, Kumar B M, Palaksha K J, Sridhar K R, Sanjeev G, Shetty A V. Effect of electron-beam irradiation on antimicrobial, antibiofilm activity, and cytotoxicity of mouth rinses. Indian J Dent Res [serial online] 2016 [cited 2020 Feb 26];27:145-50. Available from: http://www.ijdr.in/text.asp?2016/27/2/145/183116


Oral health diseases are common in all regions of the world. Oral diseases such as dental caries, periodontal diseases, and tooth loss are among the major public health problems.[1] Dental caries are infectious diseases caused by the dynamic and extremely complex oral biofilm on tooth which is called dental plaque. It results in demineralization of hard tissue of tooth as a response to the microbial challenge.[2] Periodontal diseases are initiated by the accumulation of microbial plaque above the gingival margin, which extends into the sub-gingival environment. It induces an inflammatory response in the tissue, redness, swelling, and pain.[3]

Mouth rinses are often used by the general population in conjunction with brushing and flossing as a part of daily oral care regimen to prevent or minimize microbial accumulation.[4] Electron-beam (E-beam) radiation, a form of ionizing energy, has been introduced as a means of sterilizing single-use, disposable health-care products. Ionizing radiation induces structural changes in the native pharmaceutical compound resulting in altered physico-chemical, microbiological, and toxicological properties.[5] It has also been reported to be an effective tool to decompose the organic substances and reduce the toxicity.[6]

In this present study, modulatory effect of E-beam on antimicrobial and cytotoxicity of mouth rinses was evaluated on selected oral persistent pathogens Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutans, and Candida albicans, and human gingival fibroblast (HGF) cell line.


   Materials and Methods Top


Micro-organisms and maintenance

The type strains of E. faecalis (ATCC 29212), S. aureus (ATCC 29213), S. mutans (MTCC 890), and C. albicans were obtained from Nitte University Centre for Science Education and Research, Nitte University, India. Bacterial culture media Mueller Hinton Broth (MHB), Sabouraud Dextrose Broth (SDB), Tryptone Soya Broth (TSB), Mueller Hinton Agar (MHA), and Sabouraud Dextrose Agar (SDA), were procured from HiMedia, Mumbai, India.

Cell line and maintenance

HGF cell line was obtained through explants culture of healthy gingival tissue.[7] The cell line was sub-cultured and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml of amphotericin B at 37°C and 5% CO2 in humidified incubator. Cell culture medium DMEM, fetal bovine serum, antibiotics, and chemicals were procured from HiMedia. Petri plates, tissue culture flasks, and microtiter plates were obtained from Tarsons, Mumbai, India.

Mouth rinses

Commercially available Colgate Plax (CP) and Listerine (LS) were used without modifications in this study.

Electron-beam irradiation of irrigants

The irrigants were subjected to E-beam radiation at Microtron Centre, Mangalore University, Karnataka, India. The mouth rinses (10 ml) packed in sterile polythene pouches were exposed to 250 Gy, 500 Gy, 750 Gy, and 1000 Gy of E-beam irradiation at a dose rate of 500 Gy/min. The irradiated and nonirradiated mouth rinses were evaluated for their physico-chemical, antimicrobial, and cytotoxic effects.

Antimicrobial activity by disc diffusion method

In disc diffusion method,[8] the suspension culture of E. faecalis and S. aureus was grown in MHB, S. mutans was grown in TSB, and C. albicans was grown in SDB. Suspension culture of micro-organism matching 0.5 McFarland standards was used to get uniform lawn of micro-organisms in the respective agar media. The E. faecalis, S. aureus, and S. mutans were grown in MHA, and C. albicans was grown in SDA. Bacterial and fungal culture was uniformly spread over the solidified agar media using sterile swabs. About 20 µL of mouth rinses were incorporated aseptically to sterile paper disc (6 mm, HiMedia) and placed over the solidified media. Then, culture plates were incubated for overnight at 37°C. The antimicrobial activity of mouth rinses was recorded by measuring the zone of inhibition (ZOI).

Antibiofilm assay

Biofilm was grown in microtiter plate as described previously by O'Toole.[9] The wells of the microtiter plates were seeded with 100 µl of 18 h old bacterial cultures diluted at 1:100 ratios in respective broths and incubated at 37°C for 24 h. Then, the excess broth was discarded carefully using a multichannel pipettor and washed in 3 times with phosphate buffer saline (PBS, pH 7) and air dried. Following the air drying, 20 µL of irradiated and nonirradiated mouth rinses were added to each wells containing biofilm and incubated for 10 min at 37°C. Then, the test materials were discarded, washed with distilled water for 3 times, and air dried at room temperature. The biofilm was stained by incubating with 125 µl of 0.1% crystal violet stain for 10–15 min at room temperature. Stain was removed by three washes in doubled distilled water and air dried. Destaining of the biofilm was done by incubating with 30% acetic acid for 15 min at room temperature. Quantification of the biofilm was performed at 600 nm using Lisa chem plate reader.

Cytotoxicity

3-(4, 5 Dimethythiazol-yl)-2,5-Diphenyl-tetrazolium bromide (MTT) assay [10] was employed to study the cytotoxic effect of mouth rinses. About 100 µL of media containing 10,000 cells were seeded to each well of 96-well microtiter plates. Then, the cells were allowed to grow under 5% CO2 at 37°C in humidified incubator. After 24 h of incubation, the media was removed and the cells were treated with 20 µl of mouth rinses for 10 min. After the removal of mouth rinses, the cells were washed in PBS (pH 7). The cytotoxicity of mouth rinses was evaluated by incubating the cells with 100 µL of MTT dye (0.05 mg/ml) in PBS for 4 h at 37°C in 5% CO2 incubator. The intensity of the color was measured by adding dimethyl sulphoxide at 545 nm using Lisa chem plate reader.

Physico-chemical parameters

The color, sedimentation, precipitation of control, and irradiated mouth rinses were recorded immediately after E-beam irradiation. The pH of mouth rinses was recorded using pH strips (Fisher Scientific) of range 2–10.

Statistical analysis

The antimicrobial activity and cytotoxicity data were statistically analyzed by one-way ANOVA and post hoc tests using GraphPad Prism (version 3.02, GraphPad Software Inc., San Deigo). The significance of the results was considered when P < 0.05, <0.01, and <0.001.


   Results Top


Antimicrobial activity of Colgate Plax against oral pathogens before and after irradiation

The data describing the antimicrobial activity of CP against oral pathogens are presented in [Table 1] as mean ± standard deviation (SD) CP was found to be more active against S. mutans and moderately active against other pathogens. A significant (P< 0.05) increase in the antibacterial activity of irradiated CP at 750 Gy was observed against S. aureus. However, E-beam irradiation of CP did not cause any significant (P > 0.05) difference in antimicrobial activity against E. faecalis and C. albicans after radiation. Although there was a significant (P< 0.001) decrease at lower doses, the antimicrobial activity against S. mutans was found to be insignificant (P > 0.05) at 1000 Gy compared to nonirradiated group.
Table 1: Zone of inhibition by Colgate Plax before and after irradiation at different doses

Click here to view


Antibiofilm activity of nonirradiated and irradiated mouth rinses

The antibiofilm activity of nonirradiated and irradiated CP and LS is presented in [Table 2] and [Table 3], respectively. In nonirradiated groups, the suppression of C. albicans biofilm by CP and S. mutans and C. albicans biofilms by LS was found to be highly significant (P< 0.001). However, no significant (P > 0.05) differences were observed against E. faecalis biofilm by both CP and LS. Further, the suppression was significant (P< 0.05 and P < 0.01) against S. aureus and S. mutans biofilms by CP.
Table 2: Suppression of biofilm by nonirradiated and irradiated Colgate Plax

Click here to view
Table 3: Suppression of biofilm by nonirradiated and irradiated listerine

Click here to view


E-beam irradiated CP and LS were highly effective in reducing the activity of E. faecalis biofilm at all doses, and the observed differences were statistically significant (P< 0.001). S. aureus biofilm was significantly (P< 0.01) suppressed by irradiated LS at 250 Gy and 500 Gy, whereas in CP, significant differences were observed at 500 Gy onward. The irradiated CP significantly (P< 0.001) suppressed the S. mutans and C. albicans biofilm at all doses. The LS irradiated at 250–750 Gy resulted in a highly significant (P< 001) suppression of C. albicans biofilm. Moreover, both irradiated CP and LS were highly effective in suppressing the E. faecalis biofilm compared to nonirradiated counterparts, and the differences were found statistically significant (P< 0.001).

Viability of human gingival fibroblasts in irradiated and nonirradiated mouth rinses

The viability results of HGF by irradiated and nonirradiated CP and LS are presented in [Figure 1] and [Figure 2]. Overall, a decrease in mean + SD of HGF was observed upon treating with irradiated and nonirradiated CP and LS. Both mouth rinses were highly effective in significantly (P< 0.001) reducing the number of viable HGF. Particularly, both irradiated CP and LS were found to be more effective in their activity against HGF, and the differences were highly significant (P< 0.001). Although there was no significant (P > 0.05) difference in nonirradiated and irradiated mouth rinses, the data showed a slight increase in the viability of cells treated with mouth rinses irradiated at 750 Gy.
Figure 1: Reduction in the viability of human gingival fibroblast cells treated with Colgate Plax and irradiated Colgate Plax at different doses

Click here to view
Figure 2: Reduction in the viability of human gingival fibroblast cells treated with Listerine and irradiated Listerine at different doses

Click here to view


Discoloration of mouth rinses after electron-beam irradiation

The gradual discoloration of mouth rinses CP and LS upon E-beam irradiation is shown in [Figure 3]a and [Figure 3]b, respectively.
Figure 3: Discoloration of Colgate Plax (a) and Listerine (b) mouth rinses after electron-beam irradiation

Click here to view



   Discussion Top


It is well known that the oral diseases are a worldwide health concern with a considerable impact on public. Further, due to an increasing intake of sugars in the diet, use of tobacco, inadequate exposure to fluorides, and lack of access to dental care, it is expected that the incidence of dental caries and periodontal disease will continue to increase.[11]S. mutans is a primary etiological agent of dental caries.[2]E. faecalis is an opportunistic pathogen, S. aureus and C. albicans are also isolated from persistent apical periodontal lesions.[12] Oral microflora has been considered as a critical factor in both caries and periodontal disease, and cause the disease pathogenesis mainly by producing the biofilms.[13] Hence, the use of different types of mouth rinses has been given a due importance to act against the harmful micro-organisms associated with oral diseases or infections. However, concerns regarding the development of antibiotic-resistant strains and adverse effects of contemporary mouth rinses have led to the interests in the use of nonconventional or alternative medicines and plant extracts. A few reports have also suggested the possible use of plant extracts in oral care for effective and efficient inhibition of microflora by natural antimicrobials.[14],[15] However, the potential benefits of chemotherapeutic formulations in mouth rinses provide impetus for research in finding effective mouth rinses for oral care.

The present study evaluated the modulatory effect of E-beam on antimicrobial effect of mouth rinses, CP and LS on oral persistent pathogens E. faecalis, S. aureus, S. mutans, and C. albicans. Further, the cytotoxic effect of two mouth rinses was also determined on the cultured HGF. The results showed that CP was more effective against all tested pathogens by producing a clear ZOI. This may be due to the presence of active component cetylpyridinium chloride, which is a quaternary, monocationic surfactant with broad-spectrum antimicrobial activity.[16] Whereas the mouth rinse LS did not show a ZOI against the tested micro-organisms. The inefficacy of LS against oral pathogens in our study is in concurrence with the earlier report by Aneja et al.,[17] suggesting that different formulations of constituents in mouth rinses might be responsible for varied effects.

Biofilm experiments were performed to evaluate the effect of antimicrobial components in selected mouth rinses. The results showed a greater antibiofilm activity of LS compared to CP against the tested oral pathogens. These data are in consistence with published report where the efficacy of mouth rinses containing essential oils was observed to be more than cetylpyridinium chloride formulations.[18],[19]

E-beam is a form of ionizing radiation used in food and pharmaceutical industries as a mean of sterilization. Several studies have reported the chemical stability of pharmaceutical drugs or antibiotics after irradiation.[20],[21] However, there are no reports on the effects of ionizing radiation on biological activities (antimicrobial and cytotoxicity) of pharmaceuticals. Therefore, in this study, value addition of E-beam radiation on biological properties of mouth rinses was evaluated.

The mouth rinses were exposed to lower doses of E-beam irradiation because higher doses might degrade the active component.[22] A significant increase in the antimicrobial activity of CP was observed against S. aureus upon irradiation at 750 Gy. Irradiation induced the higher efficacy of biofilm suppression by CP and LS. Irradiated CP and LS suppressed E. faecalis biofilm more effectively than the nonirradiated counterparts. Further, irradiation increased the efficacy of biofilm suppression by mouth rinses.

Any antimicrobial agents are expected to have minimal cytotoxic effect on host cells. In this study, the results demonstrated the cytotoxic effect of both CP and LS on cultured HGF. These results are in accordance with the earlier reports on CP and LS.[23],[24] However, no significant differences were observed in the reduction of HGF cells by irradiated and nonirradiated mouth rinses. Importantly, an earlier report on the effect of E-beam on sodium dodecyl sulfate demonstrated the reduction in toxicity of the surfactant.[6]

During technological processing, the majority of drugs and therapeutic substances can be negatively affected by radiation. This can be manifested by change in color or precipitation or sedimentation of the irradiated substance.[20] A gradual discoloration in mouth rinses irradiated from 250 Gy to 750 Gy and a complete discoloration at 1000 Gy of irradiation indicated the changes in chemical properties of mouth rinses upon E-beam radiation [Figure 3]. However, there were no changes in pH, as it was observed to be 6 for CP and 4 for LS before and after irradiation. These results indicate that radiation did not cause the formation of acidic or basic intermediates. These observations are in contrary with the reported work on sulfonamides,[5] where color stability and change in pH were observed.


   Conclusion Top


The present study demonstrated that the mouth rinse CP had the highest antimicrobial property against the selected oral pathogens, whereas LS was effective in suppressing the tested biofilms. E-beam component did not alter the antimicrobial properties of the mouth rinses much, but it enhanced the antibiofilm activity without modifying the cytotoxic effects on fibroblast cells. Thus, E-beam irradiation can be a useful processing tool to enhance the antimicrobial activities of contemporary mouth rinses. Further studies may be directed to investigate the cytotoxicity of these mouth rinses underin vivo conditions.

Acknowledgment

The authors gratefully acknowledge the financial support extended by the Department of Atomic Energy, Board of Research in Nuclear Sciences.

Financial support and sponsorship

The BRNS-RTAC grant (Sanction No: 2010/35/BRNS with RTAC) of the Department of Atomic Energy, Board of Research in Nuclear Sciences, provided the financial support for this work.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Pau AK. Challenges in dental public health – An overview. IeJSME 2012;6:S106-S112.  Back to cited text no. 1
    
2.
Filoche S, Wong L, Sissons CH. Oral biofilms: Emerging concepts in microbial ecology. J Dent Res 2010;89:8-18.  Back to cited text no. 2
    
3.
Lamster IB. Antimicrobial mouthrinses and the management of periodontal diseases. Introduction to the supplement. J Am Dent Assoc 2006;137:5S-9S.  Back to cited text no. 3
    
4.
Silverman S Jr., Wilder R. Antimicrobial mouthrinse as part of a comprehensive oral care regimen. Safety and compliance factors. J Am Dent Assoc 2006;137:22S-6S.  Back to cited text no. 4
    
5.
Beteshobabrud R, Nabardi F. The stability studies of penicillin and ampicillin following γ-irradiation in the solid state. Iran J Pharm Res 2009;8:153-7.  Back to cited text no. 5
    
6.
Romanelli MF, Moraes MC, Villavicencio AL, Borrely SI. Evaluation of toxicity reduction of sodium dodecyl sulfate submitted to electron beam irradiation. Radiat Phys Chem 2004;71:409-11.  Back to cited text no. 6
    
7.
Saczko J, Dominiak M, Kulbacka J, Chwilkowska A, Krawczykowska H. A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation. Folia Histochem Cytobiol 2008;46:117-9.  Back to cited text no. 7
    
8.
Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.  Back to cited text no. 8
[PUBMED]    
9.
O'Toole GA. Microtiter dish biofilm formation assay. J Vis Exp 2011. pii: 2437.  Back to cited text no. 9
    
10.
Sargent JM, Taylor CG. Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. Br J Cancer 1989;60:206-10.  Back to cited text no. 10
    
11.
Petersen PE, Bourgeois D, Ogawa H, Estupinan-Day S, Ndiaye C. The global burden of oral diseases and risks to oral health. Bull World Health Organ 2005;83:661-9.  Back to cited text no. 11
    
12.
Bowden GH, Hamilton IR. Survival of oral bacteria. Crit Rev Oral Biol Med 1998;9:54-85.  Back to cited text no. 12
    
13.
Liljemark WF, Bloomquist C. Human oral microbial ecology and dental caries and periodontal diseases. Crit Rev Oral Biol Med 1996;7:180-98.  Back to cited text no. 13
    
14.
Aspalli S, Shetty VS, Devarathnamma MV, Nagappa G, Archana D, Parab P. Evaluation of antiplaque and antigingivitis effect of herbal mouthwash in treatment of plaque induced gingivitis: A randomized, clinical trial. J Indian Soc Periodontol 2014;18:48-52.  Back to cited text no. 14
[PUBMED]  Medknow Journal  
15.
Geethashri A, Manikandan R, Ravishankar B, Shetty AV. Comparative evaluation of biofilm suppression by plant extracts on oral pathogenic bacteria. J Appl Pharm Sci 2014;4:20-3.  Back to cited text no. 15
    
16.
Arauji DB, Campos EJ, Bastos IH, Paula DM, Tenorio Junior ER, Araujo RP. Mouthrinses: Active ingredients, pharmacological properties and indications. Rev Gaucha Odontol 2012;60:349-57.  Back to cited text no. 16
    
17.
Aneja KR, Joshi R, Sharma C. The antimicrobial potential of ten often used mouthrinses against four dental caries pathogens. Jundishapur J Microbiol 2010;3:15-27.  Back to cited text no. 17
    
18.
Pan PC, Harper S, Ricci-Nittel D, Lux R, Shi W. In-vitro evidence for efficacy of antimicrobial mouthrinses. J Dent 2010;38 Suppl 1:S16-20.  Back to cited text no. 18
    
19.
Guggenheim B, Meier A.In vitro effect of chlorhexidine mouth rinses on polyspecies biofilms. Res Sci 2011;121:431-6.  Back to cited text no. 19
    
20.
Singh B, Parwate DV, Shukla SK. Radiosterilization of fluoroquinolones and cephalosporins: Assessment of radiation damage on antibiotics by changes in optical property and colorimetric parameters. AAPS PharmSciTech 2009;10:34-43.  Back to cited text no. 20
    
21.
Mercanoglu GO, Ozer AY, Colak S. Radiosterilization of sulfonamides: I: Determination of the effects of gamma irradiation on solid sulfonamides. Radiat Phys Chem 2004;69:511-20.  Back to cited text no. 21
    
22.
Varshney L, Patel KM. Effects of ionizing radiations on a pharmaceutical compound, chloramphenicol. Radiat Phys Chem 1994;43:471-80.  Back to cited text no. 22
    
23.
Flemingson, Pamela E, Ambalavanan N, Ramakrishnan T, Vijayalakshmi R. Effect of three commercial mouth rinses on cultured human gingival fibroblast: Anin vitro study. Indian J Dent Res 2008;19:29-35.  Back to cited text no. 23
    
24.
Kim YJ, Rossa C Jr., Kirkwood KL. Prostaglandin production by human gingival fibroblasts inhibited by triclosan in the presence of cetylpyridinium chloride. J Periodontol 2005;76:1735-42.  Back to cited text no. 24
    

Top
Correspondence Address:
A Veena Shetty
Department of Microbiology, K. S. Hegde Medical Academy, Nitte University, Mangalore, Karnataka
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.183116

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed1285    
    Printed25    
    Emailed0    
    PDF Downloaded104    
    Comments [Add]    

Recommend this journal