Research Article
Volume 2 Issue 4 - 2015
RESILONTM Toxic to Oral Squamous Carcinoma Cells: A Live/Dead Assay
C Jimenez1,3 and ZS Haidar1,2,3*
1 BioMAT’X, Faculty of Dentistry, University of The Andes, Chile
2Plan de Mejoramiento Institucional Institutional Improvement Plan (PMI) en Innovación-I+D+I, University of The Andes, Chile
3Biomedical Research Center, School of Medicine, University of The Andes, Chile
*Corresponding Author: Prof. Dr. Ziyad S. Haidar, Professor and Scientific Director, Faculty of Dentistry, University of The Andes, Mons. Álvaro del Portillo 12.455, Las Condes, Santiago, Chile. Founder and Head of BioMAT’X-CIB, PMI I+D+I, Universidad de los Andes, Mons. Álvaro del Portillo 12.455, Las Condes, Santiago de Chile.
Received: September 05, 2015; Published: September 24, 2015
Citation: C Jimenez and ZS Haidar. “RESILONTM Toxic to Oral Squamous Carcinoma Cells: A Live/Dead Assay”. EC Dental Science 2.4 (2015): 337-341.
Background/Purpose: Biologic responses and biomaterial/cell-and tissue-interactions are essential of particular significance for the clinical applicability of developed products.
Purpose: To assess the toxicity of endodontic filling materials on oral cancer cells.
Materials and Methods: A LIVE/DEAD BacLight fluorescent assay was used to stain and detect Cytocompatibility after 24 hr, 48 hr and 72 hr incubation periods with aggressive salivary gland-origin squamous cell carcinoma (SCA-9) and adenocarcinoma (WR-21) lines. Quantitative analysis (in triplicate) was performed using a colorimetric method, MTT, to measure cellular viability.
Results: Resilon is significantly (ρ < 0.05) more toxic to SCA-9 cells as well as to WR-21 cells than is gutta-percha, in a time-dependent manner. This can be attributed to its resin/primer content and biodegradability of polycaprolactone and by-products, over time.
Conclusion: Concerns pertaining to safe clinical usage are valid.
Keywords: Cytocompatibility; Endodontics; Resilon; Root canal filling; Sealer; Cancer
Endodontic obturation materials should not only eliminate or minimize the ingress or egress of bacteria and their by-products. Rather, they are expected to promote healing of peri-apical tissues and encompass a favorable tissue response [1]. Despite the known shortcomings, gutta-percha remains the gold standard core root filling material [2]. With advances in polymer chemistry and dental materials, ResilonTM, a FDA-approved thermoplastic synthetic polymer was introduced into the endodontic practice and consequently challenging gutta-percha, robustly, increasingly suggested to replace gutta-percha and become the stand-of-care in root canal therapy [3]. When used along a resin-based sealer, Resilon has been shown to offer improved bonding potential, enhanced resistance to tooth fracture as well as minimal microbial leakage in filled roots, when compared with gutta-percha, among others [4]. Further, Resilon was claimed to possess superior biocompatibility where several investigators concluded that it was a non-cytotoxic and non-mutagenic material [3]. Interestingly, those efforts were recently reviewed with inconclusiveness calling for more research [2-4]. Biological responses in different cell lines have been shown to be beneficial and proposed by others to be even essential [4]. For example, Key., et al. [5] reported that Resilon was as toxic or even less toxic than gutta-percha to human gingival fibroblasts after 1 and 24 hours. On the other hand, a few recent studies have reported that Resilon points were more cytotoxic than gutta-percha cones. Indeed, the cytotoxicity of Resilon to L929 mouse skin fibroblasts as well as RPC-C2A rat pulp cells was shown to significantly increase after 48 hours of exposure using a sulforhodamine-B assay. Authors concluded that the material was more toxic than gutta-percha, more so, it was time-dependant [6]. Although the material is expected to linger within the root canal, theoretically, it is well-known that peri-apical extrusion through the apical constriction or via iatrogenic perforation is far from rare, thus raising legitimate concerns for endodontists and the general practitioner regarding safety and subsequently, use efficacy [7]. So, how is Resilon compared to gutta-percha in terms of cytocompatibility?
Herein, the aim was to evaluate and compare the (a) cytotoxicity and (b) anti-proliferative effects of Resilon points with gutta-percha cones on two murine carcinoma cell lines by means of qualitative and quantitative analysis. Those cell lines were selected for their aggressiveness in comparison to others used in previous studies. This pilot in vitro investigation predecessors further in vivo and clinical studies evaluating the biocompatibility and safety of extruded materials on the condition of peri-apical tissues.
Materials and Methods
Experimental materials were RESILONTM points (Pentron Clinical Technologies, Wallingford, CT, USA) and ACEONE-ENDO® gutta-percha cones (ACEONEDENT Korea Industrial Company, Bucheon, South Korea). Only 0.2g of each material was placed in sterile vials containing 6 mL of Dulbecco’s Modified Eagle’s Medium (DMEM), incubated at 37°C for a total period of 72 hours. The two cell lines were: (a) SCA-9 clone 15 (Mus musculus Submandibular Salivary Gland Carcinoma) cells and (b) WR-21 (Mus musculus Submandibular Salivary Gland Adenocarcinoma) cells [8,9]. Both (CRL-1734™ and CRL-2189™, respectively) were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and used for cytotoxicity evaluation. ATCC reports that both lines are adherent (growth properties) with a fibroblast-like morphology. Cell culture materials include DMEM, fetal bovine serum (FBS), Dulbecco’s Phosphate Sodium Buffer (DPBS) and TrypLE™ Antibiotic-Antimycotic; were all purchased from Gibco® BRL (Carlsbad, CA, USA). The quantitative cytocompatibility assay was performed via a colorimetric method, using MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (Sigma-Aldrich Chemical, South Korea). Ultra-pure water (UPW) and all other materials and chemicals used were of analytical grade. Briefly, cells were seeded (mono-layer) in 12-well tissue culture plates, at an initial density of 2 × 104 viable cells/mL. MTT was added after 24h to a final concentration of 0.5 mg/mL and incubated for 4h at 37°C. The measurement of formazan absorbance was then carried out using a spectrophotometer (μQuant, Bio-Tek Instruments)-microplate reader-at 570 nm. Control wells were treated with 100 μL of DMEM only (10 μL MTT stock solution was added). Alternatively, cytotoxicity was studied using a nucleic acid staining dye, Hoechst 33342 and propidium iodide. Cells/Resilon and cells/gutta-percha(at 24 hr, 48 hr and 72 hr) were washed twice with PBS and incubated with the dye for 10 min at room temperature. For qualitative purposes, attached cells were imaged following incubation with fluorescent stains (calcein AM and EthD-1) using a fluorescence microscope (Olympus, Japan) for LIVE/DEAD real-time viewing. Access to microscopy was kindly provided by CIBRO (Centro de Investigación en Biología y Regeneración Oral) of the Universidad de los Andes in Santiago de Chile. Unless otherwise mentioned, all experiments were done in triplicates. Colorimetric assays and statistical analysis of obtained data were performed at the BioMAT’X Laboratory, part of CIB (Centro de Investigación Biomédica), Faculty of Dentistry, Universidad de los Andes. Results are reported as mean ± standard deviations. Multiplet-tests (unpaired/paired) were performed to assess for statistical significance at the 95% confidence level where ρ-values ≤ 0.05 were considered statistically significant.
Results and Discussion
The present study evaluated the cytotoxicity of two common endodontic obturation materials on two cancerous cell lines, in vitro. Such simple, rapid, reproducible and inexpensive methods provide valuable information and help predict the biocompatibility of materials in pre-clinical and clinical models. Furthermore, in vivo and clinical testing of dental materials may be influenced by the skill of the dentist, technical properties of the material and uncontrollable patient factors. To the best of knowledge, no studies exposed cancer cells to such materials, thus far. SCA-9 and WR-21 cell lines were used to measure alterations in cell numbers as well as morphology over a period of 3 days. The protocols used herein were optimized previously to investigate anti-cancer drugs [10] hence the cell density used was sufficient for exponential growth throughout the duration of the experiment. Also, WR-21 cells are more aggressive than SCA-9 cells. Figure 1 displays the effect of Resilon and conventional gutta-percha points on SCA-9 cells. Evidently, after 2 days of incubation, the number of cells exposed to Resilon was much less than those exposed to gutta-percha. Likewise, the morphology of cells in the latter group seems to be preserved and more fibroblast-like than the cells in the former group. Quantitative date, plotted in Figure 2, confirmed this microscopic observation. At 72 hrs, Resilon is significantly (ρ < 0.05) more toxic to SCA-9 cells as well as to WR-21 cells than is gutta-percha. Several studies have suggested the cytotoxicity of Resilon compared to gutta-percha exhibited in rat pulp cells and mouse skin fibroblasts, especially so with set sealers [6,11]. Interestingly, gutta-percha cones showed a slight increased toxicity after a 48 hr incubation period, possible explained by its zinc oxide content. However with no statistically significant differences between cell lines or the 48 hr and 72 hr values were detected. This has been shown previously using a radio chromium release assay with toxicity at longer incubation periods attributed to the leakage of zinc ions into the culture medium, according to Pascon and Spangberg [12]. Quantifying zinc oxide content in commercially-available gutta-percha products remains a matter of investigation although it is the responsibility of manufacturers to provide such relevant information [2,6]. Regarding Resilon, the observed cytotoxicity can be attributed to its resin/primer content and material (polycaprolactone) biodegradability over time [13]. It is noteworthy that contradicting results are available in the literature. For instance, in a similar rat connective tissue model, satisfactory tissue reactions were reported with the Epiphany/Resilon root canal filling system [14]. Hence, attention to differences in cell types/lines and followed experimental methodologies are necessary. Nonetheless, a systematic review of the available literature reveals more studies (not limited to in vitro) reporting the cytotoxicity of Resilon in comparison to other materials [2,15-17]. The LIVE/DEAD assay offers easy and sensitive determination of cell viability, cell vitality and compound cytotoxicity [16,17]. Figures 3 and 4 illustrate the LIVE/DEAD auto-fluorescence images captured in real-time (24 hr and 72 hr) comparing Resilon and gutta-percha in both cell lines, respectively. It is noteworthy here in that in this type of assay, green cells are alive and red cells are dead. Evidently, Resilon points were found to be significantly more toxic than gutta-percha, irrespective of the cell line. A statistically significant difference can be clearly detected between the 24 hr and 72 hr periods, warranting time-dependency. Inserts (magnification) detects light morphological changes; indicating latent cell uptake post-72 hr of incubation.
Figure 1: Effect of Resilon and gutta-percha points on SCA-9 cells after 48 hr exposure.
Figure 2: Comparison of cell viability values over time for SCA-9 and WR-21 cells.
Figure 3: LIVE/DEAD images captured at 24 hr and 72 hr for cells exposed to Resilon points.
Figure 4: LIVE/DEAD images captured at 24 hr and 72 hr for cells exposed to gutta-percha cones.
Overall, the hypothesis was accepted, revealing an alarming cytotoxic potential of Resilon points. This in turn calls for serious pre- and -clinical research investigating its safety and biocompatibility, especially, for the leaked by-products, and over extended periods of time.
 This work was supported by funding operating grants provided to the BioMAT’X Research Group (Faculty of Dentistry), part of CIB (Centro de Investigación Biomédica)through the Plan de Mejoramiento Institucional (PMI) en Innovación-I+D+i, Universidad de los Andes in Santiago de Chile. The authors do recognize CIBRO-UAndes for their technical assistance with cell imaging.
  1. Lin LM., et al. “One-appointment endodontic therapy: biological considerations”. The Journal of the AmericanDental Association 138.11 (2007): 1456-1462.
  2. Shanahan DJ and Duncan HF. “Root canal filling using Resilon: a review”. British Dental Journal 211.2 (2011): 81-88.
  3. Shipper G., et al. “An evaluation of microbial leakage in roots filled with a thermoplastic synthetic polymer-based root canal filling material (Resilon)”. Journal of Endodontics 30.5 (2004): 342-347.
  4. Shashidhar C., et al. “The comparison of microbial leakage in roots filled with resilon and gutta-percha: An in vitro study”. Journal of Conservative Dentistry 14.1 (2011): 21-27.
  5. Key JE., et al. “Cytotoxicity of a new root canal filling material on human gingival fibroblasts”. Journal of Endodontics 32.8 (2006): 756-758.
  6. Economides N., et al. “Comparative study of the cytotoxic effect of Resilon against two cell lines”. BrazilianDental Journal 19.4 (2008): 291-295.
  7. Brackett MG., et al. “Cytotoxicity of endodontic materials over 6-weeks ex vivo”. International EndodonticJournal41.12 (2008): 1072-1078.
  8. SCA-9 cells, American Type Culture Collection (ATCC), Manassas, VA 20108 USA.
  9. WR-21 cells, American Type Culture Collection (ATCC), Manassas, VA 20108 USA.
  10. Joo VS., et al. “Nano-oncology: A State-of-Art Update. Journal of Bionanoscience 4 (2010): 1-13.
  11. Donadio M., et al. “Cytotoxicity evaluation of Activ GP and Resilon sealers in vitro”. Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology 107.6 (2009): e74-e78.
  12. Pascon EA and Spångberg LS. “In vitro cytotoxicity of root canal filling materials: 1. Gutta-percha”. Journal of Endodontics 16.9 (1990): 429-433.
  13. Grecca FS., et al. “Biocompatibility of RealSeal, its primer and AH Plus implanted in subcutaneous connective tissue of rats”. Journal of Applied Oral Science 19.1 (2011): 52-56.
  14. Garcia Lda F., et al. “Biocompatibility evaluation of Epiphany/Resilon root canal filling system in subcutaneous tissue of rats”. Journal of Endodontics 36.1 (2010): 110-114.
  15. Tortini D., et al. “Warm gutta-percha obturation technique: a critical review”. Minerva Stomatologica 60.1-2 (2011): 35-50.
  16. Hansen J and Bross P. “A cellular viability assay to monitor drug toxicity”. Methods in Molecular Biology 648 (2010): 303-311.
  17. Hadjati J., et al. “Effect of Bacterial Lipopolysaccharide Contamination on Gutta Percha-versus Resilon-Induced Human Monocyte Cell Line Toxicity”. Journal of Dentistry (Tehran) 12.2 (2015): 134-139.
Copyright: © 2015 C Jimenez and ZS Haidar. 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.

PubMed Indexed Article

EC Pharmacology and Toxicology
LC-UV-MS and MS/MS Characterize Glutathione Reactivity with Different Isomers (2,2' and 2,4' vs. 4,4') of Methylene Diphenyl-Diisocyanate.

PMID: 31143884 [PubMed]

PMCID: PMC6536005

EC Pharmacology and Toxicology
Alzheimer's Pathogenesis, Metal-Mediated Redox Stress, and Potential Nanotheranostics.

PMID: 31565701 [PubMed]

PMCID: PMC6764777

EC Neurology
Differences in Rate of Cognitive Decline and Caregiver Burden between Alzheimer's Disease and Vascular Dementia: a Retrospective Study.

PMID: 27747317 [PubMed]

PMCID: PMC5065347

EC Pharmacology and Toxicology
Will Blockchain Technology Transform Healthcare and Biomedical Sciences?

PMID: 31460519 [PubMed]

PMCID: PMC6711478

EC Pharmacology and Toxicology
Is it a Prime Time for AI-powered Virtual Drug Screening?

PMID: 30215059 [PubMed]

PMCID: PMC6133253

EC Psychology and Psychiatry
Analysis of Evidence for the Combination of Pro-dopamine Regulator (KB220PAM) and Naltrexone to Prevent Opioid Use Disorder Relapse.

PMID: 30417173 [PubMed]

PMCID: PMC6226033

EC Anaesthesia
Arrest Under Anesthesia - What was the Culprit? A Case Report.

PMID: 30264037 [PubMed]

PMCID: PMC6155992

EC Orthopaedics
Distraction Implantation. A New Technique in Total Joint Arthroplasty and Direct Skeletal Attachment.

PMID: 30198026 [PubMed]

PMCID: PMC6124505

EC Pulmonology and Respiratory Medicine
Prevalence and factors associated with self-reported chronic obstructive pulmonary disease among adults aged 40-79: the National Health and Nutrition Examination Survey (NHANES) 2007-2012.

PMID: 30294723 [PubMed]

PMCID: PMC6169793

EC Dental Science
Important Dental Fiber-Reinforced Composite Molding Compound Breakthroughs

PMID: 29285526 [PubMed]

PMCID: PMC5743211

EC Microbiology
Prevalence of Intestinal Parasites Among HIV Infected and HIV Uninfected Patients Treated at the 1o De Maio Health Centre in Maputo, Mozambique

PMID: 29911204 [PubMed]

PMCID: PMC5999047

EC Microbiology
Macrophages and the Viral Dissemination Super Highway

PMID: 26949751 [PubMed]

PMCID: PMC4774560

EC Microbiology
The Microbiome, Antibiotics, and Health of the Pediatric Population.

PMID: 27390782 [PubMed]

PMCID: PMC4933318

EC Microbiology
Reactive Oxygen Species in HIV Infection

PMID: 28580453 [PubMed]

PMCID: PMC5450819

EC Microbiology
A Review of the CD4 T Cell Contribution to Lung Infection, Inflammation and Repair with a Focus on Wheeze and Asthma in the Pediatric Population

PMID: 26280024 [PubMed]

PMCID: PMC4533840

EC Neurology
Identifying Key Symptoms Differentiating Myalgic Encephalomyelitis and Chronic Fatigue Syndrome from Multiple Sclerosis

PMID: 28066845 [PubMed]

PMCID: PMC5214344

EC Pharmacology and Toxicology
Paradigm Shift is the Normal State of Pharmacology

PMID: 28936490 [PubMed]

PMCID: PMC5604476

EC Neurology
Examining those Meeting IOM Criteria Versus IOM Plus Fibromyalgia

PMID: 28713879 [PubMed]

PMCID: PMC5510658

EC Neurology
Unilateral Frontosphenoid Craniosynostosis: Case Report and a Review of the Literature

PMID: 28133641 [PubMed]

PMCID: PMC5267489

EC Ophthalmology
OCT-Angiography for Non-Invasive Monitoring of Neuronal and Vascular Structure in Mouse Retina: Implication for Characterization of Retinal Neurovascular Coupling

PMID: 29333536 [PubMed]

PMCID: PMC5766278

EC Neurology
Longer Duration of Downslope Treadmill Walking Induces Depression of H-Reflexes Measured during Standing and Walking.

PMID: 31032493 [PubMed]

PMCID: PMC6483108

EC Microbiology
Onchocerciasis in Mozambique: An Unknown Condition for Health Professionals.

PMID: 30957099 [PubMed]

PMCID: PMC6448571

EC Nutrition
Food Insecurity among Households with and without Podoconiosis in East and West Gojjam, Ethiopia.

PMID: 30101228 [PubMed]

PMCID: PMC6086333

EC Ophthalmology
REVIEW. +2 to +3 D. Reading Glasses to Prevent Myopia.

PMID: 31080964 [PubMed]

PMCID: PMC6508883

EC Gynaecology
Biomechanical Mapping of the Female Pelvic Floor: Uterine Prolapse Versus Normal Conditions.

PMID: 31093608 [PubMed]

PMCID: PMC6513001

EC Dental Science
Fiber-Reinforced Composites: A Breakthrough in Practical Clinical Applications with Advanced Wear Resistance for Dental Materials.

PMID: 31552397 [PubMed]

PMCID: PMC6758937

EC Microbiology
Neurocysticercosis in Child Bearing Women: An Overlooked Condition in Mozambique and a Potentially Missed Diagnosis in Women Presenting with Eclampsia.

PMID: 31681909 [PubMed]

PMCID: PMC6824723

EC Microbiology
Molecular Detection of Leptospira spp. in Rodents Trapped in the Mozambique Island City, Nampula Province, Mozambique.

PMID: 31681910 [PubMed]

PMCID: PMC6824726

EC Neurology
Endoplasmic Reticulum-Mitochondrial Cross-Talk in Neurodegenerative and Eye Diseases.

PMID: 31528859 [PubMed]

PMCID: PMC6746603

EC Psychology and Psychiatry
Can Chronic Consumption of Caffeine by Increasing D2/D3 Receptors Offer Benefit to Carriers of the DRD2 A1 Allele in Cocaine Abuse?

PMID: 31276119 [PubMed]

PMCID: PMC6604646

EC Anaesthesia
Real Time Locating Systems and sustainability of Perioperative Efficiency of Anesthesiologists.

PMID: 31406965 [PubMed]

PMCID: PMC6690616

EC Pharmacology and Toxicology
A Pilot STEM Curriculum Designed to Teach High School Students Concepts in Biochemical Engineering and Pharmacology.

PMID: 31517314 [PubMed]

PMCID: PMC6741290

EC Pharmacology and Toxicology
Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese.

PMID: 31633124 [PubMed]

PMCID: PMC6800226

EC Neurology
Research Volunteers' Attitudes Toward Chronic Fatigue Syndrome and Myalgic Encephalomyelitis.

PMID: 29662969 [PubMed]

PMCID: PMC5898812

EC Pharmacology and Toxicology
Hyperbaric Oxygen Therapy for Alzheimer's Disease.

PMID: 30215058 [PubMed]

PMCID: PMC6133268

News and Events

February Issue Release

We always feel pleasure to share our updates with you all. Here, notifying you that we have successfully released the February issue of respective journals and the latest articles can be viewed on the current issue pages.

Submission Deadline for Upcoming Issue

ECronicon delightfully welcomes all the authors around the globe for effective collaboration with an article submission for the upcoming issue of respective journals. Submissions are accepted on/before February 17, 2023.

Certificate of Publication

ECronicon honors with a "Publication Certificate" to the corresponding author by including the names of co-authors as a token of appreciation for publishing the work with our respective journals.

Best Article of the Issue

Editors of respective journals will always be very much interested in electing one Best Article after each issue release. The authors of the selected article will be honored with a "Best Article of the Issue" certificate.

Certifying for Review

ECronicon certifies the Editors for their first review done towards the assigned article of the respective journals.

Latest Articles

The latest articles will be updated immediately on the articles in press page of the respective journals.