Eur Arch Paediatr Dent (2015) 16:51–55 DOI 10.1007/s40368-014-0147-7
ORIGINAL SCIENTIFIC ARTICLE
In vitro toxicity of formocresol, ferric sulphate, and grey MTA on human periodontal ligament fibroblasts S. N. Al-Haj Ali • S. H. Al-Jundi • D. J. Ditto
Received: 12 June 2014 / Accepted: 5 September 2014 / Published online: 29 October 2014 Ó European Academy of Paediatric Dentistry 2014
Abstract Aim This was to assess and compare the in vitro toxicity of formocresol, ferric sulphate and MTA on cultured human periodontal ligament (PDL) fibroblasts. Study design PDL cells were obtained from sound first permanent molars and cultured in Dulbecco’s modified Eagle’s medium. Methods PDL cells were subjected to different concentrations of formocresol, ferric sulphate, and grey MTA for 24, 48, and 72 h at 37 °C. Cells that were not exposed to the tested materials served as the negative control. In vitro toxicity was assessed using MTT assay. Statistics Statistical analysis of data was accomplished using ANOVA and Tukey statistical tests (p \ 0.05). Results The overall toxicity ranking of the tested materials was as follows: formocresol [ ferric sulphate [ grey MTA. Only grey MTA had comparable cell viability to the negative control, the other tested materials were significantly inferior at the three exposure periods (p \ 0.05). Conclusion Regarding the viability of PDL fibroblasts, MTA stands as the most promising substitute to formocresol. However, considering MTA’s unavailability and high price in Jordan, ferric sulphate may be the best alternative to formocresol in pulpotomy of primary teeth.
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
Keywords MTA Fibroblasts Ferric sulphate Formocresol
Introduction Pulpotomy is a common procedure in the treatment of acutely inflamed primary teeth (McDonald et al. 2011). It is to the surgical amputation of the coronal inflamed portion of the pulp to retain the vital surface of the radicular pulp tissue treatment with a medicament to promote healing in the underlying tissue and eventually retain the tooth (Pinkham et al. 2005). Proper selection of the pulpotomy medicament is of paramount importance as it will interact with tissue fluids (Camilleri 2006); therefore biocompatibility of pulpotomy medicaments is a prerequisite. The dental literature is replete with papers that report the success or failure of primary tooth pulpotomy using a variety of medicaments. Formocresol (FC) is one of the most studied pulpotomy medicaments; however, concerns were raised over FC as a pulpotomy medicament due to the aldehyde constituent in its composition (Casas et al. 2005). Several investigations have led to the conclusion that formaldehyde is toxic to the connective tissue; it does not stay localised to the dental pulp as it is absorbed systemically, and therefore it has the potential of having carcinogenic and mutagenic effects (Fallahinejad Ghajari et al. 2008). This conclusion has created the need for alternative pulpotomy medicaments. Ferric sulphate (FS) at a concentration of 15.5 %—a non-aldehyde chemical—has also been investigated as a possible replacement for FC and has proven to have clinically and radiographically comparable success rates (Ibricevic and Al-Jame 2000; Fuks 2008). Recently, mineral trioxide aggregate (MTA) has attracted attention as a pulpotomy medicament because of excellent sealing
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ability, biocompatiblity and stimulation of hard tissue formation. It also appears to have higher long-term clinical and radiographic success rate than FC (Noorollahian 2008). Various reports in the literature studied in vitro toxicity of FC and MTA on animal cell lines; however, only few studies investigated their toxicity on cultured human periodontal ligament (PDL) fibroblasts. Moreover, comparative in vitro toxicity of FS has never been assessed on human PDL fibroblasts. Therefore, the aim of this study was to assess and compare the in vitro toxicity of FC, FS and MTA on cultured human PDL fibroblasts.
Materials and methods The research study was approved by the institutional review board and ethical committee of the faculty of medicine at Jordan University of Science and Technology (JUST). 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 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 was diced into tissue explants of 1 mm3. The tissue explants were placed into tissue culture flasks (25 cm2) 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. Cells were grown at 37 °C in a humidified atmosphere of 5 % CO2 in air. After 5 weeks, cells reached confluence and were detached after trypsinisation (0.25 % trypsin with ethylenediaminetetraacetic acid (EDTA, Sigma–Aldrich, USA)) for 5 min and transferred to larger flasks (75 cm2) 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
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(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 were: grey MTA (GMTA) (AngelusTM, Brazil), 20 % diluted FC (Formocresol Biodinamica, Brazil) and 15.5 % FS (Ultradent Products, Jordan). On the same day of cell seeding in the 96-well plates, the tested materials were prepared using a modified method to that used in a previous report (De Menezes et al. 2009). Briefly, 50 mg of each tested material was 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. Five tenfold concentrations ranging from 0.5 to 5,000 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 five concentrations of FC, FS, and GMTA, respectively, for 24, 48 and 72 h at 37 °C in 5 % CO2 atmosphere. FC well plates were separated from those of other tested materials. In addition, within the same plate, FC wells exposed to the various concentrations tested were separated from each other. Cells that were not exposed to the tested materials (DMEM ? 10 % FBS and antibiotics) served as the negative control. Experiments were done 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 of cellulose acetate filter paper (VIVID separation and filtration, USA) to ensure sterilisation. After incubation the study materials for 24, 48, and 72 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 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
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Table 1 The percentage of viable cells (SD) for all concentrations of tested pulpotomy medicaments and of negative control after 24, 48, and 72 h of exposure Tested material FC
FS
GMTA
Concentration (lg/ml) 0.5 5 50 500 5,000 0.5 5 50 500 5,000 0.5 5 50 500 5,000
Negative control
Table 2 Calculated LD50 values for FC, FS, and GMTA after 24 h of exposure
FC
% viability 24 h 66.28 50.28 12.61 9.883 6.73 85.13 77.42 72.20 64.10 23.53 96.47 93.47 89.95 87 78.43 100
48 h (12.76) (12.0) (5.2) (4.0) (1.5) (9.1) (6.0) (5.0) (4.4) (5.8) (4.8) (4.6) (6.6) (11.4) (7.657) (2.7)
Tested material
25.57 17.88 7.50 5.25 4.65 73.12 65.66 55.08 42.50 9.08 86.86 82.25 78.50 73.53 66.08 100
FS
72 h (4.1) (6.5) (1.75) (0.8) (0.98) (5.5) (4.534) (4.2) (4.8) (4.9) (4.35) (2.8) (4.9) (3.7) (7.4) (12.6)
12.95 7.38 5.25 4.27 3.68 76.85 70.08 37.97 31.65 5.47 98.70 93.78 89.91 83.86 74.12 100
GMTA (2.6) (1.5) (0.6) (0.7) (0.5) (3.2) (3.7) (8.6) (3.7) (2.3) (7.8) (7.7) (6.5) (11.2) (8.8) (10.62)
materials was calculated as the relative absorbance of tested wells versus untreated (negative control) wells (i.e. mean absorbance of tested wells/mean absorbance of untreated wells 9100). The in vitro toxicity ranking of the tested materials The test materials were ranked according to their toxicity level which was based on the calculated LD50 values [lethal dose to 50 % of the cells compared to a control group (mg/mL)] using the rating method of 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 calculated using the probit analysis. The significance level was p \ 0.05.
Results The percentage of viable cells for all concentrations of tested materials and of negative control group after 24, 48 and 72 h of exposure is shown in Table 1. Table 2 shows the calculated LD50 values for FC, FS, and GMTA after 24 h of exposure.
LD50 value (mg/ml) 0.002 0.363 10.235
As shown in Table 1, there was a significant drop in cell viability with increase in both the concentration and exposure period up to 72 h of exposure for FC (p \ 0.05). The same finding was noticed for FS and GMTA up to 48 h of exposure only, afterwards cell viability increased significantly in almost all concentrations tested of GMTA except the highest concentration in which the difference was insignificant. In addition, cell viability increased for the lower concentrations tested of FS (0.5, and 5 lg/ml); however, the difference was insignificant (p [ 0.05). The remaining concentrations tested of FS showed a significant drop in cell viability from 48 to 72 h of exposure except for the highest concentration tested (5,000 lg/ml) in which the difference was insignificant. Only GMTA (0.5, 5, and 50 lg/ml) after 24, and 72 h of exposure had almost comparable cell viability to the negative control, the rest of the tested concentrations were significantly inferior to the negative control at the three exposure periods tested. In addition, GMTA had the lowest in vitro toxicity rating of tested materials with an overall rating of 1–2 (slightly toxic) for the three exposure periods. All concentrations of FC were severely toxic as the overall toxicity rating was 4 for the three exposure periods. On the other hand, the overall toxicity rating of FS fell in the range between 2 and 3 (slightly to moderately toxic) except for the concentration (5,000 lg/ml) which had a toxicity rating of 4 (severely toxic) for the three exposure periods. Based on the calculated LD50 values after 24 h of exposure as shown in Table 2. FC recorded the highest in vitro toxicity, whilst GMTA presented the lowest and was the most biocompatible material amongst the tested materials.
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 balb/c 3T3 mouse fibroblasts to assess toxicity of dental materials (AlHiyasat et al. 2005; De Menezes et al. 2009). In this study, a cell culture technique of human PDL fibroblasts was employed since PDL fibroblasts are more representative to
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human pulp fibroblasts than animal cell lines. They are easier to culture and they have a 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 (VanWyk et al. 2001; Amaral et al. 2009). Various literature reports used scanning electron microscopy (SEM) (Camilleri and Pitt Ford 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). In this study, cytotoxicity was tested using the MTT assay which 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 (Al-Hiyasat 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, GMTA was selected because it was less toxic than white MTA in a previous report (Al-Haj Ali et al. 2014). Furthermore, GMTA was mixed with the culture medium not with distilled water as recommended by the manufacturer and the supernatant was used which is in agreement with De Menezes et al. (2009). This study has shown that cell response to the different tested materials was dose-dependent as cell viability increased with lowering the concentration of all tested materials. In addition, cell viability for all the concentrations tested of GMTA decreased after 48 h of exposure, but increased again after 72 h. This is in agreement with a previous report in which it was found that freshly mixed GMTA induced more cell death than set GMTA which demonstrated favourable biocompatibility after 72 h of exposure (Roberts et al. 2008). This could be attributed to the difference in pH, since freshly mixed MTA has high pH which can cause denaturation of adjacent cells. As the MTA becomes set, the pH changes and the cell injuries subside, therefore, cell growth increases (Jafarnia et al. 2009). In this study, the LD50 value was calculated for FC, FS, and GMTA. 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
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or cell death (Hanks et al. 1996). Based on the calculated LD50 values, this study has shown that FC presented the highest in vitro toxicity, whilst GMTA the lowest and the most biocompatible material amongst the other tested materials. The same ranking was found by previous authors (Cotes et al. 1997; De Menezes et al. 2009). This study has also shown that FC’s vapour was equally toxic; cell numbers appeared to have a tendency to decrease in wells indirectly exposed to FC’s vapour as well as in the neighbouring negative control wells, therefore, FC well plates were isolated from those of other tested materials’. In addition, within the same plate, FC wells exposed to the various concentrations tested were separated. This finding is in agreement with a previous report in which found that indirect exposure of pulp fibroblasts to FC’s vapour resulted in cellular atrophy and less dense tissue pattern (Hill et al. 1991). The results of this study regarding the biocompatibility of MTA were in agreement with previous reports (Osorio et al. 1998; Camilleri and Pitt Ford 2006). This can be explained by the fact that GMTA was the only tested powder as the others were in liquid form, as this may make them more sensitive and more affected at a mitochondrial level to in vitro toxicity assays than the materials which are powder, since acute toxicity is correlated with water solubility i.e. the more water soluble the material is, the more toxic it will be (Issa et al. 2004). This finding is in agreement with a previous report (De Menezes et al. 2009). In this study, GMTA (0.5, 5, and 50 lg/ml) at 24 and 72 h of exposure showed almost comparable cell viability results to the negative control. Therefore, it can be hypothesised that GMTA stimulated fibroblast proliferation in a similar manner to the negative control which was the culture medium. This was also found in a previous report (Takita et al. 2006). However, one of the drawbacks of MTA is its high cost, especially in Jordan, which can make its use in paediatric dentistry practice almost prohibitive in some circumstances; therefore, considering the results of FS in this study, FS can still be considered a valid and inexpensive alternative for pulpotomy in primary teeth. Nevertheless, these findings should be carefully interpreted and extrapolated into the clinical situation since the actual clinical preparation and usage of the pulpotomy materials tested are different from the methods of preparation for in vitro testing. In addition, further in vitro studies are required utilising pulp fibroblasts since their behaviour will be the closest to clinical conditions.
Conclusion The overall in vitro toxicity ranking of the tested materials was FC [ FS GMTA; FC presented the highest toxicity
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on human PDL fibroblasts; it was severely cytotoxic in nearly all the concentrations, whilst GMTA presented the lowest toxicity. MTA stands as the most promising substitute to formocresol. However, considering MTA’s unavailability and high price, FS may be the best alternative to FC in pulpotomy of primary teeth with regard to its availability. Acknowledgments The authors sincerely thank Professor Said Ismail and his team (Molecular Biology Laboratory, Faculty of Medicine, University of Jordan) for their assistance.
References Al-Haj Ali SN, Al-Jundi SH, Ditto DJ. In-vitro toxicity of grey MTA in comparison to white MTA on human periodontal ligament fibroblasts. Eur Arch Paediatr Dent. 2014. doi:10.1007/s40368014-0134-z. 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. 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. Camilleri J, Pitt Ford TR. Mineral trioxide aggregate: a review of the constituents and biological properties of the material. Int Endod J. 2006;39(10):747–54. Casas MJK, Juddy PL, Johnston DH. Do we still need formocresol in paediatric dentistry? J Can Dent Assoc. 2005;71:749–51. Cotes O, Boj JR, Canalda C, Carreras M. Pulpal tissue reaction to formocresol vs. ferric sulphate in pulpotomised rat teeth. J Clin Pediatr Dent. 1997;21(3):247–53. 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. Fallahinejad Ghajari MM, Vatanpour M, Kharrazi Fard MJ. Comparison of pulpotomy with formocresol and MTA in primary molars: a systematic review and meta-analysis. Int Endod J. 2008;3(3):45–9. Fuks AB. Vital pulp therapy with new materials for primary teeth: new directions and treatment perspectives. Pediatr Dent. 2008;30(3):211–9.
55 Hanks CT, Wataha JC, Sun Z. In vitro models of biocompatibility: a review. Dent Mater. 1996;12(3):186–93. Hill SD, Berry CW, Seale NS, Kaga M. Comparison of antimicrobial and cytotoxic effects of glutaraldehyde and formocresol. Oral Surg Oral Med Oral Pathol. 1991;71(1):89–95. Ibricevic H, Al-Jame Q. Ferric sulphate as pulpotomy agent in primary teeth: twenty month clinical follow-up. J Clin Pediatr Dent. 2000;24(4):269–72. 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. Jafarnia B, Jiang J, He J, et al. Evaluation of cytotoxicity of MTA employing various additives. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;107(5):739–44. McDonald RE, Avery, Dean JA. Dentistry for the child and adolescent. 9th ed. New York: Mosby; 2011. p. 350–65. Noorollahian H. Comparison of mineral trioxide aggregate and formocresol as pulp medicaments for pulpotomies in primary molars. Br Dent J. 2008;204(11):E20. Osorio RM, Hefti A, Vertucci FJ, Shawley AL. Cytotoxicity of endodontic materials. J Endod. 1998;24(2):91–6. Pinkham JR, Casamassimo PS, McTigue DJ, et al. Pediatric dentistry: infancy through adolescence. 4th ed. St-Louis: Elsevier Saunders; 2005. p. 384–91. 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. 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. Van Wyk CWo, Olivier A, Maritz JS. Cultured pulp fibroblasts: are they suitable for in vitro cytotoxicity testing? J Oral Pathol Med. 2001;30(3):168–77.
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ORIGINAL SCIENTIFIC ARTICLE
In vitro toxicity of formocresol, ferric sulphate, and grey MTA on human periodontal ligament fibroblasts S. N. Al-Haj Ali • S. H. Al-Jundi • D. J. Ditto
Received: 12 June 2014 / Accepted: 5 September 2014 / Published online: 29 October 2014 Ó European Academy of Paediatric Dentistry 2014
Abstract Aim This was to assess and compare the in vitro toxicity of formocresol, ferric sulphate and MTA on cultured human periodontal ligament (PDL) fibroblasts. Study design PDL cells were obtained from sound first permanent molars and cultured in Dulbecco’s modified Eagle’s medium. Methods PDL cells were subjected to different concentrations of formocresol, ferric sulphate, and grey MTA for 24, 48, and 72 h at 37 °C. Cells that were not exposed to the tested materials served as the negative control. In vitro toxicity was assessed using MTT assay. Statistics Statistical analysis of data was accomplished using ANOVA and Tukey statistical tests (p \ 0.05). Results The overall toxicity ranking of the tested materials was as follows: formocresol [ ferric sulphate [ grey MTA. Only grey MTA had comparable cell viability to the negative control, the other tested materials were significantly inferior at the three exposure periods (p \ 0.05). Conclusion Regarding the viability of PDL fibroblasts, MTA stands as the most promising substitute to formocresol. However, considering MTA’s unavailability and high price in Jordan, ferric sulphate may be the best alternative to formocresol in pulpotomy of primary teeth.
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
Keywords MTA Fibroblasts Ferric sulphate Formocresol
Introduction Pulpotomy is a common procedure in the treatment of acutely inflamed primary teeth (McDonald et al. 2011). It is to the surgical amputation of the coronal inflamed portion of the pulp to retain the vital surface of the radicular pulp tissue treatment with a medicament to promote healing in the underlying tissue and eventually retain the tooth (Pinkham et al. 2005). Proper selection of the pulpotomy medicament is of paramount importance as it will interact with tissue fluids (Camilleri 2006); therefore biocompatibility of pulpotomy medicaments is a prerequisite. The dental literature is replete with papers that report the success or failure of primary tooth pulpotomy using a variety of medicaments. Formocresol (FC) is one of the most studied pulpotomy medicaments; however, concerns were raised over FC as a pulpotomy medicament due to the aldehyde constituent in its composition (Casas et al. 2005). Several investigations have led to the conclusion that formaldehyde is toxic to the connective tissue; it does not stay localised to the dental pulp as it is absorbed systemically, and therefore it has the potential of having carcinogenic and mutagenic effects (Fallahinejad Ghajari et al. 2008). This conclusion has created the need for alternative pulpotomy medicaments. Ferric sulphate (FS) at a concentration of 15.5 %—a non-aldehyde chemical—has also been investigated as a possible replacement for FC and has proven to have clinically and radiographically comparable success rates (Ibricevic and Al-Jame 2000; Fuks 2008). Recently, mineral trioxide aggregate (MTA) has attracted attention as a pulpotomy medicament because of excellent sealing
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ability, biocompatiblity and stimulation of hard tissue formation. It also appears to have higher long-term clinical and radiographic success rate than FC (Noorollahian 2008). Various reports in the literature studied in vitro toxicity of FC and MTA on animal cell lines; however, only few studies investigated their toxicity on cultured human periodontal ligament (PDL) fibroblasts. Moreover, comparative in vitro toxicity of FS has never been assessed on human PDL fibroblasts. Therefore, the aim of this study was to assess and compare the in vitro toxicity of FC, FS and MTA on cultured human PDL fibroblasts.
Materials and methods The research study was approved by the institutional review board and ethical committee of the faculty of medicine at Jordan University of Science and Technology (JUST). 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 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 was diced into tissue explants of 1 mm3. The tissue explants were placed into tissue culture flasks (25 cm2) 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. Cells were grown at 37 °C in a humidified atmosphere of 5 % CO2 in air. After 5 weeks, cells reached confluence and were detached after trypsinisation (0.25 % trypsin with ethylenediaminetetraacetic acid (EDTA, Sigma–Aldrich, USA)) for 5 min and transferred to larger flasks (75 cm2) 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
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(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 were: grey MTA (GMTA) (AngelusTM, Brazil), 20 % diluted FC (Formocresol Biodinamica, Brazil) and 15.5 % FS (Ultradent Products, Jordan). On the same day of cell seeding in the 96-well plates, the tested materials were prepared using a modified method to that used in a previous report (De Menezes et al. 2009). Briefly, 50 mg of each tested material was 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. Five tenfold concentrations ranging from 0.5 to 5,000 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 five concentrations of FC, FS, and GMTA, respectively, for 24, 48 and 72 h at 37 °C in 5 % CO2 atmosphere. FC well plates were separated from those of other tested materials. In addition, within the same plate, FC wells exposed to the various concentrations tested were separated from each other. Cells that were not exposed to the tested materials (DMEM ? 10 % FBS and antibiotics) served as the negative control. Experiments were done 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 of cellulose acetate filter paper (VIVID separation and filtration, USA) to ensure sterilisation. After incubation the study materials for 24, 48, and 72 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 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
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Table 1 The percentage of viable cells (SD) for all concentrations of tested pulpotomy medicaments and of negative control after 24, 48, and 72 h of exposure Tested material FC
FS
GMTA
Concentration (lg/ml) 0.5 5 50 500 5,000 0.5 5 50 500 5,000 0.5 5 50 500 5,000
Negative control
Table 2 Calculated LD50 values for FC, FS, and GMTA after 24 h of exposure
FC
% viability 24 h 66.28 50.28 12.61 9.883 6.73 85.13 77.42 72.20 64.10 23.53 96.47 93.47 89.95 87 78.43 100
48 h (12.76) (12.0) (5.2) (4.0) (1.5) (9.1) (6.0) (5.0) (4.4) (5.8) (4.8) (4.6) (6.6) (11.4) (7.657) (2.7)
Tested material
25.57 17.88 7.50 5.25 4.65 73.12 65.66 55.08 42.50 9.08 86.86 82.25 78.50 73.53 66.08 100
FS
72 h (4.1) (6.5) (1.75) (0.8) (0.98) (5.5) (4.534) (4.2) (4.8) (4.9) (4.35) (2.8) (4.9) (3.7) (7.4) (12.6)
12.95 7.38 5.25 4.27 3.68 76.85 70.08 37.97 31.65 5.47 98.70 93.78 89.91 83.86 74.12 100
GMTA (2.6) (1.5) (0.6) (0.7) (0.5) (3.2) (3.7) (8.6) (3.7) (2.3) (7.8) (7.7) (6.5) (11.2) (8.8) (10.62)
materials was calculated as the relative absorbance of tested wells versus untreated (negative control) wells (i.e. mean absorbance of tested wells/mean absorbance of untreated wells 9100). The in vitro toxicity ranking of the tested materials The test materials were ranked according to their toxicity level which was based on the calculated LD50 values [lethal dose to 50 % of the cells compared to a control group (mg/mL)] using the rating method of 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 calculated using the probit analysis. The significance level was p \ 0.05.
Results The percentage of viable cells for all concentrations of tested materials and of negative control group after 24, 48 and 72 h of exposure is shown in Table 1. Table 2 shows the calculated LD50 values for FC, FS, and GMTA after 24 h of exposure.
LD50 value (mg/ml) 0.002 0.363 10.235
As shown in Table 1, there was a significant drop in cell viability with increase in both the concentration and exposure period up to 72 h of exposure for FC (p \ 0.05). The same finding was noticed for FS and GMTA up to 48 h of exposure only, afterwards cell viability increased significantly in almost all concentrations tested of GMTA except the highest concentration in which the difference was insignificant. In addition, cell viability increased for the lower concentrations tested of FS (0.5, and 5 lg/ml); however, the difference was insignificant (p [ 0.05). The remaining concentrations tested of FS showed a significant drop in cell viability from 48 to 72 h of exposure except for the highest concentration tested (5,000 lg/ml) in which the difference was insignificant. Only GMTA (0.5, 5, and 50 lg/ml) after 24, and 72 h of exposure had almost comparable cell viability to the negative control, the rest of the tested concentrations were significantly inferior to the negative control at the three exposure periods tested. In addition, GMTA had the lowest in vitro toxicity rating of tested materials with an overall rating of 1–2 (slightly toxic) for the three exposure periods. All concentrations of FC were severely toxic as the overall toxicity rating was 4 for the three exposure periods. On the other hand, the overall toxicity rating of FS fell in the range between 2 and 3 (slightly to moderately toxic) except for the concentration (5,000 lg/ml) which had a toxicity rating of 4 (severely toxic) for the three exposure periods. Based on the calculated LD50 values after 24 h of exposure as shown in Table 2. FC recorded the highest in vitro toxicity, whilst GMTA presented the lowest and was the most biocompatible material amongst the tested materials.
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 balb/c 3T3 mouse fibroblasts to assess toxicity of dental materials (AlHiyasat et al. 2005; De Menezes et al. 2009). In this study, a cell culture technique of human PDL fibroblasts was employed since PDL fibroblasts are more representative to
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human pulp fibroblasts than animal cell lines. They are easier to culture and they have a 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 (VanWyk et al. 2001; Amaral et al. 2009). Various literature reports used scanning electron microscopy (SEM) (Camilleri and Pitt Ford 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). In this study, cytotoxicity was tested using the MTT assay which 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 (Al-Hiyasat 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, GMTA was selected because it was less toxic than white MTA in a previous report (Al-Haj Ali et al. 2014). Furthermore, GMTA was mixed with the culture medium not with distilled water as recommended by the manufacturer and the supernatant was used which is in agreement with De Menezes et al. (2009). This study has shown that cell response to the different tested materials was dose-dependent as cell viability increased with lowering the concentration of all tested materials. In addition, cell viability for all the concentrations tested of GMTA decreased after 48 h of exposure, but increased again after 72 h. This is in agreement with a previous report in which it was found that freshly mixed GMTA induced more cell death than set GMTA which demonstrated favourable biocompatibility after 72 h of exposure (Roberts et al. 2008). This could be attributed to the difference in pH, since freshly mixed MTA has high pH which can cause denaturation of adjacent cells. As the MTA becomes set, the pH changes and the cell injuries subside, therefore, cell growth increases (Jafarnia et al. 2009). In this study, the LD50 value was calculated for FC, FS, and GMTA. 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
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or cell death (Hanks et al. 1996). Based on the calculated LD50 values, this study has shown that FC presented the highest in vitro toxicity, whilst GMTA the lowest and the most biocompatible material amongst the other tested materials. The same ranking was found by previous authors (Cotes et al. 1997; De Menezes et al. 2009). This study has also shown that FC’s vapour was equally toxic; cell numbers appeared to have a tendency to decrease in wells indirectly exposed to FC’s vapour as well as in the neighbouring negative control wells, therefore, FC well plates were isolated from those of other tested materials’. In addition, within the same plate, FC wells exposed to the various concentrations tested were separated. This finding is in agreement with a previous report in which found that indirect exposure of pulp fibroblasts to FC’s vapour resulted in cellular atrophy and less dense tissue pattern (Hill et al. 1991). The results of this study regarding the biocompatibility of MTA were in agreement with previous reports (Osorio et al. 1998; Camilleri and Pitt Ford 2006). This can be explained by the fact that GMTA was the only tested powder as the others were in liquid form, as this may make them more sensitive and more affected at a mitochondrial level to in vitro toxicity assays than the materials which are powder, since acute toxicity is correlated with water solubility i.e. the more water soluble the material is, the more toxic it will be (Issa et al. 2004). This finding is in agreement with a previous report (De Menezes et al. 2009). In this study, GMTA (0.5, 5, and 50 lg/ml) at 24 and 72 h of exposure showed almost comparable cell viability results to the negative control. Therefore, it can be hypothesised that GMTA stimulated fibroblast proliferation in a similar manner to the negative control which was the culture medium. This was also found in a previous report (Takita et al. 2006). However, one of the drawbacks of MTA is its high cost, especially in Jordan, which can make its use in paediatric dentistry practice almost prohibitive in some circumstances; therefore, considering the results of FS in this study, FS can still be considered a valid and inexpensive alternative for pulpotomy in primary teeth. Nevertheless, these findings should be carefully interpreted and extrapolated into the clinical situation since the actual clinical preparation and usage of the pulpotomy materials tested are different from the methods of preparation for in vitro testing. In addition, further in vitro studies are required utilising pulp fibroblasts since their behaviour will be the closest to clinical conditions.
Conclusion The overall in vitro toxicity ranking of the tested materials was FC [ FS GMTA; FC presented the highest toxicity
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on human PDL fibroblasts; it was severely cytotoxic in nearly all the concentrations, whilst GMTA presented the lowest toxicity. MTA stands as the most promising substitute to formocresol. However, considering MTA’s unavailability and high price, FS may be the best alternative to FC in pulpotomy of primary teeth with regard to its availability. Acknowledgments The authors sincerely thank Professor Said Ismail and his team (Molecular Biology Laboratory, Faculty of Medicine, University of Jordan) for their assistance.
References Al-Haj Ali SN, Al-Jundi SH, Ditto DJ. In-vitro toxicity of grey MTA in comparison to white MTA on human periodontal ligament fibroblasts. Eur Arch Paediatr Dent. 2014. doi:10.1007/s40368014-0134-z. 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. 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. Camilleri J, Pitt Ford TR. Mineral trioxide aggregate: a review of the constituents and biological properties of the material. Int Endod J. 2006;39(10):747–54. Casas MJK, Juddy PL, Johnston DH. Do we still need formocresol in paediatric dentistry? J Can Dent Assoc. 2005;71:749–51. Cotes O, Boj JR, Canalda C, Carreras M. Pulpal tissue reaction to formocresol vs. ferric sulphate in pulpotomised rat teeth. J Clin Pediatr Dent. 1997;21(3):247–53. 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. Fallahinejad Ghajari MM, Vatanpour M, Kharrazi Fard MJ. Comparison of pulpotomy with formocresol and MTA in primary molars: a systematic review and meta-analysis. Int Endod J. 2008;3(3):45–9. Fuks AB. Vital pulp therapy with new materials for primary teeth: new directions and treatment perspectives. Pediatr Dent. 2008;30(3):211–9.
55 Hanks CT, Wataha JC, Sun Z. In vitro models of biocompatibility: a review. Dent Mater. 1996;12(3):186–93. Hill SD, Berry CW, Seale NS, Kaga M. Comparison of antimicrobial and cytotoxic effects of glutaraldehyde and formocresol. Oral Surg Oral Med Oral Pathol. 1991;71(1):89–95. Ibricevic H, Al-Jame Q. Ferric sulphate as pulpotomy agent in primary teeth: twenty month clinical follow-up. J Clin Pediatr Dent. 2000;24(4):269–72. 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. Jafarnia B, Jiang J, He J, et al. Evaluation of cytotoxicity of MTA employing various additives. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009;107(5):739–44. McDonald RE, Avery, Dean JA. Dentistry for the child and adolescent. 9th ed. New York: Mosby; 2011. p. 350–65. Noorollahian H. Comparison of mineral trioxide aggregate and formocresol as pulp medicaments for pulpotomies in primary molars. Br Dent J. 2008;204(11):E20. Osorio RM, Hefti A, Vertucci FJ, Shawley AL. Cytotoxicity of endodontic materials. J Endod. 1998;24(2):91–6. Pinkham JR, Casamassimo PS, McTigue DJ, et al. Pediatric dentistry: infancy through adolescence. 4th ed. St-Louis: Elsevier Saunders; 2005. p. 384–91. 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. 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. Van Wyk CWo, Olivier A, Maritz JS. Cultured pulp fibroblasts: are they suitable for in vitro cytotoxicity testing? J Oral Pathol Med. 2001;30(3):168–77.
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