Analytical and Bioanalytical Chemistry

, Volume 410, Issue 27, pp 7113–7120 | Cite as

Imaging of growth factors on a human tooth root canal by surface-enhanced Raman spectroscopy

  • Václav Ranc
  • Radovan Žižka
  • Zuzana Chaloupková
  • Juraj Ševčík
  • Radek Zbořil
Research Paper


Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results.

Graphical abstract


SERS Nanocomposites Growth factors Imaging 


Author contributions

The manuscript was written through contributions of all the authors.

Funding information

The authors received financial support from project NPU LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic; project IGA_PrF_2018_021, CZ.1.07/2.3.00/30.0004, and Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2015073; and project 16-02938S of the Grant Agency of the Czech Republic.

Compliance with ethical standards

The approval of the ethic committee of the Faculty Hospital Olomouc and Medical Faculty of Palacký University for the study was obtained (NV-18-08-00328).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Dhillon H, Kaushik M, Sharma R. Regenerative endodontics-creating new horizons: regenerative endodontics. J Biomed Mater Res B Appl Biomater. 2016;104:676–85. Scholar
  2. 2.
    Wigler R, Kaufman AY, Lin S, Steinbock N, Hazan-Molina H, Torneck CD. Revascularization: a treatment for permanent teeth with necrotic pulp and incomplete root development. J Endod. 2013;39:319–26. Scholar
  3. 3.
    Nosrat A, Homayounfar N, Oloomi K. Drawbacks and unfavorable outcomes of regenerative endodontic treatments of necrotic immature teeth: a literature review and report of a case. J Endod. 2012;38:1428–34. Scholar
  4. 4.
    Žižka R, Šedý J. Paradigm shift from stem cells to cell-free regenerative endodontic procedures: a critical review. Stem Cells Dev. 2017;26:147–53. Scholar
  5. 5.
    Smith AJ, Scheven BA, Takahashi Y, Ferracane JL, Shelton RM, Cooper PR. Dentine as a bioactive extracellular matrix. Arch Oral Biol. 2012;57:109–21. Scholar
  6. 6.
    Galler KM, Buchalla W, Hiller K-A, Federlin M, Eidt A, Schiefersteiner M, et al. Influence of root canal disinfectants on growth factor release from dentin. J Endod. 2015;41:363–8. Scholar
  7. 7.
    Kakaboura A. Smear layer on prepared dentin. Odontostomatol Proodos. 1989;43:211–23.PubMedGoogle Scholar
  8. 8.
    Ferracane JL, Cooper PR, Smith AJ. Dentin matrix component solubilization by solutions at pH relevant to self-etching dental adhesives. J Adhes Dent. 2013;15:407–12. Scholar
  9. 9.
    Galler KM, Widbiller M, Buchalla W, Eidt A, Hiller K-A, Hoffer PC, et al. EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells. Int Endod J. 2016;49:581–90. Scholar
  10. 10.
    Zeng Q, Nguyen S, Zhang H, Chebrolu HP, Alzebdeh D, Badi MA, et al. Release of growth factors into root canal by irrigations in regenerative endodontics. J Endod. 2016;42:1760–6. Scholar
  11. 11.
    Duncan HF, Smith AJ, Fleming GJP, Reid C, Smith G, Cooper PR. Release of bio-active dentine extracellular matrix components by histone deacetylase inhibitors (HDACi). Int Endod J. 2017;50:24–38. Scholar
  12. 12.
    Hisamoto M, Goto M, Muto M, Nio-Kobayashi J, Iwanaga T, Yokoyama A. A systematic analysis for localization of predominant growth factors and their receptors involved in murine tooth germ differentiation using in situ hybridization technique. Biomed Res. 2015;36:205–17. Scholar
  13. 13.
    Li S, Pan Y. Differential expression of transforming growth factor-beta1, connective tissue growth factor, phosphorylated-SMAD2/3 and phosphorylated-ERK1/2 during mouse tooth development. J Mol Histol. 2017;48:347–55. Scholar
  14. 14.
    Niwa T, Yamakoshi Y, Yamazaki H, Karakida T, Chiba R, Hu JC-C, et al. The dynamics of TGF-β in dental pulp, odontoblasts and dentin. Sci Rep. 2018;8:4450. Scholar
  15. 15.
    Zhao S, Sloan AJ, Murray PE, Lumley PJ, Smith AJ. Ultrastructural localisation of TGF-β exposure in dentine by chemical treatment. Histochem J. 2000;32:489–94.CrossRefGoogle Scholar
  16. 16.
    Ionita I. Diagnosis of tooth decay using polarized micro-Raman confocal spectroscopy. Rom Rep Phys. 2009;61:567–74.Google Scholar
  17. 17.
    Zavala-Alonso V, Loyola-Rodríguez JP, Terrones H, Patiño-Marín N, Martínez-Castañón GA, Anusavice K. Analysis of the molecular structure of human enamel with fluorosis using micro-Raman spectroscopy. J Oral Sci. 2012;54:93–8.CrossRefGoogle Scholar
  18. 18.
    Wang Y, Yao X. Morphological/chemical imaging of demineralized dentin layer in its natural, wet state. Dent Mater. 2010;26:433–42. Scholar
  19. 19.
    Beier BD, Quivey RG, Berger AJ. Raman microspectroscopy for species identification and mapping within bacterial biofilms. AMB Express. 2012;2:35.CrossRefGoogle Scholar
  20. 20.
    Cepeda-Pérez E, Moreno-Hernández C, López-Luke T, Monzón-Hernández D, de la Rosa E. Evaluation of bacterial presence in the root canal by Raman spectroscopy: a preliminary study. Biomed Phys Eng Express. 2016;2:065006. Scholar
  21. 21.
    Fleischmann M, Hendra PJ, McQuillan AJ. Raman spectra of pyridine adsorbed at a silver electrode. Chem Phys Lett. 1974;26:4.CrossRefGoogle Scholar
  22. 22.
    Balzerova A, Fargasova A, Markova Z, Ranc V, Zboril R. Magnetically-assisted surface enhanced Raman spectroscopy (MA-SERS) for label-free determination of human immunoglobulin G (IgG) in blood using Fe3O4@Ag nanocomposite. Anal Chem. 2014;86:11107–14. Scholar
  23. 23.
    Ranc V, Markova Z, Hajduch M, Prucek R, Kvitek L, Kaslik J, et al. Magnetically assisted surface-enhanced Raman scattering selective determination of dopamine in an artificial cerebrospinal fluid and a mouse striatum using Fe3O4/Ag nanocomposite. Anal Chem. 2014;86:2939–46. Scholar
  24. 24.
    Chaloupková Z, Balzerová A, Bařinková J, Medříková Z, Šácha P, Beneš P, et al. Label-free determination of prostate specific membrane antigen in human whole blood at nanomolar levels by magnetically assisted surface enhanced Raman spectroscopy. Anal Chim Acta. 2017; Scholar
  25. 25.
    Panacek A, Balzerova A, Prucek R, Ranc V, Vecerova R, Husickova V, et al. Preparation, characterization and antimicrobial efficiency of Ag/PDDA-diatomite nanocomposite. Colloids Surf B-Biointerfaces. 2013;110:191–8. Scholar
  26. 26.
    Marková Z, Šišková K, Filip J, Šafářová K, Prucek R, Panáček A, et al. Chitosan-based synthesis of magnetically-driven nanocomposites with biogenic magnetite core, controlled silver size, and high antimicrobial activity. Green Chem. 2012;14:2550. Scholar
  27. 27.
    Prucek R, Panacek A, Fargasova A, Ranc V, Masek V, Kvitek L, et al. Re-crystallization of silver nanoparticles in a highly concentrated NaCl environment-a new substrate for surface enhanced IR-visible Raman spectroscopy. CrystEngComm. 2011;13:2242–8. Scholar
  28. 28.
    Wang Y, Schlücker S. Rational design and synthesis of SERS labels. Analyst. 2013;138:2224–38. Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of SciencePalacký University OlomoucOlomoucCzech Republic
  2. 2.Czech Educational and Dental Research Innovative GroupBrnoCzech Republic
  3. 3.Department of Analytical Chemistry, Faculty of SciencePalacký University OlomoucOlomoucCzech Republic

Personalised recommendations