Spectrophotometric evaluation of tooth discoloration caused by ProRoot® MTA, Biodentine™, and NeoPutty® in regenerative endodontics: an in vitro study

Background: Regenerative endodontic procedures (REPs) offer biologically based treatments for immature teeth but may result in tooth discoloration, compromising esthetic outcomes. This study aimed to evaluate the color stability of three commonly used materials in REPs—ProRoot MTA (PMTA), Biodentine, and NeoPutty—with or without blood contamination. Methods: A total of 96 extracted human maxillary anterior teeth were standardized and randomly assigned into eight experimental groups (n = 12), based on the material used and presence or absence of blood contamination. Two additional control groups (saline only and blood only) were included. REP was simulated following the American Association of Endodontists protocol, including canal preparation, calcium hydroxide disinfection, and placement of the test material at the cemento-enamel junction. Color measurements (∆E) were obtained using a VITA Easy Shade® spectrophotometer at baseline and after 1, 3, 6 and 12 months. Results: Statistical analysis revealed significant effects of both time and material type on color change (p < 0.05). After 12 months, the highest discoloration was observed in the PMTA/Blood group (∆E = 7.42 ± 0.56), while the lowest discoloration occurred in the Biodentine/Saline (∆E = 2.18 ± 0.43) and NeoPutty/Saline (∆E = 2.26± 0.51) groups. Blood contamination significantly increased discoloration across all materials, with PMTA demonstrating the least color stability. Conclusions: Blood contamination adversely affects the color stability of materials used in regenerative endodontic procedures. Biodentine and NeoPutty exhibited superior esthetic outcomes compared to PMTA and may be more suitable for use in anterior teeth where esthetics is a priority.

Regenerative endodontic procedures; Color stability; Discoloration; Spectrophotometric analysis; NeoPutty

[1] Shi X, Hu X, Jiang N, Mao J. Regenerative endodontic therapy: from laboratory bench to clinical practice. Journal of Advanced Research. 2025; 72: 229–263.

[2] Taha NA, Hamdan AM, Al-Hiyasat AS. Coronal discoloration induced by calcium silicate-based cements used in full pulpotomy in mature permanent molars: a randomized clinical trial. Clinical Oral Investigations. 2023; 27: 1723–1730.

[3] Chen YZ, Yong MJ, Tan VY, Kong SLS, Elnawawy HMA, Yahya NA, et al. The effect of a setting accelerator on the physical and mechanical properties of a fast-set white Portland cement mixed with nano-zirconium oxide. European Endodontic Journal. 2023; 8: 215–224.

[4] Batista LCN, Crivelari JM, Pegoraro JVDC, Janini ACP, Marciano MA, Lopes CS, et al. Physicochemical evaluation of mineral trioxide aggregate and five mineral oxides calcium silicate-based cements. BMC Oral Health. 2025; 25: 1056.

[5] Kohli MR, Yamaguchi M, Setzer FC, Karabucak B. Spectrophotometric analysis of coronal tooth discoloration induced by various bioceramic cements and other endodontic materials. Journal of Endodontics. 2015; 41: 1862–1866.

[6] Bayraktar K, Basturk FB, Turkaydin D, Gunday M. Long-term effect of acidic pH on the surface microhardness of ProRoot mineral trioxide aggregate, Biodentine, and total fill root repair material putty. Dental Research Journal. 2021; 18: 2.

[7] Shokouhinejad N, Razmi H, Farbod M, Alikhasi M, Camilleri J. Coronal tooth discoloration induced by regenerative endodontic treatment using different scaffolds and intracanal coronal barriers: a 6-month ex vivo study. Restorative Dentistry & Endodontics. 2019; 44: e25.

[8] Palma PJ, Marques JA, Falacho RI, Correia E, Vinagre A, Santos JM, et al. Six-month color stability assessment of two calcium silicate-based cements used in regenerative endodontic procedures. Journal of Functional Biomaterials. 2019; 10: 14.

[9] Türkoğlu Kayacı Ş, Solmazgül Yazici Z, Arslan H. Spectrophotometric analysis of color stability induced by various calcium silicate cements in full pulpotomy of permanent molars: Theracal PT, Biodentine, and ProRoot MTA. Journal of Endodontics. 2024; 50: 229–234.

[10] Shokouhinejad N, Nekoofar MH, Pirmoazen S, Shamshiri AR, Dummer PM. Evaluation and comparison of occurrence of tooth discoloration after the application of various calcium silicate-based cements: an ex vivo study. Journal of Endodontics. 2016; 42: 140–144.

[11] Elnawawy HM, Kutty MG, Yahya NA, Kasim NHA, Cooper PR, Camilleri J, et al. Chemical and physical properties of radiopaque Portland cement formulation with reduced particle size. Dental Materials Journal. 2024; 43: 672–682.

[12] Prasad Kumara PAAS, Cooper PR, Cathro P, Gould M, Dias G, Ratnayake J. Bioceramics in endodontics: limitations and future Innovations—a review. Dentistry Journal. 2025; 13: 157.

[13] Bastawala DS, Kapoor S, Nathani P. A comparison of coronal tooth discoloration elicited by various endodontic reparative materials MTA Plus, Bio MTA+, and Biodentine: an ex vivo study. International Journal of Clinical Pediatric Dentistry. 2020; 13: 463–467.

[14] Elnaghy A, Elsaka S. Fracture resistance of simulated immature roots using Biodentine and fiber post compared with different canal-filling materials under aging conditions. Clinical Oral Investigations. 2020; 24: 1333–1338.

[15] de Sá MAB, Nunes E, Antunes ANDG, Brito Júnior M, Horta MCR, Amaral RR, et al. Push-out bond strength and marginal adaptation of apical plugs with bioactive endodontic cements in simulated immature teeth. Restorative Dentistry & Endodontics. 2021; 46: e53.

[16] Savaris JM, Czornobay LFM, Dotto MEP, Santos PS, Garcia LDFR, Vitali FC, et al. Tooth discoloration induced by endodontic sealers of different chemical bases: a systematic review. Brazilian Dental Journal. 2024; 35: e246021.

[17] Al-Hiyasat AS, Ahmad DM, Khader YS. The effect of different calcium silicate-based pulp capping materials on tooth discoloration: an in vitro study. BMC Oral Health. 2021; 21: 330.

[18] Carvalho JA, Franco C, Proença L, Neves JA, Polido M, Mendes JJ, et al. Spectrophotometric analysis of coronal discoloration in vitro induced by bioceramic cements. Dentistry Journal. 2023; 11: 180.

[19] Lozano-Guillén A, López-García S, Rodríguez-Lozano FJ, Sanz JL, Lozano A, Llena C, et al. Comparative cytocompatibility of the new calcium silicate-based cement NeoPutty versus NeoMTA Plus and MTA on human dental pulp cells: an in vitro study. Clinical Oral Investigations. 2022; 26: 7219–7228.

[20] Ipek I, Karaağaç Eskibağlar B, Yildiz Ş, Ataş O, Ünal M. Analysis of the bond strength between conventional, putty or resin-modified calcium silicate cement and bulk fill composites. Australian Dental Journal. 2023; 68: 265–272.

[21] Alqahtani AS, Sulimany AM, Alayad AS, Alqahtani AS, Bawazir OA. Evaluation of the shear bond strength of four bioceramic materials with different restorative materials and timings. Materials. 2022; 15: 4668.

[22] Rodríguez-Lozano FJ, Lozano A, López-García S, García-Bernal D, Sanz JL, Guerrero-Gironés J, et al. Biomineralization potential and biological properties of a new tantalum oxide (Ta2O5)-containing calcium silicate cement. Clinical Oral Investigations. 2022; 26: 1427–1441.

[23] Nagas E, Ertan A, Eymirli A, Uyanik O, Cehreli ZC. Tooth discoloration induced by different calcium silicate-based cements: a two-year spectrophotometric and photographic evaluation in vitro. Journal of Clinical Pediatric Dentistry. 2021; 45: 112–116.

[24] Madani Z, Alvandifar S, Bizhani A. Evaluation of tooth discoloration after treatment with mineral trioxide aggregate, calcium-enriched mixture, and Biodentine in the presence and absence of blood. Dental Research Journal. 2019; 16: 377–383.

[25] Chen SJ, Karabucak B, Steffen JJ, Yu YH, Kohli MR. Spectrophotometric analysis of coronal tooth discoloration induced by tricalcium silicate cements in the presence of blood. Journal of Endodontics. 2020; 46: 1913–1919.

[26] American Association of Endodontists. AAE clinical considerations for a regenerative procedure. 2016. Available at: https://www.aae.org/specialty/wp-content/uploads/sites/2/2017/06/currentregenerativeendodonticconsiderations.pdf (Accessed: 10 January 2024).

[27] Marconyak LJ III, Kirkpatrick TC, Roberts HW, Roberts MD, Aparicio A, Himel VT, et al. A comparison of coronal tooth discoloration elicited by various endodontic reparative materials. Journal of Endodontics. 2016; 42: 470–473.

[28] Shokouhinejad N, Khoshkhounejad M, Alikhasi M, Bagheri P, Camilleri J. Prevention of coronal discoloration induced by regenerative endodontic treatment in an ex vivo model. Clinical Oral Investigations. 2018; 22: 1725–1731.

[29] Yoldaş SE, Bani M, Atabek D, Bodur H. Comparison of the potential discoloration effect of Bioaggregate, Biodentine, and white mineral trioxide aggregate on bovine teeth: in vitro research. Journal of Endodontics. 2016; 42: 1815–1818.