Fibrin gel scaffolds seeded with dental pulp stem cells for regenerative endodontic treatment: A pilot study

Regenerative endodontics is a strategy aimed at the regeneration of dental pulp or at least the formation of pulp-like tissue, ideally the pulp-dentin complex; regeneration of damaged coronal dentin, resorbed root, cervical or apical dentin. Regenerative endodontic strategies include stem cell transplantation and regenerative endodontic procedures (REP). REP methods are currently used in the endodontic treatment of open-apice teeth in patients aged 7–10 years. The most likely outcome of this treatment is repair (“maturation”) of the root, rather than regeneration of its tissues.
Aim of the study: to develop the method and evaluate the efficacy of dental pulp stem cells in combination with fibrin gel for regenerative endodontic treatment of mature permanent teeth in a large animal model.
Materials and methods. The study was conducted on minipigs. While under general anesthesia (xylazine and tiletamine-zolazepam), the animals underwent an X-ray examination of their oral cavities. This examination was performed to select a tooth that most closely resembled a human tooth in terms of its position in the jaw arch, as well as the number and shape of its roots and canals. The first left two-rooted molar (P2) was selected. The crown pulp was amputated, and the root pulp was extirpated. The canals were then filled with fibrin gel containing allogeneic pig dental stem cells. The gel was mixed with fibrinogen polymerization catalysts ( thrombin and calcium chloride) immediately before being injected into the canals for polymerization. Thus, the gel (3 μL, containing 15,000–18,000 cells per canal) took the shape of the canal during polymerization. Next, the gel was isolated with mineral trioxide aggregate, and the crown was filled with chemically cured, reinforced, packable glass ionomer cement. Four months after the endodontic treatment, the tooth was removed, fixed, decalcified, and used for a standard histological examination. Stepwise serial sections, 3–5 μm thick, were prepared and stained with hematoxylin and eosin. For quantitative assessment, seven sections were selected at different levels with a step of 10 μm, the images were scanned, and morphometric calculations were performed. Absolute and relative values of lengths and areas were assessed, as well as the degree of vascularization.
Results. The length, shape and volume of the root canals of the first left premolar (P2) are similar to those in humans, which allowed us to use approaches and equipment developed for the endodontic treatment of human teeth, thus increasing the relevance of the model. Histological sections in the apical part of the canal showed the formation of vascularized pulp-like stromal tissue, and in the middle and apical thirds of the root, areas with a palisade layer of cylindrical cells — active odontoblasts producing dentin — were detected next to basophilic masses. There were no inflammatory processes in the periapical area of the endodontically treated tooth. No changes in the animals’ well-being or eating behavior were observed.
Conclusion. The results of the pilot study indicate the prospects for the development of regenerative endodontics methods for the treatment of mature permanent teeth with formed roots. Scaffolds based on fibrin gel and dental pulp stem cells are generally suitable for the purposes of regenerative endodontics, but the method needs to be further improved to form pulp architectonics.

1. Huang F, Cheng L, Li J, Ren B. Nanofibrous scaffolds for regenerative endodontics treatment. Front Bioeng Biotechnol. 2022;10:1078453.

2. Lin LM, Ricucci D, Saoud TM, Sigurdsson A, Kahler B. Vital pulp therapy of mature permanent teeth with irreversible pulpitis from the perspective of pulp biology. Aust Endod J. 2020;46(1):154–166.

3. Noohi P, Abdekhodaie MJ, Nekoofar MH, Gama M, Saadatmand M, Dummer PMH. Development of Antimicrobial Peptide-loaded Hydrogels as Potential Scaffolds for Pulp-dentine Complex Regeneration: A Comparative Study. J Endod [Internet]. 2025 Feb; Available from: https://linkinghub.elsevier.com/retrieve/pii/S0099239925000548

4. Colombo JS, Jia S, D’Souza RN. Modeling Hypoxia Induced Factors to Treat Pulpal Inflammation and Drive Regeneration. J Endod [Internet]. 2020;46(9):S19–25. DOI: 10.1016/j.joen.2020.06.039

5. Jung C, Kim S, Sun T, Cho YB, Song M. Pulp-dentin regeneration: current approaches and challenges. J Tissue Eng. 2019;10: 2041731418819263 (Article ID).

6. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative Endodontics: A Review of Current Status and a Call for Action. J Endod. 2007;33(4):377–390.

7. Lin LM, Kahler B. A Review Of Regenerative Endodontics: Current Protocols And Future Directions Rejeneratif Endodonti Üzerine Bir Derleme: Güncel Protokoller Ve Geleceğe Yönelik Öneriler. J Istanbul Univ Fac Dent [Internet]. 2017;51:41–51. Available from: https://www-ncbi-nlm-nih-gov.ez.srv.meduniwien.ac.at/pmc/articles/PMC5750827/pdf/jiufd-051-s041.pdf

8. Hasija MK, Meena B, Wadhwa D, Kumar D. Pediatric Regenerative Endodontics. J Int Clin Dent Res Organ. 2021 Jan;13(1):73–76. Available from: https://journals.lww.com/10.4103/jicdro.jicdro_47_19

9. Fukushima KA, Marques MM, Tedesco TK, Carvalho GL, Gonçalves F, Caballero-Flores H, et al. Screening of hydrogel-based scaffolds for dental pulp regeneration—A systematic review. Arch Oral Biol. 2019;98(November 2018):182–194. DOI: 10.1016/j.archoralbio.2018.11.023

10. Домбровская ЮА, Енукашвили НИ, Силин АВ. Методы регенеративной биоинженерии и аддитивные технологии в стоматологии. Санкт-Петербург: Политех-Пресс; 2024. 101 c. Available from: https://e.lanbook.com/book/413090

11. Pispa J, Thesleff I. Mechanisms of ectodermal organogenesis. Dev Biol. 2003;262(2): 195–205.

12. Lobov A, Kuchur P, Khizhina A, Kotova A, Ivashkin A, Kostina D, et al. Mesenchymal cells retain the specificity of embryonal origin during osteogenic differentiation. Stem Cells. 2023 Nov 1;6:1–11. Available from: https://academic.oup.com/stmcls/advance-article/doi/10.1093/stmcls/sxad081/7336816

13. Son YB, Kang YH, Lee HJ, Jang SJ, Bharti D, Lee SL, et al. Evaluation of odonto/osteogenic differentiation potential from different regions derived dental tissue stem cells and effect of 17β-estradiol on efficiency. BMC Oral Health [Internet]. 2021;21(1):1–14. DOI: 10.1186/s12903-020-01366-2

14. Kotova A V., Lobov AA, Dombrovskaya JA, Sannikova VY, Ryumina NA, Klausen P, et al. Comparative analysis of dental pulp and periodontal stem cells: Differences in morphology, functionality, osteogenic differentiation and proteome. Biomedicines. 2021;9(11):1–26.

15. Xiao X, Xin C, Zhang Y, Yan J, Chen Z, Xu H, et al. Characterization of Odontogenic Differentiation from Human Dental Pulp Stem Cells Using TMT-Based Proteomic Analysis. Biomed Res Int. 2020; Dec 10: 3871496 (Article ID) .

16. Volponi AA, Gentleman E, Fatscher R, Pang YWY, Gentleman MM, Sharpe PT. Composition of mineral produced by dental mesenchymal stem cells. J Dent Res. 2015;94(11):1568–1574.

17. Домбровская ЮА, Енукашвили НИ, Котова АВ, Билык СС, Коваленко АН, Силин АВ. Оценка возможности создания фибриновых скаффолдов, заселенных стволовыми клетками пульпы зуба, для замещения костных дефектов челюсти. Трансляционная Медицина. 2020 Mar 11;7(1):59–69. Available from: https://transmed.almazovcentre.ru/jour/article/view/540

18. Dombrovskaya YuA, Enukashvili NI, Banashkov RE, Semenova NY, Karabak IA, Silin AV. Prospects for the use of fibrin scaffolds populated with pulp and periodontal stem cells: an experimental study. Parodontologiya. 2021;26(2):96–103.

19. Wu Q. Regenerative endodontic treatment using autologous blood from alveolar bone for mature permanent premolar with apical periodontitis: a case report. Clin Oral Investig. 2023;27(8):4869–4874. DOI: 10.1007/s00784-023-05179-9

20. Wang S, Liu Y, Fang D, Shi S. The miniature pig: A useful large animal model for dental and orofacial research. Oral Dis. 2007;13(6):530–537.

21. Акимов ДЮ, Зиятдинова АР, Снижко ЕА, Ильинская МА, Васильев АВ. Превентивные лечебные мероприятия в доклинических исследованиях ( противопаразитарная обработка). Лабораторные животные для научных исследований (Laboratory Anim Sci). 2020;4:43–55. Available from: https://elibrary.ru/doi_resolution.asp?doi=10.29296%2F2618723X-2020-04-05

22. Enukashvily NI, Aizenshtadt AA, Bagaeva V V, Supilnikova O V, Ivolgin DA, Maslennikova II, et al. Assessing the possibility to apply the fibrin glue by cord blood plasma as a scaffold for mesenchymal stem cells transplantation. Her North-Western State Med Univ named after II Mechnikov. 2017 Jun 15;9(2):35–43. Available from: https://journals.eco-vector.com/vszgmu/article/view/8557

23. Akimov DY, Makarova MN, Khan SO, Shabanov PD, Liashenko PM. Effect of propofol and combination propofol and isoflurane on vital parameters of minipigs. Bull Vet Pharmacol. 2024;4(29):8–22. Available from: https://rucont.ru/efd/912451

24. Akimov DY, Shabanov PD, Khan SO. Anesthesia in preclinical studies. Message 2: dwarf pigs. Lab Zhivotnye dlya nauchnych Issled (Laboratory Anim Sci). 2023 2023; 6(2): 53–65. Available from: https://labanimalsjournal.ru/2618723x-2023-02-05

25. Lopes LB, Neves JA, Botelho J, Machado V, Mendes JJ. Regenerative Endodontic Procedures : An Umbrella Review. Int J Environ Res Public Health. 2021;18(754):2–17.

26. Galler KM, Brandl FP, Kirchhof S, Widbiller M, Eidt A, Buchalla W, et al. Suitability of Different Natural and Synthetic Biomaterials for Dental Pulp Tissue Engineering. Tissue Eng — Part A. 2018;24(3–4):234–244.

27. Крышень КЛ, Андреев СВ, Дейкин АВ, Дмитриева АА, Енукашвили НИ, Леонова ЕИ, et al. Особенности доклинических исследований высокотехнологичных лекарственных препаратов. Консультант GLP-PLANET 2024. Мнение фармацевтической отрасли: монография. Санкт-Петербург: НПО «ДОМ ФАРМАЦИИ»; 2024. 344 с.

28. Sych LS, Reade PC. Heterochrony of tooth root initiation in rabbits. J Evol Biol. 1990; 3(3–4):283–293.

29. Aubeux D, Renard E, Pérez F, Tessier S, Geoffroy V, Gaudin A. Review of Animal Models to Study Pulp Inflammation. Front Dent Med. 2021; 2, 10.3389/fdmed.2021.673552 . hal-04064206.

30. Dombrovskaya YA, Kravets ON, Nikolaeva AV, Kotov MI, Dombrovskaya VI, Grebnev GA, et al. Changes in the morphological characteristics of the endodontic system and parameters of human dental hard tissues in the North-Western region of Siberia during the X—XX centuries. Stomatology [Internet]. 2024 Dec 20;103(6):5. Available from: https://mediasphera.ru/issues/stomatologiya/2024/6/1003917352024061005

31. Iohara K, Utsunomiya S, Kohara S, Nakashima M. Allogeneic transplantation of mobilized dental pulp stem cells with the mismatched dog leukocyte antigen type is safe and efficacious for total pulp regeneration. Stem Cell Res Ther. 2018;9(1): 116 (Article ID).

32. Nakashima M, Iohara K, Zayed M. Pulp Regeneration: Current Approaches, Challenges, and Novel Rejuvenating Strategies for an Aging Population. J Endod. 2020;46(9):S135–142. DOI: 10.1016/j.joen.2020.06.028

33. Sui B, Wu D, Xiang L, Fu Y, Kou X, Shi S. Dental Pulp Stem Cells: From Discovery to Clinical Application. J Endod. 2020;46(9):S46–55.

34. Sharpe PT. Dental mesenchymal stem cells. Development. 2016;143(13):2273–2280. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27381225

35. Lobov A, Malashicheva A. Osteogenic differentiation: a universal cell program of heterogeneous mesenchymal cells or a similar extracellular matrix mineralizing phenotype? Biol Commun. 2022;67(1):32–48.

36. Min Q, Yang L, Tian H, Tang L, Xiao Z, Shen J. Immunomodulatory Mechanism and Potential Application of Dental Pulp-Derived Stem Cells in Immune-Mediated Diseases. Int J Mol Sci. 2023;24(9): 8068 (Article ID).

37. Meza G, Urrejola D, Saint Jean N, Inostroza C, López V, Khoury M, et al. Personalized Cell Therapy for Pulpitis Using Autologous Dental Pulp Stem Cells and Leukocyte Platelet-rich Fibrin: A Case Report. J Endod. 2019;45(2):144–149.

38. Yu S, Liu XM, Liu Y, Tang L, Lei S, Geng C, et al. Inflammatory microenvironment of moderate pulpitis enhances the osteo-/odontogenic potential of dental pulp stem cells by autophagy. Int Endod J. 2024;57(10):1465–77.