Eur Arch Paediatr Dent (2014) 15:429–433 DOI 10.1007/s40368-014-0134-z
ORIGINAL SCIENTIFIC ARTICLE
In vitro toxicity of grey MTA in comparison to white MTA on human periodontal ligament fibroblasts S. N. Al-Haj Ali • S. H. Al-Jundi • D. J. Ditto
Received: 5 April 2014 / Accepted: 26 May 2014 / Published online: 16 July 2014 Ó European Academy of Paediatric Dentistry 2014
Abstract Aim This was to define and compare the in vitro toxicity of grey MTA with that of white MTA on cultured human periodontal ligament (PDL) fibroblasts. Methods PDL cells were obtained from sound first permanent molars and cultured in Dulbecco’s Modified Eagle’s Medium. Cultures were subjected to different concentrations of grey and white MTA (0.5, 5, 50 and 500 lg/ml) for 24 h at 37 °C. Cells that were not exposed to grey or white MTA served as the negative control. In vitro toxicity was assessed using MTT assay. Statistics The results were compared using ANOVA and Tukey statistical tests (p \ 0.05). Results White MTA presented higher in vitro toxicity than grey MTA. However, the differences were almost insignificant (p [ 0.05). Conclusion Both colours of MTA are biocompatible since they were both able to preserve PDL fibroblasts for up to 24 h. MTA is as a promising alternative in pulpotomy of primary teeth. Keywords
Grey MTA White MTA Fibroblasts
S. N. Al-Haj Ali (&) Department of Orthodontics and Paediatric Dentistry, Faculty of Dentistry, Qassim University, PO Box 1126, Qassim 51431, Kingdom of Saudi Arabia e-mail: [email protected] S. H. Al-Jundi D. J. Ditto Preventive Dentistry Department, Jordan University of Science and Technology, Irbid, Jordan
Introduction One of the main objectives of operative dentistry is to maintain pulp health in compromised teeth to reduce the need for root canal treatment and the potential for unwanted sequelae such as tooth loss. Maintaining pulpally-involved primary teeth in a healthy state until the time of normal exfoliation remains one of the challenges for paediatric dentistry (Mcdonald et al. 2011; Fallahinejad Ghajari et al. 2008). In particular, a vital pulpotomy procedure has always been the topic of debate (Prabhu and Munshi 1997). Vital pulpotomy implies applying a medicament over the residual radicular pulp tissue to promote healing and to ideally allow normal tooth physiology to continue. Various medicaments and pulp dressing materials have been suggested in the dental literature; these include formocresol, (Bahrololoomi et al. 2008) glutaraldehyde, ferric sulphate, calcium hydroxide and potentially regenerative materials such as mineral trioxide aggregate (MTA) (Ranly 1994). MTA is a powder which consists of fine hydrophilic particles of Portland cement, bismuth oxide, and gypsum. Portland cement is the major component and is composed of dicalcium silicate, tricalcium silicate, tricalcium aluminate, calcium sulphate dihydrate, tetracalcium aluminoferrite and bismuth oxide (Peng et al. 2006; Roberts et al. 2008; Asgary et al. 2009). In the presence of moisture, MTA forms a colloidal gel that solidifies to form hard cement within *4 h (Noorollahian 2008; Asgary et al. 2009). After Food and Drug Administration (FDA) approval, MTA became commercially available as grey MTA (GMTA) for the therapeutic endodontic use in humans (Peng et al. 2006). Because of the potential discolouration effect of GMTA, white-coloured formula of MTA (WMTA) was introduced and was more popular in incisor teeth (Asgary et al. 2009).
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Several in vitro and in vivo studies have shown that MTA prevents microleakage, is biocompatible, has excellent sealing ability, has antimicrobial properties, (Torabinejad and Chivian 1999; Peng et al. 2006) and is able to form a dentine bridge, cementum and PDL regeneration. It was even found that MTA promotes rapid cell growth and proliferation when it is placed in contact with the dental pulp or periradicular tissues (Eidelman et al. 2001; Witherspoon et al. 2006). Many reports supported its clinical use in pulpotomy for both permanent and primary teeth based on its superior capability to induce dentine bridge formation with less pulp inflammation than formocresol and ferric sulphate (Caicedo et al. 2006; Aienehchi et al. 2007). In vitro toxicity of MTA was assessed on animal cell lines; however, only few studies investigated its toxicity on cultured human PDL fibroblasts. Moreover, the literature reports comparing white and grey MTA have yielded conflicting results in terms of toxicity levels. Therefore, the aim of this study was to define and compare the in vitro toxicity of GMTA with that of WMTA on cultured human PDL fibroblasts.
Materials and methods The in vitro cell culture model involving PDL cells was approved by the institutional review board (IRB) and the ethical committee of the faculty of medicine at Jordan University of Science and Technology. Primary culture of human periodontal ligament cells The PDL tissue explants were obtained from two fully erupted sound maxillary first permanent molars extracted from an 11-year-old patient for orthodontic reasons. Written consent was obtained before extraction of potential teeth. The teeth were extracted as atraumatically as possible and they were immediately transferred to a 50-ml centrifuge tube containing 10 ml of Dulbecco’s modified Eagle’s medium (DMEM), 10 ml of foetal bovine serum (FBS), and an antibiotic solution consisting of 300 ll of penicillin– streptomycin mixture and 300 ll of amphotericin B (all obtained from Bio Whittaker, Belgium). The extracted teeth were immediately transported to the tissue culture laboratory where the middle third of the root surface was mechanically scraped with a number 15 scalpel using aseptic techniques to obtain samples of PDL tissue which were diced into small tissue explants of 1 mm3. The tissue explants were placed into tissue culture flasks (25 cm3) and were incubated with DMEM containing glucose (4.5 g/l), penicillin (100 lg/ml), streptomycin (100 lg/ml), amphotericin B (0.25 lg/ml) and 10 % heat inactivated FBS.
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Cells were grown at 37 °C in a humidified atmosphere of 5 % CO2 in air. After 5 weeks, cells reached confluence when they were detached after trypsinisation [0.25 % trypsin with ethylene diaminetetra-acetic acid (EDTA) obtained from Sigma-Aldrich, USA] for 5 min and transferred to larger flasks (75 cm3) for continued growth. Subconfluent cultures were characterised to assure their PDL cell phenotype by the presence of alkaline phosphatase. Culture medium was renewed every 2 days until cells reached 80 % confluency. Cells used for the experiments proliferated in logarithmic phase between the 7th and 15th passages. PDL fibroblast cells were seeded at a density of 1.0 9 104 cells/well in 100-ll full-growth medium (DMEM ? 10 % FBS ? antibiotics) in 96-well plates. The plates were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 24 h to allow attachment. Preparation of tested materials The materials tested in this study were: GMTA (AngelusTM, Londrina, Brazil) and WMTA (Pro Root, DENTSPLY Tulsa Dental Products, USA). On the same day of cell seeding in the 96-well plates, the tested materials were prepared using the same method which was used in a previous report (De Menezes et al. 2009). In brief, 50 mg of each tested material was vigorously mixed under aseptic conditions with 1 ml of full-growth medium. Then, the tested materials were placed in a 24-well plate and incubated for 24 h at 37 °C in 5 % CO2 atmosphere after which the supernatant was carefully removed and submitted to serial dilutions in full-growth medium. Four concentrations ranging from 0.5 to 500 lg/ml were established for each supernatant of the tested materials and they were used immediately for cytotoxicity assay. Exposure of PDL cultures to tested materials On the same day of treatment, the culture medium was drained from each well and the cells were subjected to the four concentrations of GMTA and WMTA for 24 h at 37 °C in 5 % CO2 atmosphere. Cells that were not exposed to the tested materials (DMEM ? 10 % FBS and antibiotics) served as the negative control. Experiments were completed twice in triplicate wells. Assessing the viability of cells A 5 mg/ml solution of MTT (Sigma-Aldrich, USA) in PBS was produced by dissolving 500 mg of MTT in 100 ml of PBS, then the solution was filtered with 0.45-lm cellulose acetate filter paper (VIVID separation and filtration, USA) to ensure sterilisation. After incubation with tested
Eur Arch Paediatr Dent (2014) 15:429–433
materials for 24 h at 37 °C in 5 % CO2 atmosphere, all materials were removed and the PDL cells were washed twice with PBS, then 100 ll of DMEM and 10 ll of MTT solution were added to all wells and all plates were incubated for 4 h at 37 °C. The supernatant was then eliminated and 100 ll of Dimethyl Sulphoxide (DMSO) solvent (Sigma-Aldrich, USA) was added to each well. The plates were then placed in a waving shaker machine for 30 min. The absorbance at 570 nm was measured with a microtiter plate reader (Basic Tecan, Austria) with absorbance at 650 nm used as a reference. The in vitro toxicity of the tested materials was calculated as the relative absorbance of tested wells versus untreated (negative control) wells as follows: Percent ð%Þ cell viability ¼ mean absorbance of tested wells= mean absorbance of untreated wells 100: The in vitro toxicity ranking of the tested materials The tested materials were ranked according to their toxicity level which was based on the estimated LD50 values [lethal dose to 50 % of the cells when compared to a control group (mg/ml)] using the same rating method that was used in a previous report (Sjogren et al. 2000). Statistical analysis The mean value of the triplicate wells from two independent experiments was submitted to ANOVA and Tukey’s test for statistical analysis. The LD50 value for each tested material was estimated using the probit analysis. The significance level was p \ 0.05.
Results The mean absorbance, percentage of viable cells and cytotoxicity rating for all concentrations of tested materials and the negative control group after 24 h of exposure with the estimated LD50 values for GMTA and WMTA are shown in Table 1. Figure 1 shows the dose-related inhibition after 24 h of exposure to all concentrations of GMTA and WMTA tested. As shown in Table 1, there was a drop in cell viability with increasing the concentration of both WMTA and GMTA. The overall cytotoxicity rating of WMTA was 2 which is slightly cytotoxic. For GMTA, the lowest concentrations (0.5 and 5 lg/ml) had the highest viability values with a cytotoxicity score of 1, whereas, the cytotoxicity score was 2 for the higher concentrations. When the different concentrations of the tested materials were compared, the highest concentration of WMTA
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(500 lg/ml) was significantly worse than all other concentrations of the same material and GMTA except WMTA 50 lg/ml (p \ 0.05). For the lowest concentrations tested, WMTA (5 lg/ml) was only significantly worse than GMTA (0.5 lg/ml). The rest of the differences were insignificant. Surprisingly, following 24 h of exposure, the cell viability of the negative control was significantly superior to all concentrations of the tested materials except GMTA (0.5 and 5 lg/ml) in which the cell viability was comparable to the negative control (93.467 and 96.467 %, respectively). Based on LD50 values, the following concentrations killed 50 % of the cells: 10.235 and 3.106 mg/ ml for GMTA and WMTA, respectively. WMTA presented higher in vitro toxicity than GMTA.
Discussion The use of cell culture to test the biocompatibility of dental materials is gaining importance because it offers a significant cost effective and repeatable tool to improve knowledge of possible toxic effects of materials. Several reports used animal cell lines such as L929 or 3T3 mouse fibroblasts to assess toxicity of dental materials (Al-Hiyasat et al. 2005; De Menezes et al. 2009). However, it has been shown that primary fibroblast cell cultures established from normal tissues provide a more representative model of the in vivo fibroblast cell population (Amaral et al. 2009). In addition, the cellular changes that can result from cell culture manipulation will be less in primary cell cultures (Karimjee et al. 2006). In this study, a cell culture technique of human PDL fibroblasts was employed since PDL fibroblasts are more representative of human pulp fibroblasts than animal cell lines. They are easier to culture and they have better survival rate than pulp fibroblasts. Furthermore, they are more sensitive than animal cell lines to external environmental changes which allow the detection of subtle cell responses (Van Wyk and Maritz 2001; Amaral et al. 2009). Various literature reports used scanning electron microscopy (SEM), (Camilleri 2006) enzyme assay, cytokine expression, agar overlay, radiochromium release methods, nucleic acid count (NAC), neutral red uptake (NRU), and MTT tests to test biocompatibility and cytotoxicity (Souza et al. 2006; De Menezes et al. 2009). SEM is contraindicated to evaluate cell growth and expression over materials based on Portland cement (Camilleri 2006) and NRU test is less sensitive to MTA than MTT and NAC tests (Souza et al. 2006). Moreover, agar overlay and radiochromium release methods are the least preferable because of the use of radioactive isotopes in the latter method (Camilleri 2006).
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432 Table 1 Mean absorbance, % viability and cytotoxicity rating for all concentrations of GMTA, WMTA and the negative control with the estimated LD50 values for GMTA and WMTA
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Treatment
Concentration (lg/ml)
GMTA
0.5
0.93950
5
0.91017
50
0.87517
89.9
2
500
0.85200
87.5
2
0.5
0.89117
91.4
1
5
0.84400
86.5
2
50
0.78317
80.3
2
500
0.73467
75.2
2
0.97433
100
WMTA
Untreated cells (negative control)
Fig. 1 Dose-related inhibition after 24 h of exposure to all concentrations of GMTA and WMTA tested
In this study, in vitro toxicity was tested using the MTT assay which has been used extensively to assess toxicity of dental materials. The MTT assay indicates the effects on cell viability by alterations of mitochondrial dehydrogenase activities. In this assay, methylthiazol tetrazolium is metabolically reduced to coloured formazan. Factors that inhibit dehydrogenase activity will affect the associated colour reaction. It has been shown that activated cells produce more formazan than resting cells; therefore, it is possible to measure cell activity or enzyme activities (Issa et al. 2004). MTT assay is a highly calibrated and reliable assay with the advantages of being sensitive (detecting as few as 103 viable cells/ml) and accurate with colour development strongly correlating with cell numbers (Tipton et al. 2003). In agreement with previous reports (AlHiyasat et al. 2005; De Menezes et al. 2009) a direct method was used where the material specimens were in direct contact with the cells in a biological solution. In addition, both MTA colours were mixed with the culture
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Mean absorbance
% viability
Cytotoxicity rating
Estimated LD50 (mg/ml)
96.4
1
10.235
93.4
1
3.106
medium and not with distilled water as recommended by the manufacturer and the supernatants of grey and white MTA were used since it was found in a previous report (Karimjee et al. 2006) that white MTA mixed with water or with KY Jelly had similar biocompatibility regarding the effects of their elutes on human PDL cells; therefore, it is unlikely that mixing MTA with a culture medium which is generally used to grow cells will affect its biocompatibility. Literature reports comparing white and grey MTA have yielded conflicting results in terms of biocompatibility and ability to induce tissue regeneration; few reports revealed that the two MTA colours have similar properties and cytotoxicity levels (Souza et al. 2006; Koulaouzidou et al. 2008). In this study, the LD50 value was calculated for both colours of MTA. The LD50 value is one of the most reliable indicators of toxicity because it defines the toxic concentration that is exactly in the middle point between the first signs of toxicity and the complete absence of metabolism or cell death (Ratanasathien et al. 1995; Hanks et al. 1996). Based on the calculated LD50 values, this study has shown that WMTA presented higher in vitro toxicity than GMTA which is in agreement with the finding of a previous report (Perez et al. 2003). Furthermore, 0.5 and 5 lg/ml GMTA showed comparable cell viability to the negative control. It can be hypothesised from the findings of this study that GMTA stimulated fibroblast proliferation in a similar manner to the negative control group, which is in agreement with the finding of a previous report (Takita et al. 2006). However, both colours of MTA tested can be considered biocompatible since the maximum concentration tested of both MTA colours was only slightly cytotoxic. Furthermore, the reported difference between grey and white MTA can be attributed to differences in composition, surface texture, chemical and physical properties of the two brands used; Angelus and ProRoot MTA. This could be true since it was reported that Angelus MTA showed greater release of calcium in the first 24 h of activation and a lower concentration of bismuth in the grey version. In
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addition, the manufacturer has claimed the solubility of Angelus MTA to be lower than ProRoot MTA (Saghiri et al. 2010), therefore, it could be that grey Angelus MTA dissolved less in DMEM than in white ProRoot MTA and in this case the toxic effect on the cells would be less.
Conclusion White MTA presented higher in vitro toxicity than grey MTA. However, both colours of MTA are considered biocompatible since all tested concentrations were able to preserve PDL fibroblasts for up to 24 h. MTA stands as a promising alternative in pulpotomy of primary teeth. However, this finding needs to be carefully extrapolated into the clinical situation since the actual clinical preparation and usage of MTA is different from the methods of preparation for in vitro testing. Further in vitro studies are required utilising longer periods of exposure to MTA and utilising pulp fibroblasts since their behaviour will be the closest to clinical conditions.
References Aienehchi M, Dadvand S, Fayazi S, Bayat-Movahed S. Randomised controlled trial of mineral trioxide aggregate and formocresol for pulpotomy in primary molar teeth. Int Endod J. 2007;40:261–72. Al-Hiyasat AS, Darmani H, Milhem MM. Cytotoxicity evaluation of dental resin composites and their flowable derivatives. Clin Oral Investig. 2005;9(1):21–5. Amaral M, Gomes PS, Lopes MA, et al. Cytotoxicity evaluation of nanocrystalline diamond coatings by fibroblast cell cultures. Acta Biomater. 2009;5(2):755–63. Asgary S, Eghbal MJ, Parirokh M, et al. Comparison of mineral trioxide aggregate’s composition with Portland cements and a new endodontic cement. J Endod. 2009;35(2):243–50. Bahrololoomi Z, Moeintaghavi A, Emtiazi M, Hosseini G. Clinical and radiographic comparison of primary molars after formocresol and electrosurgical pulpotomy: a randomised clinical trial. Indian J Dent Res. 2008;19(3):219–23. Caicedo R, Abbot PV, Alongi DJ, Alarcon MY. Clinical, radiographic and histologic analysis of the effects of mineral trioxide aggregate used in direct pulp capping and pulpotomies of primary teeth. Aust Dent J. 2006;51:297–305. Camilleri J. A review of the methods used to study biocompatibility of Portland cement-derived materials used in dentistry. MMJ. 2006;18(3):9–13. De Menezes JV, Takamori ER, Bijella MF, Granjeiro JM. In vitro toxicity of MTA compared with other primary teeth pulpotomy agents. J Clin Pediatr Dent. 2009;33(3):217–21. Eidelman E, Holan G, Fuks AB. Mineral trioxide aggregate vs. formocresol in pulpotomised primary molars: a preliminary report. Pediatr Dent. 2001;23(1):15–8. Fallahinejad Ghajari MM, Vatanpour M, Fard M. Comparison of pulpotomy with formocresol and MTA in primary molars: a
433 systematic review and meta-analysis. Int Endod J. 2008;3(3):45–9. Hanks CT, Wataha JC, Sun Z. In vitro models of biocompatibility: a review. Dent Mater. 1996;12(3):186–93. Issa Y, Watts DC, Brunton PA, Waters CM, Duxbury AJ. Resin composite monomers alter MTT and LDH activity of human gingival fibroblasts in vitro. Dent Mater. 2004;20(1):12–20. Karimjee CK, Koka S, Rallis DM, Gound TG. Cellular toxicity of mineral trioxide aggregate mixed with an alternative delivery vehicle. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102(4):115–20. Koulaouzidou EA, Economides N, Beltes P, Geromichalos G, Papazisis K. In vitro evaluation of the cytotoxicity of ProRoot MTA and MTA Angelus. J Oral Sci. 2008;50(4):397–402. McDonald RE, Avery DR, Dean JA. Dentistry for the child and adolescent, 9th ed. Mosby; 2011. pp. 343–63. Noorollahian H. Comparison of mineral trioxide aggregate and formocresol as pulp medicaments for pulpotomies in primary molars. Br Dent J. 2008;204(11):E20. Peng L, Ye L, Tan H, Zhou X. Evaluation of the formocresol versus mineral trioxide aggregate primary molar pulpotomy: a metaanalysis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102(6):e40–4. Perez AL, Spears R, Gutmann JL, Opperman LA. Osteoblasts and MG-63 osteosarcoma cells behave differently when in contact with ProRoot MTA and white MTA. Int Endod J. 2003;36(8):564–70. Prabhu NT, Munshi AK. Clinical, radiographic and histological observations of the radicular pulp following ‘‘feracrylum’’ pulpotomy. J Clin Pediatr Dent. 1997;21(2):151–6. Ranly DM. Pulpotomy therapy in primary teeth: new modalities for old rationales. Pediatr Dent. 1994;16(6):403–9. Ratanasathien S, Wataha JC, Hanks CT, Dennison JB. Cytotoxic interactive effects of dentin bonding components on mouse fibroblasts. J Dent Res. 1995;74(9):1602–6. Roberts HW, Toth JM, Berzins DW, Charlton DG. Mineral trioxide aggregate material use in endodontic treatment: a review of the literature. Dent Mater. 2008;24(2):149–64. Saghiri ML, Daliri M, Aeinehchi M, Behnamghader A. A comparative study of ProRoot MTA and Angelus MTA (in vitro). SRM J Res Dent Sci. 2010;1:60–5. Sjogren G, Sletten G, Dahl JE. Cytotoxicity of dental alloys, metals, and ceramics assessed by Millipore filter, agar overlay, and MTT tests. J Prosthet Dent. 2000;84(2):229–36. Souza NJ, Justo GZ, Oliveira CR, Haun M, Bincoletto C. Cytotoxicity of materials used in perforation repair tested using the V79 fibroblast cell line and the granulocyte–macrophage progenitor cells. Int Endod J. 2006;39(1):40–7. Takita T, Hayashi M, Takeichi O, et al. Effect of mineral trioxide aggregate on proliferation of cultured human dental pulp cells. Int Endod J. 2006;39(5):415–22. Tipton DA, Lyle B, Babich H, Dabbous M. In vitro cytotoxic and anti- inflammatory effects of myrrh oil on human gingival fibroblasts and epithelial cells. Toxicol In Vitro. 2003;17(3):301–10. Torabinejad M, Chivian N. Clinical applications of mineral trioxide aggregate. J Endod. 1999;25(3):197–205. Van Wyk CWO, Maritz JS. Cultured pulp fibroblasts: are they suitable for in vitro cytotoxicity testing? J Oral Pathol Med. 2001;30(3):168–77. Witherspoon DE, Small JC, Harris GZ. Mineral trioxide aggregate pulpotomies: a case series outcomes assessment. J Am Dent Assoc. 2006;137(5):610–8.
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ORIGINAL SCIENTIFIC ARTICLE
In vitro toxicity of grey MTA in comparison to white MTA on human periodontal ligament fibroblasts S. N. Al-Haj Ali • S. H. Al-Jundi • D. J. Ditto
Received: 5 April 2014 / Accepted: 26 May 2014 / Published online: 16 July 2014 Ó European Academy of Paediatric Dentistry 2014
Abstract Aim This was to define and compare the in vitro toxicity of grey MTA with that of white MTA on cultured human periodontal ligament (PDL) fibroblasts. Methods PDL cells were obtained from sound first permanent molars and cultured in Dulbecco’s Modified Eagle’s Medium. Cultures were subjected to different concentrations of grey and white MTA (0.5, 5, 50 and 500 lg/ml) for 24 h at 37 °C. Cells that were not exposed to grey or white MTA served as the negative control. In vitro toxicity was assessed using MTT assay. Statistics The results were compared using ANOVA and Tukey statistical tests (p \ 0.05). Results White MTA presented higher in vitro toxicity than grey MTA. However, the differences were almost insignificant (p [ 0.05). Conclusion Both colours of MTA are biocompatible since they were both able to preserve PDL fibroblasts for up to 24 h. MTA is as a promising alternative in pulpotomy of primary teeth. Keywords
Grey MTA White MTA Fibroblasts
S. N. Al-Haj Ali (&) Department of Orthodontics and Paediatric Dentistry, Faculty of Dentistry, Qassim University, PO Box 1126, Qassim 51431, Kingdom of Saudi Arabia e-mail: [email protected] S. H. Al-Jundi D. J. Ditto Preventive Dentistry Department, Jordan University of Science and Technology, Irbid, Jordan
Introduction One of the main objectives of operative dentistry is to maintain pulp health in compromised teeth to reduce the need for root canal treatment and the potential for unwanted sequelae such as tooth loss. Maintaining pulpally-involved primary teeth in a healthy state until the time of normal exfoliation remains one of the challenges for paediatric dentistry (Mcdonald et al. 2011; Fallahinejad Ghajari et al. 2008). In particular, a vital pulpotomy procedure has always been the topic of debate (Prabhu and Munshi 1997). Vital pulpotomy implies applying a medicament over the residual radicular pulp tissue to promote healing and to ideally allow normal tooth physiology to continue. Various medicaments and pulp dressing materials have been suggested in the dental literature; these include formocresol, (Bahrololoomi et al. 2008) glutaraldehyde, ferric sulphate, calcium hydroxide and potentially regenerative materials such as mineral trioxide aggregate (MTA) (Ranly 1994). MTA is a powder which consists of fine hydrophilic particles of Portland cement, bismuth oxide, and gypsum. Portland cement is the major component and is composed of dicalcium silicate, tricalcium silicate, tricalcium aluminate, calcium sulphate dihydrate, tetracalcium aluminoferrite and bismuth oxide (Peng et al. 2006; Roberts et al. 2008; Asgary et al. 2009). In the presence of moisture, MTA forms a colloidal gel that solidifies to form hard cement within *4 h (Noorollahian 2008; Asgary et al. 2009). After Food and Drug Administration (FDA) approval, MTA became commercially available as grey MTA (GMTA) for the therapeutic endodontic use in humans (Peng et al. 2006). Because of the potential discolouration effect of GMTA, white-coloured formula of MTA (WMTA) was introduced and was more popular in incisor teeth (Asgary et al. 2009).
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Several in vitro and in vivo studies have shown that MTA prevents microleakage, is biocompatible, has excellent sealing ability, has antimicrobial properties, (Torabinejad and Chivian 1999; Peng et al. 2006) and is able to form a dentine bridge, cementum and PDL regeneration. It was even found that MTA promotes rapid cell growth and proliferation when it is placed in contact with the dental pulp or periradicular tissues (Eidelman et al. 2001; Witherspoon et al. 2006). Many reports supported its clinical use in pulpotomy for both permanent and primary teeth based on its superior capability to induce dentine bridge formation with less pulp inflammation than formocresol and ferric sulphate (Caicedo et al. 2006; Aienehchi et al. 2007). In vitro toxicity of MTA was assessed on animal cell lines; however, only few studies investigated its toxicity on cultured human PDL fibroblasts. Moreover, the literature reports comparing white and grey MTA have yielded conflicting results in terms of toxicity levels. Therefore, the aim of this study was to define and compare the in vitro toxicity of GMTA with that of WMTA on cultured human PDL fibroblasts.
Materials and methods The in vitro cell culture model involving PDL cells was approved by the institutional review board (IRB) and the ethical committee of the faculty of medicine at Jordan University of Science and Technology. Primary culture of human periodontal ligament cells The PDL tissue explants were obtained from two fully erupted sound maxillary first permanent molars extracted from an 11-year-old patient for orthodontic reasons. Written consent was obtained before extraction of potential teeth. The teeth were extracted as atraumatically as possible and they were immediately transferred to a 50-ml centrifuge tube containing 10 ml of Dulbecco’s modified Eagle’s medium (DMEM), 10 ml of foetal bovine serum (FBS), and an antibiotic solution consisting of 300 ll of penicillin– streptomycin mixture and 300 ll of amphotericin B (all obtained from Bio Whittaker, Belgium). The extracted teeth were immediately transported to the tissue culture laboratory where the middle third of the root surface was mechanically scraped with a number 15 scalpel using aseptic techniques to obtain samples of PDL tissue which were diced into small tissue explants of 1 mm3. The tissue explants were placed into tissue culture flasks (25 cm3) and were incubated with DMEM containing glucose (4.5 g/l), penicillin (100 lg/ml), streptomycin (100 lg/ml), amphotericin B (0.25 lg/ml) and 10 % heat inactivated FBS.
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Cells were grown at 37 °C in a humidified atmosphere of 5 % CO2 in air. After 5 weeks, cells reached confluence when they were detached after trypsinisation [0.25 % trypsin with ethylene diaminetetra-acetic acid (EDTA) obtained from Sigma-Aldrich, USA] for 5 min and transferred to larger flasks (75 cm3) for continued growth. Subconfluent cultures were characterised to assure their PDL cell phenotype by the presence of alkaline phosphatase. Culture medium was renewed every 2 days until cells reached 80 % confluency. Cells used for the experiments proliferated in logarithmic phase between the 7th and 15th passages. PDL fibroblast cells were seeded at a density of 1.0 9 104 cells/well in 100-ll full-growth medium (DMEM ? 10 % FBS ? antibiotics) in 96-well plates. The plates were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 24 h to allow attachment. Preparation of tested materials The materials tested in this study were: GMTA (AngelusTM, Londrina, Brazil) and WMTA (Pro Root, DENTSPLY Tulsa Dental Products, USA). On the same day of cell seeding in the 96-well plates, the tested materials were prepared using the same method which was used in a previous report (De Menezes et al. 2009). In brief, 50 mg of each tested material was vigorously mixed under aseptic conditions with 1 ml of full-growth medium. Then, the tested materials were placed in a 24-well plate and incubated for 24 h at 37 °C in 5 % CO2 atmosphere after which the supernatant was carefully removed and submitted to serial dilutions in full-growth medium. Four concentrations ranging from 0.5 to 500 lg/ml were established for each supernatant of the tested materials and they were used immediately for cytotoxicity assay. Exposure of PDL cultures to tested materials On the same day of treatment, the culture medium was drained from each well and the cells were subjected to the four concentrations of GMTA and WMTA for 24 h at 37 °C in 5 % CO2 atmosphere. Cells that were not exposed to the tested materials (DMEM ? 10 % FBS and antibiotics) served as the negative control. Experiments were completed twice in triplicate wells. Assessing the viability of cells A 5 mg/ml solution of MTT (Sigma-Aldrich, USA) in PBS was produced by dissolving 500 mg of MTT in 100 ml of PBS, then the solution was filtered with 0.45-lm cellulose acetate filter paper (VIVID separation and filtration, USA) to ensure sterilisation. After incubation with tested
Eur Arch Paediatr Dent (2014) 15:429–433
materials for 24 h at 37 °C in 5 % CO2 atmosphere, all materials were removed and the PDL cells were washed twice with PBS, then 100 ll of DMEM and 10 ll of MTT solution were added to all wells and all plates were incubated for 4 h at 37 °C. The supernatant was then eliminated and 100 ll of Dimethyl Sulphoxide (DMSO) solvent (Sigma-Aldrich, USA) was added to each well. The plates were then placed in a waving shaker machine for 30 min. The absorbance at 570 nm was measured with a microtiter plate reader (Basic Tecan, Austria) with absorbance at 650 nm used as a reference. The in vitro toxicity of the tested materials was calculated as the relative absorbance of tested wells versus untreated (negative control) wells as follows: Percent ð%Þ cell viability ¼ mean absorbance of tested wells= mean absorbance of untreated wells 100: The in vitro toxicity ranking of the tested materials The tested materials were ranked according to their toxicity level which was based on the estimated LD50 values [lethal dose to 50 % of the cells when compared to a control group (mg/ml)] using the same rating method that was used in a previous report (Sjogren et al. 2000). Statistical analysis The mean value of the triplicate wells from two independent experiments was submitted to ANOVA and Tukey’s test for statistical analysis. The LD50 value for each tested material was estimated using the probit analysis. The significance level was p \ 0.05.
Results The mean absorbance, percentage of viable cells and cytotoxicity rating for all concentrations of tested materials and the negative control group after 24 h of exposure with the estimated LD50 values for GMTA and WMTA are shown in Table 1. Figure 1 shows the dose-related inhibition after 24 h of exposure to all concentrations of GMTA and WMTA tested. As shown in Table 1, there was a drop in cell viability with increasing the concentration of both WMTA and GMTA. The overall cytotoxicity rating of WMTA was 2 which is slightly cytotoxic. For GMTA, the lowest concentrations (0.5 and 5 lg/ml) had the highest viability values with a cytotoxicity score of 1, whereas, the cytotoxicity score was 2 for the higher concentrations. When the different concentrations of the tested materials were compared, the highest concentration of WMTA
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(500 lg/ml) was significantly worse than all other concentrations of the same material and GMTA except WMTA 50 lg/ml (p \ 0.05). For the lowest concentrations tested, WMTA (5 lg/ml) was only significantly worse than GMTA (0.5 lg/ml). The rest of the differences were insignificant. Surprisingly, following 24 h of exposure, the cell viability of the negative control was significantly superior to all concentrations of the tested materials except GMTA (0.5 and 5 lg/ml) in which the cell viability was comparable to the negative control (93.467 and 96.467 %, respectively). Based on LD50 values, the following concentrations killed 50 % of the cells: 10.235 and 3.106 mg/ ml for GMTA and WMTA, respectively. WMTA presented higher in vitro toxicity than GMTA.
Discussion The use of cell culture to test the biocompatibility of dental materials is gaining importance because it offers a significant cost effective and repeatable tool to improve knowledge of possible toxic effects of materials. Several reports used animal cell lines such as L929 or 3T3 mouse fibroblasts to assess toxicity of dental materials (Al-Hiyasat et al. 2005; De Menezes et al. 2009). However, it has been shown that primary fibroblast cell cultures established from normal tissues provide a more representative model of the in vivo fibroblast cell population (Amaral et al. 2009). In addition, the cellular changes that can result from cell culture manipulation will be less in primary cell cultures (Karimjee et al. 2006). In this study, a cell culture technique of human PDL fibroblasts was employed since PDL fibroblasts are more representative of human pulp fibroblasts than animal cell lines. They are easier to culture and they have better survival rate than pulp fibroblasts. Furthermore, they are more sensitive than animal cell lines to external environmental changes which allow the detection of subtle cell responses (Van Wyk and Maritz 2001; Amaral et al. 2009). Various literature reports used scanning electron microscopy (SEM), (Camilleri 2006) enzyme assay, cytokine expression, agar overlay, radiochromium release methods, nucleic acid count (NAC), neutral red uptake (NRU), and MTT tests to test biocompatibility and cytotoxicity (Souza et al. 2006; De Menezes et al. 2009). SEM is contraindicated to evaluate cell growth and expression over materials based on Portland cement (Camilleri 2006) and NRU test is less sensitive to MTA than MTT and NAC tests (Souza et al. 2006). Moreover, agar overlay and radiochromium release methods are the least preferable because of the use of radioactive isotopes in the latter method (Camilleri 2006).
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432 Table 1 Mean absorbance, % viability and cytotoxicity rating for all concentrations of GMTA, WMTA and the negative control with the estimated LD50 values for GMTA and WMTA
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Treatment
Concentration (lg/ml)
GMTA
0.5
0.93950
5
0.91017
50
0.87517
89.9
2
500
0.85200
87.5
2
0.5
0.89117
91.4
1
5
0.84400
86.5
2
50
0.78317
80.3
2
500
0.73467
75.2
2
0.97433
100
WMTA
Untreated cells (negative control)
Fig. 1 Dose-related inhibition after 24 h of exposure to all concentrations of GMTA and WMTA tested
In this study, in vitro toxicity was tested using the MTT assay which has been used extensively to assess toxicity of dental materials. The MTT assay indicates the effects on cell viability by alterations of mitochondrial dehydrogenase activities. In this assay, methylthiazol tetrazolium is metabolically reduced to coloured formazan. Factors that inhibit dehydrogenase activity will affect the associated colour reaction. It has been shown that activated cells produce more formazan than resting cells; therefore, it is possible to measure cell activity or enzyme activities (Issa et al. 2004). MTT assay is a highly calibrated and reliable assay with the advantages of being sensitive (detecting as few as 103 viable cells/ml) and accurate with colour development strongly correlating with cell numbers (Tipton et al. 2003). In agreement with previous reports (AlHiyasat et al. 2005; De Menezes et al. 2009) a direct method was used where the material specimens were in direct contact with the cells in a biological solution. In addition, both MTA colours were mixed with the culture
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Mean absorbance
% viability
Cytotoxicity rating
Estimated LD50 (mg/ml)
96.4
1
10.235
93.4
1
3.106
medium and not with distilled water as recommended by the manufacturer and the supernatants of grey and white MTA were used since it was found in a previous report (Karimjee et al. 2006) that white MTA mixed with water or with KY Jelly had similar biocompatibility regarding the effects of their elutes on human PDL cells; therefore, it is unlikely that mixing MTA with a culture medium which is generally used to grow cells will affect its biocompatibility. Literature reports comparing white and grey MTA have yielded conflicting results in terms of biocompatibility and ability to induce tissue regeneration; few reports revealed that the two MTA colours have similar properties and cytotoxicity levels (Souza et al. 2006; Koulaouzidou et al. 2008). In this study, the LD50 value was calculated for both colours of MTA. The LD50 value is one of the most reliable indicators of toxicity because it defines the toxic concentration that is exactly in the middle point between the first signs of toxicity and the complete absence of metabolism or cell death (Ratanasathien et al. 1995; Hanks et al. 1996). Based on the calculated LD50 values, this study has shown that WMTA presented higher in vitro toxicity than GMTA which is in agreement with the finding of a previous report (Perez et al. 2003). Furthermore, 0.5 and 5 lg/ml GMTA showed comparable cell viability to the negative control. It can be hypothesised from the findings of this study that GMTA stimulated fibroblast proliferation in a similar manner to the negative control group, which is in agreement with the finding of a previous report (Takita et al. 2006). However, both colours of MTA tested can be considered biocompatible since the maximum concentration tested of both MTA colours was only slightly cytotoxic. Furthermore, the reported difference between grey and white MTA can be attributed to differences in composition, surface texture, chemical and physical properties of the two brands used; Angelus and ProRoot MTA. This could be true since it was reported that Angelus MTA showed greater release of calcium in the first 24 h of activation and a lower concentration of bismuth in the grey version. In
Eur Arch Paediatr Dent (2014) 15:429–433
addition, the manufacturer has claimed the solubility of Angelus MTA to be lower than ProRoot MTA (Saghiri et al. 2010), therefore, it could be that grey Angelus MTA dissolved less in DMEM than in white ProRoot MTA and in this case the toxic effect on the cells would be less.
Conclusion White MTA presented higher in vitro toxicity than grey MTA. However, both colours of MTA are considered biocompatible since all tested concentrations were able to preserve PDL fibroblasts for up to 24 h. MTA stands as a promising alternative in pulpotomy of primary teeth. However, this finding needs to be carefully extrapolated into the clinical situation since the actual clinical preparation and usage of MTA is different from the methods of preparation for in vitro testing. Further in vitro studies are required utilising longer periods of exposure to MTA and utilising pulp fibroblasts since their behaviour will be the closest to clinical conditions.
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