Immunohistochemical visualization of human dental pulp neural development by detection of NF-H and nestin

Cristina Retana-Lobo; Tatiana Ramirez-Mora; Jessie Reyes-Carmona

doi:10.17126/joralres.2025.024

Cristina Retana-Lobo Department of Endodontics, University of Costa Rica, Montes de Oca, (San José,) Costa Rica http://orcid.org/0000-0001-9754-5047
Tatiana Ramirez-Mora Department of Endodontics, University of Costa Rica, Montes de Oca, (San José,) Costa Rica http://orcid.org/0000-0002-1837-0590
Jessie Reyes-Carmona Department of Endodontics, University of Costa Rica, Montes de Oca, (San José,) Costa Rica http://orcid.org/0000-0003-2872-6623

DOI: https://doi.org/10.17126/joralres.2025.024

Keywords: Dental pulp, Neurogenesis, Nestin, Neurofilament proteins, Nerve fibers, Stem cells

Abstract

Objective: To descriptively assess the expression of Neurofilament heavy (NFH) and Nestin in the dental pulp (DP).
Material and Methods: Fifteen human teeth were extracted and classified according to three Moorrees tooth development stages: initial root formation (Ri), root length ½ (R1/2), and root length complete (Rc). Immunohistochemical assays were performed for NFH and Nestin and analyzed under light microscopy. Images for each antibody immunoexpression in tissue sections from each stage of root development were qualitatively analyzed to evaluate the spatial distribution for each antibody.
Results: Paraffin-embedded tooth sections stained with hematoxylin and eosin showed an apical cell-rich zone between the DP and the apical papilla. NFH was expressed in the core of the dental pulp as bundles of nerve fibers and sprouts. NFH immunostaining is closely associated with arteries and capillaries in all the developmental stages samples. Neuroepithelial stem cell protein (Nestin) was highly expressed in differentiated odontoblasts in the predentin odontoblast and odontoblast cell processes, indicating a reservoir of newly differentiated odontoblast-like cells.
Conclusions: Nestin is a crucial antibody in the early stages of dental pulp tissue development, and NFH remains in the core of mature dental pulp suggesting its role in the tissue's homeostasis. Successful tissue regeneration depends directly on forming a functional vascular network and innervation. Therefore, the full comprehension of the biological features provides valuable insights into the complex challenge of regenerating dental pulp innervation.

References

Lambrichts I, Driesen RB, Dillen Y, Gervois P, Ratajczak J, Vangansewinkel T, et al. Dental Pulp Stem Cells: Their Potential in Reinnervation and Angiogenesis by Using Scaffolds. J Endod. 2017; 43(9):S12-6.

Manolea H, Vasile N, Opri M, Fronie A, Raul Popescu M. Immunohistochemical and electron microscopy aspects of the nerve structures from the dental pulp. Rom J Morphol Embryol. 2014;55(1):147-52.

Couve E, Schmachtenberg O. Schwann cell res-ponses and plasticity in different dental pulp scenarios. Front Cell Neurosci. 2018;12.

Wei Y, Lyu P, Bi R, Chen X, Yu Y, Li Z, Fan Y. Neural Regeneration in Regenerative Endodontic Tre-atment: An Overview and Current Trends. Int J Mol Sci. 2022;23(24):15492. https://doi.org/10.3 390/ijms232415492. PMID: 36555133; PMCID: PM C9779866.

Diogenes A. Trigeminal Sensory Neurons and Pulp Regeneration. J Endod. 2020; 46(9):S71-80.

Retana-Lobo C, Reyes-Carmona J. Immuno-histochemical characterization of stem cell, vascular, neural, and differentiation markers in the apical papilla and dental pulp of human teeth at various stages of root development. J Histotechnol. 2023;46(1):17-27.

Kuhle J, Plattner K, Bestwick JP, Lindberg RL, Ramagopalan SV, Norgren N, Nissim A, Malas-pina A, Leppert D, Giovannoni G, Kappos L. A comparative study of CSF neurofilament light and heavy chain protein in MS. Mult Scler. 2013;19(12):1597-603. https://doi.org/10.1177135 2458513482374. Epub 2013 Mar 25. PMID: 235 29999.

Henry MA, Luo S, Levinson SR. Unmyelinated nerve fibers in the human dental pulp express markers for myelinated fibers and show sodium channel accumulations. BMC Neurosci 2012;13(1):29.

Bernal A, Arranz L. Nestin-expressing progenitor cells: function, identity and therapeutic impli-cations. Cell Mol Life Sci. 2018;75(12):2177-95. .

Nakatomi M, Quispe-Salcedo A, Sakaguchi M, Ida-Yonemochi H, Okano H, Ohshima H. Nestin expression is differently regulated between odontoblasts and the subodontoblastic layer in mice. Histochem Cell Biol. 2018;149(4):383-91.

Moorrees C, Fanning E, Hunt E. Age variation of formation sages for ten permanent teeth. J Dent Res. 1963;42(6):1490–502.

Goldberg M. The Dental Pulp. Goldberg M, editor. Springer-Verlag Berlin Heidelberg; 2014. 240–245

Hossain MZ, Bakri MM, Yahya F, Ando H, Unno S, Kitagawa J. The role of transient receptor potential (TRP) channels in the transduction of dental pain. Int J Mol Sci. 2019;20. .

Kirkpatrick L L, Scott T B. Molecular Compo-nents of the Neuronal Cytoskeleton. In: Siegel GJ ABAR, Siegel GABAR et al., editor. Basic Neuro-chemistry: Molecular, Cellular and Medical Aspects. 6th edition. Philadelphia: Lippincott-Raven; 1999.

Park D, Xiang AP, Mao FF, Zhang L, Di CG, Liu XM, Shao Y, Ma BF, Lee JH, Ha KS, Walton N, Lahn BT. Nestin is required for the proper self-renewal of neural stem cells. Stem Cells. 2010;28(12):2162-71. https://doi.org/10.1002.ste m. 541. PMID: 20963821.

Hasegawa T, Suzuki H, Yoshie H, Ohshima H. Influence of extended operation time and of occlusal force on determination of pulpal healing pattern in replanted mouse molars. Cell Tissue Res. 2007;329(2):259-72.

Lopez-Lozano AP, Arevalo-Niño K, Gutierrez-Puente Y, Montiel-Hernandez JL, Urrutia-Baca VH, Del Angel-Mosqueda C, De la Garza-Ramos MA. SSEA-4 positive dental pulp stem cells from deciduous teeth and their induction to neural precursor cells. Head Face Med. 2022;18(1):9

Retana-Lobo C. Dental Pulp Regeneration: Insights from Biological Processes. Odovtos - Int J Dent Sci. 2017;20(1):10-6.

Huang GTJ, Garcia-Godoy F. Missing Concepts in De Novo Pulp Regeneration. J Dent Res. 2014;717–24.

Yuan A, Rao MV, Veeranna, Nixon RA. Neuro-filaments at a glance. J Cell Sci. 2012;125(14): 3257-63.https://doi.org/10.1242/jcs.104729. PMID: 22956720; PMCID: PMC3516374.

Bernal A, Arranz L. Nestin-expressing proge-nitor cells: function, identity and therapeutic implications. Cell Mol Life Sci. 2018;75(12):2177-2195.

Mamilos A, Winter L, Wiedenroth CB, Niedermair T, Zimmer S, Schmitt VH, Keller K, Topolčan O, Karlíková M, Rupp M, Brochhausen C, Cotarelo C. Nestin as a Marker Beyond Angiogenesis—Expression Pattern in Haemangiomas and Ly-mphangiomas. Biomedicines. 2025;13(3):565.