Abstract
Aim
To investigate and compare the cytotoxicity and bioactivity of CMCR agents on stem cells derived from exfoliated deciduous teeth.
Methodology
MTT assay, flow cytometry, Alizarin Red staining and scratch assay were used to assess the cellular viability, apoptosis, calcium matrix deposits and cell migration, respectively. The gene expression of ALP and BMP-2 was measured with RT-PCR. One-way ANOVA and Bonferroni post-test was used for statistical analysis.
Results
0.5% Carisolv showed highest cell proliferation and calcium matrix formation, whereas 0.5% Papacarie reported the highest% live cells and cell migration. The highest mRNA expression of ALP and BMP-2 was reported in SHEDs cultured in 0.5% Papacarie (after 72 h incubation) and 0.5% Carisolv (after 24 h incubation), respectively.
Conclusion
CMCR agents are biocompatible and bioactive when cultured in stem cells derived from exfoliated primary teeth.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arora R, Goswami M, Chaudhary S, Chaitra TR, Kishor A, Rallan M. Comparative evaluation of effects of chemomechanical and conventional caries removal on dentine morphology and its bonding characteristics—an SEM study. Eur Ach Pediatr Dent. 2012;13:178–84.
Bastos LA, Silva F, Thome P, Arnez M, Faccioli L, Paula-Silva F. Effects of Papain-Based Gel used for caries removal on macrophages and dental pulp cells. Braz Dent J. 2019;30(5):484–90. https://doi.org/10.1590/0103-6440201902560.
Bittencourt ST, Pereira JR, Rosa AW, Oliveria KS, Ghizoni JS, Oliveria MT. Mineral content removal after Papacarie application in primary teeth: a quantitative analysis. J Clin Pediatr Dent. 2010;34:229–31. https://doi.org/10.17796/jcpd.34.3.k15t8q1805538524.
Bussadori SK, Castro LC, Galvao AC. Papain gel: a new chemo-mechanical caries removal agent. J Clin Pediatr Dent. 2005;30:115–9.
Bussadori SK, Amancio O, Martin M, Guedes C, Afaya T, Santos E, et al. Production of extra cellular matrix proteins by human pulp fibroblasts in contact with Papacarie and Carisolv. Oral Health Prev Dent. 2014;12(1):55–9. https://doi.org/10.3290/j.ohpd.a31225.
Chomczynski P, Mackey K (1995) Short technical report. Modification of trizol reagent procedure for isolation of RNA from Polysaccharide and proteoglycan rich sources. Biotechniques 19(6):942-5
Contreras R, Rogelio J, Bulnes RC, Kanda Y, Nakajima H, Sakagami H. Cytotoxicity and pro-Inflammatory action of chemo-mechanical caries-removal agents against oral cells. In Vivo. 2014;28:549–56.
Costa CA, Ribeiro AP, Giro EM, Randall RC, Hebling J. Pulp response after application of two resin modified glass ionomer cements (RMGICs) in deep cavities of prepared human teeth. Dent Mater. 2011;27:e158-170. https://doi.org/10.1016/j.dental.2011.04.002.
Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014;15:49–63. https://doi.org/10.1038/nrm3722.
Dammaschke T, Stratmann U, Danesh G, Schafer E, Ott KH. Reaction of rat pulp tissue to Carisolv ‘new gel’—a histocytological evaluation. Aust Dent J. 2006;51:57–63. https://doi.org/10.1111/j.1834-7819.2006.tb00402.x.
Ericson D, Zimmerman M, Raber H, Gotrick B, Bornstein R, Thorell J. Clinical evaluation of efficacy and safety of a new method for chemomechanical removal of caries. Caries Res. 1999;33:171–7. https://doi.org/10.1159/000016513.
Faggion CM. Guidelines for reporting preclinical in vitro studies on dental materials. J Evid Base Det Pract. 2012;12:182–9. https://doi.org/10.1016/j.jebdp.2012.10.001.
Freshney IR, Stacy GN, Auerbach JM. Culture of human stem cells. Wiley; 2007. p. 193–203.
Gandolfi MG, Spagnuolo G, Siboni F, Procino A, Rivieccio V, Pelliccioni GA, et al. Calcium silicate/calcium phosphate biphasic cements for vital pulp therapy: chemical-physical properties and human pulp cells response. Clin Oral Investig. 2015;19:2075–89. https://doi.org/10.1007/s00784-015-1443-2.
Garcia-Contreras R, Scougall-Vilchis RJ, Contreras-Bulnes R, Kanda Y, Nakajima H, Sakagami H. Cytotoxicity and pro-inflammatory action of chemo-mechanical caries-removal agents against oral cells. In Vivo. 2014;28:549–56.
Goldman M, Kronman JH. A preliminary report on a Chemomechanical means of removing caries. J Am Dent Assoc. 1976;93:1149–53. https://doi.org/10.14219/jada.archive.1976.0249.
Gregory C, Gunn W, Peister A, Prockop D. An alizarin red based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem. 2004;329:77–84. https://doi.org/10.1016/j.ab.2004.02.002.
Gu K, Smoke RH, Rutherford RB. Expression of genes for bone morphogenic proteins and receptors in human dental pulp. Arch Oral Biol. 1996;41:919–23. https://doi.org/10.1016/s0003-9969(96)00052-0.
Habib CM, Kronman J, Goldman M. A chemical evaluation of collagen and hydroxyproline after treatment with GK-101 (Nchloroglycine). Pharmacol Therapeut Dent. 1975;2:209–15.
Hamama HH, Yiu CK, Burrow MF, King NM. Chemical, morphological and microhardness change of dentine after chemomechanical caries removal. Aust Dent J. 2013;58:283–92. https://doi.org/10.1111/adj.12093.
Hench LL, West JK. Biological application of bioactive glasses. Life Chem Rep. 1996;13:187–241.
Ingram RT, Clarke BL, Fisher LW, Fitzpatrick LA. Distribution of non-collagenous proteins in the matrix of adult human bone: evidence of anatomic and functional heterogeneity. J Bone Miner Res. 1993;8:1019–29.
Jawa D, Singh S, Somani R, Jaidka S, Sirkar K, Jaidka R. Comparative evaluation of the efficacy of chemomechanical caries removal agent (Papacarie) and conventional methods of caries removal: an in vitro study. J Indian Soc Pediatr Prev Dent. 2010;28:73–7. https://doi.org/10.4103/0970-4388.66739.
Jonkman J, Cathcart J, Xu F, Bartholini M, Amon J, Stevens K, et al. An introduction to the wound healing assay using live-cell microscopy. Cell Adh Migr. 2014;8(5):440–51. https://doi.org/10.4161/cam.36224.
Jundt G, Berghauser KH, Termine JD, Schulz A. Osteonectin– a differentiation marker of bone cells. Cell Tissue Res. 1987;248:409–15. https://doi.org/10.1007/BF00218209.
Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomater. 2006;27:2907–15. https://doi.org/10.17796/jcpd.34.2.f312p36g18463716.
Kotb RM, Abdella AA, El Kateb MA, Ahmed AM. Clinical evaluation of Papacarie in primary teeth. J Clin Pediatr Dent. 2009;34:117–23.
Kuboki Y, Zimmerman M, Raber H, Cotrick B, Bornstein R. Clinical evaluation of efficacy and safety of a new method from chemo mechanical removal of carious dentin. J Dent Res. 1977;56:1233–7. https://doi.org/10.1007/s00784-012-0701-9.
Laura S, Joao Pedro T, Maya F, Lucia F, Francisco S. Effects of Papain-based gel used for caries removal on macrophages and dental pulp cells. Braz Dent J. 2019;30(5):484–90. https://doi.org/10.1590/0103-6440201902560.
Lesot H, Osman M, Ruch JV. Immunofluorescent localization of collagens, fibronectin, and laminin during terminal differentiation of odontoblasts. Dev Biol. 1981;82:371–81. https://doi.org/10.1016/0012-1606(81)90460-7.
Mardoch-Kinch CA, Ellen MM. Minimally invasive dentistry. J Am Dent Assoc. 2003;134:87–95. https://doi.org/10.14219/jada.archive.2003.0021.
Martins MD, Fernandes KP, Motta LJ, Santos EM, Pavesi SVC, Bussadori SK. Biocompatibility analysis of chemomechanical caries removal material Papacarie on cultured fibroblast and subcutaneous tissue. J Dent Child (chic). 2009;76(2):123–9.
Maru V, Shakuntala BS, Nagarathna C. Evaluation of marginal leakage and shear bond strength of bonded restorations in primary teeth, after caries removal by conventional and chemomechanical techniques. ISRN. 2014. https://doi.org/10.1155/2014/854816.
Matsumoto SF, Motta LJ, Alfaya TA, Guedes CC, Fernandes KP, Bussadori SK. Assessment of chemomechanical removal of carious lesions using Papacarie DuoTM: randomized longitudinal clinical trial. Indian J Dent Res. 2013;24:488–92. https://doi.org/10.4103/0970-9290.118393.
Mead R. The design of experiments. Cambridge, New York: Cambridge University Press; 1988. p. 620.
Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci USA. 2003;100:5807–12.
Miura M, Chen XD, Allen MR, Bi Y, Gronthos S, Seo BM, et al. A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J Clin Investig. 2004;114:1704–13. https://doi.org/10.1172/JCI20427.
Motta LJ, Martins MD, Porta KP, Bussadori SK. Aesthetic restoration of deciduous anterior teeth after removal of carious tissue with Papacarie. Indian J Dental Res. 2009;20:117–20. https://doi.org/10.4103/0970-9290.49060.
Niepel M, Hafner M, Mills C, Subramanian K, Williams E, Chung M, et al. A multicenter study on the reproducibility of drug response assays in mammalian cell lines. Cell Syst. 2019;9:35–48. https://doi.org/10.1016/j.cels.2019.06.005.
Pijuan J, Barcelo C, Moreno D, Maiques O, Siso P, Marti R, et al. In vitro cell migration, invasion and adhesion assays: from cell imaging to data analysis. Front Cell Dev Biol. 2019;2019(7):107. https://doi.org/10.3389/fcell.2019.00107.
Robbins A. Efficacy of GK101E solution (Caridex 100) for caries removal. Gen Dent. 1987;35:392–6.
Robey PG. Vertebrate mineralized matrix proteins: structure and function. Connect Tissue Res. 1996;35:131–6. https://doi.org/10.3109/03008209609029183.
Rodríguez-Lozano FJ, Bueno C, Insausti CL, Meseguer L, Ramirez MC, Blanquer M, et al. Mesenchymal stem cells derived from dental tissues. Int Endod J. 2001;44:800–6. https://doi.org/10.1111/j.1365-2591.2011.01877.x.
Romberg RW, Werness PG, Lollar P, Riggs BL, Mann KG. Isolation and characterization of native adult osteonectin. J Biol Chem. 1985;260:2728–36.
Ruch JV. Odontoblast commitment and differentiation. Biochem Cell Biol. 1998;76:923–38.
Sepet E, Bilir A, Akcin O, Aytepe Z. The effect of caries removing gel (Carisolv) on FM3A cell-line in vitro. J Dent. 2004;32:213–8. https://doi.org/10.1016/j.jdent.2003.10.005.
Stockert JC, Horobin RW, Colombo LL, Blazquez-Castro A. Tetrazolium salts and formazan products in cell biology: viability assessment, fluorescence imaging and labelling perspectives. Acta Histochem. 2018;120(3):159–67. https://doi.org/10.1016/j.acthis.2018.02.005.
Termine JD, Kleinman HK, Whitson SW, Conn KM, Mc-Garvey ML, Martin GR. Osteonectin, a bone-specific protein linking mineral to collagen. Cell. 1981;26:99–105. https://doi.org/10.1016/0092-8674(81)90037-4.
Use of International Standard ISO-10993 (2021) Biological evaluation of medical devices part1: evaluation and testing (blue book memo) (PDF). fda.gov. FDA. Retrieved 23 January 2017.
Van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 1998;31(1):1–9. https://doi.org/10.1002/(sici)1097-0320(19980101)31:1%3c1::aid-cyto1%3e3.0.co;2-r.
Vanguilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques. 2008;44(5):619–25. https://doi.org/10.2144/000112776.
Wiame I, Remy S, Swennen R, Sagi L. Irreversible heat inactivation of DNase without RNA degradation. Biotechniques. 2000;29:252–6. https://doi.org/10.2144/00292bm11.
Acknowledgements
We would like to acknowledgee Dr. Ashita Gada, FACS Operator, for her support and guidance for this study.
Author information
Authors and Affiliations
Contributions
VM contributed to the conception and design, data acquisition, and design of figures and tables, and drafted and critically revised the manuscript. MM contributed to data acquisition, interpretation and critically revised the manuscript. SS contributed to data acquisition, interpretation, and critically revised the manuscript. PK contributed to data acquisition, interpretation, and critically revised the manuscript. AD contributed to conception and design, prepared figures, and critically revised manuscript. PS contributed to conception and statistical analysis, and critically revised the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
There is no conflict of interest to declare.
Research involving human participants and/or animals
The study was approved by the ethics committee of Government Dental College & Hospital, Mumbai – GDCH-2020/2/13/390).
Informed consent
Parent signed the informed consent forms.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Maru, V., Madkaikar, M., Shabrish, S. et al. Evaluation and comparison of cytotoxicity and bioactivity of chemomechanical caries removal agents on stem cells from human exfoliated deciduous teeth. Eur Arch Paediatr Dent 23, 787–796 (2022). https://doi.org/10.1007/s40368-021-00684-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40368-021-00684-5